key: cord-352111-frk319q1 authors: woodruff, amelita title: covid-19 follow up testing date: 2020-05-11 journal: j infect doi: 10.1016/j.jinf.2020.05.012 sha: doc_id: 352111 cord_uid: frk319q1 • positive cases of sars-cov-2 were seen in the mayo clinic fl covid virtual clinic. • 70% of patients met cdc guidelines for release from quarantine & still tested (+); • the average time from onset of symptoms to negative testing was 19 days.  positive cases of sars-cov-2 were seen in the mayo clinic fl covid virtual clinic.  70% of patients met cdc guidelines for release from quarantine & still tested (+)  the average time from onset of symptoms to negative testing was 19 days dear editor, there is some uncertainty regarding the incubation period of the sars-cov-2 virus. there is also some uncertainly on the proportion of infected individuals who are asymptomatic carriers, and the timeframe from when a patient is infectious until becoming non-infectious. 1 we provide care for covid-19 patients in the outpatient setting through a virtual clinic. our patients have tested positive via nasopharyngeal swabs and rna detection with rt-pcr. they are followed throughout their illness with visits at intervals based on the severity of their symptoms using telemedicine technology. the cdc has two strategies to determine when a patient with covid-19 can discontinue self-isolation. one is a "test-based" strategy, and the other is a "non-test-based" strategy. the non-test-based strategy recommends that covid-19 patients can discontinue self-isolation when they have been afebrile for 72 hours without anti-pyretic medications, have improvement in respiratory symptoms, and have at least 10 days elapse since symptoms started, recently increased from 7 days. the test-based strategy requires resolution of fever without the use of anti-pyretics, improvement of respiratory symptoms, and two consecutive negative covid-19 nasopharyngeal swabs collected ≥24 hours apart. 2 we decided as part of our covid-19 virtual clinic to use the test-based strategy for all of our patients to better ensure that they were not contributing to the spread of disease. our organization manufactures the test, so we had ample testing supplies and laboratory capacity. as this disease is a reportable condition, these patients were also followed by the respective county health departments. the county health departments were using the test-based strategy only for healthcare workers, or those with essential public service jobs. as of april 17, 2020, we have enrolled 97 patients in our covid virtual clinic. of these, 72 have been tested after being afebrile for at least 72 hours, and had 7 days pass since symptoms started, along with symptom improvement. that is, 72 patients met criteria for the original release from self-isolation with the non-test-based strategy, but were tested using the test-based strategy. of these, twenty-two (30.1%) tested negative upon the first two tests, while the vast majority of patients (69.9%) tested positive at this interval. of the 69.9% who failed, thirty-six (72%) were positive on the first test, while fourteen (28%) had a negative first test but were positive on the second test. in our patient population, the average time from the onset of symptoms to negative testing is 19 days. this data shows that the cdc non-test-based strategy may cause early release from isolation for covid-19 patients and result in additional community transmission. given this, it may be beneficial to prolong the self-isolation time to greater than 14 days after symptom onset. the incubation period of coronavirus disease from publicly reported confirmed cases: estimation and application discontinuation of isolation for persons with covid-19 not in healthcare settings mh&view=epic footnote: 1. the authors do not have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership key: cord-350473-f47i7y5h authors: sen-crowe, brendon; mckenney, mark; elkbuli, adel title: covid-19 laboratory testing issues and capacities as we transition to surveillance testing and contact tracing date: 2020-05-27 journal: am j emerg med doi: 10.1016/j.ajem.2020.05.071 sha: doc_id: 350473 cord_uid: f47i7y5h nan j o u r n a l p r e -p r o o f as of may 19 th , 2020, 11,834,508 covid-19 tests have been performed in the us resulting in 1,523,534 (12.9%) confirmed cases 1 . the actual number of infected americans is much larger. antibody seroprevalence testing in santa clara county, california, estimates those infected between 2.49%-4.16% implying actual infections 50-85-fold larger than confirmed cases 2 . another study concluded that undiagnosed covid cases represent the infection source of 79% of documented cases 3 . accurate testing will be crucial to controlling and understanding this pandemic. estimation relies on testing kit accuracy (sensitivity/specificity). low sensitivity will underestimate disease prevalence, while low specificity will overestimate. 2 testing comes in two broad types, testing for nasopharyngeal viral rna and serologic testing for antibodies, which occur in response to the disease. rna testing is done with polymerase chain reaction (pcr) is cost-effective, easy to perform, and now available 4 . however, the pcr test has accuracy issues. sensitivity of fda-approved viral rna tests range from 63%-95% (table 1) [5] [6] [7] [8] . sensitivity of rna tests is dependent on the site of specimen collection. sensitivity was highest in bronchioalveolar lavage (93%), then sputum (73%), nasal swab (63%), feces (29%) and blood (1%). 5 another study found that patients with pneumonia often have negative nasopharyngeal samples, but positive lower airway samples 9 . the sensitivity of pcr tests have been estimated at 71%, resulting in ~30% of infected patients having a negative finding. another drawback is the presence of viral rna does not mean the virus is live, therefore, detection does not necessarily mean the virus can be transmitted 9 . rna-based tests are limited to the setting of acute illness. saliva-based tests offer promising results as a non-invasive and non-aerosol generating method of specimen collection 10 . compared to nasopharyngeal tests, saliva specimens have high sensitivity (84.2% 10 ) and can be self-administered. 10 one study reported greater sensitivity in saliva samples as compared to nasopharyngeal swabs and less variability. 11 reduced variability in samples taken from self-administered tests is helpful for mass testing because it preserves collection reliability and allows patients to send in their own samples from the comfort of their home. the second type of test is serologic, which detects immunoglobulins (igg and igm) specific for sars-cov-2 and provides an estimation of population virus exposure 4 . one drawback of serologic testing is the lag period between symptoms and antibody formation-one analysis found patients do not begin to seroconvert until 11-12 days post-symptom onset 12 .the sensitivity and specificity of fda-approved serologic tests ranges from 61.1%-98% and 90%-100% 13 . many fda-approved serologic tests have high sensitivity and specificity. for example, cellex inc. developed a rapid diagnostic test with 93.8% sensitivity and 95.6% specificity. bio-rad manufactured an elisa test with sensitivity and specificity of 98% and 99%, respectively (table 1) 13 . there are also clinical associations with confirmed covid-19 patients. an analysis of 119 patients with covid-19 at from wuhan university revealed an association with low urine specific gravity and increased ph 14 . in addition, the urine glucose and proteinuria correlated with severe/critical cases compared to mild/moderate 4 . the results imply that certain urinalysis profiles can be used to predict the severity of disease and possibly testing of asymptomatic patients that could be quarantined until a definitive test can be completed 14 . to address the development of a reliable test, the department of health & human services (hhs) provided funding for the development of simplexa covid-19 direct assay and to qiagen to accelerate development of their rps2 test 15 . additionally, hhs is purchasing the id now covid-19 rapid point-of-care test (abbott diagnostics scarborough inc.) for public health labs (table 1) 16 . the fda is issuing emergency use authorizations to expedite distribution 17 . states have differing amounts of laboratories authorized for testing ( figure 1 ). the targeted distribution of tests to areas of high density (figure 1-black diamonds) is paramount to ensure that resources are not undersupplied. the road back to normalcy is contingent on accurate tests, allowing suppression of spread. when a localized outbreak occurs, it will be important to have reliable testing methods to promptly contain it. random serologic testing can be used to surveil populations at high-risk for an outbreak. pcr tests can be used to assess those with active infection who may be asymptomatic. targeted distribution of tests needs to be to areas where covid is more prevalent and where people are at higher risk. in addition to distribution, the quality of the tests require improvement. many prospective tests in development report promising results in under 60 minutes, such as mammoth bioscience's crispr-based lateral flow assay (sensitivity:90%, specificity:100%) and united biomedical's kit (sensitivity:100%, specificity:100%) (table 1) . 13, 18 in the present era, technology allows diagnostics to be readily available. understanding the current disease state in communities' plays a role in the acceptance of control measures that require individual actions. now is the time to ensure systematic and coordinated efforts between the clinical, commercial and public sectors to leverage the power of testing to address the pandemic at our door. covid-19 map. johns hopkins coronavirus resource center covid-19 antibody seroprevalence diagnostic testing for severe acute respiratory syndrome-related coronavirus-2: a narrative review from mitigation to containment of the covid-19 pandemic: putting the sars-cov-2 genie back in the bottle detection of sars-cov-2 in different types of clinical specimens smart detect sars-cov-2 rrt-pcr kit. inbios covid-19 rt-digital pcr detection kit respiratory sars-cov-2 panel instructions for use (handbook) report from the american society for microbiology covid-19 international summit saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease-2019 (covid-19): a cross-sectional study saliva is more sensitive for sars-cov-2 detection in covid-19 patients than nasopharyngeal swabs the promise and peril of antibody testing for covid-19 serology-based tests for covid-19. johns hopkins -center for health security the value of urine biochemical parameters in the prediction of the severity of coronavirus disease 2020;/j/cclm.ahead-of-print hhs funds development of covid-19 diagnostic tests. u.s. department of health & human services territorial and tribal public health labs with covid-19 rapid point-of-care test covid-19) -laboratory capacity crispr-cas12-based detection of sars-cov-2 key: cord-337462-9mvk86q6 authors: nan title: humanity tested date: 2020-04-08 journal: nat biomed eng doi: 10.1038/s41551-020-0553-6 sha: doc_id: 337462 cord_uid: 9mvk86q6 the world needs mass at-home serological testing for antibodies elicited by sars-cov-2, and rapid and frequent point-of-care testing for the presence of the virus’ rna in selected populations. h ow did we end up here? two ways. gradually, then suddenly. ernest hemingway's passage is a fitting description for humanity's perception of the exponential growth of covid-19 cases and deaths (fig. 1) . the worldwide spread of a highly infectious pathogen was only a matter of time, as long warned by many epidemiologists, public health experts, and influential and prominent voices, such as bill gates. yet most of the world was unprepared for such a pandemic; in fact, most western countries (prominently the united states 1 ) fumbled their response for weeks. singapore, hong kong and taiwan have shown the world that, to contain the propagation of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), governments need to quickly implement aggressive testing (by detecting the viral rna through polymerase chain reaction (pcr)), the isolation of those infected and the tracing and quarantining of their contacts, while educating their citizens about the need for physical distancing and basic public health measures (in particular, frequent hand-washing and staying at home if feeling unwell). when outbreaks are not detected and acted upon sufficiently early, drastic physical distancing -of the sort implemented by china at the end of january and maintained for months -can eventually suppress the outbreak (fig. 1 ). it is however unclear whether western countries that have implemented strict physical-distancing measures later in their infection curve will be able to gradually release such lockdowns, let alone see their outbreaks controlled. such non-pharmacological interventions aim to 'flatten' the infection curve by reducing the number of transmission chains and thus the virus' basic reproduction number -that is, the average number of new cases generated by a case in an immunologically naive population. in the absence of a safe and effective vaccine -which, if current efforts end up being successful, is unlikely to become widely available within the next two yearsnon-pharmacological interventions will need to remain in place to reduce the threat of secondary outbreaks by maintaining the basic reproduction number below 1. however, the type and degree of the interventions could be better tailored if governments knew who are currently infected and who have been infected and recovered. for this, the world needs to see the mass deployment of serological testing for sars-cov-2 antibodies (which appear to be highly specific 2 ), and frequent testing for sars-cov-2 rna in those likely to be exposed to the virus (especially healthcare workers) or at a higher risk for severe respiratory disease (such as the elderly and younger individuals with relevant comorbidities). medical-device companies and government and research laboratories around the world have rushed to adapt and scale up nucleic acid tests (mostly employing pcr, but also crispr-based detection and loop-mediated isothermal amplification) to detect the virus' rna, and government agencies are scrambling to assess them via emergency routes (such as the emergency use authorization program 3 by the united states food and drug administration (fda)). point-of-care pcr kits -based on lateral-flow technology or cartridge-based instruments for sample preparation, nucleic acid amplification and detection -also require rna extraction from nasal or throat swabs (or both) but can speed up the time-to-result from a few hours to roughly 30 minutes 4 (and in one test, positive results can be obtained in five minutes 5 ), with nearperfect sensitivity and specificity if sample acquisition and preparation and device operation are carried out appropriately by trained personnel. this limits the usefulness of these kits for at-home use, which would significantly raise the fraction of false negatives. immunoassays incorporating monoclonal antibodies specific for sars-cov-2 antigens (for instance, a domain of the virus' spike protein) should be amenable to home use, yet they are more difficult to develop (the antibodies are typically obtained via the immunization of transgenic animals) and are less accurate than nucleic acid testing. lateral flow immunoassays (akin to the pregnancy test) and enzyme-linked immunosorbent assays to detect antibodies elicited by the virus are also being rapidly developed (mostly by chinese companies thus far). tens of at-home lateral-flow devices 6 are already being commercialized, having obtained the european union's ce mark or been authorized for emergency use by the fda or the chinese fda. in many of these kits, the recombinant viral antigens bind to sars-cov-2specific immunoglobulin m (igm) and immunoglobulin g (igg) within 15 min; hence, these tests can also detect early-stage infection (of which igm levels are a marker), but at the expense of sensitivity and accuracy (which can exceed 90% and 99% for igg 7 . the real-world performance of such serology tests, which is currently unknown, will depend on the actual prevalence of covid-19 in the population. for example, at a 5% pre-test probability of having the disease, a test with 99% sensitivity and 95% specificity would lead to as many true positives as false positives. hence, before wide deployment, governments need to ensure that these finger-prick antibody tests are clinically validated 8 . the world should roll out both antibody and nucleic acid tests on a wide scale. widely available and inexpensive serological testing would help governments to tailor non-pharmacological interventions to specific locations and populations, to decide when to relax them and to permit citizens immune to the virus to help those who remain susceptible to it. mass testing would also provide valuable data to pressing unknowns: what are the infection rates across locations and populations? what fraction of the population is immune? how long does immunity last and how does it depend on age and on the severity of infection? wider deployment of nucleic acid tests would also provide clues about the prevalence of a wider range of covid-19 symptoms, the role of children in spreading the disease, and the epidemiological characteristics of superspreaders 9 and of those who were infected and asymptomatic. testing should be complemented by privacyminded digital surveillance, via phone apps, aiding contact tracing and permitting lighter levels of physical distancing -as done in singapore, south korea and taiwan. the downside is that any invasion of privacy via the tracking of people can last longer than necessary. de-identified and aggregated health data, such as heart rate and activity levels collected via commercial wearables, might also predict (https://detectstudy.org) the emergence and location of outbreaks. in our globalized world, the risk of further waves of covid-19 outbreaks, and thus of prolonged drastic economic consequences, will remain substantial as long as any outbreak anywhere remains. it is in the world's best interest that richer countries provide test kits, technical and publichealth knowledge, personnel, personal protective equipment and, eventually, the necessary vaccine doses to poorer countries to assist them in their efforts to reduce and contain the spread of sars-cov-2. this is humanity's next test. ❐ the lost month: how a failure to test blinded the u.s. to covid-19 food & drug administration accula test: sars-cov-2 test. u.s. food & drug administration covid-19 coronavirus rapid test casette. surescreen diagnostics virus test results in minutes? scientists question accuracy today's data on the geographic distribution of covid-19 cases worldwide (european centre for disease prevention and control coronavirus disease (covid-19) -statistics and research (our world in data tracking covid-19 cases and deaths key: cord-330721-hmnrnem6 authors: chambliss, allison b; tolan, nicole v title: contingency planning in the clinical laboratory: lessons learned amidst covid-19 date: 2020-04-21 journal: j appl lab med doi: 10.1093/jalm/jfaa068 sha: doc_id: 330721 cord_uid: hmnrnem6 nan global transmission of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has faced clinical laboratories with many challenges in continuing to offer critical services. round-the-clock laboratory testing remains essential to support patient care, both those with and without 2019 coronavirus disease . this pandemic is leading to an influx of hospitalized patients, while simultaneously yielding virus exposures and selfquarantines for the laboratory workforce. thus, laboratories should prepare to operate with limited staff and may need to prioritize laboratory tests according to clinical necessity. all laboratories will recognize the need to pay particular attention to those sections involved in sars-cov-2 viral testing; upstaffing areas that receive, test or send out samples, and report/call-back results. however, the laboratory should consider various staffing models to maintain healthy workers, such as altering shift hours, or even alternating staffing groups (1) . preemptive scaling back of laboratory staff and enabling them to work from home will allow for creation of a reserve labor pool that can be engaged as staff are required to quarantine with exposure. this is only possible when laboratory testing volumes for tests not relevant to covid-19 precipitously decrease as hospitals cancel all non-emergent and elective procedures that would otherwise require maintaining higher volumes of comprehensive testing. the laboratory should begin contingency planning by assessing baseline operational status, which benches can be offered less frequently (batched as sample stability allows), which can be closed altogether, and the resultant minimum number of staff required to support emergent testing ( tests that will need to be maintained include complete blood counts, metabolic panels, routine coagulation, troponin, liver function tests, blood gases, and inflammatory markers such as c-reactive protein, lactate dehydrogenase, and procalcitonin (4, 5) . with laboratory automation, it may be best to prioritize ftes by assay bench or analyzer as prioritization of individual tests would require additional work of scrutinizing and separating orders, and sorting, storing, and re-running a large number of samples. it may be most efficient to simply allow an automation line to run the complete battery of tests ordered unless analyte-specific technical issues arise. in times of particularly critical shortages of staff and/or reagents, with proper agreement of hospital leadership and use of mass notification mechanisms, non-emergent tests could be temporarily masked from providers in the test ordering system and eliminate the laboratory from receiving them in the first place. the laboratory should also evaluate reagent and supply inventory and consider increasing supplies on-hand in preparation for higher test volumes and/or possible lapses in vendor supplies or delivery mechanisms. this will need to be considered in relation to the number of tests anticipated in both critical care and general care patient populations (https://covidprotocols.org) and the likelihood of filling covid-19 expansion beds as part of surge planning ( table 2 ). the lab should prepare for an increased number of mechanically ventilated patients. hospital leadership can provide details about the plans to expand patient care areas for covid-19 patients and the expected testing volumes. it may also be valuable to preemptively evaluate the potential benefit of increased point-of-care testing to ease the burden of samples sent to the laboratory. however, it is essential to consider the entire workflow, including interface work that may be required for new tests. as elective surgical procedures are postponed, staff across the department may be available to provide support and back-up to the essential functions of the lab, particularly on offshifts. cross-training amongst the various core laboratory areas, ideally in advance of significant absenteeism, will yield flexibility of assignments. as universities are increasingly scaling back research operations, other able-bodied personnel such as research scientists, medical students, or pathology residents may help the clinical laboratory as long as institutional policies and regulatory requirements are met. non-certified personnel may assist the laboratory with, for example, internal specimen courier services, specimen accessioning, inventory, or the assembly of covid-19 test collection kits. finally, open and continuous communication, both among the laboratory department and healthcare providers, should be maintained with regards to the status of laboratory services. electronic 'daily huddles' can help with assessing the number of staff available, the benches that will operate each day, and where additional staff can be relocated to support intradepartmental needs. daily assessment and communication can be automated via e-mail templates to inform the hospital of real-time lab staffing capacity and tests that will be unavailable or delayed. in summary, there are a number of steps the laboratory can preemptively take as part of disaster planning that involve cross-specialty collaboration within laboratory medicine and with the support of hospital leadership ( table 3) . table 1 . example contingency planning fte assignment tool. using the chemistry section as an example, a similar contingency planning tool can be used across core clinical lab specialties to assess benches/testing that can be performed depending upon available staffing. its design affords managers to use this tool daily to assign benches, considering priority of assays and specimen stability for assays that are batched. notably, increased risks of staffing concerns are seen on off-shifts (weekend days, evenings, and nights) and may be addressed by identifying staff who would volunteer to be on-call to cover these shifts as needed. a similar tool can be used to automate communication within the department and help reallocate staffing where it is needed, while also providing updates to clinical care teams. data for the chemistry section is offered as an example of information to collect, which is dependent on testing volumes, breadth of testing offered, as well other lab-specific needs. lab control/receiving, hematology, and lab management sections are provided as a place holder, with blank, shaded cells indicating additional data to be entered. a downloadable excel file of this table is available as supplemental table 1 . fte: baseline full-time equivalent (fte) staff number; ds: preemptive down-staffing to create alternating labor pools; min: minimum number of fte required to support only emergent testing; %min: minimum percentage of full staffing capacity to perform testing; float: no dedicated staff, staff from other benches to cover as able; d/c: discard and cancel; 1+: requires supervisor review and sign-off. t a b l e 3 . s t r a t e g i e s f o r c o n t i n g e n c y p l a n n i n g i n t h e c l i n i c a l l a b o r a t o r y a m i d s t t h e c o v i d -1 9 p a n d e m i c v a r y s t a f f i n g m o d e l s a l t e r s h i f t h o u r the critical role of laboratory medicine during coronavirus disease 2019 (covid-19) and other viral outbreaks world health organization. second who model list of essential in vitro diagnostics. geneva: world health organization planning for laboratory operations during a disaster clinical characteristics of coronavirus disease 2019 in china clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected. interim guidance key: cord-323476-rb9n5wc0 authors: poole, stephen; townsend, jennifer; wertheim, heiman; kidd, stephen p.; welte, tobias; schuetz, philipp; luyt, charles-edouard; beishuizen, albertus; jensen, jens-ulrik stæhr; del castillo, juan gonzález; plebani, mario; saeed, kordo title: how are rapid diagnostic tests for infectious diseases used in clinical practice: a global survey by the international society of antimicrobial chemotherapy (isac) date: 2020-09-09 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-020-04031-2 sha: doc_id: 323476 cord_uid: rb9n5wc0 novel rapid diagnostic tests (rdts) offer huge potential to optimise clinical care and improve patient outcomes. in this study, we aim to assess the current patterns of use around the world, identify issues for successful implementation and suggest best practice advice on how to introduce new tests. an electronic survey was devised by the international society of antimicrobial chemotherapy (isac) rapid diagnostics and biomarkers working group focussing on the availability, structure and impact of rdts around the world. it was circulated to isac members in december 2019. results were collated according to the un human development index (hdi). 81 responses were gathered from 31 different countries. 84% of institutions reported the availability of any test 24/7. in more developed countries, this was more for respiratory viruses, whereas in high and medium/low developed countries, it was for hiv and viral hepatitis. only 37% of those carrying out rapid tests measured the impact. there is no ‘one-size fits all’ solution to rdts: the requirements must be tailored to the healthcare setting in which they are deployed and there are many factors that should be considered prior to this. electronic supplementary material: the online version of this article (10.1007/s10096-020-04031-2) contains supplementary material, which is available to authorized users. rapid diagnostic tests (rdts) are increasingly used in clinical practice to provide actionable information for patient care in a timely manner, ideally at the time and location of the patient's interaction with health care systems. rdts (often referred to as point-of-care tests (poct) when deployed near-patient) are often simple to use and therefore can offer diagnostic support in resource-limited settings or away from more sophisticated diagnostic laboratory support, for example in primary care. the treatment of many infectious diseases is time-critical. a test that facilitates early-directed therapy increases the chance of good patient outcomes and promotes good antimicrobial stewardship. furthermore, the early identification of highly transmissible illnesses allows healthcare services in high-income countries to rapidly isolate patients and limit the spread of disease: a benefit which has been particularly highlighted with the emergence of sars-cov-2. the last decade has seen a boom in rapid diagnostic products, with many developed and approved by healthcare authorities around the world [1] for a variety of different infections including gastroenteritis [2] , bloodstream infections [3] , pneumonia [4] and respiratory viruses [5] . formats of these tests include lateral flow assays and polymerase chain reaction (pcr). there are potential pitfalls around the implementation of rdts. many are expensive, and robust evidence for tangible clinical benefit to justify this outlay can be lacking. for some, sensitivity and specificity may be lower than established laboratory tests and therefore require that these can only be applicable to specific situations (e.g. when the pre-test probability is high). governance, quality control and assurance can be challenging, particularly when rdts are not sited within a traditional laboratory setup. these challenges differ around the world depending on local health diagnostic regulations, availability of resource, local epidemiology and patient expectation. the international society of antimicrobial chemotherapy (isac)'s rapid diagnostics and biomarkers working group conducted this international survey aiming to identify and highlight some key issues related to rdts and their impacts in clinical practice and provide a number of key points to consider while adopting a rdt. a questionnaire (survey monkey®) was devised and approved by the working group (supplementary materials). the survey included 9 questions about the experience of rdts: the questionnaire was circulated to 400 isac members via isac secretaries and respondents were given 4 weeks to respond during december 2019 and january 2020 with at least two reminders. everyone surveyed was either in a position to request or deliver tests. among the questions, there was an optional question asking responders to provide their specific role and institution. the location of institutions was linked to a united nations (un) human development index (hdi) ranking (very high, high, medium or low) [6] . this is a widely used, blunt representation of a nation's development which considers life expectancy, income per capita and education. responses were received from 81 isac members representing 31 countries (fig. 1) . this represented 20% of those initially surveyed. six respondents did not disclose their nationality. 81% of those who did disclose their nationality were received from countries classified as very highly developed on the un hdi, 11% were from highly developed nations and the remaining 8% from countries classified as having medium or low levels of development. 13/81 (16%) respondents reported no available rdts. the proportion of those who have these available is reported in fig. 2 . the main barrier reported for not adopting rdts was financial (64%), and other reasons were a lack of expertise (6%) or lack of applicability to their clinical setting (6%). 4% cited a lack of interest in the tests. only 37% of those with rdts reported measuring the impacts of their tests in any way (fig. 3) . 91% of those with rdts reported the laboratory carrying out the test. 28% reported the emergency department performing them. other clinical settings rarely carried out the tests (5% in clinics and 7% inwards). the governance structure for rdts is presented in fig. 4 . the most common way for reporting was in the electronic patient file (51%); fewer institutions generate the report in real time (36%). 47% of institutions directly phone the result to the requesting clinician. one respondent reported still generating paper reports, one reporting by email, one by sms and one not generating any specific laboratory reports as the test is done in the assessment area. the survey has given us an insight into what is happening globally with rdts. many respondents reported 24/7 availability of tests. very high-income countries had higher proportional availability of rapid influenza and respiratory virus tests. in lower-income countries, however, a lower proportion of respondents reported the availability of these tests, but hiv and hepatitis testing were available in greater proportions. the explanation for this pattern is likely multifactorial. in general, the epidemiology of chronic viral hepatitis and hiv is such that they are more prevalent in developing countries where public health interventions are less likely to identify and treat patients early in the course of illness [7] . the priorities for treatment are also different: influenza other resp multiplex, other respiratory multiplex; hep, hepatitis; gi, gastrointestinal tests management in secondary care is a less pressing need in resource-restricted settings where patient isolation facilities are less readily available. furthermore, the clinical impact relative to the cost of identifying a case of influenza is less than hiv or viral hepatitis where early identification and treatment make a greater difference [8, 9] . the relative cost of each test is likely to also be a factor in the difference of availability, with multiplexed assays generally being considerably more expensive and requiring more complex logistical support. methods for reducing the costs of many rdts are lacking, which limit their availability in low-income settings. there are still major gaps in capturing the impact of rdts on decision making in a systematic manner. only 37% of users measure impact. 64% of those surveyed reported that lack of money was the major barrier to bringing in rdts in their institution. developing robust impact recording systems, such as regular audit cycles, coupled with cost-effectiveness analyses are crucial to support business cases for new rdts. the current setup of rdts appears to be more laboratory centred: governance and quality control are the responsibility of laboratories in the vast majority of those surveyed. 90% of those who responded to the survey said tests were carried out in their institution by laboratory staff. simpler tests lend themselves more towards near-patient deployment and a clia waiver is often a good indicator of this. while there are a number of existing international regulatory processes for drugs and medications, providing safeguards for their safety and efficacy, they are often lacking for rdts [10, 11] . as a result, diagnostic tests are often sold and used in the developing world without any evidence of effectiveness. for example, mak et al. [12] reported the sensitivity of an rdt for sars-cov-2 of 11.1-45.7% when the manufacturer had claimed it was 98%. the benefit of rdts can be lost if not coupled with rapid pre-and post-analytical phases. the survey identified that less than half of the results are communicated to the requester directly, and only 35% of reports are generated in real-time on computers. this means delays are introduced as clinicians look up results. interestingly in some institutions, results are sent out by sms or email to requesting clinicians which would optimise the reporting process. identification of certain infectious organisms may have wider public health implications, for example, legionella; therefore we advocate real-time connection for these results to systems that allow rapid reporting to responsible public health authorities. a limitation to the method we should consider is the selection bias towards isac members who would be motivated to respond to the survey: potentially those who have the greatest interest in rdts or who are highly critical of them. there is also a bias towards respondents with greater resources suggested by the fact that at least 90% of tests had a laboratory involvement. furthermore, the survey size is relatively small and certain world regions (especially southeast asian nations and sub-saharan african nations) are poorly represented. the main aims of rdts are to improve patient care most efficiently within well-managed healthcare systems. we therefore suggest a number of best practices for implementation of rdts (table 1) . for rdts there is no 'one-size-fits-all' model; modelling of tests and costs are wildly different for different healthcare systems. our survey highlights the availability of these tests in different resource settings, as well as the current models for governance, quality control and reporting. point-of-care testing for infectious diseases: past, present, and future multiplex molecular panels for diagnosis of gastrointestinal infection: performance, result interpretation, and cost-effectiveness a review of novel technologies and techniques associated with identification of bloodstream infection etiologies and rapid antimicrobial genotypic and quantitative phenotypic determination rapid syndromic molecular testing in pneumonia: the current landscape and future potential routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial human development index (hdi) (n.d.) human development reports hepatitis b virus burden in developing countries cost-benefit analysis of real-time influenza testing for patients in german emergency rooms the economic burden of late entry into medical care for patients with hiv infection a guide for diagnostic evaluations point-of-care tests for diagnosing infections in the developing world evaluation of rapid antigen test for detection of sars-cov-2 virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors would like to thank the isac secretariat, key: cord-295126-lz2jbmcn authors: toresdahl, brett g.; asif, irfan m. title: coronavirus disease 2019 (covid-19): considerations for the competitive athlete date: 2020-04-06 journal: sports health doi: 10.1177/1941738120918876 sha: doc_id: 295126 cord_uid: lz2jbmcn nan all major sports leagues and tournaments have been suspended or canceled due to covid-19 since early march 2020. initially, some sporting events were to be held without spectators to reduce transmission through close contact among fans. 12, 13 in the case of the national basketball association, the season was suspended soon after a player tested positive for covid-19. 16 other sporting events were forced to cancel when local and state governments restricted the sizes of gatherings. 3 on march 24, 2020, the international olympic committee announced that the olympic and paralympic games tokyo 2020 would be postponed to summer 2021. 14 while the typical athlete may only experience mild symptoms as a result of covid-19, prevention strategies are necessary for multiple reasons. first and foremost, preventing the transmission of covid-19 is needed to reduce the risk of spread to individuals within a community who are most at risk of severe infection or death, which includes older individuals and the immunocompromised. 27 prevention of covid-19 is also important for the competitive athlete to minimize interruptions in training and the adverse effects that it could have on his or her respiratory tract and aerobic capacity in both the short and long term. while the first cases of covid-19 were associated with a seafood market in wuhan, the virus has since spread person-toperson primarily via respiratory droplets. 15, 26 this mode of transmission occurs when the virus, in the form of respiratory secretions from coughing or sneezing, contacts another person's mucous membranes. according to chinese data, the rate of secondary covid-19 infections ranges from 1% to 5%. 26 transmission can also occur if a person touches his or her eyes, nose, or mouth after touching a surface containing respiratory droplets with the virus, which can remain viable for hours to days. 7 presymptomatic/asymptomatic carriers, which comprised 48% of the 531 cases on the diamond princess cruise ship, are also capable of transmitting covid-19. 2, 17, 28 currently, there is no evidence that the virus is spread through the shipment of food or other products from overseas. sports medicine providers can support athletes and teams during the covid-19 pandemic by advocating the following preventative measures: hand hygiene: general guidelines include washing hands often with soap and water for at least 20 seconds or using hand sanitizer (at least 60% alcohol) if soap and water are not available. as the virus can survive for days on surfaces, frequently touched objects and surfaces should be regularly cleaned and disinfected. 22 social distancing: the centers for disease control and prevention (cdc) describes social distancing as remaining out of congregate settings, avoiding mass gatherings, and maintaining distance (approximately 6 feet) from others when possible. 9 this practice is being advocated by governments and promoted by professional athletes as well. 4, 19 travel: to slow transmission, many countries have imposed travel restrictions. measures have ranged from suspending flights, to banning travelers from affected countries, to in-home isolation for 14 days after returning from specific destinations. countries are also performing entry screening, including measuring body temperature and assessing for signs and symptoms of covid-19. domestic travel has become challenging as busy airports can be a common site of person-to-person spread. however, as a result of the sweeping suspensions and cancelations of sports leagues and tournaments, many athletes are not needing to travel beyond returning home from where they were training or competing. face mask: asymptomatic athletes should not be advised to wear a mask to prevent becoming infected with covid-19 in the community setting or while traveling since it does not significantly reduce the risk of infection. 8 inappropriate use of masks can affect supply and demand to the point where health care workers will have inadequate protection, as we are currently seeing. prolonged and strenuous training has been suggested to be associated with temporary immune system depression lasting hours to days. 21 a conservative approach would be to advise athletes to limit training sessions to <60 minutes and to <80% of maximum ability during this time to prevent covid-19. however, this "open window" theory of infection susceptibility that follows a bout of vigorous exercise has been challenged. 5 vaccines are in the early stages of development but are unlikely to be available until early to mid-2021. the incubation period is typically within 14 days from exposure, with 95% of cases occurring within 5 days. the most common symptoms include fever (99%), fatigue (70%), dry cough (59%), and myalgias (35%). 23 some may also experience anosmia (loss of smell), dysgeusia (altered taste), a sore throat, rhinorrhea, or gastrointestinal manifestations. pneumonia is the most common serious manifestation, with bilateral infiltrates seen on chest imaging. of nearly 50,000 cases in china, 81% were mild (did not require hospitalization), 14% were severe (dyspnea, hypoxia, or >50% lung involvement on imaging within 24-48 hours), and 5% were critical (respiratory failure, shock, or organ failure). influenza and bacterial pneumonia should be considered when evaluating an athlete with fever, cough, and/or shortness of breath. testing for influenza can be done either prior to testing for covid-19 or simultaneously. a complete blood count to look for leukocytosis can help determine whether the symptoms are caused by a bacterial pneumonia. conversely, lymphopenia and leukopenia have been seen in covid-19 infections, which may assist in diagnosis. 2 during the early course of the spread of covid-19, availability of outpatient testing for the virus has lagged behind clinical needs. with these limitations, testing algorithms offered preference to patients with symptoms (fever, cough, or shortness of breath), an immunocompromised state, or close contact with someone with covid-19. as more tests are developed and approved in the united states, including those with faster turnaround times, testing criteria are expected to expand and may include testing asymptomatic individuals, as was done in south korea. 11 testing is done with a nasopharyngeal swab using an rna detection polymerase chain reaction (pcr) test. retesting may be needed in those with a negative initial test and a high probability of disease. a chest computed tomography scan can also be used to evaluate for signs of viral pneumonia as reverse transcription pcr may not detect covid-19 early in the course of the infection. 1 the management of covid-19 infection depends on the severity of symptoms. in new york city, 10% of individuals age 18-45 who tested positive for covid-19 required hospitalization. 18 however, given the limited access to testing and variable symptomatology, the total number of individuals with covid-19 may be much higher so the true risk of hospitalization among this age group is likely lower. therefore, for an otherwise healthy athlete under age 45 who becomes infected with covid-19, he or she would likely experience a self-limited flu-like illness. managing symptoms in an athlete primarily involves symptomatic management with rest and overthe-counter antipyretics. in-home isolation is recommended for athletes with confirmed or suspected covid-19 who do not show severe symptoms. other members of the household should minimize time in the same room as the affected individual, who should wear a mask when others are present. the health minister of france recently advocated for use of acetaminophen to treat fever associated with covid-19 and suggested that ibuprofen could worsen the infection. 24 this appeared to be based on a theoretical concern that the antiinflammatory effects of nonsteroidal anti-inflammatory drugs (nsaids) could adversely affect the immune system. however, the who currently does not recommend against using nsaids when clinically indicated in the treatment of a covid-19 infection. the who recommends that corticosteroids not be used in patients with covid-19 pneumonia unless there are other indications, such as the exacerbation of chronic obstructive pulmonary disease. 25 corticosteroids have been associated with an increased risk for mortality in patients with influenza and delayed viral clearance in patients with mers. there has also been good evidence for short-and long-term harm in sars patients treated with corticosteroids. 20 the following agents are being investigated as potential treatment options. it is important to note that there are currently no controlled data supporting the use of these medications and their efficacy is unknown. remdesivir: randomized clinical trials are under way assessing this investigational antiviral nucleotide analog in hospitalized adults. it has shown promise in in vitro as well as in animal studies. lopinavir-ritonavir: there have been case reports of treatment with this protease inhibitor used in hiv treatment, which has shown in vitro activity against mers and sars. however, 1 trial of nearly 200 patients with severe covid-19 infection showed no difference in time to symptom resolution or mortality when compared with standard supportive treatment. 6 hydroxychloroquine/chloroquine: studies are ongoing to investigate these 2 agents, which have shown activity against covid-19 in vitro. hydroxychloroquine may have more potent antiviral activity. published clinical data are limited, and caution should be used given potential side effects, such as qt prolongation. the cdc recommends discontinuing home isolation using either a test-based strategy or non-test-based strategy, depending on availability of testing resources. 10 if a test-based strategy is used, home isolation can be discontinued when the following criteria are all met: • no fever is present without the use of fever-reducing medications • resolution of respiratory symptoms • two consecutive negative covid-19 tests collected ≥24 hours apart when a non-test based strategy is used, the following criteria must be met: • at least 7 days have passed since the appearance of symptoms • at least 72 hours (3 days) have passed since recovery of symptoms without the use of fever-reducing medications suspending seasons and canceling competitions can cause significant grief, stress, anxiety, frustration, and sadness for an athlete. the psychological impact of covid-19 on a competitive athlete is potentiated by the removal of his or her social support network and normal training routine, which for some is a critical component of managing depression or anxiety. sports medicine providers should anticipate the need for additional mental health support for athletes, which could include ensuring regular check-ins with athletes, facilitating telehealth consultation with a sports psychologist, and encouraging maintenance of social interactions with family, friends, and teammates by phone or video chat. if an athlete on a sports team develops symptoms consistent with covid-19, teammates, coaches, and other staff who had close contact with the athlete (within 6 feet) in the preceding 14 days should begin in-home isolation. if the athlete undergoes testing, contacts can discontinue isolation if the test result is negative for covid-19. however, if the test result is positive for covid-19 (or if testing is not pursued and the athlete is treated presumptively), close contacts will need to continue their in-home isolation for 14 days from the last contact with the athlete. there will likely be requests for testing from asymptomatic teammates, coaches, and other staff. testing availability will likely dictate whether these individuals can be tested. during this time, any symptoms experienced by other athletes or staff should be reported to the team physician to determine whether they are legitimate signs of covid-19. team physicians may also consider implementing daily temperature checks. for athletes with confirmed or presumed covid-19, training can begin once symptoms completely resolve and energy levels return to normal. since in-home isolation is necessary for at least 72 hours after resolution of symptoms, low-intensity indoor training may be attempted during that time. after discontinuing in-home isolation, an athlete can gradually return to training as tolerated. for asymptomatic athletes who are isolated due to recent travel or close contact with an individual with covid-19, maintaining cardiovascular fitness may be difficult. exercise that is recommended during the in-home isolation period is dependent on the available equipment, which may include a stationary bike, treadmill, and resistance training. guidance and monitoring by a strength and conditioning coach or exercise physiologist can be provided remotely. as of march 2020, covid-19 has become a global pandemic, halting athletic competition worldwide. current focus is on the prevention of viral spread through social distancing and other common hygiene measures. sports medicine providers should know the most common symptoms of covid-19, work within their environments to learn and develop testing protocols as indicated by local resources, and minimize spread among teams. treatment in the outpatient setting is mainly supportive and includes home isolation, although several treatment drugs are under clinical investigation. correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases presumed asymptomatic carrier transmission of covid-19 washington state bans public gatherings, impacting mlb, mls and xfl games nike releases new campaign to promote social distancing amid coronavirus pandemic debunking the myth of exercise-induced immune suppression: redefining the impact of exercise on immunological health across the lifespan a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 centers for disease control and prevention. interim recommendations for us households with suspected/confirmed coronavirus disease 2019 covid-19) protect yourself centers for disease control and prevention. discontinuation of home isolation for persons with covid-19 south korea took rapid, intrusive measures against covid-19-and they worked. the guardian international olympic committee. joint statement from the international olympic committee and the tokyo 2020 organising committee genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding nba to suspend season following wednesday's games field briefing: diamond princess covid-19 cases coronavirus disease 2019 (covid-19) daily data summary coronavirus disease 2019 clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury olympic textbook of medicine in sport aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan anti-inflammatories may aggravate covid-19, france advises. the guardian clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected report of the who-china joint mission on coronavirus disease characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention a familial cluster of infection associated with the 2019 novel coronavirus indicating potential person-to-person transmission during the incubation period for article reuse guidelines, please visit sage key: cord-339122-7vvqtk84 authors: deb, chaarushena; moneer, osman; nicholson price, w title: covid-19, single-sourced diagnostic tests, and innovation policy date: 2020-07-07 journal: j law biosci doi: 10.1093/jlb/lsaa053 sha: doc_id: 339122 cord_uid: 7vvqtk84 the united states’ disastrous response to the onset of the covid-19 pandemic has arisen in large part by an utter failure to provide adequate diagnostic tests for the presence of sars-cov-2. the centers for disease control were the sole testing source authorized by the food and drug administration, and when the cdc failed to provide reliable tests in sufficient volume, it took weeks for other providers to be approved and to ramp up testing. revised policies should decrease the likelihood of sole-sourcing tests in pandemic contexts, which results in a fragile system. the pandemic sole-sourcing failure, however, not only accelerated the pandemic, but also provides lessons for innovation policy about diagnostic testing more generally. sole-sourcing hurts clinical practice by limiting confirmatory testing and systemic robustness, whether in a pandemic or in regular practice. we thus argue against relying too heavily on exclusivity-creating patents as innovation incentive for diagnostic tests—including the proposed coons-tillis patent reform bill which would increase patentability for many such tests. instead, we propose the use of reformed reimbursement to create better incentives for diagnostic test innovation. in both pandemics and elsewhere, single-sourcing creates too great a point of failure, but targeted innovation policy can help at the heart of the united states' disastrous response to the covid-19 pandemic is a failure of diagnostic testing. that something so fundamental to medical care could be so botched evokes incredulity. from primary care offices to critical care settings, every patient encounter begins with a diagnostic workup. diagnostic testing tools are key parts of the physician's toolkit, and confirmatory testing is essential. but in the greatest public health challenge of the 21st century, the failures of diagnostic testing have been laid entirely bare. the story of this failure in diagnostic testing has been told in detail and will be unpacked for years to come. briefly, the u.s. pandemic response lacked high quality diagnostics, failed to deliver tests in sufficient quantity, and was too slow in deploying developed tests when initially needed. 1 this lack of appropriate testing resulted in inaccurate patient identification and poor epidemiological characterization of viral spread. 2 from there, the errors further multiplied and rendered the rest of the governmental pandemic response similarly sluggish and ineffective. this article seeks to highlight an implication of the failure that applies broadly to diagnostic testing in general: the problems inherent in relying on a sole source for a diagnostic test. though we focus on the question of single-sourcing in this exploration, we do not claim this factor as the only failure characterizing u.s. covid-19 testing policy. 3 beyond single-sourcing, the fda stumbled through a series of missteps around its application of emergency use authorization (eua) strictures and its issuance of euas. 4 nationwide testing further suffered from the cdc's initial promotion of stringent criteria (e.g., travel history, symptom severity, etc.) to determine whether patients should even be allowed to receive tests. the constellation of these factors resulted in fewer patients being tested and rapid spread of the virus. 5 on the other end of the spectrum, the fda's antibody-testing policy was likely too lax for sars-cov-2 serology tests (designed to determine viral antibody presence in blood), leading to too many poor-quality marketed tests that could lead individuals to falsely believe they were immune to covid-19. 6 the test generated a high proportion of false positives due to initial development with faulty reagents. 10 beyond quality control issues, the diagnostic test further suffered from a slow reporting of results as clinicians had to send samples back to labs beyond the clinical practice setting for analysis. 11 these shortcomings exacerbated the pandemic's toll by preventing quick containment of the virus. crisis may have been averted had there been any alternatives to the cdc's test, but under the fda's restrictions, there were no other approved tests available to perform any confirmatory testing and receive second opinions. labs were left to sit powerless until other tests were at long-last approved. this de facto diagnostic test monopoly proved critical and pushed back pandemic control efforts by several weeks. 12 even after the fda permitted other labs to conduct testing, they were all required to follow the cdc's chemical formulation. 13 non-cdc labs all required the same reagents, effectively rendering the additional approvals useless because the small reagent manufacturer could not keep up with the instantaneous jump in demand. the approval of several competing tests earlier in the pandemic response that are not chemically identical, such as the roche sars-cov-2 test, 14 could have also alleviated some of the initial supply chain pressure and underscores the need to prevent monopoly testing. of course, single-sourcing does not always lead to problems; the sars-cov-2 test adopted by the world health organization has worked well in the countries that used it. 15 but single-sourcing sharply raises the possibility of problems leading to catastrophic failure down the line. the problems with single-sourcing, though, are not limited to pandemics. diagnostic tests are crucial to quotidian clinical practice. and within that practice, the abilities to access high-quality diagnostics rapidly and to perform confirmatory testing are crucial. single-sourcing diagnostic tests jeopardizes the ability to confirm test results, but also may impact innovative efforts to develop new diagnostics based on old tools, supply robustness, and cost-effective development. 16 an appropriate model for diagnostic testing development, including innovation incentives, should go beyond simply discouraging test monopolies and promoting confirmatory testing, by also allowing for low r&d costs for test development and yielding fast delivery of high quality tests-preventing the errors highlighted in the covid-19 testing response from occurring in both emergency and non-emergency situations. unfortunately, policy efforts ongoing since before the pandemic are pointing exactly the wrong direction. after an eight-year stretch of unpatentability for certain diagnostics, efforts are afoot to change the law: recent legislative action would allow such tests to be patented again, 17 raising concerns about limitations on initial use and confirmatory testing. 18 in this paper, we first consider how to address the problems with single-sourcing diagnostic tests in emergency medical contexts like the ongoing covid-19 pandemic. we then turn to applying those lessons for standard diagnostic test development, in particular considering how we can create adequate incentives for development without relying on a problematic single-source or quasimonopoly model. the key diagnostic testing roadblock during the covid-19 pandemic response in the u.s. appears to have been the fda's decision to permit only the cdc to offer diagnostic testing for sars-cov-2. without approval to test, none of the many other potential testing providers could offer confirmatory testing when the cdc's test offered ambiguous results, provide testing to make up for the cdc's breathtaking shortfall in testing volume, or offer innovative advances on the basic test to improve the substantial time required to get results back from the cdc. 19 though the fda ultimately did relax its regulatory structure in approving diagnostic tests, 20 the policy-driven delay had profound consequences. accordingly, the most straightforward intervention would involve updates to the fda's emergency use authorization process. the 2013 pandemic and all-hazards preparedness reauthorization act (pahpra) passed under the obama administration contemplated a pandemic, but focused primarily on streamlining the administrative process necessary for the fda to start issuing "emergency use authorizations" (eua) of unapproved medical countermeasures (mcm) and expanding the emergency uses of already fda-approved mcms. 21 we suggest updating the eua model so that safety and efficacy of novel diagnostic tests can be established as quickly as possible. thus, in order to facilitate early deployment of multiple tests in a pandemic scenario, we suggest considering whether the eua model be updated to: (1) streamline the eua application process, perhaps through an alternative, temporary route administered through cms, a regulatory body that clia-approved labs are already familiar with, (2) make the regulatory regime more flexible, such that a more lenient structure can be quickly put in place to combat the early stages of an epidemic, and (3) initially institute a limited liability model that would incentivize labs, like those at stanford and the university of washington, to begin using their test if they have good scientific data backing their safety and efficacy, especially if eua processing delays are expected. an absence of any regulatory oversight brings its own problems-witness antibody testing-but overly tight entry rules can also be disastrous. fda eventually adopted a more lenient policy incorporating some of these points, but the delay was costly. perhaps most significantly, therefore, we think it important for fda to consider the potential danger of single-sourcing when shaping its early pandemic responses to avoid a situation like that faced in early 2020. we recognize these changes are not a panaceasome labs lacked clia approval, creating a parallel barrier to testing-but think they deserve careful consideration. the failure of diagnostic testing during the u.s. response to the covid-19 pandemic provides a warning and a lesson for policy surrounding diagnostic tests more generally. good policy for diagnostic testing in non-emergent times requires both protecting clinician access to diagnostic testing and creating appropriate incentives to support diagnostic test development. an incentive model for diagnostic tests should thus focus on three main aims: (i) promote the typical use case for tests, including allowing physicians to obtain second opinions, (ii) minimize the cost of research and development, and (iii) ensure rapid deployment of safe and effective tests. the third and especially the first of these aims are hindered by single-sourcing. and thus any sort of incentive model that relies on granting market exclusivity -such as the traditional patent system, fda approval exclusivity, or trade secret protection -raises the same sorts of issues, if on a smaller scale, as those grimly demonstrated by the failure of u.s. sars-cov-2 testing at the onset of the covid-19 pandemic, which ultimately resulted in the loss of many lives. 30 by preventing access to second opinions or incentivizing fast, but ultimately ineffective science, we risk putting patients in harm's way. 31 a better incentive system would allow for an increased number of players in the market, all vying to be the best diagnostic for each indication. such a system would promote diagnostic creation and implementation that mirrors actual diagnostic usage. we first summarize (a) a recent history of diagnostic test patentability, since patents have both created incentives but hampered access and clinical practice; (b) describe current efforts to increase diagnostic test patents; and then (c) propose improved reimbursement mechanisms as a solution to the balancing act of managing appropriate economic incentives and allowing for optimal clinical practice during non-emergent conditions. diagnostic testing patentability has once again entered public debate, this time in congress rather than the courts. in the summer of 2019, senators thom tillis (r-n.c.) and chris coons (d-del.) proposed draft legislation to modify existing patent law, rendering many diagnostic tests patentable. 35 the proposal no longer considers "abstract ideas," "laws of nature," or "natural phenomena" to be exceptions to patent eligibility, explicitly overturning the three supreme court decisions. 36 under the proposal, courts would no longer be able to invalidate patents because they cover underlying biological relationships-precisely the stuff of diagnostic tests. 37 the desire to incentivize diagnostic test development through exclusivity rights granted by patents drives the biotech industry's support of the bill. 38 while single gene patents are unlikely to reemerge due to the critical patent requirement of novelty-the human genome has been sequenced many times over, so genes are unlikely to be patentably "new"-patenting in other areas of molecular diagnostics (e.g., polygenic risk scoring or autoantibody detection) remains a concern. 39 32 robert although the pandemic has sidelined unrelated legislative efforts, there remains interest in revising patent law. if the tillis-coons bill passes, we may see a return to the pre-mayo era in which certain diagnostic tests existed as patent-protected monopolies, making it hard to verify quality or to obtain confirmatory tests. 40 outside the pandemic's recent horrifying exemplar, confirmatory tests have been stymied by patent-based single-sourcing in the past. for instance, until 2009 pgxhealth was the sole provider of genetic testing services for long qt syndrome (lqts). 41 independent assessment by clinicians suggested discrepancies in test accuracy and quality. 42 a competing diagnostic test could have allowed clinicians to discover these shortcomings and further validate false negatives. 43 good clinical practice also requires the availability of multiple diagnostic test options, including ideally by alternate test providers, as physicians frequently rely on second opinions to choose the best care for their patients. previous studies exploring the practice of second opinions in diagnostics have suggested that the ability to verify the results of an initial diagnosis may lead to a change in diagnosis or treatment. 44 before the supreme court intervened, myriad's exclusivity for brca1/2 genetic variant testing prevented precisely this ability. 45 the tillis-coons bill relies on patents to provide an incentive in the form of market exclusivity, but exclusivity is especially problematic for diagnostic tests. (there are other problems with relying on patents to drive biomedical innovation, especially in relation to equity, but we do not focus on those challenges here). 46 though patent holders may not always exercise their monopoly rights, the danger lies in their potential to do so. instead, we should create incentives for diagnostic test development by leveraging non-patent policy levers. a more effective incentive system would allow an increased number of players in the market, all vying to be the most accurate and cost-effective test for each indication. such a system would promote diagnostic creation and implementation that mirrors actual physician usage. increasing reimbursement rates to reflect the expected value of diagnostic tests could help. public and private insurance providers use the current procedural terminology (cpt) system to determine the level of reimbursement warranted by new diagnostics. 47 the cpt system, in which diagnostics are often treated as commodities, is based on cost and procedure instead of the diagnostic's value. 48 to incentivize diagnostic development without creating monopolies, health insurance reimbursement strategies should be updated to provide a monetary reward for the potentially substantial cost-savings and health improvements of diagnostics. for administrative simplicity, the cpt shoehorns new tests into established reimbursement categories ("codes") that have established prices, generally evaluating new diagnostics only in comparison to existing diagnostics. 49 new tests found analogous to old tests may be "cross-walked" to the old test's price, even if the new test is far more effective-and provides much more value. 50 in december of 2019, 70% of new diagnostics were cross-walked. 51 completely novel tests with no adequate comparison may require new pricing determinations that can take years to implement. 52 thus, new diagnostics face relatively cheap prices that may not offset research and development costs, decreasing incentives to create new tests. in an attempt to promote value-based pricing, some diagnostic test manufacturers have opted for a "miscellaneous" cpt code. 53 such coding requires an assignment of value from each payer, which effectively bypasses traditional cost-based pricing but represents a logistical nightmare that dissuades most manufacturers. effective diagnostic tests, however, can shift healthcare spending from therapeutic to preventative care, promote precision medicines responding specifically to a patient's disease state, lower physician trial and error, and reduce hospital stays 54 -all of which reduce health-care system costs and improve patient care. shifting to a reimbursement model that recognizes these substantial savings could properly incentivize diagnostic tests' true value. cms has recently acquired a new set of tools that may facilitate a leadership role in reimbursement model changes. the clinical laboratory fee schedule underwent a major overhaul as part of the protecting access to medicare act of 2014 (pama). though the system had not been updated in three decades, many cumbersome but necessary changes, such as a national fee schedule and private market data collection, were implemented in order to save medicare nearly $4 billion over 10 years. 55 while there are likely to be bureaucratic challenges to implementing value-based reimbursement for diagnostics via cms, some of the provisions of the new pama fee schedule, such as the shift to national pricing, could ease simpler regulatory updates rather than a complete overhaul of the fee schedule and thus make change easier. to be sure, some diagnostics will recommend more expensive care; getting the incentives right across the board will be complicated, but reimbursement is a better and more nuanced tool than bringing back broad patent exclusivity. 56 we recognize reimbursement will not create a complete incentive regime, and may need supplementing with grants or prizes. 57 and in some instances, patents will still be present, especially for more technically complex diagnostics. 58 but as a general level, reimbursement policy reform shows substantial potential for creating incentives without relying on single-sourcing. sars-cov-2 did not create new problems in biomedical innovation; instead, it dramatically exposed underlying issues, including in the development and deployment of diagnostic tests. in particular, single-sourcing, whether through regulatory restrictions or patent quasi-monopolies, may seem like an attractive way to centralize control and to create incentives. but for diagnostic tests which rely on confirmatory testing, innovative improvements, and robust access to supply, single-sourcing creates a host of serious problems. creating the right incentives demands careful attention to these problems, and ideally structuring a regime that avoids them. reimbursement, especially structured through public involvement, provides a promising option for such a regime. both to avoid future pandemic-related disasters and to improve the normal practice of medicine, it is worth rethinking how we drive and shape the development of diagnostic tests. 2019 icd-10-pcs conversion table (zip)" hyperlink; then select "icd10pcs_conversion_table.xlsx" file) (last visited apr office of the inspector general health and human services, hhs oig data brief medicare payments for clincial diagnostic laboratory test in 2015: year 2 of baseline data rev. 1115rev. (2015. 58 we acknowledge the existence of the rare cases in which early patent protection may be necessary to secure enough resources for development, most famously theorized by edmund w. kitch in the nature and function of the patent system, 20 j.l. & econ 265, (1977) (discussing the "prospect theory" of patents). the judicious use of grants and prizes may minimize the number of such cases. key: cord-325956-1kxxg0s9 authors: potluri, rahul; lavu, deepthi title: making sense of the global coronavirus data: the role of testing rates in understanding the pandemic and our exit strategy date: 2020-04-11 journal: nan doi: 10.1101/2020.04.06.20054239 sha: doc_id: 325956 cord_uid: 1kxxg0s9 the coronavirus disease 2019(covid-19) outbreak has caused havoc across the world. subsequently, research on covid-19 has focused on number of cases and deaths and predicted projections have focused on these parameters. we propose that the number of tests performed is a very important denominator in understanding the covid-19 data. we analysed the number of diagnostic tests performed in proportion to the number of cases and subsequently deaths across different countries and projected pandemic outcomes. we obtained real time covid-19 data from the reference website worldometer at 0900 bst on saturday 4th april, 2020 and collated the information obtained on the top 50 countries with the highest number of covid 19 cases. we analysed this data according to the number of tests performed as the main denominator. country wise population level pandemic projections were extrapolated utilising three models 1) inherent case per test and death per test rates at the time of obtaining the data (4/4/2020 0900 bst) for each country; 2) rates adjusted according to the countries who conducted at least 100000 tests and 3) rates adjusted according to south korea. we showed that testing rates impact on the number of cases and deaths and ultimately on future projections for the pandemic across different countries. we found that countries with the highest testing rates per population have the lowest death rates and give us an early indication of an eventual covid-19 mortality rate. it is only by continued testing on a large scale that will enable us to know if the increasing number of patients who are seriously unwell in hospitals across the world are the tip of the iceberg or not. accordingly, obtaining this information through a rapid increase in testing globally is the only way which will enable us to exit the covid-19 pandemic and reduce economic and social instability. the coronavirus disease 2019(covid-19) outbreak has caused havoc across the world after it was first reported in wuhan, china 1,2 . subsequently, research on covid-19 has exploded to understand the new disease and its impact on mankind [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . however, the number of baseless articles resulting in fake news articles has also gone up exponentially [17] [18] [19] . a number of models have been adapted by policymakers to predict the course of covid-19 across the world 4, 6, 13, 20 . the reason for such models is to ensure that healthcare systems can plan services to help them cope with the demands of this new disease which is resulting in serious cases leading to hospitalisation 3, 8 . core elements of the prediction models have been the number of cases and deaths reported and these studies extrapolated the numbers forward to the population over time 4, 6, 13, 20 . given the pandemic course of covid-19, it has become common practice to compare its spread in different countries using case fatality rates 3, 4, 7, 13 . however, such methods only tell us part of the story. vast differences amongst countries in their testing policies for varied reasons including availability of testing equipment, infrastructure, resources and local governing policies affect case fatality rates. in addition, comparing case fatality rates between countries which are at different stages of the epidemic in their region would be erroneous as rates at the beginning and end would be lower compared to rates at the peak when healthcare services are stretched to their limits. therefore, the search for a common yardstick or denominator is necessary to compare different countries so that the data can be extrapolated for global comparison. over the past four weeks, as covid-19 spread further around the world, testing rates have picked up in most countries. we propose that analysis of the number of diagnostic tests performed in proportion to the number of cases and subsequently deaths in the underlying populations of different countries is the best way to predict what might happen next. we analysed this from the acalm big data research unit. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint we obtained real time covid-19 data from the reference website worldometer at 0900 bst on saturday 4 th april, 2020 and collated the information obtained on the top 50 countries with the highest number of covid-19 cases 21 . from this source, we obtained many parameters including the number of country wise covid-19 cases, deaths, tests performed, cases per million population, deaths per million population and tests per million population. china and saudi arabia were excluded due to lack of data on number of diagnostic tests performed, therefore numbers 51 and 52 were included in the compiled top 50 list. we obtained case fatality rates by dividing the number of deaths by the number of cases represented as a percentage. next, tests per positive case were calculated by dividing the number of tests by the number of cases. we then calculated the number of cases per test and number of deaths per test by dividing the number of cases and deaths respectively, by the number of tests represented as a percentage (a case per test rate and a death per test rate). subsequently, we obtained the population of these countries (in millions) from the number of cases divided by the number of cases per million. we can obviously obtain more accurate country population statistics from other sources but to maintain our consistency of the data source and methodology (for all countries), we derived the information from this data only. we then analysed the above in three steps. firstly, we extrapolated the population level pandemic data for each country in terms of cases and number of deaths according to each country's case per test rate and death per test rate as calculated as a snapshot at the time of obtaining the data. there are a number of limitations to the methodology used when taking a snapshot of these countries at a point in time, as done above, especially because each country is likely to be on a different part of the pandemic curve and extrapolating to the population level data is not likely to be . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint accurate. therefore, we further undertook a consistent adjustment according to countries which performed the most tests; in favour of larger countries with bigger populations we chose an arbitrary cut off of 100,000 tests per country. 15 countries had undertaken more than 100,000 tests and as all these countries showed differences in their cases/test and deaths/test we took the 15 country group as a whole to obtain an adjustment factor according to the cases/test and deaths/test. using this we derived a case per test rate of 13.53% and a death per test rate of 0.77% for the 15 country group. based on the above numbers, we extrapolated figures at the population level for all 50 countries to calculate the predicated number of cases and deaths. we felt it necessary to undertake further analysis, the third analysis, to adjust the data to a country which is progressing towards the latter half of the pandemic curve -south korea [22] [23] [24] . ideally, undertaking this adjustment with data from china would be most appropriate but data for the number of diagnostic tests performed in china was not available. the adjustment factor for south korea was a case per test rate of 2.23% and 0.04% death per test rate. hence our country wise population level pandemic projections were based on 1) inherent case per test and death per test rates at the time of obtaining the data (4/4/2020 0900 bst) for each country 2) rates adjusted according to the countries who conducted at least 100000 tests and 3) rates adjusted according to south korea. our analyses are shown in the tables and figures. no additional analyses were performed. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint full data obtained on 4/4/2020 are shown in table 1 for the top 50 countries with highest number of covid-19 cases in the world. table 2 shows the countries according to number of tests performed per positive diagnosed covid-19 cases. table 3 shows population level pandemic projections for cases and deaths according to each individual country's case per test and death per test rate on 4/4/2020 0900 bst. table 4 shows population level pandemic projections adjusted for the combined case per test and death per test rate of the 15 countries group that have performed at least 100000 tests. table 5 shows the population level pandemic projections adjusted for the case per test and death per test rate of south korea. figure 1 show a scatter plot to show the relationship between the case fatality rate and the testing rate as a percentage of the total population of the country for the countries which have tested at least 1% of their total population. italy was excluded from this scatter plot as it was an outlier with a case fatality rate of 12.25%. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint discussion covid-19 statistics are complex and comparing different countries based on number of total cases, deaths and/or case fatality rate does not show the complete picture (table 1) . a common denominator is required to make senses of these numbers and we propose that this denominator is the number of diagnostic tests performed. in our analyses we showed the deaths and cases in relation to the number of tests performed and presented population level pandemic projections based on these. this is particularly relevant in the current environment where testing parameters vary across different countries leading to non-uniformity in projections. it is important to discuss each of our different analyses in turn, the rationale, drawbacks and what it means for different countries. as table 2 shows, the number of tests per positive case is an important parameter because it is an indication of how widely the testing policy of the respective country has followed the advice from the world health organisation (who) 1 high as the raw data obtain from this source but for consistency in dealing with all the raw data in the same way, we analysed according to the data obtained from worldometer. of course similar bias could be inherent for the testing data for all countries but in our defence we have treated all the raw data obtained in the same way for consistency and have opened up the data for scrutiny. another important factor to consider here is the testing policy followed in these countries. are the countries at the top of this table testing a cohort of people who have a low possibility of carrying this . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint infection? if only those with symptoms are tested then individuals are more likely to test positive for covid-19 leading to a low test per positive number. if these countries test the sickest of patients as you would expect in countries with the largest populations and limited testing kits to do, then high testing rates per positive case is even more remarkable as it may suggest lower virus rates compared to other countries but this cannot be concluded from this study. furthermore, the reliability of local testing kits is an important factor as there are a number of reports of covid-19 patients testing negative numerous times before a positive test 11 . south korea can be considered as an exception to this because following an explosion of cases initially, they embarked on an extensive testing policy along with isolation policies combined with the utility of mobile tech and applications to inform the public about real time locations of positive cases. as such, it is widely accepted that south korea are further along the pandemic curve and the rates of new cases and deaths have significantly reduced [22] [23] [24] . projections for the pandemic on an individual population level are very important for governments to plan and organise healthcare systems in response. covid-19 presents a unique problem because there is no immunity for this in the community, nor a vaccination or targeted medical treatment. given that this is a highly contagious disease that spreads very quickly, if a large part of the population suffer from the disease in a short space of time, even if majority of cases are mild, a small minority of severe/critical cases will still lead to significant pressures on healthcare systems as now seen in italy, spain and the usa (particularly new york). globally lockdowns have been instated to reduce the spread of infection, allow the healthcare systems to cope with the condition and "flatten the curve" of the pandemic. these were not enforced all at once and the projections in table 3 are based on the data available on 4/4/2020 and a snapshot depending on the actions, policies of individual countries. much more complex models have been undertaken by different groups which included time as a variable 20 . however, we propose that testing rate is a very important parameter in projecting the outcomes of the pandemic. therefore, whilst it seems far-fetched to suggest that indonesia may end up with over 7 million deaths from less than 2000 cases reported so far, we have . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint to note that only just over 7000 tests have been performed for a country of 283 million. there are a number of factors for low testing rates such as local policies, lack of resources and equipment and it is impossible to discuss them all; we point out that testing rates are extremely important in projecting the outcomes of the pandemic particularly in countries with large populations. in this context, if we look at countries such as the uk and india both of which have tested over 100000 tests, given the large populations and their case per test rate and death per test rate on 4/4/2020, both countries have projections for over 1000000 deaths. as mentioned the position of any given country on the pandemic curve is important in determining population level projections and since we proposed that testing rates have an impact on projections we adjusted all projections to the combined case per test and death per test rates of all the countries that have performed over 100000 tests. this analyses is shown in table 4. we also felt that projections should be done on the case per test and death per test rate for south korea given the countries position on the pandemic curve and are shown in table 4 [22] [23] [24] . both these analyses are biased in terms of predications for total deaths for countries with larger populations. for example in spite of the case per test and deaths per test rate being low in india, as the population of the country is large, the projections are still over 10 millions deaths as per table 4 (adjustment according to countries which have performed more than 100,000 tests) and 500,000 deaths as per table 5 (south korea adjustment). it is also no coincidence that none of the top 10 countries in table 4 or table 5 have tested at least 1% of the total population. we looked at the countries that have tested at least 1% of their populations and looked at their cases fatality rate. we excluded the 10 th country on the list -italy because of its high case fatality rate of 12.25%. all other countries had a case fatality rate of 3% or lower. we then correlated the case fatality rate with percentage of the population tested as shown in figure 1. this approach showed higher percentage of population tested in countries with lower populations who have tested a higher proportion of their total population, but not in all cases. both . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint germany (population 83 million) and to a lesser extent australia (population 25million) and have tested more than 1% of their population and showed low case fatality rates (1.4% germany and 0.54% australia). provided that their testing criteria is reliable, these figures may serve as early indicators of the actual mortality rate for covid-19 and these low figures are encouraging. none of these methods used for projections are likely to hold true in reality. if we go back to the analyses for south korea and its projections of approximately 20,000 deaths, there have been only 177 deaths in south korea so far. it seems highly improbable that for a country where the number of cases and deaths have significantly tailed off would end up with 19,952 deaths. furthermore, the herd immunity concept has been a strategy to contain disease spread not only for covid-19 but across a number of pandemics such as swine flu 26 . although it is widely debated as to what percentage of the population would need to be affected by the disease to confer herd immunity, a figure of 60% has been widely used [27] [28] [29] . even if we adjust the south korea figures (table 5) to 60%, we will probably still over estimate the number of deaths. where does all of this leave us and what is the point of all these statistics and analyses? clearly from the example of south korea we can contain covid-19 and in spite of differences of the specific policies of lockdown between countries, social distancing and limiting spread are the broad themes to take forward. the analyses in this study highlight the importance of testing as the relevant denominator for which all the covid-19 data should be related to. the testing policy is advocated strongly by the who in their covid-19 statements 1 . the suggested early indication of a low mortality rate from our analyses, coupled with the fact that covid-19 is a new disease affecting the globe in a short time, it is highly plausible that the serious cases and deaths we are seeing in the some countries may be the tip of the iceberg of a disease that has spread widely. if we look at influenza data there are millions of cases and up to half a million deaths worldwide every year due to flu and these tend to be seasonal in spite of vaccination programmes and herd immunity to some extent [27] [28] [29] [30] . in the case of covid-19 we might be experiencing the full whammy of a disease without . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint immunity, globally all at once resulting in deaths. the magnitude of these deaths in perspective to other diseases such as influenza may not be high 30 . our analyses in this study do not prove this theory but the only thing that can is continued extensive and rapid testing across the globe. this may be the only exit strategy to prevent covid-19 related economic and social breakdown. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint who. coronavirus disease 2019 (covid-19) situation report-43 clinical features of patients infected with 2019 novel coronavirus in wuhan european centre for disease prevention and control ecdc public health emergency team. rapidly increasing cumulative incidence of coronavirus disease (covid-19) in the european union/european economic area and the united kingdom retrospective analysis of the possibility of predicting the covid-19 outbreak from internet searches and social media data, china correlation of chest ct and rt-pcr testing in coronavirus disease estimating the unreported number of novel coronavirus (2019-ncov) cases in china in the first half of january 2020: a data-driven modelling analysis of the early outbreak nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint novel coronavirus infection (covid-19) in humans: a scoping review and meta-analysis opportunities and threats (swot) analysis of china's prevention and control strategy for the covid-19 epidemic coronavirus disease 2019: the harms of exaggerated information and nonevidence-based measures coronavirus disease 2019: the harms of exaggerated information and nonevidence-based measures effects of media reporting on mitigating spread of covid-19 in the early phase of the outbreak the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint estimates of the severity of coronavirus disease 2019: a model-based analysis korean society of infectious diseases; korean society of pediatric infectious diseases korean society for antimicrobial therapy; korean society for healthcare-associated infection control and prevention covid-19) outbreak in the republic of korea from estimating the reproductive number and the outbreak size of novel coronavirus disease (covid-19) using mathematical model in republic of korea. epidemiol health how is covid-19 affecting south korea? what is our current strategy? disaster med public health prep covid-19) testing: status update history and epidemiology of swine influenza in europe the vaccination coverage required to establish herd immunity against influenza viruses back to the future for influenza preimmunity-looking back at influenza virus history to infer the outcome of future infections pandemic dynamics and the breakdown of herd immunity y the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.06.20054239 doi: medrxiv preprint key: cord-303539-gimz41yb authors: goudouris, ekaterini s. title: laboratory diagnosis of covid-19() date: 2020-08-31 journal: j pediatr (rio j) doi: 10.1016/j.jped.2020.08.001 sha: doc_id: 303539 cord_uid: gimz41yb objectives: this was a non-systematic review of the literature on the laboratory diagnosis of covid-19. data sources: searches in pubmed and google scholar for articles made available in 2020, using the terms "diagnosis" or "diagnostic” or "diagnostic tests" or "tests" and "covid-19" or "sars-cov-2" in the title. summary of findings: tests for the etiological agent identify genetic material of sars-cov-2 or humoral responses to it. the gold standard for diagnosis is the identification of viral genome targets by real-time polymerase chain reaction (rt-pcr) in respiratory tract materials during the first week of symptoms. serological tests should be indicated from the second week of symptoms onwards. a wide range of different tests is available, with variable sensitivity and specificity, most of which require validation. laboratory tests such as complete blood count, c-reactive protein (crp), d-dimer, clotting tests, lactic dehydrogenase (ldh), ferritin, and procalcitonin identify risk of disease with greater severity, thromboembolic complications, myocardial damage, and/or worse prognosis. imaging tests may be useful for diagnosis, especially when there is a compatible clinical picture, and other tests presented negative results or were unavailable. conclusions: the identification of genetic material of the virus by rt-pcr is the gold standard test, but its sensitivity is not satisfactory. the diagnosis of covid-19 should be based on clinical data, epidemiological history, tests for etiological diagnosis, and tests to support the diagnosis of the disease and/or its complications. new diagnostic methods with higher sensitivity and specificity, as well as faster results, are necessary. since december 2019, humanity is once again facing a pandemic, this time caused by a betacoronavirus, the sars-cov-2. the disease caused by this infection was named coronavirus disease 2019 . 1 sars-cov-2 is a respiratory transmitting virus that causes a flu-like condition and, in some cases, severe acute respiratory syndrome (sars). 1 however, the follow-up of covid-19 patients has shown that the virus is capable of causing symptoms outside the respiratory tract, in addition to complications of an inflammatory nature in several organs, expanding the spectrum of associated clinical manifestations. 2 early and accurate diagnosis of sars-cov-2 infection is essential for prevention and pandemic containment. the heterogeneity of the clinical presentation, from asymptomatic individuals to severe cases, and the relevant diversity of non-specific clinical manifestations of covid-19, reinforce the need for complementary tests with good sensitivity and specificity. 3 the results of diagnostic tests have serious implications: return to work of a health professional, transfer to a covid-19 area of an inpatient unit, or the reverse, possible contamination of family members, among other delicate situations. as with any other infection, the gold standard for diagnosis is the identification of the infectious agent. in the case of viral infections, this identification can be made by visualizing viral particles at electron microscopy or identifying intracellular viral inclusions at light microscopy. tissue cultures are necessary for the study of in vitro virus replication. these methods require technology that is usually available only in research centers. in commercial laboratories, immunoenzymatic assays or agglutination tests are available for detection of viral antigens and nucleic acid amplification tests for detection of virus genetic material. 4, 5 an indirect way to diagnose viral infections is the identification of a specific immune system response. the humoral response, or antibody production, is the simplest way to diagnose infectious conditions. there are different techniques for identifying antibodies that are directed against different parts of viruses. 4, 5 however, it is important to note that the immune response to viral microorganisms occurs primarily by innate immunity, particularly by nk cells, and cellular immunity, especially cytotoxic t cells (tcd8+). 6 to date, pubmed features over 35,000 articles on covid-19. many of them are presented as preprint, without peer review; some of these studies were conducted with poor methodology, providing unreliable results. moreover, during the pandemic, knowledge has advanced greatly, and initially established concepts were modified, demonstrating that certain specificities of sars-cov-2 infection are not comparable with previously known viral infections. this was a non-systematic review of the literature on the laboratory diagnosis of covid-19, drawing attention to the knowledge already established, as well as the doubts that still need to be clarified. a non-systematic review of the literature was carried out in pubmed, searching for articles submitted in 2020, with the termsdiagnosisördiagnostic'' orẗestsördiagnostic testsänd ''covid-19örsars-cov-2ïn the title. since many manuscripts have been made available in preprint version, without peer review, google scholar searches have also been performed, using the same terms. this study included articles in english, portuguese, french, or spanish, using the checklists proposed by the user's guide to medical literature (jama evidence) as inclusion criteria. 7 the complementary tests used in the diagnosis of covid-19 can be divided into tests for etiological diagnosis and support tests, which help in the diagnosis or indicate the risk or presence of complications. tests for etiological diagnosis may be direct, identifying genetic material of sars-cov-2, or indirect, determining the humoral immune response to sars-cov-2. the most commonly used method for identifying genetic material from sars-cov-2 is real-time polymerase chain reaction (rt-pcr). this method involves reverse transcription of the genetic material of the virus (rna) to complementary dna (cdna), followed by amplification of some regions of the cdna. probes (dna/rna marked sequences to identify the genetic target in the material) and primers (dna/rna sequences that promote replication of the genetic material found in the sample) were created after the sars-cov-2 genome was sequenced. several serial amplification cycles are performed to identify these targets: the more cycles are needed, the lower the viral load of the material under study. 8 four regions of the sars-cov-2 genome have been targeted: rdrp gene (rna-dependent rna polymerase), genes from structural proteins e (virus envelope) and n (virus nucleocapsid), and orf1ab gene (open reading frame 1a and 1b). 3, 8 kits using different regions of the genome are commercially available. the sequential use of different probes and primers for the rdrp, e and n genes, known as the charité-berlin institute protocol, presents good sensitivity and specificity. 9 there are other proposed protocols that follow the same logic of sequential use of probes and primes for different genetic targets. 10 regardless of the method used, the sensitivity and specificity of the different rt-pcr kits are not 100%. this is considered the gold standard for diagnosis of sars-cov-2 infection, but its sensitivity is estimated to be approximately 70% and specificity, 95%. 11, 12 many factors can interfere with the results, whether related to the virus, to the method itself (the collection procedure and handling of the material), or even to the viral load of the sample (type of material collected, duration of symptoms, and disease severity). 13 mutations in the virus genome can render the probes and primers obsolete, producing false negative results. to date, sars-cov-2 has undergone mutations, but without implications for the rt-pcr detection. mismatch between primers and probes can also lead to false negative results, and ideally more than one region of the virus genome should be simultaneously or sequentially amplified. 13 factors related to the collection procedure and handling of the material are often responsible for false negative results. dacron or polyester swabs should be used and immersed immediately after collection in appropriate and refrigerated storage medium. the material should be kept under refrigeration and quickly sent to the laboratory. 10, 13 a low viral load, usually found in asymptomatic individuals or in those with mild clinical conditions, may also be responsible for a false negative result. 14, 15 individuals with more severe clinical conditions have greater elimination of viruses. 16 although it has been described that there may be elimination of viruses from two to three days before to up to six weeks after the onset of symptoms, 8 very early (before three days of symptoms) or late material collection (after the seventh day) may produce false negative results, due to lower viral load. 1, 13, 15 the type of material and the collection technique also interfere with the result. in several studies, bronchoalveolar lavage was the material with the highest positivity, followed by sputum, nasopharyngeal swabs, and nasal swabs. oropharyngeal swabs did not present good positivity. 15, 17 the identification of genetic material of the virus in feces is less common and has uncertain significance, since infecting virus was not detected in this material. 18 viral particles were not isolated in urine or blood. 18 saliva tests have also been implemented, but have lower sensitivity than the nasopharyngeal swab and require validation. 19 false positive results are most commonly related to errors in sample handling during or after swab collection, leading to inadvertent contamination. 13 tests to identify genetic material of the virus using simpler techniques, which do not require personal and sophisticated devices and that produce faster results, have been developed. 3 one example is the qualitative detection of the e and n proteins genes through the genexpert (cepheid company) platform, in which the amplification process takes place within a cartridge and provides results in 45 min. 20 point-of-care tests for sars-cov-2 proteins, most commonly using lateral flow assays, are useful for diagnosis in regions where there are no specialized laboratories. 3, 21 the presence of genetic material in respiratory tract secretions has no direct relationship with virus viability or infectivity, since inactive or dead virus particles can be identified. 8 therefore, a patient with positive rt-pcr test is not always able to infect other people. the viability of sars-cov-2 and consequent infectivity can be assessed directly, in vitro, by its ability to contaminate cells and, indirectly, through the threshold cycles (the lower the ct, the higher the viral load) or identification of sub-genomic rna (which are transcribed only by viable viruses). 18 serological tests identify the presence of humoral response to sars-cov-2. antibodies of iga, igm, and igg isotypes specific to different virus proteins are detected by enzyme-linked immunosorbent assay (elisa) or chemi-luminescence immunoassays (clia), and the latter has been shown to be more sensitive. 21 it is known that the priority immune response to the virus is related to the cytotoxic activity of nk cells and cd8 + t lymphocytes. there is evidence of robust cellular response to sars-cov-2, regardless of the results of serological tests; 22 however, tests to evaluate the specific cellular immune response for sars-cov-2 are not yet commercially available. antibodies against s protein, where the receptor-binding domain (rbd) is located, are very specific for sars-cov-2; 10 their levels presented a good correlation with the virus's neutralization capacity. 23 however, the role of antibodies directed to other proteins in the pathogenesis of covid-19, even promoting a greater penetration of the virus into cells, still need to be elucidated. 24 sensitivity and specificity of serological tests vary according to the testing technique, specificity of the antibody studied, duration of symptoms at the time of collection, and immunocompetence of the individual. 4 however, actual sensitivity and specificity values for these tests are difficult to define considering that a gold standard for diagnosis with high sensitivity is not yet available. 11 most of the tests in use were not evaluated in scientific publications. 21 the assessment of specific antibodies to n protein is more sensitive and less specific, since this protein is more abundant in coronaviruses. antibodies directed to s protein are more specific to sars-cov-2, because in this protein is rdb. 8 in addition, other factors that interfere with the results are duration of symptoms when the blood is collected and severity of the clinical picture. igm is identified from the fifth day of symptomatology, and more significantly, from the eighth day onwards. the specific iga dosage appears to be more sensitive and the values seem to increase earlier than those of igm. 21 specific igg values begin to be detectable from the tenth day of symptom onset, and more significantly, from the 14th day onwards. 21 these tests are therefore not appropriate for the early diagnosis of covid-19. they are, however, relevant when rt-pcr is not available or is negative in the face of a suggestive clinical picture, when the patient has been symptomatic for over 14 days, 8,21 or to assist in the diagnosis of covid-19-related multisystemic inflammatory syndrome. 25 some studies report patients with mild (or even asymptomatic) covid-19 present lower levels of sars-cov-2-specific antibodies or may even do not develop detectable levels, while patients with more severe conditions have higher levels of these. 26---28 these data raise questions about the protective capacity of antibodies and may suggest the participation of specific antibodies in the pathogenesis of covid-19. 14, 24 one study demonstrated that the positivity of serological tests was not accompanied by a rapid drop in virus elimination, which may indicate that the positivity of these tests does not necessarily imply prompt resolution of the disease or absence of infectivity. 18 it has recently been shown that specific igg levels suffer significant decline after two/three months. 27 considering that the immune response to the virus is primarily cellular, it is not yet known what are the implications of this reduction in the protection against the virus. regardless of the test used for diagnosis, either identification of genetic material of the virus or serologic test, the interpretation of the results is based on the accuracy of the test itself, and also on the estimated risk of the disease before the results. this risk is modified by the prevalence of covid-19 in a given region. 11 this means that tests developed in regions where the prevalence of sars-cov-2 infection is high tend to have lower sensitivity when used in regions where the prevalence is lower. a single negative test in an individual with a characteristic clinical picture should not discard the possibility of covid-19. 11 in turn, a positive rt-pcr has greater strength to confirm the diagnosis than a negative test has to discard it, since it presents high specificity, with only moderate sensitivity. 11 point-of-care tests for antibodies against sars-cov-2 using lateral flow assays (usually immunochromatography) are quite numerous and many of them have not been adequately validated. 20 moreover, they were tested in the laboratory using plasma or serum, but have been applied with whole blood, which can greatly modify their sensitivity. 21 they are not recommended to be used for the individual diagnosis of covid-19, but may be useful in implementing public policies. 29 these are laboratory or imaging tests that demonstrate characteristic manifestations of covid-19, its complications, and/or risk factors for complications. complete blood count ---lymphopenia, eosinopenia, and neutrophil/lymphocyte ratio ≥ 3.13 are related to greater severity and worse prognosis. thrombocytopenia is related to a higher risk of myocardial damage and a worse prognosis. 2 lymphopenia results from a multifactorial mechanism that includes the cytopathic effect of the virus, induction of apoptosis, il1-mediated pyroptosis, and bone marrow suppression by inflammatory cytokines. 30 high values of c-reactive protein (crp), ferritin, d-dimer, procalcitonin, lactic dehydrogenesis (dhl), prothrombin time, activated partial thromboplastin time, amyloid serum protein a, creatine kinase (ck), glutamic-pyruvic transaminase (sgpt), urea, and creatinine are risk factors for more severe disease, thromboembolic complications, myocardial damage, and/or worse prognosis. 2,30---32 immunological markers that may also represent risk factors for greater severity and/or worse prognosis are: decreased values of cd4 + t and cd8+ lymphocytes, and nk cells and increased values of il6, il-8, il-10, ifn-␥, tnf-il-2r, tnf-␣, gm-csf, and il-1 ␤. 2, 32 imaging tests imaging tests for the diagnosis of covid-19 have gained relevance, given the unavailability of tests for etiological diagnosis. [ 3] the alterations described in these tests can also be found in influenza or mycoplasma infections, in inflammatory processes of different origins, or in eosinophilic lung diseases. 33 although the findings in these tests are not specific to covid-19, given a compatible clinical picture and/or the presence of confirmed or possible history of contact, they may help in the diagnosis. plain chest x-rays are less sensitive than computed tomography, but may evidence sparse bilateral consolidations accompanied by ground glass opacities, peripheral/subpleural images, predominantly in the lower lobes. 33 computed tomography of the chest presents greater sensitivity and reveals multifocal, bilateral, peripheral/subpleural ground glass opacities, generally affecting the posterior portions of the lower lobes, with or without associated consolidations. 33, 34 children have a similar presentation to that found in adults, albeit with a milder involvement. 33 the halo sign, described as a consolidation area involved by ground glass opacities, was identified in 50% of the children. 33 an inverted halo sign, in which areas of ground glass opacities are surrounded by condensation halo, has also been described. 35 pulmonary ultrasonography has good sensitivity; the typical findings are b-lines, consolidations and pleural thickening. 36 the advantages of this method are its lower cost, absence of radiation exposure, and the fact that it does not require sedation or transportation of unstable patients. 37 most studies on diagnostic methods presented here refer to adults; however, studies specific to the pediatric age group show very similar data. 38 the data presented suggest that the diagnosis of covid-19 should be based on clinical manifestations, contact history, imaging tests, laboratory tests, and not only on serological tests and the search for the genetic material of the virus. in addition, strategies to increase sensitivity, specificity, and speed of diagnosis are fundamental. 11 the gold standard for the diagnosis of sars-cov-2 infection is the identification of viral genetic material by rt-pcr, in different samples, with greater sensitivity in bronchoalveolar lavage and nasopharyngeal swab. many factors related to the individual, the collection procedure, and the test technique interfere with the sensitivity of these tests. therefore, a negative test in a patient with a characteristic clinical picture should not discard the possibility of covid-19. the available serological tests are different from each other and many factors influence their sensitivity and specificity. not all patients who have sars-cov-2 infection will have detectable levels of antibodies, particularly if they have milder symptoms. the absence of antibodies does not imply the absence of contact or protection against the virus, since there may be an efficient specific cellular immune response. in turn, the presence of antibodies does not rule out the possibility that the individual is still infectious, as no immediate reduction in the elimination of the virus has been identified. the support laboratory and imaging tests show alterations that are characteristic of covid-19, but they lack specificity. the diagnosis of covid-19 should be based on clinical and epidemiological history, tests for etiological diagnosis, and tests to support the diagnosis of infection and/or its complications. pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (covid-19): a review immunology of covid-19: current state of the science diagnosing covid-19: the disease and tools for detection mandell, douglas and bennett's principles and practice of infectious diseases diagnosis of viral infections host defense to viruses user's guide to the medical literature ---essentials of evidence-based clinical practice interpreting diagnostic tests for sars-cov-2 detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr sars-cov-2 and the covid-19 disease: a mini review on diagnostic methods interpreting a covid-19 test result reconstructed diagnostic sensitivity and specificity of the rt-pcr test for covid-19. medrxiv. 2020, preprint real-time rt-pcr in covid-19 detection: issues affecting the results the first, holistic immunological model of covid-19: implications for prevention, diagnosis, and public health measures evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections. medrxiv. 2020, preprint viral dynamics in mild and severe cases of covid-19. the lancet infectious diseases clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan virological assessment of hospitalized patients with covid-2019 saliva as a noninvasive specimen for detection of sars-cov-2 covid-19: laboratory diagnosis for clinicians. an updating article antibody tests for identification of current and past infection with sars-cov-2 robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19. biorxiv. 2020, preprint an affordable anti-sars-cov-2 spike elisa test for early detection of igg seroconversion suited for large-scale surveillance studies in low-income countries dissecting antibody-mediated protection against sars-cov-2 multisystem inflammatory syndrome in children (mis-c) related to covid-19: a new york city experience the dynamics of humoral immune responses following sars-cov-2 infection and the potential for reinfection longitudinal evaluation and decline of antibody responses in sars-cov-2 infection. medrxiv. 2020, preprint clinical and immunological assessment of asymptomatic sars-cov-2 infections point-of-care diagnostic tests for detecting sars-cov-2 antibodies: a systematic review and meta-analysis of real-world data immune response to sars-cov-2 and mechanisms of immunopathological changes in covid-19 the role of biomarkers in diagnosis of covid-19 -a systematic review clinical and immunological features of severe and moderate coronavirus disease 2019 international expert consensus statement on chest imaging in pediatric covid-19 patient management: imaging findings, imaging study reporting and imaging study recommendations chest ct features of covid-19 in rome covid-19 pneumonia and the reversed halo sign thoracic ultrasound and sars-covid-19: a pictorial essay lung ultrasound findings in patients with coronavirus disease (covid-19) covid-19 in 7780 pediatric patients: a systematic review the author declares no conflicts of interest. key: cord-276577-06boh550 authors: schanzer, dena l.; garner, michael j.; hatchette, todd f.; langley, joanne m.; aziz, samina; tam, theresa w. s. title: estimating sensitivity of laboratory testing for influenza in canada through modelling date: 2009-08-18 journal: plos one doi: 10.1371/journal.pone.0006681 sha: doc_id: 276577 cord_uid: 06boh550 background: the weekly proportion of laboratory tests that are positive for influenza is used in public health surveillance systems to identify periods of influenza activity. we aimed to estimate the sensitivity of influenza testing in canada based on results of a national respiratory virus surveillance system. methods and findings: the weekly number of influenza-negative tests from 1999 to 2006 was modelled as a function of laboratory-confirmed positive tests for influenza, respiratory syncytial virus (rsv), adenovirus and parainfluenza viruses, seasonality, and trend using poisson regression. sensitivity was calculated as the number of influenza positive tests divided by the number of influenza positive tests plus the model-estimated number of false negative tests. the sensitivity of influenza testing was estimated to be 33% (95%ci 32–34%), varying from 30–40% depending on the season and region. conclusions: the estimated sensitivity of influenza tests reported to this national laboratory surveillance system is considerably less than reported test characteristics for most laboratory tests. a number of factors may explain this difference, including sample quality and specimen procurement issues as well as test characteristics. improved diagnosis would permit better estimation of the burden of influenza. although influenza virus infection is associated with considerable morbidity and mortality [1] [2] [3] , laboratory confirmation of clinical illness is the exception rather than the rule. clinicians do not routinely seek laboratory confirmation for several reasons: diagnosis will often not alter patient management, a paucity of real-time, accurate, inexpensive testing methods [4] and because influenza is not recognized as the etiology of the clinical presentation [5] . accurate diagnosis of influenza-like illness, however, could improve clinical care through reduced use of antibiotics and ancillary testing, and more appropriate use of antiviral therapy [6] . although rapid influenza tests such as pointof-care tests are purported to generate results in a timely fashion to influence clinical care, the performance characteristics of the currently available tests are sub-optimal [7] . new technologies with improved sensitivity such as reverse-transcriptase polymerase chain reaction (rt-pcr) [8] as well as the use of more effective collection systems such as the flocked nasopharyngeal swab compared to traditional rayon wound swabs, and the recommendation to collect more ideal specimens, such as nasopharyngeal swabs rather than throat swabs are likely to improve diagnostic sensitivity [9] [10] [11] [12] . the performance characteristics of currently available tests for influenza vary considerably and the overall sensitivities of these tests when used in routine practice are also dependent on the type of specimen collected, the age of the patient and point in their illness in which they are sampled [4, 9, [13] [14] [15] . we sought to estimate the sensitivity of influenza testing based on results of a national respiratory virus surveillance system using a model-based method [1, 2, [16] [17] [18] . weekly respiratory virus identifications from september 1999 to august 2006 were obtained from the respiratory virus detection surveillance system (rvdss), public health agency of canada [19, 20] . the rvdss collects, collates, and reports weekly data from participating laboratories on the number of tests performed and the number of specimens confirmed positive for influenza, respiratory syncytial virus (rsv), para-influenza virus (piv), and adenovirus. specimens are generally submitted to laboratories by clinicians in the course of clinical care, and by clinicians participating in one of our national influenza surveillance programs, (fluwatch [20] ). indicators of influenza activity are reported year round on a weekly basis to the fluwatch program. the rvdss is supplemented by case reports of influenza positive cases [19, 21] . from the case reports, influenza a was confirmed in all age groups and sporadic cases were confirmed in the off-season months of june through september. infants and children under the age of 5 years accounted for 25% of the influenza a positive tests, and persons over the age 65 years another 35%. unfortunately, fluwatch surveillance data does not provide the total number of tests by age. testing practices are known to be varied [22, 23] . the predominant testing methods used for influenza detection varied considerably by province or laboratory and over time. for the 2005/06 season a survey of laboratory techniques in current use indicated that culture accounted for 44% of the diagnostic tests with rt-pcr, rapid antigen tests and direct fluorescent-antibody assay (dfa) accounting for 21%, 19%, and 16% respectively [23] . the weekly number of tests negative for influenza was modelled, using poisson regression, as a function of viral identifications for influenza, rsv, adenovirus and piv as well as a baseline consisting of seasonality, trend and holiday variables. the estimated baseline implicitly accounts for influenza tests on specimens taken from patients with respiratory infections due to respiratory pathogens other than the four viruses captured in the rvdss, as long as both the testing behaviour of clinicians and respiratory illnesses caused by other respiratory pathogens follow a consistent seasonal pattern as prescribed by the model (see below, the poisson regression model with a linear link function was estimated using sas [24] proc genmod: coefficients b 5 to b 9 are multipliers. the weekly number of influenza negative tests estimated to be falsely negative is given by b 5 infla w +b 6 inflb w . the weekly number of influenza negative tests attributed to rsv is given by b 7 rsvp w. , and similarly for adenovirus and piv. for each positive influenza a test, an additional b 5 tests above baseline were performed and found to be negative. by specifying a linear link, a value of 0.33, say, for coefficient b 5 , means that for every test for which influenza a was confirmed, 0.33 additional tests, on average, were performed on truly influenza a positive specimens and found to be negativewhich corresponds to a sensitivity of 75%. sensitivity was calculated as the number of influenza positive tests divided by the number of influenza positive tests plus the model-estimated number of false negative tests, or equivalently, the estimates of sensitivity for influenza a and b are given by 1/ (1+b 5 ) and 1/(1+b 6 ) respectively. the false negative rate is 1 minus sensitivity. while the null value for b 5 is zero, which indicates no statistical association between the number of influenza positive tests and the number of influenza negative tests, the corresponding null value for sensitivity is 1. for each test confirmed positive for rsv, on average b 7 tests were performed for influenza and found to be negative for influenza. these b 7 tests are attributed to an rsv infection, however the number of influenza-negative tests that actually tested positive for rsv is unknown. if all specimens had been tested for the same viruses (panel tests), 1/b 7 would correspond to the sensitivity for rsv testing, and the sensitivity for adenovirus and piv given by 1/b 8 and 1/b 9 respectively. some laboratories are known to test for viruses sequentially [22] , and so 1/b 7 -1/b 9 were not interpreted as estimates of the sensitivity for other viruses. sequential testing may occur if a rapid test for influenza is negative and the laboratory then performs pcr or culture testing. similarly in young children with a respiratory illness in the winter, rapid tests for rsv infection may be performed first, and only specimens with negative results submitted for subsequent testing for influenza or other respiratory viruses [25] . by contrast, many laboratories conduct panel tests for multiple viruses for ease of handling, decreased patient sampling, and recognition that coinfection can occur. either form of sequential testing would not bias the estimate of sensitivity applicable to test results reported to rvdss, though significant use of rapid antigen tests in the laboratories reporting to rvdss would reduce the overall sensitivity. as a single specimen may undergo multiple tests, the false-negative rate applicable to a specimen that has undergone multiple tests would be expected to be much lower than the system average for individual tests. parameters b 1. to b 4 account for trends and the seasonality of truly negative specimens (patients presenting with other acute respiratory infections). over 50,000 tests for influenza were reported to the rvdss each year, peaking in 2004/05 at 101,000. overall 10% of the influenza tests were positive for influenza, ranging from 4% to 13% depending on the season. the proportion positive for rsv, parainfluenza and adenovirus averaged 9%, 3% and 2% respectively. as seen in figure 1 , no virus was identified in 75% of specimens submitted for testing (white area under the curve). even for the winter months of december through april, one of these 4 viruses was identified on average in no more than 30% of the specimens. the strong and consistent synchronization of negative tests with influenza positive tests, as seen in figure 1 , is suggestive that false negative results contributed to the large number of negative tests during periods of influenza activity. the sensitivity for influenza a testing averaged 33.7% (with model-estimated 95% confidence intervals of 33.3-34.1) for the 1999/2000-2005/06 period. influenza b testing had a similar estimated sensitivity at 34.7 (95% ci 33.4-36.1). estimated sensitivities varied somewhat from season to season, generally ranging from 30%-40% (table 1) , and provincial level estimates, as well, were within a similar range. stratifying by province or season produced similar estimates for the sensitivity of influenza a testing: 32% (95% ci 30-34) and 36% (95% ci 33-41) respectively. estimates of sensitivity based on test results reported to the rvdss for individual laboratories with sufficient data to fit the model showed significant variation, with estimates of sensitivity ranging from 25-65%. as expected, laboratories using primarily rapid antigen tests had lower estimated sensitivities, and laboratories that used pcr methods had higher sensitivity estimates. however, information on testing procedures is limited primarily to the 2005/06 survey. as well, additional irregularities were noticed in the laboratory data and not all laboratories provided sufficient data to fit the model. figure 2 illustrates a good model fit where the weekly number of influenza negative tests is well explained by the model covariates, with a few exceptions. firstly, it is evident that additional specimens were tested during the sars period, as indicated by the period where the number of weekly influenza negative tests exceeded the expected number, or equivalently, a period of successive positive residuals. residuals typically capture random variation; hence represent tests that can not be allocated based on the specified model. in addition to the sars period, testing appears to have been elevated for a number of weeks in january 2000 during the peak of the 1999/2000 a/ sydney/05/97 (h3n2) season in which respiratory admissions were unusually elevated [26, 27] , and in december 2003, when an elevated risk of paediatric deaths associated with the a/fujian/411/02 (h3n2) strain [28] was identified in the us. as these periods corresponded to a period of heightened public awareness due to severe influenza outbreaks, parameter estimation was repeated without these data points. exclusion of these data points did not alter the sensitivity estimate for influenza. the attribution of influenza negative test results to influenza and other viruses is illustrated in figure 3 . the baseline curve is the model estimate of the number of tests that were likely truly negative for all four viruses tested. a reduction in specimen collection and testing, primarily for viruses other than influenza, is also evident over the christmas period ( figure 3) . the weekly proportion of tests confirmed positive for influenza peaked each season at 15 to 30%. accounting for the model estimated false negative rate suggests that during periods of peak influenza activity, 40-90% of tests were performed on specimens taken from persons recently infected with influenza. influenza was confirmed in only 14% of specimens sent for testing over the winter period, whereas the sensitivity estimate would imply that up to 40% of influenza tests could be attributed to an influenza infection. the corresponding figures for the whole year indicate that 10% of specimens were confirmed positive for influenza and 30% of influenza tests could be model-attributed to an influenza infection annually. despite a relatively large number of tests in the off-season, the number of influenza positive tests was almost negligible; suggesting that the false positive rate applicable to rvdss influenza testing is minimal. the model estimated sensitivity based on influenza test results reported to the rvdss of 30-40% is much lower than the standard assay sensitivities documented in the literature. standard sensitivities for diagnostic procedures used by participating laboratories ranged from 64% for rapid antigen tests to 95% for rt-pcr tests, averaging 75% for the study period [23] . as performance characteristics of specific tests are generally based on high quality specimens, the difference of approximately 40% is likely linked to any one of many operational procedures that affects the quality of the specimen and its procurement. unlike validation studies, our samples are taken from a variety of clinical settings and processed with a variety of procedures across the country. as well, variation in the indications for diagnostic testing may vary across the country. as there are many other respiratory pathogens that are not routinely tested for, or reported to the rvdss, including human metapneumovirus (hmpv), coronaviruses, and rhinoviruses for which patients may seek medical care and present with influenza like illness [29] [30] [31] [32] , a large proportion of negative test results was expected. the overall model fit, and the general consistency of the sensitivity estimates, suggests that these many respiratory viruses were reasonably accounted for by the seasonal baseline and that the strong association between the number of influenza positive and influenza negative tests on a weekly basis is indicative of a significant number of false negative results, rather than the activity of another virus or viruses exactly synchronous with influenza. the latter would bias the estimated sensitivity of the system downwards. however, to significantly and consistently bias the estimate, the degree of synchronization would have to be fairly strong, persist over the whole study period, and occur in all provinces. synchronization was not observed among the rvdss viruses (influenza a, influenza b, rsv, adenovirus and piv), and elsewhere other viruses such as rhinovirus, coronavirus and hmpv accounted for only a small proportion of the viral identifications and were not found to be synchronized with influenza [33] . as well, patients may present for care due to a secondary bacterial infection. while any specimen would likely test negative as the virus, at this point, is likely not detectable, the model would statistically attribute a negative test in this case to the primary infection; one of the four rvdss viruses or to the seasonal baseline that represents other respiratory infections, depending on the level of viral activity at the time of the test. this is not considered a source of bias. the large variation in false negative rates estimated for individual laboratories reporting to the rvdss suggests that standardization of sample procurement, testing and reporting procedures would likely reduce the overall false negative rate. the accuracy of diagnostic tests is known to be affected by the quality of the specimen [10, 11] , its handling, the timing of collection after symptom onset, and the age of the patient [14, 15] . even with the most sensitive molecular methodologies, yield was shown to be strongly related to the time since onset of symptoms [9, 14] , with a 3-fold decline in proportion positive within 3 to 5 days after onset of symptoms for both rt-pcr and culture procedures. for most laboratory tests, specimen procurement within 72 hours of from the onset of symptoms is recommended [6] , yet patients often present much later in the course of illness. estimates of the median time since onset of symptoms suggest a delay of 3 and 5 days for outpatient and inpatients respectively [15] , however these estimates are limited to patients with laboratory confirmed influenza. in addition, there are inherent differences in the performance characteristics of the currently used diagnostic tests [4, 6, 8, [34] [35] [36] [37] [38] . lack of standardization between diagnostic tests and algorithms used in different laboratories reporting to the rvdss adds to this complexity. the routine use of rt-pcr testing has only recently become available in canada (only 20% of tests used rt-pcr methods as of 2005/06 [23] ), but increased use of this modality is expected to improve accuracy. population or system level sensitivity estimates that include the effects of sample quality are limited. grijalva and colleagues [39] estimated the diagnostic sensitivity in a capture recapture study of children hospitalized for respiratory complications at 69% for a rt-pcr based system and 39% for a clinical-laboratory based system (passive surveillance of tests performed during clinical practice, and using a variety of commercially available tests). though the expected proportion of influenza tests that were due to influenza infections is unknown and variable, our model estimate of 30% appears plausible. cooper and colleagues [33] attributed 22% of telephone health calls for cold/flu to influenza over two relatively mild years, and elsewhere 20% of admissions for acute respiratory infections (including influenza) in adults aged 20-64 years were attributed to influenza, and 42% for seniors [1] . while there are limitations with this approach, there are no other simple alternatives to assist in the interpretation of the rvdss data. it would have been helpful to analyze data based on each specimen sent for testing. with only the number of weekly tests and number of positive results, we were unable to calculate the number of specimens that were actually found to be negative for all four viruses, or to estimate the extent of co-infection. coinfection, which was not accounted for in our model, could result in an under-estimation of the number of falsely negative tests, as the attribution of an influenza negative test that was actually coinfected with influenza and another respiratory virus would have to be split between the viruses. with auxiliary information associated with each specimen, model estimates of false negative rates based on, for example, test type, time since onset of symptoms, age of the patient, or clinical presentation would have allowed us to explore the reasons for the high false negative rates. as the false negative rate appears to be laboratory dependant (data not shown), this estimated range is applicable only to the rvdss for the study period. a significant reduction in the false negative rate is anticipated as methods become standardized and with the uptake of the new rt-pcr methods. as positive results, particularly for culture, are often obtained a week or more after the specimen was received, some positive results may have been reported in a different week than the test. multiple test results for a single specimen may have also contributed to reporting irregularities. these irregularities would tend to bias the estimated parameter towards zero, and hence the estimated sensitivity towards 1. considering the overall model fit and the relative severity of influenza [1] , we conclude that our estimate of sensitivity may be slightly over-estimated (number of false negatives under-estimated). poor test sensitivity contributes to the chronic underestimation of the burden of influenza in the general population. since estimates of the burden of illness drive planning for preventive and therapeutic interventions, it is important to improve all aspects leading to improved diagnostic accuracy. we have illustrated a simple method that uses the surveillance data itself to estimate the system wide sensitivity associated with the weekly proportion of tests confirmed positive. although our estimate of sensitivity is only applicable to the interpretation of the rvdss data over the study period, similar estimates for specific cohorts or laboratory procedures may help guide 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specimens tested for influenza to influenza (a and b), and adenovirus, parainfluenza virus, and rsv combined is shown along with the numbers confirmed positive. the total is the number of weekly tests for influenza (most were likely panel tests). the baseline accounts for routine tests in the hypothetical absence of influenza, rvs, adenovirus and parainfluenza activity, and corresponds to the model estimate of the number of tests that were truly negative for all tested viruses. the blue area (light plus dark) corresponds to tests attributed to influenza, with the light blue area corresponding to tests confirmed positive for influenza. the purple area (light plus dark) corresponds to tests attributed to rsv, adenovirus or parainfluenza influenza-associated hospitalizations in the united states influenza in canada: 2005-2006 season influenza in canada: 2003-2004 season antiviral therapy and outcomes of influenza requiring hospitalization in ontario impact of changing laboratory diagnostics on influenza surveillance sas/stath 9 user's guide strategy for efficient detection of respiratory viruses in pediatric clinical specimens prescription for excellence: how innovation is saving canada's health care system emergency department overcrowding: ambulance diversion and the legal duty to care influenza-associated deaths among children in the united states characterization of viral agents causing acute respiratory infection in a san francisco university medical center clinic during the influenza season human metapneumovirus infections in adults: another piece of the puzzle human metapneumovirus infection in adults human metapneumovirus infection in the canadian population the contribution of respiratory pathogens to the seasonality of nhs direct calls superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children real-time pcr in clinical microbiology: applications for routine laboratory testing evaluation of three immunoassay kits for rapid detection of influenza virus a and b performance of six influenza rapid tests in detecting human influenza in clinical specimens comparison of the directigen flu a+b test, the quickvue influenza test, and clinical case definition to viral culture and reverse transcription-pcr for rapid diagnosis of influenza virus infection estimating the undetected burden of influenza hospitalizations in children the authors acknowledge the support of the national fluwatch network and all those involved in the collection and compilation of this data. special thanks to the anonymous reviewers for valuable comments. key: cord-345454-r1ymzk6n authors: levesque, j.; maybury, d. w. title: a note on covid-19 seroprevalence studies: a meta-analysis using hierarchical modelling date: 2020-05-06 journal: nan doi: 10.1101/2020.05.03.20089201 sha: doc_id: 345454 cord_uid: r1ymzk6n in recent weeks, several seroprevalence studies have appeared which attempt to determine the prevalence of antibodies against sars-cov-2 in the population of certain european and american locations. many of these studies find an antibody prevalence comparable to the false positive rate of their respective serology tests and the relatively low statistical power associated with each study has invited criticism. to determine the strength of the signal, we perform a meta-analysis on the publicly available seroprevalence data based on bayesian hierarchical modelling with markov chain monte carlo and generalized linear mixed modelling with prediction sampling. we examine studies with results from santa clara county (ca), los angeles county (ca), san miguel county (co), chelsea (ma), the comte de geneve (switzerland), and gangelt (germany). our results are in broad agreement with the conclusions of the studies; we find that there is evidence for non-trivial levels of antibody prevalence across all study locations. however, we also find that a significant probability mass exists for antibody prevalence at levels lower than the reported figures. the results of our meta-analysis on the recent seroprevalence studies point to an important and strongly suggestive signal. infections with mild symptoms. moreover, high antibody prevalence indicates that a significant fraction of the population has already been exposed, lowering estimates of the infection fatality rate, and providing possible clues about herd immunity. given the importance of determining the societal-wide exposure to sars-cov-2, critically understanding the results from the seroprevalence studies represents a pressing concern. the recent serology study [1] from santa clara county, in which the authors report that the santa clara area has an antibody prevalence of 1.5% (exact binomial 95ci 1.11-1.97%), has received sharp criticism [2, 3] . in particular, the combination of the small sample size, small effect size, and a competitive false positive rate led to a study with relatively low statistical power. but, given the potential importance of serology analysis to our understanding of covid-19, we must extract all the available information that the data contain. we simply cannot afford to dismiss weak signals. along with the santa clara study, a number of other studies have recently appeared using different types of tests [4, 5, 6, 7, 8] . results are disparate. for example, the study in chelsea, massachusetts suggests an antibody prevalence as high as 30%, in marked contrast to the low levels detected by the santa clara study. given the early days of this type of research, and the unevenness of the infection rates across regions, a noisy picture does not surprise us. in this note we perform a meta-analysis based on the data from seven different studies. we apply two methods: 1) bayesian hierarchical modelling with markov chain monte carlo, 2) generalized linear mixed modelling (glmm) with prediction sampling. both methods lead to similar results. we find that there is evidence for non-trivial levels of antibodies in the populations across all the studies-in broad concordance with the seroprevalence study conclusions-but that smaller than publicly stated levels are also probable. the studies we analyze were conducted in the last few weeks; peer-reviewed scientific publications are not yet available. we construct our datasets from scientific preprints, manufacturers' specifications, and transcripts from interviews with study authors published in the news media. each study differs in its sampling strategy, and data on sample demographics is not yet available for most studies. in each study, the authors aim to collect a representative sample of their population. we did not attempt to post-stratify any of the results, and our analysis is therefore limited by our lack of knowledge of the demographics associated with the samples in each study. in this sub-section we summarize the data we found on each study. the studies are highly varied with differing levels of publicly available information. antibodies in the german town of gangelt. on april 10, 2020 the authors published preliminary results [4] , stating that "around" 500 individuals had been investigated, with 14% testing positive for igg antibodies. we assume this figure means that 70 individuals tested positive. the preprint does not mention which serology test the authors use, but the principal author is quoted in die zeit on april 10, 2020, stating that the study uses the igg elisa test produced by euroimmun ag [9] . the authors do not include demographic information, but they do refer to the who recommendation of investigating a random sample of 100-300 households. the authors state that they investigated "approximately" 1000 individuals from "approximately" 400 households, and that the preliminary results are for "approximately" 500 individuals. geneva, switzerland. this ongoing study started in early april 2020 in the comté de genève [10, 5] . the study releases results on a weekly basis. according to their study protocol, researchers use the same euroimmun elisa test for igg antibodies as the gangelt study. in the week of april 6 to 10, researchers found that 3.5% of the 343 participants tested positive, which we interpret as 12 individuals. in the week of april 14 to 17, they found that 5.5% of the 417 participants tested positive, which we interpret as 23 individuals. this study aims to test most residents from a county in colorado, with a population of approximately 8,000. the study is conducted in partnership with the serological test manufacturer ubi group. in a progress update published on april 21, the county announced that out of 4,757 antibody tests processed, 26 were positive, and 70 were "borderline". the county's web-page notes that a borderline result on the first test indicates that the result produced a "high-signal flash" which is not enough to produce a positive result. a borderline result means that the individual may have been recently exposed to covid-19 and may be in the early stage of producing antibodies. in our analysis, we include the borderline cases (96 total positive tests, out of 4,757). covid-19 antibody test manufactured in china by hangzhou biotest biotech co., ltd., and distributed in the united states by premier biotech, inc. this study was conducted between april 10, and april 11, 2020 [7, 12] , involving some of the same authors from the santa clara study. a preprint of this study is not yet available, but the raw numbers were announced in communiques made by la county public health, and university of southern california. out of 863 participants, 4.1% tested positive, which we interpret as 35 individuals. the researchers reported the 95% confidence bounds on the prevalence of 2.8%, and 5.6%. the sample was generated by a random draw from a marketing firm's database, made to be representative of the county's demographics. they used the antibody test sold by premier biotech, the same used for the santa clara study. we do not included due to a lack of data about the case counts and the serology test specifications. given the size of these studies and the potential impact of their stated results, we feel that releasing the underlying data is of paramount importance. • new york. on april 24, 2020 the state of new york announced the results of a covid-19 antibody study conducted over the preceding week. in the sample of 3,000 individuals, the state reported that 13% tested positive. within those, the subset of new york city residents (1,300 individuals) tested positive at 21%. we could not find any specifications about the antibody test used. • netherlands. the netherlands' central blood bank (sanquin) tracks covid-19 antibodies by sampling the blood donations they collect on a weekly basis. however, the test they use is unknown to us (one of the study leads is quoted in a science news piece [2] : hans zaaijer, a virologist at sanquin, the dutch national blood bank, who helped lead the study, says the team used a commercial test, which "consistently shows superior results" in validation studies, but didn't provide more details. . in their first set of results [8] , • denmark. denmark is conducting a study similar to the netherlands in which they are sampling weekly donations at their central blood bank. in an update published on april 7, 2020, the danish health authority [13] indicates that among 1,000 blood donors, 2.7% had tested positive for antibodies. they also reported that the sensitivity of their test is 70% (there was no information on test specificity). this sensitivity figure is the same reported by danish researchers for the euroimmun igg test [14] . however we could not confirm this association. the seven studies we consider use four different test types. each test has a different false positive rate and a different false negative rate. we include data on each test to build our models. this test is used in the gangelt [4] , and geneva studies [10, 5] . its performance was assessed independently by a team of danish researchers (lassaunière et al., [14] ). in their assessment, 20 out of 30 confirmed positive samples tested positive, and 3 out of 82 confirmed negative samples tested positive. the authors note that "borderline data were considered negative". these results mitigate the manufacturer's claim that their test has >99% specificity. one reason for the lower specificity seems to be the test's cross-reactivity with other coronaviruses, as found by okba et al. [15] . the manufacturer's own assessment [11] found that out of 397 confirmed positive samples, 352 tested positive, and out of 128 confirmed negative samples, 12 tested positive. the manufacturer claims that 100% of the blood samples collected at day 10 or later after infection from sars-cov-2 from patients who tested positive to covid-19 by other methods were also found to be positive using the ubi r sars-cov-2 elisa [16] . in the absence of an actual figure for the number of tests, we assumed a conservative n = 30. in addition, ubi indicates that out of "over 900" blood samples collected before the covid-19 outbreak, none tested positive. again, we took a conservative approach, and assumed n = 900. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 6, 2020. . out of 75 clinically confirmed covid-19 patients with positive igg tested positive, and 78 out of 85 confirmed igm-positive samples tested positive. the manufacturer also found that 369 out of 371 confirmed negative samples tested negative. the santa clara study authors found that in a set of 37 samples confirmed to be pcr-positive, and iggor igm-positive, 25 tested positive. they also found that out of 30 confirmed negative samples, 30 tested negative. our first approach uses a hierarchical bayesian model, which we solved by markov chain monte carlo using stan [17] . there are three observables in the model: • n obs (i), the number of positive antibody tests out of n obs (i) individuals tested in each location i; • n fpos (j), the number of positive antibody tests out of n neg (j) confirmed negative samples in the validation for each test brand j (false positives); • n fneg (j), the number of negative antibody tests out of n pos (j) confirmed positive samples in the validation for each test brand j (false negatives). from these observables, we infer, p prev (i), the prevalence of antibodies at each location, i. the number of positive antibody tests n obs (i) observed in each study are random variables that depend on p prev (i), but also on p fpos (j(i)) and p fneg (j(i)), which are the 6 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 6, 2020. . false positive and false negative rates of each serology test type j, indexed as a function of study i, namely n obs (i) ∼ binomial(n obs (i), p obs (i)), p obs (i) = p prev (i)(1 − p fneg (j(i))) + (1 − p prev (i))p fpos (j(i)), n fpos (j) ∼ binomial(n neg (j), p fpos (j)) n fneg (j) ∼ binomial(n pos (j), p fneg (j)). (1) our prior construction reads, with hyper-priors of, µ prev, fpos, fneg ∼ normal(0, 10) σ prev, fpos, fneg ∼ exponential(1). we run the model in stan using 4 chains each with 10,000 samples, discarding the first 5,000 as part of the mcmc warm-up. the 20,000 post warm-up samples result inr values above 0.9999 for all parameters in the model, and effective sample counts of approximately 10,000. we plot the marginal posterior density functions for the antibody prevalence at each location in figure 1 . the santa clara study shows a density function consistent with a high probability of a non-zero antibody prevalence, with a mean and a mode slightly greater than 1%, although we note that the posterior distribution does include zero. los angeles stands out-the mode of the distribution sits at 4% with a 95% credible interval that does not include zero. the german town of gangelt was particularly hard hit by covid-19 and the subsequent serology study suggests an antibody prevalence of 14%. our bayesian analysis is consistent with the study's result, but we see that the posterior distribution has a tail that includes a prevalence below 10%. the contours in figure 2 show a two dimensional slice through the posterior distribution revealing the probability density in the prevalence-false positive plane. while the santa clara study has a false positive rate competitive with the implied prevalence, our bayesian result clearly shows the mode located substantially greater than zero prevalence. figures 1 and 2 show that geneva has an implied prevalence close to zero. the euroimmun test used in the geneva studies has a relatively high false positive rate, creating tension in the resulting marginal posterior distributions. a significant probability mass sits at zero for both weeks of the geneva study. 7 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the chelsea (ma) study uses a serology test (biomedomics) with a relatively high false positive rate. however, the strength of the signal coupled to the bayesian learning across all the studies strongly suggest a highly non-zero antibody prevalence level. the 95% credible interval around the mode excludes a prevalence level below 10%. as a check on the the bayesian implementation, we use a set of binomial generalized linear mixed models (glmm) with prediction sampling. mixed models provide 8 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 6, 2020. figure 2 : the two dimensional marginal posterior distribution functions for antibody prevalence with the false positive rate at each study location from bayesian hierarchical model. estimations in situations which have have sub-population specific effects by borrowing strength from population averages (see, for example, [18] ). the borrowing effect or "shrinkage" tempers those sub-populations which have relatively less data but otherwise allows the data to speak for itself. glmm provides an optimal compromise between complete pooling and no pooling of the sub-populations in a regression analysis. in that sense glmm is a precursor to bayesian approaches which permit greater flexibility through priors and hyper-priors and afford more control over shrinkage effects. using [19, 20] , we build separate glmms for the empirical count observation process across the study locations, for the test type false positive rates, and for the test type false negative rates. in total, we have three glmms. we then build monte carlo prediction samples [21] for the means of each glmm and compute the implied prevalence. the glmm specification is: we estimate the implied prevalence distribution at each location from, where k represents the k-th monte carlo sample from the glmm prediction. the density function of p loc [k] is the prevalence density function at each location. we estimate the three separate glmms in eq.(4) using [20] and we display the fixed and random effects with their respective the 95% confidence intervals in figures 4, 5, and 6. to build our monte carlo estimate of the prevalence density function in eq.(5), we sample the predictions from the models over the uncertainty in each parameter with each realization representing the mean of the model, namely where i denotes the glmm model for the k-th sample. figure 7 shows the resulting density functions for the antibody prevalence in each location from the glmm prediction sampling. notice the similarity with figure 1; both of our implementations lead to similar density functions and are in broad agreement. the santa clara study suggests a prevalence of 1.5% and we see in the figure that the quoted value is just beyond the mode of the distribution. we also notice that santa clara has a heavy tail towards zero, just as we found in the bayesian analysis. yet, in part by relying on "borrowing strength" from the across all the studies, we also see that the santa clara result clearly suggests a non-zero antibody prevalence. again, we also see a strong effect in los angeles county, with a mode of 4%; the 95% confidence interval around the mean does not include zero. in figure 8 we show the 95% confidence intervals with the mean for all locations computed from the glmm sampling. in figure 9 we show a contour density map of the prevalence with the false positive rate. we can clearly see the tension between the false positive rate and prevalence but we also see a clear signal associated with each region. again, notice the similarity to figure 2. in particular, note that san miguel county (co) again shows a strong prevalence result, but that the glmm shrinks the false positive rate away from zero. the glmm is in some sense "semi-bayesian"-we have a hierarchical model with a random effect on the intercept term in logit space which we sample over, but we do not have a formal set of priors with hyper-parameters. while the glmm provides a basis for learning across the data, our full bayesian specification with markov chain monte carlo provides a more complete result. however, by implementing the glmm we see that our results are robust to different approaches and model specifications. our results are not sensitive to the specifics of the prior and hyper-prior constructions in our bayesian model. both methods demonstrate that there is significant evidence for non-trivial antibody prevalence in the populations associated with these studies. while the individual statistical power of each study is not high, using bayesian techniques and glmm constructions on the data from all the studies reveal a definite signal that we should take seriously. seroprevalence studies are an important tool in combating covid-19 since public policies are dependent on how far the disease has already penetrated into the general population. not only does serology testing help us understand the overall infection fatality rate of the disease, but testing also helps us design targeted strategies such as contact tracing. furthermore, serology studies can provide insight into the dynamics of the disease propagation. while we do not correct for possible population sample bias or other demographic issues, our analysis points to an important signal: the seroprevalence studies to date show that a significant fraction of the populations examined have antibodies against sars-cov-2 in their bloodstream. the exact prevalence levels are highly region dependent. as more serology studies appear, they will sharpen our understanding of antibody prevalence in the general population. the quality of antibody prevalence estimates depend on sample size and on the specificity/sensitivity of the antibody test. high test specificity increases statistical power. thus, there is a natural trade-off between investing in tests of high quality, the human resources required to generate large sample sizes, and the speed at which society needs results. the societal and economic consequences between type i and type ii errors are not equal. covid-19 antibody seroprevalence in santa clara county, california. medrxiv antibody surveys suggesting vast undercount of coronavirus infections may be unreliable how (not) to do an antibody survey for sars-cov-2. the new scientist, 2020 vorläufiges ergebnis und schlussfolgerungen der covid-19 case-clusterstudy séroprévalence covid-19 : première estimation de la prévalence d'anticorps anti-sars-cov-2 igg dans la population genevoise county covid-19 information usc-la county study: early results of antibody testing suggest number of covid-19 infections far exceeds number of confirmed cases in los angeles county slides from technical briefing given to the netherlands government on sars-cov-2 elisa test systems from euroimmun enquête de séroprévalence répétée des anticorps igg anti-sars-cov-2 dans la population du canton de genève covid-19 igm/igg rapid test early antibody testing suggests covid-19 infections in l.a. county greatly exceed documented cases covid-19 i danmark: status ved indgang til 6. epidemiuge status-og-strategi/covid19_status-6-uge.ashx?la=da&hash= 6819e71bfeaab5aca55bd6161f38b75f1eb05999) evaluation of nine commercial sars-cov-2 immunoassays. medrxiv united biomedical group's c19 company partners with san miguel county, colorado to be first in nation to test an entire county for covid-19 with new antibody diagnostic test stan: a probabilistic programming language generalized linear mixed models r: a language and environment for statistical computing. r foundation for statistical computing fitting linear mixed-effects models using lme4 mertools: tools for analyzing mixed effect regression models key: cord-354006-j1y42oxu authors: ozdemir, vural; williams-jones, bryn; glatt, stephen j; tsuang, ming t; lohr, james b; reist, christopher title: shifting emphasis from pharmacogenomics to theragnostics date: 2006 journal: nat biotechnol doi: 10.1038/nbt0806-942 sha: doc_id: 354006 cord_uid: j1y42oxu what will be the role of theragnostic patents in upstream and downstream biomarker research? what will be the role of theragnostic patents in upstream and downstream biomarker research? p harmacogenomics aims to identify the genetic basis of variability in drug efficacy and safety, and ultimately develop diagnostics that can individualize pharmacotherapy. theragnostics, a term denoting the fusion of therapeutics and diagnostics, is receiving increasing attention as pharmacogenomics moves to applications at point of patient care. in contrast to pharmacogenomics, theragnostic tests focus not on a singular marker set, such as genetic polymorphisms, but rather on the integration of information from a diverse set of biomarkers (e.g., genomic, proteomic, metabolomic). although it remains to be seen whether theragnostics reflects a form of hyperbole in biomarker research, it is grounded in both established (e.g., genomics) and exploratory (e.g., metabolomics) technologies that can offer, respectively, mechanistic and heuristic insights for therapeutics ( fig. 1) . recent social science analyses suggest that in some cases, bio-hype can be an integral component or driver for the establishment of biotechnologies, particularly in the early stages of development of a new concept or idea [1] [2] [3] [4] [5] . the synthesis of both types of technologies (established and exploratory) will likely have a differential impact on the regulation and economic promise of pharmaceuticals developed under the overarching theme of theragnostics. moreover, we suggest that advances in this field may shape, in potentially unexpected ways, the pursuit of theragnostic patents depending on whether the research is conducted in an upstream drug discovery-oriented context or towards downstream point-of-care applications 6, 7 . as biomarker applications move towards point-of-care to individualize drug therapy, a number of qualitatively different concerns arise relating to gene patents and ethical and therapeutic policy aspects of theragnostic testing 4, 8, 9 . for example, a fear is that theragnostic tests could be adopted without regard for the particular research context in which they are being applied, or be understood as a homogeneous category, a ubiquitous set of biotechnologies with similar implications for therapeutic policy 6, [9] [10] [11] . in effect, this may result in a predicament where the patents on biomarkers are conceptualized in a one-size-fits-all manner (as in drug prescriptions) thereby precluding the equitable implementation of emerging biomarker technologies and informed critique of their societal implications. additionally, the effects of theragnostic patents on 'knowledge commons'-that is, a space (usually in universities) where knowledge is shared without undue restriction-have not been adequately evaluated 11-13 . in the present analyses, we 'unpack' and contrast the motivations at play that are driving the pursuit for theragnostic patents and its bioethical corollaries in: (1) fundamental upstream basic research oriented to the discovery of genes for human diseases; and (2) downstream clinical applications at point-of-care as theragnostic tests to stratify patient populations for individualization of pharmacotherapy. we emphasize the need for integration, as well as the risk for excessive compartmentalization, of various biomarker technologies, and evaluate the subtle distinctions between dna-and protein-based theragnostic tests. the search for genetic determinants of common complex human diseases reflects one of the pivotal upstream applications of pharmacogenomics. as dna samples are being increasingly archived in pharmaceutical clinical trials, a sizable proportion of research resources are devoted to identifying the genes causally related to human diseases 4 . for many rare congenital monogenic human diseases (e.g., duchenne muscular dystrophy), pre-or postnatal cytogenetic analysis of chromosomes or conventional clinical chemistry tests have existed for several decades. more recently, highthroughput genomic technologies spun off from the human genome project (hgp) and the decreasing cost of genotyping have made possible the adoption of molecular genetic tests that can pinpoint the precise etiology or predisposition for a few adult-onset human diseases such as huntington disease, certain familial forms of breast cancer, and alzheimer disease 14 . while genetic tests hold the promise of a more rational clinical forecast and management of disease risk in the future, they are also raising concerns about the provision of public healthcare services (reduced access) and the impact of market forces on the products of research (commercialization of technologies) and academic freedom. some of these concerns have crystallized around the issue of commercial genetic testing for disease risk, as exemplified by the case of testing for hereditary breast cancer (brca testing) 15 . in the mid-1990s, two genes (brca1 & brca2) that greatly increase the risk for hereditary breast cancer were identified and sequenced. the brca genes were given broad patent protection in the us (and subsequently internationally in canada, europe, australia, etc.), and granted to the biopharmaceutical company myriad genetics (salt lake city, ut), which had been involved in much of the initial research. this strong intellectual property protection has allowed myriad to effectively control the brca testing market in the us; the commercial bracanalysis test is available only from myriad or their licensees. most recently, myriad has licensed their test to san franciscobased dnadirect, in order to provide services direct to consumers 16 . basic research into the function of the brca genes or resulting proteins would be permissible without infringing on myriad's patent rights, although there is still contention about what precisely constitutes basic research exclusions 17 . myriad has, for example, signed agreements with the us national institutes of health and national cancer institute to provide sequencing at cost (us $1,200) for research purposes 18 . however, research that results in a commercial or clinical service, defined by myriad as any research in which a fee is charged for testing or in which results are provided to patients, would infringe on their patents. notably, technology assessment research by third parties, for example to evaluate test performance metrics such as sensitivity, specificity, or positive predictive value, is particularly jeopardized. moreover, research aimed at comparing the bracanalysis test against other testing methodologies would prove difficult, as the clinical nature of such a trial would constitute an infringement on myriad's patents. hence, the brca patents give myriad the ability to constrain research-oriented applications of brca patents and particularly head-to-head comparisons of which genotyping methodology or test product is most informative for clinical management of the susceptibility to breast cancer. in part due to myriad's broad patent rights and the attendant concerns to ensure patients' access to affordable genetic testing for breast cancer risk, the european brca patents have been constrained or not enforced in recent years 15, 19 . in some sense the myriad case may be thought of as an extreme scenario, and one from which industry and governments have learned. one specific consequence is that the initial enthusiasm for granting such broadly defined upstream patents on any and all forms of biological material has waned due to concerns for public good and scientific progress. more generally, there is a growing awareness that patents on genes and other biological materials can have an unfavorable effect on downstream genetics research and knowledge commons 13, 20 . thus, without an adequate conceptual framework on patents relating to theragnostic technologies, upstream patents on potential drug target genes or genetic methodologies for molecular definitions of human diseases (as in familial breast cancer) may lead to a monopoly on theragnostic tests. this may also dissuade some research laboratories from investigating otherwise potentially promising lines of inquiry for technology transfer towards downstream theragnostic products in the clinic 20 . the myriad patent case remains relevant since, in part, the current trends at the us patent and trademark office (uspto), and in patent offices in other countries, to grant more narrowly defined patents on biological materials were driven by this case 21 . according to some commentators, "no other event has had as big an impact on the human gene patent debate…and the case [myriad] has thus become a 'harbinger' of the policy challenges created by gene patents" 21 . more recent examples also support the idea that upstream theragnostic patents can limit translational applied clinical research. for instance, the sars-associated coronavirus genome patents were filed by the us centers for disease control (cdc) and the british columbia cancer agency (bcca) [22] [23] [24] , officially to ensure continued public access to the viral genome and pre-empt other entities from exerting restrictive or controlling rights. notably, it is suggested that this type of patent is symptomatic of what is wrong with the [gene patent] system, when pre-emptive patents have to be filed to protect science and the public good 22, 23 . a more worrying example is illustrated by the australian company genetic technologies' patents on the non-coding regions of the human genome. formerly conceived to be 'junk dna' , the biological role of non-genic segments of the genome is receiving increasing attention, and their patenting may significantly constrain the free design of primers for pcr analysis of coding regions of the genome 25, 26 . a common thread in these two recent examples, however, is that broad theragnostic patents, particularly those granted on dna and other biological materials, may serve as tollbooths that impede downstream research or discourage competition and innovation due to the broad exclusive rights granted to an individual scientist, institution or company (e.g., as in the case of myriad). this 'anticommons' effect of upstream theragnostic patents is now increasingly being recognized. patents with a broad scope may actually enclose the 'knowledge-commons' and inhibit technology transfer and development at a societal or macro level (i.e., as a contrast from the viewpoint of an individual investigator), such that the promises of new diagnostics and therapeutics are not realized 6, 11, 13 . pharmacogenomic-guided drug development represents a fundamental conceptual departure from conventional 'one-size-fits-all' clinical trifigure 1 hierarchy of biomarkers and their integration into theragnostic tests, from gene sequence (upstream or static marker) to downstream (dynamic) markers on gene and protein expression or cellular metabolites. a theragnostic profile is depicted as a synthesis of various biomarker tests that characterize an individual patient and her/his drug treatment outcome. the theragnostic profile may be heuristic in nature when only a singular biomarker is associated with treatment outcomes while more mechanistic insights can be achieved when biomarkers from different levels of the biological hierarchy corroborate and complement each other. 27 . this is a favorable advance for rational therapeutics and optimal patient care but it also engenders varying degrees of trepidation among pharmaceutical companies and financial investors about proactive implementation in the clinic: pharmacogenomics may inevitably result in smaller economic markets for drugs introduced with an attendant genetic test predictive of drug efficacy or toxicity 4,6,9 . the pharma companies fervently respond that only drugs with large-scale markets allow recovery of the r&d costs for new medications 8, 9 , which can range from $400-800 million according to different estimates 28 . while r&d costs per se are not necessarily prohibitive to pursue targeted therapies, the pharmaceutical industry will still need to find mechanisms to maintain their growth rates in such niche markets defined by theragnostic tests. the ways in which upstream and downstream theragnostic patents are sought may play a decisive role in the development of focused therapeutic interventions in smaller markets that can benefit public good and industry growth equally. as a contrast to arguments of market fragmentation, pharmacogenomics and related theragnostic technologies may enhance therapeutic differentiation and market penetration of new medicines [29] [30] [31] . many of the currently marketed drugs, however, fall under the 'metoo' designation with comparable efficacy and safety profiles differing only in terms of slight changes in their chemical structures or pharmacophore composition 32 . hence, in diseases or therapeutic areas characterized by me-too drugs, the diagnostic companies without a pharmaceutical pipeline may be more inclined to develop theragnostic tests that can impact more than one drug by virtue of being in the same therapeutic or chemical class. conversely, in the case of large drug manufacturers, a theragnostic test for a me-too drug may be equally predictive of treatment outcomes for most if not all drugs within the same me-too category, redistributing the financial gains on the theragnostic test from an individual pharma company holding the theragnostic patent to multiple firms who manufacture similar me-too drugs. consider, for example, a patient with major depression receiving the result of a theragnostic test on the serotonin transporter gene in relation to antidepressant response to paroxetine, a selective serotonin reuptake inhibitor (ssri). in this case, the patient has the freedom afterwards to choose from among a host of comparable ssri drugs without necessarily having to commit to paroxetine co-developed with the hypothetical theragnostic test. therefore, the pursuit of theragnostic patents can also be shaped by the type of industry setting (e.g., diagnostic sector versus large pharma) as well as the type of pharmaceutical (e.g., me-too drugs) associated with theragnostic tests. regardless of the 'true' cost of drug development or the varied perceptions of the impact of pharmacogenomics or theragnostic tests on the economic promise of pharmaceuticals, the fact is that the blockbuster model of drug development with large-scale markets is increasingly less viable 8, 33 . when new technologies such as pharmacogenomics and theragnostics enter the market, they can become 'paradigm-disruptive' forces that significantly undermine the traditional broadly defined market model of drug development and commercialization. diverse and divergent diagnostic tests, multiple actors (e.g., biotech diagnostic companies, small and large pharma companies) seeking to create and protect their intellectual property, and changing social and political contexts (global demands for patent reform and licensing of drugs in the developing world) create an unstable environment for drug manufacturers. despite the often very public proclamations about an interest in integrating pharmacogenomic research into drug development strategies, prospective stratification of patients using genetic tests in advanced stages of drug development with a view to proactive incorporation of pharmacogenomic data into drug labels is still rare 33, 34 . this illustrates that there is a great uncertainty about how pharmacogenomic and theragnostic tests ought to be developed as functional commercial products. it is noteworthy that multiplicity of diagnostic patents anticipated by the introduction of theragnostic technologies may also result in an 'anticommons effect' since most pharmacotherapeutic outcomes are polygenic or multifactorial in nature. if each segment of this expanding sphere of patentable biological elements along the biological dogma is held by different individuals, academic researchers or commercial firms, scientific advances in theragnostics can again be stifled, as in the case of broadly defined upstream gene patents. this is further supported by at least two seemingly divergent but complementary lines of evidence. first, the uspto and patent offices in other countries increasingly favor narrowly defined gene patents, in part as a response to the myriad case. secondly, theragnostics is now introducing the need to characterize (and motivations to patent) downstream gene products such as mrna, proteins or cellular metabolites to individualize drug therapy. because time-dependent changes in gene expression or encoded proteins cannot always be accurately inferred from the upstream gene sequence, it is conceivable that there will be many more narrowly defined patents granted in the near future along the biological dogma from gene sequence to proteins and metabolites. coupled with trends in patent offices in favor of narrowly defined gene patents, there will likely be a fragmentation of the diagnostic sector, as in the case of the blockbuster drugs and the niche therapies guided by theragnostic tests. many of today's most common diseases (including most forms of cancer, heart disease and psychiatric disorders) are known to arise not exclusively from either genes or environmental factors, but through a combination of the two (along with a significant amount of incalculable stochastic factors). moreover, certain environmental exposures may only evoke illness when experienced during a critical period and in concert with a high-risk genetic background. therefore, theragnostic tests predicated on genetic information alone would have a low a priori likelihood of capturing all of the predictable variance in a particular response outcome. ideally, genetic tests could be fashioned to capture the entire heritable portion of a response variable (distributed across one or many genetic polymorphisms). in this context, genetic polymorphisms impart a constant or 'static' state of responsiveness that can be assayed once in each individual and presumed not to change over the course of the lifespan (barring de novo somatic mutations). the residual non-heritable portion of a given response phenotype must then be assayed by other means. to the extent that environmental (i.e., non-heritable) exposure influences response phenotypes by impinging on biological systems, this additional proportion of variance can be assayed through more 'dynamic' biomarker platforms, such as transcriptomics, proteomics, and metabolomics, each of which may be influenced by both genetic and environmental factors (fig. 1) . thus, through a combination of static genetic and other dynamic biological '-omic' technologies (i.e., theragnostics), the potential to identify a more comprehensive set of predictors is maximized. there are certain unique aspects of pharmacogenomic (and theragnostic) tests that differ from genetic testing for disease susceptibility. for all the parallels between genetic tests for disease risk and drug response, the latter are applied in reference to a drug that will be administered to patients in the immediate foreseeable future, while genetic testing for disease susceptibility usually predicts a risk in the distant future, often several years or decades away. this 'temporal dissociation' between the genetic test and the future disease occurrence may permit the estimation of the attendant cumulative disease risk with use of genetic data only; there is also a functional disconnect because the disease susceptibility test provides information which is rarely accompanied by effective treatment options. in contrast to genetic testing for disease risk, pharmacogenomic tests are envisioned as being both temporally and functionally proximal. the purpose of a pharmacogenomic test is not to provide risk information as such, but to aid in the individualized prescription of a particular drug. further, most drug effects are elicited within a matter of minutes, hours or days which may require a more precise estimation of the present or acute state of the pathophysiological pathway whose function is inferred through a genetic test. hence, because the only barrier between the patient and drug safety or efficacy may be reliance on the accuracy of a pharmacogenomic test, clinicians need to know both the genetic variants in patients' dna as well as the corresponding proteins encoded by the same genes. this is essential because (1) proteins are responsible for the eventual functional or clinical significance of genes and, (2) there may be marked differences or fluctuations in protein function (than what is predicted solely by gene structure) due to environmental factors or physiological feedback mechanisms that may influence posttranscriptional/posttranslational modification of gene products and proteins. further, an accurate prediction of drug effects may require a two-step complementary strategy involving, for example, both genetic and proteomic tests for the same gene and its protein product. this may create unprecedented challenges for patents and their legal defense. for instance, what are the implications of a biotechnology company attempting to develop a metabolomics-based, non-genetic 'dynamic phenotyping biomarker' for a gene patented hitherto for a static genotyping test to predict drug response or toxicity? there are presently no definitive answers to such emerging novel intellectual property issues associated with theragnostic tests. since its first appearance in the research literature in september 1997 (refs. 2,35) , the term 'pharmacogenomics' has been hailed as a revolutionary enabling technology that can deliver highly customized drug therapies in the short term. now, nearly a decade after its introduction, the more realistic expectation is that pharmacogenomics will complement efforts for rational individualization of drug therapy in conjunction with existing therapeutic monitoring tools and other novel biotech-nologies (e.g., proteomics and metabolomics). there is increasing support for the view that the human genome is highly dynamic and that gene expression, as well as the regulation of gene function, is subject to poorly understood plasticity. to achieve the much hoped for provision of personalized medicines, the role of environmental and social factors on both drug response and the human genome (and its expressed products, mrna and proteins) requires detailed consideration. there is also growing recognition that the search for genetic biomarkers of outcomes associated with therapeutic interventions may carry the risk for compartmentalization among biomarkers through excessive reliance on a singular biotechnology. it is against this background that theragnostics is slowly emerging as a new concept to synthesize information from various biotechnologies directed at different levels of the biological dogma ranging from dna (genomics), mrna (transcriptomics), proteins (proteomics) or cellular metabolites (metabolomics). unlike mainstream genetic tests for disease susceptibility, the commercialization of theragnostic tests is inextricably linked with the deployment of patented pharmaceuticals. we suggest that theragnostic patents, particularly in the case of downstream applications at point-of-care, can be at variance with the traditional blockbuster model of drug development which stipulates the development of drugs for the entire population even though this approach yields modest therapeutic response and suboptimal drug safety 8,10,33 . hence, a very different and unprecedented story is evolving for theragnostic patents at point-of-care: the traditional tight 'coupling' between patents and their subsequent commercialization may not always occur in clinical trials designed for the registration of new therapeutic candidates under the blockbuster model 6 . experts in biotechnology patent law have thus slowly begun to point to this potential 'uncoupling' between the discovery of biomarkers on treatment outcomes and the necessary technology transfer to develop theragnostic products in the clinic. instead, the downstream theragnostic patents on biomarker discoveries may remain primarily as in-house discoveries within the pharmaceutical industry to benefit future drug discovery efforts but without the accompanying translational clinical research for their development as a diagnostic kit for prediction of treatment response, failure or drug toxicity 6, 9 . this uncoupling of biomarker discovery and necessary technology transfer towards their clinical application poses a threat to theragnostic product development. it is thus con-ceivable that academic research initiatives for biomarker discovery that consider both drug efficacy and broader functional treatment outcomes 29, 36 will play a critical role in the development of theragnostic-guided personalized medicine. in contrast to downstream patents on biomarkers associated with drug efficacy and safety, upstream patents in drug discovery and the identification of novel drug targets may be particularly welcomed by the pharmaceutical industry. this raises concerns over such patents becoming tollbooths that can increase costs for theragnostic tests in the clinic and slow or block downstream applied research. these nuanced contextual differences in applications of theragnostic patents in upstream research or at point-of-patient-care can shape the motivations at play, the strategies behind the patenting of genes, and the subsequent commercialization into theragnostic tests that may (or may not) become available to patients and consumers. the theoretical and practical framework on patents needs to incorporate implications of theragnostics on both upstream and downstream biomarker research while ensuring technology transfer by more than one stakeholder, to prevent future market monopoly and excessive premium pricing of theragnostic tests 15 . as the pharmaceutical industry transitions from the blockbuster model towards targeted therapies with market shares that resemble orphan drugs, there is a parallel need to offer incentives to stakeholders who pursue theragnostic-guided drug development. the discipline of science and technology studies (sts) is already focused on the complex issues at the intersection of emerging biotechnologies, genetic research, bioethics, market forces and the pharmaceutical industry 2,7,37 . unfortunately, the expertise in the sts research community does not always find its way into the mainstream medical research literature 38, 39 . such collaboration among geneticists, ethicists, applied pharmacologists and social scientists is essential for the equitable implementation of commercial theragnostic testing in the clinic, but also to prevent the risk of bioethics being used as a 'rubber stamp' that can 'deal with the issues' prior to the development of anticipated theragnostic-guided customized therapies. as witnessed during the initial planning and implementation stages of the hgp, we suggest that adequate attention and research resources should be made available to resolve these and similar policy and patent issues associated with theragnostic testing at the point-of-care. additionally, these efforts should parallel the development of much needed prospective clinical investigations, designed primarily for the purpose of biomarker discovery, which can importantly contribute to development of targeted therapeutic interventions in the near future. we thus believe there is reason for guarded optimism that theragnostics may allow the synthesis of different types of biomarker data, dna, protein or metabolomicbased, to achieve individualized therapeutics in medicine. the politics of personalised medicine-pharmacogenetics in the clinic ethics and law of intellectual property: current problems in politics world health organization resource on patentability of the sars virus genome we have never been modern science in action: how to follow scientists and engineers through society all authors contributed to the ideas, critique and synthesis of the data discussed in the present review, as well as the specific considerations involving the role of gene patents on theragnostic tests and nuanced distinctions among different types of biomarkers. nature biotechnology volume 24 number 8 august 2006 key: cord-310195-am3u7z76 authors: waller, j.; rubin, g. j.; potts, h. w. w.; mottershaw, a.; marteau, t. m. title: immunity passports for sars-cov-2: an online experimental study of the impact of antibody test terminology on perceived risk and behaviour date: 2020-05-10 journal: nan doi: 10.1101/2020.05.06.20093401 sha: doc_id: 310195 cord_uid: am3u7z76 objective: to assess the impact of describing an antibody-positive test result using the terms immunity and passport or certificate, alone or in combination, on perceived risk of becoming infected with sars-cov-2 and intention to continue protective behaviours. design: 2 by 3 experimental design. setting: online with data collected between 28th april and 1st may 2020. participants: 1,204 adults registered with a uk research panel. intervention: participants were randomised to receive one of six descriptions of an antibody test and results showing sars-cov-2 antibodies, differing in the terms used to describe the type of test (immunity vs antibody) and the test result (passport vs certificate vs test). main outcome measures: the primary outcome was the proportion of participants perceiving no risk of becoming infected with sars-cov-2 given an antibody positive test result. other outcomes include intended changes to frequency of hand washing and physical distancing. results: when using the term immunity (vs antibody), 19.1% of participants [95% ci: 16.1 to 22.5] (vs 9.8% [95% ci: 7.5 to 12.4]) perceived no risk of catching coronavirus at some point in the future given an antibody-positive test result (aor: 2.91 [95% ci: 1.52 to 5.55]). using the terms passport or certificate, as opposed to test, had no significant effect (aor: 1.24 [95% ci: 0.62 to 2.48] and aor: 0.96 [95% ci: 0.47 to 1.99] respectively). there was no significant interaction between the effects of the test and result terminology. across groups, perceiving no risk of infection was associated with an intention to wash hands less frequently (aor: 2.32 [95% ci: 1.25 to 4.28]) but there was no significant association with intended avoidance of physical contact with others outside of the home (aor: 1.37 [95% ci: 0.93 to 2.03]). conclusions: using the term immunity (vs antibody) to describe antibody tests for sars-cov-2 increases the proportion of people believing that an antibody-positive result means they have no risk of catching coronavirus in the future, a perception that may be associated with less frequent hand washing. the way antibody testing is described may have implications for the likely impact of testing on transmission rates. at the height of the first wave of the covid-19 pandemic, about a third of the world's population is estimated to have been in lockdown, with all but essential workers largely confined to home (1) . without an effective treatment or vaccine, testing for infection combined with contact tracing and isolation will be central to effective strategies to ease populations out of lockdown while keeping the basic reproduction number (r0) below one (2) . testing for antibodies to sars-cov-2 is a possible complement to testing for active infection to identify those who have developed antibodies to the virus and so may be able to return to work and other activities without significantly increasing transmission rates (3) . these tests have been variously described in the media as immunity passports (4, 5) , immunity certificates (6,7) immunity cards (8) and release certificates (9) . unfortunately, the use of these terms implies a certainty unmatched by current evidence about antibody tests (10) . uncertainties inherent in tests for antibodies to sars-cov-2 include the extent and duration of immunity conferred (11) . they also include the uncertainties inherent in any test regarding the proportion of those who would be correctly identified. this depends upon the test performance -its sensitivity and specificity -as well as the population prevalence of the tested condition (12) . given these uncertainties, those who receive a test result indicating the presence of antibodies will have a residual risk of becoming infected by sars-cov-2 in the future. understanding that there is this residual risk -albeit one that is difficult to quantify at present -will be important to minimise transmission that could arise from those receiving "antibody positive" test results. if people testing positive perceive that they have no risk of becoming infected by the virus, they may ignore any future symptoms of infection and facilitate transmission if they fail to self-isolate appropriately. such a perception may also overgeneralise to a belief that they are unable to transmit infection through contact with contaminated surfaces. regardless of antibody status, all individuals can indirectly transmit the virus between surfaces by touch. hand washing or sanitising therefore need to remain frequent. evidence from other testing programmes suggests that interpreting a low risk result to mean no risk can be reduced by verbal and numerical expressions of residual risk when presenting test results (13, 14) . but even before testing programmes are in place, the terms commonly used to describe these tests -immunity passport or certificates -may inadvertently be fuelling a misplaced sense of certainty about their results. it is unknown whether describing these tests as being for immunity -as opposed to antibodies -or their results as passports or certificates increases misunderstanding of the residual risk inherent in an antibody-positive . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 10, 2020. . https://doi.org/10.1101/2020.05.06.20093401 doi: medrxiv preprint test result and thereby reducing adherence to protective behaviours and increasing risk of transmission (15) . this study was designed to test two hypotheses: describing a test indicating the presence of antibodies using the term immunity (vs antibody), and describing test results as passports or certificates (vs test), increases the likelihood that those with this test result erroneously perceive they have no risk of becoming infected in the future with coronavirus. ethical approval for this study was granted by the king's college london research ethics committee (reference: mra-19/20-18685). the protocol was preregistered on the open science framework https://osf.io/tjwz8/ study 2 the statistical analysis plan was pre-specified and uploaded to the open science framework prior to receipt of the data https://osf.io/tjwz8/ study 2 an initial study with similar methods was conducted https://osf.io/tjwz8/ study 1 but, due to an error, the intervention was not correctly programmed. this study is therefore not reported. the study was an online experiment using a 2 × 3 factorial design, with participants randomised, with an equal allocation ratio, to one of six groups varying in the description of an antibody test and a result showing the presence of antibodies. these descriptions differed only in the term used for what was being tested (immunity vs antibody) and the term used for the test result (passport vs certificate vs test). a quota sample of 1,204 adults was recruited via predictiv, the behavioural insights team's online experimentation platform (https://www.bi.team/bi-ventures/predictiv/) comprising 500,000 adults in the uk. quotas were based on age, gender and uk region to achieve a sample broadly representative of the uk population. 1373 clicked on the link to enter the study of whom 1214 subsequently completed the study. ten were excluded for failing to meet quality checks. participants were reimbursed in points (equivalent to £1) which could be redeemed in cash, gift vouchers or charitable donations. participants did not know the topic of the study prior to participation. due to the rapid nature of this research, the public was not involved in the development of the study. the sample size was chosen pragmatically without reference to a specific power calculation. we are fitting a full model with an interaction. conservatively, we then had an 80% chance of detecting, at a 5% significance level, an increase in the primary outcome measure from 50% in a baseline group to 64% in another group. participants were randomised to groups by random number generation. a random number between 1 and 6 was generated for every participant upon entry to the study to determine which description they saw, with each of the six numbers corresponding to one of the six description. as this is based on true randomness, the number of participants within each group can vary due to chance. the intervention comprised a description of antibody testing and test results indicating the presence of antibodies [see box 1 for one example and s1 for wording of all six descriptions]. these differed across six groups in test name of results indicating the presence of antibodies. all descriptions included the information that the result would mean a lower risk of future infection and transmission, and that people with this result could return to work earlier. wording of the items used for each measure is shown in supplementary materials (s2). scientists are developing tests to see who has already had coronavirus. no test is 100% effective. this means that those who test 'positive' would have: • lower risk of catching coronavirus in the future, and therefore also • lower risk of passing it on to others those who test 'positive' would get an immunity passport. they could return to work early. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 10, 2020. . https://doi.org/10.1101/2020.05.06.20093401 doi: medrxiv preprint primary outcome proportion of participants perceiving an antibody-positive test result to mean no risk of catching coronavirus in the future, assessed in response to a question with four response options. perceived likelihood of catching coronavirus in the future, assessed on a visual analogue scale from 0% to 100%. intention to engage in handwashing less or more frequently than now, given an antibodypositive test result: assessed in response to a question with five response options. intention to avoid physical contact with others outside the home more or less frequently than now, given an antibody-positive test result: assessed in response to a question with five response options. interest in undergoing the test if offered today: assessed in response to a question with four response options. demographic characteristics: age, gender, level of education and geographical region of residence. employment status, planned to be included, was omitted due to a technical error. a detailed statistical analysis plan is available on the open science framework, specified prior to receipt of the data https://osf.io/tjwz8/ study 2. binary logistic regression was used to assess the impact of test type (immunity/antibody) and result type (passport/certificate/test) on the odds of believing the antibody test result means there is no risk of future infection. an interaction term was included in the model (16) . the analysis was repeated adjusting for age (including a quadratic function to model a non-linear relationship), gender, education and region based on prior results showing these are predictors of risk beliefs. binary logistic regressions were run (as above) for the secondary outcomes: intention to wash hands less, intention to engage less in social distancing and intention to undergo the test. unadjusted and adjusted odds ratios and 95% confidence intervals are reported. logistic regression was run to assess the extent to which intentions to engage in less frequent handwashing or social distancing measures is predicted by perceiving the test result to mean no risk of being infected in the future by coronavirus. as only a very small proportion of participants gave a 'zero' response on the sliding scale of future risk, we used a linear regression model to examine this outcome, rather than a binary (zero vs. other) logistic regression as pre-specified in the analysis plan. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 10, 2020. . https://doi.org/10.1101/2020.05.06.20093401 doi: medrxiv preprint data were collected using an online survey platform, predictiv. upon entry to the study, participants were informed that they were to be asked some questions about coronavirus and that it would take about five minutes to complete. participants were then shown one of six brief descriptions of an antibody test for coronavirus (see s1 for full text for each of the six descriptions). they were then asked five questions, assessing the primary and secondary outcomes. participants' demographic characteristics were accessed from the survey platform. the sample comprised 606 women and 598 men with a median age of 36 years. around a quarter had some graduate-level education (24.2%) and there was good representation of all uk regions (see table 1 ). distribution of sample characteristics by exposure group is shown in table 1 . responses to the five outcome questions for the whole sample and by experimental group are shown in table 2 perceived level of future risk (on a scale of 0 to 100%) showed a complex, trimodal distribution. the median was 35% with an interquartile range from 18% to 51%. only 5% of respondents put their risk at 0%. overall, 63% put their risk below 50%. 10% put their risk at 50%, which was the modal response. 24% put their risk at greater than 50%, but below 100%. 3% of respondents put their risk at 100%: that is, they said they were certain to contract the virus. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 10, 2020. proportion answering 'no risk' in each sub-group % (95% ci) (n=1204) odds ratio (95% confidence interval) mutually adjusted (n=1204) figure 1 ]. there was no significant effect of result type and no significant interaction. we analysed the continuous measures of future perceived risk of infection using a linear model (anova) with two levels for test type, three levels for result type, and an interaction term. overall, there was no significant effect: f5,1198 = 1.46, p = 0.20, adjusted r2 < 1%. we repeated the analysis adjusting for demographic factors as covariates. overall, there was a significant effect: f13,1165 = 1.88, p = 0.03, adjusted r2 = 1%. this was because of a significant effect of age: as age increased, perceived risk decreased. there remained no significant effect of the experimental variables. logistic regression analyses examining the impact of test type, result type and their interaction on intentions to wash hands and avoid physical contact less frequently and on willingness to have the test are shown in supplementary tables 1 and 2. neither test type, result type nor their interaction were significantly associated with these behavioural outcomes. logistic regression analyses were used to examine belief that the result meant 'no risk' as a predictor of intention to wash hands and avoid physical contact less frequently, given a positive result (see figure 2 and supplementary . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 10, 2020. using the term immunity -as opposed to antibody -to describe antibody tests for sars-cov-2 doubled the proportion who erroneously perceived they would have no risk of becoming infected with the virus in the future if they were given an antibody-positive test result, from 9.8% for antibody to 19 these was no significant association with intended frequency of avoiding physical contact with others outside of the home. interest in undergoing the test was high -with 85.2% saying they would probably or definitely have it if offered -and was unaffected by the terms used to describe the tests. this study was designed to test two hypotheses, providing strong support for the first, that describing a test indicating the presence of antibodies using the term immunity (vs antibody) increases the likelihood that those with this test result erroneously perceive they have no risk of becoming infected in the future with coronavirus. this likely reflects a certainty about risk of future infection implicit in lay understandings of the term immunity that is not implied by the term antibody (17) . qualitative studies could explore this and other potential mechanisms for the effect observed. the results of this study did not support the second hypothesis that describing test results as passports or certificates increases the likelihood that those with this test result erroneously perceive they have no risk of becoming infected in the future with coronavirus. this does not mean that these terms are unproblematic however, only that they did not influence the specific perceptions that we explored. qualitative studies are warranted to understand the broader meanings these terms have in the context of testing for antibodies for sars-cov-2 and other contexts. responses on the sliding scale of future risk showed a high variability and were largely unexplained by the experimental intervention or other variables measured. this may point to considerable uncertainty in the public as to how to interpret test results. it also likely reflects the well-described tendency of people to use a 50% response to indicate uncertainty rather than a true judgement of probability (18) . we also saw that about a quarter of respondents on the first question stated their risk was "average" or "higher". this may point to considerable uncertainty in the public as to how to interpret test results use of the top end of the scale is hard to interpret but may either reflect a failure to read the information carefully and . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 10, 2020. . https://doi.org/10.1101/2020.05.06.20093401 doi: medrxiv preprint therefore a misunderstanding of the meaning of the result, or participants' using information beyond the experiment to assess their risk and not adequately considering the hypothetical test result when making their response. while we found no evidence for a direct effect on protective behaviours of the terms used to describe antibody tests results, there was indirect evidence that perceiving no risk of future infection might reduce frequency of handwashing. this finding is tentative, given it is based on behavioural intentions in response to a hypothetical antibody-positive result. nonetheless the potential for antibody testing to increase viral transmission must be considered alongside the potential benefits the tests might have in allowing the easing of lockdown restrictions. clear communication about the ongoing need for handwashing, in particular, will be essential and raising public awareness of the main mechanisms through which sars-cov-2 is transmittedthrough air and surfacesmight help improve adherence. this, in addition to acknowledgment of the imperfect nature of the tests, will give the public a more accurate representation of the meaning and implications of an antibody test result and a better understanding of how to reduce the risk of transmission. such communications need to emphasise that transmission can occur through contact regardless of antibody status. such communications also need to be rigorously evaluated to ensure their effectiveness at communicating these points both to those undergoing antibody tests as well as to general populations that are now having to learn to live with sars-cov-2. this study provides the first experimental evidence for the potentially adverse impact on risk perceptions and protective behaviours of commonly used terms to describe sars-cov-2 antibody tests and their results. as such, it provides timely evidence to inform policy and research to mitigate these effects to realise the potential benefits of such tests. the study has several limitations. first, participants were responding to a hypothetical test and asked to imagine that they had received a test result that had detected antibodies. findings from such studies can generalise to clinical settings (19, 20) but some caution is warranted. second, the protective behaviours of handwashing and physical distancing were measured using single items assessing behavioural intentions following a hypothetical test result. third, the sample size was insufficient to detect effect sizes that could be important at a population level. it is possible, for example, that the use of the terms certificate or passport might impact on risk perception, but the current study lacked the power to detect this. fourth, while quotas were used to achieve a sample broadly representative of the uk population, research panels are not representative of the general population (21, 22) . we found no evidence that the impact of the interventions in this study was modified by demographic characteristics of the participants, providing some reassurance about the generalisability of results across age groups, gender, educational level and geographical region of the uk. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 10, 2020. the results of this study have several implications for research and policy. the effectiveness of antibody tests for sars-cov-2 will depend not only on the extent and duration of any immunity conferred and the performance of a test, but also upon a good understanding of the meaning of tests results among those offered them. first, the use of the term immunity should be avoided in phrases to describe antibody tests, whether described as passports, certificates or tests. second, research is needed to evaluate different ways of informing those offered tests and receiving tests results to minimise the proportion erroneously perceiving an antibody-positive test result to mean no risk of becoming infected with the virus. it should also focus on maximising understanding that -regardless of antibody-status -anyone can indirectly transmit the virus by touching a contaminated surface and infecting the next surface they touch. hand washing or sanitising therefore need to remain frequent. research is also needed with those undergoing actual tests, powered to detect effects judged meaningful in the context of a population-based testing programme and involving measures of actual behaviour. interest in sars-cov-2 antibody testing is high -across many countries, employers and populations. while such testing could contribute to wider strategies to ease lock-down restrictions, their use may have an adverse impact on transmission-related behaviour. this appears to vary with the way the tests are described. using the term immunity (vs antibody) to describe antibody tests increases the proportion of people believing that an antibodypositive result means they have no future risk of coronavirus, a perception that may be associated with less frequent handwashing and hence increased risk of transmission. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 10, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 10, 2020. scientists are developing tests to see who has already had coronavirus. no test is 100% effective. this means that those who test 'positive' would have: • lower risk of catching coronavirus in the future -and therefore also • lower risk of passing it on to others those who test 'positive' would get an immunity passport. they could return to work early. scientists are developing tests to see who has already had coronavirus. no test is 100% effective. this means that those who test 'positive' would have: • lower risk of catching coronavirus in the future -and therefore also • lower risk of passing it on to others those who test 'positive' would get an immunity certificate. they could return to work early. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 10, 2020. scientists are developing tests to see who has already had coronavirus. no test is 100% effective. this means that those who test 'positive' would have: • lower risk of catching coronavirus in the future -and therefore also • lower risk of passing it on to others those who test 'positive' would get a result showing immunity. they could return to work early. scientists are developing tests to see who has already had coronavirus. no test is 100% effective. this means that those who test 'positive' would have: • lower risk of catching coronavirus in the future -and therefore also • lower risk of passing it on to others those who test 'positive' would get an antibody passport. they could return to work early. scientists are developing tests to see who has already had coronavirus. no test is 100% effective. this means that those who test 'positive' would have: • lower risk of catching coronavirus in the future -and therefore also • lower risk of passing it on to others those who test 'positive' would get an antibody certificate. they could return to work early. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 10, 2020. scientists are developing tests to see who has already had coronavirus. no test is 100% effective. this means that those who test 'positive' would have: • lower risk of catching coronavirus in the future -and therefore also • lower risk of passing it on to others those who test 'positive' would get a result showing antibodies. they could return to work early. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 10, 2020. oxford covid-19 government response tracker, blavatnik school of government world health organization. critical preparedness, readiness and response actions for covid-19. world health organization disease control, civil liberties, and mass testing -calibrating restrictions during the covid-19 pandemic immunity passports' could speed up return to work after covid-19. the guardian delta's ceo said he would support an 'immunity passport' program or other steps to jumpstart travel as the airline reports its first quarterly loss in more than 5 years. business insider people could be given coronavirus 'immunity certificates' to leave lockdown early. the independent mass coronavirus antibody tests have serious limits. bloomberg.com [internet coronavirus immunity cards for americans are 'being discussed'. politico chile to push ahead with coronavirus 'release certificates' despite who warning. reuters evaluation of antibody testing for sars-cov-2 using elisa and lateral flow immunoassays. medrxiv prepr what policy makers need to know about covid-19 protective immunity. the lancet understanding sensitivity and specificity with the right side of the brain numbers or words? a randomized controlled trial of presenting screen negative results to pregnant women women's understanding of a "normal smear test result": experimental questionnaire based study immunity passports' in the context of covid-19 factorial versus multi-arm multi-stage designs for clinical trials with multiple treatments fuzzy' virus: indeterminate influenza biology, diagnosis and surveillance in the risk ontologies of the general public in time of pandemics verbal and numerical expressions of probability: 'it's a fifty-fifty chance'. organ behav hum decis process the impact of genetic testing for crohn's disease, risk magnitude and graphical format on motivation to stop smoking: an experimental analogue study effect of communicating dna based risk assessments for crohn's disease on smoking cessation: randomised controlled trial office for national statistics we thank steve reicher for comments on an earlier draft of the study protocol . cc-by-nc-nd 4.0 international license it is made available under a all authors have completed the unified competing interest form (available on request from the corresponding author) and declare: no support from any organisation for the submitted work; and no financial relationships with any organisations that might have an interest in the submitted work in the previous three years. hwwp declares consultancy fees from babylon health; all authors declare no other relationships or activities that could appear to have influenced the submitted work. the authors affirm that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as originally planned have been explained. anonymised data will be made available upon reasonable request. the study was conceptualised by tmm, jw & gjr. am completed data collection. jw & hp analysed the data. all authors contributed to, and approved, the final manuscript. key: cord-320864-k9zksbyt authors: remes-troche, j. m.; valdovinos-diaz, m. a.; viebig, r.; defilippi, c.; bustos-fernández, l. m.; sole, l.; hani-amador, a. c. title: recommendations for the reopening and activity resumption of the neurogastroenterology units in the face of the covid-19 pandemic. position of the sociedad latinoamericana de neurogastroenterología date: 2020-11-01 journal: nan doi: 10.1016/j.rgmxen.2020.07.004 sha: doc_id: 320864 cord_uid: k9zksbyt the covid 19 pandemic has forced the establishment of measures to avoid contagion during diagnostic and therapeutic tests in gastroenterology. gastrointestinal motility studies involve a high and intermediate risk of transmission of infection by this virus. given its elective or non-urgent indication in most cases, we recommend deferring the performance of these tests until there is a significant control of the infection rate in each country, during the pandemic. when health authorities allow a return to normalcy and in the absence of effective treatment or a preventive vaccine for covid 19 infection, we recommend a strict protocol to classify patients according to their infectious-contagious status through the appropriate use of tests to detect the virus and its immune response, as well as the use of protective measures to be followed by health personnel to avoid contagion during the performance of a gastrointestinal motility test. from the very start of the outbreak, sars-cov-2, or covid-19 infection, has been highly contagious. the virus spread rapidly across the globe, and by may 2020, infected more than 5 million persons in 188 countries. 1 due to its high infection and fatality rates, along with the lack of prior immunity, this new infection has been perceived as a great threat to the life and health of the worldwide human population. the first case of covid-19 in latin america was reported in brazil on february 26, 2020 , and the first death on march 7 in argentina. 2 in mexico, the first case was reported on february 25 and the first death on march 18, and in colombia, the first case was reported on march 6 and the first death of a physician on april 11. 3 thus, at the time of this writing (may 2020), latin america is considered to be the epicenter of the pandemic. confronted with that situation, and following the recommendations of the world health organization (who), the governments of the latin american countries have declared health emergencies and implemented actions (on different dates and in accordance with the epidemiologic trend of each country), such as restrictions in the public, private, and social sectors that include voluntary quarantining, shutting down schools, closing borders, suspending international air travel, carrying out physical distancing, and limiting nonessential activities. as in other parts of the world, medical care has changed dramatically in relation to non-urgent diseases that involve the performance of diagnostic and therapeutic procedures, such as those carried out at neurophysiology and/or digestive motility units. positions have already been established on how to work and/or resume activities at those units (e.g., those issued by the american neurogastroenterology and motility society [anms] 4 and the grupo español de motilidad digestiva [gemd]) 5 but due to the fact that the epidemiologic behavior, protective equipment avail-ability, serologic diagnostic test performance capacity for corroborating immunity, and socioeconomic context are different throughout latin america, a group of experts that are members of the sociedad latinoamericana de neurogastroenterología (slng) had a virtual meeting to formulate a consensus document with recommendations for the performance of gastrointestinal motility tests. the present document establishes a series of guidelines for prioritizing, selecting, and performing the most frequently indicated neurogastroenterology procedures at digestive units in the face of covid-19, such as manometry and esophageal reflux measurement, anorectal manometry and biofeedback, and breath tests. summoned by the presidency and scientific committee of the slng, a group of experts in the area of neurogastroenterology from several latin american countries had a virtual meeting on april 15, 2020, to formalize the creation of a document that would serve as a guide on how to resume and perform gastrointestinal motility procedures in the scenario of the covid-19 pandemic. at that first reunion, three working groups were organized to revise and establish the recommendations for the areas of: 1) esophageal function tests, 2) anorectal function tests, and 3) breath tests. the recommendations established were based on currently available recommendations, consensuses, and evidence, making the necessary adjustments according to the settings of each latin american country during the different phases of the pandemic. two other virtual meetings were carried out to analyze, revise, and modify the recommendations of each working group. the final virtual meeting took place on may 27, 2020, at which the seven members of the expert group unanimously approved the recommendations that follow below. high risk: due to the nature of the virus and its transmission route, it is clear that all aerosol-generating procedures involve a very high risk of infection for the personnel exposed to them. therefore, esophageal manometry, impedance ph monitoring, wireless capsule ph monitoring, antroduodenal manometry, and breath tests are the gastrointestinal motility tests that should be considered high risk. intermediate risk: the fact that the nucleocapsid protein of the virus has been detected in gastrointestinal epithelial cells and rna of the virus has been found in stool points to the possibility of fecal transmission of sars-cov-2. 6, 7 in a meta-analysis on the subject, tian et al. 6 showed that fecal pcr became positive two to five days later than positive sputum pcr in 36-53% of the infected patients, and the fecal excretion of viral particles persisted up to 11 days after sputum excretion in 23-82% of the patients. in another metaanalysis, cheung et al. 7 estimated that the prevalence of viral rna in stool in patients with covid-19 was 48.1% (95% ci 38.3---57.9), reporting the case of a 78-year-old patient in whom viral rna persisted up to 33 days. importantly, the fact that viral particles are detected in stool does not necessarily mean there is transmission by that route, but in the face of uncertainty and following the colonoscopy guidelines, anorectal manometry (arm), biofeedback, balloon expulsion test, electromyography, colonic manometry, and barostat study should be considered procedures of intermediate risk. 8 colonic transit measurements utilizing radiopaque markers, or a wireless motility capsule, are considered noninvasive procedures but involve exposure to subjects that could be asymptomatic virus carriers. thus, we suggest that those tests also be considered intermediate risk. strictly speaking, it must be understood that none of the abovementioned procedures can be considered urgent to the extent that their performance would be required during the epidemic phase of the covid-19 pandemic. according to numerous guidelines and recommendations, postponing all ''elective'' motility procedures should be managed in the context of the clinical indication and should be adapted to the reality of each latin american country. the dialogue between physicians and patients is encouraged for explaining and understanding the situation. the reopening of the motility laboratories in each latin american country will depend on several factors, including: a) the epidemiologic situation of the region. that situation is established according to the health authority of each country, considering the phases (table 1 ) determined by the who 9 for the covid-19 pandemic: • phase 1 (case importation): in this first setting, the disease arrives at a country through one person or a small number of people that acquire the virus abroad, thus the number of cases is limited to a few dozen. • phase 2 (community contagion): for this stage of the pandemic, outbreaks of the disease begin to occur in persons that have not been traveling. the first persons with covid-19 that arrived at the country infected others they came in contact with, and in turn, those persons continue to propagate the disease. confirmed cases begin to surpass the hundreds and containment becomes more complicated. • phase 3 (epidemic contagion): this is the most critical stage in the advancing of an epidemic because it means the disease is now present in the entire country and there is an elevated number of community outbreaks (thousands of persons). the main risk is that the number of patients increases exponentially, overloading the healthcare facilities and medical services. the who also recognizes other possible settings, once the situation begins to stabilize and the contagion curve begins to flatten. • phase 4 (second wave): once local contagion is reduced, imported cases are likely to present again, producing a second wave of infected patients. that can occur three to nine months after phase 3 has ended. • phase 5 (end of the epidemic): the who is in charge of declaring the end of the pandemic once the majority of countries are safe, with contagion under control. in countries with adequate epidemiologic surveillance systems, the declaration of phases is established, based on the number of new infections expected to occur as a result of infection by a single individual (r0), which expresses the speed at which the disease can propagate in a given population. 10 if the r0 is close to a value of 2, the country is experiencing exponential growth. an r0 of 1 indicates that the infection rate is remaining constant, and when it is below 1 (e.g., two infected persons would infect fewer than two individuals), the number of infected persons is decreasing. b) availability of personal protective equipment. obviously, the risk of infection in healthcare professionals is higher if they do not have the adequate personal protective equipment (ppe) (see further ahead). however, it is important to recognize that there is a worldwide shortage of material, resulting in possibly limited availability in some of the latin american countries. in fact, the inter-american society of digestive endoscopy 11 and the asian-pacific society for digestive endoscopy (apsde-covid declarations) 12 issued their recommendations based on ppe availability. we believe that could play a key role in the reopening of gastrointestinal physiologic procedures in our region (table 1) . c) type of institution at which motility studies will be performed: outpatient centers that specialize in those techniques have a lower risk than hospitals or clinics because they have a lower circulation of patients. such centers also aid in decongesting the demand at hospitals. once the return to activities or the reopening of activities is being contemplated, it is important to consider each of the following aspects: according to the gemd, 5 at present there is no conclusive scientific evidence that supports performing microbiologic tests before a motility procedure to diagnose sars-cov-2 for the purpose of modifying the protective measures related to the infection. however, it is important to recognize that increasingly more subjects infected with sars-cov-2 are recovering, but there is also a high number of subjects that can be asymptomatic carriers. rt-pcr testing determines if the patient is infected, whereas serologic testing (igm and igg against sars-cov-2) determines the immune status. depending on the availability of those tests in each country, their performance before the patient undergoes neurophysiologic studies is recommended. in line with that, patients indicated for a digestive motility study should be classified as follows: in cases 1 and 2, the motility test will not be programmed and should be re-scheduled for at least four weeks later (fig. 1) . corroboration of active infection resolution (a negative rt-pcr 24−48 h before the motility study) is recommended. in case 3, the motility test can be performed with no problem. importantly, availability of and access to those serologic and molecular biologic tests can vary between the latin american countries. therefore, in treating case 4 patients, a potential risk of infection must be assumed, and the motility test must be performed utilizing all the ppe (that scenario is probably the most common in our countries). it is very important to take into account that even though the rt-pcr test is the ''gold standard'', there is variability in its diagnostic accuracy. for example, false negatives have been reported at 30%, if the test is performed during the first days of infection or the pre-symptomatic phase. 13 therefore, the recommendations must be adjusted to the sensitivity and specificity of each test and the laboratories that perform those assays must be encouraged to use the commercial tests that have shown the best diagnostic accuracy. the esophageal physiologic studies, including esophageal manometry (conventional or high resolution), 24-h ph study, or ph-impedance study are diagnostic tests usually performed on ambulatory patients in the context of gastroesophageal reflux symptoms or nonobstructive dysphagia. they have a broad spectrum of clinical indications that includes the evaluation of antisecretory treatmentrefractory esophageal symptoms (dysphagia, regurgitation, or chest pain) not explained by upper gastrointestinal endoscopy assessment, the evaluation of esophageal motor function prior to antireflux surgery, the evaluation of persistent reflux symptoms despite medical therapy, or the development of postoperative dysphagia. 14, 15 in general, they are elective tests that in exceptional cases can have an urgent indication (table 1) . certain medical indications can have a degree of urgency, as recently described by the anms. 4 the presence of probable achalasia with severe symptoms (significant dysphagia making oral food intake and hydration impossible), the presence of large hiatal hernias (with risk of aspiration or volvulus), or the impossibility of maintaining oral hydration and nutrition are relatively urgent, or semi-urgent, indications. in the case of giant hiatal hernias, if manometry cannot be carried out due to the covid-19-related limitations, surgery should be performed without previous manometry. all other indications, such as dysphagia with no weight loss, reflux studies prior to antireflux surgery, studies due to refractory reflux symptoms, and suspicion of supragastric belching or rumination syndrome, can be postponed. 16 for as long as there is no return to performing functional tests, the recommendation is to support achalasia diagnosis or dysphagia evaluation through barium swallow tests, which can be useful. regarding patients with reflux symptoms, continuing, changing, or adjusting the medication dose is recommended during the time in which it is not possible to complete their diagnostic evaluation. according to the international anorectal physiology working group (iapwg) and the london classification, the conventional indications for performing anorectal function tests (primarily anorectal manometry [arm]) 17 are: 1) constipation and/or defecation disorder symptom evaluation, 2) fecal incontinence (fi) evaluation, 3) painful anorectal disorder evaluation, 4) preoperative and postoperative evaluation of ileorectal anastomoses, rectopexy, fistulotomies, etc., and 5) evaluation of obstetric trauma. it should also be pointed out that arm, in addition to having a diagnostic purpose, is utilized in many centers for biofeedback therapy in patients with constipation and/or fecal incontinence. in that respect, and reviewing the indications, we emphasize the fact that anorectal function tests are not urgent procedures, and thus should be postponed during the exponential phase of the pandemic. when laboratories are reopened, certain semi-urgent situations should be prioritized (table 1) . returning first to the performance of arm in patients that had already received biofeedback therapy or had been programmed for it before the pandemic, is sustained by ia and iib grades of evidence in the management of constipation and fi, respectively, according to the anms. 18 in addition, its resumption in patients with fi is supported by the fact that arm provides a pathophysiologic approach in more than 90% of cases. for example, sphincter hypertonia (low resting baseline pressure) is associated with passive fi, 19, 20 whereas hypocontractility (the inability to reach an increase in pressure during voluntary contraction) suggests that fi, specifically urge fi, can be secondary to external anal sphincter (eas) lesions. in a prospective study conducted by rao et al., arm not only confirmed the clinical impression but also contributed new clinically undetected information on patients with fi and influenced the treatment decision in the majority of cases. 21 until the return to normality, recommendations for patients that have not completed their biofeedback sessions include: continue, at home, carrying out the pelvic floor exercises learned during training or use the electronic devices. 22 if patients had learned how to perform those types of exercises before the pandemic, they should continue doing them on a regular basis. with respect to constipation and chronic proctalgia, we recognize that they considerably compromise patient quality of life, but given their chronic nature, we feel that the performance of arm can wait, as long as patients are offered symptomatic medical treatment (laxatives, antispasmodics, etc.) to mitigate symptom intensity. if defecatory dyssynergy is suspected in relation to inappropriate straining, abdominal breathing exercises can be useful. 23 if defecation alterations related to posture are suspected, its correction, including the use of a device that favors the opening of the anal right angle (a 6-inch-high footstool to favor knee flexion) can also be helpful. 24 the performance of other anorectal tests involving the placement or manipulation of probes or devices in the anorectum, e.g., surface electromyography, barostat, or pudendal nerve latency, should follow the same recommendations for arm. none of those tests are considered urgent and they should be programmed once neurogastroenterology units have returned to normality. regarding colonic transit with radiopaque markers or with a wireless capsule, even though they do not involve an aerosol-generating process, they should be postponed, given that patients and physicians are frequently going to the health units for study follow-up and surveillance. finally, colonic manometry, which requires colonoscopy-assisted probe placement, is considered a procedure with intermediate risk that should be postponed until the final stage of the pandemic. breath tests are studies that are widely used to evaluate different function alterations, such as bacterial overgrowth and intolerance to different carbohydrates (mainly lactose), and are the standard for corroborating eradication of helicobacter pylori infection. 25, 26 they are programmed studies and are not to be considered a medical emergency under any circumstance during the covid-19 pandemic. even though the filter mouthpieces of some systems incorporate a unidirectional valve and an infection control filter that have been shown to eliminate 99% and 96.5% of the bacteria and viruses in the air, respectively, specific tests have not been carried out to demonstrate whether they are capable of preventing sars-cov-2 infection. the general steps to follow are detailed below, taking into account that there can be certain variations according to the test that is performed. 4, 5 patient preparation a) a telephone interview with the patient should be carried out 24 h before a motility test to identify symptoms consistent with covid-19 (cough, fever, myalgia, anosmia, ageusia, diarrhea). if any of those symptoms are identified, the recommendation is to cancel the appointment and postpone it until the case is reevaluated. the patient should be sent to a service (clinic, emergency room, infectious diseases unit, etc.) that can perform a rt-pcr test and provide appropriate management. that recommendation will depend on the protocol for covid-19 care established in each country. b) patients should preferably go to their appointment alone, but if not possible, be accompanied by only one person, ideally under 65 years of age. the patient and companion should each be given a facemask if they entered the unit without one. the patient and companion should then apply hand sanitizer or wash their hands. c) the companion should not enter the unit, unless the patient requires specific assistance, and should stay in the waiting room. d) before entering the room in which the procedure will be performed, all patients must be asked again about the presence of respiratory symptoms or fever, to stratify their transmission risk, and their body temperature should be measured. if there is any suspicion, the procedure should be canceled and rescheduled. during the procedure a) personnel care • promote the application of basic hygiene measures, among the entire personnel, for the prevention of infection. • given that the large majority of latin american countries are in a community transmission phase, in which spread via asymptomatic individuals is reported, ppe use by all the healthcare personnel involved in the procedures is recommended. • fig. 2 shows the ppe recommended for each procedure. • the procedure should not be performed if the ppe necessary for guaranteeing the performance safety of the manometric studies is not available. • learn how to adequately put on and take off all ppe. b) equipment care and use recommendations • equipment preparation and probe calibration should be performed before the patient is admitted to the procedure room, to decrease the amount of exposure time. • if informed consent is utilized, promote the use of ''verbal or recorded'' consent, when permitted by local committees. otherwise, consider disinfecting the material involved (ballpoint pens or pencils) and insist on handwashing after contact with said material. • the patient will be asked to enter the procedure room with no personal belongings (cellphone, glasses, keys, etc.). • regarding arm, the patient will change clothes in a specific bathroom (whose cleaning should follow the specific protocol for covid-19). • all the material utilized (syringes, trays in case of vomiting) should be disposable. • the test should be performed by an expert and not more than 2 persons are recommended to be in the room during the procedure. • the manometric system, as well as the computer keyboard, can be covered in plastic film during each examination. • with respect to ph study and ph/impedance, consider whether it is possible to favor the use of disposable or single-use probes. • when using high-resolution solid-state equipment without impedance, consider whether utilizing a disposable case could be useful. • with respect to old model ph study equipment that has a leather carrying case, an alternative could be a plastic bag to cover the case and prevent damage during post-examination disinfection. • utilizing the chicago protocol and avoiding unnecessary maneuvers that prolong study duration are recommended. • to the degree possible, if there is more than one procedure room, studies could be carried out in alternating rooms to provide sufficient time for sanitization, if more than 2 studies are to be carried out per day. • if fecal material is encountered during rectal examination, and prior to the introduction of the manometry probe, an enema is customarily given, waiting 30 min to perform the test. in the context of the covid-19 pandemic, we do not recommend that measure because it can increase the risk for exposure. if an enema is to be used, it can be applied at home, before the performance of the motility study. • the london protocol is recommended because the duration of the test, which should be performed by an expert, is 15 min. • the use of high-resolution systems is promoted, given that perfusion systems have the disadvantage of the probes needing constant water perfusion, resulting in the continuous outflow of water into the anal canal, increasing the risk for contact with body fluids. • systems that have a disposable case for the probe are recommended because they are safer for the patient. • if a balloon expulsion test is to be performed at the end of the study, a disposable probe is recommended. the post-procedure measures are subject to constant review, depending on the overall situation of each hospital, daily necessities, and material availability, and are adapted to them and to the recommendations of the acting authorities in each country. • carry out the disinfecting and reprocessing of the probes according to the customary protocol. • do not reuse single-use devices. • assign cleaning personnel that exclusively work at the physiology unit. • apply the protocols for the cleaning and disinfecting of materials that come into contact with patients or their secretions, such as the examining table, keyboard, and screens. • disinfecting and cleaning are to be performed with a disinfectant included in the institutional cleaning and disinfection policy. the covid-19 virus is inactivated after five minutes of contact with disinfectants such as bleach, alcohol at 70%, and a sodium hypochlorite solution containing 1000 ppm of active chloride. • manage residuals following the local protocols of each center for category b (un3291) high-capacity ineffective material. • maintain physical distancing of 1---2 meters, basic hygiene measures, and the independent flow of patients in the recovery rooms. • consider the implementation of patient follow-up programs 7---15 days after the procedure, to evaluate the appearance of symptoms consistent with sars-cov-2 infection. • promote the delivery of unprinted reports and recommendations, i.e., making them online. the covid-19 pandemic has forced the establishment of measures to prevent contagion during the performance of therapeutic and diagnostic tests in gastroenterology. digestive tract motility tests involve intermediate and high risks for transmission of covid-19 infection. given their elective and nonurgent indication in the majority of cases, we recommend postponing those tests until there is significant control of the infection rate in each latin american country during the pandemic. when the health authorities allow the return to normality, and in the absence of an effective treatment for covid-19 infection, or a preventive vaccine, we recommend a strict protocol for classifying patients according to their infectious-contagious status through the appropriate use of tests for detecting the virus and the immune response to it, as well as the use of protection measures that the healthcare personnel should follow to prevent contagion during the performance of a gastrointestinal motility test. finally, we recognize that the recommendations contained herein can change in the future, as more evidence with respect to safety measures is being produced. no financial support was received in relation to the present article. covid-19 in latin america: the implications of the first confirmed case in brazil american neurogastroenterology and motility society (anms) task force recommendations for resumption of motility laboratory operations during the covid-19 pandemic recomendaciones de asenem para la reanudación de la actividad en los laboratorios de motilidad digestiva durante la pandemia por covid-19 review article: gastrointestinal features in covid-19 and the possibility of faecal transmission gastrointestinal manifestations of sars-cov-2 infection and virus load in fecal samples from a hong kong cohort: systematic review and meta-analysis recomendaciones generales de la asociación española de gastroenterología (aeg) y la sociedad española de patología digestiva (sepd) sobre el funcionamiento en las unidades de endoscopia digestiva y gastroenterología con motivo de la pandemia por sars-cov-2 world health organization. coronavirus disease (covid-19) pandemic el número reproductivo básico (r0): consideraciones para su aplicación en la salud pública recomendaciones para las unidades de endoscopia durante la pandemia de coronavirus (covid-19). version 3.1 español. 2020 practice of endoscopy during covid-19 pandemic: position statements of the asian pacific society for digestive endoscopy variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure utility of esophageal high-resolution manometry in clinical practice: first, do hrm clinical characteristics and outcomes of patients with postfundoplication dysphagia recommendations for essential esophageal physiologic testing during the covid-19 the international anorectal physiology working group (iapwg) recommendations: standardized testing protocol and the london classification for disorders of anorectal function anms-esnm position paper and consensus guidelines on biofeedback therapy for anorectal disorders anorectal function investigations in incontinent and continent patients. differences and discriminatory value relationship between symptoms and disordered continence mechanisms in women with idiopathic faecal incontinence long-term outcome and objective changes of anorectal function after biofeedback therapy for faecal incontinence randomized controlled trial of biofeedback for fecal incontinence effectiveness of pelvic physiotherapy in children with functional constipation compared with standard medical care influence of a defecation posture modification device (squatty potty ® ) in healthy volunteers and dyssynergic patients acg clinical guideline: small intestinal bacterial overgrowth hydrogen and methane-based breath testing in gastrointestinal disorders: the north american consensus jose maría remes troche is a member of the advisory board of takeda, asofarma, and biocodex. he has given talks for takeda, asofarma, medtronic, carnot, and alfasigma.miguel ángel valdovinos díaz is a member of the advisory board of takeda. he has given talks for takeda, asofarma, medtronic, carnot, and grünenthal.laura sole has given talks for roemmers, casasco, asofarma, temis lostaló, and raffo albis cecilia hani amador has given talks for medtronic, takeda, abbott, biopas, and astra zeneca.claudia defilippi has given talks for pharma investi, ferrer, and axon pharma.luis maría bustos fernández declares that he has no conflict of interest.ricardo veibig declares that he has no conflict of interest. key: cord-312477-2y88gzji authors: mlcochova, p.; collier, d.; ritchie, a. v.; assennato, s. m.; hosmillo, m.; goel, n.; meng, b.; chatterji, k.; mendoza, v.; temperton, n.; kiss, l.; ciazyns, k. a.; xiong, x.; briggs, j. a.; nathan, j.; mescia, f.; zhang, h.; barmpounakis, p.; demeris, n.; skells, r.; lyons, p.; bradley, j.; baker, s.; lee, h. h.; smith, k. g.; goodfellow, i.; gupta, r. k. title: combined point of care nucleic acid and antibody testing for sars-cov-2: a prospective cohort study in suspected moderate to severe covid-19 disease. date: 2020-06-18 journal: nan doi: 10.1101/2020.06.16.20133157 sha: doc_id: 312477 cord_uid: 2y88gzji abstract background rapid covid-19 diagnosis in hospital is essential for patient management and identification of infectious patients to limit the potential for nosocomial transmission. the diagnosis is complicated by 30-50% of covid-19 hospital admissions with negative nose/throat swabs negative for sars-cov-2 nucleic acid, frequently after the first week of illness when sars-cov-2 antibody responses become detectable. we assessed the diagnostic accuracy of combined rapid antibody point of care (poc) and nucleic acid assays for suspected covid-19 disease in the emergency department. methods we developed (i) an in vitro neutralization assay using a lentivirus expressing a genome encoding luciferase and pseudotyped with spike protein and (ii) an elisa test to detect igg antibodies to nucleocapsid (n) and spike (s) proteins from sars-cov-2. we tested two promising candidate lateral flow rapid fingerprick test with bands for igg and igm. we then prospectively recruited participants with suspected moderate to severe covid-19 and tested for sars-cov-2 nucleic acid in a combined nasal/throat swab using the standard laboratory rt-pcr and a validated rapid nucleic acid test. additionally, serum collected at admission was retrospectively tested by in vitro neutralization, elisa and the candidate poc antibody tests. we determined the sensitivity and specificity of the individual and combined rapid poc diagnostic tests against a composite gold standard of neutralisation and the standard laboratory rt-pcr. results 45 participants had specimens tested for nucleic acid in nose/throat swabs as well as stored sera for antibodies. serum neutralisation assay, sars-cov-2 spike igg elisa and the poc antibody test results were concordant. using the composite gold standard, prevalence of covid-19 disease was 53.3% (24/45). median age was 73.5 (iqr 54.0-86.5) years in those with covid-19 disease by our gold standard and 63.0 (iqr 41.0-72.0) years in those without disease. median duration of symptoms was 7 days (iqr 1-8) in those with infection. the overall sensitivity of rapid naat diagnosis was 79.2% (95ci 57.8-92.9%) and 50.0% (11.8-88.2) at days 8-28. sensitivity and specificity of the combined rapid poc diagnostic tests reached 100% (95ci 85.8-100) and 94.7% (95ci 74.0-99.0) overall. conclusions dual point of care sars-cov-2 testing can significantly improve diagnostic sensitivity, whilst maintaining high specificity. rapid combined tests have the potential to transform our management of covid-19, including inflammatory manifestations where nucleic acid test results are negative. a rapid combined approach will also aid recruitment into clinical trials and in prescribing therapeutics, particularly where potentially harmful immune modulators (including steroids) are used. rapid covid-19 diagnosis in hospital is essential for patient management and identification of infectious patients to limit the potential for nosocomial transmission. the diagnosis is complicated by 30-50% of covid-19 hospital admissions with negative nose/throat swabs for sars-cov-2 nucleic acid, frequently after the first week of illness when sars-cov-2 antibody responses become detectable. we assessed the diagnostic accuracy of combined rapid antibody point of care (poc) and nucleic acid assays for suspected covid-19 disease in the emergency department. we developed (i) an in vitro neutralization assay using a lentivirus expressing a genome encoding luciferase and pseudotyped with spike protein and (ii) an elisa test to detect igg antibodies to nucleocapsid (n) and spike (s) proteins from sars-cov-2. we tested two promising candidate lateral flow rapid fingerprick test with bands for igg and igm. we then prospectively recruited participants with suspected moderate to severe covid-19 and tested for sars-cov-2 nucleic acid in a combined nasal/throat swab using the standard laboratory rt-pcr and a validated rapid nucleic acid test. additionally, serum collected at admission was retrospectively tested by in vitro neutralization, elisa and the candidate poc antibody tests. we determined the sensitivity and specificity of the individual and combined rapid poc diagnostic tests against a composite 'gold' standard of neutralisation and the standard laboratory rt-pcr. results 45 participants had specimens tested for nucleic acid in nose/throat swabs as well as stored sera for antibodies. serum neutralisation assay, sars-cov-2 spike igg elisa and the poc antibody test results were concordant. using the composite gold standard, prevalence of covid-19 disease was 53.3% (24/45). median age was 73.5 (iqr 54.0-86.5) years in those with covid-19 disease by our gold standard and 63.0 (iqr 41.0-72.0) years in those without disease. median duration of symptoms was 7 days (iqr [1] [2] [3] [4] [5] [6] [7] [8] in those with infection. the overall sensitivity of rapid naat diagnosis was 79.2% (95ci 57.8-92.9%). sensitivity and specificity of the combined rapid poc diagnostic tests reached 100% (95ci 85. and 94.7% (95ci 74.0-99.0) overall. dual point of care sars-cov-2 testing can significantly improve diagnostic sensitivity, whilst maintaining high specificity. rapid combined tests have the potential to transform our management of covid-19, including inflammatory manifestations where nucleic acid test results are negative. a rapid combined approach will also aid recruitment into clinical trials and in prescribing therapeutics, particularly where potentially harmful immune modulators (including steroids) are used. as of the 5 th of june 2020, 6.7 million people have been infected with sars-cov-2 with over 390 000 deaths [1] . the unprecedented numbers requiring sars-cov-2 testing has strained healthcare systems globally. there is currently no gold standard for diagnosis of covid-19. detection of sars-cov-2 by nucleic acid amplification testing (naat), is largely done by real time rt-pcr on nose/throat swabs in centralised laboratories. rt-pcr specimens need to be handled in biosafety level 3 category laboratory (bsl3) and then batch analysed. given these bottlenecks, the turnaround time for this test is in the order of 2-4 days [2] . naat tests from a single nose/throat swab are negative in up to 50% in patients who have ct changes consistent with covid-19 and/or positive antibodies to sars-cov-2 [3] [4] [5] . the lack of detectable virus in upper airway samples is not only a serious barrier to making timely and safe decisions in the er, but also leads to multiple swab samples being sent, frequently from the same anatomical site, leading to strain on virology laboratories. additionally, recruitment into clinical trials for covid-19 treatments has moved towards 'clinical diagnosis' for eligibility. multiple factors contribute to negative results by naat, including sampling technique and timing of the sampling in the disease course. the viral load in the upper respiratory tract frequently wanes by this point [6] and as seen in a case series from france, was undetectable in nose and throat swabs from 9 days of illness in 4 out of 5 patients [7] . similarly, a case series from germany found the detection rate by rt-pcr was <50% after 5 days since onset of illness [8] . a proportion of patients develop a secondary deterioration in clinical condition requiring hospitalisation and respiratory support, at a time when immune pathology is thought to be dominate rather than direct pathology related to viral replication [7, 9] . the antibody response to sars-cov-2 is detectable 6 days from infection [10] . antibody based diagnosis of covid-19 shows increasing sensitivity in the latter part of the disease course when naat testing on nose/throat samples is more likely to be negative [11] [12] [13] [14] . one study reported that combined lab based rt-pcr with lab based antibody testing could increase sensitivity for covid-19 diagnosis from 67.1% to 99.4% in hospitalised patients [15] . however, lab antibody testing also has a turnaround time of a day or more, and rapid diagnosis and triage of patients requiring hospitalisation is needed in order to avoid overwhelming the diagnostic and isolation capacities of hospitals, especially during periods when influenza is co-circulating. we previously evaluated the diagnostic accuracy of the samba ii sars-cov-2 rapid test compared with the standard laboratory rt-pcr and found similar accuracy and a turnaround time of 2-3 hours even in real world settings [2] . several studies have now performed headto-head comparisons of immuno-chromatographic lateral flow immunoassays (lfias) [12] [13] [14] 16] . these assays are cheap to manufacture and give a binary positive/negative result, thereby lending themselves well to point of care (poc) testing. however, they have variable performance and in general they are negative in the early phase of illness, but highly sensitive in the later stage of illness [12] [13] [14] 16] . in this study we evaluated the diagnostic performance of a poc combination comprising naat and lfa antibody testing against a composite gold standard of laboratory rt-pcr and a serum neutralisation assay. 45 prospectively recruited participants with suspected moderate to severe covid-19 disease had specimens tested for nucleic acid in nose/throat swabs as well as stored sera for antibodies. samples at hospital admission were collected at a median of 7 (iqr 7-13) days after illness onset. the sera from 42.2% (19/45) participants showed strong neutralising antibody response against sars-cov-2 spike protein pseudotyped virus infection in a neutralization assay ( figure 1a ). 26 participants' sera showed no neutralising response ( figure 1b) . the neutralisation ability of participants' sera was compared with an elisa igg assay (supplementary figure 1) detecting spike antibodies. figure 1c is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 18, 2020. . https://doi.org/10.1101/2020.06.16.20133157 doi: medrxiv preprint and procalcitonin were significantly higher in confirmed covid-19 patients and 'classical' chest radiograph appearances were more common in confirmed covid-19 patients (table1, p<0.001). however, 6/24 (25%) had normal or indeterminate chest radiographs in the confirmed covid-19 group. the overall positivity rate of the rapid nucleic acid test was 79.2% (95% ci 57.8-92.9), decreasing from 88.9% (95% ci 65.3-98.6) in days 1-7 of illness to 50.0% (95% ci 11.8-88.2) in days 8-28 of illness (table 2 ). when the covidix igg/igm rapid test was combined with naat, the positivity rate increased to 100% (95% ci 81.5-100) in days 1-7 of illness and 100% (95% ci 54.1-100) in days 8-28 of illness. specificity was 90.0% (95% three participants had stored samples available for testing at multiple time points in their illness ( figure 2 ). two individuals were sampled from early after symptom onset and the third presented three weeks into illness. in the first two ( figure 2a -f), we observed an increase in neutralisation activity over time that was mirrored by band intensities on the rapid poc antibody test. as expected igm bands arose early on with igg following closely. in the individual presenting 21 days into illness ( figure 2g -i), only igg was detected with the rapid poc antibody test and as expected band intensity did not increase with time. given the need for multiple options under current demand for such tests, we next decided to use an alternative rapid lfa antibody test in combination with the samba ii naat. surescreen sars-cov-2 igg/igm test (derby, uk) was recently validated with elisa igg and demonstrated a very good sensitivity and specificity profile compared to five other tests on stored sera from acute infection [17] . we compared elisa igg and serum neutralisation (table 2) . . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . https://doi.org/10.1101/2020.06.16.20133157 doi: medrxiv preprint here we have shown that naat testing with antibody detection can improve diagnosis of covid-19 in moderate to severe suspected cases, but more importantly that accurate diagnosis can be achieved with combined rapid tests. overall positivity in nose/throat swab samples was around 80% with naat testing alone and 100% with a combined approach of rapid naat testing and either of two fingerprick blood/serum rapid antibody tests. specificity of the combined approach was 85-95% overall. as expected, nucleic acid detection in nose/throat samples was highest in the first few days (100% for samba ii sars-cov-2 test in the first 3 days after symptom onset). conversely antibody detection by lfa increased over time. a strength of this study is the use of serum neutralisation, a phenotypic test for functionality of antibodies, as part of a composite gold standard for defining covid-19 disease. this assay was carefully validated against a recently described elisa method for sars-cov-2 igg detection that is now used globally [18] . we also demonstrated that sera from participants did not neutralise sars-cov-1. use of antibody tests for covid-19 diagnosis in hospitals have been limited for a number of reasons. firstly, we know from sars-cov-1 that previous humoral immunity to hcov oc43 and 229e can elicit a cross reactive antibody response to n of sars-cov-1 in up to 14% of people tested in cross-sectional studies [19] , and previous exposure to hcov can rarely elicit an antibody response cross reaction to the n and s proteins of sars-cov-2 [17, 20] . secondly, antibody tests do not achieve the same detection rates as nucleic acid based tests early in infection, as humoral responses take time to develop following viral antigenic stimulation. however, later in disease igg reaches 100% sensitivity by day 6 [10] and this is useful in cases with immune mediated inflammatory disease where rt-pcr on respiratory samples is often negative, for example in the recently described kawasaki-like syndrome named pims (paediatric inflammatory multi-system syndrome) [21] . ct scanning has previously been shown to be highly sensitive [5] , though few countries have the resources for large scale ct based screening. in our study chest radiographs were statistically more likely to show changes associated with covid-19, but a quarter of chest radiographs in the confirmed covid-19 group were normal or indeterminate. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . this study had limited numbers of participants, though patients were distributed well by symptom onset and part of a clinical trial with complete data. we tested stored sera rather than whole finger prick blood, though this was intentional given the caution needed in interpreting antibody tests and potential cross reactivity of antibodies. although sars-cov2 elisa testing of our pre 2020 sera did reveal occasional n and s reactivity to sars-cov-2 (supplementary table 2), these samples were negative on the rapid antibody testing. in light of our data, prospective evaluation on a finger prick sample is now warranted on a larger scale in patients with moderate to severe disease. at present we cannot speculate on the diagnostic accuracy of the antibody or naat tests in mild disease. we envisage a deployment approach whereby both test samples, finger prick blood and nose/throat swab, are taken at the same time on admission to hospital. the finger prick antibody test result is available within 15 minutes and is highly specific; therefore in an individual with classical features and a positive antibody test result can be acted upon confidently, for example movement to a covid-19 area, or recruitment into a clinical treatment study. the naat result following shortly after will assist in diagnosis for early infections where antibody testing is negative. naat is also expected to be more valuable than antibody tests in milder cases given severity appears to correlate with magnitude of antibody responses [17, 22] . a combined rapid testing approach may have significant benefits in low resource settings where centralised virology laboratories are scarce and the epidemic is expanding. in addition, it removes the need for repeated nose/throat swabbing which may generate aerosols and lead to transmission. we envisage the combined rapid testing approach being important for safe and quick patient recruitment to clinical trials for covid-19, specifically where potentially harmful treatments such as immune modulators are being tested. rapid combined tests could be transformative in diagnosis and management of moderate to severe covid-19 disease requiring hospitalisation, particularly as diverse manifestations of disease emerge. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . https://doi.org/10.1101/2020.06.16.20133157 doi: medrxiv preprint 293t cells were cultured in dmem complete (dmem supplemented with 100 u/ml penicillin, 0.1 mg/ml streptomycin, and 10% fcs). viral vectors were prepared by transfection of 293t cells by using fugene hd transfection reagent (promega) as follows. confluent 293t cells were transfected with a mixture of 11ul of fugene hd, 1ug of pcaggs_sars-cov-2_spike, 1ug of p8.91 hiv-1 gag-pol expression vector [23, 24] , and 1.5ug of pcsflw (expressing the firefly luciferase reporter gene with the hiv-1 packaging signal). viral supernatant was collected at 48 and 72h after transfection, filtered through 0.45um filter and stored at -80ë�c. the 50% tissue culture infectious dose (tcid 50 ) of sars-cov-2 pseudovirus was determined using using steady-glo luciferase assay system (promega). spike pseudotype assays have been shown to have similar characteristics as neutralization testing using fully infectious wild type sars-cov-2 [25] .virus neutralization assays were performed on 293t cell transiently transfected with ace2 and tmprss2 using sars-cov-2 spike pseudotyped virus expressing luciferase. pseudovirus was incubated with serial dilution of heat inactivated human serum samples from covid-19 suspected individuals in duplicates for 1h at 37ë�c. virus and cell only controls were also included. then, freshly trypsinized 293t ace2/tmprss2 expressing cells were added to each well. following 48h incubation in a 5% co 2 environment at 37â°c, the luminescence was measured using steady-glo luciferase assay system (promega). the 50% inhibitory dilution (ec 50 ) was defined as the serum dilution at which the relative light units (rlus) were reduced by 50% compared we developed an elisa targeting the sars-cov-2 spike and n proteins. trimeric spike protein antigen used in elisa assays consists of the complete s protein ectodomain with a c-terminal extension containing a tev protease cleavage site, a t4 trimerization foldon and . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . https://doi.org/10.1101/2020.06.16.20133157 doi: medrxiv preprint a hexa-histidine tag. the s1/s2 cleavage site with amino acid sequence prrar was replaced with a single arginine residue and stabilizing proline mutants were inserted at positions 986 and 987. spike protein was expressed and purified from expi293 cells (thermo fisher). n protein consisting of residues 45-365 was initially expressed as a his-tev-sumo-fusion. after ni-nta purification, the tag was removed by tev proteolysis and the cleaved tagless protein further purified on heparin and gel filtration columns. the elisas were in a stepwise process; a positivity screen was followed by endpoint titre as previously described [18] . briefly, 96-well eia/ria plates (corning, sigma) were coated with pbs or 0.1î¼g per well of antigen at 4â°c overnight. coating solution was removed, and wells were blocked with 3% skimmed milk prepared in pbs with 0.1% tween 20 (pbst) at ambient temperature for 1 hour. previously inactivated serum samples (56â°c for 1 hour) were diluted to 1:60 or serially diluted by 3-fold, six times in 1% skimmed milk in pbst. blocking solution was aspirated and the diluted sera was added to the plates and incubated for 2 hours at ambient temperature. diluted sera were removed, and plates were washed three times with pbst. goat anti-human igg secondary antibody-peroxidase (fc-specific, sigma) prepared at 1:3,000 in pbst was added and plates were incubated for 1 hour at ambient temperature. plates were washed three times with pbst. elisas were developed using 3,5,3â�²,5â�²-tetramethylbenzidine (tmb, thermoscientific); reactions were stopped after 10 minutes using 0.16m sulfuric acid. the optical density at 450 nm (od450) was measured using a spectramax i3 plate reader. the absorbance values for each sample were determined by subtracting od values from uncoated wells. all data analyses were performed using prism 8 version 8.4.2 (graphpad). this colloidal-gold lateral flow immunoassay is designed to detect igg and igm to sars-cov-2. it was used according to the manufacturer's instructions. 10âµl of serum was added to the test well followed by 2 drops of the manufacturer's proprietary buffer. results were read as the presence or absence of a colored band the results window as igm positive-control and igm test bands present, as igg positive-control and igg test bands present or negative-control band only. in order to rule out cross reactivity of this test with seasonal coronavirus antibodies we tested 19 stored specimens from before 2020, some of which had n and s protein sars-cov-2 cross reactivity (supplementary table 2) . for quantification of igg and igm band density in covidix 2019 ncov igg/igm test, high resolution images of completed poc antibody test cassettes were acquired using chemidoc mp imaging system (bio-rad) at 20min post-. cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . https://doi.org/10.1101/2020.06.16.20133157 doi: medrxiv preprint addition of the human serum. band intensities were analysed using image lab software (bio-rad). ltd, derby, uk) . this colloidal-gold lateral flow immunoassay is designed to detect igg and igm to sars-cov-2. it was used according to the manufacturer's instructions. 10âµl of serum was added to the test well followed by 2 drops of the manufacturer's proprietary buffer. results were read as the presence or absence of a colored band the results window as igm positive-control and igm test bands present, as igg positive-control and igg test bands present or negative-control band only. it has been previously validated against historical controls and in serum from confirmed pcr positive covid-19 cases [17] . the study participants were part of the covidx trial [2] , a prospective analytical study which this was later expanded to include any adult requiring hospital admission and who was symptomatic of sars-cov-2 infection, demonstrated by clinical or radiological findings. [2] . 48 participants who had available stored sera were included in this sub-study and underwent further antibody testing. the laboratory standard rt-pcr test, developed by public health england (phe), targeting the rdrp gene was performed on a combined nose/throat swab on parallel. this test has an estimated limit of detection of 320 copies/ml. samba ii sars-cov-2 testing was performed on a combined nose/throat swab collected by dry sterile swab and inactivated in a proprietary buffer at point of sampling. samba ii sars-cov-2 targets 2 genes-orf1 and the n genes and uses nucleic acid sequence based amplification to detect sars-cov-2 rna, with limit of detection of 250 copies/ml. the sensitivity and specificity of samba ii sars-cov-2 test and covidix sars-cov-2 igg/igm test or surescreen sars-cov-2 igg/igm test for diagnosing covid-19 were . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . https://doi.org/10.1101/2020.06.16.20133157 doi: medrxiv preprint calculated alone and then in combination along with binomial 95% confidence intervals (ci). a composite gold standard was used -standard lab rt-pcr and a neutralisation assay. descriptive analyses of clinical and demographic data are presented as median and interquartile range (iqr) when continuous and as frequency and proportion (%) when categorical. the differences in continuous and categorical data were tested using wilcoxon rank sum and chi-square test respectively. the correlation between elisa and neutralisation assay was determined using the pearson correlation coefficient. statistical analysis were conducted using stata (version 13), with additional plots generated using graphpad prism. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 18, 2020. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 18, 2020. comparison between ec50 dilution titre from neutralizing assay and positive/negative poc antibody test results (covidix sars-cov-2 igm igg test). n=44, p=0.0025. an interactive web-based dashboard to track covid-19 in real time rapid point of care nucleic acid testing for sars-cov-2 in hospitalised patients: a clinical trial and implementation study. medrxiv false-negative results of initial rt-pcr assays for covid-19: a systematic review. medrxiv detection of sars-cov-2 in different types of clinical specimens sensitivity of chest ct for covid-19: comparison to rt-pcr temporal dynamics in viral shedding and transmissibility of covid-19 clinical and virological data of the first cases of covid-19 in europe: a case series virological assessment of hospitalized patients with covid-2019 covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal antibody responses to sars-cov-2 in patients with covid-19 a preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in 238 admitted hospital patients evaluation of nine commercial sars-cov-2 immunoassays. medrxiv comparative assessment of multiple covid-19 serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings test performance evaluation of sars-cov-2 serological assays. medrxiv antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 antibody testing for covid-19: a report from the national covid scientific advisory panel. medrxiv comparative assessment of multiple covid-19 serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings a serological assay to detect sars-cov-2 seroconversion in humans false-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid enzyme-linked immunosorbent assay due to hcov-oc43 and hcov-229e rectified by western blotting with recombinant sars-cov spike polypeptide evaluation of commercial and automated sars-cov-2 igg and iga elisas using coronavirus disease (covid-19) patient samples an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study sars-cov-2 neutralizing antibody responses are more efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector full-length hiv-1 gag determines protease inhibitor susceptibility within in vitro assays measuring sars-cov-2 neutralizing antibody activity using pseudotyped and chimeric viruses . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity (which was not certified by peer review)the copyright holder for this prep this version posted june 18, 2020. . https://doi.org/10.1101/2020.06. 16.20133157 doi: medrxiv preprint spike protein pseudotyped viral particles were incubated with serial dilutions of heat inactivated human serum samples from covid-19 suspected individuals (#15,16,32) in duplicates for 1h at 37ë�c. 293t ace2/tmprss2 expressing cells were added to each well. following 48h incubation in a 5% co2 environment at 37â°c, the luminescence was measured using steady-glo luciferase assay system (promega). percentage of neutralization was calculated with non-linear regression, log (inhibitor) vs. normalized response using graphpad prism 8 (graphpad software, inc., san diego, ca, usa). (c) the 50% inhibitory dilution (ec50) was defined as the serum dilution at which the relative light units (rlus) were reduced by 50% compared with the virus control wells (virus + cells) after subtraction of the background rlus in the control groups with cells only. the ec50 values were calculated with non-linear regression, log (inhibitor) vs. normalized response using graphpad prism 8 (graphpad software, inc., san diego, ca, usa). . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity (which was not certified by peer review)the copyright holder for this prep this version posted june 18, 2020. . https://doi.org/10.1101/2020.06.16.20133157 doi: medrxiv preprint key: cord-349161-4899cq99 authors: whiting, penny f; sterne, jonathan ac; westwood, marie e; bachmann, lucas m; harbord, roger; egger, matthias; deeks, jonathan j title: graphical presentation of diagnostic information date: 2008-04-11 journal: bmc med res methodol doi: 10.1186/1471-2288-8-20 sha: doc_id: 349161 cord_uid: 4899cq99 background: graphical displays of results allow researchers to summarise and communicate the key findings of their study. diagnostic information should be presented in an easily interpretable way, which conveys both test characteristics (diagnostic accuracy) and the potential for use in clinical practice (predictive value). methods: we discuss the types of graphical display commonly encountered in primary diagnostic accuracy studies and systematic reviews of such studies, and systematically review the use of graphical displays in recent diagnostic primary studies and systematic reviews. results: we identified 57 primary studies and 49 systematic reviews. fifty-six percent of primary studies and 53% of systematic reviews used graphical displays to present results. dot-plot or box-andwhisker plots were the most commonly used graph in primary studies and were included in 22 (39%) studies. roc plots were the most common type of plot included in systematic reviews and were included in 22 (45%) reviews. one primary study and five systematic reviews included a probability-modifying plot. conclusion: graphical displays are currently underused in primary diagnostic accuracy studies and systematic reviews of such studies. diagnostic accuracy studies need to include multiple types of graphic in order to provide both a detailed overview of the results (diagnostic accuracy) and to communicate information that can be used to inform clinical practice (predictive value). work is required to improve graphical displays, to better communicate the utility of a test in clinical practice and the implications of test results for individual patients. readers of a research report evaluating a diagnostic test may wish to assess the test's characteristics (diagnostic accuracy) or evaluate the impact that its use has on diag-nostic decisions (predictive value) for individual patients. graphical displays of results of test accuracy studies allow researchers to summarise and communicate the key findings of their study. we discuss the types of graphical dis-play commonly encountered in primary diagnostic accuracy studies and systematic reviews of such studies, and systematically review the use of graphical displays in recent diagnostic systematic reviews and primary studies. table 1 defines the various measures of diagnostic accuracy used. primary studies figure 1 illustrates four types of graphical display commonly used to present data on diagnostic accuracy for primary diagnostic accuracy studies. we used data from a study of the biochemical tumour marker ca-19-9 antigen to diagnose pancreatic cancer to construct these graphs [1] . dot plots (figure 1a) and box-and-whisker plots (figure 1b ) dot plots are used for test results that take many values, and display the distribution of results in patients with and without the target condition. box and whisker plots summarise these distributions: the central box covers the interquartile range with the median indicated by the line within the box. the whiskers extend either to the mini-mum and maximum values or to the most extreme values within 1.5 interquartile ranges of the quartiles, in which case more extreme values are plotted individually [2] . sometimes an indication of the threshold used to define a positive test result is included, for example by adding a horizontal line or shading at the relevant point. such plots can be used to clearly summarise a large volume of data, but are only able to display differences in the distribution of test values between patients with and without the target condition; they do not directly display the diagnostic performance of the test. although the ca-19-9 antigen test to diagnose pancreatic cancer (used to construct figure 1 ) is an example of continuous data, it is also possible to construct similar graphs for categorical test results providing that the number of categories is reasonably large. alternatively, for smaller numbers of categories, similar information can be conveyed using paired bar charts/histograms. paired histograms show the distribution of test results in patients with the target condition above the x-axis and the distribution in patients without the target condition below the x-axis. these types of graphical display are less commonly used. used as an overall (single indicator) measure of the diagnostic accuracy of a diagnostic test. it is calculated as the odds of positivity among diseased persons, divided by the odds of positivity among non-diseased. when a test provides no diagnostic evidence then the dor is 1.0. [33] this measure has a number of limitations: by combining sensitivity and specificity into a single indicator the relative values of the two are lost i.e. the dor can be the same for a very high sensitivity and low specificity as for very high specificity and low sensitivity [33] further, tests that are effective for classifying persons as having or not having the target condition have dors that whose magnitude is much greater (e.g. 100) than usually considered as indicating strong associations in epidemiological studies. [ predictive values depend on disease prevalence, the more common a disease is, the more likely it is that a positive test result is right and a negative result is wrong. [35] it is not possible to construct any of these graphs for truly dichotomous test results. however, truly dichotomous tests rarely occur in practice. examples of dichotomous tests include dipstick tests that change colour if the target condition is said to be present (although these are based on an underlying implicit threshold) or the presence/ absence of certain clinical symptoms. receiver operating characteristic (roc) plot (figure 1c ) roc plots show values of sensitivity and specificity at all of the possible thresholds that could be used to define a positive test result [3] . typically, sensitivity (true positive rate) is plotted against 1-specificity (false positive rate): each point represents a different threshold in the same group of patients. stepped lines are used for continuous example graphical displays for primary study data test results while sloping lines are used for ordered categories. roc curves may be derived directly from the observed sensitivity and specificity corresponding to different test thresholds, or by fitting curves based on parametric [4] , semi-parametric [5, 6] , or non-parametric methods [7] . the area under the roc curve (auc) is a summary of diagnostic performance, and takes values between 0.5 and 1. the more accurate the test, the more closely the curve approaches the top left hand corner of the graph (auc = 1). a test that provides no diagnostic information (auc = 0.5) will produce a straight line from the bottom left to the top right. roc curves may be restricted to a range of sensitivities or specificities of clinical interest. roc plots show how estimated sensitivity and specificity vary according to the threshold chosen, and can be used to identify suitable thresholds for clinical practice if the points on the curve are labelled with the corresponding threshold as in figure 1c , which shows for example that the sensitivity and specificity corresponding to a threshold of 39.3 are 74% and 90%, respectively. confidence intervals can be added to indicate the uncertainty in estimates of test performance at each point. roc plots also allow comparison of the performance of several tests independently of choice of threshold, by plotting data sets for multiple tests in the same roc space. however, they are thought to be difficult to interpret as they describe the characteristics of the test in a way which does not relate directly to its usefulness in clinical practice; research has shown that roc plots are generally poorly understood by clinicians [8] . flow charts (figure 1d ) these depict the flow of patients through the study: for example how many patients were eligible, how many entered the study, how many of these had the target condition, and the numbers testing positive and negative. such charts require categorisation of test results, for example as "positive" and "negative". although flow charts do not directly present diagnostic accuracy data, addition of percentages to the test result boxes (as in figure 1d ) can be used to report test sensitivity (68/90 = 76%) and specificity (46/51 = 90%). charts that first separate individuals according to test result before classification by disease status may similarly be used to depict positive and negative predictive values. the stard (standards for reporting of diagnostic accuracy) statement, an initiative to improve the reporting of diagnostic test accuracy studies similar to the consort statement for clinical trials, recommends the inclusion of a flow diagram in all reports of primary diagnostic accuracy studies [9] . this should illustrate the design of the study and provide information on the numbers of participants at each stage of the study as well as the results of the study. the example flow chart in figure 1d is not a full stard flow diagram as we do not have data on numbers of withdrawals or uninterpretable results from this study. it does, however, show the design (diagnostic case-control) and results of the study. figure 2 illustrates two graphical displays commonly used to present data on diagnostic accuracy in diagnostic systematic reviews. data from a systematic review of dipstick tests for urinary nitrite and leukocyte esterase to diagnose urinary tract infections were used to construct these graphs [10] . forest plots (figure 2a ) forest plots are commonly used to display results of metaanalysis. they display results from the individual studies together with, optionally, a summary (pooled) estimate. point estimates are shown as dots or squares (sometimes sized according to precision or sample size) and confidence intervals as horizontal lines [11] . the pooled estimate is displayed as a diamond whose centre represents the estimate and tips the confidence interval. for diagnostic accuracy studies, measures of test performance (sensitivity, specificity, predictive values, likelihood ratios or diagnostic odds ratio) are plotted on the horizontal axis. diagnostic test performance is often described by pairs of summary statistics (e.g. sensitivity and specificity; positive and negative likelihood ratios), and these are depicted side-by-side. between-study heterogeneity can readily be assessed by visual examination. results may be sorted by one of a pair of test performance measures, usually that which is most important to the clinical application of the test. a disadvantage of paired forest plots is that they do not directly display the inverse association between the two measures that commonly results from variations in threshold between studies. roc plots can be used to present the results of diagnostic systematic reviews, but differ from those used in primary studies as each point typically represents a separate study or data set within a study (individual studies may contribute more than one point). a summary roc (sroc) curve can be estimated using one of several methods [12] [13] [14] [15] and quantifies test accuracy and the association between sensitivity and specificity based on differences between studies. as with forest plots, roc plots provide an overview of the results of all included studies. however, unless there are very few studies, it is not feasible to display confidence intervals as the plot would become cluttered. results for several tests can be displayed on the same plot, facilitating test comparisons. it is also possible to display pooled estimates of sensitivity and specificity together with associated confidence intervals or prediction regions. roc plots may also be used to investigate possible expla-nations for differences in estimates of accuracy between studies, for example those arising from differences in study quality. figure 3 shows results for a recent review that we conducted on the accuracy of magnetic resonance imaging (mri) for the diagnosis of multiple sclerosis (ms) [16] . by using different symbols to illustrate studies that did (diagnostic cohort studies) and did not (other study designs) include an appropriate patient spectrum we were able to show that studies that included an inappropriate patient spectrum grossly overestimated both sensitivity and specificity. various other graphical methods have been developed to display the results of systematic reviews and meta-analyses [17, 18] . although not generally developed specifically for diagnostic test reviews these can be adapted to display the results of such reviews. funnel plots [19] and galbraith plots [20] are often used to assess evidence for publication bias or small study effects in systematic reviews of the effects of medical interventions assessed in randomized controlled trials. however, their application to systematic reviews of diagnostic test accuracy studies is example graphs for systematic review data figure 2 example graphs for systematic review data. a. paired forest plots of sensitivity and specificity for le dipstick. b. roc plot with sroc curves. problematic [20] . diagnostic odds ratios are typically far from 1, and it has been shown that, for data of this type, sampling variation can lead to artefactual associations between log odds ratios and their standard errors [21] . it is therefore recommended that the effective sample size funnel plot be used in reviews of test accuracy studies [20] . a number of graphical displays aim to put results of diagnostic test evaluations into clinical context, based either on primary studies or systematic reviews. two graphical displays commonly used for this purpose are the likelihood ratio nomogram ( figure 4a ) and the probabilitymodifying plot (figure 4b) . each allows the reader to estimate the post-test probability of the target condition in an individual patient, based on a selected pre-test probability. to use the likelihood ratio nomogram, the reader needs an estimate of the likelihood ratios for the test. he then draws a line through the appropriate likelihood ratio on the central axis, intersecting the selected pre-test probability, to derive the post-test probability of disease. the probability-modifying plot depicts separate curves for positive and negative test results. the reader draws a vertical line from the selected pre-test probability to the appropriate likelihood ratio line and then reads the post-test probability off the vertical scale. both graph types are based on a single estimate of test accuracy (likelihood ratio), although it is possible to plot separate curves on the probability-modifying plot or lines on the nomogram to depict confidence intervals around the estimated likelihood ratios. each assumes constant likelihood ratios across the range of pre-test probabilities. however, this assumption may be violated in practice [22] , because populations in which the test is used may have different spectrums of disease to those in which estimates of test accuracy were derived. example graphs for interpreting diagnostic study result figure 4 example graphs for interpreting diagnostic study result. a. likelihood ratio nomogram. b. probability modifying plot. sensitivity plotted against specificity, separately for cohort studies and for studies of other designs for mri for diagnosis of multiple sclerosis figure 3 sensitivity plotted against specificity, separately for cohort studies and for studies of other designs for mri for diagnosis of multiple sclerosis. we systematically reviewed how graphical displays are currently incorporated in studies of test performance. we included primary diagnostic accuracy studies published in 2004, identified by hand searching 12 journals (table 2) , and diagnostic systematic reviews published in 2003, identified from dare (database of abstracts of reviews of effects) [23] . searches were conducted in 2005 and so these years were the most complete available years for searching (there is a delay in adding studies to dare). diagnostic accuracy studies were studies that provided data on the sensitivity and specificity of a diagnostic test and that focused on diagnostic (whether the patient had the condition of interest) rather than prognostic (disease severity/risk prediction) questions. journals were selected to provide a mixture of the major general medical and specialty journals. we particularly aimed to select journals that clinicians read. we extracted data on the different graphical displays used to summarise information about test performance, defined as any graphical method of summarising data on diagnostic accuracy or the predictive value of a test (table 1) . we located 56 primary studies and 49 systematic reviews (web appendix). fifty-seven percent of primary studies and 53% of systematic reviews used graphical displays to present results. in publications using graphics, the number of graphs per publication ranged from 1 to 51 (median 2, iqr 1 to 3 for primary studies and median 4, iqr 2 to 7 for systematic reviews). table 3 summarises the categories of tests evaluated in the primary studies and systematic reviews. none of the tests evaluated in any of the primary studies were truly dichotomous: they all gave continuous or categorical results. three of the eight systematic reviews that assessed clinical examination looked at whether a variety of signs or symptoms were present or absent: these can be considered as truly dichotomous tests. all other reviews evaluated continuous or categorical tests. dot-plots or box-and-whisker plots were the most commonly used graphic and were included in 22 (39%) studies. generally the plots showed individual test results separately for patients with and without the target condition, with four including an indication of the threshold used to define a positive test result. three studies included both a dot plot and a box-and-whisker plot on the same figure. other variations included separate plots for different patient subgroups, different symbols to indicate different stages of disease, or separate plots for different tests. the majority of studies using these types of plots were of laboratory tests. an roc curve was displayed in 15 (26%) studies. all of these plotted full roc curves; only two provided any indication of the thresholds corresponding to one or more of the points. thirteen studies included separate roc curves for different tests, either on the same plot (10 studies) or on separate plots (3 studies). five studies included separate roc plots for different patient subgroups. although all the primary studies were published in 2004, after the publication of the stard guidelines, only one included a stard flow diagram. roc plots were included in 22 (45%) reviews. twenty showed individual study estimates of sensitivity and specificity, 14 fitted sroc curves, and two displayed a summary point. one study, which did not fit an sroc curve, added a box and whisker plot to each axis to show the distributions of sensitivity and specificity. one study plotted only summary estimates of sensitivity and specificity in roc space, with no sroc curves. some reviews included separate plots for different tests, for different patient subgroups, or for different thresholds used to define a positive test result. ten reviews (20%) used forest plots to display individual study results. one study provided a plot of diagnostic odds ratios, while all others displayed paired plots of sensitivity and specificity (8 reviews), positive and negative likelihood ratios (3 reviews), or positive and negative predictive values (1 review). several studies displayed more than one set of forest plots, including plots for more than one summary measure, for different stages of diagnosis, different test thresholds or for different tests. one study included a forest plot of summary data only, showing how pooled estimates of positive and negative likelihood ratios varied for different patient subgroups. none of the studies included a likelihood ratio nomogram. one primary study and five systematic reviews included a probability-modifying plot. research in the area of cognitive psychology suggests that sensitivity and specificity are generally poorly understood by doctors [8, 24] and are often confused with predictive values [8, 25, 26] . doctors tend to overestimate the impact of a positive test result on the probability of disease [27, 28] and this overestimation increases with decreasing pre-test probabilities of disease [29] . this research suggests that the most informative measures for doctors may be estimates of the post-test probability of disease (predictive value), which can be presented as a range corresponding to different pre-test probabilities. however, graphical displays that facilitate the derivation of post-test probabilities, such as likelihood ratio nomograms, are usually based on summary estimates of test characteristics (positive and negative likelihood ratios) without allowing for the precision of the estimate, or its applicability to a given population. use of summary estimates in this way is questionable in the context of reviews of diagnostic accuracy studies, which typically find substantial between-study heterogeneity [30] . it is particularly problematic if the summary estimate is the only information conveyed in a graphic and the graphic is taken as the key message of the paper. the inclusion of some form of graphical presentation of test accuracy data has a number of advantages compared to not using such displays. it allows fuller reporting of results, for example (s)roc plots can display results for multiple thresholds whereas reporting test accuracy results in a text or table generally requires the selection of one or more thresholds. in addition, (s)roc plots depict the trade-off between sensitivity and specificity at different thresholds. use of such displays also have the advantage of presenting all of the results of a primary study or systematic review without the need for selected analyses, which may be biased depending on the analyses selected. the inclusion of graphical displays, such as sroc plots or forest plots, in systematic reviews of test accuracy studies allows a visual assessment of heterogeneity between studies by showing the results from each individual study included in the review. there is also a suggestion that graphical displays may be easier to interpret than text or tabular summaries of the same data. diagnostic accuracy studies will usually need to include more than one graphic in order both to provide a detailed description of results (diagnostic accuracy) and to communicate appropriate summary measures that can be used to inform clinical practice (predictive value); the more detailed graphic provides context for the interpretation of summary measures. further work is required to improve on existing graphical displays. the starting point for this should be further evaluation of the types of graphical display most helpful to assessing the utility of a test in clinical practice and the implications of test results for individual patients. we hope that this paper will contribute to an increase in the use and quality of graphical displays in diagnostic accuracy studies and systematic reviews of these studies. include references to the stard flow diagram. stard itself does not comment on how graphical displays should be used to convey results of test accuracy studies other than to recommend the inclusion of a flow diagram and to provide an illustration of a dot-plot as a suggestion for how individual study results may be displayed. guidelines on the type of graphical displays that should be included in reports of test accuracy studies could be considered when stard is next updated, and should be considered by journals in their instructions for authors. our review suggests that graphical displays are currently underused in primary diagnostic accuracy studies and systematic reviews of such studies. graphical displays of diagnostic accuracy data should provide an easily interpretable and accurate representation of study results, conveying both diagnostic accuracy and predictive value. this is not usually possible in a single graphic: the type of information presented in the most commonly used graphs does not directly allow clinicians to assess the implications of test results for an individual patient. the author(s) declare that they have no competing interests. all authors contributed to the design of the study and read and approved the final manuscript. pfw and mew identified relevant studies and extracted data from included studies. pfw carried out the analysis and drafted the manuscript with help from jd and rh. venous doppler in the prediction of acid-base status of growth-restricted fetuses with elevated placental blood flow resistance detection of human polyomaviruses in urine from bone marrow transplant patients: comparison of electron microscopy with pcr magnetic resonance imaging of the breast prior to biopsy diagnosis of pancreatic cystic neoplasms: a report of the cooperative pancreatic cyst study rapid hiv-1 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prognostic, or screening marker statistics notes: diagnostic tests 2: predictive values association of coronary heart disease with pre-{beta}-hdl concentrations in japanese men this work was supported by the mrc health services research collaboration. jonathan deeks is funded by a senior research fellowship in evidence synthesis from the department of health. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/8/20/prepub key: cord-307500-2jwuzfan authors: gray, nicholas; calleja, dominic; wimbush, alex; miralles-dolz, enrique; gray, ander; de-angelis, marco; derrer-merk, elfride; oparaji, bright uchenna; stepanov, vladimir; clearkin, louis; ferson, scott title: "no test is better than a bad test": impact of diagnostic uncertainty in mass testing on the spread of covid-19 date: 2020-04-22 journal: nan doi: 10.1101/2020.04.16.20067884 sha: doc_id: 307500 cord_uid: 2jwuzfan background: the cessation of lock-down measures will require an effective testing strategy. much focus at the beginning of the uk's covid-19 epidemic was directed to deficiencies in the national testing capacity. the quantity of tests may seem an important focus, but other characteristics are likely more germane. false positive tests are more probable than positive tests when the overall population has a low prevalence of the disease, even with highly accurate tests. methods: we modify an sir model to include quarantines states and test performance using publicly accessible estimates for the current situation. three scenarios for cessation of lock-down measures are explored: (1) immediate end of lock-down measures, (2) continued lock-down with antibody testing based immunity passports, and (3) incremental relaxation of lock-down measures with active viral testing. sensitivity, specifcity, prevalence and test capacity are modified for both active viral and antibody testing to determine their population level effect on the continuing epidemic. findings: diagnostic uncertainty can have a large effect on the epidemic dynamics of covid-19 within the uk. the dynamics of the epidemic are more sensitive to test performance and targeting than test capacity. the quantity of tests is not a substitute for an effective strategy. poorly targeted testing has the propensity to exacerbate the peak in infections. interpretation: the assessment that 'no test is better than a bad test' is broadly supported by the present analysis. antibody testing is unlikely to be a solution to the lock-down, regardless of test quality or capacity. a well designed active viral testing strategy combined with incremental relaxation of the lock-down measures is shown to be a potential strategy to restore some social activity whilst continuing to keep infections low. the uk government's covid-19 epidemic management strategy has been influenced by epidemiological modelling conducted by a number of research groups [1, 2] . the analysis of the relative impact of different mitigation and suppression strategies has influenced the current approach. the "only viable strategy at the current time" is to suppress the epidemic with all available measures, including school closures and social distancing of the entire population [3] . these analyses have highlighted from the beginning that the eventual relaxation of lock-down measures would be problematic [3, 4] . without a considered cessation of the suppression strategies the risk of a second wave becomes signficant, possibly of greater magnitude than the first as the sars-cov-2 virus is now endemic in the population [5, 6] . although much attention has been focused on the number of tests being conducted [7, 8] , not enough attention has been given to the issues of imperfect testing. whilst poorly performing tests have not been prominent in public discourse, evidence suggests they are epidemiologically significant. the failure to detect the virus in infected patients can be a significant problem in high-throughput settings operating under severe pressure [9] . evidence suggest that this is indeed the case [10, 11, 12, 13] . everyone seems to agree that testing will be a pillar of whatever approach is employed to relax the current social distancing measures [14] . the public are rapidly becoming aware of the difference between the 'have you got it?' tests for detecting active cases, and the 'have you had it?' tests for the presence of antibodies, which imply some immunity to . what may be less obvious is that these different tests need to maximise different test characteristics. to be useful in ending the current social distancing measures, active viral tests need to maximise the sensitivity. how good the test is at telling you that you have the disease. high sensitivity reduces the chance of missing people who have the virus who may go on to infect others. there is an additional risk that an infected person who has been incorrectly told they do not have the disease, when in fact they do, may behave in a more reckless manner than if their disease status were uncertain. the second testing approach, seeking to detect the presence of antibodies to identify those who have had the disease would be used in a different strategy. this strategy would involve detecting those who have successfully overcome the virus, and are likely to have some level of immunity (or at least reduced susceptibility to more serious illness if they are infected again), so are relatively safe to relax their personal social distancing measures. this strategy would require a high test specificity, aiming to minimise how often the test tells someone they have had the disease when they haven't. a false positive tells people they have immunity when they don't, which is even worse than when people are uncertain about their viral history. 2 introduction to test statistics: what makes a 'good' test? in order to answer this question there are a number of important statistics: sensitivity σ -out of those who actually have the disease, that fraction that received a positive test result. specificity τ -out of these who did not have the disease, the fraction that received a negative test result. the statistics that characterise the performance of the test are computed from a confusion matrix (table 1) . we test n inf ected people who have covid-19, and n healthy people who do not have covid-19. in the first group, a people correctly test positive and c falsely test negative. among healthy people, b will falsely test positive, and d will correctly test negative. from this confusion matrix the sensitivity is given by (1) and the specificity by (2) . 2 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 22, 2020. . sensitivity is the ratio of correct positive tests to the total number of infected people involved in the study characterising the test. the specificity is the ratio of the correct negative tests to the total number of healthy people. importantly, these statistics depend only on the test itself and do not depend on the population the test is intended to be used upon. computing the statistics require a definitive way to determine the true viral status of a patient; a so-called gold standard. if there is doubt about in which column a patient falls, the confusion matrix cannot be constructed. when novel tests are employed the confusion matrix can be very challenging to rigorously assess in the midst of a fast moving epidemic [15, 16, 17] . when the test is used for diagnostic purposes, the characteristics of the population being tested become important for interpreting the test results. to interpret the diagnostic value of a positive or negative test result the following statistics must be used: prevalence p -the proportion of people in the target population that have the disease tested for. positive predictive value p p v -how likely one is to have the disease given a positive test result. negative predictive value n p v -how likely one is to not have the disease, given a negative test result. the p p v and n p v depend on the prevalence, and hence depend on the population you are focused on. this may be the uk population, a sub population with covid-19 compatible symptoms, or any other population you may wish to target. the p p v and n p v can then be calculated using bayes' rule: to improve the diagnostic performance of tests they are often repeated to increase the aggregate p p v or n p v . to do this an assumption of independence between the two tests needs to be made. this assumption could be questionable in some circumstances. for instance, it would be questionable if samples are analysed at the same time in the same lab by the same technician, or if the same method for extracting the sample from the patient is employed, it may be unsuccessful at detecting virus for the same reason. a plethora of other possible errors are imaginable. many of these errors may be truly random, and independent, but many may not be so the independence assumption may be weakly justified. the rapid development and scaling of new diagnostic systems invites error, particularly as labs are converted from other purposes and technicians are placed under pressure, and variation in test collection quality, reagent quality, sample preservation and storage, and sample registration and provenance. assessing the magnitude of these errors on the performance of tests is challenging in real time. point-ofcare tests are not immune to these errors and are often seen as less accurate than laboratory based tests [18, 19] . the prevalence of the disease matters. the p p v can vary drastically for different populations with different prevalence. the idea that prevalence depends on the population may seem counterintuitive to some audiences. for example, if we were to select 100 people from a respiratory ward this week from any hospital in the uk, and 100 people from a street outside the building, what proportion of each population have covid-19? if one tests both populations with the same test and found positives in each population, which would have the higher p p v ? 3 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 22, 2020 to illustrate the impact of prevalence on p p v , for a test with σ = τ = 0.95 if prevalence p = 0.05, then the p p v ≈ 0.48. figure 1 shows why, for 1000 test subjects there will be similar numbers of true and false positives even with high sensitivity and specificity of 95%. in contrast, using the same tests on a sample with a higher prevalence p = 0.5 we find the p p v = 0.8, see figure 2 . similarly, the n p v is lower when the prevalence is higher. the number of active cases exceeding the test capacity may not be the only discrepancy between the true cases and reported cases. the impact of uncertainty in testing may also be contributing to the discrepancy, even in the tested population. more testing will not reduce this uncertainty. the director of the who suggests that testing is a crucial part of any strategy [20] , but even testing the entire country every day would not give an accurate tally of infections. to explore the effect of imperfect testing on the disease dynamics when strategies are employed to relax the current social distancing measures the sir model described in the supplimentary material was modified. three new classes were added to the model, the first is a quarantined susceptible state, q s , the second is a quarantined infected state, q i , and the third is people who have recovered but are in quarantine, q r . to model the current lock-down, the model evaluations begin with a majority of the population in the q s (quarantined but susceptible) state. whilst in this state the transmission rate of the disease is totally suppressed. the model evaluates each day's average population-level state transitions. there are two possible tests that can be performed: an active virus infection test that is able to determine whether or not someone is currently infectious. this test is performed on some proportion of the un-quarantined population (s + i + r). it has a sensitivity of σ a and a specificity of τ a . an antibody test that determines whether or not someone has had the infection in the past. this is used on the fraction of the population that is currently in quarantine but not infected (q s + q r ) to test whether they have had the disease or not. this test has a sensitivity of σ b and a specificity of τ b . these two tests are used on some of those eligible for testing each day limited by the test capacity, ρ and φ respectively. a person (in any category) who tests positive in an active virus test transitions into the (corresponding) quarantine state, where they are unable to infect anyone else. a person, in q s or q r , who tests positive in an antibody test transitions to s and r respectively. for this parameterisation the impact of being in the susceptible quarantined state, q s , makes an individual insusceptible to being infected. similarly, being in the infected quarantined state, q i , individuals are unable to infect anyone else. in practicality there is always leaking, no quarantine is entirely effective, but for the sake of exploring the impact of testing uncertainty these effects are neglected from the model. the participation in infection propagation of individuals in either quarantine state are idiosyncratic, and on average are assumed to be negligibly small for the sake of this analysis. if the tests were almost perfect, then we can imagine how the epidemic would die out very quickly by either widespread infection or antibody testing with a coherent management strategy. a positive test on the former and the person is removed from the population, and positive test on the latter and the person, unlikely to contract the disease again, can join the population . more interesting are the effects of incorrect test results on the disease dynamics. if someone falsely tests positive in the antibody test, they enter the susceptible state. similarly, if an infected person receives a false negative for the disease they remain active in the infected state and hence can continue the disease propagation and infect further people. 4 what part will testing play in relaxing current social distancing measures? in order to explore the possible impact of testing strategies on the relaxation of current social distancing measures several scenarios have been analysed. these scenarios are illustrative of the type of impact, 5 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 16.20067884 doi: medrxiv preprint and the likely efficacy of a range of different testing configurations. immediate end to social distancing scenario: this baseline scenario is characterised by a sudden relaxation of the current social distancing measures. immunity passports scenario: a policy that has been discussed in the media [21, 22, 17] . analogous to the international certificate of vaccination and prophylaxis, antibody based testing would be used to identify those who have some level of natural immunity. incremental relaxation scenario: a phased relaxation of the government's social distancing advice is the most likely policy that will be employed. to understand the implications of such an approach this scenario has explored the effect of testing capacity and test performance on the possible disease dynamics under this type of policy. under the model parameterisation this analysis has applied an incremental transition rate from the q s state to the s state, and q r to r. whilst the authors are sensitive to the sociological and ethical concerns of any of these approaches [23, 24] , the analysis presented is purely on the question of efficacy. under the baseline scenario, characterised by the sudden and complete cessation of the current social distancing measures, we explored the impact of infection testing. under this formulation the initial conditions of the model in this scenario is that the all of the population in q s transition to s in the first iteration. as would be expected the model indicates the second wave is an inevitability and as many as 20 million people could become infected within 30 days, figure 4. to illustrate the sensitivity of the model to testing scenarios an evaluation was conducted with a range of infection test sensitivities, from 50% (i.e of no diagnostic value) to 98%. the specificity of these tests has a negligible impact on the disease dynamics. a false positive test result would mean people are unnecessarily removed from the susceptible population, but the benefit of a reduction in susceptible population is negligibly small. it's also very likely the infection testing would be heavily biased toward symptomatic carriers, where the prevalence of the disease is high so fewer false positives would be expected. two evaluations have been conducted. the first using the stated government goal of 100,000 tests per day (left graphs in figure 4 ). it remains unclear whether this aim is feasible, or if this testing capacity would include both forms of tests (antibody and active virus). the second evaluation looks at a very optimistic case where we could conduct as many as 150,000 tests per day (right graphs in figure 4 ). the authors draw no conclusions about the feasibility of achieving these levels. however the authors do wish to encourage caution that with a capacity for testing of the order targeted by the uk government, testing in isolation is not sufficient to allow any rapid cessation of the current social distancing measures without a resurgence of the virus. this caution is irrespective of test performance, even very good tests with a sensitivity of 98%, and effective isolation of cases that have tested positive, the outcome is broadly invariant. the immunity passport is an idiom describing an approach to the relaxation of the current social distancing measures that focuses heavily on antibody testing. wide-scale screening for antibodies in the general population promises significant scientific value, and targeted antibody testing is likely to have value for reducing risks to nhs and care-sector staff, and other key workers who will need to have close contact with covid-19 sufferers. the authors appreciate these other motivations for the development and roll-out of accurate antibody tests. this analysis however focuses on the appropriateness of this approach to relaxing current social distancing measures by mass testing the general population. antibody testing has been described as a 'game-changer' [25] . some commentators believe this could have a significant impact on the relaxation of social distancing measures [22] . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. much of the discussion around antibody testing in the media has focused on the performance and number of these tests. the efficacy of this strategy however is far more dependent on the prevalence of antibodies in the general population. without wide-scale antibody screening it's impossible to know the prevalence of antibodies in the general population, so there is scientific value in such an endeavour. however, the prevalence is the dominant factor to determine how efficacious antibody screening would be for relaxing social distancing measures. presumably, only people who test positive for antibodies would be allowed to leave quarantine. the more people in the population with antibodies, the more people will get a true positive, so more people would be correctly allowed to leave quarantine (under the paradigms of an immunity passport). the danger of such an approach is the false positives. we demonstrate the impact of people reentering the susceptible population who have no immunity. we assume their propensity to contract the infection is the same as those without this the false sense of security a positive test may engender. on an individual basis, and even at the population level, behavioural differences between those with false security from a positive antibody test, versus those who are uncertain about their viral history could be significant. the model parametrisation here does not include this additional confounding effect. to simulate the prevalence of antibodies in the general population the model is preconditioned with different proportions of the population in the q s and q r states. this is analogous to the proportion of people that are currently in quarantine who have either had the virus and developed some immunity, and the proportion of the population who have not contracted the virus and have no immunity. of course the individuals in these groups do not really know their viral history, and hence would not know which state 8 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. each column corresponds to a different antibody test sensitivity in figure 5 as titled. the specificity for each test in these evaluations was fixed to 90%. in figure 6 each column corresponds to a different antibody test specificity as titled. the sensitivity for each test in these evaluation was fixed to 95%. for all model evaluations in figures 5 and 6 we modelled a continuing and constant ability to conduct targeted active virus testing which continued to remove individuals from the infected population. the infection testing continued throughout each model run with a fixed capacity of 10,000 tests per day, similar to the number of unique individuals that are currently being tested. each of the graphs in the two figures shows the effect of different prevalences of antibodies in the population. to be clear, this is the proportion of the population that has contracted the virus and recovered but are in quarantine. sir patrick vallance, the uk government chief scientific advisor, in the daily press briefing on the 9 april 2020 stated his belief that this prevalence is likely to be less than 10%, possibly much less. the analysis has explored a range for prevalence from 0.1% to 50%. figure 5 explores the impact of a variation in sensitivity, from a test with 50% sensitivity (i.e no diagnostic value) to tests with a high sensitivity of 98%. it can be seen, considering the top half of the graphs, that the sensitivity of the test has no discernible impact on the number of infections. the prevalence entirely dominates. this is possibly counter intuitive, but as was discussed in section 2.1, even a highly accurate test produces a very large number of false positives when prevalence is low. in this case that would mean a large number of people are allowed to re-enter the population, placing them at risk, with a false sense of security that they have immunity. the bottom row of figure 5 shows the proportion of the entire population leaving quarantine over a year of employing this policy. at low prevalence there is no benefit to better performing tests. this again may seem obscure to many readers. if you consider the highest prevalence simulation, where 50% of the population have immunity, higher sensitivity tests are of course effective at identifying those who are immune, and gets them back into the community much faster. however this is not the case currently in the uk because, as sir patrick stated, the prevalence of antibodies is likely to be very low at least during the lock-down. a more concerning story can be seen when considering the graphs in figure 6 . now we consider a range of antibody test specificities. going from 50% (no value in ruling people out) to 98%. when the prevalence is low, a lower specificity of 75% not only leads to an initial large increase in the number of infections, but also, if employed throughout the year would lead to repeated peaks. this is because the active virus testing would still be employed along side the antibody testing. falsely-diagnosed susceptible people leaving quarantine leads to a sharp rise in the number of infections. as the prevalence of virus in the non-quarantined population increases the active virus testing becomes more effective and subdues the rise in infections because the testing is more targeted on active virus cases. this would be followed by additional waves as further false positive tests for antibodies are observed. the number of people in quarantine with antibodies declines over the length of the simulation, so naturally the prevalence of immunity in the quarantined population declines. as the prevalence declines the n p v of the test declines. when we consider the bottom half of figure 6 and look at the impact on the proportion of the population able to leave quarantine, unlike previously, the number of false positives dominates when there is a lower specificity. so there are many more people leaving the quarantine, even when the prevalence is very low (0.1%). this may be desirable to some who favour increasing economic and social activity, but it is of course at the cost of further infections. decision makers and the public need to be aware of the trade-off being made. the dangers of neglecting uncertainties in medical diagnostic testing are pertinent to this decision [26] , particularly if immunity passports become prominent in the strategy to end the current social distancing measures. at this point, some form of incremental relaxation of the current government social distancing advice seems highly likely. this could take many forms, it could be an incremental restoration of certain activities such as school openings, permission for the reopening of some businesses, the relaxation of the stay-at-home messaging, etc. under the parameterisation chosen for this analysis the model is not sensitive to any particular policy change. we consider a variety of rates of phased relaxations to the current quarantine. to model these rates we consider a weekly incremental transition rate from q s to s, and q r to r. in figure 7 , three weekly transition rates have been applied: 1%, 5% and 10% of the quarantined population. whilst in practice the rate is unlikely to be uniform as decision makers would have the ability to update their timetable as the impact of relaxations becomes apparent, it is useful to illustrate the interaction of testing capacity and release rate. the model simulates these rates of transition for a year, with a sensitivity and specificity of 90% for active virus tests. the specifics of all the runs are detailed in table 2. figure 7 shows five analyses, with increasing capacity for the active virus tests. in each, the 3 incremental transition rates are applied with initial population split σ a τ a β γ q s s q i q i q r r 0.9 0.9 0.32 0.1 0.95 0.034 0.004 0.01 0.001 0.001 table 2 : fixed parameters used for figure 7 analysis. a range of disease prevalences in the population being tested. the p p v , as discussed in section 2.1, has a greater dependence on the prevalence (at lower values) in the tested population than it does on the sensitivity of the tests, the same is true of the specificity and the n p v . it is important to notice that higher test capacities cause a higher peak of infections for the 10% quarantine release rate. this has a counterintuitive explanation. when there is the sharpest rise in the susceptible population (i.e., high rate of transition), the virus rapidly infects a large number of people. when these people recover after two weeks they become immune and thus cannot continue the spread of the virus. however, when the infection testing is conducted with a higher capacity up to 120,000 units per day, these tests transition some active viral carriers into quarantine, so the peak is slightly delayed providing more opportunity for those released from quarantine later to be infected, leading to higher peak infections. this continues until the model reaches effective herd immunity after which the number of infected in the population decays very quickly. having higher testing capacities delays but actually worsens the peak number of infections. at 10% release rate, up to a capacity of testing of 120,000 these outcomes are insensitive to the prevalence of the disease in the tested population . this analysis indicates that the relatively fast cessation of social distancing measures and stay-home advice would lead to a large resurgence of the virus. testing capacity of the magnitude stated as the goal of the uk government would not be sufficient to flatten the curve in this scenario. at the rate of 5% of the population in lock-down released incrementally each week the infection peak is suppressed compared to the 10% rate. the number of infections would remain around this level for a significantly longer period of time, up to 6 months. there is negligible impact of testing below a capacity of 50,000 tests. however if the test capacity were 80,000 tests, at a quarantine release rate of 5% the duration of the elevated levels of infections would be reduced, reducing the length of necessary wide-scale social distancing. this effect is only observed with the more targeted tests, where a prevalence of the disease in the targeted population is over 30%. any less well targeted testing and the testing would have a negligible impact compared to the untested scenario. the 1% release rate scenario indicates that a slow release by itself is sufficient to lower peak infections, but extends the duration of elevated infections. the first graph of the top row in figure 7 shows that the slow release rate causes a plateau at a significantly lower number of infections compared to the other release rates. poorly targeted tested at capacities less than 100,000 show similar consistent levels of infections. however, with a targeted test having a prevalence of 30% or more, the 1% release rate indicates that even with 50,000 tests per day continuous suppression of the infection may be possible. this analysis does support the assertion that a bad test is worse than no tests, but a good test is only effective in a carefully designed strategy. more is not necessarily better and over estimation of the test accuracy could be extremely detrimental. this analysis is not a prediction; the numbers used in this analysis are estimates, and therefore, when such policies are devised and implemented this analysis would need to be repeated with more up-to-date numerical values. as such, the authors are not drawing firm conclusions about the absolute necessary capacity of tests. nor do they wish to make specific statements about the necessary sensitivity or specificity of tests or the recommended rate of release from quarantine. the authors do, however, propose some conclusions that would broadly apply to the present situation, and therefore believe they should be considered by policy makers when designing strategies to tackle covid-19. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . 12 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . diagnostic uncertainty can have a large effect on the epidemic dynamics of covid-19 within the uk. and, sensitivity, specificity, and the capacity for testing alone are not sufficient to design effective testing procedures. great caution should be exercised in the use of antibody testing. under the assumption that the proportion of people in the uk who have had the virus is still low, it's unlikely antibody testing at any scale will significantly support the end of lock-down measures. and, the negative consequences of un-targeted antibody screening at the population level could cause more harm than good. antibody testing, with a high specificity may be very useful on an individual basis, it certainly has scientific value, and could reduce risk for key workers. but any belief that these tests would be useful to relax lock-down measures for the majority of the population is misguided. at best it is a distraction, at worst it could be dangerous. the incremental relaxation to lock-down measures, with all else equal, would significantly dampen the increase in peak infections, by 1 order of magnitude with a faster relaxation, and 2 orders of magnitude with a slower relaxation. the capacity for infection screening needs to be significantly increased if it is to be used to relax quarantine measures, but only if it is well targeted, for example through effective contact tracing. untargeted mass screening would be ineffectual and may prolong the necessary implementation of lock-down measures. one interpretation of these results is that countries that had mass testing regimes early in the pandemic but had much lower case fatality rates may have been reporting a large number of false positives. the results of this paper may explain what is being observed in nations such as singapore as they continue to employ less-targeted mass testing and after a rapid cessation to their lock-down measures are now experiencing a second peak in infections [27] . this work has been partially funded by the epsrc iaa exploration award with grant number ep/r511729/1, epsrc programme grant "digital twins for improved dynamic design", ep/r006768/1, and the epsrc and esrc centre for doctoral training in quantification and management of risk and uncertainty in complex systems and environments, ep/l015927/1 . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. sir models offer one approach to explore infection dynamics, and the prevalence of a communicable disease. in the generic sir model, there are s people susceptible to the illness, i people infected, and r people who are recovered with immunity. the infected people are able to infect susceptible people at rate β and they recover from the disease at rate γ [28] . once infected persons have recovered from the disease they are unable to become infected again or infect others. this may be because they now have immunity to the disease or because they have unfortunately died. figure 8 shows a schematic of the generic model formulation, and how people move between the states. figure 9 demonstrates the typical disease dynamics, the infected corresponding to the now well known curve that we are trying to flatten. the sir model has two ways in which the number of new infections falls to zero. either the number of susceptible people reduces to a point at which the disease can no longer propagate, perhaps because of a vaccine or natural immunity, or the epidemic stops if the basic reproduction rate of the disease falls below 1 due to social distancing or effective viral suppression. . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 16.20067884 doi: medrxiv preprint b binomial sir model the sir model used in this paper uses discrete-time binomial sampling for calculating movements of individuals between states. for a defined testing strategy, with an active viral test having sensitivity, specificity and capacity of σ a , τ a and c a respectively, an antibody test with sensitivity, specificity and capacity σ b , τ b and c b respectively and a testing prevalence of p, these rates are defined as follows: n a = min (c a , bin (s + i, ρ)) , (6a) n b = min (c b , bin (q s + q r , φ)) , (6b) t i = min (n a p, ip) , (6c) t s = min(s, n a − t i ), (6d) at each time step t, the model calculates the number of persons moving between each state in the order defined above. the use of a binomial model was prompted by a desire to incorporate both aleatory and epistemic uncertainty in each movement. the current approach does not make use of epistemic uncertainty, fixing the model parameters σ a , τ a , σ b , τ b , φ, ρ, c a , c b and p. a discrete time model was selected to allow for comparisons against available published data detailing recorded cases and recoveries on a day-by-day basis. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 16.20067884 doi: medrxiv preprint covid-19: government announces moving out of contain phase and into delay phase scaling up our testing programmes. department of health and socal care impact of nonpharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand. imperial college covid-19 response team pm address to the nation on coronavirus the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study. the lancet public health first-wave covid-19 transmissibility and severity in china outside hubei after control measures, and second-wave scenario planning: a modelling impact assessment. the lancet offline : covid-19 and the nhs -"a national scandal offline: covid-19-bewilderment and candour. the lancet coronavirus and the race to distribute reliable diagnostics why the coronavirus test gives so many false negatives difficulties in false negative diagnosis of coronavirus disease 2019: a case report assume you have the illness, even if you test negative are coronavirus tests accurate? world report developing antibody tests for sars-cov-2 labs scramble to produce new coronavirus diagnostics medical product alert no3/2020: falsified medical products, including in vitro diagnostics, that claim to prevent, detect, treat or cure covid-19 britain has millions of coronavirus antibody tests, but they don't work; 2020 molecular and antibody point-of-care tests to support the screening , diagnosis and monitoring of covid-19. oxford covid-19 evidence service point-of-care versus lab-based testing: striking a balance who director-general's opening remarks at the media briefing on covid-19 -16 why it's too early to start giving out "immunity passports"; 2020 immunity passports' could speed up return to work after covid-19 the covid-19 coronavirus is now a pandemic -can we ethically deal with lockdowns? we cannot leave our coronavirus exit strategy to the experts boris johnson and donald trump talk up potential 'game-changer' scientific advances on coronavirus calculated risks: how to know when numbers deceive you singapore coronavirus surge raises fears of post-lockdown breakouts key: cord-296306-xcomjvaa authors: rivett, lucy; sridhar, sushmita; sparkes, dominic; routledge, matthew; jones, nick k; forrest, sally; young, jamie; pereira-dias, joana; hamilton, william l; ferris, mark; torok, m estee; meredith, luke; curran, martin d; fuller, stewart; chaudhry, afzal; shaw, ashley; samworth, richard j; bradley, john r; dougan, gordon; smith, kenneth gc; lehner, paul j; matheson, nicholas j; wright, giles; goodfellow, ian g; baker, stephen; weekes, michael p title: screening of healthcare workers for sars-cov-2 highlights the role of asymptomatic carriage in covid-19 transmission date: 2020-05-11 journal: elife doi: 10.7554/elife.58728 sha: doc_id: 296306 cord_uid: xcomjvaa significant differences exist in the availability of healthcare worker (hcw) sars-cov-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. over a 3 week period (april 2020), 1032 asymptomatic hcws were screened for sars-cov-2 in a large uk teaching hospital. symptomatic staff and symptomatic household contacts were additionally tested. real-time rt-pcr was used to detect viral rna from a throat+nose self-swab. 3% of hcws in the asymptomatic screening group tested positive for sars-cov-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (covid-19)>7 days prior to testing, most self-isolating, returning well. clusters of hcw infection were discovered on two independent wards. viral genome sequencing showed that the majority of hcws had the dominant lineage b∙1. our data demonstrates the utility of comprehensive screening of hcws with minimal or no symptoms. this approach will be critical for protecting patients and hospital staff. despite the world health organisation (who) advocating widespread testing for sars-cov-2, national capacities for implementation have diverged considerably (who, 2020b; our world in data, 2020) . in the uk, the strategy has been to perform sars-cov-2 testing for essential workers who are symptomatic themselves or have symptomatic household contacts. this approach has been exemplified by recent studies of symptomatic hcws (hunter et al., 2020; keeley et al., 2020) . the role of nosocomial transmission of sars-cov-2 is becoming increasingly recognised, accounting for 12-29% of cases in some reports . importantly, data suggest that the severity and mortality risk of nosocomial transmission may be greater than for community-acquired covid-19 (mcmichael et al., 2020) . protection of hcws and their families from the acquisition of covid-19 in hospitals is paramount, and underscored by rising numbers of hcw deaths nationally and internationally (cook et al., 2020; cdc covid-19 response team, 2020) . in previous epidemics, hcw screening programmes have boosted morale, decreased absenteeism and potentially reduced long-term psychological sequelae (mcalonan et al., 2007) . screening also allows earlier return to work when individuals or their family members test negative (hunter et al., 2020; keeley et al., 2020) . another major consideration is the protection of vulnerable patients from a potentially infectious workforce (mcmichael et al., 2020) , particularly as social distancing is not possible whilst caring for patients. early identification and isolation of infectious hcws may help prevent onward transmission to patients and colleagues, and targeted infection prevention and control measures may reduce the risk of healthcare-associated outbreaks. the clinical presentation of covid-19 can include minimal or no symptoms (who, 2020a). asymptomatic or pre-symptomatic transmission is clearly reported and is estimated to account for around half of all cases of covid-19 (he et al., 2020) . screening approaches focussed solely on symptomatic hcws are therefore unlikely to be adequate for suppression of nosocomial spread. preliminary data suggests that mass screening and isolation of asymptomatic individuals can be an effective method for halting transmission in community-based settings (day, 2020) . recent modelling has suggested that weekly testing of asymptomatic hcws could reduce onward transmission by 16-23%, on top of isolation based on symptoms, provided results are available within 24 hr (imperial college covid-19 response team, 2020). the need for widespread adoption of an expanded screening programme for asymptomatic, as well as symptomatic hcws, is apparent (imperial college covid-19 response team, 2020; black et al., 2020; gandhi et al., 2020) . challenges to the roll-out of an expanded screening programme include the ability to increase diagnostic testing capacity, logistical issues affecting sampling and turnaround times and concerns about workforce depletion should substantial numbers of staff test positive. here, we describe how we have dealt with these challenges and present initial findings from a comprehensive staff screening programme at cambridge university hospitals nhs foundation trust (cuhnft). this has included systematic screening of >1000 asymptomatic hcws in their workplace, in addition to >200 symptomatic staff or household contacts. screening was performed using a validated real-time reverse transcription pcr (rt-pcr) assay detecting sars-cov-2 from combined oropharyngeal (op) and nasopharyngeal (np) swabs (sridhar et al., 2020) . rapid viral sequencing of positive samples was used to further assess potential epidemiological linkage where nosocomial transmission was suspected. our experience highlights the value of programmes targeting both symptomatic and asymptomatic staff, and will be informative for the establishment of similar programmes in the uk and globally. between 6 th and 24 th april 2020, 1,270 hcws in cuhnft and their symptomatic household contacts were swabbed and tested for sars-cov-2 by real-time rt-pcr. the median age of the hcws was 34; 71% were female and 29% male. the technical rt-pcr failure rate was 2/1,270 (0. 2% see materials and methods); these were excluded from the 'tested' population for further analysis. ultimately, 5% (n = 61) of swabs were sars-cov-2 positive. 21 individuals underwent repeat testing for a variety of reasons, including evolving symptoms (n = 3) and scoring 'medium' probability on clinical covid-19 criteria (tables 1-2) (n = 11). all remained sars-cov-2 negative. turn around time from sample collection to resulting was 12-36 hr; this varied according to the time samples were obtained. table 3 outlines the total number of sars-cov-2 tests performed in each screening group (hcw asymptomatic, hcw symptomatic, and hcw symptomatic household contact) categorised according to the ward with the highest anticipated risk of exposure to high; 'amber', medium; 'green', low; . in total, 31/1,032 (3%) of those tested in the hcw asymptomatic screening group tested sars-cov-2 positive. in comparison, 30/221 (14%) tested positive when hcw symptomatic and hcw symptomatic household contact screening groups were combined. as expected, symptomatic hcws and their household contacts were significantly more likely to test positive than hcws from the asymptomatic screening group (p<0. 0001, fisher's exact test). hcws working in 'red' or 'amber' wards were significantly more likely to test positive than those working in 'green' wards (p=0. 0042, fisher's exact test). all users of ffp3 masks underwent routine fit-testing prior to usage. cleaning and re-use of masks, theatre caps, gloves, aprons or gowns was actively discouraged. cleaning and re-use of eye protection was permitted for certain types of goggles and visors, as specified in the hospital's ppe protocol. single-use eye protection was in use in most scenario 1 and 2 areas, and was not cleaned and re-used. all non-invasive ventilation or use of high-flow nasal oxygen on laboratory-confirmed or elife digest patients admitted to nhs hospitals are now routinely screened for sars-cov-2 (the virus that causes covid-19), and isolated from other patients if necessary. yet healthcare workers, including frontline patient-facing staff such as doctors, nurses and physiotherapists, are only tested and excluded from work if they develop symptoms of the illness. however, there is emerging evidence that many people infected with sars-cov-2 never develop significant symptoms: these people will therefore be missed by 'symptomatic-only' testing. there is also important data showing that around half of all transmissions of sars-cov-2 happen before the infected individual even develops symptoms. this means that much broader testing programs are required to spot people when they are most infectious. rivett, sridhar, sparkes, routledge et al. set out to determine what proportion of healthcare workers was infected with sars-cov-2 while also feeling generally healthy at the time of testing. over 1,000 staff members at a large uk hospital who felt they were well enough to work, and did not fit the government criteria for covid-19 infection, were tested. amongst these, 3% were positive for sars-cov-2. on closer questioning, around one in five reported no symptoms, two in five very mild symptoms that they had dismissed as inconsequential, and a further two in five reported covid-19 symptoms that had stopped more than a week previously. in parallel, healthcare workers with symptoms of covid-19 (and their household contacts) who were self-isolating were also tested, in order to allow those without the virus to quickly return to work and bolster a stretched workforce. finally, the rates of infection were examined to probe how the virus could have spread through the hospital and among staff -and in particular, to understand whether rates of infection were greater among staff working in areas devoted to covid-19 patients. despite wearing appropriate personal protective equipment, healthcare workers in these areas were almost three times more likely to test positive than those working in areas without covid-19 patients. however, it is not clear whether this genuinely reflects greater rates of patients passing the infection to staff. staff may give the virus to each other, or even acquire it at home. overall, this work implies that hospitals need to be vigilant and introduce broad screening programmes across their workforces. it will be vital to establish such approaches before 'lockdown' is fully lifted, so healthcare institutions are prepared for any second peak of infections. clinically suspected covid-19 patients was performed in negative-pressure (à5 pascals) side rooms, with 10 air changes per hour and use of scenario 2 ppe. all other aerosol generating procedures were undertaken with scenario 2 ppe precautions, in negative-or neutral-pressure facilities. general clinical areas underwent a minimum of 6 air changes per hour, but all critical care areas underwent a minimum of 10 air changes per hour as a matter of routine. surgical operating theatres routinely underwent a minimum of 25 air changes per hour. viral loads varied between individuals, potentially reflecting the nature of the sampling site. however, for individuals testing positive for sars-cov-2, viral loads were significantly lower for those in the hcw asymptomatic screening group than in those tested due to the presence of symptoms (figure 1) . for the hcw symptomatic and hcw symptomatic contact screening groups, viral loads did not correlate with duration of symptoms or with clinical criteria risk score (figure 1 -figure supplement 1 and data not shown). three subgroups of sars-cov-2 positive asymptomatic hcw each individual in the hcw asymptomatic screening group was contacted by telephone to establish a clinical history, and covid-19 probability criteria ( table 1) were retrospectively applied to categorise any symptoms in the month prior to testing ( figure 2 ). one hcw could not be contacted to obtain further history. individuals captured by the hcw asymptomatic screening group were generally asymptomatic at the time of screening, however could be divided into three sub-groups: (i) hcws with no symptoms at all, (ii) hcws with (chiefly low-to-medium covid-19 probability) symptoms commencing 7 days prior to screening and (iii) hcws with (typically high covid-19 probability) symptoms commencing >7 days prior to screening ( figure 2 ). 9/12 (75%) individuals with symptom onset >7 days previously had appropriately self-isolated and then returned to work. one individual with no symptoms at the time of swabbing subsequently developed symptoms prior to being contacted with their positive result. overall, 5/1032 (0.5%) individuals in the asymptomatic screening group were identified as truly asymptomatic carriers of sars-cov-2, and 1/1032 (0.1%) was identified as pre-symptomatic. box 1 shows illustrative clinical vignettes. for the hcw asymptomatic screening group, nineteen wards were identified for systematic priority screening as part of hospital-wide surveillance. two further areas were specifically targeted for screening due to unusually high staff sickness rates (ward f), or concerns about appropriate ppe usage (ward q) ( figure 3 ). interestingly, in line with findings in the total hcw population, a significantly greater proportion of hcws working on 'red' wards compared to hcws working on 'green' wards tested positive as part of the asymptomatic screening programme ('green' 6/310 vs 'red' 19/ 372; p=0.0389, fisher's exact test). the proportion of hcw with a positive test was significantly higher on ward f than on other wards categorised as 'green' clinical areas (ward f 4/43 vs other 'green' wards 2/267; p=0.0040, fisher's exact test). likewise, amongst wards in the 'red' areas, ward q showed significantly higher rates of positive hcw test results (ward q 7/37 vs other 'red' wards 12/335; p=0.0011, fisher's exact test). ward f is an elderly care ward, designated as a 'green' area with scenario 0 ppe (tables 4-5) , with a high proportion of covid-19 vulnerable patients due to age and comorbidity. 4/43 (9%) ward staff tested positive for sars-cov-2. in addition, two staff members on this ward tested positive in the hcw symptomatic/symptomatic contact screening groups. all positive hcws were requested to self-isolate, the ward was closed to admissions and escalated to scenario 1 ppe ( table 5) . reactive screening of a further 18 ward f staff identified an additional three positive asymptomatic hcws (figure 4 ). sequence analysis indicated that 6/9 samples from hcw who worked on ward f belonged to sars-cov-2 lineage b. 1 (currently known to be circulating in at least 43 countries [rambaut et al., 2020] ), with a further two that belonged to b1. 7 and one that belonged to b2. 1. this suggests more than two introductions of sars-cov-2 into the hcw population on ward f (figure 4-figure supplements 1-2, table 6 ). it was subsequently found that two further staff members from ward f had previously been admitted to hospital with severe covid-19 infection. ward q is a general medical ward designated as a 'red' clinical area for the care of covid-19 positive patients, with a scenario 1 ppe protocol (tables 4-5). here, 7/37 (19%) ward staff tested positive for sars-cov-2. in addition, one staff member tested positive as part of the hcw symptomatic screening group, within the same period as ward surveillance. reactive screening of a further five staff working on ward q uncovered one additional infection. 4/4 sequenced viruses were of the b. 1 lineage (figure 4-figure supplements 1-2, table 6 ; other isolates could not be sequenced due to a sample ct value >30). all positive hcws were requested to self-isolate, and infection control and ppe reviews were undertaken to ensure that environmental cleaning and ppe donning/doffing practices were compliant with hospital protocol. staff training and education was provided to address observed instances of incorrect infection control or ppe practice. ward o, a 'red' medical ward, had similar numbers of asymptomatic hcws screened as ward f, and a similar positivity rate (4/44; 9%). this ward was listed for further cluster investigation after the study ended, however incorrect ppe usage was not noted during the study period. the majority of individuals who tested positive for sars-cov-2 after screening due to the presence of symptoms had high covid-19 probability ( table 7) . this reflects national guidance regarding self-isolation at the time of our study (uk government, 2020a). through the rapid establishment of an expanded hcw sars-cov-2 screening programme, we discovered that 31/1,032 (3%) of hcws tested positive for sars-cov-2 in the absence of symptoms. of 30 individuals from this asymptomatic screening group studied in more depth, 6/30 (20%) had not experienced any symptoms at the time of their test. 1/6 became symptomatic suggesting that the true asymptomatic carriage rate was 5/1,032 (0.5%). 11/30 (37%) had experienced mild symptoms prior to testing. whilst temporally associated, it cannot be assumed that these symptoms necessarily resulted from covid-19. these proportions are difficult to contextualise due to paucity of table 4 . the hospital's traffic-light colouring system for categorising wards according to anticipated covid-19 exposure risk. different types of ppe were used in each ( table 5) . red (high risk) amber (medium risk) green (low risk) areas with confirmed sars-cov-2 rt-pcr positive patients, or patients with very high clinical suspicion of covid-19 areas with patients awaiting sars-cov-2 rt-pcr test results, or that have been exposed and may be incubating infection areas with no known sars-cov-2 rt-pcr positive patients, and none with clinically suspected covid-19 point-prevalence data from asymptomatic individuals in similar healthcare settings or the wider community. for contrast, 60% of asymptomatic residents in a recent study tested positive in the midst of a care home outbreak (arons et al., 2020) . regardless of the proportion, however, many secondary and tertiary hospital-acquired infections were undoubtedly prevented by identifying and isolating these sars-cov-2 positive hcws. amber + red wards, for example intensive care unit, respiratory units with non-invasive ventilation facilities. all operating theatres, including facilities for bronchoscopy and endoscopy. 12/30 (40%) individuals from the hcw asymptomatic screening group reported symptoms > 7 days prior to testing, and the majority experiencing symptoms consistent with a high probability of covid-19 had appropriately self-isolated during that period. patients with covid-19 can remain sars-cov-2 pcr positive for a median of 20 days (iqr 17-24) after symptom onset (zhou et al., 2020) , and the limited data available suggest viable virus is not shed beyond eight days (wö lfel et al., 2020) . a pragmatic approach was taken to allowing individuals to remain at work, where the hcw had experienced high probability symptoms starting >7 days and 1 month prior to their test and had been well for the preceding 48 hr. this approach was based on the following: low seasonal incidence of alternative viral causes of high covid-19 probability symptoms in the uk (public health england, 2018), the high potential for sars-cov-2 exposure during the pandemic and the potential for prolonged, non-infectious shedding of viral rna (zhou et al., 2020; wö lfel et al., 2020) . for other individuals, we applied standard national guidelines requiring isolation for seven days from the point of testing (uk government, 2020b). however, for hcw developing symptoms after a positive swab, isolation was extended for seven days from symptom onset. our data clearly demonstrate that focusing solely on the testing of individuals fitting a strict clinical case definition for covid-19 will inevitably miss asymptomatic and pauci-symptomatic disease. this is of particular importance in the presence of falling numbers of community covid-19 cases, as hospitals will become potential epicentres of local outbreaks. therefore, we suggest that in the setting of limited testing capacity, a high priority should be given to a reactive asymptomatic screening programme that responds in real-time to hcw sickness trends, or (to add precision) incidence of positive tests by area. the value of this approach is illustrated by our detection of a cluster of cases in ward f, where the potential for uncontrolled staff-to-staff or staff-to-patient transmission could have led to substantial morbidity and mortality in a particularly vulnerable patient group. as sars-cov-2 testing capacity increases, rolling programmes of serial screening for asymptomatic staff in all box 1. clinical vignettes. self-isolation instructions were as described in table 2 . case 1: completely asymptomatic. hcw1 had recently worked on four wards (two 'green', two 'amber'). upon testing positive, she reported no symptoms over the preceding three weeks, and was requested to go home and self-isolate immediately. hcw1 lived with her partner who had no suggestive symptoms. upon follow-up telephone consultation 14 days after the test, hcw1 had not developed any significant symptoms, suggesting true asymptomatic infection. case 2: pre-symptomatic. hcw2 was swabbed whilst asymptomatic, testing positive. when telephoned with the result, she reported a cough, fever and headache starting within the last 24 hr and was advised to self-isolate from the time of onset of symptoms ( table 2) . her partner, also a hcw, was symptomatic and had been confirmed as sars-cov-2 positive 2 days previously, suggesting likely transmission of infection to hcw2. case 3: low clinical probability of covid hcw3 developed mild self-limiting pharyngitis three days prior to screening and continued to work in the absence of cough or fever. she had been working in' green' areas of the hospital, due to a background history of asthma. self-isolation commenced from the time of the positive test. hcw3's only contact outside the hospital, her housemate, was well. on follow-up telephone consultation, hcw3's mild symptoms had fully resolved, with no development of fever or persistent cough, suggesting pauci-symptomatic infection. case 4: medium clinical probability of covid hcw4 experienced anosmia, nausea and headache three days prior to screening, and continued to work in the absence of cough or fever. self-isolation commenced from the time of the positive test. one son had experienced a mild cough~3 weeks prior to hcw4's test, however her partner and other son were completely asymptomatic. upon follow-up telephone consultation 10 days after the test, hcw4's mild symptoms had not progressed, but had not yet resolved. case 5: high clinical probability of covid. hcw5 had previously self-isolated, and did not repeat this in the presence of new high-probability symptoms six days before screening. self-isolation commenced from the date of the new symptoms with the caveat that they should be completely well for 48 hr prior to return to work. all household contacts were well. however, another close colleague working on the same ward had also tested positive, suggesting potential transmission between hcws on that ward. areas of the hospital is recommended, with the frequency of screening being dictated by anticipated probability of infection. the utility of this approach in care-homes and other essential institutions should also be explored, as should serial screening of long-term inpatients. the early success of our programme relied upon substantial collaborative efforts between a diverse range of local stakeholders. similar collaborations will likely play a key role in the rapid, de novo development of comprehensive screening programmes elsewhere. the full benefits of enhanced hcw screening are critically dependent upon rapid availability of results. a key success of our programme has been bespoke optimisation of sampling and laboratory workflows enabling same-day resulting, whilst minimising disruption to hospital processes by avoiding travel to off-site testing facilities. rapid turnaround for testing and sequencing is vital in enabling timely response to localised infection clusters, as is the maintenance of reserve capacity to allow urgent, reactive investigations. there appeared to be a significantly higher incidence of hcw infections in 'red' compared to 'green' wards. many explanations for this observation exist, and this study cannot differentiate between them. possible explanations include transmission between patients and hcw, hcw-to-hcw transmission, variability of staff exposure outside the workplace and non-random selection of wards. it is also possible that, even over the three weeks of the study, 'red' wards were sampled earlier during the evolution of the epidemic when transmission was greater. further research into these findings is clearly needed on a larger scale. furthermore, given the clear potential for pre-symptomatic and asymptomatic transmission amongst hcws, and data suggesting that infectivity may peak prior to symptom onset (he et al., 2020) , there is a strong argument for basic ppe provision in all clinical areas. the identification of transmission within the hospital through routine data is problematic. hospitals are not closed systems and are subject to numerous external sources of infection. coronaviruses generally have very low mutation rates (~10 à6 per site per cycle) (sanjuán et al., 2010) , with the first reported sequence of the current pandemic only published on 12 th january 2020 (genbank, 2020). in addition, given sars cov-2 was only introduced into the human population in late 2019, there is at present a lack of diversity in circulating strains. however, as the pandemic unfolds and detailed epidemiological and genome sequence data from patient and hcw clusters are generated, realtime study of transmission dynamics will become an increasingly important means of informing disease control responses and rapidly confirming (or refuting) hospital acquired infection. importantly, implementation of such a programme would require active screening and rapid sequencing of positive cases in both the hcw and patient populations. prospective epidemiological data will also inform whether hospital staff are more likely to be infected in the community or at work, and may identify risk factors for the acquisition of infection, such as congregation in communal staff areas or inadequate access to ppe. our study is limited by the relatively short time-frame, a small number of positive tests and a lack of behavioural data. in particular, the absence of detailed workplace and community epidemiological data makes it difficult to draw firm conclusions with regards to hospital transmission dynamics. the low rate of observed positive tests may be partly explained by low rates of infection in the east of england in comparison with other areas of the uk (cumulative incidence 0.17%, thus far) (public health england, 2020). the long-term benefits of hcw screening on healthcare systems will be informed by sustained longitudinal sampling of staff in multiple locations. more comprehensive data will parametrise workforce depletion and covid-19 transmission models. the incorporation of additional information including staffing levels, absenteeism, and changes in proportions of staff self-isolating before and after the introduction of widespread testing will better inform the impact of screening at a national and international level. such models will be critical for optimising the impact on occupationally-acquired covid-19, and reducing the likelihood that hospitals become hubs for sustained covid-19 transmission. in the absence of an efficacious vaccine, additional waves of covid-19 are likely as social distancing rules are relaxed. understanding how to limit hospital transmission will be vital in determining (table 4) . hcws working across >1 ward were counted for each area. the left-hand y-axis shows the percentage of positive results from a given ward compared to the total positive results from the hcw asymptomatic screening group (blue bars). the right-hand y-axis shows the total number of sars-cov-2 tests (stars) and the number positive (pink circles). additional asymptomatic screening tests were subsequently performed in an intensified manner on ward f and ward q after identification of clusters of positive cases on these wards (figure 4) . asymptomatic screening tests were also performed for a number of individuals from other clinical areas on an opportunistic basis; none of these individuals tested positive. results of these additional tests are included in summary totals in table 1 , but not in this figure. infection control policy, and retain its relevance when reliable serological testing becomes widely available. our data suggest that the roll-out of screening programmes to include asymptomatic as well as symptomatic patient-facing staff should be a national and international priority. our approach may also be of benefit in reducing transmission in other institutions, for example carehomes. taken together, these measures will increase patient confidence and willingness to access healthcare services, benefiting both those with covid-19 and non-covid-19 disease. two parallel streams of entry into the testing programme were established and managed jointly by the occupational health and infectious diseases departments. the first (hcw symptomatic, and hcw symptomatic household contact screening groups) allowed any patient-facing or non-patientfacing hospital employee (hcw) to refer themselves or a household contact, including children, should they develop symptoms suggestive of covid-19. the second (hcw asymptomatic screening group) was a rolling programme of testing for all patient-facing and non-patient-facing staff working in defined clinical areas thought to be at risk of sars-cov-2 transmission. daily workforce sickness reports and trends in the results of hcw testing were monitored to enable areas of concern to be highlighted and targeted for screening and cluster analysis, in a reactive approach. high throughput clinical areas where staff might be exposed to large numbers of suspected covid-19 patients were also prioritised for staff screening. these included the emergency department, the covid-19 assessment unit, and a number of 'red' inpatient wards. staff caring for the highest priory 'shielding' patients (haematology/oncology, transplant medicine) were also screened, as were a representative sample of staff from 'amber' and 'green' areas. the personal protective equipment (ppe) worn by staff in these areas is summarised in table 5 . inclusion into the programme was voluntary, and offered to all individuals working in a given ward during the time of sampling. regardless of the table 6 continued on next page route of entry into the programme, the process for testing and follow-up was identical. wards were closed to external visitors. we devised a scoring system to determine the clinical probability of covid-19 based on symptoms from existing literature giacomelli et al., 2020; table 1 ). self-referring hcw and staff captured by daily workforce sickness reports were triaged by designated occupational health nurses using these criteria ( table 2) . self-isolating staff in the medium and low probability categories were prioritised for testing, since a change in the clinical management was most likely to derive from results. self-isolation and household quarantine advice was determined by estimating the pre-test probability of covid-19 (high, medium or low) in those with symptoms, based on the presence or absence of typical features (tables 1-2) . symptom history was obtained for all symptomatic hcws at the time of self-referral, and again for all positive cases via telephone interview when results became available. all individuals who had no symptoms at the time of testing were followed up by telephone within 14 days of their result. pauci-symptomatic individuals were defined as those with low-probability clinical covid-19 criteria ( table 2) . testing was primarily undertaken at temporary on-site facilities. two 'pods' (self-contained portable cabins with office, kitchen facilities, generator and toilet) were erected in close proximity both to the laboratory and main hospital. outside space was designed to enable car and pedestrian access, and ensure !2 m social distancing at all times. individuals attending on foot were given pre-prepared self-swabbing kits containing a swab, electronically labelled specimen tube, gloves and swabbing instructions contained in a zip-locked collection bag. pods were staffed by a team of re-deployed research nurses, who facilitated self-swabbing by providing instruction as required. scenario 1 ppe ( table 5 ) was worn by pod nurses at all times. individuals in cars were handed self-swabbing kits through the window, with samples dropped in collection bags into collection bins outside. any children (household contacts) were brought to the pods in cars and swabbed in situ by a parent or guardian. in addition to pod-based testing, an outreach hcw asymptomatic screening service was developed to enable self-swabbing kits to be delivered to hcws in their area of work, minimising disruption to the working routine of hospital staff, and maximising pod availability for symptomatic staff. lists of all staff working in target areas over a 24 hr period were assembled, and kits pre-prepared accordingly. self-swabbing kits were delivered to target areas by research nurses, who trained senior nurses in the area to instruct other colleagues on safe self-swabbing technique. kits were left in target areas for 24 hr to capture a full cycle of shift patterns, and all kits and delivery equipment were thoroughly decontaminated with 70% ethanol prior to collection. twice daily, specimens were delivered to the laboratory for processing. the swabbing, extraction and amplification methods for this study follow a recently validated procedure (sridhar et al., 2020) . individuals performed a self-swab at the back of the throat followed by the nasal cavity as previously described (our world in data, 2020). the single dry sterile swab was immediately placed into transport medium/lysis buffer containing 4m guanidine thiocyanate to table 7 . distribution of positive sars-cov-2 tests amongst symptomatic individuals with a positive test result, categorised according to test group and covid-19 symptom-based probability criteria (as defined in table 2 ). inactivate virus, and carrier rna. this facilitated bsl2-based manual extraction of viral rna in the presence of ms2 bacteriophage amplification control. use of these reagents and components avoided the need for nationally employed testing kits. real-time rt-pcr amplification was performed as previously described and results validated by confirmation of fam amplification of the appropriate controls with threshold cycle (ct) 36. lower ct values correspond to earlier detection of the viral rna in the rt-pcr process, corresponding with a higher copy number of the viral genome. in 2/1,270 cases, rt-pcr failed to amplify the internal control and results were discarded, with hcw offered a re-test. sequencing of positive samples was attempted on samples with a ct 30 using a multiplex pcr based approach (quick et al., 2017) using the modified artic v2 protocol (quick, 2020) and v3 primer set (artic network, 2020). genomes were assembled using reference based assembly and the bioinformatic pipeline as described (quick et al., 2017) using a 20x minimum coverage as a cut-off for any region of the genome and a 50.1% cut-off for calling of single nucleotide polymorphisms (snps). samples were sequenced as part of the covid-19 genomics uk consortium, cog-uk), a partnership of nhs organisations, academic institutions, uk public health agencies and the wellcome sanger institute. as soon as they were available, positive results were telephoned to patients by infectious diseases physicians, who took further details of symptomatology including timing of onset, and gave clinical advice ( table 2) . negative results were reported by occupational health nurses via telephone, or emailed through a secure internal email system. advice on returning to work was given as described in table 2 . individuals advised to self-isolate were instructed to do so in their usual place of residence. particularly vulnerable staff or those who had more severe illness but did not require hospitalisation were offered follow-up telephone consultations. individuals without symptoms at the time of testing were similarly followed up, to monitor for de novo symptoms. verbal consent was gained for all results to be reported to the hospital's infection control and health and safety teams, and to public health england, who received all positive and negative results as part of a daily reporting stream. swab result data were extracted directly from the hospital-laboratory interface software, epic (verona, wisconsin, usa). details of symptoms recorded at the time of telephone consultation were extracted manually from review of epic clinical records. data were collated using microsoft excel, and figures produced with graphpad prism (graphpad software, la jolla, california, usa). fisher's exact test was used for comparison of positive rates between groups defined in the main text. mann-whitney testing was used to compare ct values between different categories of tested individuals. hcw samples that gave sars cov-2 genomes were assigned global lineages defined by rambaut et al., 2020 using the pangolin utility (o'toole and mccrone, 2020). as a study of healthcare-associated infections, this investigation is exempt from requiring ethical approval under section 251 of the nhs act 2006 (see also the nhs health research authority algorithm, available at http://www.hra-decisiontools.org.uk/research/, which concludes that no formal ethical approval is required). written consent was obtained from each hcw described in the anonymised case vignettes. the citiid-nihr covid-19 bioresource collaboration ravi gupta harmeet gill; iain kean; mailis maes; nicola reynolds; michelle wantoch; sarah caddy anita furlong nathalie kingston; sofia papadia anne meadows naidine escoffery; heather jones; carla ribeiro nick brown; surendra parmar ; hongyi zhang; ailsa bowring; geraldine martell; natalie quinnell stefan grä f aloka de sa; maddie epping; andrew hinch conceptualization, data curation, formal analysis, investigation, methodology, project administration, writing -review and editing conceptualization, data curation, formal analysis, validation, methodology, project administration, writing -review and editing data curation, formal analysis, writing -original draft, project administration, writingreview and editing writing -original draft, project administration, writingreview and editing data curation, investigation, methodology, writing -original draft, project administration, writing -review and editing data curation, validation data curation, formal analysis, investigation data curation, writing -original draft conceptualization, writing -original draft, project administration, writing -review and editing data curation, supervision, writing -review and editing; the citiid-nihr covid-19 bioresource collaboration, conceptualization, data curation, formal analysis, funding acquisition, investigation, writing -original draft data curation, software; ashley shaw, supervision, project administration project administration, writing -review and editing data curation, formal analysis, supervision, project administration, writing -review and editing conceptualization, data curation, formal analysis, methodology, writing -original draft, project administration, writing -review and editing writing -original draft, project administration, writing -review and editing author orcids lucy rivett ethics human subjects: as a study of healthcare-associated infections, this investigation is exempt from requiring ethical approval under section 251 of the nhs act 2006 (see also the nhs health research authority algorithm presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility artic network. 2020. artic-ncov2019 / primer_schemes covid-19: the case for health-care worker screening to prevent hospital transmission characteristics of health care personnel with covid-19 -united states exclusive: deaths of nhs staff from covid-19 analysed covid-19: identifying and isolating asymptomatic people helped eliminate virus in italian village asymptomatic transmission, the achilles' heel of current strategies to control covid-19 wuhan seafood market pneumonia virus isolate wuhan-hu-1 complete genome self-reported olfactory and taste disorders in sars-cov-2 patients: a crosssectional study temporal dynamics in viral shedding and transmissibility of covid-19 first experience of covid-19 screening of health-care workers in england report 16: role of testing in covid-19 control roll-out of sars-cov-2 testing for healthcare workers at a large nhs foundation trust in the united kingdom immediate and sustained psychological impact of an emerging infectious disease outbreak on health care workers epidemiology of covid-19 in a long-term care facility in king county software package for assigning sars-cov-2 genome sequences to global lineages to understand the global pandemic, we need global testing -the our world in data covid-19 testing dataset surveillance of influenza and other respiratory viruses in the uk to coronavirus (covid-19) in the uk 2020 multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples ncov-2019 sequencing protocol v2 a dynamic nomenclature proposal for sars-cov-2 to assist genomic epidemiology viral mutation rates a blueprint for the implementation of a validated approach for the detection of sars-cov2 in clinical samples in academic facilities stay at home advice covid-19: management of exposed healthcare workers and patients in hospital settings clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in report of the who-china joint mission on coronavirus disease who. 2020b. covid-19 strategy update virological assessment of hospitalized patients with covid-2019 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study additional files . source data 1. asymptomatic sars-cov-2 screening programme source data.. transparent reporting form sequencing data have been deposited in gsaid under accession codes epi_isl_433989-epi_-isl_433992, epi_isl_434005, epi_isl_433489-epi_isl_433497. researchers will be prompted to register and log on to the website to access the datasets (https://www.epicov.org/epi3/ frontend#1f1442). key: cord-343340-zi0rfidc authors: aragón‐caqueo, diego; fernández‐salinas, javier; laroze, david title: optimization of group size in pool testing strategy for sars‐cov‐2: a simple mathematical model date: 2020-05-03 journal: j med virol doi: 10.1002/jmv.25929 sha: doc_id: 343340 cord_uid: zi0rfidc coronavirus disease (covid‐19) has reached unprecedented pandemic levels and is affecting almost every country in the world. ramping up the testing capacity of a country supposes an essential public health response to this new outbreak. a pool testing strategy where multiple samples are tested in a single reverse transcriptase‐polymerase chain reaction (rt‐pcr) kit could potentially increase a country's testing capacity. the aim of this study is to propose a simple mathematical model to estimate the optimum number of pooled samples according to the relative prevalence of positive tests in a particular healthcare context, assuming that if a group tests negative, no further testing is done whereas if a group tests positive, all the subjects of the group are retested individually. the model predicts group sizes that range from 11 to 3 subjects. for a prevalence of 10% of positive tests, 40.6% of tests can be saved using testing groups of four subjects. for a 20% prevalence, 17.9% of tests can be saved using groups of three subjects. for higher prevalences, the strategy flattens and loses effectiveness. pool testing individuals for severe acute respiratory syndrome coronavirus 2 is a valuable strategy that could considerably boost a country's testing capacity. however, further studies are needed to address how large these groups can be, without losing sensitivity on the rt‐pcr. the strategy best works in settings with a low prevalence of positive tests. it is best implemented in subgroups with low clinical suspicion. the model can be adapted to specific prevalences, generating a tailored to the context implementation of the pool testing strategy. in late december of 2019, several cases of pneumonia of apparent viral origin were reported in wuhan, china. 1,2 subsequently, a novel coronavirus was identified as the causative pathogen, 3 this new pathogen was identified as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the disease (coronavirus disease ) rapidly spread to neighboring countries and overseas, reaching pandemic proportions and was declared by the world health organization (who) as a public health emergency of international concern on 30 january, 2020. 4 as of 19 april, 2020, the who has reported 2 241 359 confirmed cases with 152 551 deaths worldwide, 5 a total of 185 countries affected, while 10 still remain with no reported cases. 6 the main diagnostic test that has been implemented worldwide to confirm the infection by this novel coronavirus is the real-time reverse transcriptase-polymerase chain reaction (rt-pcr) from respiratory samples with satisfactory levels of sensibility and specificity. 7 however, there might be other clinical specimens where the virus could be detected as well, using the same technique. [8] [9] [10] the procedure takes about a day to come up with a result 11 ; however, more efficient methods are being developed as the pandemic progresses. a crucial part of the public health response to this new threat is to rapidly diagnose and isolate infected individuals to prevent further spreading. 12, 13 therefore, amplifying the testing capacity of a country experiencing a massive outbreak, is a key strategy for facing this new public health emergency. 14 nowadays, the united states is the country with a greater number of confirmed cases worldwide and performs as of 19 april, 2020, 167 330 tests daily, with a total of 3 865 864 tests performed since the beginning of the outbreak 15 with all states currently testing. 16 other largely affected countries are also performing thousands of confirmatory tests on a daily basis. 17 however, due to the overwhelming number of rapidly growing cases, a considerably large number of suspected cases cannot be properly tested and isolated due to the lack of logistics of a progressively collapsing healthcare system. therefore, it becomes urgent to optimize the standard operating procedures to confirm the infection by sars-cov-2. 18 since the clinical presentation of the disease is often mild or asymptomatic, 19, 20 and that it has been reported that asymptomatic individuals could transmit the virus, 21, 22 it becomes crucial to implement an efficient testing strategy to screen that population and properly isolate them to prevent the further spread of the virus. however, as the healthcare systems around the world are progressively collapsing due to the increasing demand of moderate to severe patients that every day present to the emergency room, the testing of individuals with low clinical suspicion has been left behind, in order to prioritize the available resources for the patients with moderate to severe symptoms. although it becomes quite logical to prioritize testing for patients with higher clinical suspicion, there is a considerable segment of the population that is not being screened and become vectors of the virus, contributing even more to the spread of the disease and further collapse the healthcare system with the new cases yet to come. 23 on the other hand, as proposed by seifried and ciesek, 24 25 however, some studies suggest that the pooling of the sample should be kept as low as possible to reduce dilution and maintain the sensitivity of the test. 26, 27 since the scope of this strategy could potentially increase multiple times the testing capacity of a country, it becomes prudent to explore how to optimize the implementation of it in the healthcare setting. therefore, the aim of this study is to provide a mathematical model to estimate the optimum number of pooled samples according to the specific prevalences of positive tests in a particular country context, in order to save as many tests as possible and cover as many people as possible, knowing that if a group tests out positive, all the individuals of the sample would have to be individually tested. it is important to highlight that this model is based on the prevalence of positive tests and can be adapted to each country's specific prevalence. however, it is best implemented for countries with a large number of confirmed cases and relatively large number of tests performed on a daily basis, since more data on the specific prevalence of positive yielding results are available and more accurate estimations can be done based on this; rather than countries with a low number of confirmed cases or where the implementation of testing the population has not been the most adequate. the manuscript is arranged in the following way: in section 2, the materials and methods are introduced. in section 3, the results are given together with the discussion. finally, the final remarks are presented in section 4. thoughtful description of the process and reasoning for obtaining a formula that represents the benefit of performing a pool test of the most optimum size assuming in advance that if a group tests out positive, all the subjects in the group have to be individually tested, in order to track down the positive case or cases, while if a group tests out negative, then no further testing in that specific group is needed. all the computations were performed with the software wolfram mathematica. 28 considering that the sample of each suspected individual tested for the infection of sars-cov-2 with the rt-pcr could yield either a negative or positive result, and that performing a pool testing strategy could yield a negative result only when all the samples included in the pool sample are negative, and that it will yield a positive result when at least one of the individual samples is positive, the possible diagnostic scenarios for the pool test can be expressed by the binomial expression of where x represents the probability of subjects with an individual positive test (prevalence of positives), y represents the probability of subjects with an individual negative test (prevalence of negatives), and n is size of the pool group. such that n > 1, 0 < x < 1, and y = 1 − x. under these assumptions, we obtain that d = 1. note that the breakdown of this expression will hold all the possible events. this will be represented by the addends, and the combination present in these will be determined by x and y and its respective exponent, which will indicate the number of subjects with a positive or negative sample, respectively. the distribution of the possibilities will depend on the prevalence of the disease, in this case, being the percentage of positive test results obtained from the recent historical data available. for this reason, the probability of each expressed event occurring, will be determined by the substitution of x and y by the respective prevalences of positive and negative tests. now, let us separate equation (1) in two parts x y x y y y 1 . here, the negative groups for the pool test and its probability will be represented by y n , while the pool tests that yield a positive will correspond to all the other cases where there is at least one individual positive sample in the pool, having, therefore, a 1 − y n probability of becoming true. to facilitate the use of equation (3), it will be expressed as a function of x, which relates to the direct prevalence of positive historical testing for each country, so that it can be inputted in the equation. therefore, considering that every time a pool test yields a negative result, no further testing will be performed to that group, the saved tests of the otherwise individually tested subjects, will be ex to obtain the optimum group size given the prevalence of positive tests (x) in a determined setting, the minimal global of equation (5) must be obtained. this minimum value is calculated using x as the input, because x is a continuous variable, while n is a discrete one. let us remark that, knowing the average minimum number of tests per subject needed to diagnose one subject, then the population covered by one test using a pool testing strategy according to the optimal pool size previously calculated (and addressing the fact that when a group yields a positive result, the whole group has to be individually tested), can be expressed as = /z subjects covered per test 1 . with the model proposed above, different scenarios were tested according to different prevalences of positive tests. this was done to address the fact that each country presents a unique distribution of daily performed tests and positive results. according to this, the optimum size and average minimum tests per subject to detect a positive for the diverse chosen prevalence scenarios were calculated. then, it was further compared to the individual testing strategy and how many more positive results could be detected using pool testing, f i g u r e 1 contour plot of the average minimum number of tests per subject to diagnose one subject. horizontal-axis: prevalence of positive tests, x, the interval ranges from 0 to 0.4. vertical-axis: group size, n. the interval ranges from 2 to 100. the average minimum number of tests per subject to diagnose one subject is represented by the colors, where higher and better values go from green to orange, being orange the closest to the optimum with the same amount of tests, thus, addressing the efficiency of the strategy over individual testing, as shown on table 1 . on the other hand, given the optimum group sizes calculated for the chosen prevalence scenarios, the population covered by a 100 tests was calculated using the average minimum test per subject to detect a positive, and was compared the 100 subjects that an individual testing strategy would cover, as it is exposed in table 2 . as exposed in the results, the lower the prevalence of positive tests for a particular country is, the more tests that can be saved and the larger the pool groups will be. from prevalences ranging from 0.03 to 0.07, the testing capacity of a country using a pool testing strategy is increased by a factor of two or by a factor three, rather than using individual testing. this could bring unprecedented advances in better understanding the disease and how it distributes on a particular population. from prevalence ranging from 0.08 to 0.2, the net saving of test kits using pool testing strategy is still significant, saving around 46.6% to 17.9% of the tests if an individual testing strategy were to be performed in the same number of subjects, thus, covering a greater portion of the population. however, as prevalence rises, the efficiency of the strategy flattens. reaching a prevalence over 0.25, the net saving of tests is still significant. however, separating the samples, creating pool groups, tracking individuals in the groups that yielded a positive result, and retesting all those subjects individually, suppose logistical challenges that every healthcare center must weigh to implement this strategy over the most likely already implemented individual testing strategy. finally, reaching a prevalence near 0.3, the pool testing strategy becomes similar to the individual testing strategy, thus losing its effectiveness and becoming a logistical problem, rather than optimizing the testing protocols. this is mainly because in the model proposed, whenever a group tests positive, all the individuals of the group should get retested to track the positive subject or subjects in the pooled sample. therefore, the more positive individuals there are in the population, the highest positive pool tests there will be. thus, more tests will be lost, and more tests will be used in retesting the positive pool samples. notice that for large positive groups, further subgrouping and pool testing of those subgroups could be implemented. this could potentially save even more tests; however, it is believed that this approach might suppose a difficult logistical challenge that the progressively collapsing healthcare systems worldwide might not be able to cope for now. as of 19 april 2020, most countries have prevalences of positive tests that range around 0.1 to 0.2 of all the tests daily performed 29 so a pool testing strategy is still a plausible strategy to implement on a national level. however, for the analysis, the overall historical prevalence was used as a country scenario, but when subjects are further stratified according to clinical suspicion, a lower prevalence of positive tests are expected in lower clinical suspicion groups, so the pool testing strategy could be best implemented in this stratified subgroup rather than the whole population. as it was previously exposed, lower prevalence of positive tests, show greater efficiency in the test use, however, with larger group sizes. one of the main critiques to the pool testing strategy, is the dilution that occurs when pooling the samples together, and how this dilution might affect the test sensitivity. previous studies have shown that there is no decrease in sensitivity for rt-pcr in detecting other viruses when using pool samples of 10 and 20 subjects, 30 however, as far as the available evidence on sars-cov-2 show, samples of five subjects do not affect sensibility of rt-pcr for detecting the virus. 24 the model proposes optimum group numbers that range from 11 to 3 subjects, depending on the individual prevalence. this exquisitely copes with the possibility that larger groups might decrease rt-pcr sensitivity due to the dilution of the pooled sample and it has been proposed that to effectively implement poll testing strategy, the pooled samples should be kept as low as possible. 26, 27 further developing on this, the model predicts optimum groups of four and three subjects for the prevalence of positive tests that range from 0.1 to 0.2, which are the prevalence that most countries are reporting nowadays. therefore, it adapts to the clinical reality that the frontline workers all over the world are experiencing on a daily basis. this article proposed a simple and landed model to estimate the most optimum group number to implement pool testing strategy for sars-cov-2, according to the specific historical positive tests prevalence for a determined healthcare context. the aim of this model is to be implemented in different levels of healthcare facilities fighting the pandemic, given its flexibility to estimate the optimum group number, according to specific prevalence. these particular prevalences might differ from a healthcare facility to another, from one a city to another and might also differ from the country's overall outbreak status. therefore, it helps to create a tailored to the context implementation of the pool testing strategy for testing individuals with suspected infection by sars-cov-2. one of the main limitations of this study is that it assumes that the rt-pcr for detecting sars-cov-2 has a 100% sensitivity to the viral arn, when the evidence available shows sensitivity to be around 70%. 31 however, astonishing work is currently being done to improve test sensitivity; and addressing this non perfect sensitivity would greatly increase the complexity of the model. finally, it is worth mentioning the social implications that implementing pool testing might have. as the pandemic grows and more people get tested, implementing this testing strategy might not be well received by the general public, since patients most likely will want to know if their particular test yielded a positive or a negative result as soon as possible and will likely not accept their particular sample to be mixed with other samples. therefore, it becomes crucial to develop a strong public health policy to inform the population, secure equal access, and best implement the strategy for the greater good. the authors are thankful to dr ricardo segovia, md (hospital a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov-2 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding world health organization. statement on the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus (2019-ncov) situation report -90. geneva: who johns hopkins coronavirus resource center. covid-19 map detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr detection of sars-cov-2 in different types of clinical specimens detection of sars-cov-2 by rt-pcr in anal from patients who have recovered from coronavirus disease 2019 the presence of sars-cov-2 rna in feces of covid-19 patients reverse-transcription pcr (rt-pcr) disease control, civil liberties, and mass testing-calibrating restrictions during the covid-19 pandemic improved early recognition of coronavirus disease-2019 (covid-19): single-center data from a shanghai screening hospital laboratory testing strategy recommendations for covid-19 centers for disease control and prevention, cdc. testing in the u.s. available from to understand the global pandemic, we need global testing -the our world in data our world in data combination of rt-qpcr testing and clinical features for diagnosis of covid-19 facilitates management of sars-cov-2 outbreak characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention the clinical feature of silent infections of novel coronavirus infection (covid-19) in wenzhou transmission of 2019-ncov infection from an asymptomatic contact in germany epidemiological analysis of covid-19 and practical experience from china impact of nonpharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand pool testing of sars-cov-02 samples increases worldwide test capacities many times over. aktuelles aus der goethe universitt frankfurt pooling rt-pcr or ngs samples has the potential to cost-effectively generate estimates of covid-19 prevalence in resource limited environments optimizing screening for acute human immunodeficiency virus infection with pooled nucleic acid amplification tests data. covid-19: confirmed cases vs. tests conducted evaluation of saliva pools method for detection of congenital human cytomegalovirus infection antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 optimization of group size in pool testing strategy for sars-cov-2: a simple mathematical model the authors declare that there are no conflict of interests. http://orcid.org/0000-0001-7233-960x key: cord-289461-bnusv816 authors: droste, m. c.; stock, j.; atkeson, a. title: economic benefits of covid-19 screening tests date: 2020-10-27 journal: nan doi: 10.1101/2020.10.22.20217984 sha: doc_id: 289461 cord_uid: bnusv816 we assess the economic value of screening testing programs as a policy response to the ongoing covid-19 pandemic. we find that the fiscal, macroeconomic, and health benefits of rapid sars-cov-2 screening testing programs far exceed their costs, with the ratio of economic benefits to costs typically in the range of 4-15 (depending on program details), not counting the monetized value of lives saved. unless the screening test is highly specific, however, the signal value of the screening test alone is low, leading to concerns about adherence. confirmatory testing increases the net economic benefits of screening tests by reducing the number of healthy workers in quarantine and by increasing adherence to quarantine measures. the analysis is undertaken using a behavioral sir model for the united states with 5 age groups, 66 economic sectors, screening and diagnostic testing, and partial adherence to instructions to quarantine or to isolate. the effectiveness of a screening testing program hinges on whether those who test positive adhere to the instruction to self-isolate. using survey data from the united kingdom covering march through august 2020, smith et al. (2020) found that, of individuals reporting covid symptoms, only 18% report self-isolating; among those who were told by the national health service that they had been in close contact with a confirmed covid-19 case, only 11% reported quarantining for the recommended 14 days. these findings suggest that adherence will be low in response to other signals that have low information contentin particular, a low positive predictive value (ppv) 2about whether the individual is actually infected. we therefore allow the rate of adherence to depend on the specificity of the screening test. table 1 presents results for three representative testing programs. the programs are calibrated to existing or proposed tests and are designed to be representative of ones that might be deployable with adequate resources and effort. for cost-benefit purposes, we assume all incremental testing is federally funded. panel a considers a $5 screening test with 97.1% sensitivity and 98.5% specificity, 3 in which half of those who test positive on the screening test take a $50 confirmatory pcr test with a 48hour mean turnaround time. we suppose that adherence is high (75%) for those with a positive pcr test and low (25%) for those testing positive on the screening test but not taking a confirmatory pcr test. for random population screening testing at a weekly frequency, the total incremental cost of the program is $51 billion over the june 1 -december 31, 2020 simulation period. using our epidemiological-economic model, we project 66,000 deaths averted, an increase in gdp of $248 billion, and an increase in federal tax revenues of $68 billion over the counterfactual period of the program, june 1 -december 31, 2020, relative to a baseline with diagnostic but not screening testing. table 1 modifies this screening program so that everyone testing positive on the screening test receives a confirmatory pcr test. at a weekly testing cadence, this increases demand for pcr tests by approximately 630,000 tests per day, relative to the no-screening baseline. testing costs are somewhat higher, but because the effective adherence rate is higher under this program than in panel a, deaths averted rise to 153,000 and the increase in gdp is larger, $544 billion, for weekly testing. 2 the positive predictive value is the probability of being infected conditional on testing positive. by bayes law, the ppv depends on the specificity and sensitivity of the test and on the population rate of infection. 3 these costs and accuracy rates are those of the abbot laboratories binaxnow tm antigen test (fda (2020) ). additional estimates of test performance and costs are available in table 2 of silcox et. al. (2020) . . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 notes: counterfactual simulations suppose that the testing program was put in place on june 1, 2020. the simulations end december 31, 2020. entries are relative to a baseline with diagnostic testing at rates comparable to the summer of 2020 and no screening testing. deaths are as of january 1, 2021, and monetary values are current dollars for june 1, 2020 -december 31, 2020. panel c considers a different screening testing program, with two-step testing with a combined two-step specificity of 99.7%. as an illustration, one way to achieve this specificity is by independent two-step rapid antigen tests. the first step is a low-specificity (80%) $2 test, for example an inexpensive hypothetical paper-strip antigen test; if there is a positive test, the confirmatory test is the $5, 98.5% specificity test used in panels a and b. 4 there is no confirmatory pcr testing. we suppose the 99.7% specificity evokes a 50% adherence rate. the incremental testing costs under this program are less than for programs a or b. in part because turnaround is rapid, it averts 118,000 deaths and increases gdp by $397 billion at a weekly testing frequency, despite assumed lower adherence than to a pcr-based regime. these results and the additional sensitivity analysis below lead to four main conclusions. first, even with partial compliance, screening testing induces large net economic benefits. for the cases in table 1 , economic benefits exceed costs by a factor of 5-10 for weekly testing. if all the tests were paid for by the federal government, the additional tax revenues generated by the induced gdp growth would more than pay for the testing costs. net benefits rise if one additionally monetizes deaths averted using a statistical value of life. second, the signal value of a single positive screening test is low: in our simulations, the ppv is typically less than 5% for a test with 98.5% specificity. this low signal value could lead to low adherence and, among those who do adhere, imposes economic costs because of healthy workers isolating. introducing confirmatory testing into the program increases the signal value, reduces unnecessary isolation, and arguably would lead to greater adherence, increasing net benefits. third, screening test sensitivity is of secondary importancea finding that is consistent with, for example, larremore et al (2020) and paltiel, zheng, and walensky (2020) . for example, the results in table 1 are very similar if screening test sensitivity is reduced from 97.1% to 85%: even at 85% sensitivity, the vast majority of the tested infected are detected and, if they adhere, isolated. fourth, we find that targeting testing to younger and middle-aged adults can improve both economic and mortality outcomes, holding constant the number of screening tests. although targeting those ages retards activity by sending workers into isolation, it breaks the chain of transmission to the elderly. the model is summarized in section 2. section 3 presents the results for uniform testing, and age-based testing is examined in section 4. related literature. this paper is related to a growing literature synthesizing epidemiological models of disease transmission with macroeconomic dynamics, 5 some of which considers testing and quarantine. berger, herkenhoff, and mongey (2020) consider the effects of testing and quarantine in a sir model with a single perfect test and imperfect adherence to quarantine; the 5 an early focus of this literature concerned the macroeconomic and epidemiological effects of lockdown and re-opening policies. eichenbaum, rebelo, trabant (2020a) augment a standard new keynesian macroeconomic model with a sir-type model of disease transmission, characterize the relationship between consumption/labor supply decisions and disease transmission, and study the effects of simple lockdown policies. acemoglu, chernozhukov, werning, and whinston (2020) study a multi-group sir model where infection, hospitalization, and fatality rates vary between groups and characterize optimal age-varying lockdown policies. this literature has expanded to include other non-pharmaceutical interventions, see baqaee, farhi, mina, and stock (2020a) . see bfms (2020b) for additional references. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 authors show that testing can reduce the severity of lockdowns required to achieve a given reduction in the spread of disease. cherif and hasanov (2020) study the costs and returns of a test-and-quarantine strategy in a sir model, paying particular attention to 'smart' testing strategies that take advantage of spatial heterogeneity in disease prevalence and population density. brotherhood, kircher, santos, and tertilt (2020) and eichenbaum, rebelo, and trabandt (2020b) consider age-varying diagnostic testing and quarantine; in both models, the role of testing is primarily to resolve individual uncertainty about infection status. other papers that address testing, contact tracing, and/or quarantine are acemoglu, makhdoumi, malekian, and ozdaglar (2020) , augenblick, obermeyer, kolstad, and wang (2020) , bmfs (2020b), gans (2020) , and piguillem and shi (2020) . a closely related paper in the epidemiological literature is paltiel, zheng and walensky (2020) consider college coronavirus testing and incorporate costs of tests and of housing the quarantined. also see, among others, larremore et al (2020) , taiaple, romer, and linnarsson (2020) and peto et al (2020) . relative to this literature, our main contribution is to provide carefully calibrated and estimated model for assessing the net economic, fiscal, and total (including mortality) benefits of multistep imperfect screening testing in conjunction with diagnostic testing. by combining a 66-sector economic model with a five-age behavioral sir model, we are able to consider age-based strategies and the effect of temporary isolation on employment and output. our starting point is the bfms behavioral sir model, which connects a sir model of disease transmission to economic activity. the bfms model has five age groups (ages 0-19, 20-44, 45-64, 65-74, and 75+) and 66 private economic sectors plus the government. pre-pandemic contact matrices are estimated from polymod (mossong et. al. (2017) ) for work, home, and other activities. work contact matrices vary by sector depending on sectoral worker proximity (mongey, pilossoph and weinberg (2020) ). the behavioral aspect of the model arises from a feedback rule in which activity depends on the current weekly death rate and the slope of the weekly death rate. in addition, the behavioral rule has a lockdown-fatigue component in which high unemployment rates and cumulative past unemployment rates contribute (all else equal) to a desire to resume activity. epidemiological parameters, including age-based death rates, are taken from the epidemiological literature, from the cdc, or, for the transmission rate and initial infection rate, estimated from us data on daily deaths. for additional details, see appendix 1 and bfms. this paper extends the bfms model to incorporate explicit screening and diagnostic testing with partial adherence. the key elements of this extension are: . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10. 1101 /2020 1. individuals are selected at random, at a daily rate , for rapid screening testing. 2. a fraction ν of individuals testing positive in the screening test take a confirmatory diagnostic (pcr) test; the remaining fraction 1-ν of those who test positive are instructed to self-isolate. 3. symptomatic individuals can receive a diagnostic test. 4. those awaiting diagnostic test results are instructed to quarantine. 5. the isolation pool consists of those with a "terminal" positive test result: a positive test among the pcr-tested symptomatic, a positive pcr test among the fraction ν of screening-test positives who take a confirmatory test, or a positive screening test among the fraction 1-ν who do not. 6. adherence to instructions to quarantine or to isolate is partial. the extended sir model is illustrated in figure 1 (equations are given in appendix 1). the horizontal flows represent the disease progression from susceptible to exposed to infected to recently recovered to fully recovered, or from infected to deceased. the distinction between recently recovered and fully recovered is that the recently recovered test positive on a pcr test but are not contagious, e.g. see larremore et al. (2020) . the screening test is assumed to be less sensitive than the pcr test so detects the virus among the infected but not among the recently recovered. those instructed to isolate enter the isolation compartment (q, to distinguish it from the infected i). the extent to which they adhere with that instruction depends on whether they arrived by testing positive on the diagnostic test, which has high signal value, or on the screening test, . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint which has lower signal value. (although we describe this as, say 25% of those arriving from a positive screening test as adhering, because of the homogenous structure of the model this is equivalent to all those arriving by this channel reducing their contacts by 25%.) with full adherence, the susceptible in quarantine and in isolation would not become infected, however because adherence is partial some of the isolated susceptibles (sq) and quarantined susceptibles (sd) can become exposed. table 2 describes our baseline parameter values. the sensitivity and specificity of diagnostic test are intended to be in the range of laboratory pcr tests. the sensitivity and specificity of the baseline screening test are calibrated to analytical estimates corresponding to the binaxnow rapid test, although we consider alternative values. the rate of uptake of diagnostic testing among the non-infected (ρ0) and among the infected (ρ1) were calibrated to match the total number of tests and the positivity rate in the us during july and august 2020. we allow the frequency of screening tests (governed by μ) to vary between 0 (no screening tests at all) to 0.3 (each person is screened on average every three days). our baseline isolation adherence rate is 25% for those testing positive on the screening test with 98.5% specificity, is . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint 50% for those testing positive on the two-stage screening test with 99.7% specificity, and is 75% for those testing positive on the diagnostic test (either after qualifying by being symptomatic or as the second stage following a positive screening test). bfms has two baseline scenarios, both exhibiting a second wave of infections starting midsummer 2020. in bfms, the second wave was induced by a relaxation of social distancing, masks, and other protections, combined with a full return to school in the fall. the difference between the two scenarios was the strength of the feedback from deaths and the growth rate of deaths to activity. the baseline here uses feedback parameters that are a mid-point between the two baseline scenarios considered in bfms. we estimate the model using data through june 12, 2020. the simulation period begins june 1, 2020 and ends on january 1, 2021. thus, the simulations reflect alternative, counterfactual paths for the virus and the economy for the final seven months of 2020. notes: quarterly gdp (green step function) is shown in real levels, indexed to 1 in 2019q4. total deaths (actual in black dashed, simulated in red) under the baseline simulation are 359,000 by january 1, 2021. bands denote 67%, 90%, and 95% confidence bands using standard errors for the estimated model parameters. figure 2 shows the time path of actual deaths (black dashed), simulated deaths (red), and the level of gdp (green) indexed to its level in february 2020, under our baseline calibration with . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10. 1101 /2020 no screening testing. the bands for deaths and gdp are standard error bands based on estimation uncertainty for the model parameters. although the baseline scenario was constructed in june, it closely tracks the path of deaths through mid-august. subsequently, simulated deaths exceed actual deaths, in part because the simulation presumes a full return to school whereas many school districts chose remote or hybrid reopenings. under the baseline, there are 359,000 deaths by january 1. under the baseline, there are no screening tests ( = 0), however there are diagnostic tests at rates that match the volume and positivity rates of actual testing in july and august. under the screening testing counterfactuals, diagnostic testing is augmented by screening testing, holding constant all model parameters except for those describing the screening tests. the incremental costs and benefits of testing are computed from the number of tests and the model-implied economic and mortality outcomes under testing and no-testing scenarios. we assume the cost of the 98.5% specific screening test is $5. the 80% specific screening test is assumed to cost $2, but is packaged for use with the $5 test with 98.5% specificity at a 5-to-1 ratio for an average cost of $3/test. the price of a diagnostic pcr test varies considerably in the united states; we use $50 for our baseline. we compute three measures of benefits of tests: incremental gdp, incremental federal government revenues, and the monetized value of deaths avoided. gdp is measured in 2020 dollars. because the simulations start on june 1, gdp is the same under baseline and testing alternative have the same values for gdp for the first five months of the year, so any differences in gdp under the two scenarios occurs only from june through december; these incremental dollars of gdp are dollars for those seven months only, not at an annual rate. the effect of an increase in gdp on government revenues is computed using elasticities of income taxes, corporate profits taxes, fica, and the self-employment contributions tax from the congressional budget office (russek and kowalewski (2015 , table 3 ) and cbo (2019)). like gdp, these incremental revenues are for june-december only and are not annualized. to compute net fiscal benefits, we assume that all incremental testing is paid for by the federal government. deaths avoided are the cumulative number of deaths from covid-19 on january 1, 2020 under the testing scenario, minus the total number of deaths in the baseline scenario. deaths are . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint monetized using the value of statistical life is from the us epa (2020), converted to 2020 dollars, which is $9.3 million per life. this section provides full results for the three programs in table 1 , then provides sensitivity checks and time paths of the virus and gdp for illustrative programs. we begin with program a in table 1 , a single-stage screening test with 98.5% specificity with 50% confirmatory pcr testing. the adherence rates are 25% for those instructed to isolate based on the screening test alone and 75% for those instructed to isolate based on the diagnostic test. mortality and economic outcomes for program a in table 1 are shown in figure 3 . in all figures, the outcome of interest is plotted as a function of the screening test intensity . the multiple lines in the figures represent different screening test sensitivities, from 80% to 98.5%. relative to the no screening test baseline, testing biweekly is estimated to avert approximately 37,000 deaths, and testing weekly averts 66,000 deaths, when screening test sensitivity is 97%. the number of days that individuals are told to isolate (upper right) increases approximately linearly with the amount of screening testing (there is some curvature because symptomatic testing falls as screening intensity increases). for weekly testing, there are approximately 930 million proscribed isolation days, which amounts to 1.3% of the total of 70 billion person-days during the june-december simulation period. (because of partial adherence, only some of those isolation-days are actually observed.) because only half of the screening-testing positives receive confirmatory pcr testing, the preponderance of those instructed to isolate are false positives. the screening test ppv (middle left) is low, approximately 6% for weekly testing. because the virus is increasingly suppressed as  increases, gdp (middle right) increase with  for low and moderates testing rates ; although the increase is held back by the large number of healthy workers who are isolating. cost-benefit results screening program a are shown in the bottom panel of figure 3 and in figure 4 . additional testing costs rise approximately linearly with the testing rate. for these parameter values, net economic benefits (figure 3 , lower left) are in the range of $75-120 billion for biweekly testing and $150-200 billion for weekly testing, depending on the screening test sensitivity. when the value of life is included as a benefit (figure 3 , lower right), net benefits are in the range of $320-470 billion for biweekly testing and $650-820 billion 215 for weekly testing, depending on the screening test sensitivity. in nearly every case considered, the screening program pays for itself (figure 4, middle right) , under the assumption that all additional testing is paid for by the federal government. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10. 1101 /2020 in all these figures, increasing the sensitivity of the screening test improves outcomes, but those improvements are typically small relative to the gains from introducing the screening program in the first place. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint figure 5 and figure 6 are the counterparts of figure 3 and figure 4 for program b, in which has the same screening test but with universal confirmatory pcr testing. expanding confirmatory testing from 50% to 100% substantially reduces deaths and increases gdp considerably, because . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint of the assumed greater adherence rate for highly specific pcr testing than for the screening test alone. in addition, because universal confirmatory testing reduces the number of healthy individuals in isolation; avoiding isolating the healthy allows them to work, increasing gdp. in fact, despite the increase in testing, isolation days are less under program b than under the noscreening baseline because the prevalence of the virus is substantially reduced, reducing the total . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint number of positive diagnostic tests despite the inflow from positive screening tests. universal confirmatory testing increases testing costs, so it is not obvious a-priori whether offering universal confirmatory testing increases or decreases net economic benefits; for the values considered here, the economic benefits of universal testing dominate and net economic benefits increase. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint results for the two-step screening test of program c are shown in figure 7 and figure 8 . this program has a $3 two-step screening test with specificity of 98.5%, adherence of 50%, and no confirmatory pcr testing. mortality gains, proscribed isolation days, employment gains, and . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint gdp gains fall between the tests in programs a and b, a consequence of the assumed lower adherence rates. although net economic benefits are less for program c than for program b, the benefit-cost ratios are greatest for program c because the tests are less expensive. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint table 3 summarizes the results of various sensitivity checks. a common critique of widespread screening is that low specificity can lead to a large number of health individuals, including health workers, needlessly entering isolation (e.g., pettengill and mcadam (2020) ). panel d in table 3 considers this case, for the 98.5% specificity screening test. eliminating confirmatory pcr testing entirely from program a increases the number of healthy people, including healthy workers, in isolation. it also reduces overall adherence because the low-ppv screening test has no follow-up diagnostic testing. these two effects substantially reduce the gains from the screening testing program. in fact, without any confirmatory pcr testing the screening testing program does not pay for itself in most of the cases considered. with . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 no confirmatory testing, there are approximately 1.8 billion proscribed isolation-days with weekly testing, approximately 2.5% of all person-days. single-stage screening test with 97% specificity with partial confirmatory testing. panel e modifies program a by considering a screening test with twice the false positive rate of the test in program a. for comparison purposes we hold adherence constant although plausibly it would be lower for the panel e test. the reduced specificity increases the number of healthy individuals proscribed to isolate. testing costs increase because there are more screening false positives that need confirmation, and isolating so many healthy workers provides an additional drag on gdp. as a result, net economic benefits are less than for program a. single-stage screening test with 98.5% specificity, partial confirmatory testing, and increased adherence. this scenario, shown in panel f, modifies program a by increasing adherence from 25% to 50% for those receiving a positive screening test but not taking a confirmatory test. higher adherence substantially increases deaths averted, gdp, and revenues, and slightly decreases total testing costs because the greater suppression of the virus reduces symptomatic testing costs. net economic benefits are large, even for biweekly testing. table 3 considers program b (98.5% specificity, universal confirmatory testing) except with a more expensive confirmatory test. the cost of the diagnostic testing is borne by the federal government so in the model does not affect private decisions and thus does not affect mortality, employment, or gdp. despite the doubling in the cost of the pcr test, the overall increase in testing cost reduction is modest, for example rising from $56 million for weekly testing in program a (table 1) to $63 million. the reason is that, with universal pcr confirmatory testing, the expected cost of administering the combined test to an uninfected individual increases only slightly from $5 + .015×$50 = $5.75 for a $50 confirmatory test to $6.50 for a $100 confirmatory test. figure 9 displays the simulated time path of deaths and quarterly gdp, along with standard error bands and actual deaths, for four counterfactual scenarios: parts (a), (b), and (c) show respectively programs a, b, and c for a weekly testing rate, and panel (d) shows program c for a four-day testing rate. all cases in figure 9 have a lower path for deaths and higher path for gdp than the no-screening baseline in figure 2 . program a slows the spread of the virus but does not suppress it. the other panels, however, approach suppression and two-step testing (program c) at a 4-day cadence essentially suppresses the virus, supporting a strong economic recovery. at a weekly testing cadence, programs b and c could have avoided the second wave of the summer and fall. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. it might be more efficient to target screening testing based on individual characteristics than having population-wide random screening. because contacts and mortality differ by age, this section considers screening that is random within an age category with testing rates differing across categories. specifically, we calculate the age-based testing rates that maximizes net total benefits (economic plus monetized mortality) of the screening test, subject to the constraint that the population-wide screening testing rate equals a given value. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10. 1101 /2020 the results of the first calculationoptimized age-specific testing ratesfor screening program b (screening test with 98.5% specificity and universal confirmatory pcr testing) are shown in figure 10 , where each line is the probability of testing for a given age. the optimal age-varying testing rates are highest for young adults (ages 20-44) followed by ages 45-64, followed by ages 65-74. these results indicate that the screening testing and isolation is being used to break the chain of transmission from middle-aged adults to the elderly, either through family or service workers serving the elderly. the mortality benefits of this targeting outweigh the economic costs of isolating relatively higher fractions of the working-age population than other ages. note: dots are optimization estimates for given overall population testing rate , lines are smoothed through the estimate by age group. figure 11 shows the total net benefits for age-targeted screening testing and, for comparison, for random population screening testing. for small testing rates, there are substantial gains from targeting testing using the unconstrained allocations in figure 10 . those gains diminish at higher testing rates as the virus is suppressed, however net benefits are always higher with the agetargeted strategy. we note, however, that the costs here do not include developmental and educational costs of children missing school, and including such costs could provide an additional reason to test the young and thus allowing schools to reopen and stay open. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101/2020.10.22.20217984 doi: medrxiv preprint figure 11 . total net benefits from age-specific and age-blind screening testing there are six main arguments against widespread screening testing (e.g., pettengill and mcadam (2020) ). first, low specificity undercuts the program validity and leads to low adherence with the proscription to isolate if positive. second, low specificity unnecessarily pulls many healthy workers out of the workforce. third, because antigen tests have lower sensitivity than pcr tests, many infected individuals would slip through the cracks and undercut the effectiveness of the program. fourth, if paid for federally, their expense would be massive at a time that the federal deficit is already at a postwar high. fifth, to be effective they would need to be done at an infeasible scale, such as daily or every other day. sixth, having a screening program could change behavior, in particularly making individuals who test negative less cautious, for example reducing their willingness to wear a mask. our analysis addresses the first five of these concerns. our results underscore the importance of these first two concerns: in our analysis, the most important parameter is screening test specificity. a screening testing program must have high specificity to be credible and to evoke high adherence. this high specificity can be achieved by two-step testing if the tests are sufficiently independent. the additional costs of two-step testing, even if the second test is a pcr test, are small compared to the benefits, and screening testing with universal pcr . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 confirmatory testing generates large net benefits. test specificity is typically estimated in a laboratory using a small number of samples, so test specificity in the field could differ substantially from laboratory estimates. because low specificity undercuts the testing program, this uncertainty underscores the importance of confirmatory testing to increase specificity. the third concern, sensitivity, is legitimate in theory, but our modeling (like larremore et al (2020) ) finds that even large drops in sensitivity, say to 90%, have a small effect on the epidemiological and economic dynamics. the fourth concern, fiscal sustainability, also is legitimate in theory, but our estimates suggest that the economic gains from suppressing the virus are so large that the testing pays for itself through increased revenue. regarding the fifth concern, scale, we find that weekly testing in a regime with high compliance comes close to suppressing the virus, and moving to a four-day cadence is highly effective. weekly testing with a 98.5% specific screening test and universal confirmatory pcr testing would require increasing the number of pcr tests by roughly three-quarters of what they are today; a four-day testing would require more than doubling pcr testing capacity. our analysis does not tackle the final concern, that testing could induce more risky behavior. with that caveat, it is not self-evident that this must be the case. individuals undertake social distancing and masking to self-protect, to protect others, and to conform to local norms and laws. testing negative in the morning does not reduce the incentive to self-protect during the day. the effect on behavior of testing positive is ambiguous: altruism would lead one to reduce contacts even if not isolating, but no longer worrying about one's own health while caring little about the health of others could increase risky behavior. empirical research on this effect is needed. given the ambiguous nature of this effect, we do not consider it a reason to postpone the deployment of inexpensive rapid high-specificity screening testing. finally, our study of the economic benefits of covid-19 screening tests does not consider the public health benefits of the data generated from such a testing program for disease surveillance purposes. (we note that testing for both diagnostic and public health surveillance purposes is already routinely employed for both seasonal influenza and detection of novel strains of influenza a.) although at-home screening test results would not get into a public data system, universal confirmatory pcr testing would increase data coverage by overcoming the current selection into diagnostic testing of the symptomatic. thus, our proposed testing regimes would allow for much more timely and fine-grained analysis of the response of covid-19 prevalence and transmission to a wide range of public health interventions and disease mitigation strategies than is possible with current diagnostic testing data. presumably, consideration of the utility of the data generated from a widespread screening testing regime in shaping the design of effective and low-cost mitigation measures would add to the economic benefits that we have calculated here. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 appendix 1 this appendix provides more details on the model, which extends the model developed in baqaee, farhi, mina, and stock (2020) . our model departs from bfms in several important respects. first, we extend the model to include both a screening test regime and a diagnostic test regime. second, we assume that individuals are instructed to isolate upon receiving a terminal positive test, either a screening test with no confirmatory test or a positive diagnostic test. individuals awaiting diagnostic test results are instructed to quarantine. third, we distinguish between individuals who have recently recovered and fully recovered from the disease to capture that individuals may still test positive on a pcr test after they are no longer infectious. finally, we allow for imperfect adherence to quarantine and isolation. there are five age groups indexed by a, representing ages 0-19, 20-44, 45-64, 65-74, and 75+. there are 66 private sectors in the economy indexed by i. individuals are either s (susceptible), e (exposed), i (infected), r (recently recovered), f (fully recovered), or d (dead). in addition, individuals who are not dead are either actively circulating (a), awaiting diagnostic test results (d), awaiting screening test results (s), or in isolation following a positive test (q). thus, the population is partitioned into 21 states. for example, 2 2 , 2 , and 2 denote the number of persons aged 20-44 that are susceptible and actively circulating, susceptible and awaiting screening test results, susceptible and awaiting diagnostic test results, and susceptible and in isolation, respectively. we assume that the recovered (either recently recovered or fully recovered) are immune through the end of our simulation period. the rates of screening and diagnostic testing are given by the parameters , 0 , and 1 , described in table 2 . we assume that these parameters are equal to zero in the estimation period of our model, which runs through june 1 st , and thereafter calibrated according to the main text of this paper. the state variables (i.e. sa) are all five-dimensional vectors. let denote the a th element of any state x (the a th age group). the epidemiological side of the model has 21 transition equations: . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10. 1101 /2020 where denotes the number of individuals of age a (summing across all 21 states) and denotes the effective number of infected individuals actively circulating, = + + + . in this final expression we treat the signal value of taking a diagnostic test as being the same as receiving a positive screening test (these would be the same for the screeningtest positives taking a confirmatory pcr test), so non-adherence with quarantine is the same as non-adherence with a terminal positive screening test. the parameter is a weighted average of the parameters and , which are the isolation adherence rates for those who received screening tests and diagnostic tests, respectively. the weights are endogenously determined and given by the relative share of those instructed to . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10. 1101 /2020 isolate who arrived from screening tests versus diagnostic tests. thus, is the effective adherence rate of those in isolation. if those in isolation mainly through the screening test regime, then will be close to , the quarantine adherence rate for the screened population. given the parameters appearing in equations (1) through (21) above and a set of initial conditions, the model is straightforward to solve in discrete time by forward iteration. the unit of time is a single day and the model is solved 12 steps per day. the contact matrix c describes the expected number of contacts between each age group in the population. an actively circulating individual of age who interacts with an individual of age has an instantaneous infection probability of times the probability that the age-individual is infected. the probability that an individual of age a is infected in a given period is therefore given by summing across all their contacts. we distinguish between contacts that are made at home, at work, and elsewhere. the contact matrix is time-varying, and can change due to, for instance, npis put in place by the government or personal behavioral adaptations to avoid contracting the virus. we have: where ℎ , ℎ , indicate the expected number of contacts in each of home, work, and other environments, conditional on being at home, at work, or elsewhere. the parameters ℎ , ℎ , and , indicate the probability that an age-a individual is at home, at work, or elsewhere. we note that , is the fraction of employed in the indicated sector: where , is the number of workers of age a employed in sector i. see sections 1.2 and 2 of bfms (2020) for more information on the construction and historical estimation of the contact matrix. the behavioral component of this model endogenously determines the contact matrix in our simulation period (i.e. after june 1). this portion of the model is unchanged from bfms 2020. for completeness, we will briefly describe the key elements of this control rule here. in our simulation period (june 1 st through december 31 st ), we assume that the contact matrix responds endogenously to changes in the course of the pandemic. we formalize this by implementing a linear proportional-integral-derivative (pid) control rule, in which feedback depends on current deaths, the 14-day change in deaths, the current unemployment rate and the . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10. 1101 /2020 integral of the unemployment rate. the linear pid control rule can be expressed as: where is the unemployment rate and ̇ is the time derivative of the death rate. both and ̇ are generally unknown, available only with time aggregation and/or with reporting lags. we therefore use the 14-day average of the unemployment rate, the cumulative daily unemployment rate since march 7 th , deaths over the previous two days, and the 14-day change in the two-day death rate for the various terms on the right-hand side of this equation. the pid controller determines a sequence of sectoral labor supply shocks, shifted by the gdpto-risk index: where is the workforce in sector i at date t as a fraction of the workforce prior to the pandemic (i.e. february 2020), is the date of the beginning of the simulation period (june 1 st ), and φ is the cumulative gaussian distribution (which plays no role except as a sigmoid to constrain the controller between 0 and 1). the term is the gdp-to-risk index: the gdp-to-risk index can be interpreted as measuring the ratio of the marginal contribution to output, relative to the marginal contribution of 0 , from an additional worker of age a returning to work in sector i. up to scale, the gdp to risk index does not depend on epidemiological parameters except the contact matrix. the units of are not meaningful, so we standardize it to mean zero and unit variance across sectors (equally weighted). thus, the controller effectively alters the work contacts component of the contact matrix. similarly, we can think of the controller as generating a sequence of labor supply shocks that can be used to back out gdp using hulten's theorem as a first-order approximation: where the subscript • denotes summation over ages and ψ denotes the labor income share for sector i. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 27, 2020. ; https://doi.org/10. 1101 /2020 testing, voluntary social distancing and the spread of an infection emergency department study group on respiratory v immunochromatographic rapid test for influenza a and b virus among adult patients in the emergency department epidemiological and economic effects of lockdown group testing in a pandemic: the role of frequent testing, correlated risk, and machine learning policies for a second wave. forthcoming an seir infections disease model with testing and conditional quarantine an economic model of the covid-19 epidemic: the importance of testing and age-specific policies accuracy of rapid influenza diagnostic tests: a meta-analysis a tip against the covid-19 pandemic how did covid-19 and stabilization policies affect spending and employment? a new real-time economic tracker based on private sector data the accuracy of cbo's baseline estimates for fiscal year 2019 the macroeconomics of epidemics the macroeconomics of testing and quarantining test sensitivity for infection versus infectiousness of sars-cov-2 (2020). nber working paper 27780 fear, lockdown, and diversion: comparing drivers of pandemic economic decline 2020 national coronavirus response: a road map to reopening mandated and voluntary social distancing during the covid-19 epidemic beat covid without a vaccine test sensitivity is secondary to frequency and turnaround time for covid-19 surveillance. medrxiv preprint which workers bear the burden of social distancing policy? manuscript roadmap to recovery: a public health guide for governors assessment of sars-cov-2 screening strategies to permit the safe reopening of college campuses in the united states unnecessary obstacles to covid-19 mass testing can we test our way out of the covid-19 pandemic? optimal covid-19 quarantine and testing policies national covid-19 testing & tracing action plan roadmap to responsibly reopen america how cbo estimates automatic stabilizers a national decision point: effective testing and screening for covid-19. duke margolis center for health policy adherence to the test, trace and isolate system: results from a time series of 21 nationally representative surveys in the uk (the covid-19 rapid survey of adherence to interventions and responses population-scale testing can suppress the spread of covid-19. medrxiv preprint a realistic blueprint for reopening the economy by sector while ramping up testing binaxnow tm covid-19 ag card instructions for use key: cord-328320-1f3r80r5 authors: kim, edward title: drawing on israel’s experience organizing volunteers to operationalize drive-through coronavirus testing centers date: 2020-04-16 journal: disaster medicine and public health preparedness doi: 10.1017/dmp.2020.104 sha: doc_id: 328320 cord_uid: 1f3r80r5 to increase the country’s capacity to test and track suspected coronavirus disease 2019 (covid-19) cases, israel launched drive-through testing centers in key cities, including tel aviv, jerusalem, be’er sheva, and haifa. this article examines the challenges that the national emergency medical services and volunteers faced in the process of implementing drive-through testing centers to offer lessons learned and direction to health-care professionals in other countries. i srael's ministry of health confirmed their first case of coronavirus disease 2019 (covid-19) on february 21, 2020, following the repatriation of 1 of the 11 passengers aboard the diamond princess cruise ship. 1, 2 one month later, the first related death was made public along with details of over 700 confirmed cases. 3 by the end of march, the number of cases had increased to over 4500. 4 to respond to the increasing prevalence and spread of coronavirus throughout the country, there was an urgent need to increase testing of suspected individuals and trace community spread. 5 working alongside the ministry of health, magen david adom (mda)-israel's emergency medical, disaster, ambulance, and blood bank service-operationalized drive-through coronavirus testing centers, mirroring the models used by china and south korea. 6 drive-through testing centers, referred to in israel as "drive and test" facilities, allow for increased testing throughput while maintaining social distancing guidelines. 7 the process starts after people with symptoms of covid-19 are instructed to contact mda's dispatch center, where they are screened by a physician, given an appointment if necessary, and then instructed to fill out electronic medical forms. following this registration process, patients receive a qr code to their mobile device to be scanned at the facility on the day of testing. those who lack access to a car or are unable to travel receive a home visit, where they are tested by mda staff. after the first "drive and test" facility was piloted on march 23 in tel aviv to test capacity and refine operations, additional facilities were opened in jerusalem, be'er sheva, and haifa. the following week, mobile testing centers were set up in tamra, modi'in-maccabim-re'ut, wadi ara, and rahat, the largest bedouin community in israel. 8 facilities were set up in large open spaces, such as the parking lots of convention centers, stadiums, parks, and markets. the testing centers are staffed with medical teams, police officers, security personnel, and volunteer students, and can process 6 lanes of cars in parallel, taking on average 3-5 min per car. the medical teams consist primarily of paramedics who oversee operations, police officers and security personnel who direct traffic, and volunteers who don personal protective equipment (ppe) while testing individuals. volunteer efforts were coordinated by the federation of israel medical students (fims), which enlisted volunteers from a combined pool of approximately 700 medical students in their clinical studies. 9 while the initial setup and deployment of the "drive and test" facilities were executed swiftly and strategically, there were outstanding issues associated with preparation, implementation, and ongoing operations that needed to be addressed. anticipating an increased demand on the country's emergency call center, which typically receives 5500 calls per day, other independent call centers were repurposed and additional centers were set up in schools to expand capacity and respond to 25,000 calls per day. concurrently, over 200 volunteers signed up and underwent training to staff the multiple "drive and test" facilities and scale operations. however, to comply with health policies, students were trained in small groups of less than 10, which created time lags and constraints on the quality of the training they received. in more detail, ppe protocol reviews were limited to demonstrations only, leaving many students to don ppe for their first time on-site. a further layer of confusion arose from the fact that "drive and test" facilities and home visits used different ppe equipment and protocols. with respect to communication, several days after the launch of the "drive and test" facilities, fims changed the platform volunteers use to sign up for and manage their shifts. ultimately, this left many volunteers unable to log into the new system and view available shifts. this problem was compounded by the fact that the team relied on a single whatsapp group, of over 250 participants, to disseminate all information related to volunteering. although it was helpful to access a centralized resource, unless volunteers were checking their messages constantly, information was quickly lost due to the steady stream of new messages. not only did volunteers face challenges related to training and staffing during the preparation and early implementation phases, they also confronted operational issues during the first 2 wk of launch. for example, volunteers were not given a script to reference before testing individuals. in the first week, a driver stopped their car during testing using only the brake pedal with their car still in gear. startled during the swabbing process, the driver jerked pressing the accelerator instead and caused an accident. after the incident, volunteers were given a script that, among other things, instructed drivers to put their vehicle in park and engage their parking brake. more broadly, the rush to set up "drive and test" facilities and coordinate volunteers has had unintended downstream consequences. laboratories have received viral cultures that are unlabeled or contain inaccurate identification stickers; bags and cultures with identification stickers that do not match; as well as missing or illegible patient information on handwritten forms. coolers have also been left open for extended periods of time between sample collections. if not quickly addressed, these quality control issues will result in wasted resources, repeat testing, and inaccurate results, putting patients and others at risk if left unresolved. recognizing and responding to shortcomings is critical to successful operations in times of crisis. accordingly, an anonymous survey was disseminated to collect complaints and recommendations from volunteers to gain insights on outstanding issues. fortunately, mda addressed many of the operational issues quickly and decisively, in some cases overnight. for problems related to limited training, mda shared updated internal standards and protocols in-person and pushed the same updates to the "mda teams" app to improve the onboarding process for new volunteers. subsequently, new volunteers were paired with experienced ones for at least 1 full shift. this was a feasible solution, considering each station already required 2 volunteers: 1 to prepare and log the sample and the other to take the sample from the patient. the pairing strategy helped with oversight between the volunteers without fostering inefficiencies. it is worth noting that the continued tightening of governmentmandated travel restrictions and stricter enforcement of such policies did not reduce mda and fims' ability to organize volunteers. both organizations were quick to offer guidance on how to travel to testing sites. while there was no mechanism to provide volunteers with official certificates to expedite travel exceptions, a separate hotline was set up for those who faced any difficulty with law enforcement and to dismiss any accrued fines. in times of crisis, communication and organizational challenges occur more frequently. even with ample experience in emergency response and pilot testing, it is not possible to anticipate and prevent every hurdle. an appropriate balance between speed and thoroughness was achieved by mda's handling of the "drive and test" facilities. to quickly operationalize "drive and test" facilities while allowing for process improvements, mda used the plan-do-check-act methodology. this enabled the steering team to quickly identify and address areas of improvement while maintaining a balance between iterative refinement and consistency. 10 testing center processes evolved over the first 2 wk of implementation and resulted in a largely successful operation. by the end of march, "drive and test" centers had collected over 2000 tests daily, which accounted for 18,000 of the 53,000 tests completed nationwide. 11 world health organization disaster medicine and public health preparedness news-releases/first-confirmed-coronavirus-case-in-israel-at-sheba-medicalcenter-tel-hashomer-301009210.html world health organization world health organization substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) drive-through screening center for covid-19: a safe and efficient screening system against massive community outbreak drive-through medicine: a novel proposal for rapid evaluation of patients during an influenza pandemic the fight against corona the quality toolbox complex arrives to modi'in the author has no potential conflicts of interest to disclose. key: cord-292274-upwn9o2m authors: ghaffari, abdi; meurant, robyn; ardakani, ali title: covid-19 serological tests: how well do they actually perform? date: 2020-07-04 journal: diagnostics (basel) doi: 10.3390/diagnostics10070453 sha: doc_id: 292274 cord_uid: upwn9o2m in only a few months after initial discovery in wuhan, china, sars-cov-2 and the associated coronavirus disease 2019 (covid-19) have become a global pandemic causing significant mortality and morbidity and implementation of strict isolation measures. in the absence of vaccines and effective therapeutics, reliable serological testing must be a key element of public health policy to control further spread of the disease and gradually remove quarantine measures. serological diagnostic tests are being increasingly used to provide a broader understanding of covid-19 incidence and to assess immunity status in the population. however, there are discrepancies between claimed and actual performance data for serological diagnostic tests on the market. in this study, we conducted a review of independent studies evaluating the performance of sars-cov-2 serological tests. we found significant variability in the accuracy of marketed tests and highlight several lab-based and point-of-care rapid serological tests with high levels of performance. the findings of this review highlight the need for ongoing independent evaluations of commercialized covid-19 diagnostic tests. coronavirus disease 2019 (covid19) was first discovered in a cluster of patients with severe respiratory symptoms in hubei province, china, in december 2019. the early nucleic acid analysis of known pathogen panels led to negative results, suggesting the causative agent was of unknown origin. by early january 2020, analysis of bronchoalveolar lavage (bal) fluid from infected patients revealed a pathogen, later named sars-cov-2, with 50%, 80%, and 96% genetic sequence overlap to the genome of the middle east respiratory syndrome virus (mers-cov), the severe acute respiratory syndrome virus (sars-cov), and bat coronavirus ratg13, respectively [1, 2] . like sars-cov and mers-cov, sars-cov-2 is a single-stranded rna virus belonging to the beta genus coronavirus in the coronaviridae family [3] . as sars-cov-2 can be transmitted from human to human, the disease has spread swiftly to over 200 countries, infecting nearly 6 million people and resulting in at least 350,000 deaths worldwide (as of 27 may 2020) [4] . an unprecedented and rapidly growing global effort is underway to develop covid-19 vaccines and therapeutics, but at the time of this review, there are no vaccines, and only one antiviral drug (remdesivir) with modest clinical benefit has been approved under the u.s. food and drug administration (fda) emergency use authorization (eua) [5, 6] . under these circumstances, countries were forced to implement physical distancing measures to control the outbreak and, in the process, place approximately 3 billion people under lockdown. in any infectious disease outbreak, accurate and accessible diagnostic testing must be one of the pillars of control-measure policies to understand and minimize the spread of disease. the epidemiological studies of the outbreak in china estimated the proportion of undetected covid-19 cases to be as high as 86% [7] . as asymptomatic or mild cases could play a significant role in the transmission and spread of the sars-cov-2 virus [7, 8] , symptoms alone are not reliable diagnostic markers. there are two major types of diagnostic technologies available to address this: molecular and serological tests. currently, much of the focus is on the sars-cov-2 molecular test, which can detect, with high accuracy, the virus-specific rna molecules circulating in the host body. the gold-standard molecular test is based on reverse transcriptase polymerase chain reaction (rt-pcr) technology. however, the pcr test is not useful in distinguishing between highly infective viruses versus ones that have been neutralized by the host, and it cannot assess immunity status against sars-cov-2 [9] . serologically based antibody tests can complement molecularly based tests by providing a more accurate estimate of sars-cov-2 incidence and by potentially detecting individuals with immunity against the disease, as these tests detect markers of the immune response. in humoral immune response to infection, pathogen-specific antibodies, produced by b cells, neutralize and prevent further spread of the disease. the activation and differentiation of b cells into antibody-secreting plasma b cells are triggered by a cascade of events involving virus digestion by antigen-presenting cells (e.g., dendritic cells, macrophages) and presentation of virus-specific antigens to helper t cells ( figure 1 ). antibodies protect the host by binding to specific antigens (proteins) on the virus to neutralize its fusion and entry into the host cell and facilitate recognition and killing by phagocytic immune cells [10] . in humans, three types of antibodies or immunoglobulins have been the target of covid-19 serological tests: igm, igg, and iga. although the dynamics of the immune response in covid-19 are not fully understood, typically igm antibodies are produced by host immune cells during the early stages of a viral infection. igg is often the most abundant antibody in the blood and plays a more prominent role in the later stages of infection and in establishing long-term immune memory [11] . while igm and igg antibodies have been the leading candidates in covid-19 serological test development, recent studies show that iga, predominately present in the mucosal tissue, may also play a critical role in the immune response and disease progression [12] . in any infectious disease outbreak, accurate and accessible diagnostic testing must be one of the pillars of control-measure policies to understand and minimize the spread of disease. the epidemiological studies of the outbreak in china estimated the proportion of undetected covid-19 cases to be as high as 86% [7] . as asymptomatic or mild cases could play a significant role in the transmission and spread of the sars-cov-2 virus [7, 8] , symptoms alone are not reliable diagnostic markers. there are two major types of diagnostic technologies available to address this: molecular and serological tests. currently, much of the focus is on the sars-cov-2 molecular test, which can detect, with high accuracy, the virus-specific rna molecules circulating in the host body. the goldstandard molecular test is based on reverse transcriptase polymerase chain reaction (rt-pcr) technology. however, the pcr test is not useful in distinguishing between highly infective viruses versus ones that have been neutralized by the host, and it cannot assess immunity status against sars-cov-2 [9] . serologically based antibody tests can complement molecularly based tests by providing a more accurate estimate of sars-cov-2 incidence and by potentially detecting individuals with immunity against the disease, as these tests detect markers of the immune response. in humoral immune response to infection, pathogen-specific antibodies, produced by b cells, neutralize and prevent further spread of the disease. the activation and differentiation of b cells into antibody-secreting plasma b cells are triggered by a cascade of events involving virus digestion by antigen-presenting cells (e.g., dendritic cells, macrophages) and presentation of virus-specific antigens to helper t cells ( figure 1 ). antibodies protect the host by binding to specific antigens (proteins) on the virus to neutralize its fusion and entry into the host cell and facilitate recognition and killing by phagocytic immune cells [10] . in humans, three types of antibodies or immunoglobulins have been the target of covid-19 serological tests: igm, igg, and iga. although the dynamics of the immune response in covid-19 are not fully understood, typically igm antibodies are produced by host immune cells during the early stages of a viral infection. igg is often the most abundant antibody in the blood and plays a more prominent role in the later stages of infection and in establishing long-term immune memory [11] . while igm and igg antibodies have been the leading candidates in covid-19 serological test development, recent studies show that iga, predominately present in the mucosal tissue, may also play a critical role in the immune response and disease progression [12] . proteins. (2,3) following replication and release from the host cells, a subset of viruses will be engulfed and digested by antigen-presenting cells (apcs) like macrophages or dendritic cells. (4) fragmented sars-cov-2 antigen(s) will be presented to t helper cells, which in turn will interact and activate b cells. (5) activated b cells will proliferate and differentiate into plasma or memory b cells with high-affinity binding receptors for the original sars-cov-2 antigen. plasma cells secrete their sars-cov-2-specific receptors in the form of igm, igg, or iga antibodies. (6) antibody-mediated neutralization occurs when sars-cov-2-specific antibodies bind to viral antigen(s) and prevent virus interaction and entry into host cells. serological, or antibody, tests detect immunoglobulins produced by the host's plasma b cells following exposure to foreign antigens. the sars-cov-2 genome encodes approximately 25 proteins that are required for infection and replication, including four major structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n) (figure 1 ). the s protein plays a critical role in fusion and entry into the host cell, and it comprises an n-terminal s1 receptor-binding domain (rbd), n-terminal domain (ntd), and a c-terminal s2 subunits. the primary function of the sars-cov-2 n protein (np) is binding and packing of the viral rna genome into a helical nucleocapsid structure during viral replication [13, 14] . studies on the serum of recovered covid-19 patients suggest that host-neutralizing antibodies primarily work against s and n proteins [15, 16] . consequently, the likelihood of predicting immunity status could increase in serological tests that target various regions of s or n proteins. therefore, the characterization of specific sars-cov-2 antigen domains targeted by the humoral immune response becomes an integral part of the serological test development. there are four major types of serological diagnostic tests: the rapid diagnostic test (rdt), enzyme-linked immunosorbent assay (elisa), chemiluminescence immunoassay (clia), and neutralization assay. the neutralization assay is a lab-based test that uses live virus and cell culture methods to determine if patient antibodies can prevent viral infection in vitro. this test must be performed in laboratories with designated biosafety certificates to culture sars-cov-2-infected cells and has a time-to-result of 3-5 days. an rdt is a simple and rapid test based on lateral flow immunoassay (lfia) technology, commonly found in pregnancy test kits, for example. rdt can potentially be administered as a point-of-care (poc) test or self-test. typically, rdt test strips use a drop of blood to detect the presence of patient antibodies (igg, igm, or iga) produced against a specific sars-cov-2 antigen ( figure 2 ). an rdt is simple to use with a time-to-result anywhere between 10 and 30 min. therefore, it has the potential to be deployed in large-scale serological surveys. elisa assay, currently the most commonly used format of the serological test, is a lab-based test with an average time-to-result of 2-5 h. elisa typically uses a surface coated with specific viral antigen(s) to bind to and detect the corresponding patient antibodies (igg, igm, iga) in blood, plasma, or serum samples. the bound antigen-antibody complex is then detected by using a second antibody and a substrate that produces a color-or fluorescent-based signal. elisa assays can be found in different formats including direct, competitive, and, the most commonly used, sandwich or double-antigen-bridging assay (daba) (figure 3 ). clia technology follows a similar concept to elisa by taking advantage of high binding affinity between the viral antigen(s) and host antibodies but uses chemical probes that yield light emission through a chemical reaction to generate a positive signal. clia has an average time-to-result of 1-2 h. clia and elisa are both high-throughput laboratory-based assays with high level of analytical agreement [17, 18] . overview of rapid diagnostic serological test. rapid diagnostic tests (rdts) are typically based on colorimetric lateral flow immunoassay, in which host antibodies migrate across an adhesive pad (e.g., nitrocellulose) and interact with bound virus-specific antigens and secondary antibodies (antihuman igm/g antibodies). conjugated sars-cov-2-specific antigen(s) (labeled with gold here) will bind with the corresponding host antibodies. as antibody-antigen complexes travel up the membrane, bound anti-sars-cov-2 igm antibodies interact with fixed anti-igm secondary antibodies on the m line, and anti-sars-cov-2 igg antibodies interact with anti-igg antibodies on the g line. if the blood sample does not contain sars-cov-2-specific antibodies, the m or g lines do not appear in the final test results; only the control (c) line will be revealed. overview of enzyme-linked immunosorbent assay (elisa)-based diagnostic test. elisa can be presented in different formats based on differences in antigen immobilization and antibody labeling. in direct elisa, sars-cov-2 antigen(s) bound to a plastic solid phase is detected by the addition of a conjugated antibody. in sandwich elisa, the capture antibody is attached to the plastic solid phase. antigen(s) in the sample will bind to the capture antibody and then be detected by a second enzyme-labeled antibody. in competitive elisa, sample sars-cov-2 antigen is preincubated with the primary antibody and then added to a well coated with a secondary antibody along with an enzyme-conjugated antigen that competes with the sample antigen for binding with the primary antibody. the more sars-cov-2 antigen in the sample, the less conjugated antigen will be bound and the lower the signal will be. knowledge of virus and host immune response dynamics are essential in formulating diagnostic testing and treatment strategies. studies of covid-19 suggest that seroconversion, when antibody levels become detectable in the blood, may take place days after the viral load has peaked [19] . therefore, serological tests would be less effective in the early stages of covid-19. wolfel and colleagues further confirmed these findings by reporting igm and igg seroconversion in 50% of patients at 1 week after the onset of symptoms [20] . the median time for the detection of igm and igg in covid-19 patients was reported to be 5 and 14 days, respectively [21] . yu and colleagues detected the seroconversion of iga in direct elisa, sars-cov-2 antigen(s) bound to a plastic solid phase is detected by the addition of a conjugated antibody. in sandwich elisa, the capture antibody is attached to the plastic solid phase. antigen(s) in the sample will bind to the capture antibody and then be detected by a second enzyme-labeled antibody. in competitive elisa, sample sars-cov-2 antigen is preincubated with the primary antibody and then added to a well coated with a secondary antibody along with an enzyme-conjugated antigen that competes with the sample antigen for binding with the primary antibody. the more sars-cov-2 antigen in the sample, the less conjugated antigen will be bound and the lower the signal will be. knowledge of virus and host immune response dynamics are essential in formulating diagnostic testing and treatment strategies. studies of covid-19 suggest that seroconversion, when antibody levels become detectable in the blood, may take place days after the viral load has peaked [19] . therefore, serological tests would be less effective in the early stages of covid-19. wolfel and colleagues further confirmed these findings by reporting igm and igg seroconversion in 50% of patients at 1 week after the onset of symptoms [20] . the median time for the detection of igm and igg in covid-19 patients was reported to be 5 and 14 days, respectively [21] . yu and colleagues detected the seroconversion of iga on day 2 and igm/igg on day 5 after onset of symptoms. furthermore, the study reported that 100% of cases had detectable levels of iga, igm, and igg on day 32 after onset of symptoms [12] . their findings also revealed igm and igg levels to be significantly higher in severe covid-19 cases than in patients with mild or moderate disease [12] , suggesting that serological tests require high sensitivity to detect lower levels of antibodies in mild cases. studies on the persistence of antibodies in blood suggest that high levels of igg are detectable for at least 49 days after the onset of symptoms, while igm levels declined rapidly on day 35 postinfection [22] . the diagram in figure 4 depicts the timelines and peak levels for sars-cov-2 viral load relative to blood igm, igg, and iga antibodies. improved understanding of humoral antibody response time kinetics in covid-19 is crucial to the correct application of serological tests. diagnostics 2020, 10, x for peer review 6 of 13 on day 2 and igm/igg on day 5 after onset of symptoms. furthermore, the study reported that 100% of cases had detectable levels of iga, igm, and igg on day 32 after onset of symptoms [12] . their findings also revealed igm and igg levels to be significantly higher in severe covid-19 cases than in patients with mild or moderate disease [12] , suggesting that serological tests require high sensitivity to detect lower levels of antibodies in mild cases. studies on the persistence of antibodies in blood suggest that high levels of igg are detectable for at least 49 days after the onset of symptoms, while igm levels declined rapidly on day 35 postinfection [22] . the diagram in figure 4 depicts the timelines and peak levels for sars-cov-2 viral load relative to blood igm, igg, and iga antibodies. improved understanding of humoral antibody response time kinetics in covid-19 is crucial to the correct application of serological tests. the urgent need for the development of serological diagnostic tests in response to the covid-19 outbreak has compelled regulatory bodies to implement emergency use authorization programs to expedite the commercialization process of these tests. in light of this, independent and robust postmarket evaluations of covid-19 serological tests are needed to confirm manufacturers' performance claims. the basic measures of quantifying diagnostic test performance are sensitivity and specificity. sensitivity is the ability of a test to detect the disease agent or the host's response to the disease (i.e., antibodies) when it is truly present, whereas specificity is the ability of a test to correctly return a negative result when disease or host response is absent [23] . we conducted a systematic review of independent studies that assessed the performance of currently available sars-cov-2 serological tests. we included studies that reported sensitivity and specificity, stage of disease (early, intermediate, or late), the test format (clia, elisa, rdt), and antibody target (iga, igg, igm, or igg + igm) [24, 25] . if available, the sars-cov-2 antigens used for antibody detection was recorded. the studies that did not specify the disease stage of test samples were grouped under the "overall" category and assessed separately. in total, we reviewed the urgent need for the development of serological diagnostic tests in response to the covid-19 outbreak has compelled regulatory bodies to implement emergency use authorization programs to expedite the commercialization process of these tests. in light of this, independent and robust post-market evaluations of covid-19 serological tests are needed to confirm manufacturers' performance claims. the basic measures of quantifying diagnostic test performance are sensitivity and specificity. sensitivity is the ability of a test to detect the disease agent or the host's response to the disease (i.e., antibodies) when it is truly present, whereas specificity is the ability of a test to correctly return a negative result when disease or host response is absent [23] . we conducted a systematic review of independent studies that assessed the performance of currently available sars-cov-2 serological tests. we included studies that reported sensitivity and specificity, stage of disease (early, intermediate, or late), the test format (clia, elisa, rdt), and antibody target (iga, igg, igm, or igg + igm) [24, 25] . if available, the sars-cov-2 antigens used for antibody detection was recorded. the studies that did not specify the disease stage of test samples were grouped under the "overall" category and assessed separately. in total, we reviewed performance data on 5 serological clia tests, 15 serological elisa tests, and 42 serological rdts currently on the market (see supplementary materials) . the distribution plot of the data shows a higher degree of variability in test sensitivity values compared to specificity ( figure 5 ). this level of variability further emphasizes the need for independent evaluations of serological tests on the market. the sensitivity/specificity plots highlight tests at various stages of covid-19 and confirm the expectation that serological tests are more effective in later stages of the disease when higher igg and igm levels are present in the blood ( figure 6 ). the heatmap of tests in the "overall" category ranks the highest-performing test in each target antibody category based on sensitivity, followed by specificity (figure 7) . top-performing covid-19 serological tests (>95% sensitivity and specificity) from the xy plots and heatmap are summarized in table 1 . diagnostics 2020, 10, x for peer review 7 of 13 effective in later stages of the disease when higher igg and igm levels are present in the blood ( figure 6 ). the heatmap of tests in the "overall" category ranks the highest-performing test in each target antibody category based on sensitivity, followed by specificity (figure 7) . top-performing covid-19 serological tests (>95% sensitivity and specificity) from the xy plots and heatmap are summarized in table 1 . . independent evaluation of sars-cov-2 serological test overall performance. sensitivity and specificity data from studies that did not specify the covid-19 stage ("overall" group) are represented in a heatmap. the heatmap is ordered according to the antibody target (igg, igm, and igg/igm) followed by sensitivity and specificity values. all analyses were conducted using software r version 3.6.3. heatmaps were generated using the gplots and rcolorbrewer packages. elisa: enzyme-linked immunosorbent assay; clia: chemiluminescence immunoassay; rdt: rapid diagnostic test. figure 7 . independent evaluation of sars-cov-2 serological test overall performance. sensitivity and specificity data from studies that did not specify the covid-19 stage ("overall" group) are represented in a heatmap. the heatmap is ordered according to the antibody target (igg, igm, and igg/igm) followed by sensitivity and specificity values. all analyses were conducted using software r version 3.6.3. heatmaps were generated using the gplots and rcolorbrewer packages. elisa: enzyme-linked immunosorbent assay; clia: chemiluminescence immunoassay; rdt: rapid diagnostic test. in light of policies to ease the lockdown and reopen the economy, large-scale seroprevalence studies to screen for immunity status are being implemented in several jurisdictions. critics point to gaps in our understanding of immune response to covid-19 infection, including the ability of serological tests to detect neutralizing antibodies and the capacity of the immune system to provide long-term immunity against sars-cov-2. however, some argue that in the context of a global viral outbreak with a relatively high mortality rate, inaction due to uncertainty can have negative consequences compared to the harm caused by false-positive and false-negative serological test results [31] . several jurisdictions have initiated seroprevalence studies to provide a more accurate estimate of cases with positive sars-cov-2-specific antibodies, irrespective of disease symptoms. in los angeles county, the prevalence of sars-cov-2 antibodies in the community was estimated to be 4.65%, equivalent to 367,000 adults, which was substantially greater than the 8430 confirmed cases in the same county at the time of the study [32] . in new york city, 19.9% of the population has been estimated to have sars-cov-2 antibodies, compared to 2.1% confirmed cases as of 2 may 2020 [33]. similar studies from germany, the u.k., singapore, and china show significantly higher estimates of positive sars-cov-2 antibody cases compared to symptomatic cases confirmed by molecular tests [34] . as undetected cases with mild or no symptoms can transmit the virus, it is not surprising that countries (e.g., south korea, germany, and singapore) with large-scale and well-organized testing programs, combined with extensive isolation and contact tracing for infected individuals, have had some success in minimizing covid-19-related death in their populations [35] . as serological tests are in high demand, in part due to an increase in large-scale seroprevalence studies, it is imperative for national and regional governments to continue coordinated efforts to independently validate serological test performance and partner with industry to scale up manufacturing and production capacity. existing emergency authorization programs, intended to accelerate the manufacturing of diagnostic tests, must also be accompanied by clear and informed guidelines on preferred and minimally acceptable profiles of covid-19 serological tests designed for specific indications. despite the unprecedented response to the outbreak, major gaps remain in our understanding of the interaction between sars-cov-2 and the immune system, which can negatively impact serological testing utilization. coordinated research efforts are urgently needed to investigate some of the key gaps in our knowledge, including: which serological tests can identify sars-cov-2-neutralizing antibodies? 2. is there cross-reactivity between neutralizing antibodies and other coronaviruses? 3. which sars-cov-2 antigens are optimal for the detection of neutralizing antibodies? 4. what is the correlation between sars-cov-2-specific antibodies and protective immunity status? 5. how long does protective immunity last in recovered patients? are individuals susceptible to reinfection with sars-cov-2? 6. is humoral antibody response the best indicator for protective immunity, or are there other immune-cell-based mechanisms? in the context of the covid-19 outbreak and the execution of return-to-work policies, failing to take advantage of available diagnostic tools due to uncertainty can have profound consequences. medical professionals frequently rely on imperfect evidence with the possibility of false positives and false negatives. it is, however, important to clearly understand the limits and potential of serological tests to make informed decisions based on risk and benefit assessment in each specific situation. in the words of tedros adhanom ghebreyesus, director-general of the world health organization, "countries cannot fight this pandemic blindfolded. countries should know where the cases are." a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding world health organization covid-19 situation report 127 remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial compassionate use of remdesivir for patients with severe covid-19 substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) presumed asymptomatic carrier transmission of covid-19 the important role of serology for covid-19 control the immune system in health and disease immune response to sars-cov-2 and mechanisms of immunopathological changes in covid-19 distinct features of sars-cov-2-specific iga response in covid-19 patients how to discover antiviral drugs quickly nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals neutralizing antibodies against sars-cov-2 and other human coronaviruses in vitro diagnostic assays for covid-19: recent advances and emerging trends laboratory testing of sars-cov, mers-cov, and sars-cov-2 (2019-ncov): current status, challenges, and countermeasures temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study virological assessment of hospitalized patients with covid-2019 profiling early humoral response to diagnose novel coronavirus disease (covid-19) viral kinetics and antibody responses in patients with covid-19. medrxiv (preprint) 2020 simple statistical measures for diagnostic accuracy assessment food & drug administratin. independent evaluations of covid-19 serological tests evaluation of nine commercial sars-cov-2 immunoassays evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov-2 serology characteristics of sars-cov-2 infection since the exposure and post symptoms onset. medrxiv (preprint) 2020 serological detection of 2019-ncov respond to the epidemic: a useful complement to nucleic acid testing performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in waiting for certainty on covid-19 antibody tests-at what cost? seroprevalence of sars-cov-2-specific antibodies among adults the world is still far from herd immunity for coronavirus countries test tactics in 'war' against covid-19 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-331617-1ytcd0ax authors: horvath, karl; semlitsch, thomas; jeitler, klaus; krause, robert; siebenhofer, andrea title: antikörpertests bei covid-19 was uns die ergebnisse sagen date: 2020-05-15 journal: z evid fortbild qual gesundhwes doi: 10.1016/j.zefq.2020.05.005 sha: doc_id: 331617 cord_uid: 1ytcd0ax introduction: in the context of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic, the detection of virus-specific antibodies (ab) will play an increasing role. the presence or absence of such antibodies can potentially lead to considerations regarding immunity and infection. issue: how reliable are inferences from positive or negative test results regarding the actual presence of sars-cov-2 specific antibodies? methods: calculation of the probability that, depending on the pretest probability (prevalence of sars-cov-2 infection) and test properties, antibodies are present or absent in the case of positive or negative test results. results: sensitivity and specificity of different sars-cov-2 ab test systems vary between 53 % and 94 % and between 91 % and 99.5 %, respectively. when using a test with high test quality, the positive predictive value (ppv) is 42 % and 7 9%, respectively, with a pre-test probability of 1 % to 5 %, as can currently be assumed for the general population in austria or germany. for persons with an increased pre-test probability of 20 %, e. g. persons from high-risk professions, the ppw is 95 %, with a pre-test probability of 80 % the ppw is almost 100 %. the negative predictive value (npv) is at least 99.7 % for persons with a low pre-test probability of up to 5 % and 79.1 % for persons with a pre-test probability of 80 %. when using test systems with lower sensitivity and specificity, the reliability of the results decreases considerably. the ppv is 5.9 % with a pre-test probability of 1 %. conclusions: a sufficiently high sensitivity and specificity are prerequisites for the application of antibody test systems. positive test results are often false if the pre-test probability is low. depending on the assumed prevalence of a sars-cov-2 infection, there are substantial differences in the significance of a concrete test result for the respective affected persons. introduction: in the context of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic, the detection of virus-specific antibodies (ab) will play an increasing role. the presence or absence of such antibodies can potentially lead to considerations regarding immunity and infection. issue: how reliable are inferences from positive or negative test results regarding the actual presence of sars-cov-2 specific antibodies? methods: calculation of the probability that, depending on the pretest probability (prevalence of sars-cov-2 infection) and test properties, antibodies are present or absent in the case of positive or negative test results. positive predictive value negative predictive value pre-test probability results: sensitivity and specificity of different sars-cov-2 ab test systems vary between 53 % and 94 % and between 91 % and 99.5 %, respectively. when using a test with high test quality, the positive predictive value (ppv) is 42 % and 7 9%, respectively, with a pre-test probability of 1 % to 5 %, as can currently be assumed for the general population in austria or germany. for persons with an increased pre-test probability of 20 %, e. g. persons from high-risk professions, the ppw is 95 %, with a pre-test probability of 80 % the ppw is almost 100 %. the negative predictive value (npv) is at least 99.7 % for persons with a low pre-test probability of up to 5 % and 79.1 % for persons with a pre-test probability of 80 %. when using test systems with lower sensitivity and specificity, the reliability of the results decreases considerably. the ppv is 5.9 % with a pre-test probability of 1 %. conclusions: a sufficiently high sensitivity and specificity are prerequisites for the application of antibody test systems. positive test results are often false if the pre-test probability is low. depending on the assumed prevalence of a sars-cov-2 infection, there are substantial differences in the significance of a concrete test result for the respective affected persons. [2] . potenziell können aus dem vorhandensein oder fehlen von virusspezifischen ak auch überlegungen zur immunität oder infektion einer person angestellt werden [3] . ak tests spielen auch in den überlegungen zur implementierung von zukünftigen teststrategien und daraus abgeleiteten maßnahmen im weiteren verlauf der sars-cov-2 pandemie eine rolle. maßnahmen, die auf falschen annahmen hinsichtlich einer bestehenden infektion oder immunität beruhen, können zu falschem oder sorglosem verhalten führen. dies wiederum kann eine verstärkte ausbreitung der infektion und gefährdung betroffener personen zur folge haben. ziel dieser arbeit ist es daher, darzustellen, wie sicher, unter berücksichtigung von testeigenschaften und vortestwahrscheinlichkeit, positive bzw. negative ak testresultate auf das tatsächliche vorhandensein oder fehlen von sars-cov-2spezifischen ak schließen lassen. darüber hinaus soll diskutiert werden, welche schlüsse aus den testresultaten auch unter berücksichtigung weiterer testunabhängiger faktoren hinsichtlich einer potenzielle immunität oder infektion gezogen werden können. da weitgehend alle zur diagnose eingesetzten tests nicht vollständig fehlerfrei funktionieren, ist auch bei der testung auf vorliegen von sars-cov-2 spezifischen ak damit zu rechnen, dass es einen anteil von personen gibt, der vom test falsch klassifiziert wird. d.h. es wird personen geben, bei denen keine ak vorliegen, die aber dennoch ein positives testresultat erhalten (falsch positives testresultat). genauso wird es einen anteil von personen geben, bei denen trotz eines negativen testresultats dennoch ak vorhanden sind (falsch negatives testresultat). eine übersicht zu den möglichen testergebnissen in bezug auf das vorliegen einer erkrankung findet sich in abbildung 1. wie groß dieser jeweilig falsch klassifizierte anteil an allen getesteten personen ist, d.h. wie sicher ein positives oder negatives testresultat ist, ist abhängig von der sensitivität und spezifität des jeweiligen tests sowie von der gegebenen vortestwahrscheinlichkeit. die sensitivität berechnet sich aus dem anteil der personen mit positivem testresultat an der gesamtzahl der personen, bei denen ak vorliegen. sie gibt an wie gut der test personen mit sars-cov-2 spezifischen ak erkennt. würde die sensitivität 100% betragen, würde der test alle personen mit virusspezifischen ak erkennen und keine einzige person mit virusspezifischen ak übersehen. die spezifität berechnet sich aus dem anteil der personen mit negativem testresultat an der gesamtzahl der personen, bei denen keine ak vorliegen. sie gibt an wie gut der test personen, bei denen keine sars-cov-2 spezifischen ak vorliegen, erkennt. würde die spezifität 100% betragen, würden nur personen, bei denen tatsächlich ak vorliegen, und keine einzige person, bei der keine ak vorliegen, ein positives testresultat erhalten. die vortestwahrscheinlichkeit ist jene wahrscheinlichkeit, mit der bereits vor durchführung des tests davon auszugehen ist, dass bei einer person virusspezifische ak vorliegen. sie entspricht der häufigkeit, mit der in einer bestimmten personengruppe mit einer (aktiven oder durchgemachten) sars-cov-2 infektion zu rechnen ist. die vortestwahrscheinlichkeit ist z.b. bei personen mit covid-19 kompatiblen symptomen, aufenthalt in einer region mit hoher durchseuchung (jeweils aktuell oder in den letzten wochen) und die ggf. in einem risikoberuf tätig sind als höher anzunehmen als bei symptomlosen personen im homeoffice außerhalb eines risikogebiets. mit anderen worten beschreibt die vortestwahrscheinlichkeit das risiko, dass bei einer bisher ungetesteten person eine sars-cov-2 infektion vorliegt bzw. bestand. über die wahrscheinlichkeit, mit der bei einem positiven testresultat davon ausgegangen werden kann, dass die gesuchte erkrankung tatsächlich vorliegt, gibt der positiv prädiktive wert (ppw) auskunft. er berechnet sich aus dem anteil der richtig positiven testresultate an allen positiven testresultaten (abbildung 1). der negativ prädiktive wert (npw) gibt darüber auskunft, wie hoch die wahrscheinlichkeit ist, dass eine person mit einem negativen testresultat tatsächlich nicht erkrankt ist. der npw berechnet sich aus dem anteil der richtig negativen testresultate an allen negativen testresultaten (abbildung 1). der ppw ist umso höher, je höher die sensitivität und spezifität und je höher die prävalenz ist. der npw ist umso höher, je höher sensitivität und spezifität und je niedriger die prävalenz ist. bei der interpretation der testresultate sind neben den angeführten testeigenschaften auch weitere faktoren wie z.b. der testzeitpunkt im verlauf einer infektion zu berücksichtigen. verschiedene hersteller und distributoren geben ihrerseits sensitivitätswerte zwischen 80% und 100% und spezifitätswerte zwischen 92,5% und 100% an [4] [5] [6] [7] [8] [9] [10] . bei etablierten ak tests zu anderen virusinfekten (z.b. epstein-barr-virus, cytomegalie-virus, herpes-simplex-viren) werden werte für die sensitivität von rund 86% bis 100% und für die spezifität von rund 83% bis 100% angegeben [11] . in einer rezenten untersuchung zu den testeigenschaften von neun kommerziell erhältlichen sars-cov-2 ak tests -sowohl enzyme-linked immunosorbent assay (elisa) als auch point-of-care-testing (poc) -konnten für die sensitivität hingegen werte von 67% bis 93% und für die spezifität werte von 80% bis 100% ermittelt werden [12] . ein aktueller systematischer review gibt für elisa testsysteme sensitivitätswerte von 72,2% bis 94,4% und spezifitätswerte von 96,7% bis 99,5% an. für poc testsysteme werden werte von 52,8% bis 82,8% für die sensitivität bzw. werte von 91,4% bis 99,4% für die spezifität angeführt [13] . die prävalenz von sars-cov-2 infektionen in unterschiedlichen personengruppen kann aus derzeit verfügbaren daten nur abgeschätzt werden. aus den resultaten der aktuell vorliegenden querschnittsstudien in österreich kann abgeleitet werden, dass die prävalenz von sars-cov-2 infektionen in der allgemeinen bevölkerung zum zeitpunkt der untersuchung (1. bis 4. april 2020) wahrscheinlich bei rund 1%, zumindest aber unter 5% liegt (abgesehen von ausnahmen in tirol) [14] . eine zweite prävalenzstudie, die ende april 2020 durchgeführt wurde bestätigte diese niedrigen werte [15] . für deutschland kann von einer ähnlichen prävalenz ausgegangen werden. diese annahmen gelten auch für die prävalenz aller bisher durchgemachten infektionen, die für die überlegungen zur bedeutung der testergebnisse zum nachweis von igg-ak relevant ist. in der letzte märzwoche betrug der anteil positiv getesteter personen an der zahl aller getesteten personen (jeweils pcr test) in österreich rund 20% [16] . die bedeutung eines positiven testresultats für eine symptomlose person, die sich nicht in einer region mit hoher durchseuchung aufhält (und für die beides auch in der vergangenheit nicht der fall war) und die auch nicht der gruppe der risikoberufe angehört, unterscheidet sich somit deutlich von der bedeutung eines ebenfalls positiven testresultats für eine person mit covid-19 kompatiblen symptomen in einem risikogebiet (jeweils aktuell und in der näheren vergangenheit) und/oder tätigkeit in einem risikoberuf. negative testresultate sind bei personen mit geringer vortestwahrscheinlichkeit weitgehend verlässlich. sie bringen aber keine wesentliche zusätzliche sicherheit, da die wahrscheinlichkeit, dass keine sars-cov-2 infektion vorliegt, schon vor durchführung des tests hoch ist. das fehlen von sars-cov-2 spezifischen ak kann unterschiedliche ursachen haben. die ak können fehlen, weil tatsächlich keine infektion stattgefunden hat oder weil zwar eine infektion vorliegt, diese jedoch noch nicht lange genug besteht. die bildung von ak benötigt zeit, deshalb sind sie in der frühphase der infektion noch nicht nachweisbar. entsprechend einer untersuchung erfolgt die serokonversion bei 50% der infizierten innerhalb der ersten 7 tage. erst nach 14 tagen kann bei allen mit sars-cov-2 infizierten personen mit dem ak nachweis gerechnet werden [17] . bei der sars-cov infektion, die in den jahren 2002/2003 auftrat, konnten bei infizierten personen nach etwa 3 bis 6 tagen igm-ak und nach 8 tagen igg-ak nachgewiesen werden [18, 19] . da sars-cov-2 zur gleichen viren-familie gehört und genomstudien gezeigt haben, dass sars-cov-2 zu etwa 80% mit sars-cov identisch ist, kann davon ausgegangen werden, dass der prozess der antikörperbildung ähnlich verläuft. eine aktuelle untersuchung zum ak profil von 34 an covid-19 erkrankten personen in china bestätigt diese annahme [20] . aus einem negativen ak test bzw. dem fehlen von ak kann somit nicht sicher auf eine nicht vorhandene infektion geschlossen werden. der alleinige einsatz von ak testsystemen zum nachweis einer infektion würde daher einer fehlverwendung gleichkommen [21] . die beurteilung der bedeutung von testergebnissen für eine konkrete person wird auch dadurch limitiert, dass eine ausreichende datengrundlage zur einschätzung der vortestwahrscheinlichkeit fehlt. dies ist aber für die einschätzung des ausmaßes einer potenziellen fehlannahme essenziell. zu bedenken ist auch, dass sich die prävalenz einer sars-cov-2 infektion und damit die vortestwahrscheinlichkeit mit fortschreiten der pandemie ändern wird. die zukünftig zu erwartende zunahme bedingt, dass jeweils die für den testzeitpunkt bestehende prävalenz grundlage der beurteilung sein muss. ob selbst bei vorhandenen sars-cov-2 spezifischen ak auch eine immunität besteht, gilt derzeit als nicht gesichert [22] . ergebnissen von tierversuchen sowie erkenntnissen zu sars-cov geben jedoch hinweise darauf, dass genesene personen nur ein sehr geringes reinfektionsrisiko haben. beobachtungen von bisherigen coronaviren-infektionen deuten darauf hin, dass bis zu drei jahre nach erstinfektion eine immunität bestehen könnte [23, 24] . in wie weit das ausmaß einer potenziellen fehlannahme akzeptabel ist, hängt vom kontext der fragestellung ab. die fehlerhafte annahme einer immunität bei angehörigen von pflege-oder medizinberufen kann schwerwiegende auswirkungen haben. für epidemiologische fragestellungen kann der fehler aber möglicherweise hingenommen oder in modellen berücksichtigt werden. gezielte testungen ausgewählter personengruppen mit vermutlich sehr hoher vortestwahrscheinlichkeit könnten, eingebettet in strukturierte prozesse mit nachfolgenden handlungsanleitungen, sinnvoll sein. die verfügbaren angaben zu den testeigenschaften können nicht als gesichert angenommen werden. so basieren die aktuell vorhandenen test-evaluierungen vorwiegend auf fall-kontroll-studien und weisen somit ein erhöhtes verzerrungspotenzial auf. zudem zeigen unabhängige publikationen geringere werte für sensitivität und spezifität als die von den herstellern bzw. distributoren angegebenen. wie die berechnungen zu ppw und npw zeigen, ist jedoch eine ausreichend hohe testgüte voraussetzung für einen sinnhaften einsatz von ak testsystemen. dementsprechend sind qualitativ hochwertige evaluierungsstudien der vorhandenen ak testsysteme erforderlich. ebenso werden weitere studien zur prävalenz einer sars-cov-2 infektion in unterschiedlichen populationen benötigt. eine unkontrollierte anwendung von ak tests zum aktuellen zeitpunkt kann zu unerwünschten nachteiligen effekten führen, wenn aus den testresultaten falsche schlussfolgerungen und daraus abgeleitete handlungen resultieren. personen, bei denen fälschlicherweise eine immunität angenommen wird, können z.b. aus einem falschen sicherheitsgefühl wieder vermehrt kontakte aufweisen oder die hygieneregeln weniger beachten. das kann in folge zu einer gefährdung anderer und der eigenen person führen und die verbreitung von covid-19 wieder erhöhen. ebenso kann z.b. der fälschliche ausschluss einer akuten infektion bei personen im pflegeberufen zu weitreichenden negativen konsequenzen führen. anwendungen von ak tests sind daher nur im rahmen von organisierten, strukturierten testprogrammen zu spezifischen fragestellungen mit begleitender evaluation der daten zu empfehlen. ausreichend hohe sensitivität und spezifität sind unabdingbare voraussetzung für eine anwendung von ak testsystemen. auch bei nahezu idealen testeigenschaften sind bei geringer vortestwahrscheinlichkeit (wie sie bei der testung von personen ohne symptome und risikofaktoren für eine sars-cov-2 infektion besteht) positive testresultate häufig falsch. abhängig von der anzunehmenden prävalenz für eine sars-cov-2 infektion zeigen sich wesentliche unterschiede in der bedeutung eines konkreten testresultats für die jeweils betroffenen personen. zum nachweis bzw. ausschluss einer infektion sind ak tests alleine nicht geeignet. von einem positiven testresultat kann nicht sicher auf das vorliegen von ak und damit auf eine potenzielle immunität geschlossen werden. antibody responses to viral infections: a structural perspective across three different enveloped viruses igm in microbial infections: taken for granted? distributor: technomed gmbh. clinical data for sample correlation hersteller: intec products inc, distributor: nanorepro ag. sars-cov-2 antikörper-schnelltest (igm/igg). rev. 03 distributor: szabo-scandic handelsgmbh. wantai sars-cov-2 ab rapid test. rapid test for detection of total antibodies to sars-cov-2 distributor: szabo-scandic handelsgmbh. wantai sars-cov-2 ab elisa. diagnostic kit for total antibody to sars-cov-2 (elisa) distributor: szabo-scandic handelsgmbh. wantai sars-cov-2 igm elisa. diagnostic kit for igm antibody to sars-cov-2 (elisa) product catalogue evaluation of nine commercial sars-cov-2 immunoassays antibody tests in detecting sars-cov-2 infection: a meta-analysis ? idcservice = get pdf file&ddocname = 123050 coronavirus in österreich: daten und karten virological assessment of hospitalized patients with covid-2019 production of specific antibodies against sars-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229e and oc43 ifa in testing specific antibody of sars coronavirus profile of specific antibodies to sars-cov-2: the first report the promise and peril of antibody testing for covid-19 immunity passports'' in the context of covid-19 immunity after sars-cov-2 infection. rapid review 2020. oslo: norwegian institute of public health covid-19 (coronavirus sars-cov-2) key: cord-300520-vxn7uh41 authors: baunez, c.; degoulet, m.; luchini, s.; pintus, p.; teschl, m. title: tracking the dynamics and allocating tests for covid-19 in real-time: an acceleration index with an application to french age groups and departments date: 2020-11-07 journal: nan doi: 10.1101/2020.11.05.20226597 sha: doc_id: 300520 cord_uid: vxn7uh41 an acceleration index is proposed as a novel indicator to track the dynamics of the covid-19 in real-time. using french data on cases and tests for the period following the first lock-down from may 13, 2020, onwards our acceleration index shows that the ongoing pandemic resurgence can be dated to begin around july 7. it uncovers that the pandemic acceleration has been stronger than national average for the [59-68] and [69-78] age groups since early september, the latter being associated with the strongest acceleration index, as of october 25. in contrast, acceleration among the [19-28] age group is the lowest and is about half that of the [69-78], as of october 25. in addition, we propose an algorithm to allocate tests among french departments, based on both the acceleration index and the feedback effect of testing. our acceleration-based allocation differs from the actual distribution over french territories, which is population-based. we argue that both our acceleration index and our allocation algorithm are useful tools to guide public health policies as france enters a second lock-down period with indeterminate duration. the current covid-19 pandemic not only caught most of the world by surprise, it also highlighted the crucial aspect of uncertainty under which all societies live since the first mentioning of the new viral infection at the end of 2019. this uncertainty however not only concerns the novel sars-cov-2 pathogen and how it spreads, its effects on human health, the best possible treatments, when a vaccine will be available and how efficient it will be, to just cite some of the concerns. there are at least two more uncertainties which receive less attention. one is parameter-uncertainty of typical sir (susceptible-infected-removed)-models that are widely used to predict the evolution and consequences of the pandemic, and the other is data-uncertainty. 1 while a continuous effort is put into remedying and improving data collection, because this is, after all, the first and foremost important source of empirical knowledge on which all else is built, the parameter-uncertainty is intrinsic to modelling strategies and thus much less prone to any easy solutions. 2 a key point of this paper therefore is to say that given these uncertainties, what is of crucial importance to know, here and now, for public health specialists and decision makers and other people alike, is whether harm is accelerating or whether measures that are put in place to control the pandemic are contributing to curbing the spread and thus to decelerate harm (see taleb [11] ). in the case of a pandemic, harm can be defined, for example, as the number of confirmed cases. however, simply plotting the number of positive cases over time, as it has been done on many governmental sites, is not the right way of understanding the dynamics of harm. for example, the knowledge of positive cases depends on testing and hence cases will very much depend on the underlying testing strategy put in place. but this is exactly our point: to say something meaningful, we need to put cases and tests in relation to each other. that is, to understand whether harm, defined as the number of cases, is accelerating or decelerating, we need to plot cases against tests and not to consider them separately. although this might be seen as just another tool to visualize the data, we show that such a way to organize the data is extremely useful to uncover an index of acceleration, which turns out to be a simple and plain scale elasticity at the end date of the sample. it tells us how much additional cases are detected given additional tests, in percentage of the total of both variables attained at the date for which one has the last vintage of data. we argue that updating such an acceleration index in real time provides very valuable information and is an essential tool to apprehend the pandemic uncertainties with which we currently live. it is also dealing with parameter-uncertainties and this simply because we do not use any. instead of engaging in assumptions and probability attributions to ex-ante uncertain events, we use the data that is currently available, which is the best we have, to shed light onto the dynamics of the pandemic and the harm it produces. our aim strategy combines two aspects: information about the virus circulation and the expected feedback that testing implies, in terms of isolation and contact tracing most importantly. we propose a parsimonious algorithm to allocate tests across age groups and space, based on both our acceleration index and the average positivity rate, and on the extent to which tests reduce the virus propagation. because the latter feature can never be measured with certainty, we argue that any allocation of tests should also reflect the beliefs of (benevolent) public health authorities and experts and we add a parameter to the analysis to take into account this dimension explicitly. we show that our acceleration-based allocation of tests differs from the actual distribution of tests, which has essentially been determined by population size. but our analysis shows that population size is not necessarily the criterion that affects acceleration, at least not all the time. hence, to the extent that testing is accompanied by contact tracing and efficient isolation, this observation means that allocating tests according to where the virus accelerates the most would be a better way to control the pandemic. accurate and reliable information about how pathogens spread over space and time is of firstorder importance to fight epidemics and to properly design efficient public health policies. what is striking in the case of covid-19 is that, at least in north american and european countries, is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint in figure 1 the problem with the information contained in the graphs of the first row in figure 1 is that they would be a fine summary of the pandemic's dynamics if the number of tests performed over a unit of time was roughly constant. in that case, the evolution of the number of new cases, given a constant over time number of new tests, would capture well how fast the virus spreads. however, as shown 4 all the data used in this paper is publicly available from the web page "données relatives aux résultats des tests virologiques covid-19 si-dep" https://www.data.gouv.fr/fr/datasets/donnees-relatives-aux-resultats-destests-virologiques-covid-19/. 5 santé publique france decided at some point to report also the positivity rate, that is, the ratio of positive cases to tests at weekly frequency. however, as we will argue, even this indicator does not convey enough information and possible early warning signals that public health authorities could rely on. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint in panels (c) and (d) in figure 1 (bottom row), this assumption is far from correct in practice, and this is not only true for france because of several reasons that has concerned most countries. 6 in the period after the end of the generalized lock-down in france, when the who message to "test, test, test" 7 was becoming effectively received in france, one sees from panel (c) in figure 1 that the number of tests performed each day is far from constant and has in fact been trending up most of the time, not surprisingly given the level of unpreparedness of the french government at the beginning of the pandemic. it is obvious that the dynamics of the number of cases is strongly related to the dynamics of performed tests. after all, diagnostics are the only, albeit imperfect, way to confirm that people are positive to covid-19. 8 however, observing each independently (side by side) may give only a blurring view at best, and even a confusing sense, of the extent to which the pandemic accelerates. a manifestation of how confusing that coarse piece of information can be, if not properly organized, is the statements often voiced that one should not be worried to observe more positive cases as long as more tests are performed as time passes. after all, more tests imply more cases, almost by definition and this basic fact should not be taken at face value to indicate that the pandemic is worsening. we argue that such reasoning is wrong and that the correct understanding, in terms of measuring the acceleration/deceleration of the pandemic, is gained if a scatter-plot of the number of positive cases against the number of the tests is used in real time, instead of the panels in figure 1 . this is the first contribution of this paper, in our view, and it is presented in figure 2 . before presenting our preferred way to organize the data, a few remarks are in order. first, we use the cumulated numbers of daily cases and of daily tests as a way to keep track of the size and overall prevalence of the pandemic. second, our proposed scatter-plot has merit only if it is updated in real time, say, at daily frequency, so as to add information about additional tests and cases. this immediately raises the question of how data should be normalized, given that the number of data points obviously increases with time. we choose a simple and rather innocuous re-scaling method known as min-max normalization, 9 which in our case amounts to divide all historical values by the last data point figure. as a result, our (all positive) normalized data values are contained within the unit interval (0, 1) so that our scatter-plot lies in the unit square. 6 an important reason behind the observed non-constancy of tests over time in most countries is undoubtedly the fact that the pandemic caught most of the world by surprise in the first quarter of 2020. as a consequence, the widespread goal to progressively increase the capacity to perform tests on a large scale has led many countries to increase the number of tests since march. 7 see https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefingis the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. in figure 2 we present our proposed scatter-plot, which is in our view a very useful way to visualize the dynamics over time of the number of cases in relation to the number of tests. as alluded to above, there is a causality arrow going from the latter to the former, so we plot cases against tests, all in cumulated values. for example, panel (a) of figure 2 graphs all data points from march 13 to june 13, 2020, each point/dot representing a particular date. on the x-axis, all numbers are between zero and one due to min-max normalization and a particular value gives the fraction of tests performed that day, say, t, out of the total cumulated number of tests performed up to end date t , say. for example, 0.8 means 80% of the total amount of tests cumulated from date 0 (may 13, 2020, in our case) to date t (june 13, 2020). similarly, along the y-axis we report the cumulated number of cases for each day t ≤ t , as a fraction of the total number of cases at date t . all such normalized data points fill in a solid black curve which then represents the actual data over time. starting at the origin and moving along the solid curve in the north-east direction means moving forward through time, since we use values that add up over time. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint the dashed black line in panel (a) of figure 2 , on the other hand, is the first diagonal and it turns out to be an interesting benchmark if one is interested in capturing the acceleration/deceleration of the pandemic. counter-factually, if the dashed diagonal would represent the real data, that would tell us that the virus spreads according to a linear pattern whereby, say, 20% of the total amount of tests accounts for 20% of the total number of positive cases, and so on. and this coincidence would hold at all dates between zero and t . in other words, the pandemic neither accelerates nor decelerates since a given proportion of tests account for the same proportion of detected cases for all the data period, that is, over time. panel (a) of figure 2 shows that this has in reality not been the case for the period may 13-june 13, 2020, or for all the period covered by our analysis for that matter. more precisely, the solid line, which represents actual data, turns out to be entirely lying above the dashed diagonal, which should be interpreted in the following manner. first, the blue line representing the tangent to the black curve at the end point (1, 1) turns out to have a slope smaller than one (this is easy to see since the slope of the dashed diagonal equals one by definition). this means that between the day right before t and day t , the observed fraction of new tests (out of the total number of tests to this day) has been associated to a smaller fraction of new cases (out of the total of cases to this day). we then want to say that at date t the pandemic is decelerating. now going backward from date t into the past (that is, moving from the farthest north-east corner toward the origin along the solid curve), one sees the "slope" of the tangent to the solid curve going up as time moves backward. in other words, the slope of the tangent to the solid curve has been decreasing from date 0 to date t , which is an equivalent way of saying that the solid curve is concave: for a given fraction of tests, lower and lower fractions of cases have been detected over time. this indeed captures in a visual fashion the fact that the pandemic has been continuously decelerating between may 13 and june 13. this observation indicates that france has somehow benefited from the lock-down period in the sense that the circulation of the virus, in the aggregate, slowed down. unfortunately, this state of affairs was not to persist. panel (b) in figure 2 shows, using color coding, that a month later, on july 13, the slope of the solid curve at end date (the green line) was about one -the green line almost coincides with the dashed diagonal. in other words, the pattern of the pandemic over time switched from decelerating to roughly linear, between mid-june and mid-july. quite importantly, comparing panel (a) to panel (b) suggests that the slope at end date had started to increase and has crossed unity between june 13 and july 13. while comparing panels (a) and (b) suggests the end of deceleration, panel (c), dated august 13, clearly shows that acceleration was then taking place: the slope at end date is about two. in addition, the scatter-plot at later dates reveal that acceleration is still underway, to this day (october 25, 2020). one important conclusion is that figure 2 delivers an indicator in real time of the pandemic's dynamics, in the sense that it helps to visualize whether the pandemic accelerates or decelerates at any date. the property that the pandemic accelerates when the slope of the solid curve at end date is larger than one, and that it decelerates when the slope is smaller than one, is in fact no is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint surprise. as shown in appendix a, this slope is essentially an elasticity: its value gives the variation of cases detected by a given variation of tests, in proportion to levels of (cumulated) cases and tests attained at end date. 10 for example, a value about two as in panel (c) of figure 2 means that a given proportional increase in the amount of tests accounts for a proportional increase in the number of cases that is twice as much. one can therefore think of this elasticity as an acceleration index: acceleration happens when the elasticity is larger than one, otherwise the pandemic decelerates. in the razor's edge case when the elasticity is exactly one, a linear pattern emerges so that the pandemic neither accelerates nor decelerates. figure 2 , we also report the points corresponding to the previous dates associated with the other panels. for example, the blue dot in panel (b) corresponds to the situation at june 13. similarly, the red dot in panel (d) corresponds to august 13. interestingly, our scatterplot helps also visualizing the pandemic's dynamics in a retrospective fashion, to the extent that one can visually track not only the slope of the derivative to the solid line, but also the slope of the cord that links the origin to the point corresponding to the date under scrutiny and we now turn to this subtle point. more specifically, although figure 2 and the depiction of our acceleration index are very helpful to visualize in real-time whether the pandemic accelerates at end date, those two visualization tools have their own limitations and must be refined if one is to do a retrospective analysis of the pandemic using the information in the scatter-plot and not only the elasticity at end point. 11 the way we see it is that the scatter-plot should be updated in real time to visualize where the pandemic goes in terms of acceleration/deceleration. we can in particular infer each day, from the most recent data point, the slope of the tangent to the solid curve, the value of which is what we called the acceleration as explained above. but such an index by definition combines two types of information: variations (between two successive dates in our example) and levels at final date. to use the notation of appendix a and if we denote the elasticity/acceleration index at end date ε t , the following decomposition holds: where p t and d t denote respectively the cumulated numbers of positives/cases and diagnosis/tests at date t , and similarly for date t − 1. if we define from equation (1) the daily positivity rate to be the ratio between additional positives and additional tests, that is, what we have called 10 more generally, the (local) elasticity of a given function is the ratio between its derivative and its average value, taken at a particular point. see arrow et al. [1] . the empirical counterpart that we use in this paper is conceptually similar. 11 mathematically speaking, the normalization and updating of the data means that data points are subject to a rotational homothety over time. for instance, while the end point at t has coordinates (1, 1), it is subject at date t + 1 to a mapping which is a composition of a contraction, due to the new values of cases and tests at end point, and of a rotation, due to the new value of the ratio between the latter. this is why the colored points move over time in figure is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint earlier the tangent's slope, then the above decomposition relates the elasticity, the average positivity rate and the daily positivity rate. more precisely, the elasticity is defined as the ratio of the daily positivity rate to the average positivity rate (much as the elasticity of a function is the ratio of its derivative to its average value). this fact suggests that looking at the average positivity rate alone is not informative enough, in the sense that this indicator does not say how much additional tests translate into additional cases, even though it is of course a fine measure of the average detection rate. it is a proxy for the prevalence of the virus in the population, a key parameter for herd immunity (see britton et al. [3] , fontanet and cauchemez [5] ). that said, there might be situations in which any pair of those indicators summarize all there is to know, the third indicator being somehow redundant. for instance, the case depicted in panel (a) in figure 2 is simple in the sense that, at all dates, both the tangent's slope and the average positivity rate go down over time, and that the former looks, on close inspection, smaller than the latter. from this property, one concludes that the elasticity is smaller than one. this is of course another way to express the property that the solid line is concave (hence above the dashed diagonal since the solid line goes through the origin and the upper-right edge of the unit square). in contrast, panel (c) in figure 2 reveals that there has been a change in curvature, that is, a reversal from deceleration to acceleration, between june 13 and august 13. this can be seen from the fact that the least recent part of the solid curve is above the dashed line, while the most recent part is below it. in other words, the convex part indicates acceleration of the pandemic, while the concave part indicates deceleration at earlier dates. one difficulty that arises from such a change in curvature/motion is that the daily and average positivity rates may vary in different directions, with ambiguous implications about whether the elasticity stays smaller than one or, on the contrary, becomes persistently larger than one. in panel (c) of figure 2 , one notices an inflexion point, when the solid curve intersects the dashed diagonal in its interior. at the date when this happens, the tangent's slope/daily positivity rate starts to rise, whereas it was declining over time before that date. the average positivity rate, however, continues falling right after the inflexion point is attained, and it starts to rise only later. eventually, both the daily and the average positivity rate do coincide, and when this happens the elasticity equals one. after that date, the elasticity stays larger than one. more specifically, imagine that the elasticity is smaller than unity and that the daily positivity starts increasing while the average positivity rate is still declining over time. it then becomes possible that the elasticity starts rising and eventually crosses the unit value and possibly stays persistently larger than one. the latter situation occurs whenever the daily positivity rate rises faster (or declines more slowly) than the average positivity rate. note that all this is of course reminiscent of what happens when the graph of a continuous and increasing function changes curvature, from concave to convex. we therefore conclude that while the colored line's slope is an estimate of the elasticity at end point, looking back in time at past events from the perspective of date t through the lens of our scatter-plot requires to examine how is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint both the daily and the average positivity rates vary over time, as shown in figure 3 . in figure 3 we report over time the magnitudes of the three terms that can be inferred from the decomposition in equation (1) . remember that our objective is to detect whether the pandemic decelerates, accelerates or switches between those two types of motion. a striking feature in figure 3 is that the acceleration index -in panel (b) -leads the average positivity rate -in panel (a) -in the sense that the former starts rising about two weeks before the latter starts rising as well. in other words, the evolution of the acceleration over time appears to be an early warning of a future rise in the average positivity rate. eventually, in view of the reasoning above, one expects of course that the rising acceleration index crosses the unit value when the (rising) average positivity rate equals the (rising) daily positivity rate. the bottom line is that the decomposition that follows from the scatter-plot in figure 2 may be useful to detect the early reversal from deceleration to acceleration. in the case of france, the elasticity starts rising about july 7, while the average positivity rate starts rising about two weeks later, on july 23, as indicated by the orange bars in figure 3 . 12 to sum up, the important piece of information that one can derive from figure 3 is that a reliable measure of the pandemic's dynamics is the elasticity, which we can think of as an acceleration index. this elasticity leads the average positivity rate and is more dynamic than the daily positivity rates, 12 our acceleration index has, to our knowledge, not been studied before, but many health agencies around the world report the positivity rate over time. in particular, santé publique france started, during the second quarter of 2020, to report the weekly positivity rate on top of the number of cases. the first time a rise of such an indicator is noted is july 23, in the "point épidémiologique hebdomadaire du 23 juillet 2020", whereas our acceleration index starts rising on july 7 as shown above. this two-week gap may suggest that using daily data rather than weekly data is more appropriate. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint which are other key statistics to follow. in practice, therefore, panels (a) and (b) should be used as real-time indicators of the pandemic's dynamics. from the acceleration index and average positivity rate then follows the daily positivity rate, which is is still useful to translate the transmission dynamics back into levels (as opposed to normalized ratios). from panel (b) in figure 3 , one infers that the period since the end of the first lock-down in france can roughly be split into four sub-periods. from may 13 to july 7, our acceleration index fluctuates around a value slightly lower than one, indicating that the pandemic is in a deceleration regime. our indicator suggests that july 7 is the start of an ongoing acceleration regime in which the acceleration index increases, crosses the unit value on july 23, and continues to increase until about mid-august, when our acceleration index equals about 2. in that sense, this second sub-period can be seen as the fact that the pandemic worsens more and more over time. then a new plateau occurs until around october 1st, after which the acceleration index rises again to reach a value of about 3 on october 25. of course, the challenge that the country faces as the second lock-down period starts, on october 30, is to reverse that pattern to ensure that the acceleration index starts decreasing and eventually reaches values below one, which would indicate deceleration of the pandemic. the simple case whereby both cases and tests grow exponentially, as detailed in section b of the appendix, is perhaps useful to interpret the four sub-periods just described. the first plateau associated with deceleration is compatible with a situation where cases have been growing at a slower rate than tests for a while. in that situation, the acceleration index is roughly constant over time, below unity. then if the growth rate of positives becomes larger than the growth rate of tests, then the acceleration index starts rising and eventually crosses unity, to converge to a new and higher plateau associated with a value larger than one. finally the most recent rise, starting early october, can be understood as a new jump of the growth rate of positives. although the above interpretation stresses changes in the growth rate of positives, what matters more generally is how the ratio of growth rates -that of positives divided by that of tests -varies over time. in addition, the scatter-plot also directly offers a measure of "global acceleration/convexity": in panels (d)-(e) of figure 2 , the solid curve is located entirely below the dashed diagonal, which indicates that the persistent acceleration somehow "dominates" the earlier and short-lived deceleration. 13 based on the analysis in the previous section, we propose to use the acceleration index, calculated from the end point of our scatter-plot, as a parsimonious way to track the pandemic's dynamics. in this section, we show how useful such an index is to decompose the evolution of acceleration over age groups, first, and space, in a second step. 13 we conjecture that such a global measure of convexity relates to δ-convexity as defined by hyers and ulam, 1952. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint in figure 4 , we report the evolution over time of our acceleration index for nine age groups, all aggregated over space. in each panel, the blue line is a replica of panel (b) in figure 3 . for comparison against such an average benchmark, the orange line represents the acceleration index corresponding to the age group of each panel. one striking feature is that acceleration is underway for all age groups except people older than 79 years, as of october 25, 2020. in addition, the acceleration for each age group tracks rather closely that of the average for some groups but much less so or not all for others. to start with the former groups, the middle row in figure 4 shows that people aged 29 to 58 experience acceleration in much the same way as the average over all age groups. this is not the case for younger and older groups. children aged 10 to 18 and, quite spectacularly, young adults aged 19 to 28 seem to face a noticeable but slower acceleration since summer than the general acceleration. the latter group, in particular, is associated with an acceleration index that is maintained at intermediate levels around 2 in the last quarter of the data sample. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint the fact that the share of confirmed cases is the second highest (22.9%) for the [20-29] age group (see table 1 in appendix c for incidence rates among age groups) may contribute to the view that youngsters such as college students have contributed a lot to the resurgence of the virus since last summer. this view does not agree with the data in figure 4 . the age group [0 − 9] has experienced ups and downs since last summer. in particular, the acceleration index has started to rise again quickly in early september across france, after it went down all the way to unity at the end of summer. the question here of course is whether the rapid increase has to do with the beginning of the academic year and the fact that young pupils did not wear face masks at school. however, another reason may be that younger kids have again been in more contact with their grand-parents, who take care of them some of the time while parents are working since the beginning of the school year. yet in figure 4 , it is striking that the acceleration index for the age group [69 − 78] has risen importantly since the beginning of august, to attain the highest level at the end of the data sample, around 4! a similar, though slightly less strong rise, can be seen for people aged [59 − 68]. hence maybe it is not the young kids who infect their grand-parents, but it is the other way around? studies suggest that the "engines of the sars-cov-2 spread" are household transmissions, which may support this hypothesis (see lee et al. [9] )). equally remarkable is the group of people older than 79, which has experienced a reduction in acceleration since the end of the first lock-down, in sharp contrast with the dynamics of all other age groups. this suggests that, in spite of the large number of deaths among the very elderly at the beginning of the pandemic, the continued presence of covid-19 has been handled quite effectively in specialized nursing homes (the "ephad"). our analysis can also be applied to shed light on the local evolution of the pandemic. in figure 5 , we report in a map of france the acceleration index at the level of administrative division which is intermediate between the city and the region, called "département". red entries represent places where the pandemic accelerates (the elasticity is larger than one), while green entries represent decelerating départements. our map does not necessarily coincide with the map very recently introduced by the european centre for disease prevention and control (ecdc) for europe. 14 in that map, green-orange and red lights depend on the incidence rate and the test positivity rate of a country but their combination of factors is not dynamic and does not have any clear conceptual foundation such as our acceleration-based index has. their demarcation criteria between the different colours are set at a certain level, but it is not clear whether that level is sound from the perspective of our acceleration index. the upper left panel in figure 5 depicts the level of the acceleration index for each département on may 15, that is, on the third day after the end of the lock-down period in france. while at that date, the pandemic is decelerating over almost all the country, the situation starts to gradually but dramatically change within the next two months and to reverse course over the summer break. 14 https://www.ecdc.europa.eu/en/covid-19/situation-updates/weekly-maps-coordinated-restriction-freemovement 14 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint between july 15 and august 15 (panels in second row), the country switch to a situation such that acceleration takes over the country. as of october 25, most département face an acceleration index larger than two, with no sign of reversal. at the time when france enters a second lock-down period, lessons should be drawn about what went wrong in the period following the end of the first lock-down and we argue that it is important to analyse closely the reasons for local and group-specific increased accelerations. knowing about local and group specific accelerations can be an important guide in investigating further possible transmission channels and networks, which would then allow to intervene specifically at a network and structural level if needed. to give an example of such a fine-grained analysis, we dig a little deeper into the heterogeneity that is visible in figure 5 , and the accelerations we spotted in the different age groups. we report in in all panels, the blue line stands for the evolution of the acceleration index for all the other départements, while the second colored lines in each row represents the dynamics of acceleration for all age groups in panel (a) and the specific age group in panels (b) − (c). looking first at panel (a), one sees that the dynamics in all four départements, resemble that of the others, with perhaps more volatility in the case of bouches du rhône and rhône. note, however, that the latter have experienced a period when the acceleration index decreased, roughly from mid-august to mid-september. this is not really the case for paris and nord. in panel (b), we see considerable volatility, largely due to the fact that the absolute numbers of tests and positives are rather low for this age group at the level of the département, the acceleration index for the [0 − 9] age group hover around a plateau below one from mid-may to early july. what is striking, however, is that the acceleration index drops a lot before the end of august to levels close to unity, only to starts rising sharply again in the first half of september. as we asked before, why is there this rapid acceleration in this age group? does it have to do with the dynamics of acceleration for the age group of grand-parents, depicted in panel (c). the rapid acceleration in this age-group is especially the case in paris and nord, where the acceleration index for the elderly rises continuously from the beginning of august, and may be less so for bouches du rhône and rhône. in contrast, deceleration was pronounced both for kids and for grand-parents before the beginning of summer. although the above comments in relation to figure 5 are merely descriptive, and deliberately so, they illustrate how our acceleration index can be used at any levels of granularity that is allowed by the available data and how it may also guide further analysis about transmission channels to 16 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint complement contact tracing. an interesting avenue for research would obviously be to perform a panel data analysis, but this is beyond the scope of this paper. test strategies have many dimensions, including where to test and to allocate staff, locating groups at risk. we show in this section how to design an algorithm to allocate covid-19 test resources. we focus, for the sake of illustration, on the spatial distribution of tests based on our acceleration index. although testing is acknowledged as an efficient tool to curb epidemics 15 , little is known on the context of covid-19 about how to geographically allocate a limited quantity of test. imagine now that public health authorities would like to decide how tests should be allocated to region i at date t . according to the decomposition stated in equation (1), a small increment of the per-period fraction of tests ∆d i would be expected to lead to a proportional increase of positives the logic of our criterion to allocate tests across regions has two steps. firstly, we attribute a weight that equals w i = p i t d i t ε t to each region i. note that the weight w i attributed to each region is the product of two terms: (i) the first term is the average rate of cumulated positives as a fraction of cumulated tests, at end date t , that is, p i t /d i t ; (ii) the second term is the acceleration index ε t , which measures whether the pandemic accelerates (i.e. ε t > 1) or decelerates (i.e. ε t < 1). this means that regions that are allocated more tests relative to others are those where either the overall positive rate is larger at end date, the pandemic accelerates more (or decelerates less), or both, relative to others. second, we propose to allocate to region i a share of national test capacity that is given by: where β is a parameter which we assume takes positive real values: therefore, s i goes up with w i so that a region with a larger weight is allocated a larger share. our premise here is that any test strategy combines two aspects: information about the virus circulation and the expected feedback that testing implies, in terms of isolation and contact tracing most importantly. we therefore propose a parsimonious criterion to allocate tests across age groups and space, based on both our acceleration index and the positivity rate, and depending on the extent to which tests reduces the virus propagation. because the latter feature cannot be measured with certainty, we argue that any allocation of tests should reflect the beliefs that public health authorities have about it and we therefore add a parameter to the analysis to take this key dimension explicitly into account. obviously, we are assuming here a benevolent public health authorities, who act on objective grounds and are not trying to manipulate the indicator for political purposes. 15 the importance of testing is most clearly spelled out in an official document that has been produced by the south korean government for the international community. see "flattening the curve on covid-19 -how korea responded to a pandemic using ict", dated april 15, 2020. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint therefore, the allocation scheme that we propose builds upon the following logic. when β = 0, each and every region is allocated an equal fraction 1/n of the total amount of tests available at the national level. the other extreme configuration occurs when β tends to ∞ and it is easy to check that, in that extreme case, the region with the highest w i is allocated all national tests. one way to interpret our allocation rule for intermediate values of β is as follows. parameter β measures the extent to which testing is believed to exert a negative feedback effect on the pandemic, for example because detecting positives entails isolating them and contact tracing, thereby reducing the number of contacts that produce additional contaminations. in the limit case when β = 0, there is no such feedback and a natural benchmark arises, which consists in allocating tests equally across regions. this corresponds to a situation where information about the spatial distribution of the pandemic is maximized, since each and every region has the same input in terms of testing. in this case, the objective is to gather information about where the pathogen is currently circulating. on the other hand, when β tends to ∞, all tests are allocated to the region with the highest w i and this amounts to maximizing the number of positives so as to curb the pandemic, since in this case the negative feedback is believed to be the strongest. 16 middle cases happen when β is between zero and infinity, reflecting the belief held the public authorities deciding over the allocation of tests across regions about the strength of the feedback effect. 17 we use figure 7 to visually compare the allocation of tests, as computed from our algorithm based on the acceleration index, to the observed allocation and to the population distribution, as of october 25, 2020. in panel (a) and (b), we report the observed shares of tests and of total population in each département, respectively. comparing the two upper panels reveals that the observed allocation of tests at that date reflects the geographical distribution of population to a large degree: in short, more tests have been done in more populated areas. figure 7 depict the allocation of tests over french départements, for different weights β put on the acceleration index. comparing the upper and the lower panels makes clear that the spatial distribution of tests observed at october 25, 2020, has little connection to where the pandemic accelerates, which is somewhat paradoxical to the extent that detecting positives should help to curb the virus circulation. more precisely, while in panel (a) one sees that tests have been concentrated, especially in about roughly three sub-regions (mediterranean south east, north, and rhône), panels (c) − (d) shows that the allocation proposed by our algorithm based on the acceleration index is somewhat less concentrated or concentrated in different areas. this 16 in reinforcement learning terminology, that would lead to stop exploring others aera and only to exploit the region with highest w i when the algorithm starts much like a greedy algorithm would do -see sutton and barto [10] . this is however not a desirable property in the case of a pandemic and acceleration weights should always lead to some exploration to gather information about the virus circulation in other areas, such as in -greedy algorithms. 17 note that in theory, β could be negative. this would the case, for instance, if positives would have even more contacts with susceptible persons, generating even more contaminations, compared to a situation with no test. in this case, the share of tests a i would go down when w i goes up. although such a situation is hypothetically possible -think about bio-terrorism -we rule it out for the sake of realism. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint is particularly clear when β = 1. when β is higher, our algorithm allocates more tests where the acceleration index is larger than unity (and less tests where it is smaller than one) so that the allocation of tests becomes more concentrated but panel (d) shows how more tests should go to the south-east quarter of france. in contrast, panel (a) shows that this is not what has actually happened. the difference between the actual distribution of tests over départements, which is very much population-driven, and our proposed allocation, which acceleration-centered can be established a it more formally by looking at the following measures. in appendix d, we report in tables 2-4 the numbers behind figure 7 and we use those to compute two indicators that shed light on the differences between the actual and proposed spatial distributions of covid-19 tests. we first carry out a comparison between the different distributions using jensen-shannon (js) divergence, which is based on kullback-leibler divergence and is normalized between 0 and 1, to measure similarities between the actual distribution of tests, population shares and our accelerationbased allocaton of tests. from tables 2-4 in appendix d, we find that js divergence between the observed distribution of tests and the distribution of population equals 0.016, hence very small. this confirms our suggestion that relative population size has been an important determinant of the distribution of tests across french départements. in contrast, js divergence between the observed distribution of tests and our proposed distribution based on acceleration is higher: it equals 0.115 when β = 1 and 0.194 when β = 3. 18 not surprisingly in view of the above discussion in relation to figure 7 , we conclude that observed and proposed distributions differ in a quantitative sense, but also that putting more weight on acceleration -that is, increasing β -further enlarges thus discrepancy. obviously, the allocation of tests and the pandemic's dynamics are both endogenous and interactions between the two form a complex system. our analysis is so far limited to tracking such dynamics and proposing an algorithm to allocated tests across space, according to the acceleration index. as such, it has little to say about the extent to which testing impacts negatively the virus circulation. for instance, it is reasonable to think the weights our algorithm put on acceleration should necessarily be the same in all départements. perhaps isolation and contact tracing is more efficient in some regions that in others. in addition, additional constraints should arguably be introduced, such as the fact that the number of tests allocated to a département should not be larger than its population. 19 despite all these limitations, we still argue that our algorithm to allocate tests according to where the pandemic accelerates could be a valuable input to public health policies, not only in france of 19 while such constraint is unlikely to bind at the département level, it could at more granular levels at which our algorithm could operate as well. for example, some little populated cities could be allocated too much tests in view of their populations. in that case, our algorithm could easily be amended to account for such constraints, by capping test and reallocating the surplus of test according to the next cities where acceleration is the highest. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint course but also in all countries facing covid-19. of particular importance, at this point, is how to reallocate the national testing resources and effort within département to specific age groups, and of course when the pandemic will start to decelerate, which one hopes is not too far ahead in time. the purpose of this paper is to show that plotting the number of cases, of tests and even the positivity rate against time is far from the best way to measure the acceleration/deceleration of the virus. we propose instead a simple yet novel way to look at the data in real time. our premise is that looking solely at the number of cases over time to measure acceleration of the pandemic is not accurate and hence problematic as a foundation for public health policies. this is because the amount of tests is far from constant per unit of time and this for a number of reasons. we thus argue that a much better understanding is gained by plotting the number of cases against the number of tests, that is, a simple and plain scatter-plot. from such a scatter-plot we derive an acceleration index, which we propose, is a useful indicator to track the dynamics of pandemics like covid-19 in real-time. using french data on confirmed cases and tests for the period following the first lock-down -from may 13, 2020, onwards -our acceleration index shows that the ongoing pandemic resurgence can be dated to begin around july 7. rather surprisingly, it helps to underline the fact that the pandemic acceleration has been stronger than national average for the [59 − 68] and [69 − 78] age groups since early september, the latter being associated with the strongest acceleration index, as of october 25. in contrast, acceleration among the [19 − 28] age group is the lowest and is about half that of the [69 − 78]. we also propose an algorithm to allocate tests among french départements, based on both the acceleration of the pandemic and the feedback effect of testing. our acceleration-based allocation differs significantly from the actual distribution over french territories, which is population-based. we would like to stress that our approach has admittedly limitations, mostly due to the fact that it relies on actual data only and abstracts away from any theoretical model like those that have been stressed and used by some governments from early on. yet, we do so deliberately and this for two reasons. first and foremost, covid-19 has clearly put at the forefront the importance of admitting that the novelty of a new pathogen has implications for modelling. any estimates obtained through, say, sir or any other models, are subject to considerable and often unknown parameter uncertainty. this problem is particularly acute in the face of a novel pathogen, when public health authorities must act swiftly to prevent the virus from spreading in an uncontrolled way. our analysis offers a real-time analysis that builds only on raw data and is, as such, subject to data measurement issues as any other approaches, but not to parameter uncertainty. second, our real-time analysis is scalefree and can therefore be used at very granular levels, say cities or counties or age groups, where any model becomes rapidly either untractable or not transparent enough. in that sense, we think 22 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint that our acceleration index carries important information, for instance compared to the ubiquitous reproduction numbers r 0 and r e . in fact, the results in this paper could be used to go in the direction of advocating more diversity in the tools that should be used in an emergency crisis, that is, a type of "method averaging" (in the same sense that model-averaging is used) that seems to us necessary to take the best policy actions given the available information. the index that we have exposed in this paper could also be used in the following weeks to monitor the pandemic and to detect positive signs of deceleration and to respond rapidly to new accelerations. the point is that our acceleration index might better track any reversal than looking solely at the average or daily positivity rate. when deceleration is under way, our index gives a very clear target for an all-clear: the acceleration rate has to be lower than one independently of where we look through our "acceleration lenses", that is a spatial and group based analysis for example. it is also in this sense that our acceleration index can help assessing and modulating health policies to respond to those areas or groups where an acceleration takes place. arguably, this would help avoiding generalized lock-downs, which are extremely costly, or other policies that affect the general society independently of whether they face an acceleration or not. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint all of the above implies that, given the estimate of the first-order derivative, ε t ≡ (f i ) (1), equation (3) can be rewritten in terms of the numbers of positive and tested persons, that is: in other words, equation (4) can be used to decompose the effect of tests on positives in levels, that is, how many additional positives are detected given additional tests, between t and t + dt: equation 5 is identical to equation 1 in section 2.1, where ε t is estimated as the ratio of variations of cases and tests between t and t − 1. similarly, the above decomposition in levels holds for any date t < t , as follows: from equation (6), the effect of tests on positives in percentage terms from the perspective of date t is therefore written as: the elasticity of the number of positives with respect to the number of tests is now, because it is evaluated at date t as opposed to end date t , the product of the derivative at the relevant point, times the ratio of average positive rates -that of date t over that of date t. this section explores what the decomposition stated in section a reveals when time is assumed to be continuous and when the number of cases grows exponentially over time, as usually assumed in epidemiological models, of sir type and related for example. although typically absent in the latter strand of literature, we have to introduce tests and we assume that they also grow exponentially. more formally, using the notation in the previous section, suppose that the number of cases per unit of time is denoted by p(t) = αe βt while the number of tests per unit of time is d(t) = γe νt , where the growth rates β and ν are assumed to be positive for the sake of illustration. cumulated cases and tests are then noted p (t) = t 0 p(τ ) dτ and d(t) = t 0 d(τ ) dτ , respectively. it is easy to derive, by straight integration, the expressions: it follows that our acceleration index is given, as function of time, by: 25 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint from equation (9) , then, one infers that two cases occur. when β = ν, that is, when both cases and tests grow at the exact same rate, then our acceleration index equals 1 at all dates. when the two growth rates differ, however, ε(t) converges, when t goes to infinity, to the ratio of growth rates β/ν, independently of the scale parameters α and γ. as an illustrative example, suppose that β > ν, so that positives grow faster than tests. then the pattern of our acceleration index ε(t) over time will have two regimes: it first grows almost linearly and eventually reaches the upper bound β/ν > 1. obviously, in that case both the daily positivity rate p(t)/d(t) and the average positivity p (t)/d(t) grow over time, and the latter quantity exceeds the former all the time so that acceleration prevails. this closely resembles the pattern following early august to early october in figure 3 , as underlined in the main text. in table 1 we report a few statistics for all age groups, as of october 25, 2020. in the second and third columns we report the numbers of cumulated cases and tests, respectively. the fourth column depicts that average positivity rate, defined as the ratio of cumulated cases and cumulated tests, while the actual test shares appear in the fifth column. finally, the last column shows the share of cases by age group, which is defined as the ratio of cumulated cases. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226597 doi: medrxiv preprint capital-labor substitution and economic efficiency towards controlling of a pandemic. the lancet s0140-6736(20)30673-5 a mathematical model reveals the influence of population heterogeneity on herd immunity to sars-cov-2 sensitivity to rare and extreme events in rats: the black-swan-avoidance bias. forthcoming biorî §iv and amse covid-19 herd immunity: where are we? on the fallibility of simulation models in informing pandemic responses data mining: concepts and techniques on information and sufficiency the engines of sars-cov-2 spread reinforcement learning, an introduction antifragile, things that gain from disorder equation (3) key: cord-320970-ru2iw0py authors: peeling, rosanna w; wedderburn, catherine j; garcia, patricia j; boeras, debrah; fongwen, noah; nkengasong, john; sall, amadou; tanuri, amilcar; heymann, david l title: serology testing in the covid-19 pandemic response date: 2020-07-17 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30517-x sha: doc_id: 320970 cord_uid: ru2iw0py the collapse of global cooperation and a failure of international solidarity have led to many low-income and middle-income countries being denied access to molecular diagnostics in the covid-19 pandemic response. yet the scarcity of knowledge on the dynamics of the immune response to infection has led to hesitation on recommending the use of rapid immunodiagnostic tests, even though rapid serology tests are commercially available and scalable. on the basis of our knowledge and understanding of viral infectivity and host response, we urge countries without the capacity to do molecular testing at scale to research the use of serology tests to triage symptomatic patients in community settings, to test contacts of confirmed cases, and in situational analysis and surveillance. the who r&d blue print expert group identified eight priorities for research and development, of which the highest is to mobilise research on rapid point-of-care diagnostics for use at the community level. this research should inform control programmes of the required performance and utility of rapid serology tests, which, when applied specifically for appropriate public health measures to then be put in place, can make a huge difference. the covid-19 pandemic, now only a few months old, 1,2 has brought into sharp focus inequalities within and among countries. john nkengasong, director of the africa centres for disease control and prevention, reported that "the collapse of global cooperation and a failure of international solidarity have shoved africa out of the diagnostics market". 3 sadly, the same is true of many other low-income and middle-income countries (lmics) outside africa. why are diagnostics important? in any epidemic response, diagnostic testing plays a crucial role and this pandemic is no exception. because early clinical presentations of infected patients are non-specific, testing is needed to confirm the diagnosis of covid-19 in symptomatic patients, as soon as possible, so that these patients can be appropriately isolated and clinically managed. 1,4,5 diagnostic testing is also needed for individuals who have come into contact with someone with confirmed covid-19. some testing strategies examine only contacts who have symptoms or develop illness of any kind during the 14-day period after contact. other strategies examine all contacts when identified, regardless of whether they have any symptoms. studies have shown that a large number of infected individuals might have no symptoms at all, and there is concern that these individuals are still able to shed the virus and transmit infection through saliva droplets as they speak. [4] [5] [6] [7] [8] [9] tracking all contacts of confirmed cases and testing them for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is key to successful pandemic control. diagnostics are also needed to support rapid serosurveys that establish whether and to what extent sars-cov-2 has circulated in a community, and sur veillance systems, such as that for influenza-like illness, that monitor disease trends over time. diagnostics can also be used to identify atrisk populations and assess the effectiveness of control strategies. tedros adhanom ghebreyesus, director-general of who, urged countries to implement a comprehensive package of measures to find, isolate, test, and treat every case, and trace every contact. goodwill between countries has already been shown through the publishing of the sars-cov-2 genetic sequence and shared laboratory protocols to detect the virus. 10 however, as these molecular assays require sophisticated labora tory facilities, countries with insufficient infrastructure quickly accumulate a backlog of testing. the rapid spread of covid-19 around the world has led to a global shortage of reagents and supplies needed for testing. point-of-care molecular assays for sars-cov-2 detection are now available to enable community-based testing for covid-19 in lmics. unfortunately, the production of these test cartridges takes time and, again, global demand has outstripped supply, leaving lmics struggling for access. in march, 2020, who urged member states to "test, test, test". 11 widespread testing can help countries to map the true extent of the outbreak, including identifying hot spots and at-risk populations, and monitor the rate at which the epidemic is spreading. however, most lmics find that molecular testing, including point-of-care testing, is neither scalable nor affordable on a large scale. relying solely on centralised testing puts countries at risk of having nothing to use. what diagnostic alternatives are available to support decentralised testing that would allow countries to mount an adequate response to the pandemic? rapid antigen detection tests that are simple to do at point of care and can give results in less than 30 min would be viable alternatives to molecular testing for confirming covid-19 cases, enabling appropriate case management, and guiding public health measures, such as quarantine or self-isolation. however, although scaling up rapid antigen testing offers an effective means of triaging symptomatic individuals in community settings, early evaluations of rapid antigen detection tests show personal view suboptimal sensitivity for these tests to be recommended for clinical diagnosis or triage. 12 rapid antibody detection lateral flow tests are also simple to use, generally requiring a few drops of whole blood from a finger prick placed onto the test strip with no processing needed. these tests take 15-20 min to do with minimal training and can be done at the point of care as most do not require any equipment. rapid antibody testing is an attractive option for scaling up testing but only if these tests show satisfactory performance for a clearly specified use. the detection of sars-cov-2 infection and immune response has been described in relation to different diagnostic tests. 13 in this section, we summarise the evidence from studies to date. studies have shown that sars-cov-2 rna can be detected 2-3 days before onset of symptoms and can remain detectable up to 25-50 days after the onset of symptoms, particularly in patients who remain symptomatic for an extended period. 7,14,15 sars-cov-2 rna can be detected for longer in respiratory samples from patients with severe disease than in samples from patients with mild illness. 16 viral rna concentrations peak within the first 5 days after onset of symptoms and decrease slowly with rising antibody concentrations. 7, 17, 18 however, rna clearance is not always associated with rising antibody concentrations, particularly in patients who were critically ill. 6,18 an important question for the potential for spread of covid-19 is whether individuals who are rna-positive are shedding infectious virus. a small study in nine patients found that viral replication stopped 5-7 days after onset of symptoms but patients remained rna-positive for 1-2 weeks after this point. 6 hence, there remains some uncertainty as to whether a patient who is rna-positive is shedding live virus or not. maturation of the immune response typically takes 40 days with variations in the dynamics of the antibody response depending on disease severity and other factors still to be discovered. in most studies of laboratoryconfirmed covid-19 cases, igm antibodies start to be detectable around 5-10 days after onset of symptoms and rise rapidly. 14,18-21 igg antibody concentrations follow the igm response closely. seroconversion is typically within the first 3 weeks with the mean time for seroconversion being 9-11 days after onset of symptoms for total antibody, 10-12 days for igm, and 12-14 days for igg. 14, 18, 19, 22 antibodies against the receptor-binding domain of the spike protein and the nucleocapsid protein have been associated with neutralising activity. 14,23,24 neutralising anti bodies to these domains can be detected approximately 7 days after onset of symptoms and rise steeply over the next 2 weeks. 23, 24 several studies showed that patients can remain rna-positive despite high concentrations of igm and igg antibodies against the nucleocapsid protein and the receptor-binding domain of the spike protein. 18 whether the presence of neutralising antibodies translates into protective immunity in patients with covid-19 is unclear. some researchers speculate that antibodies can enhance infectivity as higher antibody concentrations have been observed in patients with severe disease than in those with mild disease. 18, 25 in one study (n=222), a greater proportion of patients with high igg concentrations had severe disease than did those with low igg concentrations (52% vs 32%, p=0·008). 26 the role of antibody response in the pathogenesis of covid-19 remains unclear pending further studies. who and the pan american health organization have stated that they do not currently recommend the use of immunodiagnostic tests except in research settings, 27, 28 because of scarce information on test performance and appropriate use when immunity to covid-19 is not well under stood. however, many countries are struggling to scale up testing to implement the key strategies of diagnosing all symptomatic patients and tracing all contacts. delays in confirming covid-19 cases allow continued transmission within communities and can result in failure to contain the pandemic despite other mea sures such as physical distancing and travel restrictions. countries are assessing all available testing options to address their range of needs. in settings where challenges with molecular testing exist or access to laboratories is scarce, rapid serology tests offer a needed additional option. a rapid serology test with good performance character istics is extremely important to avoid missing true cases of covid-19 and imposing unnecessary quaran tine for people with false-positive results due to cross-reactivity with seasonal coronaviruses. studies have shown more anti body cross-reactivity between the nucleo capsid proteins of sars-cov-2 and common corona viruses than between their spike proteins. 20, 22 tests that use the spike protein or fragments of the spike protein as targets might have the least amount of cross-reactivity with common coronaviruses, on the basis of sequence analysis. 22 clear articulation of the benefits and limitations of serology tests will hopefully incentivise manufacturers to improve performance. 29 when is serology testing recommended? where there is little or no access to molecular testing, rapid serology tests provide a means to quickly triage personal view suspected cases of covid-19, provided the test is highly specific for the disease. a positive result for igm in symptomatic patients fulfilling the covid-19 case definition is strongly suggestive of sars-cov-2 infection. this approach is probably most effective in individuals 5-10 days after symptom onset. in peru, public health facilities for molecular testing are sparse and only 500 beds in intensive care units exist for a population of 32 million. the ministry of health has set up a hotline and website for individuals who have symptoms to be interviewed by a health professional for possible follow-up, prioritising the visits according to age, risk factors, and severity of symptoms. a testing team visits the individual at home to do the rapid anti body test. individuals who are igm and igg positive and have mild symptoms are quarantined, whereas people who need critical care are referred to hospital. all contacts are also tested with the rapid serology test. anyone who tests negative in the antibody test has a swab collected for molecular testing. as of may 2, 2020, 355 604 people had been triaged in peru with 42 534 testing positive, 26 362 of whom were found to be positive by use of a rapid test. 30 this approach has allowed a large number of sympto matic individuals and contacts to be rapidly tested in the community, relieving the backlog, reducing waiting time for molecular testing, and preventing the health-care system from being overwhelmed. experience in china has also shown that, in symptomatic patients, the use of igm tests or total antibody tests can increase the sensitivity of covid-19 case detection. 18 further research should explore the performance and utility of rapid antigen-igm and antigen-igg combo tests and the timing of testing. in individuals who test negative for igg, research should also explore, if resources allow, the value of doing a follow-up antibody test 10-14 days later to document a definitive diagnosis through seroconversion. studies have shown that a large number of infected individuals could have only mild symptoms or no symptoms at all, but they can still transmit infection, with as much as 44% of infections being transmitted by presymptomatic individuals. 6,7,9 experience from singapore shows that tracking down all contacts of people with confirmed covid-19, testing them for evidence of infection, regardless of symptoms, and putting those contacts who test positive into isolation is an urgent priority for interrupting the chain of transmission and containing the epidemic. [31] [32] [33] this approach is particularly important in the early stages when there are only sporadic or clusters of cases, or for countries coming down from the peak to continue to reduce the extent of infection in the community. only individuals who test negative should have a throat swab collected for molecular testing, which will reduce the strain on laboratories doing these tests. in countries that have set up syndromic surveillance, such as surveillance for influenza-like illness or severe acute respiratory infections, and where blood or throat swabs are routinely collected at these sentinel sites, collected samples can be tested for covid-19 with molecular, antigen, or serology tests, either alone or in combination. if any of these samples are positive, it means covid-19 has been circulating in the community. where serial samples are available, it might be possible to date when covid-19 established itself in a community or country. in general, antibody tests can be used to establish the true extent of an outbreak, map its geographical distribution, and identify hotspots and populations that are particularly at risk. this information can in turn be used to inform public health measures and control strategies. in this case, researchers need to stick to the same serology test and test sentinel populations repeatedly, avoiding the variation in sensitivity between different rapid tests. the use of serology tests for population surveys is not recommended in low prevalence settings as this approach will probably result in more false-positive than truepositive results, even if a test with high specificity is used. for example, if the prevalence of infection is 1% in the general population, a test with 98% specificity will identify two false-positive results for every true positive result. these results could lead to a false sense of security regarding the extent of immunity in the population and premature easing of public health measures on the basis of misleading disease estimates. patients at an early stage in the disease course, or asympto matic or paucisymptomatic patients, might have low antibody concentrations that could give falsenegative results. patients' disease stage and severity are important points to consider, along with the population being tested. the estimated level of risk can be considered before using a serology test, because of the changing falsepositive rate or low positive predictive value across different populations. among the groups with the highest risk of the disease are symptomatic patients with clinical presentation of covid-19, patients with other respiratory symptoms, contacts of confirmed cases, and health-care workers in settings with little personal protective equipment. we suggest countries consider risk levels before using serology tests and creating public health guidance. scaling up testing, particularly at the community level, allows for better estimates of risks, which in turn allows more effective public health measures to be put into place than would be otherwise. in a pandemic, countries must strive to maintain a robust health-care workforce. key workers who develop personal view symptoms should be prioritised for molecular testing and receive care if infected. on recovery, should a serology test be used to decide when they can safely return to work? this strategy is based on the assumption that antibodies confer protective immunity. although antibodies against the receptor-binding domain of the spike protein and the nucleocapsid protein have been correlated with neutralising activity, 14, 23, 24 the development and duration of immunity has not yet been established. 34, 35 although it is tempting to speculate that serology tests based on the detection of neutralising antibodies can be used as markers of protective immunity, and people who test positive can get a so-called immunity passport to return to work, studies have shown that a significant proportion of patients remain rna-positive despite high concen trations of antibodies against the receptor-binding domain of the spike protein and the nucleocapsid protein. 6, 15, 18, 20, 21 wang and colleagues 36 found that elevated serum igm concentrations are correlated with poor outcomes in patients with covid-19 pneumonia, and tan and colleagues 21 found that high concentrations of igg antibodies were correlated with severe disease outcomes. hence a substantial igm or igg response is not necessarily a surrogate marker of protective immunity. to date, insufficient evidence exists to recommend the use of serology testing for health-care workers to return to work. a negative molecular test remains the safest option to establish whether health-care workers can work again safely. a policy brief by the world bank suggested that serology testing could potentially have a high net benefit if it can allow dilution of restrictions for essential workers to return to work and revive essential segments of the economy. 37 the type of tests that can be used for immunity passports remains unclear. a better understanding of the interaction between infection and immune response dynamics is needed before these passports can be considered. hospital beds are often in short supply. the recommended criteria for hospital discharge are two negative molecular tests over several days. however, molecular testing is often scarce or unavailable. can serology tests be used for discharging recovered patients when molecular testing is not available? as patients can remain positive for viral rna despite rising concentrations of antibodies against the nucleocapsid protein and receptor-binding domain of the spike protein, which are correlated with neutralising activities, antibody tests cannot be used in the place of molecular tests to confirm that the patient is virus-free or at least no longer shedding live virus. the events over the past few months have taught us that this pandemic is caused by an extraordinary pathogen that requires extraordinary measures to combat its spread and end the pandemic. the latest finding that as much as 44% of covid-19 transmission happens before index cases become symptomatic 7 means that a great deal still needs to be learnt about this novel pathogen and its spread through a population. the paucity of knowledge on the dynamics of the immune response to infection has led to much hesitation on recommending the use of rapid immuno diagnostic tests, particularly serology tests. on the basis of our current knowledge and understanding of viral infectivity and host response, we urge countries with restricted capacity for molecular testing to embark on research into the use of serology tests in triaging symptomatic patients in community settings, testing contacts of confirmed cases, and in situational analysis and surveillance. rapid and scalable tests are needed to deal with this pandemic. rapid serology tests, applied in the right situation for appropriate public health measures to be put into place, can make a huge difference. on feb 10, 2020, leading health experts from around the world identified eight research and development priorities at the who r&d blue print meeting in geneva, switzerland, of which the top priority was to "mobilize research on rapid point of care diagnostics for use at the community level". 38 in line with this decision, research on the use of rapid serology tests to inform control programmes of their required performance and utility is an urgent priority in the covid-19 pandemic response. rwp wrote the first draft of the manuscript. all authors contributed to the manuscript conception and supported manuscript revisions. we declare no competing interests. who. molecular assays to diagnose covid-19: summary table of available protocols director-general's opening remarks at the media briefing on covid-19 find evaluation update: sars-cov-2 immunoassays 2020 interpreting diagnostic tests for sars-cov-2 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china evidence summary for covid-19 viral load over course of infection antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 serology characteristics of sars-cov-2 infection since exposure and post symptoms onset profiling early humoral response to diagnose novel coronavirus disease (covid-19) viral kinetics and antibody responses in patients with covid-19 antibody responses to sars-cov-2 in patients with covid-19 neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov-2 profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid-19 advice on the use of point-of-care immunodiagnostic tests for covid-19 advice on the use of point-ofcare immunodiagnostic tests for covid-19 developing antibody tests for sars-cov-2 covid-19 in peru investigation of three clusters of covid-19 in singapore: implications for surveillance and response measures connecting clusters of covid-19: an epidemiological and serological investigation interrupting transmission of covid-19: lessons from containment efforts in singapore covid-19 immunity passports and vaccination certificates: scientific, equitable, and legal challenges what policy makers need to know about covid-19 protective immunity elevated serum igm levels indicate poor outcome in patients with coronavirus disease 2019 pneumonia: a retrospective case-control study how-two-tests-can-help-contain-covid-19-and-revive-the-economy.pdf?sequence=1&isallowed=y world experts and funders set priorities for covid-19 research we thank sergio carmona and jilian sacks of the foundation for innovative new diagnostics for helpful discussions. key: cord-325455-e464idc0 authors: atchison, christina; pristerà, philippa; cooper, emily; papageorgiou, vasiliki; redd, rozlyn; piggin, maria; flower, barnaby; fontana, gianluca; satkunarajah, sutha; ashrafian, hutan; lawrence-jones, anna; naar, lenny; chigwende, jennifer; gibbard, steve; riley, steven; darzi, ara; elliott, paul; ashby, deborah; barclay, wendy; cooke, graham s; ward, helen title: usability and acceptability of home-based self-testing for sars-cov-2 antibodies for population surveillance date: 2020-08-12 journal: clin infect dis doi: 10.1093/cid/ciaa1178 sha: doc_id: 325455 cord_uid: e464idc0 background: this study assesses acceptability and usability of home-based self-testing for sars-cov-2 antibodies using lateral flow immunoassays (lfia). methods: we carried out public involvement and pilot testing in 315 volunteers to improve usability. feedback was obtained through online discussions, questionnaires, observations and interviews of people who tried the test at home. this informed the design of a nationally representative survey of adults in england using two lfias (lfia1 and lfia2) which were sent to 10,600 and 3,800 participants, respectively, who provided further feedback. results: public involvement and pilot testing showed high levels of acceptability, but limitations with the usability of kits. most people reported completing the test; however, they identified difficulties with practical aspects of the kit, particularly the lancet and pipette, a need for clearer instructions and more guidance on interpretation of results. in the national study, 99.3% (8,693/8,754) of lfia1 and 98.4% (2,911/2,957) of lfia2 respondents attempted the test and 97.5% and 97.8% of respondents completed it, respectively. most found the instructions easy to understand, but some reported difficulties using the pipette (lfia1: 17.7%) and applying the blood drop to the cassette (lfia2: 31.3%). most respondents obtained a valid result (lfia1: 91.5%; lfia2: 94.4%). overall there was substantial concordance between participant and clinician interpreted results (kappa: lfia1 0.72; lfia2 0.89). conclusion: impactful public involvement is feasible in a rapid response setting. home self-testing with lfias can be used with a high degree of acceptability and usability by adults, making them a good option for use in seroprevalence surveys. lateral flow immunoassays (lfia) offer a rapid point-of-care (poc) approach to novel coronavirus (sars-cov-2) antibody testing. while lfias may not currently be accurate enough for individual-level clinical decisions (1, 2) , they are valuable as a public health tool. on a population level, by conducting seroprevalence surveys through widespread random sampling of the general public, and by adjusting for the sensitivity and specificity characteristics of the lfia used, it is possible to estimate the levels of past infection with sars-cov-2 in the community (3) . however, testing hundreds of thousands of people would be impractical if it required a blood sample to be drawn followed by processing in a laboratory. one solution is to use self-sampling and selftesting in the home with participants reporting results to the researchers. however, there is limited understanding of public acceptability and usability of these lfias in the home setting, as most are currently designed as poc tests performed by healthcare professionals. self-sampling and self-testing are widely used in healthcare for monitoring, for example in diabetes management (4) , and for diagnostics, for example for hiv (5, 6) . there are many advantages in terms of uptake, cost, patient activation and scale (4, 6) , but also potential disadvantages in relation to validity, usability and practicality which should be explored (6, 7) . usability research on hiv selftesting has generally found good acceptability, the devices easy to use and high validity in interpretation of self-reported test results (7) (8) (9) . however, these hiv test kits were designed for selfsampling and self-testing and went through several iterations before designs were appropriate for home use and therefore the same levels of acceptability and usability for home-based self-testing for sars-cov-2 antibody using lfias cannot be assumed. as part of the real-time assessment of community transmission (react) programme (10), we evaluated the acceptability and usability of lfias for use in large seroprevalence surveys of sars-cov-2 antibody in the community. a c c e p t e d m a n u s c r i p t 5 we evaluated two lfias with different usability characteristics from five lfias being validated in parallel in our laboratory-based study (11). both lfias required a blood sample from a finger-prick and produced a self-read test result after 10 or 15 minutes. lfia1 (guangzhou wondfo biotech co ltd) was a cassette-based system containing a "control" indicator line and a "test" indicator line (for detection of combined igm and igg antibodies). lfia2 (fortress orient gene biotech co ltd) was a cassette-based system containing a "control" indicator line and separate indicator lines for igm and igg ( figure 1 ). in early may 2020 we carried out rapid, iterative public involvement and a pilot usability study including an online forum with four discussion groups (n=37), a study of lfia1 test use with volunteers (n=44) and a broader public sample (n=234), and a nested observation and interview study (n=25). further details on the methods, including how we recruited participants from our existing involvement networks, are available online (supplement, s1). the test kits dispatched in the pilot study included one test cassette, one button-activated 28g lancet and a 2ml plastic pipette, alongside an instruction booklet also containing a weblink to an instructional video. based on findings from the pilot study, for the larger population-based usability study, the lancet and pipette were replaced with two pressure-activated 23g (larger) lancets and a smaller 1ml plastic pipette, respectively. the design and language in the instructional booklet and video were changed and an alcohol wipe was also included in the kit. a c c e p t e d m a n u s c r i p t 6 in late may 2020 we carried out a larger population-based usability study of a representative sample of the adult population (aged 18 years and over) in england. we used addresses from the postal address file to draw a random sample of 30,000 households in england to which study invitation letters were sent. we allowed up to four adults aged 18 and over in the household to register for the study. self-testing lfia kits were then posted to each registered individual. on completion of the test, participants recorded their interpretation of the result as part of an online survey, with the option of uploading a photograph of the test result. reminder letters were sent to participants who had not completed the online survey or uploaded a photograph within 10 days of test kits being dispatched. metrics to evaluate usability and acceptability were based on the hiv self-testing literature (5, 6, 8) and were measured as the percentage of participants responding to specific closed questions in the online survey. the questionnaire used is available as an online supplement (supplement, s2). the main outcome was usability of the lfia kits. this was defined as a participant's ability to complete the antibody test, and how easy or difficult it was to understand the instructions and complete each step in the process. acceptability was measured in terms of people consenting to and using the provided self-test, and the proportion who reported they would be willing to repeat a self-administered finger-prick antibody test in the future. analyses were conducted in stata (version 15.0, statacorp, texas, usa). data obtained from the questionnaires on acceptability and usability were summarised by counts and descriptive statistics, and comparisons were made between lfia1 and lfia2 using pearson's chisquared test. multivariate regression was used to identify sociodemographic factors independently associated with the proportion of participants conducting the test that achieved a valid result. variables that appeared to be associated (p<0.05) in the unadjusted analyses were considered in the adjusted analyses. adjusted odds ratios (aor) and 95% confidence intervals (ci) were estimated. associations with a p-value <0.05 in the adjusted analyses were considered statistically significant. agreement between participant-interpreted and clinician-interpreted result for test outcomes (negative, igg positive, invalid, unable to read) was assessed using the fleiss kappa statistic. a c c e p t e d m a n u s c r i p t 8 (11). therefore, we used the same operational definition of positivity for lfia2. participants were informed in the instructions to consider igm results as negative. the study obtained research ethics approval from the south central-berkshire b research ethics committee (iras id: 283787). overall, 315 members of the public contributed feedback during the involvement and pilot. this led to changes in the design and language used in the instructional video and booklet, the type and number of lancets and the size of pipette in the lfia kits (further details available online, supplement s1). for the national study, 25,000 household invitation letters were sent, and 17,411 individuals registered for the study from 8,508 households. due to the maximum number of kits we had available for the study, 14,400 participants were selected at random from those who registered. thus, 10,600 lfia1 kits were distributed, with 8,754 individual user surveys completed (82.6% response rate), and 3,800 lfia2 kits were distributed, with 2,957 individual user surveys completed (77.8% response rate). most commonly, two adults participated per household (table 1 ). baseline characteristics of study participants are shown in table 1 . the median age of participants across lfia1 and lfia2 was 51.0 years (range 18 to 95). there were some differences between lfia1 and lfia2 participants by ethnicity, region and household size. acceptability of self-testing was high ( a c c e p t e d m a n u s c r i p t 9 as in the pilot, most respondents were willing to perform another finger-prick antibody test in the future (lfia1: 98.3%; lfia2: 97.0%). only a minority preferred to do the antibody test in a clinical care or community setting than at home. as with the pilot study, respondents with children showed a high willingness to perform the antibody test on them, and this proportion increased with the age of the children (table 2) . in the pilot study most people (86.5%, 225/260) who attempted the test managed to complete it. however, significant usability issues were identified, including challenges with the lancet to obtain a blood drop and the pipette to transfer the blood to the sample well. the problems with the lancet led to some participants using alternative objects to draw blood, including pins and sewing needles, while others opened the lancet casing to access the blade. some people reported minor problems putting buffer into the buffer well. this led to the inclusion of two lancets and changes to the instructions for the national study. in the national study, almost all participants who attempted the antibody test reported completing it (97.5% for lfia1 and 97.8% for lfia2) ( table 2) . reasons for not completing the test are shown in the table. of lfia1 participants who reported damaging the test, the majority reported either accidentally removing the entire lid off the buffer bottle and spilling the solution all over the test cassette or putting the blood and buffer in the wrong well. for lfia2, few participants damaged the test and they all reported putting the blood and buffer in the wrong well. about one in four participants asked someone to help them to administer the test. most found the instructions easy to understand (figures 3), but as in the pilot, participants reported some difficulties in performing the test. for lfia1, difficulties with using the pipette were reported by 17.7% (1,512/8,521) of participants. in addition, 10.6% (908/8,556) had difficulties applying the blood to a c c e p t e d m a n u s c r i p t 10 the sample well ( figure 3 ). therefore, for lfia2 the instructions were changed to omit the use of the pipette and instead directly transfer blood from the finger-prick site to the sample well. however, participants still found creating a blood drop from the finger-prick site (23.2%; 664/2,862) and then applying the blood to the well (31.3%; 894/2,858) difficult. lfia2 was deployed after lfia1 because there was a delay in arrival of lfia2 from the supplier. this differential timing in dispatch of the kits had the unexpected benefit of allowing us to make iterative changes to the instructions. overall, 7.4% of lfia1 and 4.8% of lfia2 participants reported an invalid result (table 3 ). there was some variation in the proportion of participant-reported invalid results using lfia1 by age and gender. the higher the number of participants registered for the study in the same household, the lower the odds of the participant reporting an invalid result. no sociodemographic factors were associated with a participant-reported invalid result using lfia2 (table 4 ). after adjusting for sociodemographic differences between lfia1 and lfia2 participants, there was no difference between lfia1 and lfia2 in terms of being able to read the result (1.1% vs. 0.81%; aor 0.76 (95% ci: 0.48-1.2); p 0.24). but a lower percentage of invalid test results were reported by lfia2 participants (7.9% vs. 4.8%; aor 0.64 (95% ci: 0.53-0.77); p <0.001). table 5 shows concordance between participant and clinician interpreted results in the national study. for lfia1, there was substantial agreement overall (kappa 0.72 (95% ci: 0.71-0.73); p<0.001), however there were important differences: while there was 100.0% agreement for results reported as negative, and 98.5% agreement for invalid results, a clinician confirmed only 62.8% of participantreported positives. visible reasons (from the photograph) were insufficient blood volume to cover the bottom of the sample well, or insufficient movement of the blood and buffer solution across the result window. in addition, the clinician was able to interpret the results of all but four out of 66 a c c e p t e d m a n u s c r i p t 11 (6.1%) tests from the photographs of results participants reported as "unable to read". the four results unreadable by the clinician were because blood had leaked out of the sample well and obscured the result window. of participant-reported unable to read results, 78.8% were clinicianinterpreted as invalid (for all these tests, the control and "test" lines were both absent). table 5 ). the clinician could not interpret the results from 10 photographs reported as readable results by participants, reasons included blurred photographs and shadowing obscuring the indicator lines. overall, we found that self-testing with the two lfia kit designs used in this study was highly acceptable among adults living in england. high acceptability of in home self-testing is in keeping with self-sampling and self-testing studies in diabetes management (4), and hiv diagnostics (5, 6) . the majority of participants who attempted the test successfully completed it despite some continued difficulties with using the pipette (lfia1), creating a drop of blood from the finger-prick site (lfia2) and applying the blood to the sample well (lfia1 and lfia2). based on these findings, we proportion of invalid tests for lfia2 could reflect the better designed buffer bottle (which was a significant issue for lfia1), better performance characteristics of lfia2 over lfia1 (e.g. easier movement of the blood and buffer solution across the result window), or improved ability to get sufficient blood in the sample well using the direct blood transfer technique over using a pipette. participants' ability to obtain a valid test result using lfia1 varied marginally by age and gender and increased with the number of participants registered for the study in the same household. the latter observation is not surprising as observing another household member performing the test is likely to improve the performance of others in the same household. no sociodemographic differences in obtaining a valid result were found for lfia2. of note, about one in four participants reported that they had help administering the test, irrespective of lfia used. this could put individuals living alone at a disadvantage in terms of usability. however, comparing those that had help to those that did not, we found no difference in ability to complete the test (97.7% vs. 98.2%; p 0.08) or reported invalid results (6.1% vs. 7.0%; p 0.06). overall, there was good agreement between self-reported results and those reported by a clinician. therefore, our findings broadly support self-reporting of home-based test results using lfias. but the public and individual health impact of misinterpreting a test result that is negative but read as positive is a concern as an individual could falsely conclude that they have antibodies for sars-cov-2 and may change their behaviour as a result. to mitigate against this, and given the scientific our study is original because focusing on the acceptability and usability of lfias for self-testing for sars-cov-2 antibody in a home-based setting has not been done at such scale in the general population. it provides an attractive solution for conducting large seroprevalence surveys. the study has, however, some limitations. study participants may not be representative of the general adult population of england. however, we had data on the england population profile (2011 census data (13)), as well as the study registration profile and survey completion profile of the study participants which gave us an indication of response bias. our sample was broadly similar to the england population profile. in addition, the usability study was conducted in parallel with our laboratorybased study of performance characteristics of lfias. as such, we did not know the accuracy of the lfias chosen for the usability study at the time, or whether either would perform well enough in the laboratory to be considered for the large national seroprevalence study planned as part of the react programme (10). however, given that the majority of commercially available lfias have a similar cassette-based design and test result read out to lfia1 or lfia2 we were confident that our results would be generalisable and applicable to whichever lfia was finally selected. findings from our laboratory-based study, including the performance characteristics of lfia1 and lfia2 are forthcoming (11). overall, our study has demonstrated that home-based self-testing lfias for use in large communitybased seroprevalence surveys of sars-cov-2 antibody are both acceptable and feasible. although this study identified a few usability issues, these have now been addressed. lfia2, fortress orient gene, has been selected for a large national seroprevalence study as part of the react programme. this decision was based on criteria including the usability and acceptability determined in this study, a c c e p t e d m a n u s c r i p t 21 years where asked whether they would carry out the test on children of that age living in their households. denominator is th e number of participants who reported having children of that age living in their household. m a n u s c r i p t 23 antibody testing for covid-19: a report from the national covid scientific advisory panel evaluation of nine commercial sars-cov-2 immunoassays prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study self-monitoring of blood glucose in type 2 diabetes: recent studies acceptability, feasibility, and individual preferences of blood-based hiv self-testing in a population-based sample of adolescents in kisangani, democratic republic of the congo reliability of hiv rapid diagnostic tests for self-testing compared with testing by health-care workers: a systematic review and meta-analysis interferences and limitations in blood glucose self-testing: an overview of the current knowledge usability assessment of seven hiv self-test devices conducted with lay-users in we thank key collaborators on this work --ipsos mori: stephen finlay, john kennedy, duncan key: cord-324373-mgdtb98z authors: antonelli, andrea; guilizzoni, dario; angelucci, alessandra; melloni, giulio; mazza, federico; stanzi, alessia; venturino, massimiliano; kuller, david; aliverti, andrea title: comparison between the airgo™ device and a metabolic cart during rest and exercise † date: 2020-07-15 journal: sensors (basel) doi: 10.3390/s20143943 sha: doc_id: 324373 cord_uid: mgdtb98z the aim of this study is to compare the accuracy of airgo™, a non-invasive wearable device that records breath, with respect to a gold standard. in 21 healthy subjects (10 males, 11 females), four parameters were recorded for four min at rest and in different positions simultaneously by airgo™ and sensormedics 2900 metabolic cart. then, a cardio-pulmonary exercise test was performed using the erg 800s cycle ergometer in order to test airgo™’s accuracy during physical effort. the results reveal that the relative error median percentage of respiratory rate was of 0% for all positions at rest and for different exercise intensities, with interquartile ranges between 3.5 (standing position) and 22.4 (low-intensity exercise) breaths per minute. during exercise, normalized amplitude and ventilation relative error medians highlighted the presence of an error proportional to the volume to be estimated. for increasing intensity levels of exercise, airgo™’s estimate tended to underestimate the values of the gold standard instrument. in conclusion, the airgo™ device provides good accuracy and precision in the estimate of respiratory rate (especially at rest), an acceptable estimate of tidal volume and minute ventilation at rest and an underestimation for increasing volumes. respiratory rate measurement has been shown to be able to predict adverse clinical events, such as admission to the intensive care unit (icu). under specific circumstances, it is more effective than pulse or blood measurements at discriminating between stable patients and patients at risk [1, 2] . however, the number of trials in which the respiratory rate has been studied remains limited and mostly confined to the use of spot measurements. the work by yañez et al. [3] is based on a direct measurement of the flow to assess the respiratory frequency in copd patients. in this study, it was observed than the mean respiratory rate was raised 15 days prior to hospitalization (two days before, 15% more with respect to the baseline; the day before, the goal of the presented work is to compare a metabolic cart, considered a gold standard, with airgo™ (myair inc, boston, ma, usa; myairgo italy srl, milan, italy), a resistance-based wearable device able to derive breathing parameters from body surface motion detection acquired at the level of the lower ribcage. a study protocol has been conducted on a population of twenty-one healthy subjects comparing the output of the airgo™ system with the sensormedics 2900 metabolic cart (sensormedics inc., yorba linda, ca, usa) [22] . the research has been approved by the ethical committee of the azienda ospedaliera s. croce e carle in cuneo (opinion number 3-17, 7 june 2017) and was part of the clinical trial nct03368612 ("comparative study between the airgo™ system and standard tests in the assessment of the respiratory function"). in the first part of the protocol, the healthy subjects were asked to perform quiet breath maneuvers in five different standardized postures while wearing the airgo™ device: standing position, sitting position with back against chair, supine position, right lateral decubitus and left lateral decubitus. in the second part of the test, after the completion of the test at rest, a symptom-limited incremental exercise was performed on the electronically braked cycle ergometer ergoline 800s (ergoline gmbh, bitz, germany). the obtained data have been processed to allow the extraction of relevant respiratory parameters. the comparison between the two systems was done considering the following four parameters: normalized tidal volume, respiratory rate, normalized minute ventilation and duty cycle. the airgo™ band measures the thoracic circumference changes with a stretchable knitted matrix of nylon and spandex with a knitted-in silver coated yarn. the system employs an electrically conductive, the system calculates resistance changes continuously. each variation in resistance values, caused by each expansion and volume reduction of the chest wall during breathing activity, reflects a measurable variation in current in the silver-coated wire. the stretchable band is coupled to a microprocessor, embedded in a sintered nylon shell. the microprocessor includes an sd memory card and monitors the respiratory activity by collecting raw data at a frequency of 10 hz. the microprocessor includes: an analog-to-digital converter (adc) that converts the analog resistance level of the girth band into a 10-bit number ranging from 0 to 1023 and corresponding to the amplitude of the torso expressed in arbitrary units; a battery for operating the microprocessor and for charging the band; paths to transfer data to the sd card for on-board data storage; a bluetooth module to wirelessly communicate with a computational device (laptop computer or smartphone). the device's on-board microprocessor is connected to a nine-axis inertial measurement unit (imu). the motion detection circuit provides movement and postural orientation datapoints with the final aim to associate breathing information to the patient's posture or activity. an activity recognition algorithm based on raw accelerometer data has been developed and is described by qi and aliverti [23] . the girth band is operably coupled at the first end to the microprocessor and then encircles the torso at the level of the lower rib cage. it is then coupled at the second end to the microprocessor from the other side. when in use, the band is pre-tensioned to ensure a fit around the torso that does not dislocate when the band is worn and can record data without interruption. the part in direct contact with the skin is made of a silicon rubber with a thickness of about 0.35 mm called gecko ® (gottlieb binder gmbh & co. kg, holzgerlingen, germany), or gecko tape, because it emulates the adhesion of gecko feet and can be attached to both wet and dry vertical surfaces. this material can be detached easily and quickly from a surface, while it shows exceptional adhesion properties when attached to it. figure 2 shows a typical stretch metallic knit static resistance qualitative curve. initially, the resistance rapidly changes with elongation (l0 to l1). following this initial phase, there is an approximately linear increase in resistance with respect to length (l1 to l2). resistance continues to increase with length but with less marginal increment, as shown from l2 to lt, which is the threshold above which resistance decreases with increasing length until the elastic band reaches its maximum length (lm). the operative range of the band is therefore within the range l1-l2 in order to obtain accurate measurements without significant calibration difficulties. the system calculates resistance changes continuously. each variation in resistance values, caused by each expansion and volume reduction of the chest wall during breathing activity, reflects a measurable variation in current in the silver-coated wire. the stretchable band is coupled to a microprocessor, embedded in a sintered nylon shell. the microprocessor includes an sd memory card and monitors the respiratory activity by collecting raw data at a frequency of 10 hz. the microprocessor includes: an analog-to-digital converter (adc) that converts the analog resistance level of the girth band into a 10-bit number ranging from 0 to 1023 and corresponding to the amplitude of the torso expressed in arbitrary units; a battery for operating the microprocessor and for charging the band; paths to transfer data to the sd card for on-board data storage; a bluetooth module to wirelessly communicate with a computational device (laptop computer or smartphone). the device's on-board microprocessor is connected to a nine-axis inertial measurement unit (imu). the motion detection circuit provides movement and postural orientation datapoints with the final aim to associate breathing information to the patient's posture or activity. an activity recognition algorithm based on raw accelerometer data has been developed and is described by qi and aliverti [23] . the girth band is operably coupled at the first end to the microprocessor and then encircles the torso at the level of the lower rib cage. it is then coupled at the second end to the microprocessor from the other side. when in use, the band is pre-tensioned to ensure a fit around the torso that does not dislocate when the band is worn and can record data without interruption. the part in direct contact with the skin is made of a silicon rubber with a thickness of about 0.35 mm called gecko ® (gottlieb binder gmbh & co. kg, holzgerlingen, germany), or gecko tape, because it emulates the adhesion of gecko feet and can be attached to both wet and dry vertical surfaces. this material can be detached easily and quickly from a surface, while it shows exceptional adhesion properties when attached to it. figure 2 shows a typical stretch metallic knit static resistance qualitative curve. initially, the resistance rapidly changes with elongation (l0 to l1). following this initial phase, there is an approximately linear increase in resistance with respect to length (l1 to l2). resistance continues to increase with length but with less marginal increment, as shown from l2 to lt, which is the threshold above which resistance decreases with increasing length until the elastic band reaches its maximum length (lm). the operative range of the band is therefore within the range l1-l2 in order to obtain accurate measurements without significant calibration difficulties. figure 2 . stretch metallic knit "static" resistance qualitative curve. the graph shows the non-linear resistance/stretch curve of the resistive fabric and the different ranges of resistance (non-linear, approximately linear, flat and reverse, with lt the length threshold above which resistance decreases and lm indicating the maximum length). the section in evidence indicates the operative range. this study is a monocentric, prospective, observational, single arm clinical trial involving 21 healthy volunteers. it was conducted at the azienda sanitaria ospedaliera (aso) s. croce e carle, allergologia e fisiopatologia respiratoria, cuneo (cn), piemonte. the 21 healthy subjects comprised 11 females and 10 males with a mean age of 36 (age range from 24 to 51), mean weight of 67 kg (standard deviation ±16 kg) and mean height of 170 cm (standard deviation ± 8 cm). in order to be enrolled in the study, the following inclusion criteria have been used: this study is a monocentric, prospective, observational, single arm clinical trial involving 21 healthy volunteers. it was conducted at the azienda sanitaria ospedaliera (aso) s. croce e carle, allergologia e fisiopatologia respiratoria, cuneo (cn), piemonte. the 21 healthy subjects comprised 11 females and 10 males with a mean age of 36 (age range from 24 to 51), mean weight of 67 kg (standard deviation ±16 kg) and mean height of 170 cm (standard deviation ± 8 cm). in order to be enrolled in the study, the following inclusion criteria have been used: • the acquisition protocol was divided into three main phases: (1) preparation: measurement of the subject's thoracic oblique circumference length (c) at the end of a forced expiration maneuver. knowing this measurement, the initial length of the elastic band (l0) was decided to be 7% shorter than c in order to obtain a pre-tensioned girth band to ensure an effective fit around the torso. after being cut, the band was coupled with the airgo™ electronic device and positioned against the subject's skin; (2) test at rest: recording of respiratory parameters while letting the subject breathe quietly in five different standardized positions (standing, seated, supine, right lateral decubitus, left lateral decubitus) for 4 min. the supine position required an elevation of the subject's head not greater than 10 • with a pillow under the subject's head. tidal volume, respiratory rate, minute ventilation, inspiratory time, expiratory time and duty cycle (explained in table 1 ) were simultaneously recorded by the airgo™ system and the sensormedics 2900 metabolic cart. in order to facilitate the off-line synchronization between the two systems, subjects were asked to perform a big, deep breath at the beginning of the test for each position; (3) test under physical exercise: execution of a cardiopulmonary exercise test on the ergoline cycle ergometer 800s, followed by a recovery period. a symptom-limited incremental exercise test was performed and designed to achieve a maximum load in 10 ± 2 min in each subject wearing the airgo™ device. the physical exercise test followed a linear incremental protocol (with a slope between 15 and 20 watts per minute) was identical for men and women and chosen based on the level of training of each subject so that each test lasted approximately between 6 and 12 min, as recommended in the guidelines. subjects were asked to cycle at a velocity of 60 revolutions/minute for 2 min with no load, then to pedal at incremental workload maintaining the same speed as before until the maximal effort was reached. once the subject reached his/her maximal effort and he/she was not able to ride anymore, the exercise test was interrupted, and the recovery phase started. this phase consisted of cycling for 2 min without any resistance followed by 2 min at rest. the same respiratory parameters as test at rest have been acquired with the big-breath synchronization maneuver. airgo™'s processing unit acquires a respiratory signal based on the change in girth band resistance over time. many other factors not related to the breath cycle may influence the stretched length of the band, causing girth measurement inflections at a much higher frequency than that of the breath. therefore, raw data have been processed to filter out motion and heartbeat artefacts. the latter are characterized by a smaller amplitude and a higher frequency with respect to the respiratory signal. the system samples data at a frequency of 10 hz, averages the acquired data over 9-34 reading, blurs the averaged data from 0.3 to 1 s to filter out artefacts, determines the beginning and the end of a breath and records an adverse event if a predetermined period of time has elapsed without detecting a new breath, as is described in the related patent (number us201462007142p). filtering has an effect on knit motion artefacts, too. the total measured breath cycle resembled an upside-down "w" (figure 3 ): this non-correspondence is not only due to the heartbeat but also to the motion and acceleration artefacts of the signal coming from the knitted metallic band. a portion of the resistance is static and related to the static length of the stretched knit (the overall bell shape), but part of the resistance is also due to the spontaneous motion of the fabric. this accounts for the slight hump at the beginning of the total measured breath cycle and the double hump in the middle. sensors 2020, 20, x for peer review 6 of 19 latter are characterized by a smaller amplitude and a higher frequency with respect to the respiratory signal. the system samples data at a frequency of 10 hz, averages the acquired data over 9-34 reading, blurs the averaged data from 0.3 to 1 s to filter out artefacts, determines the beginning and the end of a breath and records an adverse event if a predetermined period of time has elapsed without detecting a new breath, as is described in the related patent (number us201462007142p). filtering has an effect on knit motion artefacts, too. the total measured breath cycle resembled an upside-down "w" (figure 3 ): this non-correspondence is not only due to the heartbeat but also to the motion and acceleration artefacts of the signal coming from the knitted metallic band. a portion of the resistance is static and related to the static length of the stretched knit (the overall bell shape), but part of the resistance is also due to the spontaneous motion of the fabric. this accounts for the slight hump at the beginning of the total measured breath cycle and the double hump in the middle. once raw data are filtered, they are processed in order to obtain the proper breath signal. the main steps of this process, shown in figure 4 , can be summarized as follows: identification of maximum and minimum of each breathing cycle;  representation of each breath by means of a vector that connects the maximum and the minimum;  automatic reconstruction of segmented breath, e.g., due to obstructions or movement;  automatic removal of fake breaths;  computation of tidal volume, respiratory rate and minute ventilation. breath data can be then transmitted to a computer via bluetooth low energy, stored, visualized and further analyzed using airgo™'s dedicated software. once raw data are filtered, they are processed in order to obtain the proper breath signal. the main steps of this process, shown in figure 4 , can be summarized as follows: • identification of maximum and minimum of each breathing cycle; • representation of each breath by means of a vector that connects the maximum and the minimum; • automatic reconstruction of segmented breath, e.g., due to obstructions or movement; • automatic removal of fake breaths; • computation of tidal volume, respiratory rate and minute ventilation. the signal acquired by the girth band is expressed in arbitrary units ranging from 0 to 1023 (10bit analog to digital converter) and expresses the amplitude of the signal recorded in the range of differential potential levels between 0.5 and 3.6 v, which is the microprocessor supply voltage. this signal represents the signal amplitude variation due to the trunk expansion and reduction during breathing. in figure 5 , an explicative diagram of the feature extraction process is reported. breath data can be then transmitted to a computer via bluetooth low energy, stored, visualized and further analyzed using airgo™'s dedicated software. the signal acquired by the girth band is expressed in arbitrary units ranging from 0 to 1023 (10-bit analog to digital converter) and expresses the amplitude of the signal recorded in the range of differential potential levels between 0.5 and 3.6 v, which is the microprocessor supply voltage. this signal represents the signal amplitude variation due to the trunk expansion and reduction during breathing. in figure 5 , an explicative diagram of the feature extraction process is reported. the signal acquired by the girth band is expressed in arbitrary units ranging from 0 to 1023 (10bit analog to digital converter) and expresses the amplitude of the signal recorded in the range of differential potential levels between 0.5 and 3.6 v, which is the microprocessor supply voltage. this signal represents the signal amplitude variation due to the trunk expansion and reduction during breathing. in figure 5 , an explicative diagram of the feature extraction process is reported. figure 5 . box scheme of the raw signal processing and extraction process of the parameters of interest: amplitude (amp), which represents airgo™'s signal variation and models sensormedics' tidal volume; respiratory rate (rr); airgo™'s minute ventilation (amp×rr), which models sensormedics' minute ventilation; duty cycle, which is the ratio between inspiratory time and total breath period. figure 5 . box scheme of the raw signal processing and extraction process of the parameters of interest: amplitude (amp), which represents airgo™'s signal variation and models sensormedics' tidal volume; respiratory rate (rr); airgo™'s minute ventilation (amp×rr), which models sensormedics' minute ventilation; duty cycle, which is the ratio between inspiratory time and total breath period. as the breath period (t) is computed as the temporal distance between two consecutive minima, the respiratory rate is the inverse of the breath period expressed as breaths per minute and the expiratory time is computed as the difference between breath period and the inspiratory time. among sensormedics' data, outliers, fake breaths and missing data were detected and deleted. these errors may be due to the incorrect positioning of the mouthpiece and the consequent air leakage. in these cases, it is necessary not to consider the entire breath. in order to validate the airgo™ system, a breath-by-breath comparison with the sensormedics outputs was performed. since the amplitude (amp) represents the same features as the tidal volume read by the metabolic cart and expressed in liters, the values obtained with the two devices had to be normalized in order to be compared. for each position of each subject, the mean of the first twenty values of quiet breathing after the initial big breath was computed and used as a reference. this leads to normalized values for airgo™'s amplitude and minute ventilation and the corresponding sensormedics' volume and minute ventilation in all conditions, allowing to derive four relative parameters: the alignment of the two signals covered a fundamental role. in order to facilitate the process of identification and extraction of the correct signal window referring to a given position to be compared in the two instruments, subjects have been asked to perform a deep, big breath at the beginning of each test in each position. the comparison between the breath signals recorded by the airgo™ device and the sensormedics metabolic cart is based on the number of breaths in the same period and on the big-breath maneuver correspondence. after this phase, in order to obtain the correct number of breaths in the same time span, all airgo™ values representing heartbeat artefacts or fake breaths have been filtered out according to the airgo™ signal processing algorithm explained in section 2.4. with the airgo™ software it was possible to reconstruct two segmented contiguous breaths that are recognized activities by linking the minimum of the first breath to the maximum of the second. thanks to the synchronization with the big breaths, the same number of breaths as in sensormedics file in the same period was determined. however, when performing the alignment, a progressive alignment shift between the two measurements after the big breath was noticed. this was due to a temporal delay in the airgo™ system's sampling process that led to a delay of about 3-4 s at the end of each position test. to overcome this problem and visualized the two sequences of values correctly, it was necessary to represent the outputs of airgo™ and sensormedics with respect to the number of breaths of each position test instead of the breath period. an example of synchronization of the amplitude outputs in a static position is shown in figure 6 , while an example of synchronization of the respiratory rate outputs is shown in figure 7 . in exercise test recordings, because of movement artefacts, noises coming from changes in posture, air losses through the mouthpiece caused by the increasing level of physical effort and the greater amount of breaths than the test at rest, the alignment process was based only on the initial big breath synchronization, so the final shift in alignment was not eliminated. an example is reported in figure 8 , where another common problem is shown: for volumes higher than the tidal volume, airgo™'s normalized amplitudes tend to underestimate the sensormedics' normalized volume. in order to improve the effectiveness of the statistical analysis and to allow comparisons between subjects with different levels of maximum workload, the entire exercise test was divided in four different regions according to the increasing physical stress intensity: low intensity ("l"), medium intensity ("m"), high/maximum intensity ("h" and "i max ") and recovery phase ("rp"). to identify the maximum of exercise intensity for each subject, the sensormedics' maximum value of normalized value of minute ventilation was considered as reference. in exercise test recordings, because of movement artefacts, noises coming from changes in posture, air losses through the mouthpiece caused by the increasing level of physical effort and the greater amount of breaths than the test at rest, the alignment process was based only on the initial big breath synchronization, so the final shift in alignment was not eliminated. an example is reported in figure 8 , where another common problem is shown: for volumes higher than the tidal volume, airgo™'s normalized amplitudes tend to underestimate the sensormedics' normalized volume. in order to improve the effectiveness of the statistical analysis and to allow comparisons between subjects with different levels of maximum workload, the entire exercise test was divided in four different regions according to the increasing physical stress intensity: low intensity ("l"), medium intensity ("m"), high/maximum intensity ("h" and "imax") and recovery phase ("rp"). to identify the maximum of exercise intensity for each subject, the sensormedics' maximum value of normalized value of minute ventilation was considered as reference. to equally divide the four regions, two points of reference were identified, one at one third and the second at two third of the normalized minute ventilation maximum value of the subject. within the first three regions characterized by an increasing ventilation, four smaller sections constituted by at least twenty breaths were identified: the first one referring to the low intensity region ("l"), the second one referring to the medium intensity region ("m"), while the third and fourth referring to the high and maximum intensity region ("h" and "imax"). because each subject reached a different maximum load according to his/her physical capacity, the four sections were centered differently from subject to subject. the section "l" was constituted by the central breaths of the first region, the section "m" by the central breaths of the second region; the section "h" by the central breaths of the third region and the section "imax" by the last twenty breaths of the exercise text track before the section "rp". in this last region, two other smaller sections were identified: the first one ("rp1") containing at least ten breaths centered around the closest normalized minute ventilation central value within the "h" section and the second one ("rp2") containing at least ten breaths centered around the closest ventilation central value within the "m" section. an example of this division into sections in the case of the minute ventilation can be seen in figure 9 . to equally divide the four regions, two points of reference were identified, one at one third and the second at two third of the normalized minute ventilation maximum value of the subject. within the first three regions characterized by an increasing ventilation, four smaller sections constituted by at least twenty breaths were identified: the first one referring to the low intensity region ("l"), the second one referring to the medium intensity region ("m"), while the third and fourth referring to the high and maximum intensity region ("h" and "imax"). because each subject reached a different maximum load according to his/her physical capacity, the four sections were centered differently from subject to subject. the section "l" was constituted by the central breaths of the first region, the section "m" by the central breaths of the second region; the section "h" by the central breaths of the third region and the section "i max " by the last twenty breaths of the exercise text track before the section "rp". in this last region, two other smaller sections were identified: the first one ("rp1") containing at least ten breaths centered around the closest normalized minute ventilation central value within the "h" section and the second one ("rp2") containing at least ten breaths centered around the closest ventilation central value within the "m" section. an example of this division into sections in the case of the minute ventilation can be seen in figure 9 . sensors 2020, 20, x for peer review 11 of 19 in order to verify the goodness-of-fit of values distribution, the kolmogorov-smirnov test of normality has been run both on single distributions and on the differences between the two methods. since the test returned that data did not come from a normal distribution, a non-parametric statistical analysis has been conducted. to have a general overview of airgo™'s estimate error in each position both during quiet breath and during the exercise test for each subject, relative error medians, interquartile ranges (iqrs) and limits of agreement (loas) were calculated for each parameter. then, to find a significant difference between different positions and intensity levels (within the exercise test), the one-way kruskal-wallis test has been conducted with a level of significance of p < 0.05. in the next paragraphs, box and whiskers plots of the differences between the two methods are reported for each position and for the two conditions (static postures and exercise test). an example of a bland-altman plot for a subject is reported to graphically show the agreement between the two instruments. the relative error median percentage, the interquartile range and the limits of agreement of the parameters during the test at rest and the exercise test are reported in tables 2 and 3 , respectively. negative signs in the percentages stand for an understimation of the airgo™'s computed parameters with respect to those of sensormedics 2900, and negative values indicate the amount of the understimation. in the case of the respiratory rate, the overall relative error median percentage in all positions was of 0%, with the highest value of interquartile range of 15.0 in the supine position. the second best result was the overall relative error median percentage in the case of the normalized amplitude parameter: in fact, in the supine position the percentage of 0.8% was the lowest relative error for the test at rest after the results of the respiratory rate. moreover, by looking at changes in the relative error median between positions for the normalized amplitude parameter it was possible to notice a difference in values between standing and horizontal positions (supine, right lateral decubitus, left lateral decubitus). in particular, for horizontal positions the overall percentage in order to verify the goodness-of-fit of values distribution, the kolmogorov-smirnov test of normality has been run both on single distributions and on the differences between the two methods. since the test returned that data did not come from a normal distribution, a non-parametric statistical analysis has been conducted. to have a general overview of airgo™'s estimate error in each position both during quiet breath and during the exercise test for each subject, relative error medians, interquartile ranges (iqrs) and limits of agreement (loas) were calculated for each parameter. then, to find a significant difference between different positions and intensity levels (within the exercise test), the one-way kruskal-wallis test has been conducted with a level of significance of p < 0.05. in the next paragraphs, box and whiskers plots of the differences between the two methods are reported for each position and for the two conditions (static postures and exercise test). an example of a bland-altman plot for a subject is reported to graphically show the agreement between the two instruments. the relative error median percentage, the interquartile range and the limits of agreement of the parameters during the test at rest and the exercise test are reported in tables 2 and 3 , respectively. negative signs in the percentages stand for an understimation of the airgo™'s computed parameters with respect to those of sensormedics 2900, and negative values indicate the amount of the understimation. in the case of the respiratory rate, the overall relative error median percentage in all positions was of 0%, with the highest value of interquartile range of 15.0 in the supine position. the second best result was the overall relative error median percentage in the case of the normalized amplitude parameter: in fact, in the supine position the percentage of 0.8% was the lowest relative error for the test at rest after the results of the respiratory rate. moreover, by looking at changes in the relative error median between positions for the normalized amplitude parameter it was possible to notice a difference in values between standing and horizontal positions (supine, right lateral decubitus, left lateral decubitus). in particular, for horizontal positions the overall percentage values were lower (0.8%, 4.4% and 1.1%) compared to the seated position (9.0%) with the standing position percentage value standing between the two (6.8%). the normalized minute ventilation parameter, calculated as the product of tidal volume and respiratory rate, gained similar results to the normalized amplitude parameter and it was affected by similar errors in the same positions. to obtain representative error values for each respiratory parameter independently from the position, considering the mean error in all positions, the relative errors were 4.4% for normalized amplitude, 0% for respiratory rate, 4.2% for normalized ventilation and −0.3 for duty cycle. table 3 . exercise test and recovery, all intensities, all parameters. aggregated data are expressed as relative error median percentage, interquartile range (iqr) and limits of agreement (loas), computed as ±1.96sd. iqr and loas are presented in brackets (iqr/loas). as shown in figure 2 , airgo™'s estimates tend to underestimate with increasing volumes and consequently with increasing ventilation during exercise, as was graphically shown in figure 8 . in the normalized amplitude values reported in table 3 , the relative error increased drastically from −11.1% at low intensity to −39.7% at maximum intensity. in the second recovery phase, the overall median relative error was −24.8%, a value that stands in between the low-intensity and medium-intensity values. this result was also more evident by looking at the overall normalized ventilation relative error medians. in this case, the respiratory rate was confirmed as the best estimate with an overall relative error median percentage of 0% in all physical effort conditions, while the duty cycle parameter had the highest error of −0.27 in the medium intensity level of the exercise. the results of one way kruskal-wallis analysis are reported in table 4 for the test at rest in different positions and for the exercise test at different intensity levels. for the test at rest, normalized amplitude, normalized ventilation and respiratory rate parameters were independent on position (p > 0.05). on the other hand, duty cycle was influenced by posture, with a statistically significant difference between standing and supine positions (p < 0.05). for the exercise test, the respiratory rate was the only parameter which resulted to be independent from different levels of exercise intensity (p > 0.05). in the cases of normalized amplitude (p < 0.01), normalized ventilation (p = 0.001) and duty cycle (p < 0.01) parameters, there was a significant effect of the intensity level of exercise on the error. in particular, the post hoc pairwise comparison highlighted that the first two parameters presented a statistically significant difference between conditions l and h (p < 0.05 for normalized amplitude and p < 0.01 for normalized ventilation), and conditions l and i max (p = 0.014 for normalized amplitude and p < 0.01 for normalized ventilation). the duty cycle presented, instead, a significant difference between m and rp1 conditions (p < 0.01), and m and rp2 conditions (p < 0.05). in figure 10 , the four parameters in the different positions for all subjects are reported in boxplots, where the different positions of the test at rest are indicated with roman numbers. the same analysis has been performed on the parameters during the exercise test and the result is shown in figure 11 . the bland-altman plot allows us to appreciate how much the system being validated differs from the reference system. the results obtained in the case of a healthy female subject (38 years old, 166 cm, 52 kg; same subject shown in figures 6, 7 and 9 ) are reported in figure 12 in the case of exercise and recovery tests for normalized ventilation and respiratory rate parameters. the obtained results are not significantly different from the results obtained in the previously illustrated representation methods. focusing on normalized ventilation (figure 12 , left), points tended to decrease with increasing ventilation, thus displaying the behavior of a proportional error. respiratory rate (figure 12 , right) showed a constant variability of the error. the bland-altman plot allows us to appreciate how much the system being validated differs from the reference system. the results obtained in the case of a healthy female subject (38 years old, 166 cm, 52 kg; same subject shown in figures 6, 7 and 9 ) are reported in figure 12 in the case of exercise and recovery tests for normalized ventilation and respiratory rate parameters. the obtained results are not significantly different from the results obtained in the previously illustrated representation methods. focusing on normalized ventilation (figure 12 , left), points tended to decrease with increasing ventilation, thus displaying the behavior of a proportional error. respiratory rate (figure 12 , right) showed a constant variability of the error. bland-altman plots of the aggregated results of respiratory rate at rest. each dot represents the value obtained from a single subject. the yellow line represents the upper limit of agreement (mean error +1.96× standard deviation); the red line represents the mean error; the purple line represents the lower limit of agreement (mean error −1.96× standard deviation). bland-altman plots of the aggregated results of respiratory rate at rest. each dot represents the value obtained from a single subject. the yellow line represents the upper limit of agreement (mean error +1.96× standard deviation); the red line represents the mean error; the purple line represents the lower limit of agreement (mean error −1.96× standard deviation). the comparison of the airgo™ device with the metabolic cart showed that the respiratory rate was the most accurate parameter, in all positions, for both rest and exercise tests in all conditions of physical effort: medians were always positioned on the zero error line with low dispersion around this value. normalized amplitude and normalized ventilation showed similar trends and similar error changes both in different positions and during exercise. at rest, the overall error was small and remained almost constant in different positions, with the supine position characterized by better results compared to the others, even though this aspect did not have statistical significance. on the other hand, airgo™'s underestimation of increasing breath volumes during exercise was confirmed and it was also more evident in normalized minute ventilation. additionally, the recovery phase was characterized by a progressive reduction in absolute errors, which tend to assume initial median values. figure 16 . bland-altman plots of the aggregated results of respiratory rate during exercise. each dot represents the value obtained from a single subject. the yellow line represents the upper limit of agreement (mean error +1.96× standard deviation); the red line represents the mean error; the purple line represents the lower limit of agreement (mean error −1.96× standard deviation). the comparison of the airgo™ device with the metabolic cart showed that the respiratory rate was the most accurate parameter, in all positions, for both rest and exercise tests in all conditions of physical effort: medians were always positioned on the zero error line with low dispersion around this value. normalized amplitude and normalized ventilation showed similar trends and similar error changes both in different positions and during exercise. at rest, the overall error was small and remained almost constant in different positions, with the supine position characterized by better results compared to the others, even though this aspect did not have statistical significance. on the other hand, airgo™'s underestimation of increasing breath volumes during exercise was confirmed and it was also more evident in normalized minute ventilation. additionally, the recovery phase was characterized by a progressive reduction in absolute errors, which tend to assume initial median values. in the results of the exercise tests, it was previously observed that in the second recovery phase, the overall median relative error was −24.8%, a value that stands between the low-intensity and medium-intensity values. the reason for this error is still a subject of research. several possible explanations might have contributed separately or in combination to this result, however it is not possible to understand the magnitude of the contribution of each of these single explanations. the first one regards the pre-tensioned length of the girth elastic band obtained during the preparation phase of the test protocol: by cutting the band at a length slightly shorter than the real thoracic circumference (−7%) to ensure an effective fitting, it is likely that, for high volumes, the band reached the saturation region of the stretch metallic knit resistance qualitative curve in figure 2 . in this case, the resistance changes with elongation fail to have a correct dynamics, exiting from the linear region. the second explanation is related to the different relative contributions of the abdomen and the chest wall to the breathing pattern according to the posture and the trunk position assumed during exercise. in fact, it is known from the literature that a progressively increased inclination of the trunk determines a progressive reduction in the chest wall contribution and a progressive increase in abdominal contribution to the tidal volume [24] , while the airgo™ band only detects changes in circumference at the level of the abdominal rib cage, thus losing information related to the abdominal contribution. finally, as can be seen in the box and whiskers plots, there was a constant underestimation of duty cycle in all positions at rest and also for all intensity levels during exercise. in particular, sensormedics' values were always within an interval between 0.4 and 0.6, while airgo™'s estimates were between 0.2 and 0.4. this means that, in the first case, inspiratory and expiratory time have a proportion of about 1:1, while in airgo™'s duty cycle estimation, this proportion is about 1:2. this explains the error underestimation and it could be due to imperfections in the airgo™ processing algorithm. specifically, the ending of a breath is determined by computational means when the girth is either smaller than what it was when the breath began or when the girth is smaller than the maximum girth in a given breath cycle by a certain amount, as specified in the related patent. the fact that the end of expiration is detected by means of a threshold might cause errors in the precise identification of the minimum. in fact, the duty cycle is computed as described in table 1 and an underestimation of inspiratory time with respect to expiratory (and total) time causes an underestimation of the duty cycle as well. the overall error remains constant between the test at rest and the exercise test, suggesting that duty cycle could be very accurate in both conditions when this issue is solved. the overall performance of airgo™ in comparison with the sensormedics metabolic cart was satisfactory and it must be noted that the research on this device, both from the point of view of the hardware and of the signal processing software, is still ongoing. respiratory rate is a vital sign used to monitor the progression of several illnesses, to predict adverse clinical events and to discriminate between patients at risk and stable patients. given that airgo™'s respiratory rate estimates had a good correspondence with the analyses performed with an instrument that can be considered the gold standard, it can be concluded that the airgo™ device could be useful to monitor respiratory rate in a non-invasive and non-intrusive way during everyday activities and sleep. applications of this device can be found in chronic respiratory diseases as well as acute pathologies, such as the novel covid-19, where the respiratory rate is predictive of the worsening of the disease. further research is needed on the estimations of tidal volume, minute ventilation and duty cycle, however the obtained results are encouraging and research works combining activity recognition and estimation of respiratory parameters are needed to assess the validity of the system in a non-controlled environment. the airgo™ device is partially described in the us patent number us201462007142p, title "breath volume monitoring system and method" by david kuller for myair llc. respiratory rate predicts cardiopulmonary arrest for internal medicine inpatients respiratory rate: the neglected vital sign monitoring breathing rate at home allows early identification of copd exacerbations exacerbations in chronic obstructive pulmonary disease: identification and prediction using a digital health system measurement of heart rate and respiratory rate using a textile-based wearable device in heart failure patients lower mortality of covid-19 by early recognition and intervention: experience from jiangsu province monitoring of physiological parameters to predict exacerbations of chronic obstructive pulmonary disease (copd): a systematic review remote respiratory monitoring in the time of covid-19 validation of the hexoskin wearable vest during lying, sitting, standing, and walking activities qualitative and quantitative evaluation of a new wearable device for ecg and respiratory holter monitoring respiration rate and volume measurements using wearable strain sensors smart vest for respiratory rate monitoring of copd patients based on non-contact capacitive sensing assessment of breathing parameters using an inertial measurement unit (imu)-based system estimation of respiration rate from three-dimensional acceleration data based on body sensor network smart textile for respiratory monitoring and thoraco-abdominal motion pattern evaluation reliability of a wearable wireless patch for continuous remote monitoring of vital signs in patients recovering from major surgery: a clinical validation study from the tracing trial towards continuous respiration monitoring research on non-contact monitoring system for human physiological signal and body movement wearable technology: role in respiratory health and disease telemonitoring systems for respiratory patients: technological aspects respiratory frequency during exercise: the neglected physiological measure validation study of the airgo™ device for continuous monitoring of respiratory function a multimodal wearable system for continuous and real-time breathing pattern monitoring during daily activity effects of gender and posture on thoraco-abdominal kinematics during quiet breathing in healthy adults this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-315077-i1xjcuae authors: branda, john a.; lewandrowski, kent title: utilization management in microbiology date: 2014-01-01 journal: clin chim acta doi: 10.1016/j.cca.2013.09.031 sha: doc_id: 315077 cord_uid: i1xjcuae the available literature concerning utilization management in the clinical microbiology laboratory is relatively limited compared with that for high-volume, automated testing in the central core laboratory. however, the same strategies employed elsewhere in the clinical laboratory operation can be applied to utilization management challenges in microbiology, including decision support systems, application of evidence-based medicine, screening algorithms and gatekeeper functions. the results of testing in the microbiology laboratory have significant effects on the cost of clinical care, especially costs related to antimicrobial agents and infection control practices. consequently many of the successful utilization management interventions described in clinical microbiology have targeted not just the volume of tests performed in the laboratory, but also the downstream costs of care. this article will review utilization management strategies in clinical microbiology, including specific examples from our institution and other healthcare organizations. clinical microbiology includes bacteriology and antimicrobial susceptibility testing, virology, parasitology, mycobacteriology, mycology, serology and molecular microbiology. unlike the core laboratory (chemistry/hematology), where the majority of testing is performed on highly automated analyzers, most testing in microbiology is performed manually or on semi-automated platforms. many microbiology tests also require interpretation by a skilled microbiology technologist, including visual interpretation of culture results and microscopic examinations. for these reasons the unit cost of microbiology testing is usually greater than that for routine automated testing. the results of microbiology tests have a significant impact on the overall cost of clinical care, most notably in the use and selection of antimicrobial therapy. therefore, when approaching utilization management in microbiology, it is important to consider not only the cost of testing within the microbiology laboratory but also the downstream costs resulting from clinical decisions based on the test results. the published literature on utilization management in microbiology is relatively limited when compared to reports on managing utilization of routine automated testing in the chemistry and hematology laboratories. this article will outline a number of utilization management interventions in microbiology that have been reported in the literature. we will also describe several unpublished initiatives that have proven successful in our institution. the specific interventions to be discussed are outlined in table 1 , and will be described in more detail in the text that follows. in a number of cases, the initiative's success arose not only from a reduction in laboratory testing per se, but rather also from its impact in the clinical care arena (for example, a reduction in antibiotic use or hospital length of length-of-stay). this observation highlights the importance of the clinical microbiology director in forming collaborative, interdepartmental teams to improve quality and reduce the cost of medical care. tests for cytomegalovirus (cmv) include antigenemia testing, viral load testing by quantitative polymerase chain reaction (qpcr), viral genotyping, shell vial culture, or serologic tests for the detection of a host immunologic response (cmv igm and igg antibody tests and antibody avidity tests). for an individual patient, the most appropriate test depends on the clinical indication. it is difficult for clinicians to keep upto-date with esoteric tests in rapidly evolving specialties, especially when there are numerous tests that can be ordered. in these situations, the use of a decision support tool is an effective mechanism to assist physicians in proper test selection, potentially avoiding inappropriate test selection. as one example, fig. 1 shows a screen display from the on-line laboratory handbook at the massachusetts general hospital. when the clinician types "cytomegalovirus" or "cmv" into the handbook's search function, the available tests and their appropriate indication are presented. in addition, the same decision-support information is provided in the electronic physician order entry (poe) system when a clinician views cmv-related test options. an advantageous feature of this approach is that when new tests become available, or outdated ones are removed from the test menu, the decision-support function can be updated accordingly. for example, the mgh microbiology laboratory recently discontinued the cmv antigenemia assay in favor of the cmv qpcr test. the information provided in the on-line handbook makes it clear that the preferred test has changed. this approach can be applied to many other tests in microbiology, particularly in areas such as molecular microbiology where new assays are supplanting more traditional assays at a rapid rate. the topic of decision support is covered in more detail in another chapter of this special edition. the problem of contamination of blood cultures from improper or poor technique is well known. it has been estimated that up to 5% of positive blood cultures may represent contaminants [1] , resulting in significant increases in resource utilization. consequently many hospitals have engaged in ongoing efforts to reduce blood culture contamination by improving staff training, or designating specific types of employees to collect the blood culture specimens. for example, blood cultures collected by medical house officers are more likely to be contaminated than those collected by phlebotomists [2] . in a retrospective study, bates et al. studied the impact of contaminated blood cultures on hospital length-of-stay and hospital charges [3] . in patients with falsely positive blood cultures, there was a 4.5-day increase in the median length-ofstay and an increase in hospital charges of 33.4%. false-positive episodes were associated with increased pharmacy charges for intravenous antibiotics (39% increase) and laboratory charges (20% increase). in another study, segal and chamberlain assessed the impact of false-positive blood cultures in a pediatric emergency department [4] . the authors reported an increase in phone calls, return visits to the emergency department, unnecessary laboratory tests, inappropriate antibiotic administration and hospital admissions. finally, tabriz et al. evaluated the practice of repeating blood cultures serially [5] . blood cultures were repeated in 31.6% of cases and amounted to approximately one-third of all blood cultures handled in the laboratory. the results of the repeated cultures showed no growth in 83.4% of cases, the same pathogen in 9.1% of cases, and a new pathogen or contaminant in 2.5% and 5.0% of cases respectively. the authors concluded that repeating blood cultures provides little additional yield and that guidelines for when to repeat blood cultures might decrease utilization. laboratory reports that are not optimally designed can lead to confusion among clinicians, with the potential for misdiagnosis or unnecessary requests for additional testing. ackerman et al. evaluated the interpretation of 5 typical microbiology reports by physicians in a teaching hospital [6] . the investigators found that reports were often misinterpreted. for example, one report of "isolation of a gram negative rod from sputum was misinterpreted by 4 out of 5 physicians." the reasons for misinterpretation were reported to be the use of jargon, unfamiliar names of bacterial species, or ill-defined reporting conventions, and the omission of a clear-cut conclusion in many reports. the misunderstandings resulted in both inappropriate use of antibiotics and orders for unnecessary testing in the laboratory. this study highlights the importance of developing clear, concise, standardized reporting formats in microbiology, and the need for the laboratory to work closely with physicians in designing and communicating microbiology reports. 2.4. rapid identification of bacteria using matrix-assisted laser desorptionionization time of flight mass spectroscopy (maldi-tof ms) to improve clinical decision making and guide antibiotic use it has long been known that rapid bacterial identification and susceptibility testing lead to more appropriate use of antibiotics and a reduction in antimicrobial utilization [7] . in the past, rapid identification and susceptibility testing were mainly accomplished using automated instrumentation to perform conventional tests. more recently, manufacturers have developed maldi-tof ms for rapid organism identification, a method that has been demonstrated to reduce turnaround time for the identification of bacteria and yeasts by 1.45 days compared with conventional methods [8] . another advantage of maldi-tof ms is its simplicity and the relatively low cost of consumables [9] . for table 1 examples of utilization management initiatives in clinical microbiology (see text for details). 1. decision support: test selection for cytomegalovirus testing 2. reducing blood culture contamination 3. proper formatting of microbiology reports to avoid misinterpretation 4. use of maldi-tof mass spectroscopy for rapid identification of pathogens 5. antimicrobial stewardship of carbapenems and other expensive antimicrobial agents 6. rapid point-of-care testing for influenza a and group a streptococcus: impact on test ordering and antibiotic utilization 7. rapid molecular diagnostic testing for patients previously colonized with methicillin resistant staphylococcus aureus (mrsa) 8. use of screening methods to reduce low-yield urine cultures 9. restricting stool examinations in hospital acquired diarrhea 10. rapid testing for respiratory viruses: impact on inpatient bed management 11. application of evidence based medicine: discontinuation of fungal blood cultures 12. selection and oversight of molecular diagnostics in microbiology example, one study performed at a large, academic medical center demonstrated potential cost savings to the laboratory (for reagents and labor) of $100,000 per year [8] . although maldi-tof ms does not provide antimicrobial susceptibility data, rapid organism identification may help clinicians earlier to select an effective empirical antimicrobial strategy [10] . carbapenems including imipenem, meropenem, ertapenem and doripenem are broad spectrum antimicrobial agents active against many gram-positive, gram-negative and anaerobic organisms. they are highly effective when used appropriately but are also very expensive relative to potential alternative agents. indiscriminant use of carbapenems will also contribute to antimicrobial resistance, decreasing the effectiveness of the drug class. many hospitals have established antimicrobial formularies in the pharmacy to assist in management of expensive antibiotic drugs. in our institution some antibiotics are restricted and can only be obtained after approval from the division of infectious diseases. however, once approved there was no requirement or formal mechanism in place to re-evaluate the ongoing need for a restricted antibiotic once the results of microbiology culture and antimicrobial susceptibility testing became available. the clinical pathology staff in our microbiology laboratory recently began an antimicrobial stewardship program related to carbapenems. each day, a microbiology fellow and laboratory director review the clinical history, culture results and susceptibility test results for all patients newly started on a carbapenem, to determine appropriate versus inappropriate use of the drugs. if objective data indicate that a patient's infection can be treated using a non-restricted agent, an email is sent to the clinician as shown in the example below. "dear dr.________ your patient ________ is currently receiving a restricted antibiotic: ertapenem. the use of this restricted drug is being monitored by the mgh antimicrobial stewardship program. recent culture and antimicrobial susceptibility data from your patient reveal that the organism(s) is/are susceptible to other, nonrestricted antibiotics (see sensitivity report below). given these data, if clinically appropriate, please consider discontinuing the restricted carbapenem and/or changing to a non-restricted antimicrobial option. this may help reduce both the development of future resistance to these broad spectrum drugs and costs of therapy. if you have not already done so, you may request an infectious disease consult in order to obtain assistance on the choice of antimicrobial agents". after implementation of this stewardship effort, a decrease in carbapenem use was observed, and carbapenems have been removed from the "top 10" list of money spent on antimicrobials. the microbiology group is now extending the program to include other high cost antimicrobials. point-of-care testing (poct) performed at the patient's bedside provides rapid, real-time results. in some cases this facilitates clinical decision making and improves the efficiency of clinical operations [11] . a number of rapid point-of-care tests are available for the diagnosis of infectious diseases [12] . among these are single use, visually read, lateral flow tests for influenza a and b. in a randomized, prospective controlled study by bonner et al., the use of a rapid poc influenza test in a pediatric emergency department was associated with a significant reduction in laboratory tests ordered (complete blood count, blood cultures, urinalysis and urine cultures), a decrease in chest radiographs performed and a reduction in emergency department length-of-stay [13] . in another study of pediatric patients presenting to the emergency department with acute pharyngitis, the authors compared antibiotic use in patients who received a rapid streptococcus a test to those who were managed by conventional throat culture alone. they reported a decrease in antibiotic use of 50% when the rapid test was employed (22.45% versus 41.38%) and concluded that the rapid test significantly reduced unnecessary prescription of antibiotics [14] . patients who are colonized with mrsa require contact precautions when admitted to the hospital. this entails placing them in a private room or cohorting the patient in a semi-private room with another colonized patient. in addition, hospital staff must wear gloves and gowns, and use dedicated equipment when interacting with the patient. some of these patients also have a longer hospital length of stay due to delays in discharge to other health care facilities. collectively, these features of mrsa colonization result in greater use of hospital resources compared with non-mrsa colonized patients. because mrsa colonization can be transient, protocols have been put in place to identify previously mrsa colonized patients who are no longer colonized, and can be managed without contact precautions. in our institution, patients with a history of mrsa are eligible for discontinuation of contact precautions if for either screening method, the patient cannot have been received antibiotics active against mrsa during the 48 h prior to screening. discontinuation of contact precautions on the basis of a single negative mrsa pcr is faster than screening using the culture-based method, and could result in an increase in discontinuation of contact precautions because of a reduction in the number of samples needed [15] . uncomplicated urinary tract infection is very common, especially among women. various strategies have been employed to make (or rule out) a diagnosis of community acquired uti in adult women without the need for urine culture. some sources suggest that uncomplicated uti in outpatients can be diagnosed and managed without culture, unless the patient fails treatment or has had recurrent utis [16, 17] . others have even suggested that suspected uti can be managed over the telephone in women with typical symptoms of cystitis and without vaginal symptoms or major co-morbidities [18] . this approach eliminates both office visit and any subsequent laboratory testing. another approach is to limit urine culture utilization by pre-screening urine using various methods. for example, dipstick urinalysis for leukocyte esterase and nitrites has a high negative predictive value, and may be used to exclude bacteriuria without a culture step when the results are negative. studies have also demonstrated that urine can be screened for significant bacteriuria prior to culture using automated urine sediment examination [19] or flow cytometry [20] . diarrhea is a common complaint among hospitalized patients. although it is common for clinicians to request a routine stool culture and ova and parasites (o&p) examination for patients with diarrhea, these tests are designed to detect agents of community-acquired rather than hospital-acquired infection. a number of studies have indicated that routine stool culture or stool o&p examination is usually not warranted in adult patients who develop diarrhea more than three days after admission to the hospital [21] [22] [23] . in contrast, testing for clostridium difficile should be considered, as this is a major cause of nosocomial diarrheal illness. new molecular diagnostic tests offer the promise of providing rapid and reliable testing for c. difficile. highly sensitive molecular testing could potentially permit rapid ruleout of c. difficile, obviating the need for unnecessary antibiotics and contact precautions in many patients. upper respiratory viral infections in the united states are frequently caused by rhinoviruses, coronaviruses, influenza a and b, parainfluenza, respiratory syncytial virus, adenovirus or metapneumovirus. while many of these illnesses can be managed on an outpatient basis, patients with severe illness or major comorbidities are often admitted to the hospital. usually they present first to the emergency department, where the initial challenge is to confirm the presence of a respiratory viral infection and then to identify the specific offending virus in order to direct specific therapy (if available) and aid in hospital bed assignment. often, respiratory viral infections occur in seasonal epidemics (especially influenza a and b), resulting in hospital overcrowding and a shortage of hospital beds. when this occurs, managing the availability of hospital beds becomes a priority. in cases where the offending virus has been identified, contact and/or droplet precautions must be instituted to prevent transmission between patients as shown in table 2 . patients on contact or droplet precautions must be placed in a private room. alternatively, if two patients are infected with the same respiratory virus, they can be cohorted together in one hospital room. in our hospital, we offer rapid molecular diagnostic testing for influenza a and b for patients who require hospital admission for a flu-like illness. testing is performed 24 h per day, 7 days per week, in order to facilitate bed management and timely initiation of anti-viral therapy. we also offer a respiratory viral panel by direct immunofluorescence for the many viruses listed above, 7 days per week during peak respiratory virus season. the ability to identify the specific virus causing the infection greatly assists in managing our inpatient beds and maintaining effective infection control measures. the savings to the hospital from improved bed management during epidemics greatly exceeds the cost of the testing in the laboratory. many clinical laboratories offer "fungal blood cultures" that employ specialized media designed to enhance the detection of yeast or mold fungemia. however, it is well-established that specialized fungal blood culture media are not superior to routine blood culture media for the detection of candida fungemia (candidemia) [24, 25] . thus, the main rationale for the use of fungal blood cultures is to improve detection of cryptococcal yeast, endemic fungi (histoplasma capsulatum, coccidioides spp., etc) and filamentous fungi (e.g., aspergillus spp.) in the blood. until recently, our institution offered fungal blood cultures using myco/ f lytic bottles (becton dickinson) designed for use with the bactec automated culture monitoring system. this approach was costly, not only in terms of reagent costs but also in terms of technical labor. the highly-enriched and non-selective culture medium needed to be incubated for up to 30 days before a negative result could be obtained, and this frequently promoted the growth and eventual detection of skin contaminants that would not have been detected using routine blood culture bottles and a 5 to 7 day incubation period. thus, we reviewed our experience with this approach to understand whether or not it provided a significant clinical benefit. we reviewed the results of all fungal blood cultures over a 44 month period. during this time period, 5544 myco/f lytic fungal blood cultures were performed. our review revealed the following: â�¢ no dimorphic fungi were recovered by fungal blood culture. â�¢ mold (fusarium sp.) was recovered twice from a single patient using fungal blood culture. however, in this case fusarium was also recovered from 2 sets of routine blood cultures (2 out of 4 bottles), several days prior to recovery from fungal blood culture. â�¢ cryptococcus neoformans was recovered from 3 patients by fungal blood culture. two of the patients had cryptococcal meningitis, and in both cases the organism was detected in numerous other ways (csf and blood cryptococcal antigen tests, csf gram stain, routine and fungal csf cultures, and routine blood cultures). the third patient had cryptococcal fungemia (but not meningitis); a cryptococcal antigen test on blood was positive, and the organism was recovered twice from routine blood cultures. based on these findings, we concluded that fungal blood cultures had failed to detect any dimorphic fungi, molds or cryptococcal yeast that were not otherwise detected by routine blood culture. furthermore, blood culture is not a useful method for the detection of invasive infection caused by molds or dimorphic fungi. therefore, blood culture specifically designed for the detection of fungi was discontinued at our institution. many microbiology tests suffer from drawbacks that limit their clinical utility. for example, results from traditional culture methods may take several days to become available, and antibiotic treatment prior to specimen collection may degrade sensitivity. serologic studies often cannot distinguish current from past infection unless acute and convalescent specimens are available. techniques for specialized cultures such as for viruses are beyond the capability of most hospital laboratories. finally many microbiology tests do not yield quantitative results which may be desirable in some situations. recent advances in molecular diagnostic techniques for microbiology offer promise to resolve some of these issues. some molecular tests, such as those for influenza a and b, hepatitis b virus dna and hiv viral load are requested in sufficient volume that many hospital laboratories are able to offer the tests in-house. in these examples, the molecular diagnostic test is either superior to alternative testing methods or provides unique information of clinical importance (e.g. viral load). however, in many cases the volume of requests for molecular microbiology tests is too low to warrant performing the test in the hospital laboratory. typically these tests are sent to outside reference laboratories for analysis which, over time, can prove very expensive. intuitively a molecular diagnostic test is expected to be highly sensitive and specific, but this is not always the case. in some situations, conventional microbiology tests are more appropriate [26] . for this reason, it is important for the clinical microbiology director to work with infectious disease specialists to scrutinize the send out budget for potential inappropriate test ordering. further, such analysis may reveal opportunities for in-sourcing the testing at significant savings. for example, our microbiology laboratory recently insourced nucleic-acid testing for epstein bar virus (ebv) saving over $100,000 per year and providing superior turnaround time. tests performed in the clinical microbiology laboratory are ripe for utilization management efforts. clinicians are frequently confused about which test to order, and appropriate test selection can be guided through decision support mechanisms and gatekeeper functions. the diagnostic yield of routine cultures can be improved by facilitating proper specimen collection and transport, or screening specimens prior to culture. clinical decision making, particularly in selecting appropriate antibiotics and implementing (or discontinuing) infection control measures, can be aided by efforts to reduce turnaround time and by antimicrobial stewardship efforts directed by microbiologists. finally, errors can be prevented by carefully formatting reports to avoid miscommunication. analysis of strategies to improve cost effectiveness of blood cultures pathology tests: is the time for demand management ripe at last contaminant blood cultures and resource utilization: the true consequences of false positive results resource utilization and contaminated blood cultures in children at risk for occult bacteremia repeating blood cultures during hospital stay: practice pattern at a teaching hospital and a proposal for guidelines consumer survey on microbiology reports rapid identification and antimicrobial susceptibility testing reduce antibiotic use and accelerate pathogen-directed antibiotic use prospective evaluation of a matrix-assisted laser desorption ionization-time of flight mass spectrometry system in a hospital clinical microbiology laboratory for identification of bacteria and yeasts: a bench-by-bench study for assessing the impact on time to identification and costeffectiveness maldi-tof mass spectroscopy: transformative proteomics for clinical microbiology impact of matrixassisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with gram-negative bacteremia: a prospective observational study implementing point-of-care testing to improve outcomes infectious disease testing at the point-of-care impact of the rapid diagnosis of influenza on physician decision making and patient management in the pediatric emergency: results of a randomized, prospective controlled study impact of rapid streptococcal test on antibiotic use in a pediatric emergency department discontinuation of contact precautions for methicillin-resistant staphylococcus aureus: a randomized controlled trial comparing passive and active screening with culture and polymerase chain reaction management of urinary tract infections in adults laboratory diagnosis of urinary tract infections in adult patients urine dipstick for diagnosing urinary tract infection bacteriuria screening by automated whole-field-image-based microscopy reduces the number of necessary urine culture evaluation of the sysmex uf-100 urine cell analyzer as a screening test to reduce the need for cultures for community-acquired urinary tract infection role of the microbiology laboratory in the diagnosis of nosocomial diarrhea rational testing for faeces in the investigation of sporadic hospital-acquired diarrhoea when should a stool culture be done in adults with nosocomial infections optimal use of myco/f lytic and standard bactec blood culture bottles for detection of yeast and mycobacteria principles and procedures for blood cultures; approved guideline. clsi document m47-a. wayne, pa: clinical and laboratory standards institute introducing a molecular test into the clinical laboratory: development, evaluation, and validation key: cord-332481-y0rd70ry authors: ljubic, t.; banovac, a.; buljan, i.; jerkovic, i.; basic, z.; kruzic, i.; kolic, a.; kolombatovic, r. r.; marusic, a.; andjelinovic, s. title: the effect of serological screening for sars-cov-2 antibodies to participants' attitudes and risk behaviour: a study on a tested population sample of industry workers in split-dalmatia county, croatia date: 2020-06-17 journal: nan doi: 10.1101/2020.06.15.20131482 sha: doc_id: 332481 cord_uid: y0rd70ry rapid serological tests for sars-cov-2 antibodies have been questioned by scientists and the public because of unexplored effects of negative test results on behaviour and attitudes, that could lower the level of adherence to protective measures. therefore, our study aimed to investigate the changes in personal attitudes and behaviour before and after negative serological test results for sars-cov-2 antibodies. we conducted a survey questionnaire on 200 industry workers (69% males and 31% females) that have been previously tested negative. the survey examined participants' self-reported general attitudes towards covid-19, sense of fear, as well as their behaviour related to protective measures before and after the testing. the participants perceived the disease as a severe health threat and acknowledged the protective measures as appropriate. they reported a high level of adherence to measures and low level of fear both before and after the testing. although those indicators were statistically significantly reduced after the test (p < 0.004), they did not result in risk behaviour. therefore, the serological tests are not an additional threat regarding the risk behaviour in an environment where protective measures are efficient. in contrast, they might contribute to reducing the fear in the society and working environment. since november 2019, the coronavirus (sars-cov-2) has been spreading around the globe leading to a pandemic with more than 4,5 million recorded cases and more than 300 000 deaths worldwide recorded on 15 may 2020 (worldometer, 2020) . countries worldwide are testing their populations to estimate the number of people with active virus infection and the number of those who have recovered from it. it is highly recommended to prioritise testing hospitalised patients, healthcare facility workers, workers in congregate living settings, first responders, residents in long-term care facilities and generally persons with symptoms of (potential) covid-19 infection using rt-pcr tests (centers for disease control and prevention, 2020) . to estimate the number of people previously exposed to and/or infected with the virus, serological immunoassay tests are currently the best option, especially due to lower cost and shorter amount of time needed to obtain the results (johns hopkins center for health security, 2020). the first case of covid-19 in the republic of croatia was reported in late february. to prevent the spreading and exponential growth of the disease, restrictive measures were introduced by the croatian government on 19 march 2020 (koronavirus.hr, 2020a (koronavirus.hr, , 2020b . from 23 march 2020, leaving the place of residence was also prohibited (koronavirus.hr, 2020c) . with such restrictive measures, croatia earned first place on the stringency scale provided by the oxford covid-19 government response tracker on 26 march (hale & webster, 2020) . among mandatory protective measures, citizens were continuously provided with recommendations for personal protection, including social distancing (1 meter in open areas, 2 meters in closed areas), wearing face masks, maintaining personal hygiene, etc. along with the national measures (koronavirus.hr, 2020a (koronavirus.hr, , 2020b (koronavirus.hr, , 2020c , many companies introduced additional measures to protect the health of employees further and to maintain the manufacture. such is the case for div group, specialised in shipbuilding and production and trade of screws and mechanical parts, which introduced serological testing for employees using rapid serological immunoassay, as a health protection element within their corporate security system (div group, 2020; jerković et al., 2020) . although the findings of serological testing for covid-19 can be an essential part of investigating the disease, the tests vary in sensitivity and specificity and also produce false negative and false positive results (long et al., 2020; west et al., 2020) . these issues impose a danger to not only the health of tested individuals and communities but can also reduce the positive effects of national . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 17, 2020 . . https://doi.org/10.1101 health policies and protective or restrictive measures necessary for the containment of the disease (lippi et al., 2020) . this can be especially devastating as some health experts worry that testing populations and providing them with a knowledge of their health and/or immunity status regarding covid-19 could lead to yet unexplored psychological and behavioural effects (green et al., 2020) . the aforementioned psychological and behavioural effects have already been investigated concerning negative test results received in various screenings. the concerns regarding these effects showed that people would, after receiving negative test results in a screening program, perceive they have a lower risk of developing the disease they were tested for and thus less likely take precautions not to get sick in future (larsen et al., 2007; marteau et al., 1996) . a systematic review included eight studies on screening programs for diseases linked to lifestyle behaviours (type 2 diabetes, breast, bowel, lung and cervical cancer, and abdominal aortic aneurysm) to determine the post-screening changes in behaviours, attitudes, and emotions. the study showed that negative screening results are unlikely to cause changes in observed characteristics and to have a negative impact on behaviour (cooper et al., 2017) . nevertheless, since covid-19 is a novel disease whose spread is most effectively prevented by maintaining social distancing, community consciousness, personal protection and hygiene practices (lakshmi priyadarsini & suresh, 2020)behaviours that are most dependent on the conscientiousness and self-control of all individuals, it is of utmost importance to examine the behaviours and attitudes of people who received negative test results. furthermore, these factors are vital in a specific working environment where interpersonal contact cannot be avoided entirely due to production characteristics. in these settings, changes in behaviour and attitudes of workers could impact the general psychological environment and, most importantly, the health of company workers and their families. thus, this study aims at investigating the changes in personal attitudes and behaviour of div group industry workers before and after receiving negative serological test results for sars-cov-2 antibodies. from may 10 to may 15, 2020, we conducted a survey of div group industry workers in split-dalmatia county, croatia, who were previously tested for sars-cov-2 antibodies by rapid immunoassays. the previous serological screening was conducted from april 23 to april 28, 2020, in collaboration with the clinical department for pathology, forensic medicine and cytology, university hospital centre split and university department of forensic sciences, university of split (split, croatia). the named testing comprised 1316 participants and it was the first mass testing in the republic of croatia, and, to the authors' knowledge, one of the first and largest studies on the corporative level in the world at that time (jerković et al., 2020) . that study analysed the test results of 1316 participants, revealing that only 0.99% of participants (95% ci 0.53-1.68) were positive for sars-cov-2 antibodies (jerković et al., 2020) . the div group facility in split employs about 2200 people, which makes them the second largest employer in the county. the split facility employee structure includes those working in production, as well as management and administration (div group, 2020; jerković et al., 2020) . to examine if the test results affected participants' attitudes and behaviour, we constructed a short questionnaire and surveyed the employees, with the permission of the management of the company. the companies' occupation safety officers distributed the questionnaire to the different company departments, including the management, administrative, and production staff. they visited different departments separately and offered employees that participated in the screening voluntary participation in the study. as only a small proportion (0.99%) of employees were tested positive for antibodies, we included only those with negative test results. the questionnaire had six parts: (1) information of the study and informed consent; (2) general demographic data and test results; (3) participants' general attitudes towards covid-19; (4) participants' protective behaviour and fear from the disease prior to testing; (5) participants' protective behaviour and fear from the disease after the testing; and (6) the factors related to compliance with personal protection measures. the general and demographic questions included gender, age, test results (negative / igg positive/ igm positive / igm + igg positive), and participants level of education. other personal data were not included to ensure the participants' anonymity. the third part of the questionnaire included the questions on participants' perception of the disease and its severity, as well as their attitudes on the protective and restrictive measurements given on the national and the company level. this question section provided seven statements that participants should have rated on a five-level likert scale for agreement (1 = strongly disagree; to 5 = strongly agree). in the fourth and the fifth part of the questionnaire, the participants were asked about their anxiety and fear of the covid-19, compliance with restrictive measures and application of protective equipment before and after the testing. this section was composed of two sub-sections. the first one included nine statements regarding the participants' fear and perception of their environment, that participants were asked to rate on a five-level likert scale for agreement (1 = strongly disagree; to 5 = strongly agree). in the second sub-section, participants were asked to rate their frequency of obeying the restrictive measurements and applying the personal protective equipment. it included four statements with responses on a five-level likert scale for frequency (1 = never; to 5 = very often). in the final section, the participants were provided with four statements about factors that influence their adherence to the restrictive and protective measures, including the serological test results and level of actual restrictive measures and recommendations. they were also asked to select the one four statements that best suited their views. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131482 doi: medrxiv preprint categorical variables, including the gender, education level, and factors affecting adherence to the protective measures, were given as frequencies and percentages. for the remaining variables, we provided the mean values with 95% confidence intervals. differences in categorical variables were examined using the chi-squared test, while the differences in participants' responses before and after the testing were examined using a paired-samples t-test. due to the increased number of multiple comparisons (n=14), we set statistical significance at p ≤ 0.004 (bonferroni correction). all analyses were performed with jasp 0.12.1 (jasp team, 2020). the sample comprised 200 participants (68% men; median age = 43, interquartile range = 21). most of them had undergraduate or graduate education (47.7%) or completed secondary education (32.7%), while fewer participants completed non-university college or professional studies (18.6%). there were only two participants with primary education (1%), and one answer was missing. most participants perceived covid-19 as a dangerous disease and reported that restrictive measures and protective guidelines conducted on a national and company level were efficient and appropriate (table 1) . *the response to the statements ranged from 1strongly disagree; 2disagree; 3neutral; 4agree; 5strongly agree. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10. 1101 on average, low levels of fear related to infection or infecting others with covid-19 were observed both before and after the testing (table 2 , statements 1-6). adherence to protective measures was also high prior to and post-testing (table 2, statements 7-9). nonetheless, changes in participants' behaviour and attitudes before and after the testing were statistically significant for most variables. suspicions and fear that a person or people in their physical vicinity were infected were significantly reduced. however, participants' perception of other peoples' adherence to measures did not change significantly (table 2, statements 7-8). the response to the statements ranged from 1strongly disagree; 2disagree; 3neutral; 4agree; 5strongly agree. *paired-samples t-test. †statistically significant values are in bold. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10. 1101 the participants on average showed a high frequency of adherence to protective measures and restrictions (table 3) . when they were asked about their pre-test and post-test adherence frequencies, they reported maintaining the application of personal protective equipment on the almost same level, but lower adherence to social distancing (table 3 , statements 2-4). although the participants reported changes in behaviour and attitudes before and after receiving the test results, most of the participants did not attribute their behaviour to the test itself but rather to the level of company and national protective measures (table 4) . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020 . . https://doi.org/10.1101 the results of the present study showed that negative serological test result is associated with changes in the behaviour and attitudes of participants, but not to the extent that would lead to irresponsible or dangerous behaviour. to the best of our knowledge, this is the first study that investigates the changes before and after receiving negative serological covid-19 test results on behaviours and attitudes connected to this disease. the results of this study indicate that the levels of fear of being infected or infecting others with covid-19, as well as behaviours regarding adherence to protective measure, changed significantly in the timeframe after receiving negative test results. however, the subjects' fear of infection and/or infecting was initially at the lower moderate to low level and dropped to an even lower level after the testing. although the disease had a pandemic character and was at that time relatively unexplored, the situation at the company was under control as the company introduced protective measures at the end of february. this was influenced by the experience of their partners . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10. 1101 in china and italy, which were at the time global pandemic hotspots. these measures, along with national protective measures, introduced by the second half of march (koronavirus.hr, 2020a (koronavirus.hr, , 2020b (koronavirus.hr, , 2020c , were probably beneficial for the participants' lower level of fear. the frequency of positive behaviour related to social distancing also reduced after the testing, but still remained high. in contrast, results indicate no significant change in behaviours related to wearing protective equipment, masks, and gloves which were highly adherent. both of the findings could be attributed to the stimulating climate in the company and society that raised awareness of protective and restrictive measures. we also did not find changes in the perception of colleagues' compliance with protective measures pre-and post-testing. these findings might additionally support the participants' responsibility and conscientiousness, regardless of their test results. this is also evident from the fact that most of them attributed their behaviour less to the tests results but more to the current level of restrictive measures. studies on screening for various diseases such as different types of cancer, sexually transmitted diseases (stds), diabetes, etc. have been conducted to determine their psychological and behavioural effects but also the perception of ones' health and future risk of getting sick (ashraf et al., 2009; berstad et al., 2015; collins et al., 2011; eborall et al., 2007; sznitman et al., 2010) . the recent review on these types of studies shows a small decrease in perceived risk of the disease screened for, slightly lower levels of anxiety or worry in the screen negative group, and highlights that out of 28 studies only five showed an unfavourable change in the negatively screened groups' health-related behaviours (cooper et al., 2017) . although our study findings indicate changes of similar direction and extent, it is difficult to compare its results to abovementioned studies. this is due to the very nature of covid-19, an infectious disease spread primarily by human contact and interaction. the other diseases populations are usually screened for are (ashraf et al., 2009; berstad et al., 2015; collins et al., 2011; eborall et al., 2007; sznitman et al., 2010) , except for stds, not transmittable. but the comparison is not possible even with stds since their transmittance is usually restricted to the most intimate of human interactions and thus limited. the cessation of infectious disease spreading such as covid-19 is impossible without necessary changes in human interactions and behaviour, which must be applied to all members of society. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10. 1101 the limitation of this study is that compared pre-and post-testing self-ratings were all given after the testing, thus potentially introducing a reporting bias. to obtain pre-testing measurements, it was only possible to survey the participants on the day of voluntary serological testing. from the organisational and protective standpoint, it was of utmost importance to minimise the time participants spent at the testing station to the time required for serological testing and completing a mandatory accompanying questionnaire on disease-related factors (jerković et al., 2020) . this would result in not only the prolonged absence of participants from their workplace but also their potentially increased exposure to the virus. since the period between testing and completing the survey questionnaire lasted a maximum of 21 days for each participant, by testing at a single point in time we relied on the participants' ability to recall recent behaviours and attitudes. while other studies on the impact of negative screening results repeated measurements after several months or years (cooper et al., 2017) , this was not possible for this study due to the very nature of covid-19 as well as differing levels of national restrictive measures. the additional limitation of this study was the lack of a control group. the covid-19 screening in div group in split (jerković et al., 2020) resulted in an insufficient number of positive participants to represent a separate group of subjects in research. therefore, due to the extremely low seroprevalence in the tested sample (about 1%), including positive participants would not provide relevant information for the scope of the study. also, having an adequate control group of non-tested participants was not possible since almost all div group industry workers in split were screened. surveying the general population for that purpose would not be appropriate, as div employees were immersed in an all-encompassing working atmosphere with special and more severe protection measures prescribed by the employer, that were introduced considerably earlier than the national measures. however, even if we would have detected that general population control group had adhered less to the protective measures, due to social climate influenced by the smaller number of newly infected or the current level of national restrictions, it could have only implied that test results had even fewer negative consequences on behaviour related to protective measures. in conclusion, our study results indicate that covid-19 serological testing does not impose an additional threat regarding the potentially irresponsible or risk behaviour in an environment where . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 17, 2020. . in front of you is a 4-page questionnaire which aims to examine the impact of the results of serological testing for sars-cov antibodies on personal attitudes and behaviour of participants. this survey is conducted by the university department of forensic sciences in cooperation with the medical faculty of the university of split and the clinical department of pathology, forensic medicine and cytology of the clinical hospital centre split. the research was approved by the ethics committee of the university department of forensic sciences (2181-227-05-12-19-0003; 024-04 / 19-03 / 00007). the survey is conducted anonymously, and your personal data will not be available to researchers or your employer, and the results will be used exclusively for research purposes. it takes a maximum of 10 minutes to complete the questionnaire. effect of ct screening on smoking habits at 1-year follow-up in the danish lung cancer screening trial (dlcst) long-term lifestyle changes after colorectal cancer screening: randomised controlled trial evaluating and testing persons for coronavirus disease emotional impact of screening: a systematic review and meta-analysis do negative screening test results cause false reassurance? a systematic review psychological impact of screening for type 2 diabetes: controlled trial and comparative study embedded in the addition (cambridge) randomised controlled trial molecular and antibody point-of-care tests to support the screening, diagnosis and monitoring of covid-19 oxford covid-19 government response tracker sars-cov-2 antibody seroprevalence in industry workers in split-dalmatia and serology-based tests for covid-19 odluka o mjerama ograničavanja društvenih okupljanja, rada u trgovini, uslužnih djelatnosti i održavanja sportskih i kulturnih događanja odluka o privremenoj zabrani prelaska graničnih prijelaza republike hrvatske odluka o zabrani napuštanja mjesta prebivališta i stalnog boravka u rh factors influencing the epidemiological characteristics of pandemic covid 19: a tism approach impact of colorectal cancer screening on future lifestyle choices: a three-year randomized controlled trial potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (covid-19) diagnosis of the coronavirus disease (covid-19): rrt-pcr or ct? the psychological impact of cardiovascular screening and intervention in primary care: a problem of false reassurance? british family heart study group the impact of communitybased sexually transmitted infection screening results on sexual risk behaviors of african american adolescents covid-19 testing: the threat of falsenegative results covid-19 coronavirus pandemic the authors would like to thank div group company and tomislav debeljak, along with all study participants. we are especially thankful to boško ramljak, marija čečuk and ivica sinovčić for their assistance in organisation and data collection. protective measures are efficient. additionally, they might be beneficial to reducing the level of fear in society and the working environment. this article does not represent in whole or in part the views of the authors' institutions. however, it does express those of the authors. key: cord-334274-4jee19hx authors: waelde, k. title: how to remove the testing bias in cov-2 statistics date: 2020-10-16 journal: nan doi: 10.1101/2020.10.14.20212431 sha: doc_id: 334274 cord_uid: 4jee19hx background. public health measures and private behaviour are based on reported numbers of sars-cov-2 infections. some argue that testing influences the confirmed number of infections. objectives/methods. do time series on reported infections and the number of tests allow one to draw conclusions about actual infection numbers? a sir model is presented where the true numbers of susceptible, infectious and removed individuals are unobserved. testing is also modelled. results. official confirmed infection numbers are likely to be biased and cannot be compared over time. the bias occurs because of different reasons for testing (e.g. by symptoms, representative or testing travellers). the paper illustrates the bias and works out the effect of the number of tests on the number of reported cases. the paper also shows that the positive rate (the ratio of positive tests to the total number of tests) is uninformative in the presence of non-representative testing. conclusions. a severity index for epidemics is proposed that is comparable over time. this index is based on covid-19 cases and can be obtained if the reason for testing is known. background. statistics have gained a lot in reputation during the covid-19 pandemic. almost everybody on this globe follows numbers and studies "the curve"on recorded cases, on daily increases or on incidences of cov-2 infections. the open question. what do these numbers mean? what does it mean that we talk about "a second wave"? intuitive interpretations of "the curve" suggest that the higher the number of new infections, say in a country, the more severe the epidemic is in this country. is this interpretation correct? when the number of infections increases, decision makers start to discuss additional or tougher public health measures. is this policy approach appropriate? our message. reported numbers of cov-2 infections are probably not comparable over time. when public health authorities report x new cases on some day in october 2020, these x new cases do not have the same meaning as x new cases in april, may or june 2020. the bias results from di¤erent testing rules that are applied simultaneously. private and public decision making should not be based on time series of cov-2-infections as the latter do not provide information about the true epidemic dynamics in a country. if the reason for testing was known, a unbiased measure of the severity of an epidemic could be computed easily. our framework. we present a theoretical framework that allows one to understand the link between testing and the number of reported infections. we extend the classic sir model (kermack and mckendrick, 1927, hethcote, 2000) to allow for asymptomatic cases and for testing. 2 our fundamental assumption states that the true numbers of susceptible, infectious and removed individuals are not observed. results. the reason for the intertemporal bias consists in relative changes of test regimes. if a society always employed only one rule when tests are taken, e.g. "test for sars-cov-2 in the presence of a certain set of symptoms", then infection numbers would be comparable over time. if tests are undertaken simultaneously, e.g. "test in the presence of symptoms"but also "test travellers without symptoms", and the relative frequency of tests changes, a comparison of the number of reported infections over time bears no meaning. the paper illustrates the bias by a "second wave"in reported cases which -by true epidemiological dynamics -is not a second wave. understanding this bias also provides an answer to one of the most frequently asked question when it comes to understanding reported infection numbers: what is the role of testing? do we observe a lot of reported infections only because we test a lot? should we believe claims such as "if we test half as much, we have half as many cases". this paper will provide a precise answer to what extent the reported number of infections is determined by the number of tests in a causal sense. the answer in a nutshell: if tests are undertaken because of symptoms, there is no causal e¤ect from the number of tests on the number of reported infections. if tests are undertaken for other reasons (travellers, representative testing), the number of reported infections go up simply because there is more testing. 3 we show that time series on the number of tests and time series on reported infections do not allow one to obtain information about the true state of an epidemic. we also study the positive rate as the ratio of the number of positive tests to the total number of tests. the positive rate is informative if we undertook representative testing only. the positive rate is not informative about true epidemiological dynamics when there are several reasons for testing. understanding the biases also allows us to understand how to correct for it. the paper presents a severity index for an epidemic that is unbiased. one can obtain this index in two ways: record the reason why a test was undertaken or count only the covid-19 cases. such an index should be used when thinking about relaxing or reimposing public health measures. testing is important for detecting infectious individuals, counting covid-19 cases is important for private and public decision making. structure of paper. the next section presents the model. section 3 shows biased and unbiased measures of the true but unobserved dynamics of an epidemic. it also studies the (lack of) informational content of time series on reported infections and time series on the number of tests, and the properties of the positive rate. it …nally presents an unbiased severity index. the conclusion summarizes. the basic assumption of our extension of the susceptible-infectious-removed (sir) model consists of the belief that true infections dynamics are not observable. simultaneous testing of an entire population or weekly representative testing is not feasible -at least given current technological, administrative and political constraints. this section therefore …rst describes the true but unobserved infection dynamics, then introduces tests into this framework and …nally computes the number of reported infections within this framework. the classic sir model we study a population of …xed size p: individuals can be in three states as in a standard sir model. the number of individuals that are susceptible to infection is denoted bys (t) : this number is unobservable to the public and to health authorities. the numbers of infectious and removed (i.e. recovered or deceased) individuals are denoted byĩ (t) andr (t) ; respectively. we assume that individuals are immune and non-infectious after being removed. let the (expected) number of individuals in the state of being susceptible at a point in time t be denoted bys (t) : 4 the number of susceptible individuals falls according to where r is a constant and c (t) rĩ (t) can be called the individual infection rate. it captures the idea that the risk of becoming infected is the greater, the higher the number of infectious individuals. 5 merging individual recovery rate and death into one constant , the number of on the bias due to tests but discuss perceptions brie ‡y below. 4 we write expected number as ordinary di¤erential equations in sir models could or should be understood as kolmogorov backward equations describing means of continuous time markov chains. see karlin and talyer (1998) or ross (1996) for an introduction. 5 in the tradition of diamand-mortensen-pissarides search and matching models in economics (diamond, 1982 , mortensen, 1982 , and pissarides, 1985 , this individual infection rate can be expressed capturing similar ideas as in a matching function: it should not only increase in the number of infectious individuals but also fall in the number of susceptible individuals. the latter reduces the probability that a random contact is infectious. see donsimoni et al. (2020a) for an implementation. 3 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . infectious individuals changes according to finally, as a residual, the number of removed individuals rises over time according to dr (t) =dt = ĩ (t). we illustrate the dynamics in the following …gure, employed also later on. the true infection dynamics (a simple sir model) we can easily integrate asymptomatic cases into this framework. we split the true number of infectious individuals described in (2) into symptomatic and asymptomatic cases, this allows us to capture the infection process in (2) by two distinct di¤erential equations. when hold, (2) holds as well. individual infection rates are now de…ned as symp c the epidemiological idea behind these equations is simple. the rate with which one individual becomes infected is the same for everybody and given by rĩ (t) : the higher the number of infectious individuals in society,ĩ (t) ; the higher the rate with which one individual gets infected. it then depends on various, at this point partially unknown, physiological conditions of the infected individual whether they develop symptoms or not. we denote the share of individuals that develop symptoms by s: we assume this share is constant. 6 epidemiological dynamics this completes the description of the model. let us now describe how we can understand (unobserved) epidemiological dynamics. we start with some initial condition fors (t). a good candidate would bys (0) = p; i.e. the entire population of size p is susceptible to being infected and become infectious. initially, there are very few infectious individuals, say, there are two,ĩ symp (t) =ĩ asymp (t) = 1: given infection rates (6a) and (6b) and parameters, the number of infectious symptomatic and asymptomatic cases evolves according to (4) and (5). 6 the model neglects the e¤ect of quarantine. if infectious individuals know about their status and therefore stay in quarantine, they should be removed fromĩ (t) or at least get a lower weight in (6). infectious individuals are removed from being infectious at a rate : 7 the number of susceptible individuals follows (1). the epidemic is over with herd immunity, i.e.s = 0 at some point (far in the future) or when recovery is su¢ ciently fast relative to in ‡ows such thatĩ = 0: 8 the epidemic is heading towards an end when d dtĩ symp (t) < 0 and d dtĩ asymp (t) < 0; i.e. the number of infectious individuals falls. 9 we abstract from public health measures and their e¤ects (as studied e.g. by dehning et al., 2020 or donsimoni et al., 2020a . if we wanted to include them, we could allow public health measures to a¤ect r in the individual infection rate in (2). 10 to understand the e¤ects of tests, we now introduce testing into our sir model. the following …gure displays all unobserved quantities in the model by dashed lines. the red circles represent the standard sir model illustrated in …gure 1. testing can take place for a variety of reasons described in test strategies adopted by various countries. the reasons for tests we take into account at this point is testing due to the presence of typical symptoms, representative testing and testing travellers. while testing by symptoms and representative testing is well-de…ned, testing travellers is really only an example for a larger type of test. this example covers all tests that are applied to a group de…ned by certain characteristics which, however, are not representative of the population as a whole. other examples of this non-representative testing include testing of soccer players, testing in retirement homes or their visitors, testing in hot spots or testing contact persons of infected individuals. the sir model with testing 7 it would be straightforward to assume, e.g. asym p > sym p . this would capture the idea that asymptomatic cases recover faster than symptomatic cases. we ignore this extension as this distinction would not a¤ect our main argument. 8 for analytical solutions of the classic sir model, showing this aspect most clearly, see harko et al. (2014) or toda (2020) . 9 this condition is related to the widely discussed reproduction number. 10 current applications of the sir model also badly neglect the non-exponential distribution in various states. it is well-known (e.g. linton et al., 2020 or lauer et al., 2020 that incubation time is (approximately) lognormally distributed. it is now also understood that the reporting delay per se and added to incubation time is also non-exponentially distributed (mitze et al., 2020, app. a.3) . the "chain trick" (hurtado and kirosingh, 2019) would allow to implement this numerically. meyer-herrmann (2018) employed a related struture but did not focus on densities of duration explicitly. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . https://doi.org/10.1101/2020.10.14.20212431 doi: medrxiv preprint testing by symptoms individuals can catch many diseases (or maybe better sets of symptoms) indexed by i = 1:::n. for simplicity, the above …gure displays only two diseases (1 and 2) and covid-19. the number of individuals that have a disease i and go to a doctor on day t is d i (t) : an individuals becomes sick with an arrival rate i and recovers from this speci…c sickness i with a rate of i . for clarity, we add a symptomatic sars-cov-2 infection to this list of diseases. the individual is infected and develops symptoms with rate symp c (t) ; which we know from (6a), and is removed with rate c : the number of symptomatic sars-cov-2 individuals isĩ symp (t) from (4). there is a certain probability p i that a doctor performs a test, given a set of symptoms i: this probability re ‡ects the subjective evaluation of the general practitioner (gp) whether certain symptoms are likely to be related to sars-cov-2. the probability to get tested with symptomatic sars-cov-2 infection (which the gp of course does cannot diagnose without a test) is denoted by p c : hence, the (average or expected) number of tests that are performed at time t due to consulting a doctor is given by the second equality replaces the number of tests by the number of sick individuals per disease times the probability that this individuals is tested. 11 note that, apart from population size p; the number of tests taken because of the presence of symptoms, t d (t) ; is the …rst variable that is observed. if health authorities collected information why a test was performed (set of symptoms that can be observed by a gp), we would observe t d i (t) and t d c : if not, we observe t d (t) only. tests can be performed for a variety of reasons. one consists in testing travellers, another consists in tests for scienti…c reasons and so on. theses tests are not related to symptoms. taking the example of representative tests, the tests are applied to the population as a whole. the number of tests is chosen by public authorities, scientists, available funds, capacity considerations and other. in any case, it is independent of infection-characteristics of the population. concerning representative testing, we denote the number of tests of this type undertaken at t by t r (t) : when it comes to travelers, we denote the number of tests by t t (t) : summarizing, the total number of tests being undertaken in our model is given by the sum of tests due to symptoms, t d (t), representative tests t r (t) and testing travellers, t t (t) ; the second equality employs the number of tests by symptoms from (7). the equation thereby reemphasizes the endogeneity of the number of tests by symptoms, t d (t) is determined by the number of symptoms occurring in a country or region, and the exogeneity of other reasons for testing, t r (t) and t t (t) : the latter are not determined by symptoms. the number of reported infections at time t is given by the sum of reported infections split by the reasons for testing introduced above, 11 in a broader interpretation, one could understand p i and p c as the probabilities that an individual gets tested and that they go to the doctor. no test is ever performed if individuals with symptoms stay at home. 6 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . https://doi.org/10.1101/2020.10.14.20212431 doi: medrxiv preprint testing by symptoms as we are perfectly informed in our theoretical world about the (expected) number of cov-2 infections and other diseases, we know that the number of positive cov-2 tests is zero for all diseases, 12 i i = 0: individuals have covid-19 related symptoms because they caught a cold, they have the ‡u or other. the probability that a cov-2 infected individual has a positive test is set equal to one (ignoring false negative tests). the number of positive tests for individuals that are infected with cov-2 is therefore identical to the number of tests, testing for other reasons the probability that a representative test is positive is denoted by p r (t) : this probability is a function of the true underlying and unobserved infection dynamics. if the sample chosen is truly representative, then the probability for a positive test is given by hence, representative tests make the true numberĩ (t) of infectious individuals visible for the moment at which the tests are undertaken. this true number includes symptomatic and asymptomatic cases as in (3). the probability that a test of travellers is positive depends on a multitude of determinants among which region traveled to and behaviour of the traveller. we denote the probability that such a test is positive by p t (t). we consider this probability to be exogenous to our analysis. a …rst step towards the total number of reported infections starts from (9) and takes (10) and (11) into account, this is also the expression displayed in …gure 2 between 'cov-2 tests'and 'con…rmed infections'. reported infections come from testing cov-2 individuals with symptoms, from representative testing and from other sources such as travellers. employing t d c (t) = p cĩsymp (t) from (7) and p r (t) from (12), the number of reported infections can be written as 3 unbiased and biased reporting is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . https://doi.org/10.1101/2020.10.14.20212431 doi: medrxiv preprint our central equation is (13). the reported number of infections would be unbiased if only tests by symptoms were undertaken. reported infections (with t r (t) = t t (t) = 0) would by (13) amount to when the reported number of infections i (t) goes up, one would be certain that the unobserved number of symptomatic cov-2 infectionsĩ symp (t) would go up as well. the more infections are reported, the more severe the epidemic is. this equation also shows under which circumstances the number of tests does not have a causal e¤ect on the number of reported infections. if tests are undertaken according to a rule that makes testing dependent on something else (e.g. the presence of symptoms), the number of tests itself is determined by the number of symptoms. hence, while the number of tests and reported infections are correlated, the causal underlying factor is the number of patients visiting a physician with cov-2 related symptoms. a second example of unbiased testing is (exclusive) representative testing. when only representative testing is undertaken, the number of reported infections (with t d c = t t (t) = 0) from (13) amounts to here, the number of reported infections, i (t) ; does rise in the number of tests, t r (t). the more we test, the higher the number of cases. yet, representative testing is (of course) the gold standard of testing. the ratio of positive cases to the number of tests yields the share of infections in the population, 13 this share is driven byĩ (t) which shows that (i) representative testing provides a snapshot at this point in time t of the current epidemic dynamics and that (ii) representative testing provides a measure of overall infections, i.e. symptomatic and asymptomatic ones. we have seen two examples of unbiased reporting, one for symptomatic infections, one for all infections. they show that the question whether the number of reported cases rises in the number of tests is not as important as the question whether the type of testing provides useful information. in the …rst example, the claim that more tests increase the number of reported infections is meaningless as the number of tests is not chosen. in the second example, the number of positive cases rises in the number of tests but the ratio of these two quantities is highly informative. illustrating a bias now imagine several types of testing are undertaken simultaneously. the number of reported infections at t is then given by the full expression in (13). consider …rst the case of symptomatic and representative testing. the number of reported cases (with t t (t) = 0) is then 13 this ratio is an example of the 'positive rate'. we will study it in more detail below. 8 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . imagine someone (the government, researchers, other) decide to undertake more representative testing, i.e. t r (t) goes up. this means that i (t) increases even though there is no change in the true numberĩ symp (t) of symptomatic cases. there is also no change in the true number i (t) of symptomatic and asymptomatic cases. whoever perceives the reported number i (t) is led to believe that something fundamental has changed within the epidemiological dynamics. but this is of course not true. the reported number goes up simply because more tests were undertaken. 14 can we gain some information out of this expression if we divide it by the number of tests t r (t) as it had turned out to be very useful in the case of exclusive representative testing in (15)? we would obtain which does contain the informative infection shareĩ (t) =p as the second term on the right hand side. but the …rst term does not have a meaningful interpretation and neither does the entire term. let us illustrate the potential bias by looking at the third type of testing considered heretesting travellers. the number of reported infections according to (14) in the case of testing by symptoms and testing travellers reads we assume that no testing of travellers took place at the beginning of the pandemic. at some later point (as of t = 60 in our …gure below), the number of tests per day, t (t) ; increases linearly in time. to make this example as close to public and common displays of infection dynamics, let us look at "the curve" represented in …gure 3 by numbers of infected individuals taking recovery into account. 15 looking at (16) shows that a further source of bias, brie ‡y mentioned earlier, can easily be identi…ed. imagine the general perception of gps changes over time concerning . then a gp might be initially sceptical, i.e. p c is low, then become more aware of health risks implied by cov-2, p c goes up, to then maybe during some other period become more reluctand again. if these changes in individual perceptions are not entirely idiosyncratic but driven by the overall attention in society to an epidemic, the number of reported infections would change independently of the true number of infections,ĩ sym p (t) orĩ (t) : 15 one could draw similar …gures with new infections per day or the number of individuals ever infected. the basic argument would remain the same. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . looking at …gure 3 we …rst focus on the blue dashed curve forĩ symp (t) ; the true number of symptomatic sars-cov-2 infections. we chose parameters such that the epidemic is coming to a halt after around 120 units of time (plotted on the horizontal axis). the green curve plots the number of reported infections i d (t) from (14) where testing takes place only in the presence of symptoms. finally, the red curve is an example of a bias in the reported number of infections. it occurs as positive tests from testing travellers are added to tests by symptoms as in (17). we see that this example displays what looks like a "second wave": reported numbers of infections go up again as of t = 100: by construction, however, this second wave is caused by misinterpretation of the reported number of infections. let us stress that we do not claim that the second wave is a statistical artefact due to testing strategies. it could be a statistical artefact, however. the conclusion shows how to obtain a severity index for an epidemic that is not prone to causing arti…cial results and which data is needed to compute such an index. a non-application to germany consider the case of germany. figure 4 shows the number of tests per week and the number of reported infections. when we look at the time series for all tests in this …gure, it corresponds to t (t) from (8). when we consider the reported number of infections per week in germany, it looks as displayed in the right panel of the above …gure. this time series corresponds to i (t) from (13). can we conclude anything from these two time series about the true dynamics of the epidemic, i.e. can we draw conclusions aboutĩ symp (t) orĩ (t)? 16 technically speaking, we have two equations, (8) and (13), reproduced here for convenience, about which the public has access to two variables, i (t) and t (t) : it seems obvious thatunless we want to make a lot of untested assumptions -o¢ cial statistics do not allow to draw 16 one might be tempted to argue that data on the positive rate in (19) should also be useful. as the positive rate is simply i (t) divided by t (t) ; it does not provide additional information. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . any conclusion about the severity of the epidemic. the right-hand side contains at least three unknowns (e.g. tests classi…ed by reason of testing, t d (t) ; t r (t), t t (t)) and two equations with three unknowns usually do not have a solution. hence, from currently available data, true epidemic dynamics,ĩ symp (t) orĩ (t) ; cannot be understood. 17 the positive rate the positive rate is the ratio of con…rmed infections to number of tests, s pos (t) i (t) =t (t) : this statistic is often discussed in the media and elsewhere (see e.g. our world in data, 2020). in our model, (13) and (8) imply what does this positive rate tell us? some argue that a rising positive rate is a sign of the epidemic 'getting worse'. if we understand the latter by a rise in the number of unobserved infections,ĩ (t) ; or the number of infections with symptoms,ĩ symp (t) ; this statement is true if we undertake representative testing only, t d c = t t (t) = 0 as in (15). in this case, when the observed positive rate s pos (t) rises, this clearly indicates that the number of unobserved infectionsĩ (t) is higher. and so would beĩ symp (t) ; whether individuals with symptoms go to a doctor or not, given the constant share s of symptomatic cases in (6a). 18 does this conclusion hold more generally, i.e. for the full expression (19) when tests are undertaken for many reasons? let us assume we only undertake tests due to symptoms and due to travelling, t r (t) = 0. 19 then the positive rate (19) reads when we increase the number of tests for travellers, we …nd (see appendix) this result is easy to understand technically and has the usual structure: when we increase a summand (t t (t) here) that appears in a fraction in numerator and denominator, the sign of the derivative depends on the other summands ( n i=1 p i d i (t) and p cĩsymp (t) in this case). as the summand is multiplied by p t (t) in the numerator, this probability appears in the condition as well. in terms of epidemiological content, the derivative says that the positive ratio can rise or fall when we increase the number of tests for travellers (or related reasons mentioned below …gure 2). testing increases the positive rate if the number of tests undertaken due to symptoms 17 this paper is about conceptional issues related to the …nding an unbiased estimator for an unobserved time series. we ignore practical data problems. the latter include the fact that the number of tests displayed in …g. 4 is not coming from the same sample of tests that yields the number of infections in this …gure. this would have to be taken into account in any application. 18 the appendix shows that the positive rate is also informative and identical toĩ (t) =p if travellers (or visitors of retirement homes, or contact persons of a positively tested individual or visitors of public events etc) are representative. this assumption is questionable, however. 19 quantitatively speaking, representative testing is probably very small relative to other reasons for testing. that are not cov-2 related, n i=1 p i d i (t) ; exceeds the number of tests undertaken because of symptoms related to cov-2, p cĩsymp (t) ; corrected for the probability that a traveller test is positive. while an intuitive interpretation of this condition seems to be a challenge, the condition nevertheless conveys a clear message: it contradicts that a rising positive rate implies a 'worse' epidemic state. we see that when t t (t) goes up and the positive rate goes up, this does not mean anything regarding the dynamics ofĩ symp (t) orĩ (t) : the same is true, of course, when t t (t) goes up and the positive rate goes down. the positive rate is not informative. this …nding also applies to a somewhat more precise statement of the above conjecture. some claim that a rising positive rate in the presence of more tests does show that infections must go up. when we increase t t (t) ; the number of tests goes up. when (21) holds, the positive rate goes up. however, we do not learn anything about infections with or without symptoms. tests go up and the positive rate goes up simply because we test more. we now propose an index for the severity of an epidemic which is comparable over time. the model illustrated in …gure 2 tells us what is needed: the index should be closely related to the number of symptoms in society. as tests that capture these symptoms are those that are undertaken because of symptoms, the index is simply i d (t) as in (14). an alternative would consist in representative testing. while the number of reported cases depends causally on the number of tests, the ratio of reported cases to number of tests is an unbiased estimator of the true epidemic dynamics as shown in (15). as regular representative testing, say with a weekly frequency, is not feasible, the only realistic severity index is i d (t) from (14). very simply speaking: if a severity index for an epidemic is desired that is comparable over time, we should test for cov-2 but count covid-19 cases. this should be done at all levels starting from the gp, through hospital admissions and patients in intensive care and, …nally, counting deaths associated with covid-19. what do these …ndings mean in practice? data which is currently available for the public (see e.g. rki, 2020 for germany or our world in data, 2020, for many other countries in the world) does break down the total numbers of tests by origin (gp, hospital and other) and region. unfortunately, this classi…cation does not relate to the reason for testing and the latter is required to infer the true infection dynamics. what should be done to quantify the relevance of the bias? local health authorities in germany collect the names of individuals with con…rmed cov-2 infections. if additional information on symptoms, that is already being collected (the reporting form 20 allows for ticks on fever, coughing and the like), was made available to the public or scientists, the bias could be computed easily. 21 we currently know covid-19 cases for intensive care in hospitals, but this data is not yet easily accessible (see https://www.intensivregister.de). while only a fraction of covid-19 cases ends up in intensive care, this number might be more informative than cov-2 infections. the number of deaths associated with covid-19 is a further measure as would be excess mortality. while these are only partial measures of covid-19 dynamics, covid-19 measures (positive cov-2 tests with symptoms, number of all covid-19 patients in hospitals, not just intensive care) would provide a better basis for regional and local decision makers than cov-2 infection measures. if one day we know how strong the quantitative bias is, we could now hope that the bias is small. then the guidance given to society by the focus on cov-2 infections would have been correct. but even with a small bias, the focus on cov-2 infections should stop. we know that it is not the perfect measure. it rather biases expectation building (and emotional reactions) of individuals. hence, as soon as better covid-19 measures are available, the cov-2 measure should be replaced. the candidates are estimates of informative positive rates and (regional) time series on covid-19 cases (and not cov-2 infections). this would allow local politicians to base their decisions on intertemporally informative data, i.e. on local covid-19 cases. true epidemic dynamics are unobserved. no country, no health authority and no scientist knows the true number of cov-2 infections with or without symptoms for a given country. this is why testing is undertaken. testing is a means to measure true but unobserved epidemic dynamics. the counted number of cov-2 infections are not relevant for decision making, what matters is the true number of cov-2 infections. infections and the corresponding disease spreads when the true number of infections is high, not when the counted number of infections is high. we extend the classic sir model to take symptomatic and asymptomatic cases into account. more importantly, we treat cov-2 infections as unobserved in the sir model and model testing. we allow for various reasons for testing and focus on testing due to symptoms, representative testing and testing travellers. testing travellers is an example of non-representative and nonsymptom related testing and includes the testing of sports professionals, in retirement homes or their visitors, in hot spots or contact persons of infected individuals. we show that the presence of various reasons for testing biases the number of con…rmed cov-2 infections over time. the number of cov-2 infections cannot be compared intertemporally. we might observe more cov-2 infections today simply because we test more. however, the true number of infections might stay constant or even fall. we do not claim, in any sense, that our …ndings have empirical relevance. we simply do not know, at least given the data that are easily accessible to the public and given the data everybody observes (number of tests and number of reported infections) and on which all public health decisions are based, what the true epidemic dynamic is. we all look at a watch and we know that it is wrong. but we do not know how much it is wrong. it may be seconds, but it can also be hours. what are the positive lessons from this analysis? we propose an index which is unbiased over time. it is deceptively simple. count the number of covid-19 cases, not the number of cov-2 infections. if we knew the number of covid-19 cases, i.e. cov-2 infections with severe acute respiratory symptoms (sars), then we would know at least one part of epidemic dynamics (ĩ symp (t) in our model). let us stress that our …ndings are not an argument against testing. testing is important for identifying infectious individuals. they need to stay in quarantine in order to prevent the further spread of cov-2 infections. this helps to reduce covid-19 cases. testing is important -but adding up con…rmed infections from all sorts of tests is misleading. as long as the public focuses on all sources of positive cases, decisions by private individuals, …rms, journalists, scientists and politicians are badly informed. emotions, decisions and behaviour are misguided. this cannot be good for public health. decisions must be based on the number of covid-19 cases. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . https://doi.org/10.1101/2020.10.14.20212431 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . https://doi.org/10.1101/2020.10.14.20212431 doi: medrxiv preprint this sections contains derivations for the main text. when we ignore testing by symptoms, the positive rate (19) reads t r (t) + t t (t) : imagine travellers were representative, then p t (t) =ĩ (t) p and the positive rate would read s pos (t) =ĩ (t) p as in (20) for representative testing. under the assumption that travellers (or visitors of retirement homes, or contact persons of a positively tested individual or visitors of public events) are representative, the positive rate would re ‡ect the true epidemic dynamics as measured bỹ i (t) =p: the derivative of the positive rate in (21) we only take tests due to symptoms and due to travelling into account, t r (t) = 0. then the positive rate (19) reads s pos (t) = p cĩsymp (t) + p t (t) t t (t) n i=1 p i d i (t) + p cĩsymp (t) + t t (t) a + p t t t b + t t ; where the second equality de…nes a and b (for this appendix only) and suppresses time arguments to simplify notation. we compute ds pos dt t = p t b + t t a + p t t t (b + t t ) 2 > 0 , p t b + t t > a + p t t t , p t b > a: when we employ the de…nition of a and b; we obtain adding time arguments gives the condition in the main text. 16 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 16, 2020. . https://doi.org/10.1101/2020.10.14.20212431 doi: medrxiv preprint e¤ects of nonpharmaceutical interventions on covid-19 cases, deaths, and demand for hospital services in the uk: a modelling study inferring covid-19 spreading rates and potential change points for case number forecasts aggregate demand management in search equilibrium projecting the spread of covid19 for germany should contact bans have been lifted more in germany? a quantitative prediction of its e¤ects impact of non-pharmaceutical interventions (npis) to reduce covid19 mortality and healthcare demand exact analytical solutions of the susceptible-infected-recovered (sir) epidemic model and of the sir model with equal death and birth rates the mathematics of infectious diseases generalizations of the linear chain trick: incorporating more ‡exible dwell time distributions into mean …eld ode models an introduction to stochastic modeling proceedings of the royal society of london series a, containing papers of a mathematical and physical character the incubation period of coronavirus disease 2019 (covid-19) from publicly reported con…rmed cases: estimation and application incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data uses and abuses of mathematics in biology coronavirus (covid-19) testing mathematical models to guide pandemic response estimation of the cancer risk induced by therapies targeting stem cell replication and treatment recommendations face masks considerably reduce covid-19 cases in germany -a synthetic control method approach property rights and e¢ ciency in mating, racing, and related games estimating unobserved sars-cov-2 infections in the united states short-run equilibrium dynamics of unemployment vacancies, and real wages laborbasierte surveillance sars-cov-2 stochastic processes susceptible-infected-recovered (sir) dynamics of covid-19 and economic impact competing interests: there are no competing interests. data and materials availability: all data and software code key: cord-354005-q5nj0ku1 authors: richaud, m.; vermeersch, o.; dolez, p.i. title: specific testing of textiles for transportation date: 2017-09-29 journal: advanced characterization and testing of textiles doi: 10.1016/b978-0-08-100453-1.00015-5 sha: doc_id: 354005 cord_uid: q5nj0ku1 the use of textiles in transportation may be associated with the need to combine comfort and functionality. the evolution of each mean of transportation along with the increased awareness about the importance of passengers' safety pushed research and development forward towards a larger use of lightweight, fire-resistant, nontoxic, and durable textiles in transportation. this in turn led to the development of test methods assessing the different aspects of textiles in transportation. this chapter starts with a presentation of the transportation textile market. then, different aspects of textile testing relevant to the transportation industry are discussed: safety, flammability, hygiene, performance of composite parts, and durability. the chapter ends with considerations regarding future trends for textiles in transportation. the use of textiles in transportation may be associated with the need to combine comfort and functionality. sails were used in ships transporting goods; cushions and curtains were used in carriages or trains for the comfort of passengers; and airplane wings were made out of woven canvas for instance. the evolution of each means of transportation along with the increased awareness about the importance of passengers' safety pushed research and development forward towards a larger use of lightweight, fire-resistant, nontoxic, and durable textiles in transportation. this chapter starts with a presentation of the transportation textile market. then, different aspects of textile testing relevant to the transportation industry are discussed. the chapter ends with considerations regarding future trends for textiles in transportation. the international trade fair for technical textiles, techtextil, has set up a classification for technical textiles according to their area of application (techtextil, 2016) . one of these twelve areas-which also include agrotech, buildtech, medtech, and oekotech-is mobiltech symbolized by a tire symbol, which covers textiles for ship and aircraft construction as well as all aspects of automobile, railway, and space travel. the mobiltech sector is in full growth due to the rapid development of all means of transportation and increased safety requirements for passengers (bertrand, 2005) . for instance, the need for fire-retardant materials in the case of a plane or train crash or a car accident is critical to limit the rate of ignition of fabrics and flame propagation; the extra time it will give passengers to exit the wreck will definitely increase their chances of survival. reinforcements, filters, fiber-reinforced composites, thermal and acoustic insulators, etc. they are used in cars; in recreational vehicles such as motorcycles, snowmobiles, campers, and quads; in public transportation such as buses and streetcars; and in commercial vehicles such as trucks, fire trucks, ambulances, army vehicles, and construction trucks. textile exports, when considered separately from clothing, accounted for $314 billion in 2014 (wto, 2015) . rising concerns for road traffic safety have motivated governments to impose stricter legislations and apply new standards in automotive transportation, such as the compulsory use of 3-point seatbelts and the presence of airbags for drivers as well as front and rear passengers. a study performed by markets and markets analysis predicts that the increase in the demand for premium cars in regions such as europe, north america, and asia pacific should drive the airbag and seatbelt markets upward to $23.6 and $9.0 billion respectively by 2019 (m&m, 2014a) . in the area of composites, the value of the automotive market is expected to reach more than $7 billion by 2019, with a compound annual growth rate of 22% for carbon fiber reinforced polymer composites (m&m, 2014b) . indeed, textile-reinforced composites offer an interesting opportunity to help solve the weight challenge (wilson, 2015) . in 2014, the automotive industry produced 58,000 tons of carbon fiberreinforced composites, 1.6 million tons of natural fiber-reinforced composites, and 3 million tons of glass fiber-reinforced composites. the use of technical textiles in aircrafts has long contributed to the reduction in weight and the associated reduction in fuel expenses. but even though the introduction of mandatory seatbelts and the presence of life vests under the seats have contributed to saving many lives, the presence of synthetic fabrics in airplanes has also been the cause of many deaths. according to a study led by the national transportation safety board on 26 plane crashes that occurred between 1983 and 2000, among the 45% passengers who did not survive the accidents, 26% lost their lives because of the violence of the impact while almost 5% died of fire-related causes (ntsb, 2001) . in addition, it was observed that, in crashes with a higher survivability rate after impact, the rate of fire-related causalities was higher. as a matter of fact, many polymers used in seats, curtains, cushions, carpets, etc. do not display a high enough flame retardancy and/or they generate toxic fumes while burning (bertrand, 2005) . furthermore, in the last 20 years, the proportion of fiber-reinforced composites used in the aerospace industry has increased from 10% to 50%, such as in the airbus 350 xwb or the boeing 787 dreamliner (airbus, 2016) . the objective is to reduce the overall weight of the structure and cut fuel consumption. these materials also require less maintenance thanks to the larger resistance to corrosion. however, the cost of the carbon fiber raw material is still higher than steel or aluminum, for instance, despite the twofold drop over the last decade made possible by the increased production volume and the development of new manufacturing processes. carbon fiber-reinforced composites are gaining ground and becoming competitive thanks to the reduction in defects and cycle time as well as the development of new resin systems and new fiber placement and composite manufacturing processes (rao, simha, rao, & ravi kumar, 2015) ; this provides interesting perspectives for aircraft manufacturers willing to push innovation forward in this sector of industry. textiles also hold a large place in marine transport (singha & singha, 2012) : sails, inflatable crafts, life jackets, personal flotation devices, hovercraft skirts, reinforcement for composite parts, securing and lifting webbings, upholstery, etc. in fact, some even wonder if, with a compound annual growth rate expected to exceed 6% by 2020, marine composites would not be one of the key components of the future of the textile industry (technavio, 2016) . indeed, marine vessels made of textile-reinforced composites are about 50% lighter than those made of steel and more than 30% lighter than those made of aluminum. in terms of performance, the technical textiles used in the marine industry are subjected to strict requirements to ensure the safety of the passengers and merchandise that are transported. flame resistance may thus be a critical criterion for some applications. in addition, requirements may include resistance to puncture and tearing as well as to ultraviolet rays, sea salt, and humidity exposure. these highly demanding requirements have pushed innovation forward within this sector; the use of new materials has been investigated in order to insure the durability of the structures in particular. as a matter of fact, boat hulls are now made of glass fiber reinforced composite with a vinylester matrix, which provides an increased resistance to uv exposure and salt corrosion (miyano & nakada, 2009 ). other high performance materials used in marine transportation include spectra®, a high strength/high modulus extended chain polyethylene fiber whose chemical and abrasion resistance is superior to aramids; treviria, a heat treated polyester fiber fabric; and carbon fibers (singha & singha, 2012) . the railway industry is another important market for technical textiles. it comprises trains and subways. the growth is fueled by the sustained demand in asia and western europe as well as rising markets in africa/middle east and eastern europe (statista, 2016) . textiles are used in flooring (carpets), seats, ceilings, curtains, as well as reinforcement for composite parts. the main driver for textile use in rail transport is, once again, weight. requirements for textiles in trains include nontoxicity of smoke, flame resistance, low abrasion, and durability. in particular, the toxicity of smoke in the case of a fire and emissions of volatile organic compounds (voc) as a result of use are of large concern. examples of requirements for fabrics and composites used in passenger rail transportation may be found in the standard nfpa 130 (2017) relative to fixed guideway transit and passenger rail systems. it covers seat upholstery, covers, window shades, woven seat cushion suspensions, and other fiber reinforced composite body parts. mass transportation is subject to very high standards in terms of passengers' safety. these translate into high requirements set by national and international regulations on fabric and composite properties and performance. on the other hand, recreational motor vehicles such as snowmobiles, motorcycles, jet skis, and sailing ships are not bound to such strict specifications, for instance, in terms of safety or durability. requirements on constitutive materials are thus less demanding. the aerospace industry has also benefited from the significant improvements in terms of mechanical properties of technical textiles. for instance, the thermal protection system of space rockets/shuttles may include fibrous silica batting, graphite rayon fabric-reinforced phenolic resin composite, aramid felts, and/or alumina fiber cloth (milgrom, 2013) . on the other hand, solar sails, made of aluminized carbon fiber fabrics, for instance, may open up new regions of the solar system to exploration by allowing the development of propellant-free interplanetary space crafts (johnson, 2012) . for most applications, the performance of technical textiles used in mass transportation is highly regulated for the sake of the passengers' safety. for that purpose, standards and specifications have been developed and are enforced by national regulations. tight control of raw materials and components by manufacturers of finished products is also observed. over the years, the technical textile market has experienced a steady growth, increasing from less than 15 million tons and about $80 billion in 1995 to more than 23.5 million tons and about $130 billion in 2010 (david rigby associates, 2003) . the trend is not slowing down: a technavio analysis has forecasted that the global technical textile market will reach a compound annual growth rate of 3.71% over the period -19 (technvio, 2014 . given the broad use of technical textiles on all five continents, this large growth rate has led to a need for manufacturers of technical textiles worldwide to standardize their testing methods for transportation applications. in particular, the requirements of manufacturers of transportation equipment have increased over time as regulations towards passengers' safety have become more stringent (pamuk & çeken, 2009) . part and component suppliers are also experiencing an increase in competition and a request for the improvement in their product quality in order to keep their original partnerships and comply with the market expectations. being able to rely on the suppliers' constant quality is a concern for manufacturers of transportation equipment. some original equipment manufacturers (oem) and their clients have built strong partnerships based on the sharing of common values regarding quality and safety. the key is to offer clients the best products available on the market at the desired price. since the creation of the international organization for standardization (iso) in 1947, a series of technical committees have been formed to support the development of standards in the various transportation means, including the following (iso 2016): • iso/tc22 for road vehicles, which was formed in 1947 and has published 839 standards; • iso/tc 20 for aircraft and space vehicles, which was also formed in 1947 and has published 649 standards; • iso/tc 8 for ships and marine technology, formed in 1947 as well and has published 288 standards; • iso/tc 269 for railway applications, which was formed in 2012 and has published 2 standards; the establishment of these reference testing methods has helped engineers develop products that can easily be exported worldwide. at the state level, standardization activities in the field of transportation equipment have also been conducted within the american society for testing and materials (astm), the canadian standards association (csa), the association française de normalisation (afnor), the deutsches institut für normung (din), the british standards institution (bsi), the italian organization for standardization (uni), the japanese engineering standards committee (jesc), and the standardization administration of china (sac) for instance. in addition, the society of automotive engineers (sae) is a us-based association whose mission includes the development of standards. initially limited to the automobile industry, its scope has been broadening in 2016 to include engineers in all types of mobility-related professions. passengers' safety is the major concern for mobiltech textile manufacturers and has to be ensured by a tight control of the fire performance of the textile components and assemblies. great efforts have been devoted over the last years to developing fire-resistant and nontoxic smoke generating textiles for airplanes, trains, ships, and road vehicles. in parallel, standards were established to confirm the level of performance of these materials. for instance, the national fire protection association (nfpa) in the united states has published a series of standards for textiles used in transportation. these documents include good practices in terms of testing methods of the fire behavior on fibers, fabrics, and composites, and the measurement of the level of toxicity of the fumes generated upon burning. the use of these standards is not limited to the united states and has found a large echo among manufacturers of transportation equipment. a certain hierarchy may be observed regarding fire protection and smoke toxicity requirements. in the case of aircrafts, the us federal aviation administration is the leading authority (horrocks, 2013) . at a second level, aircraft manufacturers (boeing, bombardier, airbus, etc.), as well as part and component suppliers set the performance thresholds along with the certification procedures. for trains, the nfpa is a leader for urban communities (for subways and commuter trains) and states (for other rolling stocks) in north america. in europe, the standard series en 45545 relative to fire protection on railway vehicles, which was published in 2010, is gradually being adopted and should prevail over state authorities as well as rolling stock manufacturers. some textiles used for mobiltech applications are also subject to various mechanical constraints. fiber reinforced composites used for structural applications such as in airplane fuselages or wings, car doors, roofs and bodywork parts have to maintain their mechanical properties for the entire lifetime of the structure. it is therefore critical, especially when passengers' safety is at stake, to assess the durability of structural parts and observe the damages induced by mechanical stressors, including impact, fatigue, and abrasion. in addition, technical textiles used for carpets, curtains, upholstery, and headliners may also have to resist use and abuse, including acts of vandalism. in europe, fabrics are increasingly used for railway seat manufacturing in order to reach passengers' expectations in terms of comfort but also reduce the overall weight of the train. these fabrics have to be particularly resistant to tearing, cutting, marker and paint, chewing gum stains, etc. resistance to these aggressors has to be assessed by laboratory tests prior to introducing the product on the market. many textiles used inside private or public means of transportation also serve an aesthetic function; thus, this aspect has to be maintained as much as possible over the lifetime of the product. for that purpose, color fastness to ultraviolet or perspiration exposure along with crocking tests are conducted on dyed fabrics, and resistance to pilling is assessed on carpets, curtains, and upholstery. all of these tests are required by car, airplane, train, and ship manufacturers and must be conducted by mobiltech textile suppliers. these tests may also be stipulated by competent authorities such as the national highway traffic safety administration (nhtsa) in the united states, motor vehicle safety (mvs) in canada, or the european council for automotive r&d (eucar) in europe. the testing of automotive equipment safety has been implemented for a long time by the car manufacturers themselves. some major groups such as ford motor co. and general motors corp. in the united states have invested millions of dollars to evaluate safety accessories in high-tech research centers (johnson, 2005; white, 2015) . for instance, ford invested $65 million in 2005 to improve its safety testing facilities. efforts include engineering adaptive airbags and seatbelts. similarly, the suppliers of safety components and materials are expected to design and manufacture very high quality products with no room for failure, especially when it comes to tires, airbags, seatbelts, emergency handles, oxygen masks in airplanes, and life vests in airplanes and boats. this implies a high safety factor in the design and no manufacturing defects. the first airbag system for cars was developed in 1951 (hetrick, 1953) . it consisted originally of bladders inflated with compressed air. nowadays, they are made of plain weave super high tenacity nylon 6,6 or polyester (pet) fabrics (orme, walsh, & westoby, 2014) . some are coated with silicone and equipped with vents in the back as a way to allow a controlled deflation after impact. in addition, a large increase in the airbag inflation rate upon impact has been obtained. they now inflate through a chemical reaction generating nitrogen gas in <50 ms (tan & yu, 2012) . during a car crash, the kinetic energy of the car is released into the bodies of the passengers who are propelled toward the windshield. their acceleration relative to the car depends on the speed of the vehicle prior to the impact and the accumulation of energy during the crash (sobhani, young, logan, & bahrololoom, 2011) . as a complement to seatbelts, the presence of airbags is now considered as a key factor for survival during a crash. airbags are controlled by a central unit which monitors a series of sensors, including accelerometers that can detect variations in the vehicle speed indicative of a crash. due to the forces involved and the speed of the airbag deployment and inflation, it has to be constructed out of very high strength fabrics so that it does not explode, distort, or tear during the two most critical phases of its action: inflation and impact of the body. the airbag has thus to be designed so that it does not fail catastrophically, doesn't allow tear propagation, and includes very high safety factors. a first step in assessing airbag requirements consists of evaluating the different speeds at which bodies will be propelled toward the airbags depending on the weight of the body and the initial car velocity. in the case of a crash involving two moving vehicles, the kinetic energy of the crash may double if the collision is frontal. to help car manufacturers determine the requirements of their airbags, sobhani et al. (2011) developed a model allowing the estimate of the injury severity in the case of a twovehicle crash based on the calculation of the collision kinetic energy using the car speed, total mass, angle of collision, etc. tests assessing the performance of airbags in the united states may be conducted on the entire system using unbelted 50th-percentile size and weight male instrumented crash dummies seated in the front of a vehicle impacting a fixed rigid barrier perpendicular to its axis of travel at speeds of up to 48 km/h (fmvss 208). injury criteria are set in terms of head acceleration, thoracic acceleration, chest deflection, force transmitted axially through the upper leg, and level of neck injury. in addition, all portions of the dummy shall remain inside the passenger compartment. by comparison, the corresponding european test method involves belted crash test dummies (ece 94, 2003) . the airbag module may also be evaluated using a test stand that simulates deployment conditions in a vehicle (astm d5428, 2008) . the performance is assessed in terms of the variation in the airbag pressure and geometry over time as well as final conditions of the airbag module components. the requirements may also be set on the airbag fabric, yarn, and sewing thread (fung & hardcastle, 2001) . efforts to provide standardized test methods to that extent have been carried out by the astm subcommittee d13.20 on inflatable restraints, the society of automotive engineers (sae), and car manufacturers, for instance. table 14 .1 provides a list of fabric properties relevant to airbag with examples of associated test methods. in addition, the fabric may be inspected for imperfections using astm d5426 (2012). the presence of seat belts in cars dates back to 1930 after two american doctors found that crash related injuries in cars were largely related to the motion of passengers under the effect of their kinetic energy and could be prevented by an appropriate retention system (imre & cotetiu, 2014) . lap or 2-point seat belts became compulsory in europe for front car seats in 1965. a further improvement was the introduction of 3-point seat belts that restrain the motion of the chest toward the steering wheel or the front panel, which proved to be fatal in many cases: they are now mandatory in front and back seats of cars in many countries and are also used in a series of other road vehicles such as trucks and in the front seats of other commercial vehicles such as buses and ambulances. lap seat belts are still found in buses and aircrafts for passenger seats. in the case of child safety seats as well as for aircraft and racing car pilots for instance, 4-, 5-, or 6-point seat belts are used with additional buckling belts over the other shoulder and between the legs. current 3-point seat belt systems comprise a piece of webbing, a retractor, a pillar-loop, a tongue, a buckle, and a 3rd point anchor. the webbing is generally a multiple layer woven made of high-tenacity continuous filament polyester yarns (fung & hardcastle, 2001) . it has to have very high strength and good abrasion resistance, be soft and flexible in the longitudinal direction and rigid in the transverse direction, and resist uv degradation. according to the current process in the car industry, the information about seat belt components is provided to the component manufacturer in a design goal document (dgd) (imre & cotetiu, 2014) . this includes the positioning of each seat belt component, which depends on the inside layout of the car, as well as the vehicle destination market, which will dictate which standards the seat belt system has to comply with. for instance, europe refers to ece r16 (restraints and safety belts) and ece r44 (child restraints in power driven vehicles), while requirements for seat belts used in the united states are provided in fmvss 208 (occupant crash protection), fmvss 209 (seat belt assemblies), fmvss 210 (anchorages for seat belt assemblies), and fmvss 213 (child restraint system). if a car manufacturer decides to develop a new component for its seat belts, it will go through several steps of validation before having its component accepted by the competent committees and organisms. computer aided design (cad) will help model the component and choose the raw materials. it is also used for running virtual failure analysis through a failure mode and effects analysis (fmea) and constraint accumulation calculation by finite elements analysis (fea). then tests will be run on prototypes, including abrasion, puncture resistance, stiffness, elongation at break, ultimate tensile strength, accelerated fatigue testing, and uv, heat, and humidity aging. finally, a serial process control (spc) will determine the stability and predictability of the process. as in the case of airbags, seat belts may be tested as a system using a 50th percentile adult male dummy in the driver and passenger front seats of a vehicle subjected to frontal barrier crash test, lateral moving barrier crash test, and rollover test (fmvss 208). the frontal barrier crash test involves the vehicle impacting a fixed rigid barrier perpendicular to its axis of travel at speeds up to 48 km/h. in the lateral moving barrier crash test, the vehicle is impacted laterally on either side by a barrier moving at 32 km/h. the rollover test is conducted at 48 km/h over a concrete surface. depending on the test performed, injury criteria may be set in terms of head acceleration, thoracic acceleration, chest deflection, force transmitted axially through the upper leg, and/ or level of neck injury. in addition, all portions of the dummy shall remain inside the passenger compartment. requirements may also apply to the webbing materials. the performance factors to be assessed include the following (tc tsd 209, 2013): • width after conditioning for at least 24 h in an atmosphere having a temperature of 23°c and a relative humidity between 48% and 67%; • breaking strength measured with a rate of grip separation between 51 and 102 mm/min; • elongation when subjected to a force of 11.120 n; • resistance to abrasion, measured in terms of residual breaking strength; • resistance to light, measured in terms of residual breaking strength and color retention after 100 h exposure to type e carbon-arc at 60°c; and • resistance to microorganisms, measured in terms of residual breaking strength. if they cannot be considered as a safety accessory per se, tires play a very large role in ensuring the safety of the vehicle. at the interface with the ground, they are a key component that determines the vehicle behavior while it ramps up and brakes. developed and patented in the mid-19th century by a scottish engineer, r. w. thompson, pneumatic tires take advantage of the exceptional performance of vulcanized rubber (fung & hardcastle, 2001) . a scottish-born veterinary surgeon, john boyd dunlop, rediscovered pneumatic tires at the end of the nineteenth century while trying to improve the rolling comfort of his son's tricycle. his design involved the use of a fabric as reinforcement for the rubber. another large step was made in 1946 by michelin, who invented the radial tire method of construction. the more stable structure it produces allows a large increase in the tire longevity, driving safety, and fuel efficiency compared to the original cross-ply construction. they now have taken over most of the passenger road vehicle market, and the use of cross-ply tires is limited to some applications for trucks, trailers, farm equipment, and emerging markets (lindenmuth, 2006) . car and light truck radial tires contain about 5% polymer textiles for the carcass and 10%-12% of steel for the belt, which provide strength to the tire (mcdonel, 2006) . the amount of textile in cross-ply tires is larger with about 21% (fung & hardcastle, 2001) . textiles in radial carcasses are primarily composed of polyester, while they are mostly nylon for cross-ply structures (mcdonel, 2006) . aramids are also used when high strength-to-weight ratio and temperature resistance are required, for instance, in aircrafts and racing cars (fung & hardcastle, 2001) . a small amount of aramid and nylon may be found as well in belt overlays (mcdonel, 2006) . other tire components include natural and synthetic rubber, reinforcing fillers such as carbon black and silica, and various additives including vulcanization agents, plasticizers, stabilizers, antioxidants, and antiozonants (lindenmuth, 2006) . some tests are performed on the whole tire structure as described for instance in the us federal motor vehicle safety standard (fmvss 139) or in its european counterpart (ec 661, 2009 ). in the case of pneumatic tires, they cover the tire dimensions, high speed performance, endurance, low inflation pressure performance, and strength. in addition, the tire textile components may also be tested at the yarn, cord, and fabric scale (astm d885, 2010). when goods are transported by road, boat, train, and air, they have to be strongly secured to keep them from tilting, sliding, or moving around, which would be a source of danger and/or might damage them (dvsa, 2017). straps and slings are continued generally used to ensure this function. they may also be employed as a way to lift objects to load them on and unload them from the transportation mean. they are generally made with high-tenacity polyamide, polyester, or polypropylene multifilament. a series of european standards cover the safety aspects of textile slings: flat woven webbing slings made of manmade fibers (en 1492-1, 2008) , roundslings made of manmade fibers (en 1492 (en -2, 2008 , and lifting slings made from natural and manmade fiber ropes (en 1492 (en -4, 2008 . a fourth standard for disposable flat woven slings is in preparation. properties and performance of slings assessed in these standards comprise the type of polymer and yarn; the webbing construction, width, thickness, and tenacity; the change in webbing width under load; the working load limit and minimum failure force; and the interaction of the sling with fittings. a description of requirements and test methods may also be found in asme b30.9 (2010) for slings used in conjunction with cranes and ansi b77.1a (2012) for passenger ropeways (aerial tramways, aerial lifts, surface lifts, tows and conveyors), for instance. 14.3 testing related to flammability, smoke generation, and toxicity tests concerning flammability, smoke generation, and toxicity are very common for a large number of transportation applications because reduced flammability is one of the main safety requirements of almost all textiles used for passenger transportation. however, test methods may vary depending on the conditions the textile will be exposed to while in service. in addition, performance thresholds may also depend on the type of transportation means. for instance, the same textile flammability test may be associated with a more constraining threshold for aerospace applications than in the for automotive applications, standards concerning flame resistance of materials have been prepared by international and national organizations as well as car manufacturers. for instance, volvo has broadened the scope of the nhtsa flammability test for motor vehicle interior materials, fmvss 302, by conducting the test on specimens aged for 14 days at 38°c at 95% rh and at 70°c in addition to conditioned specimens (vcs 5031,19, 2004) . some countries have also adopted modified versions of standards issued by organization such as nfpa, the eu council, and nhtsa in the united states. the most common flammability test for road vehicle interior materials measures the horizontal burning rate of specimens exposed to a low-energy flame for 15 s in a combustion chamber (fmvss 302; iso 3795, 1989; astm d5132, 2011; vcs 5031,19, 2004) . the test also determines if and when the flame self-extinguishes. it applies to textiles situated within 13 mm of the occupant compartment air space in passenger cars, multipurpose passenger vehicles, trucks, and buses, as well as for tractors and agriculture and forestry machinery. other flammability test methods may involve a test chamber replicating the section of a school bus to assess the burning behavior of upholstered seating used in school buses (astm e2574, 2012). the chamber is equipped with two ventilation openings at each end and holds three rows of seats. the ignition source consists of a propane gas burner installed either on top or under one of the seats. the flammability performance is assessed in terms of the time elapsed between ignition and flame extinguishment, mass loss of the seat assembly, occurrence of flame spreading to other seats, and seat material melting or dripping. in europe, the european aviation safety agency (easa) has regulatory authority and executive tasks in the field of civil aviation safety. it works jointly with the national aviation authorities (naas) of the different european country members of the esea in order to ensure that airplane manufacturers and oems from these different countries fulfill the various standardization requirements and regulations. easa also works on technical agreements with its counterparts in the world such as the federal aviation administration (faa) in the united states or the canadian aviation regulation (car). the faa publishes federal aviation regulations (far) and advisory circulars (ac), which provide guidance for compliance with airworthiness standards for airplane manufacturers. for instance, the faa standard on airworthiness of airplanes (far/cs 25.853) includes several flammability test methods that apply to textile components: easa for its part is currently reviewing its legislation on civil aircrafts to take into account the increased air traffic and arrival of new technologies such as drones (juul, 2016) . in particular, it intends to better standardize the way flammability testing is conducted (easa cmcs-004, 2013 the nfpa standard for fixed guideway transit and passenger rail systems (nfpa 130, 2014) provides guidelines on tests to be performed on materials used in trains. their behavior in the case of a fire is characterized by their flame resistance and smoke emission. tests may be carried out on component materials or complete seat or mattress assembly. table 14 .3 lists test methods recommended in the nfpa 130 standard (2014) as well as in other us standards such as astm e2061 (2015) and fra 216 (1999) of the federal railroad administration for the different types of textiles or textile-based items. the permanence of the surface flammability and smoke emission characteristics of the materials is also verified after dynamic fatigue testing using roller shear or constant force pounding (astm d3574, 2016), after washing according to the manufacturer's recommended procedure or astm e2061 (2015), and, if relevant, after dry cleaning according to astm d2724 (2007). in europe, a common strategy in terms of fire requirements and test methods for materials used in railway vehicles has been set with the recent publication of the standard en 45545-2 (2013). requirements depend on the amount of material, its location, its use, and if it is in contact with another material. three main categories of performance are measured: the international maritime organization (imo) has the worldwide authority to set new standards for safety, minimum official requirements, and environmental performance of naval transportation ships. for instance, it has published fire test procedures (ftp) for testing flammability of materials including textiles used onboard. table 14 .4 provides a list of these different tests. the flammability test for vertically suspended textiles and films is also performed on specimens subjected to accelerated aging by dry-cleaning, laundering, water leaching, and weathering (imo ftp code, 2010). indications about fire test methods for textiles used in marine ships may also be found in the us code of federal regulations 46 cfr part 72.05-55 subsection relative to structural fire protection for furniture and furnishings for shipping. in addition, ship manufacturers may have to assess the fire characteristics of mattresses and bedding assemblies according to nfpa 267 (1998). the test method uses an open calorimeter environment to determine the heat release, smoke density, weight loss, and generation of carbon monoxide of mattresses and bedding assemblies exposed to a flaming ignition source. part as closed environments, transportation vehicles may facilitate the transmission of infectious diseases, in particular airborne ones (santos o'connor, 2012) . for instance, cases of influenza, severe acute respiratory syndrome (sars), meningococcal disease, tuberculosis, and measles transmission onboard planes are seen relatively frequently. cruise ships, trains, and school buses have also been documented as potential vehicles for the spreading of various diseases. in an effort to improve air quality, cabin interior air filter systems equipped with nonwoven filters have now become a requirement for many transportation vehicles. textile filters are also used in other parts of vehicles for air, oil, and fuel filtration. a complementary strategy aimed at limiting the transmission of diseases is based on the use of antimicrobial textiles. transportation was the leading segment of nonwoven filter media applications in 2014 with 21.2% of the total market revenue (james, 2015) . this trend has been maintained since then and may be attributed in part to tougher regulations towards the reduction in carbon emissions from automobiles. further, with respect to cars, large concerns have also been raised about the air quality inside the passenger compartment (fung & hardcastle, 2001 ). indeed, research has shown that, because of the tunnel effect, the exhaust gas concentration inside a car may be six times higher than outside. this phenomenon is amplified when a car is driven as a closed distance from the preceding one. the question of inside air filtration is also critical in public transportation, especially for long distance travel segments (santos o'connor, 2012) . cabin air filters may combine three mechanisms (fung & hardcastle, 2001) : mechanical filtration of the solid particles through the pores of the nonwoven, electrostatic attraction of the solid particles on the charged nonwoven fibers, and adsorption of gases and removal of odors by activated carbon granules distributed across the nonwoven filter. the performance of air filters may be tested for particulate and gas filtration. for instance, standard iso/ts 11155-1 (2001) allows assessing the pressure loss, fractional filtration efficiency, and accelerated particulate holding capability of filter elements for road vehicle passenger compartments using standardized laboratory particulate challenges larger than 0.3 μm. the dynamic gas adsorption of the passenger compartment air filters of road vehicles may be characterized according to the test methods described in iso/ts 11155-2 (2009). the air pressure loss as well as the gas and vapor removal characteristics are measured for a series of relevant contaminants. textiles used for seats, handles, carpets, etc. may also be treated to provide them with an antimicrobial function. the antimicrobial agent may be applied as a finishing treatment on the yarn or the fabric, or it may be incorporated into the polymer extrusion solution or the spinning bath (zhao & chen, 2016) . in the latter case, it has to slowly migrate towards the fiber surface to provide its function during use. finishing treatments may use conventional exhaust and pad-dry-cure methods as well as newly developed padding, spraying, coating, foam finishing, and microencapsulation techniques. for instance, silver nanoparticles have recently generated interest because they appear as a more health and environmentally friendly antimicrobial alternative to halogenated phenols such as triclosan. in addition, bacteria are less prone to develop resistance to silver nanoparticles than conventional antibiotics (chernousova & epple, 2013) . other strategies involve the use of natural compounds such as chitosan to limit the occurrence of side effects on health and the environment (lim & hudson, 2004) . the antibacterial activity of textile products may be quantified by directly inoculating fabric swatches with gram-positive staphylococcus aureus and/or gram negative klebsiella pneumoniae organism cultures (aatcc tm100, 2012). the antibacterial activity value of the tested textile is computed using bacteria counts on the samples immediately after inoculation and after the desired contact period. other standard test methods provide alternative transfer and printing techniques for inoculation (iso 20743, 2013) . more details about antibacterial efficiency test methods are available in chapter 6. fiber reinforced composites have taken an increasing share of transportation applications. glass reinforced plastics were developed in the 1920s. one of their first use in transportation can be dated back to the late 1940s, with boat hulls made of glass fiber reinforced polyester resin (bunsell & renard, 2005) . composites were introduced in military aircrafts in the 1960s. the renault 5 was the first car in 1972 to have a bumper made of glass fiber reinforced polyester. since then, the development of new fibers, for instance carbon and aramid fibers, has allowed an increase in the performance of composites and in the ratio of composite parts in all types of vehicles. for instance, the new airbus a350 xwb contains 53% of fiber reinforced composites that can be found in the wings, fuselage, empennage, and belly fairing as well as in the skin panels, doublers, joints, and stringers (airbus, 2016) . this has allowed airbus to save 20% in mass compared to aluminum and 25% in fuel consumption. it has also seen a strong improvement in the resistance of parts to corrosion and fatigue. the aston martin one-77 structural core is made of a carbon fiber composite monocoque, making the car weight only 1500 kg (aston martin, 2008) . in railway applications, alstom teamed up with france's national railways (sncf) to develop train noses made of carbon fiber reinforced composite in order to reduce the overall weight and improve the aerodynamics of high speed trains (mason, 2004) . however, all of these composite parts have to go through intense testing in order to fulfill the requirements on which the passengers' lives depend. these may be conducted at the preform and/or composite stage and may involve destructive and nondestructive test methods. textile reinforcements for composites include yarns, strands, tows or rovings (strong, 2008) . they may be used directly, for instance, in filament winding or fiber placement processes. they may also be transformed into more complex textile structures such as wovens, knits, and braids with anisotropic properties in all three directions. on the other hand, nonwovens or mats are manufactured directly using fibers or chopped strands that are more or less randomly distributed in the plane of the structure. recently, noncrimp fabrics (ncf) have been developed to combine the strength and perfect fiber placement of wovens with the flexibility, ease of manufacture, and absence of fiber crimp of nonwovens (schnabel & gries, 2011) . the 2-d sheet materials may then be assembled by stitching, z-pinning, or tufting, for instance, to create complex shaped, 3-d structures that will be cut to shape to be fitted in the composite part mold (mouritz, 2011) . near net-shape preforms may also be prepared directly using 3-d textile manufacturing techniques, which include 3-d interlock and orthogonal noncrimp weaving as well as 3-d braiding. this allows improving the pace of the process and the quality of the composite. the 2-d reinforcements and 3-d preforms may also be delivered as prepregs, i.e., with the textile being coated with the resin only partially cured (strong, 2008) . prepregs need to be stored in cold conditions to prevent premature complete polymerization. the properties of the textile reinforcement as well as its compatibility with the matrix play a major role in the performance of the composite material (strong, 2008) . indeed, the reinforcement gives the composite its strength and stiffness because it bears the stress applied on the composite part, which is transferred by the matrix. properties measured on the reinforcement include the following: • density of the 1-d raw material (fiber/filament/yarn/strand/tow/roving), e.g., using archimedes' method (astm d3800, 2016) or a pycnometer (astm d70, 2009; astm d5550, 2014); • elastic modulus, strength, and elongation at break of the 1-d raw material, e.g., using astm d4018 (2017) for continuous filament carbon and graphite fiber tows; • dry uniaxial bending of the reinforcement, e.g., based on standard test astm d1388 (2014) or using the apparatus developed by jldain (2015) . buckling of inner yarns may be observed using a translucent bending surface. • draping behavior of the reinforcement, e.g., using the double curvature technique developed by harrabi et al. (2008) ; • in-plane shear of the reinforcement using a bias extension or picture frame technique (long, boisse, & robitaille, 2005) . in-plane shear is considered to be the main deformation mechanism taking place when the reinforcement is formed to a 3-d geometry. this test also provides a measurement of the maximum deformation, with the yarn locking angle; • biaxial in-plane tension of the reinforcement, e.g., using the biaxial tensile device with cruciform specimens described in long et al. (2005) . it allows characterizing the warp and weft yarn interaction in woven fabrics. • dry compaction of the reinforcement, e.g., adapted from standard test iso 5084 (1996) . the test involves the application of compression cycles and provides a measurement of the preform thickness under a certain level of normal stress as well as the through-thickness rigidity of the material. it also gives an estimate of the maximum fiber volume fraction that can be achieved (robitaille & gauvin, 1998 ); • pore size distribution in the reinforcement, e.g., using microscopy, x-ray microtomography, and capillary flow porometry (bonnard, causse, & trochu, 2017) . this last technique allows accessing the dual scale structure of fibrous reinforcements; • resin permeability of the reinforcement using unidirectional injection or bidirectional flow measurement from a pointwise injection gate (demaría, ruiz, & trochu, 2007) . the test may be conducted with 100 cp silicon oil that behaves as a newtonian fluid. in addition, the type of reinforcement also has a large impact on the manufacturing process, which in turn controls the performance of the final composite part. defects in the textile reinforcement may also induce reduced performance and/ or premature failure in the composite part. these defects include fiber misorientation (saboktakin, dolez, & vu-khanh, 2011) , broken fibers (mouritz, 2011) , wrinkling (zhu, yu, zhang, & tao, 2011) , and local strain concentration due to stitching (chen, endruweit, harper, & warrior, 2015) . preform inspection may be conducted using x-ray microtomography (desplentere et al., 2005) or by reconstructing geometries from sections obtained by electronic microscopy (blanc, germain, da costa, baylou, & cataldi, 2006) . depending on its functions, the composite part will have to meet different requirements. these requirements include the following (mallick, 2007) : various destructive test methods exist to characterize the properties as well as the short and long-term performance of composite parts. some tests are conducted on composite specimens (table 14 .5). oems have also developed some applicationspecific test procedures that they may perform on full-scale parts (kia, 2012; perret, mistou, fazzini, & brault, 2012) . as an alternative to destructive testing, nondestructive test (ndt) methods allow looking for defects and damages inside composite parts without cutting them apart or even decreasing their performance. these techniques are critical for in-service inspection but may also be useful for production quality control as well as rapid sample testing. they are categorized as contact and noncontact methods in table 14 .6. for instance, x-ray computed tomography and ultrasound-based techniques were successfully used to detect two significant process-induced defects, namely fiber breakage and ply misorientation, in woven-reinforced composites manufactured by vacuum assisted resin transfer molding (saboktakin rizi, 2013) . in addition, new ndt techniques are continuously developed. for instance, a micro-vibrothermography device was designed to detect deep submillimeter flaws in stitched t-joint carbon fiber reinforced polymer (cfrp) composites (zhang et al., 2016) . a burst of ultrasound waves is delivered to the specimen with a 200 pa ultrasound excitation transducer. the temperature profile is captured with an infrared camera equipped with a microlens. the same team also developed a microlaser line thermography technique, which was successfully used to detect internal microporosities in the same stitched t-joint cfrp composites. if materials and parts used in transportation have to display a certain level of performance when they come out of the production line, they also have to maintain these performance as much as possible over the entire life of the vehicle. for instance, cars today are built to last about 250,000 miles (gorzelany, 2013) . on the other hand, aircraft lifespan is determined by the number of takeoff and landing cycles they experience (maksel, 2008) ; aircrafts that only make long flights may last more than 20 years because of the lower number of pressurization cycles. however, textiles used in floorings, upholstery, and draperies in the interior cabin will not last as long and may need to be cleaned, repaired, and/or replaced at regular intervals. the performances of textile and textile-based materials related to durability for transportation applications include the following (national research council, 1995 pulse echo ultrasonic testing through transmission ultrasonic testing acoustic emission radiography (e.g. x-ray tomography) electromagnetic testing (e.g., eddy current) thermography (e.g., infrared testing) liquid penetrant testing holography magnetic particle testing shearography visual inspection table 14 .6 nondestructive test methods for textile-reinforced composites (gholizadeh, 2016) a first set of aging agents are environmental. indeed, temperature, humidity, and uv radiations may induce changes in the materials properties and performance over time. for instance, some polymers like polyolefins are prone to thermo-and photooxidation degradation, while polyester and polyamide are especially sensitive to hydrolysis and polyvinylchloride may discolor and become brittle at high temperatures (verdu, 1984) . polymers and natural fibers may also be degraded by microorganisms. the resistance of textiles and textile-based materials to heat, moisture, water spray, and uv aging is usually tested using conditions that accelerate the aging process to reduce the duration of the test. weathering programs simulating climatic conditions to which the material is likely to be subjected in service will combine cycles with variations in temperature, water, and/or radiation conditions. resistance to microorganisms may also be assessed to evaluate the effect of bacteria, mildew, and rot. the durability is generally characterized in terms of color change as well as effect on mechanical and other physical properties. table 14 .7 provides a list of test methods used in the transportation industry to characterize the resistance of materials to environmental aging. some of them have been developed by private companies, e.g., peugeot citroën (psa test methods in table 14 .7) and general motors (gm test methods). aging may also result from normal wear as well as accidental or intentional damage during service. damage may be generated by a mechanical action, for instance crocking, pilling, abrasion, snagging, tear, laceration, etc. (kern, 2014) . it may also be of chemical nature, for example, resulting from a water leak, fluid spill, perspiration, soiling by a sick passenger, etc. if the test item is designed to be laundered, colorfastness and resistance to laundering cycles may also have to be assessed. the effect may be visual with a change in color or a stain. a loss in performance and/or physical integrity of the material may also observed. table 14 .8 provides a list of test methods used in the transportation industry to characterize the resistance of materials to aging due to service conditions. some of them have been developed by private companies like volkswagen (pv test methods in (fung & hardcastle, 2001; ul, 2016) fatigue is a specific aspect of material aging where the part is subjected to loading/ unloading cycles. this mechanism is critical for the durability of rigid materials such as textile-reinforced composites. but it is also relevant for textiles and coated textiles which are more flexible. first of all, fibers themselves exhibit fatigue failure (miraftab, 2009) . loading may be applied in tension, lateral compression, flexion, and torsion. the damage may come during the manufacture of the woven or braided structure for instance as well as during use. one typical example is the cyclic fatigue experienced by a tire cord. in addition to the mechanical stress mode and conditions of application (frequency, amplitude, offset, etc.), other parameters may affect the fatigue failure of a fiber: its composition and manufacturing process/conditions, its dimensions, the presence of impurities, its environment (temperature, humidity, uv, ph, bacteria), etc. the measurement is conducted by applying cyclic loading conditions in a specified environmental. the result is expressed in terms of stress vs. number of cycles to failure (s-n) curves and survival diagrams. fatigue may also be experienced at the textile structure scale. yet, not much research appears to have been done in that field. in the case of flexural fatigue of woven (fung & hardcastle, 2001; ul, 2016) fabrics, it was shown that the performance depends on the structure of the fabric, the position and structure of the yarn, and the yarn material (schiefer & boyland, 1942) . surface fatigue also contributes to wear behavior upon abrasion (özdil, kayseri, & mengüç, 2012) . fatigue cracks form at the surface of the material due to alternating compression-tension stresses and propagate to subsurface regions where they may rejoin. fatigue failure performance is a major component in composite part design and has been the focus of several studies (carvelli & lomov, 2015) . a series of test methods for textile-reinforced composites has also been developed by various standardization organizations (table 14 .9). in addition, it was shown in a study that the s-n curve should be combined with the time-temperature superposition principle to take into account the effect of the environmental conditions, in that case temperature and water absorption (miyano & nakada, 2009 since the beginning of the 21st century, the rising concern about global warming has led the scientific community to integrate sustainable development in its research projects and look for more eco-friendly solutions in the design of new products: improvement in process manufacturing (lower energy consumption and reduction of wastes), product lifetime (lightweight fabrics, low voc emission, maintenance-free systems), and product afterlife (reuse or recycle). as a result, green fabrics and composites are becoming an interesting alternative to many synthetic products, with the use of natural fibers, biosourced resins, and nontoxic and biodegradable dyes and finishes (chard, creech, jesson, & smith, 2013) . it also pushes research forward into the reduction of greenhouse gases through the development of new lightweight and durable materials in transportation. natural fibers have been used for some time as a replacement for glass and carbon fibers in composites for nonstructural applications. indeed, in addition to their low cost, they display a low density which can lead to reduced energy consumption as well as competitive specific mechanical properties. they are also biodegradable. in the automotive industry, for instance, jute was used by mercedes-benz in 1996 for its e-class vehicle door panels (koronis, silva, & fontul, 2013) . a blend of flax and sisal later found its way as reinforcement into audi's 2000 a2 midrange car door trim panels; kenaf in toyota's 2003 spare tire cover; bamboo fibers in mitsubishi motors' interior components; and wheat straw in ford 2010 flex crossover vehicle storage bin and inner lid. natural fibers are now considered for more high-performance applications thanks to improvements in their compatibility with polymer matrices (pickering, efendy, & le, 2016) . for instance, a green hydrophobic treatment based on zinc oxide nanorods and stearic acid was developed for recycled jute fibers (arfaoui, dolez, dubé, & david, 2017) . however some issues remain, including the inherence variability in their physical properties as well as their poor moisture resistance and limited thermal stability (koronis et al., 2013) . this thus requires some adjustments in testing programs to ensure that they perform as required by the application. it may be noted that, to the authors' knowledge, natural fiber-based textiles have not found an application in transportation by themselves, i.e., without being combined with a polymer matrix. this may be eventually attributed to their short life resulting from their biodegradability. biosourced resins provide an interesting alternative to the recycling dilemma of composites. for instance, toyota has been using polylactic acid (pla), a biodegradable thermoplastic polyester derived from renewable resources, in the spare tire cover of its raum 2003 (koronis et al., 2013) ; it was sugar cane and sweet potato in that case. other bioderived resins foreseen as a matrix for green composites include poly-l-lactide (plla), polyhydroxybutyrate (phb), poly(3-hydroxybutyrate-co-3hydroxyvalerate) (phbv), and thermoplastic starch. current issues that limit the use of biosourced resins in the transportation industry include their propensity to biodegrade and their high price (koronis et al., 2013) . a debate also exists on whether or not they represent a real sustainable alternative to conventional plastics. indeed, they should not reduce the amount of cultivated food available to humans and animals, for instance, by decreasing the amount of fertile lands for edible crops, or increasing land clearing. many dyes or finishes currently used in the textile industry are highly pollutant and require very important amounts of water and energy for processing. for instance, brominated flame retardants still constitute the most common solution used for seating, curtains, carpets, etc. in transportation vehicles (icl, 2012 ). yet, their effects on health and the environment have been clearly demonstrated (see section 7.6 in chapter 7 on toxicity testing of textiles). other toxic chemicals that may be found in textiles include heavy metals, toxic dyes, pesticides, phthalates, nonylphenol ethoxylates, dioxins, and furans. in addition, some finishes like polybrominated diphenyl ether (pbde) used in airplane carpets are responsible for the emission of vocs in confined environments (allen et al., 2013) . large efforts are currently deployed to develop eco-friendly additives and finishes for textiles. for instance, phosphorous-based compounds (salmeia, gaan, & malucelli, 2016) as well as nanoclay and carbon nanotube composites (arao, 2015) are considered as an alternative to halogenated fire retardants. natural dyes may also ultimately replace the toxic synthetic dyes currently used in the textile industry (bechtold, turcanu, ganglberger, & geissler, 2003) ; in addition, their application does not require the use of solvents or other chemicals, and they lead to a reduction in the chemical load released with waste waters. tougher regulations are being set to better control the amount of chemicals used in textile processes and limit those that are the most toxic. for instance, nonylphenol ethoxylates (npe), which had been banned from use within its borders by the european union for 20 years, have also recently been voted by all eu member states to be excluded from textile imports (flynn, 2015) . in response to the largest interest of consumers for green products and sustainable development, a majority of textile companies are now including environmental management systems (ems) such as iso 14001 and/or the adhesion to voluntary eco-labels as part of their business model (see chapter 7 on toxicity testing of textiles). conferring multifunctionality to fabrics is one of the main contemporary goals of technical textile manufacturers. a fabric that can be made fire-resistant with integrated nanotechnologies such as carbon nanotubes (cnt) or nanoclays while being hydrophobic, self-cleaning, and antibacterial at the same time is gold for public transportation manufacturers (alongi, carocio, & malucelli, 2013) . cnt may also be added to composites to provide them with electrical conduction capabilities, thus reducing the use of cables for carrying information or electricity. in addition, they can be used for the in-situ detection of defects, cracks or delamination (nofar, hoa, & pugh, 2009) . they even displayed an increased sensitivity compared to strain gauges. finally, smart textiles are opening new paths in the transportation industry. for instance, smart seat belts for airplanes have been developed by ctt group in partnership with belt-tech (decaens & vermeersch, 2016) . this smart belt sends a signal if it is not buckled. this technology and the others imply the need to develop new testing methods or adapt existing ones in order to take into account the new material or functionality. in that case, the durability of the connective wire inside the seat belt should be assessed against environmental and service aging among others. the remarkable evolution of technical textiles for transportation through the last century has been driven by a constant concern for passenger's safety. insuring a comfortable and secure journey is also a key marketing strategy for aerospace, railway, marine, and automotive manufacturers, as well as a path towards bigger market shares. these companies are thus pushing research forward into developing state-of-the-art textile products respecting specifications but also featuring unique performances in order to be a step ahead of their competitors. this has led to the development of test methods assessing the different aspects of textiles in transportation. this includes performance related to safety for airbags, seat belts, tires, and slings; flammability, smoke generation, and toxicity, with test methods and specific requirements for each type of application; hygiene for filters and antimicrobial textiles; destructive and nondestructive tests conducted on textile-reinforced composites at the textile and composite level; and durability. technical textiles are promised to a bright future in transportation, with new developments involving natural fibers, biosourced resins, eco-friendly additives and finishes, and multifunctional and smart materials among others. more information about test methods may be obtained from dedicated committees in various organizations, including the following: antibacterial finishes on textile materials: assessment of american association of textile chemists and colorists flammability lab a350 xwb-cost-effectivness exposure to flame retardant chemicals on commercial airplanes smart (nano) coatings. update on flame retardant textiles: state of the art, environmental issues and innovative solutions passenger ropeways-aerial tramways, aerial lifts, surface lifts, tows and conveyors-safety requirements flame retardancy of polymer nanocomposite development and characterization of a hydrophobic treatment for jute fibres based on zinc oxide nanoparticles and a fatty acid safety standard for cableways, cranes, derricks, hoists, hooks, jacks, and slings-slings standard test method for density of semi-solid bituminous materials (pycnometer method) standard test methods for tire cords, tire cord fabrics, and industrial filament yarns made from manufactured organic-base fibers standard test method for stiffness of fabrics standard test methods for bonded, fused, and laminated apparel fabrics standard test methods for flexible cellular materials-slab, bonded, and molded urethane foams standard test method for density of high-modulus fibers standard test methods for properties of continuous filament carbon and graphite fiber tows standard guide for testing polymer matrix composite materials test method for horizontal burning rate of polymeric materials used in occupant compartments of motor vehicles standard practices for visual inspection and grading of fabrics used for inflatable restraints standard practice for accelerated aging of inflatable restraint fabrics standard practice for evaluating the performance of inflatable restraint modules standard practice for determining physical properties of fabrics, yarns, and sewing thread used in inflatable restraints standard test method for specific gravity of soil solids by gas pycnometer standard guide for testing fabric-reinforced "textile" composite materials standard guide for fire hazard assessment of rail transportation vehicles standard test method for fire testing of school bus seat assemblies aston martin-one-77 natural dyes in modern textile dyehouses-how to combine experiences of two centuries to meet the demands of the future moving fabric fiber orientation measurements in composite materials experimental characterization of the pore size distribution in fibrous reinforcements of composite materials fundamentals of fibre reinforced composite materials 2016 top markets report-technical textiles-a market assessment tool for fatigue of textile composites green composites: sustainability and mechanical performance inter-ply stitching optimisation of highly drapeable multi-ply preforms silver as antibacterial agent: ion, nanoparticle, and metal technical textiles and nonwovens: world market forecasts to wearable technologies for ppe: embedded textile monitoring sensors, power and data transmission, end-life indicators in-plane anisotropic permeability characterization of deformed woven fabrics by unidirectional injection. part i: experimental results micro-ct characterization of variability in 3d textile architecture load securing: vehicle operator guidance notification of a proposal to issue a certificate memorandum on flammability testing of interior materials type-approval requirements for the general safety of motor vehicles, their trailers and systems, components and separate technical units intended therefor textile slings-safety-part 1: flat woven webbing slings made of man-made fibres for general purpose use textile slings-safety-part 4: lifting slings for general service made from natural and man-made fibre ropes railway applications. fire protection on railway vehicles. requirements for fire behaviour of materials and components airworthiness standards: transport category airplanes. 14 cfr part dc: united states department of transportation, federal aviation administration eu countries agree textile chemical ban. the guardian occupant crash protection. 49 cfr 571.208-federal motor vehicle safety standard no. 208 flammability of interior materials. 49 cfr part 571.302-federal motor vehicle safety standard no. 302 passenger equipment safety standards. 49 cfr part 216 textiles in automotive engineering a review of non-destructive testing methods of composite materials cars that can last for 250,000 miles (or more) characterization of protective gloves stiffness: development of a multidirectional deformation test method safety cushion assembly for automotive vehicles. united states patent and trademark office flame retardant textiles for transport applications fire protection for automotive and transportation international code for application of fire test procedures contribution to validation and testing of seatbelt components about iso-what are standards? road vehicles, and tractors and machinery for agriculture and forestry-determination of burning behaviour of interior materials textiles-determination of thickness of textiles and textile products reaction to fire tests-spread of flame-part 2: lateral spread on building and transport products in vertical configuration plastics-smoke generation-part 2: determination of optical density by a single-chamber test reaction-to-fire tests-heat release, smoke production and mass loss rate-part 1: heat release rate (cone calorimeter method) and smoke production rate (dynamic measurement) reaction to fire tests for floorings-part 1: determination of the burning behaviour using a radiant heat source reaction to fire tests-ignitability of products subjected to direct impingement of flame-part 2: single-flame source test textiles-assessment of the ignitability of bedding items-part 2: ignition source: match-flame equivalent textiles-determination of antibacterial activity of textile products reaction-to-fire tests-full-scale room tests for surface products-part 2: technical background and guidance road vehicles-air filters for passenger compartments-part 1: test for particulate filtration road vehicles-air filters for passenger compartments-part 2: test for gaseous filtration nonwoven filter media market to reach $7.18 billion by 2022 behaviour and inspection of novel non-crimp dry thick reinforcement fabrics ford beefs up its safety testing facilities solar sail propulsion new civil aviation safety rules interior care and maintenance. aviationpros. retrieved from www.aviationpros.com (accessed focal project 4: structural automotive components from composite materials. automotive composites consortium green composites: a review of adequate materials for automotive applications application of a fiber-reactive chitosan derivative to cotton fabric as an antimicrobial textile finish an overview of tire technology. the pneumatic tire design and manufacture of textile composites automotive airbag and seat belt market for passenger cars-a global analysis: by geography-trends and forecasts automotive composites market by type (glass fiber composites, carbon fiber composites, natural fiber composites, metal matrix composites, and ceramic matrix composites) and by application (interior components, exterior components, chassis & powertrain components and others)-global trends & forecast to what determines an airplane's lifespan? fiber-reinforced composites: materials, manufacturing, and design composites aboard high-speed trains tire cord and cord-to-rubber bonding. the pneumatic tire space shuttle thermal protection system. ibook (18 p.) basic principles of fatigue accelerated testing for long-term durability of various frp laminates for marine use three-dimensional (3d) fiber reinforcements for composites fire-and smoke-resistant interior materials for commercial transport aircraft standard for fixed guideway transit and passenger rail systems standard method of test for fire characteristics of mattresses and bedding assemblies exposed to flaming ignition source self sensing glass/epoxy composites using carbon nanotubes survivability of accidents involving part 121 u.s. air carrier operations equivalency or compromise? a comparative study of the use of nylon 6,6 and polyester fiber in automotive airbag cushions analysis of abrasion characteristics in textiles research on the breaking and tearing strengths and elongation of automobile seat cover fabrics global behaviour of a composite stiffened panel in buckling a review of recent developments in natural fibre composites and their mechanical performance carbon composites are becoming competitive and cost effective compaction of textile reinforcements for composites manufacturing. i: review of experimental results integrity assessment of preforms and thick textile reinforced composites for aerospace applications damage identification of thin textilereinforced composite plates using vibration-based testing methods recent advances for flame retardancy of textiles based on phosphorus chemistry emerging and re-emerging infectious diseases note on flexural fatigue of textiles production of non-crimp fabrics for composites applications of textiles in marine products a kinetic energy model of two-vehicle crash injury severity rail industry-statistics & facts fundamentals of composites manufacturing. materials, methods and applications new device design and performance test on gas generator in automobile airbag technical standards document no. 209, revision 2r-seat belt assemblies global marine composites market global technical textiles market application areas automotive testing and engineering services flammability of interior materials le vieillissement des plastiques gm paving way to smarter and safer driving at all-new active safety test area vehicle weight is the key driver for automotive composites. reinforced plastics international trade statistics comparative study of microlaser excitation thermography and microultrasonic excitation thermography on submillimeter porosity in carbon fiber reinforced polymer composites halogeneted phenols and polybiguianides as antimicrobial textile finishes experimental investigation of formability of commingled woven composite preform in stamping operation the authors wish to thank mr. valério izquierdo, amir fanaei, and jonathan levesque for their assistance in preparing the manuscript. key: cord-290251-ihq8gdwj authors: hasell, joe; mathieu, edouard; beltekian, diana; macdonald, bobbie; giattino, charlie; ortiz-ospina, esteban; roser, max; ritchie, hannah title: a cross-country database of covid-19 testing date: 2020-10-08 journal: sci data doi: 10.1038/s41597-020-00688-8 sha: doc_id: 290251 cord_uid: ihq8gdwj our understanding of the evolution of the covid-19 pandemic is built upon data concerning confirmed cases and deaths. this data, however, can only be meaningfully interpreted alongside an accurate understanding of the extent of virus testing in different countries. this new database brings together official data on the extent of pcr testing over time for 94 countries. we provide a time series for the daily number of tests performed, or people tested, together with metadata describing data quality and comparability issues needed for the interpretation of the time series. the database is updated regularly through a combination of automated scraping and manual collection and verification, and is entirely replicable, with sources provided for each observation. in providing accessible cross-country data on testing output, it aims to facilitate the incorporation of this crucial information into epidemiological studies, as well as track a key component of countries’ responses to covid-19. across the world, researchers and policymakers look to confirmed counts of cases and deaths to understand and compare the spread of the covid-19 pandemic. however, data on cases and deaths can only be meaningfully interpreted alongside an accurate understanding of the extent and allocation of virus testing 1 . two countries reporting similar numbers of confirmed cases may in fact have very different underlying outbreaks: other things being equal, a country that tests less extensively will find fewer cases. many countries now publish official covid-19 testing statistics, but the insights offered by these numbers remain relatively unexplored both in public discourse and scientific research. this may be because of barriers limiting access to this data: the statistics are scattered across many websites and policy documents, in a range of different formats. no international authority has taken on the responsibility for collecting and reporting testing data. we developed a new global database to address this lack of access to reliable testing data, thereby complementing the available international datasets on death and case counts 2 . the database consists of official data on the number of covid-19 diagnostic tests performed over time across 94 countries (as of 31 august 2020). we rely on figures published in official sources, including press releases, government websites, dedicated dashboards, and social media accounts of national authorities. we do not include in our database figures that explicitly relate to only partial geographic coverage of a country (such as a particular region or city). the resulting database is (i) updated regularly through a combination of automated scraping and manual collection and verification, and (ii) entirely replicable, with sources provided for each observation. in addition, the database includes extensive metadata providing detailed descriptions of the data collected for each country. such information is essential due to heterogeneity in reporting practices, most notably regarding the units of measurement (people tested, cases tested, tests performed, samples tested, etc). series also vary in terms of whether tests pending results are included, the time period covered, and the extent to which figures are affected by aggregation across laboratories (private and public) and subnational regions. the comprehensiveness of our database enables comparisons of the extent of testing between countries and over time -in absolute terms, but also relative to countries' population, and to death or confirmed case counts (fig. 1) . such variation offers crucial insights into the pandemic. at the most basic level, it is clear that a country that tests very few people -such as the democratic republic of congo, or nigeria (fig. 1a ) -can only have very few (2020) 7:345 | https://doi.org/10.1038/s41597-020-00688-8 www.nature.com/scientificdata www.nature.com/scientificdata/ confirmed cases. the number of performed tests should be seen as an upper limit for the number of confirmed cases. further, high positive test rates ( fig. 1 -see reference lines) may help identify severe underreporting of cases. the relationship between test positivity rate and case underreporting has been explored in the context of other infectious diseases 3 . in terms of covid-19, this link is discussed by ashish jha and colleagues at the harvard global health institute, who provide a sketch of the relationship between cases, deaths and the positivity rate in the united states (see https://globalepidemics.org/2020/04/18/why-we-need-500000-tests-per-d ay-to-open-the-economy-and-stay-open). in a more formal analysis, golding et al. 4 find that their modelling estimates of the case ascertainment rate are weakly correlated (kendall's correlation coefficient of 0.16) with the number of tests per case -the inverse of the test positivity rate -derived from our database, with a positive relationship evident in the range of 10-35 tests per case 4 . the institute for health metrics and evaluation (ihme) www.nature.com/scientificdata www.nature.com/scientificdata/ include testing data sourced from our database in their covid-19 models (www.healthdata.org/covid/faqs#dif-ferences%20in%20modeling). in bringing this data together, our hope is that we will facilitate future research in this direction. more generally, our aim is to provide an essential complement to counts of confirmed cases and deaths. these are the figures that guide public policy, both in the initiation of control measures and as they start to be relaxed. but without the context provided by data on testing, reported cases and deaths may offer a very distorted view on the true scale and spread of the covid-19 pandemic. the database consists of two parts, provided for each included country: (1) a time series for the cumulative and daily number of tests performed, or people tested, plus derived variables (discussed below); (2) metadata including a detailed description of the source and any available information on data quality or comparability issues needed for the interpretation of the time series. for most countries, a single time series is provided: either for the number of people tested, or the number of tests performed. for a few countries for which both are made available, both series are provided. in such cases, metadata is provided for each separate series. data collection methods. the time series data is collected by a combination of manual and automated means. the collection process differs by country and can be categorized into three broad categories. firstly, for a number of countries, figures reported in official sources -including press releases, government websites, dedicated dashboards, and social media accounts of national authorities -are recorded manually as they are released. secondly, where such publications are released in a regular, machine-readable format, or where structured data is published at a stable location, we have automated the data collection via r and python scripts that we execute every day. these are regularly audited for technical bugs by checking their output against the original official sources (see 'technical validation' , below). lastly, in some instances where manual collection has proven prohibitively difficult, we source data from non-official groups collecting the official data, most often on github. these are also regularly audited for accuracy against the original official sources (see 'technical validation' , below). any available information on data quality or comparability issues needed for the interpretation of the time series is gathered and summarized manually into detailed metadata for each series, guided by a checklist of data quality questions (see data records, below). general been the basis for covid-19 case confirmation, in line with who recommendations 5 . since the primary purpose of the database is to provide information on testing volumes specifically to aid the interpretation of data on confirmed cases, it is exclusively this category of testing technologies that the database aims to include. in order to be included, a data point for a given country must report an aggregate figure that includes both negative tests (or negatively tested individuals) plus positive tests (or confirmed cases). the units (whether the number of tests or individuals is being counted) must be consistent across positive and negative outcomes. the aggregate figure must refer to a known time period -for instance, the number of tests performed in the last day or week. however, where a cumulative total is provided, it is not a requirement that the specific start date to which the cumulative count relates must be specified, provided that it is clear that the figure aims to capture the whole of the relevant outbreak period. figures relating to testing 'capacity' or to rough indications of average testing output, or to the number of tests that have been distributed (rather than actually performed) are not included in the database. where figures for pending tests are provided separately by a source these are excluded from our counts. where they cannot be separated, the figures including pending tests are reported. details concerning pending tests for individual countries can be found in the metadata. the database provides a time series for both the cumulative number of tests (or people tested) and for daily new tests. exactly how these series are derived depends on the way the raw data is reported by the source. where a source provides a complete time series for daily tests, we derive an additional cumulative series as the simple running total of the raw daily data. where a source provides cumulative figures, we derive an additional daily series as the day-to-day change observed in consecutive observations. in many cases the source data is not available at a daily frequency (fig. 2) . in order to facilitate cross-country comparisons over time, we derive an additional 'smoothed' daily testing series calculated as the seven-day moving average over a complete, linearly interpolated daily series (described in more detail in the data records section below). retrospective revisions in the source data. due to the efforts to produce timely data, official testing figures are subject to frequent retrospective revisions. this can occur for instance where some laboratories have longer reporting delays than others, and previously uncounted tests are then subsequently included. this issue presents no difficulties where sources provide an updated time series within which such revisions are appropriately incorporated; for instance, by backdating the additional tests to the date they were performed. however, a number of the sources we rely on provide only a 'snapshot' of the current cumulative figure, with no time series. we construct our cumulative and daily testing time series from the sequence of these 'snapshots' . for these cases, retrospective revisions do impact our data since revisions to the data are included on the day the revision is made, not when the revised tests occurred. typically, this results in only small deviations in the www.nature.com/scientificdata www.nature.com/scientificdata/ cumulative figure in proportional terms, but the derived daily testing series can be impacted more meaningfully. at the extreme, in a few cases, such revisions result in a fall in the cumulative total from one day to the next, implying a negative number of tests for that day. this issue is mitigated in two ways. firstly, given that much of retrospective revision relates to testing conducted over the last few days, the 'smoothed' daily time series we derive reduces some of the artificial volatility introduced. secondly, we alert the user as to which data is subject to such concerns as part of the information included in the metadata (see below). a copy of the database has been uploaded to figshare 6 . this provides a version of the database as it stood at the time of submission, on 31 august 2020. a live version of the database, which continues to be updated, can be downloaded from a public github repository (https://github.com/owid/covid-19-data/tree/master/public/data/testing) in csv, xlsx, and json formats, which may be imported into a variety of software programs. structure. the database consists of two components: a time series file including observations of cumulative and daily testing (covid-testing-all-observations.csv), and metadata (covid-testing-source-details.csv). each row in the metadata table provides source details (discussed below) corresponding to a given country-series (i.e. the combination of country and series fields make up a unique id within covid-testing-source-details.csv). the time series for cumulative and daily testing for each country-series is then provided in the covid-testing-all-observations.csv file. in addition, we provide the raw data (raw-collected-data.csv), as collected from the source, in order to make it plain how our time series data is constructed from the original observations. we also provide the united nations population data for 2020 (un-2020-population.csv) used to derive the per capita measures included in the time series. raw-collected-data.csv. country. each observation relates to testing conducted within the indicated country. we do not include in our database figures that explicitly relate to only partial geographic coverage of a country (such as a particular region or city). the country's 3-letter iso 3166-1 code is also provided as a separate field. units. a short description of the unit of observation of the collected testing figures, selected out of three possible categories: "people tested", "tests performed", "samples tested". series for which it was not possible to discern the category are labelled as "units unclear". series. multiple series (e.g. people tested and samples tested) are included for some countries, and are demarcated by this field. common to covid-testing-all-observations.csv and raw-collected-data.csv. date. depending on the source, this may relate to the date on which samples were taken, analyzed, or registered, or simply the date they were included in official figures (see 'retrospective revisions in the source data' , above). in general, sources try to provide testing data relating to a given, stable cut-off time each day. where significant changes in reporting windows have been found, these have been noted in the notes field (see below). www.nature.com/scientificdata www.nature.com/scientificdata/ cumulative total. the reported cumulative amount of testing as of date. the specific date to which the cumulative figures date back to, if known, is provided in the metadata (see below). in many cases this is not explicitly stated by a source, but only figures that appear to intend to capture the entire period of the testing response to covid-19 outbreak within the country are included in the database. in covid-testing-all-observations.csv, for those sources only providing daily testing figures, this field is derived as the running total of the raw daily data, and is also provided per thousand people of the country's 2020 population. daily change in cumulative total. broadly, this field may be interpreted as the number of new tests (or people tested) per day. for sources that report new tests per day directly, this field in covid-testing-all-observations. csv is identical to the raw data presented in raw-collected-data.csv. for sources that report only cumulative testing figures, the field is derived as the day-to-day change observed in consecutive observations of the raw cumulative total data. this may fail to correspond to the true number of new tests for that date where the source has included retrospective revisions in the cumulative totals (see 'retrospective revisions in the source data' , above). in covid-testing-all-observations.csv, this series is also provided per thousand people of the country's 2020 population. source url. a url at which the specific observation of the corresponding raw data can be found. source label. the name of the source for the observation. notes. contains any notes to aid the interpretation of this specific observation (above and beyond details that apply to the whole series, which are provided in covid-testing-source-details.csv). specific to covid-testing-all-observations.csv. 7-day smoothed daily change. as an outbreak progresses, flows of new tests per day, rather than cumulative figures, become more relevant for understanding trends. daily testing figures however suffer from volatility created by reporting cycles. moreover, since many sources do not provide data at daily intervals, figures for new tests per day are available with more limited coverage. to aid the cross-country analysis of testing volumes over time, we provide this short-term measure of testing output that aims to mitigate these two problems. it is calculated as the right-aligned rolling seven-day average of a complete series of daily changes. for countries for which no complete series of daily changes is available because of the reporting frequency of our source, we derive it by linearly interpolating the daily cumulative totals not available in the raw data, up to a maximum interval of 21 days. the exact code used to derive the 7-day smoothed daily change is available online (see 'code availability' , below). specific to covid-testing-source-details.csv. number of observations. the number of days for which raw observations are available. detailed description. a written summary of available information concerning the nature and quality of the source data needed for proper interpretation and cross-country comparison. the collation of this information is guided by a 'checklist' of data quality questions regarding: the unit of observation; which testing technologies figures relate to; whether tests pending results are included; the time period covered; and the extent to which figures are affected by aggregation across laboratories (private and public) and subnational regions. in practice the documentation we are able to provide is limited by that made available by the official source. we aim to include any information provided by the original source needed for the interpretation and comparison with other countries. coverage. the database includes observation for 94 countries, covering 69% of the world's population. because of differences in the frequency at which countries publish testing data, coverage is somewhat lower for more recent periods: 62% of the world's population is covered with figures relating to 30-31 august 2020; 45% is covered with figures relating to 24-31 august 2020 (fig. 2 ). the database represents a collation of publicly available data published by official sources. as such, the key quality concern for the database itself is whether it represents an accurate record of the official data. we employ four main strategies for ensuring this. firstly, all automated collection of data, whether obtained from official channels or from third-party repositories of official data, is subject to initial manual verification when it is added to our database for the first time. secondly, we employ a range of data validation processes, both for our manual and automated time series. we continually check for invalid figures such as negative daily test figures, out-of-sequence dates, or test positivity rates above 100% (by comparing testing data to confirmed case data), and we monitor each country for abrupt changes in daily testing rates. abrupt positive or negative daily changes are sometimes the result of data corrections in the official data, in which case our database includes them without alteration. these changes can be due, for example, to the deduplication of double-counted tests, or the addition of testing data that was previously not captured by the national system (see table 1 ). in order to mitigate against large impacts due to reporting lags, we automatically exclude the most recent observation for a country if its daily number of new tests is less than half that of the previous observation. this is only applied to the most recent day in each time series: as soon as data for subsequent days becomes available, the data point is reinstated if the sharp fall is still present. www.nature.com/scientificdata www.nature.com/scientificdata/ thirdly, to monitor the ongoing reliability of third-party repositories of official data, we apply a continuous audit process, which will remain active as long as this dataset is updated. each day, three observations are randomly drawn out of all observations in the database that have been obtained via third-party sources. for each selected observation, the recorded figure is manually checked against the direct official channel from which the repository purports to obtain the data. the sampling rate means that each third-party source we make use of is checked around once a week. given that any discrepancies with official channels are likely to be clustered within particular sources, this provides a high degree of quality control on these sources on a timely basis. where any discrepancies are noticed, we switch sources (for the entire time series) to either a different repository or to manual data collection directly from the official channel. finally, the testing data included in the database is viewed by tens of thousands of people every day, including many health researchers, policymakers and journalists, from which we receive a large amount of feedback concerning the data. this serves as a final, 'crowd-sourced' method of verification that has proven very effective, enabling any discrepancies between our data and that published in official channels to be flagged and resolved quickly. code used for the creation of this database is not included in the files uploaded to figshare. our scripts for data collection, processing, and transformation, are available for inspection in the public github repository that hosts our data (https://github.com/owid/covid-19-data/tree/master/scripts/scripts/testing). changes observed in the source data as of 31 august 2020 case-fatality rate and characteristics of patients dying in relation to covid-19 in italy an interactive web-based dashboard to track covid-19 in real time malaria burden through routine reporting: relationship between incidence and test positivity rates reconstructing the global dynamics of under-ascertained covid-19 cases and infections advice on the use of point-of-care immunodiagnostic tests for covid-19: scientific brief this project was funded from multiple sources, including general grants and donations to our world in data. the following is a list of funding sources and affiliations. grants: our world in data has received grants from the bill and melinda gates foundation, the department of health and social care in the united kingdom, and the german philanthropist susanne klatten. sponsors: in addition to grants, our world in data has also received donations from several individuals and organizations: center for effective altruism -effective altruism meta fund, templeton world charity foundation, effective giving, the camp foundation, the rodel foundation, the pritzker innovation fund diana beltekian -data recording, verification and analysis. charlie giattino -data recording, verification and analysis. bobbie macdonald -data recording, verification and analysis. esteban ortiz-ospina -data recording, verification and analysis the authors declare no competing interests. correspondence and requests for materials should be addressed to j.h.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.the creative commons public domain dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the metadata files associated with this article. key: cord-318570-wj7r6953 authors: xiao, yinzong; thompson, alexander j.; howell, jessica title: point-of-care tests for hepatitis b: an overview date: 2020-10-02 journal: cells doi: 10.3390/cells9102233 sha: doc_id: 318570 cord_uid: wj7r6953 despite the heavy disease burden posed by hepatitis b, around 90% of people living with hepatitis b are not diagnosed globally. many of the affected populations still have limited or no access to essential blood tests for hepatitis b. compared to conventional blood tests which heavily rely on centralised laboratory facilities, point-of-care testing for hepatitis b has the potential to broaden testing access in low-resource settings and to engage hard-to-reach populations. few hepatitis b point-of-care tests have been ratified for clinical use by international and regional regulatory bodies, and countries have been slow to adopt point-of-care testing into hepatitis b programs. this review presents currently available point-of-care tests for hepatitis b and their roles in the care cascade, reviewing evidence for testing performance, utility, acceptability, costs and cost-effectiveness when integrated into hepatitis b diagnosis and monitoring programs. we further discuss challenges and future directions in aspects of technology, implementation, and regulation when adopting point-of-care testing in hepatitis b programs. more than 257 million people, or 3.2% of the world's population, are estimated to be living with chronic hepatitis b virus infection, with the greatest disease burden in low-resource countries in the asia-pacific and sub-saharan africa [1] . without treatment, one in every four persons infected with chronic hepatitis b will develop liver cirrhosis over 20-30 years, and 2-5% of people with cirrhosis will develop liver cancer annually [2] . globally, over 800,000 deaths annually are attributable to hepatitis b infection [1, 3] . most of this disease burden is preventable by appropriate guideline-based treatment [4] [5] [6] [7] [8] . given the magnitude of the global public health burden from hepatitis b, the world health organization (who) has outlined ambitious hepatitis b elimination targets of a 65% reduction in mortality and a 90% reduction in incidence from baseline (2015) by 2030 [9] . however, current estimates suggest that we are a long way from achieving these goals unless investment and care cascade are scaled up [1, 10] . the hepatitis b vaccination has greatly contributed to preventing transmission and reducing hepatitis b incidence globally; however, vaccination coverage is still suboptimal in resource-limited regions [11] , and most countries in africa have been unable to implement the hepatitis b birth-dose vaccine due to multiple logistical and cost barriers [12, 13] . meanwhile, for people who are already living with hepatitis b, receiving early diagnosis and clinical care is the key to reducing morbidity and mortality. however, in 2016, the who estimated that only 11% of people living with hepatitis b were diagnosed, among whom only 17% of those eligible were on treatment [1]. the hepatitis b cascade of care involves multiple steps: screening, diagnosis, linkage to care, assessment of liver disease stage and treatment eligibility, then treatment and/or monitoring, including surveillance for hepatocellular carcinoma (hcc) (figure 1) . laboratory blood tests are required at every step of the care cascade, including blood tests for hepatitis b serology, quantitative hepatitis b virus (hbv) dna level by polymerase chain reaction (pcr) and liver function tests (figure 1 ). these tests require laboratory resourcing, technology and expertise beyond existing peripheral laboratory capabilities in many low-resource and geographically isolated regions [14] [15] [16] . in many countries, laboratory services are centralised due to high costs and limited skilled technician capacity; however, transport of blood samples from regional to centralised laboratories presents its own challenges in geographically isolated or insecure regions, particularly if cold chain supply must be preserved [14] . cost is another major limitation: price reductions for diagnostics have fallen slowly over time compared with medication costs, and hepatitis b diagnostic tests cost more than therapy in many low-income countries [16, 17] . moreover, the requirement for lifelong monitoring for most people living with hepatitis b that involves regular blood tests [4, 5] , combined with barriers to timely healthcare access such as hepatitis b-related stigma [18, 19] , healthcare costs for users and providers [20] and the logistics of accessing consistent, high-quality, affordable healthcare services in a timely manner are major barriers for people to receive guideline-based care [16] . these barriers lead to significant attrition from every step of the hepatitis b care cascade over time, and those lost from care represent missed opportunities for treatment and liver cancer prevention [16, 21] . laboratory-based blood tests are required at every stage of the hepatitis b cascade of care for diagnosis, assessment of liver disease stage, treatment eligibility and long-term monitoring of disease progression. diagnostic testing for hepatitis b involves detection of hepatitis b surface antigen (hbsag) in blood, which indicates active infection with the virus. standard laboratory electro-chemiluminescence immunoassay-based hbsag testing is performed on serum or plasma samples derived from whole blood. if active infection is confirmed, subsequent blood tests are performed to determine the stage of disease and need for treatment, including a hepatitis b virus (hbv) polymerase chain reaction (pcr)-based quantitative dna level or viral load, a hepatitis b eag and eab assay and liver function tests to determine whether an elevated aminotransferase (alt) indicative of liver inflammation or other signs of impaired liver function are present. further assessment for the presence of liver fibrosis and cirrhosis is also required, most commonly by transient elastography and/or liver biopsy. all patients irrespective of treatment require ongoing disease monitoring, including at minimum an hbv dna level, hbeag and hbeab (if not already seroconverted from hbeag positive to hbeab positive) and alt levels every 3-6 months. point-of-care tests (pocs; also known as rapid diagnostic tests, rdts) are simplified versions of laboratory-based tests that have the potential to circumvent major barriers people face to accessing hepatitis b blood-based testing in various settings. pocs usually require small amounts of body fluids (for example, a finger-prick blood sample or oral swab), short turn-around time, and are generally easy to use with minimal required training and therefore can be provided to people in a variety of community and outreach settings by a broad range of trained workers [22] and are scalable to rapidly reach large populations as has been seen with the highly successful egyptian national hepatitis c screening program [23] . the simple collection process (finger-prick or mouth swab) is also highly acceptable, feasible and attractive to people undergoing testing [22, 24, 25] . a key benefit of pocs in the field of hepatitis b is to engage hard-to-reach communities for testing, such as using hbsag poc tests for hepatitis b screening in remote areas, or harm reduction programs [24] [25] [26] [27] . pocs also have great potential for retaining patients in care when used in the community for chronic hepatitis b stage evaluation and disease monitoring [26, 27] . figure 2 outlines the key phases of disease in chronic hepatitis b infection and the indicators for blood testing in each stage. the who recommends that an ideal poc test needs to meet the assured criteria of being "affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users" [28] . since 1998, the who has implemented an evaluation and performance assessment program for all pocs in viral hepatitis to report on accepted quality parameters for widespread clinical use [29] . many pocs have been developed in the field of hepatitis b, particularly for screening and diagnosis; however, only three pocs for detecting hbsag have been prequalified by the who [29] . there is currently a lack of pocs for hepatitis b stage assessment or monitoring that have been endorsed to use by the who; however, several novel tests are now in clinical trials that may fill this important care delivery gap. typically, poc tests have lower accuracy than traditional laboratory-based tests, but they facilitate the triage of people who require more complex and expensive laboratory assays to confirm a positive poc test result and thereby reduce costs. regulatory and economic constraints are additional barriers to transferring pocs to field use. in different settings, they therefore require a comprehensive appraisal of factors including testing performance, feasibility (such as storage requirements, power supply), acceptability and cost-effectiveness when using pocs to scale up access to hepatitis b diagnosis and management under real-life conditions. in this review, we outline the accuracy of available poc tests for hepatitis b and explore the evidence for utility and cost-effectiveness when integrated into hepatitis b diagnosis and monitoring programs. we also describe future technologies and explore how poc tests might best be used to achieve who 2030 hepatitis b elimination goals. practically, the three key clinical requirements for poc hepatitis b assays in the field are for diagnosis of current infection, determining treatment eligibility and also monitoring, as well as diagnosis of hepatitis b immunity and the need for hepatitis b vaccination in the uninfected ( figure 2 ). detection of hepatitis b surface antigen (hbsag) is the primary step to diagnose current hepatitis b infection, and multiple hbsag pocs are commercially available. most are qualitative lateral-flow chromatographic immunoassays which are one-step, easy to use, can be used with a variety of different specimens (whole blood, serum and plasma) and provide rapid semiquantitative visible results (usually within 15-30 min). to date, three hbsag rapid tests (determine hbsag 2, alere medical co. ltd, chiba-ken, japan; vikia hbsag, biomérieux sa, marcy-l'étoile, france; and sd bioline wb, abbott diagnostics korea inc. giheung-gu, republic of korea) have met who prequalification criteria [29] , with multiple studies showing their high accuracy for determining hbsag positivity in various populations, particularly in moderate-high-prevalence populations (table s1) . determine hbsag poc test is the one of the most widely-used hbsag poc tests [30] with the most published data on clinical performance. a 2017 meta-analysis [31] including 9 studies with 7730 samples showed a pooled sensitivity of 90.8% and specificity of 99.1% using determine. though most studies [32] [33] [34] [35] [36] [37] [38] showed high clinical sensitivity of 89-100% in the general population, the reported sensitivity varied widely in hiv-infected populations (56-100%) [39] [40] [41] [42] [43] [44] . the cause of the reported lower sensitivity in hiv-coinfected populations [39, 40, 43, 44] is unclear, but potential reasons may include the cross reaction of hiv-reverse transcriptase inhibitors and hepatitis b virus, a higher rate of occult hepatitis b infection in early hiv cohorts, a higher reported rate of hbsag loss in both untreated and treated hiv-infected populations and the use of tenofovir-based hiv regimens that effectively suppress hepatitis b virus dna levels and a large decline in hbsag titres [31, 45, 46] . sd bioline hbsag [38, [47] [48] [49] and vikia hbsag poc test [32, 33, 38, 50, 51] have also been shown to have good sensitivity (above 90%) and excellent specificity (above 99%) in general populations; however, lower sensitivity was also reported in hiv-infected populations [40] . a common application of these hbsag pocs is to measure seroprevalence in general or specific subpopulations in low-resource settings [52] [53] [54] [55] [56] . they have also been used in mass screening programs for hepatitis b in both community outreach [24, 57] and health-facility-based screening [52, 53, [58] [59] [60] [61] [62] in low-resource settings and shown great public health benefits. for example, in a community-based outreach screening program conducted in 75 camps in southern india [63] , the "screen and vaccinate/linkage to care" strategy led to over 7700 vaccinations in the camps and 162 people with high viral load getting treatment. the program increased the accessibility of hepatitis b diagnostic testing in a low-resource setting, and the timely results of pocs contributed to people's engagement in post screening interventions [63] . the hbsag pocs were also used in programs to engage hard-to-reach populations such as people who inject drugs, sex workers [64] , disadvantaged groups or some ethnic groups [65, 66] by providing self-testing, community or health-facility-based testing services. in a randomised control study conducted in a clinic engaging mostly african immigrants in france [65] , people without health cover attending a clinic were provided free testing for hepatitis b, hepatitis c and hiv using either pocs or prescriptions of testing at a pathologist; a higher rate of testing and linkage to care was observed among people allocated to receive pocs. however, another multicentre randomised control study in france [66] found no difference in effectiveness of linkage to care using the approach of an hbsag rapid test plus a standard lab-based confirmatory serology test versus lab-based standard serology in five clinics. in this study, it was described that participants received testing results via mail or phone call, but it was unclear whether participants received testing results at the same visit if they were in the poc testing group [66] . other than the who-prequalified hbsag pocs, emerging new brands of hbsag have been reported in field studies including the drw-hbsag assay, diagnostics for the real world ltd., (ce-marked) [35, 67] , first response hbsag card test, premier medical corporation [68] (ce-marked), naosign(r) hbs poc strips, bioland [69] , and one step hbsag test, general biologicals corporation [70, 71] (non-exhaustive list). a study in mongolia [72] which tested 19 commercially available hbsag pocs using a serum sample showed the average sensitivity and specificity being 100% and 99%, respectively. whilst most hbsag pocs of various brands have shown promising clinical performance [35, 38, 67, 70, 73] , available validation data are limited and further studies with large sample size and in diverse populations including different ethnicity and hepatitis b prevalence populations and people living with hiv are needed. multiplex diagnostic pocs can be highly attractive for low-middle-resource settings with the capacity to detect multiple pathogens using a single testing strip. some multiplex pocs which detect hbsag are commercially available and some are ce-marked (hbsag/hcv/hiv/syphilis combo test, euro genomas; hbsag and hcv combo test, euro genomas; artron detect 3 hiv/hcv/hbv combo, artron laboratories; hiv, hbsag and hcv rapid test, maternova inc., providence, ri, usa), but none have been listed by who prequalification [16, 29, 74] . accuracy of hbsag detection using multiplex has been shown to be high [75] , but limited clinical validation data are available. innovations in sampling technique have provided more convenient specimen collection methods, such as using oral fluid as specimen collected by an oral swab [25, 48, 51] . the simplified process was highly acceptable to individuals [25, 48] , but testing accuracy is a challenge to overcome [48] , and it may additionally require trained technicians or lab-based enzyme immunoassays or equipment for sample preparation such as requiring a centrifuge for target analyte separation [51] . future development needs to consider combining sample preparation steps together with detection and readout into one single device, without sacrificing testing accuracy. although many studies of different hbsag pocs showed very good sensitivity [31, 38, 73] , false negativity is still among the biggest concerns: around one in ten negative test results on average could be hbsag positive [31] . most cases with false negative results were reported to have low titres of hbsag, such as in studies using determine/vikia hbsag pocs, where most false negative cases had hbsag titres lower than 30 iu/ml [32, 33] . as hbsag level does not correlate with severity of liver damage, there is a chance that people with advanced liver disease may be missed. other potential factors affecting the accuracy of hbsag pocs may include hbv dna level, different genotypes, co-infection with hepatitis c or hiv and hepatitis b variants with s gene mutations that are not detected by the poc hbsag test [31, 33, 76, 77] . as only a few studies have obtained comprehensive serological and genetic profiles of false negative cases, more data are needed to explore these associations and determine the implications for clinical practice. specimen type is unlikely to affect the efficacy of hbsag poc tests: a meta-analysis showed similar pooled sensitivity of studies using whole blood sample compared to plasma or serum [31] , and studies evaluating hbsag pocs using capillary whole blood collected by finger-prick all showed reasonably high sensitivity (88-90%) [32, 44, 78] . in practice, there is no absolute cut-off for testing performance when choosing pocs for hepatitis b programs, and the increased access to testing might mitigate the harm caused by reduced accuracy; however, sensitive pocs that have been validated in similar contexts to their planned use should be prioritised [79] . hepatitis b surface antibody (anti-hbs) is the key marker to determine an individual's immunity status to hepatitis b virus and triage the need for vaccination. a few anti-hbs pocs are commercially available, but most have poor reported sensitivity ranging from 20% to 70% [33, [80] [81] [82] . one study reported a sensitivity of 91.8% using an anti-hbs rapid test card among 1272 samples [70] ; however, these findings require further validation. a study [66] showed using hbsag/ anti-hbs pocs was not effective in increasing vaccination rate due to poor sensitivity of anti-hbs poc and high reliance on confirmatory enzyme immunoassay. though a poc test for anti-hbs can help with triage vaccination need, given hepatitis b vaccination is relatively cheap, context-specific cost-effectiveness analyses would be needed to determine settings where the use of pocs of anti-hbs would be cost-effective. treatment decisions in hepatitis b are guided by patient age, hepatitis b dna viral load and the degree of liver inflammation and fibrosis, as measured by alanine aminotransferase (alt) levels and either transient elastography or liver biopsy, respectively [4, 5, 83] . however, there are few poc tests currently available for these parameters, and none have been widely validated and who prequalification approved. hepatitis b virus (hbv) dna level is the critical indicator when deciding an individual's management plan as per clinical guidelines. polymerase chain reaction (pcr) platforms for nucleic acid detection are still the main technique of quantitative assessment hbv dna levels; conventional pcr platforms are usually built in laboratories and require high manual input and pose barriers for accessibility in remote areas and other resource-limited areas areas [12] . a rapid molecular test, xpert ® hbv viral load (cepheid inc., sunnyvale, ca, usa, ce-marked, approved by american fda and tga in australia), is commercially available for hbv dna quantification that provides test results in less than one hour [84, 85] . the test is a cartridge-based, real-time pcr assay which is run on the genexpert instrument, a molecular diagnostic platform. the processing unit of the system is around the size of a coffee machine, and it also runs a range of other rapid molecular tests such as who prequalified xpert hcv viral load, hiv-1 qual and hiv-1 viral load tests [29] , which poses an opportunity for hepatitis b viral load test to be adopted in areas with existing platforms at a low additional cost. so far, limited data are available on the analytical performance of the assay [84, 85] . two recent studies [84] using serum samples showed a good correlation between hbv dna quantification by using xpert hbv viral load assay with the results of the laboratory reference assay; they also have a low limit of detection (lod) of 7.5 iu/ml, which is similar to most commonly used hbv dna platforms (usually with lod of 10iu/ml) [84] . in practice, xpert testing for hbv dna led to a faster workflow with a mean time to result being 6-8 h, which provided a near-poc solution [84, 86] . however, as a new unit, genexpert facilities are still expensive; the operation requires uninterrupted power supply, as well as technician training and skills for system running, services and reagent maintenance. hepatitis e antigen (hbeag) is a key indicator to determine phase of chronic hepatitis b infection (figure 2) , treatment initiation and is used as a surrogate of hbv dna measurement for evaluating risks of maternal-to-child-transmission [2, 4, 83, 87] . several hbeag pocs are commercially available; however, published data show the accuracy of hbeag pocs has a wide range, with sensitivity of 30-82% and specificity of 67-100% [33, 80, 88] . similarly, anti-hbe pocs are reported to have poor sensitivity but excellent specificity in studies [33, 81] . given the high costs and challenges in accessing hbv dna testing in low-resource settings, the who recommends hbeag to triage treatment [83, 89, 90] ; therefore, the low testing accuracy of hbeag pocs is an urgent issue to be addressed. novel serum biomarkers such as hbv core antigen (hbcrag) have been shown to correlate with serum hbv dna levels and intrahepatic cccdna levels, a marker of hepatitis b-related hcc risk, and have therefore been explored as a potential indicator for treatment determination, off-therapy virologic suppression and hcc risk evaluation [91, 92] . however, there is no rapid lateral flow assay for hbcrag so far. another novel biomarker, serum hbv rna [93] , has been shown to be positively correlated with hbv dna level, and levels were higher than hbv dna in patients on nucleos(t)ide analogues and can thus be a potential marker for off-therapy hbv suppression. however, its clinical predictive utility is not yet well defined, and the measurement of serum hbv rna presents its own challenges even in routine lab-based testing; thus, further studies are needed to guide defining the clinical role and development of hbv rna pocs. alt is part of the liver function test assay panel and is a key marker of liver inflammation, used to determine hepatitis b treatment eligibility [4, 5, 83] (figure 2 ). alt has been proposed as an indicator for treatment in people with positive hbsag in low-resource settings where hbv dna testing is unavailable. a semiquantitative poc using alt 40u/l as the cut-off (biopoint ® alt-1) has been developed and manufacturer data suggest a high sensitivity of 94% and specificity of 85% [94] . several serological biomarkers have been combined in algorithms to offer indirect non-invasive assessment of liver fibrosis and have been validated to varying degrees in hepatitis b populations, such as the ast to platelet ratio index (apri) and fibrosis 4 index (fib-4) [95, 96] . these indices require quantitative testing results of ast, platelets with or without alt, unfortunately none of which are currently available in a poc test format. dried blood spot (dbs), while not a poc test, is a sampling method which offers viable solutions for mass screening or testing in low-resource settings where testing capacity or access are limited. in practice, a single finger-prick blood sample is applied to a chemically modified paper card which collects and store serological markers and nucleic acid; specimens obtained in the field can be transported to a laboratory at ambient temperatures, where the blood sample is processed following a dbs protocol and tested using immunoassays or molecular techniques [83, 97] . dbs samples have a relatively long shelf-life at ambient temperature without sample degradation [98] , which is attractive for regions that are geographically isolated or have varying security situations precluding rapid transport to a central laboratory. this method is now recommended by the 2017 who hepatitis b testing guidelines [83] in settings where no access to venous blood sampling or quality-assured testing assays is available. dbs testing has been used to detect hbsag, hbeag, anti-hbc, hbv dna and even for viral genotyping [99, 100] . a meta-analysis [101] evaluating dbs for hbv dna quantification showed pooled sensitivity of 95% (83-99%) and specificity of 99% (53-100%); however, most of the included studies used cold chain to store samples, which might limit the generalisation of the accuracy estimates in field conditions. although dbs testing increases testing access in low-resource and geographically isolated settings, it still requires high technical expertise and standard laboratory assays that may not be routinely available. cost-effectiveness and affordability are key considerations when adopting poc tests in hepatitis b programs. quoted costs of lateral flow-based hbsag pocs are generally lower than laboratory-based immunoassays, with the estimated procurement costs being us$0.2-0.95 and us$0.4 to 2.8 per test, respectively [72, 83] . conventional lab-based testing usually requires additional costs such as a reading machine, professional laboratory staff and technical training; therefore, the total costs for testing are often much higher than using pocs in low-resource settings. multiplex poc testing is expected to be cheaper than multiple poc tests. for example, the manufacturing costs of a hiv/hcv/hbsag poc is around us$1 [102] ; thus, using multiplex in high-risk populations who require broad spectrum pathogen screening is expected to be resource-saving. costs for conventional hepatitis nucleic acid testing are estimated to range from us$30 to 120 [15] , and the cost can be up to us$400 per assay in resource-limited countries and regions [12] . in 2018, a viral load testing program was introduced in sub-saharan african countries to access an integrated molecular diagnostics instrument (hologic panther system), at an all-inclusive ceiling price of us$12 per patient sample [103] . the foundation for innovative new diagnostics (find) has negotiated the price of xpert hbv viral load assay for 145 developing countries, and it costs us$14.9 per cartridge excluding shipment; however, the testing instrument costs between us$11,530 to us$64,350 depending on the throughput capacity of the processing unit [104, 105] . however, these costs may still be higher than what programs could afford in some settings. in addition, due to reduced accuracy compared with standard assays, diagnostic poc testing is often used as a screening tool to triage those requiring more expensive laboratory-based testing confirmation [15] , which means many of the costs for centralised laboratory services are only partially offset by poc test use. while novel poc testing may have increased testing performance, costs usually fall slowly due to patent protection laws [16] . even for countries that could afford these pocs, it may cost more than lab-based testing where well-established laboratory services are available; therefore, the main demand for pocs is limited to self-testing or outreach programs to improve testing uptake. the cost-effectiveness of using pocs for hepatitis b can therefore be different in different settings. using hbsag pocs as a screening tool was found to be cost-effective in community-based approach in hbv-endemic but low-resource settings. nayagam et al. [106] assessed a community-based hbv screening and treating program in the gambia where hbsag pocs were provided to adult participants door-to-door at a total screening cost of us$7.4 per person. the program was found to be highly cost-effective, with an icer of us$540 per daly averted compared to status quo where no publicly provided hbv screening or treatment was available. integrating low-cost hbv pocs into existed healthcare services such as antenatal screening [52, 58, 107] , blood donor screening [62] and hiv clinics [59] [60] [61] can be another solution to achieve scale-up of hbv testing [108, 109] . zhang et al. [109] showed the integration of hbv screening within the existing antenatal care in cambodia was highly cost-effective. in their model, the unit cost of hbsag and dna test (estimated to be us$1 and us$30) was one of the key parameters driving cost-effectiveness; in such cases, cheap pocs could potentially improve the cost-effectiveness of such an integration program even further. studies in low hbv endemicity countries showed programs offering hepatitis b screening followed by vaccination or linkage to clinical care among people with increased risks are likely to be cost-effective [110] ; however, there is a lack of programs adopting pocs in hepatitis b screening strategy, and thus, a lack of evidence suggesting economic impacts using pocs for hepatitis b in populations who have regular access to healthcare services. however, a few studies have shown rapid hepatitis c or hiv testing nested in harm reduction programs or among priority populations can be cost-effective [111] [112] [113] . more evidence on using pocs in hbv screening or monitoring programs in the field is needed, especially covering the implementation costs and the effects of broader testing access compared to standard testing services or no testing services (where there being no access to testing is the current practice). whilst poc testing theoretically circumvents many test access barriers, acceptability from targeted population remains a key determinant of successful implementation of hepatitis b programs. however, limited data are available on the satisfaction appraisal from users and stakeholders. in general, pocs are highly acceptable to customers due to their easy-to-use nature, short turnaround time, minimal bio sample requirement and provision of testing capacity to familiar staff in contexts where people want to be tested [114] . in a survey conducted among implementers and users of hep b and c testing services from 43 countries, almost half of respondents from low-and middle-income countries preferred a poc test method using capillary whole blood [83] . while there is no agreement on what accuracy would be considered acceptable, half of the respondents would accept an assay with a minimal sensitivity of 95% [83] . acceptability of rapid testing for hepatitis b or other blood-borne viruses and sexual transmitted diseases can be varied in different populations. a survey done in a prison setting showed hcv pocs were highly accepted [115] . another study showed that people may find it stressful when testing hiv, hcv and syphilis using a poc test [116] . when using pocs for blood-borne virus screening in public events or community outreach programs, the acceptance rate varied widely in customers with different socioeconomic status, ethnic or geographic backgrounds [63, 117, 118] . in health facility settings, healthcare providers find pocs generally speed up decision making and improve patients' compliance with chronic management plans requiring repeat testing over time [30] ; however, there are also general concerns such as suboptimal testing accuracy and increased workload for healthcare workers [119] . other than getting tested from healthcare providers or trained personnel, pocs also have the potential to be a self-testing tool with universal access. in some countries, rapid tests for hepatitis b and/or hiv and hepatitis c can be purchased online or over the counter. while self-testing offers a confidential testing solution for customers, a standard approach will be needed to ensure that people having accessible pre-and post-counselling, as well as pathways of linkage to care. a general limitation of pocs for hepatitis b is reduced accuracy compared to standard lab-based testing. there are also specific limitations for individual poc tests which were highlighted above (section 2), such as hbsag poc tests which were shown to have varied sensitivity in hiv-infected populations. in addition, there is still a lack of poc tests for liver cirrhosis and hbv dna levels required to determine treatment eligibility for patients with hepatitis b. there are also limitations in the aspects of regulatory process, procurement and storage management for poc tests, as well as costs when implementing pocs for hepatitis b in different settings. the who prequalification process for in vitro diagnostic tests for diseases with a high individual or public health risk, including hepatitis b, assesses both the test's performance and manufacturing quality. for countries without regulatory procedures in place, this provides a thorough review of potential diagnostic tests they could select based on specific needs; however, the process of getting prequalified approval by the who can be slow. for low-resource settings, stock-out and supply issues can be a barrier for use of poc tests; lack of scale may also mean they can be more expensive than high throughput assays in some settings; testing accuracy as well as instrument maintenance can be impacted by extreme weather conditions (heat, humidity) in the field; novel testing platforms such as genexpert can still be expensive, and the use of the instruments can be subject to field conditions such as power supply. on the other side, for high-income countries, a main challenge for the introduction and implementation of poc tests is the regulatory and reimbursement approval process for new diagnostics, which require demonstration of analytic and clinical validity, as well as clinical usefulness and cost-effectiveness data. as an example, the fda regulatory process can be long and expensive [120] , and the return for investment in high-income countries where poc tests will compete for market with standard diagnostic pathways can be challenging. increasing testing accuracy is the major challenge for poc tests that are already compact and easy to use. when developing poc diagnostics, features targeting resource-limited settings without basic infrastructures or cold chain need to be included; tests with high quality need to be validated across populations and specimen type. rapid affordable serology tests of high accuracy for novel biomarkers which could be alternatives for molecular testing are a major need. technology is needed to integrate convenient sampling and specimen preparation into a one-step testing assay. inter-user variability is another challenge to address if poc tests require technique training or multiple steps; a standard protocol or mobile apps can be used to overcome this problem where suitable. miniaturisation of testing instruments is the trend, especially for instruments that could perform molecular analysis such as portable hand-held devices, without sacrificing testing accuracy. dry blood spot kits for hiv and hepatitis c can already be ordered online to be sent to a home address as a private way to test for infection [121] . faecal occult blood tests are mailed out to all older adults in some regions as a public health initiative to screen for bowel cancer [122] . a similar approach could be evaluated to screen for hepatitis b among populations that are disengaged from traditional health services. in resource-limited settings, adopting hepatitis b testing in existing platforms or programs can be more cost-effective than starting a new initiative [109] . mobile phone technology has the potential to be used for screening and monitoring health conditions [123] . mobile phones are now being used around the world for contact tracing for sars-cov-2, an approach that is immediately applicable to hepatitis b. recently, google searches for anosmia have been linked to the epidemiology of sars-cov-2 [124] . there is a need for the streamlining of regulatory and reimbursement approval processes in high-income countries where the traditional approval process is expensive and slow, particularly for poc diagnostics suitable for use as public health tools to promote the engagement of marginalised individuals, including people who inject drugs, migrants and culturally and linguistically diverse communities affected by hepatitis b. in low-and middle-income countries where regulatory processes can be less demanding, the key is to ensure the quality and performance of tests as they come to market. more than 60 products have been prequalified since the who prequalification process started in 2010 [29] . it has been proposed that a model list of essential diagnostics be developed, comparable with the model list of essential medicines maintained by the who. such a list would help in the selection of diagnostic methods and would facilitate improvements in the regulation and affordability of in vitro diagnostic tests and in training in their use. the who has set ambitious goals for the elimination of hepatitis b as a public health threat by 2030. birth dose vaccination is the most important public health intervention to reduce incidence and will also reduce mortality level long term. for the individual already infected with hepatitis b, the key to preventing liver-related harm is the maintenance of sustained viral suppression. this requires diagnosis and linkage to care; in some people, antiviral therapy will be necessary. hepatitis b is typically asymptomatic until advanced disease has developed. therefore, screening is required. the risk factors and epidemiology of hepatitis b are well described, but screening rates are suboptimal and often occur in the context of opportunistic doctor-patient consultations following presentation with an unrelated problem. testing typically involves venesection followed by centralised testing in a laboratory with batch processing and automation to improve efficiency. this system works well for the engaged individual being cared for by a motivated health care practitioner. however, even in high-income countries, up to 80% of infected patients remain unaware of their infection [125] . thus, there is a need to scale up screening for hepatitis b in high-risk populations, and 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exploring the barriers and facilitators to use of point of care tests in family medicine clinics in the united states innovation under regulatory uncertainty: evidence from medical technology do you need a dbs test? secondary do you need a dbs test national bowel cancer screening program secondary national bowel cancer screening program will an innovative connected aidesmart! app-based multiplex, point-of-care screening strategy for hiv and related coinfections affect timely quality antenatal screening of rural indian women? results from a cross-sectional study in india use of google trends to investigate loss-of-smell-related searches during the covid-19 outbreak new virological tools for screening, diagnosis and monitoring of hepatitis b and c in resource-limited settings this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-226956-n5qwsvtr authors: arbia, giuseppe title: a note on early epidemiological analysis of coronavirus disease 2019 outbreak using crowdsourced data date: 2020-03-13 journal: nan doi: nan sha: doc_id: 226956 cord_uid: n5qwsvtr crowdsourcing data can prove of paramount importance in monitoring and controlling the spread of infectious diseases. the recent paper by sun, chen and viboud (2020) is important because it contributes to the understanding of the epidemiology and of the spreading of covid-19 in a period when most of the epidemic characteristics are still unknown. however, the use of crowdsourcing data raises a number of problems from the statistical point of view which run the risk of invalidating the results and of biasing estimation and hypothesis testing. while the work by sun, chen and viboud (2020) has to be commended, given the importance of the topic for worldwide health security, in this paper we deem important to remark the presence of the possible sources of statistical biases and to point out possible solutions to them the paper by sun, chen and viboud (2020) (henceforth scv) is an important example of the use of crowdsourced data in monitoring the spread of covid-19. indeed, crowdsourcing data can prove of paramount importance in monitoring and controlling the spread of infectious diseases as it is also remarked e. g. by leung and leung (2020) among the many others. the paper relies on an innovative source (potentially obtainable in real time) derived from social media and news report collected in china from 13 th to 31 st of january 2020 during the first outbreak of the corona virus epidemics. the data collected referred to 507 cases. in the paper the crowdsourced data, coming from different sources, are used to estimate several epidemiological parameters of tremendous importance in the process of surveillance and control of the diffusion of the disease such as: the relative risk by age group, the mean age and skewness of infected people, the time of delays between symptoms and seeking care at hospital, the mean incubation period. scv also use the crowdsourced data to test theoretical hypotheses using the wilcoxon test and the kruskal-wallis test 2 these test lead them to conclude that the delay between symptoms onset and seeking care at hospital or clinic decreased significantly after january 18 th and that the delay was significantly longer in hubei with respect to tianjin and yunnan and between international travelers and local population. there are two main statistical problems connected with the use of crowdsourced data in general and with those presenting a spatial configuration in particular (such as those employed by scv), namely: 1. the lack of a precise sample design 2. the presence of spatial/network correlation among the individuals in the sample we will briefly discuss the two problems in more details in the following two sections. 1 catholic university of the sacred heart, milan (italy) 2 as is it well known the wilcoxon test (or the mann-whitney u test) is a nonparametric test used to test the hypothesis of equality between two independent samples. the kruskal-wallis test is also non-parametric which extends the wilcoxon test in comparing two or more independent samples of equal or different sample sizes and can be also seen as the non-parametric version of the one-way analysis of variance (anova). see wilcoxon (1945) and kruskal and wallis (1952) a general characteristics of crowdsourced data, , likewise other unconventionally collected big data 3 , is the lack of any precise sample design (arbia, 2020 ). this situation is described in statistics as a "convenience sampling", in which case it is known that no probabilistic inference is possible (hansen et al, 1953) . as fisher (1935) says "if we aim at a satisfactory generalization of the sample results, the sample experiment needs to be rigorously programmed". indeed, while in a formal sample design the choice of sample observations is guided by a precise mechanism which allows the calculation of the probabilities of inclusion of each unit (and, hence, probabilistic inference), on the contrary with a convenience collection no probability of inclusion can be calculated thus giving rise to over-under-representativeness of the sample units. the advantages of using a convenience sampling are the obvious ease of data collection and cost-effectiveness. however, the disadvantages are that the results cannot be generalized to a larger population because the under-(or over-) representation of units produce a bias. furthermore, convenience sampling-based estimates are characterized by larger standard error and as a consequence by insufficient power in hypothesis testing. in this situation the estimation of parameters (like mean, median, proportions) based on the principle of analogy and the calculation of p-values to take decisions in hypothesis testing is not theoretically motivated. scv, indeed, acknowledge the fact that the collection criterion used could have generated possible biases in their sample. they list problems like the fact that a substantial proportion are travelers (who are predominantly adults), that data are captured by the health system and so are biased toward more severe cases, that geographical coverage is heterogeneous with an under-representation of provinces with a weaker health infrastructure. however, they do not take any action to reduce such biases. the problem raised by the convenience collection of data emerges dramatically in the big data era when we increasingly avail data which, almost invariably, do not satisfy the necessary conditions for probabilistic inference. in recent years researchers are becoming aware of this problem trying to suggest solutions to reduce the distorting effects inherent to non-probabilistic designs (fricker and schonlau, 2002) . one possible strategy consists in transforming crowdsourced datasets in such a way that they resemble a formal sample design. this procedure has been termed post-sampling (arbia et al., 2018) and represents a particular form of poststratification (holt and smith, 1979; little, 1993) . to implement a post-sampling analysis, we need to calculate, in each geographical location (e.g. the chinese provinces), a post-sampling ratio (ps), defined as the ratio between the number of observations required by a reference formal sample design (e.g. random stratified with probability of inclusion proportional to size) and those collected through crowdsourcing. more reliable estimations of population parameters can then be obtained by considering a weighted version of the dataset using the post-sampling ratio as weights. thus, crowdsourced observations will have to be over-weighted if ! > 1 and, on the contrary, down-weighted when ! < 1. this strategy has been adopted in arbia et al. (2018) in order to estimate the food price index in nigeria using data crowdsourced through smartphones and in arbia and nardelli (2020) to estimate spatial regression models. after post-sampling, estimates display less bias and lower standard errors and the reduction in the power of the tests is moderated. the dataset used by scv refer to 507 individuals (364 from china and the rest from abroad). both in the estimation of the epidemiological parameters reported in section 1 and in hypothesis testing the authors treat their crowdsourced data as if they were independent. indeed, both the wilcoxon and the kruskal-wallis test are based on the assumptions that all the observations are independent of each other. however, even if data were collected obeying a formal sample design, a further potential source of bias is the fact that the observational units could display a certain degree of spatial/network correlation (cliff and ord, 1973; arbia, 2006) . observed units that are close in space or in network interaction, may display similar values in the observed variables (e. g. age, incubation period, delays between symptoms and seeking care at hospital) due to the interaction between individuals or/and to presence of some unobserved latent variable with a geographical component. the effects of spatial correlation in the geography of epidemics is well documented in the book by cliff et al. (1981) . when observed data are not independent and display a positive spatial/network correlation, the standard errors are underestimated leading to inefficient estimation of the various parameters (mean, median, proportion etc.) . but the consequence on hypothesis testing can be even worse. due to the underestimation of the standard errors, indeed, the statistical test become artificially inflated leading to lower p-values and, as a consequence, to the rejection of the null hypotheses more frequently than we should. as a result, the significance of the statistical test can become very poor with very artificially high probability of type i error. this could explain the very low levels of p-values (of the order of 10 -4 ) reported in the scv paper despite the relatively small dataset used. standard statistical textbooks like shabenberger and gotway (2004) and cressie and wikle (2011) document how to calculate the level of spatial/network correlation between geographically located individuals and how this parameter can be used in the process of estimation and hypothesis testing in order to obtain more reliable inferential conclusions. the work by sun, chen and viboud (2020) has to be commended, given the absolute relevance of the topic for health security and the timeliness with which results are presented in a period of great uncertainty related to the worldwide diffusion of the new corona virus covid-19. their results are of invaluable help in the process of surveillance, monitoring and control of the disease. in this comment we draw the attention of the authors and of all the researchers and health operators on the fact that the crowdsourced dataset they use can lead to biases in the estimation of the epidemiological parameters and in hypothesis testing procedures. we hope that this note may help future studies in the area, so as to obtain even more reliable estimation and more grounded tests of theoretical hypotheses thus progressing rapidly and rigorously in the knowledge about covid-19 and any possible future new epidemics. a primer for spatial econometrics 2020) statistics, new empiricism and society in the era of big data, forthcoming, springerbrief on spatial lag models estimated using crowdsourcing, web-scraping or other unconventionally collected data, under revision for spatial economic analysis post-sampling crowdsourced data to allow reliable statistical inference: the case of food price indices in nigeria, paper presented at the lx conference of the italian statistical society advantages and disadvantages of internet research surveys: evidence from the literature post stratification, series a use of ranks in one-criterion variance analysis post-stratification: a modeler's perspective crowdsourcing data to mitigate epidemics, the lancet digital health early epidemiological analysis of coronavirus disease 2019 outbreak using crowdsourced data: a population level observational study statistical methods for spatial data analysis individual comparisons by ranking methods key: cord-307187-5blsjicu authors: missel, malene; bernild, camilla; dagyaran, ilkay; christensen, signe westh; berg, selina kikkenborg title: a stoic and altruistic orientation towards their work: a qualitative study of healthcare professionals’ experiences of awaiting a covid-19 test result date: 2020-11-11 journal: bmc health serv res doi: 10.1186/s12913-020-05904-0 sha: doc_id: 307187 cord_uid: 5blsjicu background: extensive measures to reduce person-to-person transmission of covid-19 are required to control the current outbreak. special attention is directed at healthcare professionals as reducing the risk of infection in healthcare is essential. the purpose of this study was to explore healthcare professionals’ experiences of awaiting a test result for a potential covid-19 infection. methods: qualitative interviews with 15 healthcare professionals were performed, underpinned by a phenomenological hermeneutical analytical framework. results: the participating healthcare professionals’ experiences of awaiting a covid-19 test result were found to be associated with a stoic and altruistic orientation towards their work. these healthcare professionals presented a strong professional identity overriding most concerns about their own health. the result of the coronavirus test was a decisive parameter for whether healthcare professionals could return to work. the healthcare professionals were aware that their family and friends were having a hard time knowing that the covid-19 infection risk was part of their jobs. this concern did not, however, cause the healthcare professionals to falter in their belief that they were doing the right thing by focusing on their core area. the threat to own health ran through the minds of the healthcare professionals occasionally, which makes access to testing particularly important. conclusion: the participating healthcare professionals had a strong professional identity. however, a discrepancy between an altruistic role as a healthcare professional and the expectations that come from the community was illuminated. a mental health coronavirus hotline for healthcare professionals is suggested. the covid-19 pandemic puts healthcare professionals (hcp) under pressure both physical and psychological [1] . the challenges include increased workload created by the outbreak but also fears of contagion for themselves, their families and patients. particularly psychological health outcomes and distress are highlighted in current research regarding the initial stage of the covid-19 outbreak in terms of anxiety, depression and post-traumatic symptoms [1] [2] [3] [4] [5] . across these studies hcps working during the epidemic report frequent concerns regarding their own health. based on our knowledge, little information is however available regarding the impact on hcps awaiting a test result for potential covid-19 infection or interventions for supporting them during this waiting time. therefore, this study aim to shed light on hcps' experiences of awaiting a test result for a potential covid-19 infection through individual interviews. this qualitative investigation will thus highlight what is at stake for hcps while in quarantine and awaiting a response as to whether they are infected with the coronavirus. the study offers an in-depth understanding of the meaning of the waiting for the test result for covid-19 infection from the hcps' perspective and should be of interest to a broad readership and add knowledge to the growing covid-19 evidence base and in developing supportive inetrventions targeted hcps in such a pandemic. while hcps, e.g. nurses, physisians, porters and healthcare workers, are caring for some of the most vulnerable groups of people both in hospital but also in primary care, they are currently also facing an unprecedented disease caused by the outbreak of a previously unknown virus [6] . this new coronavirus that can cause covid-19 disease [7] puts hcps in a position where they must avoid exposing themselves to infection but also avoid transmitting the infection to the vulnerable patients and citizens to whom they have a caring responsibility. because an infected hcp is a potential vehicle for virus dissemination, research suggests that reducing the risk of infection amongst hcps is essential [8] . spread of virus has been reported during the ebola outbreak resulting in a compromised healthcare system [9] as well as during the severe acute respiratory syndrome (sars) [10] and the middle east respiratory syndrome (mers) epidemics [11] . experiences from these previous outbreaks highlight fear among hcps in transmitting the disease and the importance of screening for the virus. on 30th january 2020, the world health organization declared the chinese outbreak of covid-19 to be a public health emergency of international concern. the emergency committee stated that the spread of covid-19 may, among other preventive efforts, be interrupted by early detection and isolation [7, 12] . general hygiene precautions are crucial in order to minimize the risk of contamination [8] . hcps have always played an important role in infection prevention, infection control, isolation, containment and public health, which for nurses initially was advocated for by florence nightingale [13] . there are studies that define the pathophysiological characteristics of covid-19 however, the mechanism of spread is uncertain. current knowledge is derived from similar coronaviruses, which are transmitted from human-to-human through respiratory infection [7] . typically, respiratory viruses are most contagious when a patient is symptomatic. however, increasing evidence suggests that human-to-human transmission may be occurring during the asymptomatic incubation period of covid-19 [14, 15] . the disease is reported to be very contagious, and measures to reduce person-to-person transmission of covid-19 are therefore required to control the outbreak [14] [15] [16] . special attention and efforts to prevent or reduce transmission is applied in susceptible populations including hcps in order to reduce transmission to patients or other vulnerable groups of people in the community [17] [18] [19] . hcps are thus among those groups of people who are being rapidly tested for coronavirus in denmark. considering the severity of infection and illness [20] , the test result might be of great importance for the healthcare system but also for the individual hcp. a sudden decrease in the number of hcps because of quarantining or isolation due to covid-19 infection would potentially overload the healthcare system and the capacity to treat either patients with coronavirus or patients with other serious conditions would be challenged [8] . for the individual hcp, it might furthermore be a threat to their own health. as far as we are aware, no research has so far focused on how hcps might perceive this test situation. therefore, the purpose of this study is to explore hcps' experiences of awaiting a test result for a potential covid-19 infection. such knowledge from the hcps' perspective are expected to increase the awareness of potential needed support while awaiting a crucial test result from a contagious and rare virus. furthermore, the study will help hospital managers to establish strategies to ensure the best possible working conditions for hcps during the pandemic. this study used a phenomenological hermeneutical methodology inspired by ricoeur's narrative philosophy [21] . in this study phenomenology was apllied as an epistemological stance for exploring first-person accounts of what it is like to wait for a test result for potential covid-19 infection. pre-reflexive experiences from the participant's lifeworld is the starting point, while hermeneutics was focused on interpreting the surplus meaning contained in this lifeworld. as human beings we leave traces when we express ourselves, and these traces are formed by the meanings and traditions to which we belong. often, it is impossible to directly understand individual's experiences because the sense in the traces is hidden. therefore, reflection on an individual's lived experiences takes place via the narratives expressed by the individuals [21, 22] . the threefold mimesis is central in ricoeur's narrative philosophy and can be seen as an epistemological approach for understanding the participants' lived experiences [23] , which, in this study, has inspired the research process as a three-fold process [22] : mimesis i (prefiguration): the life lived before it is formulated as spoken or written narrative (data collection); mimesis ii (configuration): the language stage, formulating a narrative (from speech to text); and mimesis iii (refiguration): the comprehension stage, when the text is interpreted (analysis and interpretation) [21] [22] [23] . participants in this study were recruited from a population of hcps who had been tested for coronavirus but who did not necessarily care for covid-19 patients. if they had symptoms of covid-19 infection, hcps in denmark were offered testing for the virus. we used a convenience sampling strategy [24] by encouraging tested hcps to approach the research team by e-mail if they were willing to attend an interview. the interviews were conducted by telephone based on ethical accountability for not contributing to the spread of the virus and they were scheduled in the gap between test and its result. the result of the test was during the study period given to a tested person within 24 h. the society of denmark was on lockdown due to the threat of coronavirus on march 11th 2020. coronavirus was in this period still relatively new in denmark, and 300-500 patients were hospitalized and 77 patients died due to covid-19 during week three of the epidemic. fifteen hcps agreed to participate in the study and were interviewed in march and april 2020. thereafter data saturation was achieved, making further interviewing unnecessary [24] . we included hcps with different professional backgrounds and different responsibilities from both primary care and hospitals. the characteristics of the participants are shown in table 1 . data were collected through individual interviews. human events are characterized by unreflecting preunderstanding, which ricoeur calls prefiguration (mimesis 1) [21, 22] . with the aim of gathering the participants' indepth narrative accounts of their experiences of awaiting a covid-19 test results, open questions were used. each interview began with a broad opening question, such as; "could you please tell me what led you to being tested for a potential covid-19 infection and your experiences while awaiting the test result?" table 2 lists the interview questions. the interviews lastet on average 30 min (range 9-55 min). the interviews were separately conducted by three experienced qualitative researchers who all had a professional background as registret nurses, and interviews were audio-recorded and transcribed into 217 pages. the participants' stories were thus transcribed into a textual configuration of their unarticulated experiences (from prefiguration to configuration) [21, 22] . according to ricoeur, people's narratives contain surplus meaning and hermeneutics is concerned with interpreting this surplus meaning (from configuration to refiguration). the study was undertaken in accordance with the guidelines of the danish ethical research committee and was approved by the danish data protection agency (p-2020-276). the investigation conforms with the principles outlined in the declaration of helsinki [25] . the participants received written information about the purpose of the study and their right to withdraw at any time. written informed consent was obtained from each of the participants before the interview. data were anonymized by means of identification codes. the participants were informed that interview data would be treated confidentially. according to ricoeur, interpretation is the central methodology in phenomenological research. interpretation involves a process consisting of naive interpretation, structural analysis, and comprehensive understanding [26] . naive interpretation is superficial interpretation, whereby the narratives are read and re-read to see what the texts mean to the researchers, giving an overall view of the narratives. structural analysis deals with patterns in the text that can explain what it is saying. explaining what the text expresses means moving from what the text says to what it is talking about. during the structural process, we analyzed and structured the narratives based on units of meaning, extracting meaning or themes that recurred in the narratives. the units of meaning were condensed such that the essential meaning was expressed. these units of meaning were then further condensed and gathered into themes [22, 26] . the comprehensive understanding continues with a discussion of the themes that were identified in the structural analysis, the purpose being to reach a new understanding of the possible dimensions of the participants' experiences while awaiting a covid-19 test result. the deeper interpretation of the narratives is a process of understanding in which theoretical perspectives are drawn on to help clarify and comprehend phenomena in the participants' experiences [22, 26] . see fig. 1 . throughout the study methodological rigor was attained by using the qualitative concepts of relevance, validity, and reflexivity, as described by malterud [27] . this study is one of only a few qualitative studies exploring the lived experiences of hcps during the covid-19 pandemic and to our knowledge this is the first qualitative study exploring hcps' experiences of awaiting a test result for a potential covid-19 infection. the qualitative interview method was selected in order to gain insight into these individuals' perspectives in order to understand the meaning of the investigated phenomena, i.e. the transition from experience to meaning [26] . the relevance of the study and the chosen methodology thus seems appropriate. several strategies were employed to demonstrate internal validity, including collecting indepth data, prolonged involvement with the data and use of the participants' own words to formulate and illustrate themes. the participants are quoted in order to ensure transparency and substantiate the findings of the study. ricoeur's steps in the analytical process are clearly set out and have been stringently followed. the process from prefiguration through configuration to refiguration reflects the shift from lived life to narrative accounts of lived life to the final interpretation, which provides an insight into the individual hcps' concrete experiences and into universal phenomena of life for hcps awaiting a test result. thus other researchers are able to judge and validate the extracted themes. reflexivity was ensured by discussions between the authors, both during the data collection phase and in the analysis. the fact that all interviewers were registered nurses meant that a certain agreement but also equality between participant and interviewer was present. this meant that the conversation was relatively easy and straightforward. in order, however, to prevent blind spots in relation to the research purpose, the interviewers were particularly aware of their role as researchers and qualitative interviewers and tried to bridle preunderstandings from their background as hcps and adapting a curious stance. the comprehensive understanding illuminated the meaning of the participants' experiences of awaiting a covid-19 test result as a stoic and altruistic orientation towards their work. these hcps presented a strong professional identity overriding most concerns about their own health. the result of the coronavirus test was a decisive parameter for whether healthcare professionals could return to work. experiences related to the test situation as well as the strong sense of professional identity will be described in more detail in the following. what led the participants to the test for coronavirus were their experiences of mild to moderate symptoms, which aroused suspicion of possible infection. they described the importance of protecting patients, vulnerable citizens and colleagues from the risk of infection and therefore stayed away from work until they were certain that they were not contributing to the spread of the virus. this distance from work, however, had an impact on participants who described a dilemma in terms of both feeling responsible and hypochondriac at the same time. as hcps they already knew the usual workload and therefore described feelings of failing colleagues by not taking part in the work, "we are busy in healthcare, so if there is one who is sick, then the others just have to run faster" (participant k). thus, the test result was extremely important in terms of whether one could return to work and help one's colleagues. the participants, furthermore, talked of particular responsibilities in being prepared to care for and treat patients with covid-19. they watched what is going on in the rest of the world in other healthcare settings where the epidemic of covid-19 exceeded the healthcare systems' resources. they were very concerned about their colleagues in other countries but at the same time had an altruistic view that they themselves must also be prepared. in this context, coronavirus tests are also particularly important for the participating hcps. they did, however, describe an ambivalence around the test response; if you are tested positive, then hopefully you will form some kind of immunity and thus be able to go to work after a period of quarantine without being infected again. if, on the other hand, you are tested negative, you can return to your job immediately, "i hope i don't have corona, but on the other hand, then you have had it …" (participant c). participants describe concerns and fears that many hcps will be infected at the same time, and that there will be no one to take care of the ill patients or vulnerable citizens. therefore, it was necessary to have the hcps tested so that an overview of the workforce can be maintained as hcps cannot easily be replaced. the way to being tested could, however, be quite obscure for some of the participants. for participating hcps working in the hospital, access to testing is easy and straightforward. they noticed symptoms, they discussed it with their boss, and they got tested. however, working in primary care posed major problems in figuring out access to being tested. those hcps narrated experiences of not being taken seriously, which produced a kind of powerlessness, "all of us who work in healthcare, we are there to make a difference, but you just feel that we sometimes are banging our head against the wall [experiencing lack of understanding] … it gives a sense of powerlessness" (participant e). they furthermore described frustrations of wasting precious time waiting to get to the test; time that could have been spent usefully in continuing their work. the particular commitment to caring for vulnerable and ill people was evident when participating hcps were just waiting to be tested. even though being tested for coronavirus when experiencing symptoms was strongly preferred by the participants in this study, the test situation, however, reminded and confronted them with the seriousness of the pandemic. they described their experiences of coming into the interimistic tents outside the hospital and meeting with test staff in protective equipment. the participants, being hcps, were prepared for this scenario but are anyway confronted with feelings of being part of a surreal experience or a science fiction movie but also that this new virus was real, "it is a peculiar experience to meet another person who is covered from head to toe. you suddenly feel very dangerous" (participant f). they also, however, told of a professional set-up and that being tested provided certainty, tranquility and direction. the participating hcps in this study presented a strong sense of professional identity and were highly oriented towards their work. they talked about how they were preparing for battle against the coronavirus despite the risk of being infected themselves. the frontline hcps with the critical task of caring for covid-19 patients told how for a long time and with no evidence of even having the disease, they had isolated themselves at home, "i already decided 14 days ago that we should stop sleeping in the same room and avoid physical contact completely. i have also written on my wife's and my behalf to family and friends that we will not be able to see anybody for a while" (participant b). they were tremendously aware of their specific role and duty and that nobody could stand-in for them and explained it as just being a part of their job and with a fatalistic attitude. these participants expressed a paramount need to know if they were contagious. common to the participants was that, by virtue of their profession, they had important professional knowledge about drop infections, hygiene, symptoms and pathways of infection, all of which gave them a readiness to act. they narrated how they were extremely aware of not transmitting the infection to others, as well as how to take distance and hygiene measures when they noticed symptoms of potential covid-19. these measures seemed to be integrated as an almost natural act in the participants' lives with them not questioning the necessity of doing so, "i've locked myself inside a room now and told the others in the family to stay away. and if i'm going to the toilet ..., our apartment is quite small ... but then i just shout that now i go to the toilet. and then i have hand sanitizer and cleansers and wipe it all off afterwards" (participant c). the situation thus appears to have been tackled with stoic calm by the participants as they awaited answers as to whether their possible symptoms are related to covid-19. despite their professional knowledge, participants also told of chaotic and conflicting information from the healthcare system expressed as an information flow that had become incomprehensible and overwhelming. this resulted in uncertainty and difficulty in keeping up with guidelines. the participants' social network was marked by the possible threat of covid-19 from the hcps who were just doing their job in healthcare. the participating hcps were highly aware that their family and friends were having a hard time knowing that the covid-19 infection risk was a necessary condition of their job, while they at the same time are forced to keep a distance. this concern did not, however, cause participants to falter in their belief that they were doing the right thing by focusing on their core area, which was caring for ill and vulnerable people. the threat to their own health ran though the minds of the participants once in a while, "that people who take care of their work and do what they can to make others survive can end up getting infected with covid-19 themselves, i think that's a little hard, but that's just how it is" (participant g). the participating hcps express a need to share such thoughts with somebody and ask for some kind of follow-up or a hcp corona hotline, e.g. after being tested for the virus, "when you are nervous and scared, it would be helpful if you could go to one specific place where knowledge and expertise about corona was gathered -a mental health corona hotline" (participant d). being oriented towards their job was described as a natural part of the participating hcps approach to life. they had a strong passion for and pride in their work and in this epidemic context showed solidarity across professional boundaries. they did question if they may be too uncritical but explained it with the fact that they are in a time when it is necessary to do as one is told. the participants, however, described how they have experienced the community tribute e.g. public applause for them as on the edge of hypocrisy. they rejected more applause from society and express how genuine societal recognition would be more resources in hospitals to solve problems and to give the hcps a tolerable everyday life and a decent salary. awaiting a covid-19 test result for the participating hcps was associated with a stoic and altruistic orientation towards their work in which the result of the test was crucial. this study illuminated how hcp prepare and get ready for battle against covid-19 in a devoted and solidarity-based way. this war metaphor as a response to the pandemic might illuminate the hcps' stoic and altruistic work identity. seeing the coronavirus as an enemy that should be defeated and as a part of one's job require hcps who approach their work with a stoic calm and an altruistic attitude. a similar commitment to supporting their health system and communities has been reported during the ebola epidemic [28] . the participants in our study presented a strong professional identity and their attention was directed to caring and protecting patients and vulnerable citizens while also preventing the spread of infection among colleagues. being stoic in their approach to work does not mean that hcp are cold and distant, it is rather an attitude of remaining calm and carrying on and may also involve having a certain degree of self-control and maintaining a sense of conscious self-awareness [29] . the altruistic attitude or behavior of the participants was characterized by the fact that the individual sought to promote the well-being of others without thought for their own interests and needs. according to hume, altruism is a character trait of humans that normally extends to strangers only in a weakened form and it is rare to meet with one in whom the affections of altruism do not over-balance the selfish [30] . altruism was, however, a strong moral part of the participants' professional identity which seems to be based on the inner logic of the hcp discipline. understanding of the roles altruism might play in the social and medical response to an epidemic and the stories about the nature of hcps' moral obligations has been discussed and implies the willingness to take personal risks in the line of duty [31] . a professional identity can be defined as a social identity that relates to people's understanding and presentation of themselves as professionals [32] . it is seen as the identity a person has developed through learning and practicing a given profession and thus can fulfill a particular employment function designed and integrated into a given work and professional culture. according to goffman, identities are not created individually, but rather the individual gains his or her professional identity through the attribution of certain characteristics that have the character of normative expectations [33] . in addition to performing the expected functions associated with a specific field, the individual thus supports and supplements his or her position by simultaneously playing the normatively expected role associated with that group [33] . to follow goffman [33] , the stoic and altruistic orientation towards their work presented by hcp in the present study might also point to these hcp acting in accordance with a specific role within a given social context, such as healthcare. society's normative expectations of hcp may influence their perception of their own professional identity. our study, however, illuminates a discrepancy between an altruistic role as hcps and the normative expectations that come from the community that pays tribute to them, and then an experience of working conditions and salaries that do not indicate recognition. altruism has been reported to be declining in the face of economic and pragmatic motivation [34] which might threaten healthcare practice during an epidemic such as covid-19. another threat to our study participants' stoic and altruistic orientation towards their work was also experiences of receiving chaotic, conflicting and an overwhelming information flow resulting in difficulties in keeping up with best practice guidelines. research from the a/h1n1 influenza pandemic have demonstrated how perceived sufficiency of information was associated with reduced degree of worry and how hcps less frequently felt unprotected [35, 36] . these points highlight that hospital managers should try to provide and direct information for hcps according to what is needed during the different and specific phases of a pandemic based on the affected hcps' perspectives in order to offer favourable working conditions in times of extreme distress. being tested for coronavirus for the hcps in our study was significant in order to maintain their professional identity and continue working. they did, however, also describe experiences of uncertainty and fear for own health and expressed a need to share such thoughts with somebody. a threat to the mental health of hcps during epidemics has been reported [4, 5, 37] , and interventions to promote mental well-being in hcps exposed to covid-19 are suggested to be immediately implemented [37] . a hotline for patients during the current covid-19 outbreak has been established in some places, e.g. in new york where citizens are guided to assess their own symptoms at home and can discuss any psychological impact from the disease [38] . similar initiatives directed at hcps are needed. recomandations from a recent systematic review also suggest to establish a forum for medical personnel to voice their concerns as well as a psychological assistance hotline comprised of volunteers who have received relevant psychological training to be able to provide telephonic guidance to personnel to help effectively tackle mental health problems [39] . telephone interviews in this study were unavoidable due to the risk of virus transmission between participants and interviewers. such interviews do, however, have some disadvantages. they are more impersonal in that it is not possible to have eye contact, and as an interviewer, it is difficult to show that you are interested and included in what is being said. in addition, breaks are generally less acceptable [24] . despite this, we found that participants were willing to participate in the study and appreciated talking about their experiences. the sample included in this study consisted of more female hcps (n = 11) and most were nurses (n = 8) which might be an uneven distribution of participants. women, however dominate the nursing profession, and nurses are the largest professional group in healthcare [40, 41] and the sample thus represents the general healthcare workforce. what is worth noting is that this study was conducted during the first phase of the pandemic. this means that the stoic and altruistic orientation as well as the war metaphor that we have found and described may change over time as the pandemic progresses and hpcs may experience burnout. the perspectives of hcps awaiting a test result for coronavirus provide an important contribution to the growing body of literature about covid-19. these hcps had a strong professional identity with their attention directed towards caring and protecting patients and vulnerable citizens while also preventing the spread of infection among colleagues. a discrepancy between an altruistic role as a hcp and the normative expectations that come from the community was also illuminated. the clinical implications of this study is thus, that as a stoic and altruistic attitude dominated hcps' identity, access to testing for covid-19 for these professionals is crucial. furthermore, a mental health corona hotline for hcps should be established. abbreviations hcp: healthcare 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working experiences of nurses during the middle east respiratory syndrome outbreak world health organization. novel coronavirus (2019-ncov), situation report-3. 2020 covid-19: emerging compassion, courage and resilience in the face of misinformation and adversity transmission of 2019-n-cov infection from an asymptomatic contact in germany early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) retningslinjer for håndtering af covid-19 i sundhedsvaesenet (guidelines for managing covid-19 i the healthcare system). copenhagen; 2020 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges ricoeur's narrative philosophy: a source of inspiration in critical hermeneutic health research en hermeneutisk brobygger. tekster af paul ricoeur nursing research: generating and assessing evidence for nursing practice declaration of helsinki discource and the surplus of meaning qualitative methods in medical research we and the nurses are now working with one voice": how community leaders and health committee members describe their role in sierra leone's ebola response the therapy of desire. in: theory and practice in hellenistic ethics altruism in hume's treatise diminishing returns? risk and the duty to care in the sars epidemic bankmedarbejderen-splittet mellem varnaes og scrooge (bank employees -split between varnaes and scrooge) two studies in the sociology of interaction. united states: martino fine books professional nursing values: a concept analysis general hospital staff worries, perceived sufficiency of information and associated psychological distress during the a/h1n1 influenza pandemic psychological impact of the pandemic (h1n1) 2009 on general hospital workers in kobe health professionals facing the coronavirus disease 2019 (covid-19) pandemic: what are the mental health risks? a phone call away: new york's hotline and public health in the rapidly changing covid-19 pandemic factors affecting the psychological well-being of health care workers during an epidemic: a thematic review sundhedsvaesen og sundhedspolitik (healthcare and healthcare politics) closing the gap in indigenous health inequity -is it making a difference? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the research team wishes to thank all those people who collaborated and participated in this study by sharing their experiences. without them, this study would not have been possible. we also thank anne alexandrine øhlers, camilla rotvig jensen, christina jensen, mette skriver and miriam bianca besser biyai for their help in relation with the transcription of the interviews. all authors conceived and contributed to the design and conduct of the study. skb, cb and mm conducted the collection of data material and led the analysis together with swc and id. all authors were involved in the analysis and the writing of the manuscript. all authors contributed to the preparation of this manuscript and read and approved the manuscript. this work was supported by the novo nordisk foundation (grant number nnf20sa0062831), and centre for cardiac, vascular, pulmonary and infectious diseases, rigshospitalet, copenhagen university hospital, denmark.availability of data and materials all authors have full control of all primary raw data (interview transcripts) and allow the journal to review our data if requested. all raw data are written in danish. data are stored in a locked file cabinet in a locked room at the copenhagen university hospital as requested by the danish data protection agency. the data material used in this study are available from the corresponding author on reasonale request which will not conflict with the anonymity and confidentiality of the data.ethics approval and consent to participate registration and permission was received from the authorities in the danish data protection agency under the capital region of denmark: (p-2020-276) and the study were undertaken in accordance with the guidelines of the danish ethics research committee. the participants received verbal and written information about the study prior to the study. written consent was obtained from the participants. given the qualitative nature of the study, the local ethics committee in the capital region of denmark ruled that no formal ethical approval was required in this particular case. not applicable. the authors have no conflicts of competing interest to declare.author details 1 clinical nurse specialist at the department of cardiothoracic surgery, centre for cardiac, vascular, pulmonary and infectious diseases, rigshospitalet, key: cord-342181-x14iywtr authors: taipale, j.; romer, p.; linnarsson, s. title: population-scale testing can suppress the spread of covid-19 date: 2020-05-01 journal: nan doi: 10.1101/2020.04.27.20078329 sha: doc_id: 342181 cord_uid: x14iywtr we propose an additional intervention that would contribute to the control of the covid-19 pandemic, offer more protection for people working in essential jobs, and help guide an eventual reopening of society. the intervention is based on: (1) testing every individual (2) repeatedly, and (3) self-quarantine of infected individuals. using a standard epidemiological model (sir), we show here that by identification and isolation of the majority of infectious individuals, including those who may be asymptomatic, the reproduction number r0 of sars-cov-2 would be reduced well below 1.0, and the epidemic would collapse. we replicate these observations in a more complex stochastic dynamic model on a social network graph. we also find that the testing regime would be additive to other interventions, and be effective at any level of prevalence. if adopted as a policy, any industrial society could sustain the regime for as long as it takes to find a safe and effective cure or vaccine. our model also indicates that unlike sampling-based tests, population-scale testing does not need to be very accurate: false negative rates up to 15% could be tolerated if 80% comply with testing every ten days, and false positives can be almost arbitrarily high when a high fraction of the population is already effectively quarantined. testing at the required scale would be feasible if existing qpcr-based methods are scaled up and multiplexed. a mass produced, low throughput field test kit could also be carried out at home. economic analysis also supports the feasibility of the approach: current reagent costs for tests are in the range of a dollar or less, and the estimated benefits for population-scale testing are so large that the policy would be cost-effective even if the costs were larger by more than two orders of magnitude. to identify both active and previous infections, both viral rna and antibodies could be tested. all technologies to build such test kits, and to produce them in the scale required to test the entire world's population exist already. integrating them, scaling up production, and implementing the testing regime will require resources and planning, but at a scale that is very small compared to the effort that every nation would devote to defending itself against a more traditional foe. number r0 remains greater than 1, the virus spreads rapidly until most people have been infected (fig. 1a) , creating a temporary surge of infected individuals. if, using pharmacological or social interventions, r0 can be reduced below 1, then the epidemic collapses (fig. 1b) , and most people remain uninfected (but still susceptible). because of the exponential nature of epidemics, the outcomes are nearly binary. even when r0 exceeds one by only a small amount the disease spreads at an accelerating pace, whereas as soon as r0 falls just below one it rapidly collapses. these two outcomes correspond to two distinct strategies for epidemic control, suppression and mitigation, close variants of which are currently attempted by several asian countries (with different political systems) and western democracies, respectively. in the mitigation model, the goal is to reduce r as much as possible but not below 1.0, hoping to end up with a population that is largely immune, without overwhelming the healthcare system in the process (as in fig. 1a , but attempting to flatten the temporary surge of infected individuals). this could (but is not guaranteed to) lead to "herd immunity" (see, for example refs. 3, 4 ), which would limit spread in future epidemics caused by variants of the same virus. however, exponential processes are notoriously difficult to control, particularly in the absence of accurate real-time data and when the effect of policy changes is uncertain. the choice is stark: allowing the disease to spread to a large fraction of a population, however slowly, greatly increases the total number of infected people and would cause a loss of life that most societies will not accept. furthermore, given the difficulties in controlling exponential processes using limited information, even a strongly enforced mitigation strategy runs the risk of overwhelming the health care system and significantly increasing the mortality rate due to the failure to treat every patient optimally (primarily due to the lack of intensive care capacity and sufficient numbers of ventilators). if the healthcare system is overwhelmed, patients must be triaged as in wartime, potentially for extended periods of time. notably, both suppression and mitigation are unstable: the mitigation model might first wreck the health-care system and then (as the public demands harsher controls when mortality rises) also wreck the economy. the suppression model might first wreck the economy and then as public pressure forces a relaxation of control, the virus re-emerges. for many months, both approaches are likely to force a large fraction of the population into quarantine. this is because of the large number of asymptomatic carriers of covid-19; in the absence of population-scale testing, the measures need to be implemented in an indiscriminate manner, affecting the whole population. over time, this will result in severe and unequal economic deprivation. our estimate is that in the united states, gdp per capita is already lower by about 1000 usd per month. redistribution can offer some protection for the most vulnerable families, but if a loss of income of this magnitude persists for six or twelve months, it could generate a backlash against the social distance measures that are currently our only weapon for fighting the disease. as a result, epidemiologists are giving serious consideration to scenarios that alternate between lockdown and relaxation that will lead to more loss of life and add to even more economic uncertainty. we can use what we know about the dynamics of disease to suppress this pandemic in a way that is far less disruptive than indiscriminate lockdowns and social distancing. because it is less disruptive, a nation can sustain this approach for as long as it takes to find a safe and effective vaccine or a cure. reducing the disruption will thereby save lives. we know that at low levels of prevalence, testing, contact tracing and quarantine (ttq) is a very effective means of suppression 3,5 , because it reduces the effective rate of reproduction close to zero. it is not a feasible strategy for suppressing the virus in the current, higher prevalence conditions faced by most countries because it would demand resources that would overwhelm any health department. in addition, the ttq approach suffers from its own instability. unless it identifies every single person who becomes infected, asymptomatic individuals that are not identified will generate clusters that will not be detected until someone develops a severe infection that requires medical care. if only 10% of cases are severe enough to be tested after two weeks, a single missed case will lead to an average cluster of 100 new cases before it is found. as a result, once the rate of new cases exceeds the capacity of tracing, even briefly, the epidemic runs out of control and the exponential dynamics make it almost impossible to catch up without imposing a lock-down. some have suggested that an updated version of ttq that relies on modern surveillance technology could be viable because it will not make the same resource demands on the public health system. to be sure, it would be useful for innovators to work toward a working prototype that members of the public could voluntarily adopt. but because no such system has been deployed, even as a prototype, it would be dangerous for policy makers to count on the availability in the coming months of a system that is both effective in slowing the spread of disease and acceptable to most members of society. here, we propose a radically simpler strategy: just test everyone, repeatedly. when someone tests positive, ask them to self-quarantine and provide them public assistance that reduces the burden this imposes on them. this approach relies on a key observation that has not been widely appreciated, namely that what matters is the fraction of all individuals that are identified and quarantined. it follows that testing a small number of individuals with a highly accurate test can be much less effective than testing everyone with a less accurate test. in fact, there is a quantifiable relationship between the reproduction number of a virus, and the efficiency of a population-scale testing strategy that brings the effective reproduction number below 1. below we use analytical models to derive both an upper and a lower bound on the effectiveness of testing, and demonstrate their real-world relevance using more realistic stochastic models. the approach has several important advantages. first, it will work no matter how high the prevalence of infection might be. second, it does not suffer from the inherent instability of contact tracing. the offsetting disadvantage is that it is a challenge to test at the required scale, but this is not as difficult as it might at first seem. it could be implemented using mass distribution (e.g. regular mail) without returning samples to a central testing site. in fact, the tests required do not even have to be properly "diagnostic." they will not be the basis for any decision about medical care. they only influence the decision to self-quarantine. in the worst case, they may cause people who are not infectious to be quarantined, but this is already true for most people (including the authors) in the baseline lockdown scenario. this is an important feature, as it relaxes the demands on the quality of the test. the test can tolerate many false positives, because the result of a provisionally positive test is that someone self-quarantines for two weeks when they did not have to. false negatives are also acceptable as long as people are retested frequently. although this strategy should be introduced alongside of existing measures, it is a useful exercise to ask what level of testing would be required for this strategy by itself to contain any level of infection. clearly, if a perfectly accurate test were applied to the entire population at once, and those who tested positive were fully quarantined, the epidemic would immediately collapse with no new infections (fig. 1c) . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04. 27.20078329 doi: medrxiv preprint to examine the effects of false negatives and noncompliance, we first make the best case assumption about the timing of the tests: every person who is infected is tested before encountering someone who is susceptible. this limit can be approached, for example, by a very effective form of contact-tracing. for coronavirus, it has been estimated 7 that r0 = 2.4 and uncertain data from quarantine in wuhan 8 suggests that rq = 0.3. using the standard (continuous, deterministic) sir model, the equations in fig. 2a and methods show that the optimal population-scale testing strategy will succeed if at least two thirds of all new covid-19 cases are immediately identified and quarantined. if is the true positive rate of the test and is the fraction of the public that complies in the sense that they agree to be tested and follow any instruction to go into quarantine, this bound means that the product must be greater than 2/3. next we do the opposite -assume that the test and compliance are perfect, so = = 1, and consider the worst-case assumption on the timing of the tests: each day, a randomly selected fraction of the population is tested. under that strategy, we find that testing at a rate equal to ( ! − 1) percent of the population per infectious period will ensure that r < 1 (fig. 2b, methods) . using 0 = 2.4 and a two-week infectious period for covid-19, this implies that at least 10% of the population would have to be tested each day. real-world testing strategies could do much better than test at random. for example by implementing procedures that test individuals concurrently within a region; that run the screen as a sweep across a country; that slice the population into groups that are tested in a cycle; or use other variables to predict who is more likely to be infected and to test them more frequently (see methods). because herd immunity and other interventions -including the use of masks or reliance on social distancing -are additive with respect to the testing, any of these effects can lower the required frequency of the tests. for example, fig. 1d shows the required compliance rate as a function of the strength of other interventions, assuming a fixed false negative testing rate of 15%. the standard but simple and deterministic susceptible-infectious-recovered (sir) models used to calculate these bounds is based on strong assumptions and approximations, such as random mixing of all individuals. to relax those assumptions, we implemented a more realistic numerical simulation using a stochastic model on a social interaction graph (i.e. a stochastic network seir model) to model two realistic scenarios. we focused on the initial exponential growth phase of an epidemic. fig. 3a shows a simulation that starts with 100 infected individuals and assumes that the product of the compliance and true positive rate = 0.8. population-scale testing using random weekly tests was started on day 20, and immediately suppressed the epidemic, which was fully stopped by day 100. in contrast, without testing, viral spread caused a surge of infections. death rates were 0.19% with testing, and 0.66% without, i.e. a more than three-fold improvement corresponding to 1.5 million lives saved in a us-sized population. this demonstrates the power of populationscale testing and quarantine for the suppression of novel viruses. the second scenario modeled a country that exits from a lockdown that has suppressed the growth of a pandemic and a with a small fraction of individuals who are immune (fig. 3b) . in such cases, new outbreaks will inevitably happen. ttq and periodic lockdowns can suppress these outbreaks, but so can an ongoing process of testing and isolating. in the model, a lock-down was applied from day 20 which nearly extinguished the virus by day 100. at that point, the lockdown was lifted and social interactions returned to normal, but population-scale testing and quarantine was applied as above. once again, the epidemic was suppressed indefinitely, with total deaths limited to 0.16% of the population. if . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04. 27.20078329 doi: medrxiv preprint the lockdown was lifted without population-scale testing, a powerful second wave was generated leading to the death of 0.57% of the population overall. the decreased mortality due to testing after lifting lockdown corresponds to more than one million lives saved in a us-sized population. this demonstrates that population-scale testing can be an effective replacement for periodic lockdown as a sustainable way to prevent the resurgence of the virus. finally, systematic simulation of the parameters of the stochastic model (supplemental figure 1) showed that with = 2/3 (for example, a compliance of 80% and test sensitivity of 85%) testing at least every 11 days on average was sufficient to suppress the epidemic, whereas testing less frequently was not. the scale of screening required for the approach is not unimaginable, as it is within an order of magnitude of the level of screening that is critical for protection of the segment of the workforce that is employed in essential sectors such as healthcare, elderly care, public order and delivery of foods and medicines. furthermore, building a test that could be applied at population scale is clearly feasible using current technology. it could be based on detection of antibodies to the virus 9 . however, as it takes time for antibodies to build, the antibody-test cannot detect cases early. a single test also does not discriminate between current and past infection. making sure that someone is not currently infected requires that an antibody test is performed twice over a period of three weeks, during which the individual must be held in strict quarantine. alternatively, the test can be combined with an rna or antigen test. despite these drawbacks, an antibody test will clearly be part of the solution, as it can detect immune individuals that can continue to work safely in health care or with risk groups. however, in countries where the epidemic is successfully suppressed, the fraction of immune individuals is far too small (e.g. 1-10%) for restoring normal levels of economic activity. furthermore, deploying antibody test at a population scale will be more difficult than using an rna test, as the current approach requires blood samples, which decreases compliance and makes self-testing more difficult. a population-scale test can also be based on viral proteins (technically more difficult but possible 10 ), or viral rna, like the current state-of-the-art diagnostic tests (for example ref. 11 ). technically, few measurements are easier and/or cheaper in biochemistry than determining whether a particular rna species is present in a particular sample. the main technical concern relates to false positives caused by contamination of input samples by the amplified dna from previous tests; this can be simply prevented by well-established procedures. despite the technical simplicity, detecting viral rna in the field at populationscale is difficult to achieve using the same design and strict regulatory framework that is used for tests designed for medical diagnostic purposes. current diagnostic tests for sars-cov-2 are qrt-pcr assays that require (1) nasopharyngeal swab collected by a trained nurse, (2) sample collection in viral transport media, (3) rna purification, (4) reverse transcription and quantitative pcr. the test is highly accurate, and the total cost is in the order of $100. such highly accurate testing is critical for accurate diagnosis of cases in a hospital setting. however, due to the very detailed and specific regulation, specialized staff and equipment, and centralized testing facilities, such tests have proven difficult to rapidly scale above thousands of assays in each location. a distributed system of sample collection and testing could, however, conceivably be used to scale qrt-pcr to population levels, particularly when using a regional sweeping approach to limit the number of simultaneous tests needed. the capacity could also be increased 10 to 100-fold by group testing 24 , a method with a long history of use in public health that was originally designed for syphilis tests, and now commonly also used for optimally efficient detection of defective components in industrial . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04. 27.20078329 doi: medrxiv preprint production. although some components of tests are currently limiting due to sudden high demand, scaling up their production is not difficult, as the methodology is based on raw materials that are not scarce (e.g. plastic, sand) and biological molecules (enzymes, nucleotide triphosphates and short nucleic acid primers) that are easily produced either industrially or locally using simple biotechnological processes. we also note that qpcr instruments are currently in short supply, but isothermal tests are available that require only a waterbath (see below). a parallel relatively centralized testing method based on existing dna sequencing technology could also be fielded rapidly. in this approach, viral rna in the samples is used to generate dna sequences containing the virus sequences, a sample dna barcode (to identify each case) and two unique molecular identifiers 23 at both ends of the resulting dna fragment (to count the number of virus rnas per sample and to ensure that patient samples do not get mixed in the reaction), and then sequenced using a massively parallel sequencer. this approach is very scalable, as in principle, a single sequencing instrument that is routinely used in scientific research can report more than a billion results per day. furthermore, in the future, a test based on sequencing 19-21 that covers many acute infections could also be used to suppress or even eradicate a large number of infectious diseases simultaneously. this would have significant benefits to humanity, and would be very difficult to achieve using vaccines or drugs that target each infectious agent separately. alternatively, we envisage supplementing the current testing regime with a massproduced home test kit that could be used by anyone, result in a simple easily-understood readout, and be performed without specialized equipment. the test should be as easy to use as a pregnancy test, to ensure maximal compliance. boxes of e.g. 50 tests would be massmailed to all citizens, and a national information campaign would encourage everyone to test themselves weekly. in an infected individual, viral rna is present at reasonably high levels in nasopharyngeal swabs, throat swabs, sputum, and stool for up to two weeks 12 , with the greatest amounts in sputum and stool. sputum might be the ideal source for a home test kit, given the ease of sampling. compliance of the home testing could be increased by both rewards and penalties, and potentially enforced by adding a serial number to each test that needs to be reported together with the test result to collect the rewards. the test result can be open in such a way that the result is clear to everyone. it can also be designed so that it maintains privacy. here, the result (e.g. resulting color, number of bars that are visible) needs to be reported together with the serial number to a central facility to get the answer and/or the cash reward. the open and private approaches can also be combined, to design a test that is open and contains an encoded part that needs to be reported to collect a reward. such designs may complicate the approach, but would allow the healthcare system to obtain data that would facilitate monitoring of the outbreak and large-scale contact tracing. the cash rewards could also be contingent on being regularly tested. anyone found positive would be compelled to self-quarantine, possibly under monetary or criminal sanctions, or using additional rewards for compliance. provided that the test is sufficiently quick, testing could be performed in workplaces, or even in checkpoints exiting areas with high infection rate that are currently under lockdown. our approach is not something that can only be fielded in the far future. in fact, tests suitable for home use have already been developed. in contrast to the commonly used polymerase chain reaction (pcr), which optimally requires an instrument that repeatedly changes the temperature of the reaction, many other "isothermal" detection methods have been developed that operate using a single set temperature, and do not require special equipment beyond what is available in every kitchen (e.g. hot water). for example, an . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04.27.20078329 doi: medrxiv preprint isothermal and colorimetric test has been described 13, 14 , based on reverse transcription-loop mediated amplification (rt-lamp) technology. this test has several desirable properties: unlike pcr, it does not require temperature cycling; the readout is binary and can be achieved by simple observation; and it can start from crude samples 15 . many other technologies also have the potential to detect viral rna rapidly and isothermally [16] [17] [18] ; these include recombinase polymerase amplification (rpa), transcription mediated amplification, nicking enzyme amplification reaction (near), rolling circle replication, and in vitro viral replication assays. although a population-scale test does not need to be as accurate as a clinical-grade qrt-pcr test (see above), apart from a potential increase in errors due to sample collection, there is no theoretical reason why a self-test based on isothermal amplification would not achieve the false negative and positive rates that are equivalent to the current state-of-the-art methodology. making the necessary reagents at scale is also not difficult. per 10 million people, a 100 µl test requires 1,000 liters of reagents, consisting of primers, nucleotides, ph-sensitive dye and enzyme, all of which are easy to make at the required scale. a population-scale strategy has the potential to save many lives, and to buy precious time for a vaccine or an effective drug to be developed. it's important to recognize that in contrast to vaccine and drug development, scaling up testing does not depend on any new scientific discoveries; it is a matter of engineering and logistics only. a field test would synergize with drug treatment, as many antivirals act more effectively when they are given at an early stage of the infection. furthermore, development of field-applicable tests needed for rapid population-level screening will have great benefits in combating epidemics in countries with less developed healthcare systems, and would also help in responding to future epidemics, or variants of the current one. the costs of mobilization of scientific and industrial resources for rapid development of such a test are considerable; however, in our opinion, they are still orders of magnitude lower than the costs of the current suppression and mitigation strategies. although balancing collective goods such as economic activity and public health commonly involve very difficult trade-offs, we believe that such a trade-off is not relevant in this case, and that a population-scale screening policy can be implemented in such a way that will both save more lives and cause less economic and social disruption than the current approach. as there is little overlap with other industrial mobilization efforts, such as scaling up current testing regime, building of ventilators or developing drugs or a vaccine, the increased effort for the development of tests would also have very limited opportunity cost. the development of capacity for population-scale testing would also be an important and relatively inexpensive insurance against other pandemics, or re-emergence of covid-19 once herd immunity is lost or the virus mutates to evade immunity, vaccines and/or antiviral drugs. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04. 27.20078329 doi: medrxiv preprint the authors declare no competing interests. the epidemic was first modelled with a standard (continuous, deterministic) susceptible, infected, removed (sir) model. in addition to the very general assumption that there are a relatively large number of cases, which allows modeling of a partially discrete system using a continuous model, the sir model is based on the following standard assumptions: (1) the population is fixed, (2) it mixes homogenously, (3) the only way a person can leave the susceptible group is to become infected, (4) the only way a person can leave the infected group is to recover from the disease, (5) recovered persons become immune, (6) age, sex, social status, genetics etc. do not affect the probability of being infected, (7) there is no inherited immunity, and (8) the other mitigation strategies and testing are independent of each other (for fig. 1d ). the assumption (2) leads the sir model to overestimate viral spread, as in reality population has substructure (e.g. families, workplaces) and is geographically separated and contacts are more likely between subsets of the population; this is not expected to materially affect our analysis as our conclusions are not based on the absolute rate of the spread, only on its exponential nature. in addition, we modeled the effect of testing in two ways. the first, maximally effective testing strategy assumed that every individual was tested before they infected another person, leading to the upper bound on testing performance in fig. 2a-b . under this model, the requirement for collapsing the epidemic is that the weighted average of the basic reproduction number 0 and the reproduction number in quarantine ! must be less than one: here, is the true positive rate of the test and is the compliance (fraction of all tested individuals who actually self-quarantine). using 0 = 2.4 and = 0.3 for covid-19, the product of the true positive rate and compliance must be greater than two thirds: . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 1, 2020. the second, lower bound testing strategy (fig. 2c-d) was modelled by adding an additional 'detected' state to the model, and adding transitions from infected to detected (with rate ) and from detected to recovered (with rate ). this corresponds to continuous random testing of the population at a fixed rate per person per day. here, the requirement for successful collapse of the epidemic is given by the basic reproduction number (assuming perfect quarantine; fig. 2d ), as follows. first, the rate equations for the sir model with testing are: rewriting the second equation above as follows: makes it clear that / will be negative (i.e. the epidemic will collapse) only if: note that the ratio / is the basic reproduction number 0 , so that the previous inequality can be rewritten as follows: in other words, the testing rate must exceed a threshold given by the recovery rate and the fraction of susceptible individuals / . in the beginning of an epidemic in a naïve population, when all individuals are susceptible, this reduces further to for sars-cov-2, assuming = 1/14 (i.e. an infectious interval of two weeks) and 0 = 2.4, the required minimal testing rate would be 10% of the population per day. as the . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04. 27.20078329 doi: medrxiv preprint epidemic progresses, the required testing rate drops as fewer and fewer individuals remain susceptible and herd immunity kicks in. to understand the difference between (1) testing everyone at the same time, (2) testing everyone in a time separated manner, or (3) testing the population by random sampling, it is helpful to consider an extreme case of certainly and completely collapsing an epidemic by testing and quarantine, using a perfect test that detects all infected individuals, and complete quarantine. for optimally achieving this, it is necessary to identify everyone who is infected before they have infected anyone else (denoted efficiency, e = 1) and quarantining every infected individual (c = 1). this requires obtaining a minimum of n bits of information for a population of size n. in case (1), this is achieved by testing everyone at the same time with a perfectly accurate test that returns one bit (positive or negative). in this case, e = 1 and c = 1. however, when tests are separated in time (2), the order of testing becomes important. the optimal strategy discussed in the preceding section, testing everyone at different times, but before they have had the chance of infecting anyone else also works optimally and collapses the epidemic over a single infectious period. however, most strategies for testing n individuals during time ttest_interval before t0 have e < 1, and are not sufficient to completely collapse the epidemic using one testing round, as e depends on the relationship between the order of testing and the order of infections. for example, using a random order of testing allows some individuals that have already been tested negative to become infected during the ttest_interval (the mutual information between test results and person being infected at t0 is less than one bit). however, some other regimens using a perfectly sensitive test can collapse the epidemic (but not always prevent all future infections): for example, a geographical sweep where infections (individuals) are prevented from crossing a moving test front can be used to identify every infected individual in the population by performing a single round of n tests. in case (3), random sampling of n individuals, e is always less than 1. the testing becomes less efficient than testing each of the n individuals at the same time, because some individuals are tested twice, and some not at all; some information is thus not obtained, and some tests do not return information that is completely independent of information returned by other tests (sum of mutual information between all pairs of tests is not 0 bits). in other words, if individuals are selected randomly, during a given time interval, the tests will miss some individuals, and some individuals are tested more than once (this increases true positive rate for those individuals, but this does not make up for failing to catch some individuals entirely). considering the extreme case of immediate collapse, it may appear that testing in a time separated manner or by using random sampling will not work because non-concurrent testing can permit infections to cross the testing boundary, and random sampling clearly leaves some cases undetected. however, this very intuitive idea is incorrect, as collapsing an epidemic only requires that the rate of generation of new cases per current case is less than one. the limit for random testing can be obtained using the sir model extended with testing (sir+t), which abstracts away individuals and thus can (only) be used to investigate the effect of random, time-separated testing. analytically from this model, as shown above, the < 1 condition is true when tests are performed at a rate that is higher than 0 − 1 tests per mean infectious period. the same limit results from the following consideration: reducing 0 to less than one using the method representing the lower bound -a completely random testing regime -requires that an infected individual has less than an equal probability of (a) infecting another individual over (b) being tested and quarantined or recovering from the infection (analogously, in sir+t, the combined testing and recovery rate needs to be higher than the rate of new infections). events (a) can recur, but either event (b) terminates the chain. therefore, at r = 1 there will be on average one (a) event, which requires that the order of the infectious and protective events are randomly ordered with respect to each other, with equal density. this yields 0 − 1 tests and one recovery per 0 infections per infectious period, and an upper limit of 0 tests per infectious period at infinity (because as 0 → ∞ the expectation value for becomes the geometric series ∑ 2 %& outside of the theoretical consideration of = 1, multiple population-scale tests are always required to collapse the epidemic in the absence of other interventions that achieve the same aim. performing multiple tests over time imposes an additional constrain on optimality -the allocation of tests to each transmission interval. as described above, best performance of continuous testing and quarantine is thus achieved when testing is performed immediately after infection for each individual, or as requirement for exiting quarantine. testing blood before transfusion to prevent transmission of hiv or hepatitis c, testing at border crossings, conditional opening of lockdown, or some regimes that apply contacttracing may come close to approximating this limit, which for covid-19 is = ( " − 1)/ " = 0.57 per mean infectious period; fig. 2 ). however, in most scenarios, such testing efficacy is difficult to maintain over time (because contact is lost, and the unknown infectious intervals rapidly become randomly distributed over time). this level can thus be considered an upper limit of performance of any scenario applied at population scale. using a test whose true positive rate of 1 and testing everyone at the same time performs as well as the optimal strategy. as test sensitivity decreases, the performance of the concurrent regime becomes lower than optimal. however, concurrent testing still performs well above the lower limit obtained from the random testing model. the required pc rate to bring r0 < 1 using concurrent tests has a simple relationship with the exponential growth of infectious cases. over interval t-t0, pc > 1-(infectious cases at t0)/(infectious cases at t). however, it is not as simple to relate this to original r0, because the relationship between r0 and growth rate is a function of the distribution of the generation intervals (ref. 28 ). estimating at r0 = 2.4 using even probability distribution of infections over time, the infected population becomes approx. eight times larger in a single 14 day infectious period. this means that a testing regime that is regularly spaced at 14 day intervals should have pc value of > 7/8 = 0.875 to bring r0 < 1. this is confirmed using empirical simulations to assess the rate of exponential growth in the complete absence of immunity and all other types of interventions; the limit = 1 at 0 = 2.35 with testing every infectious period (14 days) is reached when ≈ 0.85 (compared to 0.58 for testing each individual directly after infection). the required testing interval at 0 = 2.35 and = 0.8 in the absence of other interventions and immunity is 11, 8 and 5.5 days for concurrent testing, testing each individual randomly once during each testing period, and continuous random testing, respectively. these considerations can be summarized as follows: the order of testing efficacies is: everyone before they have had a chance to infect anyone > everyone at the same time > everyone once during a period > testing by random sampling -with population-scale testing . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04. 27.20078329 doi: medrxiv preprint remaining feasible and cost-effective by one or more orders of magnitude across all these regimens. the sir model fails to account for several key properties of real epidemics, such as social and geographical population structure, the discrete and stochastic nature of infection and disease progression, and the fact that testing cannot be instantaneous. to account for such more complex real-world phenomena, we implemented a stochastic network model using the gillespie algorithm for accurate numerical simulation of the stochastic dynamics. we used the seirsplus python package (https://github.com/ryansmcgee/seirsplus), which models an epidemic on a social graph, where each individual transitions between six states: susceptible, exposed, detected-exposed, infectious, detected-infectious, and recovered. the two detected states are used to model the effectiveness of testing and quarantine, and social distancing is modelled by removing edges from the initial social graph. we used a random social graph of mean degree 13 (median 10) and two-sided exponential tails, which was reduced to mean degree 2 for social distancing (lockdown) and quarantine. the population consisted of 10,000 individuals. in both shown scenarios we assumed that the test had 80% sensitivity, and epidemic parameters were modelled loosely after covid-19. detailed source code with comments and parameter settings for each model are available in the accompanying jupyter notebook at https://github.com/paulromer149/ubiquitous-testing. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 1, 2020. inequality that must be true to suppress transmission. for the epidemic to collapse, the weighted average of the natural reproduction number " and the reproduction number in self-quarantine ! must be less than one. here, represents the test true positive rate (fraction of all infectious individuals detected), and the rate of compliance. (c) parameters for a sir model with testing and a detected state. (d) requirements for testing to collapse an epidemic in the sir model with testing, expressed in terms of the testing rate required in a population where all individuals are susceptible, with inverse infectious interval . (e) parameters for the discrete, stochastic seir model on a social graph. each compartment was modelled for every individual on the social graph. (f) outcomes of ten simulation runs of the stochastic seir model on a social graph, showing total number of deaths as a function of the fraction tested every day, assuming compliance and true positive rate = 2/3. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 1, 2020. severe social distancing (lockdown) is applied after 20 days, and then lifted after 100 days. population-scale testing is implemented after day 100 (left) or not implemented (right). in all panels, shaded colored regions indicate policy regimes, and the total number of dead individuals is indicated in the sub-panel titles. for both panels, the product of compliance and test efficacy was set to = 0.8 and the testing rate was set to = 1/7. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04. 27.20078329 doi: medrxiv preprint supplemental figure 1 | successful testing strategies under the stochastic seir model on a social graph. growth curves (daily new cases) showed that given = 2/3, testing at least every 11 days successfully flipped the sign of the exponential growth curve from day 20 (when testing started), whereas testing every 13 days was insufficient. red dashed curves, piecewise exponential fits for days 0 -20, 20 -100, and 100 -250. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 1, 2020. . https://doi.org/10.1101/2020.04. 27.20078329 doi: medrxiv preprint coronavirus disease 2019 (covid-19) situation report -59 impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand pandemics: risks, impacts, and mitigation the basic reproduction number (r0) of measles: a systematic review emerging infectious diseases and pandemic potential: status quo and reducing risk of global spread scientists say mass test in italian town have halted covid-19 there substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) evolving epidemiology and impact of non-pharmaceutical interventions on the outbreak of coronavirus disease 2019 in wuhan serological assays for emerging coronaviruses: challenges and pitfalls platinum nanocatalyst amplification: redefining the gold standard for lateral flow immunoassays with ultrabroad dynamic range detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr. euro surveillance : bulletin europeen sur les maladies transmissibles clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in travel-associated transmission cluster rapid colorimetric detection of covid-19 coronavirus using a reversetranscriptional loop-mediated isothermal amplification (rt-lamp) diagnostic platform rapid molecular detection of sars-cov-2 (covid-19) virus rna using colorimetric lamp development and validation of reverse transcription loop-mediated isothermal amplification (rt-lamp) for rapid detection of zikv in mosquito samples from brazil isothermal nucleic acid amplification techniques and their use in bioanalysis isothermal exponential amplification techniques: from basic principles to applications in electrochemical biosensors isothermal amplification of nucleic acids metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance a vision for ubiquitous sequencing loeffler 4.0: diagnostic metagenomics contributions to the mathematical theory of epidemics--i. 1927 counting absolute numbers of molecules using unique molecular identifiers the detection of defective members of large populations test everyone, repeatedly to defeat covid-19. medium medium.com/@sten.linnarsson/to-stop-covid-19-test-everyone-373fd80eb03b covid-19 mass testing facilities could end the epidemic rapidly infectious disease model with testing and conditional quarantine how generation intervals shape the relationship between growth rates and reproductive numbers we thank many colleagues for comments on the early version of the work. we are especially grateful to drs. minna taipale, mikko taipale and paul pharoah for review of the draft of manuscript. a draft of this paper was initially released as a public preprint (ref. 25) , and supporting, independently developed model reported by p.r. on www.paulromer.net. we also note that during the writing of this work, we became aware of two independent analyses, one by julian peto (ref. 26), and the other by a team consisting of david berger, kyle hirkenhoff and simon mongey that report similar conclusions (ref. 27) . key: cord-283517-7gd0f06m authors: deak, eszter; marlowe, elizabeth m. title: right-sizing technology in the era of consumer-driven health care date: 2017-08-01 journal: clinical microbiology newsletter doi: 10.1016/j.clinmicnews.2017.07.001 sha: doc_id: 283517 cord_uid: 7gd0f06m abstract technology for modern clinical and public health microbiology laboratories has evolved at an impressive rate over the last two decades. contemporary diagnostics can rapidly provide powerful data that can impact patient lives and support infectious disease outbreak investigations. at the same time, dramatic changes to health care delivery are putting new pressures on a system that is now focusing on patient-centric, value-driven, convenient care. for laboratories, balancing all these demands in a cost-contained environment remains a challenge. this article explores the current and future directions of diagnostics in our dynamic health care environment. the affordable care act (aca) in the united states was enacted by president obama in march 2010. the goal of the aca was to improve the quality of and access to health care by transforming insurance coverage and lowering health care costs. we have seen shifts in health care plans (i.e., account-based health plans) that have the consumers of the health care opting for lower monthly premiums with higher deductibles. these deductibles are often paid for by personal health savings accounts, thus pushing the costs of health care onto the individual consumer. couple this with an unprecedented boom in technology, which in some cases can offer on-demand diagnostics within the time of an office visit, and the result is consumer-driven health care, particularly for those who can afford it. despite recent administrative changes in washington and the uncertainty of "repeal and replace" in the republican agenda for the current aca, the trend toward consumer-driven health care, with an emphasis on pre-budgeted spending, is likely to continue. for consumers of a product who will continue to pay more of the bill, the bright side of this trend is a movement to value-based care delivery from the perspective of the affluent consumer. valuebased care is defined as safe, appropriate, and effective care at a reasonable cost, which is predicated on evidence-based medicine and proven outcomes. patients are looking for more pricing transparency and more options for efficient care delivery (i.e., telemedicine, retail care providers, and mobile health solutions). health care providers are trying to better understand consumer wants and needs, measure performance, and improve the patient experience; this is a distinct change from the historical fee-for-service system that did little to incentivize providers to produce value. from the laboratory's perspective, there continue to be operational challenges to lead these changes. emerging and re-emerging pathogens demand rapid responses at an unprecedented level. the skilled workforce continues to shrink, while the work demands go up. there are legislative influences on testing. at the same time, reimbursement and budgets are contracting. yet still, at the end of the day, the laboratory is expected to produce quality results for improved patient care. initiatives like antibiotic stewardship are helping to drive better outcomes with laboratory results, but many of these programs are dependent on post-analytical variables for the optimal impact on patient care to be realized [1, 2] . another key laboratory issue is the breath and scope of the technology that is now available. today, we have molecular point-ofcare (mpoc) devices that can provide a rapid diagnostic answer within 20 minutes in a clinic, multiplex pcr sample-to-answer devices that can screen for >20 analytes in a single specimen in about an hour, high-volume automation that can enhance throughput and efficiency in the clinical microbiology laboratory with digital imaging, and next-generation sequencing (ngs) that can reveal a treasure trove of information in a single test. combined, the changes in health care and technology have left many laboratories asking how to "right-size technology" for routine care while transforming practice. ultimately, change will depend on the goals that are driving the conversion and utilization of the technology into daily laboratory practice. factors may include syndrome-specific diagnostic needs, ease of use, the need for rapid results, improved sensitivity and specificity, operational needs (such as staffing and expertise), laboratory design (such as centralized versus decentralized models), cost, consumer demand, and the potential for improved patient outcomes. the laboratory must weigh all these factors while trying to make a business case to improve service despite the fact that there are few or no outcome data available to support the use of new technology. technology comes at a cost that is often shifted to the consumer, the patient. while consumer choice can help push innovation, one also has to wonder to what extent the market will allow the significant increases in testing costs that can come with technology. for example, in the case of acute gastroenteritis which is typically a self-limiting infection with the majority of specimens coming from an outpatient setting, traditionally a stool culture would be ordered that would cost a patient less than $100. the newer multiplex stool pcr panels can result in a charge that can cost a patient over $1,000. will patients be willing to bear paying this increased cost for such a diagnostic test long term? while one can agree that there is improved turnaround time, sensitivity and pathogen coverage in a sophisticated multiplex diagnostic assay, it must be used in conjunction with diagnostic algorithms that prevent needless additional downstream testing as well as excess costs. clearly, there is a need for diagnostic stewardship alongside antibiotic stewardship to improve quality and the prudent use of health care dollars. this article explores the impact of technology on the clinical and public health microbiology laboratory in the age of consumer-driven health care. testing considerations "right-sizing technology" means that the right test is offered at the right time for the right patient with maximal operational efficiency and cost-effectiveness. the outcome of right-sizing is to provide results with the potential to inform therapeutic and infection control decisions for improved care and, ultimately, reduced downstream costs. the diagnostic testing needs of a medical institution such as kaiser permanente in northern california, which is comprised of 21 hospitals and over 200 medical offices spread out over a wide geographical area with over 3.5 million members and serviced by a central laboratory, are significantly different than those of a 500-bed county hospital with an on-site laboratory. advances in technology have provided flexibility in diagnostic testing to address the differing needs of health care systems and the laboratories that serve them. for any given analyte, there are a number of highly sensitive and specific tests available from which to choose. considerations that go into the selection of a test or instrument platform for implementation include perceived turnaround time needs for improved patient care, sample volume requirements, number of tests expected, suitability for the intended laboratory based on available expertise and desired workflow, as well as cost. in the past decade, manufacturers have targeted their research toward development of more sensitive and specific mpoc diagnostic infectious disease platforms and tests. such mpoc tests have evolved for more practical use at the bedside. manufacturers have appreciably simplified tests by removing the need for sample manipulation and handling. instrumentation has become more automated and/or involves fully integrated systems that are portable or significantly smaller and more modular. instruments have also incorporated mechanisms for recording and transmitting results. all the while, tests have become faster while demonstrating improved sensitivity and specificity [3] . these modifications in technology have enabled molecular testing to migrate from large central laboratories. most poc tests are still moderately complex, which is defined by the clinical laboratory improvement amendments of 1988 (clia) as one requiring basic laboratory knowledge and training for personnel performing the test. users of these tests must adhere to clia regulatory requirements, which includes quality assurance, along with appropriate documentation, validation of analytical performance, proficiency testing, and ongoing competency training. clia director oversight is still required [4]. increasingly, diagnostic molecular tests are being designed and submitted for clia-waived status. clia-waived tests are defined by the food and drug administration (fda) as being "so simple and accurate as to render the likelihood of erroneous results negligible; or pose no reasonable risk of harm to the patient if the test is performed incorrectly" [4, 5] . based on this definition, non-laboratorians can perform the test without clia director oversight if they are following the manufacturer's instructions [4]. the first clia-waived mpoc test to receive fda approval was the alere i influenza a&b in 2015. to better ensure quality results are being reported, some of the new mpoc tests have incorporated internal electronic and reagent quality control (qc) and have built in a shut-down mechanism in the event of failed qc. since the waived testing program began in 1992, the number of approved clia-waived diagnostics has increased from 9 to over 100, with more than 20 analytes approved for infectious disease testing [6] . there are over 200,000 laboratories in the united states that now hold a certificate of waiver, which enables them to perform any clia-waived test [4]. however, just because anyone can perform the test does not mean they should. there must be an understanding of test limitations by all testing personnel. few non-laboratorians realize that the central laboratory filters out many inappropriate specimens, and nonlaboratorians require extensive training to understand the testing complexities of even waived tests. laboratories frequently receive incorrectly collected specimens and are asked to test them because there is a lack of appreciation of why these specimens would not be tested. for example, clostridium difficile testing is not performed on a formed stool specimen or for patients less than 1 year old due to the confounding issues of potential colonization, and as in a central laboratory, pre-analytical knowledge and conditions would need to exist to prevent misuse of testing. one question is whether we would be needlessly treating people in these cases if left to the facility performing the waived testing. also, testing a specimen type that is not included in the intended use of an fda cleared test will result in an off-label use of an assay. application of diagnostics in medicine is a balancing act between what we can do, what we need, and what we can afford. diagnostics will continue to evolve. it will become faster, cheaper, and easier to perform, but technology comes at a price, and implementing new technologies with faster turnaround times nearer the patient requires careful thought about placement within the flow of the patients. moving a rapid molecular test closer to the patient has the potential to have an immediate impact on therapeutic decisions. a prospective cohort study examined the potential cost benefit of near-patient mpoc testing for chlamydia trachomatis (ct) and neisseria gonorrhoeae (ng) in a clinic based on reduction of contact attempts [7] . as part of the study, 1,356 patients who had ct/ng nucleic acid amplification tests (naat) also completed a questionnaire to ascertain the maximum time patients were willing to wait after consultation for ct/ng test results and thus the potential for immediate treatment of individuals testing positive while preventing unnecessary treatment of patients who tested negative. the study determined that of the 1,356 patients, 26.2% were unwilling to wait even 20 minutes for the results of an mpoc test. based on the results from a questionnaire, of 129 patients who tested positive by a naat, use of a 20-minute mpoc test would have resulted in immediate treatment of 71.9% of the individuals, whereas a 90-minute test would have influenced the immediate treatment time of only 3.1% of these positive patients. of 1,227 patients who tested negative for ct/ng by naat, use of a 20-minute mpoc test would have prevented 3.2% of empirical treatments, while a 90-minute mpoc test would have prevented 0.3% of the empirical treatments. another study looked at the impact of the 90-minute xpert ct/ng test when sample collection was performed on arrival of the patient, with the intention being that the patients receive their results and treatment as needed during the appointment [8] . actual wait times were evaluated. only 21.4% of the patients received their results before leaving the clinic with the 90-minute xpert test. it was determined that a test turnaround time greater than 30 minutes would likely not be effective, given that it took 48 minutes from the time of sample collection to the clinical consultation. for such mpoc tests to affect patient management, results will need to be available at the time of consultation to maintain patient flow. there are limited studies examining the clinical impact of mpoc tests for other infectious diseases [9] . the infectious disease society of america's practice guidelines for group a streptococcus (gas) currently recommends two-tiered testing for pediatric patients [10] . it is recommended that rapid antigen detection tests (radts) be performed on throat swabs due to the rapid turnaround time of the test (<10 minutes). however, due to the low sensitivity, bacterial cultures are recommended for confirmatory testing of negative radts. clia-waived mpoc gas diagnostic tests with turnaround times comparable to those of the radts are becoming more readily available. these pcr tests do not detect group c or group g streptococci. however, they have been shown to have improved sensitivity for detection of gas, even compared to culture [3, 11] . additional studies are needed to assess the clinical value of these tests and the potential for detection of low-level colonization. in a retrospective study, blaschke et al. [12] examined visits to u.s. emergency departments (eds) using data from the national hospital ambulatory medical care survey. they found that rapid influenza diagnostic tests (ridt) were performed during 4.2 million visits and that 42% of influenza diagnoses were made in association with ridt. test results did suggest that some influence on physician behavior occurred, as patients diagnosed with influenza had fewer ancillary tests ordered (45% versus 53% of visits), fewer antibiotic prescriptions (11% versus 23%), and increased antiviral use (56% versus 19%) when the diagnosis was made in association with ridt. thus, diagnosis of influenza made in conjunction with ridt resulted in fewer tests and antibiotic prescriptions and more frequent use of antivirals. early influenza virus antigen-based poc tests lacked sensitivity [13, 14] . the newer mpoc tests are significantly more reliable and have the potential for improved outcomes in the poc environment [15] . however, as more mpoc options become available, it will be important for laboratories to continue to assess their performance, as not all mpoc tests may demonstrate the same sensitivity and specificity [16] . a recently published open-label, randomized, controlled trial looking at the routine use of mpoc testing of respiratory viruses in adults presenting to hospital with acute respiratory illness enrolled 720 patients (362 assigned to poc testing and 358 to routine care). the authors found that routine use of mpoc for respiratory viruses did not reduce antibiotic usage. however, many patients in the study were already started on antibiotics before the mpoc results were available. mpoc was also associated with a reduced length of stay and improved antiviral use [17] . the clinical laboratory and diagnostic effectiveness (clade) study was a prospective observational cohort study undertaken to assess the impact of a highly sensitive (97%) 20-minute cliawaived mpoc influenza test on patient management in the emergency department (ed) and associated economic benefit [18] . the study indicated that 57% of the ed physicians changed their management of patients, primarily of patients who tested influenza virus negative. the influenza test results impacted decisions about hospital admissions and discharges, ordering of additional medical procedures, and laboratory tests, as well as antimicrobial and antiviral usage. this model, applied to 2,000 ed visits, revealed a cost savings of nearly $800,000 [19] . the study reiterated that getting the right information to the right people at the right time has the ability to impact clinical care. community pharmacies have also become effective players in infectious disease management through provision of vaccinations and are increasingly offering poc tests. over 5% of the laboratories with a certificate of waiver are in pharmacies [20] . a physician-pharmacist collaborative practice agreement (cpa) can be set up to delegate prescriptive authority to pharmacists for treatment of infectious diseases based on clia-waived poc test results. the use of this model has been shown to be effective for influenza virus and gas [21, 22] . in a pilot study conducted at 55 pharmacies in 3 states using the cpa model, pharmacists performed a clia-waived poc influenza test to screen individuals presenting with influenza-like symptoms [22] . pharmacists provided oseltamivir to all individuals who tested positive for influenza virus by the poc test within an hour of the initial encounter. meanwhile, individuals who tested negative for influenza virus did not receive inappropriate antiviral therapy. in a similar pilot study, pharmacists performed a clia-waived poc gas diagnostic test to screen individuals coming into the pharmacies with symptoms of pharyngitis [22] . about 13 million physician office visits are due to acute pharyngitis every year. rates of antimicrobial use as high as 80% have been reported in the literature to treat pharyngitis, although gas has been shown to be associated with only 10% to 30% of pharyngitis cases. of the individuals screened in the study, about 18% tested positive for gas and were thus treated with an antimicrobial consistent with prevalence studies. this study indicates a significant potential of poc tests in pharmacies to decrease inappropriate antibiotic usage in the outpatient setting, although it must be emphasized that moving testing from a central laboratory to a medical unit or more accessible location does not guarantee improved outcomes without systematic changes in management. additionally, when poc testing is performed by clinical staff, errors can arise from a lack of understanding of the importance of qc and quality assurance [23] . the american academy of microbiology recently convened a colloquium of industry thought leaders and subject matter experts to evaluate the role of "near-patient testing," as well as the impact of this diagnostic "paradigm shift" for microbiology [24] . the report from this colloquium was recently published, with thoughtful recommendations. these recommendations were divided into three categories: (i) implementation, (ii) oversight, and (iii) evaluation. key recommendations included (i) rethinking patient flow in the clinical setting to optimize poc utilization, (ii) retaining proper oversight by the microbiology laboratory, and (iii) the need for better outcome data which includes health economics data [24, 25] . syndromic testing has gained popularity in recent years. these multiplex tests detect most common and some uncommon pathogens associated with a syndrome based on similar signs and symptoms. in 2008, the luminex xtag respiratory viral panel was the first multiplex molecular panel to receive fda clearance in the united states. since then, a number of large syndromic multiplex panels have been fda cleared for use in clinical diagnostics. multiplex panels currently exist for gastroenteritis (gastrointestinal [gi]), bloodstream infections, and meningitis/encephalitis. although some instrument platforms still require offline extraction, many platforms have evolved into sample-to-result assays requiring less than 5 minutes of hands-on time with a turnaround time of 1 to 2 hours. for the most part, the sensitivity and specificity of these multiplex tests are comparable; however, sensitivity and specificity of the individual targets can vary by platform. multiplexed molecular panels that can target up to 27 pathogens have the potential to simplify ordering for the physician, as well as workflow in the laboratory, and require less expertise on both ends as a single automated test. as new faster and simpler technologies are introduced for multiplexed platforms, there has been continued growth in adoption of these tests for clinical diagnosis. however, there are limitations to this shotgun approach that are associated with high financial costs as reimbursements continue to decrease, as well as test interpretation dilemmas, especially in the context of low prevalence rates. a point-counterpoint paper was recently published on large multiplex panels as first-line tests for respiratory and gi pathogens [26] . a proposed advantage was the potential to provide timely results for targeted therapy. however, detecting more pathogens might not impact treatment at all, as low sensitivity for certain targets can result in missed diagnoses with additional consequences. also, low prevalence rates for many of the targets may lead to false positives followed by unnecessary treatment and potentially delayed diagnosis. diagnostic errors caused by inappropriate ordering can cause delays in care or harm patients [27] . pre-test probability is important with sensitive molecular assays. a tuberculosis meningitis case that was misdiagnosed as herpes simplex virus 1 (hsv-1) infection presented by gomez et al. [28] underscored the risk of using syndromic multiplex assays without fully understanding the limitations associated with them. the patient's true diagnosis was delayed because of an initial hsv-1-positive filmarray meningitis/encephalitis (me) panel result, which ultimately contributed to severe neurological sequelae. on the other end of the spectrum, another recent article reported that the meningitis panel demonstrated reduced sensitivity for hsv detection from pediatric cerebrospinal fluid specimens [29] . positive results due to panel detection of colonization in a gastrointestinal panel with c. difficile and long-term shedding of organisms such as norovirus or rotavirus can also lead to inappropriate therapeutic decision making [26, 30, 31] . the selection of the platforms that laboratories implement is usually based on accuracy, cost, hands-on time, level of complexity, staffing, throughput, and convenience. however, we also need to think about how we are going to use the test once we implement it, whether to restrict ordering of these tests to only the sickest patients or to offer them to everyone as a first-line test. for a highvolume laboratory, using a costly multiplex platform as a first-line test is not feasible. patient outcome data based on large multiplex tests has been slow to evolve [9, 32] . additional data are needed to determine which patients will benefit from this type of testing. for respiratory infections, testing needs may vary by season and geography. during flu season, it may be more cost effective to perform a targeted influenza/rsv panel on patients presenting with respiratory symptoms before testing for a broad panel of organisms. panels may be better suited to the critically ill or immunocompromised populations. implementation of a testing algorithm for laboratory utilization of molecular multiplex panels with decision support built into ordering may be needed to avoid substituting one set of unintended consequences for another. education and mandated improved test utilization will hopefully improve economic outcomes for the laboratory and decrease the financial burden on the patient. clia-waived status is being obtained for multiplex platforms, with a number of implications. in october 2016, biofire diagnostics received fda clearance and clia waiver for the filmarray respiratory panel ez, which requires only 2 minutes of hands-on time and has a run time of 1 hour. the ez panel is the clia-waived version of the fda-cleared respiratory panel, which tests for 14 viral and bacterial pathogens, adenovirus, coronavirus, human metapneumovirus, human rhinovirus/enterovirus, the influenza viruses, parainfluenza virus, respiratory syncytial virus (rsv), bordetella pertussis, chlamydia pneumoniae, and mycoplasma pneumoniae. there are numerous questions that arise from the availability of these expensive multiplex tests for placement outside the central laboratory without required oversight by technical experts. it will be necessary to determine what algorithms will be used by physicians to decide which patients to test and how results will be interpreted, particularly if multiple targets are positive. until recently, multiplexed molecular panels have been one size fits all. panels with fixed prices based on fixed targets may be excessive and may not necessarily include all the pathogens being considered by the physician. multiple platforms may be required in order to address the needs of the physician in such cases. this scenario becomes a very expensive approach to diagnostic testing. testing needs to fit the medical center and be tailored to the population that the laboratory services. the diagnostic needs of a children's hospital can be very different from those of a medical center that caters to a large elderly population. likewise, a cancer center or a transplant center may have very specific diagnostic needs. nanosphere has fda clearance for its verigene respiratory pathogens flex nucleic acid test (rp flex) on the automated, sample-to-result verigene system, which allows flexibility in testing and is the first multiplex test that is scalable. each rp flex cartridge contains 16 viral and bacterial targets. the physician can order any combination of targets for testing. laboratories pay for only the targets that are ordered. results for other targets not initially ordered on the panel can be reflexed at an additional cost without having to re-run the test. for example, one possible scenario during influenza season is to first order only influenza virus targets or influenza virus plus rsv from the panel. if the result is negative, adenovirus, human metapneumovirus, rhinovirus, and parainfluenza virus can be ordered and the results released. bordetella sp. targets can be ordered separately based on clinical suspicion. medicare recently proposed universal non-coverage for respiratory multiplex panels, which will make it even more challenging for laboratories to utilize the technology. there has been a lot of discussion surrounding multiplex gi panels and whether this is clinically meaningful testing. in may 2017, medicare administrative contractor palmetto gba posted draft local coverage determinations (lcd) for two types of multiplex infectious disease tests [33] . this decision would provide limited coverage for nucleic acid amplification-based gi pathogen panels and a non-coverage decision for multiplex pcr respiratory viral panels. the lcd proposed coverage for molecular panels to detect gi pathogens would be limited to 5 targets (salmonella, campylobacter, shigella, cryptosporidium, and shiga toxin-producing escherichia coli), which represent the majority of foodborne pathogens. current infectious diseases society of america guidelines for infectious diarrhea suggest a selective approach to workup based on whether the patient has traveler's diarrhea with fever or blood, hospital-acquired diarrhea, or persistent diarrhea [34] . a flex platform may be more suitable for testing of diarrheal illnesses. regardless of the number of analytes on a gi panel, the cost when reimbursement is limited to a maximum of 5 targets, may be affected only by the actual cost of the panel itself. the different approach for multiplex pcr testing for respiratory viruses, apart from influenza a/b viruses, with or without inclusion of rsv, is being applied. the reasoning for non-coverage included the fact that the pathogen targets in such panels do not represent a common syndrome and that targets can be very rare. the notice said that a "one size fits all testing approach is screening and not a medicare benefit" and went on to say that "one size fits all panels contribute to test over-utilization, and increased cost to health care without specific benefit to a given patient. testing should be limited to organisms with the greatest likelihood of occurrence in a given patient population, and if results are negative, with a reflexive testing to more exotic organisms." examples are c. pneumoniae or b. pertussis in combination with rhinovirus, influenza viruses, and rsv [33]. telemedicine and remote diagnostics can take on several roles. today with total laboratory automation (tla) and digital microbiology, laboratories have the capability to read and review slides and plates from facilities that are miles or oceans away. a recent clinical microbiology newsletter article highlighted the impact of telemedicine on gram stains in the health care system in arizona [35] . telemedicine companies like vsee (www.vsee.com) have set up field kits with multiple devices that enable remote diagnosis. through the use of software like ehealth opinion, rural patients and physician experts in the u.s. and china are connected through the virtual doctor project [36] . such projects are expanding in many parts of the developing world [37] . telemedicine companies like doctor on demand (www.doctorondemand.com) offer virtual doctor's visits through tablet computers or smartphones. other areas of remote diagnostics being explored are internetbased programs and self-collected specimens for mail-in testing. there are currently fda-approved ct/ng naat assays for self-collected specimens in clinical settings. internet-based mailin programs for sexually transmitted infection (sti) screening has been successfully implemented in a public health system (www. iwantthekit.com), as well as through private companies (mylabbox.com) [38] . public health england in 2015 published a guidance document on commissioning an internet-based chlamydia screening program [39] . such strategies are aimed at diagnostic testing and improving access over the continuum of care. with this new way of delivering care comes the question of validating at-home self-collected specimens and the stability of a specimen mailed through the post. while sti programs may be a starting point for specimen self-collection, it begins a conversation that would expand the realm of consumerism in health care to a new level. one could argue that no one is better able to properly collect a specimen than the person with the greatest interest in the results, the patient. clinical studies comparing clinician-collected and self-collected specimens in a clinical setting for ct/ng have demonstrated that self-collected vaginal specimens have equivalent performance with acceptable patient satisfaction [40, 41] . for a recent review of self-collected specimens for infectious disease testing, see tenover et al. [42] . internet-based programs have the potential to triage non-critical medical needs while reducing visits to traditional brick-and-mortar clinics. at kaiser permanente, virtual visits have been used for several years through secure e-mails, telephone calls, and some video encounters. in the northern california kaiser permanente region, which has over 8,000 physicians and over 3.5 million members, virtual visits grew from 4.1 million in 2008 to 10.5 million in 2013, with projections that virtual visits will soon exceed physical visits [37] . near-patient testing for sti programs, such as the dean street clinic in london, offering walk-in sti testing and treatment with an short message service or sms text on a cell phone to let patients know their results (www.dean.st/testing), are also available. investment in such programs is evolving, yet what is lacking is the return on investment (roi) analysis, which is needed to further policies that could help provide financial support for the evolution of technology in daily practice. ngs has had one of the most significant impacts on microbial sciences since the advent of pcr. through initiatives like the cdc's advanced molecular diagnostics (amd) and response to infectious disease outbreaks, public health microbiology has started to transform into the next generation of thinking for the investigation, prevention, and control of infectious diseases. ngs has provided insight into questions that just was not possible through previous technology. for a review of ngs technologies and amd see maccannell [43] . ngs platforms like the minion (oxford nanopore) can provide portable real-time ngs analysis. the system is miniature in size, plugs into the usb port of a laptop, and offers minimal sample preparation at a low cost (https://nanoporetech.com/products/ minion). the potential of such technology is just beginning to be realized. applications such as the direct detection of mycobacterium tuberculosis from sputum for identification and antimicrobial susceptibility prediction available the same day have been described [44] . barriers are bioinformatics, interoperability of results, and building a workforce with a new skill set and infrastructure to support it. given the debate around reimbursement and multiplex panels, it will be interesting to see where the conversation leads with ngs. ngs will provide much more information than a 20-plex respiratory or stool panel. the technology is already changing practice in the public health laboratory, and the clinical microbiology laboratory is following [45, 46] . twenty years ago, the cdc created pulsenet, a molecular-subtyping network of federal, state, and local public health laboratories designed to facilitate the identification of and response to outbreaks caused by bacterial foodborne pathogens. the specific objectives of pulsenet are to detect foodborne disease case clusters through comparison of pulsed field gel electrophoresis (pfge) "fingerprint" patterns, to facilitate early identification of commonsource outbreaks, and to help food regulatory agencies identify areas where implementation of new measures is likely to improve the safety of the food supply. at the time, pfge was considered cutting-edge technology. to celebrate the 20th anniversary of puslenet, the economic impact of the program was recently published [47] . pulsenet costs roughly $7.3 million to operate but saves more than $500 million annually in medical and productivity costs avoided [47] . this roi is impressive, but the fact that it was 20 years before this economic analysis was published is surprising given the program's success. with federal budget cuts to critical programs that support national infrastructure, evaluating and communicating the value of technology to the health economics of the nation should be part of the national strategy. looking ahead, it is clear that it is only a matter of time before pfge will be replaced by ngs for such foodborne outbreak investigations in the pulsenet system. the economic impact of ngs should be analyzed in a timely manner so that the roi of this powerful technology can be communicated to the appropriate funding agencies. the same is true for the clinical microbiology laboratory. while it sounds very appealing to place an mpoc influenza virus platform nearer to the patient in the ed or in urgent care, the initial investment to place and maintain that testing may be a daunting sell to administrators. the question around this roi is where the cost avoidance is over the continuum of care. for patients coming through the ed during influenza season the greatest impact to patient management would be avoiding a hospital admission. the average cost of a hospital admission due to pneumonia is $14,143, according to the agency for healthcare research and quality (rockville, md) [48] . compared to the cost of a tamiflu prescription, which is roughly $100, the avoidance of hospital admission would clearly have the greatest financial impact. when one adds the implementation costs of the mpoc instrument and reagents over the course of a flu season, the roi can become a more comprehensive sell to the c-suite or the corporation's senior executives. tables 1 to 3 demonstrate the estimated cost of implementing an mpoc influenza assay in a hospital system with 14 medical centers, each with an ed. the total cost of implementation for instrument and reagents over 3 months of the respiratory disease season adds up to $420,00 ( table 1 ). the number of admissions that would need to be avoided to break even on the cost of implementation is 30, or roughly 2 per ed ( table 2 ). this number is equal to 0.5% of the estimated 6,000 tested patients over the course of the respiratory disease season, which would equate to an roi of <3 months (table 3) . another factor to consider when thinking about a rapid mpoc test is that placing testing correctly in the system may actually be cost neutral, because testing is being shifted without the need to bring on any additional testing. outcome studies looking at technology placement are forthcoming [9] . in 1987, the original patent for pcr was issued, with kary mullis listed as the inventor. in 1993, he was awarded the nobel prize for pcr. these accolades came only after the article reporting the invention was rejected by both nature and science. it was finally published in methods in enzymology [49] . as one reflects on the impact of technology like pcr, it is easy to lose sight of how far technology has come. the conversation is now focused on where we need to go. in the world of instant gratification that we have become so accustomed to living in, it is important to remember that changes in medicine take time and require data built on evidence. shifting practice also requires buy-in by stakeholders that the laboratory may not be considering. during the lifespan of technology, the stakeholders will range from biotechnology companies to laboratories, physicians, regulators, policymakers, guideline committees (steered by industry thought leaders), payers, and patients. these stakeholders will influence the maturity of technology application over time. thus, the laboratory community must assess and factor in key drivers that address need, satisfy administration, and influence health economics. properly designed pilot studies remain an important step in assessment. publishing these results is key to moving the field forward. sharing information that may be initially considered only for internal quality improvement projects is essential. laboratories could benefit from more coordinated collaboration from stakeholders, who have a vested interest in such data and the impact on patient care and health economics. at the end of the day, we are all consumers of health care. we should all be looking around asking, "what are our expectations?" with the aca, we have more patients to take care of and fewer health care dollars to do it with in an imperfect health care system. we have improved and expanded technology that allows us to ask how we improve access, overcome barriers, and provide smarter care. technology can help get us there, but thoughtful approaches to technology placement and health care delivery will make it a reality. impact of an antimicrobial stewardship program on antimicrobial utilization, bacterial susceptibilities, and financial expenditures at an academic medical center the confounding role of antimicrobial stewardship pro grams in understanding the impact of technology on patient care accurate detection of streptococcus pyogenes at the point of care using the cobas liat strep a nucleic acid test clia waived testing in infectious diseases the waiting game': are current chlamydia and gonorrhoea near-patient/point-of-care tests acceptable to service users and will they impact on treatment? impact of deploying multiple point-of-care tests with a 'sample first' approach on a sexual health 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of respiratory and intestinal pathogens improving diagnosis in health care delayed diagnosis of tuberculous meningitis misdiagnosed as herpes simplex virus-1 encephalitis with the filmarray syndromic polymerase chain reaction panel comparative evaluation of the filmarray meningitis/encephalitis molecular panel in a pediatric population norovirus and medically attended gastroenteritis in u.s. children optimum diagnostic assay and clinical specimen for routine rotavirus surveillance impact of a rapid respiratory panel test on patient outcomes practice guidelines for the management of infectious diarrhea telemicrobiology: focusing on quality in an era of laboratory consolidation telemedicine in primary health: the virtual doctor project zambia the patient will see you now: the future of medicine in your hands internet-based screening for sexually transmitted infections to reach nonclinic populations in the community: risk factors for infection in men internet-based chlamydia screening guidance for com missioning. london: phe publications internet-based screening for chlamydia trachomatis to reach non-clinic populations with mailed self-administered vaginal swabs self-collected versus clinician-collected sampling for chlamydia and gonorrhea screening: a systemic review and meta-analysis self-collected specimens for infectious disease testing next generation sequencing in clinical and public health microbiology same-day diagnostic and surveillance data for tuberculosis via whole-genome sequencing of direct respiratory samples detection of cytomegalovirus drug resistance mutations by next-generation sequencing the role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the eucast subcommittee an economic evaluation of pulsenet: a network for foodborne disease surveillance health cost and utilization project dancing naked in the mind field: vintage books key: cord-300930-47a4pu27 authors: beigel, r.; kasif, s. title: rate estimation and identification of covid-19 infections: towards rational policy making during early and late stages of epidemics date: 2020-05-24 journal: nan doi: 10.1101/2020.05.22.20110585 sha: doc_id: 300930 cord_uid: 47a4pu27 pandemics have a profound impact on our world, causing loss of life, affecting our culture and historically shaping our genetics. the response to a pandemic requires both resilience and imagination. it has been clearly documented that obtaining an accurate estimate and trends of the actual infection rate and mortality risk are very important for policy makers and medical professionals. one cannot estimate mortality rates without an accurate assessment of the number of infected individuals in the population. this need is also aligned with identifying the infected individuals so they can be properly treated, monitored and tracked. however, accurate estimation of the infection rate, locally, geographically and nationally is important independently. these infection rate estimates can guide policy makers at both state, national or world level to achieve a better management of risk to society. the decisions facing policy makers are very different during early stages of an emerging epidemic where the infection rate is low, middle stages where the rate is rapidly climbing, and later stages where the epidemic curve has flattened to a low and relatively sustainable rate. in this paper we provide relatively efficient pooling methods to both estimate infection rates and identify infected individuals for populations with low infection rates. these estimates may provide significant cost reductions for testing in rural communities, third world countries and other situations where the cost of testing is expensive or testing is not widely available. as we prepare for the second wave of the pandemic this line of work may provide new solutions for both the biomedical community and policy makers at all levels. covid-19 is a deadly disease caused by the sars-cov-2 rna virus. this novel coronavirus created an epidemic of global proportions killing over 200,000 people worldwide (as of april 28 th , 2020) and infecting millions. it also caused a medical crisis and unprecedented disruptions that long-term are likely to increase the risk for multiple-socio-economic downturns that are associated with both mortality and chronic disease. the pandemic created many urgent problems such as the development of antiviral medications, vaccines, and inexpensive and widely available testing capability, expanding er and icu capabilities and much more. however, proper response to the pandemic requires estimates of the rate of infection via testing. the challenge of rapid testing has created an outstanding community response from industry and academic centers producing tests to diagnose and identify infected patients. the majority of these tests rely on pcr based techniques that are very well established. newer tests are based on isothermal amplification and most recently crispr based methods using cas-13. these tests can deliver a result in minutes to hours. high throughput sequencing is also an option for large scale testing. in addition to the biomedical crisis, the virus has also created major challenges for policy makers that need to make life and death decisions based on ethical, clinical and economic factors of unprecedented importance. a full shutdown of the economy and forced social distancing create a colossal stress on the economy, unemployment, and collapse of many business sectors. opening the economy would increase risk for the aging population and people with pre-existing conditions such as diabetes, cancer, cardiovascular disease, respiratory disease, and immunodeficiency. the decisions facing policy makers are very different at the beginning of an emerging epidemic where the infection rate is low vs middle stages (when the rates are rapidly climbing) and at the later stages where the epidemic curve flattened to a low and relatively sustainable rate. during the early stages it is relatively inefficient to test millions of patients who might be suffering from symptoms caused by rsv or influenza. at the same time is it important to monitor any unexpected turns in the progression. in many rural communities the rates usually remain low except local bursts that need to be contained. similarly in third world countries it is economically and practically impossible to perform many tests early on. mathematically, the problems of identifying infected individuals ( identification ) and estimating the total number of infected individuals in a given population ( infection rate ) are related but in fact can be addressed by subtly different algorithms to reduce the number of tests needed and thereby the total cost of doing testing. these methods generally rely on a well-studied area called combinatorial group testing. however, as we will demonstrate in this brief communication, estimating the number of infected individuals can be solved by novel adaptation of methods developed in theoretical computer science aimed at approximate counting. here we refer to these methods as aca (approximate counting algorithms). more specifically, we describe comparatively efficient methods enabling estimation of the total number of infected individuals. intuitively, we pool samples from multiple individuals and repeatedly test these pools for the virus with a single test (or perhaps a small constant number of tests to achieve better sensitivity). we provide a detailed analysis of the accuracy of the approximate counting procedure with both theoretical analysis and simulation. as a simple example, if the infection rate in a population of 1,000,000 is around 1% we can produce an unbiased estimate of the infection rate with variance approximately 7·10 −5 by making 20 group tests. in contrast, testing a sample of 20 individuals to estimate the infection rate would produce an estimate whose variance is 4.95·10 -4 . our variance is approximately 7 times smaller. in addition to rate estimation we provide a review and analysis of several identification algorithms that can be deployed in communities with low infection rates that achieve reasonable improvement over the standard algorithms for group testing that have been previously explored. we first abstract the key computational problems as follows: • estimate the rate of the infection in the population or approximately count how many people test positive in a population of a given size with as few partially pooled tests as possible. • identify the people who are positive with as few partially pooled tests as possible. we focus on these problems in the context of the population that are actively suffering from the disease, rather than post-infection and recovery. we assume the testing is done using established genomic testing procedures. while similar methods are feasible to implement to detect individuals that have or already had the disease (e.g. via antibody presence testing) we expect this number to be significantly higher and our methods are comparatively more effective for lower fractions of individuals testing positive. batch testing assumption: given a set of samples from a set s of people (infected or not), it is technically feasible to form a single batch consisting of all samples and test that batch with a single test. the batch will test positive if and only if at least one person in s is positive. this assumption is reasonable for small batch sizes (e.g. 100 ≤ |s| ≤ 1000). we will provide methods to alleviate this technical restriction on batch size in the discussion. henceforth, n will refer to the (known) number of people (total size of the population) and k will stand for the (possibly unknown) number of people who will test positive. thus k/n is the infection rate. we study two problems: identification of infected individuals and estimating k by approximate counting . the formulas used throughout the methods section are provided in the supplement for convenience. typically, one estimates the infection rate by sampling individuals. this estimate of the rate using the sample mean, is known to be highly inaccurate when the probability of infection p is small, because the variance of the estimator is much larger than p 2 . using group testing, we will produce estimates whose variance is asymptotically proportional to p 2 . therefore our proposed methods are superior to sampling individuals when p is small. we will define a random variable y such that • y can be calculated by making ⌈logn⌉ batch tests • e(2 y ) = θ(k), i.e., e(2 y ) is provably asymptotically proportional to k • e(2 y ) can be computed exactly given n and k • using linear regression we can find constants a and b such that p ≈ e((a2 y +b)/n) • the variance of the estimator (a2 y +b)/n is o(p 2 ). in contrast, the variance of the sample mean estimator is p(1−p)/⌈logn⌉. when p is small, our estimate is much more accurate than sampling individuals. we will also define a random variable w such that • w can be calculated by making m⌈logn⌉ batch tests, where m is a parameter • w is the arithmetic mean of m independent copies of y • we will use w similarly to obtain a nearly unbiased estimator for p whose variance is o(p 2 /m). • in practice this is better than using the arithmetic mean of m independent copies of 2 y . some identification algorithms based on batch testing are already known. we design two new highly parallel algorithms that are efficient for small p. the batch size for these algorithms is not fixed, but instead can be chosen optimally by making a calculation based on the estimated infection rate. we will describe how to choose an algorithm and its batch size given n and an estimate for k. one particularly favorable aspect of these algorithms is the fact that they use very few rounds of group testing which makes them easier to implement in practice than competing methods. to provide an intuitive example illustrating the principle of group testing methodologies we begin with a magic trick which is folklore in popular mathematics (figure1). we ask the reader to think of a number x between 0 and 31, e.g., x = 5. we now perform five binary tests on groups we carefully design. each test returns 1 if the number x is in the group and 0 otherwise. in this case the result of the tests would be 00101 = 5, thereby identifying the hidden number. the tests and their results are listed below in figure 1 for completeness. _________________________________________________________________________ is your number in this set: {16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31}? _0_ is your number in this set: {8,9,10,11,12,13,14,15,24,25,26,27,28,29,30,31}? _0_ is your number in this set: {4,5,6,7,12,13,14,15,20,21,22,23,28,29,30,31}? _1_ is your number in this set: {2,3,6,7,10,11,14,15,18,19,22,23,26,27,30,31}? _0_ is your number in this set: {1,3,5,7,9,11,13,15,17,19,21,23,25,27,29 ,31}? _1_ _________________________________________________________________________ figure 1 : the magic trick: the answer is 00101 = 5 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020. . https://doi.org/10. 1101 the magic trick algorithm is described in full below. number the people 0 through n−1. write those numbers in binary. number the bits right to left starting from 0. (the i-th bit is in position 2 i in the binary representation. alternatively, bit i (x) = (x div 2 i ) % 2.) • let m = ⌈logn⌉. all logarithms are base 2 unless otherwise specified. • let x be the number whose binary representation is b m b m−1 ···b 0 person number x is the one who is testing positive, as revealed by the magic trick above. _________________________________________________________________________ complexity analysis: each person sample is divided into ⌈logn⌉ samples. exactly ⌈log⌉ tests are performed, and they can all be performed in parallel. the logistics of the process of producing the pools (batches) is not considered in this paper and can be performed by robotics or multi fluidic platforms. the size of the batches is n/2, which may potentially pose sensitivity and engineering challenges. however, when k = n/2, information theory tells us that at least n − 0.5logn − 1 tests are required, so we cannot do significantly better than just testing every individual. we now describe approximate counting algorithms that use pools of samples to estimate accurate infection rates. we also provide sketches of the complexity analysis that provide bounds on the number of tests needed to estimate these rates. as alluded to before we are focusing on testing populations with a relatively low disease rate. approximate counting algorithm (aca1): number the samples randomly 1 through n. choose independently subsets of size ⌈n/2⌉, ⌈n/4⌉, ⌈n/8⌉, ⌈n/16⌉, …, 1. let y = the number of subsets that test positive. then ey ≈ log(k) where k is the number of infected individuals. complexity analysis: each person provides a single sample. ⌈log 2 n⌉ tests are performed, and they can all be performed in parallel. the largest batch size is ⌈n/2⌉, which may pose some challenges as mentioned above. if k is not very small it is still possible to deal with the large batch size issue. for example, if the maximum allowed batch size is b then we could assume that all batches larger than b individuals would give the same test result as the largest batch. if all batches test negative, then we would estimate that the infection rate is less than 1/b. we can run aca1 several times to produce a more accurate estimate w, which is discussed in the results section. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020. . https://doi.org/10.1101/2020.05.22.20110585 doi: medrxiv preprint in this section we will follow up on the rate estimation algorithms from the previous section and develop exact identification algorithms (both probabilistic and deterministic) of infected patients in a population with a low infection disease rate. we will present three algorithms , analyze their comparative performance and use each judiciously for the appropriate infection rate we estimated in the previous section. we note that algorithm 0 is the natural approach that has been recently used in nebraska with pool size s=5 without optimizing the choice of the pool size to reduce the number of tests. to be fair to this algorithm, sensitivity of testing in very large pools may be reduced without proper optimization and therefore we present algorithm 0 (small pool size) below for completeness (fig 2) . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020. . https://doi.org/10. 1101 our first new identification algorithm (pia1) is given in fig 3. analysis: the analysis of algorithm pia1 is given in the short summary below. • the probability that a pool contains 0 positives is . as an example consider this analysis with n = 10000, k = 100, and s=50. this is roughly a population of a small town with infection rate 0.01. we test 200 pools to start. on average 120.85 pools will contain 0 positives, 61.34 will contain exactly 1 positive, and 17.81 will contain more than 1 positive. we apply the magic trick algorithm on 79.15 pools of size 50, which takes 79.15log(50) = 474.9 tests. we check the results of the magic trick algorithm with 79.15 · 2 = 158.3 tests. then we test the remaining 890.5 people. on average, the number of tests performed is 1723.7. on average, 120.85*50 = 6042.5 people are classified in the 1st round, 61.34*50 = 3067 people are classified in the 3rd round, and the remaining 890.5 people are classified in the 4th round. on average, we obtain an individual's test result in 1.88 rounds. we might test the remaining people recursively, but that is harder to analyze because the expected number of remaining tests is not linear in the number of remaining people. recursion also increases the average and worst-case time to obtain an individual's test result. we would therefore use smaller pools than 50. for example, if we use pool size 25, we make only 1278.54 tests on average. in fact, pool size 24 is optimal. we calculated optimal pool size for several (n,k) pairs. empirically, optimal pool size seems to depend primarily on k/n. when k/n = 0.01, optimal pool size is 24 or 25 in the examples we tried. we now improve on the previous algorithms described in this section and present an improved probabilistic identification algorithm 2 (pia2) for a specific range of infection rate. the algorithm and its analysis are given below in figure 4 . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020. . run algorithm 2.5 on s to identify two people y and z test y, z, and the analysis of the pia2 algorithm is provided in the list below. • the probability that a pool contains 0 positives is . to summarize, on the average, the number of tests performed by algorithm 2 is: example 1: consider: n = 10,000 and k ≤ 100. pia2 with s=32 is the best choice. the expected number of tests is 1107.27. for comparison, algorithm 0 performs 1956.59 tests on average with its optimal pool size. example 2: n = 100,000 and k ≤ 100. pia2 with s=88 is the best choice. the expected number of tests is 2074.99. for comparison, pia0 performs 6276.37 tests on average. we now provide a deterministic identification algorithm applicable to populations with small infection rate (fig 5) . . a brief outline of the efficiency of the methods is provided in the list below. • identification given k = 1: ⌈logn⌉ tests in parallel (magic trick algorithm) • identification given k ≤ 1: ⌈log(n+1)⌉ tests in parallel (left to the reader) • identification given k ≤ 2: 2.5logn − 1 tests in loglogn − 1 rounds (described below) . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020. analysis: let t(n) denote the number of tests made by da2.5. assume logn is a power of 2. • ) + 1 (n) t ( t ≤ 2 √n • t(4) = 4 by solving the recurrence we find t(n) ≤ 2.5logn − 1 when logn is a power of 2. example. what is t(36)? • t(9) = t(3) + t(3) + 1 = 7 • t(12) = t(3) + t(4) + 1 = 8 • t(36) = t(3) + t(12) + 1 = t(4) + t(9) + 1 = 12 note that t(36) < 2t(6) + 1. additional examples: • t(16) = t(4) + t(4) + 1 = 9 • t(32) = t(2) + t(16) + 1 = t(3) + t(12) + 1 = t(4) + t(9) + 1 = 12 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020. . we first present a few empirical findings of approximate counting algorithms for infection rate estimation that generally support our ability to produce relatively accurate estimates using the methods provided in the previous section. : this graph displays the nearly linear relationship between k and e(2 y ). in order to exploit this relationship in practice, we simply run a program that calculates e(2 y ) for a known value of n and various values of k, and then perform a linear regression. figures 3-4 display the comparative accuracy of our pooling algorithms for estimating infection rates. in particular, figure 4 demonstrates the reduction in variance obtained by running aca1 multiple times. in order to estimate the infection rate we run aca1 m times. let w be the average of those results. the number of infected individuals k is linear in 2 w . we compute a linear regression to determine constants such that k ≈ a2 w + b. then the infection rate, p, is approximately (a2 w +b)/n. we refer to this algorithm as maca1. (in practice it is better to perform linear interpolation using two adjacent data points. the linear formula, however, is needed in order to estimate variance.) . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020. . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020 . . https://doi.org/10.1101 we now summarize the results of our probabilistic identification algorithms that we refer to as pia#. we start by providing a sample of our empirical findings using a few selected examples in table 1 . the full graph is provided in figure 9 for a relatively large population size. population size best algorithm p = 0.061 1000 ≤ n ≤ 10 6 pia2 p = 0.062 5500 ≤ n ≤ 10 6 pia1 p = 0.069 1000 ≤ n ≤ 10 6 pia1 p = 0.07 1200 ≤ n ≤ 10 6 pia0 p = 0.3 40 ≤ n ≤ 10 6 pia0 p = 0.31 300 ≤ n ≤ 10 6 individual testing (n tests) in figure 9 , we graphed the behavior (expected number of tests) of pia0, pia1, and pia2 when n = 10 6 and 0 < p < 0.07. we also observe that the optimal pool size seems to depend mostly on p. an information-theoretic lower bound on the number of tests required is . when p ≥ 0.005, we found that the number of tests performed is usually less then log ( ) k n . .4 1 log ( ) k n figure 9 : the expected number of tests performed by different algorithms for different infection rates p. pia2 is graphed in yellow. pia1 in red and pia0 in blue. pia2 generally outperforms the other algorithms when p ≤ 0.06. in this graph n = 1,000,000. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 24, 2020. . https://doi.org/10.1101/2020.05.22.20110585 doi: medrxiv preprint the covid-19 pandemic produced a very high toll worldwide and created technological, biomedical and clinical challenges. in this paper we described a cost effective application of approximate counting methods to estimate infection rates. these methods can help make crucial and rapid policy decisions with reduced investment, especially in early or late stages of the epidemic or in underserved communities, e.g., rural counties or third world countries. we also reviewed several identification algorithms and introduced new ones that provide good to modest improvements in the number of tests for different infection rates. in some cases our methods reduce the number of tests by two fold which is significant. estimating the infection rate without identification has many applications. these estimates can help policy makers determine appropriate guidelines for opening or closing the economy or implementing different types of social distancing procedures. the new method lends itself naturally to performing a very small number of tests on autopsy samples and is potentially useful in counting the number of individuals who were infected prior to death. we also note that our proposed counting method can speculatively be extended to large scale vaccine trials. a large population can be vaccinated and another population can be used for placebo. we can use our cost efficient estimates to compare the rates in these two cohorts. in fact, we expect our variance estimates to be even better than we report above. our specific identification procedures of infected individuals improved on the number of tests that must be conducted saving in costs, especially when the infection rates are low and most tests are negative. notably, we use a very small number of rounds as compared to the best competing algorithms, enabling quicker turnaround. in this paper we primarily focused on using established methods such as pcr or isothermal amplification that have been shown to produce the most reliable diagnostics in the past. we have not considered multiplex-pcr as an alternative. there are newer diagnostic procedures based on crispr (cas-13) under development. these methods vary in sensitivity, availability and cost. in theory, it is also possible to barcode every sample with a genomic tag, pool the tagged sequences into a very large multiplexed dna sample and submit the sample for high throughput sequencing. standard procedures would allow us to read the sequences and allocate any detected viral dna to the appropriate individual using tags. error correcting procedures can be deployed to produce an efficient library of tags. however, high throughput sequencing presents its own challenges and therefore we focused on pooling samples and testing using more traditional approaches. these established methods might also be easier to deploy in third world countries or rural communities that do not have access to high throughput solutions. this work can be extended in multiple useful directions both mathematically and technologically. it is highly timely for a careful consideration and possible deployment given the expected flattening of the infection curve as we approach the summer of 2020 and the potential need to detect the disease in the fall of 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 24, 2020. . https://doi.org/10. 1101 the formulas in this section were used in 1. proving our theoretical results about expected value and variance of 2 y and 2 w 2. creating tables of e[2 y ] for a given n (and all k) and e[2 w ] for given n and m (and all k) 3. creating tables of e[4 y ] and e[4 w ] the tables of e[2 y ] and e[2 w ] make it possible to estimate k via linear interpolation. the tables are also used with linear regression to obtain our empirical findings about expectation and variance. formulas used with aca1 formulas used with maca1: • for any c > 0, [c ] (1 c )p ) . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 24, 2020. . https://doi.org/10. 1101 one test. this is how you get there evaluation of group testing for sars-cov-2 rna survey, foundations and trends in communications and information theory an optimal procedure for gap closing in whole genome shotgun sequences multi-node graphs: a framework for multiplexed biological assays muplex: multi-objective multiplex pcr assay design rapid molecular detection of sars-cov-2 (covid-19) virus rna using colorimetric lamp yinhua zhang massively multiplexed nucleic acid detection using cas13 the complexity of approximate counting counting large numbers of events in small registers key: cord-328294-gii1b7s7 authors: doty, richard l.; mishra, anupam title: olfaction and its alteration by nasal obstruction, rhinitis, and rhinosinusitis date: 2009-01-02 journal: laryngoscope doi: 10.1097/00005537-200103000-00008 sha: doc_id: 328294 cord_uid: gii1b7s7 the sense of smell has been largely ignored by otorhinolaryngologists, even though 1) its medical stewardship falls within their specialty's purview, 2) olfactory dysfunction is not uncommon in the general population, and 3) disorders of olfaction have significant quality of life, nutritional, and safety consequences. this report provides a succinct overview of the major intranasal neural systems present in humans (namely, cranial nerves o, i, and v, and the nonfunctional accessory [vomeronasal] organ system), along with a summary of notable findings resulting from the application of modern olfactory tests to patient populations, emphasizing diseases of the nose. such tests have led to the discovery of significant influences of age, gender, smoking, toxic exposure, and genetics on the ability to smell. within the field of otorhinolaryngology, they have revealed that 1) surgical and medical interventions in patients with rhinosinusitis do not, on average, lead to complete recovery of olfactory function, despite common beliefs to the contrary, and 2) associations are generally lacking between measures of airway patency and olfactory function in such cases. these findings have thrown into question the dogma that olfactory loss in rhinosinusitis is attributable primarily to blockage of airflow to the receptors and have led to histopathological studies demonstrating significant olfactory epithelial compromise in sinonasal syndromes. the sense of smell largely determines the flavor of foods and beverages and serves as an early warning system for the detection of environmental hazards, including spoiled foods, leaking natural gas, smoke, and various airborne pollutants. this primary sensory system contributes significantly to the quality of life, allowing for the full appreciation of flowers, perfumes, spices, and a vast array of foods and beverages, as well as the seashore, the mountains, and the seasons of the year. thus, it is no wonder that losses or distortions of smell sensation are of considerable significance to patients, particularly those dependent on this sense for their livelihood or safety (e.g., cooks, homemakers, plumbers, firefighters, perfumers, fragrance sales persons, wine merchants, food and beverage distributors, and employees of numerous chemical, gas, and public works industries). indeed, altered smell function can adversely influence food preferences, food intake, and appetite. in this report, we review the influences of nasal obstruction, rhinitis, and rhinosinusitis (as well as well as their medical and surgical treatments) on the ability to smell. because this neglected sensory system receives so little attention in most medical textbooks, including those of clinical allergy, otolaryngology, neurology, and immunology, an overview of olfactory anatomy, physiology, and measurement is also presented. in humans, three specialized neural systems are present within the left and right nasal chambers: 1) the main olfactory system (cranial nerve i [cn i]), 2) the trigeminal somatosensory system (cranial nerve v [cn v]), and 3) the nervus terminalis or terminal nerve (cranial nerve o [cn o]). cn i mediates odor sensations (e.g., chocolate, strawberry and apple), whereas cn v mediates, through both chemical and nonchemical stimuli, somatosensory sensations, including those of burning, cooling, irritation, and tickling. the coolness of menthol and peppermint are mediated by cn v, as, for example, are the sharp sensations induced by ammonia vapors and various acids. the function of cn o, a ganglionated neural plexus that spans much of the nasal mucosa before traversing the cribriform plate to enter the forebrain medial to the olfactory tract, is unknown in humans. this nerve, whose disruption in some rodents alters reproductive behavior, 1 was discovered after the other cranial nerves had been named and is highly conserved among the vertebrates, including humans. 2, 3 despite the fact that nearly all adult humans possess, in the lower recesses of each nasal chamber, a rudimentary vomeronasal (jacobson's) organ (vno) and a vno duct approximately 15 to 20 mm from the posterior aspect of the external naris, they lack an accessory olfactory bulb, a structure necessary for its function. thus, in adult humans this system is nonfunctional, and no neural connection from the vno to the brain has been established. 4 nonetheless, local electrophysiological responses have been recorded within the human vno lumen. 5 the olfactory neuroepithelium, which harbors the sensory receptors of the main olfactory system and some cn v free nerve endings, lines the upper recesses of the nasal chambers, including the cribriform plate, superior turbinate, superior septum, and sectors of the middle turbinate. this epithelium loses its general homogeneity postnatally, and as early as the first few weeks of life metaplastic islands of respiratory-like epithelia begin to appear, presumably as a result of insults from environmental viruses, bacteria, and toxins. such islands increase in extent and number throughout life. surprisingly, the exact size of the olfactory neuroepithelium in humans is still not well established, and there is recent suggestion that it may extend further onto the middle turbinate than previously believed. on the basis of morphological and biochemical criteria, the mature olfactory epithelium comprises at least six distinct cell types (fig. 1) . 6 the first, the bipolar sensory receptor neuron, is estimated to number approximately 6,000,000 cells in the adult, exceeding the number of receptor cells in any other sensory system except vision. the olfactory receptors are located on the ciliated dendritic ends of these cells, whose surface area probably exceeds 22 cm 2 in the human. the receptor cell axons coalesce into ϳ40 bundles (termed the olfactory fila), which are ensheathed by schwann-like cells. the fila traverse the cribriform plate of the ethmoid bone to enter the anterior cranial fossa and collectively constitute cn i. the second cell type, positioned near the surface of the epithelium, is the microvillar cell. these cells are said to number approximately 600,000 in the adult. each microvillar cell, whose function is unknown, contains microvilli. the third cell type, the supporting or sustentacular cell, also projects microvilli into the mucus. these cells are believed to 1) insulate the receptor cells from one another, 2) regulate the local ionic composition of the mucus, 3) deactivate odorants, and 4) help protect the epithelium from damage from foreign agents. the supporting cells contain xenobiotic-metabolizing enzymes (e.g., cytochrome p-450), a feature shared with the fourth cell type, the cell that lines the bowman glands and ducts. the bowman glands are a major source of mucus within the region of the olfactory epithelium. the fifth and six cell types are the globose (light) basal cell and horizontal (dark) basal cell, cells that are located near the basement membrane from which the other cell types arise. the same type of basal cell, probably a globose cell, can give rise to both neurons and nonneural cells when the olfactory epithelium is damaged, expressing a multiple potency rarely observed in stem cells. it is noteworthy that the olfactory ensheathing cells, which form the bundles of axons that make up the olfactory fila, enhance remyelination and axonal conduction in demyelinated spinal tract nerves, as well as in severed rat sciatic nerves, 7 exhibiting both schwann celllike and astrocyte-like properties. the cilia of the olfactory receptor cells lack dynein arms (hence, intrinsic motility). odorant transport through the mucus to the cilia is aided by "odorant binding proteins." approximately 1000 classes of odorant receptors are currently believed to exist, reflecting the expression of the largest known vertebrate gene family, a family accounting for approximately 1% of all expressed genes. in general, the olfactory receptors are linked to the stimulatory guanine nucleotide-binding protein g olf . when stimulated, they activate the enzyme adenylate cyclase to produce the second messenger adenosine monophosphate (camp) and subsequent events related to depolarization of the cell membrane and signal propagation. although a given receptor cell seems to express only one type of receptor derived from a single allel, each cell is electrophysiologically responsive to a wide but circumscribed range of stimuli. this implies that a single receptor accepts a range of molecular entities and that coding occurs via a complex cross-fiber patterning of responses. the olfactory bulb is a complex processing center, receiving both afferent and efferent input. this ovoid structure has clear concentric layers discernible using light microscopy. the layers are, in succession, the outermost olfactory nerve layer, the glomerular layer, the external plexiform layer, the mitral cell layer, the internal plexiform layer, and the innermost granule cell layer. in the human, the receptor cell axons of the olfactory fila, after traversing the cribriform plate, form the olfactory nerve layer and synapse in the second bulbar layer within the spherical glomeruli. in general, receptor neurons expressing a given receptor type project to one or, at most, two glomeruli, making the glomeruli in effect functional units. thus, a given odorant activates a spatially defined or restricted set of glomeruli. hence, the olfactory code is reflected, at this early stage, not only as different patterns across the mucosa, but across the glomeruli as well. the major second-order neurons of the olfactory bulb (the mitral and tufted cells) project their axons centrally to elements of the olfactory cortex. the olfactory cortex comprises 1) the anterior olfactory nucleus ([aon], which in the human has a large segment in the posterior olfactory bulb), 2) the olfactory tubercle (poorly developed in humans), 3) the prepiriform cortex, 4) the lateral entorhinal cortex, 5) the periamygdaloid cortex, and 6) the cortical nucleus of the amygdala. the afferent olfactory signal is modulated at all levels of the system, from the olfactory bulb to the olfactory cortex. olfaction is unique in that information from the olfactory bulb goes directly to cortical structures without passing through the thalamus. however, thalamic connections are present for relays between various elements of the primary and secondary olfactory cortices. the most widely used tests for assessing the ability to smell are those of odor threshold and odor identification. because these are the only tests routinely used in clinical settings, and because such tests are available commercially, the current discussion focuses on these measures. the reader is referred elsewhere for discussions of the comparative reliability, sensitivity, and validity of various types of modern olfactory tests. 8, 9 olfactory threshold tests the lowest concentration of an odorant that can be reliably detected is termed the detection or absolute threshold. usually, at lower perithreshold odorant concentrations, no odor quality can be discerned, only something different from air or the comparison diluent blank or blanks. in modern olfactory detection threshold testing, the subject is asked to report which of two or more stimuli (i.e., an odorant and one or more blanks) smells strongest, rather than to simply report whether or not an odor is perceived. such "forced-choice" procedures are less susceptible to contamination by response biases (e.g., the conservatism or liberalism in reporting the presence of an odor under uncertain conditions) than non-forced-choice procedures. in addition, they are more reliable and produce lower threshold values. 8 the instructions provided to a subject are critical in measuring a detection threshold because, if the subject is instructed to report which stimulus produces an odor rather than which stimulus is stronger, a spuriously high threshold value may result because the subject's attention is diverted away from subtle differences in the presented stimuli (odor quality is present only at higher perithreshold concentrations). the recognition threshold is the lowest concentration where odor quality is reliably discerned. however, it is nearly impossible to control criterion biases in recognition threshold measurement. thus, in a forced-choice situation, guesses are not randomly distributed among alternatives, potentially leading to a spuriously low recognition threshold for the preferred alternative. a classic example of this problem comes from taste psychophysics, in which some subjects report "sour" much more frequently than the other primary qualities in the absence of a clearly discernible stimulus, resulting in a erroneously low sour taste recognition threshold measure. two types of threshold stimulus presentation procedures have received the most use in modern times: the ascending method of limits procedure (ams) and the single staircase procedure (ss). in the aml procedure, an odorant (and comparison blanks) is sequentially presented from low to high concentrations and the point of transition between detection and no detection is estimated. in the ss method, the concentration of the stimulus is increased following trials on which a subject fails to detect the stimulus and decreased following trials in which correct detection occurs. an average of the up-down transitions ("reversals") is used to estimate the threshold value. in both the aml and ss procedures, the direction of initial stimulus presentation is made from weak to strong in an effort to reduce potential adaptation effects of prior stimulation. in general, the ss procedure is preferred to the aml procedure because it is more reliable, since most investigators who employ the aml technique present only a single ascending stimulus series. unfortunately, widespread use of the single-series aml procedure has led to the erroneous conclusion that threshold measures exhibit a high degree of intrasubject variability, a conclusion not borne out when thresholds are determined using the ss procedure. 8 a modern, commercially available threshold test kit that employs an ss procedure is shown in figure 2 . this kit uses squeeze bottles containing various half-log step concentrations of an odorant known to stimulate primarily cn i, namely, the rose-like smelling odorant phenyl ethyl alcohol (pea). norms based on hundreds of subjects spanning the entire age range allow for the practical application of this test in medical and industrial applications. the development and proliferation of easy-to-use, self-administered "scratch and sniff" tests of odor identification have significantly increased our understanding of smell function in humans, including the influences of such factors as age, gender, exposure to toxic agents, smoking behavior, and various disease states. such quantitative tests, derived from test measurement theory, focus on the comparative ability of individuals to identify a number of odorants at the suprathreshold level. the most popular of these tests are the 40-odorant university of pennsylvania smell identification test ([upsit], known commercially as the smell identification test™ [or sit]) 10, 11 ; the 12odor brief-smell identification test ([b-sit], also known as the cross-cultural smell identification test™), 12 and the 3-odor pocket smell test™ (pst) (sensonics, inc., haddon heights, nj). 8 the upsit has been used most widely, having been administered to approximately 200,000 people in europe and north america since 1985. this test, shown in figure 3 , employs norms based on nearly 4000 persons and is available in english, french, german, and spanish language versions. for a given item, the patient simply scratches open a microencapsulated label with a pencil tip, smells the label, and signifies the odor quality from four alternatives provided. even if no smell is perceived, a response is required (i.e., the test is forced-choice). in addition to indicating the level of absolute smell function (i.e., normosmia, mild hyposmia, moderate hyposmia, severe hyposmia, total anosmia), this test provides a percentile rank for each age and gender group. malingering is detected on the basis of improbable responses. in general, when equated for test length, tests of odor identification are more reliable than tests of odor detection threshold and require less administration time. furthermore, most identification tests can be self-administered and tend to correlate better with a patient's complaint than measures of detection threshold. nonetheless, tests of odor identification and detection are typically correlated with one another. 8 among major nonclinical findings derived from modern sensory tests, primarily the upsit, are the following: first, the ability to identify odors has a strong genetic basis, as determined from twin studies 13, 14 ; second, women, on average, are better able than men to identify odors, and this superiority is noticeable as early as 4 years of age and is culture independent [15] [16] [17] [18] ; third, significant loss of olfactory function occurs after the age of 65 years, with more than half of persons between 65 and 80 years of age and more than three-quarters of those 80 years of age and older having such loss 16, 18, 19 ; fourth, women, on average, retain the ability to smell longer than men 16 ; fifth, the decreased smell ability associated with smoking is present in prior cigarette smokers, and recovery to presmoking levels, while possible, can take years, depending on the amount and duration of prior smoking 20 ; and sixth, olfactory function is compromised in urban residents and in workers in some industries, including the paper and chemical manufacturing industries. [21] [22] [23] [24] [25] clinical studies employing such methodology during this period have found decreased smell function relative to matched controls in dozens of diseases and disorders (see table i ). the straightforward ability to quantify olfactory function, along with recent advances in in vivo medical imaging, has made it possible to better understand the physiological basis of a number of chemosensory deficits. for example, it is apparent today that congenital anosmia is nearly always associated with markedly deformed or absent olfactory bulbs and stalks. furthermore, head trauma-related smell loss is typically accompanied by decreased bulb and tract size that presumably reflects mitigation of trophic factors from the olfactory receptor neurons, which are often sheared off or otherwise altered in head trauma. the smell loss associated with chronic alcoholism has been found to be correlated with magnetic resonance imaging (mri)-determined 1) increased cortical and ventricular cerebral spinal fluid volumes and 2) reduced volumes of the thalamus and of cortical and subcortical gray matter. 26 the smell loss of multiple sclerosis is directly associated with the number of active plaques in central brain regions, 27, 28 and that of schizophrenia with diminished olfactory bulbs and tracts. 29 the presence of hypertrophied adenoid tissue can significantly block the nasal airflow of children whose airways are otherwise patent. crysdale et al. 30 noted a 43% reduction in nasal resistance following adenoidectomy in a group of 67 children ranging in age from 4 to 17 years before surgery, and fielder 31 reported a 19% postoperative reduction in such resistance in a group of 19 children admitted for adenoidectomy and myringotomies (with or without tonsillectomy) who had at least 1 g of adenoid tissue removed. in 1983, ghorbanian et al. 32 evaluated the degree to which nasal obstruction influences the olfactory sensitivity of children. this work, which has been subsequently replicated by others, 33 determined phenyl ethyl alcohol detection thresholds in 65 children with varying degrees of nasal obstruction and in 13 children without such obstruction. the threshold values were directly related to clinical ratings of the degree of nasal obstruction. these findings, shown in figure 4 , suggest that in this subject population the degree of nasal obstruction is associated with commensurate impairment in the ability to smell and that reduction in the degree of nasal obstruction results in commensurate recovery of smell function. it is well documented that the common cold can result in permanent loss of smell function. however, such loss usually occurs in later life after the olfactory membrane has presumably undergone considerable cumulative damage. for this reason, temporary smell loss following an upper respiratory infection is much more common. in general, virus-related acute rhinitis or rhinosinusitis follows three predictable phases; namely, a predromal phase, a cathartic phase, and a viscous phase. 34 the predromal phase is characterized by sweating, shivering, headaches, loss of appetite, and other nonspecific feelings of being ill. during this phase, tickling, burning, or dryness within the nose is common and the mucosa typically appears pale. the cathartic phase follows a few hours after the predromal phase and is characterized by increased mucosal redness and swelling, nasal obstruction, and secretion of watery mucus. a few days later, during the viscous phase, the nasal secretions thicken and the intensity and frequency of the aforementioned decline, disappearing after about a week. two studies have quantitatively assessed olfaction following the onset of the common cold, with an attempt to establish whether changes in smell function are coincident with changes in nasal congestion and secretion. in the first of these studies, akerland et al. 35 measured 1-butanol odor detection thresholds in a group of student volunteers before and 4 days after nasal inoculation with the coronavirus 229e. the nine individuals who developed a cold had impaired olfactory thresholds on the postinoculation test relative to the controls. whereas the change in smell function correlated with the degree of nasal congestion, it did not correlate with the amount of nasal discharge. the second study on this topic led to the conclusion that the common cold may, in fact, affect olfactory function independent of nasal congestion. 34, 36 in this experiment, whose main purpose was to evaluate the potential doserelated effects of oxymetazoline (administered unilaterally) on olfactory function, both psychophysical (intensity ratings, odor discrimination, butanol detection threshold) and electrophysiological (event-related potentials to h 2 s and co 2 ) data were obtained. nasal volume was assessed by acoustic rhinometry. thirty-six subjects (18 women, 18 men) were evaluated soon after they experienced the natural onset of a cold. after rhinitis onset (day 0), sensory and airway measurements were obtained on days 2, 4, 6, and 35. the cold produced a decrease in the volume of the anterior nasal cavity and an increase in mucus secretion, an increase in olfactory thresholds, a decrease in intensity ratings, and a decrease in n1 evoked potential amplitudes to both olfactory and trigeminal stimuli. when mucus secretion of the contralateral nasal cavity was controlled with oxymetazoline, n1 amplitudes to olfactory stimuli were still affected by the cold, as indicated by the significant increase of amplitudes as subjects recovered; however, this phenomenon was not found for any of the other test measures or for the responses to the trigeminal stimuli. overall, the olfactory test scores tended to improve during the viscous phase. a number of studies have sought to determine the influences of acute or chronic rhinitis on olfactory function. some such studies have differentiated between rhinitis and secondary nasal and sinus disease (i.e., sinusitis and/or polyposis), although this distinction is often difficult to maintain. currently, the term rhinosinusitis is preferable to the term sinusitis because invariably inflammation of the sinuses coexists with inflammation of the nose. rhinosinusitis can be further divided into acute, subacute, recurrent acute, and chronic types, during which acute exacerbation of chronic symptoms can occur. however, no studies have comparatively evaluated olfactory function in these various forms or stages of nasal disease. nonetheless, as described below, there is strong suggestion from numerous quarters that the degree of olfactory loss is correlated with disease severity. among the first nonquantitative studies in the english literature on olfaction in rhinosinusitis and/or polyposis were those of hotchkiss 37 in the mid 1950s and fein et al. 38 in the mid 1960s. hotchkiss evaluated selfreported olfactory function in 30 patients with nasal obstruction secondary to polyposis who reported smell loss. all were treated with a total dose of 70 mg of prednisone over a 6-day period. restoration of smell was said to follow the systemic steroid therapy, with the magnitude of the restoration being proportional to the amount of polyp shrinkage. the restoration was reportedly unrelated to the duration of the loss of olfactory function. however, the self-rated improvement lasted only (on average) 10 days after the discontinuation of therapy. fein et al. examined 18 patients who reported loss of smell associated with allergic rhinitis. of these patients, 14 had other diseases, including sinusitis, polyposis, and bronchial asthma. again, no objective sensory testing was performed. on the basis of self-report, the severity of the smell dysfunction was classified as mild, moderate, or severe. of the four patients who had only allergic rhinitis, two were said to have had mild smell loss, and two, moderate smell loss. of the 14 patients with other diseases, two reportedly had mild loss, six moderate loss, and six severe loss. in the latter group, severe loss was said to be associated with the presence of both polyposis and sinusitis. although improvement in some of the subjects from hyposensitization, antibiotics, polypectomy, or various combinations of treatments was noted, a lack of a well-defined experimental protocol employing quantitative olfactory tests and the introduction of the treatments in various combinations without control for their order or time precludes a determination of the relative efficacy of the interventions. more recently, tos et al. 39 had 91 patients with polyposis rate their olfactory function on a 0 -3 scale (0 ϭ normal, 1 ϭ slight impairment, 2 ϭ moderate impairment, and 3 ϭ anosmia) before and after 6 weeks of twice-daily nasal corticosteroid treatment. before treatment, the mean rating of the 44 patients who were to receive the corticosteroid sprays was 2.09, whereas that of the 47 patients who were to receive the placebo was 2.19. following treatment, the self-ratings were 1.86 for the corticosteroid-treated subjects and 2.19 for the placebotreated subjects. even though a statistically significant change occurred in the ratings after the administration of the corticosteroid, the degree of average self-rated im-provement was not marked and, for all practical purposes, moderate loss of smell function was still reported. in contrast to these largely subjective reports are a number of studies, most appearing since 1990, that have quantitatively assessed smell dysfunction in patients with rhinosinusitis. among the first of these studies was that of goodspeed et al. 40 in this work, systemic prednisone 50 mg was administered each day for 7 days to 20 anosmic or severely hyposmic patients of several types whose olfactory function was monitored using butanol thresholds and odor identification tests. loss of smell function following the cessation of prednisone treatment was variable and was not quantitatively tested after the discontinuance of the therapy. another early empirical study 41 sought to determine the efficacy of flunisolide nasal spray in restoring olfactory function in a selected set of patients with perennial rhinitis and nasal polyposis. in this report, flunisolide and nasal decongestant sprays were introduced, with the decongestant being discontinued a week later. the olfactory testing was performed at home, and the selfadministration of the nasal sprays was performed in the moffett position to enhance delivery. daily subjective ratings and a self-administered smell test revealed a return of smell function to the mid hyposmic range in five of the seven patients after approximately 2 weeks of the flunisolide treatment. the first large-scale empirical study of olfaction in allergic rhinitis was that by cowart et al. 42 in 1993. quantitative detection threshold measures for the rose-like smelling odorant phenyl ethyl alcohol were obtained in this well-designed and carefully executed study from 91 patients with symptoms of allergic rhinitis and from 80 nonatopic control subjects. the allergy patients exhibited significantly higher detection thresholds than did the controls, with 23.1% of the patients demonstrating a clinically significant loss (i.e., a threshold at or above the 2.5 percentile of control values). clinical or radiographic evidence of rhinosinusitis or nasal polyps or both in allergy patients was found to be associated with hyposmia: 14.3% of the allergy patients with no associated rhinosinusitis exhibited hyposmia, whereas 42.9% of the allergy patients with associated rhinosinusitis did so. no association between the smell test scores and nasal resistance was seen in either the patient or control groups, although nasal resistance was higher among patients than among control subjects. two years later, apter et al. 43 reported that 28 patients with chronic rhinitis and no associated polyposis or rhinosinusitis had an average olfactory test score (based on a composite of odor identification and detection tests) indicative of moderate hyposmia. thirty-four such patients with polyps and/or chronic sinusitis were found to be generally anosmic. these results were interpreted to mean that chronic rhinitis without associated sinusitis could result in some degree of olfactory loss, but that severe loss was usually associated with the presence of polyposis and/or rhinosinusitis. in the first study on this topic to employ the upsit, golding-wood et al. 44 evaluated olfactory function once before and once after 6 weeks of betamethasone treatment in 25 well-documented patients with perennial rhinitis. the patient group was initially divided into two groups: those who answered the question "is your sense of smell impaired?" affirmatively (n ϭ 15) and those who did not (n ϭ 10). the upsit scores of each of the 15 members of the former group were higher after the betamethasone treatment (respective group means [sd], 18.93 (9.4) and 33.4 (4.01)]. this was not the case for those who initially thought that they had no problems smelling (respective pretreatment/post-treatment means [sd], 33.40 (4.01) and 32.8 (4.94)]. as in earlier studies, however, the average post-treatment upsit score was still indicative of a mild hyposmic condition. in general, the upsit scores of the patients retained a similar rank order before and after treatment (spearman's correlation coefficient [r] ϭ 0.75). moderate correlations were found between the upsit scores and the self-ratings of olfactory function both before (r ϭ ϫ0.52) and after (r ϭ ϫ0.58) treatment. a year after this study, mott et al. 45 sought to determine the efficacy of topical corticosteroid nasal spray treatment after 8 weeks in severe olfactory loss associated with severe nasal and sinus disease. on average, the objective measures of olfaction improved significantly, and a decrease in the signs of nasal and sinus disease were noted on rhinoscopic evaluation. two-thirds of the patients noted a subjective improvement in smell function. these data, along with those of golding-wood et al., 44 imply that in many patients topical corticosteroid nasal spray, when administered in a head-down-forward position, mitigates, at least to some degree, the olfactory loss associated with severe nasal and sinus disease. in perhaps the most extensive study of olfaction in rhinitis and rhinosinusitis to date, simola and malmberg 46 compared odor detection thresholds obtained from 105 rhinitis patients to those of 104 healthy control subjects. age and rhinitis were found to be associated with smell dysfunction. both the proportion of hyposmic persons and the degree of the impairment of the sense of smell were significantly higher in the rhinitis group than in the control group. the nonallergic rhinitis patients' sense of smell was found to be poorer than that of the patients with seasonal or perennial allergic rhinitis. a history of operations for nasal polyposis was associated with hyposmia, but operations for chronic maxillary sinusitis were not. two other studies appeared more or less contemporaneously with the study by simila and malberg. in the first, kondo et al. 47 administered the upsit to 36 japanese patients with a history of sinusitis/polyposis and to 131 control subjects. the mean upsit score of the patients was significantly (p ͻ.001) lower (23.80, sd ϭ 7.12) than that of the controls (32.08, sd ϭ 3.57), despite some culture-related attenuation in the test scores of both groups. detection and recognition thresholds showed a similar association. as in the case of the study by golding-wood et al., 44 moderate correlations emerged between the odor test scores and the scores on a smell ability questionnaire (spearman's r ranging from 0.58 to 0.69). in the second study, apter et al. 48 assessed odor detection and identification in 1) 60 patients who presented to a smell and taste clinic with self-described olfactory loss and were found to have allergic rhinitis and 2) 30 patients with allergic rhinitis from an allergic clinic who had no chronic rhinosinusitis or polyposis. as might be expected, the patients presenting to the smell and taste clinic with olfactory dysfunction had significantly lower olfactory test scores than those who came from the allergy clinic and who were not specifically presenting with olfactory loss. in accord with the findings of several of the prior studies, olfactory function was inversely associated with the severity of the disease. however, no meaningful relationship was apparent between the visibility of the olfactory clefts (determined from endoscopic rhinoscopy) and smell function, regardless of the disease status. self-reported fluctuations in function were less frequent in the groups without chronic rhinosinusitis than in those with chronic rhinosinusitis and/or polyposis. interestingly, self-reported distortions in smell function were generally associated with a history of upper respiratory tract infections and were more apparent in individuals with less severe disease. duration of nasal symptoms alone was not meaningfully correlated with the degree of olfactory loss. recently, rydzewski et al. 49 assessed olfactory function using the elsberg blast-injection procedure in 240 patients with perennial rhinitis, seasonal rhinitis, and bronchial asthma. of their patients, 13.8% were hyposmic, and 7.6% anosmic. surprisingly, using electrogustometry, these authors found taste disorders in even a larger percentage of these patients (30.7%). the olfactory component of this work, however, must be viewed with caution in light of the methodological problems with elsberg olfactometry. this procedure has been criticized on numerous grounds, including 1) the lack of a forced-choice response, 2) the confounding of pressure with the number of molecules in the stimulus, 3) the introduction of a very unnatural stimulus pulse into the nose, and 4) the production of highly unreliable sensitivity measures. 50, 51 in aggregate, the studies reviewed above suggest that the degree of olfactory loss is usually associated with the severity of nasal sinus disease, with the greatest loss occurring in patients who have rhinosinusitis and polyposis. employing quantitative tests, smell function has been shown to improve in some patients following systemic administration of corticosteroids, as well as topical administration of corticosteroid sprays when administered in a head-down-forward position. nonetheless, no study has compared the latter mode of delivery to that of a standard mode, and no one has administered such drugs in a blind, placebo-controlled study. importantly, the limited data available suggest that only rarely has corticosteroid treatment restored function to normal levels, implying that either 1) some chronic permanent loss of olfactory function is present or 2) such treatments are not 100% effective in reversing the disease processes responsible for the olfactory loss. interestingly, no study has been able to document in rhinitis patients an association between olfactory test scores and intranasal airway access factors save total or near-total blockage, whether measured by rhinoscopy, rhinomanometry, or acoustic rhinometry. there is currently considerable support for the hypothesis that factors other than, or in addition to, nasal airflow are responsible for many instances of smell loss in patients with rhinosinusitis, in support of the notion that chronic inflammation may be toxic to olfactory neurons. for example, kern 52 presented data, albeit preliminary, that the severity of histopathological changes within the olfactory mucosa of patients with chronic rhinosinusitis is positively related to the magnitude of olfactory loss, as measured by the upsit. in addition, authors have shown that olfactory secretions are probably regulated by both mineralcorticoids and glucocorticoids. 53, 54 feron et al. 55 reported, in a study group of 33 subjects, that nasal biopsy specimens from the posterior superior turbinate, posterior medial turbinate, and posterodorsal septum of patients with nasal disease were less likely to contain olfactory neuroepithelium than analogous biopsy specimens from patients with no such disease. lee et al. 56 have demonstrated that biopsy specimens from the region of the olfactory epithelium of anosmic patients with rhinosinusitis were less likely to contain olfactory epithelial tissue than those from rhinosinusitis patients who were not anosmic (27% vs. 61% positive biopsy results, respectively). although detailed examination by lee et al. of the epithelium from rhinosinusitis patients with normal smell function did reveal islands of respiratory-like epithelium interspersed throughout the biopsy samples, such islands were much less prevalent than in the anosmic patients for whom olfactory epithelium could be found. abnormalities in the arrangement of the epithelial cell types was common in the anosmic biopsy specimens, and in cases where olfactory epithelium was identified, it was typically atrophic and thin, often comprising mainly sustentacular and basal cells. hilberg 57 evaluated the effect of the oral antihistamine terfenadine (a histamine type 1 [h 1 ] blocker) on an allergen challenge in subjects with nasal allergy uncomplicated by polyposis and compared these results with those obtained using the topical steroid budesonide. although both drugs had an effect on the hay fever symptoms during the nasal pollen challenge, only the budesonide improved the challenge-related decrement in olfactory sensitivity. this steroid also was more effective in increasing nasal volume. however, the improvement in olfactory function occurred in less than half of the patients (7/17 [41%]). lane et al. 58 employed an abbreviated 20-item version of the upsit and acoustic rhinometry to assess olfactory and nasal function, respectively, in the immediate response to a nasal allergen challenge in eight pollensensitive subjects. a significantly greater decrease in the cross-sectional nasal airway measure occurred following allergen challenge relative to a control challenge (70% vs. 22%). as in the case of other allergic rhinitis and rhinosinusitis studies, no association was found between the olfactory and acoustic rhinometric test measures. despite the small sample size and the use of an abbreviated upsit, a modest decrease in odor identification performance was seen following the allergen challenge (16%, p ϭ .08). in 1997, hinriksdottir et al. 59 evaluated odor detection thresholds in 20 patients with known allergic rhinitis to birch pollen before and after a topically applied birch pollen challenge during a nonsymptomatic period. following the provocation, olfactory function decreased. the change in threshold was related to the measured amount of nasal secretion but not to the patients' report of nasal obstruction or measures of nasal resistance. analogous findings were subsequently noted in a study by klimek and eggers 60 in which measures of odor identification, discrimination, and detection threshold were obtained in 17 patients with allergic rhinitis to grass pollen. in this work, testing was performed preseasonally and 3, 7, 14, and 21 days into the grass pollen season. after 2 weeks of pollen exposure, most subjects were hyposmic; by 3 weeks, all patients, without exception, had mild to severe hyposmia. in another study examining patients with grassrelated allergic rhinitis, moll et al. 61 examined the same olfactory measures as those used by klimek and eggers 60 in 28 patients with allergic rhinitis to grass pollen preseasonally and 3 weeks into the grass pollen season. in addition, 47 patients with allergic rhinitis to mites and 66 healthy control subjects were evaluated on a single test occasion. the mite-sensitive patients performed more poorly than the controls on all three olfactory tests. however, they outperformed the grass-sensitive patients (tested preseasonally) on the odor detection threshold test, but not on the other two measures. the intraseasonal test results of the grass-sensitive patients were decreased for all measures relative to the preseasonal tests. nevertheless, the grass-sensitive patients in the preseason period performed more poorly than the controls only on the odor detection threshold test. the intraseasonal grasssensitive patients outperformed the mite-sensitive patients on the identification and detection threshold tests, but underperformed the mite-sensitive patients on the odor discrimination test. this finding is paradoxical, however, because these three types of olfactory measures are typically positively correlated in a wide range of test situations. the authors of the study concluded, "therefore, the different kind of allergen exposure seems to result in a different pattern of allergic olfactory dysfunction." to our knowledge, only two studies have sought to determine empirically whether septoplasty improves olfactory function, 62,63 despite the widespread use of this procedure by otolaryngologists attempting to correct smell deficits. in the first of these studies, stevens and stevens 62 measured the olfactory thresholds of 100 patients before and after surgery. of the 100 patients examined, the primary surgical procedure of 63 patients was nasal septoplasty; of 24, septorhinoplasty; of 3, turbinate resection; and of 10, polypectomy. although the authors concluded that the surgical procedures, including septoplasty, improved olfactory function, the data for each type of operation was not provided separately, and their general conclusion is weakened by methodological considerations. in addition to not having a control group to examine the influences of repeated testing on the dependent measure, the questionable elsberg blast injection procedure 64 was used to determine olfactory sensitivity. in the second study to provide data on this topic, kimmelman 63 administered the upsit before and after septoplasty to 34 patients, 31 of whom had septal deformity and 3, nasal septal perforations. again, no control group for repeated testing effects was provided, although it is known that upsit scores on average change little on repeated testing. the mean (sem) upsit scores of these largely normally functioning patients were essentially equivalent before and after the operation (36.0 [0.4] versus 35.8 [0.4] ). in perhaps the first published study to specifically address the effects of rhinoplasty on olfactory function, champion 65 questioned 200 patients who had undergone rhinoplasty about their ability to smell. ten percent of patients reported temporary anosmia lasting from 6 to 18 months after the operation, and all apparently reported regaining normal smell function. because no empirical olfactory testing was performed, the accuracy of these observations is unknown. two years later, using ground coffee, oil of peppermint, and oil of clove as test stimuli, goldwyn and shore 66 performed both preoperative and postoperative olfactory tests on 64 patients who had undergone rhinoplasty alone, 22 who had undergone rhinoplasty in combination with submucous resection, and 11 who had undergone submucous resection alone. in addition, 57 control subjects were tested. the subjects were simply asked to identify the odors that were presented. apparently, no clear benefits of the operations on smell function were found, as the findings of this study were interpreted as supporting champion's conclusion that none of these types of operations have any long-term deleterious influences on smell function. however, this work is severely limited by not differentiating between patient types and by the use of a brief non-forced-choice identification test. in 1994, kimmelman 63 administered the upsit before and 2 to 4 weeks after surgery to 15 rhinoplasty patients. a small but statistically significant increase in performance was noted postoperatively (respective preoperative and postoperative means [sem] ϭ 33.9 [0.5] and 35.7 [0.6]). however, again no control group was tested to what degree repeated testing, per se, may have accounted for this improvement. in 1989 gross-isseroff et al. 67 obtained threshold and upsit measures in children with choanal atresia before and after surgical repair at relatively advanced ages (8 -31 y). the three patients who had bilateral atresia had permanent olfactory deficits, whereas the one patient who had unilateral atresia appeared to have normal function. these findings suggest that early sensory exposure may be needed for the normal development of olfactory function, although, as the authors pointed out, the small number of cases involved necessitates additional research on this point. the most common operative procedures impacting on the ability to smell are performed in patients with chronic rhinosinusitis and/or polyposis after more conservative treatments (e.g., allergen avoidance, nasal corticosteroids) have failed. most recent studies have administered corticosteroids both preoperatively and postoperatively, although some have used such medication only after surgery, confounding the interpretation of the findings. given the variation in olfactory measurement techniques used in such studies, this section is divided into 1) studies that have employed the standardized upsit (and in some cases additional tests); 2) studies that have employed a standardized combination of identification, discrimination, and detection threshold procedures 68 ; and 3) studies that have used other types of olfactory tests. studies using the university of pennsylvania smell identification test. in perhaps the first report of the influences of nasal surgery on smell function of the modern era, jafek et al. 69 in 1987 noted dramatic improvement in upsit and butanol detection threshold scores in one patient 4 months after an intranasal sphenoethmoidectomy (and intranasal antrostomies) and a continued 5-mg-daily regimen of prednisone (upsit scores increased from 10 to 31, the latter still indicative of mild microsmia; threshold values decreased by 4%). in another patient, even greater improvement was evidenced on the apparently sole post-treatment test performed a year after bilateral intranasal sphenoethmoidectomy and a regimen of triamcinolone acetonide (upsit scores increased from 9 to 38, the latter being normal; threshold values decreased by 45%). the authors concluded that these patients had received no benefit from either prior surgery or corticosteroid treatment alone, noting that "the results of this report raise an interesting question: why was the combined treatment with corticosteroids and surgery effective in long-term reversal of anosmia, whereas individual treatment with either modality had proved ineffective?" quantitative testing had not been performed after the earlier surgeries, which were not as extensive as those subsequently performed by jafek et al., and the duration and dosage of prior steroid treatment were not noted. in 1988, seiden and smith 70 examined olfactory function in five patients before and after endoscopic intranasal surgery within the osteomeatal complex. specifically, endoscopic intranasal ethmoidectomy and antrostomy were performed. on average, the degree of smell loss before surgery was indicative of total anosmia (mean upsit score ϭ 15.8, sd ϭ 8.73), although apparently some individuals had moderate hyposmia. four weeks to 8 weeks after surgery, all five patients exhibited marked improvement in their olfactory function, which fell, on average, within the microsmic range (mean upsit score ϭ 33.4, sd ϭ 4.02). in 1994, eichel 71 administered the upsit before and after intranasal surgery to 10 patients complaining of anosmia who had advanced obstructive bilateral nasal polyposis and pansinusitis. the surgery included bilateral nasal polypectomies, bilateral sphenoethmoidectomies, and bilateral nasal antral windows. all patients received testing 6 and 12 months postoperatively; four received an additional test at 18 months. postoperatively, all were treated with a topical corticosteroid nasal spray. the surgery was associated with improved upsit scores in 7 of the 10 patients (respective median preoperative and 6and 12-mo postoperative upsit scores: 10.5, 28, and 25.5), although average postoperative function was in the severe microsmic range. kimmelman 63 administered the upsit to nine patients undergoing ethmoidectomy and nine patients undergoing polypectomy before and 2 to 4 weeks after their surgeries. a small nonsignificant increase in upsit scores was noted postoperatively in the ethmoidectomy group, although, as in the study of eichel, average postoperative performance was in the moderate (nearly severe) microsmic range (respective mean [sem] scores ϭ 25.56 [3.47] and 27.89 [3.13] ; p ϭ .07). although a statistically significant improvement in upsit scores occurred in the polypectomy group (p ϭ . el nagger et al. 72 assessed olfactory function in 29 patients with bilateral nasal polyps before and after a polypectomy. following the operation, the patients received a 6-week course of beclomethasone nasal spray (beconase) to one nostril only, with the other acting as a control. although the upsit scores were higher for most individuals on the postoperative than on the preoperative tests, the changes in the observed upsit scores were modest and essentially of the same order of postoperative severity as seen in the study of kimmelman. one arrives at the following preoperative and postoperative mean upsit scores, respectively, from the data presented by these authors in the first two of their figures: 17.08 and 19.84 for the beconase nostrils and 16.44 and 21.42 for the control nostrils. from this perspective, neither the operative procedure nor the beclomethasone spray had much of an effect on overall smell function which, on average, fell within the anosmic or severe microsmic range. lund and scadding 73 evaluated the olfactory function of 50 hyposmic (upsit scores ͻ31) patients with chronic rhinosinusitis for 3 months before and after endoscopic nasal surgery. the postsurgical evaluations were performed at 1 year. the endoscopic procedure included uncinectomy, anterior ethmoidectomy, and perforation of the ground lamella of the middle turbinate in all cases, with posterior ethmoidectomy, sphenoidectomy, clearance of the frontal recess, and enlargement of the maxillary ostium in some cases. intranasal steroids were used up to the time of the surgery and for at least 3 months afterward. significant preoperative/postoperative improvement in upsit scores and in threshold values were observed in this group of patients (respective mean upsit scores ϭ 19.5 and 25.0), although, again, on average, the postoperative upsit scores were indicative of marked microsmia. in 1996, downey et al. 74 administered the upsit before and after endoscopic sinus surgery to 50 patients with subjective anosmia and symptoms of progressive sinusitis. after surgery, 52% of patients self-reported significant improvement in smell and had higher upsit scores. of the remaining patients, some had intermittent improvement, but most remained hyposmic or anosmic despite clinically well-healed ethmoid surgical beds. a relationship was observed between upsit scores and the severity of the disease, as defined using the kennedy staging system. thus, the mean upsit scores were 35, 31, 26, and 23 for stages i to iv of the disease, respectively. disease extending beyond the ethmoids, as determined by preoperative computed tomography, was typically associated with persistent anosmia. recently, friedman et al. 75 administered the upsit to 50 patients before and 5 weeks after endoscopic sinus surgery with middle turbinate medialization and preservation. iatrogenic synechia formation was produced by initially abrading the caudal end of the middle turbinate and the opposing septal mucosa using a microdebrider. no statistically significant differences in preoperative/postoperative upsit scores were found (respective mean upsit scores ϭ 35.18 and 35.57), leading the authors to conclude that middle turbinate medialization has no discernible adverse effect on olfaction. test. in 1988, leonard et al. 76 administered odor detection and threshold tests to 25 patients known to have olfactory dysfunction. the tests were administered before and after unilateral or bilateral transantral ethmoidectomy. smell function reportedly returned to normal in nine of the patients in one or both nasal chambers after surgery (36%), whereas four evidenced mild hyposmia (16%), five moderate to severe hyposmia (20%), and the remainder no improvement (28%). surgery on one side of the nose appeared in some cases to improve smell function on the contralateral side. five years later, hoseman et al. 77 administered a "qualitative and a semiquantitative olfactory function test" to each side of the nose of 111 patients before and after intranasal surgery for chronic polypoid ethmoiditis. eighty-seven of these patients received a complete sphenoethmoidectomy, and 24 a partial resection of the ethmoidal cell system. the olfactory test comprised a nonforced-choice odor identification component, in which the subject was required to report the quality of vanillin (or cinnamon oil), peppermint oil, menthol, and acetic acid with "corrective feedback, when needed" and an odor detection threshold component. in the threshold component, non-forced-choice detection thresholds to three stimuli (phenylethanol, benzylacetate, and formic acid) were established. before surgery, 65% of the patients were reportedly hyposmic or anosmic, whereas after surgery only 8% were similarly smell deficient. no association was noted postoperatively between the size of the middle turbinate and smell ability. however, the authors concluded that their results largely reflected improvement of airflow to the receptors and that "an inflammatory affection of the sense organ itself could not be responsible [for the loss]." a year after the study of hoseman et al., 77 delank and stoll 78 evaluated odor detection thresholds to 2-phenylethanol and dimethyldisulfide before and after nasal endoscopic sinus surgery in 78 patients with chronic sinusitis with nasal polyposis. employing an ascending threshold procedure, they noted preoperative hyposmia or odor discrimination problems in 40% of their sample, and anosmia in another 36%. however, only 22% of their patients complained spontaneously of smell dysfunction. following endoscopic surgery, 71% of the smell-deficient patients reportedly improved. postoperative thresholds for 2-phenylethanol and dimethyldisulfide worsened in 9% of the patients. postoperative olfactory discrimination deteriorated in 11%. preoperative and postoperative olfactory function was not predictable in individual cases when nasal polyposis was limited. in a similar subsequent study, these authors extended the patient group to 115 patients with chronic sinusitis. 79 preoperatively, only about half of the patients (58%) were aware of or complained of an olfactory deficit. however, the threshold testing found 83% to be either hyposmic (52%) or anosmic (31%). after surgery, 70% of the patients reportedly exhibited some improvement in olfactory function; normosmia, however, was relatively rare, being achieved in only 25% of the hyposmic patients and 5% of the anosmic patients. the olfactory function of 8% of the patients was worse after surgery than before surgery. therefore, the authors concluded that "the prevalence of olfactory dysfunction in chronic sinusitis is preoperatively higher, and the rate of [postoperative] improvement is lower, than generally assumed." the authors also noted that "resections of the middle turbinate may have a negative effect on olfaction, due to damage to the olfactory fila or alteration of the normal aerodynamic pattern within the olfactory cleft." as noted by earlier investigators, the degree of olfactory dysfunction was associated with the degree of intranasal polyposis. min et al. 80 determined, in 1995, butanol thresholds before and after functional endoscopic nasal surgery in 80 patients with chronic sinusitis. patients with prior surgery, asthma, aspirin intolerance, nasal allergy, or cystic fibrosis were screened from the study group. in accord with other studies (e.g., downey et al., 74 ) preoperative dysfunction was associated, in general, with the severity of sinusitis (determined in this case from computed tomography scans of the ostiomeatal-unit complex). the percentages of persons with normosmia, hyposmia and anosmia before surgery were reported as 22%, 45%, and 33%, respectively. after surgery, these percentages were 36%, 48%, and 16%. although a postoperative average reduction in threshold values was noted, the postoperative mean threshold value remained within the range considered indicative of hyposmia. no association was present between the degree of postoperative olfactory improvement and either the severity or duration of the sinusitis. more recently, klimek et al. 81 tested the odor identification, discrimination, and identification ability of 31 patients with nasal polyps 1 to 3 days before endonasal polyposis surgery and six postoperative times thereafter (approximately 1 wk and approximately 1, 2, 3, and 6 months). on average, the olfactory function, as measured by all three tests, fluctuated postoperatively, with best recovery (i.e., mild hyposmia) occurring approximately 3 months after surgery. six months after surgery moderate hyposmia was noted to about the same degree as was observed before the surgery. the authors concluded: "this study demonstrates that olfactory function is impaired in patients with nasal polyps. endonasal sinus surgery might improve olfactory function with best results within 3 months after surgery." studies using ratings of self-perceived olfactory function. in 1997, jankowski et al. 82 asked patients to remember what their sense of smell was like before and after either 1) a radical ethmoidectomy in which all the bony lamellae and mucosa within the labyrinth were removed, including a large antrostomy, sphenoidotomy, frontal sinusotomy, and middle turbinectomy (n ϭ 39) or 2) a less systematic ethmoidectomy adapted to the extent of the disease (n ϭ 37). they were also asked to remember what their sense of smell seemed like at 6-month intervals after the operation up to the time of filling out the questionnaire. the patients were required to mark their remembrances on a 10-point scale ranging from no functional improvement to complete recovery. in general, the ratings suggested similar improvement in olfactory function in both groups 6 months after surgery, and a maintenance of the same level 36 months after nasalization. some decrement was noted in reported smell function 24 months after the less extensive ethmoidectomy. however, this study suffers from many problems, not the least of which was the lack of an actual sensory measure, the requirement of a patient remembering function retrospectively over long periods, and demand characteristics attendant on being asked to report the effectiveness of an operative procedure to which they had subjected themselves. remarkable progress has been made in the last decade in understanding the function of the olfactory system. at the transduction level, the discovery of the gene family that controls the expression of olfactory receptors has been a monumental event. at the measurement level the development and proliferation of practical and reliable olfactory tests has spawned hundreds of studies that otherwise would not have been made, demonstrating olfactory dysfunction in a wide range of clinical disorders and leading to the discovery that olfactory loss is a very early clinical sign of several major neurodegenerative diseases. the comparatively few studies that have employed modern psychophysical tests to patients with rhinitis or rhinosinusitis have generally found an association between the degree of smell loss and the severity of nasal disease, although, except in cases of marked obstruction, no relationship is apparent between airway patency and olfactory dysfunction. this observation, along with recent histopathological studies of the olfactory mucosa in these disorders and the fact that even after nasal surgery and corticosteroid treatments, smell function rarely returns, on average, to normal levels, suggests that airflow access is not the only factor determining smell loss in such patients. although the weight of the evidence suggests that nasal steroid sprays, when appropriately administered, can improve olfactory function in some patients, not a single double-blind study employing placebos has evaluated the efficacy of such procedures in restoring smell function. it is hoped that the widespread availability of easy-to-use tests of olfactory function will lead to such controlled studies in the not-too-distant future. nervus terminalis lesions, ii: enhancement of lordosis induced by tactile stimulation in the hamster structure and function of the nervus terminalis nervus terminalis (cranial nerve zero) in the adult human vomeronasal organ in bats and primates: extremes of structural variability and its phylogenetic implications the human vomeronasal system: a review adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells olfactory bulb ensheathing cells enhance peripheral nerve regeneration a study of the test-retest reliability of ten olfactory tests tests of human olfactory function: principal components analysis suggests that most measure a common source of variance development of the university of pennsylvania smell identification test: a standardized microencapsulated test of olfactory function the smell identification test administration manual development of the 12-item cross-cultural smell identification test (cc-sit) a twin study of odor identification and olfactory sensitivity twin analysis of odor identification and perception gender and endocrine-related influences upon olfactory sensitivity smell identification ability: changes with age sex differences in odor identification ability: a cross-cultural analysis performance on a smell screening test (the modsit): a study of 510 predominantly illiterate chinese subjects age, gender, medical treatment, and medication effects on smell identification dose-related effects of cigarette smoking on olfactory function solvent-associated olfactory dysfunction: not a predictor of deficits in learning and memory solvent-associated decrements in olfactory function in paint manufacturing workers olfactory function in chemical workers exposed to acrylate and methacrylate vapors work related impairment of nasal function in swedish woodwork teachers long-term effects on the olfactory system of exposure to hydrogen sulphide olfactory loss in alcoholics: correlations with cortical and subcortical mri indices olfactory dysfunction in multiple sclerosis olfactory dysfunction in multiple sclerosis: relation to plaque load in inferior frontal and temporal lobes olfactory bulb volume is reduced in patients with schizophrenia cephalometric radiographs, nasal airway resistance, and the effect of adenoidectomy the effect of adenoidectomy on nasal resistance to airflow odor perception in children in relation to nasal obstruction die olfaktorische sensitivitat bei der rachenmandelhyperplasie olfactory function in acute rhinitis olfactory threshold and nasal mucosal changes in experimentally induced common cold effects of the nasal decongestant oxymetazoline on human olfactory and intranasal trigeminal function in acute rhinitis influence of prednisone on nasal polyposis with anosmia the loss of smell in nasal allergy efficacy of an aqueous and a powder formulation of nasal budesonide compared in patients with nasal polyps corticosteroids in olfactory dysfunction topical corticosteroids can alleviate olfactory dysfunction hyposmia in allergic rhinitis allergic rhinitis and olfactory loss the treatment of hyposmia with intranasal steroids topical corticosteroid treatment of anosmia associated with nasal and sinus disease sense of smell in allergic and nonallergic rhinitis a study of the relationship between the t&t olfactometer and the university of pennsylvania smell identification test in a japanese population fluctuating olfactory sensitivity and distorted odor perception in allergic rhinitis assessment of smell and taste in patients with allergic rhinitis a test of the validity of the elsberg method of olfactometry techniques in olfactometry: a critical review of the last one hundred years chronic sinusitis and anosmia: pathologic changes in the olfactory mucosa mineralocorticoid receptors in the mammalian olfactory mucosa expression of glucocorticoid receptor mrna and protein in the olfactory mucosa: physiologic and pathophysiologic implications new techniques for biopsy and culture of human olfactory epithelial neurons olfactory mucosal findings in patients with persistent anosmia after endoscopic sinus surgery effect of terfenadine and budesonide on nasal symptoms, olfaction, and nasal airway patency following allergen challenge acoustic rhinometry in the study of the acute nasal allergic response olfactory threshold after nasal allergen challenge olfactory dysfunction in allergic rhinitis is related to nasal eosinophilic inflammation comparison of olfactory function in patients with seasonal and perennial allergic rhinitis quantitative effects of nasal surgery on olfaction the risk to olfaction from nasal surgery the sense of smell, i: a new and simple method of quantitative olfactometry anosmia associated with corrective rhinoplasty the effect of submucous resection and rhinoplasty on the sense of smell olfactory function following late repair of choanal atresia clinical evaluation of olfaction steroid-dependent anosmia endoscopic intranasal surgery as an approach to restoring olfactory function improvement of olfaction following pansinus surgery effect of beconase nasal spray on olfactory function in post-nasal polypectomy patients: a prospective controlled trial objective assessment of endoscopic sinus surgery in the management of chronic rhinosinusitis: an update anosmia and chronic sinus disease effects of middle turbinate medialization on olfaction surgical correction of olfactory disorders olfaction after endoscopic endonasal ethmoidectomy die riechfunktion vor und nach endonasaler operation der chronisch-polyposen olfactory function after functional endoscopic sinus surgery for chronic sinusitis recovery of nasal physiology after functional endoscopic sinus surgery: olfaction and mucociliary transport olfactory function after microscopic endonasal surgery in patients with nasal polyps comparison of functional results after ethmoidectomy and nasalization for diffuse and severe nasal polyposis olfactory function in chronic alcoholics assessment of olfactory deficits in detoxified alcoholics olfactory dysfunction in amyotrophic lateral sclerosis olfactory disorder in motor neuron disease are there cognitive subtypes in adult attention deficit/hyperactivity disorder? presence of both odor identification and detection deficits in alzheimer's disease olfaction in neurodegenerative disease: a meta-analysis of olfactory functioning in alzheimer's and parkinson's diseases impaired olfaction as a marker for cognitive decline: interaction with apolipoprotein e epsilon 4 status olfactory dysfunction in anorexia and bulimia nervosa abnormally diminished sense of smell in women with oestrogen receptor positive breast cancer olfactory function in painters exposed to organic solvents smell and taste function in subjects with chronic obstructive pulmonary disease: effect of longterm oxygen via nasal cannulas olfactory deficits in cystic fibrosis: distribution and severity olfactory function in young adolescents with down's syndrome olfactory dysfunction in down's syndrome olfactory deficits and down's syndrome pre-and post-operative studies of olfactory function in patients with anterior temporal lobectomy olfactory functioning before and after temporal lobe resection for intractable seizures contribution of medial versus lateral temporal-lobe structures to human odour identification odor identification deficit of the parkinsonism-dementia complex of guam: equivalence to that of alzheimer's and idiopathic parkinson's disease olfactory dysfunction in guamanian als, parkinsonism and dementia long-term follow-up of olfactory loss secondary to head trauma and upper respiratory tract infection olfactory dysfunction in patients with head trauma olfactory identification deficits in hiv infection odor identification in huntington's disease patients and asymptomatic gene carriers olfactory function in huntington's disease patients and at-risk offspring kallmann syndrome: mr evaluation of olfactory system multimodal sensory discrimination deficits in korsakoff's psychosis olfactory disturbances as the initial or most prominent symptom of multiple sclerosis olfactory dysfunction in multiple sclerosis: relation to longitudinal changes in plaque numbers in central olfactory structures olfactory function in atypical parkinsonian syndromes olfactory function in patients with nasopharyngeal carcinoma following radiotherapy olfactory dysfunction in parkinsonism: a general deficit unrelated to neurologic signs, disease stage, or disease duration the olfactory and cognitive deficits of parkinson's disease: evidence for independence bilateral olfactory dysfunction in early stage treated and untreated idiopathic parkinson's disease olfactory testing as an aid in the diagnosis of parkinson's disease: development of optimal discrimination criteria olfactory function in parkinson's disease subtypes is parkinson's disease a primary olfactory disorder? [review olfactory dysfunction in type i pseudohypoparathyroidism: dissociation from gs alpha protein deficiency ventral frontal deficits in psychopathy: neuropsychological test findings olfactory function in restless legs syndrome olfactory identification deficits in schizophrenia: correlation with duration of illness olfactory identification and stroop interference converge in schizophrenia olfactory identification and psychosis olfactory deficits in schizophrenia regional metabolism in microsmic patients with schizophrenia olfactory deficits in schizophrenia are not a function of task complexity olfactory identification deficit in relation to schizotypy monorhinal odor identification and depression scores in patients with seasonal affective disorder odor identification ability among patients with sjogren's syndrome touch and taste in the mouth: presence and character of sapid solutions the fine structure of the olfactory mucosa in man key: cord-307123-h48uwj93 authors: kiechle, frederick l.; arcenas, rodney c.; rogers, linda c. title: establishing benchmarks and metrics for disruptive technologies, inappropriate and obsolete tests in the clinical laboratory date: 2014-01-01 journal: clin chim acta doi: 10.1016/j.cca.2013.05.024 sha: doc_id: 307123 cord_uid: h48uwj93 benchmarks and metrics related to laboratory test utilization are based on evidence-based medical literature that may suffer from a positive publication bias. guidelines are only as good as the data reviewed to create them. disruptive technologies require time for appropriate use to be established before utilization review will be meaningful. metrics include monitoring the use of obsolete tests and the inappropriate use of lab tests. test utilization by clients in a hospital outreach program can be used to monitor the impact of new clients on lab workload. a multi-disciplinary laboratory utilization committee is the most effective tool for modifying bad habits, and reviewing and approving new tests for the lab formulary or by sending them out to a reference lab. laboratory test overutilization is estimated to represent 2.9% to 56% of all laboratory tests internationally. efforts have been made to reduce the demand for or utilization of these over utilized tests [1] [2] [3] [4] [5] [6] . the most efficient outcomes have involved the formation of a laboratory utilization committee [2, 6] or a laboratory formulary committee [5] based on the hospital pharmacy and therapeutics committee's organizational structure. this committee evaluates the clinical value of laboratory tests using an evidence-based review of the appropriate medical literature. this same literature is reviewed by numerous professional specialty medical organizations as well as healthcare insurance carriers to determine what tests or procedures should be performed and reimbursed. the conclusions based on these reviews need to be updated on a regular basis. "the quality of guidelines is only as good as the published studies on which they are based" [7] . often relevant studies evaluating laboratory tests demonstrate negative findings and are not published [7, 8] . this phenomenon is referred to as positive publication bias or publication bias. tzoulaki et al. [8] demonstrated publication bias during a review of reports evaluating emerging cardiovascular biomarkers. therefore, misinterpretation is a potential impact of failing to publish studies with negative results during a review of evidence-based literature. readers beware. tests may be obsolete and should be retired from clinical use, while others may be inappropriately used for specific disease categories. the playing field is not level. there are at least six newer game-changing disruptive technologies being evaluated [9] [10] [11] which will result in modifications of clinical practice and laboratory testing modalities. these newer disruptive technologies may replace obsolete or inappropriate tests. lab utilization benchmarks and metrics are under continuous flux as a consequence. in the case of evolving newer technology, it is imperative to explore their impact early in their development to anticipate and monitor their impact on laboratory testing. the three authors have reviewed the current literature related to laboratory test utilization with an emphasis on where do the definitions of obsolete or inappropriate test utilization originate. we evaluated whole genome sequencing, next generation sequencing and proteomics as examples of high impact disruptive technologies that generate large quantities of data that need software to reduce to clinically useful results. practical examples of obsolete and inappropriate tests are reviewed as potential metrics to monitor improvement in test utilization. another useful metric is test utilization by clients in a hospital outreach program which can be used to monitor the impact of new clients on laboratory workload. finally, the result of published data from the work of laboratory utilization committees is summarized. benchmarks and metrics for laboratory utilization will be reviewed for three disruptive technologies as well as obsolete and inappropriately used tests. medical practice as well as pathology is in the midst of the rapid development of at least six major game-changing disruptive technologies. they include genetics, proteomics, digital pathology, informatics, therapeutic pathology and in vivo diagnostics [9] [10] [11] . all six of these disruptive technologies share similar issues like resolution of best applications for routine clinical use, paucity of evidence-based outcome literature for review, education of practitioners and physician users of the clinical information generated and software to convert big databases the method generates into clinical useful information [9] [10] [11] . the utilization of these techniques will increase as these barriers or obstacles to clinical use are overcome. an example of a disruptive technology is next generation sequencing or massively parallel sequencing [12] [13] [14] . this technique is currently not cleared by the u.s. food and drug administration [13] . it has been used to generate genome wide sequences and one of the authors (flk) has had his genome sequenced at the clia approved laboratory at illumina (san diego, ca). the results revealed 3.23 million variants compared with the reference method and 20,426 of these variants were in the exome or in the coding elements. the study interrogated 344 genes causally associated with 140 conditions as recommended by the american college of medical genetics. in that limited number of genes, 1,254 variants were detected and classified as clinically significant (0), carrier status (1), variants of unknown significance (255), likely benign variants (356) and benign variants (642). the definition of these variants calls and the failure of this technique to detect deletions, insertions, interspersed repeats and tandem repeats (repeats adjacent to each other like triplet repeats [15] ) may lead to inappropriate interpretation of the results and expensive follow up clinical and laboratory evaluation. for example, a clinically significant pathogenic variant reported in at least 3 unrelated cases with control data may be found in additional genome studies in other populations [16] to be a benign variant that is also found with a new variant which contains the mutation that leads to the most significant deleterious effect on gene function. the software application for variant significance assignment, like datagenno [17] , will need to be up-to-date with the latest genotype/phenotype associations to prevent false positive findings and inappropriate follow-up testing. in 2009 the highest rate of reported cancers was prostate, lung and bronchus and colon and rectum for men with female breast replacing prostate for women in the u.s. [18] . the annual incidence rate was 459 cases per 100,000 individuals. comprehensive sequencing of numerous human cancers have revealed driver genes, 2 to 8 such genes per tumor, which alter intracellular signal transduction pathways related to the cells future death or survival and/or genome maintenance [19, 20] . there are at least 10 fda approved cancer therapies based on the inhibition of these tumor-activated intracellular pathways [19] . for example, the braf kinase inhibitor, vemurafenib, has shown a response rate in 50% of patients with metastatic melanoma that have the braf valine to glutamic acid mutation at codon 600 (v600e) [21] . this v600e mutation is associated with aggressive clinical course in patients with thyroid papillary microcarcinoma [22] . in one study of a hybrid score composed of one molecular diagnostic (v600e) and 3 histopathologic parameters were used to predict this tumor's clinical course with a sensitivity of 96% and specificity of 80% [22] . the selection of the correct molecular diagnostic tests for specific tumors is aided by published guidelines. immunohistochemistry detection of estrogen and progesterone receptors in breast cancer from american society of clinical oncology and college of american pathologists [23] and selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors from the international association for study of lung cancer, association for molecular pathology and college of american pathologists [24] . whole genome sequencing of a tumor will provide access to all known and unknown variants related to the tumor's survival skills [25] . the development of software [26] which will convert the patient's raw genome sequence into a medically relevant assessment of therapeutic targets and drug metabolism based on the tumor's body site will be very useful. from this genome analysis, the clinician wants to know what anticancer drug or drugs will this patient respond to as well as the dose. maldi-tof (matrix assisted laser desorption ionization-time of flight) spectroscopy is a relatively new technology to the clinical microbiology laboratory. pathogen identification has always relied on visual and biochemical interrogation where the summary of results may point to a specific identification (genus and species) or sometimes to at least the genus level. visual and biochemical results can sometimes yield variable results meaning in some cases the id may change depending on the result. the use of maldi-tof allows the clinical microbiology laboratory to identify bacteria once an isolate has been cultured potentially without performing any biochemical testing [11, 27, 28] . the implications are quicker pathogen identifications to clinicians and the potential to affect antibiotic treatment before susceptibility results are available. the ability to obtain a quicker answer will disrupt the testing workflow and require a re-evaluation of that workflow to optimize the use of maldi-tof and antibiotic susceptibility testing [11, 27, 28] . benchmarks and subsequent metrics for monitoring laboratory test utilization have been developed by professional subspecialty medical organizations in the format of recommendations and guidelines [29] . examples include guidelines for hypothyroidism in adults from the american association of clinical endocrinologists and the american thyroid association [30] , definition of myocardial infarction from the american college of cardiology foundation and american heart association [31] , definition of diabetes mellitus from the american diabetes association [32] , pharmacogenetics as well as follow-up testing for metabolic diseases identified by expanded newborn screening using tandem mass spectrometry from the national academy of clinical biochemistry [33, 34] , and use of bone metabolic markers from the japan osteoporosis society [35] . thirty-five of these specialty societies have joined the choosing wisely project organized by the american board of internal medicine. societies are asked to provide five specific, evidence-based recommendations on when tests and procedures may be appropriate or inappropriate for patient care (www.choosingwisely.org). a review of the lists from 26 specialty societies revealed 135 recommendations. laboratory tests were referenced in 25 items or 18.5% of the total. only one organization, american society of clinical pathology, had a list of 5 laboratory test-related recommendations [36] . kale et al. [37] reviewed the national annual savings if outpatient visits to the primary care physicians did not include unnecessary or inappropriate laboratory tests including cbc ($32.7 million), urinalysis ($3.3 million) and basic metabolic panel ($10.1 million). those three procedures yield an annual cost savings of $46.1 million compared to the elimination of inappropriate pap tests at an annual savings of $47.8 million. these figures illustrate the magnitude of healthcare savings by implementing simple laboratory test ordering practices which reduce duplication and/or inappropriate testing. collaboration by subspecialty medical societies in disruptive technology development and improvements in routine clinical laboratory test utilization will be a fertile area for the development of benchmarks and metrics for future laboratory test utilization. the appropriateness of laboratory tests and the appropriate utilization of laboratory tests are always important for patient care, but require increased scrutiny in the era of containment of healthcare costs. objective criteria for the judging of appropriateness of tests and their utilization have not been universally developed or applied, so it is not always easy to define these terms [38] . insurance companies are recognizing the medical and the financial burden of unnecessary testing and are taking action. many companies have posted information on their websites defining obsolete and unreliable lab tests which are readily accessible on the internet, including aetna [39] , united healthcare [40] and amerihealth [41] . one criterion for judging the appropriateness of a test is to determine if it is obsolete. the definition of "obsolete" as noted in the merriam-webster on-line dictionary at http://www.merriam-webster. com/dictionary/ is "no longer in use or no longer useful". synonyms include: antiquated, archaic, dated, démodé, demoded, fossilized, and kaput. over time, with advances in medical technology, laboratory tests become outdated. although it is difficult to remove a test from a laboratory's formulary, there are good reasons to do so. reasons include the availability of a more sensitive, specific, or accurate test or new guidelines recommend the elimination of a test with the replacement of another. there are tests in clinical pathology that must be considered for obsolescence. these include t3 uptake and lactic acid dehydrogenase (ldh) isoenzymes in the clinical chemistry lab and bleeding time in hematology. obsolete tests in the microbiology laboratory include bacterial antigen detection tests, group b streptococcus antigen (gbs) testing and hiv-1 western blot. t3 uptake and free thyroxine index (fti) are still ordered by physicians, despite the fact that alternative tests have been available for many years. t3 uptake is an old test designed with a purpose of indirectly measuring free thyroxine (t4). it was developed before the availability of direct assays able to accurately measure free t4 levels [42] . standardization of free t4 assays has been reported using the time-consuming equilibrium dialysis in combination with isotope dilution-liquid chromatography/tandem mass spectrometry [43] . t3 uptake is an assessment of unsaturated (unbound to thyroxine) thyroid binding proteins in serum and is used with total t4 to calculate fti. the fti is obtained by multiplying the (total t4) times (t3 update). there is no longer a need to estimate free t4 when there are assays for the direct measurement of free t4 in every laboratory. supporting evidence for the obsolescence of t3 update and fti has been available for decades [44] . current guidelines for the diagnosis and management of hypothyroidism [30] , hyperthyroidism [45] , and thyroid disease in pregnancy [46] , no longer include the assessment of t3 update or fti. the analysis of lactic acid dehydrogenase (ldh) isoenzymes by electrophoresis has been utilized as an aid in the diagnosis of myocardial infarction. with the development of a more specific test for myocardial damage, troponin, there is little use for this insensitive and time-consuming electrophoretic assay. current guidelines clearly establish that the preferred marker for cardiac injury is troponin [31] . bleeding time is a crude test of hemostasis (the arrest or stoppage of bleeding). it is an indication of how well platelets interact with blood vessel walls to form blood clots. indirectly assesses platelet function. it is performed by making a small incision on the skin and measuring, in seconds, the time taken for bleeding to stop. the test was designed to assess platelet function or exclude von willebrand disease. this test is labor intensive, invasive, poorly reproducible, and insensitive [36] . historically it was performed because screening tests with a higher sensitivity for platelet dysfunction and von willebrand disease (vwd) were unavailable. bleeding time has been replaced by instrumentation that can assess platelet function in whole blood by aggregation studies [47, 48] . available instrumentation includes the pfa-10 (platelet function analyzer, siemens usa), the verifynow (accumetrics), the plateletworks (helena), the impact (diamed) and the thromboelastograph (teg) (haemonetics). initial tests for a bleeding disorder rule out more common causes of bleeding. these tests include complete blood and platelet counts, ptt, pt, and possibly fibrinogen level or thrombin time. additional tests for von willebrand disease (vwf: antigen, ristocetin cofactor activity. factor viii clotting activity) can confirm the disease [48] . bacterial antigen detection tests should be considered obsolete. they have historically been used as an adjunct to other laboratory tests for the diagnosis of bacterial meningitis. the test's purported advantages were the rapid detection of h. influenza, n. meningitides, s. pneumoniae, and s. agalactiae. overall, the sensitivity is essentially the same as that of a gram-stained smear of a cyto-centrifuged csf specimen [49, 50] . with the advent of vaccines to h. influenza type b and n. meningitides (a, c, y, and w-135) the antigen testing is even less useful. the literature confirms that the use of direct antigen testing from the csf is neither sensitive nor specific [49, 50] . more importantly, the gram stain and cultures still need to be performed regardless of the initial antigen test result. based on the data reviewed, our laboratory has discontinued this testing in-house. this is an example of testing that was removed from the market based on recommendations from the centers for disease control (cdc) stating that the rapid antigen detection tests for gbs are not sensitive enough to replace the culture based prenatal screening or to use in place of the risk-based approach when culture results are unknown at the time of labor [51] . because of the poor performance of the rapid antigen testing, cdc has recommended intrapartum chemoprophylaxis antibiotics be administered to women who have certain risk factors [51] . our laboratory looked at internal data for our patient population and found the sensitivity of the rapid antigen test that was being used was 28%. forty-one patients were missed on the rapid antigen test were detected only by culture. data from the literature show an average sensitivity of 25.7% among various labs surveyed [52] . this is an example where cdc recommendations and assessment of testing performance within your laboratory supports moving a test into obsolescence. the hiv-1 western blot (wb) has been a constant as one of the confirmatory tests for hiv antibody testing. however, the wb is moving its way into obsolescence as newer generation antigen/antibody and molecular assays become part of the new hiv testing algorithms. hiv-1 wbs have always had issues with indeterminate results due to a myriad of factors (false positives and/or lack of specificity, kit design, etc.), which required either re-testing at a later time and/or molecular testing for hiv-1 nucleic acid [53] . the cdc/aphl, who, and france each have different interpretation criteria for defining a positive confirmatory result which indicates a lack of standardization for defining a patient as positive for hiv-1 [54] . the immune response to hiv-2 is well known to produce antibodies that cross react on the hiv-1 wb which could lead to a false positive hiv-1 result [54] . the lack of improvements or advancements of the wb compared to other antibody based assays has been nicely shown by masciotra et al. [55] . they demonstrate that the rapid hiv tests actually detect hiv-1 antibodies several days earlier before the wb becomes positive [55] . the introduction of 4th generation antigen/antibody testing has allowed clinicians to detect reactive patients earlier in their disease course compared to 3rd generation testing effectively narrowing the window of serological detection by approximately 4-8 days [56] . because of these new developments, clsi and aphl have recommended new algorithms that incorporate antigen/antibody combination tests, rapid tests, and molecular testing [57, 58] . the wb should be a test that will be obsolete as laboratories adopt the newer algorithms for hiv testing. in addition to obsolete tests, tests may be inappropriately used. inappropriate utilization includes the failure to follow current practice guidelines for the diagnosis and management of disease, thus ordering the incorrect test, panel of tests, or algorithm of tests; ordering tests too frequently or lack of medical rationale for the test. the problem and the resolution need to be reshaped as a way to "improve patient outcomes and lower costs" [59] . the acknowledgement of the laboratory test utilization problem has been known and published for more than 2 decades [1-6,60] studies have been done to estimate and document the percentage of unnecessary tests [37, 61] . a typical scenario for inappropriate test ordering occurs with thyroid function testing in the diagnosis and management of hypothyroidism. the guidelines are clear on the appropriate tests [30] . thyroid stimulating hormone (tsh) is regarded as the best screening test, followed by free thyroxine (free t4) if the tsh is abnormal. additional tests are often ordered including total t4. there is no need for a total t4 measurement if a free t4 is provided. furthermore, adding a total t4 level may confuse the diagnosis if changes in binding proteins via disease or drug therapy result in a total t4 that is inconsistent with other test results. tests may be ordered inappropriately or at other times, the wrong test is ordered. vitamin d testing is known to result in both inappropriate and erroneous ordering [62] . the correct test for the routine assessment of vitamin d status or deficiency is 25-hydroxy vitamin d. the test 1,25-dihydroxy vitamin d is often mistakenly ordered. the dihydroxy form of vitamin d is occasionally ordered in patients with kidney disease (decreased levels are one of the earliest changes to occur in persons with early kidney failure). however, most of the orders for 1,25-dihydroxy vitamin d are simply erroneous. tests for both vitamin d2 and d3 are unnecessary for the assessment of vitamin d status. there is no need to differentiate between the d2 and d3 forms other than in the research setting. viral cultures have traditionally been the gold standard for virological detection in the clinical microbiology laboratory. direct viral detection direct from patient samples have also been utilized with monoclonal antibodies to detect viral antigen(s) with the intent of obtaining a result in a timelier manner than viral cultures. molecular technologies have now become established in the clinical microbiology laboratory. with the increased sensitivity and specificity [63] and almost always a shorter turn-around-time, it is reasonable to ask "why do viral cultures?" [63, 64] . one example of molecular testing that helps to answer this question are panels developed for respiratory viruses. there are several commercial companies that offer their version of an rvp (respiratory virus panel). our laboratory offers a rvp assay that detects 20 viral targets. not only are results obtained sooner than traditional virology testing, but our molecular rvp offers greater sensitivity and specificity than our prior direct fluorescence assay (dfa) that was sent out to a reference laboratory. because of the increased sensitivity, the rvps have allowed us to document co-viral infections, which have not been fully appreciated before with dfa or culture based testing [64] . there have been reports of increased severity of disease, as well as reports of decreased severity of disease in the setting of co-viral infections [65] . much more research must be done to understand the potential interactions of different respiratory viruses in the setting of a respiratory infection. our laboratory publishes a "virogram" during the respiratory virus season and related viral coinfections (figs. 1 and 2 and table 1 ). the "virogram" charts the respiratory virus prevalence among the patient populations tested to give an idea what is circulating in the community. another example of molecular testing replacing viral cultures is the detection of enterovirus (ev) from csf. ev pcr has been shown to have greater sensitivity over culture [66] . a study (unpublished) performed within our institution looked at the utility of an in-house ev pcr and its affect on length of stay (los) and cost on 20 ev pcr negative and 20 ev pcr positive patients. those with an ev pcr negative result had an average los of 2.1 days greater than those that were ev pcr positive and had an estimated $187,992 of additional cost related to in-patient care. viral cultures still have importance in growing the virus for the purposes of subtyping, identifying new strains, or antiviral testing. however, these should remain in specialty or research labs and not be routine for clinical diagnostic testing. pcr testing for the causative agent of lyme disease may initially make sense, but the life cycle of b. burgdorferi is such that pcr detects borrelia dna in the blood in less than half of patients that are in the early acute stage of disease when the characteristic erythema migrans is present [67] . therefore, pcr is not recommended as a first line test for making the initial diagnosis of lyme disease. a review article showed that the median percent sensitivities of pcr testing from blood, skin biopsy, csf, and synovial fluid are 10-48%, 64-76%, 23-73%, and 66-83%, respectively [67] . the current recommendation for diagnostic testing involves a two-tiered algorithmic approach involving antibody testing [68] . there may be clinical utility for pcr, but only if the serology testing is negative or inconclusive and clinical history and symptoms strongly suggest lyme disease. hospitals have introduced outreach programs that market laboratory services to physician offices, nursing homes, and other hospitals [69, 70] to increase test volumes and reduce unit costs per test. this effort generates increased laboratory test utilization which should be monitored by average number of specific tests ordered per subspeciality physician per month ( [69] , table 40 .10). the data is collected from patient requisitions that are processed each day in the accessioning area of the outreach specimen receiving area. the sales force will introduce outreach administration to the projected number of new client accounts to be opened each month. the impact of the laboratory can be estimated by multiplying the volume of a specific test per physician for an office practice of multiple physicians. these volumes will estimate the utilization of tests per laboratory section and predict the future need for additional analyzers and/or personnel to perform the laboratory test procedures as the outreach client number increase. utilization data varies by medical subspeciality. for example, urology had 94 requisitions/physician, 1.4 procedures/requisition and 132 procedures/physician with the psa being the highest test volume. compare that to the nursing homes with 189 requisitions/physician, 3.6 procedures/requisition and 688 procedures/physician with electrolytes being the highest test volume [69] . this data is used primarily for planning the best strategies to absorb the increased workload new clients will bring to the outreach business. it can also be used to monitor the use of obsolete and inappropriate lab tests to develop educational efforts to improve test utilization practices by subspeciality among outreach clients. an excellent review of laboratory test utilization, understanding the many factors involved, and steps to implement changes are provided in a recent publication [4] . the author provides insight and guidelines to assist the laboratory in initiating improvements in laboratory utilization. ultimate goals include: developing and adopt more-effective testing algorithms, reducing testing costs, use new technologies cost-effectively, and shorten the time to diagnosis. interventions for hospitals and laboratories focus on changing physicians' test ordering behavior and include: 1. eliminating obsolete tests and modifying requisition forms. the laboratory can alter test-requisition forms to steer clinicians in the right direction. one such option is an "out of sight, out of mind" approach in which certain tests simply don't appear on the menu. 2. assisting in the education to promote appropriate lab testing can be part of a hospitals continuing medical education (cme) program for clinicians through grand rounds, newsletters, and cme lectures. 3. reinforcing positive changes by auditing clinicians' use of new protocols and offering feedback. 4. a two tier review process for molecular send out tests is a useful tool for pathologists to learn and advice on the wisdom of molecular assays [71] . finally, all hospitals should implement a laboratory utilization or formulary committee to help in overseeing testing and promoting good testing practices, similar to pharmacy and therapeutics committees. this approach has been met with great success [2, 5, 6] . the use of molecular methods for respiratory virus testing has allowed us to detect patients with more than 1 virus present. the presence of multiple viruses within a patient sample is currently underappreciated and the effect on the overall disease presentation is currently unknown. managing utilization of new diagnostic tests clinical laboratory tests not performed in a central hospital laboratory managing the demand for laboratory testing: options and opportunities demand management and test request rationalization laboratory test utilization program. structure and impact in a large academic medical center changing physician behavior in ordering diagnostic tests biomarkers in cardiovascular medicine. the shame of publication bias bias in association of emerging biomarkers with cardiovascular disease pathology resident and fellow education in a time of disruptive technologies a national agenda for the future of pathology in personalized medicine current status of matrix-assisted laser desorption ionisation-time of light mass spectrometry in the clinical microbiology laboratory national human research institute, charting a course for genomic medicine from base pairs to bedside assuring the quality of next-generation sequencing in clinical laboratory practice developing genome and exome sequencing for candidate gene identification in inherited disorders. an integrated technical and bioinformatics approach mechanisms of trinucleotide repeat instability during human development the 1000 genomes project consortium datagenno: building a new tool to bridge molecular and clinical genetics the role of high-throughput technologies in clinical cancer genomics cancer genome landscapes improved survival with vemurafenib in melanoma with braf v600e mutation a combined molecular-pathologic score improves risk stratification of thyroid papillary microcarcinoma american society of clinical oncology/college of american pathologists guideline recommendations for immunochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version) molecular testing guideline for selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors personalized medicine in a phase 1 clinical trials program: the md anderson cancer center initiative snver gui: a desktop tool for variant analysis of next-generation sequencing data mass spectrometry and tandem mass spectrometry characterization of protein patterns, protein markers and whole proteomes for pathogenic bacteria maldi-tof-ms-based species identification and typing approaches in medical mycology implementing clinical practice guidelines american association of clinical endocrinologists and american thyroid association taskforce on hypothyroidism in adults, clinical practice guidelines for hypothyroidism in adults accf 2012 expert consensus document on practical considerations in the interpretation of troponin elevations. a report of the american college of cardiology foundation task force on clinical expert consensus documents diagnosis and classification of diabetes mellitus laboratory analysis and application of pharmacogenetics to clinical practice follow up testing for metabolic diseases identified by expanded newborn screening using tandem mass spectrometry guidelines for the use of bone metabolic markers in the diagnosis and treatment of osteoporosis when less is more for patients in laboratory testing top 5" lists top $5 billion do we know what inappropriate laboratory utilization is? a systematic review of laboratory clinical audit aetna clinical policy bulletin: obsolete and unreliable tests and procedures united healthcare's policy number 300.1. obsolete or unreliable tests amerihealth's medical policy bulletin #00.01.24d. obsolete or unreliable diagnostic tests and medical services comparison of three free t4 (ft$) and free t3 (ft#) immunoassays in healthy subjects and patients with thyroid diseases and severe non-thryoid illnesses use of frozen sera for ft4 standardization: investigation by equilibrium dialysis combined with isotope dilution-mass spectrometry and immunoassay a reappraisal of the free thyroxine index hyperthyroidism and other causes of thyrotoxicosis: management guidelines of the american thyroid association and guidelines of the american thyroid association for the diagnosis and management of thyroid disease during pregnancy and postpartum standardization of platelet function testing by aggregometry through new clsi guideline diagnosis and management of von willebrand disease: guidelines for primary care comparison of bacterial antigen test and gram stain for detecting classic meningitis bacteria in cerebrospinal fluid rapid bacterial antigen detection is not clinically useful prevention of perinatal group b streptococcal disease infections in international pregnancy study: performance of the optical immunoassay test for detection of group b streptococcus frequency, causes, and new challenges of indeterminate results in western blot confirmatory testing for antibodies to human immunodeficiency virus comparison of alternative interpretive criteria for the hiv-1 and hiv-2 infections evaluation of an alternative hiv diagnostic algorithm using specimens from seroconversion panels and persons with established hiv infections evaluation of the performance of the abbott architect hiv ag/ab combo assay criteria for laboratory testing and diagnosis of human immunodeficiency virus infection; approved guideline, clsi document m53-a silver spring, maryland: the association of public health laboratories order patrol -how to rein in test requests professional review of laboratory utilization ready, fire! aim! an enquiry into laboratory test ordering vitamin d testing-what's the right answer? role of cell culture for virus detection in the age of technology detection of respiratory viruses by molecular methods an analytical comparison of four commercial respiratory virus panels. clinical virology symposium poster diagnosis of enteroviral meningitis by use of polymerase chain reaction of cerebrospinal fluid, stool and serum specimens diagnosis of lyme borreliosis notice to readers: recommendations for test performance and interpretation from the second national conference on serological diagnosis of lyme disease outreach implementation requirements: a case study hospital laboratory outreach: benefits and planning clinical requests for molecular tests. the 3-step evidence check clinical labs of hillview 3375 hillview avenue phd university of alberta hospital 481.24 walter mackenzie centre 8440 112 th street edmonton ab t6g 2b7 canada key: cord-310714-kqzlwka0 authors: braz, lucia maria almeida; tahmasebi, roozbeh; hefford, philip michael; lindoso, josé angelo lauletta title: visceral leishmaniasis diagnosis: a rapid test is a must at the hospital bedside date: 2020-06-16 journal: clinics (sao paulo) doi: 10.6061/clinics/2020/e2036 sha: doc_id: 310714 cord_uid: kqzlwka0 nan at the time of the widespread availability of rapid diagnostic tests for sars-cov-2-the causative virus of the covid-19 pandemic-from drugstores throughout brazil, there is a distinct lack of use of rapid diagnostic tests for visceral leishmaniasis (vl) at the bedsides of hospitalized patients. these tests are mainly distributed by the ministério da saúde do brazil-ms (ministry of health) only to the laboratórios centrais de saúde publica -lacens (central public health laboratories) and are predominantly provided to public hospitals. vl is the most severe form of leishmaniasis and it causes high morbidity and mortality in developing countries (1, 2) . leishmania infantum chagasi is responsible for vl in the new world, with typical clinical signs and symptoms of splenomegaly, hepatomegaly, and fever (3) . vl can be lifethreatening, and because 90% cases of vl occur in brazil, reliable and rapid diagnosis of vl is required (4) . as stated by the ms, vl case confirmation is based on clinical suspicion and positive laboratory diagnosis via either parasitological tests (pts), which are dependent on invasive procedures such as bone marrow aspiration or biopsy, or serological tests such as indirect immunofluorescence (ifi) or immunochromatographic tests (its) using rk39 recombinant antigens (5) . the serological tests ifi and it-rk39 have the advantage of being minimally invasive and they can be performed in large numbers (6) . however, ifi requires a fluorescence microscope and is time consuming. the procedure of it-rk39 takes only 10-15 minutes and requires only 10-20 ml of the peripheral blood. it is a rapid and low-cost bedside test. the rk39 dipstick used for its is the product of a gene cloned from the leishmania genus containing a 39-amino acid repeat conserved among viscerotropic leishmania species (7). the main brands of it-rk39 that were previously provided by the brazilian public health system consisted of kalazar detectt (inbios international, seattle, wa, usa), it leish s (bio-rad laboratories inc., france), and onsitet leishmania igg/igm combo test (ctk biotech, usa), which have now been replaced with the lsh ab eco test (eco diagnóstica, nova lima, mg, brasil). kalazar detectt was the first rapid test for vl diagnosis that was adopted by the brazilian public health system in 2009. it has a sensitivity and specificity of 88.1% and 90.6%, respectively. in 2015, it leish s replaced the kalazar detectt and showed an improved sensitivity and specificity of 93% and 97%, respectively (8) . however, these it-rk39 tests would usually present a lower accuracy when tested in patients coinfected with hiv (9,10). in 2017, the onsitet leishmania igg/igm combo test replaced the it leish s (8). today, ms recommends using a new brand, the lsh ab eco test, a qualitative immunoassay for the detection of antibodies (rk39) against vl in human serum (11) . the specificity for this test is equal to 100% (95% ci 0.93-1), indicating that it has high specificity for the rk39 protein. the sensitivity presented by the lsh ab eco is 92% (95% ci 0.82-0.97) (11) . lsh ab eco test was declared by the agência nacional de vigilância sanitária-anvisa (national health surveillance agency) as a criterion for the laboratory confirmation of suspected cases of the disease. therefore, suspicious patients, including those presenting with clinical signs compatible with disease and those coming from a region with known occurrence of transmission, alongside a positive rapid test, can be considered confirmed cases of vl based on clinical laboratory criteria. the lsh ab eco test has technical specifications and execution methodology similar to those of the brands used before. according to the manufacturer, lsh ab eco, a lateral flow chromatographic immunoassay used to detect class g immunoglobulin for leishmania donovani, uses recombinant antigens in the test line and chicken anti-protein a in the control line. it is easy to use and interpret. in accordance with the manufacturer's instructions and technical orientation from sdp/iom/funed n o 001/2019 (12) , the procedure of the test is as follows: add 20 ml of serum/plasma or 1 drop of blood (10 ml) to the test strip pad, below the arrows. if serum, plasma, or blood is applied to the test strip horizontally on a flat surface, take the strip by the green label and place it vertically, with the arrow pointing downwards, in a test tube or microwell containing 2-3 drops (150 ml) of the diluent buffer. if serum, plasma, or blood is applied to the test strip vertically, add 2-3 drops (150 ml) of the diluent doi: 10.6061/clinics/2020/e2036 copyright & 2020 clinics -this is an open access article distributed under the terms of the creative commons license (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. no potential conflict of interest was reported. received for publication on may 27, 2020. accepted for publication on may 29, 2020 buffer to the base of the microwell or test tube and read the test result after 10 minutes. it is important to highlight that this it for rk39 is produced by a brazilian biotechnology industry located in the state of minas gerais, brazil, as the previously used brands were produced by industries situated outside of brazil. this is an important achievement for the brazilian health system with regard to vl diagnosis. in summary, the test is suitable for use at the bedside, requires a minimal amount (10 ml) of peripheral blood, with no need of special equipment, and is simple to perform and read, with the results being available in 10 minutes. however, this simple dipstick test for rk39, distributed by the ministério da saúde do brazil to public laboratories, is not available in some public hospitals, including hospital das clinicas da faculdade de medicina da universidade de são paulo (hcfmusp). rapid test-rk39 is not even offered by the majority of private laboratories and private hospitals for vl diagnosis. even if it is provided, the turnaround time between sending the sample and receiving results is typically a minimum of 24 hours; thus, it can hardly be deemed as a 'rapid test' with any conviction. it is time to change the narrative and alter the distributive flowchart of this test. it is necessary to use the rk39 it at the bedside of suspected vl patients across hospitals to the greatest effect. why not employ the technical skills of a team who usually attend patient needs, such as nurses and nursing technicians, thereby ensuring that the rk39 it truly does indeed become a rapid diagnostic bedside test? vl can be lethal and patients simply cannot afford to wait for diagnoses/treatments. braz lma and lindoso jal designed the study, drafted and reviewed the manuscript. tahmasebi r and hefford pm reviewed the manuscript, also for english language. all of the authors have read and approved the content of the manuscript. visceral leishmaniasis in brazil: revisiting paradigms of epidemiology and control diagnosis of leishmaniasis unusual clinical manifestations of leishmania (l.) infantum chagasi in an hiv-coinfected patient and the relevance of its1-pcr-rflp: a case report a pcr and rflp-based molecular diagnostic algorithm for visceral leishmaniasis guia de vigilância em saúde. leishmaniose visceral diagnosing visceral leishmaniasis with the recombinant k39 strip test: experience from the sudan diagnostic and prognostic value of k39 recombinant antigen in indian leishmaniasis evaluation of a new brand of immunochromatographic test for visceral leishmaniasis in brazil made available from 2018 field evaluation of rk39 tests and direct agglutination test for diagnosis of visceral leishmaniasis in a population with high prevalence of human immunodeficiency vírus in ethiopia comparison of parasitological, serological, and molecular tests for visceral leishmaniasis in hiv-infected patients: a cross-sectional delayed-type study controle de qualidade de kits para diagnóstico da leishmaniose visceral humana. avaliac¸ão do teste rápido imunocromatográfico lsh ab eco da eco diagnóstica para o imunodiagnóstico da leishmaniose visceral humana (lvh). relatório técnico. belo horizonte key: cord-225183-6rusimb5 authors: boukai, ben; wang, jiayue title: bayesian modeling of covid-19 positivity rate -the indiana experience date: 2020-07-09 journal: nan doi: nan sha: doc_id: 225183 cord_uid: 6rusimb5 in this short technical report we model, within the bayesian framework, the rate of positive tests reported by the the state of indiana, accounting also for the substantial variability (and overdispeartion) in the daily count of the tests performed. the approach we take, results with a simple procedure for prediction, a posteriori, of this rate of 'positivity' and allows for an easy and a straightforward adaptation by any agency tracking daily results of covid-19 tests. the numerical results provided herein were obtained via an updatable r markdown document. the indiana state department of health, (isdh), as any other similar entity across the nation and world-wide, is closely monitoring nowadays a pandemic of the 2019 novel coronavirus, aka: covid-19. according to the world health organizing, (who), this highly contagious respiratory virus was first identified and reported in the city of wuhan in china in early january 2020. since then, this virus continues to spread and infect people around the world, including the united states. on march 11, 2020, the who published an assessment that covid-19 can be characterized as a pandemic. as of the date of this report (july 7, 2020), the john hopkins university's coronavirus resource center, reported over 11,500,000 infected people and 538,000 deaths due to the coronavirus, globally, of which over 2,938,000 infections and 130,000 deaths are in the usa alone. the state of indiana was not spared of the contagious impact of the virus. on march 6, isdh confirmed the first case of covid-19 in a hoosier with recent travel. on march 16, the isdh reported the first death in indiana due to . it subsequently worked with federal and local partners, including the centers for disease control and prevention, (cdc), to respond to this pandemic and the grave public health situation. among other responses, the isdh created a dashboard and a data depository for tracking and reporting the daily number of covid-19 deaths in the state as well as the daily number of tests performed and the daily number of positive cases. while there is substantial (and urgent) effort being made, worldwide, for modeling of the the death rate (or the infection fatality rate, ifr) of covid-19, (see for example basu (2020) ), there has been very little attention given to modeling the rate of reported positive tests (often referred to rate of 'positivity'). in this report we model, within the bayesian framework, the rate of positive tests reported by the the state of indiana, accounting also for the substantial variability of the daily number test performed. the bayesian approach has been used successfully in other covid-19 related studies, e.g.: dana, at. el. (2020), or bayes, at. el. (2020). however, to the best of our knowledge, none of the available studies (to date) have direct relevancy to the modeling of the rate of positivity. the approach we take, provides a method for a valid prediction of this rate and allows for an easy and a straightforward adaptation by any agency tracking daily results of covid-19 tests. the indiana covid-19 data are available for retrieval through the isdh data hub [10] as are reported for the state in the file covid report date.xlsx. it includes the daily records of the number of covid-19 deaths cases, the total (daily) number of testings performed and the count of the positive cases, (see appendix b for additional information). for the purpose of this report, we focus attention only on two of the quantities reported; the daily reported number of test performed, covid test and the daily reported number of positive tests, covid count as were reported for the (successive) 127 daily records to date. their ratio, the daily percentage of positive tests (ppt), is used for tracking and monitoring the pandemic's progression by the state and localities. the ppt is also an important indicator of the scope and extent of the state's testing enterprise; its high value suggests that testing is conducted primarily of the sickest patients and less so of the mild or the asymptomatic cases. a lower ppt may suggest that testing has been extended to cover patients with milder or no symptoms at all. according to the cdc guidelines [7] , among other stipulations, a ppt ≤ 15% serves as a threshold for entering phase ii of the reopening plans for the state, whereas achieving a ppt ≤ 10% serves a threshold of entering phase iii of the reopening plans. at present, the 7-day average of this rate (of positive tests) for indiana is 7.61% and cumulatively, the indiana ppt stands at 9.18% (i.e. the total number of positive tests relative to the total number of tests performed to date, see also the isdh dashboard, which enabled the state to enter phase iii of the reopening plans. thus, the importance of the appropriate modeling and tracking this percentage of reported positive tests cannot be overstated, as it has tremendous economic implications for the state and its people. in particular, constructing a valid predictive model, which predicts reasonably well the 'next-day' ppt, would provide the state with an early sign of a looming surge in positive cases, and would allow it to implement corrective measures and policies. since the available records for the latest few days is still updating, we included records up to the last 3 days in the series. thus, the data series includes m = 109 data points out of the available 127 records. also marked in the figure are two horizontal lines indicating the 10% and 5% thresholds. in appendix a below, we provide basic descriptive statistics concerning the daily reported number of tests and the daily reported number of positive tests as well as of their ratio, the ppt. in figure 2 above, we present the cumulative ppt rate for indiana over these days. we also super-imposed on the daily chart the 0.95 (or 95%) posterior predictive interval of [0.07981, 0.1001] for the current indiana (cumulative) ppt rate, as resulted from the bayesian predictive modeling we developed and present in this report (see illustrations 1 and 2 below). as can be seen, the calculated posterior prediction interval contains the cdc's 10% threshold, which could serve as an early warning flag to the state and may prompt it to initiate some mitigating measures (such as mandating masks in public) in an effort to maintain the rate of positivity below it. having retrieved the data as described above, we labeled them as y i ≡covid count and k i ≡covid tests for i = 1, . . . , m where m = 109 is the total number of observations included in the analysis. thus, we denote the given data as ordinarily, when modeling the count of positive test results y i recorded out of the k i tests performed (assuming independence, risk homogeneity of the tested population and no lagging information), the binomial model would be appropriate. so that conditional on the number of tests performed, and a given p ∈ (0, 1) accordingly, given k 1 , k 2 , . . . , k m , the daily counts of the positive tests, y 1 , y 2 , . . . , y m are conditionally independent binomial random variables with p = pr(t est = +). in a similar fashion, we model the reported number of tests performed k 1 , k 2 , . . . , k m as (independent) negative binomial random variables. that is, for given (fixed) integer r > 0, and θ ∈ (0, 1), as we can see from appendix a, the observed marginal distribution of the reported daily number of test performed is exhibiting over-dispersion features that are characteristic to mixed-poisson or to negative binomial counts. thus, combining (1)(2), we obtain that given (p, θ), the joint probability (mass) function of the m pairs, (y i , k i ), i = 1, . . . , m, (i.e. the likelihood function), is where, ∝ indicates proportionality of terms, up to a constant, and where n m = m j=1 k j and x m = m j=1 y j are the cumulative reported number of tests performed and the cumulative reported positive tests. we note in passing that the cumulative ppt rate mentioned in the introduction is merely the ratio,p m := x m /n m . the standard bayesian predictive model in the case of binomial-negative binomial counts (as in (1)-(2)), is that with the conjugate beta-beta joint prior distribution on (p, θ) (see for example gelman et. al. (2014) ). that is, in the case of the likelihood function f (d m |p, θ) given in (3) above, we consider the bayesian model that assumes that for given p, and n m , for some a > 0 and b > 0 and where, given θ > 0, for some fixed integer r > 0, and some c > 0, and d > 0. thus, the joint prior pd f of (p, θ) is, accordingly, and since the joint posterior pd f for (p, θ) **given** the data d m , is we immediately obtain from (3)-(6) (due to the conjugacy) that **given** the data, x m and n m , the (marginal) posterior distribution of p, denoted as π(p |x m , n m ), is also a beta distribution and the (marginal) posterior distribution of θ, denoted as π(θ |n m ), is also a beta distribution. specifically, it follows that given x m and n m , where these four updated parameters are given by: hence, the bayes estimates of θ and p given the data (x m , n m ), are the respective posterior means:p remark: the choice in (2) for using the negative binomial distribution to model the reported daily number of tests k i , could be seen as specific to the indiana covid-19 testing data, which might reflect testing capacity limitation and daily variability unique to that state. other models that account for the observed overdispersion characteristics of the data (see appendix a) could be used instead. for instance, one may alternatively consider the related mixed-poisson distribution in (2). having observed (x m , n m ), the posterior predictive distribution , under the bayesian model (3)-(6) and given (x m , n m ), of a 'new' (or 'future') observation on the number of positive cases, y * , out of a given k * = k * new tests is the the beta-binomial distribution, for y * = 0, 1, . . . , k * . we denote this (predictive) distribution for y * , given k * = k * , as with a m and b m as in (7). the corresponding posterior predictive mean and variance of y * are given by in a similar manner we obtain the posterior predictive distribution under this bayesian model and given (x m , n m ), of a 'new' (or 'future') number of tests k * is the beta-negative binomial distribution. in fact, with k * | θ ∼ negbinom(r, θ), we have for k * = 0, 1, 2, . . . . we denote this (posterior predictive) distribution for k * as with c m and d m as in (7). the corresponding posterior predictive mean and variance of k * are given by it is straightforward to see that the joint posterior predictive probability (mass) function of (y * , k * ), can easily be obtained from expressions (7) and (8) is clearly, along with the value of y * as the number of positive tests predicted out of the predicted number of tests, k * , one may obtain their ratio, p * := y * /k * , as the rate of positive tests predicted next, given the data. while an explicit expression for the posterior predictive distribution of p * is not readily available, it may be estimated, quite accurately, through monte-carlo simulations. towards that end, we denote by q * m (·) the posterior predictive cd f of p * , given the data d m . that is, for any t ∈ r, and recall that the α th percentile (α ∈ (0, 1)), of this distribution, is defined as thus, when available, the interval, [t * 1−α , t * α ] serves as a 1 − 2α posterior prediction interval for p * given the data d m , so that as was mentioned in the previous section, while explicit expression for q * m (·), the posterior predictive cd f of p * , given the data d m , is not available, it may be estimated via monte-carlo simulations which exploit the explicit expression, in (9) , for the joint posterior predictive distribution of (y * , k * ). given the data, d m and with parameters a m , b m , c m and d m and with r as above, generate a random sample of a large size b (b = 5, 000, say) from q * m (·) as follows: 1) given n m , draw k * ∼ betanegbinom(r, c m , d m ); 2) given x m , n m and k * = k * , draw y * ∼ betabinom(k * , a m , b m ) to obtain the pair (y * , k * ); 3) calculate the simulated predicted ppt as p * = y * /k * . b times so as to form p * 1 , p * 2 , . . . , p * b as a random sample from q * m (·). having obtained the random sample p * 1 , p * 2 , . . . , p * b , we estimate q * m (·) by its empirical ver-sionq * accordingly, and in similarity to (10), we estimate the α st percentile of q * m (·) bŷ a simple r script (see appendix c) which utilizes the built-in functions rbnbinom() (for the betanegbinom(·) distribution) and rbbinom() (for the betabinom(·) distribution), of the extradistr package produces the simulated sample from the (respective) posterior predictive distribution of (y * , k * ) and the corresponding predicted ppt, p * = y * /k * . we continue with the same prior parameterization used in illustration 1, of a = b = 1, c = d = 1 and r = 3. recall that the given data d m , yields, m = 109, n m = 5.22946 × 10 5 and x m = 4.6907 × 10 4 . we first simulated b = 5000 sample values for the respective predictive distributions of (y * , k * ) and of p * and used these simulated values to estimate the posterior predictive distribution of p * , which in turn, was used it to obtain the 95% posterior prediction interval, [0.07981, 0.1001] for the 'next-day' indiana's ppt as was displayed in figure 2 . the means and standard deviations of the (estimated) posterior predictive distributions of y * , k * and p * , along with the corresponding 95% prediction interval are presented in table 1 figure 3 : the estimated (monte-carlo) posterior predictive distribution of indiana ppt, p * , along with the marked (in blue) the 95% prediction interval figure 3 above, displays the monte-carlo histogram of that predictive distribution, along with a nonparametric and a normal density (in red) approximations. also marked are the corresponding bound for the 95% posterior prediction interval for p * . the monte-carlo marginal (posterior) distribution of k * is displayed in figure 4 and that of y * in figure 5 . we conclude this illustration with figure 6 , where we display the posterior prediction intervals for the ppt as were calculated for each report day in the series. that is, based on the given data on the n th day, d n , we calculated the 95% posterior predicted interval for the ppt on the (n + 1) th , day (the "next" day), for each n = 2, 3, . . . , m of the m = 109 days available in the data set. as can be seen, each of the daily calculated ppt (as in figure 2 ), fell well within the corresponding prediction interval (marked in red in figure 6 ) as was calculated based on the previous' days data, thus providing also a partial validation for the applicability of this bayesian approach (with its underlying assumptions) to these covid-19 count data. the indiana covid-19 data are available for retrieval through the isdh data hub[10] as are reported for the state in the file covid report date.xlsx. it includes the daily records (as columns) on: • date: date of the event, it is equal to the investigation starting date for positive cases; the date of death for deaths; the coalesce of specimen date and report date for testings (if specimen collected date is unknown, use report date), respectively • covid test: total number of testings (i.e. number of new people tested on the date. indiana residents only) • daily delta tests: the number of most recent (i.e latest report) new testings that are reported into the testing pool. the date of specimen collected is typically earlier than the report date • daily base tests: the number of tests from the last report. records might be removed due to information correctness • covid count: total number of positive cases (i.e. number of patients who started investigation for their positive report) on the date • daily delta cases: the number of most recent(i.e latest report) new positive cases that are reported into the positive case pool. the investigation starting date could be earlier than the report date due to necessary process • daily base cases: the number of positive cases from the last report. records might be removed due to information correctness • covid deaths total: number of deaths on the date • daily delta deaths the number of most recent (i.e latest report) new death cases that are reported into the death case pool. the date of death could be earlier than the report date due to necessary process and confirmation • daily base deaths: the number of deaths from the last report. records might be removed due to information correctness • covid count cumsum: the cumulative number of positive cases as of the report date • covid deaths cumsum: the cumulative number of deaths as of the report date • covid test cumsum: the cumulative number of tests as of the report date a simple r script to obtain the monte-carlo sample from q m (·), bayesian data analysis chapman & hall/crc texts in statistical science estimating the infection fatality rate among symptomatic covid-19 cases in the united states health affairs 39, no. 7. project hopethe people-to-people health foundation coronavirus covid-19 global cases covid-19 positive cases, evidence on the time evolution of the epidemic or an indicator of local testing capabilities? a case study in the united states available modelling death rates due to covid-19: a bayesian approach available online at brazilian modeling of covid-19(bram-cod): a bayesian monte carlo approach for covid-19 spread in a limited data set context available online at medrxiv preprint doi cdc guidelines-cdc activities and initiatives supporting the covid-19 response and the presidents plan for opening america up again -centers for disease control and prevention available online as indiana covid-19 data report-indiana state department of health available online key: cord-319436-mlitd45q authors: brinati, d.; campagner, a.; ferrari, d.; locatelli, m.; banfi, g.; cabitza, f. title: detection of covid-19 infection from routine blood exams with machine learning: a feasibility study date: 2020-04-25 journal: nan doi: 10.1101/2020.04.22.20075143 sha: doc_id: 319436 cord_uid: mlitd45q background the covid-19 pandemia due to the sars-cov-2 coronavirus, in its first 4 months since its outbreak, has to date reached more than 200 countries worldwide with more than 2 million confirmed cases (probably a much higher number of infected), and almost 200,000 deaths. amplification of viral rna by (real time) reverse transcription polymerase chain reaction (rrt-pcr) is the current gold standard test for confirmation of infection, although it presents known shortcomings: long turnaround times (3-4 hours to generate results), potential shortage of reagents, false-negative rates as large as 15-20%, the need for certified laboratories, expensive equipment and trained personnel. thus there is a need for alternative, faster, less expensive and more accessible tests. material and methods we developed two machine learning classification models using hematochemical values from routine blood exams (namely: white blood cells counts, and the platelets, crp, ast, alt, ggt, alp, ldh plasma levels) drawn from 279 patients who, after being admitted to the san raffaele hospital (milan, italy) emergency-room with covid-19 symptoms, were screened with the rrt-pcr test performed on respiratory tract specimens. of these patients, 177 resulted positive, whereas 102 received a negative response. results we have developed two machine learning models, to discriminate between patients who are either positive or negative to the sars-cov-2: their accuracy ranges between 82% and 86%, and sensitivity between 92% e 95%, so comparably well with respect to the gold standard. we also developed an interpretable decision tree model as a simple decision aid for clinician interpreting blood tests (even off-line) for covid-19 suspect cases. discussion this study demonstrated the feasibility and clinical soundness of using blood tests analysis and machine learning as an alternative to rrt-pcr for identifying covid-19 positive patients. this is especially useful in those countries, like developing ones, suffering from shortages of rrt-pcr reagents and specialized laboratories. we made available a web-based tool for clinical reference and evaluation. this tool is available at https://covid19-blood-ml.herokuapp.com. the pandemic disease caused by the sars-cov-2 virus named covid-19 is requiring unprecedented responses of exceptional intensity and scope to more than 200 states around the world, after having infected, in the first 4 months since its outbreak, a number of people between 2 and 20 million with at least 200,000 deaths. to cope with the spread of the covid-19 infection, governments all over the world has taken drastic measures like the quarantine of hundreds of millions of residents worldwide. however, because of the covid-19 symptomatology, which showed a large number of asymptomatics [12] , these efforts are limited by the problem of differentiating between covid-19 positive and negative individuals. thus, tests to identify the sars-cov-2 virus are believed to be crucial to identify positive cases to this infection and thus curb the pandemic. to this aim, the current test of choice is the reverse transcriptase polymerase chain reaction (rt-pcr)-based assays performed in the laboratory on respiratory specimens. taking this as a gold standard, machine learning techniques have been employed to detect covid-19 from lung ct-scans with 90% sensitivity, and high auroc ( 0.95) [25, 18] . although chest cts have been found associated with high sensitivity for the diagnosis of covid-19 [1] , this kind of exam can hardly be employed for screening tasks, for the radiation doses, the relative low number of devices available, and the related operation costs. a similar attempt was recently performed on chest x-rays [4] , which is a low-dose and less expensive test, with promising statistical performance (e.g., sensitivity 97%). however, since almost 60% of chest x-rays taken in patients with confirmed and symptomatic covid19 have been found to be normal [40] , systems based on this exam need to be thoroughly validated in real-world settings [6] . the public health emergency requires an unprecedented global effort to increase testing capacity [29] . the large demand for rrt-pcr tests (also commonly known as nasopharyngeal swab tests) due to the worldwide extension of the virus is highlighting the limitations of this type of diagnosis on a large-scale such as: the long turnaround times (on average over 2 to 3 hours to generate results); the need of certified laboratories; trained personnel; expensive equipment and reagents for which demand can easily overcome supply [26] . for instance in italy, the scarcity of reagents and specialized laboratories forced the government to limit the swab testing to those people who clearly showed symptoms of severe respiratory syndrome, thus leading to a number of infected people and a contagion rate that were largely underestimated [34] . for this reason, and also in light of the predictable wide adoption of mobile apps for contact tracing [14] , which will likely increase the demand for population screening, there is an urgent need for alternative (or complementary) testing methods by which to quickly identify infected covid-19 patients to mitigate virus transmission and guarantee a prompt patients treatment. on a previous work published in the laboratory medicine literature [13] , we showed how simple blood tests might help identifying false positive/negative rrt-pcr tests. this work and the considerations made above strongly motivated us to apply machine learning methods to routine, low-cost 2 blood exams, and to evaluate the feasibility of predictive models in this important task for the massscreening of potential covid-19 infected individuals. in what follows we report this feasibility study in detail. the aim of this work is to develop a predictive model, based on machine learning techniques, to predict the positivity or negativity for covid-19. in the rest of this section we report on the dataset used for model training and on the data analysis pipeline adopted. the dataset used for this study was made available by the irccs ospedale san raffaele 3 and it consisted of 279 cases, randomly extracted from patients admitted to that hospital from the end of february 2020 to mid of march 2020. each case included the patient's age, gender, and values from routine blood tests, as well as the result of the rt-pcr test for covid-19, performed by nasopharyngeal swab. the parameters collected by the blood test are reported in table 1 . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 25, 2020. . https://doi.org/10.1101/2020.04. 22.20075143 doi: medrxiv preprint the dependent variable "swab" is binary and it is equal to 0 in the absence of covid-19 infection (negative swab test), and it is equal to 1 in the case of covid-19 infection (positive to the swab test). the number of occurrences for the negative and positive class was respectively 102 (37%) and 177 (63%), thus the dataset was slightly imbalanced towards positive cases. figure 1 shows the pairwise correlation of the features used for this study, while figure 2 focuses on variables "age", "wbc ", "crp", "ast " and "lymphocytes". first of all, the categorical feature gender has been transformed into two binary features by one-hot encoding. further, we notice that the dataset was affected by missing values in most of its features (see table 2 ). to address data incompleteness, we performed missing data imputation by means of the multivariate imputation by chained equation (mice) [5] method. mice is a multiple imputation method that works in an iterative fashion: in each imputation round, one feature with missing values is selected and is modeled as a function of all the other features; the estimated values are then used to impute the missing values and re-used in the subsequent imputation rounds. we chose this method because multiple imputation techniques are known to be more robust and better capable to account for uncertainty compared with all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. single imputation ones [33] (as they employ the joint distribution of the available features), and mice in particular can also handle different data types. we developed and compared different classes of machine learning classifiers. in particular, we considered the following classifier models: -decision tree [35] (dt); -extremely randomized trees [16] (et); -k-nearest neighbors [2] (knn); -logistic regression [20] (lr); -naïve bayes [23] (nb); -random forest [21] (rf); -support vector machines [36] (svm). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 25, 2020. . https://doi.org/10.1101/2020.04.22.20075143 doi: medrxiv preprint we also considered a modification of the random forest algorithm, called three-way random forest classifier [7] (twrf), which allows the model to abstain on instances for which it can express low confidence; in so doing, a twfr achieves higher accuracy on the effectively classified instances at expense of coverage (i.e., the number of instances on which it makes a prediction). we decided to consider also this class of models as they could provide more reliable predictions in a large part of cases, while exposing the uncertainty regarding other cases so as to suggest further (and more expensive) tests on them. from a technical point of view, since random forest is a class of probability scoring classifiers (that is, for each instance the model assigns a probability score for every possible class), the abstention is performed on the basis of two thresholds α, β ∈ [0, 1]: if we denote with 1 the positive class and 0 the negative class, then each instance is classified as positive if score(1) > α and score(1) > score(0), negative if score(0) > β and score(0) > score (1) and, otherwise, the model abstains. in these models the performance is usually evaluated only on the non-abstained instances [15] , and the coverage is a further performance element to be considered. the models mentioned above have been trained, and evaluated, through a nested cross validation [19, 9] procedure. this procedure allows for an unbiased generalization error estimation while the hyperparameter search (including feature selection) is performed: an inner cross-validation loop is executed to find the optimal hyperparameters via grid search and an outer loop evaluates the model performance on five folds. models were evaluated in terms of accuracy, balanced accuracy 4 , positive predictive value (ppv) 5 , sensitivity, specificity and, except for the three-way random forest, the area under the roc curve (auc). after discussing this with the clinicians involved in this study, we considered accuracy and sensitivity to be the main quality metrics, since false negatives (that is, patients positive to covid-10 which are, however, classified as negative, and possibly let go home) are more harmful than false positives in this screening task. 4 we recall that balanced accuracy is defined as the average of sensitivity and specificity. if accuracy and balanced accuracy significantly differ, the data could be interpreted as unbalanced with respect to class prevalence. 5 we recall here that ppv represents the probability that subjects with a positive screening test truly have the disease. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. tables 3 and 4 show the 95% confidence intervals of, respectively, average accuracy and average balanced accuracy (that is, the average of sensitivity and specificity) of the models (on the nested cross-validation) trained on the two best-performing sets of features: the first one, dataset a, includes all the variables, while the second one, dataset b, excludes the "gender " variable, as this was found of negligible predictive value. figure 3 shows the performance of the traditional models (i.e., the twrf model was excluded) on the nested cross-validation. to further validate the above findings, the entire dataset has been splitted into training and test/validation sets, respectively the 80% and the 20% of the total instances. the best performing model, i.e. the random forest classifier, trained on dataset b, achieved the following results on the test/validation set: accuracy = 82% , sensitivity = 92%, ppv = 83%, specificity = 65%, auc = 84%. figures 4 and 5 show the performance of this model in the roc and precision/recall space, respectively. the optimal hyperparameters found are shown in table 5 . similarly, for the best three-way random forest classifier on the validation set we observed: accuracy = 86%, sensitivity = 95%, ppv = 86%, specificity = 75%, coverage = 70% (that is, for 30% of the validation instances the model abstained). the feature importance assessed for the the best performing model (random forest on dataset b), are shown in figure 6 . in order to provide an interpretable overview (in the sense of explainable ai [17] ) of the predictive models that we developed, we also developed a decision all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. tree model, which is shown in figure 7 . although the depicted decision tree is associated with a lower discriminative performance than the two former (inscrutable) models, such a tree can be used as a simple decision aid by clinicians interested in the use of blood values to assess covid-19 suspect cases. we have developed two machine learning models to discriminate between patients who are either positive or negative to the sars-cov-2, which is the coronavirus causing the covid-19 pandemia. in this task, patients are represented in terms of few basic demographic characteristics (gender, age) and a small array of routine blood tests, chosen for their convenience, low cost and because they are usually available within 30 minutes from the blood draw in regular emergency department. the ground truth was established through rt-pcr swab tests. we presented the best traditional model, as it is common practice, and a three-way model, which guarantees best sensitivity and positive predictive value: the former is the proportion of infected (and contagious) people who will have a positive result and therefore it is useful to clinicians when deciding which test to use. on the other hand, ppv is useful for patients as it tells the odds of one having covid-19 if they have a positive result. the performance achieved by these two best models (sensitivity between 92% and 95%, accuracy between 82% and 86%) provides proof that this kind of data, and computational models, can be used to discriminate among potential covid-19 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. infectious patients with sufficient reliability, and similar sensitivity to the current gold standard. this is the most important contribution of our study. also from the clinical point of view, the feature selection was considered valid by the clinicians involved. indeed, the specialist literature has found that covid-19 positivity is associated with lymphopenia (that is, abnormally low level of white blood cells in the blood), damage to liver and muscle tissue [42, 39] , and significantly increased c-reactive protein (crp) levels [10] . in [27] a comprehensive all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. list of the most frequent abnormalities in covid-19 patients has been reported: among the 14 conditions considered, they report increased aspartate aminotransferase (ast), decreased lymphocyte count (wbc), increased lactate dehydrogenase (ldh), increased c-reactive protein (crp), increased white blood cell count (wbc) and increased alanine aminotransferase (alt). these parameters are also the most predictive features identified by the best classifier (random forest), all together with the age attribute. also other studies confirm the relevance of these features and their association with the covid-19 positivity [8, 30, 32, 44] , compared to other kinds of pneumonia [43] . this also gives confirmation that our models ground on clinically relevant features and that most of these values can be extracted from routine blood exams. the interpretable decision tree model provides a further confirmation (see figure 7 ) of the soundness of the approach: the clinicians (ml, gb) and the biochemist (df) involved in this study found reasonable that the ast would be the first parameter to consider (i.e., mirrored by the fact that ast was the root of the decision tree) and that it was found to be the most important predictive feature. indeed, values of ast below 25 are good predictors of covid-19 positivity (accuracy = ppv = 76%), while values below 25 are a good predictor of covid-19 negativity (accuracy = negative predictive value = 83%). similar observations can also be made about crp, lymphocytes and general wbc counts. no statistically significant difference was found between the accuracy and the balanced accuracy of the models (as mirrored by the overlap of the 95% confidence intervals), as a sign that the dataset was not significantly unbalanced. moreover, we can notice that the best performing ml classifier (random forest) exhibited a very high sensitivity (∼ 90%) but, in comparison, a limited specificity of only 65%. that gives the main motivation for the three-way classifier: this model offers a trade-off between increased specificity (a 10% increment compared all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 25, 2020. . https://doi.org/10.1101/2020.04.22.20075143 doi: medrxiv preprint fig. 7 an interpretable decision tree, developed in order to support the interpretation of the predictions from the other models. color gradients denote predictivity for either classes (shades of blue correspond to covid-19 negativity, shades of orange to positivity). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 25, 2020. . https://doi.org/10.1101/2020.04. 22.20075143 doi: medrxiv preprint with the best traditional ml model) and reduced coverage, as the three-way approach abstains on uncertain instances (i.e., the cases that cannot be classified with high confidence neither as positive nor negative ). this means that the model yields more robust and reliable prediction for the classified instances (as it is mirrored by the increase in all of the performance measures), while for the other ones it is anyway useful in suggesting further tests, e.g., by either a pcr-rna swab test or a chest x-ray. in regard to the specificity exhibited by our models, we can further notice that even while these values are relatively low compared with other tests (which are more specific but slower and less accessible), this may not be too much of a limitation as there is a significant disparity between the costs of false positives and false negatives and in fact our models favors sensitivity (thus, they avoid false negatives). further, the high ppv (> 80%) of our models suggest that the large majority of cases identified as positives by our models would likely be covid-19 positive cases. that said, the study presents two main limitations: the first, and more obvious one, regards the relatively low number of cases considered. this was tackled by performing nested cross-validation in order to control for bias [38] , and by employing models that are known to be effective also with moderately sized samples [3, 31, 37] . nonetheless, further research should be aimed at confirming our findings, by integrating hematochemical data from multiple centers and increasing the number of the cases considered. the second limitation may be less obvious, as it regards the reliability of the ground truth itself. although this was built by means of the current gold standard for covid-19 detection, i.e., the rrt-pcr test, a recent study observed that the accuracy of this test may be highly affected by problems like inadequate procedures for collection, handling, transport and storage of the swabs, sample contamination, and presence of interfering substances, among the others [28] . as a result, some recent studies have reported up to 20% false-negative results for the rrt-pcr test [41, 24, 22] , and a recent systematic review reported an average sensitivity of 92% and cautioned that "up to 29% of patients could have an initial rt-pcr false-negative result". thus, contrary to common belief and some preliminary study (e.g., [11] ), the accuracy of this test could be less than optimal, and this could have affected the reliability of the ground truth also in this study (as in any other using this test for ground truthing, unless cases are annotated after multiple tests. however, besides being a limitation, this is also a further motivation to pursue alternative ways to perform the diagnosis of sars-cov-2 infection, such as our methods are. future work will be devoted to the inclusion of more hematochemical parameters, including those from arterial blood gas assays (abg), to evaluate their predictiveness with respect to covid-19 positiveness, and the inclusion of cases whose probability to be covid-positive is almost 100%, as they resulted positive to two or more swabs or to serologic antibody tests. this would allow to associate a higher weight with misidentifying those cases, so as, we conjecture, improve the sensitivity further. moreover, we want to investigate the interpretability of our models further, by both having more clinicians validate the current decision tree, and possibly construct a more accurate one, so that clinicians can use it as a convenient decision aid to interpret blood tests in regard to covid-19 suspect cases (even off-line). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 25, 2020. . https://doi.org/10.1101/2020.04. 22.20075143 doi: medrxiv preprint finally, this was conceived as a feasibility study for an alternative covid-19 test on the basis of hematochimical values. in virtue of this ambitious goal, the success of this study does not exempt us from pursuing a real-world, ecological validation of the models [6] . to this aim, we deployed an online web-based tool 6 by which clinicians can test the model, by feeding it with clinical values, and considering the sensibleness and usefulness of the indications provided back by the model. after this successful feasibility study, we will conceive proper external validation tasks and undertake an ecological validation to assess the cost-effectiveness and utility of these models for the screening of covid-19 infection in all the real-world settings (e.g., hospitals, workplaces) where routine blood tests are a viable test of choice. not applicable not applicable research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the helsinki declaration (as revised in 2013), and was approved by the authors' institutional review board on the 20th of april. individuals signed an informed consent authorizing the use of their anonymously collected data for retrospective observational studies (article 9.2.j; eu general data protection regulation 2016/679 [gdpr]), according to the irccs san raffaele hospital policy (iog075/2016). the developed web tool is available at the following address: https://covid19-blood-ml. herokuapp.com/ the complete dataset will be made available on the zenodo platform as soon as the work gets accepted for publication. 6 the tool is available at the following address: https://covid19-blood-ml.herokuapp.com/. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 25, 2020. . https://doi.org/10.1101/2020.04. 22.20075143 doi: medrxiv preprint correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases an introduction to kernel and nearest-neighbor nonparametric regression model selection for support vector machines: advantages and disadvantages of the machine learning theory covid-19: automatic detection from x-ray images utilizing transfer learning with convolutional neural networks mice: multivariate imputation by chained equations in r the proof of the pudding: in praise of a culture of real-world validation for medical artificial intelligence the three-way-in and three-wayout framework to treat and exploit ambiguity in data di napoli r (2020) features, evaluation and treatment coronavirus (covid-19) on over-fitting in model selection and subsequent selection bias in performance evaluation epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr covid-19: identifying and isolating asymptomatic people helped eliminate virus in italian village routine blood tests as a potential diagnostic tool for covid-19 quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing. science all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted extremely randomized trees explainable ai: the new 42? coronavirus detection and analysis on chest ct with deep learning the elements of statistical learning: data mining, inference, and prediction applied logistic regression random decision forest insufficient sensitivity of rna dependent rna polymerase gene of sars-cov-2 viral genome as confirmatory test using korean covid-19 cases naive (bayes) at forty: the independence assumption in information retrieval false-negative results of real-time reverse-transcriptase polymerase chain reaction for severe acute respiratory syndrome coronavirus 2: role of deep-learning-based ct diagnosis and insights from two cases artificial intelligence distinguishes covid-19 from community acquired pneumonia on chest ct development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis laboratory abnormalities in patients with covid-2019 infection potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (covid-19) diagnostic testing for severe acute respiratory syndrome-related coronavirus-2: a narrative review time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia random forest for bioinformatics dysregulation of immune response in patients with covid-19 in wuhan multiple imputation for nonresponse in surveys as covid-19 cases, deaths and fatality rates surge in italy, underlying causes require investigation a survey of decision tree classifier methodology learning with kernels: support vector machines, regularization, optimization, and beyond stabilizing classifiers for very small sample sizes bias in error estimation when using cross-validation for model selection clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china chest x-ray findings in 636 ambulatory patients with covid-19 presenting to an urgent care center: a normal chest x-ray is no guarantee chest ct for typical 2019-ncov pneumonia: relationship to negative rt-pcr testing liver injury in covid-19: management and challenges a comparative study on the clinical features of covid-19 pneumonia to other pneumonias functional exhaustion of antiviral lymphocytes in covid-19 patients the complete code will be made available on the zenodo platform as soon as the work gets accepted for publication. key: cord-308021-cnf4xljc authors: kohns vasconcelos, malte; renk, hanna; popielska, jolanta; nyirenda nyang’wa, maggie; burokiene, sigita; gkentzi, despoina; gowin, ewelina; donà, daniele; villanueva-medina, sara; riordan, andrew; hufnagel, markus; eisen, sarah; da dalt, liviana; giaquinto, carlo; bielicki, julia a. title: sars-cov-2 testing and infection control strategies in european paediatric emergency departments during the first wave of the pandemic date: 2020-10-13 journal: eur j pediatr doi: 10.1007/s00431-020-03843-w sha: doc_id: 308021 cord_uid: cnf4xljc between february and may 2020, during the first wave of the covid-19 pandemic, paediatric emergency departments in 12 european countries were prospectively surveyed on their implementation of sars-cov-2 disease (covid-19) testing and infection control strategies. all participating departments (23) implemented standardised case definitions, testing guidelines, early triage and infection control strategies early in the outbreak. patient testing criteria initially focused on suspect cases and later began to include screening, mainly for hospital admissions. long turnaround times for test results likely put additional strain on healthcare resources. conclusion: shortening turnaround times for sars-cov-2 tests should be a priority. specific paediatric testing criteria are needed. european reference laboratories established widespread capacities for testing for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) within a matter of days after a diagnostic test was made publicly available [1, 2] . world health organization (who) and public health authorities in europe issued case definitions, testing and infection control recommendations for covid-19 in january 2020. the current understanding of covid-19 in paediatric patients is that children more often have mild disease compared to adults [3, 4] . current knowledge suggests that the peak of infectiousness of sars-cov-2 infection occurs a few days before and after the onset of symptoms, meaning that presymptomatic people are able to transmit the infection [5] . transmission unobserved by public health authorities occurs frequently, with at least one-third of cases having been undetected during the early epidemic [6, 7] . the aim of this study was to describe the implementation of testing and infection control strategies and their evolution in paediatric emergency departments in europe. from mid-february to the first week of may 2020, we surveyed all european paediatric collaboration sites within the penta id paediatric research network weekly for developments in their testing and infection control strategies. portable document format (pdf) survey forms were sent out by email to contact officers at 78 paediatric departments across europe. the survey form is available as an online supplement to this article. completed and signed survey forms were handed in by return email. missing weekly replies were imputed as last observation carried forward. paediatric departments of 23 mostly tertiary care hospitals in 12 european countries (belgium, germany, france, italy, poland, portugal, the uk, the netherlands, greece, spain, lithuania and switzerland) participated in the surveys (response rate 29%). multiple sites participated in the uk (5, 3 tertiary and 2 secondary level), germany (5, 4 tertiary and 1 secondary level), spain (3, 1 network of sites representing the madrid region, 1 tertiary and 1 secondary level) and poland (2, both tertiary level). in each of the remaining countries (belgium, france, italy, portugal, the netherlands, greece, lithuania and switzerland), one site participated. by the end of february 2020, all hospitals had implemented standardised case definitions for suspected covid-19 cases, with the majority (16 out of 21 participating at that point in time, 76%) following national government or public health authority guidelines and three directly following who guidelines. standardised definitions of suspected cases showed high similarity between sites. all definitions consisted of a clinical component of acute respiratory infection and an epidemiological component of possible exposure to the virus. the latter changed between february and april: initially, definitions at all sites required contact within 14 days with a confirmed case or travel to specified geographic areas; in time, this changed to staying in any area with ongoing community transmission. twenty participating sites used suspected case definitions from the beginning that did not exclude patients on detection of an alternative pathogen. two of the spanish sites and one site in poland initially excluded patients with confirmed alternative diagnoses of respiratory infections from being suspect cases for covid-19. this changed by april at all three sites, so that afterwards detection of another pathogen that could explain the respiratory symptoms no longer excluded a patient from being a suspect case and from undergoing sars-cov-2 testing. ten sites (43%) reported that they strictly only tested patients for sars-cov-2 if they matched the definition of a suspected case. another 12 (52%) had a policy to only test patients matching the case definition but reported that exceptions occurred regularly. until april, no site reported that their decision to test patients was based on separate local guidelines. in april, several german and uk sites started broader testing, first with testing of patients admitted to oncology or intensive care units and from the end of april with routine screening of all admitted patients. table 1 shows example developments of testing guidelines at four participating children's emergency departments. in terms of sampling site, 52% of the participating hospitals restricted testing to upper respiratory samples, while 48% obtained upper and lower respiratory samples if the latter could be obtained. although discharge and infection control strategies after admission relied heavily on test results, by the end of the survey period only 9 hospitals (39%) received test results multiple times daily, while another 7 (30%) had waiting times of more than 24 h before test results would be back. at all but two sites, where faster turnaround times could be achieved, expected time to test results did not change over the survey period. by the beginning of march, 9 (43%) were discharging patients with pending test results when they were clinically stable. three of the seven hospitals where results took more than 24 h to come back would not discharge patients while test results were pending, regardless of whether the patients were clinically stable. by mid-march all hospitals were discharging patients with pending test results. until the beginning of march, sites saw up to 30 suspected cases per week. while only one site (in germany) had a child positive for sars-cov-2, 67% of sites had already provided care to suspected cases of sars-cov-2. at the different sites, the highest number of patients tested per week differed widely between 7 per week for a secondary care hospital in western germany and 112 for a specialised tertiary care children's hospital in north england. community test centres for covid-19 opened across germany in early to mid-march and in the uk only in april. opening of community test centres in proximity to the surveyed sites coincided with varying stages of development of overall case numbers in the areas. therefore, our data allow no firm conclusion on whether opening of community test centres alleviated patient pressure on paediatric emergency departments. most departments used early clinical triage at the emergency department to separate suspected cases from other patients from the beginning of the survey period. only one site (in greece) initially did not triage but changed this in the last week of february. two uk hospitals had plans to refer patients with positive test results to other hospitals for admission, and eight hospitals were planning to place multiple patients tested positive in cohort isolation if limited capacity for individual isolation occurred. at most hospitals, staff used respirators, i.e. filtering face piece (ffp) masks, when treating suspected cases in the emergency department. in contrast, staff at four uk sites, in the netherlands and at one site in poland used surgical masks only. this did not change over the survey period. in the early stages of the covid-19 pandemic, paediatric emergency departments implemented standardised case definitions, testing guidelines and infection control measures rapidly. while this is an important and reassuring finding regarding the preparedness of paediatric emergency care in europe, it may be a limitation of this survey that our sample of hospitals was biased towards tertiary care hospitals with strong international research links. although infection control strategies and even discharge of patients relied heavily on receiving sars-cov-2 test results, most hospitals only received these after considerable delay, often more than 24 h. shortening turnaround times for tests should be a priority. prior to discharge, infection control measures on uninfected patients awaiting test results place a huge burden on emergency care resources. most departments rightly responded by discharging patients while test results were pending. this does not, however, mitigate against the public health impact of delayed result reporting on efficient contact tracing and subsequent isolation or quarantine of contacts in the community. the guidelines for testing focused on two aims: establishing aetiology in children with symptoms of ari and excluding infection for inpatient infection control purposes. this was a necessary restriction while numbers of new infections were high and capacities for testing were limited. we believe that in the current situation with vastly expanded laboratory capacities, a broader approach with more testing of mildly symptomatic patients or asymptomatic contacts may be warranted. to allocate testing resources responsibly, we believe that specific testing criteria for the paediatric population are needed because both the individual risk of children to suffer from severe disease and to sustain transmission in the community differ from that in adults [10, 11] . children and adolescents suffer serious consequences from school closures and allowing schools to re-open has positive social, psychological and economic implications [12] . benefits of broader access to testing may include the ability to detect outbreaks in day care facilities and schools earlier in order to limit spread of infections while maintaining as much normality as possible for children and adolescents. authors' contributions mkv, cg and jab designed the study, all authors commented on the design; cg provided resources for the survey; mkv received and analysed the survey forms; mkv, hr, jp, mnn, dd and jab wrote the manuscript; sb, eg and ar revised the manuscript; all authors commented on the manuscript and approved the final version. funding open access funding enabled and organized by projekt deal. conflict of interest the authors declare that they have no conflict of interest. ethical approval the survey was considered a clinical audit. this article does not contain any studies with human participants or animals performed by any of the authors. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. laboratory readiness and response for novel coronavirus (2019-ncov) in expert laboratories in 30 eu/ eea countries coronavirus infections in children including covid-19: an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children systematic review of covid-19 in children shows milder cases and a better prognosis than adults temporal dynamics in viral shedding and transmissibility of covid-19 genetic structure of sars-cov-2 reflects clonal superspreading and multiple independent introduction events modelling the covid-19 epidemic and implementation of population-wide interventions in italy coronavirus country profiles. www. ourworldindata.org/coronavirus. accessed aktueller lage-/situationsbericht des rki zu covid-19 transmission of sars-cov-2 by children covid-19 in children and adolescents in europe: a multinational, multicentre cohort study coronakrise: kinder haben das recht auf bildung acknowledgements the authors would like to thank the following people for the kind contribution of survey data: key: cord-281495-beb164oy authors: charpentier, charlotte; ichou, houria; damond, florence; bouvet, elisabeth; chaix, marie-laure; ferré, valentine; delaugerre, constance; mahjoub, nadia; larrouy, lucile; le hingrat, quentin; visseaux, benoit; mackiewicz, vincent; descamps, diane; fidouh-houhou, nadhira title: performance evaluation of two sars-cov-2 igg/igm rapid tests (covid-presto and ng-test) and one igg automated immunoassay (abbott) date: 2020-09-03 journal: j clin virol doi: 10.1016/j.jcv.2020.104618 sha: doc_id: 281495 cord_uid: beb164oy the aim of this study was to assess the analytical performances, sensitivity and specificity, of two rapid tests (covidpresto® test rapid covid-19 igg/igm and ng-test® igm-igg covid-19) and one automated immunoassay (abbott sars-cov-2 igg) for detecting antisars-cov-2 antibodies. this study was performed with: (i) a positive panel constituted of 88 sars-cov-2 specimens collected from patients with a positive sars-cov-2 rt-pcr, and (ii) a negative panel of 120 serum samples, all collected before november 2019, including 64 samples with a cross-reactivity panel. sensitivity of covid-presto® test for igm and igg was 78.4% and 92.0%, respectively. sensitivity of ng-test® for igm and igg was 96.6% and 94.9%, respectively. sensitivity of abbott igg assay was 96.5% showing an excellent agreement with the two rapid tests (κ = 0.947 and κ = 0.936 for ngtest ® and covid-presto® test, respectively). an excellent agreement was also observed between the two rapid tests (κ = 0.937). specificity for igm was 100% and 86.5% for covid-presto® test and ng-test®, respectively. specificity for igg was 92.0%, 94.9% and 96.5% for covid-presto®, ngtest ®, and abbott, respectively. most of the false positive results observed with ng-test® resulted from samples containing malarial antibodies. in conclusion, performances of these 2 rapid tests are very good and comparable to those obtained with automated immunoassay, except for igm specificity with the ng-test®. thus, isolated igm should be cautiously interpreted due to the possible false-positive reactions with this test. finally, before their large use, the rapid tests must be reliably evaluated with adequate and large panel including early seroconversion and possible cross-reactive samples sars-cov-2 antibodies. this study was performed with: (i) a positive panel constituted of 88 sars-cov-2 specimens collected from patients with a positive sars-cov-2 rt-pcr, , in particular patients presenting strong covid-19 suspicions with negative pcr. serological tests also make it possible to catch up later with undiagnosed people at time of active infection, since antibodies have been found in almost all people who have been in contact with sars-cov-2 within a variable period depending on the severity of the infection [1, 2] . furthermore, studies showed that the kinetics of appearance of igm and igg were relatively close [3] . two types of tests are available to detect anti-sars-cov-2 antibodies: rapid lateral flow tests and automated immunoassays. several studies have assessed analytical performances of the automated immunoassays [4] [5] [6] [7] . on the other hand, although a very large number of rapid tests have been developed, few of them have been reliably evaluated with a suitable serum panel. however, this is very important to have data about the efficacy of these rapid tests to reliably detect anti-sars-cov-2 antibodies, since their increasing use in the world. the aim of this study was to assess the analytical performances (sensitivity and specificity) and agreement of two rapid tests and one automated immunoassay for detecting antibodies against sars-cov-2. in addition, we assessed five samples containing autoantibodies (four rheumatoid factor and one systemic lupus erythematosus). we also assessed the serum of 54 health-care workers who presented clinical symptoms during the epidemic period for whom sars-cov-2 rt-pcr was negative or not carried out. we evaluated two lateral flow tests: covid-presto ® test rapid covid-19 igg/igm (aaz, boulogne-billancourt, france) and ng-test ® igm-igg covid-19 (ng biotech, guipry, france) according to the manufacturer's instructions. five and ten microliters of serum for covid-presto ® test and ng-test ® , respectively, were added and results were read and interpreted 10 minutes after depositing serum. abbott sars-cov-2 igg kit (chemiluminescent microparticle immunoassay) (abbott, il, usa) was performed according to the manufacturer's instructions. the assay cut-off is an index of 1.40 and the assigned grey zone is comprised between 1.12 and 1.68. all statistical analyses were performed using excel. to assess sensitivity, rt-pcr results were chosen as gold standard. cohen kappa statistics and absolute agreement were calculated to evaluate the agreement between the different tests. all participants were not opposed to the collection of their data. sensitivity of covid-presto ® test was assessed on 88 samples collected between day 4 and day 42 after onset of symptoms and sensitivity of the ng-test ® was assessed on a subgroup of 59 samples among the 88 samples tested with covid-presto ® test, collected between days 7 and 28 after onset of symptoms ( table 1) . sensitivity of covid-presto ® test for igm was 67% (n=12/18), 88% (n=29/33) and 76% (n=28/37) for samples collected between days 4 and 9, between days 10 and 14, and after 14 days after onset of symptoms, respectively. sensitivity of covid-presto ® test for igg was 72% (n=13/18), 94% (n=31/33) and 100% (n=37/37) for samples collected between days 4 and 9, between days 10 and 14, and after 14 days after onset of symptoms, respectively. when combining igm and igg, sensitivity of covid-presto ® test was 83% (n=15/18), 97% (n=32/33) and 100% (n=37/37) for samples collected between days 4 and 9, between days 10 and 14, and after 14 days after onset of symptoms, respectively. sensitivity of ng-test ® for igm was 83% (n=5/6), 100% (n=22/22) and 97% (n=30/31) for samples collected between days 7 and 9 after, between days 10 and 14, and after 14 days after j o u r n a l p r e -p r o o f onset of symptoms, respectively. sensitivity of ng-test ® test for igg was 83% (n=5/6), 96% (n=21/22) and 97% (n=30/31) for samples collected between days 7 and 9, between days 10 and 14, and after 14 days after onset of symptoms, respectively. when combining igm and igg, sensitivity of ng-test ® test was 83% (n=5/6), 100% (n=22/22) and 97% (n=30/31) for samples collected between days 7 and 9, between days 10 and 14, and after 14 days after onset of symptoms, respectively. among the 59 serum samples of this pcr positive panel tested by the two rapid tests, 57 were compared with abbott sars-cov-2 igg automated immunoassay. sensitivity of abbott igg test was 67% (n=4/6), 100% (n=22/22) and 100% (n=29/29) for samples collected between days 7 and 9, between days 10 and 14, and after 14 days after onset of symptoms, respectively. agreement between abbott assay and rapid tests (igm/igg combined) was of 96.5% (n=55/57). in one case, the two rapid tests detected igg that were not detected by abbott (index=0.94), this sample was collected between days 7 and 9 after symptoms onset. for the second case, igg were detected in the greyzone of abbott (index=1.45) but not by ng-test ® . this latter sample was collected between days 10 and 14 after symptoms onset and igm were positive with the two rapid tests. specificity of covid-presto ® test was assessed on 120 samples described in the methods section. specificity of ng-test ® and abbott assay was assessed on a subgroup of 52 samples among the 120 samples tested with covid-presto ® test ( table 1) . npv was 97.5%, 95.9% and 94.3% for covid-presto ® , ng-test ® and abbott, respectively. in the present study, we evaluated two different lateral flow tests (covid-presto ® and ng-test ® ) and compared their performances to that of the automated abbott immunoassay using the same samples panel. sensitivity has been assessed using a panel of 88 serum samples of covid-19-infected patients (confirmed with a positive pcr), serum was collected between day 4 and day 42 after symptoms onset. sensitivity for igm, among the samples collected before day 9 after symptoms onset, was 67% and 83% for covid-presto ® test and ng-test ® , respectively. in the recent study of nicol et al., they found sensitivity of ng-test for igm of 43.8% for the samples collected before day 7 after symptoms onset and of 81.8% among all samples [5] . the excellent sensitivity of covid-presto ® test observed in our study confirmed the findings of the prazuck et al. study showing 100% of sensitivity in samples collected more than 15 days after symptoms [8] . among some samples collected before day 10 after symptoms onset, a simultaneous detection of igm and igg antibodies has been detected. these findings are in line with the antibodies kinetics described for igm and igg also using lateral flow rapids, as previously described with other techniques [3] . in the present study for covid-presto ® test, it allowed to increase the sensitivity from 67% when only igm are taken into account to 83% when both igm and igg are taken into account, highlighting the important added value to interpret the rapid tests by combining igm and igg antibodies. sensitivity for igg in samples collected later than 10 days after symptoms onset was excellent with the different tests being equal to 97.1%, 96.2% and 100% for covid-presto ® , ng-test ® , and abbott, respectively. thus, both rapid tests showed an excellent sensitivity for igg with a very good agreement with abbott. a previous study assessing abbott test performance j o u r n a l p r e -p r o o f showed sensitivity of 100% for igg for samples collected after 15 days after symptoms onset and of 69% for samples collected between 9 and 14 days after symptoms onset [6] . in this latter study, results sensitivity for igg were similar using ng-test ® [6] . in another study, igg sensitivity of abbott test was 91.8% for patients hospitalized 15 days after symptoms onset and 95.7% for patients non-hospitalized 20 days after symptoms onset. a limitation of our study could be that most of the patients of the positive panel presented severe infections, since 74% of them were hospitalized in infectious disease unit or in intensive care. interestingly, among the 14 out-patients, samples were collected for 9 of them 10 days after symptoms onset, showing positive igm and/or igg in seven cases with covid-presto ® test. insufficient quantity of serum for these patients was available to also test with ng-test ® and abbott. previous studies have reported that the kinetics and intensity of immune response could differ depending on the disease severity [1, 2] , thus it will be needed to also evaluate rapid tests in mild and pauci-symptomatic patients. another limitation is the difference in the number of tested samples for the early panel (serum samples collected before 9 days after symptoms onset) between the two rapid tests that which can bias the comparison between these tests for this group. a limitation is that we make this evaluation from serum samples and not from capillary blood specimens. regarding specificity evaluation, a crucial point for rapid tests, we used a large panel with 120 pre-endemic samples including 64 representatives of different profiles that can generate possible cross-reactivity. in our study, we showed an excellent specificity, above 96% in all cases and equal to 100% for igm with covid-presto ® test. the excellent specificity of covid-presto ® test was also observed in the study of prazuck et al. [8] . in our study, the only issue regarding specificity is for igm with ng-test ® , since specificity is only of 86.5%. however, this low specificity is mainly due to cross-reactivity with sera containing reactivity malarial antibodies. in the study of nicol et al. igm specificity with ng-test ® was 95.3% [5] , higher j o u r n a l p r e -p r o o f than in our study, however their negative panel contained no serum with malaria antibodies. regarding automated immunoassay, we showed a very good specificity of 96.2% for igg with abbott, confirming previous results of 99.3%, 99.6% and 100% [9] . serum samples containing malarial antibodies are absent or underrepresented in the negative panel of the other studies, although they are known to generate possible cross reactivity. this is very important to include it in the negative panel, since this is a differential diagnosis in patients returning from malaria endemic region with flu-like symptoms. overall, in our study, we observed a very good ppv and npv for both rapid tests. in conclusion, analytical performances for detection of anti-sars-cov-2 igg antibodies by two lateral flow rapid tests are very good and quite comparable to those obtained with automated immunoassay. however, serological tests should be used after day 10 following symptoms onset. before this, rt-pcr is the gold standard test for covid-19 diagnosis. the interpretation by combining igm and igg increased sensitivity of rapid tests. the presence of isolated igm should be cautiously interpreted due to the possible false-positive reactions. finally, the rapid tests must be reliably evaluated with adequate and large panels including early seroconversion and possible cross-reactive samples, before their large use and particular interest in low-resource settings. antibody responses to sars-cov-2 in patients with covid-19 different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid-19 patients interpreting diagnostic tests for sars-cov-2 inmicovid-19 laboratory team, performance evaluation of abbott architect sars-cov-2 igg immunoassay in comparison with indirect immunofluorescence and virus microneutralization test assessment of sars-cov-2 serological tests for the diagnosis of covid-19 through the evaluation of three immunoassays: two automated immunoassays (euroimmun and abbott) and one rapid lateral flow immunoassay (ng biotech) immunoassays in comparison with microneutralisation clinical evaluation of serological igg antibody response on the abbott architect sars-cov-2 infection evaluation of performance of two sars-cov-2 rapid whole-blood finger-stick igm-igg combined antibody tests saint-louis core (covid research) group, evaluation of covid-19 igg/igm rapid test from orient gene biotech key: cord-017359-zr0bo9el authors: pfannschmidt, karlson; hüllermeier, eyke; held, susanne; neiger, reto title: evaluating tests in medical diagnosis: combining machine learning with game-theoretical concepts date: 2016-05-10 journal: information processing and management of uncertainty in knowledge-based systems doi: 10.1007/978-3-319-40596-4_38 sha: doc_id: 17359 cord_uid: zr0bo9el in medical diagnosis, information about the health state of a patient can often be obtained through different tests, which may perhaps be combined into an overall decision rule. practically, this leads to several important questions. for example, which test or which subset of tests should be selected, taking into account the effectiveness of individual tests, synergies and redundancies between them, as well as their cost. how to produce an optimal decision rule on the basis of the data given, which typically consists of test results for patients with or without confirmed health condition. to address questions of this kind, we develop an approach that combines (semi-supervised) machine learning methodology with concepts from (cooperative) game theory. roughly speaking, while the former is responsible for optimally combining single tests into decision rules, the latter is used to judge the influence and importance of individual tests as well as the interaction between them. our approach is motivated and illustrated by a concrete case study in veterinary medicine, namely the diagnosis of a disease in cats called feline infectious peritonitis. different types of tests, such as measuring serum antibody concentrations, are commonly used in medical diagnostics in order to reveal the health condition of an individual. the effectiveness of a single test is typically determined by correlating the test outcome with the true condition. moreover, classical statistical hypothesis testing can be used to compare different test procedures in terms of their effectiveness. in this paper, we tackle the problem of evaluating or selecting a test procedure from a slightly different perspective using methods of (semi-)supervised machine learning. roughly speaking, the idea is that, by learning a model in which various candidate tests play the role of predictor variables, information about the usefulness of individual tests as well as their combination is provided by properties of that model. an approach of that kind has at least two important advantages: -first, it not only allows for judging the usefulness of single tests but also of combined tests, i.e., the combination of different tests into one overall (diagnostic) decision rule. thus, it informs about possible synergies (as well as redundancies) between individual tests and the potential to improve diagnostic accuracy thanks to a suitable combination of these tests. -second, going beyond the standard setting of supervised learning, a machine learning approach suggests various ways of improving the selection of tests by taking advantage of additional sources of information. an important special case is the use of semi-supervised learning to exploit "unlabeled" data coming from individuals for which tests have been made but the true health condition is unknown. this situation is highly relevant in medical practice, because tests can often be conducted quite easily, whereas determining the true health condition is very difficult or expensive. our approach is motivated by a concrete case study in veterinary medicine, namely the diagnosis of a disease in cats called feline infectious peritonitis (fip). complete certainty about whether or not a cat is fip-positive, and eventually will die from the disease, requires a necropsy [1, 10] ; unfortunately, no test performed in a cat while still alive has a 100 % sensitivity or 100 % specificity. consequently, while different tests can be applied to cats quite easily, "labeling" a cat in the sense of supervised learning is expensive, difficult and time-consuming. in addition to the use of (semi-supervised) machine learning methodology in medical diagnosis, we propose a game-theoretical approach for measuring the usefulness of individual tests as well as model-based combinations of such tests. roughly speaking, the idea is to consider a combination of tests as a "coalition" in the sense of cooperative game theory, and the "payoff" of the coalition as the diagnostic accuracy achieved by the test combination. this approach will be detailed in the next section, prior to elaborating more closely on our case study in sect. 3, presenting experimental results in sect. 4 and concluding the paper in sect. 5. suppose a set of tests x 1 , . . . , x k to be available. we consider the outcome of each test as a random variable x k : ω −→ r, where ω is the population of individuals to which the test can be applied. jointly, the k tests thus define a random vector the health state is a dichotomous variable y ∈ y = {−1, +1}. typically, each test is a positive indicator in the sense that p(y = +1 | x k ) increases with x k , i.e., the larger x k , the larger the probability of the positive class. using machine learning terminology, each test corresponds to a feature or predictor variable. moreover, x is the instance space, each x ∈ x is an instance, and y is the (binary) output or response variable. if a diagnostic decisionŷ ∈ {−1, +1} is not necessarily based on a single test x k alone, but possibly uses a combination of several tests, a first question concerns the way in which such a combination is realized. from a machine learning point of view, this question is related to the choice of an underlying models class (hypothesis space) where j ≤ k is the number of tests included in the decision rule. formally, we specify a combined test in terms of the subset the model class h could be defined, for example, as the class of linear threshold functions of the form where w 1 , . . . , w j , t ∈ r + and · maps true predicates to +1 and false predicates to −1; moreover, σ(j) is the j-th test included in the combination, i.e., i.e., a decision rule that minimizes the loss in expectation. we denote the expected loss of this model, which corresponds to the bayes predictors in h |a| , by e * (a) = l y, h * a (x) d p(x, y).(2) in practice, of course, neither the bayes predictor h * a nor the ideal generalization performance e * (a) are known. instead, we only assume a data set d = d l ∪ d u to be given, which consists of a set of labeled instances and possibly another set of unlabeled instances (test results without ground truth) d u = {x j } u j=1 ⊂ x . from a machine learning point of view, it is then natural to estimate the generalization performance on the basis of d for each a ⊆ [k] . to this end, models (1) can be fitted and their generalization performance can be estimated, for example, using cross-validation techniques or the bootstrap. more specifically, what can be estimated in this way is the generalization performance of a model that is trained on a combination a and data in the form of l labeled and u unlabeled examples. therefore, we shall denote a corresponding estimate byê(a, l, u ) or simplyê(a) (assuming the underlying data to be given). needless to say, the estimatesê(a) thus obtained are not necessarily monotone in the sense thatê(b) ≤ê(a) for a ⊆ b. in fact, while e * (a) is the generalization performance of the bayes predictor, i.e., the model that is obtained in the limit of an infinite sample size (provided the underlying learner is consistent), the estimatesê(a) are obtained from models trained on a finite (and possibly small) data set. therefore, practical problems such as overfitting become an issue, i.e., including additional tests may deteriorate instead of improve generalization performance. how can the ideal generalization performances be estimated? starting with the finite-sample estimates our proposal is to correct these estimates so as to assure monotonicity. in fact, monotonicity is the main difference between the ideal and finite-sample scores. apart from that, the ideal scores (3) should not differ too much from the estimates (4), i.e., e * (a) ≈ê(a), at least if the training data is not too small. these considerations suggest the following estimation principle: find a set of values (3) that satisfy monotonicity while remaining as close as possible to the corresponding scores (4). this principle can be formalized as an optimization problem of the following kind: the above problem can be tackled by means of methods for isotonic regression. more specifically, since the inclusion relation on subsets induces a partial order on 2 [k] , methods for isotonic regression on partially ordered structures are needed [3, 14] . consider the set function ν : obviously, ν is a monotone measure (of the usefulness of combined tests). moreover, this measure can be normalized by setting where ν (∅) is the performance of the best (default) decision rule that does not use any test, i.e., which either always predictsŷ = +1 or alwaysŷ = −1. the measure ν * (·) thus defined satisfies the following properties: thus, ν * is a normalized, monotone (but not necessarily additive) set function, referred to as fuzzy measure or capacity in the literature [5] . for each combined test a, ν * (a) is a reasonable measure of the usefulness of this test. in a similar way, a measure v • can be defined on the basis of the finite-sample scores (4), that is, by normalizing ν where ν min = 1− max b⊆[k]ê (b) and ν max = 1− min b⊆[k]ê (b). note, however, that this measure is not necessarily monotone. which of the two measures is more meaningful, ν * or ν • ? the answer to this question depends on practical considerations and what the measure is actually supposed to capture. when being interested in the potential asymptotic usefulness of a test combination, then ν * is the right measure. otherwise, if a model induced from a concrete set of training data is supposed to be put into (medical) practice, ν • is arguably more relevant. from the point of view of (cooperative) game theory, each (test) combination a ⊆ [k] can be seen as a coalition and ν ∈ {ν * , ν • } as the characteristic function, i.e., v(a) is the payoff achieved by the coalition a. thanks to this view, we can take advantage of various established game-theoretical concepts for analyzing the importance of individual players, which correspond to tests in our case, as well as the interaction between them. in particular, the shapley value, also called importance index, is defined as follows [17] : the shapley value of ν is the vector ϕ(ν) = (ϕ(1), . . . , ϕ(k)). for monotone measures (such as ν = ν * ), one can show that 0 ≤ ϕ(k) ≤ 1 and k k=1 ϕ(k) = 1; thus, ϕ(k) is a measure of the relative importance of the test x k . the interaction index, as proposed by [13] , is defined as follows: this index ranges between −1 and +1 and indicates a positive (negative) interaction between the tests x i and x j if i i,j > 0 (i i,j < 0). it is worth mentioning that the approach put forward in this section is quite in line with the idea of shapley value regression [11] , which makes use of the shapley value in order to quantify the contribution of predictor variables in (linear) regression analysis (quantifying the value of a set of variables in terms of the r 2 measure on the training data). feline infectious peritonitis (fip) is a disease with an affinity to young cats, a predisposition to involve cats living in larger groups. as it exhibits typical physical examination and clinical laboratory findings, it appears to be easy to diagnose. however, while a presumptive diagnosis is quickly established, a definite diagnosis is difficult to impossible to obtain without gross and histopathological evaluation including immunohistochemistry [1, 10] . the seroprevalence is high, especially in catteries where up to 90 % of the cats are positive [2] , but also up to 50 % of cats living in single-cat households have coronavirus-specific antibodies [4] . of these, 5-10 % will develop the deadly form of fip. a characteristic symptom of fip is body cavity effusion, which also appears in other diseases [8] . several treatment options exist for some of these diseases while fip is deadly and no reliable effective therapy is known so far [16] . therefore, it is important to diagnose the correct disease early. several diagnostic tests are available that diagnose fip, for which sensitivity, specificity, positive and negative predictive value vary between different studies, presumably because different forms of fip (effusive and dry) were investigated and because various clinical signs, geographic locations, years of investigation, prevalence and combination of tests were used [4, 6, 7, 9, 15, 18] . in studies so far, no cat had all available tests performed. the data underlying our study includes the following diagnostic tests: -albumin to globulin ratio, plasma (x 1 ) and effusion (x 2 ) -rivalta test (x 3 ) -presence of antibodies against feline coronavirus (fcov, x 4 ) -reverse transcriptase nested polymerase chain reaction (rt-npcr) to detect fcov-rna in edta-blood (x 5 ) and in the effusion (x 6 ) -immunofluorescence staining (ifa) of fcov antigen in macrophages in the effusion (x 7 ) our dataset consists of 100 cats in total. for 29 of these cats, a necropsy was performed to establish the gold standard diagnosis; 11 of the 29 cats were diagnosed with feline infectious peritonitis (fip). additionally, the above 7 diagnostic tests were performed on all cats (i.e., k = 7, l = 29 and u = 71). to estimate the generalization accuracy (in terms of the simple 0/1 loss function) of each of the 2 7 = 128 combined diagnostic tests, we employ a semisupervised classification technique called maximum contrastive pessimistic likelihood estimation (mcpl) [12] . logistic regression with l 2 penalization is used as the base learner in mcpl, i.e., individual tests are combined using a linear model of the form (1). estimatesê(a) of the (finite-sample) classification errors are obtained as follows: we resample the set of 29 labelled cats and split the resulting sample into 16 training and 13 test examples. the remaining 71 cats without label information are added to the training set. this procedure is repeated 501 times for each of the 128 combinations of tests, and the results are averaged. to obtain estimates e * (a) of the ideal generalization performances, the finite-sample estimates are subsequently corrected using isotonic regression [3, 14] as described in sect. 2.4. to further illustrate the importance of the diagnostic test rt-npcr, fig. 2 shows the mean validated classification accuracy for all 128 test combinations. the 80 % empirical percentiles are indicated by the vertical lines, and the subsets are sorted in decreasing order of their mean validated accuracy. moreover, the results for those subsets including rt-npcr (measured in blood) are highlighted in blue. evidently, the concentration of subsets containing rt-npcr (blood) is systematically higher to the left of the plot, which confirms that the inclusion of the test improves diagnostic accuracy. the effect of isotonic regression on the finite-sample estimates is shown in fig. 3 . here, each blue dot corresponds to an estimateê(a) for a particular subset a of diagnostic tests. since partial monotonicity, which is assured by isotonic regression, cannot be visualized in a two-dimensional plot, the data points are sorted by their corrected classification accuracy (and ties are broken at random). the green line shows the isotonic regression fit. the corrected performance estimates ν * (a) can subsequently be used to calculate the shapley values for each diagnostic test. the results are shown in fig. 4 . due to the monotonicity of ν * , all values are now positive. again, the rt-npcr tests achieve the highest shapley values, but fcov antibody titer and ifa (effusion) obtain values > 0.15, too. note that the relative order of the rt-npcr tests changed from the one in fig. 1 , probably due to their accuracy being very similar and the random nature of the bootstrap validation. figure 5 shows the accuracy estimates for all subsets. the dots indicate the corrected accuracies ν * (a) and are used to sort subsets in decreasing order, while fig. 2) , the subsets containing rt-npcr (blood) can mostly be found on the left side of the plot; this trend is now even more pronounced. an important question for a veterinary physician is which combination a of tests to perform, taking into account both diagnostic accuracy and effort. figure 6 shows the corrected accuracies ν * (a) (green dots) of all subsets of tests and their combined monetary cost in euro. the pareto set, consisting of those combinations that are not outperformed by any other combination in terms of both accuracy and cost at the same time, is indicated as a blue line. from a practical point of view, the result suggests to use a single diagnostic test, namely rt-npcr (blood or effusion), because the inclusion of more tests yields only minor improvements. this is confirmed by the pairwise interaction indices shown for both measures ν • and ν * in table 1 . all these measures are negative, suggesting that the tests are more redundant than complementary. note that, once a decision in favor of using a single test is made, the shapley value, as a measure of average improvement achieved by adding a test, is no longer the best indicator of the usefulness of a test. instead, a selection should be made based on the tests' individual performance. with a validated accuracy of 87 %, rt-npcr (effusion) appears to be the best choice in this regard. in this paper, we proposed a method for measuring the importance and usefulness of predictor variables in (semi-/supervised) machine learning, which makes use of concepts from cooperative game theory: subsets of variables are considered as coalitions, and their predictive performance plays the role of the payoff. although our approach is motivated by a concrete application in veterinary medicine, namely the diagnosis of feline infectious peritonitis in cats, it is completely general and can obviously be used for other learning problems as well. for the case study just mentioned, our method produces results that appear to be plausible and agree with the medical experts' experience. roughly speaking, there are two strong diagnostic tests that are significantly more accurate than others; practically, it suffices to use one of them, since a combination with other tests yields only minor improvements. there are several directions for future work. for example, the principle we proposed in sect. 2.4 for inducing ideal generalization performances e * (a) from finite-sample estimatesê(a) is clearly plausible and, moreover, seems to be indeed able to calibrate the original estimates thanks to an ensemble effect. nevertheless, it calls for a more thorough analysis and theoretical justification. recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium prevalence of feline coronavirus types i and ii in cats with histopathologically verified feline infectious peritonitis structure algorithms for partially ordered isotonic regression performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases fundamentals of uncertainty calculi with applications to fuzzy inference comparison of different tests to diagnose feline infectious peritonitis using direct immunofluorescence to detect coronaviruses in peritoneal in peritoneal and pleural effusions sensitivity and specificity of cytologic evaluation in the diagnosis of neoplasia in body fluids from dogs and cats positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population a comparison of lymphatic tissues from cats with spontaneous feline infectious peritonitis (fip), cats with fip virus infection but no fip, and cats with no infection analysis of regression in game theory approach contrastive pessimistic likelihood estimation for semi-supervised classification techniques for reading fuzzy measures (iii): interaction index algorithms for a class of isotonic regression problems using direct immunofluorescence to detect coronaviruses in peritoneal and pleural effusions effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis a value for n-person games detection of ascitic feline coronavirus rna from cats with clinically suspected feline infectious peritonitis key: cord-001253-3jnkki5z authors: mohammad, fahim; theisen-toupal, jesse c.; arnaout, ramy title: advantages and limitations of anticipating laboratory test results from regressionand tree-based rules derived from electronic health-record data date: 2014-04-14 journal: plos one doi: 10.1371/journal.pone.0092199 sha: doc_id: 1253 cord_uid: 3jnkki5z laboratory testing is the single highest-volume medical activity, making it useful to ask how well one can anticipate whether a given test result will be high, low, or within the reference interval (“normal”). we analyzed 10 years of electronic health records—a total of 69.4 million blood tests—to see how well standard rule-mining techniques can anticipate test results based on patient age and gender, recent diagnoses, and recent laboratory test results. we evaluated rules according to their positive and negative predictive value (ppv and npv) and area under the receiver-operator characteristic curve (roc aucs). using a stringent cutoff of ppv and/or npv≥0.95, standard techniques yield few rules for sendout tests but several for in-house tests, mostly for repeat laboratory tests that are part of the complete blood count and basic metabolic panel. most rules were clinically and pathophysiologically plausible, and several seemed clinically useful for informing pre-test probability of a given result. but overall, rules were unlikely to be able to function as a general substitute for actually ordering a test. improving laboratory utilization will likely require different input data and/or alternative methods. laboratory testing is the single highest-volume medical activity [1] . its main role is to help adjust the level of clinical suspicion of a diagnosis to help rule it in or out; it is also used for disease monitoring. in practice, the level of clinical suspicion and the probability of a given test result can be correlated: the higher the suspicion, the more likely it is that the result will confirm the diagnosis. information that feeds into the clinical suspicionincluding the age and gender of the patient, prior diagnoses, and prior laboratory results-thus may also influence the test result. in principle, this relationship can be used to improve laboratory testing by making it possible to estimate the pre-test probability of getting a given test result before ordering the test, and, in the limit, to reduce test utilization without adversely affecting patient outcomes. indeed, ordering fewer tests, where warranted, might benefit outcomes by saving the patient the burden of following up false positives (or negatives) [2] [3] [4] . conceptually, the relationship between clinical suspicion and pre-test probability is used routinely to help set guidelines regarding when and when not to order a given test. for example, the pre-test probability of lyme serology being positive given a targetoid rash is high enough that, given the test's sensitivity and specificity, ordering the test is contraindicated [5] . because of the large number of tests and clinical scenarios that exist, and in light of evidence from across medicine that utilization of laboratory testing can be improved [1, 6] , it is of interest to understand whether analyzing large clinical databases using the robust application of standard statistical techniques can turn this relationship into actionable decision-support rules-or whether progress toward better laboratory utilization might instead lie elsewhere. we sought to test the limits of rule-mining for this purpose. to what extent can laboratory results be anticipated computationally based on data available to the clinician, or a clinical decision support system, at the time of the order? we addressed this question using generalized linear modeling (glm), a generalized form of linear regression [7] , and, for comparison, classification trees (ct) [8, 9] . we used four types of input-age, gender, diagnoses (three-digit icd-9 codes), and results of laboratory tests on blood samples added to the record in the seven days before a given test was ordered-to build simple, robust models for whether the result of a test would be within the reference interval (''normal'') or outside of it in a given direction (''abnormal''), treating high and low results separately. we based our study on 10 years of records from the beth israel deaconess medical center (bidmc), a 585-bed tertiary care center in boston, ma. we first anonymized records and reconciled test names (work approved by bidmc committee on clinical investigation's institutional review board for research involving human subjects, protocol 2012-p-000229/ 01). informed consent was not obtained because patient records/ information was anonymized prior to analysis. each blood test (the test of interest), over 69.4 million in all, was marked as an in-house test (performed at the hospital) or a sendout (performed off-site). for each test, we compiled a list of all instances in which the test was ordered and performed. for each instance, we recorded the patient's age, gender, and any diagnoses or other blood-test test results from the seven days prior to the result of interest. when a test was ordered multiple times within a seven-day period, we considered only the most recent one (i.e., the one closest in time to the sendout order) as input data. for relevance, we considered only those tests that were ordered at least 1,000 times over the entire 10-year period, for an average of at least twice a week. we randomly divided the resulting instances into a training set and a test set (see below for details). all tests had either two (reference vs. abnormal) or three (low, normal, or high) possible response values. for tests with three values, we performed two separate rule searches: one for high vs. not high-i.e., grouping normal and low-and one for low vs. not low. we sought to identify simple, robust subsets of our input data to evaluate as linear predictors (''rules'') for whether a test result would be normal or abnormal. to do this, we used glm twice: first to find rules based on a particular training set and a second time to find rules based on just those items that were common to rules found from a number of different training sets (to avoid overfitting any one training set). we did this as follows, for each test of interest (the response variable or ''response''). we first excluded those input variables (''features'') that appeared with fewer than 5 percent of the response. we then temporarily set aside the most common features (those of the complete blood count and basic metabolic panel) as well as age and gender, and searched the remaining items for frequent featuresets (using the apriori algorithm [10, 11] ). we then added back to each resulting featureset the common features, age, and gender (which are frequent items by definition, since they appear in all instances) with a support threshold of 0.60 (i.e., itemsets for which all items were present with at least 60 percent of instances of the response variable). this set-aside/add-back approach sped the search for featuresets without loss of comprehensiveness. we used each featureset to create a model for the test of interest using r's glm function (with the family argument set to ''binomial''). we used backward feature elimination to remove non-significant features one at a time from the featureset (using a significance threshold p-value of 1610 25 ; see below) until the only features that remained were all significantly correlated with the response. we also removed features that are used to calculate the result for the test of interest-e.g., cd4 and cd8 count for t-cell count, which is the sum of cd4 and cd8-for all but proof-ofprinciple runs. the significance threshold was corrected for multiple comparisons by dividing the traditional threshold of p = 0.05 by the product of the total number of tests considered and the average number of rules generated for each test. the combined total number of features (in-house tests plus sendout tests plus diagnoses) was 170+81+434 = 685. the average number of rules after application of glm for the first time for each test is 6. thus our threshold p-value was 0.05/(6*685) = 1.2610 25 , which we rounded to 1610 25 . we constructed a model for the result by running glm a second time on a training set (see below) based on this reduced featureset. of note, there was no guarantee that any feature would be significantly correlated (p#1610 25 ) or that there would be enough instances (glm's threshold was 200) of the test appearing with all features of even the reduced featureset for glm to produce a model. when feature elimination resulted in no significant features or too few instances, no model was constructed. we scored models using ppv, npv, and roc auc. we were interested only in models that were robust to the size and choice of training set. therefore we repeated the above process for a range of training set-test set splits (80-20, 70-30, 60-40, 50-50, 40-60, 30-70, and 20-80 percent). for each split, we ran the above process 10 times and found the number of rules with auc$0.75. we decided on using a 60-40 split for downstream analyses as this split generated a total number of rules comparable to 70-30 and 80-20 splits but with less training data (fig. 1) . finally, for each test of interest, we selected features that appeared in a strict majority of rules for that test and reran glm using only those features. this made rules both simpler and more robust by removing features whose presence was contingent on a particular choice of training or test set. for each of the inhouse and sendout tests we used cart, implemented as rpart in r (rpart v3.1-50; cran.r-project.org/ package = rpart), to predict the response from all input features, using 60:40 training:test-set splits. we fixed some of the metrics (see below) that were used in building the final tree. the cart grows classification tree in two stages. in stage one, a tree is grown by finding a feature which best splits the data into two groups. splitting is done only if the overall ''impurity,'' the number of outcomes different from the majority (e.g., a ''low'' response alongside many ''normal'' responses), decreases, above some threshold (the ''complexity parameter;'' 0.01). then, in top-down fashion, these two subgroups are further divided in a recursive manner until the subgroups reach a minimum size (minsplit = 20 records) or until no further improvement can be made. the resulting tree may overfit the training data. to avoid this, crossvalidation (xval = 10; 10-fold cross-validation) was used in the second stage by pruning the tree. we fixed the maximum depth (maxdepth) of the tree, i.e., the maximum number of branchings from stem to leaf, to be 20. the final models were tested on the test data and performance statistics are found. we repeated modelbuilding 10 times for each test and summarized the statistics. data-processing was performed in python (enthought canopy python version 2.7.3. r (version 2.15.3) was used for statistical analysis and reports generation. to determine how well sendout and in-house test results can be anticipated based on basic information available in the medical record, we used two independent methods-generalized linear modeling (glm) and classification and regression trees (cart)to build simple, robust test-result predictors and then evaluated the performance of these predictors according to the standard clinical metrics of positive predictive value (ppv) and negative predictive value (npv), as well as sensitivity and specificity via the receiveroperator curve (roc) area under the curve (auc). as proof of principle for glm, we first tested it on the anion gap, a result calculated by subtracting the serum concentrations of the anions chloride and bicarbonate from those of the cations sodium and potassium, and confirmed that our methods found a rule for elevated anion gap based on these four items. we next applied glm to 81 sendout tests ordered regularly at our hospital. glm generated rules for just 11 of these tests. for the remaining tests, either no recent diagnosis or in-house test result (or age or gender) was sufficiently correlated with the sendout test result, or there were not enough instances in which correlated items appeared with the result, to generate a rule. only two tests-for high corticotropin (acth) and for low ceruloplasmin-had npv$0.95. of these, ceruloplasmin had a ppv$0.94. the mean auc for all rules was 0.69, with models for only three tests having an average auc$0.75 over 10 repeat runs. removal of features that did not appear in a majority of rules had essentially no effect on these aucs (difference in mean auc#0.02). cart generated rules for 60 tests. however, the auc for most of these rules was low, with only five tests having auc$0.75: free t3, alpha-macroglobulin, ca27-29, hyaluronic acid, and alpha fetoprotein (auc 0.75-0.79). we next applied glm to in-house tests. a total of 170 in-house tests were analyzed. a number of rules exhibited a high ppv (the probability of seeing an abnormal value given a prediction of an abnormal value by the rule) or npv (the probability of seeing a normal value given prediction of a normal value). these were mostly components of the complete blood count (cbc) and metabolic panels. interestingly, the predictive power of these rules was almost exclusively based on a previous measurement of the test in question: in other words, the best rules were for repeat tests, and the best predictor of a result being normal or abnormal was whether it had been normal or abnormal within the previous seven days. for example, the npv for a low red blood cell count was 0.95 (with ppv = 0.75), with a rule that depended most on the previous red blood cell count also having been low, and the ppv for high total calcium was 0.98 (npv = 0.76) and based exclusively on the previous total calcium having been high. for comparison, we applied cart to in-house tests, again including in the input data the most recent result for that test if performed within a week of the order. again, a number of rules exhibited a high ppv ($0.95), and again these were often tests of the cbc and metabolic panels, with rules based almost exclusively on a previous abnormal value. examples included low white blood cell count (wbc; ppv = 0.97, npv = 0.79), platelet count (0.95, 0.88), and serum sodium (0.96, 0.65), and high total calcium (0.99, 0.67), mean corpuscular volume (0.98, 0.84), and iron (0.97, 0.56) all of which were determined almost exclusively from the previous value being low or high (table 1) . overall, there was good agreement in ppv between glm and cart for tests for which both methods found rules, but cart outperformed glm noticeably in npv (fig. 2 ). the growing availability of large clinical databases has raised interest in the possibility of using systematic rule-mining for clinical decision support [12] [13] [14] [15] . one popular and well characterized approach has been logistic regression [16] [17] [18] , a special case of generalized linear modeling (glm). researchers have applied these approaches for diverse health-related purposes including prediction of cardiovascular risk [19] , mortality in head trauma [18] , texture analysis of magnetic resonance images [16] , and many other applications [17, 20, 21] . however, we note that glm does not easily incorporate missing values, as it removes records with missing features; a feature will be ''missing'' for any record in which that test (the feature) was not performed. other methods, such as classification and regression trees (cart) and artificial neural networks [18] , have also been applied. most of these studies were limited in scope to predicting risk of a particular diagnosis. harper [22] compared four classification techniques (regression, cart, artificial neural networks, and discriminant analysis) on four different datasets and concluded that there was no obvious best choice for their data; while cart performed best, regression was fastest and nearly as good. similar comparative studies on coronary artery disease [20] and alzheimer disease [23] indicated that newer algorithms such as ann and random forests [24] have little advantage over simpler, more traditional approaches. also, the utility and limitations of these approaches for predicting laboratory results (as opposed to diagnoses) are unclear. however, while cart is both a top performer and overcomes glm's problem with missing values, it is also more computationally intensive and potentially less sensitive to simple algebraic relationships among features (e.g., among sodium, chloride and bicarbonate and the anion gap). therefore we chose glm as a well-understood approach with strong performance and excellent speed, and cart as the bestperforming complementary approach for purposes of comparison. given the importance of laboratory testing, we asked how much information regression-or classification tree-based rules could provide in assessing the pre-test probability of a test result being abnormal for 251 commonly ordered in-house and sendout tests at our hospital. data-mining can sometimes find spurious correlations, artifacts of the particular partitioning of the data into training and test set. to avoid such artifacts, we repeated our regression on multiple independent partitions of the data and kept only items that appeared in a majority of the resulting rules. this safeguard also had the effect of simplifying rules by making each rule dependent on a smaller number of items. as expected, the effect on performance was negligible and dependence on the resulting items was more often clinically and pathophysiologically plausible than rules derived from each run. when data-mining it is also important to consider the setting. the rules we found do not exist in a vacuum but are ''contingent'' in the sense that they depend on current clinical practice. certain tests and panels are ordered in patterns. in a sense, contingency is a form of selection bias: there may well be other diagnoses or test result results that correlate with the result for the test of interest that are not routinely measured according to current best practices. however, as long as the setting in which such rules would be applied is the substantially similar to that in which they were found, selection bias would have little if any effect on finding rules. as long as one is clear that one is looking for relationships in a current practice process, and not among all things that could possibly be measured, any rules that are discovered will by construction be setting-appropriate. but while our rules appear to be plausible and settingappropriate, the motivating question behind this study is whether the rules we found could be useful clinically. one way to approach this question is by considering the positive and negative predictive value of each rule (ppv and npv). these metrics are in contrast to sensitivity and specificity, by which rules are often measured but which do not incorporate disease prevalence in spite of its importance to clinical decision-making. a ppv of 0.95 means that when a rule suggests that the test result will be abnormal, the result actually will be abnormal 95 percent of the time. a npv of 0.95 means that when a rule suggests that the test result will be normal, the result actually will be normal 95 percent of the time. we found rules with ppv and/or npv$0.95 (by glm) for only two tests that are sendouts at our hospital-one of which is ceruloplasmin, which we have previously suggested is overordered via chart review [25] . in contrast, for in-house tests we found over a dozen such rules. interestingly, the main determinant for rules for in-house tests was a normal or abnormal result for the same test within the previous seven days. although in this study we did not set out explicitly to make a statement about repeat laboratory testing, the appropriateness of which has been investigated elsewhere [4] , these results suggest that repeat laboratory testing within one week does not always add information that could not have been anticipated from the previous result. refining this observation using the same unbiased approach we have followed here is potentially an area for future investigation. our results should not be taken as a categorical criticism of repeat testing. first, while the ppv was $0.95 in several cases, the npv was more typically 0.70-0.85. thus, while prediction that a result will be abnormal may be correct 95 percent of the time, which may be good enough to discourage repeat ordering, prediction that a result will be normal may not be so dependable. therefore use of a rule depends on the subtle distinction of whether the clinical question is ''will the result be abnormal'' vs. ''will the result be normal.'' second, we note that no rules with such strong performance were found for the majority of our sendout or in-house tests by either of our two complementary approaches. thus while the rules we found can inform clinical decision-makers, the information they provide rarely replaces the information obtained from actually performing these tests. it is interesting to note that on average, our simple rules yielded a ppv of 0.84 and an npv of 0.75. this means that on average, rules will correctly predict an abnormal laboratory result 5 times out of 6 (5/6<0.84) and correctly predict a normal result 3 times out of 4. while not good enough to replace testing (especially for rules that depend on previous test results), these observations raise the question of how much better prediction can get. integration of information not considered in the present study, including vital signs, chief complaints, and physical findings, may improve prediction by these methods. the ulysses syndrome the dangers of false-positive and false-negative test results: false-positive results as a function of pretest probability the landscape of inappropriate laboratory testing: a 15-year systematic review and meta-analysis laboratory evaluation in the diagnosis of lyme disease elementary, my dear doctor watson generalized linear models electronic health record surveillance algorithms facilitate the detection of transfusion-related pulmonary complications using classification trees to assess low birth weight outcomes fast algorithms for mining association rules fast discovery of association rules data mining and clinical data repositories: insights from a 667,000 patient data set mining association rules from clinical databases: an intelligent diagnostic process in healthcare machine learning for personalized medicine: predicting primary myocardial infarction from electronic health records predictive data mining in clinical medicine: current issues and guidelines textural analysis of contrast-enhanced mr images of the breast establishing a clinical decision rule of severe acute respiratory syndrome at the emergency department comparison of artificial neural network and logistic regression models for prediction of mortality in head trauma based on initial clinical data improved cardiovascular risk prediction using nonparametric regression and electronic health record data comparing performances of logistic regression, classification and regression tree, and neural networks for predicting coronary artery disease predicting improvement in urinary and bowel incontinence for home health patients using electronic health record data a review and comparison of classification algorithms for medical decision making application and comparison of classification algorithms for recognition of alzheimer's disease in electrical brain activity (eeg) random forests the overuse of serum ceruloplasmin measurement key: cord-316047-d9cpe9yl authors: gonzalez, t.; de la rubia, m. a.; hincz, k. p.; comas-lopez, m.; subirats, laia; fort, santi; sacha, g. m. title: influence of covid-19 confinement on students’ performance in higher education date: 2020-10-09 journal: plos one doi: 10.1371/journal.pone.0239490 sha: doc_id: 316047 cord_uid: d9cpe9yl this study analyzes the effects of covid-19 confinement on the autonomous learning performance of students in higher education. using a field experiment with 458 students from three different subjects at universidad autónoma de madrid (spain), we study the differences in assessments by dividing students into two groups. the first group (control) corresponds to academic years 2017/2018 and 2018/2019. the second group (experimental) corresponds to students from 2019/2020, which is the group of students that had their face-to-face activities interrupted because of the confinement. the results show that there is a significant positive effect of the covid-19 confinement on students’ performance. this effect is also significant in activities that did not change their format when performed after the confinement. we find that this effect is significant both in subjects that increased the number of assessment activities and subjects that did not change the student workload. additionally, an analysis of students’ learning strategies before confinement shows that students did not study on a continuous basis. based on these results, we conclude that covid-19 confinement changed students’ learning strategies to a more continuous habit, improving their efficiency. for these reasons, better scores in students’ assessment are expected due to covid-19 confinement that can be explained by an improvement in their learning performance. the coronavirus covid-19 outbreak disrupted life around the globe in 2020. as in any other sector, the covid-19 pandemic affected education in many ways. government actions have followed a common goal of reducing the spread of coronavirus by introducing measures limiting social contact. many countries suspended face-to-face teaching and exams as well as placing restrictions on immigration affecting erasmus students [1] . where possible, traditional classes are being replaced with books and materials taken from school. various e-learning platforms enable interaction between teachers and students, and, in some cases, national television shows or social media platforms are being used for education. some education systems announced exceptional holidays to better prepare for this distance-learning scenario. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in terms of the impact of the covid-19 pandemic on different countries' education systems many differences exist. this lack of homogeneity is caused by such factors as the start and end dates of academic years and the timing of school holidays. while some countries suspended in-person classes from march/april until further notice, others were less restrictive, and universities were only advised to reduce face-to-face teaching and replace it with online solutions wherever practicable. in other cases, depending on the academic calendar, it was possible to postpone the start of the summer semester [2] . fortunately, there is a range of modern tools available to face the challenge of distance learning imposed by the covid-19 pandemic [3] . using these tools, the modification of contents that were previously taught face-to-face is easily conceivable. there are however other important tasks in the learning process, such as assessment or autonomous learning, that can still be challenging without the direct supervision of teachers. all these arguments end in a common topic: how to ensure the assessment's adequacy to correctly measure students' progress. thus, how can teachers compare students' results if they differ from previous years? on one hand, if students achieve higher scores than in previous years, this could be linked with cheating in online exams or with changes in the format of the evaluation tools. on the other hand, lower grades could also be caused by the evaluation format change or be attributable to autonomous learning as a less effective teaching method. the objective of this article is to reduce the uncertainty in the assessment process in higher education during the covid-19 pandemic. to achieve this goal, we analyze students' learning strategies before and after confinement. altogether, our data indicates that autonomous learning in this scenario has increased students' performance and higher scores should be expected. we also discuss the reasons underneath this effect. we present a study that involves more than 450 students enrolled in 3 subjects from different degrees from the universidad autónoma de madrid (spain) during three academic years, including data obtained in the 2019/2020 academic year, when the restrictions due to the covid-19 pandemic have been in force. e-learning has experienced significant change due to the exponential growth of the internet and information technology [4] . new e-learning platforms are being developed for tutors to facilitate assessments and for learners to participate in lectures [4, 5] . both assessment processes and self-evaluation have been proven to benefit from technological advancement. even courses that solely offer online contents such as massive open online courses (moocs) [6, 7] have also become popular. the inclusion of e-learning tools in higher education implies that a greater amount of information can be analyzed, improving teaching quality [8] [9] [10] . in recent years, many studies have been performed analyzing the advantages and challenges of massive data analysis in higher education [11] . for example, a study of gasevic et al. [12] indicates that time management tactics had significant correlations with academic performance. jovanovic et al also demonstrated that assisting students in their management of learning resources is critical for a correct management of their learning strategies in terms of regularity [13] . within few days, the covid-19 pandemic enhanced the role of remote working, e-learning, video streaming, etc. on a broad scale [14] . in [15] , we can see that the most popular remote collaboration tools are private chat messages, followed by two-participant-calls, multiperson-meetings, and team chat messages. in addition, several recommendations to help teachers in the process of online instruction have appeared [16] . furthermore, mobile learning has become an alternative suitable for some students with fewer technological resources. regarding the feedback of e-classes given by students, some studies [17] point out that students were satisfied with the teacher's way of delivering the lecture and that the main problem was poor internet connection. related to autonomous learning, many studies have been performed regarding the concept of self-regulated learning (srl), in which students are active and responsible for their own learning process [18, 19] as well as being knowledgeable, self-aware and able to select their own approach to learning [20, 21] . some studies indicated that srl significantly affected students' academic achievement and learning performance [22] [23] [24] . researchers indicated that students with strongly developed srl skills were more likely to be successful both in classrooms [25] and online learning [26] . these studies and the development of adequate tools for evaluation and self-evaluation of learners have become especially necessary in the covid-19 pandemic in order to guarantee good performance in e-learning environments [27] . linear tests, which require all students to take the same assessment in terms of the number and order of items during a test session, are among the most common tools used in computerbased testing. computer adaptive test (cat), based on item response theory, was formally proposed by lord in 1980 [28] [29] [30] , as is the case with linear testing. some platforms couple the advantages of cat-specific feedback with multistage adaptive testing [38] . the use of cat is also increasingly being promoted in clinical practice to improve patient quality of life. over the decades, different systems and approaches based on cat have been used in the educational space to enhance the learning process [39, 40] . considering the usage of cat as a learning tool, establishing the knowledge of the learner is crucial for personalizing subsequent question difficulty. cat does have some negative aspects such as continued test item exposure, which allows learners to memorize the test answers and share them with their peers [41, 42]. as a solution to limit test item exposure, a large question bank has been suggested. this solution is unfeasible in most cases, since most of the cat models already require more items than comparable linear testing [43]. the aim of this study is to identify the effect of covid-19 confinement on students' performance. this main objective leads to the first hypothesis of this study which can be formulated as h1: covid-19 confinement has a significant effect on students' performance. the confirmation of this hypothesis should be done discarding any potential side effects such as students cheating in their assessment process related to remote learning. moreover, a further analysis should be done to investigate which factors of covid-19 confinement are responsible for the change. a second hypothesis is h2: covid-19 confinement has a significant effect on the assessment process. the aim of the project was therefore to investigate the following questions: 1. is there any effect (positive or negative) of the covid-19 confinement on students' performance? 2. is it possible to be sure that the covid-19 confinement is the origin of the different performance (if any)? 3. what are the reasons for the differences (if any) in students' performance? 4. what are the expected effects of the differences in students' performance (if any) in the assessment process? we have used two online platforms. the first one is e-valuam [44] , an online platform that aims to increase the quality of tests by improving the objectivity, robustness, security and relevance of assessment content. e-valuam implements all the cat tests described in the following sections. the second online platform used in this study is the moodle platform provided by the biochemistry department from universidad autónoma de madrid, where all the tests that do not use adaptive questions are implemented. adaptive tests have been used in the subjects "applied computing" and "design of water treatment facilities". traditional tests have been used in the subject "metabolism". 2.1.1 cat theoretical model. let us consider a test composed by n q items. in the most general form, the normalized grade s j obtained by a student in the j-attempt will be a function of the weights of all the questions α and the normalized scores ψ (s j = s j (α, φ)), and can be defined as: where the φ i is defined as where δ is the kronecker delta, a i the correct answer and r i the student's answer to the i-question. by using this definition, we limit φ i to only two possible values: 1 and 0; φ i = 1 when the student's answer is correct and φ i = 0 when the student gives a wrong value. this definition is valid for both open answer and multiple-choice tests. in the case of multiple-choice test with n r possible answers, φ i can be reduced to consider the random effect. in this case: independently of using eqs 2 or 3, to be sure that s j (α, φ) is normalized (i.e. 0< = s j (α, φ)< = 1), we must impose the following additional condition on α: in the context of needing a final grade (fg) between 0 and a certain value m, which typically takes values such as 10 or 100, we just need to rescale the s j (α, φ) value obtained in our model by a factor k, i.e. fg j = k s j (α, φ). we will now include the option of having questions with an additional parameter l, which will be related to the level of relevance of the question. l is a number that we will assign to all the questions included in the repository of the test (i.e. the pool of questions from where the questions of a j-test will be selected). the concept of relevance can take different significances depending on the context and the opinion of the teachers. in our model, the questions with lower l values will be shown initially to the students, when the students answer correctly a certain number of questions with the lower l value, the system starts proposing questions from the next l value. by defining n l as the number of possible l values, the l value that must be obtained in the k-question of the j-test can be defined as: where trunc means the truncation of the value between brackets. it is worth noting that l k is proportional to the sum of the student's answers to all the previous questions in the test. this fact means that, in our model, the l k depends on the full history of answers given by the student. l k is inversely proportional to n q , which means that it takes a higher number of correct answers to increase l k . once l k is defined, a randomly selected question is shown to the student. another important fact that implies the use of eq 5 in the adaptive test is that we will never have l k <l k-1 . in other words, once a student starts answering questions from a certain l value, they will never go back to the previous one. in fig 1 we show a simple example of the multiple possible paths that a student can follow when facing a test that uses our model. in the example shown in fig 1 we have used n q = 6 and n l = 3. in this very straightforward example, the l value changes every time a student gives 2 correct answers (following diagonal arrows on the diagram). wrong answers imply a vertical drop on the diagram. in this case, the l value will not change (l k = l k-1 ). the final student's grade g is shown on the lower part of the diagram. the lowest grade (g = 0) corresponds to a test where the student has failed all the n q questions. in this case, l = 1 for all the possible j values. the highest grade (g = 6) implies that the student has faced questions from all the possible l values (l = {1,2,3} in fig 1) . ma-ts are used for evaluation and self-evaluation of theoretical contents. in this case, we use a standard format where a single correct answer must be chosen from a pool of possible answers shown by the system. since students can sometimes answer correctly by a random selection, eq (3) must be used when evaluating the score of any item. the format of the ma-t in our cat method requires the following elements: in addition, the e-valuam platform, where the method proposed in this article has been implemented, allows optional use of images or sounds for both the statement and the answers. e-valuam shows the statement and the possible answers. all other information relating to the cat method such as the level of the item is hidden from the student. the interface also shows optional feedback information about the performance of the learner in the previous item. finally, it shows the time remaining to finish the test. to train the contents of numerical problems, an oa-t was developed in which the statements include at least one parameter that will change its value with each execution of the application. this kind of question requires the following elements to be created: • statement with explicit indication of the modifiable parameter(s). • minimum and maximum values of each modifiable parameter. • programming code (matlab in e-valuam) that calculates the solution to the problem. • level of the item. as with ma-t, there is also a possibility of including multimedia files in the item. however, in this type of question, the multimedia option is only available in the question as the answer is numerical and included by the user in all cases. in this case the interface has a space for entering the numerical answer. in this figure, we also show the feedback of the application when an incorrect previous answer has been introduced. traditional tests have been used in the subject "metabolism" from the human nutrition and dietetics degree. the course contents are divided into 6 parts corresponding to different aspects of human metabolism. each part is taught with face-to-face lectures followed by different on-line activities that the students perform in the moodle platform and that are later discussed between them and with the professor in face-to-face workshops. all 6 parts of the course have a similar format, being a lecture followed by a selection of online activities in this order: "exercises workshop", "discussion workshop", "self-assessment" and "test". except for the discussion workshops outlined below, all activities are on-line questionnaires (closed, multiple-choice questions) that the students must perform in moodle, achieving a score that is automatically calculated and showed to them as part of their continuous assessment. exercises workshops and self-assessments are completed by the students without tutor supervision, whereas the tests are done on-line but under controlled conditions, just in the same way as a regular examination. each student should perform a total of 15 online activities: 5 exercises workshops, 6 self-assessment activities and 4 tests. discussion workshops are practical cases that must be debated by the students and sent to the tutor on moodle. as these activities do not obtain an automatic score but require a correction by the professor, they have not been considered in this study. the control group of our study is formed by the students of "applied computing" and "metabolism" from academic years 2017/2018 and 2018/2019 and by students of "design of water treatment facilities" from academic year 2017/2018. in the case of "design of water treatment facilities", a longitudinal study has been performed in academic year 2017/2018 to analyse the effect of rewards in the students' learning strategies, especially those related to time management. the experimental group of our study are students of "applied computing" and "metabolism" from academic year 2019/2020. in the longitudinal study of "design of water treatment facilities", experimental group corresponds to the third stage of the study. to answer our research questions, we first set up an experiment that obtains accurate measurements of the autonomous learning activities both in the control and the experimental groups. high accuracy in the measurements of the autonomous learning activities is achieved by using the previously described adaptive tools both in learning and assessment. in our experiment, the evaluation format and the e-learning tools are known to the students from the start, which motivates them to use the tools and perform the tests more consistently. in fig 2 we show the procedure used in our experiments to measure autonomous learning. there are two kinds of contents in the subjects included in the experiment: theory and numerical problems. in the case of theory, students can use the e-valuam platform through ma-t. the final evaluation is performed by oa-t. in the case of numerical problems, both learning and evaluation use oa-t. in this case, student motivation for using the platform is even higher because their final scores will be obtained by the same tool they use for their autonomous learning. this system is applied in all the experiments related to analysing the autonomous learning activity. in the case of "design of water treatment facilities", an additional longitudinal study has been performed and is shown in fig 2. this study has been performed in three stages. in the first stage, the self-evaluation material was presented to the students right before the evaluation test. in the second stage, the self-evaluation material was available 3 weeks before. to manage the theory contents, a ma-t with three possible answers to each question and only one correct has been created. in the first stage, with the test made available only one day before the exam, students had at their disposal a ma-t composed of 16 questions and with a time limit of 15 minutes. in the second stage, the time available was increased to 30 minutes and the question number to 18. these changes are related to the nature of the subject contents and do not have a significant impact on the study. the oa-t tests had 6 and 9 questions in the first and second stage, respectively. in order to maintain consistency in the subsequent study, an additional effort has been made to keep the difficulty of the questions as similar as possible. stage 3 had no new material added, instead, it used all the material created in stages 1 and 2. stage 3 corresponded to the time window between the end of classes and the last exam. in this stage, all the material was available from the beginning. the protocol used in this stage only varied from stage 2 in the inclusion of a reward for students who used the application on 3 or more different days. the reward was related to a bonus in the final grades. once the correct measure of the autonomous learning is ensured, as explained in the previous section, we must develop an experiment to describe the effect of covid-19 confinement. this experiment compares results of control and experimental groups in the whole assessment process. there are two stages that must be considered to determine the effect of the covid-19 confinement. the first one corresponds to the period without confinement in the year 2019/2020 (before march 11), when all the measurable activities are performed in similar conditions for experimental and control groups. the second stage corresponds to the period of covid-19 confinement (after march 11), where some measurable activities were performed in a different format and statistical differences can be found by comparing experimental and control groups. in the case of "applied computing", students use the e-valuam application on a continuous basis as it is available from the beginning of the course. they use the same oa-t for training and for their final exam, ensuring often and consistent use of the platform throughout the course. for this reason, in "applied computing", data analysis is performed over a continuous self-evaluation process over a single test along the whole academic year. the test performed by the students did not change its format or available questions in the whole three years under study. in the case of "metabolism", the confinement started just before the beginning of part iv of the subject. therefore, in this second stage, the scheduled in-person lectures from part iv were recorded on video by the tutor and provided to the students. afterwards, the students had an opportunity to perform the corresponding on-line continuous assessment activities, just in the same way as they would have done under normal conditions (as these activities were always not face-to-face). as this subject has already had an important number of on-line activities programmed prior to the suspension of face-to-face teaching, it is an extremely valuable tool for analysing the effect of the confinement on students' performance. in fig 3, we show the full experiment described in this section. first, the two studies performed in years prior to covid-19 confinement. in these first studies, we focus on the learning strategies of the students and their response to reward scenarios. information from these first studies will be useful for later discussion of the reasons for the changes caused by the covid-19 confinement. the second part of our experiment is related to the effect of covid-19 confinement, where we compare results of similar assessment activities between control and experimental groups. in fig 3 we include information about all control and experimental groups in the different interventions. this work involves data that has been collected from 458 students of the universidad autónoma de madrid. this data has been collected in the following manner: 1. it does not involve minors. 2. it has been collected anonymously. students have been identified by a numerical code, avoiding gathering of any personal information. 3. students have been informed by the lecturers that some information about their activity could be anonymously collected for statistical purposes. authors of this study did not receive any objections. 4. the tasks related to this study were completely voluntary and they did not in any form alter students' activities, classes, or the assessment process. considering these circumstances, we have received confirmation from the research ethics committee of universidad autónoma de madrid that we do not need to apply for ethics approval from our university since no personal data, minors or potentially hazardous activities were involved in the study. teachers involved in the study (coauthors of the present manuscript) who were responsible for the subjects taught also gave consent to carry out the study. we obtained verbal consent from the participants in the study. experiments have been performed in the subjects "applied computing", "metabolism" and "design of water treatment facilities". 2.3.1 applied computing. this study with real students took place during the 2017/2018, 2018/2019 and 2019/2020 academic years in the first-year subject "applied computing". 97, 73 and 91 students were involved each academic year, respectively. this subject was taught through theory lessons and practical classes in the computer laboratory. this course corresponds to 6 ects and belongs to the chemical engineering degree in the faculty of sciences from universidad autónoma de madrid, spain. due to covid-19 pandemic, the face-to-face teaching was cancelled on march 11, having a strong impact on the 2019/2020 course since it started in the first week of february and lasted until mid-may. the data for this study has been obtained from real students enrolled on the "metabolism" course in the human nutrition and dietetics degree from universidad autónoma de madrid, spain. this is a 6 ects compulsory subject taught during the second semester of the first year of the degree. the study comprises data from 2017/2018 and 2018/ 2019 academic years, with 64 and 63 enrolled students, respectively, and from the present 2019/2020 academic year (47 students), which has been strongly affected by the restrictions caused by the covid-19 pandemic. this year, the course started on january 28 and finished on april 30. face-to-face teaching cancellation and beginning of confinement occurred approximately halfway through the semester. the experiment with real students took place during the 2017/2018 academic year. 23 students were involved in the optional fourth-year subject "design of water treatment facilities". this subject was taught through theoretical and practical classes in the classroom and laboratory, respectively. our study focused on the theoretical part of the subject. this course corresponds to 6 ects and belongs to the chemical engineering degree in the faculty of sciences of the universidad autónoma de madrid, spain. 2.4.1 applied computing. in this subject we have expressed student performance as a 0-10 score and analyzed it across all tests performed in self-evaluation. those oa-t format tests were available from the beginning of the academic year and students used them on a continuous basis. data from this subject imply results from an adaptive test. this imply that there are some points in the test where students get stacked and some scores are repeated more often than others. we tested if data followed a gaussian distribution using a d'agostino and pearson omnibus normality test [45] . as the data did not pass the normality test, we used nonparametric statistical tests to compare the data from all 3 academic years. means were compared by a kruskal-wallis test [46] and, when differences were found, by a mann-whitney post hoc test [47] to determine which pairs of means were different. all statistical analysis was performed using graphpad prism 6 (graphpad software, la jolla, california, usa). data is presented as mean±sd and statistical significance was set at p<0.05. here, we have analysed 2 variables, the score (0-10) obtained by the students in the different activities and the proportion of students that passed the activity (score�5), from 2019/2020 and the 2 previous academic years. firstly, we checked if the results from the previous 2 years (2017/2018 and 2018/2019) were similar, thus allowing them to be compared with results from the present year. to perform the analysis, we tested the score data for normality using a d'agostino and pearson omnibus normality test [41], later comparing them with an unpaired 2-tailed t-test, if they were normal, or with a non-parametric mann-whitney test, if they were not normal. afterwards, we proceeded similarly to compare the results from the present year with those from the previous 2 years. when data from each activity followed a normal distribution, we compared the 3 means by 1-way anova followed by an unpaired 2-tailed t-test. when the data did not follow a normal distribution, we compared the 3 means with a kruskal-wallis test followed by a mann-whitney post hoc test. in order to compare the proportion of students that passed the activities (score�5) in different years, we performed a z-test (after confirming that our data met the central limit theorem). all statistical analysis was performed using graphpad prism 6. data is presented as mean±sd and statistical significance was set at p<0.05. in order to substantiate related samples of results in our experiment, we performed a wilcoxon signed rank test which compares two related samples that cannot be assumed to be normally distributed. in the experiments related to this subject, the same students were involved in both samples. data is presented as mean ±sd and statistical significance was set at p<0.05. in this section, we analyze students' self-learning strategies in the subject "applied computing" in the years that were not influenced by covid-19 (2017/2018 and 2018/2019). however, since the data is very similar in both courses, we will focus con 2017/2018 for clarity. all statistical results can be also applied to 2018/2019. in both years, theoretical lessons were taught in the classroom and practical lessons in computer laboratories with the teacher physically present. practical lessons always used the e-valuam platform and the adaptive test described previously. in fig 4a, we show the scores obtained by all 2017/2018 students in all the attempts made with adaptive tests. attempts are ordered chronologically. a score of -1 in the figure implies that the student did not finish the test and did not obtain a numerical score. those tests have been included in the figure as a part of student's autonomous work and must be considered as learning time; however, we will exclude them from score data analysis. there are some patterns in the figure that must be considered. first, certain scores repeat more than others (2.5, 5 and 7.5), which corresponds to a jump in question level (l) (level 1: 0-2.5, level 2: 2.5-5, level 3: 5-7.5 and, level 4: 7.5-10), meaning that students were not yet able to answer more difficult questions. another pattern that can be easily seen in the figure is a region where a huge number of tests had a score of -1 (between 600 and 700 approximately). on those days teachers asked the students to find the most difficult questions in the test. for that reason, students interrupted the tests when they reached those questions. however, the most interesting pattern, which can be observed in fig 4a, is the distribution of the tests in time, with a much bigger number of tests done in may, being the last month of the academic year for this subject. students used the platform 688 times in total in may and only 486 times in the period between february and april. it is worth noting that the academic year for this subject ended with the final exam on may 21. in fig 4b we show the distribution of attempts between february and may. we found 62 in february, which is a low but reasonable number since students did not yet have enough knowledge to face tests properly. they generally started using the platform in the middle of the month. in march, we noted an increase in the number of attempts followed by a slight decrease in april. this difference can be justified by the easter holidays (one week) in april. the number of attempts strongly increased in may. even when counting only the first two weeks of the month, we have more attempts than in previous months (292 from may 1-15). in the period of may 16-21, we have recorded 396 attempts (469 if we count the attempts in the final exam). in the inset of fig 4b we show the attempt distribution in the last period (may 16-21). as we can see, students used the platform 127 times the day before the exam and 173 times the day of the final exam (95 for studying and 78 in the exam itself). more than 33% of tests were performed in the last 6 days before the final exam (and more that 50% of those tests were performed the day before the exam). to study the effects of a rewarded scenario, we have developed an experiment in the academic year 2017/2018 for students from the subject "design of water treatment facilities". we have used 3 stages (fully described in the materials and methods section): stage 1, where the self-evaluation tests have been made available for 1 day; stage 2, where the self-evaluation tests have been made available for 3 weeks; and stage 3, where self-evaluation tests were available for a longer period with a reward related to their use. fig 5 shows the attempts distribution in all the stages. focusing on the ma-t, 13/20 students made more attempts in the first stage than in the second, which is striking considering that in the second stage application was available for longer. three students have not made any attempts in either of the 2 stages. as for the group of students who have used the application for more than one day, we noted only 3 individuals working for 3 days or more. considering that the application was available for 3 weeks in this second stage, these results show the very low willingness of the students to work continuously. focusing on the oa-t, this effect is even more significant, as only two cases in which students used the application for more than one day were recorded. increasing student motivation and analyzing the results of a more persistent use of the tool was the objective of stage 3. in fig 5 we can see the distribution of attempts made by the students in the whole study (3 stages) . there is a fourth stage in the figure that corresponds to an additional exam taken by the students that did not pass the previous ones. since only a few of them were involved in this fourth stage, it was excluded from the analysis. as we can see in the figure, attempts from stage 1 were limited to a couple of days, which corresponds to the short period when the test was available. some attempts can be found in a wider window in stage 2. however, a much higher number of attempts extended in time can be found in stage 3, which corresponds to the rewarded stage. in order to measure the use of the tool over time, we summarized the use of the system by students in stage 2 and 3 by comparing the number of days the students attempted the stage 2 ma-t and oa-t tests during the 3 weeks leading up to the exams given after each stage. it is interesting to note that, during stage 3, six more students utilized the system than in stage 2; however, the total attempts reduced between the 2 stages from 123 to 102. this translates to a 23% increase in the average use of the application over time from 1.9 days in stage 2 to 2.4 days in stage 3 combined with a reduction of attempts per student from 9.5 in stage 2, to 5.4 in stage 3. in the earlier stage, fewer students used the application intensively by making multiple attempts a day. students who logged in at the later stage were more likely to use the application once or twice a day over several days because of the reward related to this behavior. out of a total sample of 19 students who used the tool in stage 3, 5 were eliminated by the test as they accessed the tests the same number of days in both stages. then, for the use of the application by the remaining 14 students the w-value was of 15.5, with the critical value of w = 25 at p.0.05. therefore, the result is significant. we have studied two cases related to two different and common scenarios in covid-19 distance learning. the first one is related to subjects where teachers increased the number of tasks that students should perform autonomously. the second one is related to subjects where all the autonomous tasks were the same as in previous courses. in this case, only face-to-face sessions were replaced by distance learning activities such as online sessions or recorded classes. 3.2.1 additional autonomous activities during confinement. in fig 6 we show the scores obtained by all the students in the 3 courses analysed in all the e-valuam tests performed in the subject "applied computing". in the figure we indicate the period of the covid-19 confinement during the 2019/2020 course (from march 11 to march 30). for clarity, we also indicate the equivalent period in the two previous years. as we can see in the figure, the number of tests performed before confinement was similar (225 and 246) in the two previous years (control group), but there is a remarkable increment in the number of tests (328) performed in the experimental group. these differences can be easily explained normalizing these numbers to the number of students each year (97, 73 and 91 respectively). by doing that, we obtain 2.31, 3.37 and 3.6 test/student, respectively. thus, differences between courses 2018/ 2019 and 2019/2020 are reduced, although there is still some difference with respect students from course 2017/2018, that can be explained by the fact that some of these students were retaking the subject and, therefore, did not use the e-valuam application often until the contents were more difficult. if we count the number of tests finished until the end of the academic year in courses 2017/2018 and 2018/2019, we find 1345 and 1175 tests respectively. normalizing with the number of students, we find 13.87 and 16.1 test/student respectively. this is a difference of 2.23 test/student in a period of 3.5 months, which means a difference of less than 1 test/student each month. the conditions after the covid-19 confinement radically changed in the course 2019/ 2020 because students were required to perform a higher number of tests each week. as we can see in fig 6, there are still small differences between courses 2017/2018 and 2018/2019. however, the number of tests taken in 2019/2020 is almost 5 times higher. in the inset of fig 6 we show a histogram with the data for course 2019/2020. adaptive tests used in this experiment induce the effect of higher number of attempts at scores 2.5, 5 and 7.5, which are the points where the level of the questions increases. for that reason, the curve is not normal. this fact is statistically tested in the following analysis. when comparing the mean scores from the period before confinement (fig 7) , we found that they were statistically different between the 3 academic years. differences between the mean score from the 2019/2020 (experimental group) and both the other 2 years (control group) were . the mean scores are significantly different both in the control and in the experimental groups between both periods (before and after confinement). we have also seen an increase in the mean score differences after the confinement. this study has been performed with students from the subject "metabolism", which is a subject where no additional tasks have been imposed to students because of the covid-19 confinement. we have analysed students' scores and the proportion of students that passed the activity (score�5). as it can be observed in fig 8a, the scores obtained by students in the different activities during the course were consistently similar in both years of the control group, with the only exception in the second activity where there was a slightly decrease in year 2018/2019 (p<0.05). similarly, the proportion of students with a score�5 was very similar in the control group (p>0.05) ( fig 8b) . therefore, students' performance in the previous 2 years (control group) was not significantly different, allowing us to compare these results to the results from 2019/2020. scores obtained by the students in the 2019/2020 academic year before confinement were similar to those obtained by students from the control group, although some differences were found at the beginning of the course (activities number 2, 3 and 4, p<0.05). we think that this could be due to the adaptation of the students to the course and to the new assessment methodology, being afterwards similar again. however, after the end of face-to-face teaching and at the beginning of the confinement, students' scores were significantly higher than in the previous academic years (fig 8a) . the most remarkable differences are evident in activities 10 and 11, that have always been on-line activities. thus, in activity 10, mean score in the present year (2019/2020) was 8.1±0.2 vs. 6.5±0.2 (p<0.0001) and vs. 6.7±0.3 (p = 0.0005) in 2017/2018 and https://doi.org/10.1371/journal.pone.0239490.g008 significant differences when comparing students' performance in confinement with the performance in previous periods where activities were not limited to distant learning. at this point, it is clear that the variable that correlates with the change in students' performance is the beginning of the confinement. however, we cannot yet establish if the difference is due to either: • the new learning methodology, or • the new assessment process. for those reasons, we should now answer research question 2 and establish whether the differences found when answering research question 1 are only due to the covid-19 confinement. the problem with confinement is that not only the learning, and teaching strategies should be modified, but also the assessment process as it cannot be done face-to-face. there are some concerns related to these new assessment methods such as the opportunity for cheating by the students. this is the reason why we have chosen only subjects that include several tests that have not been modified because of the confinement. the results of our study show that students also achieved significant improvements in their scores even in tests that were performed in the on-line format in previous years. moreover, this improvement is only significant when comparing data after the confinement (i.e. there are no significant differences in on-line tests that were performed before the confinement). these findings reveal that the new assessment process cannot be the reason for the improvement in students' performance because the learners also achieved better scores when the format of the assessment did not change. for these reasons, we establish that the new learning methodology is the main reason for the change in students' performance during the confinement. now, we shall discuss research question 3 (what are the reasons of the differences (if any) in students' performance?). we have proven that something has changed in students' learning methodology. the question is: what is the common element in those methodologies that caused the improvement in the learning process? we have analyzed data from two different subjects that used two very different learning strategies during the confinement (section 3.2). in one of them (section 3.2.1), additional elearning tasks were imposed on the students. theoretical lessons were replaced by written documents. in the other one (section 3.2.2), no additional e-learning tasks were given, but theoretical lessons were replaced by multimedia classes. in both cases, we have found a significant increment in students' performance in the evaluation tests after the confinement. it seems that students' performance is increased independently of the learning strategies followed by teachers. since we have established that the assessment process cannot be responsible for the differences, and we cannot find any common elements in the learning methodologies, we must conclude on a general change in the autonomous learning process. we have also analyzed data from the previous years in two subjects that demonstrate that students do not work on a continuous basis (section 3.1). in both subjects, students work hard only in the last days before the final exams. in one subject, we have found that more than 33% of the autonomous work was done in the 5 days immediately preceding the final exam (section 3.1.1). in the other subject, students only used the e-learning material in the last 2 days before the final exam, even when it had been provided three weeks in advance (section 3.1.2). in this study, we have also found that students easily change their learning behavior and study continuously when a reward is offered. this extra motivation dramatically changed their learning strategy and students worked in a much wider time window, increasing their performance. an increase in the students' performance due to adequate time management in the learning process is well-established in the literature [12, 13] . in the present covid-19 confinement, students can find by themselves many different motivations (rewards) to work on a continuous basis. first, the confinement is a new scenario that has never been faced by the students. for this reason, students do not have any previous experience to use as a reference in their learning process. without any previous reference, students need to be confident that they are following the course correctly and therefore, work continuously in order not to miss any important content. another interpretation is that they are afraid of missing the academic year because of the covid-19 confinement and they work harder to overcome any difficulty. finally, students might be motivated by their intrinsic responsibility in a very confused situation and work hard to contribute as much as they can to solve the problems that higher education is facing. most probably, different students will find different motivations in this new scenario (probably a combination of many). we conclude that there is a real and measurable improvement in the students' learning performance that we believe can guarantee good progress this academic year despite the covid-19 confinement. answering question 4, we have demonstrated that students get better grades in activities that did not change their format after the covid-19 confinement. moreover, we have demonstrated that there is an improvement in their learning performance. in conclusion, higher scores are expected due to the covid-19 confinement that can be directly related to a real improvement in students' learning. supporting information s1 data. (xlsx) better performance of students was strongly evident when analysing the proportion of students that passed these 2 activities (fig 8b). thus, 95.6% of students passed activity 10 (p<0.0047 vs tracking e-learning through published papers: a systematic review a comparative analysis of forums and wikis as tools for online collaborative learning designing videogames to improve students' motivation investigating variation in learning processes in a future-learn mooc the mooc model for digital practice. sshrc application, knowledge synthesis for the digital economy teamwork assessment in collaborative projects through process mining techniques assessment of collaborative learning experiences by graphical analysis of wiki contributions. interactive learning environments mining theorybased patterns from big data: identifying self-regulated learning strategies in massive open online courses complexity leadership in learning analytics: drivers, challenges and opportunities analytics of time management strategies in a flipped classroom predictive power of regularity of pre-class activities in a flipped classroom covid-19 epidemic as e-learning boost? chronological development and effects at an austrian university against the background of the concept of "e-learning readiness campus traffic and e-learning during covid-19 pandemic online assessment in higher education in the time of covid-19. education in the knowledge society e-learning during covid 19 pandemic comparing students' self-discipline and self-regulation measures and their prediction of academic achievement investigating self-regulation and motivation: historical background, methodological developments, and future prospects the metacognitive control components of self-regulated learning student differences in self-regulated learning: relating grade, sex, and giftedness to self-efficacy and strategy use using formative assessment to influence self-and coregulated learning: the role of evaluative judgement theorizing and researching levels of processing in self-regulated learning developing web-based assessment strategies for facilitating junior high school students to perform self-regulated learning in an e-learning environment applying a web-based training to foster self-regulated learning-effects of an intervention for large numbers of participants. the internet and higher education comparing online and blended learner's self-regulated learning strategies and academic performance. the internet and higher education the authors want to thank dr. gema perez-chacón for her valuable comments on statistical analysis. conceptualization: santi fort. the aim of this study was to identify the effect of covid-19 confinement on students' performance. therefore, we conducted an experiment among 450 students from three subjects in different degrees of higher education at universidad autónoma de madrid, spain. the results of this study answer our 4 research questions:1. is there any effect (positive or negative) of covid-19 confinement on students' performance?2. is it possible to be sure that covid-19 confinement is the origin of the different performance (if any)?3. what are the reasons of the differences (if any) in students' performance?4. what are the expected effects of the differences in students' performance (if any) in the assessment process?with respect to research question 1, the results analyzed in section 3.2 show that there is a significant positive effect of covid-19 confinement on students' performance. the results indicate that students obtained better scores in all kinds of tests that were performed after the beginning of confinement. different sources of error have been removed from our study by including only subjects that fit the following requirements for the last three academic years:• same teaching methodology and teachers in all the years.• same assessment process in all years, including both distance and on-site activities.• tests performed both before and after the covid-19 confinement in the present academicyear.• tests that only imply an objective assessment process in order to avoid any possible subjective interpretation by the lecturers.we have compared results of students from the present 2019/2020 academic year (experimental group) with results of students from the last two academic years 2018/2019 and 2017/ 2018 (control group). students from the two previous academic years had similar circumstances from the beginning to the end of their course. when compared to students of the 2019/2020 academic year, we have two periods with different conditions. the first period is taught in the same conditions as in previous years (before confinement). in the second period, those conditions dramatically changed, and all the teaching and learning activities were limited to distance learning. the results of our studies (section 3.2) clearly indicate that there are key: cord-119183-pbliko1k authors: cohen, tomer; finkelman, lior; grimberg, gal; shenhar, gadi; strichman, ofer; strichman, yonatan; yeger, stav title: a combination of 'pooling' with a prediction model can reduce by 73% the number of covid-19 (corona-virus) tests date: 2020-05-03 journal: nan doi: nan sha: doc_id: 119183 cord_uid: pbliko1k we show that combining a prediction model (based on neural networks), with a new method of test pooling (better than the original dorfman method, and better than double-pooling) called 'grid', we can reduce the number of covid-19 tests by 73%. the search for carriers of covid-19 is done primarily through testing using rt-pcr's. these tests are the most common way to empirically identify carriers of the virus, and urgently need to be conducted on a large scale. today, patients are granted a test if deemed necessary by the government and are carried out individually, i.e., every sample is tested separately. the problem is that the number of samples gathered today supersedes the amount of tests that can be conducted daily; moreover, the world-wide shortage in equipment and resources prevents a much-needed increase in the number of daily tests. as a result, the testing system today is at full capacity, and falls short of the need. two recent developments are relevant to the solution that we describe here: 1. data regarding tests and the patients behind them has been gathered (over 120,000 tests in israel as of mid. april, 2020) that enables us to build a prediction model of who is likely to be positive; 2. a new study [1] has shown that it is possible to combine up to 32 samples in one 'pool ' and identify whether at least one of them is positive with a single test. pooling in general (in the context of other tests) is an old idea due to dorfman [3] . a simple calculation shows that creating random pools of samples will not be efficient in identifying positive patients, with the current rate of positive samples in israel --~8%. with this rate, less than 7% of the pools will succeed, and the rest will have to be re-tested one by one. our method solves this problem by identifying those samples that have a much smaller chance of being positive and putting them in one pool. furthermore, it recommends the optimal size of the pool, given the probability that samples in that pool are positive. by using the meta-data of the tests, which was gathered by the ministry of health, we created an algorithm (based on machine learning, and specifically a 'neural network'), that predicts the probability of a patient being negative or positive for the virus based on his/her data: whether they cough, has a sore throat, shortness of breath, or a headache, as well as his/her age group, gender, and likely cause for the sickness. based on this data our model predicts with an accuracy of 95.5% the outcome 1 . for each patient, the outcome of the neural network is a number indicating the likelihood that this patient is positive. hence by sorting the patients according to this value, we can pool together tests with almost a uniform probability of being positive. in appendices a -c, we survey three different ways to use this data -the first-of-which is a known method due to dorfman [3] -which we'll call here `single pooling' (appendix a): according to this method, if the pool turns out to be positive, all samples in that pool need to be re-tested individually, as shown in the following diagram: the expected number of tests can be calculated, based on the probability of the samples in the pool to be positive, and the size of the pool (see appendix a for details). we show that for each value of there is an optimal pool size. for example, for patients with 1% probability of being positive, the optimal pool size is 11. for probabilities higher than some threshold, it is not cost-effective to use pooling at all. we calculated that if this method had been used on all tests carried thus far in israel (see appendix e for data), we would reach full accurate classification of all the patients with about 33% of the tests. the best method that we found is called '2d-pooling' (or 'matrix pooling') [6] [7], and is described in appendix c -it can do the same thing with only 27% of the tests. appendix d includes a table comparing the required number of tests per patient with the various methods, as a function of , and also a comparison to a recently introduced method called double-pooling [4] . 3. challenges: changing the process, and the overhead of re-testing. our solution does not require new equipment. it requires, however, a change in the current process. first, the samples corresponding to patients that are selected for pooling, will have to be duplicated for potential repetition of the test, in case the pool turns out to be positive. 1 the dataset that we used was published by the moh. it has a known bias which likely distorts the result: positive cases are much more likely to include the clinical data mentioned above than negative ones, since for negative cases this data was not always entered retroactively. in itself this may have contributed to the success of the prediction model, since the very fact that there is no clinical data is a good predictor of the result. this problem will disappear if the data collection process will improve. the numbers that we present here will likely not be as good in practice because of this reason. a detailed discussion of this matter appears in [5] . second, the tests that are currently given to the lab in some arbitrary order, will have to be resorted, based on the recommendation of our system. the total number of tests is not the only objective. one should also consider • the number of test iterations. each such test takes time (several hours on the pcr machine), which leads to a delay in the response time to the patient. • the amount of sample duplication. if a sample will potentially need to be retested, it has to be duplicated. in appendix d we consider these objectives when comparing the various methods. in appendices a -c we describe different pooling methods, with a decreasing number of expected tests. appendix d compares the efficiency of the various methods. the raw data can be downloaded from [2] . appendix a: the single pooling method [3] in this model a sample can only be part of a pool once. that is, if the pooled sample fails, then all samples in the pool are re-tested individually. let be the probability of a test to be positive, the pool size, and the population size. the expected number of tests, as a function of the probability of a test to be positive and the pool size is calculated as follows: in the case of a failure there are + 1 tests (left part of (1)) for samples, and in case of success there is a single test for samples (right part of (1)). the overall number of pools is / . let us take two numeric examples: for = 0.1, = 12, = 32 , the average number of tests is 25.62. for = 0.01 and the same values of , , the expected number of tests drops to 6.4. it is clear that from some threshold for the value of , it is not cost-effective to use pooling at all. the plots in figure 1 are based on (1), divided by , so it reflects the expected number of tests per patient. it is evident that for each probability, there is an optimal pool size -the size that brings to minimum the expected number of tests. for example, for = 0.01, the optimal pool size is 12, and we saw earlier that this implies 0.19 tests per patient. appendix b: the binary-tree method one can extend the idea presented in appendix a to multiple levels of pooling. this is sometimes called 'multi-stage pooling' [8] . that is, given a pool of size that fails, split it to two and retest, until reaching the leaves of the search tree. a small optimization is achieved as follows. suppose that a node at level in the binary tree is positive, and we then check, e.g., the left child node at level − 1. if that node is negative, not only that we can skip the whole subtree under that node, we can also skip the other node at level − 1, because it is bound to be positive. the plots in figure 2 below are based on simulations, including this optimization. p: some of the data from our simulation with = 32 appear in table 1 below, where the optimal pool size (the value of ) is highlighted for each value of . one may observe that above a certain probability ( = 0.35) pooling is not cost-effective. the main disadvantage of the binary tree method is that it imposes log iterations (the height of the binary tree). furthermore, if the optimization mention above is activated, it doubles the number of iterations (because one cannot test the two sibling nodes simultaneously). see appendix d for further discussion. another method of pooling is based on a matrix of pools. we arrange an × matrix of samples. we then pool together each column and each row, hence 2 pools of samples each. this implies that each sample participates in two pools (i.e., row and column pools). suppose that out of the 2 samples are positive. this means that in the worst case 2 pools will test positive (as each such sample makes a separate row and column positive), and correspondingly there will be 2 samples that are potentially positive and hence need to be re-tested. there is a limit, however, on the number of additional tests: it cannot surpass 2 . hence the worst case can only happen times. the grid (also can be seen as a matrix) in figure 3 is for = 5. each of the 25 junctions is a sample. correspondingly, 10 pools will be tested. for example, the 5 samples in the first row form a pool, and the 5 samples in the first column form another pool. suppose there are 2 positive cases from those 25 samples, and they are located at (2, 2) and (4,4) (marked in red). correspondingly, row-pools 2, 4, and column pools 2,4 are positive (marked by 'x'). but this leaves us with 4 candidate samples, which must be retested individually. so altogether, in this case we need 14 tests, for 25 people. a more fortunate case is when the positive cases happen to be on the same row or column. for example, if those two positive cases were on the same row, we would not need more tests at all as they would be uniquely identified. eq. (2) below expresses the expected number of tests with the 2d-pooling method, in the worst case. as before, is the probability for a positive test, is the size of the population, and , in this case, is the number of columns/rows in the matrix. recall that the worst case is when each positive sample is a singleton on its row and column. in that case the number of added tests is 2 = ( 2 ) 2 . hence, we have this expression is relevant only when ≤ . since = 2 , this happens when 2 ≤ , or ≤ 1/ . furthermore, it is relevant only as long as (# ) < , which implies that eq. (3) is a strictly tighter bound than the former ≤ 1/ . plotting (2) for different values of and where = 400 (see figure 4) , shows that here, too, for each value of , there is an optimal matrix size. we note that for these results to apply, we must have 2 ≤ (otherwise we do not have enough samples to fill the matrix). for example, for = 0.01 the optimal value of is 20, which means that we can use the result only if ≥ 400 (here the results were computed, as mentioned, with =400). we also simulated this method, in order to get the actual statistics rather than the worst-case scenario. we included the following (small) optimization in the simulation: suppose that the number of positive rows is . for each positive column, if the first − 1 samples turns out to be negative, there is no need to test the last one, as it is bound to be positive. the same argument applies to each row. in the best-case scenario all the positive cases are in the last row and last column -in such a case we save 2 − 1 tests (-1 because of the bottom corner). the downside of this optimization is that it adds an iteration. see appendix d for a discussion. there are other possible optimizations, which are discussed in [7] [8] and we did not include in our simulation. for example: test rows only; if none of them is positive -stop; if exactly one row is positive, check all samples in that row individually; otherwise -continue as usual. appendix d: comparing the methods table 2 below compares the three methods in terms of their tests per patient (tpp): the ratio between the expected number of tests and the population size . note that each cell was calculated with the optimal pool size for the given value of . for example, for = 0.01 the optimal value of in the binary-tree method is 11, whereas the optimal value of in the 2dpooling method for this probability is 20. the last line of the table refers to results of simulating the 2d-pooling method, which is expected to be better (and indeed our results show that it is) than the worst-case as formulated in (2). it is clear from the table that the actual (i.e.., simulated) 2d-pooling method is the most efficient one in terms of minimizing the number of tests. finally, let us compare the 2d-pooling method to a recently introduced method called doublepooling [4] . citing [4] : "given a probability of a positive test, pick an optimal size 2( ) for the pool size. divide the population to be tested into non-overlapping pools of size s2 (the division is assumed to be random) twice. thus, now every patient belongs to two pools and is tested in two parallel rounds, a and b. for every patient if both the pools test positive then test the patient individually. otherwise consider that patient cleared.". using their analysis (which we independently verified via simulation), the tests-per-patient of double-pooling is the following: hence both the 2d-pooling method and the binary-tree method are more efficient than double-pooling. both the 2d-pooling-and the double-pooling method require two steps. so far we only compared the methods by the expected number of tests per patient. let us now compare them by the number of iterations, and the number of sample duplication that is necessary (this represents the number of times a patient will be retested in the worst case): sample duplication single pooling 2 binary tree log , (or 2 log with the optimization). log 2d-pooling 2 (or 3, with the optimization). appendix e: the current distribution of samples in israel in figure 5 , the horizontal axis shows the value predicted by our neural network, multiplied by 100. the vertical axis shows the accumulated percentage of the population that falls under this value. for example, over 80% of the population are classified by our neural network as having less than 2% chance of being positive. the raw data is accessible from [2] . evaluation of covid-19 rt-qpcr test in multi-sample pools / idan yelin, noga aharony the data used in the analysis of the algorithms the data from the ministry of health the detection of defective members of large populations a note on double pooling tests (preliminary version improved matrix pooling the use of a square array scheme in blood testing the blood testing problem appendix c: the 2d-pooling method [6] [7] key: cord-024631-yvek5vjz authors: althaus, t.; thaipadungpanit, j.; greer, r.c; swe, m.m.m; dittrich, s.; peerawaranun, p.; smit, p.w; wangrangsimakul, t.; blacksell, s.; winchell, j.m.; diaz, m.h.; day, n.p.j; smithuis, f.; turner, p.; lubell, y. title: causes of fever in primary care in southeast asia and the performance of c-reactive protein in discriminating bacterial from viral pathogens date: 2020-05-11 journal: int j infect dis doi: 10.1016/j.ijid.2020.05.016 sha: doc_id: 24631 cord_uid: yvek5vjz objectives: we investigated causes of fever in the primary levels of care in southeast asia, and evaluated whether c-reactive protein (crp) could distinguish bacterial from viral pathogens. methods: blood and nasopharyngeal swab specimens were taken from children and adults with fever (>37.5˚c) or history of fever (<14 days) in thailand and myanmar. results: of 773 patients with at least one blood or nasopharyngeal swab specimen collected, 227 (29.4%) had a target organism detected. influenza virus type a was detected in 85/227 cases (37.5%), followed by dengue virus (30 cases, 13.2%), respiratory syncytial virus (24 cases, 10.6%) and leptospira spp. (9 cases, 4.0%). clinical outcome was similar between patients with a bacterial or a viral organism, regardless of antibiotic prescription. crp was higher among patients with a bacterial organism compared to those with a viral organism (median 18 mg/l, interquartile range [10-49] versus 10 mg/l [≤8-22], p-value 0.003), with an area under the curve of 0.65, 95% confidence interval (0.55-0.75). conclusions: serious bacterial infections requiring antibiotics are exceptions rather than the rule in the first lines of care. crp-testing could assist in ruling out such cases in settings where diagnostic uncertainty is high and routine antibiotic prescription is common. the original crp randomised-controlled trial (rct) was registered with clinicaltrials.gov, number nct02758821. fever is a common reason for seeking healthcare in southeast asia and as malaria incidence declines, bacteria and viruses now represent the main contributors to acute febrile illness [1] [2] [3] [4] [5] . identifying these pathogens is challenging, even in well-resourced laboratories with specialised staff, and most aetiological data for febrile illness originate in tertiary hospitals [6] . hospitalised patients, however, are by definition more severely ill, often with important comorbidities, implying that findings may not be applicable to febrile patients attending primary levels of care. primary care in low-middle income countries (lmics) is typically characterised by a shortage in human resources, diagnostics and evidence-based guidelines [7] . studies investigating causes of fever in this environment are few and frequently of poor quality: enrolment is often limited to a single clinical presentation and specific age category, and microbiological investigations rarely use goldstandard methods [8] [9] [10] . additionally, most primary care patients attend early after symptom onset with non-severe presentations, lowering the chances of detecting a pathogen [8] [9] [10] [11] [12] [13] [14] [15] [16] . empiric treatment guidelines are therefore based on limited epidemiological evidence and are often implemented by insufficient and poorly trained staff, contributing to irrational antibiotic prescription practices [17] [18] [19] . high prescription levels are partly driven by frequent clinical overlap between bacterial and viral infections, challenging the identification of patients who might benefit from antibiotics [2, 3, 20] . given these limitations in clinical judgment and laboratory structures, point-of-care testing (poct) to guide fever management could be beneficial in primary care settings [21] . pathogen-specific tests represent one such option but several barriers undermine their potential use: these only exist for a small number of pathogens, with inconsistent performance, and most are antibody detection-based that might preclude the distinction between active infection and past-exposure [22, 23] . a few antigen detection-based pocts exist but their integration in low-level care is unrealistic: a salmonella typhi rapid test requires laboratory infrastructure with poor detection in blood even at high concentrations and results are not available before 24-48 hours [24, 25] ; test sensitivities for influenza virus a, respiratory syncytial virus (rsv) and group a streptococcus antigen-based pocts are inconsistent j o u r n a l p r e -p r o o f 5 [26] [27] [28] [29] ; and accurate dengue antigen-based rdts have not been found to be cost-effective in resource-poor settings [30, 31] . non-specific host biomarkers measure the host-response to stimuli, and have been evaluated in the context of fever to discriminate between bacterial and viral pathogens [32] . c-reactive protein (crp) is one of the most studied host-response biomarkers of bacterial infection, consistently showing high sensitivity and moderate specificity, and crp pocts have been shown to be cost-effective in resource-poor environments [30, 32] . however, 80% studies evaluating crp performance originate from high income countries [32] . in southeast asia, these evaluations are mainly hospital-based [33] [34] [35] other than a single community-based study [36] . good diagnostic performance of crp in identifying bacterial infections was observed but generalisability was limited due to demographic, clinical and diagnostic heterogeneity of these studies. in this study, we aim to identify key organisms among acutely febrile children and adults attending primary health care in southeast asia, and to evaluate the performance of crp for discriminating between bacteria and viruses. chiang rai province is the northernmost province in thailand bordering myanmar and lao people's democratic republic. the majority of the population are thai, with approximately 15% ethnic minorities and hill-tribes. the six participating primary care sites were located within a 30-kilometre radius of chiang rai city centre, covering rural and peri-urban as well as mountainous and plateau areas. hlaing tha yar, lower myanmar, is a peri-urban township on the west side of yangon. the township has the highest rates of diseases related to hygiene and environmental conditions (e.g. diarrhoea, dysentery, and tuberculosis) in yangon [37] . four sites were included: three primary care clinics and one outpatient department from a public governmental hospital. both chiang rai and hlaing tha yar are defined by a tropical climate. specimens were collected from febrile patients recruited into a previously described multi-centre randomised-controlled trial evaluating the impact of c-reactive protein (crp) testing on antibiotic prescription in primary care [38] . febrile children and adults (defined as ≥12 years of age) were recruited between june 2016 and august 2017. inclusion criteria were age ≥1 year with a documented fever (defined as a tympanic temperature >37.5˚c) or a chief complaint of acute fever (<14 days), regardless of previous antibiotic intake and co-morbidities other than malignancies. exclusion criteria were symptoms requiring hospital referral defined as either impaired consciousness; inability to take oral medication or convulsions; a positive malaria test; the main complaint being trauma and/or injury; suspicion of either tuberculosis, urinary tract infection, local skin infection or dental abscess; any symptom present for more than 14 days; any bleeding; and inability to comply with the follow-up visit at day 5. on the day of enrolment, all patients had demographic information collected and underwent a routine clinical examination including vital signs (blood pressure, pulse, respiratory rate, temperature). patients were followed-up after their enrolment both at day 5 and day 14. of the 2,392 febrile children and adults recruited, 799 were randomly allocated to the control group with blood specimens collected for off-site crp testing (as compared with the intervention groups that had crp tests performed on-site). details are illustrated in figure 1 . antibiotics were prescribed to this group according to routine clinical practice, clinicians were not informed of the crp results or any aetiological findings. in case of co-detection in blood and np swabs, target organisms detected in blood were assumed to be the primary cause of illness. bacterial and viral aetiological groups included all cases where any bacteria or viruses were detected in blood, respectively, and only target viruses and bacteria detected in np swabs. we described organism distribution among children and adults (defined as ≥12 years of age) separately. descriptive analysis for continuous variables with normal distribution used means and standard deviations (sd) and medians with inter-quartile ranges (iqr) for non-normally distributed continuous variables. comparison between groups used t-tests for normally distributed variables, the mann-whitney test for non-normally distributed variables, and chi-squared test for categorical variables. crp values were compared across aetiological groups and clinical syndrome using the mann-whitney u test for two-group comparisons and the kruskal-wallis test for multi-group comparisons. non-parametric receiver operating characteristic (roc) curves were plotted and the wald test was used to compare areas under the curve. covariates included the following factors: patient age and prior use of antibiotics. diagnostic accuracy was assessed by calculating the areas under the roc j o u r n a l p r e -p r o o f 9 curves (auc). an auc of >0.9 was considered excellent; 0.8-0.9, very good; 0.7-0.8, good; 0.6-0.7, average; <0.6, poor [39, 40] . sensitivity, specificity, and percentage of correctly classified cases were also assessed for the two crp cut-off points used in the original trial: 20 mg/l and 40 mg/l and these were compared with the accuracy of routine prescribing practice [38] . data analyses were performed with stata version 15 (college station, texas, usa). the protocol, informed consent form and case record forms were reviewed and approved by the of the 799 patients prospectively enrolled and randomised into the trial control group, 773 (96.8%) had at least one blood or np swab specimen collected, including 371 (48.0%) children and 402 adults (52.0%). out of these 773 patients, 33 had only a np swab and no blood collected, while 146 had only a blood specimen collected without a np swab. among these 146 patients, 80 had a blood specimen obtained on a dried blood spot (dbs). as shown in table 2 , children presented significantly earlier after symptom onset than adults, with fewer comorbidities and less self-reported medication (p <0.05). antibiotic intake declaration was similar in children and adults (p = 0.237). page 10 of 31 j o u r n a l p r e -p r o o f 10 clinically, respiratory syndrome was the most prevalent presentation both in children and adults. within patients with a respiratory syndrome, the most frequent symptoms were localised in the upper respiratory tract including common cold, diagnosed in 48.2% (120/249) of children and 45.0% (108/240) of adults (p = 0.479). gastrointestinal syndrome was the second most prevalent presentation, and there were no differences between children and adults. 630 blood specimens tested for bacterial screening, using taqman array card (n=601) and bacterial singleplex polymerase chain reaction (n=626) ** 634 blood specimens tested for leptospira screening, using the taqman array card (n=601), the bacterial singleplex polymerase chain reaction (n=626) and the microagglutination test (n=134) *** 658 blood specimens tested for orientia tsutsugamushi and rickettsia spp. screening, using taqman array card (n=601), the bacterial singleplex polymerase chain reaction (n=626), and the indirect immmunofluoresence assay (n=656) **** 601 blood specimens tested using the taqman array card only (n=601) ***** 686 blood specimens tested for dengue, chikungunya and zika virus screening, using taqman array card (n=601), the viral singleplex polymerase chain reaction on fresh blood (n=626) and dried blood spot (n=245) no evidence for a difference in antibiotic prescription was observed between the bacterial and viral groups at day 0, and clinical outcomes were also not significantly different between the two groups (table 4 ). outcome characteristics by aetiological group in chiang rai, northern thailand and hlaing tha yar, lower myanmar, 2016-2017. the prescription of antibiotics at the facility was considered between the enrolment at day 0 until day 14 of the follow-up severity was ranked from 1-4 with severity=1 as the less severe presentation crp: c-reactive protein elevated crp defined as ≥50 mg/l in children and ≥100 mg/l in adults sae: serious adverse event, defined as admission to hospital or death within 14 days of enrolment broad-spectrum antibiotics include ceftriaxone, cefixime, ciprofloxacin, levofloxacin, azithromycin, and amoxicillin with clavulanic acid. among patients with a bacterial organism, two-thirds did not receive any antibiotic ( occurrence of sae, n (%) 0 (0) 0 (0) 1.000 unscheduled visits, n (%) 0 (0) 6 (3.1) 0.281 antibiotic. no evidence for a difference in clinical outcomes was observed after 14 days of follow-up, regardless of whether an antibiotic was prescribed. of we investigated the spectrum of organisms among febrile children and adults in the community and evaluated the performance of crp in distinguishing bacteria from viruses including its potential impact on antibiotic prescription compared with current practice. patients were recruited prospectively across ten sites in thailand and myanmar including urban, semi-urban and rural areas spanning over a full calendar year. in our study, leptospira spp., influenza virus and dengue virus were the leading organisms identified, which is consistent with previous reports in the region [8, 36] . the broad inclusion criteria, allowing for enrolment of all patients over 1 year old regardless of previous antibiotic intake, comorbidities, or clinical presentation, make our findings more generalisable than previous studies. investigating non-malarial acute febrile illness remains challenging in resource-poor areas [8] , and despite screening for multiple organisms on blood and respiratory specimens, we were only able to identify a probable cause of fever in 227 (29.4%) of patients. this low detection may be explained by the inclusion of only non-severe outpatients [14, 41, 42] , while other studies in southeast asia recruiting more severe and hospitalised patients identified an organism in around 50% of cases [2] [3] [4] 35] . only 15.9% (36/227) of organisms detected were bacteria, which may be explained by the lower risk of bacterial infections in non-severely ill patients, and where present, characterised by lower bacterial loads [42] . most bacteria were identified using a singleplex pcr and not the tac assay, while viruses were equally detected by these two molecular methods. this lower sensitivity in the tac assay for the detection of bacteria has been described in previous studies using multi-pathogen molecular detection platforms [43, 44] . the trade-off between advantages for screening multiple organisms at the same time with a simplified molecular platform should be weighed against potentially lower sensitivity, especially for bacteria such as o. tsutsugamushi or leptospira spp., which are considered important drivers of acute febrile illness in southeast asia [45, 46] . the tac assay to identify infections among neonates in south asia, but detected the presence of certain organisms among both controls and cases [47] . a 2019 multi-country study into causes of severe pneumonia also excluded molecular assay results positive for k. pneumoniae because of poor assay specificity [48] . furthermore, most of our patients presented with low crp regardless of whether a bacterial or viral organism was detected, and recovered regardless of whether an antibiotic was prescribed. it is likely that invasive bacterial infections requiring an antibiotic are exceptions while most primary care patients present with a self-limiting infection [49] . other primary care-based studies have recently supported restriction of antibiotic prescription to a small minority of patients: in tanzania, a clinical trial using a 80 mg/l threshold lowered antibiotic reduction to 2.3% without affecting outcomes, while a u.s. study concluded that 72% of outpatients attending a general practice for a respiratory presentation should not even require a medical consultation, let alone an antibiotic prescription [50, 51] . strategies whereby testing for crp as a predictor of clinical outcome rather than determining aetiology have been evaluated in primary care: a cluster-randomised controlled trial in belgium showed crp to rule-out serious infection using a 5 mg/l threshold, while a systematic review found crp-testing to be useful in identifying serious infections among febrile children [52, 53] . in our study, crp performance in distinguishing bacteria from viruses was average (auc 0.65) and lower than another study from the region which found (auc 0.83 among 1,372 patients with a microbiologically-confirmed diagnosis from thailand, cambodia and lao pdr [36] our study has several limitations, mostly relating to the limited scope and accuracy of the reference diagnostic tests, and the impact of even slightly less than perfect "gold-standard" reference tests on the evaluation of new diagnostic and biomarker tests can be profound [56, 57] . as mentioned above, the sensitivity and specificity of the multiplex tac assay was not optimal for bacteria detection, and the absence of convalescence specimens impeded our ability to diagnose patients based on serology, particularly with respect to bacterial zoonoses. even genuine detection of bacterial and viral dna in normally sterile sites cannot be used to conclusively determine causality, as this has been reported among healthy individuals, and in patients even weeks after recovery from infections, challenging the interpretation of molecular assays [58, 59] . blood culture was not available and this further limited our aetiological investigation. we did not recruit a concomitant control group, which precludes robust attribution of causality in the organisms we detected, particularly in np swabs. in a paediatric study in asia and africa, the inclusion of controls matched with pneumonia cases weakened the evidence of causality for almost all organisms detected [48] , and only rsv, hmpv, influenza virus a and b, parainfluenza virus type 1 and b. pertussis were considered pathogenic, consistent with other lmicbased studies [60, 61] . these organisms, however, are sometimes present in healthy individuals, with a prevalence of influenza virus among healthy children between 0-6%, rsv at 0-9% and hmpv between 0-7% [62] . on the other hand, we did not regard other organisms detected in np swabs such as rhinovirus, parainfluenza virus or s. pneumoniae as pathogenic, because these are commonly detected among healthy individuals [48, 63, 64] . all these limitations in reference diagnostic tests might explain the average performance of crp in our analysis. we presented the key organisms detected among febrile children and adults attending primary healthcare in southeast asia. the performance of crp in distinguishing between bacterial and viral organisms was limited, although the current findings suggest that crp-guided treatment would increase the appropriate use of antibiotics with respect to aetiology. this is supported by the overall reduction in prescribing compared with current practice demonstrated in the original trial. our findings also support conclusions from previous studies that even in the presence of bacterial organisms, very few ambulatory patients are likely to benefit from the extensive and poorly targeted antibiotic prescribing practices that currently prevail in most southeast asian primary care settings. the funders had no role in study design, data collection, data interpretation or writing the manuscript. the corresponding author had full access to all the data and took the final decision to submit for publication.  point-of-care diagnostic tools could guide health workers' antibiotic prescription  c-reactive protein was significantly increased in case of bacterial infections  most primary care patients recovered regardless of antibiotic prescription  antibiotic prescription 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individuals microorganisms associated with pneumonia in children< 5 years of age in developing and emerging countries: the gabriel pneumonia multicenter, prospective, case-control study the role of influenza, rsv and other common respiratory viruses in severe acute respiratory infections and influenza-like illness in a population with a high hiv sero-prevalence viral and bacterial interactions in the upper respiratory tract frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values asymptomatic shedding of respiratory virus among an ambulatory population across seasons. msphere we thank all primary care patients and health workers from chiang rai primary care centres and hlaing tha yar public hospital and mam clinics for taking part in the study. we are very grateful to the study staff and to the clinical trial support group at mahidol-oxford tropical medicineresearch unit for ensuring the successful completion of the study. we thank heiman wertheim, arjen dondorp, direk limmathurotsakul, christopher parry, and paul newton for guidance on the study design; clare ling, toni whistler, ampai tanganuchitcharncha, areerat thaiprakhong, nattapon pinthong, prapaporn srilohasin and kyaw soe for laboratory support, as well as duangjai suwancharoen from the national institute for animal health (niah) for carrying out the leptospira mat. finally, we thank elizabeth ashley, jeroen bok, joshua cohen, ni ni tun, and khin yupar soe for their assistance in study site coordination. key: cord-007047-7ty9mxa9 authors: reller, l. barth; weinstein, melvin p.; baron, ellen jo title: implications of new technology for infectious diseases practice date: 2006-11-15 journal: clin infect dis doi: 10.1086/508536 sha: doc_id: 7047 cord_uid: 7ty9mxa9 new assays for the diagnosis of infectious diseases—particularly those that use molecular technologies—will revolutionize infectious diseases practices, but the fulfillment of the promise is several years away. problems with currently available molecular assays include a lack of knowledge about the extent of microbial nucleic acid in “normal” hosts, concentration of agent material in small volume samples, lack of microbiologist expertise, lack of adequate reimbursement, and difficulty with validation based on conventional methods. clinicians must appreciate the shortcomings of new technology to use it effectively and appropriately. however, we are realizing tangible progress in our ability to detect new etiological agents; the availability of rapid, accurate diagnostic tests for previously difficult infections; and advances into new, human response—based paradigms for diagnostic testing. the promise of a star trek-inspired hand-held infectious diseases diagnostic device that scans a patient's body and immediately provides the information needed for diagnosis and treatment is probably years away. until that time, when clinicians and microbiologists become unemployed, we can use the amazing technologies that are already available. this overview will discuss a selection of technologies and provide an opinion about their utility, their pitfalls (summarized in table 1) , and their potential. although the day when all diagnostic microbiology laboratories will routinely use molecular tools has not yet come [1, 2] , many clinicians believe that a plethora of such tests is readily available. laboratorians are regularly asked to perform a pcr test to detect an agent that only a few published research studies have reported, with those studies usually describing only preliminary results. physicians often seem surprised that the laboratory cannot provide this service. a survey by the american society for microbiology published in 2003 provides a snapshot of the current environment in the united states. of the 612 laboratories (representing community, academic, and commercial laboratories) that responded to the survey, 95% performed bacteriology tests, but only 17% performed any molecular tests for infectious diseases [3] . the bulk of the latter laboratories offered only molecular testing for chlamydia/neisseria gonorrhoeae (as determined on the basis of college of american pathologists surveys participant summaries). for the foreseeable future, because of staffing and financial issues, many nonconsolidated or nonacademic hospital laboratories will not perform new, high-technology, expensive, and skill-demanding assays. there is a national shortage of clinical laboratory scientists. the number of training programs has decreased over the past decade, and qualified students are now choosing careers in medicine or computer science. nurses are paid dramatically more than clinical laboratory scientists-a further disincentive for students to consider a laboratory career-although the same level of professionalism and dedication to patient care is required. the overall vacancy rate among microbiology technologists is 57%, and almost one-half of all vacancies require at least 3 months to fill [3] . the staffing shortage in the pipeline in the united states today becomes more worrisome when combined with the aging of the current experienced staff. thirty-four percent of microbiologists working today are 150 years old [3] ; most of these will retire in the next 10 years. their competence in molecular methods is miniscule. however, these senior microbiologists can interpret a gram stain and deliver an educated guess as to the identification of a pathogen at an early stage. infectious diseases clinicians have relied on these expert workers, and reliable molecular diagnostic tests are not readily available for many infectious agents commercial tests should only be used for validated specimen types transportation problems, low concentrations of infectious agent, primer binding site genetic changes, final assay volume, inhibition, contamination, nonspecific amplification, and operator error lead to false-negative and false-positive amplification results genomic bacterial sequencing is subject to error because of sequence homology among different bacteria, database problems, and mutations unknown extent of microbial dna in "normal" host tissues lack of necessary resources (human and economic) reimbursement often not sufficient to cover costs today's infectious diseases trainees are still being trained in laboratories utilizing such resources. the new, younger laboratory workforce is not highly skilled in either the conventional or the high-tech environment, and microbiologists in general are in short supply. we are facing a knowledge and practice gap for the next 10-15 years, during which time molecular tools will gradually fulfill their promise. even in those laboratories that are staffed with skilled microbiologists during the daytime, many critical test results are needed on the evening and night shifts, when the laboratories are staffed by nonspecialized laboratorians. as a result, physicians will have less trust in test results and will need to make a greater number of empirical decisions and use a greater number of broad-spectrum antimicrobial agents. another aspect of the dearth of experienced technologists is that testing has become more instrument based than discipline based. this is apparent in the migration of hepatitis and hiv serological testing from microbiology or virology departments to the immunochemistry laboratory, where generalists or chemistry technologists perform these tests along with tests for other serum factors, such as toxins and proteins. the testers are, in general, not knowledgeable about the agents for which they are testing, which could lead to interpretation errors or failure to recognize inappropriate results. it is difficult, at best, to validate test results (assuring the clinical utility, reliability, and reproducibility of results) using new technologies. traditional culture-or antigen-based comparator methods often yield results that fail to correlate with the newer, more sensitive tests. finding clinical criteria or alternate molecular targets to verify which result is correct can be tedious, expensive, and occasionally impossible. obtaining adequate standards or positive patient samples to validate test results and to train performing technologists is also challenging. one example is pcr of csf specimens for herpes simplex virus meningoencephalitis; this is considered to be the best diagnostic assay available, because cultures are insensitive, and brain biopsy is too invasive [4] . the difficulties in finding enough samples and in creating seeded samples with known viral loads for validation studies have prevented or delayed development of this and other tests in many laboratories [5, 6] . in fact, identical problems have impeded submission and us food and drug administration (fda) approval of commercial assays for herpes simplex virus. some analyte-specific reagent test kits are available for both herpes simplex virus and enteroviruses, but they still require extensive in-house validation studies. the threat of an avian influenza pandemic may help spur the development of multiplex molecular assays for respiratory viruses [7] . the use of laboratory tests for diagnosis of enteroviral cns disease was shown to be cost effective long ago [8] , but the availability of rapid molecular tests for this purpose has been long coming. in many circumstances, tests that infectious diseases physicians have heard about (e.g., at national meetings, such as that of the infectious diseases society of america) will be available only as send-out tests to referral laboratories, accompanied by the attendant transportation problems and possible specimen degradation, delayed results, and costs. if an available molecular test is not performed in the local laboratory, one of the most important benefits-rapid results-is not realized, because the sample must be sent to a distant reference laboratory. even after the newest generation of real-time molecular test systems, such as the genexpert test for group b streptococci (cepheid), slated for fda clearance as moderately complex, becomes available [9] , their initial cost may delay widespread implementation. there are, of course, some widely performed molecular methods, such as hiv load [10] and nucleic acid amplification (naa) assays for chlamydia trachomatis and n. gonorrhoeae that use genital samples or urine specimens [11, 12] . however, many physicians assume that one can just as easily test an eye swab as a cervical swab, without appreciating that the laboratory must perform an extensive validation of a sample not included in the original fda clearance of the device if a report is to be issued (and the test is to be paid for) [13, 14] . many molecular tests are limited to reference laboratories or for research investigations only. of course, not all new technology is nucleic acid based, and i will also discuss other methods. a number of nucleic acid hybridization and amplification methods are now in use, including direct probe hybridization (advandx fish for staphylococcus aureus [advandx] and genprobe for group a streptococci [genprobe]), hybrid capture (digene for human papillomavirus; digene), pcr, branched-chain dna (bdna; bayer diagnostics), and transcription-mediated amplification (probe-tec for chlamydia and n. gonorrhoeae; becton dickinson). sample volume (which encompasses both original sample volume and number of nucleic acid sequences present in the final amplified sample portion), transport conditions, dilution factors, inhibitory factors, genome changes, instrument problems, and operator error all contribute to false-positive and false-negative test results [6, [15] [16] [17] [18] . even closed systems at the detection end do not prevent cross-contamination from positive sample processing at the front end. for example, current fda restrictions require that papanicolaou smears be first performed when a single suspension of cervical cells is received in preservative, with requests for both cytology and for detection of human papillomavirus, chlamydia species, and n. gonorrhoeae; this contributes to the common finding that insufficient sample size remains for the infectious diseases tests after the papanicolaou sample has been removed [19] . unfortunately, unlike direct visual examinations in which sample quality can be assessed, a negative result of such an assay includes no comment on the quality of the specimen. although molecular tests that use whole blood samples for detection of the agents of sepsis are not yet available, several such assays are in development. volume will be a factor in the sensitivity of these tests. the amount of bacteria in blood obtained from a patient with bacteremia is often !1 cfu/ml [20] , yet the volume used in most naa reaction vials is !10 ml. even if the sample is concentrated in some way, the chance that microbial genetic material will reach the reaction vial could be very small, resulting in false-negative results. in samples containing human neutrophils or erythrocytes-the very samples most likely to harbor the infectious agent-it is challenging to concentrate microbial dna without also adding overwhelming amounts of human somatic dna, and the available frontend methods (extraction and filtration columns, magnetic particle capture, and silica-based systems) still lack efficiency [21, 22] . manufacturers are spending most of their energy on backend technology (detection and reporting). front-end processing is urgently needed, ideally resulting in the development of a creative new method for concentrating cell-rich specimens (such as purulent fluid, blood, and sputum) from humans to allow for sensitive detection of microbial nucleic acids. diagnosis of tuberculous meningitis is another example of the problems inherent in amplification technology using pcr [23] . patients may have confounding signs and symptoms, the number of organisms in the csf sample may be very low, and conventional tests are insensitive and/or slow. direct acid-fast staining of csf specimens has a sensitivity of ∼52% [24] . the volume of csf needed for adequate culture is at least 5 ml (preferably 10 ml), because mycobacterium tuberculosis is extremely buoyant in csf as a result of its waxy cell wall; thus, concentration by centrifugation is inefficient [25] . smears for acid-fast bacilli made from the sediment must be layered and examined exhaustively-sometimes for 30 min-in the hope of detecting the rare m. tuberculosis rod [23] . naa would seem to be the answer. sadly, naa tests display an overall sensitivity of 71% (range, 25%-100%) and a specificity of 95% (range, 92%-97%), as determined by a meta-analysis of 49 published studies, 35 of which used home-brewed (individual laboratorydeveloped and -validated) methods [26] . when results from the 14 laboratories that used commercially available assays were evaluated separately, the overall sensitivity decreased to 56% (range, 46%-66%). these are the assays that are likely to be available to most clinicians. the naa results were equal to those for the acid-fast bacilli smear. clinicians must realize that unwanted reliance on new technology may lead to diagnostic errors. another highly favored technology for laboratory diagnosis of infectious diseases, whether testing tissue directly or testing isolated colonies, is identification by nucleic acid sequencing. there are also pitfalls in this approach. for example, many organisms, although they may be antigenically or biochemically distinct, are virtually genetically identical. members of the bacteroides fragilis group, for example, are highly related and cannot be separated easily by standard sequencing methods [27] . most clinicians realize that escherichia coli and shigella species are highly related [28] , but they have not considered the difficulty that this relatedness would pose if we relied on sequencing to identify these genera directly in patient samples. the extent of shared genes and taxonomically (versus phenotypically) shared traits among most microbes is unknown. another issue that vexes clinicians is the taxonomic confusion caused by genetic investigations. rhinosporidium seeberi, for example, is an agent of mass lesions in the sinus and mucous membranes. this organism has often been observed in histopathologic sections, but it has never been grown in culture. it resembles the spherule of coccidioides immitis, however, so it has long been considered an "unculturable" fungus. recently, 18s rrna sequencing placed the organism into a group of protozoan fish parasites [29] . we are constantly faced with new names for familiar organisms-and even with new organisms that may or may not be important in familiar syndromes. before more-extensive testing was performed, the newly discovered mycoplasma fermentans (originally called mycoplasma incognitus) had been implicated in both gulf war syndrome and hiv infection [30, 31] . it was later found to be quite common in humans [32] . after effective therapy, results of pcr of genital samples obtained from patients with c. trachomatis remain positive for 12 weeks [33] . nucleic acid is detectable but probably not viable. we do not know the extent of carriage of unculturable microbial or "normal" microbial dna in other healthy hosts. the cost of equipment, expensive reagents, and additional skilled personnel needed is not easy to justify. for example, the fda has cleared a real-time pcr test for group b streptococcal vaginal/rectal colonization during labor [34] . widespread use of this test could negate the need to perform cultures for pregnant women during weeks 35-37 of gestation, and it would solve the problems associated with treating women who present in labor without screening test results [35] . currently, 25% of women receive prophylactic penicillin during labor [36] . this number would decrease if only women with pcr results positive for group b streptococci at labor were treated [36, 37] . the problem, however, is that current recommendations [38] have already reduced the incidence of early-onset neonatal group b streptococcal disease to less than the 2010 national health objective of 0.5 cases per 1000 live births, and additional improvement is not cost-effective [39] . lucile packard childrens hospital johnson center, a stanford university medical center affiliate on the same campus, delivers ∼1800 babies annually. to perform the pcr assay immediately after each sample is received (a necessity to assure that prophylaxis is administered for at least 4 h before delivery), we would have to staff 1 technologist at all times. test performance takes ∼15 min of hands-on time, and the test itself runs 45 min, so a technologist must perform continuous sample management. twenty-four/seven staffing for 365 days per year would require 3.9 clinical laboratory scientists, at a labor and overhead cost of $316,875. the reagents-not counting the cost of the instrument itself-cost $46,800 annually, for a total yearly cost of $363,675. stanford university medical center (stanford, ca) patients have a colonization rate of 17% among women in labor, resulting in 306 babies at risk per year. to prevent 3 potential group b streptococcal infections using this test, the cost per infection avoided would be $121,225. this is a hard sell to hospital administrators who are focused on the bottom line. potential laboratory staff labor savings with the use of random-access, moderately complex molecular systems, such as genexpert (mentioned above), may help to bring down costs. costs for new technologies are often added onto existing testing, so overall diagnostic costs increase. centers for medicare and medicaid services reimbursement is lagging dramatically behind the use of new tests. in the northern california reimbursement area, our laboratory receives only $98.08 for a pcr test for bordetella pertussis and bordetella parapertussis, although it costs the laboratory more than $150 to test each sample, because the tests are always performed one specimen at a time, which necessitates testing an additional 3 controls simultaneously. the future is not all bleak, of course. new agents of important diseases are being discovered with the help of molecular technologies. among those to have been elucidated recently are tropheryma whipplei, bartonella henselae, ehrlichia chaffeensis, and mycoplasma genitalium [40] . infectious disease-related challenges are ready for solutions with our new molecular tools. the etiology of 42% of cases of pharyngitis is unknown [41] , and the etiology of 20%-40% of cases of diarrhea is unknown [42] , as is the etiology of 40%-60% of cases of pneumonia [43] . we cannot cultivate ∼25% of the organisms in the subgingival crevices of patients with periodontal disease [40] . all of these mysteries regarding vexing syndromes will eventually yield to molecular technologies. proteomics (i.e., profiling the proteins generated by human cells in response to various stimuli, including components and products of infectious agents) is in its infancy, but exciting developments are enticing. the spots of dna or rna on "gene chips" can be designed to be homologous with nucleic acid sequences in the sample, representing nucleic acids found in both the normal situation and the activated state of transcription or representing various genetic sequences unique to a vast array of microbes for detection of infectious agents [44] . samples are treated to break down their dna or rna into small discrete sequences, for which an identical sequence exists on the chip. if the sample nucleic acid finds its match, it binds and emits an electronic signal [45] . such a human-response chip can be used to determine a patient's response to potentially toxic antimicrobial treatments in advance of delivery. if the patient lacks enzymes to break down toxins or cannot metabolize a drug effectively, alternative therapies can be used to avoid dangerous adverse effects [46] . in another prospective microarray-based test, human leukocyte response to various infectious agents may be used to pinpoint the etiology before the agent itself can be detected [47] . development of this sort of technology is on a fast track, to facilitate detection of bioterrorism agents before the symptomatic phase of disease renders widespread preventive measurements inadequate [48] . proteomics is being used to fine-tune the development of new anti-infectives as well [49] . one aspect of microarray technol-ogy yet to be standardized and codified is the monumental problem of data analysis [50] . an entire new discipline of bioinformatics has developed for this purpose. this challenge of interpreting the vast volume of data points generated by microarray studies is acquired along with the technological advances, and its resolution will require a collective and extensive effort. in summary, the future potential utility of these incredible technologies-and of numerous others not mentioned herewill revolutionize infectious diseases diagnostics. however, the shortcomings of these technologies must be recognized. present dangers include failure to support the need for traditionally trained microbiologists, allowance of expertise to be moved to sites distant from patient care activities, inappropriate trust of new technology, and underestimation of the value of clinical diagnosis based on the acumen of experienced laboratorians and infectious diseases practitioners. dna probes for infectious diseases molecular and cellular microbiology: new tools of the trade survey of clinical microbiology laboratory workloads, productivity rates, andstaffing vacancies herpes simplex encephalitis: children and adolescents cumitech 31-verification and validation of procedures in the clinical microbiology laboratory verification of infectious disease molecular assays: a self-study module (cd-rom or web-based) a multiplex rt-pcr for detection of type a influenza virus and differentiation of avian h5, h7, and h9 hemagglutinin subtypes the clinical relevance of 'csf viral culture': a two-year experience with aseptic meningitis in a rapid and highly accurate assay for the detection of enterovirus infections in cerebrospinal fluid samples using the genexpert dx system impact of human immunodeficiency virus type 1 (hiv-1) genetic diversity on performance of four commercial viral load assays: lcx hiv rna quantitative, am-plicor hiv-1 monitor v1.5, versant hiv-1 rna 3.0, and nuclisens hiv-1 qt comparing first-void urine specimens, self-collected vaginal swabs, and endocervical specimens to detect chlamydia trachomatis and neisseria gonorrhoeae by a nucleic acid amplification test comparison of methods for detection of chlamydia trachomatis and neisseria gonorrhoeae using commercially available nucleic acid amplification tests and a liquid pap smear medium validation of molecular-diagnostic techniques in the parasitological laboratory diagnostic pcr: validation and sample preparation are two sides of the same coin failure of commercial ligase chain reaction to detect mycobacterium tuberculosis dna in sputum samples from a patient with smear-positive pulmonary tuberculosis due to a deletion of the target region reproducibility of positive test results in the bdprobetec et system for detection of chlamydia trachomatis and neisseria gonorrhoeae false-positive gen-probe direct mycobacterium tuberculosis amplification test results for patients with pulmonary m. kansasii and m. avium infections the current status and potential role of laboratory testing to prevent transfusion-transmitted malaria characteristics of apparently false-negative digene hybrid capture 2 high-risk hpv dna testing occurrence and documentation of low-level bacteremia in a community hospital's patient population comparison of 9 different pcr primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 rna extraction methods comparison of six dna extraction methods for recovery of fungal dna as assessed by quantitative pcr rapid diagnosis of tuberculous meningitis: what is the optimal method? comparison of conventional bacteriology with nucleic acid amplification (amplified mycobacterium direct test) for diagnosis of tuberculous meningitis before and after inception of antituberculosis chemotherapy the bacteriological diagnosis of tuberculous meningitis diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: a systematic review and meta-analysis rapid identification of the species of the bacteroides fragilis group by multiplex pcr assays using group-and species-specific primers sequence analysis of four shigella boydii o-antigen loci: implication for escherichia coli and shigella relationships rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites lack of serological evidence for mycoplasma fermentans infection in army gulf war veterans: a large scale case-control study mycoplasma fermentans in individuals seropositive and seronegative for hiv-1 serological responses to mycoplasmas in hiv-infected and non-infected individuals monitoring of chlamydia trachomatis infections after antibiotic treatment using rna detection by nucleic acid sequence based amplification multicenter study of a rapid molecular-based assay for the diagnosis of group b streptococcus colonization in pregnant women comparison of rapid intrapartum screening methods for group b streptococcal vaginal colonization perinatal screening for group b streptococci: cost-benefit analysis of rapid polymerase chain reaction risk factors for early-onset group b streptococcal sepsis: estimation of odds ratios by critical literature review prevention of perinatal group b streptococcal disease: revised guidelines from cdc centers for disease control and prevention. diminishing racial disparities in early-onset neonatal group b streptococcal disease-united states discovering new pathogens: culture-resistant bacteria etiology of acute pharyngitis in children: is antibiotic therapy needed? the management of acute diarrhea in children: oral rehydration, maintenance, and nutritional therapy practice guidelines for the management of community-acquired pneumonia in adults. infectious diseases society of america dna microarray technology: devices, systems, and applications molecular signatures for diagnosis of infection: application of microarray technology use of dna microarrays to monitor host response to virus and virus-derived gene therapy vectors comparative dna microarray analysis of host cell transcriptional responses to infection by coxiella burnetii or chlamydia trachomatis applications and challenges of dna microarray technology in military medical research using microarray gene signatures to elucidate mechanisms of antibiotic action and resistance iterative group analysis (iga): a simple tool to enhance sensitivity and facilitate interpretation of microarray experiments potential conflicts of interest. e.j.b. has been on the speakers' bureau for becton dickinson and cepheid and has received research support materials from cepheid, roche molecular diagnostics, and advandx. key: cord-027445-bpx4qr0i authors: eisty, nasir u.; perez, danny; carver, jeffrey c.; moulton, j. david; nam, hai ah title: testing research software: a case study date: 2020-05-25 journal: computational science iccs 2020 doi: 10.1007/978-3-030-50436-6_33 sha: doc_id: 27445 cord_uid: bpx4qr0i background: the increasing importance of software for the conduct of various types of research raises the necessity of proper testing to ensure correctness. the unique characteristics of the research software produce challenges in the testing process that require attention. aims: therefore, the goal of this paper is to share the experience of implementing a testing framework using a statistical approach for a specific type of research software, i.e. non-deterministic software. method: using the parsplice research software project as a case, we implemented a testing framework based on a statistical testing approach called multinomial test. results: using the new framework, we were able to test the parsplice project and demonstrate correctness in a situation where traditional methodical testing approaches were not feasible. conclusions: this study opens up the possibilities of using statistical testing approaches for research software that can overcome some of the inherent challenges involved in testing non-deterministic research software. research software can enable mission-critical tasks, provide predictive capability to support decision making, and generate results for research publications. faults in research software can produce erroneous results, which have significant impacts including the retraction of publications [8] . there are at least two factors leading to faults in research software: (1) , the complexity of the software (often including non-determinism) presents difficulties for implementing a standard testing process and (2) the background of people who develop research software differ from traditional software developers. research software often has complex, non-deterministic computational behavior, with many execution paths and requires many inputs. this complexity makes it difficult for developers to manually identify critical input domain boundaries and partition the input space to identify a small but sufficient set of test cases. in addition, some research software can produce complex outputs whose assessment might rely on the experience of domain experts rather than on an objective test oracle. finally, the use of floating-point calculations can make it difficult to choose suitable tolerances for the correctness of outputs. in addition, research software developers generally have a limited understanding of standard software engineering testing concepts [7] . because research software projects often have difficulty obtaining adequate budget for testing activities [11] , they prioritize producing results over ensuring the quality of the software that produces those results. this problem is exacerbated by the inherent exploratory nature of the software [5] and the constant focus on adding new features. finally, researchers usually do not have training in software engineering [3] , so the lack of recognition of the importance of the corresponding skills causes them to treat testing as a secondary activity [10] . to address some of the challenges with testing research software, we conducted a case study on the development of a testing infrastructure for the par-splice 1 research software project. the goal of this paper is to demonstrate the use of a statistical method for testing research software. the key contributions of this paper are (1) an overview of available testing techniques for non-deterministic stochastic research software, (2) implementation of a testing infrastructure of a non-deterministic parallel research software, and (3) demonstration of the use of a statistical testing method to test research software that can be a role model for other research software projects. in a non-deterministic system, there is often no direct way for the tester (or test oracle) to exactly predetermine the expected behavior. in parsplice (described in sect. 2.1), the non-determinism stems from (1) the use of stochastic differential equations to model the physics and (2) the order in which communication between the procedures occurs (note however that even though the results from each execution depends upon message ordering, each valid order produces a statistically accurate result, which is the key requirement for the validity of parsplice simulations). in cases where development of test oracles is difficult due to the nondeterminism, some potentially viable testing approaches include metamorphic testing, run-time assertions, and machine learning techniques [6] . after describing the parsplice project, the remainder of this section explains these techniques along with their possible applicability to parsplice. parsplice (parallel trajectory splicing) [9] aims at overcoming the challenge of simulating the evolution of materials over long time scales through the timewise parallelization of long atomistic trajectories using multiple independent producers. the key idea is that statistically accurate long-time trajectories can be assembled by splicing end-to-end short, independently-generated, trajectory segments. the trajectory can then grow by splicing a segment that begins in the state where the trajectory currently ends, where a state corresponds to a finite region of the configuration space of the problem. this procedure yields provably statistically accurate results, so long as the segments obey certain (relatively simple) conditions. details can be found in the original publication [9] . the parsplice code is a management layer that orchestrates a large number of calculations and does not perform the actual molecular dynamics itself. instead, parsplice uses external molecular dynamics engines. the simulations used in parsplice rely on stochastic equations of motion to mimic the interaction of the system of interest with the wider environment, which introduces a first source of non-determinism. a basic parsplice implementation contains two types of processes: a splicer and producers. the splicer manages a database of segments, generates a trajectory by consuming segments from the database, and schedules execution of additional segments, each grouped by their respective initial state. producers fulfill requests from the splicer and generate trajectory segments beginning in a given state; the results are then returned to the splicer. the number of segments to be scheduled for execution in any known state is determined through a predictor statistical model, built on-the-fly. importantly, the quality of the predictor model only affects the efficiency of parsplice and not the accuracy of the trajectory. this property is important because the predictor model will almost always be incomplete, as it is inferred from a finite number of simulations. the unavailability of the ground truth model (which is an extremely complex function of the underlying physical model) makes assessment of the results difficult. in addition, this type of stochastic simulation is not reproducible, adding to the difficulty of testing the code. therefore, in this case study we create a basis for the parsplice testing infrastructure using various methodical approaches and apply the test framework to the continuous integration process. metamorphic testing operates by checking whether the program under test behaves according to a set of metamorphic relations. for example, a metamorphic relation r would express a relationship among multiple inputs x1, x2,.., xn (for n > 1) to function f and their corresponding output values f(x1), f(x2),.., f(xn) [2] . these relations specify how a change to an input affects the output. these metamorphic relations serve as a test oracle to determine whether a test case passes or fails. in the case of parsplice, it is difficult to identify metamorphic relations because the outputs are non-deterministic. the relationship between the x 's and the f 's is therefore not direct but statistical in nature. an assertion is a boolean expression or constraint used to verify a necessary property of the program under test. usually, testers embed assertions into the source code that evaluate when a test case is executed. later testers use these assertions to verify whether the output is within an expected range or if there are some known relationships between program variables. in this way, a set of assertions can act as an oracle. in the context of parsplice, assertions can be used to test specific functions, but not to test the overall validity of the simulations, would protect only against catastrophic failures, such as instabilities in the integration scheme. machine learning is a useful approach for developing oracles for non-deterministic programs. researchers have shown possibilities of both black-box features (developed using only inputs and outputs of the program) and white-box features (developed using the internal structure of the program) to train the classifier used as the oracle [1, 4] . it is possible to test parsplice with machine learning techniques. for example, we could fake the molecular dynamics (md) engine with our own model to produce output data to use as a training set and consider the actual output data as a testing set. due to the amount of effort required to use this approach in parsplice, we determined that it was not feasible. to implement the testing framework, the first author spent a summer at los alamos national laboratory working on the parsplice project. the testing framework is based on the multinomial testing approach (described in sect. 3.2), implemented using a progress tracking card (ptc) in the productivity and sustainability improvement plan (psip) 2 methodology. the testing approach is integrated with the cmake/ctest tool for use in the runtime environment and continuous integration. in this section, we describe the psip methodology, the multinomial test approach, and results that verify the implementation of the testing framework. the psip methodology provides a constructive approach to increase software quality. it helps decrease the cost, time, and effort required to develop and maintain software over its intended lifetime. the psip workflow is a lightweight, multi-step, iterative process that fits within a project's standard planning and development process. the steps of psip are: a) document project practices, b) set goals, c) construct progress tracking card, d) record current ptc values, e) create plan for increasing ptc values, f) execute plan, g) assess progress, h) repeat. we created and followed a ptc containing a list of practices we were working to improve, with qualitative descriptions and values that helped set and track our progress. our progress tracking card consists of 6 scores with a target finish date to develop the testing framework. the scores are: -score 0 -no tests or approach exists -score 1 -requirement gathering and background research -score 2 -develop statistical test framework -score 3 -design code backend to integrate test -score 4 -test framework implemented into parsplice infrastructure -score 5 -integrate into ci infrastructure we were able to progress through these levels and obtain a score of 5 by the end of the case study. the multinomial test is a statistical test of the null hypothesis that the parameters of a multinomial distribution are given by specified values. in a multinomial population, the data is categorical and belongs to a collection of discrete nonoverlapping classes. for instance, multinomial distributions model the probability of counts of each side for rolling a k-sided die n times. the multinomial test uses pearson's χ 2 test to test the null hypothesis that the observed counts are consistent with the given probabilities. the null hypothesis is rejected if the pvalue of the following χ 2 test statistics is less than a given significance level. this approach enables us to test whether the observed frequency of segments starting in i and ending in j is indeed consistent with the probabilities p ij given as input to the monte carlo backend. our multinomial test script uses the output file of parsplice as its input and execute the test and post-processes the results by performing pearson's χ 2 to assess whether to reject the null-hypothesis. a key insight from the theory that underpins parsplice is that a random process that describes the splicing procedure should rigorously converge to a discrete time markov chain in a discrete state space. in other words, the probability that a segment added to a trajectory currently ending in state i leaves the trajectory in state j should be a constant p ij that is independent of the past history of the trajectory. one way to test parsplice would be to verify that the splicing procedure is indeed markovian (memory-less). however, taken alone, such a test would not guarantee that the splicing proceeds according to the proper markov chain. a more powerful test would assess whether the spliced trajectory is consistent with the ground-truth markov chain. a key obstacle to such a test is that this ground-truth model is, in practice, unknown and can only be statistically parameterized from simulation data. to address this issue, we replaced the molecular dynamics (md) simulation backend with a simpler monte carlo implementation that samples from a pre-specified, markov chain. that is, we replaced the extremely complex model inherent to the md backend with a known, given model of predefined probabilities. the task then becomes assessing whether the trajectory generated by parsplice, as run in parallel on large numbers of cores, reproduces the statistics of the ground truth model. in this context, this technique is the ultimate test of correctness, as parsplice is specifically designed to parallelize the generation of very long trajectories that are consistent with the underlying model. statistical agreement between the trajectory and the model demonstrates that the scheduling procedure is functional (otherwise, the splicing the of trajectory would halt), the task ordering procedure is correct, the tasks executed properly, the results reduced correctly, and the splicing algorithm was correct. the statistical assessment to test parsplice can be conducted using the multinomial test approach. our null hypothesis was that the observed counts generated by parsplice are consistent with the probabilities in the model. if the p-value from the multinomial test is less than 0.05, we reject the null hypothesis and conclude that the observed counts differ from the expected ones. conversely, if the p-value is greater than 0.05, we do not reject the null hypothesis and can conclude that the test passes. for the sake of verifying our multinomial test, we ran parsplice in different time frames and observed the result. figure 1 shows the p-values obtained from running parsplice for 1, 2, 5, 10, 20, 40, 60, and 90 min. we can see that in all cases, the p-values are greater than 0.05, which indicates that the tests passed during these instances of the execution. in this paper, we describe a case study of the parsplice project in which we followed the psip methodology to develop a testing framework to address the difficulties of testing non-deterministic parallel research software. we first considered applying traditional industrial testing approaches. however, the nondeterminism of parsplice made these approaches unusable. then we identified testing techniques specially designed for non-deterministic software. once again, those techniques did not fit parsplice. finally, we identified a statistical testing approach, multinomial testing, that would work for parsplice. the multinomial testing approach is ideal for parsplice given its constraints, i.e. time, non-determinism, and the existing continuous integration system. the lessons learned from this case study can be valuable to the larger research software community because, like parsplice, many research software projects have stochastic behavior which produces non-deterministic results. the approach we followed to develop the test framework can be a model for other research software projects. we plan to extend the testing infrastructure in a more methodological way with as many possible testing techniques installed in the system. pat: a pattern classification approach to automatic reference oracles for the testing of mesh simplification programs fault-based testing in the absence of an oracle engineering the software for understanding climate change automating image segmentation verification and validation by learning test oracles improving the development process for cse software techniques for testing scientific programs without an oracle testing scientific software: a systematic literature review a scientist's nightmare: software problem leads to five retractions long-time dynamics through parallel trajectory splicing some problems of professional end user developers software development cultures and cooperation problems: a field study of the early stages of development of software for a scientific community key: cord-021511-88xaynf7 authors: werner, linda l.; turnwald, grant h.; willard, michael d. title: immunologic and plasma protein disorders date: 2009-05-15 journal: small animal clinical diagnosis by laboratory methods doi: 10.1016/b0-72-168903-5/50017-3 sha: doc_id: 21511 cord_uid: 88xaynf7 nan occurs in glomerular disease (and may be severe; see chapter 7). albuminuria as the result of glomerulopathy is rarely associated with significant globulin loss. decreased production is due to chronic hepatic insufficiency or hyperglobulinemia. the latter may cause mild hypoalbuminemia, whereas chronic hepatic insufficiency can cause moderate to severe decreases. in hyperglobulinemia, albumin synthesis may be decreased (i.e., "down regulation"). when inflammation is associated with hypoalbuminemia and hyperglobulinemia, albumin is sometimes called a negative acute phase protein. inadequate protein intake (including poorly digestible protein), maldigestion, and malabsorption are rare causes of mild hypoalbuminemia; however, occasionally they accompany other conditions causing hepatic insufficiency or increased protein loss. significant hypoalbuminemia (i.e., albumin < 2.1 g/dl) should never be attributed solely to decreased nutrition until hepatic insufficiency and proteinlosing disorders have been eliminated by definitive tests (not just by history and physical examination). decreased intake very rarely causes serum albumin concentrations less than 2.1 g/dl, except perhaps in very young animals. plasma total protein 6.0-7.8 6.0-7.5 serum total protein 5.5-7.5 5.5-7.8 serum albumin 2.5-4.0 2.5-4.0 serum globulin 3.0-3.5 3.0-3.8 sequestration may occur in pleural or peritoneal cavities or subcutaneous (sc) tissues. thus patients with effusion caused by hypoalbuminemia may further lower their serum albumin concentration via sequestration. alternatively, sequestration can be secondary to increased hydrostatic pressure (e.g., portal hypertension, right-sided cardiac failure). immune-mediated or infectious vasculopathies (e.g., endotoxemia and bacteremia, ehrlichiosis, rocky mountain spotted fever [rmsf] ) also allow loss from the vascular compartment. hypoalbuminemia as the result of sequestration or vasculopathy is usually mild. a diagnostic approach to hypoalbuminemic patients is outlined in figure 12 -1. a urinalysis (sometimes including urine protein:creatinine ratio; see chapter 7) and measurement of serum bile acids (see chapter 9) are indicated. severe cutaneous exudative lesions may be diagnosed by physical examination, but the possibility of renal, hepatic, and alimentary disease should still be investigated. hypercholesterolemia plus hypoalbuminemia suggests protein-losing nephropathy. significant proteinuria indicates a diagnostic workup for protein-losing nephropathy (pln) (see chapter 7). hypocholesterolemia plus hypoalbuminemia is suggestive of hepatic insufficiency or ple. hypoalbuminemia associated with hepatomegaly; microhepatia; neurologic signs; icterus; decreased blood urea nitrogen (bun) with or without increased alanine aminotransferase (alt), serum alkaline phosphatase (sap), or both; or abnormal hepatic function test results (e.g., serum bile acids) requires a diagnostic workup for hepatic insufficiency (see chapter 9). note: alt and sap are normal in many patients with severe hepatic disease. a portosystemic shunt is more likely in young animals; however, congenital shunts can be diagnosed in animals more than 10 years old. acquired hepatic disease is more common in adults and requires hepatic biopsy for diagnosis; however, some dogs less than 1 year old have severe, acquired hepatic disease. hypoalbuminemia with normal liver function tests and absence of proteinuria or cutaneous lesions allows one to diagnose ple by exclusion (see chapter 9), even if feces are normal. if the patient has renal or hepatic disease and ple is still a concern, then measurement of fecal alpha-1 protease inhibitor concentrations (chapter 9) may allow diagnosis of ple by inclusion. intestinal biopsy may then provide a definitive diagnosis of which intestinal disease is causing ple. endoscopic biopsies are safer, but it is critical that excellent quality tissue samples be obtained (many endoscopically-obtained samples are nondiagnostic). exploratory laparotomy is acceptable. if laparotomy is performed, hepatic biopsy should generally be performed along with intestinal biopsies. it is important to 292 small animal clinical diagnosis by laboratory methods obtain biopsy specimens at several sites along the small intestine, even when no apparent gross lesions are found. edematous sc fluid accumulations associated with hypoalbuminemia are usually transudates: pln or ple, chronic hepatic insufficiency, and immune-mediated or infectious vasculitis may be responsible. however, one should always sample fluid accumulations to be sure that they are in fact transudates, as opposed to unexpected modified transudates or exudates. • see following discussion of protein electrophoresis. occasionally indicated • protein electrophoresis is performed when hyperglobulinemia is not caused by hemoconcentration and either (1) the globulin concentration is high enough to make monoclonal gammopathy a reasonable possibility or (2) humoral immunodeficiencies are suspected. a paleblue background on stained blood or bone marrow smears can represent increased plasma protein and may be an indication for protein electrophoresis. disadvantages • specific diagnosis is seldom obtained. although a specific diagnosis is seldom obtained from electrophoresis, electrophoretic patterns can be valuable when interpreted with clinical signs and other laboratory data. electrophoresis is quantitative. immunoelectrophoresis is qualitative, identifying specific proteins (e.g., immunoglobulins) but not detecting slight increases or decreases. immunoelectrophoresis is the method of choice to detect urinary and serum bence jones protein, a monoclonal protein equivalent to immunoglobulin light chains which occasionally occurs in multiple myeloma and macroglobulinemia. a routine protein electrophoresis performed on a concentrated urine sample occasionally detects an isolated monoclonal peak in the urine (e.g., bence jones protein). finding a urine electrophoresis pattern mimicking that of serum indicates a glomerular lesion substantial enough to allow leakage of most serum proteins, including the serum monoclonal heavy chain peak; therefore, it is not evidence of bence jones light chains. canine bence jones proteinuria is only rarely detected by heat precipitation. positive results for bence jones proteins by an acid precipitation screening test should be confirmed by concentrated urine electrophoresis or immunoelectrophoresis because of the possibility of false-positive results. analysis • serum or urine may be analyzed, and it may be refrigerated or frozen. analysis • the cellulose acetate technique is the method of choice. interpretation of electrophoretograms is based on densitometric measurements of intensity of staining of protein bands on cellulose acetate strips. the serum separates into four fractions: (1) albumin, (2) alpha (α) globulins, (3) beta (β) globulins, and (4) gamma (γ) globulins. canine and feline α-, β-, and γ-globulins are usually divided into two subfractions each: α 1 , α 2 ; β 1 , β 2 ; and γ 1 , γ 2 (table 12 -4). normal-appearing electrophoretograms from dogs and cats are presented in figures 12-2 and 12-3. artifacts • albumin concentration is usually underestimated by electrophoresis compared with a chemical determination. therefore the albumin:globulin ratio (a:g) is usually higher by chemical determinations than by electrophoretic determination. analysis • after electrophoresis in agar gel, polyclonal antiserum to specific proteins (including immunoglobulins) is added to a trough parallel with the separated serum proteins. the reagents are allowed to diffuse. to obtain quantitation of the individual immunoglobulins, radial immunodiffusion (rid), electroimmunodiffusion (i.e., rocket electrophoresis), or laser nephelometry is performed. these procedures, when available, can also be used to quantitate immunoglobulin subclasses. (feldsburg, 1994) . it is likely that breedspecific variations also occur. staining are overestimated. igg migrates in βand γ-globulin regions; therefore, igg concentrations determined by rid are usually higher than the γ-globulin fraction determined by electrophoresis. this discrepancy increases when igg hyperproduction occurs, such as in some myelomas, canine ehrlichiosis, feline infectious peritonitis [fip] , and other chronic infections. causes of hyperglobulinemia • polyclonal hyperglobulinemias, also called gammopathies, have a broad-based peak encompassing β and γ regions and suggest persistent antigenic stimulation and inflammation (chronic bacterial, viral, fungal, protozoal, rickettsial, or parasitic disorders), neoplasia, or immune-mediated disease (see table 12 -3). the most common causes in dogs are cutaneous parasitism, pyoderma, dirofilariasis, and ehrlichiosis, depending on geographic location (see figure 12 -2 and table 12 -3). increases are commonly in the β and γ regions. in cats the most common cause of severe polyclonal gammopathy is fip (see figure 12 -3). increases in feline globulins are commonly in the γ region. a concurrent mild decrease in albumin synthesis may occur in patients with hyperglobulinemia, perhaps to maintain oncotic pressure or viscosity. in hypoalbuminemic states, globulin may increase secondarily. monoclonal hyperglobulinemias have a narrow-based electrophoretic peak (i.e., "spike") in the β or γ region, normally no wider than the albumin peak. monoclonal immunoglobulin elevations are also called paraproteins or m proteins and are usually the result of lymphocyte and plasma cell neoplasias (e.g., multiple myeloma, macroglobulinemia, lymphosarcoma; see table 12 -3). monoclonal or oligoclonal spikes are occasionally caused by infectious (e.g., ehrlichiosis; see figure 12 -2) or idiopathic disorders. both multiple myeloma and ehrlichiosis can have monoclonal electrophoretic patterns and bone marrow plasmacytosis. in ehrlichiosis, however, the electrophoretic pattern is often a monoclonal or oligoclonal pattern superimposed on or arising within a broader-based globulin peak (see figure 12 -2, top). in such cases, examination of the stained electrophoretogram bands shows a clearly restricted monoclonal band with sharper edges within a paler, broader background band. in contrast, monoclonal spikes associated with neoplastic disorders are frequently accompanied by normal to decreased nonparaprotein globulin fractions, often reflecting impaired production of other immunoglobulins. a suggested diagnostic approach to hyperglobulinemia is outlined in figure 12 -4. in dogs with hyperglobulinemia and severe pruritic dermatitis, diagnostic evaluation may involve only a physical examination to identify fleas and ticks or skin scrapings to detect mites. demodex canis mites are usually detected easily, whereas sarcoptes scabiei are often difficult to find. for canine heartworm disease, the enzyme-linked immunosorbent assay (elisa) antigen test is the preferred screening procedure. in areas endemic for ehrlichiosis or rmsf, titers are indicated (see chapter 15), particularly if the patient has anemia, thrombocytopenia, leukopenia (or a combination thereof). testing for other infectious disorders, such as canine brucellosis, blastomycosis, histoplasmosis, coccidioidomycosis, or feline cryptococcosis, is dictated by geographic location and other physical, laboratory, or radiographic abnormalities. the possibility of more than one cause of hyperglobulinemia should be considered. gammopathies associated with immune-mediated disease and nonlymphocytic and plasmacytic neoplasia are usually mild and rarely require extensive evaluation of the gammopathy. feline corona virus (i.e., fip) titers are generally not useful in cats with signs consistent with fip. if an exudative effusion is present, fluid:serum γ-globulin ratios can be determined (see chapter 15). confirmatory testing for fip can be performed on affected tissue biopsies, usually liver, using an immunohistochemical stain for fip virus. hyperglobulinemia can be the result of dirofilariasis in cats, but the disease is less common than in dogs. hyperglobulinemia can also be present in chronic feline leukemia virus (felv) and feline immunodeficiency virus (fiv) infections. if joint pain, stiff gait, or increased joint fluid volume accompanies hyperglobulinemia, radiographs and joint fluid analysis (see chapter 10) are indicated. rheumatoid factor (rf) testing, ana testing, rickettsial titers, or a borreliosis titer (or a combination of these tests) may be helpful if the joint fluid is a nonseptic exudate. if a diagnosis is not obtained at this stage, if a patient appears inappropriately ill, if the hyperglobulinemia seems excessive (e.g., globulin > 5 g/dl), or if any signs consistent with hyperviscosity are present (see serum viscosity), one should differentiate monoclonal from polyclonal gammopathies by serum protein electrophoresis. if monoclonal gammopathy is detected in a patient with multiple myeloma or lymphosarcoma, immunoelectrophoresis or quantitation of immunoglobulins by rid (or rocket electrophoresis) identifies the class of immunoglobulin composing the paraprotein. these tests also detect nonparaprotein immunoglobulin deficiencies, which are common in patients with lymphocytic or plasmacytic neoplasias (see causes of hypoglobulinemia). polyclonal gammopathies may be caused by infectious, immune-mediated, or neoplastic disorders. if the cause of polyclonal gammopathy is unknown, thoracic and abdominal immunologic and plasma protein disorders 297 figure 12-4. diagnostic evaluation of moderate to severe hyperglobulinemia (globulin ≥ 5.0 g/dl) in dogs and cats. cbc, complete blood count; elisa, enzyme-linked immunosorbent assay; fip, feline infectious peritonitis; felv, feline leukemia virus; fiv, feline immunodeficiency virus. imaging, various titers, and/or exploratory surgery plus biopsy may be indicated. monoclonal gammopathies are usually caused by lymphoproliferative (e.g., plasma cell) neoplasia. evaluation of patients with monoclonal gammopathy may include skeletal radiographs, serum and urine immunoelectrophoresis, and bone marrow biopsy with cytologic and histologic evaluation. diagnosis of multiple myeloma in dogs requires finding at least two of the following: lytic skeletal lesions, bone marrow plasmacytosis, bence jones proteinuria, or a monoclonal spike on serum protein electrophoresis. skeletal lesions are uncommon in feline multiple myeloma. in areas endemic for ehrlichiosis, an ehrlichia canis titer should be performed in dogs with a monoclonal gammopathy. ehrlichiosis commonly causes proteinuria and bone marrow plasmacytosis, closely resembling multiple myeloma. fip rarely causes monoclonal spikes. serum viscosity can be measured. causes of hypoglobulinemia • newborn animals are physiologically hypogammaglobulinemic and have serum total protein concentrations 60% to 80% of adult values. congenital combined or selective immunodeficiencies occur but are rarely diagnosed, probably because immunodeficient puppies or kittens rapidly succumb to infections. in dogs these infections are usually distemper or parvovirus, and they occur in the postnatal period after maternal immunity wanes. immunodeficiency should be suspected when more than one pup in a properly cared for litter dies of infection in the first 2 to 6 months of life. complete blood count (cbc), serum chemistry profile, serum protein electrophoresis, and immunoelectrophoresis are typical initial tests in such situations. immunoglobulin quantitation by rid or rocket electrophoresis is recommended for confirmation and characterization of the type of immunoglobulin deficiency present. immunoglobulin quantitation techniques are more precise than qualitative methods (i.e., immunoelectrophoresis). selective (single class or subclass) and partial immunoglobulin deficiencies may not be readily detectable via serum electrophoresis and immunoelectrophoresis; all immunoglobulin classes (and igg subclasses when available) should be quantitated when a humoral immunodeficiency is suspected. humoral immunodeficiency is usually detected by finding decreased igg and iga and normal to decreased igm concentrations. selective iga deficiency and transient hypogammaglobulinemia occur in dogs; patients with selective iga deficiency may show chronic problems involving mucosal immunity (e.g., antibiotic responsive enteropathy) or be asymptomatic. serum electrophoresis in cases of selective or partial immunoglobulin deficiency may yield normal results or even show polyclonal elevations in globulins. immunoelectrophoresis of normal canine serum may show a low or absent stainable iga precipitation band because of iga's relatively low concentration or the result of quality assurance problems involving antisera or technique. when selective iga deficiency is suspected, the best diagnostic test is quantitative rid or rocket electrophoresis. iga deficiency is sometimes accompanied by elevated concentrations of igm or igg. the relationship between certain types of chronic inflammatory enteropathy (e.g., lymphocytic plasmacytic enteritis) and serum iga deficiency is unclear at this time. it is likely that deficiency of local secretory iga is not always reflected by serum iga values. in addition, low concentrations of serum iga have been associated with canine allergic and parasitic disorders, with return to normal after successful therapy. this finding suggests that those disease processes may lead to down regulation of iga as an immunomodulation event, and low iga probably has no causative role in the underlying chronic inflammatory bowel disease (hill, moriello, and deboer, 1995) . breed-specific normal ranges for immunoglobulins may provide a clearer picture of the role of selective iga deficiency in chronic enteropathies. the most common causes of hypoglobulinemia are external blood loss and ple. less common causes are pln and hepatic insufficiency (see chapters 7 and 9, respectively). in patients with paraproteinemias (e.g., multiple myeloma, macroglobulinemia, lymphosarcoma), remaining immunoglobulins are usually depressed. a complement measurement and evaluations of lymphocyte and phagocyte functions may also be indicated for patients with suspected congenital immunodeficiency (see testing for cellular immunity). rarely indicated • monoclonal gammopathy, hyperglobulinemic patients with signs of hyperviscosity (i.e., poor tissue perfusion, dilated retinal vessels, retinal hemorrhage or retinal detachment, renal disease, central nervous system (cns) dysfunction, bleeding problems), and monitoring of treatment of diseases causing hyperviscosity. hyperviscosity may be suspected based on finding high serum protein (usually > 10 g/dl) or on physical characteristics of serum (i.e., viscous). polycythemia should be included as a cause of increased blood viscosity to be eliminated in patients showing clinical signs of hyperviscosity. advantages • simple and diagnostically significant. ostwald viscosimeter or a 0.1 ml capillary pipette. time for a given volume of serum to flow from the pipette is compared with that for the same volume of water. artifacts • volume depletion causing increased serum protein concentration may increase serum viscosity but is unlikely to be clinically significant. markedly increased blood viscosity can interfere with tests using flow through devices (e.g., hematology autoanalyzers). viscosity • any drug causing volume depletion can increase serum viscosity. a relative viscosity greater than or equal to 4 is abnormal in people and probably abnormal for dogs. because of the relatively large size of igm, it has the greatest potential to cause hyperviscosity. iga (which can exist as a polymer or dimer) and very high concentrations of igg can also cause hyperviscosity. clinically significant serum hyperviscosity is almost invariably caused by lymphocyte and plasma cell neoplastic disorders (e.g., multiple myeloma, macroglobulinemia, lymphosarcoma; see table 12 -3). hyperviscosity syndrome rarely occurs in monoclonal gammopathy caused by ehrlichiosis. diagnostic approach is described in figure 12 -4 under monoclonal gammopathy. because lymphosarcoma and plasma cell myeloma are the major causes of serum hyperviscosity, aspiration of bone marrow, involved lymph nodes, masses, or other abnormal lymphoreticular organs is indicated. if results are equivocal, biopsy and histopathologic evaluation of bone marrow or involved tissues are indicated. cytologic or histologic evaluation of spleen and liver occasionally helps diagnose lymphocytic or plasmacytic neoplasia when samples from lymph nodes, bone marrow, or solid masses are not diagnostic. cryoglobulins are usually monoclonal or complexed immunoglobulins that reversibly precipitate or gel at low temperatures but dissolve when heated. cryoglobulins are rarely found in canine multiple myeloma and macroglobulinemia. rarely indicated • paraproteins that precipitate or gel when blood or serum is refrigerated at 4°c. advantage • detection of cryoglobulins is important, because failing to detect them may cause false-negative tests for hyperglobulinemia or monoclonal paraproteinemia in patients with clinical or laboratory evidence of hyperviscosity. analysis • the clinician should contact a reference lab about assaying for cryoglobulins. causes of cryoglobulinemia • macroglobulinemia or multiple myeloma of igm and iga classes may cause canine cryoglobulinemia. finding cryoglobulinemia indicates diagnostic evaluation for lymphocyte and plasma cell neoplasia as described for monoclonal gammopathies. occasionally indicated • abnormalities suggestive of systemic lupus erythematosus (sle), such as symmetric dermatitis principally distributed on the head and mucous membranes, hemolytic anemia, thrombocytopenia, nonseptic polyarthritis, myositis, proteinuria, or fever of unknown origin. less common are neuromuscular, cardiac, note: cryoglobulins are not cold agglutinins (i.e., antibodies binding antigen reversibly at temperatures < 37°c). or pulmonary abnormalities. the test can be used to monitor patients being treated for sle. advantages • simple and indicative of immune-mediated disease when positive with a relatively high titer in conjunction with compatible clinical abnormalities. disadvantages • not a disease-specific test (i.e., many diseases besides sle may be associated with a low titer; table 12-6). analysis • measured in serum by indirect immunofluorescence testing (iit). the result should be reported as the highest dilution of a patient's serum causing definite staining of nuclei. several patterns of nuclear fluorescence are recognized: homogeneous (diffuse), rim (peripheral), speckled (fine or large speckles), and nucleolar. normal values • values vary among laboratories owing to different substrates, controls, and procedures used. accuracy requires procedural consistency and experience. fetal and newborn sera do not stain nuclei. most veterinary laboratories use tissue culture monolayers of human epithelial-2 (hep-2) cells as substrate, which allows improved discernment of fluorescent patterns of staining and more standardized procedural consistency. when this test method was used in a comparative study involving 112 canine serum samples, a significant positive titer was established at a screening dilution of 1:25 for greater than 95% specificity, whereas a minimum significant ana titer of 1:100 was established as the corresponding significant titer using rat liver sections, which identified the identical group of ana-positive dogs at the same specificity of greater than 95% (hansson, turnwald-wigh, and karlsson-parra, 1996) . drug therapy that may alter antinuclear antibody titer • anything decreasing antibody synthesis (e.g., cytotoxic drugs, chronic or high-dose corticosteroid therapy) can decrease the titer. positive ana titers have been attributed to treatment with griseofulvin, hydralazine, procainamide, sulfonamides, and tetracyclines. positive ana titers can occur in cats treated with propylthiouracil or methimazole (peterson, kintzer, and hurvitz, 1988) . some of these cats develop drug-induced, immune-mediated hemolytic anemia (imha) and immunemediated thrombocytopenia (imt). artifacts • improper reagent preparation, storage, or application; inadequate controls. • a positive titer may occur in a number of infectious, inflammatory, and neoplastic disorders. a partial list of diseases (in addition to sle, in which a positive ana titer can be found) is given in table 12-6. normal dogs and cats can also have detectable ana; however, these tend to be low titers. positive titers obtained in disorders other than sle are generally not markedly elevated; therefore, it is important to consider values established by the laboratory for low, moderate, and high titers. equally important is consideration of other clinical and clinicopathologic changes consistent with a diagnosis of sle. suggested criteria for diagnosis of canine and feline sle are a positive ana titer plus one or more of the following: skin or oral cavity lesions with histopathologic and immunopathologic changes consistent with sle, polyarthritis, coombs'-positive hemolytic anemia, idiopathic or imt, pln, myositis, or (particularly in cats) neurologic disturbances. a positive ana titer is the most important criterion for diagnosis of sle, providing 300 small animal clinical diagnosis by laboratory methods established clinical criteria are met and exclusionary diagnoses are not made (e.g., felv or fip infection, cholangiohepatitis, rickettsial or systemic parasitic diseases). no well-established patterns of ana fluorescence in cats and dogs distinguish sle from other (i.e., non-sle) immune-mediated diseases or other conditions associated with positive ana titers (see table 12 -6). although ana titers in sle are frequently higher than in other disorders, there can be some overlap. magnitude of titer does not parallel severity of disease. periodic ana titers may be useful in monitoring a lupus patient's response to therapy. a positive ana titer can be an indication for performing additional tests to distinguish sle from non-sle disease: dermatitis favoring mucocutaneous junctions is an indication for biopsy, histopathologic examination, and immunohistochemical or direct immunofluorescent testing (dit; see immunostaining of tissues); hemolytic anemia is an indication for an antiglobulin (i.e., coombs') test (see chapter 3) and tests for hemoparasites; swollen, painful joints are an indication for arthrocentesis, fluid analysis (see chapter 10), and rf test (see later discussion). occasionally indicated • suspected sle (see antinuclear antibody). advantages • specific; does not require species-specific reagents, therefore more widely available. disdvantages • time-consuming and much less sensitive than the ana test; requires very fresh blood sample. analysis • depending on the laboratory, heparinized or clotted blood is used. formation of le-cells in vivo is rarely demonstrated in routinely stained bone marrow smears or in joint fluid from patients with polyarthritis (color plate 5a), but it is highly suggestive of sle when present. a laboratory experienced in performing and interpreting le cells is necessary. artifacts • le cells must be differentiated from "tart" cells, which are neutrophils that have phagocytosed intact nuclei. the le cell test is complement dependent, and low concentrations of complement, excessive heparin, or failure to use freshly drawn blood may cause false-negative results. • steroid therapy alters test results. the le cell test is more sensitive to effects of steroids than is an ana titer. • ideally a minimum of three to four le cells on a slide is necessary for a diagnosis of sle. the test should be performed at least three times before results are considered negative. the test is specific for sle but insensitive. negative results are common in sle patients that are ana positive. such patients may lack the particular autoantibodies to histone-dna involved in le cell formation but have other types of ana detectable by immunofluorescence. positive test results may rarely be obtained in other diseases (e.g., osteochondritis dissecans, nonimmunologic joint disease, neoplasia, disseminated intravascular coagulation). if a positive le cell preparation is obtained, an ana titer and tests to obtain other evidence of sle are indicated (as described earlier under antinuclear antibody). an ana titer is the preferred screening test for sle. see chapter 3. dogs and cats showing marked thrombocytopenia (< 50,000/µl) may have imt. this is typically a diagnosis of exclusion because no widely available test allows a definitive diagnosis of imt. measurement of platelet-associated antibodies and assay for antimegakaryocyte antibodies have been performed, but as of this writing, neither has been proven to have the desired sensitivity or specificity in clinical practice. a recently reported technique seems promising (scott et al, 2002) . occasionally indicated • dogs, particularly small breeds, suspected of having rheumatoid arthritis (ra) because of lameness, heat, swelling, or pain of multiple joints, particularly peripheral joints. crepitation and joint laxity may be detected in chronic cases. nonspecific signs include anorexia, fever, depression, and reluctance to move and may precede clinically or radiographically detectable evidence of joint disease. disadvantages • insensitive (i.e., many false-negative results) and not highly specific for ra in dogs because of relatively low titers. analysis • serum is submitted refrigerated but unfrozen. the rose-waaler test is the recommended test for canine rf (autoantibody reacting against a patient's own igg). canine rfs are predominantly igg but may also be mixed complexes of igg, iga, and igm. factors are detected by incubating rabbit iggsensitized sheep red blood cells (rbcs) with serial dilutions of the patient's serum (the rabbit rose-waaler test). if rf is present, agglutination occurs. because canine sera often contain naturally occurring antibodies to sheep rbcs, a control must be used with unsensitized sheep rbcs. if agglutination to an equal or higher dilution appears in the control, natural antibodies must be absorbed from the sera before testing for rf. latex agglutination tests for canine rf are not recommended because results are poorly reproducible and lack specificity. latex agglutination titers may differ substantially from rose-waaler titers. both tests are available through human laboratories; proper test controls are necessary. • for the rose-waaler test in normal animals, the differential titer (difference between titers at which agglutination of unsensitized versus sensitized sheep rbcs occurs) should be less than eight (barta, 1993) . a titer against sensitized rbcs not corrected for the actual titer of natural antibodies to sheep rbcs can be misleading because some normal animals have higher titers of naturally occurring antibodies to sheep rbcs. some laboratories perform the rose-waaler test by preabsorbing all test sera with sheep rbcs. in this case a titer of less than 16 is expected in normal dogs. artifacts • serum submitted for rf testing should not be frozen, because rf activity (especially igm) may be destroyed, causing a false-negative result. canine rf tends to self-associate, forming multimeric complexes that significantly lower the detectable titer. wide, sometimes negative, fluctuations in rf titer occur over time; these fluctuations do not appear to correlate with disease severity. false-positive results may occur in the rose-waaler test if a patient's serum has antibodies against sheep rbcs and appropriate controls or absorption of these antibodies is not performed. joints is the most reliable means of diagnosing canine ra. histopathologic examination of synovium allows an early diagnosis and therapy in patients lacking classic radiographic changes. an ana titer helps distinguish sle from ra; but it is occasionally positive for ra. finding both types of autoantibodies may represent the rare, combined occurrence of both sle and ra (a so-called overlap syndrome) or merely the appearance of multiple autoantibodies in a patient with ra or sle. in either case, clinical criteria are required for diagnosis. immunofluorescent testing of tissues uses a fluorescent dye conjugated with an antibody (i.e., fluorescein isothiocyanate [fitc] reagent) that detects antigen, immunoglobulin, or complement. several techniques are used for immunohistochemical staining involving enzyme-conjugated antibody in a chromogenic reaction system. the testing may be direct or indirect. in the direct test, tissues are assayed for deposits of antigen, immunoglobulins, or complement. occasionally indicated • suspected autoimmune skin disease or (rarely) diseases of internal organs (e.g., kidney) that may have an immunologic basis. in veterinary medicine, the test is used mainly on biopsy specimens of erosive or vesiculobullous cutaneous lesions. advantages • a definitive diagnosis can occasionally be made; formalin-fixed tissue can be used for some immunohistochemical procedures, allowing routine histopathology and immunostaining on sections from the same biopsy. disadvantages • extreme care must be taken in tissue selection and preservation. the test result is markedly influenced by corticosteroids; some immunofluorescence procedures require a special fixative and transport medium or fresh, snap-frozen tissue. sample preparation • requirements for handling and submitting biopsied tissues vary according to the procedure and reagents used, so the laboratory should be contacted. in cutaneous diseases it is imperative that early primary lesions (i.e., vesicles, bullae, pustules) and a margin of uninvolved skin be obtained. multiple specimens are recommended. obtaining primary lesions may necessitate checking a patient several times daily to identify a suitable, newly erupting lesion. ulcerated, crusted, or scarred lesions are worthless. ideally, biopsy specimens should not be obtained from the planum nasale or foot pads if other primary lesions are available because positive staining occurs at the basement membrane of these sites in many normal dogs. biopsy specimens for routine histopathology should be taken from the same, or at least a similar, lesion. larger samples can be bisected to provide comparable material for both tests. the biopsy specimen should not remain attached to the wall of the fixative container because this may cause inadequate preservation and false-negative results. analysis • tissue sections are prepared and tissue incubated with labeled antibodies to igg, igm, iga, or c3 and examined by fluorescent microscopy for immunofluorescent assays or routine microscopy for immunohistochemical assays. normal values • healthy epidermis has no detectable deposits of immunoglobulins in the intercellular spaces or at the basement membrane. nonspecific fluorescence of the stratum corneum should not be mistaken for deposits of immunoglobulins. appropriate controls are necessary to detect nonspecific background staining. artifacts • biopsy of inappropriate lesions produces false-negative results. cutaneous bacterial infections may cause immunoglobulin deposits in pustules, which should not be mistaken for autoimmune disease. • corticosteroids and immunosuppressive drugs (e.g., cyclophosphamide) may cause negative results. if an animal is receiving short-acting corticosteroids, the drug should be withdrawn for at least 3 weeks before biopsy. a longer period (1 to 2 months) may be necessary if long-acting injectable corticosteroids have been used. immunostaining results must be interpreted in light of clinical and histopathologic findings. immunoglobulin deposits can occur in other skin disorders, including mycosis fungoides, pyodermas, and acariasis. in these disorders, staining is usually patchy, focal, and present in pustules rather than in the adjacent tissues. in the indirect test, serum is assayed for circulating autoantibodies to a specific tissue component, such as cutaneous basement membrane, intercellular cement substance, or renal glomerular basement membrane. the ana test is another example of iit discussed previously. • as an adjunct test for suspected pemphigus, bullous pemphigoid, or immune-mediated glomerulonephritis. advantages • noninvasive method to detect circulating autoantibodies to tissue, especially when biopsy of these organs is problematic or nondiagnostic. disadvantages • significantly less sensitive than direct immunostaining, because a relatively large amount of antibody must be present to be detected. this test so rarely gives positive results in confirmed cases of autoimmune skin disease or immune-mediated glomerulonephritis that it is not recommended. routinely available tests are used to screen suspected cases of immunodeficiency. these tests include serial cbc examinations (total lymphocyte and neutrophil numbers), serum immunoglobulins (see causes of hypoglobulinemia), and antibody titers to common viral vaccines such as distemper or parvovirus. sustained lymphopenias (< 1000/µl) are noted in some cases of severe combined immunodeficiency, and marked neutrophilias (> 50,000/µl) are often observed in neutrophil function or chemotactic disorders (see chapter 4). routine histologic evaluation and immunophenotypic staining of lymph node tissue taken at biopsy are occasionally useful in pursuing a diagnosis of congenital or acquired immunodeficiency. possible abnormal findings include depletion or altered distribution of b-lymphocyte or t-lymphocyte populations within the nodal architecture. tests for cellular immunity include lymphocyte blastogenesis (i.e., transformation), assays for cell-mediated cytotoxicity, enumeration of t-lymphocyte and b-lymphocyte subpopulations, neutrophil function, and evaluation of leukocytic and erythrocytic histocompatibility antigens. if cellular immunity needs to be evaluated, the veterinarian should contact a college of veterinary medicine or large diagnostic laboratory to determine availability. clinically significant phagocytic function disorders tend to occur in purebreds (e.g., irish setters, weimaraners) or closely related crossbreds as a congenital or inherited defect. these animals show chronic or recurrent bacterial infections from a young age, along with markedly elevated neutrophil counts and general unthriftiness. tests include phagocytic assays, bactericidal assays, chemotactic response, and testing for cell surface markers such as leukocyte adhesion molecules lfa-1 and mo-1. the veterinarian should contact a college of veterinary medicine to determine availability. occasional indications • poor response to vaccinations, chronic unresponsive infections, persistently elevated or decreased lymphocyte counts, distinguishing between neoplastic and reactive lymphoproliferation by evaluating clonality. analysis • studies are usually performed on peripheral blood lymphocytes but can also be performed on lymph node tissue obtained by biopsy. the clinician should consult individual laboratories for sample specification. • inherited defects of the immune system, lymphosarcoma, lymphocytic leukemia, and some viral infections (especially retroviruses such as felv, fiv, and other viruses infecting lymphoid cells) change the proportions of t and b cells. occasional indications • poor vaccination response or recurrent infections. the lymphocyte transformation test is the most commonly used test for assessing functional capability of lymphocytes. disadvantages • the test is not readily available and is only a simulation of an in vivo situation; therefore, the results should be interpreted as an approximation only. analysis • isolated lymphocytes are exposed to various substances that cause their activation and division. the proliferation response of stimulated cells is quantitated by their increased uptake of radionucleotide compared with control cells from healthy animals of the same species and similar age. these tests can detect intrinsic lymphocyte function defects and serum suppressive factors, which can be found in many acquired disease states. if evaluation of lymphocyte function testing in vitro is contemplated, the reader should contact the local or regional veterinary school immunology laboratory for additional information on availability of testing. rose-waaler test laboratory techniques of veterinary clinical immunology immune complexes and rheumatoid factors in canine arthritides canine albumin results detection of antinuclear antibodies by indirect immunofluorescence in dog sera: comparison of rat liver tissue and human epithelial-2 cells as antigenic substrate kinetics of serum antiplatelet antibodies in experimental acute canine ehrlichiosis dog immunoglobulins: immunochemical characterization of dog serum, saliva, colostrum, milk and small bowel fluid concentrations of total serum ige, iga, and igg in atopic and parasitized dogs serum proteins and the dysproteinemias concurrent hypoadrenocorticism and hypoalbuminemia in dogs: a retrospective study hematologic and serum biochemical effects of long-term administration of anti-inflammatory doses of prednisone in dogs methimazole treatment of 262 cats with hyperthyroidism immunologic profiles of cats with persistent naturally acquired feline leukemia virus infection quantitation of canine immunoglobulins serum immunoglobulin levels in grey collies immunologic methods for the detection of humoral and cellular immunity immune-mediated dermatoses in domestic animals: ten years after. ii, comp cont ed 9 development of a sensitive immunoradiometric assay for detection of platelet surfaceassociated immunoglobulins in thrombocytopenic dogs diagnosis of autoimmune skin disease in the dog: correlation between histopathologic, direct immunofluorescent and clinical findings factor titer • because rf is an antibody against fc fragments of immunoglobulin molecules that become exposed only after antibody binds to antigen, any disease with long-standing immune complexes can eventually induce rf formation. in the rose-waaler test, a differential titer of greater than or equal to 1:8 is positive for rf (barta, 1993) . between 40% and 75% of dogs with ra have a positive rf test result. hence, a negative result does not eliminate rf. the rf test is rarely positive in normal dogs and occasionally positive in some patients with sle, because rf may be a part of the sle complex. incidence of rf in other arthropathies and systemic diseases has not been adequately studied via the rose-waaler method, but other methods have shown rf (in titers comparable with those of patients with ra) in these arthropathies (carter et al, 1989) . therefore a positive rf test result should never be the sole criterion for a diagnosis of canine ra.ra is a progressive, erosive, immunemediated polyarthritis that must be differentiated from other types of joint diseases, preferably before joint destruction occurs. unfortunately no test for canine rf is highly reliable in making this distinction. other routine tests indicated in making the diagnosis include joint radiographs and synovial fluid analysis. septic polyarthritis often causes erosive lesions, especially involving larger joints, plus evidence of sepsis on routine blood and synovial fluid examinations (see chapter 10), including culture. in contrast, ra typically causes erosive lesions of smaller peripheral joints before progressing to larger joints. radiographic lesions may be lacking or inconclusive early in the course of ra. furthermore, no distinguishing cytologic features can reliably differentiate among ra, sle, and other types of immune-mediated joint disease that have similar joint fluid cytology. histopathologic examination of synovium from affected key: cord-033420-pjtyv0pv authors: kalokairinou, louiza; zettler, patricia j; nagappan, ashwini; kyweluk, moira a; wexler, anna title: the promise of direct-to-consumer covid-19 testing: ethical and regulatory issues date: 2020-09-23 journal: j law biosci doi: 10.1093/jlb/lsaa069 sha: doc_id: 33420 cord_uid: pjtyv0pv widespread diagnostic and serological (antibody) testing is one key to mitigating the covid-19 pandemic. while at first, the majority of covid-19 diagnostic testing in the usa took place in healthcare settings, quickly a direct-to-consumer (dtc) testing market also emerged. in these dtc provision models, the test is initiated by a consumer and the sample collection occurs at home or in a commercial laboratory. although the provision of dtc tests has potential benefits—such as expanding access to testing and reducing the risk of exposure for consumers and medical personnel—it also raises significant ethical and regulatory concerns. this article reviews these challenges and shows how they parallel and also diverge from prior concerns raised in the dtc health testing arena. the first part of this paper provides an overview of the landscape of diagnostic and serological tests for covid-19, anticipating how provision models are likely to evolve in the future. the second part discusses five primary issues for dtc covid-19 tests: test accuracy; potential misinterpretation of results; misleading claims and other misinformation; privacy concerns; and fair allocation of scarce resources. we conclude with recommendations for regulators and companies that aim to ensure ethically marketed dtc covid-19 tests. in the wake of the covid-19 pandemic, widespread diagnostic and serological (immunity) testing is considered key in containing the spread of the disease, as well as in easing stay-at-home measures and informing policies for restarting the economy. 1 most tests, whether diagnostic or serological, are currently offered by healthcare providers who interface with the healthcare system. however, a number of companies 2 have begun to offer covid-19 testing on a direct-to-consumer (dtc) basis 3 : that is, the test is initiated by a consumer-not a healthcare professional-typically via the company's website. while a healthcare professional may be involved at some stage of the process that involvement may be as minimal as a brief review of a questionnaire that the consumer has completed as part of the purchasing process. as such, the consumer may have no direct contact with a healthcare professional (although some companies offer the option of post-test consultations in the case of positive results). in addition, the results of dtc tests do not necessarily become part of patients' health records, as the companies offering such tests operate independently of the healthcare system. the acquisition of the sample in dtc tests may occur in one of two ways: via at-home collection or in-laboratory collection. in the former, individuals order and receive an at-home collection kit, where they acquire the sample and mail it back to the company (or a lab that processes the results on behalf of the company). for inlaboratory collection, individuals make an appointment to visit a commercial laboratory where their sample is collected. both models of dtc covid-19 tests-at-home or in-laboratory collection-are distinguishable from those offered by hospitals and 3 the term dtc testing has been used in the literature to describe different provision models of health products. for the purposes of this article, our key criteria for considering a test to be dtc are (a) that it is promoted directly to consumers; (b) the testing process is initiated by consumers, who order the test online or by phone and/or book an appointment to a commercial lab in order to have their sample taken; (c) it requires little to no involvement of a healthcare provider, and whatever involvement does occur is usually not in person. see health clinics, as it is the consumer who initiates the order and completes the testing process with little to no interaction with a healthcare professional. even though the covid-19 pandemic has changed the way much healthcare is delivered by increasing the use of telemedicine to minimize in-person visits, the doctor-patient relationship remains central. in this regard, the dtc testing model is distinct. although healthcare professionals employed by dtc companies may authorize the ordering of laboratory tests, they have little, and usually no direct contact with the consumer. indeed, from the perspective of the consumer, the process of ordering a dtc covid-19 test may feel similar to purchasing other goods or services. for example, to order the labcorp covid-19 diagnostic test, an individual must click through a brief questionnaire, after which they can input their credit card information, click 'order,' and thereafter receive shipping updates via email. the dtc provision of covid-19 tests by private companies may expand access to testing and contribute to the mitigation of the present public health crisis. in particular, there are potential benefits to at-home testing, which eliminates the risks of exposure that individuals and medical personnel collecting samples may incur. in addition, at-home testing reduces the demand for personal protective equipment required by sample collectors and decreases the testing burden for the healthcare system as a whole. however, dtc tests also raise significant ethical concerns and pose regulatory challenges. although the regulatory environment for covid-19 tests is not identical to that for other types of dtc health tests (such as currently marketed genetic or hormone tests), 4 many of the concerns raised by dtc covid-19 tests parallel those wrought by other types of dtc health tests. at the same time, there are important contextual differences that make concerns over dtc covid-19 tests particularly urgent, such as the global population affected, the infectious nature of sars-cov-2, and the historic challenges to the healthcare system and economy during the pandemic. as widening the provision of diagnostic and serological covid-19 tests may be crucial to containing the pandemic, it is important to critically consider the key issues that might lie ahead for dtc covid-19 testing. in this article, we examine the main ethical and regulatory challenges of dtc diagnostic and serological tests, focusing on the us context. the first part of this article provides an overview of the landscape of diagnostic and serological tests for covid-19, anticipating how their provision models could evolve in the future. the second part identifies five primary ethical and regulatory issues for dtc covid-19 tests: uncertainty over the accuracy of test results; potential misinterpretation of test results by users; misleading product promotion and misinformation; privacy concerns; and fair allocation of scarce resources. each of these issues parallels prior concerns raised in the dtc health testing arena, but also diverges from them in important ways. we conclude with recommendations for regulators, companies, and other relevant stakeholders that can help ensure high-quality, accurate, and equitably distributed covid-19 tests, and inform the ethical provision of dtc health tests during public health crises. covid-19 diagnostic tests aim to determine whether a person is currently infected with sars-cov-2, the virus that causes covid-19. the most commonly used diagnostic tests during the covid-19 pandemic are polymerase chain reaction (pcr) tests. 5 for the purposes of testing, a swab is taken from the patient and tested in a lab for the genetic material of the virus. although the typical sample used in these tests is obtained by a nasopharyngeal swab that reaches deep into the nose, the fda recently issued emergency use authorizations (euas) for diagnostic pcr tests using samples collected from shallow nasal swabs and saliva. 6 while pcr is considered to be the 'gold standard' of molecular testing due to its high reliability, 7 this high accuracy may not be applicable to certain rapid point-of-care pcr tests, which according to a recent preprint study, may miss up to 48% of infections. 8 at present, the only non-pcr diagnostic test that has received an eua is an antigen test used on shallow nasal swabs can rapidly detect fragments of the virus. 9 however, these antigen tests have a higher probability of producing false-negative results than pcr tests, as they cannot detect all active infections. 10 although at first, the majority of covid-19 diagnostic testing in the usa took place in healthcare settings, a dtc testing market quickly emerged: first with companies marketing at-home tests without fda authorization, and more recently, with such tests authorized by the fda. specifically, in march 2020, companies such as nurx, everlywell and carbon health began marketing at-home collection kits directly to consumers. 11 the provision of tests by these companies involved healthcare professionals to various degrees (i.e. to review an eligibility questionnaire), although typically only after the consumer had initiated or completed the purchase of the test. 12 the marketing of such tests largely coincided with the release of an fda guidance explaining that the agency would permit laboratories and commercial manufacturers to develop and offer covid-19 diagnostic tests as long as they notified fda that their assays had been validated and submitted an eua. this policy, however, did not apply to at-home sample collection, which instead required that fda issue an eua before marketing. after fda clarified this policy in an update to its guidance document, 13 some companies pulled their at-home collection kits from the market. 14 at least one other did so only after facing enforcement action from local authorities. 15 dtc at-home collection kits, however, are rapidly reemerging with fda authorization. in april 2020, pixel by labcorp received an eua for the first covid-19 diagnostic test with at-home collection (via a shallow nasal swab kit). 16 currently, consumers can purchase this kit for $119 after completing a brief online survey that assesses symptom level (severe, mild or none); potential exposure to coronavirus; and whether an individual is low-or high-risk. 17 one month later, a second diagnostic athome kit that utilizes saliva samples was issued an eua. 18 at present (august 2020), there are approximately a dozen companies, including everlywell, letsgetchecked, phosphorus, hims, vault health, and 1health.io, offering authorized at-home covid-19 diagnostic tests that involve nasal swab or saliva collection. 19 notably, some of these companies have previously faced criticisms outside the covid-19 context. more specifically, commentators have raised ethical concerns about companies such as hims, whose business model permits consumers to order prescription drugs without interfacing directly with a healthcare professional. 20 concerns about everywell's dtc tests measuring the immune response to food in order to determine food sensitivity. 21 while diagnostic tests aim to detect an active infection, serological tests aim to detect antibodies in the blood, indicating that a person had been infected and recovered from covid-19. the presence of such antibodies could potentially indicate that the individual has developed some level of immunity against the virus. the blood sample for these tests is usually obtained through a finger prick. the sample can be collected by an individual and sent to be analyzed in a lab, or be analyzed with laboratory equipment available at a point-of-care location (e.g. in a pharmacy or physician's office) that can deliver results within a few minutes. although the accuracy of these tests for covid-19 antibodies is not well established, 22 they are at present widely available through numerous laboratories with or without a referral, often in a dtc context that requires consumers to initiate the testing process outside the healthcare system. the widespread availability of serological testing was made possible from a regulatory perspective because, until recently, fda exercised its discretion not to enforce requirements for laboratories and commercial manufacturers operating without an eua, as long as they provided a disclaimer that the tests had not been reviewed by fda and that results should not be used as the sole basis for confirming that someone has the disease. 23 following concerns about the quality of many tests on the market, in may 2020 fda changed its approach, aligning its policy for serological tests with that for diagnostic tests. 24 this means that like with covid-19 diagnostic tests, companies distributing at-home serological tests must obtain an eua before marketing their products. in addition, later the same month, fda published a list of serological tests that could no longer be marketed or distributed. 25 this was because while the manufacturers of these tests had notified fda that they intended to seek an eua, eventually they either voluntarily withdrew their tests from the notification list or failed to apply for an eua. currently, at least two companies offer dtc covid-19 serological tests. 26 as opposed to at-home collection kits, in this context, consumers initiate the test by making an appointment and having their sample collected in a lab. for example, the quest diagnostics serological test can be purchased online for $119 after individuals complete a brief questionnaire that assesses whether an individual is currently symptomatic for covid-19. 27 other companies offer tests specifically to employers interested in testing their employees for covid-19 antibodies. 28 although to-date there are no strictly at-home serological tests, these may be on the horizon: for example, scanwell together with lemonaid health are reportedly developing an at-home antibody test that will require consumers to extract a blood sample and share a picture of the blood testing stick with a healthcare provider, who would interpret the results in a subsequent consultation. 29 in the future, it is possible that covid-19 diagnostic and serological tests will become increasingly available both on an at-home and consumer-initiated basis, for a number of reasons. first, there is consumer demand, which provides financial incentives for companies to offer such tests. second, there are public health benefits in expanding at-home testing capacity-this type of testing may allow for more individuals to get tested without exposing themselves and others to the risk of infection. third, fda has recently expressed its explicit support for at-home covid-19 testing, clarifying that such tests may be issued an eua as long as manufacturers provide adequate data and scientific evidence supporting the safety and accuracy of such tests. 30 however, the development of such a market raises ethical concerns and regulatory challenges, which need to be addressed in order to reap the full potential of dtc testing. although covid-19 testing in general-even when not provided on a dtc basismay raise many ethical and regulatory issues, the five main concerns discussed in this section are particularly pronounced in the dtc space. more specifically, the athome covid-19 diagnostic testing model presents heightened challenges regarding the accuracy of tests compared to testing that involves sample collection from a healthcare provider. in addition, the absence (or minimal involvement) of a healthcare provider and, potentially of adequate information accompanying the tests, intensifies concerns about the possible misinterpretation of test results. furthermore, to-date, there has been a number of misleading claims made by companies advertising dtc covid-19 tests (and misinformation from other sources), as well as numerous cases of marketing of fraudulent dtc tests. privacy concerns are also more pronounced in the dtc context, since some companies may not be covered by the health insurance portability and accountability act (hipaa). finally, the provision of dtc tests by commercial providers may exacerbate inequalities in access to testing at the expense of communities that are disproportionately affected by covid-19. the major ethical and regulatory concerns that surround the actual processes of dtc testing for both active covid-19 infection and serological testing mirror existing concerns about dtc health monitoring and disease evaluation tests already widely available in the usa. many of these existing tests, such as hiv tests or the monitoring of hbac levels for diabetes management, have proven track records in medical treatment. however, many others-such as food sensitivity tests and genetic tests for susceptibility to multifactorial disorders-are of uncertain quality, particularly because of their inconclusive clinical validity (i.e. the ability of a test to correctly identify that a particular variant is correlated with increased risk of disease or condition) 31 and unproven clinical utility (i.e. the ability of the test to inform clinical management of a patient). 32 in the covid-19 context, the public health value of accurate covid-19 tests is clear. however, there remain questions regarding the accuracy of dtc tests, which is dependent on various factors, including the quality of the sample collected, proper shipment, and stability of the specimen, as well as the sensitivity and specificity of the test. indeed, fda has recently acknowledged that covid-19 tests involving at-home sample collection may present unique issues regarding accuracy, and in recognition of these challenges it recently published an eua template to help provide guidance to manufacturers of such tests. 33 regarding the quality of the sample, although many diagnostic covid-19 tests in the healthcare setting utilize nasopharyngeal swabs-which may be difficult for individuals to obtain themselves-such tests seem unlikely to come to the us dtc market. the currently available dtc at-home tests utilize shallow nasal swabs or saliva samples, which are easier for individuals to obtain. early studies have indicated that nasal swabs and saliva samples-even when self-collected-can effectively and reliably identify infections of the sars-cov-2 virus, 34 suggested that saliva may actually be more sensitive than nasopharyngeal swabs. 35 thus, while further research in this field is necessary, at-present, quality for selfcollected shallow nasal swabs and saliva samples does not appear to be a primary concern. with regard to the shipment and stability of sample, fda has noted that at-home tests may raise particular concerns due to the time lapse between the collection and the analysis. in addition, samples may be subject to conditions during shipment (e.g. high temperatures) that may compromise them. 36 however, for some nasal swabs (foam or polyester) shipped in certain ways (in a dry tube or in saline solution), fda has recently indicated that preliminary data suggest that the samples appear to be stable. 37 thus, at present, samples collected and shipped using these methods appear to alleviate stability concerns, although questions remain regarding stability for other forms of samples and shipment methods. in addition, the sensitivity (i.e. the ability of the test to detect the presence of the virus or antibodies) and specificity (i.e. the ability of the test to detect the absence of the virus or antibodies) of the test itself are also crucial for the reliability of results. while the pcr tests currently used in covid-19 diagnostic testing are considered to be of high accuracy, there is always a risk of false-positive or false-negative results, and those risks may depend on factors such as testing outside the diagnostic window, and the use of inadequately validated assays. 38 in the case of serological tests for covid-19, such concerns are more pressing, as their accuracy has not been well-established and the chance of such tests producing false-positive results may be high, especially in low prevalence populations. 39 a recent preprint study performed by a consortium of laboratories in california found that of the 12 antibody tests studied, one test produced false-positive results over 15% of the time, and three other tests more than 10% of the time. 40 given that until recently there has been only limited oversight of these tests, the reliability of serological tests remains an important concern. 41 although dtc covid tests are reviewed by fda before they enter the market this does not eliminate concerns about their accuracy. issues of unproven quality are potentially inherent to those tests that are marketed under an eua, as all fdaauthorized covid-19 tests currently are. for fda to issue an eua for a test, the federal food, drug, and cosmetic act requires, among other things, that fda conclude it is 'reasonable to believe' that the test 'may be effective in diagnosing' the disease. 42 consistent with this statutory language, fda has explained that the standard for a product being issued an eua requires 'a lower level of evidence than the 'effectiveness' standard that fda uses for [standard] product approvals.' 43 concerns about test quality have intensified following the us department of health and human services's (hhs) august 2020 statement that fda will not require premarket review for laboratory developed tests (ldts), including for covid-19 ldts, unless the agency first goes through notice-and-comment rulemaking to do so. 44 fda describes ldts as tests that are "designed, manufactured and used in a single laboratory," and historically has exercised its discretion not to enforce premarket review requirements for many ldts. 45 but fda also has enforced such requirements for dtc tests (even when they may meet the definition of an ldt), 46 and hhs's statement did not explain whether it was intended to apply to dtc products that may meet the definition of an ldt. it, thus, is unclear what hhs intended its statement to cover and how fda will treat dtc ldts going forward. 47 it is possible that fda will allow some dtc covid-19 tests that are considered ldts to be offered without an eua. 48 as a result, more tests of uncertain quality may enter the market in the near future. in addition, issues regarding the quality of dtc covid-19 tests may be heightened because of the widespread, historic nature of the pandemic and the urgent need for expanding testing capacity-and accompanying political pressure. at the same time, however, it is critical that regulators, industry, and the public recognize the need to ensure high-quality standards. the promise of direct-to-consumer covid-19 testing • 11 for all dtc health testing, the absence of a healthcare professional and, potentially of adequate information regarding the potential limitations of these products, has raised concerns about the risk of misinterpretation of results and of potentially inappropriate subsequent healthcare decision making. these concerns apply even for those tests that have met relevant quality standards. moreover, such concerns are particularly salient for covid-19 testing. for diagnostic testing, false-negative results could create a false sense of security and contribute to further spread of the virus, while false-positive test results could keep people out of work, school, or childcare, exacerbating economic and educational harms. additionally, although the interpretation of covid-19 diagnostic testing is relatively straightforward-indicating whether an individual has an acute manifestation of the infectionserological testing is far more difficult to interpret. for example, while preliminary data suggest that recovery from covid-19 might confer immunity to subsequent infection, it is still uncertain how protective such immunity is and how long it may last. 50 in addition, while many manufacturers claim that their serological tests are of high sensitivity and specificity, many have not released any data supporting their claims. 51 considering the potential unreliability of currently available serological tests and the uncertainty over the meaning of covid-19 immunity, it is of concern that several companies are marketing serological tests to employers interested in testing their employees, as such results could be used to make decisions about employees going back to work. misinterpreting or overestimating test results could expose individuals to risks of reinfection and could undermine public health mitigation efforts. 52 in this regard, dtc covid-19 tests, whether diagnostic or serological, should be accompanied by clear guidance and information about their potential and limitations. during the pandemic, with millions of individuals fearful of being sick, but also desperate to resume work, the scale, and immediacy of the adverse impact of misinterpreting covid-19 test results are far greater than other commercially available dtc health-related tests. many concerns that have been previously raised regarding dtc promotion of healthrelated tests-such as misleading or inaccurate claims, exaggeration of benefits and minimization of risks 53 pandemic, the market has been flooded by companies making unsubstantiated and often fraudulent claims, such as falsely stating that their tests have been approved by fda 54 or that their serological tests can diagnose the disease. 55 to-date, several state and local regulators have issued cease-and-desist orders to individuals and companies on the grounds that they have been illegally promoting and offering covid-19 tests. 56 at a federal level, both fda and federal trade commission (ftc) have warned companies to stop making misleading claims, including about treating and preventing the coronavirus. 57 in addition to misleading information coming from companies themselves, the public has been receiving an overwhelming amount of misleading information about serological testing from other sources. politicians in the usa and abroad have exaggerated the potential of such tests, touting widespread serological testing as a 'game changer' 58 and a key to restarting the economy. some governments have reportedly considered issuing 'immunity passports' based on positive serological tests results. 59 additionally, in a may 2020 statement, the governor of new york stated that detecting covid-19 antibodies means that, 'you can get to work, you can go back to school, you can do whatever you want.' 60 yet, as explained above, it is still uncertain what positive serological test results mean for functional immunity, how protective any immunity is, and how long it may last. misinformation about covid-19 tests, whether from companies or other sources, is particularly concerning. 61 misleading product promotion capitalizes on widespread anxiety caused by the pandemic and preys on the vulnerability of consumers, many of whom are likely concerned about their health. the massive amount of information (and misinformation) about covid-19, and the quickly changing landscape, may make it particularly challenging for consumers to differentiate between legitimate tests and fraudulent products (which might mislead consumers about their covid-19 health 54 status). moreover, misinformation regarding the potential benefits and limitations of the tests could lead individuals to misunderstand their covid-19 health status-even if companies selling the tests are not themselves providing misleading information. dtc testing by private companies in the realm of genetics has raised important questions regarding privacy and confidentiality of personal data. 62 in the usa, companies may not be subject to laws intended to protect the privacy of health information, such as the hipaa. 63 thus, the protection of personal data, including the duration of storage and the third party access to them, is largely determined by terms of service created by the companies themselves. 64 similarly, many of the companies offering at-home covid-19 tests, whether diagnostic or serological, may not be covered by hipaa and the protection of personal data may, therefore, depend on companies' individual policies. in the covid-19 context, consumers may erroneously assume, in some cases, that privacy rules governing data in the healthcare setting (such as hipaa) and doctorpatient confidentiality also apply to dtc companies, giving them a false sense of security. 65 furthermore, consumers may not realize that for infectious diseases, the limits of confidentiality may be narrower as compared to other types of tests. more specifically, companies may need to comply with relevant local and state regulations regarding the reporting of positive test results to authorities and may need to disclose protected health information without obtaining prior permission from the consumer. 66 currently, several state and county health departments require healthcare practitioners and medical laboratories to report covid-19 cases, providing, amongst other information, the name and address of the patient. 67 while the policies of some companies are more transparent than others, it is likely that consumers will click 'i agree' to terms and conditions without ever reading them. this may be especially the case with individuals ordering diagnostic tests, as it is likely that such tests will be purchased under conditions of urgency and anxiety that may not allow for careful review of the relevant contracts. in addition, given the scarcity of tests, some people may not feel that they have the option to refuse. policies regarding third-party access to data are particularly relevant, especially in view of risks of discrimination in the context of employment. more specifically, it is possible that employers may be interested in accessing serological test results, especially when they are the ones initiating the testing. this is because confirming that employees have immunity could be relevant for recruitment decisions. 68 for these reasons, it is important for consumers to understand the limits of confidentiality of their health information and the risks of a privacy breach. in recent years, dtc health testing has been marketed to consumers as an opportunity to access a wide range of health information directly, often completely bypassing the mainstream healthcare setting. by offering consumers information on genetic susceptibility to multifactorial disorders, carrier status, reproductive health, and infectious diseases, many dtc companies have presented themselves as expanding access to testing and enabling consumers to take control of their health. however, there have been longstanding concerns that such testing could eventually lead consumers back to the mainstream healthcare system for consultations regarding their test results, creating downstream costs and using resources that, in some settings, may be scarce. 69 for covid-19 tests, competition over scarce resources may be more direct and tangible. currently, there are shortages of basic elements of diagnostic tests, such as swabs and reagents, both in the usa and worldwide. such shortages are partly responsible for the inadequate testing capacity in the usa. 70 despite an increase in the number of tests performed daily since the first month of the pandemic, testing is still not scaled up sufficiently to meet public health needs or consumer demand. 71 in this regard, companies offering dtc covid-19 testing could provide an alternative for individuals and expand access to testing. however, given the scarcity of resources, they could also be in direct competition with other healthcare providers, such as hospitals. the use of scarce resources by companies offering dtc testing during a global pandemic could also raise concerns over fair allocation of such resources. previous research has indicated that consumers who purchase dtc health testing tend to be of higher socioeconomic status. for example, empirical studies of dtc genetic testing have shown that the majority of the consumer base is white, highly educated, and has a higher than average household income. 72 consistent with such findings, in the context of covid-19, it is possible that poor and marginalized communities will have less access to dtc tests, because of inadequate financial resources, limited information about the availability of such tests, companies' marketing strategies, or other reasons. this is particularly concerning given that racial and ethnic minority groups have been disproportionately affected by the pandemic. 73 currently, several state and county health departments are expanding testing capacity by launching testing sites in underserved communities. 74 in order to avoid two-tiered access to testing based on socioeconomic status or race, it is important that efforts to provide free testing to low-income communities, as well as to communities of color, are sustained and expanded across the usa. currently, issues of fair access to covid-19 testing are more pressing for diagnostic tests, as they have clear clinical utility and are considered more reliable compared to serological tests. however, considering the ongoing policy discussions in some countries regarding using serological test results as an 'immunity passport' that would allow individuals to return to work, 75 ensuring equitable access to serological testing may become crucial in the near future. as this article highlights, many of the concerns surrounding dtc covid-19 parallel those surrounding other dtc health tests that are widely available in the usa. however, the scale of the present pandemic lends an increased urgency to existing issues. given the fast-changing landscape of the covid-19 pandemic, this article cannot predict or address all issues associated with dtc covid-19 testing that are likely to arise. there are, however, several recommendations that can help inform the ethical provision of dtc health tests during this public health crisis. first, consistent with its obligations under the federal food, drug, and cosmetic act, 76 fda should reassess its euas and remove authorization for tests that are found to be of low quality. enabling testing to reach the market as quickly as possible is, of course, an important goal. but testing is not useful if consumers, healthcare professionals, and public health regulators cannot be confident in the results. ultimately, fda must ensure progress in studying and developing high-quality testing continues, and assure available testing meets the conventional-rather the lower, euastandards. second, all stakeholders should be working to educate policymakers and the public with accurate, non-misleading information about the limits and potential benefits of dtc testing. for example, companies should provide consumers adequate and clear information regarding their tests, in both communications about how to interpret test results and in promotional materials. ftc and states should continuously monitor the market and take action when necessary to protect consumers and ensure they have accurate and non-misleading information. in addition, for companies that are marketing dtc tests under an eua and that make misleading claims that have a negative public health impact, fda should make clear it will consider withdrawing the eua. 77 third, with regard to privacy, it is crucial that companies provide transparent and easy-to-comprehend privacy policies that are not buried amongst other terms and conditions. considering the vulnerability of consumers and the ongoing public health crisis, the ftc should monitor the practices of such companies closely to ensure that such terms are not disproportionate and safeguard the rights of consumers to privacy. fourth, equitable access to high-quality dtc tests is critical. at a minimum, companies should make clear whether their tests are covered by health insurance and whether they provide options for individuals who cannot afford standard prices, whether or not they are insured. but more is likely to be needed-and if mechanisms for equitable access for covid-19 testing are successfully developed, they can inform models of access for other kinds of potentially beneficial dtc testing. ensuring a market of accurate and ethically marketed dtc covid-19 tests presents significant challenges for regulators and requires the buy-in of all stakeholders, including industry. at the same time, regulators and industry may be well-equipped to address many of these challenges, drawing on past experience regulating other dtc tests. regulators, industry, and the public also have an opportunity to think carefully about the risks and benefits of different models of dtc testing, and ultimately use the lessons learned from covid-19 to improve the dtc testing market. saliva is more sensitive for sars-cov-2 detection in covid-19 patients than nasopharyngeal swabs, medrxiv food and drug administration, supra note 30 potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease test performance evaluation of sars-cov-2 serological assays antibody tests go to market largely unregulated, warns house subcommittee chair direct-to-consumer genetic testing: perceptions, problems, and policy responses all your data (effectively) belong to us: data practices among direct-to-consumer genetic testing firms the future of dtc genomics and the law hipaa and protecting health information in the 21st century privacy in the age of medical big data genomic privacy and direct-to-consumer genetics: big consumer genetic data-what's in that contract? health system implications of direct-to-consumer personal genome testing, 14 public health genomi hipaa for professionals: faq reporting covid-19/sars-cov-2 infections employers rush to adopt virus screening. the tools may not help much testing remains scarce as governors weigh reopening states testing challenge: why it's so hard to overcome testing shortages in the united states a dire warning from covid-19 test providers cities still lack testing capacity consumer perspectives on access to direct-to-consumer genetic testing: role of demographic factors and the testing experience: consumer perspectives on access to direct-to-consumer genetic testing design, methods, and participant characteristics of the impact of personal genomics (pgen) study, a prospective cohort study of direct-to-consumer personal genomic testing customers the impact of the covid-19 pandemic on marginalized populations in the united states: a research agenda covid-19: disproportionate impact on ethnic minority healthcare workers will be explored by government amid ongoing covid-19 pandemic, governor cuomo launches new initiative to expand access to testing in low-income communities and communities of color denver launching coronavirus testing to help communities of color county expands coronavirus testing in hard-hit black, latino communities louiza kalokairinou is a postdoctoral fellow at the department of medical ethics and health policy, perelman school of medicine, university of pennsylvania. louiza received a phd degree in she worked as a policy officer in the research ethics and integrity sector of the european commission. louiza's research focuses the ethical, legal, and social aspects of direct-to-consumer health products and emerging technologies zettler is an associate professor of law at the ohio state university moritz college of drug enforcement & policy center housed at the college of law, and a member of the ohio state university comprehensive cancer center in addition to professor zettler's academic work, she served as an associate chief counsel in the fda's office of the chief counsel ashwini received a ba degree in public health/sociology from nyu and master of bioethics from the university of pennsylvania. she will continue her education at ucla this fall with a phd in health policy and management kyweluk is a postdoctoral fellow at the department of medical ethics and health policy, perelman school of medicine, university of pennsylvania. moira holds a joint phd/mph from northwestern university in medical anthropology. her research specialization is reproduction in the united states with a focus on assisted reproductive technologies, queer and transgender family building, reproductive health, and peri-conception genetic testing anna wexler is an assistant professor in the department of medical ethics and health policy, perelman school of medicine, university of pennsylvania. she is the principal investigator of the wexler lab, which studies ethical, legal, and social issues surrounding emerging technology, with a particular focus on do-it-yourself and direct-to-consumer medicine and science this study was supported by the office of the director, nih, under award number dp5od026420. dr. kyweluk's work was supported by a postdoctoral training grant from the national human genome research institute grant number t32hg009496. key: cord-031567-w2676lrz authors: sabit, maureen; wong, cecil; andaya, agnes; ramos, john donnie title: pollen allergen skin test and specific ige reactivity among filipinos: a community-based study date: 2020-08-12 journal: allergy asthma clin immunol doi: 10.1186/s13223-020-00471-9 sha: doc_id: 31567 cord_uid: w2676lrz background: despite the clinical importance of pollen allergens among filipinos, few studies delve into the sensitization profiles of filipinos against pollen allergens. this study determined the sensitization profile of filipinos to pollen using skin prick test (spt) and pollen-specific elisa. methods: pollen from fifteen selected plant sources was collected and extracted for use in sensitization tests. volunteers were interviewed for their clinical history prior to blood sampling and spt. the blood samples collected were assessed using enzyme-linked immunosorbent assay (elisa). results: the best panel of pollen allergens for the skin prick test was mangifera indica (64%), acacia auriculiformis (28%), mimosa spp. (25%) amaranthus spinosus (22%), lantana camara (20%), pilea microphylla (16%) and dichanthium aristatum (15%). young adults had more sensitizations to pollen than among early childhood and elderly. there were more allergic subjects that have rhinitis (61%) than asthma (42%) and atopic dermatitis (35%). pollen-specific ige levels show low percent reactivity as compared to the skin test with cocos nucifera obtaining the highest ige reactivity (21%). conclusions: pollen allergens from both arboreal and herbaceous plants used in this study yielded positive reactivities for both skin tests and specific ige tests. allergy is a hypersensitive reaction characterized by an immune-mediated inflammatory response to common environmental protein allergens that are deemed to be harmless in non-allergic individuals [1] . the global increase in the prevalence of allergic respiratory diseases and their effect on the quality of life of allergic patients is a health issue that needs immediate attention [2, 3] . in the philippines, the reported overall prevalence of allergic rhinitis and allergic rhinoconjunctivitis is 20% and 14%, respectively [4] ; whereas, work absence due to asthma is reported at 46.6% [5] . pollen is one of the most common and important sensitizing aeroallergens [6] [7] [8] [9] that cause respiratory allergies such as allergic rhinitis, allergic asthma, and atopic dermatitis. dissemination or dispersal of pollen, which occurs during a plant's pollination or flowering period, ensures survival and continuity of its lineage. small, lightweight pollen, which is produced in copious amounts by anemophilous (wind-pollinated) plants, are the major allergens in the atmosphere. several studies have shown that the incidence of pollinosis in urban areas is higher than the countryside due to unsuitable green space construction, urban heat island effect, and traffic pollution [10] . allergy, asthma & clinical immunology *correspondence: mbsabit@ust.edu.ph; mhayen11@gmail.com 2 research center for the natural and applied sciences, thomas aquinas research complex, university of santo tomas, 1008 manila, philippines full list of author information is available at the end of the article in tropical asia, little information on pollen allergens is available [11] , and information on the sensitization profiles of filipino allergic patients to pollen allergens is limited. sensitization to grass and weed pollen among filipinos was reported earlier [12] and described anew by recent studies using immunobiological techniques [13, 14] . it is in this context that this study was conducted. aqueous extracts of tree and weed pollen found to be abundant in the atmosphere were used to generate the sensitization profile of filipinos using skin tests. this study aims to determine the total ige and pollen specific-ige profiles of skin-test positive and negative subjects and correlate skin tests with pollen-specific ige reactivity. vegetation or the "green space" in a highly urbanized city of metro manila, is found only in parks, gardens, and trees planted along the road. ten barangays near the vicinity of a pollen trap (situated within the university of santo tomas, manila) were randomly chosen for this study. prior to sampling, an approval from the ust graduate school institutional ethics committee review board was obtained. a total of 541 volunteers who have been living, working, or studying in the "university belt" for more than 2 years prior to the conduct of this study were recruited. spt-positive subjects were designated as cases while spt-negative subjects were controls. all participants gave their informed consent prior to answering a standardized questionnaire. this questionnaire was adapted and modified by de guia [15] from previous sources [16] [17] [18] , and, validated these questionnaires for filipino patients. the fifteen plants chosen as sources of pollen were previously reported as widespread in metro manila, and most are representative of the plant families with a high prevalence of airborne pollen [19] . . pollen samples were collected from the mature anthers of these plants and processed as described [20] . flowers from trees and weeds were dried then passed through reducing sizes of mesh sieves (150, 75, 50 and 25 µm, respectively). the presence of pollen was confirmed under a stereomicroscope (bs-2030t digital biological trinocular microscope). pollen was stored in a tightly sealed container with a desiccant at 4 °c. one gram (dry weight) of pollen was mixed with 10 ml diethyl ether and placed on a shaker overnight. the defatted pollen was centrifuged at 4000 rpm for 10 min, left to dry overnight, and mixed with 10 ml phosphate buffered saline (pbs). the mixture was stirred overnight at 4 °c and centrifuged at 13,000 rpm, 4 °c for 30 min. the supernatant was transferred to a dialysis tubing (6-8 kd mwco, supplied by spectrum labs, usa) and passed through a 0.2 μm millipore filter (whatman puradisc 25, pes sterile). 1 ml aliquots of the dialyzed products were transferred to microcentrifuge tubes and stored at − 20 °c until use. the total protein content of the pollen was analyzed using bio-rad protein assay kit ii (bio-rad laboratories inc, hercules, ca, usa). each crude pollen aqueous extract was diluted with the appropriate amount of pbs to get a final concentration of 10 µg/ml for use in skin prick test (spt) and pollenspecific ige elisa. volunteer selection was made carefully following the guidelines as described [1] . spt was performed on all participants using a panel of 15 crude pollen extracts, house dust mites (hdm) suidaisia pontifica (sud), and blomia tropicalis (blo), and with histamine (0.1%) in pbs and physiologic saline solution as positive and negative controls, respectively. a drop of each pollen extract was directly pricked on the participant's forearm using a 1 mm-point sterile lancet. a white wheal measuring ≥ 3 mm in diameter and a red flare around the pricked skin area was interpreted as positive spt [21] . five ml peripheral blood of study participants was collected in edta tubes and then centrifuged to separate the plasma. samples were aliquoted and stored at − 20 °c until further use. the total ige levels of cases (n = 130) and controls (n = 110) were determined following the manufacturer's protocol (human ige elisa core kit, komabiotech, south korea). for pollen-specific ige elisa, 10 μg/ml aqueous pollen extracts diluted in carbonate-bicarbonate buffer were coated overnight at 4 °c onto the wells of high-binding microtiter plates (corning costar, ny, usa). plates were blocked with 1% bsa (sigma-aldrich, saint louis, mo, usa) in pbs for 1 h at room temperature. plasma samples from both cases and controls were dispensed in duplicates onto the wells and incubated for 1 h at room temperature. plates were incubated with 500× dilution of an hrp-conjugated goat anti-human ige for 1 h at room temperature. colorimetric reactions for all immunobiological tests were performed using tmb (3,3′,5,5′-tetramethylbenzidine) and the reaction was stopped with 2 m h 2 so 4 . absorbance was read at 450 nm using bio-tek elx800 elisa reader (tecan, austria). data characteristics of test subjects (cases and controls) and positive reactions to different pollen allergens using skin prick test and specific ige elisa were presented as frequency (percentage) and compared using the chi square test of homogeneity or fisher's exact test or z-test for two sample proportions. the diagnostic performance of spt (positive and negative predictive values, specificity and sensitivity) with pollen, was computed. spearman's correlation coefficient was used to test the association of the different pollen based on pollen-specific ige elisa results. percent reactivity of pollen-specific ige and sensitization profile among age groups were graphically represented. frequency tables and graphs were created, and data analyzed using spss (v.20), ms excel and/or graphpad prism 8. five hundred forty-one study subjects from metro manila were recruited. seven percent (7%) of the study subjects were excluded because of non-cooperation, had taken antihistamine drugs before testing and backing out at the last minute. of the 41% that were positive to skin tests (n = 205), 49% (n = 101) were positive to both pollen and hdm, 14% (n = 29) were positive only to pollen, and 37% (n = 75) were positive only to hdm. subjects that tested positive to pollen (cases, n = 130), who self-reported to having allergic asthma (aa), allergic rhinitis (ar), and atopic dermatitis (ad), were referred to as allergic (79%), while those who self-reported not to have allergic diseases were asymptomatic. of the 373 skin-test negative subjects (to pollen), 110 were asymptomatic and referred to as controls. table 1 shows the characteristics of the test subjects (cases and controls). significant differences were shown in the percentage number of children (2-9 years old), young adults (20-40 years old), between gender and those with a family history of allergies between cases and controls. of asymptomatic cases (n = 27), 37% have family members that were allergic. allergic asthma (aa) and ar were commonly reported in either the father or mother of the cases. several cases and controls have pets at home, and there were no significant differences between them. the most common household pet were dogs (cases-68%, controls-88%), cats and birds (cases-10%, controls-12%) and a combination of dogs, cats, and birds (for cases only-22%). likewise, no significant differences were shown between cases and controls who self-reported to smoke or have family members who smoke. allergic rhinitis (61%) was prevalent among allergic cases, followed by aa (42%) and ad (35%). total ige values of ≥ 100 iu/ml of subjects in cases and controls were 87% (n = 113) and 55% (n = 60), respectively. based on spearman's rho, there was no significant difference in the mean ige levels between gender (cases and controls) and presence of allergic diseases in cases, although, the highest mean was obtained from male allergic subjects (284.58 iu/ml) and those with aa (344.82 iu/ml) and ad (324.89 iu/ml). of the 11 species of arboreal plants used for spt, most of the study subjects tested positive to three pollen sources: mangifera indica (man), acacia auriculiformis (aca), and lantana camara (lan) as shown in table 2 . study subjects were also positive to the pollen of herbaceous species, namely, mimosa spp. (mim), amaranthus spinosus (ama), pilea microphylla (pil), and dichanthium aristatum (dic). wheal diameters of 6-10 mm were observed in subjects who tested positive to mim and man. based on sensitizations, 40% and 31% of positive subjects from skin tests and pollenspecific ige elisa, respectively, were sensitized to one allergen (monosensitization) while the rest of the test subjects were sensitized to two or more allergens (polysensitization). figures 1 and 2 show the sensitization profile of allergic subjects across age groups. male children (2-9 years old) have early sensitization than females. however, females show high sensitizations than males in all other age groups. overall, there was an increase in the number of sensitizations to young adults (20-40 years old) and then gradually declined in the older age groups. likewise, the number of polysensitized (65% males, 56% females) subjects show similar tendencies while monosensitization (35% male, 44% female) were more evident in younger age groups. the sensitivity and specificity of the skin tests were 30% (95% ci 25-35%) and 83% (95% ci 75-88%), respectively. the prevalence of the allergic disease in the population was 69% with a positive predictive value of 79% (95% ci 71-85%), and a negative predictive value of 34% (95% ci 30-39%). significant differences in positive reaction to skin tests and pollen-specific ige elisa were shown in 10 pollen allergens ( table 2) . fifty-four percent (54%) of skin ige of ≤ 100 iu/ml (fig. 3) . as shown in table 3 recent sensitization studies in the philippines were mostly on specific-ige profiles of filipinos to selected grass species (pollen) and house dust mites [13, 22] . studies on spt using pollen from grasses and weeds have also been described [12] . after a few decades, in this study, other sources of pollen, particularly from trees were utilized for both spt and elisa tests. although m. indica, a. auriculiformis, and l. camara are entomophilous, there have also been published reports of their allergenicity [9, 23] . skin prick tests (spt), which is an essential procedure to confirm sensitization in ige-mediated allergic disease in subjects with allergic rhinitis, asthma, and atopic dermatitis, can be performed from infancy to old age [24] . likewise, spt is considered a safe diagnostic procedure as the occurrence of systemic reaction has decreased and cases of fatalities were extremely low [25] . aside from time, cost and safety, spt had a high sensitivity to aeroallergens, mainly pollen and house dust mites [26] . factors that influence the pollen threshold values for the development of allergic symptoms are as follows: time of the season, weather conditions, pollen allergenicity, air pollution and patient characteristics [27] . the recruitment of study subjects was done near the end of the flowering season (from april to june) when most of the trees (e.g. m. indica, c. nucifera, eucalyptus spp., etc.) were in full bloom and bear fruits. the flowering of some plant sources are seasonal (e.g. m. indica, eucalyptus spp., etc.) but most flower all throughout the year (e.g. c. nucifera, d. meyeniana, l. camara, etc.). as previously reported [19] , the concentration of airborne pollen decrease near the end of may up to the first weeks of june, due to the increasing amount of rainfall in the region. the high prevalence of allergic disease and high positive predictive value signify that there is a high probability that the amount of pollen in the air may have caused the allergic disease of the allergic subjects. although skin tests in this study detected only almost one-third of the study subjects with allergic disease (low sensitivity), it has a high probability (specificity) that it can discriminate those who do not have an allergic disease in any of the pollen allergens used. similarly, in another study, only 28.2% of their study subjects were sensitive to pollen allergens [8] . allergic cases who tested negative to the skin tests require another test or another panel of pollen allergen to confirm results. whereas, asymptomatics may develop allergic diseases later in life [28] . in this study, there's a discrepancy between results from spt and pollen specific-ige (sige), particularly that of m. indica. a recent study [29] made an assumption that tropical flora produces highly glycosylated allergens that hid or mask protein epitopes. as a result, ige binds to these non-allergenic, pollen-derived carbohydrate epitopes instead of the allergenic protein allergens. this ultimately resulted in a specific-ige negative response. likewise, spt-negative subjects that are positive to specific-ige elisa may be due to the presence of a crossreactive carbohydrate determinant or ccd [30] [31] [32] , or the lack of pollen coat allergens, containing allergic epitopes [33] . more than 20% of allergic patients that were asymptomatic have their ige bind to carbohydrate compounds instead of the allergen [34] . in this study, some skin test-positive subjects were asymptomatic. ideally, a subject that has a positive clinical history would also have positive allergen-specific test results [35] . instead, anomalies in having either positive or negative test results occur since the cause or presence of allergic disease are not revealed in all cases with positive clinical history. even if a patient was clinically allergic to an allergen if there were no recent exposure, then allergic symptoms may be caused by something else; or, a patient may be sensitized to an allergen but not clinically allergic to it [36] . however, a significant number of allergic sensitizations may be missed if only one type of testing was performed [37] . thus, spt and specific-ige testing should not be interpreted interchangeably but instead used as complementary [38] . among asians, exposure to pollen in urban communities is less than in rural areas. studies suggest that early/childhood exposure to pollen (and keeping pets) can protect against allergic sensitization up to adulthood [39] . in contrast, this protective effect of rural living had changed due to a shift in urban lifestyle in rural areas [40] . locally, pediatric patients showed that house dust mites and cockroaches were the main allergens, followed by sorghum jalapense, a. spinosus, and m. indica [41] . likewise, a. spinosus, m. indica, together with, mimosa and a. auriculiformis were less allergenic to children. although m. indica and a. auriculiformis belong to < 1% of the airborne pollen in manila [19] , its [42, 43] . alternatively, in this study, trees such as c. papaya and c. nucifera belonged to > 1% of airborne pollen in manila [19] but showed low sensitization in skin tests. this contrasts with other studies, wherein c. papaya, and c. nucifera elicited a high response to spt [44, 45] , which may be attributed to geographical location, climate and allergen exposure. a high percentage of test subjects in this study reported a history of rhinitis and asthma, which, are allergic diseases often associated with sensitization to aeroallergens [46] . sensitization to pollen in this study peaked at the young adult stage (20-40 years old), and male sensitization started earlier than females, as is also shown in previous studies [38, 47, 48] . a longitudinal study of an urban population in central italy [28] revealed a significant increase in skin prick test reactivity, using the same sample population, across age groups and particularly in subjects < 40 years old. at present, there is no accepted explanation for the sensitization shift between genders [38] . in a review assessing the impact of age on atopy, immunosenescence or a lower expression of ige was observed as the cause of age-related decrease of positive skin test [49] . exposure to environmental allergens increases over time as evidenced by the number of polysensitization, particularly in older age groups, as shown in this study. this polysensitization was also evident when pollen allergens were tested for cross-reactivity using an elisa inhibition assay. nearly all pollen, either closely or distantly related, had weak to strong associations and although of low percentage inhibition value, this may indicate the presence of cross-reactive proteins. cross-reactivity between closely related plants reflects phylogeny, shared antigens or epitope binding sites, while cross-reactivity in distantly related plants, is due to minor allergens (e.g., profilins, lipid transfer proteins, and pathogenesis-related proteins) [50] . these minor allergens, called panallergens [51] play a significant role in the distribution of the allergic response to conserved epitopes of different allergenic sources [52] . in conclusion, pollen allergens from both arboreal and herbaceous plants used in this study yielded positive reactivities for both skin tests and specific ige tests. additionally, for a small community-based study population, it shows that filipinos living in a highly urbanized city are allergenic to local pollen. further sensitization studies should be done to assess if there would be differences between those living in urban and rural areas. moreover, longitudinal studies comparing populations using the same tests and methods should be undertaken at different periods to have a more conclusive set of data. likewise, an investigation of the genetic factors associated with pollen sensitization and response to therapy should be made. diagnosis, treatment and prevention of allergic disease: the basics state of world allergy report 2008: allergy and chronic respiratory diseases an aerobiological perspective in allergy and asthma prevalence of allergic rhinitis in filipino adults based on the national nutrition and health survey allergic rhinitis and its impact on asthma (aria) allergic rhinitis and its impact on asthma in asia pacific and the aria update allergen skin test and total ige in adults with rhinitis in singapore role of pollen allergy in taiwanese patients with 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and increased citations maximum visibility for your research: over 100m website views per year • at bmc aerobiology: 222 aerobiological and immunological studies on coconut pollen allergy common aeroallergens in patients with asthma and allergic rhinitis living in southwestern part of iran: based on skin prick test reactivity disagreement between skin prick test and specific ige in young children variability of offending allergens of allergic rhinitis according to age: optimization of skin prick test allergens the impact of age on prevalence of positive skin prick tests and specific ige tests guidelines for using pollen cross-reactivity in formulating allergen immunotherapy types of sensitization to aeroallergens: definitions, prevalences and impact on the diagnosis and treatment of allergic respiratory disease pollen-cross allergenicity mediated by panallergens: a clue to the pathogenesis of multiple sensitizations publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank the following individuals for their assistance and technical support: nicole andreanna bulseco, vanessa maris cariño, romina bianca dinglasan, rencie espino, raymond michael baladad, ina marie de jesus, john darrel pamintuan, emmanuel tom rosales, and carol navidad. gratitude is also extended to the following institutions for the use of their facilities: university of santo tomas college of science, university of santo tomas graduate school, and university of santo tomas research center for the natural and applied sciences. ms designed the study, gathered all data, performed statistical analyses and interpretation of data, and drafted and revised the manuscript. cw contributed to data collection and interpretation of data. aa contributed to the design of the study and helped interpret data. jr contributed to the design of the study, helped in the interpretation of data and participated in the revision of the manuscript. all authors read and approved the final manuscript. there were no funding agencies used in the conduct of this study. the datasets during and/or analysed during the current study are available from the corresponding author on reasonable request. key: cord-292209-d1ty9etr authors: horta, bernardo l; silveira, mariângela f; barros, aluísio j d; barros, fernando c; hartwig, fernando p; dias, mariane s; menezes, ana m b; hallal, pedro c; victora, cesar g title: prevalence of antibodies against sars-cov-2 according to socioeconomic and ethnic status in a nationwide brazilian survey date: 2020-10-29 journal: rev panam salud publica doi: 10.26633/rpsp.2020.135 sha: doc_id: 292209 cord_uid: d1ty9etr objectives. to investigate socioeconomic and ethnic group inequalities in prevalence of antibodies against sars-cov-2 in the 27 federative units of brazil. methods. in this cross-sectional study, three household surveys were carried out on may 14-21, june 4-7, and june 21-24, 2020 in 133 brazilian urban areas. multi-stage sampling was used to select 250 individuals in each city to undergo a rapid antibody test. subjects answered a questionnaire on household assets, schooling and self-reported skin color/ethnicity using the standard brazilian classification in five categories: white, black, brown, asian or indigenous. principal component analyses of assets was used to classify socioeconomic position into five wealth quintiles. poisson regression was used for the analyses. results. 25 025 subjects were tested in the first, 31 165 in the second, and 33 207 in the third wave of the survey, with prevalence of positive results equal to 1.4%, 2.4%, and 2.9% respectively. individuals in the poorest quintile were 2.16 times (95% confidence interval 1.86; 2.51) more likely to test positive than those in the wealthiest quintile, and those with 12 or more years of schooling had lower prevalence than subjects with less education. indigenous individuals had 4.71 (3.65; 6.08) times higher prevalence than whites, as did those with black or brown skin color. adjustment for region of the country reduced the prevalence ratios according to wealth, education and ethnicity, but results remained statistically significant. conclusions. the prevalence of antibodies against sars-cov-2 in brazil shows steep class and ethnic gradients, with lowest risks among white, educated and wealthy individuals. in brazil, prominent covid-19 cases, including state governors and more recently president jair bolsonaro (https://www. bbc.com/news/world-latin-america-53319517), led to a disseminated impression that the epidemic affects brazilian society as a whole, without distinction of class or ethnic group. if true, this finding would be in sharp contrast with data from high-income countries, where the pandemic is disproportionally affecting ethnic minorities and poor populations (1) . in the united states (https://www.nytimes.com/interactive/2020/07/05/ us/coronavirus-latinos-african-americans-cdc-data.html), african-americans and latinos are suffering from higher disease incidence and mortality than whites, according to reported cases. a study carried out in the oxford royal college of general practitioners research and surveillance centre network observed that black people and those living in the more deprived areas were more likely to test positive for sars-cov-2 (2) . another study in the united kingdom (3) reported that non-white ethnicity and higher deprivation scores were strongly associated with increased covid-19 mortality. in contrast, the large national surveys carried out in spain did not find either nationality or education as risk factors for the presence of antibodies against sars-cov-2 (4). we were only able to locate a single study of ethnic or social inequalities in covid-19 in low-or middle-income countries. baqui et al (5) described that in brazil, covid-19 hospital case-fatality was higher among individuals classified with black or with mixed ancestry, compared to whites (5) . a commentary on this publication argued, without providing new data, that living conditions of brazil's poor would make them more vulnerable to covid-19 morbidity and mortality (6) . their study did not include a sufficient number of indigenous individuals for analyses. we were unable to locate any population-based study from low or middle-income countries on social and ethnic inequalities in covid-19 morbidity or mortality. the present analyses were aimed at assessing socioeconomic and ethnic group inequalities in prevalence of antibodies against sars-cov-2 in 133 sentinel cities throughout brazil, as part of the epicovid-19 study (www.epicovid19brasil.org). in this cross-sectional study, three population-based repeated serological surveys were carried out in 133 brazilian sentinel cities in brazil's 27 federative units. the cities included brasilia, 26 state capitals and the largest cities in each of the country's intermediate regions, as defined by the brazilian institute of geography and statistics (ibge). in each city, 25 urban census tracts were selected with probability proportionate to size, and 10 households were randomly sampled in each tract. in each sampled household, all residents were listed, and one was randomly selected to be tested. if the selected individual refused to provide a blood sample, a second household member was randomly selected. if this person also refused, the interviewers moved on to the next household to the right of the one that had been originally selected. the next household to the right was also selected in case of absent residents. in the present manuscript, we pooled the data from the three survey waves that took place on may 14-21, june 4-7, and june 21-24, 2020. with 250 individuals per city, the margins of error (approximately two standard errors) for estimating prevalence figures of 2%, 5% and 10% are respectively 1.77, 2.70, and 3.79 percent points, and at national level, with total sample size of 33 250, the corresponding margins of error are 0.15, 0.24 and 0.33. the wondfo sars-cov-2 antibody test (wondfo biotech co., guangzhou, china) was used to evaluate the presence of antibodies for sars-cov-2, using finger prick blood samples. at the time of the first survey, this was the only test available in the country in large numbers, and over 100 000 tests were provided to the study by the ministry of health. the test detects immunoglobulins of both igg and igm isotypes specific to sars-cov-2 antigens in a lateral flow assay. the assay reagent consists of colloidal gold particles coated with recombinant sars-cov-2 antigens. following the introduction of the blood sample, reactive antibody:antigen:colloidal gold complexes, if present, are captured by antibodies against human igm and igg present on the "test" (t) line in the kit's window, leading to the appearance of a dark-colored line. valid tests are identified by a positive control line (c) in the same window. if this control line is not visible, the test is deemed non-conclusive, which is uncommon. the rapid test underwent independent validation studies that used rt-pcr as the gold standard. according to the manufacturer, it has a sensitivity of 86.4% and specificity of 99.6% (https://en.wondfo.com.cn/product/wondfo-sars-cov-2-antibody-test-lateral-flow-method-2/). a validation study carried out by the national institute for quality control in health (incqs, oswaldo cruz foundation, rj, brazil) showed a sensitivity of 100% and specificity of 98.7%. whitman et al evaluated 10 different lateral flow assays (7) and reported that the wondfo test had a sensitivity of 81.5% and specificity of 99.1%. a validation study carried out by our research group observed a sensitivity of 77.1% and specificity of 98.0% (8) . by pooling the results from the four validation studies, weighted by sample sizes, sensitivity is estimated at 84.8% (95% ci 81.4%;87.8%) and specificity at 99.0% (95% ci 97.8%; 99.7%) (8) . participants answered short questionnaires including sociodemographic information (sex, age, schooling, skin color, household size and household assets), covid-19-related symptoms, use of health services, compliance with social distancing measures and use of masks. due to the presence of widespread multiethnic population, the official brazilian classification of ethnicity recognizes five groups, based on the question: "how do you classify yourself in terms of color or race?" the five response options are "white", "brown" ("pardo" in portuguese), "black", "yellow (asian)" and "indigenous". interviewers were instructed to check the "yellow" option when the respondent mentions being of asian descent, and "indigenous" when any of the multiple first nations are mentioned. the "brown" category reflects mixed ancestry including european, african and/or indigenous backgrounds. this system is endorsed by the afro-descendants movement, which advocates for disaggregation of all national statistics to raise their visibility (9) . socioeconomic position was assessed using a wealth index derived through principal component analyses of household assets (10). the first component was divided into quintiles. achieved schooling was recorded as the highest grade completed successfully. field workers used tablets to record the full interviews, register all answers, and photograph the test results. the questionnaire was applied before the test result was disclosed to each participant. inconclusive tests were repeated, and 35 subjects presented a non-conclusive result in the second test, which were treated as missing values. all positive or inconclusive tests were read by a second observer, as well as 20% of the negative tests. five regions of the country. in these analyses, the prevalences of seropositivity were still lower in the richest quintile, but the magnitude of the prevalence ratio decreased. indigenous individuals still showed higher prevalence than whites (prevalence ratio: 2.25; 95% ci 1.74; 2.91), as did individuals classified as black or brown. table 3 shows that in the northern region, in spite of the decrease in the magnitudes of the associations compared to the national analyses (table 2) , the inverse associations with wealth remained significant, and the higher prevalence among indigenous and brown subjects compared to whites also persisted. in the northeastern region, seroprevalence was also inversely associated with wealth, but not with ethnicity. prevalence for indigenous subjects were 2.27 times higher than for whites. in the remaining regions, where prevalence was low at the time of the surveys, we did not observe any clear pattern of association with wealth, but black and brown subjects had significantly higher risks than whites. consistent results were observed for education in all regions, with lower risk for subjects with 12 or more years of schooling than for the other groups. table 4 shows that the even after controlling for region and socioeconomic status, the seroprevalence remained significantly higher among indigenous, brown and black subjects. interviewers were tested and found to be negative for the virus and were provided with individual protection equipment that was discarded after visiting each home. we used stata 15 for the analyses. proportions of positive tests according to region, sex, wealth quintiles, achieved schooling and skin color were compared using the chi-squared test. for ordinal variables, both in bivariate and multivariate analyses, we estimated the p-value for linear trend and for heterogeneity, and presented the one with the lower p-value. we also stratified the analyses of seroprevalence according to socioeconomic variables by region of the country (north; northeast; southeast; south and center-west), using poisson regression with robust variance to estimate prevalence ratios. all analyses controlled for the cluster-sampling design using svy prefix. ethical approval was obtained from the brazilian's national ethics committee (process number caae 30721520.7.1001.5313), with written informed consent from all adult participants; for minors, written consent was provided by parents or caregivers, and assent forms were also signed by the child or adolescent (provided that they were literate). the dataset is stored in anonymous form. positive cases were reported to the municipal covid-19 surveillance systems. in the three waves of the seroprevalence survey, 89 397 subjects were tested and 35 individuals with inconclusive test results in the test and retest were excluded from the analyses. therefore, in the present study we evaluated 89 362 subjects. the response rates were 54.4%, 52.6% and 55.6% in the three waves, mainly due to the fact that the whole family was away from home when the visit took place. the prevalence of positive results was 1.4%, 2.4%, and 2.9% in the first, second and third surveys, respectively. table 1 shows that the proportion of males and young subjects in the studied population was below what was expected on the basis of the national population. concerning skin color, most of the studied subjects reported being mixed (brown) or white and only 1.4% self-identified as indigenous. the proportion of subjects who reported being white was lower than the national estimates. in the three phases of the study, there were 2 064 positive tests (2.31%) among the 89 362 subjects with valid test results. table 2 shows that the proportion of positive tests was higher in the north region (6.7%), whereas in the southern region only 0.2% of the studied subjects had a positive test. results for unadjusted analyses, and analyses with adjustment for age and sex, were very similar. antibody prevalence was inversely associated with wealth quintiles; compared to the wealthiest, the poorest were about twice as likely to present antibodies against sars-cov-2. for schooling, the association was not linear, but subjects with 12 or more years of schooling were less likely to present positive tests than any of the other groups. the largest prevalence ratio were observed in the comparison between indigenous and white individuals, with a near five-fold ratio. whites were less likely to test positive than any ethnic group, followed by asians. because the proportion of individuals with antibodies against sars-cov-2 was higher in the northern (amazon) region, where indigenous and poor populations are concentrated, we carried out additional analyses with further adjustment for the our study is the largest population-based serological survey for antibodies against sars-cov-2 in low-and middleincome countries, and only comparable to the national surveys carried out in spain (4). our findings show that the covid-19 pandemic is hitting harder at the poorest and disadvantaged groups in brazil. the proportions of individuals with positive tests was higher among indigenous, black and brown subjects compared to whites, as well as being inversely associated with socioeconomic position. concerning ethnic inequalities in health and nutrition in brazil, several studies have reported that indigenous children and adolescents show higher mortality than other ethnic with respect to the study limitations, the use of rapid serological tests for clinical decision-making and for defining individuals as immune to covid-19 has been criticized. but the use of such tests to estimate the seroprevalence is much less controversial, provided the test has been validated (16, 17) . the rapid test used in our study (wondfo sars-cov-2 antibody test) underwent different validation studies using rt-pcr as the gold standard, including one carried out by our own team. these studies estimated the test's sensitivity and specificity at 84.8% and 99.0%, respectively. it has been suggested that using capillary blood to estimate seroprevalence tends to increase the rate of false negative results (18) ; however, this finding has not been replicated by other studies (19) . furthermore, our validation study (8) used capillary blood and the observed sensitivity was similar to that reported in another studies. therefore, this should not be considered as a main study limitation as all population subgroups should be affected. recent evidence suggests that antibody levels against sars-cov-2 fall rapidly over a few weeks, regardless of the type of test used (20) ; therefore, our results correspond to relatively recent infections rather than cumulative prevalence. again, all population subgroups are likely to be similarly affected. the restriction of the sample to sentinel sites that are the larger and more developed cities should not be considered as a major limitation, as we are not trying to estimate the prevalence of the infection in the whole country, but its association with socioeconomic and demographic characteristics. due to logistic difficulties including a massive fake news campaign through social media, it was not possible to complete the first round of the study in 87 cities, so that the sample size was 25 025 instead of the planned 33 250 tests. these difficulties were overcome in the next two rounds, when the intended sample size was nearly achieved. concerning selection bias, the response rates of around 54% are similar to that reported in the spanish survey (59.5%) and higher than achieved in national surveys in iceland and austria, both of which had response rates of about one third of the intended sample (21) . the higher proportion of female in the studied sample could be due to the fact that males were less like to comply with the stay at home recommendations. the most frequent reason for non-response was the fact that the whole family was away from home when the visit took place, which may be associated with temporary moves to smaller towns or to rural area, as larger cities were more strongly hit by the pandemic in the early phases. regarding indigenous populations, it should be noted that our sample was restricted to those living in urban areas. lastly, our sample had fewer children than expected, which was probably due to their reluctance to undergo a finger prick when randomly selected within the household; in these cases, a second person was randomly selected and if that person also refused the household was replaced. in summary, the analyses of the three waves of national serological surveys in brazil showed important inequalities in the prevalence of antibodies against sars-cov-2 according to family wealth, education and ethnic groups. contrary to the initial impressions that covid-19 would strike all groups in brazilian society with similar intensity, our analyses show that individuals from poor families and with little schooling were at higher risk of having been infected. in terms of ethnicity or skin color, whites had the lowest risk, whereas indigenous subjects and those with black or brown skin color were most affected (22) . groups (11) , and that similar gaps are also observed for adult mortality (12) . indeed, there is overwhelming evidence that indigenous populations have been left behind when health conditions improved in brazil in the recent past (13) . it would be surprising if covid-19 turned out to be different from other existing health conditions. it has been reported that covid-19 is hitting hard at rural indigenous villages in reservations (14) , but there are no comparisons with other ethnic groups. as mentioned in the introduction, baqui et al (5) found that covid-19 hospital case-fatality was higher among individuals classified with black and with mixed ancestry, compared to whites. this study was based on a public dataset on hospitalizations and only a few subjects were identified as indigenous, and for this reason the case fatality among them was not estimated (5) . because the magnitude of the association of seroprevalence for covid-19 and skin color decreased after controlling for region of the country, the five-fold difference observed in antibody prevalence between indigenous and white subjects had been partly inflated by place of residence. yet, even after adjustment for region, indigenous individuals were about twice as likely as whites to present antibodies against sars-cov-2, and in the national analyses including adjustment for region of the country and socioeconomic status, the prevalence ratio remained at around two. the interpretation of these analyses suggests that indigenous subjects were at substantially higher risk than other ethnic groups. this was partly due to the fact that they were concentrated in the amazon region, where prevalence was the highest in the country at the time of the surveys, and also because their living standards were the lowest when compared to other groups. nevertheless, their increased risk persisted in the stratified and adjusted analyses for socioeconomic status. future studies will need to investigate the mechanisms behind this association. in terms of ethnicity, the "brown" or "pardo" category had the second highest prevalence among the five groups. this category includes individuals who self-report has having mixed ancestry. genomic ancestry studies (15) show that in the northern city of belém self-classified brown individuals had, on average, 69% european ancestry, followed by 21% amerindian ancestry and 11% african ancestry, while in the south they had on average 44% european, 11% amerindian and 45% african ancestries. therefore, the evidence suggests that brown subjects in the north -among whom seroprevalence was 7.1%-were genetically closer to amerindians than was the case for the same group in other parts of the country. sponsors did not influence in any way the design, the data collection, the analysis, the writing, and the decision to publish these results. prevalencia de anticuerpos contra el sars-cov-2 según el estatus socioeconómico y étnico en una encuesta nacional de brasil resumen objetivos. investigar las desigualdades socioeconómicas y entre distintos grupos étnicos en la prevalencia de anticuerpos contra el sars-cov-2 en las 27 unidades federativas del brasil. métodos. en este estudio transversal, se realizaron tres encuestas de hogares los días 14-21 de mayo, 4-7 de junio y 21-24 de junio, 2020 en 133 áreas urbanas brasileñas. se utilizó un muestreo de etapas múltiples para seleccionar 250 individuos en cada ciudad a fin de someterlos a una prueba rápida de anticuerpos. los sujetos respondieron un cuestionario sobre los bienes del hogar, la escolaridad y el color de la piel/etnia (autodeclarado utilizando la clasificación brasileña estándar de cinco categorías: blanco, negro, pardo, asiático o indígena). se utilizó el análisis de los componentes principales de los bienes para clasificar la posición socioeconómica en cinco quintiles de riqueza. se empleó la regresión de poisson para los análisis. resultados. se analizaron 25 025 sujetos en la primera encuesta, 31 165 en la segunda y 33 207 en la tercera, que mostraron una prevalencia de resultados positivos de 1,4%, 2,4% y 2,9% respectivamente. los individuos del quintil más pobre tuvieron 2,16 veces más probabilidades de presentar un resultado positivo (intervalo de confianza del 95% 1,86; 2,51) que los del quintil más rico, y los que tenían 12 o más años de escolaridad tuvieron una prevalencia menor que los sujetos con menos educación. las personas indígenas presentaron una prevalencia 4,71 (3,65; 6,08) veces mayor que las blancas, al igual que las de piel negra o parda. el ajuste por región del país redujo los índices de prevalencia según la riqueza, la educación y el origen étnico, pero los resultados siguieron siendo estadísticamente significativos. conclusiones. la prevalencia de anticuerpos contra el sars-cov-2 en el brasil muestra gradientes relacionados con la posición socioeconómica y la etnia muy pronunciados, con menor riesgo en las personas blancas, educadas y ricas. epidemiología; infecciones por coronavirus; encuestas y cuestionarios; inequidad social; brasil. objetivos. investigar as desigualdades socioeconômicas e étnicas na prevalência de anticorpos contra sars-cov-2 nas 27 unidades federativas do brasil. métodos. neste estudo transversal, três pesquisas domiciliares foram realizadas de 14 a 21 de maio, 4 a 7 de junho, e 21-24 de junho, 2020 em 133 áreas urbanas brasileiras. amostragem em várias etapas foi utilizada para selecionar 250 indivíduos em cada cidade para se submeter a um teste rápido de anticorpos. os sujeitos responderam a um questionário sobre bens domésticos, escolaridade e cor da pele/etnicidade (auto-relatada utilizando a classificação padrão brasileira de cinco categorias: branco, preto, pardo, asiático ou indígena). a análise dos componentes principais dos ativos foi utilizada para classificar a posição socioeconómica em cinco quintis de riqueza. a regressão de poisson foi utilizada para as análises. resultados. 25 025 indivíduos foram testados na primeira pesquisa, 31 165 na segunda, e 33 207 na terceira, com prevalência de resultados positivos de 1,4%, 2,4% e 2,9%, respectivamente. indivíduos no quintil mais pobre tinham 2,16 vezes (intervalo de confiança de 95% 1,86; 2,51) mais probabilidade de ter um resultado positivo do que aqueles do quintil mais rico, e aqueles com 12 ou mais anos de escolaridade tinham uma prevalência menor do que aqueles com menos educação. os indivíduos indígenas tinham 4,71 (3,65; 6,08) vezes mais prevalência do que os brancos, assim como aqueles com cor da pele preta ou parda. o ajuste regional reduziu as taxas de prevalência de acordo com a riqueza, educação e etnia, mas os resultados permaneceram estatisticamente significativos. conclusões. a prevalência de anticorpos contra a sars-cov-2 no brasil mostra gradientes relacionados com a posição socioeconómica e a etnia muito acentuados, com os menores riscos entre os indivíduos brancos, educados e ricos. society and the 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population census indigenous children and adolescent mortality inequity in brazil: what can we learn from the 2010 national demographic census? emerging health needs and epidemiological research in indigenous peoples in brazil covid-19 experience among brasil s indigenous people the genomic ancestry of individuals from different geographical regions of brazil is more uniform than expected serology for sars-cov-2: apprehensions, opportunities, and the path forward the role of antibody testing for sars-cov-2: is there one? sensitivity of the wondfo one step covid-19 test using serum samples analytical performance of lateral flow immunoassay for sars-cov-2 exposure screening on venous and capillary blood samples rapid decay of anti-sars-cov-2 antibodies in persons with mild covid-19 spread of sars-cov-2 in the icelandic population health conditions and health-policy innovations in brazil: the way forward disclaimer. authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the rpsp/pajph and/or paho.authors´ contributions. blh, mfs, pch, cgv conceived the study, analyzed the data, wrote/reviewed the manuscript. ajdb, fcb, fph, msd, ambm contributed with data analysis, interpreted the results, and reviewed the manuscript. all authors reviewed and approved the final version.funding. the study was funded by the brazilian ministry of health, instituto serrapilheira, brazilian collective health association and the jbs sa initiative fazer o bem faz bem. the key: cord-023584-yaxawqhj authors: bucknall, r.a. title: the continuing search for antiviral drugs date: 2008-04-10 journal: adv pharmacol doi: 10.1016/s1054-3589(08)60460-3 sha: doc_id: 23584 cord_uid: yaxawqhj this chapter discusses the continuing search for antiviral drugs. many virus diseases, both of humans and animals, have been successfully controlled by vaccines. these successes have naturally led to improvements in the spectrum and duration of protection offered by vaccines until, at present it is difficult to see how antiviral drugs could compete with vaccines in the control of many virus diseases. one may cite smallpox, yellow fever, polio, and recently measles among human diseases, newcastle disease, marek's disease, and infectious bronchitis among poultry diseases—an area of veterinary disease control where vaccines have been particularly important. research into the treatment of virus diseases by drugs is at present directed toward three general areas: (1) attempts to stimulate the defense mechanism of the host animal, (2) large screening programs to find drugs which directly block some virus-specific process, and (3) alleviation of the symptoms of the disease. the treatment of the symptoms, rather than the cause of a disease, has been the mainstay of medical practice from time immemorial, and this is still the case with most virus disease. the short incubation period of many virus diseases will inevitably restrict the therapeutic use of antiviral drugs and in cases where symptoms have already appeared. although research into the mechanisms of virus infection is carried out by many sections of the scientific community, the search for antiviral drugs is almost exclusively the province of pharmaceutical manufacturers. the reasons for this are partly historical, but chiefly it is because the facilities for running large-scale screening programs are expensive and can only be met by commercial and, occasionally, governmental resources. because of the need to protect their discoveries from unauthorized exploitation, a good deal of secrecy inevitably surrounds the work being carried out in commercial organizations. this secrecy is regrettable but necessary, since the survival of such organizations depends largely then on getting a fair financial return on the money invested by them in research. the security aspects of commercial research naturally restrict frank and constructive discussions between competitors as well as third parties, and this has been particularly true in antiviral research. perhaps it was a revolt against this enforced isolationism which led, at least in part, to the first conference on antiviral substance's held by the new york academy of sciences in 1965, in which manufacturers disclosed many of the details of their antiviral research and had a chance to discuss their failures and comparative successes (whipple, 1965) . 295 since then there have been a number of reviews of progress in the field of antiviral chemothrrapy, and in most of these there have appeared comments and recommendations ivhich indicate that a radical rethinking is taking place of the prospects for antiviral drugs (osdene, 1967; mcfadzean, 1969 ; goz and prusoff, 1970; swallow, 1971 ). it was natural in the early 1950s to espect that antiviral drugs would be discoverrd which would be analogous to the antibacterial antibiotics, the darlings of the prrvious decade. the experience up to date has proved that this was not to br the case, and despite prodigious efforts by the drug houses, only three clinically useful antiviral agents, which are far from perfect, have emerged. i believe thcre are lessons to be learned from this disappointing record, and i hope that the following remarks may help to continue further the critical reappraisal of this field, so that future progress may be faster. before embarking on a search for antiviral drugs, a number of points must be considered in order to assess thr technical feasibility of treating or preventing a virus disrase u-ith a drug. too often in the past massive screens have bccn set up against many viruses in the hope that a drug would turn up, and it would then find a natural place in human or veterinary medicine. although it is true that random screening still offers the best chance of discovering new drugs, unless a realistic appraisal of the whole project is made a t the outset, the products of random screens could well be useless as potential medicines. somr of the more important considcrations are listed below. a. diseases of economic importance i n ordcr to he commercially viable a drug must sell in sufficient quantities to pay for its devclopmcnt and manufacture as well as for future research. because of this, and because antiviral chemicals tend to inhibit specific viruses. diseases of low incidence or loneconomic importance are ruled out as primary targets for drug devclopment. this may seem inhumane, particularly in the field of human virus diseases, but the fact remains that it is no easier to find a drug against rabies than against influenza, and the sales from a specific antirabies drug would never cover its own developmrnt costs. diseases of loiv incidence or low cconomic importance, no matter how serious the outcome may be for the infected individual, inevitably must remain the subjects of sponsored research. nevertheless, treatments for some of these diseases may emerge as drugs are developed against major diseases. b. immunological control many virus diseases, both of humans and animals, have been successfully controlled by vaccines. these successes have naturally led to improvements in the spectrum and duration of protection offered by vaccines until, today, it is difficult to see how antiviral drugs could compete with vaccines in the control of many virus diseases. as examples one may cite smallpox, yellow fever, polio, and, more recently, measles among human diseases, and newcastle disease, marek's disease, and infectious bronchitis among poultry diseases-an area of veterinary disease control where vaccines have been particularly important. despite these triumphs, vaccines are not, and probably never will be, the complete answer to the control of certain virus diseases. it is in these areas where drugs would be useful, and it is on these diseases that efforts should be concentrated. the two most important human diseases in this class are influenza and the common cold. when a novel strain of influenza appears among the human population, as happened in 1933, 1947, and 1956, existing immunity to the previous current influenza strain is not effective, and widespread epidemics of disease occur. the disease spreads so rapidly after it first appears that it is not possible to develop, distribute, and administer a vaccine based on the new strain soon enough to protect useful numbers of the population. even after the initial overwhelming pandemic, successive epidemics will occur as the disease penetrates into pockets of the community that 'had escaped infection. the disease is then maintained partly by the continuing appearance of susceptible juveniles, partly by spontaneous antigenic modifications in the virus enabling it to overcome previous immunity, and partly by the general decline in immunity of other individuals with the passage of time. after the initial pandemic, it is theoretically possible to control the disease with widespread vaccination, but in practice this is not done. consequently the disease smoulders on in the community, appearing as isolated cases and occasional outbreaks and epidemics. these are the conditions under which the disease normally exists, and it is this situation, rather than the much publicized pandemics, which causes the greatest economic loss to industrialized countries. the overall loss in 1956-1957 in great britain due to the asian influenza pandemic was estimated at x100 million, but the continuing loss, from that time on has been at least x30 million each year. losses in europe, north america, japan, and similar industrialized communities must be romparablr, and if only a fraction of the disease could be prevented by a drug, the economic benefits would be enormous. losses due to the common cold are comparable. the careful study of lidwell and williams (1961) showed that approximately 11 x lo6 working days arc lost cach year in great britain alone from the common cold. scvertheless, the prospects for a common cold vaccine are poor because of the large number of viruses which are known to cause the disease. there are 89 known rhinoviruses (kapikian, 1971) , probably a comparable number of as yct unclassified rhinoviruses, a growing catalog of coronaviruses (kapikian, 1969) , and a selection of other viruses including myxoviruses, adenoviruses, and herpcsviruses (tyrrell, 1965) , all of which have been isolated from clinical colds. it is the serological diversity of these etiological agents which makes the prospects for a vaccine so poor, and the common cold must, therefore, be considered as a target, even though a difficult target, for antiviral drugs. besides the problems of antigenic variation, exemplified by influenza, and thc multiplicity of serotypes, exemplified by the common cold, there are two further problems associated with the control of respiratory diseases by parmterally administered vaccines. the first is that circulating antibodies appear in only small amounts in respiratory mucus, and, consequently, the degree of protection afforded to the respiratory tract is less and of shorter duration than might be expected. the second problem has been brought to light by the use of experimental vaccines against respiratory syncytial virus disease in infants. when infants who had been vaccinated parenterally against this disease contracted the natural disease, they were more ill than infants who had not received the vaccine. the reason seems to be that the circulating antibodies resulting from parenteral vaccinations not only offer little protection to the respiratory tract, but when a natural infection occurs, these antibodies combine with the virus antigens at the surface of the respiratory epithelial cclls causing an inflammatory response with a corresponding increase in the severity of clinical symptoms kim et al., 1969; kapikian et al., 1969) . chanock et a2. (1970) have pointed out that if an immunopathological process involving serum antibodies occurs during respiratory syncytial virus infection, then stimulation of local, respiratory tract, secretory antibody by intranasal instillation of live or inactivated virus may give adequate protection without unwanted hyperreactivity. a similar allergic reaction between virus antigen and preexisting antibody is a factor, possibly a major factor, in the pathological processes initiated by herpesviruses (jones and patterson, 1967) and marks the herpes diseases as possible candidates for antiviral drug development. one factor that deserves more careful consideration than it usually receives is the way in which a potential antiviral drug would be administered to the animal or patient requiring protection. clearly a common cold treatment would be unacceptable if it had to be given intravenously 3 times a day. but there are less obvious, but no less real, difficulties in dosing large herds of cattle or sheep so that effective protection is maintained. with freeranging animals the duration of protection from a single dose would need to be prolonged to offset the labor of administering the dose. also, it is easier to dose herds by injection than by mouth, so that certain veterinary antiviral drugs may not be required in an orally active formulation. conversely, oral dosing, preferably by an addition to food or drinking water, is the most convenient way of dosing poultry. the onset of symptoms in most virus diseases is acute and may be the first indication that the host has contracted an infection. because of this, it is usually assumed that antiviral drugs will only be of value in preventing and not in curing virus diseases. nevertheless, although the periods of virus growth in infected individuals may be short, they may well be long enough to allow useful therapy. for example, pate1 et al. (1964) have shown in volunteers infected intranasally with coxsackie a21 virus that maximum virus growth precedes the onset of symptoms by 24 hours. but from their work it may be seen that a considerable amount of virus growth is concurrent with the period of overt symptoms, and application of antiviral drugs during this period of 2-3 days might well prevent the full development of the disease. the work of douglas et al. (1966) with volunteers shows that a similar period exists in acute rhinovirus infections, and dawkins et al. (1968) and wingfield et al. (1969) have shown that the course of influenza in humans can be modified, even after the onset of symptoms, by treatment with 1-aminoadamantane. it hardly seems necessary to point out that before undertaking a search for a drug against a particular disease, the etiological agent of the disease should be unequivocally identified. there are a number of important virus diseases for which the causative agent is either unknown or is in doubt, for example, bovine pneumonia and human epidemic viral gasteroenteritis. it would be a mistake to set up screens against agents that were only suspected of being implicated in these diseases in case subsequent work should demonstrate that these were not in fact the causative agents. although it is difficult to predict changes in governmental legislation toward public or animal health, nevertheless, the existing and prospective legal position in various countries should be considered since these may affect the prospects for potential drugs. for example, in great britain, foot and mouth disease is controlled by the policy of slaughter and compensation, but in other countries the disease is controlled by vaccination. in the lattrr countries, a drug may find a ready market, but if legislation should change, then that market would be lost. in summary, we may say that before embarking on a search for antiviral drugs, the target disease must be carefully selected by a consideration of all the relevant factors. the more important of thesc a r t : 1. there must be an adequate market for the drug. 2. there should be no effective immunological control, and no prospects 3. due regard should be given to the practicability and the timing of 4. the etiology of the disease should be clearly established. other relevant factors, e.g., medical or veterinary legislation, should be takcn into consideration. in table i , a number of virus diseases of economic importance are listed together with some comments to illustrate how many seemingly attractive drug-target diseases are in fact precluded by other factors. for such control. dosing the patients or animals at risk. it is easy to list the properties of the ideal antiviral drug-wide spectrum of activity, nontoxic, accessible to the target organ, etc. what is not so easy is to predict what sort of properties one might expect from antiviral leads detected by screening programs. all the same, it is only by intelligent attempts to do just this that screens may be designed to detect compounds that one day may lead to the development of useful medicines. the scientific literature abounds with reports of antiviral chemicals discovered by screening random compounds in tissue culture systems, but all too frequently these compounds turn out to be false positives or active only against some relatively unimportant virus. for the products of a tissue culture screen to be of potential value, the screen must meet the requirements outlined below. first, the screen should be able to process large numbers of chemical compounds, or fermentation products, since the more that are tested, the greater the chance of success in finding active leads. perhaps the time will come when new drugs can be designed and synthesized on entirely rational grounds, but at present most active leads are chance discoveries. buthala (1965) quotes 6% of all compounds tested in a tissue screen as showing some antiviral activity, but in my experience the rate varies from 1 to o.ol$!&, the higher rate referring to influenza a viruses, and the lower rate to picornaviruses. bauer (1967) has suggested that the larger the virus, the more susceptible it is to inhibition, since, for any given intracellular concentration of drug, a large virus will encompass, both physically and in terms of synthetic requirements, more drug molecules than a small one. undoubtedly, this principle will contribute to our observed high rate of inhibition of influenza virus, but another important factor seems to be that the adsorption of influenza viruses to cellular receptors is particularly vulnerable to interference by extraneous substances. given that only a fraction of a percent of all compounds tested in tissue culture will show activity and that of these only a small proportion will show activity in animal models, a realistic screening rate would be not less than 5000 compounds a year. at rates less than this, the chances of finding useful compounds become so small as to make the whole project not worthwhile. in many of the published screening procedures, the virus with which the screens are run seem to have been chosen more for the ease with which they can be handled rather than for their relevance to any virus disease target. for example, ehrlich et al. (1965) describes a screen where the primary test viruses include parainfluenza type 3, measles, and poliovirus, and johnson (1965) describes a screen that includes pseudorabies, adenovirus 111, and mouse hepatitis virus. of course, if wide-spectrum leads appear, the choice of test virus may be irrelevant, but the antiviral compounds (as distinct from interferon inducers) known at present are characterized by their relatively limited spectrum of activity, e.g., methisazone is active only against poxviruses (bauer and sadler, 1960) and possibly adenoviruses (bauer and apostolov, 1966) ; l-aminoadamantane is active only against influenza a1 and as and not against other myxo-or paramyxoviruses (davies et al., 1964) ; guanidine and a-hydroxybenzyl benzimidazole are active only against picornaviruses and not against other small ribonucleic acid (rna) viruses (eggers and tamm, 1961) . thus, whenever possible, the viruses used in routine screens should be those that are responsible for the clinical large-scale screens use large numbers of tissue culture cells, and there has been an inevitable trend toward the use of continuous cell lines in screening procedures. such cells have many attractive features. they grow rapidly, they can easily be obtained in large quantities, they remain "the same" year after year, they can be madr to prrform useful technical tricks such as rapidly changing the ph of their medium and surviving for long periods under agar, and, perhaps most important, they will support the growth of a wide range of viruses. continuous cell lines seem to be the natural choice for running routine screens. xevertheless, it is worthwhile remembering that many of the desirable technical properties exhibited by these cells may be a direct result of their neoplastic nature, and to use a continuous rather than a primary or diploid cell in a tissue culture system is to take yet another step away from the natural disease. it is truc that antiviral agents, such as l-aminoadamantane and methisazone, which can be shown to protect humans against virus diseascs, also exert their antiviral action in neoplastic cells in tissue culture, for example hela and kb cells, but we have striking evidence that this may not always follow. we have recently discovered a family of chemical compounds that have high activity against rhinoviruses when grown in human diploid lung cells, but virtually no activity against the same viruscs growing in monkey kidney cells, hela cells, or kb cells (bucknall, unpublished results) . these compounds and any others that may exhibit this property would have been missed in tests carried out in continuous cell lines. in summary, a tissue culture screen should be able to proccss large numbers of tcst compounds, using viruses as relevant as possible to the diseases for which a drug is required, and should employ normal rather than neoplastic cells. unfortunately, in most of the published screening procedures the last two requirements have been sacrificed to technical considerations designed to increase the number of compounds tested, as the following descriptions will show. in its simplest form, a test for antiviral activity involves treating cultures of cells with a range of concentrations of a test compound. first the maximum concentration tolerated by the cclls is assessed; then, second, the growth of virus at lowcr concentrations of compound that are not cytotoxic is measured. several ingenious methods have been devised for measuring these two responses-cytotoxicity and virus growth-all designed to facilitate the screening of large numbers of compounds. for example, herrmann et al. (1960) devised a zone-inhibition test in which large flat dishes of chicken cells were infected with test virus, overlaid with agar containing a vital stain, and paper discs impregnated with test compounds placed on the surface of the agar. compounds with antiviral activity showed two concentric zones around the paper disc, the innermost being pale in color due to the destruction of host cells by cytotoxic concentrations of compound diffusing from the disc. outside this was a deeply staining zone where cells were exposed to nontoxic concentrations of compound which also protected them from the destructive effects of the virus with which they had been infected. beyond this, where the concentration of compound was too low to protect the cells, the cell sheet was destroyed by virus and stained poorly. thus, by visual inspection of the dishes after 3 to 4 days, active compounds could be quickly detected. rada et al. (1960) devised a similar agar diffusion test in which test compounds were applied to the virus-infected cell sheets in circular wells in the agar overlay. although agar diffusion tests are capable of processing large numbers of test compounds, they suffer from two drawbacks. first, they are of comparatively low .sensitivity in detecting both the cytotoxic and antiviral levels of compounds, and second, they are limited to viruses that produce plaques under agar. rightsel et al. (1956) devised a system based on the fact that if cells were damaged either by the toxic effects of a chemical compound or by virus growth, they would not swing the ph of their medium. thus, by incubating virus-infected cells in a series of concentrations of a compound and then looking for the cultures that had changed the color of the phenol red indicator in their medium from pink to yellow, active compounds could be detected. this technique has also been used to assay neutralizing antibody and interferon action (pauker, 1965). finter (1970) described a system whereby the cytotoxic effects of test compounds could be assayed by the reduction in the amount of neutral red taken up by treated cells, and, similarly, the cytopathic effects of virus growth could be quantitated by measuring the reduction in the uptake of neutral red by infected cells. the system can readily be adapted to the screening of test compounds for antiviral activity. if myxoviruses are used in this system, because their cytopathic effects may not be pronounced, their growth is best monitored, not by a reduction in neutral red uptake, but by a quantitative hemadsorption method which matches the neutral red uptake method in its accuracy and sensitivity (finter, 1964) . in contrast to the eone-inhibition and ph-swing tests, the neutral red uptake test is precise in operation and may be used to demonstrate fine differences in the relative toxicity and activity of test compounds; it is probably no more timeconsuming than the former tests. a system of testing for antiviral agents based on the inhibition of nucleic acid synthesis was dcscribcd by lliller et al. (1970) and has been used to screen compounds and mold metabolites for antiviral activity (miller et ul., 1968) . for the test, hela cells were suspended in a medium containing uridine-3h. if a test compound has toxic effects on the hela cells, then the cellular r s a synthesis, as measured by u r i d i n~-~h fixation, will be reduced. similarly, if cells are infected with an r s a virus and treated with sctinomycin d. then r s a synthesis will be due to virus growth only. thus, if test compounds reduce this virus-directed rxa synthesis at concentrations that do not affect cellular r s a synthesis, then the compound is exerting a specific effect on virus growth. the test can also be used for deoxyribonucleic acid (dsa) viruses, the cellular and virus dxa synthesis being monitored by including th~midine-~h in the medium. virus dsa synthesis is distinguished from cellular dsa synthesis by disrupting the cells at the end of the test and treating them with dcoxyribonuclease when encapsulated virus dsa is resistant to digestion and the unprotected host cell dsa is not. thus, the selective effect of test compounds on the synthesis of virus dsa can be measured. the authors claim that the system operates satisfactorily ivith a range of viruses-some of them important disease organisms-and is simple, reliable, and rapid. the chief criticism of this method is that, because nucleic acid synthesis is used as the sole measure of virus growth, test compounds that might act on subsequent stages in the virus replicative cycle may not be detected. for example, any disturbances in the sequencing of virus nucleic acid, inhibition of structural protein synthesis, or failure of assembly or release of mature virions, would provide a sound basis for a useful drug, but these phenomena may not be detected in this type of test. also, since high infecting doses of virus are used to give satisfactory operation of this test (up to 15 virus particles per cell), the test may be rather insensitive in detecting antiviral activity. all antiviral activity is, in the broadest sense, competitive, either a t the level of cellular membrane receptors or at an cnzymic or template level. thus, the more virus is used to initiate infection, the less effective an antiviral compound is likely to be. although this factor will not turn a highly active compound into an inactive one, it may well obscure low levels of activity which might be useful starting points for chemical exploitation. the real value of miller's test is the use of the important biochemical system of nucleic acid synthesis to monitor the toxic manifestations of test compounds. this subject is discussed furthrr in the following section. reference has already been made to four methods for measuring the toxicity of chemical compounds in tissue culture cells: direct cytopathic ef-fects, vital dye uptake, metabolic activity (ph-swing), and nucleic acid inhibition, and there is no shortage of other methods. nevertheless, the inadequate assessment of compound toxicity in antiviral testing probably gives rise to more false leads than any other single cause, before discussing how compound toxicity might be measured, it must first be defined. strictly, any interference with cellular metabolism by an extraneous compound is a toxic effect, and the most stringent tissue culture test of lack of toxicity is the continued normal division and growth of cells in the presence of an extraneous compound. however, this test is too cumbersome for use in rapid screening procedures, and simpler, but less critical, tests are invariably used in primary screens. undoubtedly, the simplest method of assessing compound toxicity is by direct microscopic examination of cells for cytopathic effects or more subtle morphological changes. it is necessary for the observer to be trained to detect such changes, and an arbitrary scale must be devised to record the observations, but if these simple requirements are met, the method is generally successful. it may be objected that this system would be unworkable where large numbers of compounds are being screened because of the correspondingly large numbers of microscopic examinations required; but given a good low-power microscope, an experienced reader, and the fact that most random compounds tested will show no antiviral activity and will, therefore, not require more than a cursory examination, the system is reliable, fast, and economical. in the author's laboratory a system of this kind has been in use for over 5 years, and it is possible for one worker to screen a hundred compounds against three viruses each week. we have found that with this system, compounds appear to be toxic a t lower concentrations than with either the zone diffusion or the dye uptake method. we conclude, therefore, that our method is more sensitive than the others mentioned in detecting the toxic effects of compounds. the direct microscopic assessment of toxicity is not without its deficiencies, but if the method is seen only as a preliminary determination of toxicity, these deficiencies are not serious. chief among these (and this applies even more to indirect methods) is the occasional failure to detect certain types of toxicity. for example, from time to time we have had compounds which appeared to prevent virus growth and to show no toxicity to confluent sheets of tissue culture cells, these cultures looked normal for several days in the presence of the compound, but viruses would not grow in these cells. nevertheless, further studies (see below) have shown that the compounds were exerting an inhibitory effect on some aspect of the cellular metabolism and it was this which prevented virus growth. we have investigated this effect with (a) inhibitors of nucleic acid synthe-sis and ( b ) uncouplers of oxidative phosphorylation, two classes of compound that are particularly prone to giving misleading results. nucleic acid inhibitors are often slow to produce cytopathic effects in confluent monolayers of cultured cells. the d s a synthrsis of such cells is low, and sufficient r s a synthesis is often maintained in the presence of partially effective concentrations of an inhibitor, enabling the cellular structural integrity to be sustained. all the same, an invading virus is unable to replicate in a cell under these reduced circumstances, and this will lead to an apparent antiviral specificity. this is the mechanism by which the chlorinated ribofuranosylbenzimidazoles exert their antiviral effects (bucknall, 1967) . these compounds were extensively studied as antiviral agents before their "activity" was found not to be specific for the virus (tamm et al., 1954; tamm and srmes, 1957; tamm and overman, 1957) . uncouplers of oxidative phosphorylation also often appear to be antiviral agents becausc concentrations that greatly reduce the energy-generating systems of cells in confluent monolayers are often slow to produce morphological changes. in this half-poisoned state, the cultures appear normal, but do not support virus growth, and thus another false "lead compound" is generated. as mentioned earlier, these remarks apply to all tissue culture systems to a greater or lesser extent, and tissue culture tests for antiviral activity must always be regarded as strictly preliminary. active leads from such tests must always be subjected to the closest scrutiny to determine whether the activity is truly specific for a virus-coded process or simply results from a subtle toxic effect on the host cell. the margin betwen the maximum nontoxic concentration and the minimum antiviral concentration of a test compound is conveniently expressed as thrrapcutie ratio = max. nontoxic concentration/min. antiviral concentration and will vary according to how these two concentrations are determined. the simplest and most stringent test of the maximum nontoxic concentration of a compound in zdtro is to grow cells in the presence of the compound and determine the maximum concentration a t which division and growth will proceed normally. if this concentration, and lower ones, protect the cells from virus attack, then this is an unequivocal demonstration that the compound is exerting a specific effect on some aspect of virus replicat ion. like miller et al. (1970) , we have found the inhibition of cellular nucleic acid synthesis, particularly r s a synthesis, to be a useful system for detecting the toxic effects of test compounds. cells are treated with a range of concentrations of a compound, then the uptake of ~r i d i n e -~h into acidinsoluble material is measured and compared with that of normal cells (bucknall, 1967) . the test is simple to run, and since the nucleic acid metabolism is a cardinal area in the cellular metabolism, even if a compound has no direct effect on the nucleic acid synthesis, disturbances of other synthetic or homeostatic mechanisms are quickly reflected in changes in the synthesis of rna or dna or both. in fig. 1 , the dose-response curves of one experimental compound are determined in human diploid lung cells by the three methods outlined above-direct cytopathic effect in confluent monolayers, inhibition of rna synthesis, and inhibition of cell growth in newly seeded cultures. although this compound is not a specific inhibitor of rna synthesis, the inhibition of rna synthesis and the production of cytopathic effects run close together. at concentrations that indirectly affect the nucleic acid synthesis, sufficient disturbance is caused in other areas of the cellular metabolism to lead to a general cytopathic effect. cell division is affected a t lower concentrations and in this particular case, inhibitory (and therefore toxic) effects can be detected with concentrations 10 times lower than those that cause cell destruction, nevertheless, even with cell growth as a measure of toxicity, and effects on the cellular growth rate (&-a) are higher than those that suppress virus growth, indicating that ici 65,709 is exerting a specific effect on virus growth. (50% end points: cpe, 10 pg/ml; rna synthesis, 10 pglml; cell growth, 1.5 pg/ml; virus growth, 0.05 pg/ml.) there is a clear margin between the toxic and antiviral effects, as may be seen from the virus yield curve. fusidic acid, which has been reported to show specific antiviral activity in tissue culture (acornley et al., 1967) , was also tested in the same way (fig. 2) . in this case, the concentration causing 50% cytopathic effect after 48 hours was 100 pg/ml, whereas virus yield was depressed to 50% by only 3 pg/lml, giving an apparent therapeutic ratio of 33. but when the effects of fusidic acid on cellular synthesis were studied, it was clear that cellular rka synthesis was drastically reduced by 3 pg,!ml. thus, fusidic acid probably reduces virus growth by inhibiting cellular, rather than virus, synthetic processes, and probably accounts for the fact that this compound showed no clinically useful effects in virus-infected volunteers, despite good levels of drug in blood and nasal secretions (acornley et al., 1967) . since the toxic effects of compounds in vitro usually increase with time, it is important when comparing toxicity and antiviral activity, to ensure that the compound has been in contact xvith cells for the same length of time in each case. in the above experiment, the cytopathic effect, rna synthesis, cell growth, and virus inhibition were all measured in cells that had been exposed to the compound for 48 hours. when a virus disease is limited to a particular target organ, such as the respiratory tract, it is of great value to be able to culture a portion of the organ for in vitro studies. the culture of portibns of trachea has been extensively used to study the growth of respiratory viruses (hoorn and tyrell, 1965, 1966; mcintosh et al., 1967; craighead and brennan, 1968; herbst-laier, 1970) , and recently organ cultures of human embryonic gut have been used to study the agents of human "virus" gastroenteritis ( d o h et al., 1970) . the technique could presumably be extended to the study of viruses, such as polio, rabies, smallpox, and herpes, which localize in specific organs of infected individuals. we have found the use of human embryo and animal tracheal pieces of value in studying the toxicity and the antiviral activity of leads produced by tissue culture screening programs. our technique is to excise a trachea and cut it transversely into rings 1-2 mm thick. these are placed in 3 x 3 in. tubes with 1 ml of eagle's medium and rolled exactly as conventional tissue cultures. with a low-power microscope the ciliary activity of the respiratory epithelium is assessed on an arbitrary scale of 0 to 4. in the presence of a test compound, the reduction of ciliary action is a highly sensitive measure of compound toxicity, and it is easy to determine the concentration at which full ciliary activity can be maintained (fig. 3) . at lower concentrations the effects on virus growth may be measured by harvesting the culture fluid at intervals and titrating for infectious virus. by using this technique, we have found that almost 70% of the so-called active compounds produced by a tissue culture screen against influenza a appear negative when tested in ferret tracheal cultures. in almost all cases, compounds were toxic at lower concentrations in tracheal cultures than in conventional tissue culture monolayers, as judged by a cessation of ciliary activity. in a proportion of these compounds, some data concerning their biochemical action were available, and in most cases these drugs were uncouplers of oxidative phosphorylation, nucleic acid inhibitors, or general antimetabolites. the inhibition of ciliary action in tracheal cultures is, therefore; a much more sensitive index of toxic effects than morphological changes in monolayers. in fig. 3 , the 50% cilia-inhibitory concentration of ici 65,709 is 2.0 pg/ ml. this effect is detected at a concentration 5 times lower than is necessary to cause morphological changes in conventional tissue culture cells (fig. l) , presumably because the metabolic patterns of the ciliated cells are more complex than those of static cells in culture and are, therefore, more readily disturbed. in general, the concentrations of compounds that suppress ciliary activity arc comparable to those that prevent cell growth, except in the case of specific inhibitors of dsa synthesis for which cell division and growth are usually more sensitive than ciliary activity. any conventional technique may be used to measure virus growth in antiviral tests-cytopathic effect, plaque reduction, yield of infectious virus, and ht.madsorption, bring thc most common. because of thcir simplicity, cytopathic effect and hcmadsorption are widdy used, hut these techniques must be used with some precautions if certain types of antiviral activity are not to be missed. in order to speed up the rate of antiviral testing, the quantity of challenge virus is often increased to a theoretical maximum of 1 virus particle per cell. the whole cell culture then behaves synchronously, and results are obtained in whatever time the virus takes to complete its replicative cycle-usually between 10 and 20 hours. with this procedure, however. a test compound that prevented the formation of infectious virus but was unable to protect the infected cell from destruction would not be detected. for example, in a relatively complex virus, such as influenza, it is not difficult to imagine that rna synthesis could be interrupted while hemagglutinin production continued, much as it does in the van magnus effect. the result would be a monolayer showing full hemadsorption and yet no transmissible virus would have been formed. test compounds that produce this effect would be of great interest as potential drugs but could be missed in tests where the dose of challenge virus is too high. wherever possible, tests should permit several cycles of virus growth to occur so that compounds that interrupt any part of the cycle may be detected. there is a school of thought that tissue culture testing is so artificial as to be of little value in detecting useful antiviral substances. it is argued that by testing for antiviral effects directly in animals, the activity that is detected is likely to be more valid and more useful than that detected in tissue culture. it is true that the majority of active compounds detected in tissue culture screens are not active in animals, even after full authentication of the antiviral activity by tests such as those discussed above. the reason for this is usually that the compounds do not reach the target organs in sufficient amounts to show activity rather than because of some intrinsic defects in the antiviral activity of the compounds. also, apart from interferon inducers, unless a compound manifests some activity in vitro, it is highly unlikely to do so in vivo. on the one hand, it is argued that a virus growing in a tissue culture cell is of little relevance to the processes by which that virus causes disease in the whole animal. on the other hand, it can be said that, since virus growth is the basis of the pathological processes, if virus growth can be halted, then the disease can be stopped. furthermore, the literature discloses that some highly irrelevant viruses are being used in animal test systems and that, even when human pathogens are used, e.g., influenza, they require extensive adaption to their animal host and the course of the disease is usually very different from that in humans. finally, there are no convenient animal models for studying the growth of human rhino-and coronaviruses and, unless tissue culture tests are used, there could be no screening for antiviral compounds against these important pathogens. there is, therefore, no convincing theoretical advantage in using animals for routine antiviral screens. this, together with the cumbersome nature of animal tests and the difficulties of "scaling-up" to test large numbers of compounds makes tissue culture testing a more attractive proposition for the initial screening program. the foregoing remarks apply, of course, only to the detection of compounds that have a direct effect on specific virus processes, e.g., absorption, penetration, and replication. in the field of interferon inducers, immune enhancers, and other stimulators of thr host defense mechanisms, obviously one has no choice but to use test animals; but here one is concerned to detect any overall rffcct on the course of a disease rathrr than accurately t o model a particular human or veterinary infection. accordingly, the choice of test system is less critical provided it fulfills certain requirements. for instancc, (a) thr virus and test compound should be administered a t separate sites to avoid any possible local destruction of the challenge virus by test compound; (6) the test compound should be given parenterally to give the best chance of absorption, and (c) the dose of challenge virus should be sufficiently small to allow a useful incubation period before symptoms develop. the following system has been used successfully by us. groups of 5 mice are dosed intraperitoneally with test compounds a t 50, 12.5, and 3.1 mg/ kg on four successive days. twenty-four hours after the first dose, they are challenged intramuscularly with 100 jild,, of semliki forest virus, and after the last dose they are observed for symptoms twice daily. the mcan rcciprocal day of death (mrdd) for each test group is calculated after 14 days and comparcd with that of a similar undosed group as w l l as with that of a group givcn four daily injections of a protective agent such as polyinsinie-polyrytidilir acid (poly ic) . figure 4 shows the typical response of mice treated nith poly ic and untreated controls. under these particular conditions, the t~7 0 response curves overlap. by selecting an mrdd of 0.133 as the criterion of "active" or '5nactive," then theoretically 10% of all inactive compounds tested will appear as spurious actives and would require to be retested to establish their true status. some caution is needed in interpreting the converse overlap. given 100 known active compounds, or a single active compound tested 100 times, then 6 tests in every 100 would miss such a compound. but in practice, the vast majority of compounds passing through the test will be inactive, and the chance that the occasional true active compound will by chance fall into the "6% missed" category is correspondingly reduced. even with an in vivo test reduced to such a minimum as this, it still requires a large effort in terms of manpower and facilities to test realistic numbers of compounds. although many human viruses will grow in animal hosts it is often difficult to assess the potential value of an antiviral compound for human use by using animal models. there are five main reasons for this. first, a relatively benign human virus infection will often follow a very different, and often severe course in an animal. for example, influenza virus, herpes simplex, and coxsackie viruses may cause much more serious diseases in laboratory animals than they do in man. second, human viruses often must be adapted by multiple passaging before they will grow satisfactorily in animal hosts, and, therefore, the challenge virus in the animal model may be a very different creature from the original human pathogen. third, the quantity of virus administered to an animal in order to produce some measurable effect, e.g., symptoms, virus isolation, and seroconversion, is usually vastly greater than would ordinarily be encountered by the natural host, and this can have a profound bearing on the efficacy of any curative agent. for example, finter (1967) has shown that the protection offered to mice by doses of interferon is greatly increased as the quantity of challenge virus is reduced. fourth, the fate of a drug when administered t o an animal may be very different from that seen in man. and last, a compound may show toxio effects in man which it did not show in animals. the last two points are probably the most important in determining how far the results obtained with animal models are relevant to man. in theory the above considerations should operate in both directions; that is, a compound that shows a positive result in an animal model may be positive or negative in man, and a compound that is negative in an animal may be negative or positive in man. but, since many animal models offer a greater challenge to the therapeutic potential of a drug than the natural disease in man, a positive result in an animal model always gives great hopes that a positive result might be achieved in man. this consideration often encourages the evaluation of potential antiviral drugs in man on the very slimmest of grounds. an example of the dilemma that an animal model may pose is afforded by the �ork of boyle and his colleagues (1970) on the compound skf 30097. this compound was shown to be active against a number of viruses, including a wide range of human rhinoviruses, in tissue culture. the problem arose of how to evaluate this compound in vivo. there are no smallanimal modrls of human rhinovirus infections, and the authors, therefore, decided to test the compound in chimpanzees, which are one of the few primates susceptible to human rhinoviruses. because of lack of knowledge on infectivity of human rhinoviruses for chimpanzees, the authors gave up to 10, ooo tcd,, of challenge virus to each animal to ensurc infcction. the animals were given the drug orally 3 times a day, and the course of the disease was monitored by virus shedding from the nose. the rate of antibody rise was also measured. the numbers of animals in each experiment were necessarily small-usually 2 or 3 treated with drug and 2 or 3 controls. the final results were tantalizingly inconclusive: not clearly negative, nor convincingly positive that the drug had produced a curative effect. the investigators admit that this system is far from satisfactory but conclude from their results that the compound is n-orthwhile studying further in human subjects. the same conclusion, however, would probably have been reached if the compound had been clearly inactive in the chimpanzees, on the grounds that the excessive doses of challenge virus and unknown factors in the chimpanzee metabolism could have led to this result. animal models of human virus disease must at best be regarded as poor imitations of the natural condition, and results obtained with animal models, whether they are positive or negative, encouraging or discouraging, should be interprcted cautiously and never be used as the sole basis for predicting the outcome in man. research into the treatment of virus diseases by drugs is a t present directed toward three general areas: ( 1 ) attempts to stimulate the defense mechanism of the host animal; (2) large screening programs to find drugs that directly block some virus-specific process; and (3) alleviation of the symptoms of the disease. the first approach is exemplified by the variety of interferon inducers which are a t present under intensive study. a disappointing feature of these is their uniformly low activity in man, despite highly promising results in laboratory animals, such as mice, rats, and rabbits. perhaps the interferon response in man and primates is less important in defense against virus disease than it is in other taxonomic groups. searches are being made for more general stimulants of host defense mechanisms, for example, stimulators of phagocytosis and the immune response, but very little progress has been reported so far. the search for drugs that will directly inhibit virus replication, by stopping a virus-coded synthetic process, or the absorption, penetration, uncoating, assembly, or release of virions, has been intensive and is still continuing. nevertheless, the products of this effort will find application only in a relatively small number of virus diseases for the reasons outlined above. only for those diseases of high economic importance, and for which no effective vaccines are available, will specific antiviral drugs ever be a commercial reality. the present paucity of such drugs is undoubtedly due largely to the intimate association of viruses at the molecular level with their host cells. for this reason, disease targets must be carefully defined, screening procedures made as meaningful as possible, and the limitations of animal models be clearly recognized. only by attention to these details can the maximum effort be brought to this difficult problem. again, the lack of clinically useful drugs allows no more than speculation on the possibilities of drug-resistant viruses emerging when antiviral drugs are eventually in widespread use. the phenomenon of drug resistance in viruses is well established in the laboratory (melnick et al., 1961; tamm and eggers, 1962; renis and buthala, 1965) , and, unless potential antiviral drugs are free of this serious defect, their commercial life will be embarrassingly short. the treatment of the symptoms, rather than the cause of a disease, has been the mainstay of medical practice from time immemorial, and this is still the case with most virus disease. the short incubation period of many virus diseases will inevitably restrict the therapeutic use of antiviral drugs, and in cases where symptoms have already appeared, the physician and layman alike will have recourse to the extensive armamentanurn of palliatives available for alleviating the symptoms of virus disease. all the same, this is clearly an unsatisfactory state of affairs, and the ultimate goal of antiviral research is the prevention of virus disease. as the understanding of viruses increases, so a more rational approach to the chemotherapy of virus diseases will become feasible. also, random discoveries of antiviral activity in novel chemical compounds will shed further light on those areas of virus metabolism that are susceptible to chemical attack. with increasing con-tributions from both these approaches, virus chemotherapy should soon emerge from a theoretical possibility to a practical reality, and antiviral drugs ivill make their long awaited contributions to clinical medicine. modern trends in medical virology zn "virus-induced immunopathology arch. gesanlfe virusforsch. 30 zn "diagnostic procedures for virus and rickettsia1 infections amer zn "topics in medicinal chemistry ezperientia 16, 487 virology 4, 483. tamm, j., and overman antiviral substances key: cord-144448-mqs502xm authors: theagarajan, lakshmi n. title: group testing for covid-19: how to stop worrying and test more date: 2020-04-14 journal: nan doi: nan sha: doc_id: 144448 cord_uid: mqs502xm the corona virus disease 2019 (covid-19) caused by the novel corona virus has an exponential rate of infection. covid-19 is particularly notorious as the onset of symptoms in infected patients are usually delayed and there exists a large number of asymptomatic carriers. in order to prevent overwhelming of medical facilities and large fatality rate, early stage testing and diagnosis are key requirements. in this article, we discuss the methodologies from the group testing literature and its relevance to covid-19 diagnosis. specifically, we investigate the efficiency of group testing using polymerase chain reaction (pcr) for covid-19. group testing is a method in which multiple samples are pooled together in groups and fewer tests are performed on these groups to discern all the infected samples. we study the effect of dilution due to pooling in group testing and show that group tests can perform well even in the presence of dilution effects. we present multiple group testing algorithms that could reduce the number of tests performed for covid-19 diagnosis. we analyze the efficiency of these tests and provide insights on their practical relevance. with the use of algorithms described here, test plans can be developed that can enable testing centers to increase the number of diagnosis performed without increasing the number of pcr tests. the codes for generating test plans are available online at [1]. the novel corona virus has caused a pandemic in early 2020. the reproduction rate of the corona virus is estimated to be between 1.4 and 3.9 [2] . from an infected patient, covid spreads to 1.4 to 3.9 others on an average. as the virus spreads through respiratory droplets, it spreads at an exponential rate, especially, in densely populated locations. the infected patients become carriers at an early stage even before the onset of symptoms [3] . thus, it becomes imperative to test a large number of people and identify early stage infections to contain the spread of covid-19. the pcr based testing is considered to be one of the best techniques for early stage diagnosis [4] . group testing is well known methodology in the combinatorics and compressed sensing literature [5] . in group testing(gt), multiple samples are pooled together to form groups (fewer in number than the total number of samples). tests are performed on these groups and from the outcome of these tests, the infected samples are inferred. group testing relies on the fact that not all of the tested samples may be infected. thus, by employing group testing for covid-19 diagnosis, the number of samples tested can be increased, the tests performed can be made economical and the speed of detection can be improved. this can lead to efficient containment of the spread of covid-19. in this article, we investigate group testing for covid diagnosis using pcrs from a practical view. we analytically study the effect of dilution, caused by pooling in group tests, on the accuracy of the tests. we present multiple group testing algorithms with which a test plan for diagnosis of a pool of samples can be generated. we analyze and present the scenarios in which the presented algorithms are appropriate. we show through simulations that group testing can provide considerable gains in the number of tests performed. finally, we provide some guidelines for performing covid diagnosis in practice. a practitioner can directly generate the test plans using the matlab codes that are available online at [1] . certain key findings and discussions of this article can be listed as follows: • when the infection rate is less than 33%, group test can help to reduce the number of tests significantly. some examples on the gains obtained through group testing (with a sensitivity of more than 99%) are given below. number of samples to test 16 16 16 32 32 32 infection rate 5% 10% 20% 5% 10% 20% number of tests required by group testing 6 8 12 11 16 26 • replication or repeating a test is required to improve the accuracy of any qrt-pcr test outcome. we find that replicating the test twice could be sufficient to get high accuracy. further, group testing could provide inherent replication and reduce the number of replicates required. for details refer to sections 3.1 and 5.3. • we also analyzed the effect of dilution caused by dividing a swab sample's content to smaller portions and mixing multiple samples together. this helped us to determine an upper limit on the number of samples that can be pooled together. it was found that up to 57 samples can be pooled together without reducing the test accuracy. for details refer to section 5.2. • it was found that different group testing strategies are optimal under different conditions. the diagnostician should adapt the test plan depending on the infection rate of a given cluster or local community. test plans that are optimal for a given infection rate are discussed in section 6. further guidelines to follow while testing for covid-19 through group testing are discussed in section 7. notations: . denotes the floor operation, i.e., largest integer lesser than or equal to the given number. . denotes the ceil operation, i.e., smallest integer greater than or equal to the given number. . denotes rounding off operation. |a| denotes the cardinality of the set a, i.e., the total number of elements in the set a. the real time or quantitative reverse transcription polymerase chain reaction (qrt-pcr) is the testing method used for early detection of covid. in rt-pcr, the viral rna molecules present in the test medium are first reverse transcribed into dna, which is referred to as the complementary dna (cdna). the cdna corresponding to the covid genes are amplified using pcr, which is performed through thermal cycling. in the high temperature cycle, the target cdna's double-helix strands are separated (referred to as the denaturation process). in the low temperature cycle, the primers bind onto the cdna strands (referred to as annealing) to initiate the polymerization through which the free nucleotides assemble on the dna strands to form a new double-helix dna (referred to as the elongation process). thus, in every cycle the number of dna particles present are doubled; refer fig. 1 . after several cycles, the total number of target dna present is increased to an exponentially large volume. finally, fluorescent dyes that fluoresce in the presence of the target dna are used to visually identify the presence or absence of the virus. the number of thermal cycles determine the final volume of target dna present; hence, by increasing the thermal cycles (also referred to as amplification cycle), the sensitivity or the detection accuracy of qrt-pcr can be improved. a detailed procedure and reagents used in qrt-pcr for detection of covid can be found in [6] . using qrt-pcr, it was found that 10 virus particles are sufficient to successfully detect the presence of the virus in a test sample with very high accuracy (>99.9%) [7] . however, this accuracy or goodness of test may vary with the qrt-pcr kit used. we define the following metrics to compare and analyze the goodness of tests. let x be a binary random variable that denotes the presence (x = 1) or absence (x = 0) of the virus in a test sample. let x t be a binary random variable that denotes the outcome of a test. definition -false negative rate : γ = pr(x t = 0|x = 1) the rate or probability of the outcome of a test being negative when the virus is actually present in the sample. alternatively, the sensitivity is defined as the probability of the test outcome being positive when the virus is actually present in the sample; this is given by 1−γ = pr(x t = 1|x = 1). it is desirable for the test to have a low value for γ. definition -false positive rate : β = pr(x t = 1|x = 0) the rate or probability of the outcome of a test being positive when the virus is actually absent in the sample. it is desirable for the test to have a low value for β. definition -prior : α = pr(x = 1) the rate of occurrence of the virus in a given population. the prior can be approximately computed, using previous history, as the ratio of the number of positive cases to the total number of tests performed. it is important to minimize sample bias in this heuristic computation. according to [8] , the prior (at the time of writing this article) for united states of america is about 0.1973 and for india is about 0.0379. among the countries where more than 10 5 tests were performed, the lowest prior is for uae (about 0.005) and the highest is for spain (about 0.4423). in real life, we are more concerned about the false negative rate (as opposed to false positive rate). it is very important to have the least γ to minimize the spread of covid and fatality caused by it. further, the value of these rates depend on the viral load or the number of viral rna copies present in the test (denoted by l). we denote the false negative and positive rates as a function of l using γ(l) and β(l), respectively. it was found in [7] that γ(5) ≈ 0.07. therefore, the goodness or efficiency of a testing technique is given by γ(l) and β(l). it is necessary to know these parameters for a given testing technique before we can analyze the efficiency of group testing using the same. often tests are performed multiple times on a single sample to confirm the outcome and account for any variability in the testing procedure, thereby improving the accuracy of the test. here, we shall analyze the improvement in accuracy when tests are replicated for a given sample. when the test is replicated r times, the outcome is declared based on the majority rule. to declare the final decision as negative, the apr should have a value greater than 1. the expressions to compute the sensitivity and apr due to replication are derived in appendix a. figure 2 illustrates the improvement in accuracy for double and triple replication. from fig. 2a , one can choose the value of r, i.e., how many tests to perform depending on the γ of the testing technique and the γ r that is desired. for example, when γ is small (< 0.05), double and triple replicates give very similar performance; hence, double replication will suffice. figure 2b shows us that replication is necessary when the prior is more than 8% at a false negative and positive rates of 5% and 10%, respectively, for the testing technique. group testing(gt) refers to the idea of pooling multiple samples together and performing tests on certain subsets of these samples to discern the infected samples. first, we illustrate gt through a simple example given in fig. 3 here, we have utilized only 6 tests to diagnose 8 samples -a 25% reduction in the number of tests. note that, if we had known that there was exactly one infected sample, then only 4 tests would be required (tests 4 and 6 would not be required). this gt algorithm is referred to as the binary search and is the optimal gt when there is exactly one infected sample in a given pool. in general, to test a pool of n samples with up to one infected sample, we require at most 2 log 2 n tests and at least log 2 n tests. this gain increases exponentially with n ; for example, n = 64 requires at most 14 tests with gt as opposed to 64 tests without gt. the theory of group testing deals with the design and analysis of algorithms that tell us how to choose subsets (groups) of samples to pool together and test, and identify the infected samples. in other words, gt aims to minimize the number of tests (t ) required to identify at most d number of infected samples among n number of given samples (pool size); 0 ≤ d < n and t ≤ n . in practice, the value of d is not known. nevertheless, depending on the value of the prior, an upper bound on the number of infected samples can be chosen 1 as d for a given n . there are two paradigms of gt, namely, • combinatorial group testing (cgt): the combinatorial algorithms require the exact number (or an upper bound) of the infected samples d. as long as the chosen value of d is greater than or equal to the true number of infected samples, cgt algorithms always identifies the infected samples correctly. • probabilistic group testing (pgt): the probabilistic algorithms require an upper bound on α and identify all infected samples with certain probability p d . usually, the detection probability p d is very close to 1; however, there still may be a non-zero probability of the infected cases going unidentified. for small values of α, the average number of tests performed in pgt is less than n ; however, there can exist cases for which the number of tests performed by pgt might be greater than n , albeit, the probability of occurrence of such cases will be relatively small. in pgt, there exists a trade-off between p d and the average value of t . from the above discussion, it can be seen that, due to their deterministic nature, cgt algorithms are preferred in practice to test for diseases. when an upper bound on d cannot be reliably established, pgt algorithms may prove to be more efficient as opposed to cgt. there exists pgt algorithms in which p d = 1. in the worst case (i.e., when d = n − 1), the value of t can be greater than n , but the average value of t can still be much less than n . this is further discussed in section 6. cgt and pgt algorithms are further classified into • adaptive tests: here, the tests are performed sequentially. first a group is chosen randomly based on d n or α and tested, the outcome of this test determines the next group to test and so on. thus, the size and samples of a group are chosen adaptively based on previous group and its test outcome. the gt described in figure 3 is an example of adaptive cgt. • non-adaptive tests: when the test plan is fixed for a given d and n , then it is known as the non-adaptive gt. here, a fixed number of tests are always performed irrespective of the number of infected samples present in the pool. an advantage of non-adaptive gt is that, if t n is the number of groups to be tested, then all the t n tests can be simultaneously run in parallel. the non-adaptive gt can be represented by a matrix of dimension t n ×n (referred to as the measurement matrix), and the samples can be represented by a n × 1 vector with 0's for uninfected samples and 1's for infected samples. the measurement matrix consists of 0's and 1's, the 1's in a row correspond to the samples included in a test and the 1's in a column correspond to the number of times a particular sample is tested. the boolean or operation between the measurement matrix and the samples vector gives the gt outcomes. now, designing a non-adaptive gt algorithm becomes a problem of designing efficient measurement matrices 2 and decoding algorithms to discern the positions of 1's in the sparse samples vector with the given t n test measurements. the non-adaptive test algorithms can be studied with the aid of compressed sensing literature [9] , [5] . a key drawback of the non-adaptive gt algorithms is that it requires a large value of n (in the order of 10 3 −10 6 ) to be efficient and provide reliable performance. also, when the number of testing kits are limited and the prior is small, sequential tests are more efficient and quicker in identifying and discarding uninfected samples than parallel tests. therefore, in this article, we shall limit our study only to adaptive tests. in this section, we shall discuss some limitations and surprising advantages of performing group tests for covid diagnosis in practice using qrt-pcr. a key question in the application of gt for covid testing is how small should d be, relative to n ? that is, what is the range of values of d n for which gt reduces the total number of tests required? this was first answered in [10] ; gt is said to reduce the number of tests required when d n < 3− √ 5 2 . as a general rule of thumb, considerable reduction in the total number of tests can be achieved when d n or the prior α is less than 33% ; in all other cases, it is best to perform individual tests. as discussed in section 3, typical value of α for covid is less than 0.33. thus, gt can be utilized to efficiently increase the number of samples tested for covid with minimal number of tests performed. in group testing, dilution occurs in two stages, namely, preparation and pooling. we shall discuss the effects of these dilutions on the allowable sample size and goodness of test. the following discussion will also help us to determine a practically appropriate value of the pool size n that provides reliable test outcomes. when preparing the samples for gt, each sample needs to be divided into multiple portions for usage in multiple groups. in the example in fig. 3 , each sample is required to be divided into 3 portions since any sample could be involved in a maximum of 3 groups. in general, a sample may be involved in at most log 2 n groups 3 while testing n samples [11] . if each test is replicated r times, then a sample may have to be divided into r log 2 n portions. however, we shall see in the next subsection that each test need not be replicated in gt as it could provide inherent replication. in practice, to test for covid, swabs from nasopharynx or throat [3] are taken. the swab samples can be dissolved in liquid buffer media [12] and this can be divided into multiple portions. when the samples are divided into multiple portions, the viral load gets distributed across the divided portions. therefore, each sample can be divided only into certain number of portions such that the viral load in each portion can still be detected reliably by qrt-pcr. the swab from nasopharynx contains 10 6 − 10 9 corona viral particles [3] , if l is the amount of viral load required for reliable detection, then each sample can be divided into at most v l l portions, where v l is the amount of viral particles in the swab. as discussed in section 3, the false negative rate γ(l) depends on the viral load in the test sample. if γ * is the required false negative rate for each test, then the corresponding viral load is given by the inverse function γ −1 (γ * ), and each sample can be divided into at most v l γ −1 (γ * ) . in figure 4 , using the γ(l) from [7] for qrt-pcr, we plot the number of portions into which a swab containing v l viral load can be divided to achieve a given false negative rate with three replicates. it can be seen that, when the swab has a viral load of 10 4 , we can still obtain about 220 portions for γ = 0.02. when portions of n swabs, of which d are infected, are mixed or pooled together for a qrt-pcr test, the viral load in d samples are diluted by the n − d virusfree samples. this dilution is referred to as the pooling dilution. the effect of this dilution on the performance of virus detection has been studied in [13] . based on the probit model described in [14] , the effect of pooling dilution was derived in [13] . though the model derived in [13] is for testing hiv, the authors mention that the model is applicable to other viral tests. simplifying the model in [13] for covid, the sensitivity after pooling dilution is given by where φ(.) is the cdf of the normal distribution, χ is the number of rna copies per viral particle (χ = 1 in the case of corona virus), d is the number of infected samples, v p is the viral load in each infected sample, v 50 and v 95 are the viral loads corresponding to a sensitivity of 0.5 and 0.95, respectively, for the considered testing 3 the maximum number of groups in which a sample will be involved may vary depending on the gt algorithms. it can be proved that log 2 n are sufficient to identify an infected sample among an arbitrary number of infected samples [11] . further, once identified, as infected or uninfected, that sample is not used in any subsequent groups for testing. technique, i.e., v x = γ −1 (1 − x). in fig. 5 , we show the reduction in sensitivity of the pooled test when different number of samples (n ) are pooled together with different number of infected samples (d). it can be seen that the pool size of 32 still provides a very high sensitivity after pooling dilution even when a single sample is infected. a similar conclusion on the pool size was reported through experimentation in [15] . in general, if t is the number of tests for covid that are to be performed on a swab with viral load v l with r replicates to achieve a sensitivity of 1 − γ * , then the maximum pool size can be derived as to achieve a sensitivity of 95%, the above equation can be simplified 4 to n = v l rt v95 . for t = log 2 n , we get where w 0 (.) is the lambert w function. this formulation includes the effect of dilution in both the sample preparation and pooling stages. example: for a swab containing a viral load of 10 6 viral particles, and a testing technique that requires 10 3 viral particles to achieve 95% sensitivity, the maximum pool size for 95% sensitivity of the group test should be n = 57. as seen from the above example, an efficient group test which performs the least number of total tests (t ) per sample can enable us to increase the pool size, which, in turn, increases the testing speed and cost. therefore, employing an efficient gt algorithm for covid testing is the key component in pooling tests. we shall discuss some practically relevant algorithms in section 6. in group testing, samples that belong to a group, which has a test outcome of positive, are often tested again in smaller groups. this ensures that multiple tests are performed for some samples. this can be observed in the example described in fig. 3 ; sample 5 is involved in 3 tests that are positive. in gt, almost every sample that tests positive is replicated at least twice. this inherent replication reduces drastically the false positive rates of gt without an explicit replication of the individual tests. the exact amount of reduction of the false positive rate depends on the gt algorithm. note that the group tests whose outcome are negative may not be replicated in gt and may require explicit replication to reduce false negative rates. therefore, the false negative rates or the sensitivity would suffice to be an appropriate metric to evaluate group tests and one can focus on reducing γ to improve gt. in this section, we discuss two combinatorial group tests and a probabilistic group test which could be practically useful in testing for covid-19. the generalized binary splitting (gbs) and multi-stage testing (mst) are the cgt algorithms, and nested testing (nt) is the pgt algorithm that we describe in the following subsections. the generalized binary splitting (gbs) is the most commonly used adaptive algorithm in the cgt literature [11] . gbs is the generalization of the binary splitting procedure (bsp). first, we describe bsp, and generalize it to obtain gbs. the binary splitting procedure assumes that there exists at least one infected sample in a given pool and the goal of bsp is to identify exactly one infected sample in the least number of tests. that is, bsp works on a pool of size n with d ≥ 1 and identifies an infected sample in exactly log 2 n tests. the steps in bsp are given below: • step 1 split the n samples into two halves, say groups g 1 and g 2 . • step 2 test g 1 . • step 3 when the test is positive: (i) continue performing all future tests with the samples from only g 1 , (ii) set n = |g 1 |, and (iii) if the number of samples in g 1 is 1, then one infected sample has been identified and the algorithm is terminated. • step 4 when the test is negative: (i) continue performing all future tests with the samples from only g 2 , (ii) set n = |g 2 |, and (iii) if the number of samples in g 2 is 1, then one infected sample has been identified and the algorithm is terminated. note that, since d ≥ 1, if g 1 tests negative, then g 2 must test positive. • step 5 with the updated n and samples pool, goto step 1. the gbs algorithm simply attempts to perform the bsp d times to identify at most d infected samples in a given pool of size n . the pseudocode and the steps in gbs algorithm are given in algorithm 2. analysis: since the outcome of a test performed at each step determines the next group for testing, the total number of tests varies for different inputs for a given n and d. the number of tests performed in the worst case, i.e., the maximum value that t can attain in gbs is given by t ≤ log 2 n d + d. figure 6 shows the maximum number of tests required by gbs with each test replicated twice for different values of n and d. it can be seen that gbs testing requires lesser number of tests, even in the worst case, than the conventional 5 testing, which is 2n . in gbs, each sample may be involved in up to log 2 n d + 1 tests; the sample preparation stage should provide at least log 2 n d + 1 portions for each sample. practical considerations: when employing gbs in practice, the following points need to be kept in mind. • the tests in gbs are sequential. that is, all the group tests have to be performed adaptively and in-order to obtain the final result. • the gains of gbs are higher for large values of n. from simulations, we observed that gbs is suited for d n ≤ 20% and the average number of tests required can be reduced by up to 50% of that of conventional tests. • a key thing to note in gbs is that the algorithm can fail if the value of d is underestimated. if the pool contains lesser number of infected samples than d, then gbs identifies all infected samples perfectly. • inherent replication: in gbs, when a group is tested positive, often it gets tested again in the subsequent groups, thereby providing inherent replication. however, due to bsp, there exists scenarios where no samples are involved in multiple tests. therefore, in practice, one has to explicitly watch out for such samples and replicate the test, if required. • at the end of gbs tests, the n − d samples, that are marked as uninfected, can be pooled into one group and tested. this ensures that d is not underestimated. some of the disadvantages of gbs are overcome by the multi-stage testing algorithm, which is described in the next subsection. a disadvantage in gbs tests is that the number of group tests that a particular sample undergoes is difficult to compute. to overcome this issue, we can employ c. h. li's multi-stage testing (mst) [11] . in mst, each sample undergoes exactly s number of tests. the value of s can be chosen as dictated by any practical restrictions; however, there is an optimal number of stages that would minimize the total number of tests performed, this is given by [11] s = arg min the pseudocode of the mst algorithm is given in algorithm 1. it can be explained as follows. maintain three bins: infected samples' bin (isb), uninfected samples' bin (usb) and queued samples' bin (qsb). initially, all n are in qsb. in the ith stage (i = 1, 2, · · · , s), form g i groups with all samples in qsb and test them. the samples that belong to groups which tested negative are moved to usb. if the group size is more than one, then the samples that belong to groups that tested positive are retained in qsb, else they are moved to isb. proceed to i + 1th stage and repeat the above steps till the group sizes become 1. analysis: the group size g i is computed as g i = n i−1 /k i , where k i = δ 1− i s is the average number of samples in a group. in practice, some groups of g i may contain k i samples and the rest may contain k i + 1 samples. note that, in the last stage, all groups contain exactly one sample, i.e., k s = 1. the total number of tests performed in mst is t = s i=1 g i . the value of t is not fixed for a given n and d as it depends on the group test outcomes. however, we can determine the value of t in the worst case, i.e., the maximum value that t can take for a given n and d. this is given by t ≤ ed ln δ [11] . practical considerations: when used in practice, mst has the following advantages. • all groups in each stage can be tested in parallel. thus, despite being an adaptive gt, testing can be sped up using mst. • when more than d groups test positive at any stage, then it can be inferred that the estimate of d is incorrect. in this case, the remaining samples in qsb can be tested with an mst test plan for the updated value of d and n (the number of samples in qsb will be the new value of n ). this ensures that the tests performed in all previous stages are still useful even when the value of d is incorrect. • when the true number of infected samples is less than or equal to d, mst identifies all infected samples. • since the number of stages s is known, the number of times any sample will be tested is at most s. thus, in the sample preparation, it is sufficient to divide each swab content to only sr portions, where r is the number of replicates. • inherent replication: since all the samples in qsb at the end of stage-i are tested again in stage-i + 1, the tests for infected samples are automatically replicated. therefore, only those groups that test negative at each stage needs replication. the groups that test positive are replicated inherently at least s times, thereby increasing the test accuracy and reducing false positives. this inherent replication further reduces the total number of tests required. we derived that the total number of tests reduced due to inherent replication in mst is at least k 1 • the maximum number of tests required by mst is always bounded above by n , i.e., t ≤ n . in fig. 7a , we plot the optimal number of stages required in mst for different values of d and n . note that, when the number of stages increase, the inherent replication for infected samples increase and improves the accuracy without any additional tests for replication. in fig. 7b , we plot the number of tests required by mst in the worst case scenario for different values of d and n with double replication. for n = 16, the maximum number of tests required for mst saturates at 30 as opposed to 32 in the conventional testing with double replicates. it can be seen that mst requires uniformly lesser number of tests than the conventional testing. though these numbers represent the worst case scenario, simulations show that the average number of tests required to detect infected samples could be 40% less than the conventional testing. gbs vs mst: figure 8 shows the total number of tests required in the worst case for gbs and mst at different values of n and d = 1, · · · , 6. the number of tests required by conventional testing is also indicated for baseline comparison. it can be seen that for small values of δ, gbs outperforms mst. as d increases for a fixed n , gbs may sometimes require more number of tests than the conventional testing; however, mst always requires less than or equal number of tests as conventional testing. in general, for large values of n (>24), gbs may be appropriate, and mst may be appropriate in the other regime. figure 8 can be used as a guideline to choose the best test for a given value of n and d. the value of d played an important role in the adaptive cgt algorithms discussed in the previous subsections. when the value of d is an underestimate, then the test plan provided by cgt may fail or may turn out to be suboptimal. this shortcoming can be addressed by the usage of pgt algorithms. here, we shall describe a probabilistic group testing algorithm known as the nested testing (nt) [16] . the nt algorithm takes n and α as the input and provides an adaptive test plan that minimizes the average number of total tests required to diagnose n samples. the actual number of infected samples in a pool or its estimate is not required for nt, only the prior probability of the presence of an infected sample is needed. nested testing is an optimal pgt which identifies all infected samples without failure [16] . the algorithm proceeds by testing a group of samples from ub and moving them to pib, if they test positive, then transferring the diagnosed samples to db and returning the rest to ub, and repeating the whole process till all samples are diagnosed. analysis: the nt algorithm models the presence of the virus in each sample as an independent bernoulli random variable with probability α. thus, nt can identify any number of infected samples in a pool, i.e., 0 ≤ d ≤ n . the probability pr(d = i), i = 0, · · · , n , is given by the binomial distribution; nested testing exploits this fact to identify the group that has the highest probability to contain infected samples and tests it. the nt algorithm gives the least number of tests for which pr(d = i) is the maximum. when the number of infected samples is i, such that pr(d = i) is the least, the total number of tests required by nt may be more than n . however, when the nt test plan is applied to multiple pools of size n , the average number of tests required per pool will be lesser than n . the average number of tests required by nt is given by g(0, n ) , where g() is as given in (9) 6 . figure 9 plots the average number of tests required by nt for different values of n and α. it is clear that, for smaller values of the prior probability, nt can provide large gains in terms of the average number of tests performed without any knowledge of d. practical considerations: when used in practice, nt has the following advantages. • nested testing can identify all infected samples irrespective of the actual value of d. the probability of detection of infected samples by nt is 1. • a key condition for nt to perform well in practice is the availability of independent samples. that is, the probability of each sample being infected should be independent of the other samples in the pool and equal α. this can be achieved in practice through randomization of samples. that is, constituting a pool with samples from reasonably separated geographical locations. • the value of the prior α can be estimated based on the past history as discussed in section 3. further, this value should be continuously updated based on the outcomes of the tests performed each time. • inherent replication: in nt, an infected sample has very high probability of getting tested in at least two groups. hence, nt can ensure at least double replication for samples that test negative. • to create an nt test plan, the algorithm needs to evaluate the expression in (9) group testing is a promising method that can be effectively utilized to ramp up the number of tests performed in covid-19 diagnosis. gt can help us quickly identify several early stage infections. by reducing the number of tests performed, gt can make the testing less expensive and economical. the following are some guidelines that can be followed in employing gt for covid-19 diagnosis. • before employing pooling, the sensitivity of the testing technique needs to be characterized. some qrt-pcr kits are known to provide accurate results with smaller viral load, whereas some are known to require a large amount of viral load to detect the virus. this sensitivity characteristics, i.e., γ(l), needs to be understood before the formulation of gt test plans. the knowledge of γ(l) would help us to decide the optimal pool size and number of replications required. • when the testing speed is a primary factor, it may be better to employ mst as it enables to perform parallel group tests. • in cgt algorithms such as gbs or mst, randomization is not required. the statistical relation between the samples are irrelevant in cgt group tests. however, the estimate of the upper bound on the number of infected samples in a pool is an important parameter that needs to be accurate. the value of d can be estimated by observing the history of a location or cluster. when more samples from a location test positive, then the estimate of d should be correspondingly increased and vice versa. • pgt algorithms such as nt has the advantage of not requiring the exact value or an upper bound on the number of infected samples in a pool. pgt algorithms require the prior probability values (α). once again, this can be obtained from the history of the tests performed for a location or cluster. the ratio of the number of samples tested positive to the total number of tests performed can give us this estimate (refer section 3). this estimate needs to be update continuously over time. • randomization could significantly reduce the number of tests performed in pgt. when samples from a small community or cluster are pooled together, most of them are likely to have a similar test outcome. however, pgt works best when the samples are independent. hence, the testing center should randomize by picking samples from different communities or clusters to form a pool. this randomization can bring down the average fraction of samples that are positive in a pool and the dependence among the samples. • in practice, pgt tests are suited well for locations where the infection rate is higher and the cgt tests are suited well for locations where the infection rate is small. this is because, pgt does not need the value of d and an underestimated value could cause failure of cgt tests. • when choosing a gt algorithm to perform diagnosis, the practitioner can intelligently switch between gbs, mst and nt, depending on the estimate of d and α, and their accuracy. the performance plots provided in section 6 can be utilized in choosing the best test for a given scenario. • from the study so far, it is clear that considerable reduction in the total number of tests can be achieved when d n or the prior α is less than 33%. • note that when d > n 2 or α > 1 2 , group testing can still be helpful. under this condition, the goal of gt would be reversed, i.e., to identify the uninfected samples rather than the infected samples. all our discussions so far remain applicable with this switch. • the matlab codes for simulating the results and generating group test plans are available online at [1] . where x t is the random variable denoting the outcome of the tth replicate. when the test is replicated twice, pr(x 1 = 0, x 2 = 0|x = 1) = γ 2 . for triple replication, pr(x 1 + x 2 + x 3 ≤ 1|x = 1) = 3γ 2 (1 − γ) + γ 3 and pr(x 1 = 0, x 2 = 0, x 3 = 0|x = 1) = γ 3 . for a given set of test replicate outcomes x 1 , · · · , x r , the probability of the sample actually containing the virus is given by pr(x = 1|x 1 , · · · , x r ) -this is referred to as the a posteriori probability. the a posteriori probability ratio (apr) pr(x=0|x1,··· ,xr) pr(x=1|x1,··· ,xr) is easier to compute than the individual a posteriori probabilities. the apr for a given set of outcomes is ap r = pr(x = 0|x 1 , · · · , x r ) pr(x = 1|x 1 , · · · , x r ) = pr(x 1 , · · · , x r |x = 0) pr(x 1 , · · · , x r |x = 1) where m is the number of negative outcomes (i.e., x t = 0) in r replicates. divide p i−1 into g i disjoint groups -g 1 , g 2 , · · · , g gi such that g 1 ∪ g 2 ∪ · · · ∪ g gi = p i−1 test groups g 1 , g 2 , · · · , g gi 8: discard groups that tested negative test group g ⊆ p if test outcome is positive then 6: identify an infected sample in g with bsp (since the group tested positive, it must contain at least one infected sample) update n = n − 1 − g (where g is the number of uninfected items diagnosed from bsp, remove these from the pool) test the n samples individually 15: end if available online: matlab codes for test plan generation pattern of early human-to-human transmission of wuhan 2019 novel coronavirus (2019-ncov) sars-cov-2 (covid-19) by the numbers coronavirus and the race to distribute reliable diagnostics group testing and sparse signal recovery diagnostic detection of 2019-ncov by real-time rt-pcr detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr coronavirus disease (covid-19)-statistics and research boolean compressed sensing and noisy group testing on the cut-off point for combinatorial group testing combinatorial group testing and its applications transport of viral specimens a methodology for deriving the sensitivity of pooled testing, based on viral load progression and pooling dilution refinement of a viral transmission risk model for blood donations in seroconversion window phase screened by nucleic acid testing in different pool sizes and repeat test algorithms evaluation of covid-19 rt-qpcr test in multi-sample pools born again group testing: multiaccess communications group testing to eliminate efficiently all defectives in a binomial sample the author thanks namrata m. nilavar, indian institute of science, bangalore, for useful discussions. hence, the false negative and positive rates for r tests are given by (each replicate is assumed to be independent of the other) compute h =g(0, |u|) using (8) test a group g ⊆ u of size h 6:if test is negative then 8:else 10:if h == 1 then update d = d + g and make p empty while p is not empty do compute g =g(|p|, |p ∪ u|) using (8) test a group g ⊆ p of size g 17: if test is positive then g(1, m) = g(0, m − 1), g(0, 1) = 1, g(0, 0) = 0. key: cord-026215-neqsup40 authors: grzegorzewski, przemyslaw title: two-sample dispersion problem for fuzzy data date: 2020-05-16 journal: information processing and management of uncertainty in knowledge-based systems doi: 10.1007/978-3-030-50153-2_7 sha: doc_id: 26215 cord_uid: neqsup40 the problem of comparing variability of two populations with fuzzy data is considered. a new permutation two-sample test for dispersion based on fuzzy random variables is proposed. a case-study illustrating the applicability of the suggested testing procedure is also presented. various two-sample statistical tests are designed to determine whether given two populations differ significantly. in such case we assume that the universe of discourse consists of two populations, say x and y , with cumulative distribution functions f and g, respectively. then, having a random sample of size n drawn from the x population and another random sample of size m drawn from the y population, we consider the null hypothesis that these two samples are actually drawn from the same population, i.e. h 0 : f = g. one may verify h 0 against the general alternative hypothesis that the populations just differ in some way. the kolmogorov-smirnov test or the wald-wolfowitz run test are often used in this context (see e.g. [5] ). however, they are really useful in preliminary studies only since affected by any type of difference between distributions, they are not very efficient in detecting any specific type of the difference like difference in location or difference in variablity. other tests, like the mann-whitney-wilcoxon test, the median test, etc. (see e.g. [5] ) are particularly sensitive to differences in location when the populations are identical otherwise and hence cannot be expected to perform extremely well against other alternatives. however, sometimes we need statistical procedures designed to detect differences in variability or dispersion instead of location. indeed, comparison of variability might be of interest in many areas including social sciences, biology, clinical trials, engineering, manufacturing and quality control, etc. moreover, tests for the equality of variances are often required as a preliminary tool for the analysis of variance (anova), dose-response modeling, discriminant analysis, etc. it is important to emphasize that comparing variability is much harder than comparing measures of location. the famous f test assumes that both underlying populations are normally distributed and is not robust to departures from normality even asymptotically. thus many nonparametric two-sample tests based on the ranks have been proposed for the scale problem. the best-known tests are the ansari-bradley test, the mood test, the siegel-tukey test, the klotz normal-scores test, the sukhatme test, etc. designing tests for the dispersion problem turns out to be much more difficult in the case of imprecise or vague data which appear quite often in the real-life problems. in particular, human ratings based on opinions or associated with perceptions often lead to data that cannot be expressed in a numerical scale because they consist of intrinsically imprecise or fuzzy elements. since they are also realizations of some random experiment, we are faced with random fuzzy structures that cannot be analyzed with classical statistical methods. obviously, one may try to neglect, hide or remove imprecision but the most recommended approach is to consider it as a challenge for modeling and developing new inferential tools. a general framework for such modeling is given by fuzzy random variables. however, besides mathematical elegence they also bring some fundamental difficulties. for instance, random fuzzy numbers are not linearly ordered so the aforementioned tests based on ranks cannot be directly applied in fuzzy environment. depending on the context various test constructions have been proposed in the literature (for the overview we refer the reader e.g. to [7, 8, [11] [12] [13] [14] 16, 18, 19, 21, 26] ). however, the dispersion problem with imprecise data has not beed considered very often. ramos-guajardo and lubiano [26] proposed the bootstrap generalization of the levene test for random fuzzy sets to examine homoscedasticity of k populations. grzegorzewski [15] introduced two generalizations of the sukhatme test for interval-valued data. in this paper we suggest a permutation test for fuzzy data to compare variability of two populations. for motivations we turned back to the classical inference showing that permutation tests, like the bootstrap, require extremly limited assumptions. indeed, permutation tests are totally distribution-free and require only exchangeability (i.e., under the null hypothesis we can exchange the labels on the observations without affecting the results). classical permutation test are often more powerful than their bootstrap counterparts (see [9] ). permutation test are exact if all permutation are considered, while bootstrap tests are exact only for very large samples. moreover, asymptotically permutation tests are usually as powerful as the most powerful parametric tests (see [1] ). keeping this in mind we combine the pan test [22] and the marozzi test [20] and then generalize them into the permutation testing procedure that handle fuzzy data. the paper is organized as follows: in sect. 2 we recall basic concepts related to fuzzy data modeling and operations on fuzzy numbers. section 3 is devoted to fuzzy random variables. in sect. 4 we introduce the two-sample test for the dispersion dedicated to fuzzy data. next, in sect. 5 we present some results of the simulation study and the case study with the proposed test. finally, conclusions and some indications for the futher research are given in sect. 6. a fuzzy number is an imprecise value characterized by a mapping a : r → [0, 1], called a membership function), such that its α-cut defined by is a nonempty compact interval for each α ∈ [0, 1]. operator cl in (1) denotes for the closure. thus every fuzzy number is completely characterized both by its memberschip function a(x) or by a family of its α-cuts {a α } α∈ [0, 1] . two α-cuts are of special interest: a 1 = core(a) known as the core, which contains all values which are fully compatible with the concept described by the fuzzy number a and a 0 = supp(a) called the support, which are compatible to some extent with the concept modeled by a. the most often used fuzzy numbers are trapezoidal fuzzy numbers (sometimes called fuzzy intervals) with membership functions of the form where a 1 , a 2 , a 3 , a 4 ∈ r such that a 1 a 2 a 3 a 4 . a trapezoidal fuzzy number (2) is often denoted as tra(a 1 , a 2 , a 3 , a 4 ). obviously, a 1 = inf supp(a), a 2 = inf core(a), a 3 = sup core(a) and a 4 = sup supp(a), which means that each trapezoidal fuzzy numbers is completely described by its support and core. a fuzzy number a is said to be a triangular fuzzy number if a 2 = a 3 , while if a 1 = a 2 and a 3 = a 4 we have the so-called interval (or rectangular) fuzzy number. the families of all fuzzy numbers, trapezoidal fuzzy numbers, triangular fuzzy number and interval fuzzy numbers will be denoted by f(r), basic arithmetic operations in f(r) are defined through natural α-cut-wise operations on intervals. in particular, the sum of two fuzzy numbers a and b is given by the minkowski addition of corresponding α-cuts, i.e. for all α ∈ [0, 1]. similarly, the product of a fuzzy number a by a scalar θ ∈ r is defined by the minkowski scalar product for intervals, i.e. for all α ∈ [0, 1] it is worth noting that a sum of trapezoidal fuzzy numbers is also a trapezoidal fuzzy number: if a = tra(a 1 , a 2 , a 3 , a 4 ) and b = tra moreover, the product of a trapezoidal fuzzy number a = tra(a 1 , a 2 , a 3 , a 4 ) by a scalar θ is a trapezoidal fuzzy number unfortuntely, f(r), +, · has not linear but semilinear structure since in general a + (−1 · a) = 1 {0} . consequently, the minkowski-based difference does not satisfy, in general, the addition/subtraction property that (a+(−1·b))+b = a. to overcome this problem the so-called hukuhara difference was defined as follows: hold but the hukuhara difference does not always exist. therefore, one should be aware that subtraction in f(r) generally leads to critical problems and should be avoided, if possible. at least some of the problems associated with the lack of a satisfying difference in constructing statistical tools for reasoning based on fuzzy observations could be overcome by using adequate metrics defined in f(r) -for the general overview see [2] . obviously, one can define various metrics in f(r) but perhaps the most often used in statistical context is the one proposed by gil et al. [6] and by trutschnig et al. [27] . let λ be a normalized measure associated with a continuous distribution having support in [0, 1] and let θ > 0. then for any a, b ∈ f(r) we define a metric d λ θ as follows denote the mid-point and the radius of the α-cut a α , respectively. both λ and θ correspond to some weighting: λ allows to weight the influence of each α-cut, while by a particular choice of θ one may weight the impact of the distance between the mid-points of the α-cuts (i.e. the deviation in location) in contrast to the distance between their spreads (i.e. the deviation in vagueness). in practice, the most common choice of λ is the lebesgue measure on [0, 1]), while the most popular choice is θ = 1 or θ = 1 3 . it is worth noting that assuming θ = 1 we obtain i.e. the metric which weights uniformly the two squared euclidean distances and is equivalent to the distance considered in [4, 10] . one may also notice that assuming θ = 1 3 we obtain where a aggregates uniformly the squared euclidean distances between the convex combination of points in α-cuts representing a and b. it should be stressed that whatever (λ, θ) is chosen d λ θ is an l 2 -type metric in f(r) having some important and useful properties. is a separable metric space and for each fixed λ all d λ θ are topologically equivalent. suppose that the result of an experiment consists of random samples of imprecise data described by fuzzy numbers. to cope with such problem we need a model which grasps both aspects of uncertainty that appear in data, i.e. randomness (associated with data generation mechanism) and fuzziness (connected with data nature, i.e. their imprecision). to handle such data puri and ralescu [24] introduced the notion of a fuzzy random variable (also called a random fuzzy number). in other words, x is a random fuzzy variable if and only if x is a borel measurable function w.r.t. the borel σ-field generated by the topology induced by d λ θ . puri and ralescu [24] defined also the aumann-type mean of a fuzzy random variable x as the fuzzy number e(x) ∈ f(r) such that for each α ∈ [0, 1] the α-cut e(x) α is equal to the aumann integral of x α . it is seen that to characterize dispersion of a fuzzy random variable x we can also define (see [17] ) the d λ θ -fréchet-type variance v(x), which is a nonnegative real number such that given a sample of random fuzzy numbers x = (x 1 , . . . , x n ) a natural estimator of e(x) is the average x ∈ f(r) such that for each α ∈ [0, 1] while the estimator of v(x) is the d λ θ -type sample variance s 2 ∈ r given by although aforementioned constructions preserve many properties known from the real-valued inference, one should be aware of the problems typical of statistical reasoning with fuzzy data. as it was noted in sect. 2, there are problems with subtraction of fuzzy numbers. similar problems appear in the case of division of fuzzy numbers. hence, it is advisable to avoid both operations wherever it is possible. moreover, some difficulties in fuzzy data analysis is caused by the lack of universally accepted total ranking between fuzzy numbers. another source of possible problems that appear in conjunction of randomness and fuzziness is the absence of suitable models for the distribution of fuzzy random variables. even worse, there are not yet central limit theorems for fuzzy random variables which can be applied directly in statistical inference. the disadvantages mentioned above make the straightforward generalization of the classical statistical methodology into the fuzzy context either difficult or, sometimes, even impossible. for instance, in most cases we are not able to find the null distribution of a test statistic based on fuzzy data and, consequently, to find either the critical value or to compute the p-value required for rejection or acceptance of the hypothesis under study. to break through that problem some researchers propose to use the bootstrap [7, 8, 18, 19, 21, 25, 26] . in this paper we suggest another methodology based on permutations. for motivations we turn back to the classical inference which shows that permutation tests, like the bootstrap, require extremly limited assumptions. bootstrap tests usually rely on assumption that successive observations are independent, while permutation tests require only exchangeability, i.e. under the null hypothesis we can exchange the labels on the observations without affecting the results (obviously, if the observations in a sample are independent and identically distributed then they are exchangeable). in the real-valued framework one can also indicate two advantages of the permutation tests over the bootstrap tests. firstly, permutation test are often more powerful than their bootstrap counterparts (see [9] ). secondly, permutation test are exact if all permutation are considered, while bootstrap tests are exact only for very large samples. moreover, asymptotically permutation tests are usually as powerful as the most powerful parametric tests (see [1] ). for more information on classical permutation tests we refer the reader to [9, 23] . all these reasons indicate that the permutation test applied to fuzzy random variables might be also a competitive tool useful in statistical inference for imprecise data. suppose, we observe independently two fuzzy random samples x = (x 1 , . . . , x n ) and y = (y 1 , . . . , y m ) drawn from populations with unknown distributions function f and g, respectively. we want to verify the null hypothesis that both samples come from the same distribution, i.e. against the alternative hypothesis that the dispersion of the distributions f and g differ (or against the one-sided alternative that the indicated distribution is more dispersed that the other one). most of the tests for scale assume that the distributions under study do not differ in location since possible location differences may mask differences in dispersion. otherwise, the sample observations should be adjusted by subtrating the respective location parameters, like means or medians. if the true characteristics of location are not known we usually subtract their estimators. following remarks of marozzi [20] on the resampling version of the pan test [22] and the resampling framework for scale testing described by boos and brownie [3] , we'll try to eliminate the location effects with sample means. however, keeping in mind problems with subtratiion in fuzzy environment described in sect. 2, contrary to the crisp case, we do not consider the differences but the distances between sample observations and corresponding sample means calculated as in (8) . therefeore, further on instead of x = (x 1 , . . . , x n ) and y = (y 1 , . . . , y m ) we consider the adjusted samples v = (v 1 , . . . , v n ) and w = (w 1 , . . . , w m ), respectively, where . . , m. now let us consider the following test statistics where s 2 v and s 2 w denote sample variances of v and w, respectively, calculated by (9) . obviously, too big or too small values of (11) indicate that the null hypothesis should be rejected since the considered distributions differ in dispersion. in the original pan test [22] the decision whether to reject the null hypothesis is based on the test statistic valued with respect to some quantile from the t-student distribution. however, marozzi [20] showed that the resampling version of the pan test should be rather preferred to the original one. in the case of fuzzy data any assumptions on the type of the underlying distribution of the samples are much more dubious than in the crisp case. for this reason we also consider here the permutation version of the pan test. to carry out such a test we adapt the general idea of permutation tests to our fuzzy context. the crucial idea of the proposed test construction is that the null hypothesis implies total exchangeability of observed data with respect to groups. indeed, if h 0 holds then all available observations may be viewed as if they were randomly assigned to two groups but they come from the same population. let z = x y, where stands for the vector concatenation, so that the two samples are pooled into one, i.e. now, let z * denote a permutation of the initial dataset z. more formally, if ν = {1, 2, . . . , n} and π ν is a permutation of the integers ν, then z * i = z πν (i) for i = 1, . . . , n. then the first n elements of z * is assigned to the first sample z * and the remaining m elements to z * . in other words, it works like a random assignment of elements into two samples of the size n and m, respectively. each permutation corresponds to some relabeling of the combined dataset z. please, note that if h 0 holds then we are completely free to exchange the labels x or y attributed to particular observations. as a consequence of elements' exchangeability in z * under h 0 we can estimate the distribution of the test statistic t by considering all permutations of the initial dataset z and computing a value of t (z * ) corresponding to each permutation. namely, given z = z, where z = x y, we take its permutation z * and determine its adjustment with respect to sample means, i.e. we create two samples v * = (v * 1 , . . . , v * n ) and w * = (w * 1 , . . . , w * m ) as follows next, following (11) we compute its actual value corresponding to given permutation z * , i.e. finally, assuming k denotes a fixed number of drawings (usually not smaller than 1000), we calculate the p-value of our test. in the case on the one-sided upperer-tail test, i.e. when verifying h 0 : f = g vs. h 1 stating that f is more disperded than g, we obtain where each z * k ∈ p(z), z * k = x * k y * k , and t 0 = t (x, y) stands for the test statistic value obtained for the original fuzzy samples x and y. for the one-sided lower-tail test, i.e. when verifying h 0 : f = g vs. h 1 : f is less disperded than g, we have while for the two-sided test, i.e. when verifying h 0 : f = g vs. h 1 : f and g differ in dispersion, we obtain we conducted some simulations to illustrate the behavior of the proposed test. to generate fuzzy samples from a trapezoidal-valued fuzzy random variable x = tra(ξ 1 , ξ 2 , ξ 3 , ξ 4 ), where ξ 1 , ξ 2 , ξ 3 , ξ 4 are real-valued random variables such that ξ 1 ξ 2 ξ 3 ξ 4 , the following characterization appears to be useful (see [19] ): c = 1 2 (ξ 3 + ξ 2 ) = mid 1 x, s = 1 2 (ξ 3 − ξ 2 ) = spr 1 x, l = ξ 2 − ξ 1 and r = ξ 4 − ξ 3 . conversely, we have tra c, s, l, r = tra(c − s − l, c − s, c + s, c + s + r). in our study we generated fuzzy observations x = (x 1 , . . . , x n ) and y = (y 1 , . . . , y m ) by simulating the four real-valued random variables x i = c xi , s xi , l xi , r xi and y i = c y j , s y j , l y j , r y j , respectively, with the last three ones random variables in each quartet being nonnegative. in particular, we generated trapezoidal-valued fuzzy random variables using the following real-valued random variables: c xi , c y j from the normal distribution and s xi , s y j , l xi , l y j , r xi and r y j from the uniform or chi-square distribution. an illustration how the test works, is shown in fig. 1 and fig. 2 . figure 1 shows a histogram made for the test statistic (11) null distribution obtained for two fuzzy samples of sizes n = 10 and m = 12. both samples were generated as follows: c x and c y came from the standard normal distribution n(0, 1) and s x , s y , l x , l y and r x , r y from the uniform distribution u(0.0.5). in this case we have obtained t 0 = 0.3088, which is illustrated by a vertical line, while p-value = 0.384. a decision suggested by our test is: do not reject h 0 . on the other hand, in fig. 2 we have a histogram made for the test statistic (11) null distribution obtained for two fuzzy samples of the same samle sizes as before but which differ in dispersion. namely, c x was generated from the standard normal distribution n(0, 1), but c y from n(0, 2), while s x , s y , l x , l y and r x , r y were, as befor, uniformly distributed from u(0.0.5). in this case we have obtained t 0 = −3.5373, illustrated by a vertical line, and p-value = 0.007, leading to the decision: reject h 0 . we also examined the proposed permutation test with respect to its size. therefore, 1000 simulations of the test performed on independent fuzzy samples comming from the same distribution were generated at the significance level 0.05. in each test k = 1000 permutations were drawn. then empirical percentages of rejections under h 0 were determined. the results both for equal and nonequal sample sizes are gathered in table 1 . it is seen that our test is conservative. moreover, this tendency deepens significantly as the imbalance of the sample sizes increases. these interesting results of the preliminary study of the proposed test properties indicate that further and more extensive study is highly recommended. some statistical analyses of fuzzy data related to the gamonedo cheese quality inspection was performed by ramos-guajardo and lubiano [26] and ramos-guajardo et al. [25] . the gamonedo cheese is a kind of a blue cheese produced asturias, spain. it experiences a smoked process and later on is let settle in natural caves or a dry place. to keep the quality of a cheese the experts (or tasters) usually express their subjective perceptions about different characteristics of the cheese, like visual parameters (shape, rind and appearance), texture parameters (hardness and crumbliness), olfactory-gustatory parameters (smell intensity, smell quality, flavour intensity, flavour quality and aftertaste) and their overall impression of the cheese. recently some of the tasters were proposed to express their subjective perceptions about the quality of the gamonedo cheese by using trapezoidal fuzzy numbers. these fuzzy sets were determined in the following way: the set of values considered by the expert to be fully compatible with his/her opinion led to α = 1-cut, while the set of values that he/she considered to be compatible with his/her opinion at some extent (i.e., the taster thought that it was not possible that the quality was out of this set) led to α = 0-cut of a fuzzy number. then these two α-cuts were linearly interpolated to get the trapezoidal fuzzy set representing exppert's personal valuation. for more details on the data aquisition and analysis we refer the reader to ramos-guajardo et al. [25] . here we utilize some data given in [25] to compare the opinions of the two experts about the overall impression of the gamonedo cheese (the trapezoidal fuzzy sets corresponding to their opinions are gathered in table 2 ). thus we have two independent fuzzy samples of sizes n = 40 and m = 38 comming from the unknown distributions f and g, respectively. our problem is to check whether there is a general agreement between these two experts. to reach the goal we verify the following null hypothesis h 0 : f = g, stating there is no significant difference between experts' opinions, against h 1 : ¬h 0 that their opinions on the cheese quality differ. substituting data from table 2 into formula (11) we obtain t 0 = 1.355. then, after combining samples and generating k = 10 000 random permutations we have obtained the p-value of 0.082. hence, assuming the typical 5% significant level we may conclude that there is no significant difference between the dispersion of experts' opinion on the overal impression of the gamonedo cheese. in fig. 3 one can find the empirical null distribution of the permutation test with red vertical line indicating the value t 0 of the test statistic. hypothesis testing with samples which consist of random fuzzy numbers is neither easy nor straightforward. most of statistical tests developed in this area are based on the bootstrap. in this paper another approach for constructing tests for fuzzy data is proposed. namely, the two-sample permutation test for dispersion is suggested. some simulations to illustrate its behavior and to examine its properties are given. moreover, the case study dedicated to fuzzy rating problem is performed. the results obtained seem to be promissing, but further research including power studies and a comparison with other tests are still intended in the nearest future. in particular, the behavior of the test under strong imbalance in the sample sizes is worth of further examination. next, we would like to perform an extensive simulation study to compare the performance of our permuatation test and the bootstrap test for the dispersion. moreover, some other topics related to the dispersion problem with fuzzy data seem to be of interest. firstly, we plan to design other two-sample tests for scale, like the permutation test for fuzzy data based on the classical o'brien test, as well as a permutation test for the homogeneity of more than two fuzzy samples. secondly, a permutation test for fuzzy data to compare jointly the central tendency and variability of two populations would be of desirable. asymptotic expansion for the power of distributionfree tests in the two-sample problem a distance-based statistic analysis of fuzzy numbervalued data (with rejoinder) comparing variances and other measures of dispersion metric spaces of fuzzy sets nonparametric statistical inference least squares fitting of an affine function and strength of association for interval-valued data bootstrap techniques and fuzzy random variables: synergy in hypothesis testing with fuzzy data. fuzzy sets syst fuzzy data treated as functional data. a one-way anova test approach permutation, parametric and bootstrap tests of hypotheses metrics and orders in space of fuzzy numbers statical inference about the median from vague data testing statistical hypotheses with vague data. fuzzy sets syst fuzzy tests -defuzzification and randomization. fuzzy sets syst k-sample median test for vague data two-sample dispersion tests for interval-valued data goodness-of-fit tests for fuzzy data the λ-mean squared dispersion associated with a fuzzy random variable hypothesis testing for means in connection with fuzzy rating scale-based data: algorithms and applications hypothesis testing-based comparative analysis between rating scales for intrinsically imprecise data levene type tests for the ratio of two scales asymptotic and bootstrap techniques for testing the expected value of a fuzzy random variable on a levene type test for equality of two variances multivariate permutation tests fuzzy random variables applying statistical methods with imprecise data to quality control in cheese manufacturing k-sample tests for equality of variances of random fuzzy sets a new family of metrics for compact, convex (fuzzy) sets based on a generalized concept of mid and spread key: cord-102704-wfuzk2dp authors: meza, diana k.; broos, alice; becker, daniel j.; behdenna, abdelkader; willett, brian j.; viana, mafalda; streicker, daniel g. title: predicting the presence and titer of rabies virus neutralizing antibodies from low-volume serum samples in low-containment facilities date: 2020-04-30 journal: biorxiv doi: 10.1101/2020.04.24.060095 sha: doc_id: 102704 cord_uid: wfuzk2dp serology is a core component of the surveillance and management of viral zoonoses. virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. here, focusing on rabies lyssavirus, a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent focus inhibition test (pmrffit) that overcomes these limitations. specifically, we adapted an existing micro-neutralization test to use a green fluorescent protein–tagged murine leukemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low-containment laboratories. we further used statistical analysis to generate rapid, quantitative predictions of the probability and titer of rabies virus neutralizing antibodies from microscopic imaging of neutralization outcomes. using 47 serum samples from domestic dogs with neutralizing antibody titers estimated using the fluorescent antibody virus neutralization test (favn), pmrffit showed moderate sensitivity (78.79%) and high specificity (84.62%). despite small conflicts, titer predictions were correlated across tests repeated on different dates both for dog samples (r = 0.93), and for a second dataset of sera from wild common vampire bats (r = 0.72, n = 41), indicating repeatability. our test uses a starting volume of 3.5 μl of serum, estimates titers from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. the pmrffit enables high-throughput detection of rabies virus neutralizing antibodies in low-biocontainment settings and is suited to studies in wild or captive animals where large serum volumes cannot be obtained. serology is a core component of the surveillance and management of viral zoonoses. virus 12 neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of 13 serum and high biosafety containment can limit widespread use. here, focusing on rabies lyssavirus, 14 a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent 15 focus inhibition test (pmrffit) that overcomes these limitations. specifically, we adapted an existing 16 micro-neutralization test to use a green fluorescent protein-tagged murine leukemia virus 17 pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen 18 detection and enabling use in low-containment laboratories. we further used statistical analysis to 19 generate rapid, quantitative predictions of the probability and titer of rabies virus neutralizing 20 antibodies from microscopic imaging of neutralization outcomes. using 47 serum samples from 21 domestic dogs with neutralizing antibody titers estimated using the fluorescent antibody virus 22 neutralization test (favn), pmrffit showed moderate sensitivity (78.79%) and high specificity 23 (84.62%). despite small conflicts, titer predictions were correlated across tests repeated on different 24 dates both for dog samples (r = 0.93), and for a second dataset of sera from wild common vampire 25 bats (r = 0.72, n = 41), indicating repeatability. our test uses a starting volume of 3.5 µl of serum, 26 estimates titers from a single dilution of serum rather than requiring multiple dilutions and end point 27 titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. 28 the pmrffit enables high-throughput detection of rabies virus neutralizing antibodies in low-29 biocontainment settings and is suited to studies in wild or captive animals where large serum volumes the last few decades have seen a surge in newly emerging human viruses that originate from wildlife 34 ( of gfp fluorescence. next, the command "analyze particles" was used to count the total number of 169 fluorescent cells per field (i.e. infected cells). this command grouped and counted the white 170 neighboring pixels with a predetermined size area and circularity to be a single cell (size area: 5-50 171 circularity: 0.80-1.0), so counts corresponded to the number of infected cells. cell count outputs were 172 converted into a standardized spreadsheet using a python version 3.7.2 script (python core team, 173 2019) (script available in supplementary materials). at the end of the image processing step, each 174 serum sample was described by 10 data points consisting of the number of the fluorescent cells in 175 each of 5 fields (photographs) in the 1:10 and 1:25 dilutions (figure 1 b) . 176 all statistical analyses were executed in r (r core team, 2018 (scaled to improve model convergence) and 2) the serum dilution level (two factors: 1:10 and 1:25 187 dilution). random slope and intercept terms were also considered for the date the test was run ("test 188 date") to account for observed variation in the relationships between srig titers and infected cell 189 counts across dates (figure 2 ) and for the field number (1 to 5) within each microscope well ("field") 190 to account for variation in cell counts between fields (the middle field, field 5, had more agglomerated 191 cells in particular). to evaluate whether a simpler, single dilution test produced comparable results, 192 the full dataset was then subset to the 1:10 dilution only. the binomial and log-normal models fit to 193 this data subset included only the fixed effect of the virus-infected n2a cell counts, but the random 194 effects were identical to those explained above (i.e. test date and field). models were fit using the 195 'lme4' package (bates et al., 2015) . the 'predict' function was used to generate the predicted 196 probability that a srig concentration or serum sample was seropositive (binomial model) and its 197 corresponding rvna titer (log-normal model). predictions per field were averaged to obtain results 198 per sample (figure 1 c) with a threshold of > 0.1 iu/ml (positives) were used as the benchmark reference. 234 to understand the variability of the pmrffit, replicate srig titer concentration curves were produced 236 on 6 different dates between 30/05/2019 and 27/06/2019 (hereafter "test 1" through "test 6"). as 237 expected, the number of infected cells declined at higher srig titers in all replicates; however, the 238 shape of the antibody decay curve varied across test dates (figure 2) . at the 0.1 iu/ml srig 239 concentration, infected cell counts were more dispersed in the 1:25 dilution than in the 1:10 dilution, 240 as indicated by higher interquartile range (iqr) within each of the 6 test dates. across all the srig 241 concentrations in all test dates (n = 36), 77.78% of the count comparisons were less dispersed in the 242 1:10 dilution suggesting this dilution could be more precise for downstream statistical analysis (si 2). 243 binomial glmms accurately predicted seropositive and seronegative srig concentrations (figure 3) . 245 the best random effects for the binomial model included a random slope and intercept for test date 246 ( table 1) . the models built with the 1:10 dilution data only ("one-dilution model") and from both the 247 1:10 and 1:25 dilution data ("two-dilution model") had equivalent specificity (100%), but the one-248 dilution model was more sensitive (100% versus 58.33%, figure 3 a, b) . furthermore, the two-dilution 249 binomial model failed to correctly predict the seropositive controls on 4 out of the 6 test dates, 250 confirming improved performance of the one-dilution model (figure 3 b) . 251 the log-normal glmms gave repeatable predictions of rvna titers from the datasets generated 253 through our protocol across test dates (figure 3 c) . the best log-normal model included a random 254 intercept and slope for test date. although the most complex model had the lowest aicc, the simpler 255 model (without the random intercept of field) had a δaicc < 2 ( table 1) . observed and predicted srig 256 titers were highly correlated for both the one and two-dilution models (r = 0.95, figure 3 c). when 257 comparing test dates (i.e. one-to-one comparison between correlations of the one-dilution and the 258 two-dilution model from the same test dates), the correlation coefficients were similar, suggesting the 259 simpler one-dilution model is sufficient for titer prediction (figure 3 d) . the most commonly applied serological tests to detect rvna titers challenge a range of serial dilutions 286 of serum with infectious rv. this process is labor-intensive and requires laboratory capacity to grow 287 large quantities of pathogenic rv. here, we provide an alternative serological framework that uses a 288 combination of digital image analysis and statistical analysis to estimate the presence and titer of 289 rvna from a single dilution using only 3.5 µl of serum. 290 the pmrffit differs from other lyssavirus neutralization tests in several key aspects. it uses an 291 mlv(rg) pseudotype rather than pathogenic rv, allowing the pmrffit to be performed in any low-292 containment laboratory with appropriate cell culture and microscopy facilities. the addition of gfp 293 expression is significant, since it removes the need for fitc-conjugated antibody (reducing reagent 294 costs) and the fixation and staining steps used in traditional rffit or favn. one potential drawback 295 of using gfp expression to measure infectivity is the prolonged neutralization period (66 h versus 24 296 h rffit and 48 h favn) required to gain sufficient fluorescence for image processing (aubert, 1992; 297 smith et al., 1973) . longer neutralization requirements (60 h) were also required in a favn 298 modification using a gfp expressing recombinant cvs-11-egfp but did not alter results relative to the 299 test run with cvs-11 (xue et al., 2014) . fortunately, extended incubations are unlikely to alter 300 neutralization outcomes since mlv(rg) pseudotype is replication incompetent, preventing infection 301 of additional cells during the incubation (temperton et al., 2015) . the pmrffit also uses an imaging 302 pipeline that combines systematic photography of microscope fields with automated digital image 303 processing to count infected cells. microscopy in neutralization tests is time consuming and presents 304 challenges for interlaboratory comparisons due to multiple sources of variation, especially those that 305 affect the manual readout (e.g. laboratory user, manual pipetting, uneven cell monolayer) ( the pmrffit standardized approach minimizes these sources of error while potentially reducing 308 microscope operator time. moreover, the imaging process generates traceable and permanent 309 electronic records of the raw data, eliminating the need to manually digitize records of field counts. 310 several investigators have previously incorporated image processing into rv neutralization tests. to count pixels using a microrffit but did not make full use of the quantitative nature of imaging data 315 to obtain rvna titers and used pathogenic rv rather than a viral pseudotype. a final distinction is that 316 instead of scoring microscope field or wells as virus positive or negative, the pmrffit predicts 317 serological status and rvna titer from infected cell counts in a single serum dilution using statistical 318 modelling. the efficacy of this approach highlights the value of historically underutilized quantitative 319 data on cellular infectivity for lyssavirus serology. 320 model selection indicated substantial day-to-day variation in the srig dilution series. this was 321 unsurprising, since virus neutralization tests are biologically dynamic systems that can be influenced 322 by many factors (e.g. variability in the humidity of the incubator, technical manipulation, light 323 condition of the microscope, variability in gfp expression in the cells) (briggs et al., 1998; hammami 324 et al., 1999; kostense et al., 2012) . since our statistical approach handles this variability through the 325 random effect of test date, the pmrffit is best suited for large numbers of serum samples that require 326 testing to be carried out across multiple batches. however, performance is only marginally reduced 327 when running single models for each test date, implying the pmrffit may still be useful when fewer 328 samples are available for testing (see si 5, 6). surprisingly, fitting the glmms to data from a single 1:10 329 dilution of srig predicted both seropositivity and rvna titer more accurately than models fit to both 330 the 1:10 and 1:25 dilutions. the reduced performance of the two-dilution model reflected higher 331 variability in the 1:25 dilution compared to the 1:10 dilution, as evidenced by greater iqr values (si 332 2). ultimately, this variability likely reflects both higher stochasticity in infected cell counts at lower 333 serum concentrations and pipetting error. regardless, the ability to detect low titers (< 0. the authors declare no conflict of interest. 411 perform the cell count of the fluorescent cells to construct a database to fit the statistical models. c) 668 construction of the statistical models with two different types of prediction. 669 srig data to fit models~6 6 hrs 5 fields/well antibodies to rabies virus in terrestrial wild mammals in native 414 rainforest on the north coast of são paulo state, brazil practical significance of rabies antibodies in cats and dogs rabies virus 420 exposure of brazilian free-ranging wildlife from municipalities without clinical cases in 421 humans or in terrestrial wildlife mumin: multi-model inference measurement of rabies-specific antibodies in carnivores by an 425 enzyme-linked immunosorbent assay fitting linear mixed-428 effects models using lme4 temporal and spatial 431 limitations in global surveillance for bat filoviruses and henipaviruses the use of pseudotypes to study viruses, 434 virus sero-epidemiology and vaccination resolving the roles of immunity , 437 pathogenesis and immigration for rabies persistence in vampire bats supporting online 438 material contents : s1 seroprevalence data s2 estimation of seasonal birth rate s3 methods for 439 parameter estimation use of a transient assay for studying the 442 genetic determinants of fv1 restriction estimating time of infection using prior serological and individual information can greatly improve incidence estimation of 446 human and wildlife infections a comparison of two serological methods for detecting the immune response after 450 rabies vaccination in dogs and cats being exported to rabies-free areas rabies surveillance in wild mammals in south of brazil. 454 transboundary and emerging diseases development of a fluorescent antibody virus 456 neutralisation test (favn test) for the quantitation of rabies-neutralising antibody development of a qualitative indirect elisa for the measurement of rabies virus-460 specific antibodies from vaccinated dogs and cats principles of virology. fields virology rabies in new mexico 464 cavern bats serological investigation of rabies virus 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antibodies in 501 healthy, unvaccinated individuals: what do they mean for rabies epidemiology? plos 502 neglected tropical diseases integrating landscape hierarchies 504 in the discovery and modeling of ecological drivers of zoonotically transmitted disease from 505 wildlife convergence of humans, bats, trees, and culture in nipah virus transmission emerging infectious diseases vaccination of 513 tunisian dogs with the lyophilised sag2 oral rabies vaccine incorporated into the dbl2 dog 514 bait virus isolation and quantitation between roost contact is essential for maintenance of european bat lyssavirus type-2 519 in myotis daubentonii bat reservoir: 'the swarming hypothesis printed in great britain studies on the different conditions for rabies virus 522 neutralization by monoclonal antibodies f1-46-12 and f7-1-9 journal of 526 wildlife diseases outbreak of 528 seoul virus among rats and rat owners-united states and canada validation of the rapid fluorescent focus inhibition test for 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virus neutralizing 568 sampling to elucidate the 571 dynamics of infections in reservoir hosts transmission or within-host dynamics driving pulses of zoonotic viruses in reservoir-575 host populations python: a dynamic, open source programming language. python 578 software foundation r: a language and environment for statistical computing usa: national institutes of health serologic 584 evidence of lyssavirus infection in bats lyssaviruses and rabies: current conundrums, 587 concerns, contradictions and controversies. f1000research, 6, 184 fiji: an open-source platform for biological-image analysis rabies transmitted by vampire bats to humans: an emerging zoonotic disease in latin 594 america? detection of rabies virus antibodies in brazilian free-ranging wild 598 a rapid reproducible test for determining rabies 601 neutralizing antibody prevalence of rabies specific antibodies in the mexican 604 free-tailed bat (tadarida brasiliensis mexicana) at lava cave, new mexico anthropogenic roost switching 607 and rabies virus dynamics in house-roosting big brown bats. vector-borne and zoonotic 608 diseases ecological and anthropogenic drivers of rabies exposure in vampire bats: 611 implications for transmission and control retroviral pseudotypes -from scientific tools to 614 clinical utility rapid 617 fluorescent focus inhibition test optimization and validation: improved detection of 618 neutralizing antibodies to rabies virus a conserved 621 mechanism of retrovirus restriction in mammals host immunity 624 to repeated rabies virus infection in big brown bats evaluation of elisa for detection of rabies antibodies in 627 domestic carnivores an evaluation of two commercially available 630 elisas and one in-house reference laboratory elisa for the determination of human anti-rabies 631 virus antibodies human rabies: 2016 updates and call for data who expert consultation on rabies, third report. world health organization technical 636 report series virus neutralising activity of african fruit bat (eidolon helvum) sera against 639 emerging lyssaviruses a 641 robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and 642 lyssaviruses in africa investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-645 species comparison generation of recombinant 648 rabies virus cvs-11 expressing egfp applied to the rapid virus neutralization test a pneumonia 651 outbreak associated with a new coronavirus of probable bat origin mixed effects models and 654 extensions in ecology with r tables 658 table 1 . random key: cord-012934-c6pbr64i authors: hao, weiming; zhao, liping; yu, huiqian; li, huawei title: vestibular prognosis in idiopathic sudden sensorineural hearing loss with vestibular dysfunction treated with oral or intratympanic glucocorticoids: a protocol for randomized controlled trial date: 2020-07-22 journal: trials doi: 10.1186/s13063-020-04579-6 sha: doc_id: 12934 cord_uid: c6pbr64i background: idiopathic sudden sensorineural hearing loss (issnhl) is a rapid-onset sensorineural hearing impairment with unclear etiology and unsatisfying treatment effects. vestibular dysfunction has been considered as a poor indicator in the clinical manifestations and prognosis of issnhl, which occurred in approximately 28–57% cases. glucocorticoids, administered through oral or intratympanic way, are currently regularly and standardly applied for issnhl to improve the hearing outcome. however, the vestibular prognosis of issnhl after routine treatments remains seldom explored. this study aims to compare the effectiveness of oral and intratympanic glucocorticoids in issnhl with vestibular dysfunction in terms of the pattern and trajectory of possible process of vestibular function recovery. methods/design: a randomized, outcome-assessorand analyst-blinded, controlled, clinical trial (rct) will be carried out. seventy-two patients with issnhl complaining of vestibular dysfunction appearing as vertigo or imbalance will be recruited and randomized into either oral or intratympanic glucocorticoid therapy group with a 1:1 allocation ratio. the primary outcomes will be vestibular function outcomes assessed by sensory organization test, caloric test, video head impulse test, cervical vestibular evoked myogenic potential, and ocular vestibular evoked myogenic potential; the secondary outcomes include self-reported vestibular dysfunction symptoms; dizziness-related handicap, visual analogue scale for vertigo and tinnitus; and pure tone audiometry. assessments of primary outcomes will be performed at baseline and at 4 and 8 weeks post-randomization, while assessments of secondary outcomes will be performed at baseline and 1, 2, 4, and 8 weeks post-randomization. discussion: previous intervention studies of issnhl included only hearing outcomes, with little attention paid on the prognosis of vestibular dysfunction. this trial will be the first rct study focusing on the progress and prognosis of vestibular dysfunction in issnhl. the efficacy of two commonly used therapies of glucocorticoids will be compared in both auditory and vestibular function fields, rather than in the hearing outcome alone. trial registration: clinicaltrials.gov nct03974867. registered on 23 july 2019 discussion: previous intervention studies of issnhl included only hearing outcomes, with little attention paid on the prognosis of vestibular dysfunction. this trial will be the first rct study focusing on the progress and prognosis of vestibular dysfunction in issnhl. the efficacy of two commonly used therapies of glucocorticoids will be compared in both auditory and vestibular function fields, rather than in the hearing outcome alone. trial registration: clinicaltrials.gov nct03974867. registered on 23 july 2019 keywords: randomized controlled trial, vestibular function, sudden hearing loss, glucocorticoid background sudden sensorineural hearing loss (ssnhl) is a rapidonset inner ear disease. it is defined as a sensorineural hearing loss of at least 30 db over at least three test frequencies occurring within 72 h [1, 2] . the reported incidence rate of ssnhl is about 5-27/100,000 people per year [1] , which has been widely considered underestimated because of the unregistered cases with out-ofhospital spontaneous recoveries. the etiology in about 71 to 90% of ssnhl cases remains uncertain, which is defined as idiopathic ssnhl (issnhl) [1, 3] . various postulated etiological theories have been proposed including microvascular impairment, viral infections, inner ear electrolytic disorders, trauma, autoimmune diseases, and central nervous system (cns) diseases [3] [4] [5] [6] [7] . based on the close anatomic relationship between cochlea and vestibule, approximately 28-57% of issnhl patients have also reported of co-occurring symptoms of vertigo [8] . there are two administration patterns of glucocorticoids for issnhl: systemic (oral or intravenous) and intratympanic therapies. compared with the traditional oral administration, intratympanic therapy is thought to be superior for its (1) bypassing the blood-labyrinth barrier and achieving higher drug concentrates in the inner ear and (2) avoiding most of the systemic side-effects of glucocorticoids. a well-designed randomized trial was conducted in 2011 by rauch and his colleagues, which supported the non-inferiority in hearing outcomes of intratympanic therapy compared with oral prednisone in issnhl. however, the study was failed to reject the inferiority based on the 10-db non-inferiority standards in the subgroup analysis of issnhl with dizziness [9] . considering that few investigations have been carried out on progress and prognosis of issnhl-related vestibular dysfunctions after basic treatment, we designed this randomized, assessor-and analyst-blinded, controlled trial with two interventional arms, one is the oral glucocorticoid therapy group and the other one is the intratympanic glucocorticoid therapy group. our research hypothesis is that intratympanic methylprednisolone is superior to oral prednisone on vestibular function recovery of issnhl patients with vertigo. this protocol is reported in accordance with the standard protocol items: recommendations for interventional trials guidelines (spirit) (additional file 1) [10] . this study is designed as an 8-week, single-center, randomized, assessor-and analyst-blinded, controlled trial with two parallel interventional groups in a 1:1 allocation. patients will be recruited from outpatient clinics of the eye and ent hospital of fudan university in shanghai, qualified with well-trained doctors, staff, and required facilities for this clinical trial. patients who meet all of the following inclusion criteria will be considered eligible: patients who visit doctors from the outpatients and suspected of issnhl with vestibular dysfunction will be screened for eligibility. eligible patients who consent to participate will receive either oral prednisone or intratympanic methylprednisolone randomly in a 1:1 allocation. the randomization sequence will be generated through @random.org, an internet website (https:// www.random.org) producing true random sequence according to atmosphere noise. the concealed randomization sequence file will be constructed and kept in sequentially numbered, sealed, opaque envelopes by a staff member (zhao l.) in the eye and ent hospital of fudan university outside the study team. all the potential participants will be evaluated in strict chronological order of visiting time. when a patient is officially enrolled and numbered, the staff member will be contacted by telephone and open the corresponding envelope to find the randomized treatment for this patient. assessors in examination rooms and statistician analysts are not allowed to receive any information of the group allocation. the information of participants will be referred using research code among investigators, and their personal information (like name and contacts) will be kept confidential before, during, and after the trial. following randomization, baseline information of the participants in demographic and clinical characteristics will be collected, such as age, gender, nationality, occupation, date of onset, predisposition, initial symptoms, time of diagnosis, any treatments before enrolments, body mass index (bmi), and comorbidities. the objective vestibular function evaluated by sot, videonystagmography (vng), vhit, cvemp, and ovemp and the hearing outcome assessed by pta will be performed at baseline and at 4 and 8 weeks after randomization, while the subjective vestibular dysfunction feelings reflexed by dizziness handicap inventory (dhi) and visual analogue scale for vertigo (vas-v) and the tinnitus condition assessed by vas in tinnitus (vas-t, visual analogue scale for tinnitus) will be performed at baseline and at 1, 2, 4, and 8 weeks after randomization. caloric test is routinely included in vng. acoustic impedance and magnetic resonance imaging of the internal auditory canal (mri-iac) will be performed only once at the baseline examination to exclude the potential misdiagnosed ssnhl. once a participant is randomized, the treatment procedure starts immediately ( table 1 ). the participants in group 1 will receive oral prednisone 1 mg/kg/day (maximum daily dosage is no more than 60 mg) for 7 days, followed by a 7-day taper ( table 1 ). the patients are advised to take the medicine in 30 min before every breakfast, and not to divide the doses. the participants in group 2 will receive 7 intratympanic 40 mg/ml methylprednisolone injections in 14 days, one injection every other day. the otolaryngologists in charge of the injection work are asked to inject at the posterior superior quadrant and fulfill the tympanic cavity using operating microscopes, after lidocaine spray anesthesia. patients will be asked to keep supine position with the affected ear slightly up to 30°and avoid swallow during injection and in 30 min after the injection. the drug for injection is methylprednisolone sodium succinate for injection by pfizer manufacturing belgium nv. of all the participants, the length of treatment would be extended for another week if the change in hearing threshold (average db in pta) is less than 10 db, or average of pta is worse than 55 db in the patient's affected ear, under the participant's agreement. the salvage treatment regimen is 3 injections of 40 mg/ml methylprednisolone every other day. records and effects of the salvage treatments will be reported in the study results and evaluated according to both intention-totreat (itt) and per-protocol analyses. for those with severe vertigo attacks, drugs for symptom control like mannitol or diazepam can be used temporarily. in table 2 , criteria are listed in detail to distinguish the central vestibular system (cvs) compensation and peripheral vestibular system (pvs) restoration according to the following evidence: 1) sot is a measurement for dynamic and static posturography, which may figure out the different roles of the somatosensory, visual, and vestibular systems. the vestibular scores in sot will help us to distinguish peripheral function self-restoration from cvs compensation. 2) the caloric test has been used to evaluate the lateral semicircular canal function with a good sensitivity [11] . in the caloric test, unilateral weakness (uw), directional preponderance (dp), and spontaneous nystagmus (spn) are three parameters to judge the different phases of central vestibular compensation. uw presents at all three phases of acute injury, static compensation, and dynamic compensation phase and dp presents at the acute injury phase and static compensation phase, while spn only presents at the acute injury phase [12] . meanwhile, the caloric test results of complete function restoration in pvs should be the same as those in normal individuals: absence of uw, dp, or spn. 3) vhit can be employed for evaluating horizontal and vertical semicircular canals and has been taken as a specific indicator of peripheral vestibular function [13, 14] . the decreased gains and corrective saccades are signs of the corresponding semicircular canal dysfunction. 4) cvemp is a widely used measurement for assessing the saccular and inferior vestibular pathway functions, while ovemp for evaluating the utricular and superior vestibular pathway functions [15] . based on the integrity of neural pathways, we may reasonably assume that if the dysfunction of otolith organ and its afferent pathway has not recovered in our participants, the compensation effects of cvs will not bring a normal vemp result [14, 16] . here come two possible patterns in vestibular dysfunction recovery of issnhl. the first pattern (pattern a) is that the patients undergo well central vestibular compensations with no recovery of pvs function. in this pattern, patients may show a quite normal ability of balance evaluated by subjective complaints, dhi or vas-v; however, since the peripheral vestibular organs remain dysfunctional, vestibular test battery (i.e., sot, caloric tests, vhit, and vemps) will come out with abnormal results. the second possible pattern (pattern b) is that the pvs injuries are completely or partially restored in the patients. in this condition, not only the subjective complaints will disappear, but also the objective vestibular test results will get back to normal, or at least less abnormal. the methodology details of the following tests have been reported before [17] . if the patient's weight is less than 50 kg, the administration will be stopped after the day with < 10 mg prednisone administered, for example, a patient weighs 45 kg will stop receiving glucocorticoids at the 13th day b one day early or late of injection is allowed for practicality . six sensory test modes are performed with changing supports and visual conditions. the results are analyzed comprehensively to give a score for each sensory system (somatosensory, visual, and vestibular systems) and a composite score. results are considered abnormal when the score is lower than the age-specific normative data. caloric test we deliver the caloric test using the air caloric irrigator system of ics aircal (gn otometrics, taastrup, denmark) and record eye movements using videonystagmography (vng) of synapsys vng ulmer (synapsys, inc. marseille, france). patients are placed in a supine position in a darkroom, with the head flexed at 30°. the temperature of the warm and cool air is 50°c and 22°c, respectively. the unilateral weakness (uw) and directional preponderance (dp) are used to quantify the difference between the caloric responses of the two ears. the abnormal caloric result is defined as an absolute value of uw% greater than 22% and/or an absolute value of dp greater than 27%. vhit ics impulse system (gn otometrics, taastrup, denmark) will be used for vhit in this study. the examiner performs head impulses (150 to 200°/s peak head velocity) randomly in unpredictable timing and direction in the plane of each canal (the horizontal, anterior, and posterior semicircular canal), and at least 15 impulses for each side are acquired. the software analysis algorithm calculates the vestibular-ocular gain. normal gain is defined as > 0.8 for the lateral canals and > 0.7 for the vertical canals. the pathological saccade is defined as refixation saccades categorized as either covert saccades (occurring during a head movement) or overt saccades (occurring after a head movement) [18] . the result of a semicircular canal will be considered abnormal when there are pathological saccades and the gain is out of normal range. vemp the recording device of both cvemp and ovemp is the bio-logic navigator pro (natus medical inc., san carlos, usa). cvemp patients are asked to lie in supine position on a bed, with head raised to 30 to 45°away from the bed during recording to ensure good muscle tone. electrodes are placed as operation manual. air-conducted sound with 500-hz short tone bursts (2 ms rise/fall time and 2 ms plateau time) are presented through insert headphones as stimuli. the starting stimulus intensity is 95 db nhl and decreases by 5 db nhl each time until the meaningful wave is undetectable. the lowest intensity with a characteristic waveform is defined as threshold. the result will be considered abnormal if one of the following conditions is met: (1) the amplitude asymmetry ratio (ar) is more than 37%; (2) the cvemp meaningful waveform (where the waveforms with positive-negative-positive peak, p1-n1-p2, could be recognized and well repeatable) is absent at 95-db nhl stimulus or the threshold is out of range compared with age-specific normative data; (3) delayed response: the cvemp threshold shift is out of the range: p1 range 15.66 ± 7.22; n1 range 23.42 ± 5.18 (the mean of normal range ± 2 × sd) [19] [20] [21] [22] . ovemp the recording device, software, and stimuli are the same as those in cvemp. the patients remain lying supine and are asked to look upward (approximately 30°a bove the horizontal plane) during recording. electrodes are placed as reported in previous studies [17] . the recording procedure is the same as that in cvemp, and the lowest intensity with a characteristic waveform is defined as threshold. we define the abnormal result of ovemp as (1) absence of meaningful waveform (where the waveforms with negative-positive peak, n1-p1, could be recognized and well repeatable); (2) the threshold out of range compared with age-specific normative data; and (3) amplitude asymmetry ratio (ar) ≥ 40% [19, 20] . all examinations will be performed by trained physicians skilled at neuro-otological tests. is a self-assessment questionnaire with 25 items, and the reliability of the chinese version has been verified [23] [24] [25] [26] . the 25 items can be divided into 3 subscales: physical, emotional, and functional aspects, and the total scores range from 0 to 100. the higher the score, the more severe the dizziness is in the patients. visual analogue scale is a universal psychometric scale evaluating subjective attitudes [27, 28] . when applied in vertigo or tinnitus (vas-v or vas-t), respondents specify their level of vertigoes or tinnitus by indicating a position along a continuous line between two endpoints without marked scale. a score from 0 to 10 will be made based on the length of the line. the higher the score, the more severe the symptom is. to evaluate the recovery of vestibular function, we set the recovery rates of the whole battery of vestibular function tests (sot/caloric test/vhit/vemps) as the primary outcome, which is the proportion of patients whose abnormal results of vestibular function tests at baseline recover to normal at 4-/8-week follow-up: in this study, we define a 10-db pta criterion as clinically significant difference based on a previous rct [9] . 3) safety: rates of study-related adverse events (aes), such as short-term systemic use of glucocorticoids related saes and aes, and intratympanic injectionrelated aes. the study included 5 follow-up visits in total: a baseline visit to sign the consent and to record the baseline information, 2 visits during treatment interval to record subjective vestibular questionnaires (dhi, vas-v, and vas-t) and hearing outcomes (pta) and to monitor treatment safety outcomes, and 2 follow-up visits at 4 weeks and 8 weeks to assess hearing and vestibular functions and monitor safety outcomes. to optimize examination resources and reduce burden of examiners, among all five vestibular function tests (sot, caloric test, vhit, cvemp, and ovemp), only those with previous abnormal results will be repeated at follow-up visits. the study flow diagram is shown in fig. 1 , and the spirit figure of enrolment, interventions, and assessments is presented in table 3 . any untoward medical occurrences with unfavorable symptoms in the participants are defined as adverse events (aes). ae is not necessarily to have a causal relationship with the treatment. if the aes are lifethreatening, result in death, persistent, and significant disability or incapacity, or make the participants' hospitalization, we define them as serious adverse events (saes). the investigators will routinely ask the patients if there have been any unexpected symptoms. studyrelated and non-study-related aes will be recorded in the patient's clinical history by doctors in clinics and then reported to a trial investigator (yu h.) . participants experiencing aes will be followed up until the end of the events or the end of the trial. any saes during this trial will be reported to the trial steering committee table 3 the spirit figure of enrolment, interventions, and assessments vng videonystagmography which includes the caloric test, mri-iac magnetic resonance of the internal auditory canal *a follow-up test is only repeated when the previous test results in abnormal findings (tsc) and adverse drug reaction administration (adra) of the eye & ent hospital of fudan university within 48 h at learning of the event. we list some possible study-related aes here: 1) short-term systemic use of glucocorticoids related saes: fracture, sepsis, and venous thromboembolism [29] ; 2) short-term systemic use of glucocorticoids related aes: blood glucose problem, appetite change, sleep change, weight change; and 3) intratympanic injection-related aes: ear pain, ear infection, tympanic membrane perforation, and worse vertigo after injection. the participants will be informed of the right to withdraw from the study at any time after consent. the enrolled participants who are found ineligible later, lost to follow-up, or withdraw consent for any reason will be regarded as withdrawal. as for those who decided not to receive the protocol intervention, we will ask them if they would like to continue with the follow-up visits and if they agreed, their results will be regarded as variation other than withdrawals in itt analysis. we provide an online wechat and telephone contacts to all the participants for timely communication and health education, which will promote the participants' retention and adherence to the intervention protocols. furthermore, we optimized the follow-up process by setting a specialty clinic for ssnhl, minimizing the waiting time, and waiving extra examination and treatment fees. we decide not to set blindness of the interventions to patients and doctors, because that setting blindness to patients and doctors will need placebo injections to some of the participants, which may bring unnecessary pain and tympanic membrane perforation risk to the patients. the specialists in test rooms and statistical analysts will be kept blinded during the trial until the statistical analyses are done. a data monitoring committee (dmc) has been established to store, monitor, and check the authenticity, security, and integrity of the database. all members in the dmc are independent of the study sponsors and declared no competing interests. the dmc will periodically review the accumulated data and communicate the problems of its deliberations to the study team if necessary. the frequency of the interim analyses will be judged by the chair of the dmc, based on the consultation with the tmc. we anticipate that there might be 2-3 interim analyses before the final analysis. in our previous studies, we reported the poor indicator of the vestibular system lesion patterns in ssnhl and put forward that the incidence of vestibular dysfunction [30, 31] . however, we only found the existence of vestibular recovery in clinical case reports [16] . due to lack of previous reported data on changes of vestibular function test results, this study sample size was calculated based on data from preliminary clinical practice in our hospital. among patients who were diagnosed as issn hl and treated with oral prednisone, the recovery proportion of the vestibular function tests (measured by sot, caloric test, or vemps) was 0.25, while the recovery proportion among those treated with intratympanic glucocorticoids was approximate 0.64. using the program of g*power 3.1 for fisher's exact test, we calculated a sample size of 30 per arm with 80% power at a two-tailed 5% level of significance [32] . to allow for 20% dropout, 36 participants per arm will be needed. for the outcomes, categoric variables will be expressed as rates, while numerical variables as the mean ± sd. baseline data will be compared between two groups with a chi-squared test for dichotomous samples and student's t test or mann-whitney u test for two independent samples where appropriate. for comparisons of recovery in 4 and 8 weeks from baseline, the recovery rates of vestibular function tests will be calculated by chi-squared tests, and logistic regression adjusts for potential confounders like age, initial pta, the number of involved vestibular organs, mri-iac results, and other characteristics, while the numerical variables like uw of the caloric test, pta, and scores in dhi, vas-v, and vas-t will be calculated by mixed-model with repeated measures analyses of variance (anova), with group and time as fixed effects and subject as a random effect, controlling for the potential confounders. between two intervention groups, the chi-squared test will be used to analyze the difference for dichotomous outcomes (e.g., recovery rates) and student's t test for two independent samples or mann-whitney u test where appropriate. a value of p < 0.05 of two-sided is considered statistically significant. we will calculate relative risk (rr) with corresponding 95% confidence intervals to compare dichotomous variables, and mean difference (md) for continuous variables. an itt analysis and a per-protocol analysis will be both performed at each outcome. for the missing data in itt analyses, if there is any, multiple imputation methods will be used. up-to-date versions of spss (spss, chicago, il, usa) will be used to conduct analyses. a statistician from the medical college of fudan university will perform the analyses. not until the statistician complete the analysis, will he be unblinded to the group allocations and the study hypotheses. all data sent to the analyst will be anonymized, and the study groups will be coded as groups a and b. this protocol and the template informed consent forms have been reviewed and approved by the institutional review board (irb) and the ethical committee of eye & ent hospital of fudan university (reference number: 2017047). the tmc will make safety and progress reports to the irb at least annually and within 3 months of study completion. patients and the public were not involved in the design of this study. however, the study was initially discussed with issnhl patients' representative on how best to involve patients throughout the proposed project. finally, the study results will be informed to the public via peer-reviewed journals or academic conferences. vestibular dysfunction, commonly complained by patients as vertigo, dizziness, or lateropulsion, has been considered as a risk factor of profound hearing loss and poor prognosis [30] [31] [32] [33] [34] . recently, researchers made their efforts to specify the lesion patterns of the vestibular system in issnhl and proposed that the utricle and superior vestibular pathway is the most vulnerable vestibular site in issnhl, followed by the lateral semicircular canal and superior vestibular pathway [30] . severe vertigo and static imbalance markedly improve over a couple of days or weeks in most of patients, while some may suffer from long-term residual dizziness. recovery of peripheral vestibular function and central vestibular compensation might be involved in this process. vestibular function tests, such as sot, caloric test, vhit, and vemp, are effective and objective methods to distinguish restoration of peripheral vestibular function and compensation of the central vestibular. it has been reported that in a few cases of ssnhl, function of otolith organs reflexed by vemps was absent at onset and recovered or improved after weeks to months [16] . these cases suggested the role of peripheral vestibular function restorations. when peripheral vestibular injury is failed to recover by itself, the central compensation will take place. however, due to few previous researches in this field, the roles and proportions of central compensation versus peripheral recovery remain uncertain. we assume that appropriate treatments for issnhl may have favorable effects promoting the vestibular recovery process, as those in hearing outcomes. glucocorticoid therapy is currently a regular and standard treatment for issnhl according to guidelines [1, 35] . the plausible mechanisms include anti-inflammatory, immune-suppressive, and inner-ear-homeostasis-related gene regulative effects [36] . regrettably, with numerous studies of different evidence levels delivered in the past six decades, the effectiveness of glucocorticoids in treating issnhl remains uncertain. most of the rcts and meta-analyses on this topic claimed of disappointing results with little measurable improvements in glucocorticoids over placebo arms [37] [38] [39] . it is worth noting that many confounding factors like various regimens and administration of drugs, different time points of starting treatments, and different probable causes existed in these studies. few studies have focused on the effects of glucocorticoids in vestibular recovery of issnhl. taking the previous researches in acute unilateral vestibulopathy for reference, improvements have been found evaluated by vestibular function tests, the symptom loads, and dhi scores [40, 41] . hereby, we may speculate that glucocorticoids could possibly accelerate vestibular function recovery via restoration of the peripheral vestibular system. to assess the vestibular functions, we intend to perform a battery of vestibular function tests including (1) the sot for assessing static and dynamic posture control ability of somatosensory, visual, and vestibular systems distinguishingly and comprehensively; (2) the caloric test for evaluating horizontal semicircular canal functions and superior vestibular integrity; (3) vhit for evaluating functions of horizontal and vertical semicircular canals; and (4) cvemp for investigating the saccular function and the inferior vestibular pathway and ovemp for assessing utricular function and the superior vestibular pathway [11, 13, 15, 42, 43] . moreover, we plan to perform dhi and vas-v, two qualified and widely used subjective measurements, to assess the severity of symptoms and negative influence on patients' daily lives [25, 44] . in conclusion, our study will be the first to assess and follow the vestibular function conditions of issnhl up using a whole battery of vestibular function tests. based on our clinical practice experience, we hypothesize that the effects of intratympanic glucocorticoids will be superior to those of oral therapy in terms of the outcome measurements mentioned above. moreover, we expect that participants in the intratympanic group will experience more significantly reduced dhi scores, lessened vas-v scores, and better enhanced recovery of pta. the planned date of the first enrolment is 1 sep 2019. the estimated time required for recruitment is 12 months. the total duration of this study is expected to be 18 months, including statistical analysis and article writing. this protocol version number is ver.3. clinical practice guideline: sudden hearing loss (update) national institute on deafness and other communication disorders. nidcd fact sheet: sudden deafness accessed 15 systematic review of the evidence for the etiology of adult sudden sensorineural hearing loss pathology of vascular sensorineural hearing impairment autoimmune sensorineural hearing loss the pathology of sudden deafness sudden sensorineural hearing loss: is there a connection with inner ear electrolytic disorders? a literature review idiopathic sudden sensorineural hearing loss oral vs intratympanic corticosteroid therapy for idiopathic sudden sensorineural hearing loss: a randomized trial spirit 2013 statement: defining standard protocol items for clinical trials the caloric irrigation test vestibular function examination technique. xi'an: air force medical university press the video head impulse test plasticity during vestibular compensation: the role of saccades vestibular-evoked myogenic potentials recovery of ocular and cervical vestibular evoked myogenic potentials after treatment of inner ear diseases contrasting results of tests of peripheral vestibular function in patients with bilateral large vestibular aqueduct syndrome vestibular migraine screening in a migraine-diagnosed patient population, and assessment of vestibulocochlear function assessment of balance and vestibular functions in patients with idiopathic sudden sensorineural hearing loss the potential dysfunction of otolith organs in patients after mumps infection normal values of cervical vestibular evoked myogenic potential and its influence factors prognostic factors in sudden sensorineural hearing loss the development of the dizziness handicap inventory discussion of the dizziness handicap inventory uni-and multivariate models for investigating potential prognostic factors in idiopathic sudden sensorineural hearing loss analysis of reliability and validity of the chinese version of dizziness handicap inventory (dhi) visual vertigo analogue scale: an assessment questionnaire for visual vertigo measuring pain. visual analog scale versus numeric pain scale: what is the difference? short term use of oral corticosteroids and related harms among adults in the united states: population based cohort study association of vertigo with hearing outcomes in patients with sudden sensorineural hearing loss: a systematic review and meta-analysis vestibular dysfunctions in sudden sensorineural hearing loss: a systematic review and meta-analysis gpower: a general power analysis program vertigo as a prognostic sign in sudden sensorineural hearing loss the relationship between hearing loss and vestibular dysfunction in patients with sudden sensorineural hearing loss society of otorhinolaryngology head and neck surgery, chinese medical association. guideline of diagnosis and treatment of sudden deafness (2015) corticosteroid therapy for hearing and balance disorders treatment of sudden sensorineural hearing loss: ii. a meta-analysis steroids for treatment of sudden sensorineural hearing loss: a meta-analysis of randomized controlled trials corticosteroid treatment of idiopathic sudden sensorineural hearing loss: randomized triple-blind placebocontrolled trial glucocorticoids improve acute dizziness symptoms following acute unilateral vestibulopathy methylprednisolone, valacyclovir, or the combination for vestibular neuritis characteristics and clinical applications of ocular vestibular evoked myogenic potentials learning effects of repetitive administrations of the sensory organization test in healthy young adults efficacy and safety of acupuncture for dizziness and vertigo in emergency department: a pilot cohort study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the patients for participating in the trial and our colleagues for supporting the study. supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-020-04579-6.additional file 1. spirit checklist.additional file 2. case report forms template.additional file 3. adverse event forms template. this study was supported by the science and technology commission of shanghai municipality (grant number 184119551900), contact at +86-021-23111111. this funding source played no part in the study design and will not have any role in its collection, management, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication. during the study, the datasets used in the current study are available from the corresponding author on reasonable request. after the study, the results of this trial will be published in peer-reviewed journals and presented at national and/or international conferences. the study has been approved by the ethical committee of eye & ent hospital of fudan university (reference number 2017047-1), and informed consent will be obtained from all participants of this trial before participating; the model consent form is attached in crf in additional file 2. not applicable. key: cord-281241-k1adcls8 authors: döhla, m.; boesecke, c.; schulte, b.; diegmann, c.; sib, e.; richter, e.; eschbach-bludau, m.; aldabbagh, s.; marx, b.; eis-hübinger, a.-m.; schmithausen, r.m.; streeck, h. title: rapid point-of-care testing for sars-cov-2 in a community screening setting shows low sensitivity date: 2020-04-18 journal: public health doi: 10.1016/j.puhe.2020.04.009 sha: doc_id: 281241 cord_uid: k1adcls8 objective: with the current sars-cov2 outbreak, countless tests need to be performed on potential symptomatic individuals, contacts and travellers. the gold standard is a quantitative polymerase chain reaction (qpcr)–based system taking several hours to confirm positivity. for effective public health containment measures, this time span is too long. we therefore evaluated a rapid test in a high-prevalence community setting. study design: thirty-nine randomly selected individuals at a covid-19 screening centre were simultaneously tested via qpcr and a rapid test. ten previously diagnosed individuals with known sars-cov-2 infection were also analysed. methods: the evaluated rapid test is an igg/igm–based test for sars-cov-2 with a time to result of 20 min. two drops of blood are needed for the test performance. results: of 49 individuals, 22 tested positive by repeated qpcr. in contrast, the rapid test detected only eight of those positive correctly (sensitivity: 36.4%). of the 27 qpcr-negative individuals, 24 were detected correctly (specificity: 88.9%). conclusion: given the low sensitivity, we recommend not to rely on an antibody-based rapid test for public health measures such as community screenings. objective: with the current sars-cov2 outbreak, countless tests need to be performed on potential symptomatic individuals, contacts and travellers. the gold standard is a quantitative polymerase chain reaction (qpcr)ebased system taking several hours to confirm positivity. for effective public health containment measures, this time span is too long. we therefore evaluated a rapid test in a highprevalence community setting. study design: thirty-nine randomly selected individuals at a covid-19 screening centre were simultaneously tested via qpcr and a rapid test. ten previously diagnosed individuals with known sars-cov-2 infection were also analysed. methods: the evaluated rapid test is an igg/igmebased test for sars-cov-2 with a time to result of 20 min. two drops of blood are needed for the test performance. results: of 49 individuals, 22 tested positive by repeated qpcr. in contrast, the rapid test detected only eight of those positive correctly (sensitivity: 36.4%). of the 27 qpcr-negative individuals, 24 were detected correctly (specificity: 88.9%). conclusion: given the low sensitivity, we recommend not to rely on an antibody-based rapid test for public health measures such as community screenings. © 2020 the royal society for public health. published by elsevier ltd. all rights reserved. covid-19 is rapidly spreading worldwide, and the number of cases in europe is rising with increasing pace in several affected regions. 1 while there is an urgent need to contain the pandemic to protect the elderly and vulnerable population, there are several obstacles to control the spread of new infections. the vast majority of sars-cov-2einfected individuals appear to have only mild to moderate symptoms similar to the flu or other flu-like infections, 2e6 lacking defining symptoms. thus, while we start losing the ability to trace all sars-cov-2einfected contacts, identification of potentially infected individuals becomes increasingly hard ( table 1 , fig. 1 ). to protect the vulnerable population, it is necessary to assess the infection status of potential contacts to patients with covid-19 rapidly but also to approve employees to work with at-risk individuals in the hospital or nursing homes. the current gold standard for sars-cov-2 detection is a sars-cov-2especific, quantitative real-time polymerase chain reaction (rt-qpcr) testing from a nasal or pharyngeal swab, sputum or broncoalveolar lavage. 7, 8 following standard protocols, rna needs to be extracted and the presence of viral rna confirmed by rt-qpcr. this requires several potentially erroneous steps and several hours for sampling and evaluation. even high-throughput laboratories require a minimum of 3e4 h from sampling to evaluation, and final information of the infection status may take up to 24 h. this bears the risk of a potential further spread of sars-cov-2 in the meantime and hinders widespread testing of all potential contacts. there is currently 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 no rapid method to detect potentially sars-cov-2epositive individuals that would allow an assessment of their infection status in a reliable manner. there is an urgent need for immediate targeted detection of infected individuals to slow the pandemic. we therefore evaluated a rapid antibody igg/igmebased testing system in the community setting for its ability, specificity and sensitivity to reliably identify infected individuals. the german red cross had established a covid-19 screening centre in a high-prevalence area with more than 300 confirmed cases among 12,000 inhabitants. the cluster outbreak occurred after a carnival celebration and secondary transmissions in the families and rural community. the medical personnel at the screening site perform 150e200 throat swabs for sars-cov-2 diagnostics every day on symptomatic individuals. thirty-nine randomly selected individuals at the centre were tested simultaneously using the sars-cov-2 rapid test and the gold standard rt-qpcr method (altona diagnostics). in addition, collected and stored serum samples of 10 previously diagnosed individuals with known sars-cov-2 infection were analysed. all individuals accepted testing via written informed consent. the rapid test used for evaluation is a qualitative igg/igm detection system to test for a current or past infection of sars-cov-2. the chemical coupling pad contains gold-labelled sars-cov-2 weak and strong is superior; the manufacturer's recommendation is also to interpret weak results as positive. lrþ: positive likelihood ratio; lr-: negative likelihood ratio; roc: receiver operating characteristics; ci: confidence interval. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 antigens and mouse igg controls. there are two detection bands (t1 ¼ igm and t2 ¼ igg) on the test strip, which are coated with mouse anti-human igm and igg antibodies, respectively. the control band (c) is coated with a goat anti-mouse igg antibody. after discarding the first drop of blood from a fingertip prick, two drops of blood are applied onto the rapid test chip. in addition, two drops of a provided solution are added. the test indicates positivity for igg after 15 min and for igm after 20 min. when a test sample is added to the sample-loading area, the antigen forms an immune conjugate with the gold-labelled antibodies and then move to the detection zone by a capillary action. the negative conformity rate has been described to be 100% for 68 negative controls. the positive conformity rate has been described to be 70% at early stages of infection (day 4e10) and 100% at late stages of infection (day 11e24). the study population was well balanced in terms of age (median: 46 years, interquartile range [iqr]: 28e72) and gender (24/49 female [49.0%]). the majority described symptoms including dry coughing (70.8%), fatigue (64.6%) and a runny nose (45.8%). only five individuals had no symptoms. twenty-two individuals were tested positive by repeated rt-qpcr, while 27 were tested negative. positive individuals reported five symptoms in median (iqr: 3e7), while negative individuals reported only 4 (iqr: 2e5) symptoms. we were able to identify the probable date of exposure of 22 individuals (44.9%). median time between exposure and test was 18.5 days (iqr: 15e24). all used rapid tests were valid; 38 of 49 (77.6%) tests were negative. we saw a weak response in 7 cases and a strong response in 4 cases. there was no case of a singular igm response indicating acute or recent sars-cov-2 infection. the manufacturer recommends to classify weak responses as positive which was supported via receiver operating characteristics (roc) curve analysis. therefore, we defined 11 tests as positive in our study. considering the pcr results, we found eight tests to be true-positive and 3 to be false-positive, whereas 24 tests were true-negative and 14 tests were false-negative (table) there was no statistically significant correlation between rapid test results and time from potential exposure (exact test, p ¼ 0.636), presence of symptoms (exact test, p ¼ 0.689), age (exact test, p ¼ 0.145) or gender (exact test, p ¼ 0.531). the sars-cov-2 outbreak in 2019/2020 followed an unprecedented international response to contain the pandemic. high transmission rates and the vast majority presenting with only mild to moderate unspecific symptoms complicate the ability to contain the virus. 9 moreover, laboratory methods to detect sars-cov-2 infection rely on rt-qpcr testing that require longer time for sample handling, preparation and diagnosis. while rapid point-ofcare testing is critically needed, the current evaluation of an antibody-based system demonstrates only low sensitivity and is therefore not recommendable to detect potential infections as a stand-alone test. indeed, studies demonstrated that seroconversion occurred sequentially for igm and then igg with a median time of 11 and 14 days, respectively. the presence of antibodies was <40% among patients in the first 7 days of illness and then rapidly increased to 100% at day 15 after onset of symptoms, which appear to be too late from a public health perspective. 10 in this real-life study setting at a community sars-cov-2 testing site after a cluster outbreak, we investigated the superiority of an antibody-based rapid test in comparison with the current sars-cov-2 rt-qpcr gold standard. we tested screened persons of an official screening centre that we had selected by chance. this is a scenario that already occurs and will more often occur in all european union (eu) member states within the next months. the rapid test was substantially inferior to the rt-qpcr testing and should therefore neither be used for individual risk assessment nor for decisions on public health measures. as there is an urgent need for a sufficient rapid testing system for sars-cov-2, an antigen-based system may therefore be more appropriate. we recommend accelerating the development and evaluation of effective point-of-care testing systems. the study has been approved by the local institutional review board in march 2020 (085/20). none declared . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 european centre for disease prevention and control (ecdc) clinical characteristics of coronavirus disease 2019 in china scientists are sprinting to outpace the novel coronavirus imaging and clinical features of patients with 2019 novel coronavirus sarscov-2 evidence of sars-cov-2 infection in returning travelers from wuhan, china improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19-rdrp/hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens diagnosis of sars-cov-2 infection based on ct scan vs. rt-pcr: reflecting on experience from mers-cov epidemiology and transmission of covid-19 in shenzhen china: analysis of 391 cases and 1,286 of their close contacts antibody responses to sars-cov-2 in patients of novel coronavirus disease none q3 declared. key: cord-284945-837qlk8y authors: rahmandad, h.; lim, t. y.; sterman, j. title: estimating the global spread of covid-19 date: 2020-06-26 journal: nan doi: 10.1101/2020.06.24.20139451 sha: doc_id: 284945 cord_uid: 837qlk8y limited and inconsistent testing and differences in age distribution, health care resources, social distancing, and policies have caused large variations in the extent and dynamics of the covid-19 pandemic across nations, complicating the estimation of prevalence, the infection fatality rate (ifr), and other factors important to care providers and policymakers. using data for all 84 countries with reliable testing data (spanning 4.75 billion people) we develop a dynamic epidemiological model integrating data on cases, deaths, excess mortality and other factors to estimate how asymptomatic transmission, disease acuity, hospitalization, and behavioral and policy responses to risk condition prevalence and ifr across nations and over time. for these nations we estimate ifr averages 0.68% (0.64%-0.7%). cases and deaths through june 18, 2020 are estimated to be 11.8 and 1.48 times official reports, respectively, at 88.5 (85-95.3) million and 600 (586-622) thousand. prevalence and ifr vary substantially, e.g., ecuador (18%; 0.61%), chile (15.5%; 0.57%), mexico (8.8%; 0.69%), iran (7.9%; 0.44%), usa (5.3%; 0.99%), uk (5.2%; 1.59%), iceland (1.65%, 0.56%), new zealand (0.1%, 0.64%), but all nations remain well below the level needed for herd immunity. by alerting the public earlier and reducing contacts, extensive testing when the pandemic was declared could have averted 35.3 (32.7-42.7) million cases and 197 (171-232) thousand deaths. however, future outcomes are less dependent on testing and more contingent on the willingness of communities and governments to reduce transmission. absent breakthroughs in treatment or vaccination and with mildly improved responses we project 249 (186-586) million cases and 1.75 (1.40-3.67) million deaths in the 84 countries by spring 2021. explanatory mechanisms: a) differences in population density, lifestyle, and interaction patterns create variations in reproduction number. b) risk perception, behavioral change and policy responses alter transmission rates endogenously. c) testing is prioritized based on symptoms and risk factors, so detection rates depend on both testing rates and current prevalence. d) limited hospital capacity is allocated based on symptoms, influencing fatality rates. e) weather may have a role in transmission (7) while age, socio-economic status, and pre-existing conditions impact severity (8, 9) , with the poor and minorities suffering disproportionately (10) . prior research has addressed different parts of this puzzle, including estimating the infection fatality rate (ifr) in well-controlled settings (11) or using statistical extrapolation (12) , assessing the asymptomatic fraction in smaller populations or through random testing (13, 14) , and estimating prevalence using random antibody testing in highly affected cities and regions (15) . yet we lack a global view of the pandemic that is both consistent with these more focused findings and simultaneously explains the large variance in official cases and fatality across different countries. in this paper we build and estimate a multi-country model of the covid-19 pandemic at global scale. our model captures transmission dynamics for the disease, as well as how, at the country level, transmission rates vary in response to risk perception and weather, testing rates condition infection and death data, and fatality rates depend on demographics and hospitalization. estimating this model using data from every country with more than 1000 cases for which testing data is available, we infer some of the key characteristics of covid-19 pandemic globally and inform how alternative control policies may have played out across the globe. we use a multi-country modified seir model to simultaneously estimate the transmission of covid-19 in 84 countries. the model tracks community transmission, excluding the global travel network and instead separately estimating the date of introduction of patient zero for each country. within each country, the core of the model tracks the population through susceptible, pre-symptomatic, infected pre-testing, infected post-testing, and recovered states. basic transmission dynamics reflect a classical seir process, with a baseline transmission rate, incubation period, and duration of infection. building on this basic structure the model explicitly represents the dynamics of covid testing, including demand for tests, sensitivity of pcr based testing, and allocation of test capacity between positive and negative cases, as well as the dynamics of hospital system capacity, both with concomitant effects on transmission and mortality. it also endogenously generates population-level behavioral and policy responses due to the perceived risks and government intervention. infected people and those with other risk factors (e.g. hospital workers) or similar symptoms due to other illnesses. we assume, based on prior research (16) , that infected individuals are potentially tested on average 5 days after the onset of symptoms. on any given day, individuals with more severe symptoms get priority for testing. following (17) , this prioritization exerts a selection effect that results in different average severity of covid in the tested vs. untested infected populations. we also assume a 30% false negative rate for pcr-based covid tests (4, 5) . a positive test results in reduced contact rates for tested individuals, changing the transmission rates endogenously. thus the model tracks various mechanisms generating the observable measures: a daily reported infection rate that combines the total positive results from each day's tests with daily post-mortem infection confirmations, and a daily reported death rate of confirmed cases. unobserved parameters, such as ifr and asymptomatic fraction, can then be estimated by fitting the model against data for observables. we estimate the effective hospital capacity for each country as a function of total hospital beds available, modified for population density. this modification approximates the effect of geographic heterogeneity in demand for hospital capacity -in larger and less densely populated countries, geographic heterogeneity in the progression of the epidemic means that some areas (e.g. new york city) may be overwhelmed with demand even when other parts of the country have unused hospital capacity. as with testing, hospital capacity is allocated between more severe covid cases (both those tested and those not tested) and demand from non-covid patients. hospitalization can reduce the mortality rate for covid patients, while other determinants of fatality rates are the severity of symptoms and the age distribution of the population (8, 9) . each country also exhibits a response to the perceived risk of covid. the response is endogenously driven by a weighted combination of reported and actual hazard of infection. perceived risk drives reduction in contact rate and hence transmission rates through non-pharmaceutical interventions such as social distancing measures. specific government policies are not explicitly represented, but treated as part of this endogenous risk response. different countries could vary in their risk perception, response time, as well as the magnitude of their response based on the perceived risk. overall each country is represented by a system of ordinary differential equations tracking seven population stocks (susceptible, pre-symptomatic infected, infected pre-testing, and four groups of infected post-testing -positively tested or not × hospitalized or not) and a state variable for perceived risk. the model also includes multiple additional state variables for measurement and accounting of e.g. deaths and recoveries. model parameters are specified based on prior literature and formal estimation. main parameters adopted from the literature include incubation period (5 days (8, 16) ), onset-to-detection delay (5 days (16) ), sensitivity of pcr-based testing (70% (4, 5) ), and post-detection illness duration (10 days (8) ). we estimate the remaining parameters using a maximum likelihood approach. using testing rate time series and various country-level data points (e.g. population, hospital capacity, comorbidities, age distribution), the model endogenously simulates confirmed new daily cases and deaths over time and matches them against observed data by maximizing the likelihood of observing those data given the model parameters. we use a negative binomial likelihood function to accommodate excess dispersion in infection and death flows compared to more common gaussian specifications. where data on mean) are 2.2% (9.4% for median across countries, med) and 8.4% (med: 10.2%) for cumulative infections and deaths, respectively. r-square values exceed 0.9 for 56% of 336 country-specific cumulative and incidence time series. our projections are also consistent with excess mortality data, where available (see appendix s4). main findings include estimated parameters of covid-19 epidemics across countries, as well as trajectories of key measures. for estimated parameters we report a) the mean of most likely values across countries (mean); b) the standard deviation of those values (std), which quantifies variability of the underlying concept across countries; and c) the mean of country-level interquartile ranges (miqr), which captures the inherent uncertainty in parameter estimates for each country. the model brings together infection, testing, risk perception, behavioral and policy responses, hospitalization, and fatality using data from across the globe. here we focus on three main results. first, the magnitude of epidemic is widely under-reported with much variation globally. we estimate the asymptomatic fraction to be 55% (mean; std: 0.6%; miqr: 3.7%). combined with a sensitivity of 70% (4, 5) for pcr tests, the fraction of infections that could potentially be identified without mass testing is limited to 32%. most countries do not reach this upper bound, however, and testing is the binding constraint. figure 2 -a reports the ratio of estimated to reported infections and deaths, ranging between 2.4 and 157 for infections and 0.65 to 27 for deaths. in our sample of 84 countries (total population of 4.675 billion), we estimate total infections at 88.5 (85-95.3, 95% credible interval (ci)) million and 600 (ci: 586-622) thousands by june 18 2020, 11.8 and 1.48 times larger than reported numbers respectively. under-counting has been larger earlier in the epidemic. for example, usa faced peak infection rate (of 395 thousands per day) when detection rate was less than 10% of daily new cases. the most affected countries (based on percent infected) to date include ecuador (18%), peru (16.6%), chile (15.5%), mexico (8.8%), iran (7.9%), qatar (7.3%), spain (7.1%), usa (5.3%), uk (5.2%), and netherlands (4.8%) (see figure 1-b) . despite the order-of-magnitude under-reporting of cases, however, herd immunity due to infection remains far from reach (figure 2-b) . the closest a a b . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint country has come to herd immunity is chile, which at its peak infection rate was 194 (ci: days away from infection of 80% of its total population. second, we quantify both significant heterogeneity, and notable consistency, across countries in terms of basic parameters of the epidemic. the estimated initial reproduction number re ranges between 1.24 (indonesia) and 10.28 (iran), with the median of 4.5 (iceland) (re estimates include significant uncertainty; see figure s6 for details). even absent endogenous reductions in contact due to realized risk, these initial rates may be higher than longer-term levels for most countries because of early exposures happening in denser social networks. cross-country variations also reflect differences in population density, cultural practices, hygiene, as well as the timing of infection and thus early preparedness. we find support for the impact of weather on reproduction number, with the multiplier (rw) developed by xu and colleagues (7) explaining transmission rates closely (rw 0.76 ). we estimate the global infection fatality rate (ifr) to have been 0.68% (ci: 0.64%-0.70%), with a wide range (figure 2 -c) between 0.14% (ci: 0.12-0.16; qatar) and 2.08% (ci: 1.73-2.28; italy). the variations in ifr are primarily due to demographics with elderly having much higher risks (9) . hospital availability and treatment effectiveness provide a second mechanism affecting fatality rates. we find that hospitalization can bring down risk of death to 39.5% (std: 17.8%; miqr: 7.9%) of baseline. the model explains much variability in ifr with minimum country-level variability in basic fatality parameters. nevertheless, the quality of fit deteriorates in a handful of countries. for example, a ratio of simulated to reported deaths below one in a handful of countries signals that the model expected fewer deaths than observed. notably, we find it hard to reconcile, using only the model's mechanisms, the low fatality rates in qatar, singapore, and thailand relative to infection statistics, and high fatality rates in belgium and france. third, testing regulates transmission dynamics though a few mechanisms. by providing early warning, testing sets in motion the behavioral and policy responses essential to reducing transmission rate and controlling the epidemic. the exponential nature of contagion amplifies small early differences, such that a few days' difference in response time can lead to large differences in peak infection and final epidemic size. behavioral and policy responses to changes in infection risk are adopted rapidly, on average reducing effective contact (and thus transmission) rates in 13.5 (std: 7.1; miqr: 3.8) days. those responses are relaxed when risks attenuate, though down-regulating risk perception is slower (mean: 70.0; std: 31.2; miqr: 49.2 days). given limited data on rebounds in infections to date, how quickly perceived risk is downgraded is a major source of uncertainty in projecting the future of the pandemic. we also find that those with a positive test reduce their infectious contacts to a fraction of the original level (mean: 11.8% of original contact rate; std: 2.3%; miqr: 11.0%). these reductions are especially important because asymptomatic individuals are estimated to be less infectious than symptomatic ones (mean: 28%; std: 0.68%; miqr: 2.4%). finally, by regulating infection rates through the previous two channels, testing also 'flattens the curve', allowing a larger fraction of those in need of medical care to be hospitalized. together, these mechanisms create an early race between testing and the spread of infection ( figure 3 ). when infection is ahead, detection is compromised (moving up into the darker shades in figure) , responses lag further, and exponential infection growth can overwhelm hospital capacity. high early test capacity and its rapid expansion (moving to the right in the figure), brings the epidemic to light, activates policy responses, and allow for control. 4 (panels a and b) shows one alternative testing scenario and counter-factual trajectories of pandemic to date. in this scenario, all countries shift their testing rates from baseline to 0.1% of population per day (a rate currently achieved by multiple countries, e.g. usa, australia, in figure 3 ). the shift happens on march 11, 2020 (the date covid-19 was officially designated as a pandemic). such enhanced testing would have reduced total cases from 88.5 millions to 53.2 (ci: 50.5-59.6) millions. corresponding reduction in deaths would have been from 600k to 403k(ci: 377k-438k). by making additional assumptions on future testing and responses, the model can inform future trajectories. we explore a few projections out to spring 2021 that exclude vaccine and treatment availability. figure 4 (panels c and d) shows projections under three scenarios: i) using the current country-specific testing rates and response functions moving forward; ii) if enhanced testing (of 0.1% per day) is adopted on july 1 st 2020p; and iii) if sensitivity of contact rate to perceived risk is set to 8 (approx. 70 th percentile of estimated value across countries), leaving testing at current levels. contrary to the impacts of early differences in testing ( figure 4-a) , the reductions in future cases from additional testing (ii) are rather modest (from 1.55 in i to 1.37 billions in ii), because recognition of epidemic is no longer the bottleneck to response. both these scenarios project a very large burden of new cases in the fall 2020, with hundreds of millions of cases concentrated in a few countries estimated a b c d . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06. 24.20139451 doi: medrxiv preprint to have insufficient responses given perceived risks (primarily india, but also bangladesh, pakistan, and usa). in contrast, changes in response policies would make a major difference. scenario iii brings down future cases sharply, to as low as 153 (ci: 147-170) millions (cumulatively) by the end of winter. across scenarios, we find that the infection rate in each country usually peaks and then stabilizes at a lower and slowly declining linear rate, in some cases after damped oscillations (i.e. second waves). these (approximately) steady-state infection rates are at levels that motivate just enough policy and behavioral response to bring effective reproduction number re to 1. faster rates lead to exponential growth, raise alarms and bring down contacts; slower ones lead to relaxation of policies bringing the reproduction number back up to 1. those post-peak infection rates vary widely across countries and depend on risk perception parameters, time constants, and contact sensitivity to risk, as well as weather and susceptible population size. in this scenario we shift both testing and contact sensitivity to perceived risks to arguably more realistic values between current ones and those in scenarios ii and iii in figure 4 -c. specifically, on july 1 st 2020 we shift test rates to a value 20% between the current rates and 0.1% per day (which increases testing in most countries and reduces it in a few). similarly, sensitivity of contacts to perceived risk is shifted to 20% of the way between estimated country-level values and a high value of 8. resulting infection and death rates at the end of this period reflect the post-peak burden of the disease discussed above and are estimated at 0.01% (ci: 0.005-0.045) and 7.9e-5% (ci: 4.4e . appendix s4 provides projections for all countries. note that projections are highly sensitive to assumed testing, behavioral, and policy responses in the scenario. as such they should be interpreted as indicators of potential risk and not precise predictions of future cases; more rigorous testing and reductions in contacts in response to risk perception will significantly reduce future cases while laxer response and normalization of risks can lead to overwhelming breakouts. to enable empirical estimation and considering limits to data availability at a global scale, the model includes important simplifications and uncertainties that should temper the interpretation of the results. first, by analyzing an aggregate, country-level model, we abstract away much heterogeneity, from social networks to super-spreaders and local events. moreover, we offer no explanation for the underlying heterogeneity in reproduction numbers across countries. second, lacking hospitalization data across the world, the model's hospital sector includes additional uncertainty. similarly, our estimated behavioral and policy response functions do not tease apart the impact of various determinants such as national and local policies, business and event closures, use of personal protective equipment, reductions in physical interactions, and a host of other contributors. thus, results are not informative about the effectiveness of specific policies, and extrapolation of response function in future scenarios includes unknown uncertainties. for example, if normalization of risk reduced response magnitude in the second wave, our projections would miss that and prove optimistic. finally, absent explicit travel network our results would under-estimate the risk of the reintroduction of the disease in locations that have contained the epidemic. this paper provides a systemic view of the covid-19 pandemic globally. by incorporating testing capacity and prioritization, hospitalization, and risk perception and responses into an epidemiological model, we are able to closely match widely varying country-specific trajectories of the epidemic. our model explains the observed heterogeneities primarily through three pathways. first, estimates point to much variance in initial reproduction numbers across countries. this heterogeneity is not surprising given that reproduction number is very much a function of human behaviors and interaction is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . environments. nevertheless, our study was not designed to tease out the sources of that heterogeneity, an important task that requires other types of data and analysis. second, the course of the epidemic is also significantly impacted by risk perception and behavioral and policy responses that vary notably across different countries. finally, we show that differences in testing rates play an important role in shaping those trajectories. despite those variations, we also find much that is consistent across the globe: a) we estimate that just over half of the infections are asymptomatic, consistent with detailed estimates from smaller samples (1, 13, 14) . b) we estimate that asymptomatic individuals are about a third as infective as symptomatic patients. c) in our model, hospitalization brings down fatality substantially, highlighting the crucial importance of keeping cases below hospital capacity. d) we find an average global infection fatality rate close to 0.68%. this is consistent with growing evidence on ifr (11, 28) . our estimates for ifr change predictably with age, and require few other predictors and no country-specific factors to stay consistent with the data. e) an order of magnitude under-reporting of cases is the norm across most countries, with a few percent of population already infected across many nations; nevertheless, herd immunity remains distant. f) finally, absent notable improvements in country level responses or breakthroughs in vaccination or treatment, the outlook for the epidemic remains grim, with most nations settling into a steady state of cases and deaths that, while below their peaks, are troublingly large. 20 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. the model simulates the evolution of covid-19 epidemic, risk perception and response, testing, hospitalization, and fatality at the level of a country. here we explain key equations and structures in each sector, followed by complete listing of model equations and parameters in s5. the model is a derivative of the well-known seir (susceptible, exposed, infectious, recovered) framework for simulating infection dynamics. figure s5 provides an overview of key population groups and the population movements among them 1 . . we assume demand for testing and hospitalization are driven by symptoms, so all asymptomatic patients will be in the latter category. 1 in the equations below we use short-hands to simplify mathematical notations. the full model documentation uses full variable names. table s1 provides the mapping between the short-hands and the full names, as well as the sources and equations for those variables and parameters discussed below. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint from these infectious categories, resolution flows (r…) take individuals to either recovered (r…) or dead (d…) states, with corresponding subscripts u, c, ch, and uh for stocks and uu, uhch etc. for flows. given the differences in severity and potential survival extension due to hospitalization, we distinguish between resolution delay for those in hospital (hospitalized resolution time; τh) and those not hospitalized (post-detection phase resolution time; τr). we use first order exponential delays for all lags, though sensitivity analyses showed very little impact of using higher order delays. the infection rate (rsp) controls the flow from s to p and depends on infectious contacts (ci), fraction of total population (n) that is susceptible, and weather effect on transmission (w). the latter is a function of rw, the country-level projections for impact of weather on covid-19 transmission risk year-round developed by xu and colleagues (7) and a parameter, sensitivity to weather (sw), to be estimated: infectious contacts depend on the reference force of infection (β), various infectious sub-populations (and their relative transmission rates; ma for asymptomatic and mt for confirmed), and contacts relative to normal (fc), which captures behavioral and policy responses as a fractional multiplier to baseline infectious contacts: in this equation we separate various stocks (of i and p) into asymptomatic (a superscript) and symptomatic (s superscript). that distinction is treated analytically using a zero-inflated poisson distribution that is discussed in the next section. in light of evidence on the short serial interval for covid-19, likely below the incubation period (29, 30), we do not distinguish the infectivity of pre-symptomatic individuals from those post onset. contagion dynamics start from patient zero arrival time, t0, another estimated parameter. the key mechanisms regulating the population flows among these stocks are discussed below, and a schematic of important relationships is provided in figure s6 . five parameters are estimated in the equations discussed above. one of them (sw) is global (i.e. assumed identical across countries; see the estimation section below for details on the distinction between global and country-specific parameters) and the remaining four are country-specific: β, mt, ma, and t0. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. covid-19 infection varies in acuity, from asymptomatic to life-threatening. acuity of disease affects notonly baseline fatality risk, but also testing and hospitalization decisions which impact official records of infection and fatality rates. since movement between population groups via testing or hospitalization is itself a function of acuity, to allow for consistent inference of mean acuity across different population groups, we use an analytical framework to track acuity levels. this framework, which we adapted from prior research (17) , obviates the need to disaggregate the population by different acuity levels (which would prohibitively raise the computational costs for estimation). specifically, we represent acuity using a zero-inflated poisson distribution. this distribution combines two subpopulations -one with poisson-distributed acuity levels with mean covid acuity (αc), and another additional asymptomatic fraction with zero acuity, which is the zero-inflated component. the sum of those with zero acuity from the poisson part of the population and the second group is the total asymptomatic fraction (a). we assume this asymptomatic group is not given priority in testing or hospitalization, and is not at risk of death. thus they will always follow the → → → → pathway. the pathways for the remaining population depend on acuity and its impacts on testing, hospitalization, and death. note that the concept of acuity defined here only needs to have a monotonic relationship with tangible symptoms and risk factors and it does not have a one-to-one relationship with any real world measure of acuity, and as such is better seen as a mathematical construct that informs modeling rather than a real-world variable with clinical definition. from this framework two parameters, a and αc, are estimated as country specific parameters with limited variability across countries. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint the testing sector reads the active test rate (tt) for each country as exogenous input data (see appendix s3 for pre-processing details for this data). a fraction of this total test rate, typically small, is allocated to postmortem testing of covid-19 victims who have not been previously confirmed (post mortem tests total, tpm). specifically, of the deaths of unconfirmed infectious individuals (whether hospitalized or not), a certain fraction of fatalities screened post mortem (npm) will be identified true post-mortem tests. we anchor the npm to fraction covid death in hospitals previously tested (ndch). the rationale for this anchoring is that on the margin if there are many unidentified covid patients in hospitals, the chances are that the system lacks enough testing capacity and thus post-mortem testing should also be less thorough: we experimented other functional forms with a free parameter connecting the two constructs, but following our conservative estimation principle decided against including that free parameter in the final model. we feared that absent clear observables to identify this additional parameter (e.g. on country-specific policies regulating post-mortem testing) the degree of freedom would improve the fit but potentially for the wrong reason. the where the multiplier recent infections to test (mit), captures the sensitivity of negative test demand to recent infection reports. to allocate the available tests (tnet) between these two sources of demand, we use an analytical logic that allocates testing based on acuity of symptoms. the basic idea is that through self selection and screening by testing centers, people who have more symptoms or other signals that correlate with covid infection (e.g. high exposure risk) are more likely to be tested. we assume each unit of acuity increases the likelihood that an individual gets tested, based on a variable prob missing symptom, pms. this variable represents the probability that each acuity unit fails to convince the testing decision process to test a given individual, i.e. how selectively and sparingly tests are conducted. specifically, in this model an individual with k acuity units is tested with probability: we assume the negative test demand is coming from a population with a poisson-distributed, unit average acuity level (αn=1) for symptoms of non-covid influenza-like illnesses. the test demand from covid . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint patients also comes from a poisson distribution of acuity, but with mean αc. with the poisson distribution and given a level of α and pms, one can calculate the fraction of each demand source that would be tested: we therefore need to find the pms that allows test supply to match demand that is satisfied, specifically, by solving the following equation for pms*: having solved for p*ms (numerically), we analytically calculate the average acuity level for those positively tested (αcp: average acuity of positively tested ) and those either not tested or having received a false negative result (αcn). specifically, if test sensitivity was 100%, the average acuity for those not tested would be: the acuity level for those tested could then be found based on the conservation of total acuity across those positively tested and those not. starting with this basic specification we further account for the sensitivity of covid test (st) to calculate the values of αcp and αcn. we parametrize sensitivity at 70%, which is the estimated sensitivity for the pcr-based tests used as the primary diagnosis method of current infections of covid-19 (4, 5) . overall, the testing rates that are determined by solving for * , combined with sensitivity of tests, inform the fraction of covid positive individuals transitioning from pre-detection (ip) to confirmed vs. unconfirmed states (ic or ich vs. iu or iuh), while the calculated α values inform the likelihood of hospitalization and fatality rates, as discussed next. the testing sector includes the following two country level parameters that are estimated: nst, mit. the hospitalization sector of the model starts with each country's nominal hospital capacity (hn) in total hospital beds. in practice, geographic variation in hospital density and demand creates imperfect matching of available beds with cases of covid-19 at any point in time, e.g. because some potential capacity is physically distant from current covid hotspots. this imperfect matching means some of the nominal hospital capacity is effectively unavailable at any time, especially in larger, less densely populated countries. we therefore calculate effective hospital capacity (he) by considering geographic density of hospital beds (bed per square kilometer; dh): where the * represents a large reference hospital density of 6.06 beds per km 2 (which is the value of for south korea). the parameter sdh (impact of population density on hospital availability) is estimated. effective capacity is allocated between potential hospital demand (hcd) from covid-19 cases and the regular demand for hospital beds from all other conditions (which we assume equals pre-pandemic effective hospital capacity). we assume that covid-19 patients will have higher priority for hospitalization compared to regular demand. specifically, we assume that fraction of regular demand allocated (mhr) would be the . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint square of that for covid demand (mhc), = 2 , and solve the resulting hospital capacity allocation problem analytically: we determine the covid demand for hospitalization based on a screening process similar to that for testing. two types of covid patients may seek hospitalization: those with confirmed test results and those without. the former are more likely to seek hospital treatment. we first calculate a parameter analogous to pms in the testing sector that informs the demand from confirmed covid patients for hospitalization. this parameter, the pmas confirmed for hospital demand (pmhc) is determined based on acuity level of confirmed (αct) and reference covid hospitalization fraction confirmed (rh), an estimated parameter capturing the overall need for hospitalization among covid patients: for unconfirmed covid patients we scale the analogue of this parameter (pmhu) based on how much priority non-covid patients generally receive: this formulation ensures that: 1) confirmed covid patients are more likely to be hospitalized, but also that 2) if there is ample hospital capacity (mhr~1), then confirmed and unconfirmed covid patients will receive similar priority for the same level of acuity. in short, the pm. values determine hospital demand by confirmed and unconfirmed covid patients, which add up to hcd. the latter determines the fraction of hospital demand that is met. analogous to the testing sector, this fraction along with demand determines the flow of individuals from the pre-detection (ip) state to hospitalized vs. non-hospitalized states (ich or iuh vs. ic or iu). matching demand to allocated capacity also allows us to calculate the realized probability of missing acuity signal at hospitals (p*m) for confirmed and unconfirmed patients. as in the testing sector, those probabilities let us approximate for the expected acuity levels for covid patients in and out of hospital, as well as tested vs. not-tested, i.e. αct, αch, αu, and αuh. these average acuity levels in turn inform fatality rates for each group. the hospital sector includes two country level estimated parameter with limited variation across countries: sdh and rh. for patients in each of the u, c, ch, and uh groups we specify the infection fatality rate (f), as: the parameter base fatality rate for unit acuity (fb) sets the baseline for fatality rate. sensitivity of fatality rate to acuity (sf) determines how fatality changes with estimated acuity levels; more severe cases are expected to have higher fatality rates. hospitalization reduces fatality rates, expressed as the relative impact of treatment on fatality rate (shf). the function incorporates the impact of age distribution on fatality rates. for age effect, we calculate a risk factor for each country based on its age distribution and the relative fatality risks for covid patients in different ages documented in prior work (9) . we normalize this factor . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint against its value for china where the original study was conducted. given the well-established impact of age on fatality, this factor is directly multiplied into the infection fatality equations. overall, the fatality sector includes three parameters that are estimated at the country level, with limited variance across countries, those are: , , and . note on comorbidities and fatality: we also explored including three comorbidities but found the estimates unreliable and therefore they are not included in the main specification of the model. those comorbidities include obesity, chronic disease, and liver disease. the effects we explored for each were: (.) = (.) (.) , where we used the following country-level indicators from the world health organization (23), normalized by the average across all countries (d(.)): for obesity: prevalence of obesity among adults, bmi ≥30 (age-standardized estimate) (%) for chronic health issues: probability (%) of dying between age 30 and exact age 70 from any of cardiovascular disease, cancer, diabetes, or chronic respiratory disease for liver disease: liver cirrhosis, age-standardized death rates (15+), per 100,000 population in equation 3 we noted that contacts relative to normal (fc) regulates infection rates. this factor ranges between a minimum (min contact fraction; cmin) and 1 as a function of the relative utility from normal activities (un) compared to utility from limited activities (ul) in light of additional risks associated with normal activity patterns: the utilities from normal and limited activities are specified as . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint overall, the risk perception and response sector includes the following five country-specific parameters that are estimated: cmin, τru, τrd, λ, and wr. table s1 summarizes the main equations discussed in appendix s1, providing the mapping between full variable names and the short forms. it also includes all estimated model parameters, as well as those specified based on prior research. table s1 -mapping between full variable names and their short form for the subset of variables and parameters discussed in s1. also included are equations explained above and sources for other variables. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint the model we estimate is nonlinear and complex, and any estimation framework is unlikely to have clean analytical solutions or provable bounds on errors and biases. therefore, in designing our estimation procedure, we set out to be conservative. specifically: we use a likelihood function that accommodates overdispersion and autocorrelation (negative binomial); we utilize a hierarchical bayesian framework to couple parameter estimates across different countries which reduces the risk of over-fitting the data; and we use the conceptual definitions of parameters and their expected similarity across countries to inform the magnitude of that coupling across countries. compared to more common choices in similar estimation settings, these choices tend to widen the credible regions for our estimates and reduce the quality of the fit between model and data. in return, we think the results may be more reliable for projection, more informative about the underlying processes, and better reflective of uncertainties in such complex estimation settings. the model is a deterministic system of ordinary differential equations with a set of known and unknown parameters. the known parameters are those specified based on the existing literature and do not play an active role in estimation. the unknown parameters can be categorized into those that vary across different countries and those that are the same across all countries (i.e. general parameters). the estimation method is designed to identify both the most likely value and the credible regions for the unknown parameters, given the data on reported cases and deaths (and for a subset of countries, the excess deaths). this is done through a combination of estimating the most likely parameter values in a likelihood based framework, and using markov chain monte carlo simulations to quantify the uncertainties in parameters and projections. we first introduce the 3 different components of the likelihood function we use: the fit to time series data, the random effects component coupling country-level parameters, and the penalty for excess mortality. then we explain the implementation details. define model calculations for expected reported cases and deaths for country i as μij(t) (with index j specifying cases and deaths) and the observed data for those variables as yij(t); the country-level vector of unknown parameters as and the general unknown parameters as ϕ. note that vector includes several parameters, each specifying an unknown model parameter, such as impact of treatment on fatality, or total asymptomatic fraction, for country i. the model can be summarized as a function f that produces predictions for expected cases and deaths for each country given the general and country-specific parameters: we use a negative binomial distribution to specify the likelihood of observing the y values given θ and ϕ. specifically, the logarithm of likelihood for observing the data series y given model predictions μ(θ ,ϕ) is: where (dropping time index for clarity): . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. summing the lt function over time provides the full (log) likelihood for the observed data given a parameterization of the model. the negative binomial likelihood function includes two parameters, μ and ε which determine the mean and the scaling/shape of the observed outcomes. the second parameter, ε, provides the flexibility needed fit outcomes with fat tails and auto-correlation. this parameter could itself be subject to search in the optimization process. specifically, we assume that: thus we create a (set of) country specific parameter(s) ( ) and two general parameters ( ) which should be estimated along with the conceptual model parameters. the country level scale ( ) implicitly assesses the reliability and inherent variability in country level reports, and the general ones inform the variability in case data vs. deaths. we augment the vectors ϕ and θ to include these scaling parameters as well. up to this point we have not included any relationship among country specific parameters, . this independence assumption would allow parameters representing the same underlying concept to vary widely across different countries. such treatment, by providing more flexibility, enhances the model's fit to historical data. however, it ignores the conceptual link that exists for a given parameter across countries, potentially allowing the model to fit the data for the wrong reasons (i.e. using parameter values that do not correspond to meaningful real world concepts). the result would likely be less reliable and also not robust for future projections. we therefore define a hierarchical bayesian framework to account for the potential dependencies among model parameters. specifically, we assume the same conceptual parameters (e.g. impact of treatment on fatality), across different countries, are coming from an underlying normal distribution with an unknown mean (to be estimated) and a pre-specified standard deviation. this assumption is similar to the use of "random effect" models common in regression frameworks, though we deviate from canonical random effect models by pre-specifying the standard deviation. in fact it is possible to estimate the standard deviation across countries as well (and to obtain better fits to data), but adding those degrees of freedom ignores qualitatively relevant insights about the level of coupling across different countries for each parameter, and thus results may fit the data better but for the wrong reasons. for example, some parameters, such as patient zero arrival time, could be very different across countries, whereas parameters reflecting innate properties of the sars-cov-2 virus itself (e.g. total asymptomatic fraction (a)) or those determining fatality (e.g. base fatality rate for unit acuity (fb)) should be very similar across different countries. allowing the model to determine the variance for the latter will lead to better fits: the model can find baseline fatality rates that easily match fatality variations across countries, and would expand the corresponding variance parameter accordingly. however, as a result the estimation algorithm will have too easy a job: it will not require a precise balancing between hospitalization, impact of acuity on fatality, and post-mortem testing decisions to fit fatality data. thus, the estimates may well be less informative, or further from true underlying processes and the general characteristics of the disease which we care about. the implementation of this random effect introduces another element to the overall likelihood function: here θik represents the k th parameter for country i, and ̅ is the (estimated) average across countries for the k th parameter. σk is the pre-specified allowable variability for the k th parameter across different countries. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint specifying these standard deviations adds a subjective element to the estimation process. however, we note that subjective elements are ultimately indispensable in any modeling activity: from specifying the model boundary to the level of aggregation, use of various functional forms, and choice of likelihood functions, these choices are built on subjective assessments that experts bring to a modeling project. absent our conceptually informed variability factors, we would need to make the assumption that country-level parameters are independent, or that our complex estimation process would correctly identify the true dependencies among those parameters. we think both those alternatives are inferior in the chosen method. so here we focus on transparently documenting and explaining those assumptions. table s2 summarizes the estimated model parameters, their estimated values (mean across countries and mean of inter-quartile range) and the assumed variability factor (σk) for each. sw sensitivity to weather 0.765 (global parameter so the other metrics do not apply) *given the wide range and potential long tail for these parameters the log10 transformation is used in specifying the dispersion penalty (equation 27) and variability factors are reported as 10 σ , where σ is used in equation 27. ** these parameters are expected to be less variable across countries and thus are assigned small variability allowances compared to their mean. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint finally, we include a likelihood-based penalty term to allow model predictions be informed by excess mortality data collected by various news agencies and researchers for a subset of countries in our sample. these data provide snapshots of excess mortality (compared to a historical baseline) for a window of time in each country. subtracting from total excess mortality the covid-19 deaths officially recorded in that window offers a data point for excess mortality not accounted for in official data (ei). we can calculate in the model the counter-part for this construct: the simulated mortality that is not included in the simulated reported covid-19 deaths ( ̅ ). there is uncertainty in these excess mortality data: the historical baselines used by various sources do not adjust for demographic change, excess mortality may be due to factors other than covid-19, and some of it may be due to changes in healthcare availability and utilization motivated by covid-19 but not directly attributable to the disease (for example when surgeries are delayed, hospitalization is avoided, or heart conditions are ignored). excess mortality may also be reduced due to reduced traffic accidents (in light of physical distancing policies) and pollution related deaths. given these uncertainties, we use the following penalty function to keep the simulated unaccounted excess mortality close to data: this penalty could be seen as a likelihood coming from the probability distribution ( ) = should be attributed to covid-19 deaths, but that there is significant uncertainty around this, so some 20% variation across this figure is quite plausible (70%-110% of data). however, numbers outside of this range start to impose increasingly large penalties, so that very large deviation becomes unlikely. combining these three components, we obtain the full likelihood function used in the analysis: for each country we include the lt component from the first day they have reached 0.1% of their cumulative cases to-date, or a minimum of 50 cumulative cases. this excludes very early rates that are both unreliable and which, given very small estimated model predictions for infection, could lead to unreasonably large likelihood contributions. the model includes a large number of parameters to be estimated: a general parameter for the impact of weather, 2 general parameters for , and 21 parameters for each country that are coupled together based on the random effects framework described above. out of those 1 parameter is for and the rest are informing various features of disease transmission, testing, hospitalization, and risk perception and response. with a sample of 84 countries, this would lead to about 1800 parameters to be estimated. a direct optimization approach to this problem suffers from potential risk of getting stuck in local optima, and direct use of mcmc methods to find the promising region of parameter space suffers from the curse of dimensionality. we therefore designed the following 4-step procedure to find more reliable solutions to both problems. 1) we estimate the model with the full parameter vector for a smaller number of countries with larger outbreaks (3-5 countries). we use the powell direction search method implemented in vensim™ simulation software for this step. the method is a local search approach though it has features that allows it to escape local optima in some cases. we restart the optimization from various random points in the feasible parameter space and track the convergence of those restarts to unique local peaks. we stop this process when we are repeatedly landing on the same local peaks in the parameter . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint space. this procedure showed that local peaks do exist, but they are not many; for example within 100 restarts we may find 2-4 distinct peaks. this provides a coherent set of starting points for ϕ and ̅ for next steps. 2) we go through iterations of the following two steps: a) conduct country-specific optimizations with 50 restarts to find the vector of θi given the ϕ and ̅ from first optimization or from the step b. b) conduct a global optimization, including all countries but fixing θi and optimizing on ϕ (and ̅ ; though that is simply the mean across country level parameters from previous round). we stop when iterations offer little improvement from one round to the next (less than 0.05% improvement in loglikelihood). 3) we conduct a full optimization allowing all parameters (θi, ϕ and ̅ ) to change, starting from the point found in the last iteration of step 2. this step finds the exact peak on the likelihood landscape which is the best-fitting parameter set for the model. 4) for the mcmc, theoretically one should conduct the sampling from all model parameters in the full model. however, our experiments showed that the large dimensionality of the parameter space requires an infeasible number of samples to achieve adequate mixing and ensure reliable credible regions for parameters and projections. to overcome this challenge we note that the parameters of different countries are connected to each other only through ϕ and ̅ , and these general parameters are rather insensitive to dynamics in each country. the insensitivity is due to the fact that a single country only contributes about 1% to the general parameters' values, and within a typical mcmc the country-level parameters often can't change more than 10% before the resulting samples become highly unlikely. therefore, one can conduct an approximate country-level mcmc by fixing the general parameters at those from step 3, and only sampling from the θi for each country. the mcmc algorithm used is one designed for exploring high dimensional parameter spaces using differential evolution and self-adaptive randomized subspace sampling (19) . using this method we obtain good mixing and stable outcomes (robin-brooks-gelman psfr convergence statistic remaining under 1.1) after about 600,000 samples (the burn-in period). we continue the mcmc for each country for 1 million samples and then randomly take a subsample of those points after the burn-in period for the next step. 5) the resulting subsamples for different countries from step 4 are assembled together to create a final sample of parameters for the full model to conduct projections and sensitivity analysis at the global scale. uncertainties in the handful of global parameters is not identified in this procedure, but can be quantified by assessing the sensitivity of the global likelihood surface to changes in those parameters. the process above is automated using a python script that controls the simulation software (vensim). we conduct the analysis using a parallel computing feature of vensim on a windows server with 48 cores. after compiling the simulation model into c++ code (which speeds up calculations significantly), and using a simulation time step of 0.25 days, it takes about 20 hours to complete the estimation for 84 countries. full analysis code is available online at https://github.com/tseyanglim/covidglobal. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint s3-data pre-processing getting contemporaneous, comprehensive, national-level data on covid-19 is a challenge. the most widely-cited data aggregators, such as the johns hopkins center for systems science and engineering's covid-19 database (31) , ourworldindata portal (3), and the us-focused covid tracking project (21), get their data from the same few official sources, such as the us centers for disease control and prevention (cdc), the european cdc (ecdc), and the world health organization (who). these official agencies in turn get their data from national and subnational public health authorities, which ultimately rely on reports from hospitals, clinics, and private and public health labs. as a result, idiosyncrasies in the ground-level data collection processes permeate virtually all sources of aggregate data. most notably, data collection involves time lags, which can differ from source to source. daily death counts could reflect the date of actual death or the date a death is registered or reported; different uk government sources, for instance, use each of these metrics. 2 daily infection or case counts could include the total new cases reported on a given date, or the total cases confirmed from that date; the latter would result in some 'backfill' whereby case counts for previous days can continue to increase for some time as delayed confirmations come in. daily counts of tests conducted could report samples collected, samples processed, results reported, or a mix of these; the us cdc, for instance, reports a mix of testing by date of sample collection and date of sample delivery to the cdc. 3 aside from differences in unit of measure (people vs. tests vs. samples), there may be different time lags involved as well. in addition to these idiosyncrasies, testing data in particular is also patchy for many countries, even as testing has become more widespread. the who does not report country-by-country testing, nor does the jhu covid map outside the us. furthermore, there are sometimes irregular delays in the reporting of test results, which can create occasional unexpected spikes in reported numbers of tests, infections, or both. 4 depending on the specifics of how daily infection and test counts are reported, there can in some cases be a disjunction between the two. because confirmed case counts largely depend on positive test results, test and infection counts should be correlated -ceteris paribus, a day with a lot of samples collected for testing should see more confirmed cases attributed to it, while a day with no sample collection should see no cases. but since cases may not be reported by the date of the test, and tests may not be reported by the date of sample collection, officially reported numbers can get out of sync in either direction. this problem is most salient when there are clear weekly cycles in daily rates. in most of the world, particularly western countries, daily test rates are far lower on weekends than during the week. as a result, infection numbers show a clear weekly cyclical component as well. but the weekly cycles in testing and infection numbers for a given country do not always line up. our model explicitly accounts for the effect of testing on reported infections, but we do not explicitly model the country-level idiosyncrasies of reporting and how they vary between test data and infections. instead we account for any such lags in pre-processing of the data to align testing and case data. the weekly cycle occurs in many countries' death rate data as well, where it presents a different problem. a weekly cycle in testing is a behaviourally realistic part of the data-generation process, as many labs, clinics, or other testing sites for instance may be closed on weekends. as testing provides the window on the state of confirmed infections, a comparable cycle in confirmed cases is to be expected as well. by linking case confirmations to testing, our model explicitly accounts for this limited visibility on the true state of the epidemic. however, a weekly cycle in death rates almost certainly reflects different limitations of the data-2 https://blog.ons.gov.uk/2020/03/31/counting-deaths-involving-the-coronavirus-covid-19/ 3 https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/previous-testing-in-us.html 4 see e.g. https://www.wcvb.com/article/massachusetts-coronavirus-reporting-delay-due-to-quest-lab-itglitch/32288903# . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint generation process, typically to do with hospital staffing, 5 which we do not explicitly model. as such we need to address any weekly cycle in death rates through data pre-processing as well. to deal with these challenges, we developed a multi-step algorithm to pre-process our data before feeding it into the model for calibration. the algorithm is described below. it was implemented in python, largely using the pandas and numpy packages, and the code is available in full at: https://github.com/tseyanglim/covidglobal. the algorithm proceeds country-by-country, following these steps on each country. 1) examine daily cumulative test data; if data are insufficient (6 or fewer data points), drop country from the dataset. 2) interpolate any missing daily cumulative test data points using a piecewise cubic hermite interpolating polynomial (pchip) spline. if the first reported infection is before the first reported cumulative test, also extrapolate cumulative tests back to the date of first reported infection. a. extrapolation to the date of first reported infection is necessary since both in the model and, to a large extent, in reality, reported infections require testing for confirmation. b. pchip spline interpolation yields a continuous monotonic function with a continuous first derivative, thus avoiding generating any anomalous rapid change in daily test rate. c. we used the implementation of pchip interpolation from the widely used scipy package for python. 6 3) calculate daily test rate as daily cumulative tests less the preceding day's cumulative test total: 4) examine the original daily cumulative test data to estimate how much of the calculated daily test rate is based on interpolated vs. original data. a. daily test rates calculated based on mostly original data should be expected to include any weekly cycles or occasional irregularities that would also be reflected in daily infection counts. conversely, daily test rates calculated from cumulative test counts that are largely interpolated would not be expected to fully reproduce any such cycles or irregularities, since the interpolation produces a relatively smooth function. b. as a rule of thumb, we examine the cumulative test data for the second half of the time from the first test to the latest test. if fewer than half the days in that window have original cumulative test data, we consider the test data to be 'sparse', requiring further processing. 5 it may be argued that there are weekly cycles in large-scale human behaviour that may drive some true weekly cyclicality in the true rates of infection and death, and as such it may be wrong to consider such cycles to be artefacts of the datageneration process. however, we find this unlikely for a few reasons. first, weekly cycles in human interactions, largely driven by the work and school week and weekend, will have been significantly attenuated by widespread adoption of social distancing measures around the world. second and more importantly, variation in incubation period and time before development of symptoms means that any true cyclicality in the timing of initial infection will be further attenuated in the timing of symptom development. by the same logic, wide variability in the delay from symptom development to death means there should be minimal cyclicality, if any, in the timing of deaths, meaning any such cycles visible in the data are due to measurement and reporting lags. 6 https://docs.scipy.org/doc/scipy/reference/generated/scipy.interpolate.pchipinterpolator.html . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint 5) if the test data are not sparse, account for any potential lag or other reporting delay differences between daily test rate and daily infection rate using a time-shift algorithm to estimate any such lags or delays from the data and shift the test rate time series accordingly. the time-shift algorithm ensures that any weekly cycles present in the daily infection rate data are reflected in the daily test rate data and aligned as best as possible on date, thereby accounting for the fact that model-generated reported infections depends on testing but with no time lag between test and result. a. first, identify the weekly component of the time series of daily infection rate and daily test rate using a seasonal-trend decomposition based on loess (stl) procedure. 7 i. stl deconstructs time series data into several components, including a trend and a seasonal component over a specified period (weekly, in this case) as well as a residual. stl is an additive decomposition, and has the advantage of allowing the seasonal component to change over time (rather than being a fixed pattern repeated exactly across the whole time series). ii. we used the stl implementation from the statsmodels package for python. 8 b. shift the time series over a one-week range (from -2 to +4 days of lag between test and infection reporting), calculating the cross-correlation between the weekly seasonal component of the daily infection rate data and the daily test rate data for each time shift. c. identify the time shift within this range that maximizes the cross-correlation between the infection rate and test rate data, and shift the test rate data accordingly. 6) if the test data are sparse, the spline interpolation will generally cut out some of any weekly cyclicality that may be present. visual inspection of daily test rates for countries with sparse test data also shows large, irregular spikes in reported tests are not uncommon, without necessarily having concomitant irregular spikes in reported daily infection rates. as such, rather than attempting to eliminate differences in reporting lags through the time-shift algorithm described above, we instead apply a data-smoothing algorithm to both daily test rate and daily infection rate, in order to reduce any cyclicality and irregular spikes. this smoothing allows the calibration of the main model to focus on matching the underlying trends in the data. in all cases, whether daily cumulative test data are sparse or not and whether infection and test rate data are smoothed or not, since weekly cycles in death data are reflective of reporting lags not captured in the model, daily death rate data is smoothed using the same algorithm. 8) the smoothing algorithm used is designed first to conserve the total number of reported cases (tests, infections, or deaths), and second to preserve some degree of variation in the time series, as some noise may be informative and retaining some is important to the calibration of the model. a. starting from when the time series of daily rate (test or infection) exceeds a specified minimum value (5/day), calculate the rolling mean of the daily rate, using a centred moving window of 11 days. b. calculate the residual between each day's data point and the rolling mean for that day, and divide by the square root of the rolling mean, to get an adjusted deviation value: i. dividing by the square root of the rolling mean reflects a heuristic assumption that each daily rate (of infections, deaths, or tests) behaves as a poisson process (stdev of pois() =  0.5 ). 7 is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06. 24.20139451 doi: medrxiv preprint ii. the functional result of this adjustment is that both absolute and relative magnitudes of deviations from the rolling mean are given some weight -large relative deviations when absolute values are small (and data are noisier) are not ignored, but neither do they outweigh larger absolute (but smaller relative) deviations that occur when the mean is large, which is important since most of the time series data are growing significantly over the time horizon of the model. c. calculate thresholds for identifying dips and peaks in the data based on the median of the adjusted deviations, ± one median absolute deviation (mad) of the adjusted deviations: i. using the median absolute deviation to determine thresholds for peaks and dips is robust to outliers in the deviations, which do arise occasionally in the data. ii. a threshold width of one mad is relatively narrow for outlier detection, but by inspection of the data, is about right for identifying most of the peaks and dips caused by weekly cycles in test, infection, and death rates, as well as larger outliers. d. once thresholds are calculated, iterate through the data points in the time series first forward in time from oldest to newest, filling in any 'dips' (data points with adjusted deviations below the lower threshold), then backward in time from newest to oldest, smoothing out any 'peaks' (data points with adjusted deviations above the upper threshold) that remain. repeat the process until all data points' adjusted deviations are within the originally calculated thresholds for the time series. i. we infer that the underlying processes generating dips and peaks are somewhat different. dips are generally the result of weekly cycles in the data, e.g. lower rates of testing or longer lags in death reporting that occur on weekends. peaks arise to some extent due to the same weekly processes, e.g. some deaths that occur on weekends only being recorded at the start of the next week. however, some peaks, especially larger ones, may result from irregular random delays in reporting, such as large batches of tests being held up due to logistical issues and then getting processed all at once. as such the smoothing procedure for dips vs. peaks is slightly different. e. the dip-filling step fills a fraction of each dip (specified as a smoothing factor) by redistributing data counts based on a multinomial draw from the subsequent few days following each dip. i. first, calculate the amount to fill based on the deviation and the smoothing factor specified, in this case 0.67: ii. calculate the amount redistributed from each of the following few (7) days +1 , +2 , … + , = 7, based on a multinomial distribution as follows: where +1 , +2 , … +7 are calculated as: iii. this formulation allows some redistribution from any of the subsequent few days whose adjusted deviations exceed the focal day's adjusted deviation, but with more redistribution from days with higher adjusted deviations. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint f. the peak-smoothing step similarly redistributes a fraction of each peak, specified by the smoothing factor, to the preceding several days based on another multinomial draw. i. first, calculate the amount to redistribute similarly to the dip-filling step: ii. calculate the amount redistributed to each of the preceding several (14) iii. this formulation redistributes peaks to preceding days based on the calculated rolling mean counts of those days, on the assumption that the irregular delays that generate random spikes in counts are essentially random and equally likely to affect any given unit of data over a several-day span. as such, the probability that a unit showing up in a spike due to such delays comes from a given preceding day is simply proportional to the expected count for that day, as approximated by the rolling mean. g. by filling dips first before smoothing peaks, the combined algorithm largely addresses any peaks that are due primarily to weekly cycles during the dip-filling stage, such that remaining peaks that get smoothed tend to be the larger, irregular ones. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint table s3 reports two quality of fit metrics for different countries and different time series. the first four columns report mean absolute error normalized by mean (maen) and the last four report the r-squared measures. errors for cumulative infection and deaths are followed by those for the new cases and deaths (flow variables). . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint figure s7 shows the visualization of fit between data and simulations for all the countries in our sample. these graphs include data and model outputs for reported new cases (blue; left axis in thousands per day) and deaths (red; right axis in thousands per day) starting from the beginning of the epidemic in each country until june 20. figure s7 -comparison of data and simulation. new cases in blue (left axis, in thousands per day) and new deaths (red, right axis). . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint estimates for true cumulative cases (blue; left axis in millions) and deaths (red; right axis in thousands) across different countries up to june 20 are reported in figure s8 . figure s9 shows the ratio of estimated excess deaths, i.e. covid-19 fatalities not reported as such, to reported excess deaths, i.e. deaths over historical baseline not accounted for by reported covid-19 deaths, for the countries for which such data are available. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint figure s9 -ratio of estimated excess deaths to reported excess deaths. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint figure s10 shows the initial reproduction number (re) occurring in each country. reproduction numbers are changing dynamically and transient dynamics may lead to larger than equilibrium numbers if maximum re values were used. we therefore use the 90 th percentile of simulated reproduction number in this graph. also note the large credible intervals for these estimates, partly coming from changes in what is included in the 90 th percentile (sometimes it includes the highest values and sometimes it does not), as well as the inherent uncertainty when both reproduction number and behavioral and policy responses are estimated: one can have smaller initial re and smaller response functions, or larger values for both, and stay consistent with the data. figure s10 -maximum reproduction number re for each country's outbreak . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint parameter estimates figure s11 reports most likely estimates for the vector of country-specific parameters (θi). the figure also includes 95% credible intervals for these parameter estimates. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint figure s11 -parameter estimates and 95% credible regions for country-specific parameters. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint figure s12 reports country-level projections for new cases and deaths based on the following assumptions (consistent with those reported in figure 2 -d in the main paper). we shift both testing and contact sensitivity to perceived risks to arguably more realistic values between current ones that capture some improvements but not a completely different behavior. specifically, on july 1 st 2020 we shift test rate to a value 20% between the current rates and 0.1% per day (which increases testing in most countries and reduces it in a few). similarly, sensitivity of contacts to perceived risk is shifted to 20% of the way between estimated country-level values and a high value of 8 that is. figure s12 -country level projections until spring 2021. daily cases (in thousands, blue, left axis) and daily deaths (red, right axis) are graphed. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 26, 2020. . https://doi.org/10.1101/2020.06.24.20139451 doi: medrxiv preprint spread of sars-cov-2 in the icelandic population clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (covid-19): a multi-center study in wenzhou city detection of sars-cov-2 in different types of clinical specimens sensitivity of chest ct for covid-19: comparison to rt-pcr stability issues of rt-pcr testing of sars-cov-2 for hospitalized patients clinically diagnosed with covid-19 weather conditions and covid-19 transmission: estimates and projections. medrxiv clinical characteristics of coronavirus disease 2019 in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention covid-19 exacerbating inequalities in the us suppression of covid-19 outbreak in the municipality of vo estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship universal screening for sars-cov-2 in women admitted for delivery incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data modeling the rework cycle: capturing multiple defects per task data analysis using regression and multilevel/hierarchical models accelerating markov chain monte carlo simulation by differential evolution with self-adaptive randomized subspace sampling most equations are self-explanatory. the "[…]" notation is used to subscript variables over a set of members. for example, the subscript "rgn" is used to identify different countries. therefore [rgn] indicates that a variable is defined separately for each member of the set "rgn". other subscript ranges used in the equations are: expnt: used for numerically solving the probability of missing symptoms equation. pdim: used for setting policy levels for a few variables. priors: used for implementing the random effects estimation components. each estimated parameter is mapped into an element of this subscript to simplify vector-based calculations tststs: the test status including those confirmed ('tested') and those unconfirmed complete equations and units potential test demand from susceptible population[rgn] , positive candidates interested in testing poisson subset adj active test rate[rgn] = if then else ( time < new testing time , datatestrate[rgn] , external test rate initial( inputave[priorendoave] * ( 1 -sw endoave ) + sw endoave * calcave activities allowed by government additional asymptomatic post detection[rgn] = weighted infected post detection gate[rgn] * additional asymptomatic relative to symptomatic[rgn] / ( 1 + additional asymptomatic relative to symptomatic additional asymptomatic relative to symptomatic[rgn] = zidz ( total asymptomatic fraction net[rgn] -exp ( -covid acuity[rgn] ) , 1 -total asymptomatic fraction net all recovery[rgn] = recovery of confirmed[rgn] + recovery of untested[rgn] + sum ( hospital discharges expected positive poisson covid patients[rgn] + sqrt ( expected positive poisson covid patients[rgn] ^ 2 + 4 * effective hospital capacity[rgn] * effective hospital capacity[rgn] ) ) / ( 2 * effective hospital capacity allocated fration noncovid hospitalized[rgn] = smoothi ( allocated fraction covid hospitalized = min ( maxalp , ialp * alpr = min ( 1, talp * alpr[rgn] ) units: dmnl 17) alpr area of region[rgn] = get vdf constants(constant data file average acuity of positively tested[rgn] * xidz ( ( 1 -probability of missing acuity signal at hospitals[rgn,tested] * fraction poisson not hospitalized[rgn,tested] ^ 2) , 1 -fraction poisson not hospitalized average acuity of untested poisson subset[rgn] * ( 1 -probability of missing acuity signal at hospitals[rgn,notest] * fraction poisson not hospitalized[rgn,notest] ^ 2) , 1 -fraction poisson not hospitalized average acuity not hospitalized[rgn,notest] = zidz ( average acuity not hospitalized poisson[rgn,notest] * infectious not tested or in hospitals poisson average acuity not hospitalized[rgn,tested] = average acuity not hospitalized poisson average acuity not hospitalized poisson[rgn,tested] = max ( 0, probability of missing acuity signal at hospitals[rgn,tested] * average acuity of positively tested[rgn] * fraction poisson not hospitalized probability of missing acuity signal at hospitals[rgn,notest] * average acuity of untested poisson subset[rgn] * fraction poisson not hospitalized -prob missing symptom[rgn] * fraction interested not tested[rgn] ^ 2) , 1 -fraction interested not tested[rgn] , 2 * prob missing symptom average acuity of untested poisson subset[rgn] = zidz ( poisson subset reaching test gate[rgn] * covid acuity[rgn] -positive tests of infected[rgn] * average acuity of positively tested[rgn] , poisson subset not tested passing gate positive candidates interested in testing poisson subset adj[rgn] -potential test demand from susceptible population[rgn] , positive candidates interested in testing poisson subset adj base fatality rate for unit acuity base fatality rate for unit acuity net[rgn] = initial( base fatality rate for unit acuity baseerror = 5 units: person baseline daily fraction susceptible seeking tests demographic impact on fatality relative to china[rgn] * base fatality rate for unit acuity net baseline risk of transmission by asymptomatic[rgn] = initial( baseline transmission multiplier for untested symptomatic * multiplier transmission risk for asymptomatic net baseline transmission multiplier for untested symptomatic = 1 units: dmnl bed per square kilometer[rgn] = initial( nominal hospital capacity[rgn] / area of region beds per thousand population[rgn] = get vdf constants(constant data file zidz ( lnymix deaths of symptomatic untested[rgn] -post mortem test rate[rgn] * frac post mortem from untreated chronic death rate[rgn] = get vdf constants(constant data file chronic impact on fatality[rgn] = initial cml death frac in hosp[rgn] = xidz ( cumulative deaths at hospital cml death fraction in hospitals large enough = sum ( if then else ( cml death frac in hosp cml known death frac hosp[rgn] = xidz ( cumulative deaths at hospital cmlterrpw = 2 units: dmnl cmltpenaltyscl = 0 units: dmnl constant data file :is: 'covidmodelinputs -constantdata contacts relative to normal[rgn] = min ( voluntary reduction in contacts[rgn] , activities allowed by government reaching testing gate[rgn] -symptomatic infected to testing[rgn] -untested symptomatic infected to hospital excess death start count[rgn] = :na:, 0, if then else ( time >= excess death start count flu acuity * covid acuity relative to flu net covid acuity relative to flu net[rgn] = initial( covid acuity relative to flu total covid hospitalized[rgn] , infectious not tested or in hospitals poisson[rgn] + infectious confirmed not hospitalized[rgn] + total covid hospitalized cumulative cases[rgn] = integ( new cases[rgn] , 0) units: person cumulative confirmed cases[rgn] = infectious confirmed not hospitalized[rgn] + hospitalized infectious[rgn,tested] + cumulative deaths of confirmed confirmed recovered[rgn] + cumulative recovered at hospitals cumulative death fraction cumulative deaths at hospital[rgn,tststs cumulative deaths of confirmed[rgn] = cumulative deaths at hospital[rgn,tested] + cumulative deaths of confirmed untreated cumulative deaths of confirmed untreated[rgn] = integ( deaths of confirmed cumulative deaths untested untreated cumulative fraction total cases hospitalized[rgn] = zidz ( sum ( cumulative deaths at hospital[rgn,tststs!] + cumulative recovered at hospitals[rgn,tststs!] + hospitalized infectious[rgn,tststs!] ) , cumulative cases cumulative missed death[rgn] = integ( count missed death[rgn] , 0) units: person cumulative negative tests cumulative recovered at hospitals[rgn,tststs] = integ( hospital discharges cumulative recoveries[rgn] = integ( all recovery cumulative tests conducted cumulative tests data current test rate per capita dalp = 0.1 units: dmnl data excess deaths[rgn] = get vdf constants(constant data file , 'dataconstants[rgn]', 15) units: person data pseudo case fatality raw: := datacmltinfection raw: := datacmltdeath raw: units: person/day raw: := dataflowinfection raw: := dataflowdeath raw: := datatestrate raw: units: person/day cumulative confirmed cases new testing time ) ) ) units: person/day datalimitfromtime = if then else ( time > max time data used , 0, 1) units: dmnl = 365 units: day/year death rate[rgn] = deaths of confirmed[rgn] + deaths of symptomatic untested[rgn] + sum ( hospitalized infectious deaths tested untreated resolution[rgn] * fatality rate untreated post-detection phase resolution time" * fatality rate untreated delay order = 1 units: dmnl demographic impact on fatality relative to china[rgn] = get vdf constants(constant data file discount rate annual = 0.03 units: 1/year discount rate per day = initial( discount rate annual / days per year ) units: 1/day dread factor in risk perception dread factor in risk perception net[rgn] = if then else ( response policy time on < time , dread factor policy * response policy weight[dfcp] + ( 1 -response policy weight[dfcp] ) * dread factor in risk perception[rgn] , dread factor in risk perception dread factor policy = 3000 units: dmnl effective hospital capacity[rgn] = nominal hospital capacity[rgn] * normalized hospital density[rgn] ^ impact of population density on hospital availability excess death end count[rgn] = get vdf constants(constant data file excess death mean frac = 0.9 units: dmnl excess death range frac = 0.2 units: dmnl zidz ( cumulative missed death[rgn] -excess death mean frac * data excess deaths[rgn] , excess death range frac * data excess deaths excess death start count[rgn] = get vdf constants(constant data file expected positive poisson covid patients[rgn] = sum ( potential hospital demand[rgn,tststs!] ) * "post-detection phase resolution time" units: person 122) expnt external test rate[rgn] = population[rgn] * policy test rate utility from normal activities[rgn] / ( utility from limited activities[rgn] + utility from normal activities units: dmnl baseline fatality multiplier[rgn] * impact of treatment on fatality rate[rgn] * average acuity hospitalized baseline fatality multiplier[rgn] * average acuity not hospitalized poisson[rgn,tststs] ^ sensitivity of fatality rate to acuity net final test rate per capita[rgn] = initial( current test rate per capita[rgn] + weight max in test goal * ( max test rate per capita -current test rate per capita final time = 250 units: day flu acuity relative to covid deaths of symptomatic untested fraction covid death in hospitals previously tested[rgn] = zidz ( hospitalized infectious deaths fraction covid hospitalized positively tested[rgn] = zidz ( hospitalized infectious fraction interested not tested[rgn] = 1 -zidz ( total test on covid patients[rgn] , positive candidates interested in testing poisson subset fraction interseted not correctly tested[rgn] = 1 -( 1 -fraction interested not additional asymptomatic relative to symptomatic[rgn] / ( 1 + additional asymptomatic relative to symptomatic fraction of fatalities screened post mortem[rgn] = indicated fraction post mortem testing[rgn] * switch for government response fraction of population hospitalized for covid[rgn] = total covid hospitalized fraction poisson not hospitalized[rgn,tested] = exp ( -average acuity of positively tested[rgn] * ( 1 -probability of missing acuity signal at hospitals fraction poisson not hospitalized[rgn,notest] = exp ( -average acuity of untested poisson subset[rgn] * ( 1 -probability of missing acuity signal at hospitals fraction seeking test fraction tests positive[rgn] = zidz ( positive tests of fraction tests positive data[rgn] = min ( 1, zidz ( dataflowinfection[rgn] , active test rate[rgn] ) ) units: dmnl 149) fractional value of limited activities = 0.5 units: dmnl global cases = sum ( cumulative cases[rgn!] ) units: person global deaths = sum ( cumulative deaths[rgn!] ) units: person global deaths , global cases ) units: dmnl government response start time hazard of symptomatic infection[rgn] = infection rate[rgn] / susceptible[rgn] * ( 1 -total asymptomatic fraction net herd immunity fraction = 0.8 units: dmnl hospital admission infectious[rgn,tststs] = hospital admits all hospital admit ratio[rgn,tststs] = xidz ( hospital admits all hospital admits all[rgn,tested] = hospital demand from tested[rgn] * allocated fraction covid hospitalized hospital admits all[rgn,notest] = hospital demand from not tested[rgn] * allocated fraction covid hospitalized poisson subset not tested passing gate[rgn] * ( 1 -exp ( -average acuity of untested poisson subset[rgn] * ( 1 -pmas unconfirmed for hospital demand positive tests of infected[rgn] * ( 1 -exp ( -average acuity of positively tested[rgn] * ( 1 -pmas confirmed for hospital demand = ( 1 -fatality rate treated[rgn,tststs] ) * hospital outflow covid positive hospital outflow covid positive[rgn,tststs] = hospitalized infectious[rgn,tststs] / hospitalized resolution time units cumulative deaths at hospital hospitalized infectious deaths hospitalized infectious deaths[rgn,tststs] = fatality rate treated[rgn,tststs] * hospital outflow covid positive hospitalized resolution time = 20 units: day zidz ( sum ( hospitalized infectious deaths[rgn,tststs!] ) , sum ( hospital outflow covid positive zidz ( sum ( cumulative deaths at hospital[rgn,tststs!] ) , sum ( cumulative deaths at hospital[rgn,tststs!] + cumulative recovered at hospitals units: day flu acuity * ( prob missing symptom indicated fraction post mortem testing[rgn] = fraction covid death in hospitals previously tested[rgn] ^ sensitivity post mortem testing to capacity indicated risk of life loss[rgn] = min ( 1, switch for government response[rgn] * perceived hazard of infection[rgn] * dread factor in risk perception net[rgn] * pseudocfr / discount rate per day ) units: dmnl symptomatic infected to testing[rgn] -untested symptomatic infected to hospital[rgn] , 0) units: person 180) transmission multiplier presymptomatic[rgn] + infected pre detection[rgn] * transmission multiplier pre detection[rgn] + ( additional asymptomatic post detection[rgn] + "poisson not-tested asymptomatic transmission multiplier for confirmed[rgn] + sum ( hospitalized infectious[rgn,tststs!] * transmission multiplier for hospitalized[rgn,tststs!] ) ) * reference force of infection infectious not tested or in hospitals poisson constant data file , 'dataconstants[rgn]', 1) units: person 187) initial time = 30 units known death fraction in hospitals large enough = sum ( if then else ( cml known death frac hosp[rgn!] < minhspdtreshadv , 1, 0) * advcntrs liver disease impact on fatality[rgn] = initial( ( liver disease rate liver disease rate[rgn] = get vdf constants(constant data file , 'dataconstants[rgn]', 18) units: dmnl max test rate per capita = 0.001 units: 1/day max time data used = 247 units: day 200) maxalp = 1 units: dmnl units: person maxrtresh = 8 units: dmnl constant data file , 'meanchronic', 1) units: dmnl constant data file , 'meanliver', 1) units: dmnl constant data file , 'meanobesity', 1) units: dmnl min contact fraction minadjt = 1 units: day multiplier recent infections to test multiplier transmission risk for asymptomatic multiplier transmission risk for asymptomatic net[rgn] = initial( multiplier transmission risk for asymptomatic + alp negative test results new cases[rgn] = infection rate new testing time = 250 units: day nominal hospital capacity[rgn] = initial( initial population[rgn] * beds per thousand population[rgn] / 1000 ) units: person normalized hospital density[rgn] = initial( bed per square kilometer[rgn] / reference hospital density ) units: dmnl not too few susceptibles = sum ( if then else ( suscfrac[rgn!] < minsusctresh , 1, 0) ) units: dmnl sens obesity impact net infection rate[rgn] , incubation period ,0, delay order ) units: person/day 229) onset to detection delay = 5 units overall death fraction[rgn] = zidz ( death rate[rgn] , all recovery[rgn] ) units: dmnl patient zero arrival[rgn] = if then else ( time < patient zero arrival time patient zero arrival time units: person 234) payoff = 0 units: dmnl 235) pdim : tstp,dfcp,dgtp,scup perceived hazard of infection[rgn] = ( weight on reported probability of infection[rgn] * reported hazard of infection[rgn] + ( 1 -weight on reported probability of infection[rgn] ) * hazard of symptomatic infection indicated risk of life loss[rgn] -perceived risk of life loss[rgn] ) / if then else ( indicated risk of life loss[rgn] > perceived risk of life loss pmas confirmed for hospital demand[rgn] = ( 1 -reference covid hospitalization fraction confirmed[rgn] ) ^ ( 1 / average acuity of positively tested pmas confirmed for hospital demand[rgn] + ( 1 -pmas confirmed for hospital demand[rgn] ) * untested pmas gap with tested infectious not tested or in hospitals poisson[rgn] * exp ( -average acuity not hospitalized poisson poisson subset not tested passing gate[rgn] = poisson subset reaching test gate[rgn] -positive tests of infected poisson subset reaching test gate[rgn] = reaching testing gate[rgn] / ( 1 + additional asymptomatic relative to symptomatic policy test rate[rgn] = if then else ( time < new testing time , current test rate per capita[rgn] , final test rate per capita infected unconfirmed post-detection"[rgn] + susceptible[rgn] + recovered unconfirmed[rgn] + confirmed recovered[rgn] + infectious confirmed not hospitalized[rgn] + "pre-symptomatic infected positive candidates interested in testing poisson subset[rgn] = poisson subset reaching test gate[rgn] * fraction seeking test positive candidates interested in testing poisson subset adj[rgn] = max ( 0.001 * potential test demand from susceptible population[rgn] , positive candidates interested in testing poisson subset positive testing of infected untreated[rgn] = positive tests of infected[rgn] * fraction poisson not hospitalized positive tests of infected[rgn] = positive candidates interested in testing poisson subset[rgn] * ( 1 -fraction interseted not correctly tested post mortem test rate[rgn] = post mortem tests total[rgn] * sensitivity of covid test units post mortem test untreated[rgn] = post mortem test rate[rgn] * frac post mortem from untreated deaths of symptomatic untested[rgn] + hospitalized infectious deaths[rgn,notest] ) * fraction of fatalities screened post mortem post mortem tests total[rgn] = min ( post mortem testing need[rgn] , active test rate[rgn] ) units: person/day 255 hospitalized infectious[rgn,notest] / minadjt , post mortem test rate[rgn] * ( 1 -frac post mortem from potential hospital demand[rgn,notest] = hospital demand from not tested potential hospital demand[rgn,tested] = hospital demand from recovered unconfirmed[rgn] + cumulative recovered at hospitals[rgn,notest] ) * ( baseline daily fraction susceptible seeking tests[rgn] * fraction seeking test[rgn] + multiplier recent infections to test infection rate[rgn] + patient zero arrival[rgn] -onset of symptoms[rgn] , 0) units: person 261) priorendoave : upadj mtrasym 264) priors : upadj prob missing symptom probability of missing acuity signal at hospitals[rgn,tested] = zidz ( ln average acuity of untested poisson subset[rgn] ) + 1 units: dmnl 268) pseudocfr units: dmnl effective reproduction rate[rgn] = zidz ( infection rate[rgn] , total weighted infected population reaching testing gate realistic r0 = sum ( if then else ( r effective reproduction rate recovery of untested[rgn] , 0) units: person 274) recovery of confirmed[rgn] = tested untreated resolution[rgn] * ( 1 -fatality rate untreated post-detection phase resolution time" ) -deaths of symptomatic untested reference covid hospitalization fraction confirmed units: person/(km*km) regionalinputs[rfi,rgn] = reference force of infection sensitivity post mortem testing to capacity baseline daily fraction susceptible seeking tests weight on reported probability of infection multiplier recent infections to test min contact fraction confirmation impact on contact impact of population density on hospital availability impact of treatment on fatality rate log ( dread factor in risk perception reference covid hospitalization fraction confirmed base fatality rate for unit acuity net covid acuity relative to flu net sensitivity of fatality rate to acuity net total asymptomatic fraction net sens obesity impact net sens chronic impact net sens liver impact net multiplier transmission risk for asymptomatic net relative risk of transmission by hospitalized = 1 units: dmnl relative risk of transmission by presymptomatic = 1 units: dmnl reported hazard of infection[rgn] = positive tests of infected response policy time on = 1000 units: day response policy weight[pdim] = 0 units: dmnl 307) rgn : albania saveper = 1 units: day sens chronic impact = 1e-06 units: dmnl sens chronic impact net[rgn] = initial( sens chronic impact * ( 1 -sw gen sens liver impact = 1e-06 units: dmnl sens liver impact net[rgn] = initial( sens liver impact * ( 1 -sw gen sens obesity impact = 1e-06 units: dmnl sens obesity impact net[rgn] = initial( sens obesity impact * ( 1 -sw gen senscoviduntestedadmission = 1 units: dmnl sensitivity of contact reduction to utility sensitivity of contact reduction to utility policy * response policy weight[scup] + ( 1 -response policy weight[scup] ) * sensitivity of contact reduction to utility sensitivity of contact reduction to utility policy = 10 units: dmnl sensitivity of covid test = 0.7 units: dmnl sensitivity of fatality rate to acuity sensitivity of fatality rate to acuity net[rgn] = initial( sensitivity of fatality rate to acuity sensitivity post mortem testing to capacity ^ cmlterrpw ) / ( baseerror + datacmltovertime sim pseudo case fatality[rgn] = zidz ( cumulative deaths of confirmed[rgn] , cumulative confirmed cases simcmltovertime[rgn,infection] = cumulative confirmed cases post mortem test rate[rgn] + positive tests of infected post mortem test rate total simulated tests = if then else ( flowresiduals[rgn,series] = :na:, :na:, flowresiduals units: dmnl 0 units: dmnl 342) switch for government response[rgn] = if then else ( time > government response start time symptomatic fraction in poisson[rgn] = initial( 1 -exp ( -covid acuity[rgn] ) ) units: dmnl symptomatic fraction negative symptomatic infected to testing[rgn] = positive testing of infected untreated[rgn] + hospital admission infectious testing capacity net of post mortem tests[rgn] = active test rate positive candidates interested in testing poisson subset[rgn] * symptomatic fraction in poisson[rgn] + potential test demand from susceptible population[rgn] * symptomatic fraction negative testing on living[rgn] = min ( testing capacity net of post mortem tests indicated fraction negative demand tested[rgn] * potential test demand from susceptible population[rgn] , indicated fraction negative demand tested[rgn] * potential test demand from susceptible population[rgn] + indicated fraction positive demand tested[rgn] * positive candidates interested in testing poisson subset tests per million[rgn] = cumulative tests data[rgn] / initial population time step = 0.25 units: day time to adjust testing = 30 units: day time to downgrade risk time to downgrade risk policy * response policy weight[dgtp] + ( 1 -response policy weight[dgtp] ) * time to downgrade risk time to downgrade risk policy = 300 units: day time to herd immunity[rgn] = xidz ( herd immunity fraction * susceptible time to upgrade risk total asymptomatic fraction total asymptomatic fraction net[rgn] = initial( total asymptomatic fraction total covid hospitalized[rgn] = sum ( hospitalized infectious total disease duration = onset to detection delay + "post-detection phase resolution time" + incubation period units: day total simulated tests[rgn] = post mortem tests total[rgn] + testing on living total test on covid patients[rgn] = max ( 0, min ( positive candidates interested in testing poisson subset total to official cases simulated[rgn] = zidz ( cumulative cases[rgn] , simcmltovertime[rgn,infection] ) units: dmnl pre-symptomatic infected transmission multiplier for confirmed[rgn] = initial( baseline transmission multiplier for untested symptomatic * confirmation impact on contact transmission multiplier for hospitalized[rgn,tststs] = initial( baseline transmission multiplier for untested symptomatic * relative risk of transmission by hospitalized * if then else ( tststs = 1, confirmation impact on contact transmission multiplier pre detection[rgn] = initial( baseline transmission multiplier for untested symptomatic * ( 1 -total asymptomatic fraction net[rgn] ) + total asymptomatic fraction net[rgn] * baseline risk of transmission by asymptomatic baseline transmission multiplier for untested symptomatic * relative risk of transmission by presymptomatic ) * ( 1 -total asymptomatic fraction net[rgn] ) + total asymptomatic fraction net[rgn] * baseline risk of transmission by asymptomatic[rgn] * relative risk of transmission by presymptomatic ) units: dmnl active test rate[rgn] units: person/day 378) tststs : tested untested pmas gap with tested[rgn] = ( 1 -allocated fration noncovid hospitalized[rgn] ) ^ senscoviduntestedadmission units: dmnl untested symptomatic infected to hospital[rgn] = hospital admission infectious utility from limited activities[rgn] = exp ( sensitivity of contact reduction to utility net[rgn] * fractional value of limited activities ) units: dmnl utility from normal activities[rgn] = exp ( sensitivity of contact reduction to utility net[rgn] * ( 1 -perceived risk of life loss voluntary reduction in contacts[rgn] = f[rgn] / f0[rgn] * ( 1 -min contact fraction[rgn] ) + min contact fraction zidz ( sum ( average acuity hospitalized[rgn,tststs!] * hospitalized infectious[rgn,tststs!] ) , sum ( hospitalized infectious weather effect on transmission weight max in test goal = 0 units: dmnl infected unconfirmed post-detection"[rgn] + infectious confirmed not hospitalized[rgn] + sum ( hospitalized infectious[rgn,tststs!] ) * "post-detection phase resolution time" / hospitalized resolution time units spread of sars-cov-2 in the icelandic population clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (covid-19): a multi-center study in wenzhou city detection of sars-cov-2 in different types of clinical specimens sensitivity of chest ct for covid-19: comparison to rt-pcr stability issues of rt-pcr testing of sars-cov-2 for hospitalized patients clinically diagnosed with covid-19 weather conditions and covid-19 transmission: estimates and projections. medrxiv clinical characteristics of coronavirus disease 2019 in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention covid-19 exacerbating inequalities in the us estimating the infection and case fatality ratio for coronavirus disease (covid-19) using age-adjusted data from the outbreak on the diamond princess cruise ship estimating the infection fatality rate among symptomatic covid-19 cases in the united states. health aff (millwood) suppression of covid-19 outbreak in the municipality of vo estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship universal screening for sars-cov-2 in women admitted for delivery incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data modeling the rework cycle: capturing multiple defects per task data analysis using regression and multilevel/hierarchical models accelerating markov chain monte carlo simulation by differential evolution with self-adaptive randomized subspace sampling an interactive web-based dashboard to track covid-19 in real time simulation-based estimation of the early spread of covid-19 in iran: actual versus confirmed cases estimates of the severity of coronavirus disease 2019: a model-based analysis. the lancet infectious diseases serial interval of covid-19 among publicly reported confirmed cases serial interval of novel coronavirus (covid-19) infections an interactive web-based dashboard to track covid-19 in real time key: cord-018335-4l7scdqk authors: kiechle, frederick l.; arcenas, rodney c. title: utilization management in a large community hospital date: 2016-12-01 journal: utilization management in the clinical laboratory and other ancillary services doi: 10.1007/978-3-319-34199-6_14 sha: doc_id: 18335 cord_uid: 4l7scdqk the utilization management of laboratory tests in a large community hospital is similar to academic and smaller community hospitals. there are numerous factors that influence laboratory utilization. outside influences like hospitals buying physician practices, increasing numbers of hospitalists, and hospital consolidation will influence the number and complexity of the test menu that will need to be monitored for over and/or under utilization in the central laboratory and reference laboratory. clia’88 outlines the four test categories including point-of-care testing (waived) and provider-performed microscopy that need laboratory test utilization management. incremental cost analysis is the most efficient method for evaluating utilization reduction cost savings. economies of scale define reduced unit cost per test as test volume increases. outreach programs in large community hospitals provide additional laboratory tests from non-patients in physician offices, nursing homes, and other hospitals. disruptive innovations are changing the present paradigms in clinical diagnostics, like wearable sensors, maldi-tof, multiplex infectious disease panels, cell-free dna, and others. obsolete tests need to be universally defined and accepted by manufacturers, physicians, laboratories, and hospitals, to eliminate access to their reagents and testing platforms. this chapter will review the numerous factors that infl uence laboratory test utilization in a large community hospital (table 14 .1 ). assessment of laboratory test utilization usually relies on data compiled after a utilization review or analysis of the necessity, appropriateness, and effi ciency of laboratory tests on a concurrent and/or retrospective basis. most laboratory utilization studies have been reported from academic medical centers [ 1 -8 ] , however, there are often common fi ndings in large community hospital settings [ 9 -13 ] . the major difference between an academic center and community healthcare system is usually the number of hospital-employed physicians versus independent physicians with their own offi ce practices. some large community hospitals have only employed physicians on their medical staff like kaiser permanente, henry ford hospital, and others. generally, it will be easier to assemble a group of specialists to convene a laboratory utilization committee meeting, if the physicians are accustomed to leaving their practice responsibilities and going to a hospital-oriented committee meeting in the middle of the day. since the focus of this committee is to review the evidence-based evaluation of a new or old laboratory test to rule in or out a specifi c disease, attendance and the quality of participation will vary depending on the physician's commitment to the project. in a large community hospital, it may "save time" during these meetings if homework is assigned beforehand. "this includes what do people need to read, think about, bring with them, or come prepared to discuss so the meeting will be more productive" [ 14 ] . one of the authors (flk) has worked for 23 years directing the clinical laboratory and outreach laboratory at a large community hospital (william beaumont hospital) in royal oak, mi and for the past 9 years at a six hospital county healthcare system (memorial healthcare system) in hollywood, fl with the co-author (rca). we will review several of the issues listed in table 14 .1 . in preparation for the shift from fee-for-service to a valuebased payment system [ 15 ] large community hospitals have been actively engaged in three enterprises which will impact laboratory test utilization: buying physician practices, increasing the use of hospitalists and consolidation of hospitals. in the early 1990s during the initiation of health maintenance organizations (hmos) , hospitals purchased physician practices. in general, at that time, hospitals had a diffi cult time managing the physicians and their practices. during this second more recent phase of buying, contracts are designed to enhance physician productivity [ 16 -19 ] . the "key motivation for hospital acquisition of physician practices is the ability to gain market share for inpatient admissions and outpatient services by capturing referrals from physicians employed by the owned practices" [ 16 ] . carlin et al. [ 16 ] documented a shift in inpatient admissions, outpatient ct scans and mri procedures from three large multispecialty clinic systems to a two hospital-owned integrated delivery system (ids) after the ids purchased them. this same shift of referral patterns to the new hospital owner will also be true for laboratory tests ordered by newly acquired physician practices. in the usa 57 % of physicians were independent in 2000 compared to 39 % in 2012 [ 19 ] and 36 % of male and 23 % of female physicians in 2015 [ 17 ] . in 2004, 11 % of physicians were employed by hospitals compared to 64 % in 2014 [ 18 ] . a downside of this trend is the fi nding that hospitals charge more when the doctors work for them attributing the cause to higher overall costs [ 19 ] . this current trend should drive more laboratory testing from new hospitalbased physicians to the hospital central laboratory with the consequence of potential utilization issues. the hospitalist model of inpatient care is one of the most rapidly growing forms of medical practice in the usa since its introduction in the mid-1990s [ 20 , 21 ] . in 2006, there were more than 12,000 hospitalists in the usa [ 20 ] which has increased to 34,000 in 2014 [ 21 ] . most hospitalists practice in hospitals with greater than 200 beds [ 20 , 21 ] . their average starting salary was greater than that for internal medicine or family practice physicians in 2014 [ 18 ] . hospitalists work strictly in the hospital and oversee the care of complex patients with the goal of reducing the need of transferring patients from one physician to another [ 22 ] . most large community hospitals use a voluntary hospitalist system in which primary care physicians can choose to admit to a hospitalist service or attend to their own patients [ 23 ] . large community hospitals are more likely to adopt a hospitalist model if their case mix complexity was greater than the national average for medicare's diagnosis rated group index, while high health maintenance organization market share resulted in lower interest in this model [ 22 ] . the hypothesis that the hospitalist model will lead to a reduction in the patient's length of stay and total hospital costs has been demonstrated [ 20 , 22 -24 ] . hospitalists may order excessive diagnostic tests secondary to their lack of previous knowledge of the patient [ 20 ] . the choosing wisely campaign sponsored by the american board of internal medicine foundation, consumer reports, and more than 60 specialty societies have recommended reduction or elimination of inappropriate use of radiologic, laboratory, and therapeutic procedures [ 12 , 25 -27 ] . the fi rst 25 societies provided fi ve selections each, of which 12 % were related to laboratory tests or pathology [ 27 ] . one of these lists from the society of hospital medicine recommended reducing the use of repetitive common laboratory testing when the patient is clinically stable [ 26 ] . in a quality improvement project focused on hospitalists, an effort was made through education to reduce the repetitive use of complete blood counts and basic metabolic panels [ 25 ] . these panels are often embedded in order sets established for specifi c diseases or for specifi c physicians to simplify computerized physician order entry [ 28 ] . there was a 10-month baseline period before the intervention followed by a 7-month intervention period [ 25 ] . the intervention resulted in a 10 % reduction of these two panels ordered per patient day associated with decreased direct costs of $16.19 per patient and annualized savings of $151,682 [ 25 ] . this study illustrates how a small segment of laboratory test ordering physicians can impact expenses through overutilization and by analogy underutilization of laboratory tests. like the fi rst round of hospitals buying physician practices started in the early 1990s, so did the consolidation or mergers of hospitals [ 29 ] . there has been a recent increase in both horizontal and vertical consolidation [ 29 , 30 ] . horizontal consolidation involves hospitals merging with other hospitals that supply similar services in geographic proximity [ 29 , 30 ] . these mergers are most likely to be investigated for antitrust violations [ 29 ] . vertical consolidations involve hospitals consolidating with other health care provider entities [ 16 , 29 ] . from 2007 to 2012, 432 hospital mergers and acquisitions were announced involving 835 hospitals [ 30 ] . sixty percent of hospitals are now part of health systems. the downside to these mergers has been a 10-40 % increase in prices secondary to increased market share [ 30 ] . strategies have been suggested for avoiding this market disequilibrium [ 30 , 31 ] . no us hospital markets were rated highly competitive [ 30 ] while the german market is competitive and the number of hospital systems decreased by 18 % from 2000 to 2007 [ 32 ] . there are myths associated with the latest hospital merger activity. the fi rst myth is that consolidation is equivalent to integration [ 33 ] . the second is that higher quality is associated with size rather than leadership and competition [ 33 , 34 ] . evidence supports the suggestion that hospitals in competitive markets tend to have better administrative management [ 33 ] . the combination of hospital mergers and increased hospital-employed physicians [ 35 ] will lead to increase in laboratory test volumes and the need for robust utilization management practices. the clinical laboratory improvement amendment of 1988 (clia'88) went into effect september 1, 1992. the regulations categorize laboratory procedures based on test complexity using well-defi ned criteria: waived, moderately complex, and highly complex or provider-performed microscopy . these regulations defi ne the universe of tests that a large community hospital laboratory is directly or indirectly responsible for their utilization management. there are a variety of clia-tests that have been classifi ed as waived by the fda and a list of them from 2000 to present can be found at www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfclia/ testswaived.cfm . the implementation of point-of-care testing (poct) usually involves a desire to decrease the total turn-around time for an analytical test and improve patient outcome [ 36 ] . however, decreased turn-around time does not always equal improved patient outcome [ 37 , 38 ] . two prospective studies analyzed the effect of the poct i-stat device (abbott, abbott park, il) on length of stay in the emergency department after a control period when the central laboratory was used. the i-stat cartridge that analyzed sodium, potassium, chloride, urea, glucose and calculated hemoglobin was used. neither study revealed any change in the length of stay or clinical outcome for emergency department patients, although both showed a decrease in time required to obtain laboratory results for the six tests on the cartridge when the istat was used. the blood test was not the rate-limiting step in the patient's length of stay [ 38 ] . in a similar study using fi ve different testing cartridges for the istat (inr, lactate, brain natriuretic peptide, troponin t and chemistry with hemoglobin and hematocrit), singer et al. [ 39 ] reported a reduced turn-around time for poct compared to the central laboratory which translated into a reduction in time to completion of iv contrast ct and the length of stay of those specifi c patients. it is not clear whether relocating poct tests to the patient's bedside increases [ 40 , 41 ] or decreases [ 42 ] the test volume of that test in the central laboratory. it has been reported in both adult and newborn intensive-care units that patients with indwelling arterial lines have more blood drawn for laboratory studies than patients without arterial lines [ 43 , 44 ] . the blood loss may be great enough to require a transfusion [ 43 ] . certainly, utilization management of poct programs will require investigations to determine the relationship between total laboratory turn-around time for results, patient outcome and hospital costs using cost effectiveness analyses [ 36 ] . there are at least 17 different sources of body fl uids in a human [ 45 ] . some of these are laboratory specimens that are examined under the bright-fi eld or phase contrast microscope and are classifi ed as provider-performed microscopy (ppm) by clia'88 [ 46 , 47 ] . a separate cms license is available for sites performing these assays which include koh preparation, pinworm detection, fern test, microscope urinalysis, semen analysis for presence of sperm and motility, and eosinophils in nasal smears [ 46 , 47 ] . it is wise for the laboratory poct administrative committee to assist in the initiation of a ppm testing program in collaboration with the ppm license holder for that clinically defi ned program, like fern testing in the labor and delivery area under the direction of a staff ob/gyn physician. in this model, training and utilization management is not under the direction of the hospital poct administrative committee [ 46 , 47 ] . in order to audit laboratory test utilization year to year it is "essential to understand how test volumes are actually counted" [ 1 ] . are test panels bundled and counted as one test or are the test panels unbundled and each test in the panel counted separately. in an evaluation of the total inpatient test volumes from 1978 to 2000 for the department of clinical pathology at william beaumont hospital, royal oak, mi, it was determined that the method for counting inpatient tests changed in 1992 and 1996 [ 36 , there are two categories for cost reductions : "hard" cost savings and "soft" cost avoidance. tangible "hard" cost savings are often achieved by bringing reference laboratory tests inhouse to the clinical laboratory (or eliminating the reference laboratory test altogether) [ 4 , 11 ] . the more intangible "soft" cost avoidance includes such things as decreases costs associated with the introduction of a new laboratory test with the intent to decrease costs in the future. it also can occur when a cost is lower than the original expense that would have otherwise been required if the cost avoidance exercise had not been undertaken. since processes consume overhead and overheard costs money, any signifi cant process improvement could represent signifi cant cost avoidance for an organization. total cost includes direct and indirect costs [ 48 ] . direct cost includes personnel time to prepare and perform the test, reagents, quality control, profi ciency testing, and equipment depreciation. indirect costs including reporting costs (computer) and hospital overhead. incremental or marginal costs include only variable direct costs and not the indirect costs [ 48 -50 ] . therefore, incremental costs demonstrate what it would cost to perform one more laboratory test, assuming the equipment and facility are already available. neither total cost analysis nor incremental cost analysis includes an analysis of the defect rate or failure to achieve established goals, like turn-around time [ 50 ] . they are defi ned as internal or external failure rates. internal failure costs are incurred by the testing center as a consequence of a defect in the testing system. the receiver of the test results incurs the external failure costs. over utilization of laboratory tests incurs both internal and external failure costs in the excess time spent in the laboratory to generate the result and then excess insurance charges to the patient and nursing/physician time to evaluate the results. to illustrate cost avoidance , consider the presentation of potential enterovirus meningitis in the emergency department. children and adults with detectable enterovirus in the cerebrospinal fl uid (csf) may exhibit symptoms of meningitis including photophobia, stiff neck, acousticophobia , severe headache with vomiting, confusion, diffi culty concentrating, seizure and sleepiness. a molecular test for enterovirus detection will alter the patient's length of stay in the emergency department. if the patient is positive for enterovirus in the csf specimen, the patient will be discharged for home care until the viral meningitis resolves (table 14. 2 ). if the patient does not have enterovirus in the csf, they will need further hospitalization to rule out a bac-terial source for the meningitis with culture and sensitivity studies (table 14. 2 ). romero [ 51 ] has demonstrated the cost range for hospitalization related to enterovirus testing/care of infection to be $4476-4921 with an average length of stay of 3-4 days. we used $4476 for the calculation in table 14 .2 , which illustrates a cost avoidance of $187,992 for 20 patients with or without enteroviral detection by molecular methods. "cost per unit went down if you could make longer and longer runs of identical products. this gave rise to the theory of economies of scale " [ 52 ] . the laboratory achieves economies of scale and lower unit costs per test by expanding the volume of laboratory tests it analyzes. in the early 1990s, the number of inpatient laboratory tests at william beaumont hospital began to decrease. to fi ll the gap, after a 3-year preparation period, we initiated an outreach program (beaumont reference laboratory) expanding our laboratory testing services to non-patients from physician offi ces, nursing homes, and other hospitals [ 53 , 54 ] . several years later (1992) beaumont reference laboratory joined a regional laboratory network of other hospital-based laboratory outreach programs in michigan, joint venture hospital laboratories , to accommodate the wide geographic coverage required by third party payers [ 53 , 54 ] . the participating laboratories are independently owned and operated. a central network administrator coordinates negotiations for managed care contracts. the volume of brl specimens grew to half of the total volume of clinical pathology procedures of six million tests in 2004. this increased volume permitted an expansion of the test menu in each laboratory section. in 2002, we reviewed 2,976,494 procedures ordered by 2806 physicians in nine subspecialty areas (family practice, pediatrics , internal medicine, cardiology, endocrinology, gastroenterology, nursing home, ob/gyn, and urology). the requisitions for physician, procedures per requisition and procedures/physician were calculated for each of the nine groups of physicians [ 54 ] . family practice (464 physicians) and internal medicine (831) ordered the greatest number of total procedures as well as procedures/physician while urology (126) ordered the least of these two categories [ 54 ] . the tests ordered by each of the nine groups were counted in seven laboratory sections. all nine groups ordered more chemistry tests than any other category but the percent varied from 34.5 % for ob/gyn to 90.2 % for internal medicine. the most popular individual tests in fi ve laboratory sections (chemistry, hematology, immunology, microbiology, and molecular diagnostics) were calculated as an average number of a specifi c test ordered per physician per month. using this data, a laboratory section could prepare themselves for the increased utilization from a six-member internal medicine group that the beaumont reference laboratory sales force just signed up as a new client. this type of deep dive into specifi c physician specialty ordering patterns is an invaluable resource for managing a growing outreach business [ 54 ] . the 20 million requests for chemistry, hematology, and microbiology tests were included for all physicians in calgary, canada who ordered a test in fi scal year 2013-2014 [ 55 ] . the physicians were divided into 30 subspecialties and the average yearly cost per group and average yearly cost per physician in each of the 30 groups was calculated. family practice and internal medicine had the greatest average yearly cost per group while hematology and nephrology had the highest average yearly cost per physician per group secondary to utilization of more expensive laboratory tests [ 55 ] . this cost-based approach to utilization review requires the calculation of an average median cost for each test which in the usa would be much less than the price listed on the hospital's charge master. there was a synergistic relationship between the growth of beaumont reference laboratory and test mix complexity in each laboratory section. in the molecular diagnostics laboratory started in 1992, chlamydia trachomatis (ct) nisseria gonorrhoeae (ng) were performed in urine using the ligase chain reaction in 1996 and pcr in 2002 [ 56 , 57 ] . more than 75 % of the requests for these two assays are from brl clients (table 14. 3 ). the increased volume of these two assays helped turn the molecular diagnostic section into a profi t center in 4 years. annual utilization review revealed that the outreach program contributed 33,019/43,814 = 75 % of the volume, hospital a (8624/43,814 = 20 %) and hospital b (2181/43,814 = 5 %). the multiplex assay using primarily urine specimens made a margin of $72.00 per assay billed (at that time) based on an average medicare reimbursement ($83.00) and cost/test of $11.00. why was there an exceptionally low ng volume from hospital b? after an investigation it was learned that hospital b chose to do the less sensitive ng culture assay [ 56 ] to retain laboratory test volume which was encouraged by their hospital administration. also, a myth existed at hospital b that ng would not survive the transport time (40-60 min) to hospital a. the ordering physicians at hospital b prevailed and the request for molecular detection of ng at hospital b was followed. this case illustrates just how complex problem solving in utilization management issues can be in a large community hospital. some new technologies are defi ned as disruptive innovations , when they offer new paradigms in diagnostics (table 14 .4 ) [ 12 , 83 , 84 ] . all seven of the technologies listed in table 14 .4 share similar issues including clarifi cation of the best applications for routine clinical use, paucity of evidence-based outcome literature to review, education of practitioners and physician users of the clinical information generated and software to convert big databases the method generates into useful information. the references in table 14 .4 will direct attention to these issues for the seven disruptive innovations [ 58 -82 ] . as the paradigm shifts and these strategies become incorporated into daily clinical practice, the debate about appropriate utilization will diminish. this next section will devote time to describing the impact of current changes in microbiology ( mass spectrometry for bacterial identifi cation [ 12 , 68 , 69 ] and multiplex molecular panels for infectious agent detection for respiratory viral panels and gastric pathogen panels) and future changes ( microscopy for antibacterial drug sensitivity). traditionally, the laboratory diagnosis of most infectious disease pathogens has relied on culturing and in vitro growth of the causative agent. once culture growth has been achieved, then automated and/or manual biochemical tests can be performed to identify the microbial organism(s). these methodologies are dependent on skilled medical technologists to perform the manual tasks required to determine the bacterial identifi cation (id). the approach to id and antimicrobial susceptibility testing (ast) has been dependent on testing a single pathogen at a time, regardless if the culture [ 81 , 82 ] growth yielded multiple, signifi cant pathogens. although automated id and ast systems can run multiple isolates to help streamline the workfl ow and maximize throughput, the basic testing is still individually performed for each isolate being analyzed. another limiting factor for culture based detection methods is that some bacteria do not grow well or at all in vitro adding to the potential of missing a signifi cant organism(s). viral cultures are time-consuming because cytopathic effects (cpe) must be observed before other methods can be used to determine the viral identifi cation [ 85 -87 ] . the development of viral antigen based testing [direct fl uorescence antigen (dfa) and other rapid antigen testing devices] directly from the sample shortened the culture time to obtain a faster diagnosis. however, the reliable performance of the viral antigen based testing is highly dependent on the quality of the sample collected and is less sensitive than viral cultures [ 85 ] . a suboptimal specimen could lead to a false negative antigen/dfa result. are viral cultures and antigen based testing truly needed in a clinical microbiology/virology laboratory since molecular methods are becoming the new gold standard? [ 85 -87 ] . many routine clinical microbiology/ virology laboratories do not have the capabilities to perform viral cultures lacking the physical space and expertise to interpret the cpe [ 88 ] . as technology advances, the traditionally " agrarian society " of the laboratory is becoming more industrialized with the implementation of automation, molecular based testing, and use of mass spectrometry ( maldi-tof -matrix-assisted laser desorption ionization-time of flight). many of these advances are revolutionizing how microbiology testing is performed and disrupting how traditional clinical microbiology workfl ows and processes are set up. however, all of these technological advances are shortening the time for a laboratory diagnosis and ultimately maximizing the impact to patient care and how physicians at a large community hospital will utilize the more rapid microbiology laboratory services. there is a trend in clinical microbiology to develop syndromic panels using molecular techniques. for example, positive blood culture panels have been developed to reduce time to start the most appropriate antibiotics in the patient. once a blood culture bottle is fl agged as positive, a gram stained smear is prepared to determine the presence of bacteria (and potentially yeast) in the patient's blood sample. if any organism(s) are seen, a call is made to the patient's healthcare provider so that broad-spectrum antimicrobial therapy can be initiated until the confi rmed microbiological id is resulted. a caveat for the clinician is that s/he must make their best educated guess for determining which antibiotic treatment to use. clinical microbiology laboratories typically publish an antibiogram so that clinicians and hospital pharmacists know the prevalence of susceptible and resistant phenotypes for their most common bacteria isolated. although this provides a good start and useful reference, there is a heavy emphasis on antimicrobial stewardship and tailoring therapy as soon as possible so that there is less pressure for the development of antimicrobial resistance . many institutions have developed or are in the process of developing formal antibiotic stewardship programs/committees in an effort to improve the utilization of antimicrobial treatments. molecular methods have been developed that will, within one test, identify a number of pathogens from a positive blood culture sample. these methods have decreased the amount of time to provide a more defi nitive id and an abbreviated antimicrobial resistance genetic profi le. advandx/ biomerieux, inc. utilizes pna-fish (peptide nucleic acidfl uorescent in situ hybridization) and detection of a positive fl uorescent signal directly from a positive blood culture . gram stained smear fi ndings typically are resulted as "gram positive cocci in clusters" or "gram negative rods" (table 14 .5 ). this information is useful in deciding broadspectrum therapy, but a more focused therapy is the ultimate goal to ensure that the pathogen is adequately treated. the pna-fish assays offer four basic assays [ staphylococcus (gram positive), enterococcus (gram positive), gram negative, and candida ] that complements the gram stain result so that clinicians at least have a presumptive genus identifi cation. additional pna-fish probes do have the ability to separate s. aureus /coagulase-negative staphylococcus and a meca probe for the identifi cation of mrsa. use of these pna-fish assays requires a fl uorescent microscope to visualize the results. a number of studies have shown the clinical benefi ts of pna-fish implementation as part of the blood culture workup before the confi rmatory culture growth and potentially identifying methicillin resistance genotype, in the example of s. aureus , before the full antibiotic susceptibilities can be performed [ 89 -93 ] . antibiotic therapy can, therefore, be tailored or deescalated as appropriate. similar to the pna-fish scenario, when utilizing cepheid's xpert mrsa/sa blood culture test the gram stained smear prepared from the positive blood culture smear will determine whether cepheid's assay should be run. cepheid's methodology is real-time pcr based and does not require any subjective interpretation of the results by the laboratory staff. the cartridge for the xpert mrsa/sa test houses all the reagents and is compartmentalized to accommodate the nucleic acid extraction, pcr amplifi cation, and detection in one device. the testing is automated once the sample is loaded into the test cartridge and the software analyzes the pcr amplifi cation curves to determine if a patient's blood sample is positive or negative for mrsa or mssa [ 94 -97 ] . the cepheid and advandx tests must be added to the already existing workfl ow, and serves as another laboratory tool to more quickly determine the pathogen identifi cation and a preliminary ast profi le. nanosphere, inc. and biofire diagnostics, inc. approach testing from positive blood culture bottles by targeting the most common pathogens (bacteria or yeast) that can cause sepsis. this is a unique approach due to the overlap in patient symptoms for a specifi c syndrome or condition. when the symptoms overlap, clinicians have diffi culty defi ning the causative pathogen(s) infecting their patient solely from the clinical picture, and delaying specifi c pathogen-based therapy. use of these syndromic multiplex molecular panels streamlines the testing process to more of a "one-and-done" approach. nanosphere offers different panels: (1) gram positive ( bc-gp panel ); (2) gram negative ( bc-gn panel ); and (3) yeast ( bc-y panel ) and the organism(s) observed from the gram stained smear will determine which panel(s) to run. additional testing with the gram positive and gram negative panels also includes testing for certain antibiotic resistance genes encoding for methicillin ( meca ) and vancomycin ( vana and vanb ) resistance (table 14 .5 ). there are many studies showing overall good performance for the bc-gp panel [ 98 -104 ] , gram negative species and candida spp. panels [ 105 , 106 ] . there are limitations with molecular testing as observed by buchan et al. [ 101 ] . a positive meca target was not able to be assigned due to the presence of a mixed infection. in this case the full antibiotic sensitivity testing is still recommended because the traditional methods test each bacterial pathogen individually. beal et al. [ 102 ] noted that when blood culture infections were caused by one pathogen, there was good performance of the multiplex molecular assays. when polymicrobial blood culture infections were noted, there was only 33 % agreement with the routine cultures. mestas et al. [ 103 ] also noticed a lower percentage agreement for polymicrobial infections when compared to monomicrobial infections. polymicrobial bacteremia is relatively rare, but can potentially be severe [ 107 ] . again, cultures are still required to identify the full antibiotic susceptibility profi le, and to identify pathogens that are not included in the multiplex molecular panels. the filmarray blood culture identifi cation panel (bcid) is another comprehensive panel that covers gram positive, gram negative, and yeast pathogens (table 14 .5 ) and a gram stain is not required. however, it is still good routine practice to perform the gram stain to correlate results with the molecular panel results and the eventual culture testing and antibiotic susceptibility testing. altum et al. [ 105 ] observed that certain pathogens were detected in routine cultures that were not detected in the filmarray panel because those pathogens were not in the molecular panel. so although the comprehensive panel covers the most common pathogens, clinical intuition is still ultimately needed especially when clinical symptoms and other laboratory data point to a bacteremic process in the setting of a negative filmarray panel. overall, these assays show the potential for a decreased tat and a preliminary susceptibility profi le based on the antibiotic resistance genes tested [ 104 , 105 , 108 , 109 ] . the ability to have a more rapid answer that is technically more sensitive and specifi c will have positive downstream effects on patient care and antibiotic stewardship. the impact of these rapid pcr blood culture assays on the clinical end users (infection control, pharmacy, length of stay, and overall hospital costs) is not well defi ned. one study by bauer et al. [ 95 ] demonstrated clinical benefi t after implementing the cepheid xpert mrsa/sa assay for positive blood cultures. a 4-month pre-pcr period was evaluated followed by a 4-month post-pcr period. there was an overall shorter length of stay (6.2 days shorter) and mean hospital costs were $21,387 less than what was observed in the pre-pcr period. infectious disease pharmacists were more effective in deescalating or changing to more specifi c antibiotic therapies compared to the pre-pcr period. benefi ts were gained by having a more rapid and sensitive test. when adopting newer molecular methods, the laboratory must work with their clinical counterparts to determine the clinical utility of a more rapid test. the cost and potential benefi ts of the newer tests may not be warranted if the clinical staff is not able to effectively utilize this information in their workfl ow. even though the downstream benefi ts have been documented and are almost inarguable from a clinical perspective, there are fi nancial and workfl ow impacts to the microbiology/molecular laboratories that implement these assays. as mentioned prior, the gram stain results from the positive blood cultures will help drive the culture workup. thus, there is the time required for a blood culture bottle to alarm as positive, and then the culture time waiting for growth on the culture plates. the use of these molecular methods must be introduced into the workfl ow and will add additional work because culture id and ast methods still must be performed. in the setting of having a continual decrease of incoming medical technologist graduates and an increasing number of laboratorians retiring, this puts the burden of additional testing on the existing staff. another example of a clinically benefi cial, but disruptive test within the laboratory is the development of respiratory pathogen panels. molecular multiplex panels have been developed to target the general syndrome of a respiratory illness. table 14 .6 shows that there are a variety of fda-approved assays available with a varying number of pathogens offered within their respective multiplex assay. each assay also requires varying levels of hands-on-involvement and molecular expertise required by the medical technologist. prior to the implementation of a respiratory virus panel (rvp) in our institution, the only viral testing offered were the rapid antigen immunochromatographic devices for infl uenza a/b and respiratory syncytial virus (rsv). with the addition of offering rvp testing, we are now able to provide our clinicians with a more comprehensive answer of which virus(es) are occurring in their patient. this benefi ts transplant, immunocompromised, and oncology patients who have more frequent respiratory viral infections [ 110 -112 ] . we had serendipitously brought in the rvp before the 2009 h1n1-infl uenza a outbreak which demonstrated the poor performance of the rapid antigen testing [ 113 ] . because of this observation, many laboratories have discontinued their rapid antigen test offerings and now only offer molecular tests. in our laboratory, we have observed a decline in rapid infl uenza and rsa antigen testing (fig. 14.1 ) . the spike in volumes in 2008-2009 was related to the 2009 h1n1 infl uenza a outbreak. we will eliminate these rapid antigen tests in favor of a rapid molecular test for infl uenza and rsv. an algorithm will refl ex a negative rapid molecular infl uenza a/b and rsv test to a more comprehensive viral and/or bacterial panel. when compared to the dfa and/or shell vial culture, a molecular multiplex respiratory virus panel saved time and lowered costs to the patient [ 114 , 115 ] . despite the clinical benefi ts observed, a limitation of molecular based methodologies is that they are batched and can take hours to perform in the laboratory. for example, in our laboratory the testing time is 6-7 h as compared to the 20-min it takes to run the rapid antigen testing. that trade off in time to result is offset by the increase in sensitivity, specifi city, and breadth of viral pathogens discovered. this can be a challenge for clinicians because many times they will want a fast answer for the purposes of triaging or taking action on a patient. however, the question that should be asked of them is whether they want a bad quality, rapid answer or a good quality, not-so-fast answer. biofire diagnostics, inc. has attempted to solve the testing time problem by offering a comprehensive respiratory pathogen panel that tests directly from the respiratory specimen. only one patient can be run on one instrument and the assay time is approximately 1 h. there will be certain institutional settings where this technology will have benefi ts such as an urgent care clinic, smaller community hospital, or within a laboratory that has minimal molecular testing experience. however, for those institutions with a higher volume where batch testing is more optimal, the biofire may not be the best solution. there are other commercial panels that differ in the number of pathogens offered as well as various levels of medical technologist involvement (table 14 .6 ). the decision to implement one of these panels is driven by a myriad of factors such as cost, workfl ow, physician demand, and the technical capabilities of the laboratory staff. whatever respiratory pathogen panel is introduced, remember that the sample testing volume is highly dependent on seasonal variations. figure 14. 2 shows a graph of our volumes over two respiratory virus seasons (august 2013 through april 2015) with our peak volumes occurring over the winter months. this variability can have a signifi cant impact to how microbiology/molecular laboratories are staffed. physician demand, coupled with an increase in testing volumes, may be high enough to warrant increasing the number of runs per day as the staffi ng levels allow, potentially leading to an increase in employee overtime hours. interestingly during fig. 14.1 the decline in the rapid antigen infl uenza and rsv testing volumes in response to the introduction of rvp testing in conjunction with the 2009 h1n1/infl uenza a outbreak the 2014 respiratory virus season, amidst reports of the infl uenza vaccine having suboptimal effi cacy [ 116 ] , we observed a large increase in rvp testing volumes compared to the prior season. thus, one factor that is virtually impossible to control is the antigenic drift/shift of the infl uenza a virus affecting the effectiveness of the current vaccine in use. assay performance may also be affected due to genetic mutations being introduced into the pcr targeted gene regions. with the limitations of culture and antigen based testing , co-infections were greatly under-appreciated for respiratory viral infections. one can expect an increased incidence of co-infections with multiplex molecular panels. figures 14.3 and 14 .4 show our experiences with co-infections among pediatric and adult patients. the clinical signifi cance of these co-infections is not completely understood in relationship to modulation of disease severity [ 117 -119 ] . research is needed to fully understand virus-virus and bacteria-virus co-infections and their interactions with the other pathogens present as well as the pathogen-host interactions . every respiratory virus season, our laboratory publishes a " virogram " that shows the prevalence of viruses currently circulating among the patient population (fig. 14.5a, b ) . infectious gastrointestinal illness is another syndrome targeted by commercial vendors. as of this writing, there are a number of fda-approved assays from luminex, inc. , biofire diagnostics, inc. , becton dickinson diagnostics, inc. , nanosphere, inc. , and genprobe-prodesse (table 14.7 ) . clinicians are often unaware of what pathogens are actually included when they order a stool culture and ova & parasite (o&p) testing [ 120 ] . similar to respiratory illness symptoms, the symptoms of an infectious gastrointestinal (gi) illness overlap also making it diffi cult to ascertain the true pathogen(s) causing the disease. the development of an infectious gi panel that targets bacterial , viral , and parasitic pathogens , provides a more effi cient approach to diagnosis compared to standard practices. the appeal to the clinical microbiology laboratory is the consolidation of culture, antigen, biochemical, and single-molecular analyte testing into one comprehensive panel. the implications to the state and public health laboratories that rely on culture isolates for epidemiological typing and characterization may not be immediately recognized when considering these molecular stool panel tests. because of the improved ability to detect a pathogen(s) with molecular methods as compared to culture, there will be scenarios when the molecular test is positive and the culture growth is negative. it is imperative that clinical laboratories communicate with their state/public health laboratory counterparts to come up with an amenable solution given that there will be discordant molecular and culture results if an isolate is required to be sent to the state/public health laboratory. similar to respiratory co-infections , gi co-infections are also an under-appreciated aspect of disease and pathogenicity. what potentially makes gi co-infections more confusing and would require some additional clinical scrutiny is that some bacteria can be colonizers (i.e., c. diffi cile ) and so the burden of determining clinical signifi cance is left to the clinician. maldi-tof is another technology that shortens the time to result for determining the microbial identifi cation of clinically signifi cant pathogens. the reader is referred to a number of reference review articles on the technology itself [ 121 -126 ] . recently, maldi-tof systems have been made commercially available for use in the clinical microbiology laboratory. how this technology compares with the currently available testing (culture/biochemical and dna sequencing) is outside the scope of this chapter [ 127 -132 ] . maldi-tof does outperform the conventional methods in overall accuracy and time to result. our laboratory has implemented maldi-tof as an identifi cation tool. results are available in approximately 1 day earlier when compared to our traditional culture based testing. branda et al. [ 88 ] found that maldi-tof reduced turn-around time by 1.45 days compared with their traditional testing. a side effect that we have observed is an increased number of calls from clinicians asking for the antimicrobial susceptibility results. these results are available the next day from our automated ast system. in addition laboratories will need to adapt their workfl ow processes when maldi-tof testing is implemented. the upfront cost of instrumentation is high (approximately $200,000), resulting in delayed implementation in some clinical microbiology laboratories. the fi nancial savings are realized in the cost per isolate of running the maldi-tof compared to the cost per isolate for a traditional work-up [ 127 , 133 , 134 ] . there can be reductions in the reagent and laboratory costs when compared to culture/ biochemical methodologies [ 134 , 135 ] . despite the initial capital expenditure to obtain the instrumentation, there are cost-savings after maldi-tof is implemented. if the organism is not in the maldi-tof database, it will need to be confi rmed by traditional methods and/or dna sequencing . another potential limitation is that the defi nitive speciation by maldi-tof can confuse clinician end users when they see a new bacterial genus/species name that they do not readily recognize and may pose challenges in deciding what antibiotics to prescribe. this new defi nitive identifi cation is a result of technological advancements that have a greater ability to further speciate bacteria when only a genus answer may have been given with traditional techniques (i.e., coagulase-negative staphylococcus or enterobacter cloacae complexes). from the laboratory perspective, changes in nomenclature must be updated in the laboratory information system as appropriate when microorganisms undergo taxonomic reclassifi cations. future advancements with maldi-tof technology also will affect the clinical microbiology laboratory workfl ow. studies have preliminarily shown the ability to detect the pathogen directly from a patient sample (i.e., positive blood cultures [ 92 , 135 , 136 ] and urines [ 137 -139 ] ), bypassing the current requirement for testing on a culture isolate. however, it should be stressed that direct sample testing is in the very early stages of development. antibiotic susceptibility testing has also been examined and hrabak et al. [ 140 ] provide an in-depth review of using maldi-tof for these purposes. interestingly, this technology has also been described in identifying the species of ticks potentially minimizing the ectoparasite experience normally required [ 141 ] . technological advancements are focused on shortening the time of pathogen detection so that clinical action can be taken much more quickly. two relatively new companies have been working on methodologies that will further disrupt clinical microbiology practices. t2 biosystems, inc. has recently received fda approval for the detection of fi ve candida species ( c. albicans , c. tropicalis , c. parapsilosis , c. krusei , and c. glaboratoryrata ) direct from the patient's blood sample without prior incubation within a blood culture bottle. the t2 biosystems assay claims to detect these candida species within 5 h. this technology shortens start time for appropriate candidemia treatment. patient mortality is decreased, the earlier treatment is started [ 142 ] . having the ability to identify the particular candida species is critical since c. glaboratoryrata and c. krusei have signifi cant rates of azole resistance [ 143 , 144 ] . similar to the other blood culture tests mentioned above, this technology will not eliminate the need for culture/pcr and antimicrobial susceptibility testing after a blood culture bottle becomes positive. the technology allows for processing of the whole blood sample, since a thermostable mutated dna polymerase that is not affected by inhibitors in whole blood detection is used to amplify dna. detection of any pcr product is done via t2 magnetic resonance technology. ( www.t2biosystems.com ). clinical trials data show an overall sensitivity of 91.1 % with a mean time of 4.4 h for detection and species identifi cation [ 144 ] . the limit of other studies have demonstrated earlier detection and its effect on antimicrobial stewardship [ 145 , 146 ] . because this is a relatively new technology, hospitals and other healthcare institutions are currently determining the most optimal and cost effective way to utilize this technology. this test is not intended as a screening tool, but should be used in a more targeted patient population where candidemia is more signifi cant and more likely to occur. accelerate diagnostics, inc. has developed a methodology for both rapid pathogen identifi cation and antimicrobial susceptibility testing that can purportedly be performed within 5 h ( www.accerlatediagnostics.com ). the company is conducting clinical trials on blood culture pathogen panel at the time of this writing. the technology utilizes fish dna probes to identify the panel pathogens that may be present. the antimicrobial susceptibility testing results are determined by single-cell microbiological analysis via time-lapse computerized images of the pathogen's growth characteristics in the presence of a particular antibiotic. the blood culture pathogen panel assay is intended to be the company's fi rst fda-approved assay with other sample type panels in their assay pipeline. automation in the microbiology laboratory has been a slow to make an impact unlike the other laboratory sections (i.e., chemistry, urinalysis, etc.) attributable to the inherent manual process of specimen preparation required. vendors are developing automated plate streakers for more consistent yields with culture plating. also, companies are developing automated specimen processors that can be programmed to inoculate a battery of plates. one can imagine the advantages to be gained with high volume sections of the laboratory such as urine cultures [ 147 -153 ] . the implementation of microbiology automation is in its infancy and there is debate on the utility of automation and its widespread adoption. the potential is there for a large impact on the manual workfl ow and disruption of how clinical microbiology laboratories function. obviously, there is a fi nancial aspect to the implementation of automation and the estimated total cost of a total automated microbiology solution can be in the millions of dollars [ 149 ] . it is not out of the realm of possibilities for further advancements for a total microbiology laboratory automated technological solution where clinical microbiologists may be able to function from a "virtual" bench able to work up cultures and set-ups for other downstream tests from a computer touchscreen/tablet eliminating the potential hazard of being exposed to pathogenic and/or bioterrorism organisms. the arrival of new equipment in a large community hospital laboratory, chemistry automation [ 154 ] for example, creates a lot of stress on the staff to complete the performance verifi cation of the new quantitative analytical systems [ 155 , 156 ] . the practicing physician and healthcare system depend on this equipment to perform well. the assays will require verifi cation of calibration , linearity , analytic measurement ranges, accuracy, precision, appropriateness of the reference range and quality control requirements [ 155 -157 ] . a variety of poct and main chemistry laboratory methods for hba1c have been evaluated to see if that meets the total allowable error goal set by the cap proficiency testing program and the national glycohemoglobin standardization program (ngsp) [ 158 -160 ] . "clearly, many methods, including a few poc methods, do perform well in laboratories, as seen by data from the cap proficiency surveys" [ 160 ] , our new system was not one of those good performers. we replaced immunoassays for hba1c (roche diagnostics method, siemens medical solutions diagnostics-potential new method) with a capillary electrophoresis method (sebia) [ 161 -163 ] . during the evaluation of these three hba1c methods, the roche immunoassay reported hba1c values (3.7-4.8 %) for four patients with no hba but had hbsc. during the screening of 231 random patients, 13 % had homozygous or heterozygous variants [ 163 ] . hb n-baltimore comigrates with hba1c on capillary electrophoresis while hb silver spring and 17 other hb variants did not [ 162 ] . in this case, a method for hba1c had to be quickly evaluated to replace the immunoassay originally planned for implementation to prevent repeated profi ciency testing failures and potential discontinuation of the hba1c assay. an obsolete test is a test that is no longer in use or no longer useful (table 14 .8 ) [ 12 , 164 -171 ] . an effective way to evaluate whether a test has become obsolete is to review it at the laboratory utilization committee that is responsible for the laboratory formulary [ 172 ] . the formulary concept comes from the play book of the pharmacy and therapeutics committee that approve medication for use by medical providers and under what circumstances. when a newer more effective drug is fda approved it may replace an older less effective drug in the formulary. in the laboratory, the perfect obsolete test cannot be ordered by a medical provider because the reagents are no longer provided by the in vitro diagnostics industry. for example, protein bound iodine (pbi) [ 169 ] is no longer available at any reference laboratory because the test reagents are no longer manufactured. however, t3 uptake is just as obsolete and useless; however, its reagents are still manufactured by many vendors and still offered by reference laboratories [ 12 ] . in a utilization review of our hospital's send out test volume, it was asked by the reference laboratory why physicians ordered so many t3 uptake assays from them. i said it is not on our formulary just like ck mb [ 164 -166 ] , but both of these obsolete tests and others are still ordered and performed by your reference laboratory. the response was "we offer the test because physicians order the test," however, if the reagents were not available from the manufacturer, it is unlikely the reference laboratory would develop a laboratory developed test to support obsolescence. the workaround for removal of the obsolete tests from the hospital laboratory formulary usually involves the use of an emr that has reference laboratory test ordering built for the convenience of the medical providers. if this feature is not inactivated for inpatients the hospital laboratory formulary develops a leak from which a fl ood of abuse can originate. until locally defi ned obsolete tests are universally accepted and eliminated from the test lists of hospitals, manufacturers, and reference laboratories, the discovery of ingenious work-arounds will occupy the time of the medical providers who by habit are accustomed to having the obsolete test results by their side. the utilization management of laboratory tests in a large community hospital is similar to academic and smaller community hospitals. there are numerous factors that infl uence laboratory utilization (table 14 .1 ). outside infl uences like hospitals buying physician practices, increase in the placement of hospitalists and hospital consolidation will infl uence the number and complexity of test menu that will need to be monitored for over and under utilization in the central laboratory and reference laboratory. the laboratory utilization committee and laboratory formulary stewardship are key to a successful beginning. there are numerous excellent suggestions and reports of the successful implementation of remedies that have been reviewed and arranged in generic toolkits or tool boxes [ 1 , 173 , 174 ] or with solutions for specifi c laboratory sections like microbiology [ 12 , 88 ] , toxicology [ 5 ] , chemistry [ 175 ] , transfusion medicine [ 176 ] , and molecular diagnostics [ 177 -180 ] . this useful approach provides a resource for the exploration of laboratory test utilization management issues and their potential resolution using a method that is successful in your local geographical environment. 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outcomes of older patients hospitalized for heart failure do hospitalist physicians improve quality of inpatient care delivery? a systematic review of process, efficiency and outcome measures a multifaceted hospitalist quality improvement intervention: decreased frequency of common labs choosing wisely in adult hospital medicine: fi ve opportunities for improved healthcare value choosing wisely-the politics and economics of laboratoryeling low value services default settings on computerized physician order entry system order sets drive ordering habits hospital industry consolidation-still more to come? hospitals, market share, and consolidation seeking lower prices where providers are consolidated: an examination of market and policy strategies consolidation and concentration in the german hospital market: the two sides of the coin hospital consolidation, competition, and quality. is bigger necessarily better? hospital consolidation isn't the key to lowering costs and raising quality updated data on physician practice arrangements: inching toward hospital ownership. policy research perspectives. ama the hitchhiker's guide to improving efficiency in the clinical laboratory impact of point-of-care testing on patient's length of stay in a large emergency department point of care testing: randomized controlled trial of clinical outcomes comprehensive bedside point of care testing in critical ed patients: a before and after study utilization and cost analysis of bedside capillary glucose testing in a large teaching hospital: implications for managing point of care testing assessment of a critical limit protocol for point-of-care glucose testing quality improvement in critical care laboratory phlebotomy for diagnostic laboratory tests in adults phlebotomy overdraw in the neonatal intensive care nursery principles and point-of-care testing for body fl uids provider-performed microscopy provider-performed microscopy calculating cost savings in utilization management principles and practice of point-of-care testing principles and practice of point-of-care testing reverse-transcription polymerase chain reaction detection of the enteroviruses. overview and clinical utility in pediatric enteroviral infections creating a new civilization: the politics of the third wave hospital laboratory outreach program builds success through affi liation with a laboratory network outreach implementation requirement: a case study yearly clinical laboratory tests expenditures for different medical specialties in a major canadian city recommendations for the laboratory-based detection of chlamydia trachomatis and neisseria gonorrhoeae -2014 accuracy of results obtained by performing a second ligase chain reaction assay and pcr analysis on urine samples with positive or near-cutoff results in the lcx test for chlamydia trachomatis newborn screening: adapting to advancements in whole-genome sequencing design of a genomics curriculum. competencies for practicing pathologists standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology molecular diagnostic profi ling of lung cancer specimens with a semiconductor-based massive parallel sequencing approach. feasibility, costs, and performance compared with conventional sequencing a variant detection pipeline for inherited cardiomyopathy-associated genes using next-generation sequencing non-invasive prenatal rhd genotyping using cell-free fetal dna from maternal plasma: an italian experience society for maternal-fetal medicine. cell-free dna for fetal aneuploidy. committee opinion number 640 dna sequencing versus standard prenatal aneuploidy screening noninvasive prenatal testing and incidental detection of occult maternal malignancies circulating cell-free tumour dna in the management of cancer maldi-tof mass spectrometry for microorganism identifi cation beyond identifi cation. emerging and future uses for maldi-tof mass spectrometry in the clinical microbiology laboratory disposable platform provides visual and color-based point-of-care anemia self testing a smartphone dongle for diagnosis of infectious disease at the point of care new quick and cheap bedside test for cortisol uses smartphone can hospital rounds with pocket ultrasound by cardiologists reduce standard echocardiology? point-of-care ultrasound in medical education-stop listening and look defi ning digital medicine tattoo-bases non-invasive glucose monitoring: a proof-ofconcept study all-organic optoelectronic sensor for pulse oximetry bioinformatics for beginners. genes, genomes, molecular evolution, databases and analytical tools. london: academic computational solutions for omics data confi rming variants in next-generation sequencing panel testing by sanger sequencing digital imaging in pathology guideline from the college of american pathologists pathology and laboratory quality center why disruptive innovations matter in laboratory diagnostics pathology resident and fellow education in a time of disruptive technologies role of cell culture for virus detection in the age of technology replacement of biologic by molecular techniques in diagnostic virology: thirty years after the advent of pcr technology-do we still need conventional methods? detection of respiratory viruses by molecular methods utilization management in microbiology impact upon clinical outcomes of translation of pna fish-generated laboratory data from the clinical microbiology bench to bedside in real time rapid diagnosis of staphylococcus aureus bacteremia using s. aureus pna fish an evaluation of the advandx staphylococcus aureus /cns pna fish assay emerging technologies for rapid identifi cation of bloodstream pathogens clinical consequences of using pna-fish in staphylococcal bacteraemia impact of an assay that enables rapid determination of staphylococcus species and their drug susceptibility on the treatment of patients with positive blood culture results an antimicrobial stewardship program impact with rapid polymerase chain reaction methicillin-resistant staphylococcus aureus / s. aureus blood culture test in patients with s. aureus bacteremia impact of rapid methicillin resistant staphylococcus aureus polymerase chain reaction testing on mortality and cost effectiveness in hospitalized patients with bacteraemia: a decision model le interest of realtime pcr xpert mrsa/sa on genexpert(®) dx system in the investigation of staphylococcal bacteremia evaluation of the verigene gram-positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants rapid detection of gram-positive organisms by use of the verigene gram-positive blood culture nucleic acid test and the bact/alert pediatric fan system in a multicenter pediatric evaluation evaluation of a microarray-based assay for rapid identifi cation of gram-positive organisms and resistance markers in positive blood cultures multiplex identifi cation of gram-positive bacteria and resistance determinants directly from positive blood culture broths: evaluation of an automated microarray-based nucleic acid test evaluation of the nanosphere verigene gram-positive blood culture assay with the versatrek blood culture system and assessment of possible impact on selected patients performance of the verigene gram-positive blood culture assay for direct detection of gram-positive organisms and resistance markers in a pediatric hospital evaluation of filmarray and verigene systems for rapid identifi cation of positive blood cultures clinical evaluation of the filmarray blood culture identifi cation panel in identifi cation of bacteria and yeasts from positive blood culture bottles discrimination of candida albicans from candida dubliniensis by use of the biofire filmarray blood culture identifi cation panel clinical signifi cance and outcomes of polymicrobial staphylococcus aureus prospective assessment of filmarray technology for the rapid identifi cation of yeasts isolated from blood cultures rapid identifi cation of pathogens from positive blood cultures by multiplex polymerase chain reaction using the filmarray system community respiratory virus infections in immunocomprised patients: hematopoietic stem cell and solid organ transplant recipients, and individuals with human immunodefi ciency virus infection viral diagnostics and antiviral therapy in hematopoietic stem cell transplantation respiratory viral infections in transplant recipients evaluation of multiple test methods for the detection of the novel 2009 infl uenza a (h1n1) during the new york city outbreak cost analysis of multiplex pcr testing for diagnosing respiratory virus infections implementation of filmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care early estimates of seasonal infl uenza vaccine effectiveness. united states co-infections with infl uenza and other respiratory viruses coronavirus causes lower respiratory infections less frequently than rsv in hospitalized norwegian children identifi cation of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in hong kong by multiplex pcr assays survey of physician diagnostic practices for patients with acute diarrhea: clinical and public health implications what is new in clinical microbiology: microbial identifi cation by maldi-tof mass spectrometry application of maldi-tof mass spectrometry in clinical diagnostic microbiology applications of maldi-tof mass spectrometry in clinical diagnostic microbiology maldi-tof mass spectrometry applications in clinical microbiology matrix-assisted laser desorption ionization-time of fl ight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology maldi-tof mass spectrometry for microorganism identifi cation comparison of two matrix-assisted laser desorption ionization-time of fl ight mass spectrometry methods with conventional phenotypic identifi cation of bacteria to the species level performance of the vitek ms matrix-assisted laser desorption ionization-time of fl ight mass spectrometry system for rapid identifi cation of bacteria in routine clinical microbiology high-throughput identifi cation of bacteria and yeast by matrix-assisted laser desorption ionization-time of fl ight mass spectrometry in conventional medical microbiology laboratories performance of matrix-assisted laser desorption ionization-time of fl ight mass spectrometry of identifi cation of bacterial strains routinely isolated in a clinical microbiology laboratory prospective evaluation of a matrix-assisted laser desorption ionization-time of fl ight mass spectrometry system in a hospital clinical microbiology laboratory for identifi cation of bacteria and yeasts: a bench-by-bench study of assessing the impact on time to identifi cation and cost-effectiveness matrix-assisted laser desorption ionization-time of fl ight mass spectrometry for identifi cation of clinically signifi cant bacteria that are diffi cult to identify in clinical laboratories performance and cost analysis of matrix-assisted laser desorption ionization-time of fl ight mass spectrometry for routine identifi cation of yeasts for the diagnosis of infectious diseases direct identifi cation of bacteria in positive blood cultures: comparison of two rapid methods, filmarray and mass spectrometry evaluation of three rapid diagnostic methods for direct identifi cation of microorganisms in positive blood cultures direct identifi cation of bacteria causing urinary tract infections by combining matrix-assisted laser desorption ionization-time of fl ight mass spectrometry with uf-1000i urine fl ow cytometry procedure for microbial identifi cation based on matrix-assisted laser desorption ionization-time of fl ight mass spectrometry from screening-positive urine samples direct identifi cation of urinary tract pathogens from urine samples by matrix-assisted laser desorption ionization-time of fl ight matrix-assisted laser desorption ionization-time of fl ight (maldi-tof) mass spectrometry for detection of antibiotic resistance mechanisms: from research to routine diagnosis matrix-assisted laser desorption ionization-time of fl ight mass spectrometry for rapid identifi cation of tick vectors mechanism of fl uconazole resistance in candida krusei clinical practice guidelines for the management of candidiasis: 2009 update by the infectious diseases society of america t2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial t2 magnetic resonance: a diagnostic platform for studying integrated hemostasis in whole blood-proof of concept comparison of the t2dx instrument with t2candida assay and automated blood culture in the detection of candida species using seeded blood samples automation in clinical microbiology point-counterpoint: the automated clinical microbiology laboratory: fact of fantasy? automation in the clinical microbiology laboratory emerging technologies for the clinical microbiology laboratory laboratory automation in clinical microbiology: a quiet revolution automation in clinical bacteriology: what system to choose? the future of diagnostic bacteriology the william beaumont hospital experience with total laboratory automation verifi cation of method performance for clinical laboratories verifying performance characteristics of quantitative analytical systems satisfying regulatory and accreditation requirements for quality control three of 7 hemoglobin a1c point-of-care instruments do not meet generally accepted analytical performance criteria utilization of assay performance characteristics to estimate hemoglobin a1c result reliability performance of hemoglobin a1c assay methods: good enough? evaluation of the sebia capillarys 2 fl ex piercing for the measurement of hb a1c on venous and capillary blood samples effects of 49 rare hb variants on hb a1c measurements in eight methods hemoglobin a1c: evaluation of 4 methods antiquated tests within the clinical pathology laboratory requiem for a heavyweight: the demise of creatine kinase-mb creatine kinase-mg. the journey to obsolescence should the qualitative serum pregnancy test be considered obsolete? aetna clinical policy bulletin: obsolete and unreliable tests and procedures the interpretation of the serum protein-bound iodine: a review cerebrospinal fl uid myelin basic protein is frequently ordered but has little value. a test utilization study acg clinical guidelines: diagnosis and management of celiac disease laboratory formularies the laboratory test utilization management toolbox reducing unnecessary laboratory testing using health informatics applications. a case study in a tertiary care hospital effect of population-based interventions on laboratory utilization. a time series analysis utilization management in the blood transfusion service clinical requests for molecular tests. the 3-step evidence check improving molecular genetic test utilization through order restriction, test review, and guidance genetic counselor review of genetic test orders in a reference laboratory reduces unnecessary testing differences in brca counseling and testing practices based on ordering provider type key: cord-280571-ntgt5hy9 authors: ginocchio, christine c. title: strengths and weaknesses of fda-approved/cleared diagnostic devices for the molecular detection of respiratory pathogens date: 2011-05-01 journal: clin infect dis doi: 10.1093/cid/cir046 sha: doc_id: 280571 cord_uid: ntgt5hy9 the rapid, sensitive, and specific identification of the microbial etiological characteristics of respiratory tract infections enhances the appropriate use of both antibiotics and antiviral agents and reduces the risk of nosocomial transmission. this article reviews the current nucleic acid amplification tests approved by the u.s. food and drug administration (fda) for the detection of respiratory pathogens. in addition, emergency use authorization tests for the detection of 2009 influenza a h1n1 are discussed. the advantages and limitations of the current fda-approved/cleared tests are reviewed. the identification of the causative agent(s) of respiratory tract infections is essential to provide an accurate diagnosis, appropriately manage patient care, and reduce the risk of nosocomial transmission within health care facilities. with the steady rise of antibiotic resistance and limited or no options available for the treatment of multidrug or panresistant bacterial infections, pathogen identification is a key component in restricting antibiotic use to those circumstances in which antibiotic therapy is clearly indicated [1] . initial empiric therapeutic choices may be standardized and initiated on the basis of patient clinical status, underlying disease, and/or risk for infection with a multidrug-resistant pathogen. however, subsequent bacterial pathogen identification and accurate susceptibility data should assist in promoting switches to targeted specific therapies and reduce the use of broad-spectrum antibiotics when not indicated, thereby promoting good antibiotic stewardship [2] . viral infections probably cause between 75% and 80% of respiratory tract diseases. nonetheless, it is estimated that 22.6 million (55%) of 41 million antibiotic prescriptions for respiratory tract infections, including both lower and upper respiratory infections, were for causes unlikely to have a bacterial etiology [3] . although in many cases of otitis and sinusitis and probably 20%-40% of cases of community-acquired pneumonia a bacterial superinfection may occur, respiratory infections due to a virus(es) alone are common in the adult and pediatric outpatient populations. in addition, the vast majority of respiratory tract infections in nonimmunocompromised hospitalized children (especially those ,5 years of age) are due to 1 or more viruses, without a secondary bacterial infection. the overuse of antibiotics for the treatment of outpatients is primarily due to the fact that in adults respiratory virus testing is either not performed or generally limited to rapid antigen direct tests (radts) for influenza. testing for the elderly, for persons with chronic obstructive pulmonary disease, and for pediatric patients is limited to radts for influenza and respiratory syncytial virus (rsv). radts have highly variable sensitivities (10%-75%) and specificities (50%-100%) depending on the viral target, age of the patient, sample collection, and duration of symptoms prior to testing [4] [5] [6] . in general, radts perform better when testing pediatric samples, because children shed higher titers of virus and for longer time periods than adults [4, [7] [8] [9] . in addition to radts, there are u.s. food and drug administration (fda)-approved/cleared nonmolecular-based viral diagnostic methods with a more rapid time to result, compared with traditional viral tube culture, eg, direct fluorescent antibody (dfa) testing and rapid cell culture. both methods can readily detect 7 of the common respiratory viruses (adenovirus, influenza a, influenza b, parainfluenza 1, 2, and 3 [piv-1, piv-2, piv-3], and rsv). in addition, dfa testing can detect human metapneumovirus (hmpv) [6] . the specificity of dfa testing and rapid cell culture are high, but the sensitivities of the tests can vary from a low of 50% (rsv culture) to a high of .80% (influenza a), when compared with nucleic acid amplification tests (naats) [5, 6, 10] . dfa testing can be performed in as little as 30-60 min, and shell vial and r-mix rapid cell cultures (quidel/diagnostic hybrids) generally identify respiratory viruses in 24-48 h [6] . if these tests are performed on site, the time to virus detection can be within a time frame that could affect patient management. however, these tests are not widely available outside larger hospitals and reference laboratories. although these 8 viruses are responsible for a large number of respiratory tract infections, bocavirus, selected coronaviruses (229e, oc43, nl63, and hku-1), parainfluenza 4, and rhinovirus are also important causes of respiratory disease and are generally only detected using naats. because antiviral therapies are currently limited to the treatment of influenza a, influenza b, cytomegalovirus pneumonia, and varicella zoster virus pneumonia, it is often argued that the specific identification of other viruses is not relevant, because the information would not change patient management. from a treatment standpoint, this may currently be true; however, the respiratory viruses cause similar illnesses, and diagnosis based on clinical symptoms alone can be highly inaccurate [11] . for example, a study by poehling et al revealed that physicians who used only clinical symptoms recognized influenza in only 28% of hospitalized children and 17% of nonhospitalized children with laboratory-confirmed influenza [11] . therefore, establishing the viral etiological characteristics of the illness is often highly dependent on accurate diagnostic testing. in addition, new therapeutic agents for respiratory viruses are in development, and clinical trials for these agents will need rapid diagnostics that detect a broad range of viral pathogens. once these new drugs are approved, clinical laboratories will need the tools to identify each virus so that appropriate antiviral therapy can be rapidly initiated. in addition, during the first weeks of the 2009 influenza a h1n1 outbreak in the spring of 2009, multiple influenza viruses were cocirculating [5] . it was necessary to subtype influenza a strains to differentiate seasonal influenza a/h1, seasonal influenza a/h3, and 2009 influenza a h1n1. subtyping is necessary to provide relevant information needed for appropriate selection of antiviral therapy, in particular for acutely ill patients. antiviral resistance testing of influenza isolates from 2009 (www.cdc.gov/flu) revealed that the circulating seasonal influenza a/h1 strains were resistant to oseltamivir (99.6%), and seasonal influenza a/h3 and 2009 influenza a h1n1 were resistant to the adamantanes (100%). in addition, several patients with 2009 influenza a h1n1 infections developed oseltamivir resistance [12, 13] . other clinical factors, such as the potential for the development of more severe disease on the basis of the virus etiology, may need to be considered in the management of certain patients. for example, studies from our laboratory have revealed that children with hmpv infections have a higher incidence of admission to an intensive care unit and more often require mechanical ventilation than children with rsv infections [14, 15] . especially in the treatment of inpatients, the costs of rapid viral diagnostics can be offset by the improvement in patient care and financial outcomes [16] [17] [18] [19] . hendrickson et al showed that rapid respiratory virus diagnosis can lead to benefits in several areas, including up to a 50% reduction in hospital days, 30% reduction in antibiotic use, and 20% reduction in unnecessary diagnostic tests and procedures [16] . the burden of nosocomial influenza can be high, incurring additional costs for diagnostic tests, increased morbidity, and extended hospitalization [17, 18] . therefore, rapid diagnostic tests are needed to identify patients with influenza at admission, in order to prevent nosocomial transmission by facilitating isolation and cohorting decisions [18] . studies have documented substantial nosocomial transmission of hmpv in pediatric units [20] , as well as in chronic care facilities [21] , similar to what is seen with rsv. during the height of rsv season, many institutions must cohort rsv-positive children because of a lack of private rooms. however, dual infections with rsv and hmpv do occur. limiting diagnostics to rsv alone in a cohorting scenario could put other seriously ill children at risk for acquisition of a second viral infection with hmpv. the meaning of mixed viral infections can be defined only if testing is comprehensive. broad test panels also allow for monitoring the epidemiologic patterns of respiratory disease and for identifying new or reemerging pathogens. finally, the identification of the exact respiratory virus is essential for the accurate assessment of the efficacy of vaccines. the costs for testing using fda-approved/cleared naats are highly variable and can range from approximately $30 to .$200 per test. factors that determine test cost include test volume, the price of the test kits, the number of tests per kit, size of the testing run, the number of controls required per testing run, and the type and amount of ancillary supplies. often fda-approved/ cleared kits do not contain nucleic acid extraction reagents, so additional equipment and reagents are necessary and can add $3-$15 per test. batch testing of samples may reduce the cost per test; however, often batching is not practical if the time to result is delayed beyond a period that would affect either clinical management or infection control practices. instrumentation costs for molecular tests can be substantial, with real-time instrumentation costing on average $35,000-$85,000 per instrument, and higher volume laboratories may require multiple instruments. laboratories must also calculate the cost for technical time, which is again highly variable depending on the test complexity and run size. finally, the costs for quality control, proficiency testing, and competency assessment all affect the overall cost per test. in deciding which tests are appropriate and at what cost for the patient population at a particular health care facility, all of the above factors need to be considered in light of the clinical impact of the test result. the use of naats is an intrinsic part of infectious disease diagnostics. the ability of naats to rapidly and accurately detect a novel pathogen was best exemplified during the 2009 influenza a h1n1 pandemic [5, [22] [23] [24] . naats are especially suited for the identification of respiratory pathogens that are not routinely or easily cultured (eg, hmpv, bocavirus, parainfluenza 4, and chlamydophila pneumoniae), pathogens that are dangerous to culture (eg, severe acute respiratory syndrome coronavirus), pathogens for which the time to detection by traditional means is often too delayed to affect patient care (eg, tube cell culture for influenza), and pathogens for which serologic testing is difficult to interpret (eg, c. pneumoniae). in most cases, naats offer enhanced sensitivity over culture methods, radts, and dfa testing [5, 6, 10] . the specificity of naats varies with target and assay design but is generally very high. in addition, laboratorydeveloped naats validated in accordance with the clinical laboratory improvement amendments (clia) requirements can be used to identify new pathogens until fda-approved/ cleared in vitro diagnostic (ivd) devices become available. with clinical integration of real-time polymerase chain reaction (pcr) and fda-approved/cleared simple cartridge-based naats, laboratories of all sizes are now able to perform molecular diagnostic tests. the detection of mycobacterium tuberculosis directly in a clinical sample from a patient not yet identified as m. tuberculosis positive is always clinically relevant. detection of such bacterial pathogens as bordetella pertussis, bordetella parapertussis, legionella pneumophila, mycoplasma pneumoniae, or c. pneumoniae usually indicates active infection. in contrast, the detection of other bacteria, such as streptococcus pneumoniae, may indicate infection or simply colonization. therefore, interpretation of the molecular detection of many bacterial pathogens must be viewed in light of the clinical specimen (sterile vs nonsterile body site) and determination of the quantity of organisms present. in addition, molecular methods must include all potential pathogens, even though culture is still required for antimicrobial susceptibility testing. for these reasons, there is currently a scarcity of fda-approved/cleared assays for nonviral respiratory pathogens (table 1 ). for such targets as the atypical pneumonia pathogens, the interpretation of naat results is generally not an issue, quantitative tests are not necessarily indicated, and naats could replace traditional test methods. however, the lack of fda-approved/cleared naats for these pathogens most probably relates to the ivd device manufacturers' reluctance to perform costly clinical trials for targets with a relatively low prevalence rate and for which the potential volume of testing may be minimal. in light of the rise in m. tuberculosis drug resistance worldwide, the rapid identification of patients with pulmonary tuberculosis is essential and allows for improved patient outcomes, the appropriate use of isolation facilities, the initiation of appropriate treatment, and the identification of possible infected contacts [25] [26] [27] . in addition, studies have shown that the estimated costs associated with an inaccurate diagnosis of m. tuberculosis infection are substantial ($11,576 per patient) because of institutional isolation procedures [27] . direct sample testing can detect m. tuberculosis within 24-48 h of sample collection, compared with 1-4 weeks for liquid and traditional solid media culture methods. although acid-fast bacilli (afb) smear microscopy is inexpensive and rapid to perform, the sensitivity of the test can be poor (45%-80% with culture-confirmed pulmonary m. tuberculosis colonization or infection) and cannot differentiate m. tuberculosis from mycobacteria other than m. tuberculosis [26] . the 2009 centers for disease control and prevention (cdc) guidelines recommend that naats should be performed on at least 1 respiratory sample from patients with signs and symptoms of pulmonary m. tuberculosis colonization or infection for whom a diagnosis of m. tuberculosis colonization or infection has not been established or when the results would alter case management or infection control procedures [28] . mycobacterial culture is still required, because the m. tuberculosis isolate is needed for drug susceptibility testing. currently, there are 2 fda-approved naats available for the detection of m. tuberculosis complex from respiratory samples, the amplified mycobacterium tuberculosis direct test (amtd, gen-probe) [25, [29] [30] [31] and the amplicor mycobacterium tuberculosis test (amplicor, roche diagnostics) [32] [33] [34] [35] . recently, a new assay called the xpert mtb/rif (cepheid) was developed to detect the presence of m. tuberculosis and rifampin resistance directly from processed clinical samples [reviewed in 36] . the assay uses a heminested real-time pcr with molecular beacon detection and is performed on the genexpert instrument (cepheid). benefits of this assay include low technical complexity, allowing for potential point-of-care testing; minimal hands-on time (15-20 min); test turnaround time of ,2 h; and containment of the sample, extracted nucleic acids, and amplicons in the test cartridge. when compared with culture, the sensitivity of the assay for the detection of m. tuberculosis ranged from 98.2% (1 sample tested per patient) to 99.8% (3 samples tested per patient) for afb smear-positive samples and from 72.5% (1 sample tested per patient) to 90.2% (3 samples tested per patient) for afb smear-negative samples. the specificity of the assay ranged from 98.1% to 99.2%. the assay is currently available for diagnostic testing only in europe. amplified mycobacterium tuberculosis direct test (amtd, gen-probe) the amtd is a target-amplified nucleic acid probe test for the direct detection of m. tuberculosis complex (mycobacterium bovis, m. bovis bcg, mycobacterium africanum, mycobacterium microti, and m. tuberculosis) ribosomal rna (rrna) [25, [29] [30] [31] . amtd is approved for testing both afb smear-positive and smear-negative respiratory specimens collected from patients suspected of having m. tuberculosis colonization or infection. the amtd test is based on isothermal (42 o c) transcription-mediated amplification that uses 2 enzymes, reverse transcriptase (rt) and t7 rna polymerase, and targetspecific primers. a hybridization protection detection assay that uses a chemiluminescent-labeled, single-stranded dna probe complementary to the m. tuberculosis complex-specific sequences detects the amplified rrna using the gen-probe leader luminometer. numerous studies have evaluated amtd and have revealed an average sensitivity and specificity of .95% and .98%, respectively, for the detection of m. tuberculosis complex in smear-positive respiratory samples and a sensitivity and specificity of .60% and .72%, respectively, for smear-negative respiratory samples [29] [30] [31] . additional studies have evaluated the use of the test for several types of nonrespiratory samples, such as cerebrospinal fluid and lymph nodes [29] [30] [31] . amplicor mycobacterium tuberculosis test (amplicor, roche diagnostics) the amplicor mycobacterium tuberculosis test is fda approved for use with afb smear-positive respiratory specimens from patients suspected of having m. tuberculosis infection or colonization [32] [33] [34] [35] . the amplicor mycobacterium tuberculosis test uses traditional pcr to amplify a region of the gene encoding the 16s rrna of all mycobacteria. members of the m. tuberculosis complex are identified after hybridization with a dna target-specific probe, followed by substrate addition and colorimetric detection [32] . when testing afb smear-positive samples, the amplicor mycobacterium tuberculosis test demonstrated sensitivities and specificities for the detection of m. tuberculosis complex ranging from 92.9% to 100% and from 77.3% to 100%, respectively [32, 33] . the sensitivities and specificities of amplicor mycobacterium tuberculosis for afb smear-negative specimens ranged from 51.2% to 73.1% and 99% to 99.8%, respectively [32, 33] . the amplicor mycobacterium tuberculosis test has also been evaluated for detection of m. tuberculosis meningitis [34] and for use with nonrespiratory sample types [35] . viral respiratory pathogens are particularly suited for detection using naat, since the number of targets is relatively limited, compared with the numerous potential bacterial pathogens that can cause respiratory disease (table 1) . although there is much to learn regarding the clinical relevance of mixed viral respiratory infections, the detection of a respiratory virus is generally considered diagnostic at this time. today, most laboratories do not have the facilities for comprehensive tissue culture based viral diagnostics and therefore are limited to testing with less sensitive and less specific radts for only influenza a, influenza b, and rsv. naats offer an excellent alternative to greatly expand the test menu of clinical laboratories, thereby providing rapid, accurate comprehensive diagnostics. the first multiplex naat to receive clearance by the fda was the xtag respiratory virus panel (rvp) assay in january 2008. the fda-approved version of rvp detects adenovirus, influenza a (with subtyping of seasonal influenza a/h1 and seasonal influenza a/h3), influenza b, piv-1, piv-2, piv-3, hmpv, rhinovirus, rsv a, and rsv b [37, 38] . the us/canadian research use-only version of the assay also detects 4 coronaviruses (oc43, 229e, nl63, and hku-1), parainfluenza type 4, and enterovirus. the test is approved for use with nasopharyngeal swab samples collected from persons symptomatic for a respiratory virus infection and placed in viral transport media. the xtag rvp is a multistep test that takes approximately 8-10 h to complete, depending on the number of samples to be tested (figure 1 ). viral nucleic acid and an internal control (e. coli ms2 phage) are coextracted from clinical samples using the qiaamp minielute (qiagen), the easymag (biomã©rieux), or the minimag (biomã©rieux) extraction platforms. a multiplex reverse transcription polymerase chain reaction (rt-pcr), using primer sets specific for the test targets, amplifies the viral nucleic acid and internal control nucleic acid. pcr products are treated with exonuclease i to degrade any remaining primers and with shrimp alkaline phosphatase to degrade any remaining nucleotides. the next step consists of target-specific primer extension (tspe). when a viral target(s) is present, the target-specific primer (containing a unique tag sequence) is extended and biotin-deoxycytidine triphosphate is incorporated into the extending chain. on completion of the tspe, the detection of amplified products is performed using luminex's universal tag sorting system. the tspe reaction is added directly to microwells containing spectrally distinguishable beads with antitags, which are complementary to the sequence tags on the primers. each tagged primer will hybridize only to its unique antitag complement associated with a specific colored bead. a fluorescent reporter molecule (streptavidin-phycoerythrin) will bind to the biotin on the extended primers. the beads are then analyzed with the luminex xmap 100/200 instruments. two lasers read each bead; the first identifies the virus-specific color-coded bead, and the second determines whether an amplicon is hybridized to the bead on the basis of the detection of fluorescence (mean fluorescence intensities [mfis]) above a background threshold. the clinical performance characteristics of the xtag rvp assay were established by the manufacturer through prospectively collected nasopharyngeal swab samples (n 5 544) tested during the 2005-2006 influenza season at 4 north american clinical laboratories [101] . all specimens were tested by means of viral culture and/or dfa testing for the following targets: influenza a, influenza b, rsv, piv-1, piv-2, piv-3, and adenovirus. the comparator methods for influenza a subtyping, rsv subtyping, and hmpv and rhinovirus detection were wellcharacterized rt-pcr assays followed by bidirectional sequencing. xtag rvp sensitivity for each target was determined as follows: the benefits of the xtag rvp assay include the broad spectrum of viruses detected by a single test, with a cost per test comparable to real-time assays that only target up to 3 analytes. the subtyping of influenza a viruses as seasonal influenza a/h1 or seasonal influenza a/h3 or the identification of an ''unsubtypeable'' virus has proven to be an important aid in identifying novel influenza a strains. a limitation of the assay is the decreased sensitivity for the detection of certain adenovirus strains. in-house validation studies by our laboratory have found that by reducing the positive cutoff mfi level from 300 to 150 for adenovirus, improved sensitivity for the detection of adenovirus was obtained without loss of specificity (unpublished data). additional limitations of the assay include the time to final results, number of required steps, technical handson time, and a potential for amplicon contamination. there is a second-generation assay called rvp fast (luminex) available in europe that addresses several of these issues by reducing the number of steps and the time to results by 3-4 h, making it possible to provide comprehensive results within a single shift. gen-probe/prodesse proflu,1proflu-st, pro hmpv1, and proparaflu1 assays (gen-probe/prodesse) currently, there are 3 gen-probe/prodesse fda-cleared multiplex real-time rt-pcr assays for the qualitative detection and discrimination of respiratory viruses. the proflu1 assay targets the matrix gene for influenza a, nonstructural genes ns-1 and ns-2 for influenza b, and the polymerase gene for rsv a and rsv b. the pro hmpv1 assay targets highly conserved regions of the nucleocapsid (n) gene for hmpv and a transcript derived from escherichia coli bacteriophage ms2 a-protein gene (internal control). the proparaflu1 assay targets conserved regions of the hemagglutinin-neuraminidase gene for piv-1, piv-2, and piv-3 and a transcript derived from e. coli bacteriophage ms2 a-protein gene (internal control). all 3 assays are approved for testing nasopharyngeal swab specimens obtained from symptomatic persons. viral nucleic acids from patient samples are coextracted with an internal control that monitors assay performance and the presence of amplification inhibitors that could lead to false-negative results. nucleic acids are extracted using a magna pure lc instrument (roche diagnostics corp) and the magna pure total nucleic acid isolation kit (roche) or a nuclisens easymag system and the automated magnetic extraction reagents (bio-mã©rieux). the purified nucleic acids are amplified by means of rt-pcr using target-specific oligonucleotide primers and taqman probes complementary to highly conserved regions of the target gene. the taqman probes are labeled with a quencher dye attached to the 3'-end and a reporter dye at the 5'-end. when amplified target is present, the probes bind and the 5'-3' exonuclease activity of taq polymerase cleaves the probe, thus separating the reporter dye from the quencher. because the quencher and reporter dye are now physically separated, there is an increase in fluorescent signal upon excitation from a light source. the fluorescent signal increases with each cycle as additional reporter dye molecules are cleaved from their respective probes. during each pcr cycle, the fluorescent intensity is monitored by the realtime instrument, the smartcycler ii (time to results, including extraction, is approximately 3-3.5 hr for each test run). the performance characteristics of the assays were established by the manufacturer through prospective and retrospective clinical studies used for fda clearance of the tests. proflu1 assay results obtained by testing 891 nasopharyngeal samples were compared with results of rapid shell vial culture. proflu1 sensitivities and specificities for the detection of influenza a were 100% and 92.6%, respectively; for influenza b were 97.8% and 98.6%, respectively; and for rsv were 89.5% and 94.9%, respectively [102]. a study by liao et al compared the prodesse proflu-1 assay, a previous version of the proflu1 assay, with viral culture and radts for rsv (now rsv, binax, inverness medical) and for influenza a and b (directogen a1b, bd diagnostics) [10] . the specificities of all methods were found to be .99%. the sensitivities for detection of influenza were 59% for directogen a 1 b, 54% for viral culture, and 98% for proflu-1; the sensitivities for the detection of rsv were 82% for rsv now, 57% for viral culture, and 95% for proflu-1. in another study, legoff et al evaluated the performance of proflu-1 in 353 pediatric nasopharyngeal specimens [40] . results were compared with dfa testing and viral culture. the sensitivities and specificities of proflu-1 ranged from 97% to 100%, and proflu-1 detected viruses in 9% of the samples that had negative results by conventional methods. in response to the 2009 influenza a h1n1 outbreak, an influenza a subtyping assay (proflu-st) was developed by prodesse and received emergency use authorization (eua) from the fda in july 2009 (see eua section). proflu-st is a qualitative multiplex real-time rt-pcr assay that targets the nucleoprotein gene of 2009 influenza a h1n1, the specific hemagglutinin genes of seasonal influenza a/h1 and seasonal influenza a/h3, and an internal control (ms2 phage). the identification of 2009 influenza a h1n1 is aided by an algorithm that relies on seasonal influenza a/h1 virus and seasonal influenza a/h3 virus results in nasopharyngeal swab specimens from patients who receive a diagnosis of influenza a by a currently available fda-cleared or authorized device. this assay is intended for use in only clia high-complexity laboratories. the performance of the assay was evaluated retrospectively using nasopharyngeal swab specimens that were previously tested with either the cdc rrt-pcr flu panel (ivd device) to detect seasonal influenza a/h1 and influenza a/h3 or the cdc rrt-pcr swine flu panel (eua). the positive and negative agreements for the detection of seasonal influenza a/h1were 95.8% and 100%, respectively; for seasonal influenza a/h3 were 100% and 100%, respectively; and for 2009 influenza a h1n1 were 96.2% and 100%, respectively [103] . the pro hmpv1 assay clinical trial study for fda clearance evaluated the assay's clinical performance using 1275 nasopharyngeal swab specimens tested by 4 clinical laboratories across the united states. using the luminex rvp assay as the predicate device, the sensitivity of pro hmpv1 was 94.1% and the specificity was 99.3% [104]. the performance of the proparaflu1 assay was evaluated during the clinical trials for fda clearance. using 857 nasopharyngeal swab specimens tested by 4 clinical laboratories across the united states [105], the sensitivities and specificities of the assay for the detection of piv-1 were 88.9% and 99.9%, respectively; for the detection of piv-2 were 96.3% and 99.8%, respectively, and for the detection of piv-3 were 97.3% and 99.2%, respectively [105] . at this time, no additional independent performance data are available. the benefits of the gen-probe/prodesse assays include ease of use, with approximately 1.5 h of hands-on time for nucleic acid extraction preparation and a 1-step rt-pcr setup. the overall time to results is 4.5-5.5 h. multiple smartcycler instruments can be run simultaneously, with as many units per cycler used as needed, giving flexibility to run sizes. because tubes containing amplicons are never opened, the risk of amplicon contamination is minimal. one limitation of the assays is a maximum of 3 targets plus an internal control that can be detected in a single reaction. therefore, a comprehensive viral diagnostic panel requires multiple pcrs. multiple pcrs are costly to the laboratory in both technical time and reagent cost. however, the limited panel size could provide a mix-and-match test menu, allowing clinicians the option of selecting 1 or several panels. the first-generation verigene respiratory virus nucleic acid test (vrnat) was cleared by the fda in may 2009. this test has been replaced by the automated verigene vrnat sp , a clia moderately complex test that is intended for the identification of influenza a, influenza b, and rsv (types a and b inclusive) from nasopharyngeal swab specimens placed in viral transport media. the verigene system consists of 2 instruments (the fully automated verigene processor and the verigene reader) and single-use test cartridges (figure 2 ). the entire test process only requires 1 user pipetting step, less than 5 min of technical handson time, and a sample-to-result turnaround time of about 3.5 h. the basis of vrnatsp is nanosphere's proprietary gold nanoparticle hybridization technology [41] . the gold nanoparticles contain a high density (200) of sequence-specific oligonucleotides with a high affinity for complementary dna, which allows for very efficient hybridization kinetics. the clinical sample is pipetted into a single-use extraction tray, which is loaded into the verigene processing unit. chaotropic agents are added to the sample to lyse cells, viral particles, and an internal control (ms2 phage) that is added prior to the extraction step. the released nucleic acids are then captured on magnetic microparticles (mmps). the mmp-bound nucleic acids are washed, and the purified nucleic acids are eluted from the mmps and transferred to the amplification tray. a 1-step rt-pcr is performed using primers that target the influenza a matrix gene, the influenza b matrix gene and nonstructural gene, and the rsv l gene and f gene. the rt-pcr reaction is followed by the primary hybridization step. during primary hybridization, target dna is simultaneously hybridized to target-specific capture dna oligonucleotides arrayed in replicate on a solid substrate (a microarray) and to target-specific mediator dna oligonucleotides. after removal of uncaptured target nucleic acids and unhybridized mediator oligonucleotides, the process continues with the secondary hybridization. each microarray spot, where an appropriate target is hybridized to capture both an oligonucleotide and a mediator oligonucleotide, is saturated with silver-coated gold nanoparticle probes. after hybridization, the cartridge is removed from the processor unit and the glass slide (microarray) is separated from the cartridge and inserted into the verigene reader. a light-scattering technique, in which the slide is illuminated internally with light parallel to the slide surface, is used to analyze the results. spots where silverenhanced gold nanoparticle probes are present scatter this light, and the light scatter is detected optically and translated into a measurable signal. the performance characteristics of the first-generation vrnat assay were established during the clinical trials for ivd device clearance. vrnat was compared with viral culture/dfa testing with bidirectional sequencing to resolve discordant results. test sensitivities and specificities for the detection of influenza a were 100% and 99.8%, respectively; for influenza b were 100% and 99.1%, respectively; and for rsv were 95.7% and 98.2%, respectively [106] . comparison of vrnat with vrnatsp revealed an overall positive agreement of 97.9% and a negative agreement value of 100% [107] . the benefits of the vrnatsp system include the scalability of the system (addition of multiple processors and readers), individual sample processing with random access format, minimal hands-on time, and minimal technical expertise required to perform the test. because this test is clia moderate complexity, trained laboratory technicians could perform the testing. this test would be well suited for small-to medium-sized laboratories, in particular for laboratories with little or no molecular testing experience. limitations of the assay include the single test format that requires a dedicated processor for each sample for 3-3.5 h. multiple processor units would be needed for larger volume laboratories, and the additional instrumentation would increase costs for the lab, compared with using an instrument that can run multiple tests at a time. table 2 were rescinded as of 23 june 2010. as of july 2010, only 2 of these tests have received fda approval for use as an ivd device for the diagnosis of 2009 pandemic influenza a h1n1. the first test approved was a new optimized cdc h1n1 assay that is available in cdc-qualified laboratories. the second test is the focus diagnostics simplexa influenza a h1n1 (2009), which is performed using the 3m integrated cycler (3m). the use of fda-approved/cleared naats, in contrast to laboratory-developed tests, has substantial benefits for the laboratory. approved tests have undergone extensive analytical and clinical validations during the course of the fda evaluations. therefore, performance parameters are well characterized, and the fda monitors postapproval performance. most laboratories do not have the expertise and resources to perform extensive (table 3) , thereby saving the laboratory considerable cost and technical time. the regulatory requirements for ongoing quality assurance monitoring are also fewer for fda-approved/cleared naats than for laboratory-developed tests [42] . in addition, for regulatory reasons, some health care institutions will only allow the use of fda-approved/cleared naats. because the clinical relevance of the assays has been established, the use of fda-approved/cleared naats has a higher chance of reimbursement from both federal and private insurance payors. however, fda approval/clearance does not guarantee that these tests will be reimbursed, and, if reimbursed, the actual amount of the reimbursement can vary from state to state and by payor. finally, the simple-to-use fully automated molecular platforms, such as the genexpert and genestat, which incorporate all steps of the test process in a single test cartridge and have random-access sample-in result-out reporting, enable laboratories of all sizes to perform rapid, accurate, and sensitive molecular diagnostic testing. the most important limitation of the current fda-approved/ cleared naats is the lack of tests for the detection of nonviral targets. in particular, naats are needed for the detection of the atypical pneumonia pathogens. however, the costs of clinical trials for ivd device clearance can sometimes be quite prohibitive (.$10 million, not including the costs of developing and manufacturing the ivd device) and depend on the complexity and clinical relevance of the test and what is required for fda approval (eg, number of trial sites, number of patients enrolled, clinical indications sought, need for long-term patient follow-up, associated diagnostic procedures [such as biopsy], device evaluations, predicate device comparisons, legal and regulatory components, and so forth). manufacturers must consider the potential number of tests that will be purchased and the difficulty of the trials for low-prevalence pathogens before committing the finances and resources to bring such tests through the approval process. these factors will continue to limit the scope of fda-approved/cleared naats until the approval process can be modified to encourage the submission of naats for low-prevalence targets. the current formats of the naats require laboratories to choose between real-time, easier to perform, more rapid assays that have limited targets (generally up to 3) and more highly multiplex tests (.10) that require more hands-on time and technical steps and more time to result reporting. naats with limited targets would require laboratories to run multiple tests if a broader range of target detection is indicated. multiple tests would increase both the required technical time and the cost per patient diagnosis. conversely, the use of multiple, lower multiplex tests allows for the selection of the most appropriate panels as related to age of the patient, clinical status, underlying disease, state of immune competence, and the suspected virus(es). currently in development are modified faster versions of the highly multiplex assays that have been designed to reduce technical hands-on time and the time to results. some of the fda-approved/cleared real-time naats do not allow the user to see and evaluate the actual amplification curves. the inability to see the curves makes it difficult to troubleshoot when problems occur. although the user should not be able to alter cutoff values established during the fda trials, access to all or part of the raw data is desirable. one caveat for most qualitative naats is that they cannot distinguish between live and dead organisms and therefore, depending on the time for nucleic acid clearance, can be limited in monitoring response to therapy. in addition, with multiple viral infections, determining the relevance of each virus present in the sample is difficult, because residual virus detected may have been from a previous infection and may not be contributing to the current illness. the development of quantitative assays and evidence of declining viral load may clarify or resolve both of these issues. finally, issues relating to billing and reimbursement are substantial. the lack of specific current procedural terminology (cpt) codes for each of the individual targets requires laboratories to bill for multiple targets using the same generic amplified probe cpt code (87798). although it is appropriate to bill for each target present in a multiplex assay, the use of the same cpt code multiple times for a single test can be problematic. for example, many laboratory or hospital billing systems do not recognize multiple similar cpts for a single test and will only drop 1 cpt code charge. some insurance payors will cover only the charge of the first cpt code, and there are also limitations on how many times per day a similar cpt code can be billed. as a result, reimbursement for a highly multiplexed assay could be limited to 1 charge, thereby significantly increasing the costs to the laboratory and decreasing test profitability. under these circumstances and with pressure to reduce medical costs, many administrators will not approve the implementation of this testing if reimbursement is questionable. laboratories need analyte-specific cpt codes, and payors should reimburse for medically relevant naats. the north shore-long island jewish health system (ns-lijhs) infectious diseases molecular diagnostics laboratory opened 12 years ago. we performed 3 tests: human immunodeficiency virus type 1 viral load, amtd, and chlamydia trachomatis/ neisseria gonorrhoeae naats, with a total testing volume of 5000 tests per year. today, the laboratory performs naats for 40 different pathogens (including bacterial, mycobacterial, fungal, parasitic, and viral targets), with a volume of more than 225,000 tests per year, and in space that has tripled in size. molecular diagnostics for infectious diseases, along with molecular pathology, are 2 of the fastest growing laboratory departments in the ns-lijhs. in addition, as best exemplified by the 2001 anthrax bioterrorism event and the 2009 influenza a h1n1 pandemic, laboratories must be able to respond immediately by rapidly expanding testing capabilities, including sensitive and specific molecular diagnostics [43] . at the onset of the queens, new york, area 2009 influenza a h1n1 outbreak, the ns-lijhs laboratories' respiratory virus testing volume increased from a baseline of 225 tests per day to more than 950 tests per day, within 3 days. it was clear from the first week of the outbreak that the laboratory urgently needed to switch all influenza testing to molecular methods to obtain sufficient diagnostic sensitivity and to subtype the influenza strains. fortunately, an fda-approved test, the luminex xtag rvp test, was available and met our diagnostic requirements [5, 24, 43] . laboratories must continue to plan for future pandemic outbreaks and biothreat events. therefore, laboratories are constantly under pressure to expand testing and meet the demands of their clinical staff and their diverse patient populations. to do this, laboratories must provide clinically relevant, high-quality, cost-effective naats that are preferably fda approved/cleared. as we move forward, we request that the fda work closely with such organizations as the infectious diseases society of america and the american society for microbiology, with laboratory directors, and with ivd device manufacturers so that this goal can be met. the labor-intensive, complex methods and platforms of yesterday have evolved into simpler, user friendly versions that can be applicable to all health care settings. we encourage the fda to allow the submission process to evolve in a similar manner. no longer are the old ''gold standards'' of culture applicable, and new ways to evaluate these tests must be considered [44] so that accurate performance characteristics can be determined in a cost-effective manner. the latter is especially true for low-volume but highly relevant assays, such as quantitative naats for transplantation monitoring. in a reasonable and clear regulatory environment, the ivd device manufacturers will succeed in bringing their naats through the approval process. the role of antimicrobial management programs in optimizing antibiotic prescribing within hospitals antimicrobial stewardship programs in health care systems excessive antibiotic use for acute respiratory infections in the united states 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clinical features of pediatric rsv and human metapneumovirus infections clinical evaluation of nuclisens magnetic extraction and nuclisens analyte specific reagents for the real-time detection of respiratory syncytial virus (rsv) in paediatric respiratory specimens cost-effective use of rapid diagnostic techniques in the treatment and prevention of viral respiratory infections comparison of rapid detection methods for influenza a virus and their value in health care-management of institutionalized geriatric patients why diagnose influenza in hospitalized pediatric patients? cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients an outbreak of severe respiratory tract infection due to human metapneumovirus in a longterm care facility outbreak of human metapneumovirus infection in psychiatric inpatients: implications for directly observed use of alcohol hand rub in prevention of nosocomial outbreaks outbreak of swine-origin influenza a (h1n1) virus infection-mexico swine-origin influenza a (h1n1) virus infections in a school likelihood that an unsubtypeable influenza a result in the luminex xtag respiratory virus panel is indicative of novel a/h1n1 (swine-like) influenza comprehensive evaluation of performance, laboratory application, and clinical usefulness of two direct amplification technologies for the detection of mycobacterium tuberculosis complex use of the amplified mycobacterium tuberculosis direct test in a public health laboratory: test performance and impact on clinical care evaluation and follow up of infectious tuberculosis at the university of ottawa updated guidelines for the use of nucleic acid amplification tests in the diagnosis of tuberculosis comparative evaluation of initial and new versions of the gen-probe amplified mycobacterium tuberculosis direct test for direct detection of mycobacterium tuberculosis in respiratory and nonrespiratory specimens comparison of the real-time pcr method and the gen-probe amplified mycobacterium tuberculosis direct test for detection of mycobacterium tuberculosis in pulmonary and nonpulmonary specimens evaluation of gen-probe amplified mycobacterium tuberculosis direct test by using respiratory and nonrespiratory specimens in a tertiary care center laboratory detection of mycobacterum tuberculosis by pcr amplification with pan-mycobacterium primers and hybridization to an m. tuberculosis-specific probe rapid diagnosis of pulmonary tuberculosis by using the roche amplicor mycobacterium tuberculosis pcr test use of roche amplicor mycobacterum tuberculosis pcr in early diagnosis of tuberculous meningitis application of the roche amplicor mycobacterum tuberculosis (pcr) test to specimens other than respiratory secretions xpert m. tuberculosis/ rif for point-of-care diagnosis of tb in high-hiv burden, resourcelimited countries: hype or hope? development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay principles of the xtag respiratory viral panel assay (rvp assay) xtag respiratory virus panel luminex molecular diagnostics enhanced viral etiological diagnosis of respiratory system infection outbreaks by use of a multitarget nucleic acid amplification assay wi: gen-probe/prodesse evaluation of the one-step multiplex real-time reverse transcription-pcr proflu-1 assay for detection of influenza a and influenza b viruses and respiratory syncytial viruses in children wi: gen-probe/prodesse wi: gen-probe/prodesse wi: gen-probe/prodesse verigene respiratory virus nucleic acid test il: nanosphere il: nanosphere homogeneous detection of unamplified genomic dna sequences based on colorimetric scatter of gold nanoparticle probes quality assurance in clinical virology laboratory surge response to pandemic (h1n1) 2009 outbreak discrepant analysis: how can we test a test? i sincerely thank the fda and the infectious diseases society of america for providing a forum for discussion and exchange of ideas relating to this timely and clinically relevant topic.financial support. this work was supported by us department of defense award w81xwh-08-1-0477. potential conflicts of interest. c. c. g. is a member of the scientific advisory boards of gen-probe/prodesse and luminex molecular diagnostics. c. c. g. has received research grants and honoraria from gen-probe/prodesse, luminex molecular diagnostics, and diagnostic hybrids and research grants from 3m molecular diagnostics and has been a consultant for nanosphere. key: cord-272995-yvj2pqh1 authors: bergman, christian; stall, nathan m.; haimowitz, daniel; aronson, louise; lynn, joanne; steinberg, karl; wasserman, michael title: recommendations for welcoming back nursing home visitors during the covid-19 pandemic: results of a delphi panel date: 2020-10-07 journal: j am med dir assoc doi: 10.1016/j.jamda.2020.09.036 sha: doc_id: 272995 cord_uid: yvj2pqh1 objectives nursing homes became epicenters of covid-19 in the spring of 2020. due to the substantial case fatality rates within congregate settings, federal agencies recommended restrictions to family visits. six months into the covid-19 pandemic, these largely remain in place. the objective of this study was to generate consensus guidance statements focusing on essential family caregivers and visitors. design a modified two-step delphi process was used to generate consensus statements. setting and participants the delphi panel consisted of 21 us and canadian post-acute and long-term care experts in clinical medicine, administration, and patient care advocacy. methods state and federal reopening statements were collected in june 2020 and the panel voted on these using a three-point likert scale with consensus defined as ≥80% of panel members voting “agree.” the consensus statements then informed development of the visitor guidance statements. results the delphi process yielded 77 consensus statements. regarding visitor guidance, the panel made five strong recommendations: 1) maintain strong infection prevention and control precautions, 2) facilitate indoor and outdoor visits, 3) allow limited physical contact with appropriate precautions, 4) assess individual residents' care preferences and level of risk tolerance, and 5) dedicate an essential caregiver and extend the definition of compassionate care visits to include care that promotes psychosocial wellbeing of residents. conclusions and implications the covid-19 pandemic has seen substantial regulatory changes without strong consideration of the impact on residents. in the absence of timely and rigorous research, the involvement of clinicians and patient care advocates is important to help create the balance between individual resident preferences and the health of the collective. the results of this evidence-based delphi process will help guide policy decisions as well as inform future research. visitor guidance for america's nursing homes regarding the abrogation of self-determination and clinical concerns that ongoing restrictions 24 have begun to outweigh any potential benefits. 15, 20-22 25 cms released phased reopening guidelines on may 18, 2020, instructing nursing homes 26 to reopen only when the facility had no new covid-19 cases for a 28 day period and no 27 shortages in ppe, staffing, or testing capacity. 23, 24 three months after release of these guidelines, 28 many facilities are still far from meeting these criteria. 11 residents, families, clinicians, and 29 advocates are calling for a more immediately actionable, sustainable, balanced, nuanced, and 30 resident-centered approach to reopening nursing homes that respects residents' rights to 31 autonomy, informed risk taking, access to essential family caregivers, and other face-to-face 32 interactions. a group of experts convened to develop a set of evidence-informed guidance 33 statements to welcome back visitors and essential family caregivers to america's nursing homes. in round one, participants voted on the statements, using a three-point likert scale 74 ("agree," "neutral," or "disagree") with an option to offer "absent" due to a lack of perceived 75 expertise. consensus was defined as ≥80% of participants voting "agree" (green statements). 76 we grouped non-consensus statements into ≤50% (red statements) and 51-79% (yellow 77 statements) for purposes of facilitating discussion after each round. the whole panel discussed 78 statements not reaching consensus in a videoconference, starting with statements that had the 79 highest degree of uncertainty (red statements). in preparation for round two of voting, 80 participants also suggested additional statements for consideration. following the second round 81 of voting, a final videoconference discussion collected comments on the non-consensus 82 statements to help inform the final document. we report the final count of consensus and non-83 consensus statements. descriptive statistics and tables illuminated differences in responses 84 among the expert panel. 85 86 although the present reopening statements cover a wide range of topics, we focused on the 88 statements specific to visitors in order to communicate immediately actionable recommendations 89 to policymakers. those statements reaching consensus shaped our visitor guidance document. 90 we edited the final guidance statements for clarity, aiming to capture the consensus of the delphi aspects of the following topics (see table 1 ): testing of asymptomatic staff and residents, 111 surveillance testing, visitor guidance, immunity from prior covid-19 infection and associated 112 risk of infecting others. the panel generally agreed on the need for testing of asymptomatic staff 113 (79%); but the panel discussion reflected the importance of understanding community prevalence 114 as a key factor in deciding to test asymptomatic individuals. while the panel mostly agreed 115 j o u r n a l p r e -p r o o f visitor guidance for america's nursing homes (68%) that residents should be allowed to opt out of testing for sole purposes of surveillance, 116 fewer agreed that testing of asymptomatic residents should not be done (53%). most members 117 agreed that an asymptomatic resident who has recovered from the disease need not be tested 118 within 8 weeks from the onset of symptoms (74%) but fewer agreed to extend that to 90 days 119 (58%) or to never test again (11%). this general uncertainty about the time was again reflected 120 when the panel commented on whether an asymptomatic covid-19 resident who has recovered 121 could be contagious 8 weeks after recovery (65% agreement that they are not contagious), or 122 after 90 days (53% agreement that they are not contagious). a minority of the panel members 123 (35%) agreed that a recovered covid-19 who remains asymptomatic is not contagious. 124 the delphi process reached consensus on 12 of 14 statements related to visitors. these 127 statements were then merged and expanded into guidance statements (see table 2 guidance that begins to balance the well-being and self-determination of residents and their 141 families with the very real public health concern of preventing nursing home outbreaks. 142 our panel was able to review 119 guideline statements and develop consensus around 77 143 general statements to help inform a set of suggested visitor guidance statements. the panel 144 strongly agreed on some preconditions that would be essential prior to welcoming back visitors, 145 such as universal masking for staff, sufficient disinfecting supplies, ppe, and written plans 146 around isolation, cohorting, screening, testing, and outbreak investigations. furthermore, our 147 panel had wide consensus on testing of symptomatic residents and staff, the importance of 148 contact tracing, and barring communal and group activities for symptomatic residents. a key 149 finding reinforced by the panel was the future need to assess individual residents' care 150 preferences and level of risk tolerance, something that has been missing in many of the existing 151 reopening guidelines, in part due to cohorting challenges. this was envisioned by the panel as 152 allowing some residents to participate in a risk-accepting group that could be cohorted together 153 for increased social interactions and dining. 154 however, as illustrated in table 1 j o u r n a l p r e -p r o o f agreed that limited physical contact between visitors and residents should be allowed with 185 meticulous hand hygiene before and after resident contact, and the use of masks, gowns and 186 gloves. a lack of visitor access to ppe should not preclude a visit, so nursing homes must be 187 able to provide masks, gloves and gowns when required. 188 the panel had less accord, however, regarding infection prevention strategies during a 189 visit encounter. for example, universal masking for staff was supported but the group did not 190 reach consensus on which type of mask (e.g. surgical vs. cloth) or whether all visitors had to 191 wear a mask all of the time during a visit. similarly, it was agreed that physical distancing be 192 required in public, common spaces such as the lobby, hallways, or nursing stations, but perhaps 193 not applicable during a visit encounter with an asymptomatic resident. 194 regarding visitor guidance logistics, the panel strongly recommended the use of an 195 electronic process to schedule visits and a sign-in log with contact information to aid in potential 196 contact tracing. additionally, the panel recommended allowing the designation of one or two 197 essential family caregivers by the resident or surrogate decision maker. the essential family 198 caregiver(s) and the surrogate decision maker would have priority to visit the resident. these 199 visitors, for example, might provide complex care, such as assistance with feeding or support for 200 responsive behaviors commonly encountered in residents with dementia. all visitors and 201 essential family caregivers must be provided entry during serious illness, including at the end-of-202 life, irrespective of covid-19 status of the resident, provided that the visitor dons appropriate 203 lastly, regarding visitor guidance and risk tolerance, the panel acknowledged that 205 essential family caregivers may wish to visit a resident who may be contagious such as 1) a 206 symptomatic resident with a positive covid-19 test, 2) a symptomatic resident with an 207 j o u r n a l p r e -p r o o f unknown or pending covid-19 test, or 3) an asymptomatic resident who has tested positive for 208 covid-19. after discussion, the authors recommend three steps be followed. first, a shared 209 informed consent discussion between essential caregivers and nursing leadership that would regarding immunity and cohorting, our panel agreed (83%) that cohorting asymptomatic 216 residents who have all recovered for covid-19 can be safely done and that a resident who has 217 recovered from covid-19, remains asymptomatic, and is at least 3 weeks post onset of 218 symptoms is likely not infectious (88% consensus). regarding the role of antibody testing, a 219 modest majority agreed (69%) that antibody testing could be a surrogate marker of individual 220 immunity; but all agreed that a positive immunity test does not currently inform clinical practice 221 and instead one has to rely upon recovery from prior infection. it should be noted that the delphi 222 process occurred before the cdc guidance that recovered covid-19 residents do not require re-223 testing or precautions for 90 days had been released 27 . 224 the areas of congruence leading to the suggested visitor guidance statements stem from 225 thoughtful resident-centered debates among a panel of delphi experts. the fact that there are 226 many areas with substantial variation certainly arises from the state of the science but might also 227 be a result of the interaction of certain statements with each other, difficulties with precise 228 wording or statements, or the persistent inability of guidelines to accommodate the wealth of 229 variations in clinical situations. one limitation of this study was that a rapid two-step modified 230 j o u r n a l p r e -p r o o f delphi process may not have allowed enough time for the panel to develop consensus around 231 some of the challenging language or the more controversial topics, but the panel felt the urgency 232 to produce high-quality guidance statements promptly. 233 the use of a modified delphi process to standardize the process, provide iterative 234 feedback, and consensus-gathering strengthens the findings of this study. the delphi process 235 itself limits bias but could be influenced by how panelists were selected 29 . there was some 236 degree of self-selection in the organization of this panel as the group shared a common concern 237 regarding the health and wellbeing of the vulnerable older adults living in nursing homes during 238 the covid-19 pandemic. additionally, the panel had substantial diversity but did not include all 239 important stakeholders, such as nurse leaders, direct care workers, or residents. nevertheless, the 240 panelists were chosen for their expertise in the field of geriatrics and long-term care medicine, 241 and have all been listed in the acknowledgement section. 242 243 the objective of this study was to develop a set of visitor guidance statements that could 245 be used to welcome back visitors and essential family caregivers to us nursing homes. even 246 after a structured delphi process, experts in nursing home care still had substantial discord on 247 important elements. however, through rigorous and evidence-informed discussions, a concise 248 and practical set of guidance statements was developed (table 2) in order for a nursing home to proceed with phased reopening, there should be no new nh-onset cases for 28 days. 14 (74) 1 4 testing a proportion of randomly selected asymptomatic hcp (staff) who have not previously tested positive should be done for surveillance efforts. the frequency and sample size of staff should be guided by size of the nursing home and level of local community spread. in facilities without any positive covid-19 cases, test 100% of asymptomatic hcp (staff) who have previously not tested positive weekly for 4 weeks; if no new positives may test 25% of asymptomatic hcp (staff) every 7 days such that 100% of the nursing home staff are tested each month. 10 (53) 3 6 testing a proportion of randomly selected asymptomatic residents who have not previously tested positive should not be done for surveillance efforts. instead, residents who are asymptomatic should only be tested during outbreak investigations of close contacts of a known covid-19 positive resident or staff member. residents who are asymptomatic should be allowed to opt out of testing for sole purposes of surveillance. this statement would not be applicable for contact tracing with a known exposure to a covid-19 resident or staff member. an asymptomatic resident who has previously tested positive for covid-19 and recovered does not need to be 14 (74) 2 3 tested again within an 8 week window of prior onset of symptoms. an asymptomatic resident who has previously tested positive for covid-19 and recovered does not need to be tested again within a 90 day window of prior onset of symptoms. an asymptomatic resident who has previously tested positive for covid-19 and recovered does not need to be tested again. a new or returning asymptomatic nursing home resident without a prior diagnosis of covid-19 and who has remained under isolation in a private room for 14 days since admission tests positive during nursing home testing of asymptomatic residents. not during an outbreak investigation and there has been no exposure to a covid-19 positive resident or staff. in this situation, re-test the resident only. if subsequently negative and no further suspicion of covid-19 in the building, this scenario would not warrant nursing home-wide testing or phase regression. 14 (74) 1 4 a negative covid-19 test is not a requirement prior to visiting a nursing home. 14 (70) 1 5 visitors who wish to visit a nursing home resident who is actively symptomatic but for whom covid-19 testing is pending or unknown should have an informed consent discussion with nursing leadership, demonstrate appropriate donning/doffing of ppe and agree to wear appropriate ppe during the visit. allow entry of all essential and non-essential healthcare personnel, contractors, and vendors with appropriate screening, physical distancing, hand hygiene, and face coverings. they would be subject to the same testing and surveillance requirements as the rest of the hcp (staff) cohort. visitors including non-employed caregivers and surrogate-decision makers would be subject to the visitor the nursing home should consider a designated care giver (or dedicated support person, surrogate decision-maker) an essential member of the healthcare team who would not be subject to visitor guidelines if resources (ppe, training, monitoring) are available at the time and the person is directly engaged in compassionate care to alleviate a residents psycho-social stress as a result of isolation. 15 (79) 0 4 a resident who engages in a visit with family or friends beyond the nursing home grounds, remains outside, and the visit does not involve close contact with covid+ individuals or symptomatic individuals would not be subject to isolation upon re-entry to the nursing home. after a resident returns from an outside trip beyond the nursing home grounds and prior to the resident resuming activities within a shared space, the resident should be bathed according to accepted practice with soap and have the clothes they were wearing laundered in a standard fashion. immunity a currently asymptomatic individual who has recovered from covid-19 and is post 8 weeks from onset of symptoms is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the resident is not contagious. a currently asymptomatic individual who has recovered from covid-19 and is post 90 days from onset of symptoms is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the resident is not contagious. a currently asymptomatic individual who has recovered from covid-19 is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the resident is not contagious. antibody testing can be a surrogate marker of individual immunity but does not currently inform clinical practice; recovery from prior infection does. color scheme represents level of consensus among panel. yellow represents statements in which 51-79% of members voted "agree" and red represents statements in which <50% of members voted "agree." j o u r n a l p r e -p r o o f all staff, residents, and visitors engage in basic hand hygiene and physical distancing in public, shared spaces. all staff wear a medical-grade mask while in the nursing home. all residents and visitors wear a face covering when in shared, public spaces. if a resident or visitor does not own a face covering, one must be provided by the nursing home. the facility has sufficient disinfecting supplies (hand sanitizers, soap, detergent, etc.) and adequate personal protective equipment (gloves, gowns, masks, face shields/goggles). a written isolation and cohorting plan is in place. a written screening and testing plan with adequate capacity for implementation is in place. a written contact tracing and outbreak investigation plan is in place. all persons entering the nursing home (staff, visitors, volunteers, and vendors) undergo the same entrance screening process, including a temperature check and answering an exposure and symptom questionnaire by a trained entrance screener. visitors that do not comply with the screening procedure are not allowed to enter. visitors and volunteers can sign up to visit a resident for a defined time period using an electronic process. the nursing home maintains a sign-in log that includes contact information (name, phone number, email address) of visitors and volunteers to help with contact tracing in the event of an exposure. a nursing home may need to limit the number of indoor visitors to no more than 2 visitors at one time to allow physical distancing between visitor groups. visit frequency and the number of visitors a nursing home is able to accommodate would depend on the physical space, availability to visit outdoors, and ppe availability. visitors must be guided to the designated visit area to limit interactions with patient-care areas, staff, or other residents. gloves and a gown with associated hand hygiene are required if visitors wish to engage in limited physical contact with a resident, such as hugging, hand holding, or direct resident care such as assistance with meals. the nursing home must provide gloves and gowns for this purpose. the nursing home should designate areas for indoor and outdoor visits. ideally the visits would occur outside, conditions permitting. indoor areas should be accessible without walking through a resident care area, must be disinfected between scheduled visits, and should be large enough to facilitate physical distancing between visit groups. a nursing home should allow each resident or surrogate decision maker to choose essential family caregivers who, along with the surrogate decision maker, would have priority to frequently visit a resident, e.g. to provide complex care, aid in feeding, or redirect and reassure those residents living with dementia who have responsive behaviors. visiting a resident with or without symptoms who has a positive, unknown, or pending covid-19 test result requires the following steps: 1. the visitor must participate in an informed consent discussion with leadership regarding the risks of potential exposure to covid-19 and whether they outweigh the benefits of a visit. additionally, visitors should be counseled to understand the covid-19 test status and encouraged to wait for a pending test result to return prior to a scheduled visit. 2. the nursing home must provide education and training so that the visitor can demonstrate appropriate donning/doffing of ppe, including a mask, gowns, gloves, and possibly a face shield. 3. the visitor must agree to wear the recommended ppe during the visit and follow all infection prevention and control procedures within the nursing home. the nursing home should make every attempt possible to work with visitors of residents who are seriously ill, receiving care focused on comfort, and approaching end-of-life. specifically, facilities may waive the visitor limits, offer extended hours, and offer an in-person room visit to help facilitate the psycho-social well-being of the resident and family members. j o u r n a l p r e -p r o o f  social distancing, hand washing, and disinfection practices need to continue in directpatient care areas.  residents, visitors, and volunteers wear cloth face coverings or a facemask when in a shared-space.  all persons entering the facility (including staff, visitors, volunteers, and vendors) should undergo screening to include: temperature check, exposure questionnaire, and symptom questionnaire.  entry screening is performed by a screener who has received training in basic infection control, appropriate education on questionnaires and hands-on practice with thermometer.  all persons attempting to enter the facility who have either recorded a temperature >99.5 f or report having taken a medication to treat fever (anti-pyretic such as acetaminophen) should not be permitted to enter.  all residents should undergo a daily symptom screening and have temperature monitored.  symptomatic residents/staff o test all symptomatic residents and staff but allow individual residents autonomy with an appropriate plan on how to isolate and cohort a resident who is symptomatic but does not wish to be tested. o a symptomatic staff member who does not wish to be tested would be excluded from work until they meet the return to work criteria of a presumed positive individual. o treat a symptomatic resident who does not wish to be tested as a presumed positive. isolate and cohort accordingly. o  asymptomatic residents/staff o all residents, staff should have undergone baseline testing as part of phase 1 and phase 2. o have a plan for ongoing surveillance testing of asymptomatic staff and residents.  testing a proportion of randomly selected asymptomatic hcp (staff) who have not previously tested positive should be done for surveillance efforts. the frequency and sample size of staff should be guided by size of facility and level of local community spread. -79% agreement  in facilities without any positive covid-19 cases, test 100% of asymptomatic hcp (staff) who have previously not tested positive weekly for 4 weeks; if no new positives may test 25% of asymptomatic hcp (staff) every 7 days such that 100% of facility staff are tested each month. 53% agreement  testing a proportion of randomly selected asymptomatic resident who have not previously tested positive should not be done for surveillance efforts. instead, residents who are asymptomatic should only be tested during outbreak investigations of close contacts of a known covid-19 positive resident or staff member. 53% agreement  residents who are asymptomatic should be allowed to opt out of testing for sole purposes of surveillance. this statement would not be applicable for contact tracing with a known exposure to a covid-19 patient or staff member. -68% agreement o triggers to increase testing:  a trigger to increase testing of asymptomatic individuals would be based on response to an outbreak investigation and contact tracing results.  one covid-19 + case in staff or residents should trigger the execution of a comprehensive plan addressing contact tracing, isolation/cohorting, and testing within 24 hours of positive test result.  during an outbreak investigation, there should be a low threshold to extend testing of all staff and residents to entire units, floors, buildings if the situation deems it necessary.  asymptomatic residents and staff who have previously tested positive would not be subject to repeat testing.  once one nh-onset case (case definition from cdc) has been identified within a facility, facilities should resume testing of asymptomatic hcp (staff) who have not previously tested positive o the facility should make every effort possible to secure a collection method that is least invasive and uncomfortable if testing residents and staff with a low pretest probability of covid-19 disease (asymptomatic without known exposure), such as saliva testing or nasal/oral swabs instead of a nasopharyngeal swab. o asymptomatic covid-19 recovered resident  an asymptomatic resident who has previously tested positive for covid-19 and recovered does not need to be tested again within an 8 week window of prior onset of symptoms. 74% agreement  an asymptomatic resident who has previously tested positive for covid-19 and recovered does not need to be tested again within a 90 day window of prior onset of symptoms. 58% agreement  an asymptomatic resident who has previously tested positive for covid-19 and recovered does not need to be tested again. 11% agreement  a process should be identified for how facilities will actively track staff/ resident and visitor interactions to help facilitate appropriate contact tracing in the event of an outbreak investigation.  in the event of a pui or covid-19 positive staff or resident, a list of individuals with possible exposures should be able to be generated for the prior 3 days (preferably 5 days) within 24 hours.  facilities should be aware and document individual resident and/or surrogate decisionmakers' care preferences regarding testing, cohorting, and isolation. it may be possible to cohort a certain group of individuals (ie recovered covid-19 positive patients who are asymptomatic) as long as the risks for other residents is not substantially increased.  new admissions should be placed in a dedicated area of the facility where appropriate isolation and contact precautions are maintained.  there should be a written cohorting and isolation plan for the facility. group activities  do not allow symptomatic residents with an unknown covid-19 status to participate in group activities in which proper infection control practices cannot be maintained.  make every effort possible to maintain social distancing, practice hand hygiene, and wear a mask during group activities.  try to facilitate indoor group activities in a well-ventilated space that allows for appropriate social distancing.  make an effort to offer residents the ability to join a risk-accepting group that could be cohorted together for activities, provided that the facility can manage them separately. non-medically necessary trips outside facility  residents must adhere to face coverings, hand hygiene, and social distancing during trips outside of the facility.  isolation o a resident who engages in a supervised outside visit with family or friends within the nursing home grounds, remains outside, and the visit does not involve close contact with covid+ individuals or symptomatic individuals would not be subject to isolation upon re-entry to the facility. o a resident that makes a trip outside the facility and is exposed to a covid+ individual, symptomatic individual or otherwise fails the screening questionnaire upon re-entry to the building would be subject to 14 days of isolation. o a resident who engages in a visit with family or friends beyond the nursing home grounds, remains outside, and the visit does not involve close contact with covid+ individuals or symptomatic individuals would not be subject to isolation upon re-entry to the facility. 17% agreement  infection control o after a resident returns from an outside trip beyond the nursing facility grounds and prior to the resident resuming activities within a shared space, the resident should practice hand hygiene and have their wheelchair and belongings disinfected. o after a resident returns from an outside trip beyond the nursing facility grounds and prior to the resident resuming activities within a shared space, the resident should be bathed according to accepted practice with soap and have the clothes they were wearing laundered in a standard fashion. 47% agreement  leave of absence o the facility should have a discussion regarding risks/benefits with every resident and family who requests a leave of absence with a bed hold. this would include a discussion on hand hygiene, social distancing, and mask covering as well as subsequent isolation upon return to the facility if deemed necessary at the time of the visit based on level of community spread.  phase regression and facility wide restrictions should not be imposed after one isolated covid-19 case. rather, a prompt outbreak investigation should occur with further results triggering appropriate restrictions.  response to positive resident o once one nursing home resident tests positive for covid-19, an outbreak investigation should include baseline testing of close contacts (to include roommate, neighboring rooms, and staff) o once one nh-onset case (case definition from cdc) has been identified within a facility, facilities should resume testing of asymptomatic hcp (staff) who have not previously tested positive. o during an outbreak investigation of a single case it is determined that there is 1 nh-onset case on an isolated wing with isolated staff. this scenario would warrant testing of the entire wing staff and residents but not warrant facility wide testing or phase regression. o a new snf admission who has remained under isolation in a private room becomes symptomatic within 14 days of admission and tests positive. in this situation, i would re-test and extend testing to close contacts. if no further positive cases, this situation would not warrant facility wide testing or phase regression. o a new or returning asymptomatic nursing home resident without a prior diagnosis of covid-19 and who has remained under isolation in a private room for 14 days since admission tests positive during facility testing of asymptomatic residents. not during an outbreak investigation and there has been no exposure to a covid-19 positive patient or staff. in this situation, i would re-test the resident only. if subsequently negative and no further suspicion of covid-19 in the building, this scenario would not warrant facility-wide testing or phase regression. 74% agreement  response to positive hcp o an asymptomatic hcp tests positive on routine surveillance testing and is appropriately following work-restrictions. this scenario should prompt an outbreak investigation of close contacts but should not automatically warrant a phase regression as long as the outbreak investigation does not identify new cases among staff or residents who have not previously tested positive. o a symptomatic hcp tests positive. this would warrant testing of close contacts (staff and residents) of the immediate patient care area. o once one nursing home staff member tests positive for covid-19, an outbreak investigation should include baseline testing of close contacts (to include roomate, neighboring rooms, and staff)  phase regression o during an outbreak investigation, it is determined that there is >2 nhonset cases in a building within a short time period (<14 days). there is concern for wide spread disease in the building. this scenario would warrant testing of the entire facility and phase regression with subsequent restrictions on visitors, communal dining, and group activities. immunity  a patient who has recovered from covid-19 disease and is 3 weeks post onset of symptoms is likely not infectious to another individual as long as they have not developed new symptoms.  a cohort of asymptomatic individuals who have all recovered from covid-19 can safely be cohorted together.  antibody testing can be a surrogate marker of individual immunity but does not currently inform clinical practice; recovery from prior infection does. 69% agreement  covid-19 recovered individual o a currently asymptomatic individual who has recovered from covid-19 and is post 8 weeks from onset of symptoms is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the patient is not contagious. 65% agreement o a currently asymptomatic individual who has recovered from covid-19 and is post 90 days from onset of symptoms is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the patient is not contagious. 53% agreement o a currently asymptomatic individual who has recovered from covid-19 is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the patient is not contagious. 35% agreement  hairdressers, beauticians, hospice staff and other staff members who work within a nursing home should be included in the cdc definition of healthcare personnel (hcp) and follow the same guidelines regarding screening and testing.  hairdressers and stylists should be considered direct patient care staff and be subject to the same screening and work restrictions as other healthcare facility staff.  patients that are unable to adhere to social distancing or face coverings should be allowed to visit with family in a private isolated area as long as visitors were full ppe.  dialysis patients who leave the facility regularly for hemodialysis will remain under appropriate isolation and contact precautions and not mix with covid-, asymptomatic individuals. j o u r n a l p r e -p r o o f definitions i agree with the following modification of the formal cdc definition of healthcare personnel (hcp). "hcp include, but are not limited to, emergency medical service personnel, nurses, nursing assistants, physicians, technicians, therapists, phlebotomists, pharmacists, feeding assistants, students and trainees, contractual hcp not employed by the healthcare facility, and persons not directly involved in patient care but who could be exposed to infectious agents that can be transmitted in the healthcare setting (e.g., clerical, dietary, environmental services, laundry, security, beauticians and hairdressers, engineering and facilities management, administrative, billing, and volunteer personnel)". (added beauticians to cdc definition of hcp) nursing homes are ground zero for covid-19 covid-19 nursing home data covid-19 in nursing homes: calming the perfect storm coronavirus disease 2019 in geriatrics and long-term care: the abcds of covid-19 presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility asymptomatic sars-cov-2 infection in belgian long-term care facilities presymptomatic transmission of sars-cov-2 amongst residents and staff at a skilled nursing facility: results of real-time pcr and serologic testing epidemiology of covid-19 in a long-term care facility in king county, washington cms announces new measures to protect nursing home residents from covid-19 centers for disease control and prevention. preparing for covid-19 in nursing homes html?cdc_aa_refval=https%3a%2f%2fwww.cdc.gov%2fcoronavirus%2f2019-ncov%2fhealthcare-facilities%2fprevent-spread-in-long-term-care-facilities continued bans on nursing home visitors are unhealthy and unethical. the washington post amid the covid-19 pandemic, meaningful communication between family caregivers and residents of long-term care facilities is imperative families caring for an aging america essential family caregivers in long-term care during the covid-19 pandemic detrimental effects of confinement and isolation on the cognitive and psychological health of people living with dementia during covid-19: emerging evidence. ltccovid, international long-term care policy network: care policy and evaluation centre (cpec) finding the right balance: an evidence-informed guidance document to support the re-opening of canadian nursing homes to family caregivers and visitors during the covid-19 pandemic we gratefully acknowledge the time and dedication of our delphi panel experts and other experts who have participated in this process, provided guidance, or critically appraised our manuscript. their names are listed below in alphabetical order. none received compensation, financial or otherwise, for their contributions. j o u r n a l p r e -p r o o f the facility should make every effort possible to secure a collection method that is least invasive and uncomfortbale if testing residents and staff with a low pretest probability of covid-19 disease (asymptomatic without known exposure), such as saliva testing or nasal/oral swabs instead of a nasopharyngeal swab. immunity q86 a currently asymptomatic individual who has recovered from covid-19 and is post 8 weeks from onset of symptoms is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the patient is not contagious. immunity a currently asymptomatic individual who has recovered from covid-19 and is post 90 days from onset of symptoms is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the patient is not contagious. 9 1 7 17 53% immunity a currently asymptomatic individual who has recovered from covid-19 is not considered infectious and should not be tested. if tested and the test returns positive, as long as the resident remains asymptomatic, it would not be considered a re-infection and the patient is not contagious. immunity q85 antibody testing can be a surrogate marker of individual immunity but does not currently inform clinical practice; recovery from prior infection does. key: cord-021550-evh3b7o2 authors: brokopp, charles; resultan, eric; holmes, harvey; wagner, michael m. title: laboratories date: 2007-09-02 journal: handbook of biosurveillance doi: 10.1016/b978-012369378-5/50010-7 sha: doc_id: 21550 cord_uid: evh3b7o2 nan results from laboratory testing are an important source of information for biosurveillance systems. clinical laboratory tests are vital for the correct diagnosis and treatment of individuals. clinical laboratories analyze blood, urine, mucus, saliva, respiratory secretions, cerebrospinal fluid, semen, vaginal secretions, sweat, feces, fluid aspirated from joints, and tissues from humans and animals. the tests performed include cell counts; analytical chemistries, including drug and toxin tests; and examinations to detect and identify microbes and markers of current and past infection. environmental testing is critical to the detection of outbreaks, the prevention of disease, and the monitoring of the environment. environmental laboratories analyze samples of water, food, air, soil, plant material, and unknown powders, as well as samples taken from surfaces for evidence of bacterial, viral, toxin, or chemical contamination. data produced by laboratories are important for biosurveillance of virtually every disease caused by biological agents, chemicals, or toxins. data collected during the preanalytical, analytical, and postanalytical phases of testing can be captured and incorporated directly into biosurveillance systems. preanalytical data, such as the type of test ordered and the reason for a test, can provide an early clue to the existence of an outbreak. similarly, analytic results, such as the initial gram stain of a cerebrospinal fluid specimen, can potentially confirm a diagnosis when combined with other clinical information, as it did in the first case of inhalational anthrax in the 2001 postal attack. the actual results of tests are obviously foundational to biosurveillance. the range of tests offered by an individual laboratory varies significantly among laboratories in the united states. many small laboratories perform a limited number of tests that are needed on an urgent basis or for screening purposes. larger laboratories provide more complex confirmatory analyses. the majority of clinical laboratories in the united states are small laboratories located in physician offices; however, the larger laboratories account for a high volume of all tests performed. table 8 .1 describes the clinical laboratory tests that contribute to the diagnosis of inhalational anthrax. anthrax, as well as many other infectious diseases, is diagnosed after the performance of presumptive and confirmatory tests in combination with the clinical presentation. clinical specimens, such as blood, cerebrospinal fluid, urine, sputum, throat swabs, and skin scrapings, are used to isolate a causative agent that is later subjected to further testing with confirmatory procedures to make the final identification. preliminary tests results are sometimes released before the confirmatory tests results become available. when preliminary results are reported, the report often contains a statement about when final results will be available. laboratories that produce the types of data most useful for biosurveillance include clinical laboratories operated by the human or animal health systems, commercial laboratories, and governmental laboratories. laboratories typically specialize in either human or animal testing. commercial laboratories are free-standing laboratories that are not associated with hospitals or healthcare facilities and that often provide a broad range of services over a wide geographical area. governmental laboratories exist at the federal, state, and local level and often provide testing that is not readily available from other laboratories. we describe each of these types of laboratories in this chapter. laboratories that test for biologic agents are classified as biosafety level 1, 2, 3, or 4, with biosafety level 4 providing the highest degree of protection to personnel and the environment. most clinical laboratory work is performed at level 2. these biosafety levels combine the use of laboratory safety practices, safety equipment, and laboratory facilities to provide greater levels of safety for the more dangerous organisms. each level is specifically appropriate for handling various biologic agents (cdc, 1999) . there are more than 186,000 clinical laboratories in the united states in which clinical laboratory scientists, pathologists, the centers for medicare and medicaid services (cms) registers all clinical laboratories in the united states that examine materials derived from the human body for diagnosis, prevention, or treatment. cms administers the program for the secretary of health and humans services in conjunction with the centers for disease control and prevention (cdc) and the food and drug administration (fda). cms regulates laboratories and establishes criteria for other organizations, such as state health departments, that also regulate laboratories to ensure compliance with the federal clinical laboratory improvement act (clia). clia was first enacted by congress in 1967 and set guidelines for large independent laboratories. in 1988, congress amended clia 67 to expand the type of laboratories that must comply; clia 88 further established quality standards for laboratories to ensure accuracy, reliability, and timeliness of test results. in august, 2004, 186,734 laboratories were registered with the cms (centers for medicare and medicaid services, 2004) . table 8 .2 shows the distribution of these laboratories by type. more than 55% of these laboratories are located in physician offices. skilled nursing facilities (7.9%), hospitals (4.6%) ,and home health agencies (4.4%) accounted for an additional 20% of laboratories. the remaining clinical laboratories are found in community health clinics, health maintenance organizations, blood banks, industrial facilities, medical technologists, and laboratory technicians perform 7 billion or more diagnostic tests annually (centers for medicare and medicaid services, 2004) . the american society for clinical pathology (ascp) currently certifies more than 280,000 laboratory professionals who primarily work in clinical diagnostic and research laboratories. clinical laboratory services in the united states are delivered either by commercial clinical laboratories or by "in-house" laboratories at healthcare facilities (hospitals, clinics, physician offices), departments of health, veterinary hospitals, and clinics. individual veterinarians and physicians and the staff within their offices also conduct laboratory testing and produce results that are important for biosurveillance. professional laboratorians provide services that include simple, rapid screening tests; more advanced diagnostic tests; and complex confirmatory analyses. clinicians use the information provided by laboratories to establish diagnoses and to make treatment decisions on virtually every patient. the demand for testing is increasing as the population ages and requires more health care, including analytical services. new tests are frequently introduced that improve diagnosis and care. the emergence of new diseases, the threat of bioterrorism, and the need for better biosurveillance systems have increased the demand for qualified laboratory professional in all fields, especially infectious disease testing. although the demand for more laboratory professionals is increasing, the number of established laboratory professional training programs is decreasing. test kits determined by the fda to be sufficiently simple to perform that there is little risk of operator error. laboratories that perform waived tests must enroll in the clia program, pay certification fees, and follow the manufacturers' test instructions. however, laboratories that perform only waived tests do not undergo inspections or need to comply with other clia requirements for larger laboratories. laboratories that perform tests that use a microscope to examine specimens that are not easily transportable during the course of a patient visit are required to enroll in a clia program, pay applicable fees, and maintain certain quality and administrative requirements. these laboratories, known as provider-performed microscopy providers (ppmps), represent 22% of the registered laboratories and are not subject to routine inspections. the remaining clinical laboratories must either be accredited by 1 of 6 approved clinical laboratory accrediting organizations (american association of blood banks, american osteopathic association, american society of histocompatibility and immunogenetics, college of american pathologists; commission on office laboratory accreditation, joint commission on accreditation of healthcare organizations) or obtain a compliance certificate directly from cms. in august 2004, these six organizations had accredited 15,667 (8.7%) laboratories, and cms had certified 20,758 (11.5%) laboratories. the balance of the clinical laboratories (144,022) were either waived test providers or ppmps (centers for medicare and medicaid services, 2004) . accreditation of clinical laboratories helps ensure that laboratories meet or exceed clinical standards established by governmental and nongovernmental associations. cms, insurance companies, and healthcare plans require laboratories to be certified or accredited by these organizations for reimbursement of laboratory services. table 8 .3 shows the annual test volume of the 20,758 clinical laboratories that were certified by cms in august 2004. over 84% of these laboratories perform fewer than 25,000 tests per year. only 125 of these laboratories performed 500,000 or more tests per year. these 125 laboratories represent only 0.6 % of all laboratories, yet they perform approximately 20% of all tests. two state health departments have developed and currently administer state clinical laboratory improvement programs that cms deems equivalent to the cms program. laboratories in washington and new york must meet the standards of these state programs. approximately 25 additional states have laboratory licensure programs. they receive funding from cms to implement the federal clia program. in states that do not have clinical laboratory regulatory programs, the laboratories must choose between accreditation through one of the six approved organizations or submitting to inspection and certification by cms. environmental testing laboratories perform physical, chemical, and microbiological analysis of specimens collected in the environment. for example, a water sample may undergo physical testing (temperature, turbidity, odor, color), chemical testing (nitrates, sulfates, pesticides, metals), and microbiological testing (total plate counts, coliforms, giardia, cryptosporium). environmental testing laboratories provide a wide range of testing that is in many ways similar to the testing performed in clinical laboratories. sanitarians or water quality technicians often perform basic tests (e.g., for temperature, ph, volatility, and physical appearance) at the site where samples are collected. they transmit the results of these simple tests to the laboratory along with the samples, where chemists and microbiologists perform additional presumptive and confirmatory testing. results from the simple tests may suggest the need for more definitive testing, using instruments such as atomic adsorption spectrophotometers, gas chromatographs, and mass spectrometers. laboratories perform much of the routine environmental testing in batches of 10 to 50 samples on semiautomated or fully automated instruments. the raw analytical data are captured, processed, and reported by using software that interfaces directly with the instrument and the laboratory's data management system. environmental laboratories are certified by accreditation authorities recognized by the environmental protection agency (epa) as part of the national environmental laboratory accreditation program (nelap). at least 12 states currently are recognized by the epa as environmental laboratory accrediting authorities. these state programs apply nationally recognized standards to the laboratories that they accredit so that there is some consistency in the quality of tests performed by accredited laboratories. commercial laboratories are an important component of the medical delivery system in the united states. a commercial laboratory is a laboratory that is free-standing; that is, it is not associated with a hospital or other healthcare organization. commercial laboratories may specialize in clinical specimens, environmental specimens, or both. commercial laboratories can be important partners for organizations that wish to develop biosurveillance systems because of size of the laboratories and their use of information technology. commercial laboratories have grown significantly in size during the past decade as a result of mergers and consolidations, and they may offer tests that are not readily available. many of these laboratories began as specialized reference laboratories, offering tests that could not be economically provided by smaller laboratories. small laboratories merged with larger laboratories that were, in turn, purchased by large laboratory corporations. regional consolidation of clinical laboratories that provided services to healthcare organizations led to the formation of large commercial laboratories. these commercial laboratories, such as lab corp, arur and quest, have developed service systems that allow them to provide clinical testing at their headquarters and at distributed sites around the country. large commercial laboratories have established elaborate courier systems that collect samples locally for overnight distribution to the appropriate laboratory in their network. although a sample may be transported to a distant laboratory, the results, nevertheless, frequently become available overnight. the commercial laboratories use information systems referred to as laboratory information management systems (limss) to track tests and results. these systems monitor test requests, capture test results, and electronically report the results, often within hours of the sample being received. as discussed in chapter 5, clinical laboratories are required to report notifiable diseases to local health departments. the large, multistate commercial laboratories face challenges in complying with notifiable disease reporting requirements that vary by state. further, reporting requirements frequently change as new diseases of public health interest are identified and many states have expanded laboratory reporting requirements to include suspected cases of notifiable diseases. commercial laboratories are increasingly using electronic laboratory reporting to satisfy these complex and changing requirements. commercial laboratories may specialize in environmental testing. these commercial environmental laboratories provide testing on a variety of samples, such as water, air, hazardous materials, dust, and soil. these laboratories often contract with governmental agencies, such as epa, department of defense (dod), and u.s. department of agriculture (usda) at the federal level and with environmental and regulatory agencies at the state and local level. federal, state, and local government agencies, such as health departments, operate laboratories or contract with commercial laboratories for testing related to diagnosis, regulatory compliance, investigations, and environmental monitoring. since the early 1800s, governmental laboratories have performed testing that led to the identification of outbreaks of diphtheria, cholera, smallpox, and typhoid fever. during the 20th century, these laboratories, in conjunction with academic laboratories, helped develop vaccines and contributed to the detection of polio, rubella, measles, and whooping cough. the response to west nile fever in the united states and the release of viable bacillus anthracis in 2001 required these laboratories to develop procedures quickly for diagnosis and identification of agents that they had not previously encountered. governmental laboratories are key to the recognition of new and emerging infectious diseases and are vital to surveillance efforts. the department of health and human services (dhhs), usda, department of energy (doe), dod, and departments of commerce and justice, and the epa operate or fund clinical, environmental, forensic, and research laboratories. federal laboratories provide reference testing and are often involved with the development of new technologies, as well as the transfer of these technologies to other laboratories. many federal laboratories collaborate with international partners and serve as reference centers for specialized testing. federal laboratories often provide training, confirmatory testing, and reference materials for other governmental laboratories. the dhhs laboratories that are associated with disease detection, control, and surveillance activities are found at the national institutes of health (nih), cdc, and fda. the nih, headquartered in bethesda, maryland, conducts research on acute and chronic diseases, develops new therapies and immunizations, and develops new laboratory and diagnostic technologies for many infectious and noninfectious diseases and health conditions. much of the nih work is done through extramural grants and contracts with universities, private companies, and other governmental organizations. the cdc's laboratories focus on infectious diseases, occupational diseases, and environmental causes of diseases. the cdc specialized laboratories are in colorado, ohio, west virginia, and puerto rico, in addition to its headquarters in atlanta, georgia. the cdc has led the development of rapid national laboratory reporting systems that have been successfully used to identify multistate outbreaks of diseases ( bean et al., 1992; hutwagner et al., 1997) . cdc-based scientists have developed and used new technologies to identify outbreaks of disease in cooperation with state and local public health laboratories. one such example of the successful application of a new technology is known as pulsenet (discussed in chapters 3 and 5; http:// www.cdc.gov/pulsenet/). pulsenet is a national network of public health laboratories that perform dna "fingerprinting" of foodborne pathogens and clinical isolates to allow matching of isolates. this epidemiological typing method is the basis for detecting clusters of disease that are geographically diffuse, and for linking bacteria found in a specific food to bacteria found in one or more persons with a particular disease. pulsenet permits recognition of outbreaks that previously went undetected. a multistate outbreak of listeriosis in 2000 was identified only after the isolates of listeria monocytogenes were tested by using pulsed-field gel electrophoresis and determined to all have a common pulsenet pattern (cdc, 2000) . fda laboratories focus primarily on monitoring the food supply and ensuring the purity and potency of drugs and other pharmaceuticals. these regulatory laboratories frequently become involved in the investigation of food contamination (including ground beef, poultry) and the adulteration of drugs. fda maintains regional laboratories in washington, new york, colorado, michigan, kansas, california, georgia, and arkansas, as well as specialized laboratories in ohio, pennsylvania, puerto rico, and massachusetts. the fda laboratories provide testing to support investigation and compliance activities. fda's electronic laboratory exchange network (elexnet) is a web-based system for real-time sharing of laboratory data derived from foods. this system allows public health officials to compare laboratory findings and to identify outbreaks earlier. the usda operates laboratories that support many of their regulatory, monitoring, and investigative programs. usda laboratories conduct research on animal and plant diseases and provide testing of animals and agricultural products. the usda's national veterinary services laboratories (nvsl) located in ames, iowa, tests for domestic and foreign animal diseases, and function as the primary animal disease reference laboratory. the nvsl provides diagnostic support for disease control and eradication programs, import and export testing of animals, and laboratory certification for selected animal diseases. diseases, such as anthrax, rabies, brucellosis, and bovine spongiform encephalopathy (mad cow disease), may impact both animals and humans and, therefore, constitute priorities at this laboratory. a former usda laboratory, the foreign animal disease diagnostic laboratory (faddl), which is located on plum island off long island, new york, was recently transferred to the department of homeland security after supporting years of research on some of the most dangerous animal pathogens. the doe oversees the operation of 25 doe national laboratories, many of which were established to support the production, use, and response to nuclear materials. after the end of the cold war, the focus of some of the doe laboratories shifted to other projects, including the human genome project and the development of technologies and assays to support homeland security initiatives. the doe national laboratories develop new technologies for countering biologic and chemical threats, including systems for the detection, modeling, and response to terrorist attacks (see www-ed.fnal.gov/doe/ doc_labs.html). the dod has established laboratories worldwide in locations such as peru, indonesia, egypt, thailand, and kenya. dod laboratories serve the needs of the armed forces and function as screening or sentinel laboratories for infectious diseases. the u.s. army medical research institute of infectious diseases (usamriid) has the capability to detect unusual biological agents that often require advanced testing techniques. this laboratory, located in maryland, is a member of the laboratory response network (lrn; discussed later) and one of a few laboratories worldwide that can isolate and identify the most dangerous human agents, such as ebola, smallpox and marburg viruses. the epa operates 10 regional environmental testing laboratories across the united states. these laboratories have a research and environmental monitoring mission: they analyze air, drinking water, ground water, surface water, soil, sediment, and hazardous materials for biological, chemical, and radiological materials that are toxic to the environment. epa develops standard methods for the analysis of environmental samples. epa maintains large databases of environmental monitoring data produced by its own laboratories and others. its office of research and development (ord) directs laboratory activities at 12 locations, including the national center for environmental assessment in research triangle park, north carolina. although the capability and capacity of the federal laboratories described above is large, this capacity was challenged by the volume of environmental and clinical samples generated during the 2001 anthrax postal attack. the distribution of anthrax spores in mail during october 2001 led to an unprecedented demand for quality testing throughout the united states owing to discovery of real and suspected contaminations. although few of the more than 125,000 environmental samples tested contained b. anthracis, the existing network of public and commercial laboratories was barely able to meet the demands for testing, and there were significant delays caused by the sheer volume of samples. the concept of a high-throughput laboratory capable of testing thousands of biologic, chemical, or radiological samples would require the laboratory to be equipped with the latest automated instrumentation and supported with an efficient lims (layne and beugelsdijk, 2003) . the establishment of high-throughput laboratories to support the nation's homeland security needs is a reasonable concept, especially if the major federal partners were to colocate resources on a national interagency homeland security laboratory campus. approximately 200 of the more than 186,000 laboratories in the united states are classified as state public health laboratories. included in this number are about 150 regional or branch laboratories that are administered as part of the state public health laboratory. more than 6,500 laboratory professionals are employed by state public health laboratories. each state and five territories operate a state public health laboratory. one major function of theses laboratories is to provide diagnostic and analytical services for surveillance of infectious, communicable, genetic, and chronic diseases. state (and local) public health laboratories provide testing to support many public health programs (tuberculosis, sexually transmitted diseases, hiv, immunizations, and newborn screening). areas of analysis include clinical and environmental chemistry, immunology, pathogenic microbiology, virology, and parasitology. state laboratories serve as reference laboratories, and they provide confirmatory testing of specimens submitted from other laboratories. the state public health laboratories frequently measure toxicants in human samples to document exposures to chemicals found in the diet or environment. this specialized testing requires expensive equipment (mass spectrometers, chromatographs) and well-trained chemists. the capabilities, responsibilities, and practices of the state public health laboratories vary. during recent years, many of these laboratories have received substantial federal funding to increase staff, equipment, and capabilities to respond to biologic and chemical threats, as part of the dhhs' cooperative agreement on public health preparedness and response for bioterrorism. a large percentage of the state public health laboratories have used some of the federal funding to build, remodel, and upgrade facilities. funding for state laboratories is generally a mix of state and federal funds. many states rely on fees, reimbursements, and service contracts to carry out their mission. some states have regional or district laboratories to provide the necessary services throughout an entire state. state public health laboratories partner with public health laboratories operated by counties or cities to meet the needs of their communities. state public health laboratories are generally better prepared to respond to an incident requiring biologic testing as opposed to an incident requiring chemical testing. although research is not the primary mission of public health laboratories, several state public health laboratories have developed close ties with academic institutions. the opportunity to work on surveillance projects with faculty and students from schools of public health and academic training programs for laboratory professionals has been beneficial to these laboratories. arrangements with academic centers afford opportunities to improve laboratory services and surveillance systems. flexible funding and other resources, such as grants, faculty, and students, help support special research projects and training opportunities within public health laboratories. state laboratories provide services to health departments; healthcare organizations; local, state, and federal law enforcement; local hazardous materials (hazmat) teams; civil support teams; and other private and governmental laboratories. state laboratories analyze thousands of water and air samples daily. state laboratories involved in the analysis of drinking water and other environmental samples are accredited by the nelar certified by epa office of drinking water programs, or accredited by state-specific accreditation programs. state public health laboratories are the backbone of the laboratory response network (lrn), which we discuss below. the lrn also includes laboratories under the jurisdiction of federal agencies discussed in this chapter. at the time of publication, 96 state and local public health laboratories make up the 146 laboratories that are members of the lrn. of these, 72 state, territorial, and metropolitan public health laboratories are part of the lrn's chemical testing network. more than 1,900 local public health laboratories provide support for city and county public health programs. these laboratories offer onsite testing for child health screening, tuberculosis, refugee screening, food safety, sexually transmitted diseases, and lead poisoning prevention, as well as epidemiologic investigations and environmental monitoring. local public health laboratories often forward specimens to their state laboratories for tests that are not available locally. approximately 40% of the local public health laboratories only perform waived tests. another 40% perform moderate complexity testing, and 20% perform highly complex testing (association of public health laboratories, 2004) . many state and locally operated laboratories in addition to clinical and public health laboratories produce test results that may be useful for surveillance. forensic laboratories and toxicology laboratories are frequently associated with departments of public safety or with state or local medical examiners. these forensic laboratories may be positioned at the federal, state, or local level, depending on the jurisdiction. medical examiners contribute to biosurveillance by elucidating unusual causes of death (see chapter 11). medical examiners and forensic pathologists frequently rely on laboratories to establish the exact cause of death. readers may understand laboratories as only providing test results after the analysis of samples that are submitted. this view of laboratories only focuses on one of their products and overlooks services provided by the professionals who work in the laboratories. laboratory professionals provide consultation on the selection of the most useful tests for screening, diagnosis, and verification of disease. they provide information on the proper handling and transportation of samples. development of new technologies, evaluation of technologies, and the application of technologies are other important services provided by laboratory professionals. laboratories evaluate test kits for ease of use, storage conditions, and performance characteristics. laboratories train individuals to perform testing and educate and inform users of laboratory services. laboratories may also operate laboratory-based surveillance systems for respiratory and enteric diseases. laboratories employ many analytic technologies to provide testing for diagnosis and surveillance. these tests range from simple screening tests to presumptive diagnostic tests and confirmatory tests. simple laboratory tests are used to screen biologic samples for the presence of biological agents or other substances that might indicate a disease, an infection with a biologic agent, or a contamination with a toxin or other chemical. the classical approach to identification of biologic agents involves direct examination of stained materials by microscopic examination for the presence of agents (dhhs/cdc, n.d.; york, 2003) . microscopic examination relies on staining characteristics and the size and shape of the organisms found. with few exceptions, it is rarely possible to identify an agent based only on microscopic characteristics. direct stains may help eliminate organisms from further consideration, but they are usually not sufficient without further testing to identify a pathogen. wet mounts are used for the direct microscopic examination of clinical materials for fungi and parasitic agents. additional simple assays take advantage of the ability of an organism to metabolize chemicals or produce chemical reactions that result in color changes of a substrate. recently, many assay kits have been developed for waived tests. these waived test kits can produce reliable results in most cases; however, one must understand the limitations of these kits and the need for confirmatory testing. field test kits, often referred to as handheld assays, have become popular with first responders who have a need to know if an unknown material contains a biologic agent or toxic chemical. simple immunologic reactions, coupled with a colorimetric indicator, form the detection systems for many handheld assays. validating the performance of handheld assays in comparative studies is a task generally reserved for governmental agencies or contract laboratories (emanuel et al., 2003) . the major advantage of the simple assays is that they are quick, as a result may be available in 5 to 20 minutes. the short turnaround time makes these tests ideal for use by surveillance systems provided one understands the limitations of the tests and that a method for confirmatory testing is available. presumptive diagnostic tests are procedures that, when properly performed, may indicate the presence of a particular agent or closely related agents. presumptive diagnostic tests for biologic and chemical agents are more complex and require more time to perform than do the simple tests described above. most bacterial and viral agents can readily be grown in culture media or cell culture if the appropriate conditions are met. these conditions include the appropriate temperature, ph, and a source of energy (e.g., glucose). inhibitory substances, such as antibiotics, are often placed into the growth media to prevent the growth of unwanted organisms. growth of organisms in cell culture or artificial media takes 24 hours to 30 or more days, depending on the organism. once organisms are detected on or in growth media, various techniques are used to determine the genus and species of the organism. for bacteria, the differential growth on select media, metabolism of various carbohydrates and other chemicals, and the presence of selected enzymes are often used for identification. special stains that incorporate florescent dyes coupled with antibodies to selected organisms are used for identification of bacteria and viruses. laboratories use immunologic assays to identify many biologic agents and for the subspecies typing that is needed for epidemiological purposes. immunologic assays generally involve the use of a specific antibody that can attach to or react with the outer-surface structures of an agent. other immunologic assays are used to detect antibodies in biological materials after infection with biological agents. after the isolation and identification of many organisms, drug-susceptibility testing determines the organism's susceptibility to drugs that are used to treat infections with the organism. a definitive or confirmatory test is a test that will identify with a very high degree of certainty the true identity of an agent. these tests have a very low likelihood of providing a falsepositive result. many of the definitive and confirmatory assays used today are molecular assays, which detect genetic material that is specific to a bacterium, virus, protozoa, or other organism. nucleic-acid-based assays rely on the unique differences found in the structure of single strands of dna and rna. the unique pattern of bases is specific for a single organism or closely related organisms. the nucleic-acid-based assays involve the use of probes, which are strands of dna or rna that match distinctive dna or rna patterns of the organism being tested and will bind with that dna or rna if it is in the clinical specimen. once the binding occurs, the binding can be detected by using electrochemical, colorimetric, and optical systems (committee on research and development needs for improving civilian medical response to chemical and biological terrorism incidents, 1999) . this binding provides extreme selectivity between the known probes and the material found in clinical specimens. the use of a technique known as polymerase chain reaction (pcr) makes it is possible to amplify trace quantities of dna or rna in a clinical specimen to enable the detection of as few as 1,000 bacteria or viruses. the high specificity and sensitivity of molecular assays makes them especially suited for detecting minute quantities of biologic agents and toxins. the use of genetic fingerprints has become a valuable tool for microbial forensics (murch, 2003) . by identifying distinct features of genes using sequencing techniques, it is possible to identify individual strains of organisms and to use this information as epidemiological markers. molecular techniques allow investigators to link strains from various sources and to form associations that often unravel the mystery of disease outbreaks. libraries of genetic patterns, or fingerprints, make it possible to trace the origin of many outbreaks. not only does sequencing identify dna or rna patterns unique to a particular organism, but, in many cases, these probe technologies are simpler, faster, and less technology-dependent than are other traditional assays. many biotechnology companies are pursuing production of microarray systems that will test for 100 to 100,000 or more different dna fragments simultaneously. the technology embeds the probes on a single glass or nylon substrate called a microchip. by using these arrays and various detection systems onto which the clinical specimen is applied, the individual components of the microchip will react with dna fragments in the specimen and be detected. as this technology develops, it will become more common to use the microarray systems for screening and detection of diseases. simultaneous pcr assays for multiple respiratory viruses now have sufficient sensitivity and specificity to be a valuable tool for diagnosis of respiratory viral infections (hindiyeh et al., 2001) . in the future, pcr assays will be able to screen a single specimen for a multitude of biologic agents. a lims is a computer system that a laboratory uses to track specimens and manage analytical data. the function of a lims can perhaps best be explained by tracing a single test-a white blood count--from the time that a clinician orders the test until the time the clinician receives the result of the test. a clinician requests (orders) that a laboratory perform a white blood count on a blood sample from a patient in one of several ways. the clinician writes the order on paper, gives a verbal order to a nurse, or enters the order directly into a computer system. a lims may receive this order in one of several ways: directly, if the lims provides an order-entry component that either the clinician uses directly or the nurse or ward clerk uses on her behalf; indirectly, if a paper order form accompanies the specimen; or electronically, from an order-entry system embedded in another information system (such a point-of-care system as discussed in chapter 6). we will trace the most automated path in which the lims receives the order electronically. the received order sets up a specimen-tracking process that is a central lims function. the lims (or a point-of-care system) controls a printer, which is often located in the clinical area from which the order originated. the printer generates a bar-coded label. a phlebotomist attaches the label to a "purple top" vial (containing magnesium citrate or ethylenediaminetetraacetic acid [edta] to prevent the blood from coagulating). the phlebotomist draws the blood after checking carefully that the identification on the labeled tube matches the patient, and sends the sample to the laboratory. oftentimes, the labeled specimen is transported by pneumatic chute from clinical areas, such as the emergency department, or by express overnight delivery to the laboratory. a technician in the laboratory scans the barcode of the specimen with an optical scanner, which is connected to the lims. if the laboratory has an automated analyzer for blood counts, the technician simply places the vial into the specimen carousel of the analyzer. the analyzer recognizes the bar code, communicates with the lims over an internal laboratory network to determine which tests were ordered for the spedmen, runs the tests, and transmits the results to the lims. other types of specimens may require preparation, such as centrifugation, before being placed into the carousel of an automatic analyzer, but the information processing and communication between the analyzer and the lims are otherwise identical. for tests that are done by hand, the lims provides user-interfaces into which medical technologists register specimens and enter intermediate and final results of tests. depending on the laboratory and the needs of its clinical customers, the lims may deliver the results as paper reports, via web-browser interfaces, or by e-mail. a lims typically offers all of these options. a lims invariably has an outbound computerto-computer interface that can transmit results to other clinical information and public health information systems. although our example is of a clinical test, the process is identical for environmental tests ordered by sanitarians, water quality technicians, or outbreak investigators. the vast majority of laboratory work in the united states is highly automated in the fashion just described. with the exception of tests done in the field or in office practices, limss track and manage the analytic results of most laboratory tests performed in the united states. limss are designed to make test results available to clinicians and other information systems soon after they are performed. limss are highly reliable and operate in real time. limss are developed within the laboratory by information technology staff, purchased from commercial vendors, or a combination of both. because of the importance of laboratory tests in biosurveillance, biosurveillance organizations are attempting to establish connections between limss and their own biosurveillance computers. at present, however, there are significant technical barriers to connecting a lims located in a laboratory with a computer located in a biosurveillance organization. the most difficult technical barrier is data incompatibility. most laboratories do not use standard coding systems to identify the names and results of laboratory tests. data standards exist, but few laboratories use them. most laboratories have evolved their own naming or coding systems for the tests that they perform and the results of those tests. they use these proprietary codes (or free text) to identify the laboratory test, specimen type, organism, or other results of a test. as a result, each lims-tobiosurveillance-computer interface requires significant effort to understand the data and to create means to translate the data into a standard format so that it can be integrated with data coming from other lims. we discuss standard data formats in detail in chapter 32. outbound communication standards, such as hl7, also exist and we discuss them in chapter 32. although most lims support these standards, the specific implementations vary. even if both the biosurveillance computer and the lims use the hl7 standard, they will not be able to communicate without significant effort to understand the specifics of the messages and to create a means for extracting the data from the messages. importantly, a new version of hl7 (version 3.0) will solve this problem; but its penetration in the lims market is low at present. because of the customization required to connect even a single lims to a biosurveillance organization, there are only a few regions that have integrated data from most of the limss that serve their region into biosurveillance. ultimately, these barriers will be addressed by standardization, but as we discuss in chapter 32, it takes many years to achieve widespread standardization of any component in a biosurveillance system. the organization of laboratories into collaborative networks has been ongoing for more than a decade. the concept of laboratory networks arose from the need to ensure that critical laboratory services were available throughout the country (gilchrist, 2000) . the cdc, fda, usda, and state governmental laboratories formed many of the original partnerships that grew into the laboratory networks that exist today. early networks included the national laboratory system (nls) of clinical, public health and federal laboratories (mcdade and hughes, 1998) , and the public health laboratory information system (phlis). phlis was an early dos-based system that involved voluntary reporting of selected laboratory tests directly to the cdc by 23 state and local public health laboratories and numerous military laboratories (see chapter 3). phlis was one of the earliest laboratory-based surveillance systems that became an effective tool for the identification of outbreaks of salmonellosis (bean et al., 1992; hutwagner et al., 1997) . the cdc's lrn was established in 1999 in compliance with a presidential directive that outlined federal agencies' counterterrorism goals and responsibilities. the mission of the lrn is "to maintain an integrated national and international network of laboratories that can respond quickly to acts of chemical or biological terrorism, emerging infectious diseases and other public health threats and emergencies." the lrn was first tasked to address state and local public health laboratory preparedness and response for bioterrorism. since its inception, its mission has expanded to include chemical terrorism. the scope of laboratories in the lrn has expanded beyond state and local public health laboratories in order to meet national security needs (http://www.bt. cdc.gov/lrn/). the lrn and its partners maintain a network of laboratories that can respond quickly to acts of chemical or biological terrorism, emerging infectious diseases, or other public health threats and emergencies. the network included 152 federal, state, and local public health; military; and international laboratories as of august 1, 2005. figure 8 .1 shows the distribution of lrn laboratories by type as of march 11, 2005 . the lrn employs tests that can detect biological threat agents in clinical specimens, environmental samples, food, animals, and water. the association of public health laboratories was a partner in the early development of the lrn and has played an important role in ensuring that the lrn provides the training, standardized methods, and equipment necessary for detecting biologic agents of terrorism and other threats to public health. the federal bureau of investigation (fbi) was also a key partner in establishing the network. the fbi brought its forensic expertise and evidence-gathering requirements to the program. public health and law enforcement have overlapping approaches to their investigations that required collaboration between cdc and law enforcement to both enhance and protect the integrity of their investigations. three levels of laboratories are recognized within the lrn for bioterrorism agent detection. lrn laboratories responsible for bioterrorism agent detection are designated as national, reference, or sentinel laboratories (figure 8 .2). the national laboratories include laboratories at the cdc, usamriid, and the naval medical research center. they have unique resources to safely identify highly infectious agents (biological safety level 4 agents) and the ability to provide definitive characterization of biologic agents. the national laboratories have developed standard tests and protocols, trained laboratory analysts, and established secure communications for the rapid sharing of laboratory results from reference laboratories. reference laboratories in the lrn perform tests to detect and confirm the presence of biological threat agents, such as those that cause anthrax, plague, tularemia, smallpox, or botulism. these laboratories ensure that state and local laboratories can have a timely response in the event of a bioterrorism incident or an emerging disease. rather than having to rely on confirmation testing from national laboratories, reference laboratories are capable of producing conclusive results needed by local authorities for rapidly responding to emergencies. specimens received by reference laboratories that contain threat agents are usually submitted to a national laboratory for definitive characterizations of the agent. state and local public health laboratories comprise most of the reference laboratories within the lrn. although sentinel laboratories are not counted among the lrn laboratories, they represent the thousands of clinical (human and veterinary) and environmental laboratories identified by the state lrn reference laboratory to serve as the front line of defense in detecting agents of terrorism. qualification of the sentinel laboratory is based on the experience and competency of the laboratory staff, appropriateness of facilities, and completion of training provided by the lrn. sentinel laboratories can often rule out potential bioterrorism agents based on a battery of simple tests. in a covert terrorist attack, a sentinel laboratory could be the first facility to identify a suspicious agent or an unusual cluster f i g u r e 8.2 laboratory response network (lrn) structure for bioterrorism response laboratories. of diseases. a sentinel laboratory's responsibility is to rule out other diseases and refer a suspicious sample to an appropriate reference laboratory. the lrn uses an array of presumptive and confirmatory assays to detect biological threat agents or chemical agents. presumptive assays generally include traditional microbiological assays, such as growth on special media, use of special stains, and the use of rapid molecular diagnostic assays, such as real-time pcr. confirmatory methods used by the national laboratories are considered the gold standard for detecting the target agent. methods, such as time-resolved fluorescence (trf) and molecular characterization, have replaced many of the more traditional confirmatory methods that were based on the growth and biochemical properties of the agent. reference laboratories have access to lrn protocols through a secure web site and are supplied standardized reagents needed to perform the necessary tests. uniform procedures for use by sentinel laboratories have also been developed and are used for training of sentinel laboratory staff. all laboratories that are part of the lrn must provide a safe and secure environment for performing tests and must participate in a recognized proficiency testing program. the chemical component of the lrn employs a more centralized structure, with only a few laboratories currently prepared to provide definitive analysis of specimens for chemical agents or their metabolites. lrn laboratories responsible for chemical agent detection are designated as level 1, 2, or 3 laboratories. five laboratories located in california, michigan, new mexico, new york, and virginia participate in level 1 activities. at this level, personnel are trained to detect exposure to an expanded number of chemicals in human blood or urine, including all level 2 laboratory analyses, plus analyses for mustard agents, nerve agents, and other toxic chemicals. fortyone laboratories participate in the chemical lrn by providing level 2 activities. at this level, laboratory personnel are trained to detect exposure to a limited number of toxic chemical agents in human blood or urine. analysis of cyanide and toxic metals in human samples are examples of level 2 laboratory activities. each of the 62 chemical network members participates in level 3 activities. level 3 laboratories are responsible for the following: 9 working with hospitals in their jurisdiction 9 knowing how to properly collect and ship clinical specimens 9 ensuring that specimens used as evidence in a criminal investigation are handled properly and chain-of-custody procedures are followed 9 being familiar with chemical agents and their health effects 9 training on anticipated clinical sample flow and shipping regulations 9 working to develop a coordinated response plan for their respective state and jurisdiction initial testing in a suspected chemical event will occur at cdc or i of 5 level i chemical laboratories that have been established by cdc. by use of mass spectrometry, cdc laboratories perform tests on the first 40 or more clinical specimens to measure human exposure. results of these tests would be reported to affected states, and if needed, appropriate lrn members may be asked to test additional samples. this approach is necessary because the analytical expertise and technology resources required to respond to a chemical event is expected to be high. the lrn supports secure communications on emerging and emergency issues, a secure mechanism for ordering reagents and testing protocols, and a system for electronically reporting test results. the lrn provided valuable testing after the release of anthrax spores in 2001, the identification of monkey pox in 2002, and the response to severe acute respiratory syndrome (sars) in 2003. in 2001, canada created a laboratory network, known as the canadian public health laboratory network (cphln), to strengthen the linkages between federal and provincial public health laboratories. this canadian network is modeled after the lrn in the united states. the cphln has been providing responses to naturally occurring infections and deliberate releases of biologic agents and toxins. the cphln coordinates pathogen detection and infectious disease prevention activities, as well as conducts laboratory-based surveillance and early warning systems for emerging pathogens and bioterrorism threats. the national animal health laboratory network (nahln) is part of a national strategy in the united states to coordinate the nation's federal, state, and academic animal health laboratories. the usda has taken the lead in the development of this network, which includes agriculture and animal health laboratories operated by state agricultural agencies and those associated with veterinary teaching facilities. the facilities and professional expertise of nahln members allows authorities to better respond to animal health emergencies that might include a bioterrorist event, the emergence of a new domestic animal disease, or the appearance of a foreign animal disease that could threaten the nation's food supply and public health. because many of the biologic agents that cause the greatest concern as terrorist agents infect both humans and animals, the role of veterinarians in the early detection of disease is very important. the nahln currently consists of 44 laboratories in 37 states. an effort is underway to deploy standardized testing methods in member laboratories and to improve the information technology system used by member laboratories to track test requests and report results. the u.s. animal health association (usaha) and the american association of veterinary laboratory diagnosticians (aavld) have members who participate in the nahlh and contribute expertise to protect animals and public health. the national plant diagnostic network (npdn) is an agricultural laboratory network that provides detection, identification, and reporting of pests and pathogens that have been deliberately or accidentally introduced into agricultural systems. the primary concern of this network is food security and the economic threats to the nation's food supplies. the npdn includes five regional centers located at cornell university, michigan state, kansas state, university of florida at gainesville, and the university of california at davis. the npdn recently implemented a new database that helps with required reporting to state and national agencies. a web-based plant diagnostic system using digital photography allows laboratories to share images with specialists in remote locations. the food emergency response network (fern) is a network of state and federal laboratories that are committed to analyzing food samples in the event of a biological, chemical, or radiological terrorist attack in the united states. the federal partners in the fern include the fda, usda, cdc, and epa. as of may 2005, there are 99 laboratories in fern, representing 44 states and puerto rico. twenty-six federal laboratories, 68 state laboratories, and five local laboratories are enrolled in fern. the mission of fern is to integrate the nation's food testing laboratories for the detection of threat agents in food while using standardized diagnostic protocols and procedures. of the 99 laboratories in fern, there are 64 that perform chemical tests on food, 64 that perform biological tests on food, and 25 that perform radiological tests on food. these laboratories strengthen preparedness and provide surge capacity. the fda and usda jointly share the leadership within fern and have been working to obtain federal funds that can be made available to support further development of the network. the data capture and information exchange system for fern is the elexnet, is an integrated, secure system designed for use by multiple governmental agencies involved in food safety activities. laboratories report test results and public health officials assess risks and analyze trends in foodborne diseases, elexnet has gis reporting functions and uses hl7 data exchanges between laboratories. similar to those in lrn, participants in fern receive training on the latest equipment and are required to participate in proficiency testing programs, elexnet provides the necessary infrastructure for an early warning system that can identify potentially hazardous foods and share laboratory reports in a timely manner. as of january 2005, 113 federal, state, and local laboratories in all 50 states have joined the elexnet system. about 90 laboratories actively exchange data by using elexnet. the radiological emergency analytical laboratory network (realnet) is a national network of radiological laboratories that are capable of responding to the needs for radiological testing after a terrorist attack. academic, commercial, military, federal, state, and local laboratories participate in realnet. these laboratories serve as a science and technology asset for the department of homeland security. realnet is modeled after the lrn and fern and includes a web-based database containing information on the capabilities, capacity, and competence of member laboratories. information on accreditation, certification, and performance testing is also maintained. standards, guidelines, and laboratory procedures are developed and distributed by realnet. gaps in the standards are being addressed by appropriate standards development organizations. internet-based tools, such as bulletin boards and list servers, are used to promote the exchange of information. the system provides links to other resources that would be useful during an emergency caused by the release of a radioactive material. the expansion of laboratory networks designed to produce test results in response to an act of terrorism or other public health emergency has led to increased sharing of laboratory results. coordination and integration of the various networks has not always been a priority. duplicate systems and overlapping missions suggest that an integrated consortium of laboratory networks could provide timely results for early detection and response to acts of terrorism. the networks need to agree on standardized tests and policies that would promote a timely response no matter which network is reporting results. an overall system to ensure that laboratory capacity will be available to test clinical (human and animal) specimens and environmental samples, including food and water, does not exist. laboratories that can perform screening, monitoring, and definitive testing are needed for each class of specimen. although laboratory information systems have been developed for capturing and sharing data, the diverse systems are not fully compatible with each other. failure to standardize nomenclatures and use recognized data transmission protocols make sharing of large quantities of data for surveillance purposes problematic. in an effort to improve coordination among laboratory networks, the department of homeland security is working to integrate these networks through the creation of the integrated consortium of laboratory networks (icln). the icln employs the lrn model for coordinating laboratory assets for terrorism among federal, state, local, and scientific partners. a memorandum of agreement was signed in 2005 by the usda, dod, doe, dhhs, epa, and the departments of homeland security, state, justice, and interior to form the icln. these federal agencies will collaborate to ensure that laboratory resources available within each agency can be used to respond to terrorist events and other national emergencies. clinical, commercial, and governmental laboratories are an important source of data for biosurveillance systems. test results obtained from the analysis of human and animal specimens may be early indicators of disease within the population. before the establishment of a definitive diagnosis, a sudden rise in the number of tests being requested by clinicians might be the first indication of an outbreak. the combination of laboratory data from environmental laboratories and the occurrence of illness in humans or animals might signal onset of an infectious disease or poisoning. the establishment of a lims that can rapidly capture and report laboratory data electronically will greatly contribute to the use of laboratory data in biosurveillance. challenges still exist for integrating 190,000 laboratories into a real-time network to support biosurveillance. the multitude of laboratory networks that are being formed will enhance the analytical capabilities of laboratories as well as the ability of laboratories to distribute peak loads during emergencies and quickly and efficiently share data electronically. enormous potential exists for the use of data that that is locked up in systems by the lack of standardization. although much recent emphasis has been focused on building laboratory networks in preparation for a biological, chemical, or nuclear attack, many of the enhanced laboratory capabilities will assist with the response to other public health emergencies. association of public health laboratories national plant diagnostic network (npdn) informatics for the clinical laboratory: a practical guide laboratory information management systems: development, and implementation for a quality assurance laboratory phlis: an electronic system for reporting public health data from remote sites detection and measurement of biological agents. in chemical and biological terrorism--research and development to improve civilian medical response validating the performance of biological detection equipment: the role of the federal government a national laboratory network for bioterrorism: evolution from a prototype network of laboratories performing routine surveillance evaluation of the prodesse hexaplex multiplex pcr assay for direct detection of seven respiratory viruses in clinical specimens using laboratory-based surveillance data for prevention: an algorithm for detecting salmonella outbreaks c gi ? cmd---r etriev e &db=pub med &dop t=citation high-throughput laboratories for homeland and national security the u.s. needs a national laboratory system microbial forensics: building a national capacity to investigate bioterrorism sentinel bioterrorism responders: are hospital labs ready key: cord-262846-1mhimfsf authors: gray, nicholas; calleja, dominic; wimbush, alexander; miralles-dolz, enrique; gray, ander; de angelis, marco; derrer-merk, elfriede; oparaji, bright uchenna; stepanov, vladimir; clearkin, louis; ferson, scott title: is “no test is better than a bad test”? impact of diagnostic uncertainty in mass testing on the spread of covid-19 date: 2020-10-21 journal: plos one doi: 10.1371/journal.pone.0240775 sha: doc_id: 262846 cord_uid: 1mhimfsf testing is viewed as a critical aspect of any strategy to tackle epidemics. much of the dialogue around testing has concentrated on how countries can scale up capacity, but the uncertainty in testing has not received nearly as much attention beyond asking if a test is accurate enough to be used. even for highly accurate tests, false positives and false negatives will accumulate as mass testing strategies are employed under pressure, and these misdiagnoses could have major implications on the ability of governments to suppress the virus. the present analysis uses a modified sir model to understand the implication and magnitude of misdiagnosis in the context of ending lockdown measures. the results indicate that increased testing capacity alone will not provide a solution to lockdown measures. the progression of the epidemic and peak infections is shown to depend heavily on test characteristics, test targeting, and prevalence of the infection. antibody based immunity passports are rejected as a solution to ending lockdown, as they can put the population at risk if poorly targeted. similarly, mass screening for active viral infection may only be beneficial if it can be sufficiently well targeted, otherwise reliance on this approach for protection of the population can again put them at risk. a well targeted active viral test combined with a slow release rate is a viable strategy for continuous suppression of the virus. during the early stages of the united kingdoms sars-cov-2 epidemic, the british government's covid-19 epidemic management strategy was been influenced by epidemiological modelling conducted by a number of research groups [1, 2] . the analysis of the relative impact of different mitigation and suppression strategies concluded that the "only viable strategy at the current time" is to suppress the epidemic with all available measures, including the lockdown of the population with schools closed [3, 4] . similar analysis in other countries lead to over half the world population being in some form of lockdown by april 2020 and over 90% of global schools closed [5, 6] . these analyses have highlighted from the beginning that the eventual relaxation of lockdown measures would be problematic [3] . without a considered cessation of the suppression strategies the risk of a second wave becomes significant, possibly of greater magnitude than the first as the sars-cov-2 virus is now endemic in the population [7, 8] . although much attention was focused on the number of tests being conducted and the effect that testing could have in supressing the disease [9] [10] [11] . not enough attention has been given to the issues of imperfect testing, beyond matt hancock, uk secretary of state for health and social care, stating in a press conference on 2nd april 2020 that "no test is better than a bad test" [12] . in this paper we will explore the validity of this claim. the failure to detect the virus in infected patients can be a significant problem in highthroughput settings operating under severe pressure, with evidence suggesting that this is indeed the case [13] [14] [15] [16] [17] . the public are rapidly becoming aware of the difference between the 'have you got it?' tests for detecting active cases, and the 'have you had it?' tests for the presence of antibodies, which imply some immunity to covid-19. what may be less obvious is that these different tests need to maximise different test characteristics. to be useful in ending lockdown measures, active viral tests need to maximise the sensitivity. high sensitivity reduces the chance of missing people who have the virus who may go on to infect others. there is an additional risk that an infected person who has been incorrectly told they do not have the disease, when in fact they do, may behave in a more reckless manner than if their disease status were uncertain. the second testing approach, seeking to detect the presence of antibodies to identify those who have had the disease would be used in a different strategy. this strategy would involve detecting those who have successfully overcome the virus, and are likely to have some level of immunity (or at least reduced susceptibility to more serious illness if they are infected again), so are relatively safe to relax their personal lockdown measures. this strategy would require a high test specificity, aiming to minimise how often the test tells someone they have had the disease when they haven't [18] . a false positive tells people they have immunity when they don't, which may be worse than if people are uncertain about their viral history. the successes of south korea, singapore, taiwan and hong kong in limiting the impact of the sars-cov-2 virus has been attributed to their ability to deploy widespread testing, with digital surveillance, and impose targeted quarantines in some cases [13] . this testing has predominantly been based on the use of reverse transcription polymerase chain reaction (rt-pcr) testing. during the 2009 h1n1 pandemic the rapid development of high sensitivity pcr assay were employed early with some success in that global pandemic [19] . these tests, when well targeted, clearly provide a useful tool for managing and tracking pandemics. these tests form the basis of much of the research into the incidence, dynamics and comorbidities of sars-cov-2, but few, if any, of these studies give consideration to the impact of false test results [20] [21] [22] [23] [24] . increasing reliance on lower-sensitivity tests to address capacity concerns is likely to make available data on confirmed cases more difficult to accurately utilise [19] . it may be the case that false test results contribute to some of the counter-intuitive disease dynamics observed [25] . there is evidence that both active infection [26] [27] [28] [29] [30] and antibody [31] [32] [33] tests lack perfect sensitivity and specificity even in best-case scenarios. alternative screening methods such as chest x-rays may be found to have high sensitivity based on biased data [34] or may simply perform poorly even compared to imperfect rt-pcr tests [29] . the foundation for innovative in order to answer this question there are a number of important statistics: • sensitivity σ-out of those who actually have the disease, that fraction that received a positive test result. • specificity τ-out of these who did not have the disease, the fraction that received a negative test result. the statistics that characterise the performance of the test are computed from a confusion matrix (table 1) . we test n infected people who have covid-19, and n healthy people who do not have covid-19. in the first group, a people correctly test positive and c falsely test negative. among healthy people, b will falsely test positive, and d will correctly test negative. from this confusion matrix the sensitivity is given by (1) and the specificity by (2). s ¼ a n infected ð1þ sensitivity is the ratio of correct positive tests to the total number of infected people involved in the study characterising the test. the specificity is the ratio of the correct negative tests to the total number of healthy people. importantly, these statistics depend only on the test itself and do not depend on the population the test is intended to be used upon. when the test is used for diagnostic purposes, the characteristics of the population being tested become important for interpreting the test results. to interpret the diagnostic value of a positive or negative test result the following statistics must be used: • prevalence p-the proportion of people in the target population that have the disease tested for. • positive predictive value ppv-how likely one is to have the disease given a positive test result. • negative predictive value npv-how likely one is to not have the disease, given a negative test result. the ppv and npv depend on the prevalence, and hence depend on the population you are focused on. this may an entire nation or region, a sub-population with covid-19 compatible symptoms, or any other population you may wish to target. the ppv and npv can be calculated using bayes' rule: to illustrate the impact of prevalence on ppv, for a test with σ = τ = 0.95, if prevalence p = 0.05, then the ppv = 0.5. therefore, a positive result only indicates a 50% chance that an individual will have the disease given that they have tested positive, even though the test is highly accurate. fig 1 shows why, for 1000 test subjects there will be similar numbers of true and false positives even with high sensitivity and specificity of 95%. in contrast, using the same tests on a sample with a higher prevalence p = 0.5 we find the ppv = 0.95, see fig 2. similarly, the npv is lower when the prevalence is higher. sir models offer one approach to explore infection dynamics, and the prevalence of a communicable disease. in the generic sir model, there are s people susceptible to the illness, i people is "no test is better than a bad test"? infected, and r people who are recovered with immunity. the infected people are able to infect susceptible people at rate β and they recover from the disease at rate γ [38] , fig 3 shows how people move between the different states of an sir model. once infected persons have recovered from the disease they are unable to become infected again or infect others. this may be because they now have immunity to the disease or because they have unfortunately died. to explore the effect of imperfect testing on the disease dynamics when strategies testing regimes are employed to relax lockdown measures, three new classes were added to the model. the first is a quarantined susceptible state, q s , the second is a quarantined infected state, q i , and the third is people who have recovered but are in quarantine, q r , as shown in is "no test is better than a bad test"? the present model is similar to other sir models that take into account the effect of quarantining regimes on disease dynamics, such as lipsitch et al. (2003) [39] or giordano et al. (2020) [23] . lipsitch et al. implement quarantine in their model but do not incorporate the effects on the dynamics from imperfect testing, nor do they consider how the quality and scale of an available test affect the spread of a disease. diagnostic uncertainty plays no part in the model they present. likewise, giordano et al reduce population based diagnostic strategies to two parameters which confound test capacity, test targeting, and diagnostic uncertainty. again, they do not investigate the role that diagnostic uncertainty plays in the spread of a disease. the intent of this model is not to create a more sophisticated sir model, but to investigate how diagnostic uncertainty affects the dynamics of an epidemic. the model evaluates each day's population-level state transitions. there are two possible tests that can be performed: • an active virus infection test that is able to determine whether or not someone is currently infectious. this test is performed on some proportion of the un-quarantined population (s + i + r). it has a sensitivity of σ a and a specificity of τ a . • an antibody test that determines whether or not someone has had the infection in the past. this is used on the fraction of the population that is currently in quarantine but not infected (q s + q r ) to test whether they have had the disease or not. this test has a sensitivity of σ b and a specificity of τ b . each test is defined by a number of parameters. testing each day is limited by the test capacity c, the maximum number of tests that can be performed each day. each day a population n will be submitted for testing. the targeting capability of the test, t indicates the probability that an individual submitted for testing is positive, this is effectively the ppv of the initial screening effort. this results in a number of individuals m being considered for screening who are negative, of which k will be tested. targeting must be imperfect, as if it were perfect there would be no need for testing. unless otherwise stated, scenarios consider a default targeting of t = 0.8, representing an extremely effective screening capability that is nonetheless imperfect. if daily testing targets are a goal regardless of the prevalence of the illness, t can be overruled to ensure n � c for example. this condition is referred to as strict capacity and is denoted with boolean parameter g, defaulting to true for all scenarios. tests can also be conducted periodically by changing the test interval parameter d. these default to 1, i.e. daily testing. each test has unique parameters, so for example test a (active virus infection test) has a targeting parameter t a whilst test b (antibody test) has t b . the parameters σ, τ, t, c, g and d define a test. a person in any category who tests positive in an active virus test transitions into the corresponding quarantine state, where they are unable to infect anyone else. a person, in q s or q r , who tests positive in an antibody test transitions to s and r respectively. any person within i or q i who recovers transitions to r, on the assumption that the end of the illness is clear and they will know when they have recovered. for this parameterisation the impact of being in the susceptible quarantined state, q s , makes an individual insusceptible to being infected. similarly, being in the infected quarantined state, q i , individuals are unable to infect anyone else. in practicality there is always leaking, no quarantine is entirely effective, but for the sake of exploring the impact of testing uncertainty these effects are neglected from the model. other situations may require including this effect. the sir model used in this paper uses discrete-time binomial sampling for calculating movements of individuals between states. for a defined testing strategy these rates are defined as follows: in eq 7, bin(n, p) refers to a binomial distribution with count n and rate p, h(n, k, m) refers to a hypergeometric distribution with populations n and k and a sample size m. the model must be initialised with a defined population split between the six states. at each time step t, the model calculates the number of persons moving between each state in the order defined above. the use of binomial and hypergeometric sampling was prompted by a desire to incorporate aleatory uncertainty in each movement. the current approach does not account for epistemic uncertainty, fixing the model parameters σ, τ, c, t and d. a discrete time model was selected to allow for comparisons against available published data detailing recorded cases and recoveries on a day-by-day basis. if the tests were almost perfect, then we can imagine how the epidemic would die out very quickly by either widespread infection or antibody testing with a coherent management strategy. a positive test on the former and the person is removed from the population, and positive test on the latter and the person, unlikely to contract the disease again, can join the population. more interesting are the effects of incorrect test results on the disease dynamics. if someone falsely tests positive in the antibody test, they enter the susceptible state. similarly, if an infected person receives a false negative for the disease they remain active in the infected state and hence can continue the disease propagation and infect further people. in order to explore the possible impact of testing strategies on the relaxation of lockdown measures several scenarios have been analysed. these scenarios are illustrative of the type of impact, and the likely efficacy of a range of different testing configurations. • immediate end to lockdown scenario: this baseline scenario is characterised by a sudden relaxation of lockdown measures. • immunity passports scenario: a policy that has been discussed in the media [40] [41] [42] . analogous to the international certificate of vaccination and prophylaxis, antibody based testing would be used to identify those who have some level of natural immunity. • incremental relaxation scenario: a phased relaxation of lockdown is the most likely policy that will be employed. to understand the implications of such an approach this scenario has explored the effect of testing capacity and test performance on the possible disease dynamics under this type of policy. under the model parameterisation this analysis has applied an incremental transition rate from the q s state to the s state, and q r to r. whilst the authors are sensitive to the sociological and ethical concerns of any of these approaches, the analysis presented is purely on the question of efficacy. for the purpose of the analysis we have selected a population similar in size to the united kingdom, 6.7 × 10 7 people, β and γ were set to 0.32 and 0.1 respectively, this was ensure that r 0 value of the model was broadly in line with other models [43, 44] . under the baseline scenario, characterised by the sudden and complete cessation of lockdown measures, we explored the impact of infection testing. under this formulation the initial conditions of the model in this scenario is that the all of the population in q s transition to s in the first iteration. the impact of infection testing under this scenario was analysed in fig 5 using the parameters shown in table 2 . these scenarios consider the impact of attempts to control the disease through increased testing capacity and a more sensitive test. a test capacity range between 1 × 10 5 and 2 × 10 5 was considered as representative of the capabilities of a country such as the uk. to illustrate the sensitivity of the model to testing scenarios an evaluation was conducted with a range of infection test sensitivities, from 50% (i.e of no diagnostic value) to 98%. the specificity of these tests has a negligible impact on the disease dynamics in these scenarios. a false positive would mean people are unnecessarily removed from the susceptible population, but the benefit of a reduction in susceptible population is negligibly small. as would be expected the model indicates a second wave is an inevitability and as many as 20 million people could become infected within 30 days. a high-sensitivity test has little impact table 2 . https://doi.org/10.1371/journal.pone.0240775.g005 is "no test is better than a bad test"? beyond quarantining a slightly higher percentage of the population if capacities are low. at higher capacities this patterns remains, though peak infection counts are marginally reduced. overall it is clear that reliance on infection testing, even with a highly sensitive test and high capacities, is not enough to prevent widespread infection. the immunity passport is an idiom describing an approach to the relaxation of lockdown measures that focuses heavily on antibody testing. wide-scale screening for antibodies in the general population promises significant scientific value, and targeted antibody testing is likely to have value for reducing risks to nhs and care-sector staff, and other key workers who will need to have close contact with covid-19 sufferers. the authors appreciate these other motivations for the development and roll-out of accurate antibody tests. this analysis however focuses on the appropriateness of this approach to relaxing lockdown measures by mass testing the general population. antibody testing has been described as a 'game-changer' [45] . some commentators believe this could have a significant impact on the relaxation of lockdown measures [41] , but others note that there are severe ethical, logistical and medical concerns which need to be resolved before antibody testing could support a strategy such as this [46] . much of the discussion around antibody testing in the media has focused on the performance and number of these tests. the efficacy of this strategy however is far more dependent on the prevalence of antibodies (seroprevalence) in the general population. without widescale antibody screening it is impossible to know the seroprevalence in the general population, so there is scientific value in such an endeavour. however, the seroprevalence is the dominant factor to determine how efficacious antibody screening would be for relaxing lockdown measures. presumably, only people who test positive for antibodies would be allowed to leave quarantine. the more people in the population with antibodies, the more people will get a true positive, so more people would be correctly allowed to leave quarantine (under the paradigms of an immunity passport). the danger of such an approach are false positives. we demonstrate the impact of people reentering the susceptible population who have no immunity. we assume their propensity to contract the infection is the same as those without the false sense of security a positive test may engender. on an individual basis, and even at the population level, behavioural differences between those with false security from a positive antibody test, versus those who are uncertain about their viral history could be significant. the model parametrisation here does not include this additional confounding effect. to simulate the seroprevalence in the general population the model is preconditioned with different proportions of the population in the q s and q r states. this is analogous to the proportion of people that are currently in quarantine who have either had the virus and developed some immunity, and the proportion of the population who have not contracted the virus and is "no test is better than a bad test"? have no immunity. of course the individuals in these groups do not really know their viral history, and hence would not know which state they begin in. the model evaluations explore a range of sensitivity and specificities for the antibody testing. these sensitivity and specificities, along with the capacity for testing, govern the transition of individuals from q r to r (true positive tests), and from q s to s (false positive tests). each of the plots in figs 6 and 7 show the effect of different seroprevalence in the population. to be clear, this is the proportion of the population that has contracted the virus and recovered but are in quarantine. the analysis has explored a range of seroprevalence from 0.1% to 50%. fig 6 explores the impact of a variation in sensitivity, from a test with 50% sensitivity to tests with a high sensitivity of 98%. it can be seen, considering the top row of fig 6, that the sensitivity of the test has no discernible impact on the number of infections. the seroprevalence entirely dominates. this is possibly counter intuitive, but as was discussed above, even a highly accurate test produces a very large number of false positives when seroprevalence is low. in this case that would mean a large number of people are allowed to re-enter the population, placing them at risk, with a false sense of security that they have immunity. the bottom row of fig 6 shows the proportion of the entire population leaving quarantine over a year of employing this policy. at low seroprevalence there is no benefit to better table 3 . performing tests. this again may seem obscure to many readers. if you consider the highest seroprevalence simulation, where 50% of the population have immunity, higher sensitivity tests are of course effective at identifying those who are immune, and gets them back into the community much faster. a more concerning story can be seen when considering the graphs in fig 7 . now we consider a range of antibody test specificities. going from 50% to 98%. low specificities (τ < 0.9) table 4 . https://doi.org/10.1371/journal.pone.0240775.g007 lead to extreme second peaks, and could possibly lead to more. this is due to the progressive release of false-positives from the quarantined population, which eventually swells the susceptible population to a size where the infection count can resume exponential growth. high specificities avoid this at the cost of a prolonged lockdown, which is naturally limited by the lower false-positive rate. clearly some means of release beyond immunity passports would be required to avoid this scenario. notably, a reasonably specific test (τ b = 0.9) is capable of restraining a second peak to reasonably low levels regardless of seroprevalence. this may allow for other means of reducing lockdown measures, though with very low seroprevalence this could still be a potentially risky strategy. the dangers of neglecting uncertainties in medical diagnostic testing are pertinent to this decision [47] . considering the above, some form of incremental relaxation of lockdown seems appropriate. this could take many forms, it could be an incremental restoration of certain activities such as school openings, permission for the reopening of some businesses, the relaxation of stay-athome messaging, etc. under the parameterisation chosen for this analysis the model is not sensitive to any particular policy change. we consider a variety of rates of phased relaxations to quarantine. to model these rates we consider a weekly incremental transition rate from q s to s, and q r to r. in fig 8, three weekly transition rates have been applied: 1%, 5% and 10% of the quarantined population. whilst in practice the rate is unlikely to be uniform as decision makers would have the ability to update their timetable as the impact of relaxations becomes apparent, it is useful to illustrate the interaction of testing capacity and release rate. the model simulates these rates of transition for a year, with a sensitivity and specificity of 90% for active virus tests. the specifics of all the runs are detailed in table 5 . fig 8 shows five analyses, with increasing capacity for the active virus tests. in each, the 3 incremental transition rates are applied with a range of targeting capabilities. the value of 0.8 used previously represents an unrealistically extreme case of effective targeting. the ppv, as discussed above, has a greater dependence on the prevalence (at lower values) in the tested population than it does on the sensitivity of the tests, the same is true of the specificity and the npv. it is important to notice that higher test capacities cause a higher peak of infections for higher release rates. this has a counterintuitive explanation. when there is the sharpest rise in the susceptible population (i.e., high rate of transition), the virus rapidly infects a large number of people. when these people recover after around two weeks they become immune and thus cannot continue the spread of the virus. however, when the infection testing is conducted with a higher capacity up to 150,000 units per day, these tests transition some active viral carriers into quarantine, so the peak is slightly delayed providing more opportunity for those released from quarantine later to be infected, leading to higher peak infections. this continues until the model reaches effective herd immunity after which the number of infected in the population decays very quickly. having higher testing capacities delays but actually has the potential to worsen the peak number of infections. at 10% release rate, up to a capacity of testing of 150,000 these outcomes are insensitive to the prevalence of the disease in the tested population. this analysis indicates that the relatively fast cessation of lockdown measures and stay-home advice would lead to a large resurgence of the virus. testing capacity of the magnitude stated as the goal of the uk government would not be sufficient to flatten the curve in this scenario. the 1% release rate scenario indicates that a slow release by itself is sufficient to lower peak infections, but potentially extends the duration of elevated infections. the first graph of the top row in fig 8 shows that the slow release rate causes a plateau at a significantly lower number of infections compared to the other release rates. poorly targeted tests at capacities less than 100,000 show similar consistent levels of infections. however, with a targeted test having a prevalence of 30% or more, the 1% release rate indicates that even with 50,000 tests per day continuous suppression of the infection may be possible. the per-day testing capacity is varied across the five columns of graphs. rate, the percentage of the initial quarantined population being released each week is varied among rows. the prevalence of infections in the tested population is varied among different colours. to facilitate comparison within each column of graphs, the gray curves show the results observed for other rates and prevalences with the same testing intensity. model parameters are shown in table 5 . https://doi.org/10.1371/journal.pone.0240775.g008 at the rate of 5% of the population in lock-down released incrementally each week the infection peak is suppressed compared to the 10% rate. the number of infections would remain around this level for a significantly longer period of time, up to 6 months. there is negligible impact of testing below a capacity of 100,000 tests. however, with a test capacity of 150,000 tests the duration of the elevated levels of infections could be reduced if the test is extremely well targeted (t a = 0.7), reducing the length of necessary wide-scale lockdown. if this level of targeting is not achieved, increasing capacity may again increase peak infections, so care must be taken to ensure a highly targeted testing strategy. this analysis does support the assertion that a bad test is potentially worse than no tests, but a good test is only effective in a carefully designed strategy. more is not necessarily better and over estimation of the test accuracy could be extremely detrimental. this analysis is not a prediction; the numbers used in this analysis are estimates and the sirq model used is unlikely to be detailed enough to inform policy decisions. as such, the authors are not drawing firm conclusions about the absolute necessary capacity of tests. nor do they wish to make specific statements about the necessary sensitivity or specificity of tests or the recommended rate of release from quarantine. the authors do, however, propose some conclusions that would broadly apply when testing and quarantining regimes are used to suppress epidemics, and therefore believe they should be considered by policy makers when designing strategies to tackle covid-19. • diagnostic uncertainty can have a large effect on the dynamics of an epidemic. and, sensitivity, specificity, and the capacity for testing alone are not sufficient to design effective testing procedures. policy makers need to be aware of the accuracy of the tests, the prevelence of the disease at increased granularity and the characteristics of the target population, when deciding on testing strategies. • caution should be exercised in the use of antibody testing. assuming that the prevalence of antibodies is low, it is unlikely antibody testing at any scale will support the end of lockdown measures. and, un-targeted antibody screening at the population level could cause more harm than good. • antibody testing, with a high specificity may be useful on an individual basis, it has scientific value, and could reduce risk for key workers. but any belief that these tests would be useful to relax lockdown measures for the majority of the population is misguided. • the incremental relaxation to lockdown measures, with all else equal, would significantly dampen the increase in peak infections, by 1 order of magnitude with a faster relaxation, and 2 orders of magnitude with a slower relaxation. • as the prevelence of the disease is suppressed in different regions, it may be the case that small spikes in cases could be the result of false positives. this problem is potentially exacerbated by increased testing in localities in response to small increases in positive tests. policy decisions that depend on small changes in the number of positive tests may, therefore, be flawed. • for infection screening to be used to relax quarantine measures the capacity needs to be sufficiently large but also well targeted to be effective. for example this could be achieved through effective contact tracing. untargeted mass screening at any capacity would be ineffectual and may prolong the necessary implementation of lockdown measures. epidemiological models used for policy making in real time will need to take into account the impact of diagnostic uncertainty of testing, as well as the dynamical behaviour and sensitivity analyses of modelled parameters in an appropriately complex model that may need to include quarantining, contact tracing and other surveillance strategies, test availability and targeting, and multiple subpopulations of susceptible, infected and recovered categories. covid-19: government announces moving out of contain phase and into delay phase scaling up our testing programmes. department of health and socal care impact 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case report if you have coronavirus symptoms, assume you have the illness, even if you test negative are coronavirus tests accurate? report 16: role of testing in covid-19 control detecting 2009 pandemic influenza a (h1n1) virus infection: availability of diagnostic testing led to rapid pandemic response epidemiology and transmission of covid-19 in shenzhen china: analysis of 391 cases and 1,286 of their close contacts. medrxiv early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia spread of sars-cov-2 in the icelandic population modelling the covid-19 epidemic and implementation of population-wide interventions in italy projecting the transmission dynamics of sars-cov-2 through the postpandemic period updates on covid-19 in republic of korea development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections abstract interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (covid-19) correlation of chest ct and rt-pcr testing in coronavirus disease evaluation of novel antigen-based rapid detection test for the diagnosis of sars-cov-2 in respiratory samples. available at ssrn 3569871 potential false-negative nucleic acid testing results for severe acute respiratory syndrome coronavirus 2 from thermal inactivation of samples with low viral loads performance of vivadiag covid-19 igm/igg rapid test is inadequate for diagnosis of covid-19 in acute patients referring to emergency room department rapid point-of-care testing for sars-cov-2 in a community screening setting shows low sensitivity prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal sars-cov-2 diagnostics: performance data point-of-care versus lab-based testing: striking a balance molecular and antibody point-of-care tests to support the screening, diagnosis and monitoring of covid-19. oxford covid-19 evidence service transmission dynamics and control of severe acute respiratory syndrome why it's too early to start giving out immunity passports' could speed up return to work after covid-19 britain has millions of coronavirus antibody tests, but they don't work the reproductive number of covid-19 is higher compared to sars coronavirus euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin boris johnson and donald trump talk up potential'game-changer' scientific advances on coronavirus developing a national strategy for serology (antibody testing) in the united states calculated risks: how to know when numbers deceive you key: cord-017072-qwe1ne3q authors: poritz, mark a.; lingenfelter, beth title: multiplex pcr for detection and identification of microbial pathogens date: 2018-11-10 journal: advanced techniques in diagnostic microbiology doi: 10.1007/978-3-319-95111-9_19 sha: doc_id: 17072 cord_uid: qwe1ne3q multiplexed nucleic acid-based tests for infectious disease have become a standard part of clinical laboratory practice. these tests provide a comprehensive syndrome-based approach to determine the etiological agent of disease. the technology underlying these different systems is reviewed here with a special focus on the biofire filmarray® platform. the literature on the clinical utility and cost-effectiveness of these platforms for respiratory, blood culture, and gastrointestinal infections is discussed. although there are reports showing a clear benefit to the patient or to the healthcare system from adopting a syndromic molecular approach, it is also apparent that clinical laboratories and healthcare providers are still learning how to take full advantage of the new systems. finally, some improvements to this technology that should appear in the next few years are discussed. these include automated pathogen-specific surveillance based on aggregating the data from these systems, a move toward point-of-care syndromic testing, and further decreases in time to result of the tests. over the last decade, a number of manufacturers have developed multiplexed in vitro diagnostic (ivd) platforms that can detect the nucleic acid signatures of many of the organisms responsible for infectious disease. some of these testing platforms offer limited test menus (i.e., influenza a, influenza b, and rsv), while others are designed to detect a more comprehensive set of potential pathogens that can cause a particular infectious disease syndrome (e.g., respiratory, gastrointestinal, sepsis, meningitis) [1] [2] [3] . this chapter will describe fda-cleared and/or ce-marked multiplex assays that are designed to detect a comprehensive set of pathogens associated with a particular infectious disease syndrome (≥10 assays/ test). these include the biofire (salt lake city, ut) filmarray ® system [4] , the genmark (carlsbad, ca) esensor xt-8® [5] and eplex® [6] , and the luminex (austin, tx) xtag® [7] , nxtag ® [8] , and verigene® systems [9] . in addition to being comprehensive with respect to pathogens responsible for a particular syndrome, these multiplex panels offer the advantage of superior test sensitivity and specificity. many of these panels have been designed to be easy-to-use, allowing molecular testing to be performed in moderate or low complexity settings and eliminating barriers that prevented many laboratories from being able to perform molecular assays on-site. another important benefit of multiplex panel is the fast time to result. molecular multiplex tests require hours to perform instead of the days required for culture-based methods. these systems differ in particular details (the exact turnaround time, sample throughput, cost per sample, and number of target pathogens detected). these differences are due to the underlying technologies used for the detection of the pathogen nucleic acid. however, all these systems share the common attribute that the incremental cost of each additional assay in the test cartridge (in materials, labor, and quality control (qc) testing) is small compared to the total manufacturing costs for the disposable. this has enabled ivd manufacturers to develop broad test panels that include organisms which have not been a part of standard testing protocols because of the technical limitations of existing methods. nonetheless the availability of syndromic infectious disease panels poses hard questions for clinicians and the healthcare system overall: does the wealth of information in a comprehensive test improve the treatment of an individual patient, and how can the economic value of this improved treatment be measured? commercially available fda-cleared multiplex nucleic acid-based tests for infectious agents include systems from luminex, genmark, and biofire (now a subsidiary of biomérieux) ( table 1) . at present, all such systems combine the sequential steps of: 1. nucleic acid purification from the appropriate human sample matrix (e.g., nasal swab, blood or blood culture, stool) 2. cdna synthesis (reverse transcription) to convert viral rna to dna, if necessary 3. multiplex pcr to amplify molecules of the pathogen nucleic acid 4. specific detection of the expected amplicons to confirm that the correct target nucleic acids have been identified the different systems vary mainly in whether the nucleic acid purification steps are integrated into the same cartridge that is used for amplification (verigene, eplex, and filmarray) and in how the specific detection of amplicon is achieved. the luminex xtag and nxtag systems use a fluorescent signal generated after hybridization of the amplicon to fluorescently encoded bead arrays to detect a specific amplicon [10, 11] . the verigene system uses hybrid capture of the amplicons on a microarray with detection by gold nanoparticle probes [12] . the genmark esensor xt-8 and the eplex systems use electrochemical detection of the target amplicon hybridized to a specific gold microelectrode [13] . the filmarray system is described in more detail below. the filmarray pouch and chemistry the filmarray system performs sample-to-answer multiplex nucleic acid testing for infectious disease. to accomplish this, the filmarray pouch ( fig. 1) integrates all of the steps of nucleic acid purification, nested multiplex pcr amplification, and automated data analysis into a closed system [4] . the pouch is created by welding two sheets of plastic film together with heat in such a fashion that fluid can move between the working areas of the pouch via channels left in the plastic. a hard plastic fitment attached to the film (indicated in fig. 1b) provides the enzymes and buffers needed to perform the biochemical reactions in the pouch. other reagents (#1, #2, and #3 in fig. 1a ) are inserted between the film layers during manufacture of the pouch. the filmarray reagents are lyophilized in the pouch, and the pouch is stored under vacuum before use. this benefits the end user in two ways. first the freezedrying process stabilizes the pcr reagents so that the pouch has a shelf life, at ambient temperature, in excess of 1 year -which simplifies the logistics of acquiring and storing the pouches. second vacuum storage ensures that the wells of the fitment are also under vacuum. thus the user does not need to control the volume of hydration fluid or sample that is injected into the pouch -which simplifies the steps needed to load a pouch. lysis and homogenization of bacteria, spores, and viruses occur in the filmarray pouch by means of vigorous agitation in the presence of zirconium beads (#1 in fig. 1a ) and a denaturing buffer. dna and rna in the sample are purified by binding to silica magnetic beads (#2 in fig. 1a) , the beads are washed to remove proteins and other pcr inhibitors, and the nucleic acids are eluted into a buffer compatible with reverse transcription and pcr [14] . the eluted material hydrates a pill (#3 in fig. 1a ) that contains all of the primers needed for reverse transcription (for rna targets) and first stage pcr. the primers in this pill are specific to each pathogen target (assay) contained in the pouch. the filmarray system performs nested, multiplex pcr in two stages (fig. 2) . reverse transcription and first stage pcr (pcr1) are performed in the same reaction volume and in a multiplex format, combining primer sets for all assays for a filmarray panel in one mix. following pcr1 amplification, the reaction is diluted approximately 100-fold to reduce the concentrations of the outer primers, nonspecific products, and first-stage pcr chemistry. the diluted reaction is then combined with a fresh pcr master mix and flooded over a 102-well array, where a second stage pcr occurs (pcr2). each well of the array contains assay-specific primers that anneal within the first-stage outer amplicon. at the end of pcr2, a dna melt curve analysis of the amplicons is performed using a nonspecific dna-binding dye, lcgreen® plus [15] , that fluoresces in the presence of double-stranded dna. the presence of a specific melt curve with tm (melting temperature) in the range predicted for that specific amplicon confirms the presence and identity of the products made in each well. the mechanics and chemistry of the filmarray pouch have been described in greater detail elsewhere [4] . here we highlight some of the features of the system that have contributed to the robustness of the test. the combination of a denaturing lysis buffer and "bead beating" with zirconium beads has been shown to lyse organisms and release intact total nucleic acid (dna and rna) from spores [16] as well as from the cerebral spinal fluid (csf), stool, blood culture media, and whole blood. the nested pcr of the multiplex chemistry makes the filmarray test remarkably resistant to pcr inhibitors that may remain after nucleic acid purification. even modest levels of amplification in first-stage pcr can still be detected as an amplicon in the inner, nested product of pcr2. the dilution step following the firststage amplification reduces the complexity of the input material to the second-stage reaction enough that, in most cases, the dna-binding dye detects the presence of a single amplicon in the reaction (the melt curve analysis serves as an additional filter for the correct product). the filmarray pouch and instrument contribute to the sensitivity of the overall system in several important ways. first the nucleic acid sample purification starts with a relatively large volume of the sample (100 μl for the rp and gi panels, 60 μl for the bcid), and, nominally, all of the nucleic acid purified from this volume is delivered to the combined reverse transcription -first-stage pcr reaction. unlike some benchtop protocols, there is no dilution from the purified nucleic acid into an rt step and a second dilution into a pcr. secondly the pouch is controlled by pneumatic actuators (described below) that can move liquid between blisters of the pouch in seconds. this minimizes the time during which nucleic acid could be degraded or pcr primers could bind to an incorrect dna or rna target and thus generate specific "nonspecific" amplicons. to the same end, the filmarray pouch achieves a true mechanical hot start. in both the first stage and second stage pcrs, the primers do not come in contact with the dna polymerase/mg, dntp mixture until both components are at or above the temperature at which polymerization will take place. this minimizes the formation of primer dimers, and higher-order multiplex primer structures in the first-stage pcr, which compete with the correct amplicons for pcr reagents. this enhances the specificity of the amplification reactions and thus increases the sensitivity of the individual pcr assays. all filmarray pouches have at least two internal controls that demonstrate the proper functioning of the pouch. one is a small amount of synthetic dna spotted onto the second stage pcr array along with primers to amplify this target. this "pcr2" control only monitors the function of the second stage pcr. a more important control for pouch function is generated by freeze-drying a small number of cells of the yeast schizosaccharomyces pombe into the well of the pouch fitment that receives the sample. nucleic acid purified from these yeast cells must pass through all the steps of the pouch. outer primers in the first-stage pcr and inner primers spotted onto the second-stage pcr array are designed to amplify a spliced messenger rna from the s. pombe cells (in the filmarray bcid pouch which does not contain reverse transcriptase, the s. pombe target is a genomic dna sequence). biofire has shown during the development of several different pouches that artificially induced failure modes that prevent detection of a pathogen organism in a sample also prevent detection of the yeast control. the filmarray instrument controls the movement of liquid through the pouch using pneumatically-actuated bladders and seals that force liquid from a pouch blister or prevent liquid from leaving a blister, respectively. the hydrated reagents in the pouch fitment are introduced into the pouch blisters via additional pneumatically actuated pistons. the amplification reactions are thermocycled using 1 inch square peltier devices situated adjacent to the first-and second-stage pcrs (#3 and #4 of fig. 1a , respectively). to detect the melting of the second-stage pcr amplicons, a blue led illuminates the array, and a camera with a filter to detect green light observes the signal generated by the dna-binding dye lcgreen® plus. multiplex panels are particularly attractive to clinicians because they provide a comprehensive and accurate test result in a short period of time, and the test panels have been designed to match the clinical syndrome (i.e., respiratory infection, infectious gastroenteritis). however, given the increased cost of these tests, it is important to demonstrate their impact to patient management and their cost-effectiveness. it is intuitive to believe that a rapid, accurate, and comprehensive diagnostic test should improve patient management by shortening the time to the most effective therapy, by preventing inappropriate therapy (especially empiric antimicrobials), by reducing additional diagnostic testing (e.g., imaging studies), by improving the use of infection control measures, and by reducing patient length of stay. of particular importance, these tests can reduce the unnecessary use of empiric antimicrobials by reducing the time to pathogen-directed therapy. use of broad-spectrum antibiotics for patients with serious illnesses and the inappropriate use of antibiotics in the outpatient setting "just in case" are important drivers of antibiotic resistance which is one of the major healthcare threats of our time. in this section, we will review what is known about the clinical utility and cost-effectiveness of these multiplex panels. because the clinical implication for each syndromic panel is different, the discussion is presented by syndrome. currently there are three vendors with multiplex respiratory panels that are both fda-cleared and ce marked (biofire diagnostics, salt lake city, utah; luminex, austin, texas; and genmark, san diego, california) and several more that are ce-marked (seegene, seoul south korea; curetis, holzgerlingen, germany, fast-track diagnostics, sliema, malta). these panels include assays for several viral pathogens (e.g., influenza, respiratory syncytial virus (rsv), human rhinovirus, parainfluenza viruses, adenovirus, coronaviruses, etc.) and some also include selected bacterial targets (e.g., mycoplasma pneumoniae, bordetella spp, legionella pneumophila). all of the fda-cleared test are limited to testing nasopharyngeal swab (nps) samples, while several of the ce-marked tests include a larger range of respiratory sample types (e.g., bronchoalveolar lavage, sputum, etc.). prior to the availability of multiplex respiratory panels, testing for viral respiratory pathogens relied on viral culture, direct fluorescent antigen (dfa) testing, enzyme immunoassays (eias), and traditional pcr assays. while viral culture was the gold standard, it has several limitations, including that it is a complex test requiring highly skilled laboratory workers to both set up the test and interpret the results, it is slow (taking days to complete), and only a limited number of human pathogens can be grown in viral cultures. dfa tests can be performed directly on the patient sample and can have a fast turnaround time; however, these tests are also technically complex, and the range of pathogens is limited. eia assays can be performed in a variety of ways and are used for rapid antigen tests. rapid antigen tests are fast and simple to use but are known to have poor test sensitivity and a limited test menu. traditional pcr assays are highly sensitive and specific; however, they are complex and require highly trained laboratory staff and specialized laboratory facilities. in addition, each test is ordered independently placing a large burden on the ordering clinician to select the correct test or to order multiple individual tests. due to their complexity, these tests are commonly sent to specialized reference laboratories which can increase cost and slows the time to result. the introduction of multiplex respiratory panels has allowed for comprehensive testing (ability to test viral pathogens that do not grow in cell culture and the ability to simultaneously test for bacterial and viral pathogens) in a shorter time frame. the easy-to-use systems allow the testing to be performed by laboratory workers without specialized molecular skills and in laboratories without specialized equipment and facilities. these systems allow testing to be performed closer to the patient and therefore further reduce the time to test result by reducing the need to transport samples and eliminating the delays associated with batch testing. multiplex respiratory panels have the potential to improve patient management and lower overall healthcare costs by improving use of influenza antivirals, reducing inappropriate use of antibiotics and antivirals, reducing use of healthcare resource (e.g., additional laboratory or imaging procedures), informing appropriate infection control practices, and reducing length of hospital, emergency department, and intensive care unit (icu) stay. several studies have evaluated the effect on patient and healthcare outcomes linked to the use of a multiplex respiratory panel. xu et al. [17] showed that replacing dfa testing with on-demand use of the filmarray rp for children presenting to the emergency department (ed) resulted in dramatic reductions in test turnaround time (7 vs 1.4 h), timely (defined as within 3 h of discharge from the emergency department) administration of oseltamivir for 81% of patients testing positive for influenza, effective use of cohorting for admitted patients, and a potential saving of 900 h of ed boarding time. similarly, in a pre-/post-intervention study of pediatric patients admitted through the ed, rodgers et al. [18] compared several outcome measures when the filmarray rp replaced the use of three clinicianordered traditional pcr tests (prodesse assays for, flua/b/rsv, piv 1,2,3, and hmpv). the study demonstrated that use of the filmarray rp resulted in a significant increase in the number of patients with positive test results (77.9% vs 59.8%, p < 0.001) and a 65% reduction in time to result (6.38 vs 18.65 h, p < 0.001) when compared to use of traditional pcr tests. these improvements lead to a mean reduction in length of hospital stay of 0.3 days for patients with positive test results, reduced duration of antibiotics for patients with positive test results (2.7 vs 3.2 days, p < 0.001) or when results were reported in <4 h (2.8 vs 3.2 days, p < 0.001), and an overall reduction in healthcare costs of $231/patient. another study of 4779 pediatric patients reported significant reductions in the duration of antibiotic use (4 vs 5 days, p < 0.01), use of chest radiographs (59% vs 78%, p < 0.01), and an increase in appropriate use of isolation measures [19] . similar findings have been observed in studies of adult patients. brendish et al. [20] performed a prospective randomized study of adult patients presenting to the emergency department (ed) with respiratory symptoms over two respiratory seasons. in the control arm, patients were tested for respiratory viruses at the clinician's discretion using nine traditional pcr assays performed at a reference laboratory. in the intervention arm, all patients were tested with the filmarray rp, and testing was performed in the ed. the study demonstrated the expected increase in pathogen detection (45% vs 15%, p < 0.0001) and reduction in time to result (2.3 vs 37.1 h, p < 0.0001) but failed to show the expected reduction in the proportion of patients that received antibiotics (84% vs 83%, p = 0.96). however, there was an increase in the proportion of patients that received a short course of antibiotics (< 48 h, 17% vs 9%, p = 0.0047) especially for patients with positive filmarray rp test results. patients in the intervention group also had a mean reduction in hospital length of stay of 1.1 days (5.7 vs 6.8 d, p = 0.0443) with the shortest length of stay observed in patients with positive filmarray rp results. the use of influenza antivirals was the same in both groups (18% vs 14%, p = 0.16); however, the intervention group had a significant increase in the number of influenza-positive patients that received influenza antivirals (82% vs 47%, p = 0.0001) and a reduction in the use of antivirals for patients that were influenza-negative (18% vs 53%). in another study evaluating adult patients with a positive influenza result on a multiplex respiratory panel, rappo [21] reported a significantly lower odds ratio for hospital admission (p = 0.046), a reduced length of stay (p = 0.040), reductions in antimicrobial duration (p = 0.032), and a reduction in the number of chest radiographs (p = 0.005). there is currently only one study evaluating the use of multiplex panels in an outpatient setting. greene et al. evaluated the difference in use of antibiotics and antivirals for adult outpatients tested with the filmarray rp that were [1] positive for influenza, [2] positive for non-influenza pathogen, or [3] negative for all pathogens [22] . they observed significant increases in the use of influenza antivirals (81.0% vs 5.5-2.5%, p < 0.001) and reduced use of the antibiotics (29.5% vs 48.6-49.3%, p = 0.005) for individuals with a positive result for influenza. however, detection of non-influenza viral pathogens did not lead to a reduction in the use of antibiotics when compared to those with no pathogen detected (48.6% vs 49.3%). the authors suggest that either influenza testing alone is more cost-effective for this patient population or that additional education and/or antibiotic stewardship is needed to drive appropriate use of antibiotics in the outpatient setting. blood culture panels are designed to test positive blood cultures with the aim to provide a faster time to organism identification. in addition, these panels include assays for selected antibiotic resistance genes providing important information to guide antibiotic therapy. there are currently three vendors with multiplex blood culture panels that are both fda-cleared and ce-marked (biofire diagnostics; luminex and accelerate diagnostics, tucson, arizona), while genmark (san diego, california, usa) and curetis (holzgerlingen, germany) have panels that are ce-marked. these panels include assays for gram-positive bacteria, gram-negative bacteria, yeast, and antibiotic resistance markers or, in one case (accelerate pheno™ system), antibiotic susceptibility test results. the curetis unyvero bcu blood culture application cartridge® panel is the most comprehensive (with identification of ~100 bacteria and 16 resistance markers) followed by the biofire filmarray blood culture identification panel (identification of ~27 bacteria and 3 resistance markers). the genmark eplex and luminex verigene systems provide separate panels that are specific to gram-positive bacteria, gram-negative bacteria, or yeast with selection of the appropriate panel determined by blood culture gram stain. all of these tests can be used with a variety of different blood culture media and blood culture systems. prior to the availability of multiplex blood culture panels, pathogen identification was performed using classic standard culture-based systems with phenotypic (or maldi-tof) identification followed by traditional growth-based antimicrobial susceptibility testing. these tests are the gold standard methods and are very reliable; however, they suffer from a slow time to result (1-3 days after the positive blood culture) and technical complexity. molecular assays for specific pathogen identification, such as staphylococcus aureus and enterococci, have been in use for some time and have shown improvements in patient outcomes [23] [24] [25] [26] . another method that reduces time to organism identification is using maldi-tof to identify bacteria directly from minimally processed blood cultures without the need to subculture to agar plates. the maldi-tof identification is very comprehensive, and the reduced time to bacterial identification has also been shown to improve patient outcomes [27, 28] . while individual molecular assays and maldi-tof identification have both been shown to improve patient outcomes, they are both technically complex and require specialized skills. as a result, these test methods are typically performed during standard laboratory working hours and are typically not used on night shifts when staffing is limited. molecular multiplex panels offer the advantage of a fast time to organism identification along with ease of use. all of the fda-cleared and ce-marked panels use unprocessed blood culture media and provide identification results within 1-7 h of test initiation. due to the ease of use, these panels can be used by laboratory staff without specialized molecular biology skills; however, because these panels do not identify all possible pathogens, the results must be interpreted in conjunction with the blood culture gram stain, and traditional culture and sensitives must still be performed. furthermore, appropriate adjustments to antimicrobials rely on proper test interpretation and a good understanding for the local patterns of antimicrobial resistance (e.g., antibiogram). treatment adjustments should be based on local guidelines developed by an antimicrobial stewardship program (asp) team with an in-depth understanding of the capabilities of the test being used, the local antibiogram, and the local patient populations. as with the individual molecular assays and the maldi-tof identification, numerous studies have shown that use of multiplex molecular blood culture panels dramatically reduces the time to organism identification [29] [30] [31] [32] which drives more appropriate pathogen-directed therapy. pathogen-directed therapy includes antibiotic escalation (the addition or change of dose when the current therapy is ineffective against the identified organism) and antibiotic de-escalation (discontinuation of unnecessary empiric antibiotics). a prospective randomized study conducted at the mayo clinic showed that antibiotic escalation occurred more quickly with or without real-time asp oversight (5 h with bcid and asp, 6 h with bcid only, and 24 h without bcid or asp, p = 0.04); however, optimal antibiotic de-escalation requires oversight by an asp (21 h with bcid and asp, 34 h with bcid only, and 38 h without bcid or asp, p < 0.001) [29] . the finding that multiplex molecular blood culture panels paired with an asp results in a faster time to optimal antibiotic therapy (most narrow effective therapy) has been confirmed in several additional studies [31, 33] . the use of molecular multiplex blood culture panels has also been shown to reduce unnecessary treatment due to contaminated blood cultures [29, 34] . reducing the use or duration of unnecessary antibiotics is important to reducing the incidence of antibiotic resistance. the cost of molecular multiplex blood culture panel and the fact that they do not replace existing testing have been raised as reasons to not use them; however, studies of the overall healthcare cost prove the panels to be cost neutral [29] [30] [31] or to reduce overall healthcare cost [30, 31, [34] [35] [36] [37] . while the evidence is strong that multiplex panels dramatically reduce time to organism identification and time to optimal antibiotic therapy, the evidence is inconsistent with regard to reductions in patient mortality and length of hospital stay, mostly likely because the management of patients with sepsis is complex and multifactorial. however, a recent meta-analysis [38] of studies using rapid molecular methods to test positive blood cultures found a small but statistically significant reduction in patient mortality when the results were used as part of an asp (or 0.64, 95% ci 0.51-0.79) but not when used outside of an asp (or 0.72, 95% ci 0.46-1.12). the improvements were seen for patients with gram-positive (or 0.73, 95% ci 0.55-0.97) or gram-negative bacteremia (or 0.51, 95% ci 0.33-0.78), but not for patients with candidemia (or 0.90 95% ci 0.49-1.67). the study also found that time to effective therapy decreased by a weighted mean difference of −5.03 h (95% ci −8.60 to −1.45) and that length of hospital stay decreased by −2.48 days (95% ci −3.90 to −1.06). currently there are three vendors with multiplex gastrointestinal panels that are both fda-cleared and ce-marked (biofire diagnostics; luminex, and bd, sparks, maryland) and several more that are ce-marked (seegene, soul, south korea; mobidiag, finland; serosep, limerick, ireland). some of these panels include assays for bacteria, viruses, and parasites in one test (filmarray gi panel, biofire diagnostics; luminex ggp, luminex; verigene enteric pathogens test, luminex), while others provide separate panels for bacterial, viral, or parasitic pathogens (allplex™, seegene; bd max®, bd; amplidiag®, mobidiag; entericbio® realtime dx, serosep). most tests are performed with raw stool samples or stool in transport media. the use of fecal swabs with transport media is also common, and direct testing of rectal swabs is desirable. current testing for infectious gastroenteritis includes many tests (stool culture, ova and parasite examination (o&p), enzyme immunoassays) that are technically complex and suffer from low diagnostic yield. the gold standard for stool pathogen testing is stool culture; however, stool culture has low diagnostic yield, is technical complexity, and has a long time to result. the yield for stool cultures is reported to be between 1.5% and 2.9% with a cost per positive result of $952 to $1200 [39] . another commonly ordered test is o&p, which requires the collection and testing of three different stool samples. this method is also known to be technically difficult, to have low sensitivity, to be improperly used, and to have a low diagnostic yield of 1.4% [40] . as a result of the low diagnostic yield, clinicians often order multiple tests for the same stool sample, or perform testing sequentially until a causative pathogen is identified. as an example, a study conducted at a children's hospital found that a median of three tests (range 1-10) were ordered per stool sample [41] . to make matters worse, several studies have shown that clinician test ordering practices for gastroenteritis are problematic, in part due to the complexity of which pathogens are covered by what test [40, [42] [43] [44] . the use of culture-independent molecular multiplex panels has increased the diagnostic yield for stool testing due both to increased test sensitivity and an expanded test menu. studies using the filmarray gi panel identified a pathogen in 40-50% of stool samples [41, 45, 46] . some of the assay requires specialized molecular laboratories and personnel (bd max, luminex ggp, allplex); however, some are designed to be simple to use (filmarray gi panel, verigene enteric panel) and to provide a fast time to result (as little as 1 h from test initiation). while these tests have the benefits of offering a comprehensive and accurate result in a relatively fast time period, the impact to patient care is largely unknown; however, one recent pre-/post-implementation study highlighted several important improvements when the filmarray gi panel was used to test pediatric and adult inpatients [47] . these included an increase in diagnostic yield from 6.7% to 32.8% and an improved time to result from a mean of 54.75 h to 8.94 h when compared to traditional clinician ordered tests. when compared to a matched historical control group, implementation of the filmarray gi panel led to a reduction in the number of additional stool tests (3.02 vs 0.58, p = 0.001), a trend toward shorter duration of antibiotics (2.12 days vs 1.54 day, p = 0.06), significantly fewer imaging studies (0.39 vs 0.18, p = 0.0002), and a reduction in the length of hospital stay after sample collection (3.9 days vs 3.4 days, p = 0.04). reduced length of stay was more pronounced for the adult population (4.3 days vs 3.6 days, p = 0.01). recent studies have also shown that these tests have important advantages for infection control. in a recent retrospective study, the filmarray gi panel was used to test frozen stool samples that had previously been tested for rotavirus and c. difficile for infection control purposes [48] . the study showed that 22% of the samples contained pathogens that should have required infection control measures, including norovirus, rotavirus, and c. difficile. of these patients, 60% were under no or inadequate contact precautions, for a total of 109 patient days. conversely 24.5% of the patients with negative results by the filmarray gi panel were unnecessarily placed under contact precautions for a total of 181 patient days. this study illustrates that without a rapid comprehensive test result, contract precautions are not rationally applied, leading to both an increased risk of nosocomial infections and unnecessary costs associated with inappropriately applied contact precautions. these are important factors when considering the care of patients in hospitals and in long-term care facilities. use of the luminex xtag gastrointestinal pathogen panel (gpp) has been compared with conventional laboratory testing for hospitalized patients and was found to be cost-effective because the increased cost of the laboratory testing was more than offset by the cost saving for elimination of unneeded contact precautions. importantly, the cost savings was directly related to the time to test result [49] . the national institute for health care excellence (nice) in the uk recently published a very comprehensive health economics assessment of molecular multiplex panels [50] . they reported that there was considerable uncertainty in their models; however, the luminex ggp panel was determined to be cost-effective for use in community-acquired and traveler diarrhea and both the filmarray gi panel and the luminex ggp were found to be cost-effective for use in inpatients with diarrhea. these models will be updated as new information (such as the recent study by beal [47] ) becomes available. further clinical utility studies will highlight the importance of the different pathogens detected by the multiplex panels. however, independent of this work, continuing improvements to these ivd systems are likely to increase their value in the clinical infectious disease setting. in contrast to previous generations of infectious disease ivd platforms which used cell culture, microscopy, or immunoassay technology, the current generation of multiplex systems are all highly automated and computerized [4, [6] [7] [8] 51] . this opens the possibility of exporting the results of a patient test directly to an internet (or "cloud") database. a pilot version of such a system has been achieved with development of filmarray trend [52] . trend aggregates result from geographically dispersed clinical laboratories and displays the results on a website (www.syndromictrends.com) in close to real time, resulting in a form of infectious disease "weather map." the pilot project summarized the respiratory pathogen results for the filmarray rp from >360,000 patient samples acquired over 4 years ending in july 2017 from 20 clinical laboratories in the united states (fig. 3) . similar to social media-based disease reporting systems, the data is syndrome-based [53] . however, a unique feature of the trend dataset is that analysis is pathogen-specific. the filmarray rp patient test results demonstrate that a number of viruses (rsv, hmpv, piv, and cov) show seasonal occurrence that is similar to influenza and thus can be confused with influenza when a symptomatic diagnosis of influenza-like-illness is made. this has important implications for treatment of influenza and for determining the efficacy of influenza vaccination and thus emphasizes the value of multiplex testing for respiratory symptoms. the filmarray trend data also show that 7% of the filmarray rp tests detect the presence of two or three pathogens in a single sample. the importance of this result for patient treatment is not currently clear. clinical studies that focus on patients presenting with more than one pathogen are needed. data from the filmarray gi panel are also available at the www.syndromictrends.com website. over time, additional filmarray ivd panels will be added to trend, thus enabling the tracking of the pathogens that those panels detect. in 2017 the us fda cleared the filmarray rp-ez panel [54] . this panel has clinical laboratory improvement act (clia)-waived status and thus can be used in settings close to the patient including low-complexity outpatient settings. in other work, the time to result for the rp panel has been decreased from 63 min for rp v1.7 to 45 min for the rp2 panel [55] . speed, ease of use, and a comprehensive test menu are all critical features if the possibilities of point-of-care syndromic testing are to be fully realized [56] . in the longer term, additional simplification of the test setup procedure as well as reductions in the time to result should further expand the number of outpatient settings able to use multiplex testing. the data from farrar and wittwer [57] on "extreme" pcr conditions (cycle times below 1 s) suggest that reductions in the time to result are limited by the instrument and not by the chemistry and that substantial improvements are still possible. as noted earlier, the cost of multiplex diagnostic systems is not driven by that of the primers needed for each assay, so the number of assays in a test is limited by assay format and the ability to separate the signal for each analyte. the depth of multiplex achieved in pcr multiplexes for infectious disease has steadily increased from the earliest, manually performed multiplexes, compared to the those being developed for the automated systems of today. the point will soon be reached in which the feasible number of assays in a test begins to exceed the number of distinct pathogens that need to be tested for in any particular syndrome. however, there are situations (e.g., hiv drug resistance testing or hpv serotyping) where the ability to detect a limited number of point mutations would add great value to the test result. there is also great interest in the direct detection of sepsis pathogens from blood because the hours saved by not having to culture the bacteria reduces the risk of incorrect antibiotic treatment. with a combination of direct enrichment of the bacteria from the blood and careful attention to removing endogenous bacteria from the test manufacturing process, it should be possible to achieve the necessary sensitivity to detect bacterial pathogens directly from blood, without the time and labor of culturing the sample. an infectious disease society of america policy paper from 2013 [58] made a number of recommendations for the key characteristics of future infectious disease ivd tests. the current generation of multiplex tests already meets many of these goals (direct testing from easily accessible sample types, able to rule out infection with high certainty, based on clinical syndromes). the technical improvements described above suggest that meeting many of the remaining goals (rapid testing, point-of-care syndromic testing, improved outbreak detection) will be accomplished in the next decade. emerging technologies for the clinical microbiology laboratory multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis evaluation of four commercial multiplex molecular tests for the diagnosis of acute respiratory infections an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection comparison of the genmark diagnostics esensor respiratory viral panel to real-time pcr for detection of respiratory viruses in children the eplex® respiratory pathogen panel (rp)* comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis comparison of luminex nxtag respiratory pathogen panel and xtag respiratory viral panel fast version 2 for the detection of respiratory viruses comparison of three commercial rt-pcr systems for the detection of respiratory viruses clinical evaluation of the luminex nxtag respiratory pathogen panel bead-based suspension arrays for the detection and identification of respiratory viruses selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles electronic detection of nucleic acids: a versatile platform for molecular diagnostics rapid and simple method for purification of nucleic acids high-resolution genotyping by amplicon melting analysis using lcgreen evaluation of the filmarray(r) system for detection of bacillus anthracis, francisella tularensis and yersinia pestis implementation of filmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care impact of a rapid respiratory panel test on patient outcomes impact of multiplex polymerase chain reaction testing for respiratory pathogens on healthcare resource utilization for pediatric inpatients routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients clinical utility of ondemand multiplex respiratory pathogen testing among adult outpatients a retrospective study of the impact of rapid diagnostic testing on time to pathogen identification and antibiotic use for children with positive blood cultures the impact of implementation of rapid quickfish testing for detection of coagulase-negative staphylococci at a community-based hospital impact of rapid in situ hybridization testing on coagulase-negative staphylococci positive blood cultures peptide nucleic acid fluorescent in situ hybridization for hospital-acquired enterococcal bacteremia: delivering earlier effective antimicrobial therapy the clinical impact of rapid, direct maldi-tof identification of bacteria from positive blood cultures integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs randomized trial of rapid multiplex polymerase chain reaction-based blood culture identification and susceptibility testing impact of a rapid multiplex polymerase chain reaction blood culture identification technology on outcomes in patients with vancomycinresistant enterococcal bacteremia benefits of adding a rapid pcr-based blood culture identification panel to an established antimicrobial stewardship program clinical impact and provider acceptability of real-time antimicrobial stewardship decision support for rapid diagnostics in children with positive blood culture results potential clinical impact of the film array meningitis encephalitis panel in children with suspected central nervous system infections clinical and economic impact of antimicrobial stewardship interventions with the filmarray blood culture identification panel rapid testing using the verigene gram-negative blood culture nucleic acid test in combination with antimicrobial stewardship intervention against gram-negative bacteremia stewardship approach for optimizing antimicrobial therapy through use of a rapid microarray assay on blood cultures positive for enterococcus species prospective intervention study with a microarray-based, multiplexed, automated molecular diagnosis instrument (verigene system) for the rapid diagnosis of bloodstream infections, and its impact on the clinical outcomes the effect of molecular rapid diagnostic testing on clinical outcomes in bloodstream infections: a systematic review and meta-analysis practice guidelines for the management of infectious diarrhea physician use of parasite tests in the united states from 1997 to 2006 and in a utah cryptosporidium outbreak in 2007 how well does physician selection of microbiologic tests identify clostridium difficile and other pathogens in paediatric diarrhoea? insights using multiplex pcr-based detection survey of physician diagnostic practices for patients with acute diarrhea: clinical and public health implications management of suspected infectious diarrhoea by english gps: are they right? general practitioner practices in requesting laboratory tests for patients with gastroenteritis in the netherlands multicenter evaluation of the biofire filmarray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis spectrum of enteropathogens detected by the filmarray gi panel in a multicentre study of communityacquired gastroenteritis a gastrointestinal pcr panel improves clinical management and lowers health care costs multiplex gastrointestinal pathogen panels: implications for infection control a cost benefit analysis of the luminex xtag gastrointestinal pathogen panel for detection of infectious gastroenteritis in hospitalised patients integrated multiplex pcr tests for identifying gastrointestinal pathogens in people with suspected gastroenteritis (xtag gastrointestinal pathogen panel, filmarray gi panel and faecal pathogens b assay). diagnostics guidance detection of influenza a and b with the alere i influenza a & b: a novel isothermal nucleic acid amplification assay. influenza other respir viruses automated real-time collection of pathogen-specific diagnostic data: syndromic infectious disease epidemiology infectious disease surveillance in the big data era: towards faster and locally relevant systems fda clearance for filmarray respiratory panel ez (rp ez) fda clearance for filmarray respiratory panel 2 (rp2 the point-of-care laboratory in clinical microbiology extreme pcr: efficient and specific dna amplification in 15-60 seconds better tests, better care: improved diagnostics for infectious diseases key: cord-018271-ybfxtc7x authors: van doorne, hans; roesti, david; staerk, alexandra title: microbiology date: 2015-02-09 journal: practical pharmaceutics doi: 10.1007/978-3-319-15814-3_19 sha: doc_id: 18271 cord_uid: ybfxtc7x microbial contamination of pharmaceutical preparations may cause health hazard to the patient (e.g. infection, pyrogenic or allergic reaction), altered therapeutic activity of the product, or other decrease in quality (turbidity, loss of consistency, altered ph). this chapter provides a general introduction on pharmaceutical microbiology by focusing on the essential properties of micro-organisms. first of all the basic characteristics of life and the types of biological contaminants and potentially infectious agents of pharmaceutical products will be discussed: viz. prions, viruses, mollicutes, bacteria, fungi, and endotoxins. in the next section factors affecting survival and growth of micro-organisms are discussed. in addition to well-known factors such as time, temperature, and chemical and physical characteristics of the environment, attention will be paid to biofilm formation. primary microbiological contamination is prevented by implementing an adequate microbiological quality control and quality assurance program and by following cgmps during production. microbiological quality control of pharmaceutical preparations and monitoring of production areas depend on the detection and quantification of micro-organisms. the classical, growth based, methods and some of the commercially available alternative methods are discussed. understanding essential microbiological concepts is necessary in designing both microbiologically stable pharmaceutical products and ensuring an effective quality control and monitoring program within the manufacturing or preparation facility. although there is no universal definition of life, scientists generally agree that living systems share all or at least the characteristics: organisation, interaction with the environment, adaptation, metabolism, growth, motility and communication. organisms are composed of one or more cells, which are the basic units of life. each cell must be highly organised because growth and multiplication can only occur when the individual biochemical processes are synchronised. in higher organisms, organisation within the organs, and communication with other organs are essential for the normal functioning of the body. cells respond to chemical and physical input from the environment. a response is often expressed by motion. chemotaxis, the movement of a cell in response to a concentration gradient of a substance, is an example of such an interaction. adaptation is the accommodation of a living organism to its environment. it is fundamental to the process of evolution, by which cells change their characteristics and transmit these new properties to their offspring. metabolism involves the uptake of nutrients from the environment, their conversion to energy (adenosine triphosphate: atp) and cellular components, and the deposition of waste products (e.g. carbon dioxide: co 2 ) or metabolites (e.g. toxins or antibiotics) into the environment. living things require energy to maintain internal organisation (homeostasis) and to produce the other phenomena associated with life. growth is the increase in biomass. a growing individual increases up to a point in size in all of its parts. reproduction is the result of a series of biochemical events that result in the production of a new individual (asexually, from a single parent organism, or sexually, from at least two differing parent organisms). in microbiology growth is often used as a synonym for reproduction. dormancy is a state of decreased metabolic activity in which there is no growth, i.e. no increase in biomass. it may be a dynamic state in which the number of newly formed cells balances the number of dying cells. the duration of this period is relatively short (hours, days). dormancy of bacterial spores may continue for considerably longer periods of time. many cells and organisms can move under their own power. movement to a new location may offer the cell new resources. motility however is not a characteristic of all living organisms. animals are typically motile, whereas plants are non-motile. in micro-organisms motility is dependent on the type of organism and sometimes even on the stage of the life cycle the cells have reached. throughout evolution humans and animals have developed a multitude of ways for communication. micro-organisms (bacteria, yeasts and moulds) can communicate through a process called quorum sensing. quorum sensing is the regulation of gene expression in response to fluctuations in cellpopulation density. quorum sensing exemplifies interactive social behaviour innate to the microbial world that controls features such as virulence, biofilm formation, antibiotic resistance, swarming motility, and sporulation [1, 2] . in gram-negative bacteria, small molecules (e.g. acyl homoserine lactone (alh) [3] ) serve as signals to recognise microbial cell population size. when a signal exceeds some threshold concentration the expression of specific genes is changed [4] . if a microbial cell is introduced into a pharmaceutical preparation or onto a surface it will sense whether suitable conditions (nutrients, moisture, etc.) for growth are available (interaction with the environment). if possible the cell will adapt to its new habitat, and start to metabolise the available nutrients. eventually growth will take place. motility of individual cells will facilitate colonisation of other sites. production of toxins (in case of a pathogen) is a demanding biochemical process and will occur only when quorum sensing indicates that a sufficiently large population has developed. thus the interplay between all these characteristics determine whether a cell will be able to grow in a specific product, or on a surface. survival and growth of micro-organisms in pharmaceutical preparations is governed by extrinsic factors (particularly temperature) and intrinsic factors (product composition and physico-chemical characteristics). the combination of intrinsic and extrinsic factors will determine the types and number of micro-organisms that will develop in a product or on a surface. temperature has a strong influence on whether an organism can survive or thrive. temperature exerts its influence indirectly through water (which has to be in the liquid state), and directly through its influence on the organic molecules composing the living cells. mesophilic organisms are widespread in nature. they have the potential to grow in a temperature range of roughly 8-45 c. at temperatures above 30 c some contaminants of water and air including different types of bacteria and moulds will fail to grow or grow more slowly. the optimum growth temperature for pathogenic bacteria is around 37 c with an upper range from 44 c to 48 c. legionella pneumophila (l. pneumophila) is an exception; it will grow at temperatures up to 55 c. geobacillus stearothermophilus is a thermophile and grows at temperatures between 50 c and 65 c. it is used as a test organism (biological indicator) to verify the efficacy of moist heat sterilisation processes. for reasons of chemical stability some preparations must be stored frozen. under these conditions (no liquid water) microbial growth is not possible. however, the majority of the preparations are either stored refrigerated (4-8 c) or at room temperature (20-25 c) , which will allow growth of most mesophilic micro-organisms. according to european good manufacturing practice (eu-gmp) regulations water for injection (wfi) should be produced, stored and distributed in a manner which prevents microbial growth, for example by constant circulation at a temperature above 80 c (see also sect. 27.5.2). the presence of water is essential to every form of life including micro-organisms. in the late 1930s, it was recognised that water activity (or a w ), as opposed to water content, was the more significant factor in studying the relationship of water to microbial growth. the a w value is defined as the proportion between the water vapour pressure of the product and the vapour pressure of pure water at a common temperature. lowering the water content has historically been a convenient method to protect foods from microbial spoilage. examples where the available moisture is reduced are dried fruits, syrups, and pickled meats and vegetables. low water activity will also prevent microbial growth within pharmaceutical preparations, see also sect. 22.3.3. water activity values supporting microbial growth of a number of representative micro-organisms are provided in the usp [5] . that chapter also suggests a strategy for microbial limit testing based on water activity. pharmaceutical cleaning operations usually involve a final rinse with water of suitable pharmaceutical quality. to prevent microbial growth, it is essential to dry the object as soon as possible after rinsing. although water is essential for microbial growth, a low a w value does not necessarily lead to cell death. for instance, some dry raw materials, especially those of natural origin, may be heavily contaminated with both endospores and vegetative cells. freeze drying in a suitable medium, for instance, is an excellent way to preserve pure cultures of viable micro-organisms. a water activity below 0.6 does not enable micro-organisms to grow. solid oral dosage forms such as tablets have in general an a w value lower than 0.5 which means that these products remain stable from a microbiological point of view over long periods of time if the product is stored in a waterproof blister that remains integral. micro-organisms require for their growth suitable nutrient sources of elements (e.g. c, h, o, p, s, n), minerals (e.g. na + , ca 2+ , mg 2+ ) and trace elements (e.g. cu 2+ , mn 2+ , co 2+ ). even in a relatively nutritionally poor medium such as distilled water, the number of micro-organisms may be as high as 10 5 -10 6 colony forming units (cfu)/ml, indicating that nutrients are present in sufficient quantity to allow proliferation of water-borne bacteria such as the pseudomonads. the presence of readily assimilated substances such as sugars or polyalcohols in dosage forms such as creams or syrups can lead to an increased probability of microbial adulteration of those products. a ph range of 6-7 does not exert any discernible selective influence on growth of pharmaceutically relevant types of organisms. below ph 6, growth of many bacteria will start to be inhibited. pathogenic and toxinogenic bacteria generally will not grow at ph values below 4.5, but they may survive long times of immersion in weakly acidic solutions. yeasts and moulds unlike bacteria, generally tolerate acidic media quite well. optimum ph of most fungi is about ph 5. at more alkaline conditions, pseudomonads will predominate. for example, ps. aeruginosa is known to grow at a ph range of 6-9. microbes are classified as aerobes or anaerobes. this is now defined in terms of oxidation-reduction potential (e h ). generally, the range at which different micro-organisms can grow are as follows: aerobes +500 to +300 mv; facultative anaerobes +300 to à100 mv; and anaerobes +100 to less than à250 mv. the redox potential of a normal aerobic nutrient medium is about + 300 mv. thioglycolate medium, which is used for growth of anaerobic bacteria has an e h of about à200 mv. for reasons of chemical stability, the redox potential of some pharmaceutical preparations is kept at a low level by means of reducing agents such as sulfite, tocopherol or ascorbic acid. the effect of a reduced redox potential on the microbial flora of such preparations has never been studied. the number and types of micro-organisms that may develop in various pharmaceutical dosage forms is greatly influenced by the presence of substances with antimicrobial properties. antimicrobial active substances can be divided into three groups, as follows: • the first group consists of substances used for therapeutic or preventive antimicrobial purposes, for example: antibiotics, chemotherapeutics, and disinfectants. • the second group consists of preservatives (see sect. 23.8), used to guarantee the microbiological quality of the product throughout its shelf life. for example: esters of hydroxybenzoic acid, quaternary ammonium substances and sorbic acid are widely used in pharmaceutical and cosmetic preparations. other preservatives that are used include phenol, chlorhexidine, benzoic acid and benzyl alcohol. • the third group consists of excipients with 'collateral' antimicrobial activity that are principally added to dosage forms for reasons unrelated to their (sometimes weak) antimicrobial activity. for example, sodium lauryl sulfate is known to inactivate some gram-positive bacteria. similarly, edetate has weak antimicrobial activity, and it confers synergistic antimicrobial properties when combined with quaternary ammonium substances. in the term 'prion diseases' refers to a group of neurodegenerative disorders. these include scrapie (in sheep and goat), kuru (prion disease endemic amongst cannibalistic tribes in papua new guinea), bovine spongiform encephalopathy (in cattle) and creutzfeldt-jakob disease (cjd) (in humans) [6] . prion diseases are characterised by long incubation periods ranging from months to years and are invariably fatal once clinical symptoms have appeared. in all prion diseases the infectious prions are generated in the brain of the afflicted animal. in the rare cases of interspecies transmission, such as from cattle to humans a 'template assisted replication' takes place. this means that the prions that replicate in the human brain have the amino acid sequence encoded by the dna of the host (human being) and not the sequence of the donor animal [7] . of pharmaceutical preparations bse was first diagnosed in the united kingdom in 1986 and a large number of cattle and individual herds have been affected. interspecies tse transmission is restricted by a number of natural barriers, transmissibility being affected by the species of origin, the prion strain, dose, and route of exposure. transmission of scrapie to sheep and goats occurred following use of a formol-inactivated vaccine against contagious agalactia, prepared with brain and mammary gland homogenates of sheep infected with mycoplasma agalactiae [8] . iatrogenic transmission of human prion disease can occur through medical or surgical procedures. an example is the injection of hormones such as gonadotropins extracted from cadaver pituitaries. current evidence indicates that, with respect to the risk of tse infection, urinary-derived gonadotrophins appear to be safe [9] . the risks of urinederived fertility products could now outweigh their benefits, particularly considering the availability of recombinant products [10] . cases of cjd have also been attributed to the use of contaminated instruments in brain surgery and with the transplantation of human dura mater and cornea [11] . suppliers of materials may minimise the risks of contamination of tse by ensuring [12] : • the source animals and their geographical origin • nature of animal material used in manufacture and any procedures in place to avoid cross-contamination with higher risk materials • production process(es) including the quality control and quality assurance system in place to ensure product consistency and traceability manufacturers of pharmaceutical preparations select their raw materials so they are tse free (see also sect. 23.1.7). this can be ensured either by purchasing materials from non-animal origin or from non-tse relevant animal species (e.g. porcine instead of bovine). if material from tse-relevant animal species is purchased, it should be delivered with a certificate that confirms it free of tse. regular audits verifying this assumption should be performed by the manufacturer at the material's supplier manufacturing site. prions, unlike the normal prp c proteins, are very resistant to inactivation. the only methods that appear to be completely effective under worst-case conditions are strong sodium hypochlorite solutions or hot solutions of sodium hydroxide [13, 14] . some cross contamination can be avoided by the use of disposable instruments, e.g. in tonsillectomy [15] . a virus is a non-cellular genetic element, which is dependent on a suitable host cell for its multiplication. their size generally ranges from 20 to 300 nm. it has been argued extensively whether viruses are living organisms. the majority of virologists consider them as non-living as they lack many of the characteristics of life, such as independent metabolism. viruses exist in various states throughout their life cycle. in the extracellular state a virus particle is called a virion. virions are composed of a core of genetic material, which can either be in the form of dna or ribonucleic acid (rna), and a protein coat or capsid. in some viruses (enveloped viruses) the capsid is surrounded by a lipid bilayer membrane. attached to these membranes are specific proteins, which may play a role in the attachment of the virion to the host cell, or release from the host. thus, haemagglutinin and neuraminidase are two important enzymes present in the envelope of the influenza virus. the virions are metabolically inactive because they are devoid of a self-generating energy system, transfer rna, ribosomes, and so forth. many viruses do contain enzymes that become essential in rendering these agents infectious to susceptible hosts. viruses are obligate intracellular parasites. replication occurs only inside the cell of a suitable host. replication usually leads to destruction of the host cell. sometimes the viral dna is incorporated into the genetic material of the host. this principle is successfully used in genetic engineering, where viruses are used as vectors to incorporate a new gene in a cell. viruses are causative agents of many human, animal, and plant diseases. aids, sars, and avian flu are viral diseases, which are nearly daily covered by the headlines in papers and by the news items on radio and television. in 1917-1919 a 'spanish flu' pandemic killed over 50 million people. the virus involved was most probably a mutation of some avian virus. the avian flu pandemic (caused by the h 5 n 1 variant) was, by comparison very small, as it has caused 'only' about 150 fatalities. the great concern for virologists and epidemiologists is the extremely high mortality rate (over 50 %) of infections with this virus. in the form of vaccines, viruses are inactivated or attenuated so as to prevent diseases in susceptible populations. the baltimore classification is the preferred way of classifying viruses. viruses are grouped into families depending on their type of genome (dna, rna, singlestranded (ss), double-stranded (ds) etc.) and their method of replication. a series of important medicines is derived from animal or human sources and may potentially be contaminated with undesired virus particles. such medicines include: • coagulation factors, immunoglobulins and albumin from human blood plasma • vaccines and monoclonal antibodies from cell cultures • proteins from cells altered by genetic engineering • homoeopathic preparations of animal origin vaccination is one of the most important public health accomplishments. however, since vaccine preparation involves the use of materials of biological origin, such as chinese hamster ovary cells, vaccines are susceptible to contamination by micro-organisms, including viruses [16] [17] [18] . several cases of viral vaccine contamination have been reported. for example, human vaccines against poliomyelitis were found to be contaminated with sv40 virus from the use of monkey primary renal cells. several veterinary vaccines have been contaminated by pestiviruses from foetal calf serum [19] . in 2010 the detection of fragments of a porcine circovirus was the reason for a temporary withdrawal of some commercial vaccines from the spanish market [20] . several methods are being used or in development to reduce infectivity of blood products, including solventdetergent processing of plasma and nucleic acid crosslinking via photochemical reactions with methylene blue, riboflavin, psoralen and alkylating agents. several opportunities exist to further improve blood safety through advances in infectious disease screening and pathogen inactivation methods [21, 22] . one potential way to increase the safety of therapeutic biological products is the use of a virusretentive filter [23] . plasma pools may be submitted to serological tests and/or genome amplification assays before they are released for further fractionation [24] . multidose containers and the environment were found to be the source of a number of nosocomial viral infections [25] [26] [27] [28] [29] [30] [31] [32] [33] . presence of viruses in pharmaceutical preparations may be verified by performing either in vivo tests by inoculating the product directly in animals (e.g. rabbits, mice) or in-vitro tests such as polymerase chain reaction (pcr) or cell culture safety tests. along with the archea, bacteria belong to the prokaryotic organisms, i.e. cells that do not possess a real nucleus and that reproduce asexually. they are unicellular microorganisms with a size in general ranging from 0.2 to 10 μm. their extraordinary diversity in terms of biochemical processes and metabolic characteristics enable bacteria to adapt themselves to a large variety of environments. indeed, some species have the capacity to grow in anaerobic (absence of free oxygen in the air) environments by using other electron acceptors than oxygen, such as sulphates or nitrates or by fermentation. other species may use energy and carbon sources for growth from not only organic substances but also from carbon dioxide and light energy. for this reason, bacteria have colonised most habitats on earth (soil, water, animals, plants) and even the most extreme environments such as deep-sea fumaroles or geysers. another fascinating (but critical in terms of product safety) characteristic of bacteria is their capacity to grow extremely fast if the environmental conditions in terms of nutrient availability, moisture and temperature become favourable. during growth, an individual cells first increases in size and then the cell is divided in two parts (binary fission). in nutrient media, bacteria follow four growth phases (see fig. 19 .3). the first is the lag phase, during which the bacteria adapt to their new environment by repairing damaged structures and synthesising enzymes to catabolise nutrients in the medium. the second phase, the most spectacular, is the exponential phase during which nutrients in the medium are metabolised rapidly leading to a rapid doubling of the population of bacterial cells. the population of escherichia coli cells under optimal growth conditions can multiply each 20 min. this would mean that after 8 h the population would reach one million cells and after 43 h, the volume of cells produced would be equivalent to the volume of planet earth! once nutrients start to deplete, the exponential growth is slowed down and the amounts of cells in the overall population remains stable; this is the third phase called the stationary phase. in this phase, secondary metabolites such as antibiotics are produced in higher quantities. the last phase is when no more nutrients are available and the amount of bacterial cells starts to drop. in the human microflora, there are at least 10 times more bacterial cells than human cells and most of them are harmless. human bacterial infections are mainly caused by strict pathogenic species (less than 2 % of bacterial species) or by opportunistic pathogens when the immune system of the person is depleted. bacteria may cause a large variety of infections, the most common being food poisoning, pneumonia, skin infection, urinary tract infection, throat and mouth infection, meningitis, eye infection. depending on the infectious agent, the minimum amount of microorganisms provoking an infectious dose varies greatly. for instance in some cases, only 10 cells of shigella dysentriae need to be ingested to provoke dysentry but at least a 1,000 cells of vibrio cholera to provoke cholera [34] . mollicutes, also known under the trivial name mycoplasmas, are the smallest free-living prokaryotic organisms and for years were thought to be viruses because they passed through the usual bacterial filters. they resemble protoplasts, because they lack a cell wall, but they are relatively resistant to osmotic lysis due to the presence of sterols in the cell membrane. in this respect the mycoplasmas form an exceptional group, because sterols are absent in other prokaryotic cells. mycoplasmas are widespread in nature and many are animal, plant or human pathogens. most mycoplasmas that infect humans are extracellular parasites. examples of human pathogenic mycoplasmas are mycoplasma pneumonia (infections of upper respiratory tract), and mycoplasma genitalium (nongonococcal urethritis) [35] . mycoplasma contamination is a major concern for vaccine and biotechnological industries since the organisms may cause disease and may interfere with cell culture [36] . peptones, and animal sera used as components of cell culture media may be sources of this contamination [37, 38] . mycoplasmas can be cultured in liquid or on solid media. however, in contrast with other bacteria, their growth is slow, and a microbiological assay as described in the ph. eur. is time-consuming (at least 28 days). alternative, rapid methods, based on nucleic acid technologies such as pcr, have been developed [36, 39] . under certain conditions these methods may be used as an alternative method instead of the official, growth based method [40] . bacteria may be composed of the following structural elements (see fig. 19 bacteria become motile by means of flagella [41] . bacterial flagella are protein threads which originate in a defined region of the cytoplasmic membrane and protrude through the peptidoglycan layer and the outer membrane. the number of flagella per cell and their position depends on the species. pseudomonas aeruginosa (ps. aeruginosa) has only one (polar) flagellum at the tip of the cell, whereas escherichia coli (e. coli) has numerous flagella spread over the entire cell surface (peritrichous). flagella may play an important role in pathogenicity [41, 42] . the surface of cells of some bacterial species is covered with many (10 to several thousands), thin (3-25 nm), and long (up to 12 μm) threads called pili, or fimbriae. they play a role in the initial adhesion of bacteria to host tissues and inanimate surfaces [43, 44] . attachment to a surface is the first step in biofilm formation. upon attachment on tissue cells they may trigger a number of biochemical signals from the host, which ultimately leads to the bacterial disease [45] . capsules and slime layers -collectively called glycocalixconsist of source polysaccharide material secreted by the cell. a capsule is a rigid structure, whereas a slime layer, or loose extracellular slime, is more flexible, with diffuse boundaries. the glycocalix has several functions. it is involved in cell attachment and it may protect cells from being digested, a phenomenon known as phagocytosis. whilst encapsulated strains of streptococcus pneumoniae are highly pathogenic, non-encapsulated mutants are completely avirulent [46, 47] . dextran, a slime layer product of leuconostoc mesenteroides of relatively low molecular weight can be used as a therapeutic agent in restoring blood volume [48] . the outer surface of the bacterial cell plays an important role in the adhesion of the cell to various surfaces. in addition to the factors that have been discussed, adhesion may also be mediated by so-called surface-associated adherence factors, usually designated as adhesins. adhesion, which is the first step in a series of events leading to colonisation, biofilm formation and ultimately infection, is a specific process in which the adhesin "recognises" a receptor on the host surface. this specificity explains why micro-organisms such as influenza or mycobacterium tuberculosis can cause targeted infection of the respiratory tract but otherwise are relatively harmless when contacting other host tissues. the cell wall gives the cell its shape and strength. the cell wall must resist the internal osmotic pressure of the cell that is estimated to be about 2 bar. the composition of cell walls of gram-positive bacteria is very different from those that stain gram-negative. peptidoglycan is the common cell wall component of bacteria (excluding mollicutes) and gives the wall its shape and strength. it is a polymer consisting of a backbone of alternating n-acetylglucosamine and n-acetylmuramic acid residues, cross-linked with small peptide bridges. peptidoglycan accounts for about 80-90 % of the wall of grampositive bacteria and for about 10 % of the gram-negative cell wall. the gram-negative cell wall contains only a shallow peptidoglycan layer. on the outer side of this layer is the outer membrane, a complex structure consisting of four major components: phospholipids, lipopolysaccharide, proteins (e.g., porins), and lipoprotein. lipolysaccharide (endotoxin) is responsible for the pyrogenicity of the gram-negative bacteria. the cytoplasmic membrane, or plasma membrane is a phospholipid bilayer into which proteins/enzymes are embedded. the function of the cytoplasmic membrane is to act as a selective permeability barrier between the cytoplasm and the exterior environment. a mesosome is an organelle of bacteria that appears as an invagination of the plasma membrane and functions either in dna replication and cell division, energy production, or excretion of exoenzymes. flagella (if present) originate in a special structure in the cytoplasmic membrane (see the section on flagella under structure of the bacterial cell). the cytoplasm is a viscous liquid, which contains all other essential elements for the living cell. the genetic material is mainly organised in the genome, a circular string of dna. there is no discrete bacterial nucleus. the genetic code is translated into messenger rna and then transported to the ribosomes, where the protein synthesis occurs. the building blocks of the proteins (amino acids) are transported to the ribosomes by means of transfer rna. some genetic information such as antibiotic resistance may be encoded in plasmids -dna molecules that are independent of the genome and that can replicate themselves. some plasmids contain a set of genes (in the tra region) that enable the transfer of the plasmid by cell to cell contact (conjugation). the plasmid is replicated during this process and its genetic information (e.g. antibiotic resistance) is thus transferred to the recipient cell. there are both intra-species and inter-species plasmid transfer phenomena. the cytoplasm may also contain reserve material such as polyhydroxybutyric acid, and other substances of uncertain function (e.g. polyphosphate, volutin). some gram-positive rods such as the genera bacillus, geobacillus and clostridium are capable of forming endospores that enable these genera to survive harsher conditions, such as exposure to heat, radiation, or chemicals. bacterial spores are resistant forms of life. some experts have suggested that they may remain viable (capable of life) for millions of years. the bacterial spore has a complex structure, consisting of various layers, including coat and cortex, all of which play a role in long-term survival (see fig. 19 . endospore formation is a non-reproductive process: one cell produces only one spore which, after germination, produces one vegetative cell. bacterial sporulation occurs when growth decreases due to exhaustion of an essential nutrient. it is a complicated process requiring the participation of more than 200 enzymes. conversion to a vegetative cell involves three steps: activation, germination and outgrowth. activation can be accomplished by heating the spores to a non-lethal temperature. germination can be induced by a variety of events, including exposure to nutrients (amino acids, sugars, or purine nucleosides), non-nutrient germinants (dodecylamine, lysozyme) and heating [49] . the spore loses its characteristic constituents, and heat resistance decreases dramatically. in the last stage water is taken up, and metabolism (synthesis of atp, destruction of bacterial spores is the ultimate goal of sterilisation processes. bacterial spores are typically used in biological indicators for validation and monitoring of sterilisation processes. pyrogens are substances that cause a febrile reaction. two groups of pyrogens can be distinguished: exogenous and endogenous pyrogens. the exogenous pyrogens form a heterogeneous group of substances; the most important one is lipopolysaccharide (lps) from the cell wall of gramnegative bacteria. lps, also known as endotoxin, has antigenic properties (o antigen) and causes fever when injected intravenously. lipoteichoic acid, muramyldipeptide, porins, glycans and nucleic acids, are examples of non-endotoxin pyrogens originating from bacteria (gram-positive and gram-negative), yeast and moulds. the pyrogenic activity of lps is much higher than that of most other pyrogenic substances. this is the reason why an in-vitro limit test for lps (the limulus amoebocyte lysate, or lal test) generally suffices for quality control purposes of parenteral medicines and raw materials, including water for injection. the european pharmacopoeia requires the rabbit pyrogen test for a number of vaccines, some antibiotics, and specific excipients including glucose, if intended for the preparation of large volume parenterals (see sect. 32.8). these products may be contaminated with pyrogens other than lps, or are known to inhibit the lal test. a third test, the monocyte activation test (mat) is based on the in-vitro activation of human blood cells by pyrogens. this leads to the release of pro-inflammatory cytokines tumour necrosis factor alfa (tnf-alfa), interleukin-1 beta (il-1beta) and interleukin-6 (il-6) that are determined by enzyme-linked immuno sorbent assay (elisa). consequently, the mat will detect the presence of both exogenous and endogenous pyrogens in the test sample. the mat is suitable, after a product-specific validation, as a replacement for the rabbit pyrogen test [50] . the reagent for the lal is isolated from the blood of the horseshoe crab (limulus polyphemus). the blood is collected from wild animals. many animals do not survive (mortality rates of up to 30-50 % have been reported), and this living fossil is threatened with extinction. it is to be expected that in the near future the mat test or other alternatives for the lal test and the rabbit test will be more generally introduced. the development of such new methods will significantly reduce animal testing. the commercially most successful alternative method, which replaces the rabbit pyrogen test for bacterial impurities in medicines with a test using human cells, could save the life of 200,000 rabbits a year. biofilms are multicellular, microbial communities held together by a self-produced extracellular matrix that adhere to biological or non-biological surfaces [51] . a biofilm has a defined architecture, and it provides an optimal environment for the exchange of genetic material between cells, e.g. spread of antibiotic resistance. cells within a biofilm may communicate via quorum sensing (see sect. 19.1.7), which may in turn affect biofilm processes such as detachment of cells. the ability to form biofilms is a universal attribute of bacteria and many other micro-organisms. elimination of bacteria in this mode of growth is challenging due to the resistance of biofilm structures to both antimicrobials and host defences. biofilms have great importance for public health because of their role in certain infectious diseases and their role in a variety of device-related infections. biofilm infections on indwelling devices or implants are difficult to eradicate because of their much better protection against macrophages and antibiotics, compared to free living cells, leading to severe clinical complications often with lethal outcome. fungi are widespread in nature and have considerable economic and medical importance, because: • they may contaminate and cause spoilage and deterioration of pharmaceutical preparations. mould and yeasts are the second cause of fda recalls in non-sterile pharmaceuticals [52] . • this group of organisms is used by producers of active substances, including antibiotics, such as penicillins by penicillium species, or alkaloids, such as ergotamine by claviceps purpurea. • some fungi are pathogenic to humans. they may cause infections (e.g., trychophyton sp. or candida sp.), or produce toxic substances (e.g., aflatoxin by aspergillus flavus). two groups of fungi are relevant in the context of pharmaceutical products or processes: the moulds and the yeasts. their physical differentiation is not always clear, because some fungal species (e.g., candida, histoplasma and cryptococcus) show dimorphism, a phenomenon in which a filamentous and a yeast-like stage both exist. moulds are obligate aerobic micro-organisms; they grow on the surface or in the uppermost layers of the substrate. characteristic of moulds is the filamentous body, the mycelium. vegetative growth of moulds occurs at the tip of the individual filaments (hyphae). depending on the species, hyphae may be divided into compartments by means of septa (eumycetes). each septum contains a pore, which allows flow of cytoplasmic constituents from one compartment to another. the lower fungi (phycomycetes) have aseptate (coenocytic) hyphae; the mycelium is a multinucleated cell. asexual reproduction of moulds normally occurs by means of spore formation. from the mycelium special branches reach up into the air. at the tip of these conidiophores the spores (conidiospores) are formed on a genus specific structure. the colour of mould colonies on solid substrates (e.g., different shades of green for penicillium species, or black for aspergillus niger) is entirely due to the massive production of these conidiospores. mould spores may cause significant issues in the production of pharmaceutical preparations since they survive desiccation and may be transported via air, personnel or material flow into products. the spores are readily dispersed into the environment and may form a new mycelium. because of mechanical forces, such as those exerted during vortexing, hyphae may break up into smaller fragments, which may also form new mycelia. clumps of conidiospores may also break up into smaller units. such fragmentation caused by vigorous mixing in the course of microbiological examination of pharmaceutical samples may lead to considerable uncertainty in fungal counts. yeasts are typically unicellular organisms. yeast cells are spherical or oval. growth (asexual reproduction) takes place by a process called budding. a new cell is formed as an outgrowth of the mother cell, the daughter cell enlarges and finally the two cells separate. pathogenic dimorphic fungi usually form yeast-like cells in the human body and a mycelium at room temperature (e.g. histoplasma). candida sp. is an exception because it forms hyphae in the host tissue. sexual reproduction is associated with many yeasts and moulds. a stage in which spores are formed is always involved in the sexual process. depending on the type of sexual spore formation four groups of moulds can be distinguished: ascomycetes, basidiomycetes, zygomycetes, and oomycetes. the spores are called ascospores, basidiospores, zygospores, and oospores, respectively. fungi for which no sexual reproduction has been demonstrated are classified as fungi imperfecti (deuteromycetes). the majority of fungi thus far classified fall into this category. penicillium and some aspergillus species are well-known representatives of this group. the cell wall of fungi consists of 80-90 % polysaccharides. chitin is a common constituent of fungal cell walls, but is replaced by other substances such as mannan, galactosan or chitosan in some species. peptidoglycan, the common constituent of bacterial cell walls is never present. this phenomenon explains why fungi are insensitive to antibiotics that inhibit murein synthesis, such as the penicillins and the cephalosporins. sterols are essential structural components of the fungal cytoplasmic membrane. this characteristic makes fungi sensitive to antibiotics that interact with sterols, such as nystatin and amphotericin. in the previous sections the characteristics of potential contaminants of pharmaceutical preparations and their requirements for survival and growth have been discussed. there appears to be a complicated interplay between the type and characteristics of the micro-organisms (e.g. spore formation), their ability to form biofilms, the composition of the environment, and external factors, particularly the temperature. the fate of a micro-organism in a pharmaceutical preparation depends on this interplay. four different curves may be observed when numbers of colony forming units (cfu) are plotted against time (fig. 19.3) . microbial growth follows the well-known sigmoid growth curve, with lag phase, log phase and maximum stationary phase. microbial destruction under influences of heat, radiation or chemicals is frequently a first order kinetic process. when microbial destruction is plotted on a semi-logarithmic scale, a straight line is observed. a 'shoulder' is sometimes observed at the beginning of the curve. this lower death rate is attributed to the genetic repair mechanisms of the cells, e.g. when exposed to low doses of uv radiation. bacterial spores must be 'activated' before they can germinate and grow out to become prototypical vegetative cells. this phenomenon may also cause a 'shoulder' in survival curves. at the end of the survival curve, a 'tail' may be observed, indicating the presence of resistant cells or clumps of cells. true dormancy is found only in bacterial endospores. nevertheless, even vegetative organisms can produce an effective state of dormancy because of either a relatively slow death rate or growth and kill rates that offset each other. in pharmaceutical preparations another type of curve is sometimes observed. an initial decrease in the number of colony forming units may occur, followed by an increase. this phenomenon can be observed when analysing data from preservative efficacy testing of inadequately preserved dosage forms. it is the reason why in pharmacopoeial preservative efficacy tests the number of viable cells must be followed for a period of 28 days (see sect. 32.8). microbial contamination of pharmaceutical products may result in deterioration of the product or direct hazard to the patient. whether a contaminated pharmaceutical product will trigger infection or disease in the patient depends on various factors such as: • number of micro-organisms (cfu per g or ml) • ability of the contaminant to grow and metabolise components of the product • properties of the particular strain • immunocompetence of the patient, due to disease (aids) or use of immunosuppressiva • route of administration deterioration or spoilage of the product because of microbial growth may result in several effects including: • loss of texture because of metabolism of oil/fat phase • loss of organoleptic quality because of production of olfactory products • loss of preservative efficacy because of metabolism of the preservative • loss of package integrity because of excessive gas production • loss of therapeutic activity because of metabolism of the active substance(s) • release of toxigenic substances, including toxins (e.g. aflatoxin) and pyrogens the contamination can be primary or secondary. primary contamination occurs at the premises or during preparation: • personnel. personnel account for the majority of contaminations in the clean room environments. this can be explained by the high number of micro-organisms located on or in the human body. the organisms may be introduced into the environment due to inadequate gowning or hygiene, infrequent or ineffective hand washing and disinfection procedures, unqualified behaviour (non-clean room adequate) of personnel, etc. in the aseptic production of sterile pharmaceutical preparations living micro-organisms should not enter the aseptic filling area and the product should not contain any viable microorganism. in those situations, low-level microbial contaminations of products occur mostly at critical interventions near to the product during processing. microbial contamination of non-sterile pharmaceutical preparations may not originate primarily from the human body, but raw materials, equipment, air and packaging material may also play an important role • raw materials. raw materials from natural origin may be highly contaminated with micro-organisms especially spore-forming bacteria and moulds and in some cases with more critical enterobacteriaceae. soon after a publication on salmonellosis in more than 200 persons caused by the contamination of thyroid tablets with two types of salmonella originating from the raw material [53] , proposals for the examination of non-sterile pharmaceutical preparations and acceptance criteria were published [54] . • water. water may be used to clean equipment and clean rooms as well as a product component. water contains water-borne micro-organisms that may grow under low nutrient conditions. in a recent review of fda product recalls, almost half (48 %) of them were due to contamination by water-borne micro-organisms such as burkholderia cepacia, pseudomonas species, or ralstonia pickettii [52] . • air. micro-organisms may be carried over from dust or soil particles and may be transported into manufacturing areas by personnel, material or airflow. mould spores for instance were carried over from a highly contaminated source into the production room [55] . • equipment. equipment may be contaminated if inappropriate cleaning, disinfection or sterilisation procedures have been performed. • primary packaging. the microbiological quality of primary packaging material is critical for sterile preparations. vials, ampoules and stoppers shall be sterile and free of pyrogens before filling. for non-sterile preparations the microbiological quality of the packaging material is less critical. because of the production process, bottles, tubes etc. will have only low levels of contamination, provided they have been packed, stored and handled under appropriate conditions. secondary contamination may occur during storage, transport, and administration of the product. the root cause for contamination during storage and transport is mainly insufficient closure integrity (see sect. 24.3). contamination during administration can be avoided by suitable design of the primary package (see sect. 24.1) and appropriate instruction of the medical staff or patient (see sect. 37.4). tubes are less prone to contamination than jars. in hospitals eye drops should only be used for one specific patient and preferably for one specific eye. microbiological quality assurance and microbiological quality control should be part of the pharmaceutical quality system of any production site. its main aim is to prevent microbial contamination in case of products required to be sterile (e.g. parenteral medicines), or to reduce the microbial counts and avoid objectionable micro-organisms in case of non-sterile products (e.g. tablets). the elements of a pharmaceutical quality system (pqs) that are crucial for microbiological quality regard adequate design of premises, procedures and controls. they are discussed in this section. the principles are included in current good manufacturing practices (cgmp) guidelines (see sect. 35.5.7), which are legally binding for pharmaceutical manufacturers. microbiological quality assurance (qa) refers to the procedures in the quality system that ensures that microbiological requirements of the product are fulfilled. microbiological quality control (qc) refers to the tests performed to verify that the product meets the required specifications. the following procedures and measures concerning facilities should mitigate the risk of microbiological contamination: • qualified personnel. only trained and qualified personnel should enter areas where products are manufactured or prepared. personnel should wear dedicated gowning which provides a physical barrier between the body and the working environment. the more critical the activity or product microbiological requirements, the stricter the gowning. gowning may consist for instance of overalls, face masks, gloves (which are disinfected with ethanol) or even goggles in case of aseptic processing. for basic hygiene at the preparation of non-sterile medicines the main points are given here. contact between the product and the operator is to be prevented. thus: • equipment and production processes shall be designed so that direct contact between operator and product is minimised. • under no condition shall the product be touched with bare hands. if manipulation is unavoidable use utensils, such as forceps, or wear gloves. gloves shall be changed when appropriate, particularly at every preparation and after obvious contamination such as sneezing and wiping the nose. • refrain from talking above the product. coughing and particularly sneezing are difficult to suppress. wearing a facial mask and changing it at least every 2 h will considerably reduce the risk of contamination by this route. the operator shall inform his or her superior in case of a disease such as a cold. (continued) • facial hair shall be appropriately covered; this may require the wearing of a head cover and a facial mask to cover moustaches and beards. this is also necessary from a safety point of view when operating with rotating equipment such as an ointment mill. from a pure microbiological viewpoint wearing an overall doesn't make sense other than the promotion of an attitude of working cleanly and neatly. already after 1-2 h the overall bears as much contamination as the personal clothing. directions for clothing are however also necessary to promote occupational safety and health (see sect. 26.4.3) . the overall has to remain in the preparation area (thus taken off at lunch and coffee breaks) and has to be cleaned according fixed schemes, such as daily and when visibly contaminated. the overall shall have long sleeves and cover completely personal clothing. washing hands must always occur: • at the start of preparation • if hands are visibly dirty • after using the toilets • after sneezing and wiping the nose • between two different preparation processes, because of cross contamination nails have to be kept short and proper hand washing procedures include removal of watches, voluminous rings and bracelets (remaining off during the preparation process). washing hands technique requires preferably lukewarm water, soap from a dispenser, proper attention to thumbs, sufficient duration and proper drying with a towel because that will carry off micro-organisms too. • controlled environments. clean rooms in which pharmaceutical preparations are prepared processed, and packed are controlled for room pressure, humidity and temperature. they are segregated from other operating areas and may be entered via separate air locks for personnel and material. the incoming air of the clean rooms is filtered using hepa (high efficiency particulate air) filters that may retain more than 99.995 % of 0.3 μm air particles (see table 27 .4). the higher the microbiological quality of the product or the critical the process step, the higher the clean room quality criteria. for instance in aseptic processing of sterile products for critical areas where the product is exposed to the surrounding environment, the air should not contain more than 3,520 particles of 0.5 μm size per cubic metre and should be devoid of viable micro-organisms (see air classifications in tables 27.2 and 27.3). clean room design should ensure that air, personnel and material flow is optimal to prevent microbial contamination from a less clean area to a cleaner area. isolators have been introduced as an alternative to conventional clean rooms for aseptic production. these may be large installations, in which complex operations such as large-scale aseptic filling of syringes can be performed. controlled environments also regard to aseptic handling in pharmacies, where they are achieved using laminar flow cabinets, biological safety cabinets or isolators (see sect. 28.4). • cleaning and disinfection. the procedures for cleaning and disinfection (destruction of micro-organisms -but not necessarily spores -by chemical agents, see sect. 31.4.3) of equipment parts that are in contact with the product have to be validated. in addition, for the more critical products that are required to be sterile, the equipment parts that are in contact with the product need to be sterilised. sterilisation (destruction of micro-organisms including spores by heat) process of the manufacturing lines has also to be validated. for products, which are required to be sterile, the aseptic status of the production line is regularly evaluated by performing media fill simulations that consist of replacing the product with a microbial culture medium and evaluating if filled-media containers remain sterile. • reducing bioburden. the preparation processes may reduce or even eliminate living micro-organisms. for instance on the preparation of tablets, the tableting of a granulate into a tablet may kill non-spore forming microorganisms by the shearing forces of the interparticulate movement. products required to be sterile are either sterile filtered (filter 0.2 μm pore diameter, see sect. 30.6) or terminally sterilised directly in their container or package (e.g. steam sterilisation (sect. 30.5.1), radiation (sect. 30.5.3), ethylene oxide gas (30.5.4) ). • water distribution system. the distribution and storage systems for water that is used for cleaning, sterilisation and preparation should be devoid of biofilms. a distribution system may be controlled by continuously circulating heated water (>80 c) in loops, avoiding one-way systems and dead ends, and application of disinfection steps such as adding ozone to the re-circulating water (see sect. 27 microbiological testing is performed to monitor the microbiological bioburden and to ensure that the final product complies with the regulatory microbiological specifications. it comprises: • an environmental monitoring program in order to monitor the microbiological levels of classified rooms. air, product-contacting surfaces, working surfaces, floors and personnel are sampled. frequency and sampling locations are defined based on a risk assessment. maximum microbiological count levels should be defined either based on historical data or on regulatory guidelines. if they are exceeded, this may signal a deviation from normal conditions that would require an investigation and an evaluation on the impact on the respective product produced. trending of environmental results may also be performed in order to evaluate shifts in the overall hygienic conditions over an extended period of time to define appropriate corrective actions. see also sect. 31.6.1. • testing of primary packaging materials, raw materials (excipients, active substances, water) and products according to internal or official methods and specifications. microbial limits of pharmaceutical preparations are given in relevant monographs of the european pharmacopoeia. section 19.6 provides a deeper insight on the european test methods of pharmaceutical preparations and acceptance criteria. • monitoring water distribution and storage system. • root cause investigation (see sect. 35.6.15) . when a test does not fulfil a microbiological acceptance criterion, this is considered as a deviation or out of specification result. this requires an investigation to determine the root cause of contamination, e.g. whether it occurred during laboratory testing, sampling or manufacturing. if a source of contamination has been found, corrective and preventive actions are put in place to eliminate and prevent re-occurrence of the contamination. failure to meet measures to prevent microbiological contamination of pharmaceutical preparations may have dramatic consequences. a major health issue related to microbiologically contaminated pharmaceutical preparations was the 2012 new england (continued) compounding center (necc) meningitis outbreak in the usa [56] . on october 4, 2012, the cdc (us centers for disease control) and the fda (us food and drug administration) issued a recall alert for pharmaceutical preparations produced by the necc, following a multistate outbreak of fungal meningitis and other infections among patients who received contaminated preservative-free methylprednisolone acetate epidural injections. most patients suffered infection by the fungus exserohilum rostratum and in august 2013, cdc reported 749 cases and 63 deaths. after performing microbiological testing of the unopened incriminated product lots, cdc confirmed presence of exserohilum rostratum, along with other fungi (e.g. cladosporium cladosporioide, aspergillus fumigatus) and spore-forming bacteria (e.g. bacillus subtilis) [57]. after inspecting the necc preparation area, the fda mentions in its report [58] that moulds and bacteria were found in large numbers in many air and surface samples where the products were prepared. a number of physical and chemical techniques to eliminate or to destroy micro-organisms may be employed in order to assure that the microbiological quality of the product complies with pharmacopoeial requirements, immediately after production and throughout its shelf life. since these techniques are discussed in detail in other chapters, they are mentioned only briefly. physical removal of micro-organisms (filtration, see sect. 30.6) is applied for gases and liquids. high efficiency particulate air (hepa) filters are used to remove viable and non-viable particles from the air introduced in classified working areas (see sect. 27.5.1). hydrophobic membrane filters are used as vent filters on tanks and to filter production gases. membrane filtration of liquids is applied to reduce the bioburden of raw materials and of final products (see sect. 30.6). in an aseptic process, membrane filtration is the final step before filling. during the production of a terminally sterilised product membrane filtration is applied to reduce the bioburden. micro-organisms may be physically destroyed by means of dry heat (see sect. 30.5.2), moist heat (see sect. 30.5.1) and ionising radiation (see sect. 30.5.3) . dry heat sterilisation is primarily applied for glassware and some heat stable raw materials. the standard temperature is between 160 c and 180 c. ointments and some powders may be treated at lower temperatures because they are not stable enough. higher temperatures (250 c and above) are used for the depyrogenation and sterilisation of glass vials. moist heat sterilisation at 121 c for 15 min is the method of choice for terminal sterilisation of finished products. sterilisation by means of ionising radiation of pharmaceutical preparations is not allowed in a number of countries. many active substances and raw materials are decomposed by the doses required for sterilisation. some polymers become brittle and glass may become discoloured. for these reasons there is only limited application for this sterilisation method for pharmaceutical preparations. radiation sterilisation is however widely used in the medical device industry. disinfection, sanitisation, decontamination, chemical sterilisation, are only a few terms for these processes. disinfection, defined as removal, destruction or de-activation of micro-organisms on objects or surfaces, is the term of preference in this book. disinfection of surfaces (gloves, equipment, floors and walls) is done with a range of products, including isopropyl alcohol, ethanol, quaternary ammonium compounds, biguanides and amphoteric agents. if a sporicidal activity is required (as alcohols are not sporicidal), oxidising agents such as chlorine/hypochlorite, peracetic acid, or hydrogen peroxide may be used. for medical devices a number of processes are available such as ethylene oxide and low-temperature hydrogen peroxide gas plasma sterilisation. the objective of preservation is to assure the microbiological quality throughout storage and the period of use. a number of substances, including parabens, chlorhexidine, and sorbic acid are used (see sect. 23.8). sterility tests appeared in the british pharmacopoeia for the first time in 1932 and in the united states pharmacopeia in 1936 [59] . currently sterility testing is a legally binding release test for industrially manufactured sterile products. only under specific conditions, including an excellent track record and a high level gmp, national authorities may grant a product and site-specific approval for parametric release (release without performing a sterility test) (see sect. 32.8). for many products prepared in hospital pharmacies or in institutions such as blood banks, the batch size is too small (one or only a few units) or the shelf life is too short (<14 days) to perform a complete sterility test as described in pharmacopoeias. in such instances, such as with radiopharmaceuticals (see sect. 15.6.7), the pharmacist has to rely on the aseptic precautions during preparation (see sect. 31.6). the use of rapid microbiological methods (rmms) may also be an alternative to test products with such short shelf lives. the objective of the sterility test is obviously to demonstrate that a batch is sterile, i.e. does not contain any viable micro-organism. there is an on-going debate on the rationale of the sterility test. the test is a destructive one and relatively small samples are taken from the batch. the results cannot be extrapolated to items that have not been examined. for terminally sterilised products a limit (sterility assurance level, sal of 10 6 ) has been imposed for which there is no appropriate test methodology of sufficient sensitivity. the test will only detect those micro-organisms capable of growing to detectable levels under the defined incubation conditions (media, temperatures and time). the sterility test is however the last possibility to detect any gross error in the production process. this means that applying an appropriate quality system with stable and well-controlled preparation and sterilisation processes (and not just performing a sterility test on the final product) is the key to ensure that a product that purports to be sterile remains free of microbial contamination. regulatory initiatives strengthen the view that quality cannot be assured by final product testing, but should rather be assured by appropriate design of the manufacturing process (see sect. 35.3) . consequently, these initiatives emphasise process understanding and monitoring over final product testing. for the way in which the process is monitored to ensure sterility during aseptic handling, see sect. 31.6.1. after several years of negotiation the sterility tests of the european pharmacopoeia, the united states pharmacopeia and the japanese pharmacopoeia are harmonised [59] . in the next sections the five steps of this harmonised test, as described in ph. eur. 2.6.1 will be discussed: sampling, sample preparation, inoculation, incubation, and interpretation. prior to the routine application of the test, a suitability test with a range of specified test organisms shall demonstrate that growth of these organisms is not inhibited due to residual antimicrobial activity of the product. the monograph specifies the minimum number of items that shall be examined in relationship to the batch size, and the minimum volume/amount that shall be taken from each container. the samples used should be representative for the whole batch. furthermore immediately after an intervention in an aseptic filling operation (e.g. re-adjustment of a filling needle) the probability of contamination may be increased, and specific sampling for sterility testing may be justified. this has to be specified in relevant procedures. sample preparation includes all operations necessary before the actual examination can be performed, including opening of the primary packaging, withdrawal of the required amount and, if necessary, dilution or dissolution of the sample in a suitable liquid. the technique of membrane filtration is used whenever the nature of the product permits. the membrane is transferred to the growth medium, or the medium is transferred onto the membrane. alternatively, the prepared sample is inoculated directly into the appropriate media. this method is only used when the product (e.g. some vaccines) cannot be dissolved or diluted in a nontoxic diluent. the media used are fluid thioglycolate medium (ftm) for aerobic, micro-aerophilic and anaerobic bacteria, and soybean casein digest broth (scdb) for aerobic bacteria and fungi. ftm and scdb are incubated at 30-35 c and 20-25 c respectively, both for a period of not less than 14 days. this relatively long incubation period seems to be justified, because an unacceptable proportion of contaminants would be missed by limiting incubation to 7 days [60]. the media are examined at intervals and at the end of the incubation period. if no growth is visually observed the sample passes the test. normally the sample shall be rejected if growth is observed in at least one of the media. however under certain conditions the test may be invalidated and in that case be repeated. 3) and requirements regarding specific micro-organisms. limits depend on the route of administration, the nature of raw materials, the type of micro-organism, and on the fact whether an antimicrobial treatment can be given to the product (cf. the more stringent criteria for the aqueous preparations than for non-aqueous oral preparations reflect the greater potential for microbial growth of the former. e. coli must be absent from most of the preparations because this is an indicator organism for poor hygiene during production, or poor microbiological quality of the raw materials. products intended for inhalation must be free of staphylococcus aureus, because it may cause pulmonary infections. this organism also indicates poor hygiene during production. pseudomonas aeruginosa should be absent as well, because this organism may cause serious lung infections. because of the aqueous nature of these products the presence of this organism and other bile tolerant gram-negative bacteria is a serious potential risk. bile tolerant gram-negative bacteria form a complex group of micro-organisms comprising of the enterobacteriaceae and many other strains (formerly pseudomonads), including burkholderia cepacia [63] and ralstonia pickettii [64] . burkholderia cepacia is a waterborne organism and causes great problems to the pharmaceutical industry and hospitals. it may be resistant to many commonly used disinfectants and preservatives, such as chlorhexidine, and it may cause serious lung infections, particularly in compromised hosts, such as cystic fibrosis patients. salmonella must be absent from 25 g of herbal preparations. this reflects the low infectious dose of this organism and the severity of infections. test methods for the microbiological examination are described in ph. eur. sections 2.6.12 [65] and 2.6.13 [66] respectively. section 2.6.12 describes qualitative methods for the determination of the total aerobic microbial count and the total yeast and mould count. section 2.6.13 describes tests for specified organisms. the total aerobic microbial count (tamc) is defined as the number of colonies observed on casein soya bean digest agar. the total combined yeast and mould count (tymc) is defined as the number of colonies observed on sabourauddextrose agar. the assessment consists of four steps: sampling, sample preparation/testing, incubation, and interpretation. in general the sample size shall be 10 g or 10 ml. an exception is made for those active substances for which the amount per dosage unit or per 1 g or 1 ml (for preparations not presented in dose units) is less than 1 mg or 1 ml. in these cases the amount to be tested shall be not less than the amount in 10 dosage units or in 10 g or 10 ml of the product. any sample shall be representative for the whole batch. however batch sizes may range from extremely small (e.g. for some biotechnological products) to very large (e.g. a batch of tablets). the ph. eur. does not describe how the samples shall be taken and this must be put down in local procedures. if necessary the sample is dissolved and diluted in a suitable non-toxic diluent. buffered sodium chloride-peptone solution to which an emulsifier and/or a neutraliser for antimicrobial agents may be added is widely used. three methods may be used for the enumeration: membrane filtration, plate count, and most probable number (mpn) method. the advantages of the membrane filter method are its low limit of detection (lod) of < 1 cfu/g or ml and the efficient separation of the micro-organisms from components of the product, particularly antimicrobial agents. for the pour-plate method, the sample is generally 1: 10 dissolved in the diluent, and 1 ml of the dilution is mixed with the agar. this corresponds to a lod of 10 cfu/g or ml. the lod is sometimes higher (e.g. 100 cfu/g or ml) if the product needs to be further diluted due to microbial inhibition, or lower in case of products with low microbial acceptance criteria. if the spread plate count technique is used the lod is a factor of ten higher (>100 cfu/g or ml), because only 0.1 ml of the absence of bile-tolerant gramnegative bacteria (1 g or 1 ml) special ph. eur. provision for oral dosage forms containing raw materials of natural (animal, vegetal or mineral) origin for which antimicrobial pretreatment is not feasible and for which the competent authority accepts tamc of the raw material exceeding 10 3 cfu/g or cfu/ml 10 4 10 2 not more than 10 2 cfu of bile-tolerant gram-negative bacteria (1 g or 1 ml) absence of salmonella (10 g or 10 ml) absence of escherichia coli (1 g or 1 ml) absence of staphylococcus aureus (1 g or 1 ml) a colony forming unit (cfu): one or more micro-organisms that produce a visible, discrete growth entity on a semisolid, agar-based microbiological medium dilution can be spread over the surface of the agar plate. the precision and accuracy of the mpn method is less than that of the membrane filtration and the plate count methods. unreliable results are particularly obtained for moulds. for these reasons the mpn method is reserved for the enumeration of tamc in situations where no other method is available. casein soya bean digest agar is incubated for 3-5 days at 30-35 c. sabouraud dextrose agar is incubated for 5-7 days at 20-25 c. duplicate plates are prepared for each dilution and each medium. if the mpn method is used the tubes are incubated for 3-5 days at 30-35 c. at the end of the incubation period the colonies on the plates are enumerated. the number of cfu per gram or per ml of product is calculated for each medium from the arithmetic means of the plates. because of the relatively poor accuracy and precision of microbiological enumerations, according to the ph. eur. an acceptance criterion may be interpreted as follows: 10 1 cfu: maximum acceptable count is 20 10 2 cfu: maximum acceptable count is 200; etc. the group of specified micro-organisms is a limited group of organisms which may be either pathogenic, or are indicator organisms for lack of hygiene during production. this group includes a number of organisms that have been discussed in a previous section, viz. escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, and bile tolerant gramnegative bacteria. the group also includes: salmonella, clostridium, and candida albicans. tests for the detection of these organisms are described in the pharmacopoeias (e.g. ph. eur. 2.6.13 [65] ). salmonella belongs to the family of the enterobacteriaceae. over 2,000 species are known. salmonellae are differentiated by means of biochemical reactions and by serotyping. salmonellae are pathogenic bacteria causing food poisoning. the bacterium is present in many free-living and domesticated animals. pharmaceutical raw materials that have been contaminated include carmine, pancreatic powder and thyroid powder. bacteria from the genus clostridium are anaerobic, sporeforming gram-positive rods. the spores are heatresistant and can survive in foods that are incorrectly or minimally processed. the genus contains a number of dangerous pathogens, including clostridium botulinum, clostridium difficile, and clostridium tetani. candida albicans is a fungus that grows both as yeast and filamentous cells and is a causal agent of oral and genital infections in humans. systemic fungal infections including those by candida albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., patients with aids, cancer chemotherapy, and transplantations). candida albicans biofilms may form on the surface of implantable medical devices. in addition, hospital-acquired infections by candida albicans have become a cause of major health concerns. in addition to these well-known organisms it has however been demonstrated that other organisms (e.g. burkholderia cepacia, or ralstonia pickettii) can cause infection when present in pharmaceutical preparations. for this reason the concept of 'objectionable organisms' was originally introduced in the usa. objectionable micro-organisms are defined as contaminants that, depending on the microbiological species, would adversely affect product safety and product quality. so the assessment of microbial safety of a medicine has to include the examination of absence of objectionable micro-organisms as well. one approach to determine whether an organism is objectionable is to perform a risk analysis [55] , see also chap. 21. such an analysis should address at least four issues: • potential level of microbial contamination (total aerobic microbial count, total yeast and mould count) • identity and characteristics of possible micro-organisms present (pathogenicity, ability to metabolise product components, ability to survive or even grow in the conditions of the product) • product characteristics (presence of antimicrobials, water activity, route of administration, container and closure design) • potential impact on patients (for instance: is the preparation meant for immunocompromised patients or for neonates) the examination of pharmaceutical preparations for specified micro-organisms involves generally the following steps: sampling, sample preparation, resuscitation and enrichment, incubation on diagnostic or selective media, and evaluation. sampling and sample preparation is basically the same as for tamc and tymc determination and will not be further discussed. as a result of mild heat treatment, drying, or chemical antimicrobial treatment, cells may be sublethally injured. a cell is, by definition, sublethally injured if it is unable to grow on a selective medium that is typically suitable for normal healthy cells of that type. sublethally injured cells may recover when transferred to a suitable non-selective medium and thus regain all their normal characteristics, including resistance to selective antimicrobial agents and pathogenicity. this recovery process is called resuscitation. the examination of non-sterile pharmaceutical products for the presence of specified micro-organisms involves one or more steps with selective media. to increase the likelihood of detecting sublethally injured micro-organisms, the product is initially incubated in a non-selective (enrichment) medium. this non-selective culture thus may serve a twofold purpose: to first resuscitate sub-lethally injured cells and, secondarily, to facilitate their growth for purposes of further isolation and identification. for some organisms a second enrichment step in a selective medium is include in the method. table 19 .2 gives an overview of the purposes of the media used for each (group of) specified micro-organisms. if there is no growth in the selective medium, the corresponding specified micro-organism is absent and the product complies with the requirement. if there is typical growth in the diagnostic medium a confirmation of the presence of the specified micro-organism should be performed using either biochemical tests or other identification methods (e.g. 16s rdna sequencing). in the previous sections only the conventional microbiological methods, developed in the late ninetieth and first half of the twentieth century, have been discussed. all these methods are growth-based methods that have their own limitations (long incubation periods, only viable micro-organisms that also grow on the media can be recovered, variability in nutrient media quality, etc.). in the past decades many new technologies have been developed with the first aim to reduce the time to obtain results of microbiological testing. the use of such rapid microbiological methods (rmm) is beneficial in terms of reduction of throughput time for release (especially of parenterals), early identification of product contaminations, allows for causal investigations to be carried out earlier, making it easier to find and eliminate contamination causes [67] . for short shelf life products (<14 days), rapid microbiological methods are essential for assessing microbiological safety. in addition, automation by alternative methods enables to reduce hands-on time, human error and paperless data recording. before microbiological testing can be performed with an alternative method, the user must demonstrate that the alternative method is suitable for its intended purpose. this validation is based on demonstrating at least equivalent performance of the rmm compared to the traditional method and is performed according to ph. eur. 5.1.6., usp <1,223 > or pda tr-33 [68] [69] [70] . it is beyond the scope of this chapter to discuss all available technologies, just one example of each of three detection principles will be mentioned here, viz., a growth based method, a non-growth based method and a nucleic acid determination method. for an encyclopaedic overview the reader is referred to the literature [71] . bioluminescence atp is a metabolite present in all organisms (excluding viruses). the amount of atp per cell is species-dependent and also is dependent on the metabolic state of the cell. atp can be measured semi-quantitatively with the luciferin/luciferase system. this system (naturally occurring in the firefly) emits light in the presence of atp. the sample is mixed with a special reagent, which causes lysis of the cells, liberating the atp, followed by addition of the luciferin and luciferase reagents. the amount of emitted light is measured by means of a photomultiplier system and used as an indicator of the number of cells present. this principle can be applied for numerous purposes, such as the detection of micro-organisms in products or monitoring of cleaning and disinfection procedures. its use has been suggested for microbiological examination of sterile and non-sterile pharmaceutical preparations [72, 73] . laser scanning cytometry a sample is filtered over a membrane filter and treated with a fluorogenic reagent. living cells actively take up the reagent and convert it to a fluorescent substance. the filter is scanned with a laser beam and the fluorescence is measured. the system records the position of the fluorescent item, so that it can be verified microscopically that the signal was not due to an artefact. the method is able to detect a single cell within about 2 h post sample processing and testing. it is used to monitor large water systems in pharmaceutical plants [74] . 19.6.5.3 nucleic acid based identification: ribotyping ribotyping measures the pattern that is generated when dna from an organism is treated with a restriction enzyme. dna is isolated from a pure culture. the genes encoding for 16s rrna are amplified with pcr, treated with one or more restriction enzymes, separated by means of electrophoresis and finally probed. the obtained genetic fingerprint is a stable marker that provides definitive species discrimination or even characterisation below species level. microbial chemical signaling: a current perspective bacterial quorum sensing: functional features and potential applications in biotechnology quorum sensing in gram-negative bacteria: small-molecule modulation of ahl and ai-2 quorum sensing pathways biosynthesis of peptide signals in gram-positive bacteria the united states pharmacopeial usp 37/the national formulary nf 32 (2014) <1112>, application of water activity determination to non-sterile pharmaceutical products. the united states pharmacopeial convention human prion diseases prions. in: block ss (ed) disinfection, sterilization and preservation a relevant long-term impact of the circulation of a potentially contaminated vaccine on the distribution of scrapie in italy. results from a retrospective cohort study consensus statement on the bio-safety of urinary-derived gonadotrophins with respect to creuzfeldt-jakob disease detection of prion protein in urine-derived injectable fertility products by a targeted proteomic approach how is creutzfeldt-jakob disease acquired? minimising the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products. european pharmacopoeia, 8th edn inactivation of transmissible degenerative encephalopathy agents: a review acid inactivation of prions: efficient at elevated temperature or high acid concentration use of pcr-based assays for the detection of the adventitious agent porcine circovirus type 1 (pcv1) in vaccines, and for confirming the identity of cell substrates and viruses used in vaccine production root cause investigation of a viral contamination incident occurred during master cell bank (mcb) testing and characterization-a case study swine torque teno virus detection in pig commercial vaccines, enzymes for laboratory use and human drugs containing components of porcine origin human and animal vaccine contaminations circovirus and impact of temporary withdrawal of rotavirus vaccines in spain approaches to minimize infection risk in blood banking and transfusion practice effect of viral nucleic acid testing on contamination frequency of manufacturing plasma pools improvement of virus safety of an antihemophilc factor ix by virus filtration process pathogen safety of plasma-derived products -haemate p/humate-p nosocomial outbreak of epidemic keratoconjunctivitis accompanying environmental contamination with adenoviruses the survival of adenovirus in multidose bottles of topical fluorescein hepatitis c virus infections from a contaminated radiopharmaceutical used in myocardial perfusion studies hepatitis c transmission due to contamination of multidose medication vials: summary of an outbreak and a call to action transmission of hepatitis c virus during myocardial perfusion imaging in an outpatient clinic patient-to-patient transmission of hepatitis c virus through the use of multidose vials during general anesthesia nosocomial transmission of hepatitis c virus associated with the use of multidose saline vials the survival of herpes simplex virus in multidose office ophthalmic solutions nosocomial transmission of hepatitis b virus infection through multiple-dose vials the microbiology of safe food highlights of mycoplasma research-an historical perspective the scope of mycoplasma contamination within the biopharmaceutical industry the growth and long term survival of acholeplasma laidlawii in media products used in biopharmaceutical manufacturing effect of storage conditions on detection of mycoplasma in biopharmaceutical products mycoplasma testing of cell substrates and biologics: review of alternative non-microbiological techniques mycoplasmas. european pharmacopoeia, 8th edn functions of bacterial flagella bacterial flagellar diversity and significance in pathogenesis bacterial adhesion and entry into host cells pili in gram-positive pathogens bacterial pili: molecular mechanisms of pathogenesis genetic bases and medical relevance of capsular polysaccharide biosynthesis in pathogenic streptococci conjugate vaccines-a breakthrough in vaccine development leuconostoc dextransucrase and dextran: production, properties and applications spore germination monocyte-activation test. european pharmacopoeia, 8th edn microbial diversity in pharmaceutical product recalls and environments microbiological contamination of medical preparations microbiological quality of pharmaceutical preparations. guidelines and methods objectionable micro-organisms in non-sterile pharmaceutical drug products: risk assessment and origins of contamination fungal infections associated with contaminated methylprednisolone injections fda report on new england compounding pharmacy. www.fda. gov search on ucm325980 the sterility testing of pharmaceuticals the incubation period in sterility testing microbiological quality of pharmaceutical preparations. european pharmacopoeia, 8th edn microbiological quality of herbal medicinal products for oral use. european pharmacopoeia, 8th edn burkholderia cepacia: this decision is overdue a multistate nosocomial outbreak of ralstonia pickettii colonization associated with an intrinsically contaminated respiratory care solution microbiological examination of non-sterile products: microbial enumeration tests. european pharmacopoeia, 8th edn microbiological examination of non-sterile products: test for specified micro-organisms. european pharmacopoeia, 8th edn overview of rapid microbiological methods evaluated, validated and implemented for microbiological quality control alternative methods for control of microbiological quality. european pharmacopoeia, 8th edn the united states pharmacopeial usp 37/the national formulary nf 32 (2014) <1223>, validation of alternative microbiological methods. the united states pharmacopeial convention evaluation, validation and implementation of new microbiological testing methods pda. bethesda encyclopedia of rapid microbiological methods growth promoting properties of different solid nutrient media evaluated with stressed and unstressed micro-organisms: prestudy for the validation of a rapid sterility test identification of micro-organisms after milliflex rapid detection-a possibility to identify nonsterile findings in the milliflex rapid sterility test evaluation of the chemscan system for rapid microbiological analysis of pharmaceutical water key: cord-298441-77w86l8q authors: lombardi, andrea; consonni, dario; carugno, michele; bozzi, giorgio; mangioni, davide; muscatello, antonio; castelli, valeria; palomba, emanuele; cantù, anna paola; ceriotti, ferruccio; tiso, basilio; pesatori, angela cecilia; riboldi, luciano; bandera, alessandra; lunghi, giovanna; gori, andrea title: characteristics of 1,573 healthcare workers who underwent nasopharyngeal swab for sars-cov-2 in milano, lombardy, italy date: 2020-06-20 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.013 sha: doc_id: 298441 cord_uid: 77w86l8q objectives: the management of healthcare workers (hcws) exposed to confirmed cases of covid-19 is still a matter of debate. we aimed to assess in this group the attack rate of asymptomatic carriers and the symptoms most frequently associated with the infection. methods: occupational and clinical characteristics of hcws who performed a nasopharyngeal swab for the detection of sars-cov-2 in a university hospital from february 24, to march 31, 2020, were collected. for those who tested positive and for the asymptomatic positives we checked laboratory and clinical data as of may 22 to calculate the time necessary to become test-negative and to verify whether symptoms developed thereafter. frequencies of positive tests were compared according to selected variables using multivariable logistic regression models. results: positive tests were 139 among 1,573 hcws (8.8%, 95% confidence interval [ci]: 7.5-10.3), with a marked difference between symptomatic (122/503, 24.2%) and asymptomatic (17/1,070, 1.6%) workers (p<0.001). physicians were the group with the highest frequency of positive tests (61/582, 10.5%), whereas clerical workers and technicians displayed the lowest frequency (5/137, 3.6%). the likelihood of being positive increased with the number of reported symptoms and the strongest predictors were taste and smell alterations (odds ratio [or]= 76.9) and fever (or = 9.12). the median time from first positive test to a negative test was 27 days (95% ci: 24-30). conclusions: a relevant number of hcws can be infected by sars-cov-2 without displaying any symptom. among symptomatic workers, the key symptoms to guide diagnosis are taste and smell alterations and fever. in median, almost four weeks are necessary to achieve negativity of nasopharyngeal swab. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a previously unknown 48 virus which recently jumped from a not yet identified animal host to humans and it is 49 responsible of coronavirus disease 2019 . 1 the virus has now spread worldwide 50 from china, causing the first pandemic of the xxi century, disrupting health-care services in 51 the affected countries and exacting a terrific toll of human lives. 2-3 52 healthcare workers (hcws) are a crucial actor in the pandemic. indeed, they are acting in an 53 emergency situation and are continuously at risk of being infected. at the same time, they are 54 in contact with the most fragile elements of our society, those who need health assistance. it 55 is therefore mandatory to avoid that infected hcws act as spreaders of the disease. 56 unfortunately, it is still unclear which microbiologic investigations and procedures should be 57 adopted toward hcws in covid-19 settings, especially to those exposed to confirmed cases 58 of covid-19 and at risk for infection. to answer this question, we reviewed all the 59 nasopharyngeal swab performed in hcws exposed to confirmed cases of covid-19 at the 60 foundation irccs ca' granda ospedale maggiore policlinico located in milan, the capital 61 of lombardy, by large the italian region mostly affected by we assessed 62 frequency of positive tests among symptomatic and asymptomatic hcws and evaluated the 63 association between occupation, symptoms (type and number), and presence of the infection. furthermore, we also calculated the median time between the day of diagnosis (first positive test) and the day in which the hcw became test-negative. we collected occupational and clinical characteristics of all the consecutive hcws who we tested hcws at risk for infection, which is defined as a contact with a patient or another 74 hcw with (or later diagnosed with) sars-cov-2 infection. all those at risk were, according 75 to the internal protocol, identified and contacted by the hospital infection prevention unit, 76 isolated at home and tested. hcws were subdivided into physicians (including residents), 77 nurses and midwives, healthcare assistants, health technicians, and clerical workers and 78 technicians. all the information was collected by the infectious disease notification form 79 associated to each test. workers were defined as symptomatic if presented any of the 80 following in the 14 days preceding the test: fever, cough, dyspnoea, asthenia, myalgia, 81 coryza, sore throat, headache, ageusia or dysgeusia, anosmia or parosmia, ocular symptoms, men (three physicians and two nurses) and three women (two physicians and a clerical 144 worker) were hospitalized. a minority of the hcws (81/1,537, 5.3%) reported to have had a contact with an infected 146 person outside the hospital (relatives, colleague, or friends). of these, 12/81 (14/81, 8.7%) 147 were found to be positive. in this italian group of hcws exposed to confirmed cases of covid-19, the presence of 150 symptoms, and particularly taste and smell alterations and fever, was associated with interestingly, the auc of a model considering six groups of symptoms (fever, myalgia, 155 asthenia, ocular symptoms, dyspnoea, and taste and smell alterations) was 0.83. based on 156 these results, it seems reasonable to tailor the screening approach of hcws at risk based on 157 the resources available. in low-resource settings we suggest focusing to test those with 158 symptoms to maximize efficacy, especially considering the continuous exposure of hcws to 159 at risk situations, thus requiring repeated testing sessions. nevertheless, it should be 160 underlined that in our study a non-negligible number of workers were infected but displayed 161 no symptoms, meaning that a fraction of those infected can be lost with a symptoms-based 162 screening strategy. therefore, in middle-and high-resource settings a mass screening for all 163 hcws exposed to confirmed covid-19 cases appears the best approach to limit the spread when stratified according to occupation, test-positive frequencies were clearly higher among 177 subsets with direct contact with patients (physicians including residents, nurses and 178 midwives, healthcare assistants and health technicians) than those without (clerical works and 179 technicians). consequently, careful screening of these groups of workers should be 180 mandatory. no differences in terms of infection attack rate were seen between different age 181 groups nor between men and women, suggesting that risk factors for acquiring covid-19 182 among hcws are unrelated to age and sex. another relevant point is the significant number of hcws who were negative at the first test 184 but resulted positive when tested a second time. this might represent a serious concern, as a 185 discrete fraction of those can further spread the virus unnoticed, thus hampering the efficacy 186 of the screening strategy. it should be noted, however, that the second test was performed on 187 a small number of operators and not on a routine basis, making these considerations subject 188 to several potential biases. in addition, in a relevant proportion of our population we could 189 not retrieve information about the most likely date of exposure to a documented case. thus, we cannot exclude a recent contact in which case the first test may have been performed too early (i.e. still in the incubation period which has been estimated to be five 192 days), before a sufficient amount of viral particles is detectable in the nasopharynx. 8 moreover, it has to be considered that hcws employed in covid-19 units/hospitals are at 194 risk of sars-cov-2 exposure on a daily basis and therefore repeated exposures, even 195 unnoticed, can occur also after the first one who motivated the test. moreover, technical 196 limitation can be responsible of falsely negative test, considering that the sensitivity of 197 nasopharyngeal swab for sars-cov-2 detection has been estimated to be around 71%. 9 finally, we observed a median time from first positive test to a negative test of 27 days. this our study has some limitations. first, the surveillance system was quickly set-up in a few 206 days due the virus spread in our region since february 20, when the first italian case was 207 identified in the south-east part of lombardy. therefore, data quality was imperfect and 208 extensive time-consuming data editing (through review of electronical records and, when 209 necessary, of paper forms) was required to retrieve and complete the relevant information. for the same reason, and because we wanted to provide a rapid response to concerns about 211 virus spread in the hospital, we were forced to limit the analyses to only a part of the work world health organization (who), covid-19 definitions key: cord-102817-m1l1t0e1 authors: lucas, t. c.; pollington, t. m.; davis, e. l.; hollingsworth, t. d. title: responsible modelling: unit testing for infectious disease epidemiology date: 2020-08-16 journal: nan doi: 10.1101/2020.08.14.20175216 sha: doc_id: 102817 cord_uid: m1l1t0e1 infectious disease epidemiology is increasingly reliant on large-scale computation and inference. models have guided health policy for epidemics including covid19 and ebola and endemic diseases such as malaria and tuberculosis. yet a single coding bug may bias results, leading to incorrect conclusions and wrong actions that could cause avoidable harm. we are ethically obliged to ensure our code is as free of error as possible. unit testing is a coding method to avoid such bugs, but unit testing is rarely used in epidemiology. we demonstrate through simple examples how unit testing can handle the particular quirks of infectious disease models. modelling is an important tool for understanding fundamental biological processes in infectious disease dynamics, evaluating potential intervention efficacy and forecasting disease burden. at the time of writing, infectious disease modellers are playing a central role in the interpretation of available data on the covid-19 pandemic to inform policy design and evaluation [1] [2] [3] . similarly, policy on endemic infectious diseases, such as duration and frequency of control programmes and spatial prioritisation, is also directed by models [4] . such research builds on a long history of modelling for policy [5] and a general understanding of the dynamics of infectious disease systems. given the importance of modelling results, it is vital that the code they rely on is both coded correctly and trusted. bugs can be caused by typos, code behaving in unexpected ways, or logical flaws in the construction of the code. outside of epidemiology, bugs have been found in code that had been used by many researchers [6] and may lead to retractions [7] . bugs have also been found in highly influential work; a paper that informed austerity policies globally was found to have a fatal computational mistake [8] . in engineering bugs caused the mars climate orbiter and the mariner 1 spacecraft to become lost or destroyed [9, 10] . we do not know of high profile cases of infectious disease models being found to have bugs once published, but as code is not always shared and little post-publication testing of code occurs, this likely represents a failure of detection. the issue of trust was highlighted recently when neil ferguson, one of the leading modellers informing uk covid-19 government policy, tweeted: "i'm conscious that lots of people would like to see and run the pandemic simulation code we are using to model control measures against covid-19. to explain the background -i wrote the code (thousands of lines of undocumented c) 13+ years ago to model flu pandemics. . . " [11] . the code that was released did not include any tests [12] but subsequent work has added documentation, while independent code reviews have supported the results of the study [13, 14] . the tweet and lack of tests garnered considerable backlash (some of which may have been politically motivated [15] ), with observers from the software industry noting that code should be both documented and tested to ensure its correctness [14] . it is understandable that during the fastmoving, early stages of a pandemic, other priorities were put above testing and documenting the code. however, this simply reinforces that time should be taken to test and document code when it is first written so that it is already well tested for when it is needed under short deadlines. it is also important to note that a lack of tests is not unusual in our field, or for some of the authors of this article. to guard against error, policy-makers now standardly request analyses from multiple modelling groups (as is the case in the uk for covid -19 [16] ) as a means to provide scientific robustness and reliability [17] , yet this is not enough if the models themselves lack internal validity. infectious disease modellers are rarely trained as professional programmers [14] and recently some observers have made the case that this has been due to a lack of funding [18] . it is also notable that there are few texts available which demonstrate the use of unit testing to check infectious disease models. infectious disease modellers have sought to address these issues (as in the case above) by having multiple, independently developed models to inform policy, to address structural and parameter uncertainty [17, 19] . whilst there many drivers and attempts to address this problem with code robusteness, today's models are increasingly moving from mean-field ordinary differential equation approximations to individual-based models with complex, data-driven contact processes [20, 21] . these increases in model complexity are accompanied with growing codebases. as the mathematical methods depend increasingly on numerical solutions rather than analytical pen-and-paper methods, it becomes more difficult to tell if a bug is present based on model outputs alone. traditionally, the workflow is to write the complete code, run the model and examine plots of the output. this crude ad hoc testing approach can miss many bugs. furthermore, this workflow is biased as models that show expected behaviour are assumed bug-free, whereas the opposite gets more scrutiny. unit testing is a formally-defined, principled framework that compares comprehensive output scenarios from code to what the programmer expected to happen ( [22] chapter 7, [23] , [24] ). ready-to-run frameworks for unit testing are available in r [25] , julia [26] and python [27] and are standard practice in the software industry. these testing concepts also apply to many other scientific fields but here we focus on infectious diseases. infectious disease modelling presents specific challenges, such as stochastic outputs, which are difficult to test and not covered in general unit testing literature. in this primer we introduce unit testing with a demonstration of an infectious disease model. we also outline the available testing frameworks in various languages commonly used by modellers. at the heart of every unit test is a function output, its known or expected value and a process to compare the two. for the square root function ( √ x or sqrt(x) in r), we could write a test that runs the function for the number 4, i.e. sqrt(x = 4), and compares it to the correct answer i.e. 2. however, often our function arguments will cover an infinite range of possibilities and we cannot exhaustively check them all. instead we devise tests that cover standard usage as well as corner case scenarios: what do we want our function to do if given a negative number e.g. sqrt(-1), or a vector argument containing strings or missing values e.g. sqrt(c(4,"melon",na))? in r, the testthat package [28] , provides a simple interface for testing. while a variety of test functions can make different comparisons, the two main ones are expect_true() and expect_equal(). expect_true() takes one argument: an expression that should evaluate to true. for our square root example 3 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 16, 2020. . above, we would write expect_true(sqrt(4) == 2). expect_equal() takes two arguments, an expression and the expected output; so we would write expect_equal(sqrt(4), 2). there are a number of ways to incorporate unit testing into your programming workflow. 1. each time you write code for a new, discrete chunk of functionality, you should write tests that confirm it does what you expect. these tests should be kept in the same directory as the code it is testing. 2. whenever a bug is found in the code outside of the existing testing framework, a new test should be written to capture it. then if the bug re-emerges it will hopefully be quickly flagged so that the developor can fix it. 3. all of these tests should be regularly run as you develop new code. if a change causes the older tests to break, this points to the introduction of an error in the new code, or implies that the older code could not generalise to the adapted environment. here we define a toy epidemiological model and then demonstrate how to effectively write unit tests for it in r code. consider a multi-pathogen system, with a population of n infected individuals who each get infected by a new pathogen at every time step (fig. 1 ). in this toy example, we imagine that individuals are infected with exactly one pathogen at a time. some aspects of this model could reflect the dynamics of a population where specific antibiotics are used regularly i.e. each time step an individual is infected, diagnosed and treated suboptimally, leaving the individual susceptible to infection from any pathogen, including the one they were just treated for. the aim of this model however is not to be realistic but serve as a learning tool with succinct code. each individual i, at time t, is defined by the pathogen they are currently infected with i it ∈ {a, b, c} for a 3-pathogen system. the population is therefore defined by a length n state vector i t = (i it ) i= [1,n ] . at each time step, every 4 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint individual's infection status is updated as: that is, at each iteration, the new infection status of each individual is a uniform random sample from the set of infection statuses in the previous iteration (including itself i i,t−1 ). certainly this toy model is naïve as it is governed by mass-action principles, ignoring contact and spatial dynamics. nevertheless it will serve its purpose. code 1 shows our first attempt at implementing this model. usually we would make some output plots to explore if our code is performing sensibly. plotting the time course of which pathogen is infecting one individual shows repeated infection with different pathogens as expected (fig. 2) . however, if we look at the proportion of each pathogen through time (not shown here) we quickly see that the pathogen proportions are identical through time and so 5 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . there must be a bug that we had not originally noticed. this simple example demonstrates a number of points. firstly, bugs can be subtle. secondly, it is not easy to notice an error, even in just 7 lines of code. thirdly, it is much easier to debug code when you know there is a bug. fourthly, while plotting simulation runs is an excellent way to check model behaviour, if we had only relied on fig. 2 we would have missed the bug. additionally, manually checking plots is a time consuming and non-scalable method because a human has to perform this scan every test run. in summary this ad hoc plotting approach reduces the chances that we will catch all bugs. the cause of the bug is that sample() defaults to sampling without replacement sample(..., replace = false); this means everyone transmits their infection pathogen on a one-to-one basis rather than one-to-many as required by the model. setting replace = true fixes this (code 2) and when we plot the proportion of each pathogen ( fig. 3 ) we see the correct behaviour (a single pathogen drifting to dominance). in the subsequent sections we will develop this base example as we consider different concepts in unit testing, resulting in well-tested code by the end. write small functions to ensure the unit tests are evaluating the exact code as run in the analysis, code should be structured in functions, which can be used to both run unit tests with and to generate results as part of a larger model codebase. make your functions compact with a single clearly-defined task. we have defined a function, initialisepop(), to initialise the population and another, updatepop(), to run one iteration of the simulation (code 1). organising the codebase into these bite-sized operations makes following the programming flow easier as well as understanding the structure of the code. at this stage we have also enabled the varying of the number of pathogens 6 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint if we start with a small population with few pathogens, we can then easily work out exactly what the initialised population should look like (code 2). when we initialise a population with three individuals and three pathogens, we will get the sequence "a", "b", "c" as seen in the first test. when the number of individuals is greater than the number of pathogens, the sequence will be repeated as seen in the second test. finally, when the number of individuals is greater than the number of pathogens, but is not a multiple of the number of pathogens, the sequence will have an incomplete repeat at the end as seen in the third test. in this sequence of tests, we have taken our logical understanding of what the function should do, and used it to make predictions of what the results should be. we then test that the result is the same as what we expect. 7 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint code 2: using simple parameter sets we can work out beforehand what results to expect pop1 <-initialisepop(t = 2, n = 3, pathogens = 3) expect_equal(pop1[1,], c("a", "b", "c")) pop2 <-initialisepop(t = 2, n = 6, pathogens = 3) expect_equal(pop2[1,], c("a", "b", "c", "a", "b", "c")) pop3 <-initialisepop(t = 2, n = 5, pathogens = 3) expect_equal(pop3[1,], c("a", "b", "c", "a", "b")) initialisepop() has three arguments to check. first we initialise the population, and then alter each argument in turn (code 3). arguments t and n directly change the expected dimension of the returned matrix so we check that the output of the function is the expected size. for the pathogens argument we test that the number of pathogens is equal to the number requested. we can also cover cases that expose deviations from the logical structure of the system. after initialising our population, we expect all the rows other than the first to contain na. we also expect each of the pathogens a, b and c to occur at least once on the first row if pathogens = 3 and n ≥ 3. finally, updatepop() performs a single simulation time step, so we expect only one additional row to be populated. instead of testing by their numerical values, we verify logical statements of the results within our macro understanding of the model system (code 4). code 4: test more complex cases using your understanding of the system 8 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint .na(pop1[-1,]) )) # expect all 3 pathogens at t = 1 expect_true(all(c("a", "b", "c") %in% pop1[1,])) pop2 <-updatepop(pop1, t = 2, n = 12) # after update expect 1st & 2nd row not to have nas expect_true(all (!is.na(pop2[1:2,]) )) # and also expect that rows other than the 1st & 2nd are still nas. expect_true(all(is. na(pop2[-c(1:2) ,]))) in the end an entire model only runs when its functions work together seamlessly. so we next check their connections; achieved through nesting functions together, or defining them at a higher level and checking the macro aspects of the model. we define a function fullsim() that runs both initialisepop() and updatepop() to yield one complete simulation. we would expect there to be no nas in the output from fullsim() and every element to be either a, b or c. # expect no nas expect_true(!any(is.na(pop))) # expect all 3 pathogens contained within expect_true(all(pop %in% c("a", "b", "c"))) stochastic simulations are a common feature in infectious disease models. stochastic events are difficult to test effectively because, by definition, we do not know beforehand what the result will be. we can check very broad-scale properties, like code 5, where we check the range of pathogen values. however, code could still pass and be wrong (for example the base example (code 1) would still pass that test). there are however a number of approaches that can help. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint isolate the stochastic parts of your code. for example, updatepop() performs stochastic and deterministic operations in one line (code 1). firstly, updatepop() stochastically samples who gets infected by whom at iteration t. then it takes those infection events and assigns the new infectious status for each individual. we demonstrate in code 1 how this could be split. we accept this is a fairly exaggerated example and splitting a single line of code into two functions is rare! the more common scenario is splitting a multi-line function into smaller functions which also brings benefits of code organisation so it does not feel like extra effort. x <-updateinfectionstatus(x, t, infector_pathogen) return(x) } now, half of updatepop() is deterministic so can be checked as previously discussed. we still have chooseinfector() that is irreducibly stochastic. we now examine some techniques for directly testing the stochastic parts of a model. in the same way that we used simple parameters values in code 2, we can often find simple cases for which our stochastic functions become deterministic. for example, samples from x ∼ bernoulli(p) will always be zeroes for p = 0 or ones for p = 1. in the case of a single pathogen (code 2), the model is no longer stochastic. so initialisation with one pathogen means the second time step should equal the first. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint vector with the indices of the infector for each infectee. paste0() then turns this length-2 vector into a length-1 string with two characters; we expect this to be one of "11", "22", "12" or "21". replicate() runs the expression 300 times, but in your unit test you should choose a value high enough so that you are confident that all of the distinct outcomes will have occurred at least once. # collapse each draw into a single string to make comparisons easier. manypops <-replicate(300, paste0(chooseinfector(n = 2), collapse = "")) expect_true(all(manypops %in% c("11", "22", "12", "21"))) while the previous example worked well for a small set of possible outputs, testing can conversely be made easier by using very large numbers. this typically involves large sample sizes or numbers of stochastic runs. for example, the clearest test to distinguish between our original, buggy code (code 1) and our correct code (code 2) is that in the correct code there is the possibility for an individual to infect more than one individual in a single time step. in any given run this is never guaranteed, but the larger the population size the more likely it is to occur. with a population of one thousand, the probability that no individual infects two others is vanishingly rare (code 4). as this test is now stochastic we should set the seed of the random number generator so that the test is reproducible. setting the seed with set.seed means that each time the code is run, the same pseudo-random numbers will be generated. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint set.seed(10261985) n <-1e3 infector_pathogen <-chooseinfector(n) if we have an event that we know should never happen, we can use a large number of simulations to provide stronger evidence that it does not stochastically occur. however, it can be difficult to determine how many times is reasonable to run a simulation, especially if time is short. this strategy works best when we have a specific bug that occurs relatively frequently (perhaps once every ten simulations or so). if the bug occurs every ten simulations and we have not fixed it we would be confident that it will occur at least once if we run the simulation 500 or 1000 times. conversely, if the bug does not occur even once in 500 or 1000 simulations we can be fairly sure we have fixed it. similarly, a bug might cause an event that should be rare to happen very regularly or even every time the code is run. in our original buggy code (code 1) we found that the proportions remained identical for entire simulations. we would expect this to happen only very rarely. we can run a large number of short simulations to check that this specific bug is not still occurring by confirming that the proportion of each pathogen is not always the same between the first and last time point. as long as we find at least one simulation where the proportions of each pathogen are different between the first and last iteration, we know the bug has been fixed. sapply(manysims, diffproportions) )) 12 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint as well as testing that functions work when given the correct inputs, we must also test that they behave sensibly when given wrong ones. this typically involves the user inputting argument values that do not make sense. this may be, for example, because the inputted argument values are the wrong class, in the wrong numeric range or have missing data values. therefore it is useful to test that functions fail gracefully if they are given incorrect inputs. this is especially true for external, exported functions, available to a user on a package's front-end. however, it is not always obvious what constitutes an 'incorrect value' even to the person who wrote the code. in some cases, inputting incorrect argument values may cause the function to fail quickly. in other cases code may run silently giving false results or take a long time to give an error. both of these cases can be serious or annoying and difficult to debug afterwards. often for these cases, the expected behaviour of the function should be to give an error. there is no correct output for an epidemiological model with -1 pathogens. instead the function should give an informative error message. often the simplest solution is to include argument checks at the beginning of functions. we then have to write slightly unintuitive tests for an expression where the expected behaviour is an error. if the expression does not throw an error the test should throw an error (as this is not the expected behaviour). conversely, if the expression does throw an error the test should pass and not throw an error. we can use the expect_error() function for this task. this function takes an expression as its first argument and reports an error if the given expression does not throw an error as expected. we can first check that the code sensibly handles the user inputting a string instead of an integer for the number of pathogens. because this expression throws an error, expect_error() does not throw an error and the test passes. now we contrast what happens if the user inputs a vector of pathogens to the initialisepop() function. here we are imagining that the users intent wass to run a simulation with three pathogens: 1, 2 and 3. this test fails because the function does not throw an error. instead the code takes the first element of pathogens and ignores the rest. therefore, a 13 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . population is created with one pathogen, not three, which is almost certainly not what the user wanted. here, the safest fix is to add an explicit argument check at the top of the function as implemented below. the same test now passes because initialisepop() throws an error when a vector is supplied to the pathogens argument. we can similarly check how the code handles a user inputting a vector of numbers to the t argument (perhaps thinking it needed a vector of all time points to run). in code 3, initialisepop() does not throw an error if a vector is supplied to t. however, fullsim() does throw an error if a vector is supplied to t. while it is a good thing that fullsim() throws an error, the error message is not very informative. if the code that runs before the error is thrown (in this case the initialisepop() function) takes a long time, it can also be time consuming to work out what threw the error. it is also a signature of fragile code that the error is coincidental; a small change in the code might stop the error from occurring. to remedy this we can add an additional argument check to initialisepop(). importantly, we then want to check that fullsim() errors in the correct place (i.e. in initialisepop() rather than afterwards). we can achieve this using the regexp argument of expect_error() that compares the actual error message to the expected error messages. the test will only pass if the error message contains the string provided. similarly, special cases can be triggered with parameter sets that do not match the extrema of parameter space. this is where understanding of the functional form of the model can help. consider a function divide(x, y) that divides x by y. we could test this function by noting that y * divide(x, y) should return x. if we write code that tests standard values of x and y such as 2 * divide(3, 2) == 3 we would believe the function works for nearly all values of division, unless we ever try y = 0. we checked earlier if the pathogens argument of initialisepop() worked by verifying that the returned population had the correct number of pathogens. however, if we set the pathogens argument to be greater than the number of individuals in the population we get a population with n pathogens. the function does not therefore pass the test we defined in code 3. for edge cases like this it may be rather subjective what the correct behaviour should be. it might be appropriate for the function to throw an error or give a warning if the user requests more pathogens than individuals. here however, we will decide that this behaviour is acceptable. the test above was still useful to highlight this unusual case. as our expected output from the function has changed, we should change our test; we now expect a population with n pathogens. we should however retain the test in code 3 so that we have two tests: one test checks that when pathogens < n, the number of unique 15 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . pathogens in the population is equal to pathogens; the other test checks that when pathogens > n, the number of unique pathogens is equal to n. most programming languages have established testing packages. for r, there is the testthat package as already discussed. when structuring r code as a package, tests should be kept in the directory tests/testthat; further requirements to the structure can be found in [22] chapter 7. all the tests in a package can then be run with test() from the devtools package [29] or check() for additional checks relevant to the package build. if the code is to be part of a package then these tools are essential to run the code within the context of a build environment. these tools also provide a clean environment to highlight if a test was previously relying on objects defined outside of the test script. other programming languages have similar testing frameworks. their specifics differ but the main concept of comparing a function evaluation to the expected output remains the same. in julia there is the test package [30] . the basic structure for tests with this package is demonstrated below. we name the test and write a single expression that evaluates to true or false. for a julia package, unit tests reside in test/runtests.jl and tests are run with pkg.test(). code 1: julia test example @testset "my_test_name" begin @test sqrt(4) == 2 end finally, in python we have the unittest framework [31]; tests must be written into a class that inherits from the testcase class. the tests must be written as methods with self as the first argument. an example test script is shown below. tests should be kept in a directory called lib/test, and the filename of every file with tests should begin with "test_". code 2: python test example 16 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 16, 2020. . https://doi.org/10.1101/2020.08.14.20175216 doi: medrxiv preprint import unittest import math class testmaths(unittest.testcase): def test_sqrt(self): self.assertequal(math.sqrt(4), 2) unittest.main() if your code is under version control [23, 24] and hosted on github, gitlab or bitbucket, you can automate the running of unit tests-also known as continuous integration. in this setup, whenever you push code changes from your local computer to the online repository, any tests that you have defined get run automatically. furthermore these tests can be automated irrespective of changes to your code or tests: since dependencies with other packages and knock-on changes from them into a bug in your code can be automatically found and notified to you by email. there are various continuous integration services such as travis-ci.org, github actions and gitlab pipelines. these services are often free on a limited basis, or free if your code is open source. we briefly describe the setup of the simplest case usign travis ci. setting testing up is very straightforward for r code already organised into a package and hosted openly on github. within your version-controlled folder that contains the r code, you add a one-liner file named ".travis.yml" that contains a description of which language the code uses. code 1: a basic travis yml file. this file can also be created with use_travis() from the usethis package. you then sign up to travis-ci.org and point it to the correct github repository. to authenticate and trigger the first automatic test you need to make a minor change to your code, commit and push to github. more details can be found in [22] chapter 14.3. it is vital that infectious disease models are coded to minimise bugs. unit testing is a well-defined, principled way we can do this. there are many frameworks that make aspects of unit testing automatic and more informative and these should be used where possible. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 16, 2020. . the basic principles of unit testing are simple but writing good tests is a skill that takes time, practice and thought. however, ensuring your code is robust and well-tested saves time and effort in the long run and helps prevent spurious results. these qualities are important for the reproducibility of science, but are particularly relevant where code may be shared between individuals or groups, or where you wish to publish your code as a package to be used by others. our aim in this paper was to demonstrate tailored results for infectious disease modelling. there are number of standard programming approaches to unit testing which would be good followup reading ( [22] chapter 7, [23] , [24] ). as demonstrated here, it is initially time consuming to program in this way, but over time it will become habitual and both you and the policy-makers who use your models will benefit from it. please see the fully-reproducible and version-controlled code at www.github. com/timcdlucas/unit_test_for_infectious_disease. tmp, tdh, tcdl and eld gratefully acknowledge funding of the ntd modelling consortium by the bill & melinda gates foundation (bmgf) (grant number opp1184344). views, opinions, assumptions or any other information set out in this article should not be attributed to bmgf or any person connected with them. tmp's phd is supported by the engineering & physical sciences research council, medical research council and university of warwick (grant number ep/l015374/1). tmp thanks the big data institute for hosting him during this work. all funders had no role in the study design, collection, analysis, interpretation of data, writing of the report, or decision to submit the manuscript for publication. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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consistently stifle economic growth? a critique of reinhart and rogoff. cambridge journal of economics mars climate orbiter mishap investigation board phase i report november 10 tweet from @neil_ferguson mrc centre for global infectious disease analysis impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand the chartered institute for it. professionalising software development in scientific research critiqued coronavirus simulation gets thumbs up from codechecking efforts guidelines for multi-model comparisons of the impact of infectious disease interventions pandemic response shines spotlight on coding in science learning from multi-model comparisons: collaboration leads to insights, but limitations remain lessons from a decade of individual-based models for infectious disease transmission: a systematic review strategies for mitigating an influenza pandemic r packages: organize, test, document, and share your code ten simple rules for effective computational research best practices for scientific computing r v3.6.3: a language and environment for statistical computing austria: r foundation for statistical computing julia: a fresh approach to numerical computing python core team. python v3.8.2: a dynamic, open source programming language 2: get started with testing key: cord-270935-t9pym9k0 authors: dumyati, ghinwa; gaur, swati; nace, david a.; jump, robin l.p. title: does universal testing for covid-19 work for everyone? date: 2020-08-15 journal: j am med dir assoc doi: 10.1016/j.jamda.2020.08.013 sha: doc_id: 270935 cord_uid: t9pym9k0 abstract the covid-19 pandemic has been especially devastating among nursing home residents, with both the health circumstances of individual residents as well as communal living settings contributing to increased morbidity and mortality. preventing the spread of covid-19 infection requires a multipronged approach that includes early identification of infected residents and healthcare personnel, compliance with infection prevention and control measures, cohorting infected residents, and furlough of infected staff. strategies to address covid-19 infections among nursing home residents vary based on the availability for sars-cov-2 tests, the incorporation of tests into broader surveillance efforts, and using results to help mitigate the spread of covid-19 by identifying asymptomatic and presymptomatic infections. we review the tests available to diagnose covid-19 infections, the implications of universal testing for nursing home staff and residents, interpretation of test results, issues around repeat testing, and incorporation of test results as part of a long-term response to the covid-19 pandemic. we propose a structured approach for facility-wide testing of resident and staff and provide alternatives if testing capacity is limited, emphasizing contact tracing. nursing homes with strong screening protocols for residents and staff, that engage in contact tracing for new cases, and that continue to remain vigilant about infection prevent and control practices, may better serve their residents and staff by thoughtful use of symptomand risk-based testing strategies. dr. jump reports support for this work in part through the cleveland geriatric research 50 while there is general agreement that increased access to testing is important for personal and 23 public health, the selection and use of diagnostic tests to mitigate covid-19 infections in post-24 acute and long-term care settings is complex and should be tailored to individual sites. he responded to antibiotics and his fever resolved within 12 hours. because he met the nursing 36 home's enhanced screening criteria for covid-19 (table 1) , 1 he was placed on transmission-37 based precautions and a laboratory test for sars-cov-2 was ordered. 38 39 the food and drug administration (fda) has authorized several diagnostic and serologic 41 (blood-based) tests as part of the emergency use authorization (eua) process ( table 2) . reported that rt-pcr was 70% sensitive when compared to a computed tomography (ct) scan 54 of the chest. 4 this study was conducted in the early stages of the pandemic on 1014 patients. 55 since then, the number of rt-pcr-based tests and availability of those tests, as well as the 56 prevalence and clinical recognition of signs and symptoms of covid-19 infection, has changed 57 dramatically. all of these factors influence the sensitivity and specificity of diagnostic rt-pcr 58 tests. even if the sensitivity of rt-pcr based testing has increased to an optimistic 90% to 95%, 59 it still means that 1 out of every 10 to 20 negative results will be a false negative and miss a 60 covid-19 infection. a lower level of sensitivity may not be an issue in settings or regions 61 where the disease prevalence is low. where the disease is prevalent however, such as during a 62 facility outbreak, negative results must be viewed with caution. the sensitivity of the test also depends on when testing is done during the course of illness, with 65 the peak of viral shedding occurring around the time of symptom onset. 5 a bayesian hierarchical 66 model, fit to previously published studies describing the performance of sars-cov-2 rt-pcr 67 tests, reported the probability of a false negative rt-pcr test over time. 6 from exposure (day 0) 68 to the typical time of symptom manifestation (day 5), the probability of a false negative results 69 changes from 100% on day 1, to 67% on day 4, reaching a nadir of 20% three days after 70 symptom onset on day 8. in some individuals, rt-pcr tests will remain positive for weeks after 71 symptom resolution. 10 staff, particularly in areas with mandates for serial testing of healthcare workers. this also offers 97 the possibility that staff may effectively collect their own samples. self-collected samples have 98 the advantage of decreasing staff exposure and their use of ppe. 10 99 100 antigen tests on may 9 th , 2020, the fda provided the first eua for a test designed to detect 101 an antigen specific to sars-cov-2; a second antigen-based test received an eua on july 2 nd , 102 2020. 11,12 antigen tests target markers on the outer surface of the viral capsule and do not 103 require extraction of rna. viral structures that represent good candidates for antigen-based 104 testing include nucleocapsid phosphoproteins and spike glycopeptide proteins. 13 advantages to 105 antigen-based tests are the relatively lower cost compared to rt-pcr tests, the ease of collection 106 samples from the anterior nares, the potential to scale the number of tests, and the rapid turn-107 around-time, providing answers within minutes. 12 a disadvantage to antigen-based tests, 108 however, is lower sensitivity, which can lead to false negative results. this is a particular issue 109 in the setting of outbreaks or in regions with increased prevalence rates of sars-cov-2. the 110 j o u r n a l p r e -p r o o f fda recommends that a second pcr-based test should be used to confirm negative results for 111 individuals likely to have covid19. 11 given that only 2 of the 164 diagnostic tests given euas 112 are antigen-based, 12 the role for these tests is still evolving and may be best suited for diagnosing 113 symptomatic individuals seeking medical care, rather than screening asymptomatic individuals 114 or healthcare workers. . regardless, cms recently announced a plan to deploy the two available 115 antigen test kits to all u.s. nursing homes to assist in universal screening efforts, 14 though noting 116 caution in interpreting negative results. 15 facilities using antigen tests need to make sure they 117 have obtained the required federal and/or state clinical laboratory improvement amendments 118 (clia) waivers, competency trained staff, a recording system to document and track results, and 119 are able to report results to state and/or federal agencies as required. 15 unfortunately, many nursing homes may not be able to implement universal testing for a 169 multitude of reasons, including lack of access to testing, inadequate supplies of ppe, limited 170 personnel, and insufficient resources to pay for testing. 27-30 as they attempt to respond to 171 federal 31 and state 32 recommendations and requirements, many nursing homes will need to 172 consider other approaches to testing such as unit-based testing and/or risk-based testing. 173 regardless of how they proceed, it is essential that nursing homes work with their laboratory 174 provider to prioritize residents and staff tests and ensure rapid turnaround times (48 hours or 175 less). 33 176 177 unit-based strategies, while not desirable, would be most suitable for situations in which testing 178 capacity is limited. for this approach, the building focuses testing on residents and staff members 179 who had close contact with the index case. testing can also be extended to include high risk 180 individuals such as those leaving the facility frequently (e.g., for hemodialysis, radiation therapy, 181 or off-site chemotherapy) and those recently admitted. determination of whom to test first is 182 inherently complex and may be best approached by a multi-disciplinary team that includes the 183 infection preventionist, medical director, director of nursing, and administrator. the need and frequency of staff and resident testing in that particular facility. as mentioned 277 above, however, insufficient access to rapid diagnostic testing, ppe, staff, and resources may 278 render seemingly insurmountable challenges. even under these dire circumstances, nursing 279 homes will continue to strive to provide the best care they can offer for residents entrusted to 280 their care ( table 3) . the prospect of reopening nursing homes, which includes permitting visitors, presents additional 297 complexities. efforts by states to relax stay-at-home orders, reopen businesses, and even 298 encourage tourism 38 contribute to increased spread of sars-cov-2 in the community which in 299 turn means increased risk for nursing home staff to acquire and transmit the virus to their 300 residents. as part of plans to reopen nursing homes, cms recommended "baseline" testing for 301 all nursing residents and staff, along with the capacity to continue weekly tests for staff. 31 after 302 the initial response to the cms recommendation, most, if not all, nursing homes should continue 303 surveillance testing. older adults often manifest atypical signs and symptoms of infection. while 304 it may be tempting to attribute a change in condition to exacerbation of known heart failure or 305 sinus congestion to allergies or a "summer cold", the severity of the illness experienced by some if testing many residents, consider using two teams with staff not assigned to frontline care -one to prepare each resident and room, and one to obtain the specimen. identify the ordering provider for the laboratory requisition, which can be the medical director. ensure that ppe is available for the staff obtaining np swab. ideally, ppe worn during sample collection includes a respirator, eye protection, a gown, and gloves. in settings without respirators, a surgical mask and face shield may be used instead. testing of staff identify the list of staff that will need to be tested. decide on the source of the sars-cov-2 specimen (np, nasal, oropharyngeal, oral). if using pcr, identify and train staff to perform testing including need to change ppe between staff members. chose a room for testing and ensure that staff are able to social distance while waiting for testing. if testing is done in the facility, identify the ordering provider, which might be the medical director or an employee health provider. if testing is done outside the facility, evaluate the process of obtaining the results. identify and communicate with the laboratory to ensure that the testing supplies are available and the laboratory can run the some states do not need a provider order for the staff testing but this is requested by the laboratory. some insurance providers are not covering testing of asymptomatic nursing home staff. when possible, sample collection should be in a well-ventilated area, which includes outdoors and, weather permitting, may be feasible to accomplish with staff members. j o u r n a l p r e -p r o o f 5 large number of tests and provide the results within 24-48 hours. dealing with sars-cov-2 testing results from residents create a process to communicate the test results with residents and their families or responsible party. identify how information is shared with the local or state health department. ensure that a cohorting plan is in place before testing. assess the availability of personal protective equipment (ppe). ensure that shared equipment is available to dedicate to the covid-19 cohort develop a policy for when covid-19 positive residents should be transferred to an outside covid-19 facility, another nursing home or hospital. one option is to communicate with resident's family that they will only be informed of positive test results. plan for use of multiple means of communication to residents, family members, and/or responsible parties. this may include use of recorded phone systems, email, website updates, town-hall updates, and direct phone communication. communication about resident results should ensure individual privacy. prepare for the additional works of moving residents and their belongings to a covid-19 cohorting area. dealing with sars-cov-2 results from staff create a process to communicate the test results with staff, respecting employee privacy. create a process to communicate with residents and their families or responsible party that the facility has staff that tested positive. identify a process to communicate results with the local or state health department, including plans for monitoring of isolation, return to work and contact tracing for quarantine. for staff that test positive and work at multiple facilities, inform the health department so the information can be communicated to other facilities. assign a staff member the role of monitoring staff furloughs and review of criteria to return to work. they also should monitor adverse effects such as hospitalization and death establish a process to deal with staff shortages: 1) train non-medical staff at your facility to assist with resident related tasks, 2) establish relationship with nursing agencies, 3) collaborate with hospital systems and evaluate if they can assist with staffing needs, 4) evaluate other staffing options such as reaching out to nursing schools. unprecedented solutions for extraordinary times: 343 helping long-term care settings deal with the covid-19 pandemic the food and drug administration comparison of four molecular in vitro diagnostic 350 assays for the detection of sars-cov-2 in nasopharyngeal specimens correlation of chest ct and rt-pcr testing in coronavirus disease 353 2019 (covid-19) in china: a report of 1014 cases interpreting diagnostic tests for sars-cov-2 variation in false-negative rate of 358 reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since 359 annals of internal medicine duration of isolation and precautions for adults with covid-19. centers for disease 362 control and prevention detection of sars-cov-2 in different types of clinical 365 specimens a combined 367 oropharyngeal/nares swab is a suitable alternative to nasopharyngeal swabs for the 368 detection of sars-cov-2 swabs collected by patients or health care workers for 370 sars-cov-2 testing the food and drug administration. coronavirus (covid-19) update: fda authorizes first 373 antigen test to help in the rapid detection of the virus that causes covid-19 in patients commissioner o of the. coronavirus (covid-19) update: daily roundup molecular targets for the testing of covid-19 trump administration announces initiative for more and faster 385 more-faster-covid-19-testing-nursing-homes.html 386 15. centers for medicare and medicaid services. frequently asked questions: covid-19 testing 387 at skilled nursing facilities/ nursing homes american health care association. point-of-care antigen test devices -ahca information for laboratories about coronavirus (covid-19). centers for disease 396 control and prevention the food and drug administration duration of antibody responses after severe acute 402 respiratory syndrome persistence of antibodies against middle east respiratory 405 syndrome coronavirus performing facility-wide sars-cov-2 testing in nursing homes. centers for disease 408 control and prevention covid-19): interim clinical guidance for management of 411 patients with confirmed coronavirus disease (covid-19). centers for disease control and 412 prevention the incubation period of coronavirus disease from publicly reported confirmed cases: estimation and application asymptomatic and presymptomatic sars-cov-2 419 infections in residents of a long-term care skilled nursing facility -king county evidence supporting transmission of severe acute 425 respiratory syndrome coronavirus 2 while presymptomatic or asymptomatic nursing homes continue to face critical supply and staff shortages 429 as covid-19 toll has mounted nursing home care in crisis in the wake of covid-19 centers for medicare & medicaid services. toolkit on state actions to mitigate covid-19 434 prevalence in nursing homes new state by state breakdown: covid-19 testing for nursing homes and assisted living 441 american health care association/national center for assisted living 442 the centers for medicare & medicaid services. nursing home reopening 448 recommendations for state and local officials | cms 30 continuing temporary suspension and modification of laws relating to the 453 disaster emergency guidance for infection control and prevention of coronavirus disease 2019 (covid-460 19) in nursing homes (revised) | cms discontinuation of transmission-based precautions and disposition of patients with 468 covid-19 in healthcare settings (interim guidance). centers for disease control and 469 prevention guidance for use of certain industrial respirators by health care personnel | cms how not to wear a mask. the new york times taking action to protect america's nursing home residents against 479 covid-19. your valley medicare administrative contractor (mac) 482 covid-19 test pricing 41. r14som.pdf. accessed key: cord-121057-986xoy22 authors: mahdi, esam; abuzaid, ali h. title: simultaneous diagnostic testing for linear-nonlinear dependence in time series date: 2020-08-18 journal: nan doi: nan sha: doc_id: 121057 cord_uid: 986xoy22 several goodness-of-fit tests have been proposed to detect linearity in stationary time series based on the autocorrelations of the residuals. others have been developed based on the autocorrelations of the square residuals or based on the cross-correlations between residuals and their squares to test for nonlinearity. in this paper, we propose omnibus portmanteau tests that can be used for detecting, simultaneously, many linear, bilinear, and nonlinear dependence structures in stationary time series based on combining all these correlations. an extensive simulation study is conducted to examine the finite sample performance of the proposed tests. the simulation results show that the proposed tests successfully control the type i error probability and tend to be more powerful than other tests in most cases. the efficacy of the proposed tests is demonstrated through the analysis of amazon.com, inc., daily log-returns. the problem of the analysis of nonlinear time series models has attracted a great deal of interest in finance, business, physics, and other sciences. granger and anderson (1978) and tong and lim (1980) noticed, in many time series modeled by box and jenkins (1970) , that the squared residuals are significantly autocorrelated even though the residual autocorrelations are not. this indicates that the innovation of these models might be uncorrelated but not independent. the authors suggested using the autocorrelation function of the squared values of the series in order to detect the nonlinearity. in this respect, engle (1982) showed that the classical portmanteau tests proposed by box and pierce (1970) and ljung and box (1978) , based on the autocorrelation function of the residuals, fail to detect the presence of the autoregressive conditional heteroscedasticity, arch , in many financial time series models. he introduced a lagrange multiplier statistic, based on the autocorrelations of the squared-residuals, to test for the presence of arch process. mcleod and li (1983) proposed a portmanteau test, which was also based on the squared-residuals autocorrelations, to detect nonlinearity and arch effect. their statistic is similar in spirit to the modified test of box and pierce (1970) proposed by ljung and box (1978) . since then, several authors have developed goodness-of-fit tests based on the autocorrelation function of the squared-residuals to detect nonlinear structures in time series models (e.g., li and mak (1994) ; rodríguez (2002, 2006) ; rodríguez and ruiz (2005) ; fisher and gallagher (2012) ). simulation studies demonstrate the usefulness of using these statistics to test for arch effects but they tend to lack power compared to other types of nonlinear models that do no have such effects. a possible reason for such lack of power could be the fact that none of these tests have considered the cross correlation between the residuals at different powers where the correlations at positive and negative lags might be vary. lewis (1985, 1987) introduced the idea of using the generalized correlations 1 of the residuals to detect nonlinearity without taking into account the distribution of the parameters estimation. recently, psaradakis and vávra (2019) implemented the lewis (1985, 1987) idea and they proposed four goodness-of-fit tests, based on the generalized correlations of the residuals of linear time series model, to test for linearity of stationary time series. two of these tests are similar in spirit to box and pierce (1970) where they replaced the autocorrelations of the residuals in box and pierce (1970) by the cross-correlations of the residuals at different powers getting two tests: one is associated with positive lags; and the other is based on negative lags 2 . similarly, the other two tests were proposed by replacing, respectively, the autocorrelations of the residuals in ljung and box (1978) (or square-residuals in mcleod and li (1983) ) tests by the cross-correlations of the residuals at different powers. their preliminary analysis indicated that the mcleod and li (1983) modification tests control the type i error probability somewhat more successfully than the other tests. hence, they restricted their simulation study focusing on comparing these two tests (where r, s ∈ {1, 2}, r = s) with mcleod and li (1983) test (where r = s = 2) and they suggested, in most cases, that at least one of the two cross-correlation tests tends to have more 1 generalized correlation is defined as the autocorrelation between the residuals to the power r at time t and the residuals to the power s at time t + k where r, s are positive s and k is the lag time. 2 the values of autocorrelations of the residuals at positive lags are the same values at negative lags. on the other hand the values of the cross-correlations between the residuals to the power r at lag time t and the residuals to the power s at lag time t + k when k > 0 are not the same when k < 0. power than the test based on the autocorrelation of the squared-residuals in several time series models. to our knowledge, none of the tests in the time series literature has simultaneously combined the autocorrelations of the residuals and the autocorrelations of their square values with the cross-correlation between them. in this article, we fill this gap by proposing four goodness-of-fit tests. the first part in each of the proposed tests is based on the autocorrelations of the residuals, which is used to test for linearity (adequacy of fitted arma model); the second is based on the autocorrelation of the squared-residuals, which can be used to test for arch , and the third one is based on the cross correlations between the residuals and their squared values, which can be used to test for other types of nonlinear models in which the residuals and their values are crosscorrelated. similar to psaradakis and vávra (2019) , the cross-correlations between the residuals and their squared values propose two different tests. one is based on positive lags and other is based on negative lags. in section 2 we discuss the generalized correlations of residuals and review some test statistics that have been commonly used to detect nonlinearity structure in stationary time series models. in section 3, we propose new goodness-of-fit (auto-and-cross-correlated) tests that can be used to detect, simultaneously, linear, bilinear, and nonlinear dependency in time series models, and derive their asymptotic distribution as a chi-squared distribution. in section 4, we provide a monte carlo study comparing the performance of the proposed statistics with those from the literature. we show that the empirical significance level of the proposed tests successfully controls the type i error probability and tends to have higher power than others. an illustrative application is given in section 5 to demonstrate the usefulness of the proposed test for a real world data. we finish this article in section 6 by providing a concluding remarks. 2 auto-and-cross-correlated portmanteau tests let z 1 , z 2 , · · · , z n denote a realization of real-valued stochastic process {z t } where where the polynomials φ p (b) and θ q (b) are assumed to have all roots outside the unit circle and to have no common roots and η = (φ 1 , · · · , φ p , θ 1 , · · · , θ q ). in time series literature, the notion of linearity is commonly used when the process {z t } admits the moving-average, ma , representation (1) where {ε t } are independent and identically distributed, i.i.d., random variables with a zero mean and a constant variance σ 2 . this is the notion considered by mcleod and li (1983) ; lewis (1985, 1987) and psaradakis and vávra (2019) which will also be adopted in this article. on the other hand, there are several time series that do not exhibit a linear behavior. for example, when the innovations {ε t } are uncorrelated but not independent, engle (1982) proposed the autoregressive conditional heteroscedasticity, arch , model that is widely used for analyzing financial time series. this has been generalized by bollerslev (1986) to the generalized autoregressive conditional heteroscedasticity where {ξ t } is a sequence of i.i.d. random variables with a mean value of 0 and a variance value has been proposed to model the dynamic behavior of conditional heteroscedasticity in real time series. e.g.; the exponential garch (egarch ) model proposed by nelson (1991) to allow for asymmetric effects between positive and negative financial time series. tsay (2005) and carmona (2014) provided nice reviews of the garch models. another popular nonlinear model is the threshold autoregressive (tar ) model of (tong, 1978 (tong, , 1983 (tong, , 1990 tsay, 1989) , which was generalized by chan and tong (1986) and terasvirta (1994) to the smooth transition autoregressive (star ) model. the two regime-switching star model of order (2; p, p) takes the form i ), i = 0, 1, · · · , p are the autoregressive coefficients, 0 ≤ f (.) ≤ 1 is a transition continuous function that allows the dynamics of model to switch between regimes smoothly, and s t is a transition variable. a common formulations for the transition function, which was proposed by terasvirta (1994) , is the first-order logistic function and can represented as: where γ > 0 denotes the smoothness parameter of the transition from one regime to the other, d ≥ 1 is the delay parameter, c is a threshold variable that separates the two-regimes, and σ z is the standard deviation of z t . an alternative choice, which was also proposed by terasvirta (1994) , is the exponential function given by when the value of γ increases, the transition function f (z t ; γ, c) approaches the indicator function in this case, the model in (4) reduces to the tar model of (tong, 1978) . arguably the tar models is consider to be the most popular class of nonlinear time series model. therefore, testing for tar and star models have attracted much attention (xia et al., 2020) . volterra series is another form of the nonlinear stationary process that is widely used in many applications (wiener, 1953, lecture 10) . these models have the form where µ is the mean level of z t and {ε t , −∞ < t < ∞} is a strictly stationary process of i.i.d. random variables. let β = (η, µ, σ 2 ) denote the true parameter values in (1),β = (η,μ,σ 2 ) denote the estimated values, andε i , i = 1, · · · , n denote the residuals and define the correlation coefficient at lag time k betweenε r t andε s t+k (r, s = 1, 2) aŝ whereγ rs (k) = n −1 n−k t=1 f r (ε t )f s (ε t+k ) for k ≥ 0, γ rs (k) = γ sr (−k) for k < 0, is the autocovariance (cross-covariance), at lag time k, between the residuals to the power r and the residuals to the power s for r, s = 1, 2, and f j ( (1) of the squared residuals from the linear model given in (2) have been widely used in several portmanteau statistics to test for nonlinearity. the commonly employed test, which is similar in spirit to box and pierce (1970) , isq where 0 < m < n is the maximum lag considered for a significant autocorrelation 4 . this test is asymptotically distributed as chi-square with m degrees of freedom. mcleod and li (1983) showed that theq 22 test can be improved if the autocorrelation coefficients in (8) are replaced with their standardized values r rs (k) = n + 2 n − |k|r rs (k), (r = s = 1, 2), k = ±1, · · · , ±m. mcleod and li (1983) proposed a portmanteau test, for detecting the presence of the arch effects. their test statistic is given by where the q 22 test is asymptotically distributed as chi-square with m degrees of freedom. simulation studies have showed that theq 22 and q 22 tests respond well to arch models but tend to lack power compared to other types of nonlinear models that do not have the arch effects. 3 the case for absolute residuals is beyond the scope of this article and we focus our attention to the squaredresiduals case. 4 the value of m depends on the sample size, and as a rule of thumb, we consider m ∈ {1, 2, · · · , √ n }, where n denotes the largest integer not exceeding n. a reasonable justification for the lack of powers could be neglecting the cross-correlations between the residuals at different exponent powers (generalized correlations betweenε r t andε s t+k , where k ≥ 0 and r + s > 2). in this regard, lewis (1985, 1987) proposed the idea of implementing the sample generalized correlations to detect the nonlinear dependency without considering the distribution of the parameter estimation. recently, psaradakis and vávra (2019) used the idea of lewis (1985, 1987) under the assumption in (1). they proposed portmanteau tests based on the generalized correlations and demonstrated the usefulness of using the test statistics based on the cross-correlation, between the residuals at different powers, for detecting linearity in time series. their test statistics arê and whereq rs and q rs (r = s) are asymptotically distributed as χ 2 m . psaradakis and vávra (2019) suggested that the tests based on the cross-correlations tend to be more powerful against many types of nonlinearity compared to other statistics based on squared-residual autocorrelations. motivated by the ideas of lewis (1985, 1987) , and psaradakis and vávra (2019), we propose in the next section new test statistics that can be used to detect nonlinearity in time series models. in this section we made some assumptions that are needed to derive the asymptotic distribution of the proposed statistics. in general, the limiting distribution of the portmanteau test statistic required the following assumptions: a2. the polynomial ψ ∞ (b) is differentiable with respect to η in an open neighborhood of the closed disc |b| ≤ 1. this guarantees that the 1/ψ ∞ (b) series is converge and the process {z t } admits the following autoregressive, ar, representation with order ∞ where φ j , j = 0, 1, · · · which can be found by solving . hereβ can be estimated by the least squares method or the maximum (or quasi-maximum) likelihood method (see e.g., hannan (1973) ; hosoya and taniguchi (1982) ; kuersteiner (2001)). a4. ∂γ rs (k)/∂β = o p (1/ √ n) for k = 1, 2, · · · , n − 1 and r, s ∈ n such that r + s ≥ 2 and if the model in (2) is correctly identified and the assumptions a1-a4 are held, we propose the portmanteau test statisticsĉ and where the asymptotic distribution ofĉ rs and c rs is chi-square with 3m − (p − q) degrees of freedom, where r = s ∈ {1, 2}. remark. each of the two test statistics in (16) can be seen as a linear combination of three existent tests, ljung and box (1978) , mcleod and li (1983) , and psaradakis and vávra (2019), modifying the corresponding tests in (15). theorem 1. if {z t } satisfies (1) and the assumptions a1-a4 are held, then, for any integer m < n, the asymptotic distribution of √ n(r rr (1), · · · ,r rr (m),r rs (1), · · · ,r rs (m),r ss (1), · · · ,r ss (m)) , where r, s = 1, 2, r = s, as n → ∞ is gaussian with zero mean vector and the covariance matrix equals to i 3m . proof. for a fixed m < n, where n is large, the first m sample autocorrelations of the innovations are asymptotically normal with a mean of (ρ 11 (1), ρ 11 (2), · · · , ρ 11 (m)) and covariance matrix n −1 w , where the ( , k)th element of w are given by where ρ 11 (m) is the population autocorrelation of the innovations at lag m. under the assumption a1-a3, bartlett (1946) showed that ρ 11 (m) = 0 for all m = 0; hence the asymptotic distribution of r 1 = √ n(r 11 (1), · · · ,r 11 (m)) is multivariate normal with mean vector zero and identity covariance matrix (anderson and walker, 1964) . similarly, under the assumption a1-a4 and from theorem 14 of hannan (1971, p.228 ) and the result 2c.4.12 of rao (1973) , mcleod and li (1983) showed that the limiting distribution r 2 = √ n(r 22 (1), · · · ,r 22 (m)) is multivariate normal with mean vector zero and identity covariance matrix. also, under the same assumptions, psaradakis and vávra (2019) showed that the asymptotic distribution of r 12 = √ n(r 12 (1), · · · ,r 12 (m)) is gaussian with zero mean vector and identity covariance matrix. furthermore, the previous assumptions imply that r 1 , r 2 , and r 12 are independent. therefore, we may conclude that, as n → ∞, the distribution of √ n(r 11 (1), · · · ,r 11 (m),r 12 (1), · · · ,r 12 (m),r 22 (1), · · · ,r 22 (m)) is gaussian with zero mean vector and covariance matrix equals to i 3m . similarly, it is straightforward to show that √ n(r 22 (1), · · · ,r 22 (m),r 21 (1), · · · ,r 21 (m),r 11 (1), · · · ,r 11 (m)) converges weakly to the standard normal distribution on r 3m . using the results from theorem 1, under the adequacy of the fitted arma (p, q) model, and from the theorem on quadratic forms given by box (1954) , it is straightforward to conclude that c rs and c rs will be asymptotically distributed as chi-square with 3m−(p−q) degrees of freedom. this section presents the simulation results regarding the finite-sample properties of the asymptotic results of the proposed tests. for illustrative purposes, we also consider the three statistics q 22 and (q 12 , q 21 ) given by (11) and (13). the comparative study is similar to that provided in psaradakis and vávra (2019) where the data-generating processes (dgp) are based on the following eighteen models: (2019)). for each of the m1-m18 models, we use r to simulate 1,000 independent trajectories, each is a series of length n + n/2 with n ∈ {200, 500, 1, 000}, but only the last n data points are used to carry out portmanteau tests to the residuals at different lags m ∈ {5, 10, 15, 20, 25, 30} (r core team, 2020). figure 1 illustrates the accuracy of the approximation of the empirical distribution ofĉ rs and c rs , r = s ∈ {1, 2} defined by (15) and (16) the sample size), and our preliminary analysis 5 indicate that the portmanteau tests based on the statistics c rs control the type i error probability more successfully than the tests based on the statisticsĉ rs ; hence, we recommend the use of c rs , r = s ∈ {1, 2}. in this section, we calculate the type i error probability (of nominal size 1% and 5% 6 ) based on the five test statistics, (c 12 , c 21 ), (q 12 , q 21 ), and q 22 defined by (16) of fitting a true model, for each n ∈ {200, 500, 1, 000}, are averaged across the non-gaussian linear (m1-m5) and nonlinear (m6-m14) models. in addition, the relative rejection frequencies outside the 99% significant limits are put in boldface. as shown in table 2 , the simulation results clearly indicate insensitivity to non-gaussianity noise and the empirical level (regardless of the sample size) agrees very well with the corresponding nominal size. the powers of the proposed tests c 12 and c 21 are compared with the powers of q 12 , q 21 , and q 22 statistics for nominal levels α = .01, .05, and .10 7 . similar to the simulation design in section 4.1, we generate artificial series of lengths n ∈ {200, 500, 1, 000} from m1-m18 models with innovations having either normal or non-normal distributions. for the non-normal distribution, as seen in figures 5-7 , the proposed tests c 12 and c 21 both, substantially, have higher rejection frequencies than the q 12 , q 21 , and q 22 statistics and in all cases, the test c 21 is the more powerful test. in this section, the aforementioned statistics are applied to detect nonlinear dependency in the daily adjusted log-returns of amazon.com, inc., spanning the period january 02, 2019 to decemestimation. the order of the best of fitted model is selected by minimizing the bayesian information criterion (bic) over the arma (p, q) models, where (p, q) ∈ {0, 1, · · · , 8(n/100) 1/4 } as suggested by ng and perron (2005) . the bic suggested that no arma process (linear) is detected, in the log-returns of the amazon.com, inc., neither before nor after the spread of the disease. the simulation study suggested the proposed tests, c 12 and c 21 , appeared to be as good as the mcleod and li (1983) statistic in detecting long memory processes and more powerful than psaradakis and vávra (2019). as seen in table 3 , the two tests of psaradakis and vávra (2019) fail to detect the nonlinearity structure in both series. however, when the mcleod and li (1983) and the proposed statistics are used, a clear indication of nonlinear structure appears. in this article, we propose four goodness-of-fit tests to detect various types of linear and nonlinear dependency in stationary time series models. the proposed tests are based on a linear combination of three auto-and-cross-correlation components. the first and the second components are based on the autocorrelations of the residuals and their squares, respectively, whereas the third is based on the cross-correlations between the residuals and their squares. two tests can be seen as an extended modification version to the ljung and box (1978) test, and the others might be considered as an extended modification version to the box and pierce (1970) test. our simulation study recommends to use the former tests as they are insensitive with respect to non-gaussianity assumption of the noise, controlling the type i error probability more successfully than the other tests, and almost always having more power than mcleod and li (1983) and psaradakis and vávra (2019) tests. the idea discussed in this article might be extended to detect seasonality in time series and to identify various types of nonlinearity dependency in multivariate time series. when the underlying process is assumed to be uncorrelated but yet is not independent (weak assumptions), one might be able to propose a new portmanteau test. in such a case, the monte carlo significance test as suggested by lin and mcleod (2006) and mahdi and mcleod (2012) might be used to calculate the p-values based on approximating the sampling distribution of the proposed test. on the asymptotic distribution of the autocorrelations of a sample from a linear stochastic process a single-blind controlled competition among tests for nonlinearity and chaos on the theoretical specification and sampling properties of autocorrelated time series generalized autorregressive conditional heteroskedasticity time series 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estimation of linear processes a tukey nonadditivity-type test for time series nonlinearity optimal instrumental variables estimation for arma models modelling and residual analysis of nonlinear autoregressive time series in exponential variables higher-order residual analysis for nonlinear time series with autoregressive correlation structures testing for neglected nonlinearity in time series models: a comparison of neural network methods and alternative tests on the squared residual autocorrelations in non-linear time series with conditional heteroskedasticity improved peňa-rodríguez portmanteau test on a measure of lack of fit in time series models improved multivariate portmanteau test distribution of the residual autocorrelation in multivariate arma time series models conditional heteroskedasticity in asset returns: a new approach. econometrica a note on the selection of time series models a powerful portmanteau test of lack of test for time series the log of the determinant of the autocorrelation matrix for testing goodness of fit in time series portmanteau tests for linearity of stationary time series r: a language and environment for statistical computing. r foundation for statistical computing linear statistical inference and its applications a powerful test for conditional heteroscedasticity for financial time series with highly persistent volatilities specification, estimation, and evaluation of smooth transition autoregressive models on a threshold model in pattern recognition and signal processing threshold models in non-linear time series analysis non-linear time series: a dynamical system approach threshold autoregression, limit cyclesand cyclical data (with discussion) testing and modeling threshold autoregressive processes analysis of financial time series non-linear problems in random theory coronavirus disease (covid-2019) situation reports a portmanteau test for smooth transition autoregressive models key: cord-170195-lrg11s5n authors: stoye, jorg title: a critical assessment of some recent work on covid-19 date: 2020-05-20 journal: nan doi: nan sha: doc_id: 170195 cord_uid: lrg11s5n i tentatively re-analyze data from two well-publicized studies on covid-19, namely the charit'{e}"viral load in children"and the bonn"seroprevalence in heinsberg/gangelt"study, from information available in the preprints. the studies have the following in common: they received worldwide attention and arguably had policy impact. the thrusts of their findings align with the respective lead authors' (different) public stances on appropriate response to covid-19. tentatively, my reading of the gangelt study neutralizes its thrust, and my reading of the charit'{e} study reverses it. the exercise may aid in placing these studies in the literature. with all caveats that apply to n=2 quickfire analyses based off preprints, one also wonders whether it illustrates inadvertent effects of"researcher degrees of freedom." this note takes a second look at two recent papers on covid-19 released by leading german research groups. both received widespread attention and arguably had policy impact. the first, by christian drosten's research group, was cited as evidence against opening schools; the second one, by hendrick streeck with coauthors, was cited as evidence in favor of a partial reopening of germany. both made important data collection efforts and then interpreted those data. i limit my analysis exclusively to this last step of interpretation, which is also the only aspect on which i can claim expertise. i find that, based on information available in the preprints, my own analysis would plausibly have had a neutral or even the opposite tendency in each case. i next elaborate this somewhat formally and then discuss implications. 2 the charité study: viral load in children 2.1 background jones, mühlemann, veith, zuchowski, hofmann, stein, edelmann, corman, and drosten (2020, henceforth " the charité study") analyze viral load by age in a convenience sample of covid-positive patients in berlin. the aim of the investigation was to see whether epidemiological and anecdotal evidence of children being less contagious could be corroborated. the study received massive media coverage in germany and abroad. its data are visualized in figure 1 . 1 the seemingly strong association between age and viral load is corroborated by a nonparametric omnibus test of association. however, the paper next tests for significant differences across all 45 pairwise combinations of age bins (and repeats the analysis with different binning). after adjustment for multiple hypothesis testing, no specific comparison is significant. this is reported in the abstract as follows: " [t] hese data indicate that viral loads in the very young do not differ significantly from those of adults." some disagreements with the analysis may come down to taste. the authors focus on a hypothesis test as deliverable of their analysis; i would have recommended a nonparametric mean regression with error bands, resulting in some estimated age effect. the main reason is that substantive significance does not equal statistical significance -if we found a minuscule but highly significant effect, we would not care either. 2 next, the core finding is that a certain hypothesis does not get rejected. thus, the analysis suggests absence of evidence, table 2 , panel 1, in the charité study. error bars display ±1.96 standard errors. from the study's abstract: " [t] hese data indicate that viral loads in the very young do not differ significantly from those of adults." (inspired by the image accompanying textor (2020) .) which is of course not evidence of absence. although this distinction is central to how we teach hypothesis tests, including in medicine (altman and bland, 1995) , it is at most barely maintained in the paper (see the aforecited sentence) and completely lost in its perception. as a result, the data that are visualized in figure 1 fueled headlines like "covid patients found to have similar virus levels across ages" (gale and mulier, 2020) and "children are just as contagious as adults" (kieselbach, 2020) . more troublingly, i am not convinced that the evidence is absent. to the statistically educated reader, the above headlines may suggest that the study tests, and fails to reject, h 0 : "children have the same viral load as adults." it does not. it rather splits the sample into 10 age bins and then simultaneously tests whether any of the 45 possible pairwise combinations of age groups exhibit a significant difference in load. with proper adjustment for the possibility of false discoveries from 45 tests, not one comparison shows up as significant. this is reported as main conclusion, even though it is at tension with a test reported elsewhere in the preprint that indicates some difference across age bins (kruskal-wallis test, table a3 , p = 0.8% for [3] the bins in figure 1 ). that is, the analysis finds a significant age effect -it is merely that post-hoc comparison does not single out a specific one of 45 pairs as culprit. if one's take-home message is a finding of nonsignificance, one should make a good faith effort to apply a powerful test. that did not seem to happen here. on the contrary, artificially coarsening the age variable and, especially, pooling the comparison of interest with 44 comparisons that nobody inquired about is bound to sacrifice power. what would the result have been without these choices? to take an educated guess, i will now try to recover from the paper a test of h 0 : "children have the same viral load as adults." to this purpose, i combine the first two and the remaining age bins of figure 1 to find means of 4.74 and 5.21 with 95% confidence intervals of [4.42, 5.05] and [5.15, 5.27], respectively. a simple t-test rejects the null of equal means at a two-sided p-value of 0.4%. 3 it would seem that the lower viral load in the youngest age groups is highly significant. what gives? i reiterate that this simple test would not be my preferred analysis of the raw data. however, i am willing to defend it vis a vis the preprint's multiple nonparametric hypothesis tests. specifically: • the test only considers one rather than 45 hypotheses. i consider this a feature and not a bug. the data were collected specifically to investigate the relative viral load of children, and the methodological justification for pooling this comparison with 44 others is not elaborated. indeed, one could arguably conduct a one-sided test and report a p-value of 0.2%. • the test is influenced by the authors' choice of bins and by my inspection of the visualization, so it strictly speaking involved an informal specification search. however, given the study's purpose, my own pre-data plan (conditionally on involving a simple hypothesis test to begin with) would have proposed more or less the same comparison. hence, this should not a major concern. that said, taking the above p-value entirely at face value is not recommended, but it is also not necessary for my point. • the test is exact only if relevant true distributions are normal. this is clearly not the case, neither exactly (negative values are impossible) nor approximately (the paper reports corresponding tests). however, i compare means across two samples of size 127 and 3585, and the empirical distributions appear slightly skewed but well-behaved (see figure 1 in the charité study). an appeal to the central limit theorem would seem innocuous. in fact, this consideration is more pressing for the charité study itself. they perform 45 simultaneous tests on relatively small bins and therefore encounter the one-two punch of (i) looking at the further-out-in-the-tails quantiles of sampling distributions that become relevant with bonferroni-type adjustments and (ii) rather small sample cells. indeed, 9 of the tests involve the "91-100" age bin, which contains 17 observations. this makes it harder to appeal to normal approximations, yet two of the three multiple hypothesis tests reported in the paper do so anyway and essentially differ from my test only through adjusting for multiplicity of hypotheses. 4 this simple analysis is not meant to be conclusive. my only point is that the paper's nonsignificance result seems to be driven by researcher choices. for any conclusions beyond that, i would want to see the results of a nonparametric regression analysis that undoes the age binning of data. the closest approximation to this that i am aware of is the insightful renalysis by held (2020) , which qualitatively agrees with mine. the ideal solution here would be publication of the data; while this might not be legally easy, i note that a lot could be done with literally only age and viral load. streeck, schulte, kümmerer, richter, höller, fuhrmann, bartok, dolscheid, berger, wessendorf, eschbach-bludau, kellings, schwaiger, coenen, hoffmann, stoffel-wagner, nöthen, eis-hübinger, exner, schmithausen, schmid, and hartmann (2020, henceforth "the gangelt study") is an early seroprevalence study conducted in the small german town of gangelt, which had witnessed a superspreader event. the study made headlines for estimating a local prevalence of 15% and especially an ifr of .4%, which was perceived as surprisingly low (and would certainly be considered on the very low end now). from the latter number, the authors also informally extrapolate to 1.8 million infected germans, an extrapolation that was correspondingly perceived as high, therefore arguably reassuring, but also implausible. the study received considerable attention that was apparently seeked out by the lead author, who vocally advocated for partial reopening of north rhine-westphalia. 5 reanalysis. it has been widely remarked that, even if one feels confidence extrapolating gangelt's ifr, one cannot extrapolate the ci to either nationwide ifr or nationwide prevalence. the reason is that gangelt's fatality count was considered nonstochastic, but for the purpose of extrapolating results to germany, it is a realization of 8 "successes" (scare quotes very much intended) in a large trial. for illustration, a simplistic 95% confidence interval for gangelt's expected fatality count covers all integers from 4 to 16. 6 but the point estimator itself may not be the only plausible choice either. it implies that only 10% of cases were detected nationwide, an implication that was picked up by the media (burger, 2020) but also frequently called out as implausible. indeed, the discovery rate in gangelt was estimated to be 20%. extrapolating from this number to germany would imply an ifr of .9% rather than .4%. what gives? the discrepancy stems from the fact that, if we believe gangelt to be representative of germany, we have overdetermined but not quite consistent estimating equations. for sake of argument, say we have 8 fatalities, 388 officially confirmed cases, and 1956 true infections in gangelt, while we have 7928 fatalities, 174975 confirmed cases, and an unknown true prevalence in germany. 7 by the rule of proportions, we could back out an estimate of nationwide prevalence either as 1956 × 7928/8 = 1938396 (an update of the study's preferred estimate) or as 1956 × 174975/388 = 882090 (extrapolating the undercount). the numbers differ by so much because the crude case fatality rate (cfr) equals 8/388 = 2.1% for gangelt and 7928/174975 = 4.5% for germany. thus, assuming that the ifr (interpreted as population-level expectation) is actually the same, gangelt either tested more or got really lucky. by extrapolating the local ifr, the study assumes the former but without discussing this assumption. at the same time, proponents of extrapolating the undercount could point out that the underlying binomial samples of 8/388 versus 7928/174975 are of vastly different size. should we really prioritize the former over the latter in extrapolation? or was gangelt indeed just lucky? this is marginally implausible in that the (exact binomial) two-sided p-value for the former sample being drawn from the latter urn is 2.6%. but modest demographic difference between gangelt and germany, or accounting for clustered sampling of both numerator and denominator (the sample units were households; the superspreading event apparently spared local nursing homes), could easily compensate for that. what's more, the fatality count meanwhile increased to 9. while it is not clear that this fatality was among the infected during the study's time frame, adding it would change the aforementioned p-value to 5.2%. also, it would mean that the local and nationwide numbers are consistent up to sampling error according to the more sophisticated analysis in rothe (2020, numerical claim verified in personal communication). again, my point is not that the study's implicit argument is necessarily false. gangelt's undercount might indeed be atypically low: being a hotspot, the town saw much more testing than other places. however, that does not logically imply that gangelt had more testing relative to true prevalence. my complaint is that this case was not made, and a 6 this ci just inverts the poisson distribution. it is reported to illustrate that the issue is not negligible. see rothe (2020) for a sophisticated analysis of the multilayered inference problem. note also that i use the higher (=8) of two fatality counts offered in the preprint. 7 the nationwide numbers were reported by johns hopkins university on 5/14. they implied a slightly lower case fatality rate at the time of the study. [6] different extrapolation that would align the study with other recent seroprevalence studies (and have shorter confidence intervals to boot) was not discussed. a balanced analysis might well provide both and also some form of interpolation. partial identification would be one framework close to my heart, but the primary concern is transparency about how different identifying assumptions would interact with the data at hand. this is not a "gotcha" paper. both aforecited studies are carefully and competently executed, especially considering the extreme time pressure. they are transparent about computations, the computations appear correct, and i have no doubt that they were executed in good faith. compared to some concurrent work by prominent u.s. researchers (e.g., as discussed by gelman (2020)), both studies are models of good science. also, there are obvious limitations to quickfire reanalysis without raw data, and everything i have to say is accordingly tentative. with that said, i stand by the following assessments: there are many good arguments against a quick reopening of schools, but the charité study does not add to them. neither does the gangelt study paint a substantially more optimistic picture on ifr than other recent seroprevalence studies. 8 with the due caution warranted by a sample size of n = 2, it is of note that both studies were perceived as having a certain policy thrust; in both cases, this thrust correlated with public perception of some authors' stand on covid-19 policy; and in neither case was it corroborated by my reading. that is my methodological point. specific, reasonable but not objectively forced choices of statistical analysis -what is sometimes referred to as "researcher degrees of freedom"-informed results that align with researchers' public stands. i emphasize that i do not suggest any intent. however, even at the very top of the discipline, the effects of, for example, deciding when an analysis is complete cannot be assumed to just go away. while i think that my comments on the specific studies are of interest, an important message to you -the reader-and me is to redouble consideration for such effects in our next study. on an immediately practical note, it illustrates that a data repository for covid-19related research would be invaluable. finally, it is a reminder of the importance of professional peer review. while both of the above studies were controversially discussed by experts right away, my impression is that critical voices gained any traction with the public only for the gangelt one. in sum, this may be the rare case where rapid peer review of an academic paper would plausibly have affected the news cycle. statistics notes: absence of evidence is not evidence of absence german study suggests infections are 10 times the number of confirmed cases fatal flaws in stanford study of coronavirus prevalence a discussion and reanalysis of the results an analysis of sars-cov-2 viral load by patient age kinder sind genauso ansteckend wie erwachsene (children are just as contagious as adults) open review of the manuscript: 'an analysis of sars-cov-2 viral load combining population and study data for inference on event rates infection fatality rate of sars-cov-2 infection in a german community with a super-spreading event here's a replot of their own data. means +/-2*se from their table 2. their conclusion: no sig. viral load difference between children and adult. media: children just as infectious as adults, study shows key: cord-263893-zb6h3q4k authors: nan title: what to expect from covid-19 serology in a period of deconfinement? date: 2020-06-01 journal: bull acad natl med doi: 10.1016/j.banm.2020.05.100 sha: doc_id: 263893 cord_uid: zb6h3q4k nan what to expect from covid-19 serology in a period of deconfinement? ଝ despite the significant impact of containment on the course q4 of the covid-19 epidemic, the circulation of the virus persists on the french territory, even among the least affected regions. as of may 11, the date set for the end of the containment, a model from the pasteur institute estimated that the infection would have affected 5.7% of the population, with significant regional variations, from less than 2% in brittany, new aquitaine and pays de loire to 10-13% in the grand est and île-de-france [1] . the level of immunity of the french population to sars-cov-2 thus seems very low, far from the theoretical threshold of 60% which would allow us to expect a collective level of protection. this situation makes it necessary to implement iterative population-based sero-epidemiological surveys, representative of each region, each age group and each socio-professional category to assess the spread of the epidemic in the population. in addition, there is a strong demand for serological tests from the professionally exposed workers and, more widely, from many worried people anxious to know their immune status. numerous serological tests have been developed for the detection of igg and igm antibodies against sars-cov-2 in a sample of venous or capillary blood. these are the elisa tests that can be used in large series on automatic machines and the unit tests (rapid diagnostic tests, tdr, doi of original article:https://doi.org/10.1016/j.banm.2020. 05.099. ଝ press release from the french national academy of medicine and q1 the national academy of pharmacy, 20 may 2020. and rapid diagnostic orientation tests, trod), which can be performed individually on a drop of capillary blood obtained by fingertip pricking [2] . their evaluation by the national centres of reference (cnr) for respiratory infection viruses has made it possible to select tests that meet the performance requirements of the french high authority for health (sensitivity ≥ 90% and specificity ≥ 98%), but these tests have not yet been validated by the health authorities and do not give the right to reimbursement. it should be noted, however, that even if a test with a specificity of 98% is used, the positive predictive value of seropositivity will only be 50% in all regions of the country spared by the epidemic, where seroprevalence is estimated an average of 2% [3] . furthermore, while positive serology indicates immunity to the virus, it does not allow to predict with certainty that the person will be protected in the event of a reinfection. in the current context, given the sharp increase in individual requests for serological tests without medical prescription, access to tests must remain controlled in order to avoid any behavioural drift that could be induced by misinterpretation of the results. the national academies of medicine and pharmacy recommend: • that only those tests that will be recommended by the cnrs and validated by the ministry of health and solidarity be used, whether they are unit tests or elisa tests; • that the sero-epidemiological population surveys be coordinated by the regional health authority (ars) and that each person recruited be informed personally and confidentially of his or her serological status; • that the covid-19 serological tests should be carried out neither at a simple individual request, nor at the behest of employers; • that the serological tests be carried out only on medical prescription, the general practitioner having to judge their necessity after consultation or teleconsultation; • that prescribing physicians have access to interpretation algorithms, helping them to comment on their patients' results, to request additional virological tests if necessary, and draw the possible consequences; • that a positive test result does not lead to the establishment of a ''certificate of seropositivity'' or of an ''immunological passport''; • that medical confidentiality be scrupulously preserved. q5 the authors declare that they have no competing interest. estimating the burden of sars-cov-2 in france tests covid-19 : applications collectives et individuelles place des tests sérologiques rapides (tdr, trod, autotests) dans la stratégie de prise en charge de la maladie covid-19 key: cord-278377-jgq3dz3u authors: busson, l.; bartiaux, m.; brahim, s.; konopnicki, d.; dauby, n.; gérard, m.; de backer, p.; van vaerenbergh, k.; mahadeb, b.; de foor, m.; wautier, m.; vandenberg, o.; mols, p.; levy, j.; hallin, m. title: prospective evaluation of diagnostic tools for respiratory viruses in children and adults date: 2019-01-15 journal: j virol methods doi: 10.1016/j.jviromet.2019.01.006 sha: doc_id: 278377 cord_uid: jgq3dz3u aim: to compare the performances of molecular and non-molecular tests to diagnose respiratory viral infections and to evaluate the pros and contras of each technique. methods: two hundred ninety-nine respiratory samples were prospectively explored using multiplex molecular techniques (filmarray respiratory panel, clart pneumovir), immunological techniques (direct fluorescent assay, lateral flow chromatography) and cell cultures. findings: molecular techniques permitted the recovery of up to 50% more respiratory pathogens in comparison to non-molecular methods. filmarray detected at least 30% more pathogens than clart pneumovir which could be explained by the differences in their technical designs. the turnaround time under 2 hours for the filmarray permitted delivery of results when patients were still in the emergency room. since the discovery of viruses in the twentieth century, considerable efforts have been made to improve the technics to detect and identify them. cell cultures were the first diagnostic tool to be used in the mid-1950s, and since then, new techniques have been developed to decrease the time to a result (immunofluorescence, lateral flow chromatography) or to boost the sensitivity (molecular techniques) (levine, 1996; ginocchio and harris, 2011) . many improvements have been made, and techniques combining speed and sensitivity are currently available, such as fully automated 'sample-in, result-out' multiplexed syndromic molecular tools (bluchan and ledeboer, 2014) . aside from being effective in terms of sensitivity and specificity, these latter diagnostic tools are able to recover non-cultivable viruses. as a consequence, questions regarding the usefulness of 'older' diagnostic methods regularly arise (leland and ginocchio, 2007; hodinka and kaiser, 2013) . meanwhile, important questions concerning these 'new' expensive rapid molecular techniques remain unanswered, such as their cost-effectiveness in terms of patient's management, or the clinical significance of detecting nucleic acids of micro-organisms that could be non-infectious at the time the sample is collected. the objective of this work was to compare the performances of antigen detection and cell cultures techniques routinely used since years for the diagnosis of respiratory viral infections in the setting of a tertiary care hospital to those of newer molecular techniques (clart pneumovir, genomica, coslada, spain and filmarray respiratory panel, biofire, biomérieux, marcy l'etoile, france). the study was initiated on the 1 st of february (week 5) and ended on the 15 th of march 2016 (week 11) in the saint-pierre university hospital, a tertiary general hospital with 626 beds located in downtown brussels. this was during the peak of the 2015-2016 influenza season which was moderate in belgium and lasted from week 4 to week 13. more than 90% of influenza a isolates collected in belgium were https://doi.org/10.1016/j.jviromet.2019.01.006 received 21 june 2018; received in revised form 11 january 2019; accepted 15 january 2019 a(h1n1)pdm2009. regarding influenza b, circulating strains were almost exclusively from the victoria lineage according to the belgian scientific institute of public health (belgian public health institute, 2019). the enrolment period was chosen in order to be sure to gather positive samples for influenza as it is intended to evaluate, in another article, the impact of the results on antiviral prescription. this choice obviously affects the prevalence of other viruses. adults and children attending the emergency room (er) and presenting with upper or lower respiratory symptoms were prospectively included if either intended to be maintained in the hospital or had any of the following conditions known to expose to a higher rate of complications of viral respiratory infections: chronic respiratory diseases such as cystic fibrosis or asthma, sickle-cell disease, asplenia, neuromuscular diseases, severe neurological affections, hereditary metabolic disorders including diabetes, congenital or acquired immunosuppression, heart defects, chronic nephropathies, chronic liver diseases and pregnancy. children under 3 months of age with a fever without focus of infection were also included. upon inclusion, a respiratory sample was collected. nasopharyngeal aspirate (npa) samples were typically collected from children under 2 years old, and nasopharyngeal swabs (nps) (flocked swab + utm 3 ml, copan, brescia, italy) were collected from older children and adults. the samples were immediately sent to the microbiology laboratory for testing. prior to testing, npa were diluted with 3 ml of viral transport medium composed of veal infusion broth (difco, becton dickinson, sparks, md, usa) supplemented with bovine albumin (sigma aldrich, st. louis, mo, usa). lateral flow chromatography (lfc) tests and the filmarray respiratory panel were used to test samples 24/7, whereas direct fluorescent assays (dfa) and cell cultures were performed during working hours (8:00 am to 5:00 pm) from monday to saturday. the clart pneumovir test was performed once a week. lab results as well as clinical data from patients' chart were recorded and analyzed. antigen detection tests and cell cultures are routine tests performed for patients attending the emergency departments. filmarray and clart pneumovir were performed for the study. because only 3 tests per day are reimbursed by the social welfare, the combination of lfc and dfa tests performed varied during the evaluation based on the most prevalent circulating viruses. from february 1 st to the 10 th , influenza (sofia influenza a + b, quidel, san diego, ca, usa), rsv (binaxnow rsv, alere, waltham, ma, usa) and human metapneumovirus (hmpv) dfa (argene, biomérieux, marcy l'etoile, france) tests were performed. fifty-nine samples were analyzed with this combination. from february 10 th to march 15 th 2016, metapneumovirus detection was replaced by an adenovirus detection test (adenorespi k-set, coris bioconcept, gembloux, belgium). cell cultures were performed as follows: an aliquot of the sample was inoculated on confluent vero (african green monkey kidney), mrc5 (human lung) and llc-mk2 (rhesus monkey kidney) cell cultures (vircell, santa-fé, spain) in 24-well or 6-well tissue culture plates (greiner-bio one, frickenhausen, germany). cultures were incubated at 36°c in a 5% co 2 atmosphere for 2 weeks for the vero cultures plates and llc-mk2 cells and 3 weeks for the mrc5 cells. the culture media were replaced weekly. cultures were examined every two to three days using an inverted microscope. hemadsorption was performed on the llc-mk2 cells at the end of the second week of incubation. the filmarray respiratory panel 1.7 is a closed 'sample-in, resultout' multiplex pcr system that integrates sample preparation, amplification, detection and analysis of results in approximately an hour. the panel detects the most common respiratory viruses: adenovirus, coronavirus (229e, hku1, nl63 and oc43), human metapneumovirus, human rhinovirus/enterovirus (without distinction between the two), influenza a (with differentiations of h1, h1-pdm2009 and h3 strains), influenza b, parainfluenza 1-4 and respiratory syncytial virus (rsv). the panel also detects 3 bacteria; mycoplasma pneumoniae, chlamydophila pneumoniae and bordetella pertussis which were not evaluated in this study. tests were performed according to the manufacturer's instructions. the clart pneumovir test is a microarray technique that targets the same pathogens as the filmarray respiratory panel, with the exception of coronaviruses hku1, nl63 and oc43 and the 3 aforementioned bacteria. among enteroviruses, this technique only detects echoviruses, but it can differentiate them from rhinoviruses. the extraction of nucleic acids was carried out with the qiasymphony system sp (qiagen) using the qiasymphony virus/bacteria midi kit (input volume 1500 μl, output volume 110 μl). the pneumovir assay was performed according to manufacturer's instructions, and detection and interpretation of the results were conducted by a carreader (genomica). as the molecular tests used were presumably more sensitive than the reference standard (viral culture), we constructed a "composite" reference standard to avoid bias in establishing the specificity of the evaluated tests. this "composite" reference standard was constructed as follows: when discrepant results between the 2 molecular techniques were observed, a third molecular technique was performed (argene, biomérieux, marcy l'etoile, france). a sample was considered truly positive for a pathogen if at least 2 molecular techniques were positive for this pathogen. for the targets included in the filmarray test but not in the clart pneumovir panel (coronaviruses hku1, nl63 and oc43), only the specificity of the positive results were evaluated using the argene or laboratory developed tests. data were analyzed using two sided exact chi-square fisher's tests followed in case of statistical significance by chi-square trend tests. the statistical software used were medcalc v14.12.0 and ibm spss v24.0. a total of 299 samples (93 npa and 206 nps) from 291 patients were analyzed: 149 samples were obtained from 142 children, of whom 62 were female and 80 were male (mean age: 1 years and 10 months old; median: 7 months old); and 150 samples were obtained from 149 adults, of whom 73 were female and 76 were male (mean age: 53 years old; median: 52). for children, 80% (119/149) of samples were positive for at least one pathogen. the pathogen detected, ranging from most to least, was rhino/enteroviruses (49), influenza b (32), influenza a (25), coronaviruses (22), adenovirus (18), metapneumovirus (10), rsv (7) and parainfluenza (2). for adults, 63% (94/150) of samples were positive, and the detected viruses were influenza a (32), influenza b (26), rhino/enteroviruses (13), coronavirus (12), adenovirus (8), metapneumovirus (6) and rsv (4). no parainfluenza viruses were detected in the adult patients. all influenza a isolates were a(h1n1)pdm09 with the exception of one a(h3n2) from one adult patient. influenza b strains were not subtyped. co-detection was more common in children than in adults (35.3% vs 8.5%; p < 0.001). table 1 details the sensitivity and specificity observed for the different techniques, depending on the pathogens. table 2 provides the rate of false negative results, partial agreement (meaning at least one but not all the expected pathogens were detected) and complete agreement (meaning all the expected pathogens were detected) of the non-molecular techniques and molecular techniques compared to the established standard. the filmarray test produced fewer false negative results and partial results compared to non-molecular techniques and the clart pneumovir test (p < 0.001). false negative results with molecular techniques were significantly more frequent in samples with codetections compared to those with only one pathogen: 12% vs 3% for the filmarray test (p = 0.034) and 76% vs 11% for the clart pneumovir test (p < 0.001). similar result was also observed for cell cultures when limiting the analysis to culturable viruses (100% vs 37%; p < 0.001). table 3 reports the mean turnaround time (tat) of the techniques from the reception of the sample in the laboratory to the introduction of the results into the laboratory information system. the lfc and fil-marray techniques have the shortest tat, while cell cultures and the clart pneumovir test have the longest tat. the tat for negative cell cultures is about 3 weeks as they are discarded after this delay. tables 4 and 5 provide the detection rates for different techniques for influenza a and b, depending on the duration of the cough or the age of the patients. there is an effect of cough duration for influenza a with the antigen detection test (p = 0.032) and cell cultures (p = 0.010), due to a decrease in diagnostic sensitivity with cough duration (p = 0.017 and p = 0.010, respectively). the decrease is observed as soon as the cough duration exceeds 5 days. an age effect was also observed for influenza a with the antigen detection test (p = 0.006) and cell cultures (p = 0.023), due to a decrease in sensitivity with age (p = 0.002 and p = 0.004, respectively). the decrease is observed from the first age category with the antigen detection and from 15 years for the cell cultures. no effect of cough duration or age was observed with the detection of influenza a for the filmarray and clart pneumovir tests or on the detection of influenza b with any method. as expected, molecular techniques were observed to be the most sensitive. however, the performances of the two evaluated techniques differed; lower sensitivity for the detection of several targets was observed for the clart pneumovir test, notably this technique only detects echoviruses among all enteroviruses. for adenoviruses, metapneumoviruses and rsv, the explanation probably relies more on technical considerations. indeed, false negative results with the filmarray and the clart pneumovir tests were not always for samples with a low viral load, as indicated by the cycle threshold (which correlates with the viral load) of the third molecular technique used to elucidate discrepant results between the first two techniques. as an example, the mean ct value and standard deviation of negative and positive samples for adenoviruses with clart pneumovir were 32.7 (1.6) and 31.8 (2.2) respectively (p = 0.402). the choice of primers and probes used to design tests can determine whether a certain strain of virus will be detected. although the filmarray test had a better sensitivity than the clart pneumovir test for the detection of adenoviruses, it may lack sensitivity in detecting certain strains, which could impact the management of immunocompromised patients (song et al., 2016) . a huge advantage of molecular techniques is that they allow the recovery of those pathogens that are difficult to culture, that will not grow in culture or for which no antigen detection tests are available. in the present study, the rate of complete detection of the pathogens present in the samples was far more important with molecular techniques compared to non-molecular ones. this has already been reported. (weinberg et al., 2004) viruses with poor detection using non-molecular techniques primarily include adenoviruses, metapneumoviruses and rhino/enteroviruses. there were also positive samples for coronaviruses (mahony, 2008) which, in our experience, do not commonly grow with routine cell lines, even if coronavirus nl63 was originally described in llc-mk2 (van der hoek et al., 2004) . the rate of co-detection was more important for children, which is a common finding and could be explained by notably increased, longerlasting viral shedding in children under 3 months of age due to less developed mucosal immunity (sharma et al., 2012) . the co-detection of viruses in children is apparently not linked with a more severe outcome (comerlato scotta et al., 2016) , and the molecular detection of a virus in a respiratory sample is not always associated with symptoms. the type of detected virus and the age of the patient can be helpful to decide on a care plan (self et al., 2016) . as previously stated, false negative results with molecular techniques were significantly more frequent in samples with multiple pathogens compared to those with only one pathogen; this finding could be due to possible competition for the reagents when multiple targets are to be detected (bezerra et al., 2011) . the specificity of all molecular techniques was over 98%, except that for rhino/enteroviruses with the filmarray test (96.6%). the differences observed between molecular techniques could also be due to primer and probe choices. overall, the clart pneumovir is less sensitive than the filmarray except for influenza b and its hands-on time and turnaround time are longer. only 3 positive samples were analyzed for metapneumovirus using an antigen detection test, as it was substituted for adenovirus test during the evaluation. this test nonetheless appeared more sensitive than the cell culture tests, as no metapneumovirus was recovered from cell cultures. this finding was under the expected sensitivity, which is approximately 50% according to the literature (tang and crowe, 2011) . the apparent low sensitivity of non-molecular techniques observed for adenoviruses deserves comment. indeed, prolonged shedding after the primary infection is classically described for adenovirus. adenoviruses can also be detected in tonsillar tissue or isolated from a throat sample of up to 11% of healthy children (song et al., 2016; kalu et al., 2010) . these states of prolonged shedding or "latency" are more likely to be detected with molecular techniques. it is also noteworthy that 85% of adenoviruses detected in this evaluation were associated with one or more other pathogens, supporting the hypothesis that they could be bystanders in some cases. the sensitivity of cell cultures for rhino/enterovirus was also low and could be partially attributed to the fact that group c rhinoviruses do not grow on standard cell cultures (jacobs et al., 2013) . enteroviruses grow inconsistently on cell cultures, depending on their type, and no cell line enables the detection of all strains (stellrecht et al., 2011) . unfortunately, rhinoviruses and enteroviruses in this study were not typed. the presence of multiple viruses in a sample also influences cultures because the growth of the fitter or more abundant virus can mask the growth of others (george et al., 2002) . for example, on the 46 false negative cultures for rhino/enteroviruses, 16 were positive for another virus. likewise, for adenoviruses, for the 22 false negative cell cultures, 8 recovered another virus. molecular techniques supposedly do not suffer from this drawback, although as previously discussed, a higher rate of false negatives was observed when multiple pathogens were present in the sample. cell cultures, however, can enable the detection of unsuspected viruses. this detection was the case in this evaluation for one measles virus, which was not originally suspected, and 2 herpes simplex viruses and 5 cytomegaloviruses (cmv). in one case, cmv could explain the symptoms exhibited by a 3-month-old patient with a fever without focus of infection. the other cases were more likely to be recurrences or prolonged shedding. regarding the sensitivity of antigen detection tests for influenza, influenza a is more easily detected than influenza b, which has already been reported (busson et al., 2014) . the decreased sensitivity for influenza a with antigen detection and cell cultures, which depends on the age of patients, can be explained by the higher viral shedding in children, and also because for younger children, npa were preferred to nps. aspirates usually increase the detection rate for viruses over swabs (loens et al., 2009) . the decreased sensitivity of antigen detection and cell cultures when the duration of the cough increases is expected, because viral shedding is more important in the first days of the disease (aoki and boivin, 2009 ). the reason for the lack of similar findings for influenza b is unclear. the absence of sensitivity loss for molecular techniques, depending on the age of the patients and the duration of the cough, can be attributed to the high sensitivity of the techniques. usually, antigen detection tests have a faster turnaround time than cell cultures but a lower sensitivity except for viruses growing poorly, especially metapneumovirus and rsv, for which the sensitivity of antigen detection tests can be better than the one of cell cultures. the specificity of non-molecular tests was above 98%. false positive results in cell cultures can occur due to cross-contamination between wells on culture plates. antigen detection tests based on lateral flow chromatography are usually the easiest and fastest to perform. techniques based on immunofluorescence are slightly more time-consuming. currently, totally automated techniques with a short hands-on time, such as filmarray, can be realized in a time frame comparable to that of lfc tests. however, only one can be performed at one time, and the analyzer remains occupied for slightly more than an hour. the number of required instruments is based on the test volume at each facility and the desired tat. cell cultures remain the most time-consuming techniques, and results often arrive too late to impact patient management (ginocchio, 2007) . this was also the case for clart pneumovir in our setting, as it was only performed once a week due to the complexity of the corresponding analytical process. in belgium, non-molecular techniques for the diagnosis of respiratory viruses are reimbursed by the social welfare program, which is not the case for molecular techniques which are charged to the patients. during this evaluation, molecular techniques were free of charge for patients, but they cost approximately 135 euros per patient. the necessity of molecular tests must be seriously considered before prescription. these tests should probably be reserved for the most severely ill patients, for whom rapid and comprehensive microbiological evaluation is most likely to impact patient management. as a comparison, antigen detection tests cost, including workforce, can range between 5.5 and 14 euros depending on the test and cell cultures cost about 15 euros. however, non-molecular tests are reimbursed by the social welfare in belgium permitting a broader prescription. the possibilities to implement molecular tests can vary between facilities and countries depending on local policies. using molecular rather than non-molecular techniques could impact patients' isolation strategies (richardson et al., 2016) , antibiotic/antiviral use or reduce of the length of stay (brendish et al., 2017; rogers et al., 2015) , but cost-benefit analyses are difficult to appraise because of many intertwined elements. molecular techniques have considerably increased our capacity to detect respiratory viruses in a timely manner. the main factors limiting a wide utilization of these techniques are cost and the difficulty to absorb the workload in large facilities with numerous samples to analyze per day. another drawback is that latent viruses or traces of genetic material may be detected. the interpretation of a positive result might be difficult, and it must be carefully correlated to the clinical history of the patient as the presence of a virus in the respiratory tract is a factor exposing to bacterial superinfection (vareille et al., 2011) . it is possible to quantify the viral load in a respiratory sample, but there are conflicting reports regarding correlations between viral load and outcome (granados et al., 2017; wishaupt et al., 2017) . technical improvements should be made to prevent variation in quantification caused by sample dilution with saline instilled during aspirates or by the variable quantity of sampled material with swabs. these improvements could render comparisons between studies more reliable and permit the establishment of a threshold, above which detected viruses are indeed involved in an ongoing infectious process. in addition, a positive antigen detection test usually correlates with a high viral load in a sample, and a positive cell culture can only be achieved with infective viral particles. whatever technique is used, fully understanding the benefits and limitations of each is crucial for interpretation. the choice of technique should depend, besides financial considerations, on the necessary sensitivity and speed based on symptoms, comorbidity and risk of a detrimental outcome for each patient. a tertiary care hospital should be able to offer diagnostic tools suitable for each individual case; if testing all patients with molecular techniques is not possible, the use of nonmolecular techniques is preferable over nothing at all. influenza virus shedding -excretion patterns and effects of antiviral treatment viral and atypical bacterial detection in acute respiratory infection in children under five years emerging 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role of cell culture for virus detection in the age of technology the origins of virology optimal sampling sites and methods for detection of pathogens possibly causing community-acquired lower respiratory tract infections detection of respiratory viruses by molecular methods comparison of respiratory virus shedding by conventional and molecular testing methods in patients with haematological malignancy impact of a rapid respiratory panel test on patient outcomes respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia the developing human preterm neonatal immune system: a case for more research in this area performances of filmarray respiratory panel v1.7 for detection of adenovirus in a large cohort of pediatric nasopharyngeal samples: one test may not fit all enteroviruses and parechoviruses respiratory syncytial virus and human metapneumovirus identification of a new human coronavirus the airway epithelium: soldier in the fight against respiratory viruses superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children pitfalls in interpretation of ct-values of rt-pcr in children with acute respiratory tract infection the author acknowledges the team of the virology none declared. this study was approved by the ethics committee of university hospital saint pierre (brussels). key: cord-025556-oyfx3ij5 authors: thunström, linda; ashworth, madison; shogren, jason f.; newbold, stephen; finnoff, david title: testing for covid-19: willful ignorance or selfless behavior? date: 2020-05-08 journal: nan doi: 10.1017/bpp.2020.15 sha: doc_id: 25556 cord_uid: oyfx3ij5 widespread testing is key to controlling the spread of covid-19. but should we worry about self-selection bias in the testing? the recent literature on willful ignorance says we should – people often avoid health information. in the context of covid-19, such willful ignorance can bias testing data. furthermore, willful ignorance often arises when selfish wants conflict with social benefits, which might be particularly likely for potential ‘super-spreaders’ – people with many social interactions – given people who test positive are urged to self-isolate for two weeks. we design a survey in which participants (n = 897) choose whether to take a costless covid-19 test. we find that 70% would take a test. surprisingly, the people most likely to widely spread covid-19 – the extraverts, others who meet more people in their daily lives and younger people – are the most willing to take a test. people's ability to financially or emotionally sustain self-isolation does not matter to their decision. we conclude that people are selfless in their decision to test for covid-19. our results are encouraging – they imply that covod-19 testing may succeed in targeting those who generate the largest social benefits from self-isolation if infected, which strengthens the case for widespread testing. covid-19 rapidly developed into a pandemic, and by 26 march 2020, the usa had the highest reported number of infected people in the world. a general message from public health experts is that effective control of the spread of covid-19 requires widespread medical testing (who, 2020) . the testing will serve to determine whether people are infected or not, and ideally also if they have been infected and have reached immunity status. three reasons motivate widespread testing. first, if a person learns that they are infected, they can take appropriate measures to reduce the probability of infecting others, such as the recommended 14-day self-isolation (harvard medical school, 2020) . second, the data provided by widespread testing will better inform the need for the current social distancing policies (e.g., sheltering at home, avoiding gatherings of 10 or more people, keeping at least 6 feet away from other people and temporarily closing schools, universities, daycare centers, major sports leagues, cultural events and public spaces) (stock, 2020) . third, testing provides data about the asymptomatic rate in the usa (the share of infected people who show no or very mild symptoms) and insight into how close americans are to developing herd immunity to covid-19. this information is useful in order to determine when and where it makes sense to relax these costly social distancing measures. while the usa has increased its capacity to conduct more testing, around 1.8% of the population had been tested by 30 april 2020 (covid tracking project, 2020) . the effectiveness of testing in controlling covid-19 depends largely on how the tests are conducted. the ideal scenario is to test everyone, but that is infeasible. a second-best scenario is random sample testing (stock, 2020) . but for random testing to be effective, all sampled people would either need to voluntarily agree to be tested (which is unlikely, as we explain) or be forced to do so (which is illegal in the usa). the third-best (and first-best feasible) strategy is voluntary random testing. this strategy, however, could lead to a systematic selection biaswe will only test those individuals who prefer to learn their health status regarding covid-19; a significant fraction of people might not want to know. these individuals might find that their private costs outweigh any social benefits from not infecting others. this implies that they might want to avoid testing. if their private costs include above-average opportunity costs of social interactions, then individuals who decline to be tested may also be disproportionately likely to become superspreaders. to understand why this might happen, consider the ongoing literature on willful ignorance of health information (also called strategic ignorance). while standard economic theory suggests people never ignore information that enables them to adjust behavior (stigler, 1961) , many new studies find that people willfully ignore medical diagnoses, even when such knowledge would enable them to adjust behavior to better accommodate their health condition (sharot & sunstein, 2020) . for example, we see willful ignorance in many people at risk for breast cancer (thompson et al., 2002) , alzheimer's disease (cutler & hodgson, 2003) , hiv (hightow et al., 2003) and huntington's disease (oster et al., 2013) . the study by ganguly and tasoff (2017) is particularly relevant: they observe people will avoid a costless test for herpesa disease for which there is currently no cure, but for which information is useful in that it helps adjust behavior. people have also been found to willfully ignore health risk information, such as calories in food (thunström et al., 2016; woolley & risen, 2018; sunstein, 2019; thunström, 2019; nordström et al., 2020) . willful ignorance of health outcomes is likely to arise when people are torn between what they think they should do and what they want to do (thunström, 2016; woolley & risen, 2018) , or when ignorance allows them to form optimal expectations (downplay the probability of a bad health outcome; oster et al., 2013; nordström et al., 2020) . for instance, a person may think she should eat healthy, but want to indulge in ice-cream-she might then choose to avoid learning about the exact amount of calories in the ice-cream in order to avoid either her inner pressure to reduce the ice-cream consumption or the guilt from consuming it despite being aware of the calorie content. in this paper, we explore self-selection in covid-19 testing in the usa. we examine if people willfully avoid getting tested for covid-19, and, if so, what individual or household characteristics and circumstances are associated with testing avoidance. given that random voluntary testing is not yet available in the usa, there are no observational data to rely on for our analysis. we therefore design a hypothetical randomized controlled trial (rct). we recruit a nationally representative sample of 1000 participants. the study entails two treatments, across which we vary information about the potential emotional cost of testing before asking if participants would agree or disagree to a financially costless covid-19 test. in the baseline treatment, we inform participants that if they are found to be infected, they are urged to self-isolate at home for 14 days. in the high-cost treatment, we tell participants that those who test positive are strongly urged to self-isolate, which may be in a self-quarantine site away from home. we assume the test itself is costless and that the only cost incurred from a covid-19 test is the recommended self-isolation for 14 days should the test come back positive. if people are concerned only with their private benefits and costs from taking a covid-19 test, those with large private benefits and low private costs from knowing that they are infected will be the most likely to get tested. private benefits are significant for people at elevated risk for severe health consequences if they contract the virus or with family members at higher risk, while private costs are low for those who generally live a solitary life, professionally and in private. we expect elderly and those who haveor have a family member that haspre-existing conditions to be more willing to test. furthermore, we expect those at the lowest risk of losing out financially (e.g., risk to labor income or health care costs) or emotionally from self-isolating (i.e., if they are introverts who attach a low value to social interactions) to be the most willing to get tested for covid-19. in contrast, if people are concerned only with social benefits and costs, we would expect those most at risk of exposing others to be the most likely to get tested (e.g., potential 'super-spreaders'; i.e., people with jobs that entail mixing with other people, people living in urban areas, young people and people who are extroverts and attach a high value to social interactions). people with the potential to be super-spreaders might be torn about learning whether they are infected by covid-19. both private costs and social benefits from being urged to self-isolate for the next 14 days (harvard medical school, 2020) might be high for this group, and so influence their testing decision in opposite directions. for example, consider the behavior of an extrovert who values social interactions highly. if unsure of being infected, the extrovert behaves just as if she is not infected (e.g., as found for huntington's disease, by oster et al., 2013) . self-isolation means this person needs to give up highly valued social interactionsa factor that might deter them from a voluntary test for covid-19. at the same time, their self-isolation provides particularly meaningful private and social health benefits from reduction in exposure to, and spread of, the disease. these benefits to learning whether they are infected might encourage them to voluntarily test. it is an open question if the benefits outweigh the costs, causing the extrovert to take a costless covid-19 test. similarly, imagine a store clerk who risks losing income if self-isolating for 14 days. the potential private loss of income would deter them from testing, while the social benefits from reducing disease spread would encourage taking the test. again, the decision to test becomes an open question. previous studies show that willful ignorance arises when prosocial behavior is privately costly (dana et al., 2007; conrads & irlenbusch, 2013; onwezen & van der weele, 2016; gigerenzer & garcia-retamero, 2017; grossman & van der weele, 2017) . in this study, we find that around 70% of people want to take a costless covid-19 test. as might be expected, people who worry more about their health are particularly likely to want to take the test. we also find that those most likely to want to take the test are those most likely to spread the virus if unaware of their infection, including young people and extroverts with a preference for socializing. extroverts might also be at the highest risk for being infected, but even when we control for personal risks (amount of social interactions and worry about own health), we find they are more willing to test. the ability to afford to self-isolate for 14 days does not seem to affect the willingness to test. our results suggest that there is a significant amount of selflessness in the decision to test; people appear to be highly concerned about the social benefits from testing for covid-19 and little concerned about private costs. in addition, we do not find the expected treatment effect of our experimental manipulation of the private cost to testing (i.e., the location of self-isolation (at home or in a facility away from home) does not seem to matter to the testing decision). our expectation was that willful ignorance would be higher if selfisolation might take place away from home, since we assume self-isolation away from home is perceived as more costly. one interpretation of the lack of expected treatment effect is that it lends further support to the idea that private costs play a negligible role in the decision to test for covid-19. our results matter because they underscore the value of widespread covid-19 testing, even if such testing cannot be done randomly. our findings suggest that widely available and costless voluntary testing will target rather than scare off those most likely to be 'super-spreaders'. to test people's willingness to take a financially costless covid-19 test, we designed a hypothetical field experiment. the experiment is a rct with a between-subjects design, consisting of two treatments. in the first treatment (treatment baseline), participants were told they would be urged to selfisolate at home, if having tested positive. in the second treatment (treatment high cost), they were told they might be urged to self-isolate at a special site away from home. participants (n = 1000) were recruited by the research firm qualtrics, and the sample was required to be nationally representative along the dimensions of gender, age, education, race, income and residential region (east, west, north or south). while the recruitment costs from qualtrics are higher than when recruiting from amazon mechanical turk or turk prime, qualtrics continuously quality checks participants, which enabled us to avoid many issues that may otherwise contaminate online panels (e.g., see chandler & paolacci, 2017; sharpe wessling et al., 2017) . participants received standard qualtrics compensation to participate in a survey. testing for the sequence of the experimental study was as follows: step 1: all participants were asked screening questions at the front end of the survey about their gender, age, education, race, income and region in order to ensure the sample met us national quotas for those characteristics. step 2: participants were asked whether they had already been tested for the covid-19. if 'yes', they were asked why they got tested, the outcome of the test, how many days prior to survey participation they had taken the test and if the test was costly. if 'no', they were randomized into one of the two treatments and asked about their willingness to take a test. specifically, if in treatment baseline, they received the following information: currently, us authorities are working to test more people for the coronavirus. legislators are urging people who test positive, i.e., are found to be infected by the virus, to self-isolate at home for 14 days. if in treatment high cost, they were instead told: currently, us authorities are working to test more people for the coronavirus. legislators are urging people who test positive, i.e., are found to be infected by the virus, to self-isolate for 14 days. some states have started building self-quarantine sitessites where people who have the virus would be isolated for 14 days. if people stay at those sites, it is easier to ensure they comply with the guidelines to self-isolate. thereafter, participants in both treatments were asked: if you were given the opportunity to take a coronavirus test for free within the next 3 days, would you take the test? default alternatives to get or not get information have been shown to affect observed choices of ignorance (grossman, 2014) . to avoid nudging participants toward any particular answer, there was no default alternative; participants needed to choose either "yes, i would take the test," or "no, i would not take the test." step 3: all participants were asked about their current level of social distancing (how many people outside their household they had been within 6 feet of in the last 3 days; how many gatherings with more than 10 people they had participated in; and self-assessed level of compliance with social distancing). they were also asked whether they supported the public recommendations for social distancing in general. step 4: participants were asked questions about factors that might affect the perceived cost of a positive covid-19 test (implying social isolation for 14 days), as well as the perceived benefits from being able to make behavioral adjustments. they were asked about their job situation, job security and possibility of the main income provider in the household taking sick leave; risk factors for contracting the virus (e.g., living in a urban area, working in a health care facility, working in a grocery store or pharmacy); risk factors (for self or any children) for suffering severe health consequences if contracting the virus (e.g., underlying health conditions that increases the risk, such as cancer, obesity, diabetes, etc.); level of extraversion (francis et al., 1992) ; and social lifestyle. step 5: participants were asked about religious belonging, religiosity, political affiliation and social and fiscal conservatism (everett, 2013) . the full survey can be found in the online supplementary material. of our total sample, 103 participants stated that they had already been tested, while 897 stated that they had not already been tested. we asked those who had been tested for the primary reason they had taken the covid-19 test. table 1 shows their answers. as expected, given the current prevailing strategy in the usa of focusing the limited testing on people who are symptomatic, most people got tested because they themselves showed symptoms (almost 55%) or because someone close to them either showed symptoms or was diagnosed with covid-19 (around 35%). summary statistics for the 103 participants who had been tested before participating in our study are shown in the online supplementary material. our analysis focuses on the 897 participants who stated that they had not been tested for covid-19. due to a coding error in the survey at the beginning of the data collection, seven participants did not respond to the question on whether they were a business owner, employed or unemployed. we dropped these seven participants from our analysis, and we were left with 890 observations. table 2 presents the summary statistics for these participants. unless otherwise stated, all of the remaining analysis focuses on the results for this group of participants. table 2 shows that 53% of participants who had not yet been tested for covid-19 are female. the variable age describes a participant's age in years, and the mean age in our sample is 47 years. the variable high-risk age is a dummy variable that takes the value 1 if a person is aged 65 years or older. table 2 shows that 16% of our participants are aged 65 years or older. the variable rural area takes a value 1 if participants stated that they live in a rural area and 0 if they live in an urban area. table 2 shows that 35% of our participants live in a rural area. the variables emotional tolerance and financial tolerance are dummy variables that take the value 1 if the participant answered that the maximum time (from the time of taking the survey) he/she would be able to emotionally or financially sustain social distancing was 14 days or longer, given the 14-day recommended time to self-isolate if you test positive for covid-19. these variables take the value 0 if their stated maximum time was 13 days or less. table 2 shows that 84% of participants stated that they can afford to continue their testing for covid-19 7 current level of social distancing for 14 days or more, while 87% stated that they can emotionally tolerate another 14 days or more of their current level of social distancing. the variable lifestyle impacthealthy is an index that measures the extent to which social distancing has changed participants' behavior in a healthier direction. participants were assigned a value 1 for each of the following: if they stated that social distancing had (a) increased consumption of vegetables, (b) decreased in-between-meals snacking (excluding fruits and vegetables), (c) increased time spent in green spaces, (d) increased time spent doing strenuous or (e) moderate exercising and (f) reduced stress. this variable could take a value between 0 and 6, where higher values represent healthier changes. the variable lifestyle impactunhealthy is an index that measures the extent to which social distancing has changed behavior in an unhealthy direction. participants were assigned a value 1 for each of the following: if they stated that social distancing had (a) decreased consumption of vegetables, (b) increased in-between-meals snacking (excluding fruits and vegetables), (c) decreased time spent in green spaces, (d) decreased time spent doing strenuous or (e) moderate exercising and (f) increased stress. this variable could also take a value between 0 and 6, where higher values represent a higher number of unhealthy changes. the summary statistics in table 2 suggest that social distancing has led to more unhealthy behavior than it has healthy behavior, as implied by the lower mean value of lifestyle impacthealthy. we note, however, that these variables are crude measures of the lifestyle impact from social distancing, where each change is given equal weight, although some changes might have a more important health effect than others. 1 the variable business impact takes a value 1 if a participant is a business owner whose business has experienced negative impacts due to covid-19, such as their operation losing income, going out of business or being at risk of going out of business. the variable employer impact takes a value 1 if a participant is an employee and his/her employer has experienced a negative impact due to covid-19, such as their employer losing income or being at risk for going out of business, if they had experienced pay cuts, reduced working hours or were on unpaid leave as a result of the virus. 2 table 2 shows that of participants who are business owners, or where until a month ago (n = 89), 64% had experienced a negative impact on their business from covid-19. of participants who are employees, or were until a month ago (n = 477), 57% had experienced a negative impact on their job security, payment or employer revenues. the variable social distant compliant -6 feet measures how many people a participant has been close to in the last 3 days. participants could state 'none', 1 person, 2-3 people, 4-5 people, 6-9 people, 10-15 people, 16-25 people, 26-50 people or 'more than 50 people'. we assigned participants the midpoint of the range they picked. for those in the highest range (50 and more), we assumed the same size interval as the second to highest interval (i.e., we assumed an endpoint of the last interval equal to 75 people). table 2 shows that the average number of people that participants had been close to during the last 3 days, besides their household members, was 5.76. although not reported in the table 2 , the median was 2.5. the variable social distant compliantgroups measures how many times during the last 3 days a participant has been in a room with 10 or more people. participants could state a value anywhere between zero and '5 or more times'. the median of this variable is 0. table 2 shows that participants on average had been in a room with 10 or more people around 0.5 times during the last 3 days. the variable self health risk measures the sum of 10 underlying health conditions that would put the participant at higher risk for developing severe health consequences if becoming infected with covid-19. these health conditions include chronic respiratory conditions, heart disease, neurological conditions, diabetes and obesity (cdc, 2020). the variable child health risk measures the same sum of underlying health conditions for a child in the household. the variable worry about own health is based on the stated extent to which participants worry about their own health due to covid-19, where the value 0 indicates 'not at all' and the value 3 indicates 'a lot'. the dummy variable insurance takes a value 1 if a participant states that he/she has private health insurance or is covered by medicare or medicaid and 0 if the participant stated not having any coverage. table 2 shows that 69% of participants have insurance or medicare or medicaid coverage. the dummy variables republican, democrat and other political party take a value 1 if a participant identifies as republican, democrat or neither, and 0 otherwise. about 34% of participants identify as republican, 43% as democrat and 23% as other. that they had been placed on unpaid leave; and 23% of workers said that they had had their pay reduced. the variable extrovert is based on the extraversion scale developed by francis et al. (1992) and includes participants' answers to questions such as "are you a talkative person?" and "can you easily get some life into a rather dull party?" in addition to the extraversion scale, we also asked participants to indicate their level of agreement with statements about their general social lifestyle, such as "my social life is very important to me," and "in my spare time, my favorite thing to do is to spend time with friends." if the participant answered yes to three or more of these questions, they were assigned a 1 for the extrovert dummy variable. the answers to these statements were, however, highly correlated with the extraversion scale, so they were excluded from our analysis because they provided little or no additional information. when we pool participants from both treatments who had not been tested prior to participating in our study (n = 897), we find that 69% of participants would be willing to take a costless covid-19 test. 3 we find no difference in shares of participants willing to test across treatments (pearson χ 2 (1.619); p = 0.203), suggesting that the location of self-isolation (at home or in a facility away from home), in the event the test comes back positive, is not an important determinant of people's willingness to test for covid-19. not only is the treatment effect small, it is also of the unexpected signthe share of people willing to test if self-isolation would happen at home is smaller (67%) than the share of people willing to test if self-isolation might happen at a facility away from home (71%). if anything, people might be slightly more inclined to test if a positive result could lead to isolation away from home (potentially due to this also signaling the greater severity of the covid-19 situation), but the size of the effect is too small for us to detect with our sample size. we have ruled out 3 participants who stated that they would prefer not to take the test (278/897) were asked for their reasons not to want to take the test. they were given the following alternatives and were asked to mark all that apply: "i would not change my behavior if i learned i had the virus" (11%); "i do not want to self-isolate for 14 days" (4%); "my job prevents me from self-isolating for 14 days" (6%); "i think i have already had the virus" (2%); "it would cause me emotional discomfort if i knew i had the virus" (7%); "it doesn't matter to me if i get tested or not" (30%); other (53%). the numbers in parentheses show the shares of participants who agreed with the statement. as shown, a large share of participants stated that the test would not matter, and that they would not change their behavior anyway. a potential reason for the prominence of these reasons could be that they are already highly complying with social distancing. (table 3 shows that the more people comply with social distancing, the less likely they are to want to take a covid-19 test.) however, a large share also states 'other', suggesting that the alternatives presented to our participants did not cover the full range of reasons as to why people may refrain from testing. we encourage future research to further explore these reasons. that this absence of identifiable average effect masks any potentially 'rational' heterogeneity in the population (i.e., we have explored whether there exists a treatment effect for subgroups of the population, such as those with children, higher-quality homes (as measured by income) or health anxiety (as measured by underlying health conditions)). one interpretation of the absence of treatment effect is that people assign little weight to the personal cost associated with the location of self-isolating when they decide on whether to take a covid-19 test. we pool participants from both treatments and examine the determinants of willingness to test. we estimate a probit model. table 3 shows the resulting average marginal effects. concerns about own health are captured by the variable worry about own health. the results in table 3 imply that the more a person worries about their health due to covid-19, the more likely they are to take a test. the inclusion of this variable in our model also renders the coefficient for the variable that measures underlying health conditions (i.e., self health risk) small and statistically insignificant (if worry about own health is excluded from the regression, self health risk has the expected positive, and statistically significant, effect on willingness to test). we do not find that people with children who have underlying health conditions are more likely to take the test, perhaps due to the expectation that people of young age are less affected. this result remains robust if we recode the variable child health risk into a dummy variable that takes the value 1 if any child in the household has one or more underlying health conditions. variables that affect a person's financial situation do not seem to matter to the willingness to take a covid-19 test. in particular, we do not find an effect from business impact or employer impact. we examine the robustness of these findings to alternative measures of business and employer impact. first, we instead include the multitude of variables underlying business impact and employer impact in the regression model (see footnote 2), but we do not find an effect from any of those variables that is close to statistically significant at even the 10% level. furthermore, we do not find an effect on willingness to test from financial tolerance. second, we recode the variables such that they range from little impact to severe impact (ranging from if a business owner or employee has experienced no adverse effects from covid-19, to one or multiple effects). again, we find no statistically significant effects from these variables on the willingness to test. taken together, these results suggest that people do not consider their own private costs from a positive test when deciding on taking a covid-19 test. similarly, we find no effect on the willingness to test from emotional tolerance, implying that the private emotional cost from social isolation in the event of a positive test might not affect the decision to take a covid-19 test. we find that healthy younger people are more likely to take a test than healthy older people, as implied by the negative parameter estimate for high-risk age. this age effect is consistent with findings in other studies that standard errors in parentheses. *p < 0.1, **p < 0.05, ***p < 0.01. observe older people avoid health-related information more than younger people (thunström et al., 2016; gigerenzer & garcia-retamero, 2017) . while this result could imply that older people are more likely to be willfully ignorant, it is also in line with the idea that those with more social interactions (younger people) are more likely to get tested. studies find that people below 60 years old have more social contacts, and therefore are more likely to transmit infectious diseases (mossong et al., 2008) . furthermore, we find that those who have met more people during the last 3 days are more willing to take the test, as suggested by the negative parameter estimate for social distance compliant -6 feet. furthermore, the potential 'super-spreaders'the extrovertsare more likely (by 8%) to take a test compared to the introverts. taken together, this suggests that social benefits weight heavily in people's decisions to test; those most at risk to spread covid-19 are the most willing to get tested. we find that republicans are 13% less likely than democrats to get tested. we speculate that this might be due to different information sources and because the risks of covid-19 might be portrayed differently in liberal and conservative popular and social media. we examined the robustness of this result by including a conservatism scale (everett, 2013) in the probit regression, and the result remains the same: people who are more conservative are less likely to want to take a covid-19 test. the conservatism scale is, however, not included in the final model, given its high correlation with the political dummy variables. finally, we find that people with health insurance, or coverage from medicare or medicaid, are around 8% more likely to take the test. this result might suggest that people who lack health care coverage use willful ignorance as a means to reduce anxiety about how to deal with a diagnosis. this would be in line with previous studies that suggest willful ignorance of health diagnoses may be motivated by the drive to reduce anxiety about the future (e.g., oster et al., 2013) . our results are robust to the inclusion of other explanatory variables, such as race, education, income and profession with high exposure to infected people (health care worker, store clerk, etc.). but these variables lack explanatory power or are highly correlated with other explanatory variables included in table 3 . the tests conducted prior to participating in our study were neither randomly offered to people (so far, testing for covid-19 in the usa has been primarily of individuals who showed symptoms), nor costless (52% of those who had tested prior to participating in our study stated the tests were financially costly and 60% said testing was time consuming). the value of data on observed testing is limited when it comes to helping us understand whether people might purposefully ignore such tests. while acknowledging that, we still compare our identified determinants of testing in table 3 to the determinants of having taken a test before participating in our study (see online supplementary material). while the levels of statistical significance vary, all coefficients are of the same sign as those in table 3 , except for four variables. having taken a test before participating in our study seems to be positively affected by having spent more time in groups with 10 or more people (i.e., social distance compliantgroups), as well as by a child having underlying health conditions (i.e., child health risk). employer impact has a (weakly) statistically significant positive effect on testing prior to participating in our study, while it is not statistically significant in table 3 . insurance is not a statistically significant determinant of having been tested prior to participating in our study, while it does have an effect in table 3 . widespread testing is one of the most important actions that us governments at any level can undertake to help slow down the spread of covid-19. given budget and testing supply constraints, it is likely that random, but voluntary, testing will be the most effective policy. we design a survey to examine the risks from self-selection into taking a covid-19 test. overall, we observe that around 70% of people would agree to a costless covid-19 test. we find that people who are more worried about their own health due to covid-19 are more likely to test, as are young healthy people, relative to older healthy people. ability to afford self-isolation for 14 days does not seem to affect the decision to test. furthermore, people who worry more about their health, and people with health insurance or health coverage through medicare or medicaid, are more likely to take the test, as are people identifying as democrats compared to republicans. contrary to our expectation, we also find that potential 'super-spreaders' are more likely than other individuals to agree to a costless covid-19. it could be that extroverts are more willing than expected to take a covid-19 test because their private cost of doing so is unusually low due to the broadly implemented social distancing at the time of data collection for this study. if extroverts are already relatively isolated (i.e., due to a stay-athome order and mandated closures by the state governor of public spaces, such as gyms, restaurants and bars), the personal cost of testing might be low. furthermore, extroverts might be more likely to get infected if they socialize more, which could be a 'selfish' motivation to get tested. however, we control for the current level of compliance with social distancing, which should address both of these private motivations for increased probability of testing, and we find that people who comply more are less motivated to take the test. we also control for their worry about own health due to covid-19. even so, the positive effect on willingness to test from being an extrovert persists. we therefore conclude that the positive effect of being an extrovert on willingness to test for covid-19 is likely due to social health benefits weighing more heavily in their decision than their private costs from potential self-isolation for 14 days, should the test come back positive. the importance of the prosocial motive in determining covid-19 testing is consistent with the results of the study by jordan et al. (2020) , who find that prosocial messages are more effective than self-interested messages in promoting behavior that prevent the spread of covid-19 (e.g., hand washing, hand shaking, hugging). our results suggest that the risks of adverse selection (in terms of failing to target the people most likely to spread the virus) in testing for covid-19 might be fairly low. this underscores the value of widespread testing, even if it cannot be truly random, and the importance of making such testing available nationwide in the usa as soon as possible. an important shortcoming of our analysis is that it builds on hypothetical survey data. it is well documented that survey answers may be affected by a 'hypothetical bias', meaning that people answer one way in a survey and behave in a different way when faced with real, incentivized decisions. this risk pertains to our study as well, and the hypothetical bias might be particularly pronounced if the choice to test for covid-19 is regarded as prosocial. several studies suggest that a hypothetical bias is particularly likely when measuring prosocial behaviorpeople often exaggerate the extent to which they engage in such behavior (e.g., murphy et al., 2005; vossler et al., 2012; jacquemet et al., 2013) . furthermore, it is possible that personal costs to the testing decision are less salient in a hypothetical context. once testing is more widespread in the usa, it will be important to examine who actually chooses to get tested, and the extent to which they deviate from the general population. that said, hypothetical and incentivized behavior generally correlate, such that an analysis like ours can provide important insights into the potential pitfalls of voluntary testing, prior to the actual testing. this is useful information to have on hand when designing an efficient and costeffective testing strategy. to view supplementary material for this article, please visit https://doi.org/10.1017/bpp. 2020.15 16 l i n d a t h u n s t r ö m e t a l . what you can do if you are at higher risk of severe illness from covid-19 lie for a dime: when most prescreening responses are honest but most study participants are impostors strategic ignorance in ultimatum bargaining most recent data to test or not to test: interest in genetic testing for alzheimer's disease among middle-aged adults exploiting moral wiggle room: experiments demonstrating an illusory preference for fairness the 12 item social and economic conservatism scale (secs)', plos one the development of an abbreviated form of the revised eysenck personality questionnaire (epqr-a): its use among students in england, canada, the usa and australia fantasy and dread: the demand for information and the consumption utility of the future cassandra's regret: the psychology of not wanting to know strategic ignorance and the robustness of social preferences self-image and willful ignorance in social decisions coronavirus resource center failure to return for hiv 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consequentiality: theory and field evidence on discrete choice experiments closing your eyes to follow your heart: avoiding information to protect a strong intuitive preference report of the who-china joint mission on coronavirus disease we thank the stroock fund for financial support. this study was approved by the irb at university of wyoming and was pre-registered in the aea rct registry (rct id: aearctr-0005587). key: cord-290978-e7imc11r authors: shevachman, m.; mandal, a.; mitragotri, s.; joshi, n. title: a long-lasting sanitizing skin protectant based on cage, a choline and geranic acid eutectic date: 2020-08-07 journal: nan doi: 10.1101/2020.08.04.20161067 sha: doc_id: 290978 cord_uid: e7imc11r the recent outbreak and rapid spread of coronavirus disease 2019 (covid-19) is a global pandemic and a massive public health crisis. covid-19 has also had a severe impact on the quality of life and mental health. while different health authorities such as who and cdc are encouraging adoption of strategies including hand washing and use of facemasks to reduce the spread of the pathogens and infections, adoption of these approaches requires substantial commitment. current hand sanitizers based on ethanol provide immediate protection, however, the protection rendered by such sanitizers is very short-lived due to their rapid evaporation. a long-lasting sanitizing skin protectant that can effectively inactivate sars-cov-2 and provide persistent efficacy over several hours will provide people the freedom to carry on with their activities without constant concerns about the cleanliness of their hands. herein, we describe a skin protectant, ionlasttm, based on an ionic liquid/deep eutectic solvent, formed by gras materials, choline and geranic acid (cage, cg-101), that provides protection for at least 4h after a single application. ionlasttm was formulated as a gel that facilitates easy application on the skin. tolerance of cg-101 was substantiated through a study in human volunteers. in vitro studies confirmed that ionlasttm effectively inactivates a human coronavirus hcov229e. a second human clinical study established that a single application of ionlasttm imparts protection against microbes that lasts up to several hours. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has emerged as a global pandemic that has infected >13million people worldwide with 580,000 deaths reported as of july 14, 2020[1]. the zoonotic virus, sars-cov-2 is responsible for the coronavirus disease (covid19) , which is associated with severe respiratory distress, fever or chills, cough, sore throat, congestion, nausea, and diarrhea [2] . older adults and individuals with underlying health conditions such as heart or lung diseases and diabetes patients are at a higher risk of experiencing mortality from the infection [3, 4] . the world health organization (who) declared covid-19 outbreak as a pandemic on march 11, 2020 and since then, the number of confirmed cases, deaths, and economic losses arising fromsars-cov-2 have amassed to an alarming level [5] . sars-cov-2 pandemic is not only a significant public concern in terms of human health, but its impact on the economy is also far-reaching [6] . apart from disproportionately affecting the vulnerable, especially the elderly and minorities, it has ravaged the economic wellbeing of many, and has placed an incalculable burden on local governments. in a mere few months, it has had an impact of greater than $2t on the economy, causing the largest global recession in history [7] . in fact, covid-19 is expected to result in long-term health issues straining the healthcare system more than ever before [8] . even as the number of new cases seem to plateau in certain parts of the us, the recent uptick in number of new cases in other parts of the country suggests that this is not merely an issue to be managed by the healthcare system, but a call for everyone in the society to play their part to minimize the risk of transmission [9] . sars-cov-2 is highly contagious and can be easily transmitted from human-to-human even when the subjects are asymptomatic, presymptomatic or have mild symptoms [10] . while vaccines and antiviral drug discovery remain paramount to curb this deadly pandemic, virus containment is of utmost importance and will require behavioral changes from each individual [11, 12] . regulatory agencies including the center for disease control (cdc) and who have issued guidance for behavioral changes including handwashing for 20 seconds, wearing masks when in public and social distancing [13, 14] . whilst these are simple in principle, strict compliance with these requirements is difficult to sustain. since it is neither practical nor viable to completely lockdown all aspects of the economy or daily lives, methods that can improve compliance will contribute significantly towards the outcome. since 1994, the us food and drug administration proposed ethanol at concentrations between 60% and 95% as safe and effective for hand sanitizers [15] . who recommends using formulations containing 80%v/v ethyl alcohol or 75%v/v isopropyl alcohol to inactivate pathogens effectively, including acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and most recently, sars-cov-2 [16] . however, the biggest shortcoming of such alcohol-based formulations is that their protective effect lasts only till the alcohol evaporates i.e. 15 seconds-2 minutes. once the alcohol evaporates there is no protection remaining on the hands leaving the person vulnerable to the very same risks of infection and transmission. hence, development of an effective hand sanitizer that can generate long-lasting protective effects can offer significant benefits in minimizing rampant viral transmissions and maximize virus inactivation in a pandemic situation like sars-cov-2 outbreak [15, [17] [18] [19] . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint there has been no significant innovation in hand santizers from the 1960s when alcoholbased sanitizers were first introduced [20] . here, we report a sanitizing skin protectant, ionlast tm that contains an ionic liquid/deep eutectic solvent, choline geranate (cg-101, 5% w/w) formulated in ethanol. cg-101 is non-volatile and is hypothesized to remain on the skin long after ethanol evaporates thus protecting the skin for several hours. cg-101 has already been confirmed to demonstrate broad-spectrum antimicrobial properties against 47 atcc strains of bacteria, virus, and fungi [21] [22] [23] [24] . the mechanism of antimicrobial action is mediated through lipid extraction and membrane disruption [21] . we anticipated that the same properties of cg-101 may support its efficacy against sars-cov-2. safety of cg-101 in human volunteers. safety of cg-101 (100% concentration) was tested in a 3-week human repeat insult patch test (hript) in 52 adult human volunteers. the concentration of cg-101 was 20 times higher than that used in ionlast tm . cg-101 was applied to the back of the subjects over a marked 2 x 2 cm 2 area and covered with an occlusive hypoallergenic patch to maximize penetration. distilled water was used as a negative control. the patches were removed by the subjects after 24h and a new one was applied by the site staff at the same exact site at the next visit. the subjects reported to the site for a total of 9 visits, 3 each week. during each visit the application area was observed by the study investigator and a new patch was applied. none of the subjects showed any skin reactions in the first 3 visits (7-days). 43 subjects did not display any irritation throughout the entire 21-day study. between visits 4 through 9, 8 subjects experienced some skin irritation. however, there was no follow-up treatment required for any of these 8 subjects that showed irritation. there were no other adverse events reported in the study. furthermore, upon completion of the 21-day study period, and a rest period of two weeks, cg-101 application with occlusion to a different part of the body, did not create any sensitization response at 24 and 48h. superior antibacterial efficacy of ionlast tm . in order to determine the prolonged protective effects of ionlast tm in contrast to the currently marketed products containing 70% ethyl alcohol, an in-vitro efficacy study was conducted using e. coli following modified astm e1153 methodologies. one milliliter of the test and comparator products were applied onto the precleaned glass surfaces and dried for 30min. glass slides were inoculated with 10µl of bacterial suspension at various timepoints (30min, 2h, and 4h). the test compound was chemically neutralized after an exposure duration of 5 min before transferring the samples to agar plates. the agar plates were visually compared for microbial growth after incubation for 48h at 37°c. no e. coli growth was observed for the ionlast tm -treated groups for 30min, 2h as well as 4h which clearly indicated the persistent antimicrobial activity of ionlast tm . in contrast, almost similar e. coli growth was observed for the saline-treated group and the comparator, ethyl alcohol 70% hand sanitizer treated group (fig. 1) . this indicates that alcohol-based products might have limited ability to provide long lasting protective effects against microbes. on the contrary, ionlast tm provided clear indication of long-lasting protection against pathogens and thus potentially reducing disease transmission during a pandemic. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint protection against hcov229e. based on the previously demonstrated broad-spectrum antimicrobial properties of cg-101 [23] , we anticipated that cg-101 and the skin sanitizer containing cg-101 (ionlast tm ) would effectively deactivate coronavirus likely through disruption of the phospholipid bilayer glycoproteinaceous envelope. we evaluated the virucidal effects of ionlast tm gel and the active cg-101 (5% w/w in purified water) against human coronavirus strain 229e (hcov229e) using a virucidal suspension test (in-vitro time-kill method) based on industry/regulatory-relevant global standardized methodologies (astm e1052-20). the log10 reductions from the initial population of the hcov229e following 15s and 30s exposure to ionlast tm was found to be >4.00. in fact, the percent reduction for both the time points was >99.99. similarly, we evaluated the virucidal activity of cg-101 (5% w/w) using the quantitative suspension test for exposure times, 5min and 10min. the percent and log10 reductions from the initial population of the hcov229e following 5min and 10min exposure to cg-101 (5% w/w) was found to be >99.99 and >4.00 respectively ( fig. 2 and tables s1 and 2). clinical studies approved by an irb were performed at bioscience laboratories, inc. (testing facility) in compliance with good clinical practice regulations to test the effectiveness of ionlast tm to provide extended protection against microbes. specifically, ionlast tm was applied to the forearms of human volunteers and the randomly assigned test sites on each forearm were challenged by application of staphylococcus aureus (s. aureus) at various times after the application of ionlast tm . the residual antimicrobial efficacy of ionlast tm was tested using a modification of the standardized test method described in astm e2752-10 (2015) in 12 healthy subjects. a significant (≥5.15) log10 reduction in s. aureus from the control was observed following 30min., 2h and 4h post-ionlast tm application. immediately following application and 30-min air-dry of the ionlast tm , the mean log10 microbial recovery for the treated subjects was found to be 0.86±0.00 in contrast to 6.30±0.05 for the untreated subjects. furthermore, the log10 microbial recovery, 2h following application of the ionlast tm , was found to be 0.86±0.00 for the treated subjects vs 6.24±0.07 for untreated subjects. interestingly, post 4h application of ionlast tm , the log10 microbial recovery for treated subjects was also 0.86±0.00 in comparison to 6.24±0.12 for untreated subjects. note that the lowest detectable limit of the study was 0.86 log10 cfu/cm 2 . ionlast tm thus demonstrated prolonged microbial protection for up to 4h after a single application ( fig. 3 and table s3 ). these data and the lack of any trend from 0.5h, 2h and 4h suggest that the effectiveness of ionlast tm could persist beyond 4h. sar-cov-2, the virus that causes the covid-19 disease has evolved into a global pandemic and is highly contagious affecting >25million people worldwide over a span of just 6 months. while the development of effective vaccines and antiviral therapies remain at the forefront of the global efforts to curb this deadly infection, the immediate response plan to control covid-19 should include breaking the chain of transmission of the virus and preventing associated illness and death [25] . current evidence suggests that sars-cov-2 predominantly spreads through person-. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint to-person via saliva and respiratory secretions or droplets [26] . other modes of transmission could be contaminated objects or surfaces (fomite transmission), fecal-oral, bloodborne, mother-to-child, and animal-to-human transmission [27] . the relative roles of various routes are unclear and will require additional epidemiological data as well as mechanistic information about the virus entry. for example, angiotensin-converting enzyme 2 (ace2), the cell receptor for sars-cov-2 entry is abundantly present in blood vessels/capillaries of the skin, the basal layer of the epidermis, and hair follicles [28, 29] . hand hygiene is widely recognized for playing a major role in limiting the spread and transmission of such infectious diseases [18] . in fact, in order to cope with such a pandemic situation, cdc has specifically recommended washing of hands with soap and water whenever possible. however, it has been reported that frequent washing hands with water and soap may damage skin potentially leading to dermatitis [30] . while alcohol-based hand sanitizers with at least 60% alcohol can help in avoiding sickness and spreading germs to others, unfortunately the protective effect of alcohol lasts only till the alcohol evaporates i.e. less than a couple of minutes. frequent handwashing is also burdensome and disruptive in many situations, and in some cases just not practical [31] . inspired by the fact, that cg-101 exhibits broad-spectrum antimicrobial properties against a range of bacteria, virus, and fungi, we developed an alcohol-based sanitizing skin protectant containing cg-101 as a countermeasure to restrict the rampant spread of the covid-19 infection. cg-101 inactivates pathogens by extracting lipids and disrupting the cell membrane [21] . more importantly, unlike ethanol, which is highly volatile, cg-101, being an ionic liquid/deep eutectic solvent, has a very low vapor pressure and can remain on the skin long after ethanol evaporates. this long-lasting residence of cg-101 is expected to be responsible for long-lasting protection against sars-cov-2 thus limiting the need for frequent hand washing. human volunteer studies confirmed the safety of the active, cg-101(~100% concentration). hript, an industry standard test for evaluating the potential of a test material to induce sensitization in humans after repeated exposure was adopted [32] . among 52 adult human volunteers who were treated with 20x higher cg-101 than that used in the ionlast tm , 43 subjects did not display any irritation throughout the entire 21-day study. there were no other adverse events reported in the study. furthermore, following completion of the study and a rest period of two weeks, cg-101 application to a different part of the body, did not create any sensitization response at 24 and 48 h which indicates the potential of cg-101 as a safe additive for topical products. in-vitro bactericidal assay was conducted to compare the longevity of antibacterial efficacy of ionlast tm in contrast to currently marketed alcohol-based products against e. coli. the visual difference in the number of viable test microorganisms for the alcohol-based product and ionlast tm was striking for all the time points including 30min, 2h and 4h. no notable difference in the microbial growth was observed between the untreated and alcohol-based hand sanitizer treated groups (fig. 1) indicating the short duration of protection rendered by ethanol. this signifies the persistence of protective effects of ionlast tm against e. coli and possibly other pathogens for up to 4h. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint the encouraging results obtained from the bactericidal efficacy studies, motivated us to test ionlast tm against hcov229e. as theorized, cg-101 was able to deactivate the virus, generating excellent virucidal efficacy against hcov229e. ionlast tm generated >4.00 log10 reductions in viral titers following 15s and 30s exposure. moreover, virucidal activity of the active, cg-101 (5% w/w) for exposure times, 5min and 10min generated similar log10 reductions (>4.00) from the initial population of the hcov229e as ionlast tm (fig. 2) indicating hcov229e is highly susceptible to ionlast tm . residual antimicrobial efficacy study was undertaken to measure the relative persistence of antibacterial activity under controlled clinical test conditions. the results demonstrated a significant (≥5.15) log10 reduction in s. aureus following immediate, 2h and 4h post-ionlast tm application (fig. 3) . these results further support persistent pathogen inactivation efficacy of ionlast tm . upon further clinical studies demonstrating the prolonged protection rendered against hcov229e, ionlast tm may open new opportunities in the recommended hand hygiene protocols for preventing disease transmission. with its long-lasting effect, ionlast offers a promising complement to the existing cdc/who guidelines for good hand hygiene. clinical safety study of cg-101. for the hript, skin irritation and sensitization potential of cg-101 was evaluated in a 21-day human repeat insult patch test in 52 volunteers. this cosmetic study was approved by an irb and conducted by ama laboratories inc. in new york. cg-101 (100%, liquid) was applied on the back of volunteers over a 2 x 2 cm 2 area on each of 9 visits over a period of 3 weeks. the application area was occluded with hypoallergenic tape. the subjects were required to keep the tape for 24h. at each visit, the application site was evaluated for skin irritation on a 5-point scale (0-4). any subjects that showed irritation rated at 3 or higher were withdrawn from the study and monitored for changes. after completion of the 3-week test period the subjects were given a 10 to 14-day rest, after which cg-101 solution was applied to a previously unexposed site. the subjects were then evaluated for any skin reactions after 24 and 48h. in-vitro bactericidal efficacy. in order to determine the relative bactericidal efficacy of ionlast tm in contrast to an alcohol-based product, astm e1153, standard test method for efficacy of sanitizers recommended for inanimate non-food contact surfaces was modified and employed accordingly. briefly, pre-cleaned surfaces were transferred into sterile petri plates using sterile forceps and covered with 1ml of the test substances and held for 30min at ambient temperature to dry. the inoculation with 10 µl of e. coli suspension was performed at 30 min, 2h and at 4h. after each inoculation, the exposure time was 5 min. after 5 min, the test compound was neutralized, and the viable bacteria were resuspended. a neutralization study was conducted to assure that the neutralizers used for the study quench the antimicrobial activity of each test material and were not toxic to the challenge species. after neutralization, the samples were plated on agar plates and transferred into the incubator set at 37°c for 48h. an average of at least ≥10 4 cfu of bacteria were recovered from the negative and neutralization controls. the number of viable organisms on the agar plates treated with ionlast tm were then compared visually to a . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint negative control (untreated) and the comparator (ethyl alcohol 70% hand sanitizer) following 48 h incubation. virucidal suspension test. the virucidal test was based upon astm e1052-20, standard practice to assess the activity of microbicides against viruses in suspension. all testing was performed in accordance with good laboratory practices (glp), as specified in 21 cfr part 58 at bioscience laboratories, inc. (bsl), bozeman, montana. the characterization of the identity, strength, purity, composition, stability, and solubility of the test product(s) was performed by cage bio inc. the percent and log10 reductions from the initial population of the viral strain(s) was determined following exposure to the test product, ionlast tm , for 15s and 30s, and to the test product, cg-101 (5% w/w), for 5minand 10min. testing was performed in 1 replicate. plating was performed in four replicates. coronavirus (alphacoronavirus) strain 229e (atcc #vr-740) and mrc-5 (atcc #ccl-171; human lung fibroblast cells) were used for the study. residual antimicrobial efficacy test. the residual antimicrobial efficacy testing was carried out using a modification of the standardized test method described in astm e2752-10 (2015), standard guide for evaluation of residual effectiveness of antibacterial personal cleansing products. the study was conducted in compliance with good clinical practice regulations, good laboratory practice regulations, the standard operating procedures of bioscience laboratories, inc., the study protocol, and any protocol amendments. bacterial recoveries were assayed after application of test material, using the forearms as a substrate. at least twelve test subjects (aged 18-65 years), with healthy skin were used in this study, and the test product was applied on one arm, and had the other arm untreated as the negative control. the test sites on both forearms were inoculated with suspensions containing s. aureus (atcc #6538), immediately after the 30-minute product drying time, and at approximately 2h and 4h following test material applications. the test sites were sampled using the cup scrub procedure approximately 20min following each inoculation. the log 10 microbial recoveries of treated versus untreated sites were the basis for assessing the residual antimicrobial effectiveness of the test product. a neutralization study was also performed to assure that the neutralizers used in the recovery medium quench the antimicrobial activity of each test material and were not toxic to the challenge species. study procedures were based on astm e 1054-08(2013), standard test methods for evaluation of inactivators of antimicrobial agents. s. aureus (atcc #6538) was used as the challenge species in the neutralization study. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 7, 2020. table s1 . log10 reduction and percent reduction from control of hcov229e (atcc #vr-740) following 15-and 30s by the test product: ionlast tm table s2 . log10 reduction and percent reduction from control of hcov229e (atcc #vr-740) following 5-and 10min by the test product: cg-101 (5% w/w) table s3 . log10 microbial recoveries and log10 microbial reductions from control of s. aureus (atcc #6538), by subject; immediately (30min), 2h, and 4h following application of the test product: ionlast tm author contributions: ms led characterization, formulation development; ms, am and nj designed the glp antibacterial and vircucidal studies, and data analysis; ms and nj participated in characterization, design of clinical safety study and data analysis; am performed data summary, am and sm summarized findings and wrote the manuscript with the help of all authors. funding: this study was funded by cage bio. competing interests: cage bio has a license to and ownership of patents pertaining to ionic liquids. am, ms, and nj are employees and shareholders of cage bio. sm is a shareholder and board member/consultant to liquideon, cage bio, and i2o therapeutics. data and materials availability: any data associated with this study are available from the authors upon reasonable request. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint figures fig. 1 . in-vitro antibacterial efficacy against e. coli. number of viable test microorganisms treated with the test substances, ethyl alcohol 70% hand sanitizer (comparator) and ionlast tm in comparison to the saline-treated group (negative control) the treated groups were incubated with the test substances for 30min, 2h and 4h and subsequently exposed to e coli for 5min and then neutralized. bacterial growth was assessed on agar plates after a 48-hour incubation period. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint virucidal infectivity to mrc-5 cells for (a) ionlast tm and (b) cg-101 (5% w/w) treated viral suspensions in comparison to virus control (untreated) for different exposure times. log10 reduction are included above the bar. viral titers are displayed as tcid50/ml values (n=4). lloq, lower limit of quantification; tcid50/ml, 50% tissue culture infectious dose. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint fig. 3 . residual antimicrobial efficacy against s. aureus. the means of log10 microbial recovery in comparison to untreated subjects (a) immediately (30min), (b) 2h, and (c) 4h following application of the test product: ionlast tm . data are averages ± sem, statistics by two-way anova with bonferroni's multiple comparison-test. ****p < 0.0001 (n=11). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.04.20161067 doi: medrxiv preprint a new coronavirus associated with human respiratory disease in china covid-19: a novel zoonotic disease caused by a coronavirus from china: what we know and what we don't sars-cov-2 and covid-19: a genetic, epidemiological, and evolutionary perspective the proximal origin of sars-cov-2 transmission of sars-cov-2, required developments in research and associated public health concerns covid-19: in the absence of vaccination -'mask-the-nation the socio-economic implications of the coronavirus pandemic (covid-19): a review review of the 2019 novel coronavirus (sars-cov-2) based on current evidence from sars to covid-19: a previously unknown sars-related coronavirus (sars-cov-2) of pandemic potential infecting humans -call for a one health approach. one health airborne transmission of sars-cov-2: the world should face the reality broad-spectrum coronavirus antiviral drug discovery decoding sars-cov-2 transmission and evolution and ramifications for covid-19 diagnosis, vaccine, and medicine hand hygiene and the novel coronavirus pandemic: the role of healthcare workers universal use of face masks for success against covid-19: evidence and implications for prevention policies hand sanitisers amid covid-19: a critical review of alcohol-based products on the market and formulation approaches to respond to increasing demand inactivation of severe acute respiratory syndrome coronavirus 2 by who-recommended hand rub formulations and alcohols. emerg infect dis hand sanitizers: a review on formulation aspects, adverse effects, and regulations rational hand hygiene during the coronavirus 2019 (covid-19) pandemic efficacy of various disinfectants against sars coronavirus hand sanitizers: a review of ingredients, mechanisms of action, modes of delivery, and efficacy against coronaviruses mechanism of antibacterial activity of choline-based ionic liquids (cage) choline and geranate deep eutectic solvent as a broad-spectrum antiseptic agent for preventive and therapeutic applications ionic liquids as a class of materials for transdermal delivery and pathogen neutralization scope and efficacy of the broad-spectrum topical antiseptic choline geranate current knowledge of covid-19 and infection prevention and control strategies in healthcare settings: a global analysis transmission of covid-19 virus by droplets and aerosols: a critical review on the unresolved dichotomy risk of sars-cov-2 transmission by aerosols, the rational use of masks, and protection of healthcare workers from covid-19 high expression of ace2 on keratinocytes reveals skin as a potential target for sars-cov-2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor frequent hand washing for covid-19 prevention can cause hand dermatitis: management tips. cureus overzealous hand hygiene during the covid 19 pandemic causing an increased incidence of hand eczema among general population in vivo studies of substances used in the cosmetic industry . cc-by-nc-nd 4.0 international license it is made available under a key: cord-266036-qhlo99l7 authors: axell-house, dierdre b.; lavingia, richa; rafferty, megan; clark, eva; amirian, e. susan; chiao, elizabeth y. title: the estimation of diagnostic accuracy of tests for covid-19: a scoping review date: 2020-08-31 journal: j infect doi: 10.1016/j.jinf.2020.08.043 sha: doc_id: 266036 cord_uid: qhlo99l7 objectives: to assess the methodologies used in the estimation of diagnostic accuracy of sars-cov-2 real-time reverse transcription polymerase chain reaction (rrt-pcr) and other nucleic acid amplification tests (naats) and to evaluate the quality and reliability of the studies employing those methods. methods: we conducted a systematic search of english-language articles published december 31, 2019-june 19, 2020. studies of any design that performed tests on ≥10 patients and reported or inferred correlative statistics were included. studies were evaluated using elements of the quality assessment of diagnostic accuracy studies (quadas-2) guidelines. results: we conducted a narrative and tabular synthesis of studies organized by their reference standard strategy or comparative agreement method, resulting in six categorizations. critical study details were frequently unreported, including the mechanism for patient/sample selection and researcher blinding to results, which lead to concern for bias. conclusions: current studies estimating test performance characteristics have imperfect study design and statistical methods for the estimation of test performance characteristics of sars-cov-2 tests. the included studies employ heterogeneous methods and overall have an increased risk of bias. employing standardized guidelines for study designs and statistical methods will improve the process for developing and validating rrt-pcr and naat for the diagnosis of covid-19. after its emergence in december 2019, the virus now known as sars-cov-2 was identified and sequenced in early january 2020, 1 allowing for the rapid development of diagnostic testing based on the detection of viral nucleic acid (i.e., real-time reverse transcription polymerase chain reaction [rrt-pcr]). 2 because infected patients can present with non-specific symptoms or be asymptomatic, the development of accurate diagnostic tests for both clinical and epidemiological purposes was a crucial step in the response to the covid-19 pandemic. 3 in the united states, the spread of sars-cov-2 rapidly outpaced the capacity to test for it, resulting in the food and drug administration (fda) relaxing regulatory requirements to increase testing availability. the fda granted the first emergency use authorization (eua) for a sars-cov-2 rrt-pcr diagnostic test on february 4, 2020. consequently, hundreds of tests for sars-cov-2, among them rrt-pcrs, other types of nucleic acid amplification tests (naats), and automated and/or multiplex methods based on proprietary platforms, obtained fda emergency use authorization (eua). as of august 4 th , 2020, the fda has granted euas to 203 diagnostic tests, including 166 molecular tests, 35 antibody assays, and 2 antigen tests. although the fda began requiring the submission of validation methods and results as part of eua application for sars-cov-2 diagnostic tests, these tests were not initially required to undergo the rigorous assessment that would normally be part of the fda approval process. researchers also began developing alternative nucleic-acid based methodologies to detect sars-cov-2, including reverse-transcription loop-mediated isothermal amplification (rt-lamp), and others. concurrently with rapid test production, publications emerged reporting clinical diagnostic test performance characteristics, such as "sensitivity" and "specificity", though some lacked the rigorous methodologies usually required to formally estimate diagnostic accuracy. here we present a scoping review of the literature with two main objectives: 1) to assess the methodologies used in the estimation of diagnostic accuracy of sars-cov-2 tests and 2) to evaluate the quality and reliability of the studies employing those methods. searches were performed through medline (ovid), embase (elsevier), scopus, web of science, cinahl, and pubmed following the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines 4 between december 1, 2019 and june 19, 2020. the following search string was used: (2019-ncov or sars-cov-2 or sars-cov2 or covid-19 or covid19 or covid) and ("positive agreement" or "negative agreement" or "overall agreement" or "diagnostic accuracy" or "positive rate" or "positivity rate" or "test performance" or "reference standard" or "gold standard" or sensitivity or specificity or "percent agreement" or "concordance" or "test agreement" or "predictive value" or "false negative" or "false positive") and ("polymerase chain reaction" or pcr or "reverse transcriptase" or "nucleic acid amplification test" or naat or isothermal or "rt-lamp" or "rt-pcr" or "molecular test"). the literature hub litcovid's "diagnosis" section was screened in its entirety once and then daily for relevant titles. we liberally screened articles by title and abstract for further evaluation. articles were included if they met the following criteria on screening: 1) peer-reviewed publication, 2) study evaluated diagnostic test accuracy of naat, 3) diagnostic test performed on ≥10 patients, 4) diagnostic/clinical sensitivity, specificity, other correlative statistics, or test positive rate were either identified by name or were included in the publication as a numerical value and we could reproduce the calculations. exclusion criteria included: 1) pre-print status, 2) guidelines, consensus, review, opinion, and other summary articles 3) entirely pregnant or pediatric populations, 4) overlap of study population with another included publication. four authors independently extracted data and two authors reviewed data for accuracy. for study characteristics, we extracted: first author name, country, study design, patient population, total number of patients or samples included in test performance calculations, and number of cases according to rrt-pcr (tables 1-5) or total number of cases based on positive result of any platform tested (table 6) . for patient characteristics, we extracted age and sex. for index test and reference standard characteristics, we extracted: test type (naat) or definition (clinical diagnosis, composite reference standards), specimen (naat), specimen dry/collection liquid status (for studies evaluating abbott id now), proprietary automated and/or multiplex systems -henceforth called "platforms" (naat), and target genes of primers (naat). for outcomes, we extracted the values of test performance characteristics with their designation according to the original authors, without our interpretation. for this reason, we indicate these outcomes as "reported" (r): reported sensitivity (rsn), specificity (rsp), positive predictive value (rppv), negative predictive value (rnpv), accuracy (racc), positive percent agreement (rppa), negative percent agreement (rnpa), overall agreement (roa), and kappa coefficient. additionally, we extracted "positive rate," a non-standard term used by the included studies to refer to the number of positive naats in a population of patients suspected to have covid-19 (table 1) , or to the number of positive samples in a total population of positive samples after repeat testing (table 2) . we constructed 2 x 2 contingency tables and reproduced test performance characteristic calculations to demonstrate the methods of how the original authors obtained the values (supplementary table 1) . we report additional pertinent study data in supplementary table 2 : enrollment dates, number of sites of enrollment, symptomatic status, and chest radiology status. no articles were excluded on the basis of quality in order to present the most comprehensive summary of the currently available evidence. we presented the extracted data in tabular form mirrored by a descriptive synthesis 5 in two broad categories: diagnostic accuracy studies for rrt-pcr (tables 1-3) , and diagnostic accuracy or comparative agreement studies of two naats (tables 4-6 ). tables are thematically divided based on the reference standard strategy, or approach to obtaining comparative agreement measures. diagnostic accuracy studies for rrt-pcr were arranged alphabetically in tables by first author last name (tables 1-3) . diagnostic accuracy and comparative agreement studies for two naats were arranged by decreasing order of studies per methodology, then alphabetically by methodology or platform (tables 4-6) for easy comparison. due to significant diversity in methods and reporting of results, we reported grouped summary data for study characteristics, patient characteristics, and outcomes. we used the framework of the quality assessment of diagnostic accuracy studies (quadas-2) 6 to evaluate our selected articles (supplementary table 3 ). we collected data, or noted their absence, for a narrative description of risk of bias and concerns of applicability based on the quadas domains. for assessment of bias in patient selection, we evaluated author conflicts of interest, study design type, inclusion/exclusion criteria, method of patient enrollment, and reporting of patient demographics and characteristics (i.e. symptomatic status). for assessment of bias in reference standard and index test, we evaluated the accuracy of the reference standard, the description of duration of symptoms at time of testing, whether the threshold to determine a positive test was prespecified, and researcher blinding to reference standard and index test results. for assessment of bias in flow and timing, we evaluated whether the reference standard was the same for all patients, the sequence and timing of the performance of the reference standard and index test, whether test performance characteristics were calculated based on sample numbers or patient numbers, and whether indeterminate or invalid results were included in test performance calculations. our search yielded 1537 articles, with 816 unique articles after deduplication. after screening title and abstract, 130 articles underwent full text evaluation. ultimately, 49 articles were included in our review ( figure 1 ). three studies, with 19 to 1014 patients, report a "positive rate" as the number of positive rrt-pcr out of the number of suspected cases of covid-19, with a range of 38.42% to 59% (table 1) . [7] [8] [9] the studies do not report these values as "sensitivity" directly, however these concern that their low calculated positive rates (38.42% and 47.4%, respectively) were indicative of a failure of rrt-pcr to diagnose covid-19. 8, 9 in terms of quality assessment, the studies lack specific details as to how patients were classified as having suspected covid-19 infection. the accuracy of clinical diagnosis based on case definitions is unclear but is likely not ideal for diagnosis. additionally, duration of symptoms at the time of clinical diagnosis or rrt-pcr testing was not provided (supplementary table 3 ). eight studies, with a range of 36 to 22,061 patients per study, attempted to determine the accuracy of rrt-pcr by comparing the initial rrt-pcr result to the result after multiple repeated samples from the patient submitted for rrt-pcr testing, which was called the reference standard (table 2) . [10] [11] [12] [13] [14] [15] [16] [17] three studies reported this value as a "positive rate," ranging from 51.25% to 88%, 10, 14, 17 and five reported sensitivity, with a range of 57.9% to 94.6%. [11] [12] [13] 15, 16 of these studies, only he et al included an rsp of 100%, calculated from patients who remained negative for sars-cov-2 after repeated sample testing (supplementary table 1 ). 13 green et al. included patients in their study regardless of whether they were tested once or multiple times, using data from these subsets of patients to make assumptions for estimating clinical test characteristics. in addition, this study also conducted multiple different naats and rrt-pcrs on patients, whereas other studies employing this strategy used only one type of naat. additionally, the authors do not clarify whether patients who had repeat sars-cov-2 test were consistently tested with the same naat/rrt-pcr test or a different one. they also calculated test performance characteristics differently from other studies: two estimates of sensitivity were calculated, one in which the rate of false negatives for single-tested patients was 0%, and one in which the "false negative" rate was the same as in repeat-tested patients in their study of approximately 16.8%. 12 however, the details of how they calculated test characteristics were not presented. to clarify the two assumptions made in the calculations, we in terms of quality assessment, most of the studies were performed with non-cohort design, and six consisted of only patients who were determined to have covid-19 by rrt-pcr, i.e. cases only (table 2) . 10, 11, [14] [15] [16] five of the studies had inclusion criteria which caused pertinent patients to be excluded by necessitating patients to have had a well-performed ct chest or x-ray (supplementary table 3 ). this excluded several patients who would otherwise have been pertinent to the study of test diagnostic accuracy. 10, 11, 13, 16, 17 the studies involved repeating rrt-pcr several times for a reference standard, but each patient received a different number of repeat tests over a different time period, resulting in each patient receiving a different reference standard. one study tracked negative-to-positive conversion over 1 to 49 days, 12 and another tracked over 1 to 14 days, 13 leading to concern that potentially a patient could have been infected in the time between the initial test and the final test and confounding results. one study counted invalid results as negative results and indeterminate results as positive results when calculating test performance characteristics, 12 otherwise the rationale and ways invalid and indeterminate results were handled were not reported in these studies. three studies determined the accuracy or agreement of rrt-pcr or automated rrt-pcr platforms/instruments compared to a reference standard based on the results of several tests as a "composite reference standard" (table 3) . [18] [19] [20] there were between 58 and 184 patients per study. suo et al considered a positive result of either repeated measurements of rrt-pcr or serology to indicate a positive test according to the reference standard; reported sensitivity of initial rrt-pcr result was 40%, rsp 100%, rppv 100%, and rnpv 16%. 19 zhen et al compared rrt-pcr performed according to the us cdc protocol to a composite reference standard in which the consensus result of 3 or more out of 4 molecular assays was considered the correct result. the rrt-pcr had an rppa of 100%, an rnpa of 98%, and cohen's kappa coefficient of 0.98. 20 cradic et al did not study rrt-pcr but studied three automated molecular assays and used a composite reference standard of the consensus result of two or more of the three assays. while abbott id now had a rppa of 91%, the roche cobas 6800 and diasorin simplexa assays had a rppa of 100%. 18 these studies either did not report how samples were selected for evaluation (supplementary table 3 ), 19, 20 or reported that only samples which had sufficient residual volume and had been properly stored were selected. 18 days after the initial test, after the patient had been discharged from the hospital, leading to potential exposure for initial infection or reinfection. 19 the performance other nucleic acid amplification test methods compared to standard rrt-pcr fourteen studies compared other nucleic acid amplification test methods to detect sars-cov-2 to rrt-pcr (table 4) 30 suo et al evaluated digital droplet polymerase chain reaction (ddpcr), with rsn 94%, rsp 100%, rppv 100%, rnpv of 63%, and racc 95%. 19 bulterys et al study evaluated an isothermal amplification method with rsn 82.8% and cohen's kappa 0.86. 31 wang, cai, and zhang et al evaluated one-step single-tube nested quantitative polymerase chain reaction (osn-qrt-pcr) with cohen's kappa of 0.737. 32 regarding evaluation of quality (supplementary table 3) , the majority of studies did not report how patient samples were selected for evaluation. 21, 22, 24, 25, [27] [28] [29] [30] 32 in the study conducted by bulterys et al, sample selection was a convenience selection of samples with residual volume that had been stored correctly. 31 most studies did not report symptomatic status of the patient 21-28,30-32 or patient demographics. [21] [22] [23] [24] [25] [27] [28] [29] [30] [31] [32] problematically, many of the studies did not report when the reference standard was conducted on the patient samples compared to the index test, or whether actions that could potentially alter test results (such as freeze/thaw cycles) occurred between reference standard or index test. [21] [22] [23] [24] 27, 28, 31 four studies calculated test performance characteristics based on number of samples rather than number of patients. 23, 27, 30, 32 the management of indeterminate and invalid test results went largely unreported. [21] [22] [23] [24] [25] 27, 30 the performance of naat platforms compared to rrt-pcr as the reference standard fifteen studies compared automated naat platforms to various rrt-pcr assays to determine test performance characteristics ( other studies evaluated ausdiagnostics (rsn 100%, rsp 92.16%), 43 hologic panther fusion (rppa 98.7%, rnpa 98.1%), 44 luminex nxtag (rsn 97.8%, rsp 100%), 45 mesa biotech accula (rppa 68.0%, rnpa 100%), 46 or qiastat-dx (rsn 100%, rsp 93%) 47 compared to rrt-pcr. with regards to quality evaluation (supplementary table 3 ), most studies did not report method of sample collection/patient recruitment, 33, 35, 37, [41] [42] [43] [44] [45] [46] [47] and four studies conducted a convenience selection of samples, including enrichment for positive samples. [34] [35] [36] 39 eight studies conducted test performance calculations on sample numbers instead of patient numbers. 33, 35, 36, 38, 39, [42] [43] [44] four studies conducted calculation of test performance characteristics with indeterminate or inconclusive results as "positive," 35, 38, 39, 42 and the management of indeterminate/inconclusive as well as invalid results went unreported in an additional three studies. 33, 37, 47 no study reported the blinding of researchers to the reference standard or index test results. ten studies, containing between 15 and 524 patients per study, evaluated the agreement between two different types of naat platforms ( in the studies, some platforms were identified as the "comparator" or "reference" platforms, including cepheid xpert xpress, 49 abbott realtime, 34, 48 hologic panther fusion, 50, 51 and roche cobas 6800, 52,55 and these were listed as "platform #1" in table 6 . three studies did not identify any studied platform as the "comparator" or "reference standard," and instead only reported general, non-directional measures of agreement such as overall agreement, cohen's kappa, or alternatively, the calculations of ppa and npa were identical no matter their method of calculation (supplementary table 1) . 39, 53, 54 regarding quality evaluation (supplementary table 3 ), the samples used for calculating test performance characteristics were reported to be selected for enrichment of positive samples, 34, 39 for diversity of viral load, 52,54 otherwise curated, 50 or the method of selecting samples was unreported. 49, 51, 53, 55 symptomatic status of the patients was largely unreported. 39, [49] [50] [51] [52] [53] [54] [55] five studies included samples where one test was conducted, then interim freezing, cooling, or other storage, before performance of the second test. 39, [50] [51] [52] 54 two studies did not report the sequence of testing of the two platforms or interim handling or storage of the samples. 49, 53 the status of researcher blinding to either platform result was not reported in any study. in our scoping review of 49 articles concerning test performance characteristics of rrt-pcr and other naat used for the diagnosis of covid-19, we were able to observe several overarching themes. clinical diagnosis by the case definition for covid-19 used in the early period of the pandemic does not correlate well with positive rates of covid-19 rrt-pcr ( table 1 ). the result of the initial rrt-pcr performed on a patient, if negative, may not be reflective of the result after multiple repeated rrt-pcrs for that patient ( table 2) . several alternative naat methods, many of which are easier or faster to perform, may be comparable to standard rrt-pcr (table 4 ). proprietary multiplex, automated, and/or point-of-care methods are comparable to in accuracy to rrt-pcr (table 5 ) and to each other (table 6 ), although the abbott id now sars-cov-2 test appears to have lower comparative agreement to other platforms. 34, [48] [49] [50] [51] [52] these findings should be viewed cautiously as the sars-cov-2 tests in these studies have not undergone rigorous evaluation necessary for fda approval due to the emergency state generated by the covid-19 pandemic. in addition, during our scoping review, we found substantial heterogeneity among available studies in terms of test types, reference standards, metrics, and details of study design and methodology. we categorized the included studies by four different reference standard strategies: clinical diagnosis/case definitions (table 1) , repeated index testing (table 2) , composite reference standard (table 3) , and rrt-pcr (table 4 and 5) . additionally, we identified a fifth category, where instead of using a reference standard, comparative agreement between two naat platforms was calculated (table 5 and 6 ). the main limitation of the first group of studies (table 1 ) was the use of a "case definition" as the reference standard to report a "positive rate" of rrt-pcr. during novel disease outbreaks, standard case definitions are often developed to assist clinicians in case identification before a diagnostic test is available. unfortunately, the studies included in this group were unable to use a clear case definition; instead they refer to a population of "suspected cases," for which the definition is not reported. [7] [8] [9] because this group enrolled patients prior to february 15, 2020 in china, during the time in which the chinese national guideline for diagnosis and treatment of covid-19 (ngdtc) published five different versions of the covid-19 case definition, the case definitions in use at the time of these studies varied. 56 a recent study estimated that if a single guideline (specifically, version 5 of the ngdtc) had been used to identify cases from the beginning of the outbreak to february 20, 2020, there would have been more than three times as many identified cases in hubei province. 56 this is relevant to our review because the two largest studies that evaluated the rrt-pcr positive rate of patients with a clinical diagnosis of covid-19 took place in wuhan, hubei province, and included patients evaluated before february 14, 2020 7, 8 (supplementary table 2 ). this increased case estimate due to diagnosis of covid-19 based on case definition complicates the legitimacy and reported accuracy of the "positive rate" of rrt-pcr referred to in these studies. the second group assessed rrt-pcr test performance characteristics via repeated index rrt-pcr testing ( table 2 ). most studies in this group reported "sensitivity" by dividing the number of participants with positive baseline rrt-pcrs by the total number of participants who eventually had a positive rrt-pcr after repeated measurements. while such an approach may have some advantages over the use of a case definition alone as a reference standard, this strategy is, nonetheless, an imperfect solution with its own set of inherent limitations. sars-cov-2 infection is transient and the associated viral loads are time-varying because of the natural pathophysiology of the infection. therefore, the time interval between each repeated test becomes crucially important, and even relatively small time differences (and/or lack of uniformly used intervals) could complicate the interpretation of re-test results and their quality as reference standards. furthermore, repeated use of the same test as a reference standard for itself does not eliminate the inaccuracies or limitations of the test. such comparisons ultimately reflect the reliability of the test (assuming a short, uniform time interval between tests), rather than providing a true view of test accuracy. the third group of three studies calculated test performance characteristics of rrt-pcr according to a composite reference standard (table 3 ). using arbitrary rules to combine multiple different and imperfect tests inevitably creates a reference standard with some degree of bias. 57 furthermore, all three studies in this group included the test under evaluation as part of the composite reference standard, which leads to additional bias, described below. 58 use of a biased composite standard is likely to lead to reduced sensitivity, among other errors affecting true test performance characteristics. 59 the fourth group of studies evaluated sars-cov-2 diagnostic tests that are under development as well as proprietary testing platforms (most of which are based on standard rrt-pcr methods). these studies used traditional rrt-pcr as a reference standard; results are summarized in tables 4 and 5 , respectively. importantly, while these studies were not designed to estimate the accuracy of rrt-pcr, their results indicate that the index tests did not identify significantly more positive samples than rrt-pcr. finally, the last group of studies compared sars-cov-2 naat platforms (table 6) . these comparative accuracy studies examined the agreement between two non-reference standard tests. although most of the testing platforms evaluated in these studies were based on standard rrt-pcr, the agreement between two non-reference standard tests is not equivalent to test accuracy, as mentioned previously. this scoping review is limited by the lack of reporting of several key study features in the majority of the articles evaluated, which is an important indicator of quality and potential bias. based on the quadas-2 criteria, most of the included studies had concern for bias (supplementary table 3 ). the most prominent concerns were unclear inclusion/exclusion criteria, unclear method of enrollment/selection of patients and samples, and unclear handling of indeterminate/inconclusive and invalid results. additionally, many of the studies were conducted in a so-called "two gate" (case-control) design, in which cases and controls were known and selected ahead of time, rather than performing the test on a group of patients or samples with suspected covid-19. these factors likely incorporate bias that significantly confounds the results of the studies, thus, the accuracy of the tests in other settings with different prevalences (such as asymptomatic screening, other age groups) may not be truly generalizable. furthermore, few studies were able to evaluate both the index and reference tests simultaneously or within a short period of time, which is key to avoiding biases caused by changes in the patient's true disease status; this bias can also affect the diagnostic accuracy of the index test. the best approach to determining diagnostic test performance characteristics in the absence of a "gold" standard is an open question in diagnostic accuracy methodology. while many methods have been described, there are only a few well-defined statistical approaches that use a reference standard in lieu of a gold standard, reviewed elsewhere. 60 latent class analysis is one commonly used approach in situations in which neither the true error rates of the reference standard nor the true prevalence of the disease are known. this approach uses the results of a set of imperfect tests to estimate parameters related to sensitivity, specificity, and prevalence often using maximum likelihood methods. however, this is not the only method available and every method has its own strengths and limitations. 57 therefore, careful interpretation by studies that attempt to estimate test characteristics is warranted to account for and clarify the inherent limitations of assessing accuracy-related metrics when a gold standard is unavailable. evaluation of the performance characteristics of sars-cov-2 diagnostic tests is vital to control of the ongoing covid-19 pandemic. while more than 200 sars-cov-2 molecular diagnostic tests have received fda euas, we have described in this scoping review that the performance of few of these tests has been assessed appropriately. the lack of robust test performance that we noted in many studies published to date is undoubtably due in part to the critical need for tests, which resulted in accelerated test development. however, our scoping review also uncovered imperfect methods for estimating diagnostic test performance in the absence of a gold standard and demonstrate that the accuracy of these tests should be interpreted with caution. future studies would benefit from employing statistical methods such as latent class analysis and other methods referenced above to accurately analyze their data. indeed, instituting national requirements for test performance analysis and reporting, perhaps based on the existing fda guidelines on diagnostic tests, 61 would advance the goal of standardizing the evaluation sars-cov-2 diagnostic test performance. such an initiative would lead to statistically robust conclusions regarding the accuracy of the index test, which will in turn support hospitals and clinicians as they determine the optimal test to use for covid-19 diagnosis. table 2 . abbreviations-aigs: automatic integrated gene detection system, bal: bronchoalveolar lavage, ci: confidence interval, ddpcr: digital droplet polymerase chain reaction, e: envelope, iamp: isothermal amplification, iqr: interquartile range, κ: kappa statistic, n/a: not applicable, n: nucleocapsid, no.: number, nps: nasopharyngeal swab, nr: not reported, ops: oropharyngeal swab, orf1ab: open reading frame 1ab, osn-qrt-pcr: one-step single-tube nested quantitative real-time polymerase chain reaction, racc: reported accuracy, rdrp: rna-dependent rna polymerase, ref stnd: reference standard, rnpa: reported negative percent agreement, rnpv: reported negative predictive value, roa: reported overall agreement, rppa: reported positive percent agreement, rppv: reported positive predictive value, rrt-pcr: real-time reverse transcription polymerase chain reaction, rsn: reported sensitivity, rsp: reported specificity, rt-lamp: reverse transcription loop-mediated isothermal amplification, rt-raa: reverse-transcription recombinase-aided amplification, s: spike, y: years. a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding presymptomatic transmission of sars-cov-2 -singapore preferred reporting items for systematic reviews and meta-analyses: the prisma statement synthesis without meta-analysis (swim) in systematic reviews: reporting guideline quadas-2: a revised tool for the quality assessment of diagnostic accuracy studies correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases positive rate of rt-pcr detection of sars-cov-2 infection in 4880 cases from one hospital in comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection sensitivity of chest ct for covid-19: comparison to rt-pcr clinical performance of sars-cov-2 molecular testing diagnostic performance between ct and initial real-time rt-pcr for clinically suspected 2019 coronavirus disease (covid-19) patients outside wuhan, china testing for sars-cov-2: can we stop at two? clin infect dis diagnosis of the coronavirus disease (covid-19): rrt-pcr or ct? frequency and distribution of chest radiographic findings in covid-19 positive patients detection and analysis of nucleic acid in various biological samples of covid-19 patients clinical evaluation and utilization of multiple molecular in vitro diagnostic assays for the detection of sars-cov-2 ddpcr: a more accurate tool for sars-cov-2 detection in low viral load specimens comparison of four molecular in vitro diagnostic assays for the detection of sars-cov-2 in nasopharyngeal specimens development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel sars-cov-2 evaluation of rapid diagnosis of novel coronavirus disease (covid-19) using loop-mediated isothermal amplification real-time reverse transcription loop-mediated isothermal amplification for rapid detection of sars-cov-2 a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov-2 rapid and visual detection of 2019 novel coronavirus (sars-cov-2) by a reverse transcription loop-mediated isothermal amplification assay multiple-centre clinical evaluation of an ultrafast single-tube assay for sars-cov-2 rna a reverse-transcription recombinase-aided amplification assay for rapid detection of the 2019 novel coronavirus (sars-cov-2) multiplexing primer/probe sets for detection of sars-cov-2 by qrt-pcr triplex real-time rt-pcr for severe acute respiratory syndrome coronavirus 2 development of an automatic integrated gene detection system for novel severe acute respiratory syndrome-related coronavirus (sars-cov 2) comparison of a laboratory-developed test targeting the envelope gene with three nucleic acid amplification tests for detection of sars-cov-2 novel one-step single-tube nested quantitative real-time pcr assay for highly sensitive detection of sars-cov-2 evaluation of the covid19 id now eua assay comparison of two commercial molecular tests and a laboratory-developed modification of the cdc 2019-ncov rt-pcr assay for the detection of sars-cov-2 comparison of abbott id now, diasorin simplexa, and cdc fda eua methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from individuals diagnosed with covid-19 validation and verification of the abbott realtime sars-cov-2 assay analytical and clinical performance multi-center evaluation of the cepheid xpert xpress sars-cov-2 assay for the detection of sars-cov-2 in oropharyngeal swab specimens comparison of commercially available and laboratory developed assays for in vitro detection of sars-cov-2 in clinical laboratories multicenter evaluation of the cepheid xpert xpress sars-cov-2 test rapid and sensitive detection of sars-cov-2 rna using the simplexa tm covid-19 direct assay clinical evaluation of the cobas sars-cov-2 test and a diagnostic platform switch during 48 hours in the midst of the covid-19 pandemic comparison of sars-cov-2 detection from nasopharyngeal swab samples by the roche cobas 6800 sars-cov-2 test and a laboratory-developed real-time rt-pcr test interpret with caution: an evaluation of the commercial ausdiagnostics versus in-house developed assays for the detection of sars-cov-2 virus comparison of the panther fusion and a laboratory-developed test targeting the envelope gene for detection of sars-cov-2 clinical performance of the luminex nxtag cov extended panel for sars-cov-2 detection in nasopharyngeal specimens of covid-19 patients in hong kong comparison of the accula sars-cov-2 test with a laboratory-developed assay for detection of sars-cov-2 rna in clinical nasopharyngeal specimens evaluation of the qiastat-dx respiratory sars-cov-2 panel, the first rapid multiplex pcr commercial assay for sars-cov-2 detection comparison of abbott id now and abbott m2000 methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from symptomatic patients performance of abbott id now covid-19 rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a new york city academic institution five-minute point-of-care testing for sars-cov-2: not there yet clinical evaluation of three sample-to-answer platforms for the detection of sars-cov-2 comparison of cepheid xpert xpress and abbott id now to roche cobas for the rapid detection of sars-cov-2 the detection of sars-cov-2 using the cepheid xpert xpress sars-cov-2 and roche cobas sars-cov-2 assays comparison of two high-throughput reverse transcription-polymerase chain reaction systems for the detection of severe acute respiratory syndrome coronavirus 2 clinical evaluation of a sars-cov-2 rt-pcr assay on a fully automated system for rapid ondemand testing in the hospital setting effect of changing case definitions for covid-19 on the epidemic curve and transmission parameters in mainland china: a modelling study evaluation of diagnostic tests when there is no gold standard. a review of methods using a combination of reference tests to assess the accuracy of a new diagnostic test value of composite reference standards in diagnostic research diagnostic test evaluation methodology: a systematic review of methods employed to evaluate diagnostic tests in the absence of gold standard -an update food and drug administration cfdarh. statistical guidance on reporting results from studies evaluating diagnostic tests key: cord-324944-ixh3ykrc authors: mitsakakis, konstantinos; d'acremont, valérie; hin, sebastian; von stetten, felix; zengerle, roland title: diagnostic tools for tackling febrile illness and enhancing patient management date: 2018-12-05 journal: microelectron eng doi: 10.1016/j.mee.2018.10.001 sha: doc_id: 324944 cord_uid: ixh3ykrc most patients with acute infectious diseases develop fever, which is frequently a reason to visit health facilities in resource-limited settings. the symptomatic overlap between febrile diseases impedes their diagnosis on clinical grounds. therefore, the world health organization promotes an integrated management of febrile illness. along this line, we present an overview of endemic and epidemic etiologies of fever and state-of-the-art diagnostic tools used in the field. it becomes evident that there is an urgent need for the development of novel technologies to fulfill end-users' requirements. this need can be met with point-of-care and near-patient diagnostic platforms, as well as e-health clinical algorithms, which co-assess test results with key clinical elements and biosensors, assisting clinicians in patient triage and management, thus enhancing disease surveillance and outbreak alerts. this review gives an overview of diagnostic technologies featuring a platform based approach: (i) assay (nucleic acid amplification technologies are examined); (ii) cartridge (microfluidic technologies are presented); (iii) instrument (various detection technologies are discussed); and at the end proposes a way that such technologies can be interfaced with electronic clinical decision-making algorithms towards a broad and complete diagnostic ecosystem. people in low-and middle-income countries (lmics), primarily in sub-saharan africa, south-east asia, and latin america, suffer dramatically from infectious diseases, some of which are endemic, while others occur in the form of epidemics. a combination of factors leads to the perpetuation of this situation, including the tropical climate, the simultaneous presentation of multiple diseases, innate or acquired host factors (micronutrient deficiency, immunodeficiency, co-morbidities), lack of resources (low income) to procure medicines and vaccines or other preventive tools, lack/misuse (due to lack of training or information) of (suitable) diagnostic tools, increase of pathogen resistance to antimicrobial medicines, as well as social and community factors. as a result, in 2010, 64% of deaths worldwide were still due to infectious diseases; pneumonia, malaria, and sepsis were the leading causes of death, particularly in africa [1] . moreover, while the borders between tropical diseases -thought to be confined around the equator (such as ebola) -and infectious diseases classically related to northern countries (such as influenza) were discrete, climate change and the increase in population migration contributed to a blurring of these borders, thus making the spread of febrile illnesses a global reality and concern. many people with acute infectious diseases develop fever, which is a frequent complaint, observed in approximately 80% of pediatric and adult patients presenting with an acute medical condition to health facilities. the estimated incidence of febrile episodes in malaria endemic countries varies according to studies but is generally between 2 and 7 episodes per person per year in children < 5 years [2] . despite the fact that all these infectious diseases cause fever, they can result from very different types of pathogens -parasites, viruses, bacteriaeach of which requires a different type of treatment and management. malaria, which was a major cause of fever a few decades ago, has now https://doi.org/10.1016/j.mee.2018. 10 .001 given the still high (and often increasing) numbers of febrile (and related) illness cases and accompanied mortality globally, the (re-) emergence of global threats (e.g. ebola in 2014) and the increasing resistance to antimicrobial medicines, there have been several conventions at the international level with the aim to define a roadmap to tackle these challenges. the most significant conclusions and decisions deriving from summit meetings are summarized below. the world health organization (who) has changed its policy regarding the case management of malaria, to shift from presumptive treatment to laboratory-based confirmation of malaria before treatment [8] . thus, wide access to malaria diagnosis is advocated to protect available antimalarial medicines against the development of resistance. according to the who 2014 global report on surveillance and antimicrobial resistance, the emergence of drug resistance due to misuse of antimicrobials is one of the major current public health threats worldwide. new tools that could mitigate this threat are urgently required [9] . in a summary of the lessons learned from the 2014 ebola epidemic, bill gates mentioned that: "we need to invest in better disease-surveillance and laboratory-testing capacity, for normal situations and for epidemics. routine surveillance systems should…detect early signs of an outbreak beyond their sentinel sites and be quickly scaled up during epidemics. they should be linked with national public health laboratories to enable robust monitoring and response." [10] . in an ebola-focusing summit that was convened by the paul allen foundation in april 2015, one of the conclusions stated that "the greatest unmet need…is a field-test for use in community or village settings… to help determine whether isolation and/or referral is indicated." [11] . during an expert meeting at who in june 2015 focusing on interoperability standards, it was concluded that there is a clear need for interface systems between diagnostic tools and e-health technologies, aiming at both patient management and epidemics control [12] . these conclusions and directives coming from multiple sites strongly demonstrate the need and the motivation that exists to support the development of new, innovative diagnostic tools that can efficiently resolve the aforementioned challenges related to febrile illnesses. the first step to improve the management of patients with febrile illness is to estimate the distribution of the various syndromes and pathogens that cause fever. even though the distribution of fevercausing diseases varies by geography, season, age and immunity of patients and the level of care, some findings were similar among studies [2] . these were related to cosmopolitan diseases found in most places worldwide, such as pneumonia or urinary tract infection. for other diseases, with various levels of endemicity, such as typhoid or rickettsiosis, or that occur rather in an epidemic way, such as arbovirus infections, the prevalence varied significantly according to the geographic location and, even more, according to the season or the calendar year. in studies that have recorded the clinical presentation of patients (and not only their laboratory results), the causes of fever in outpatients could be classified into four main syndromes: 1) acute respiratory infections (ari, of any type); 2) diarrhea (gastroenteritis); 3) fever with another clear focus (e.g. meningitis or skin infection); and 4) non-specific fevers [13] (each diagnostic platform described in section 5 focuses on at least one of the aforementioned cases). even though important diagnostic challenges remain regarding the first three syndromes, clear clinical guidelines have been developed by who, such as the integrated management of childhood illness -imci [14] , to help clinicians to assess their patients and to decide on their treatment. because they correspond to localized infections, these syndromes are generally diagnosed using the best available clinical predictors, while diagnostic tools are indeed not included. in contrast, regarding the 4th syndrome (non-specific fevers), very few guidelines are available. the main challenge faced by clinicians is the management of patients with fever without specific symptoms or signs to guide them to the source of infection, combined, in endemic areas, with a negative malaria test result (the "negative syndrome" as labeled by health workers in tanzania). these non-specific fevers, also called "fever without focus", are caused by a wide range of pathogens ( fig. 1 and table 1 ) that cannot be identified without accurate laboratory tests. recent studies that aimed at determining the etiologies of fever [13, [15] [16] [17] [18] have identified the following important diseases as causes of non-specific fever: (i) malaria continues to be a major global health problem. according to the 2016 world malaria report, 91 countries were still malaria endemic [19] . there were an estimated 212 million cases of malaria worldwide in 2016 and an estimated 429,000 deaths, while 90% of all malaria deaths occurred in africa. malaria prevalence varies widely, from 0 to 32% of all fevers. indeed, the decrease in malaria transmission in many places in africa [20, 21] has led to significant heterogeneity in transmission. the prevalence of malaria among patients with febrile illness has, therefore, become unpredictable and has triggered the switch of the who malaria case management policy from presumptive treatment, to treatment based on laboratory confirmation [8] . (ii) urinary tract infection was an important cause of fever in young children (generally younger than two years old) that cannot be suspected on the basis of a history of urinary symptoms at that age (and therefore, it is included in the category of non-specific fevers). the prevalence among all children under five with febrile illness was 1-6%. (iii) enteric fever was a significant cause of fever in children older than two years old and in adults, accounting for 2-10% of all fevers, with salmonella typhi being the most frequent pathogen identified, followed by salmonella non-typhi. (iv) other bloodstream infections, mainly due to streptococcus pneumoniae and escherichia coli, accounted for about 2% of all fevers. other systemic bacterial diseases, such as leptospirosis (0.2-13%), rickettsiosis (1-25%), q fever (1-8%), and brucellosis (5%) were also important causes of fever, especially in adults who were more exposed to zoonoses and other environmental-related diseases than children. (v) dengue was also an important cause and seemed to emerge in several african countries. its prevalence is extremely variable, not so much geographically but temporally (seasonal and year to year variation). (vi) cosmopolitan viruses, such as human herpesvirus 6, parvovirus b19, epstein-barr virus, and cytomegalovirus, which are transmitted mainly through close contact within family members, were mostly prevalent in children, accounting for 9% of all fevers and 22% of non-specific fevers in children [13] , while adults were more susceptible to vector-borne diseases such as chikungunya or west nile virus. acute hiv was found in a few patients. (vii) co-infection with two or even more pathogens was frequent (in one study, 23% of pediatric patients had more than one disease simultaneously, fig. 2 ), especially when an outbreak overlapped with an endemic situation, as it happened in a fever study in tanzania in 2013. suddenly, in january 2014, a significant outbreak of dengue started, and at the peak of the epidemic, almost 80% of patients presenting with fever were infected with dengue virus of serotype 2 [6] . because of the absence of diagnostic tools outside the study sites, most of these patients were considered as malaria cases and treated with antimalarial medicines or, in case that malaria tests where available, they were considered as possibly infected by bacteria and treated with antibiotics. (viii) regarding hiv, chronic hiv is an important co-morbidity, especially for adults (40% were positive in two tanzanian studies, while hiv prevalence in the corresponding community was 5 to 10%), and should, thus, be systematically screened for, which is unfortunately not the case presently in africa. whatever the etiology, the first step in assessing febrile patients is to triage them as having a severe rather than a mild illness [22] . objective point-of-care (poc) biosensors (such as oximeters), in addition to the subjective assessment of clinical danger signs (such as lethargy or severe dehydration), are helpful to correctly triage patients and should be integrated in the evaluation process [23] . when patients present with a mild disease, ambulatory management with appropriate home-based treatment is sufficient [24, 25] . the main challenge is then to identify the few patients who will benefit from antimicrobial treatment among all those with self-limited infection. presumptive treatment with antimicrobials should be avoided as most of these infections are of viral origin (fig. 2) , especially in children (except for malaria that can be ruled out by a rapid diagnostic test). follow-up of patients to detect persistent or new symptoms and signs will then allow for the detection of rare clinical failures. at the primary care level, the decision to administer an antimicrobial should be based on an initial classification of the condition in the four categories mentioned in section 2.2, combined with an accurate test specific for each syndrome that can distinguish between bacterial and viral etiology. as the existing host biomarkers lack accuracy (for example, c-reactive protein to identify the few bacterial pneumonia cases among the many viral respiratory infections), they should be combined with clinical predictors (e.g. fast breathing [26] ) to improve the overall specificity [23] . patients with a rather severe febrile illness (most of them satisfy the criteria for sepsis) should be admitted and assessed for the different etiologies of fever, irrespective of the syndrome they present with. in sepsis, the symptoms and signs are indeed often unspecific to allow for initial classification into the four syndromes mentioned in section 2.2 and all possibilities should be considered [15] . under these conditions, accurate laboratory diagnosis is important, and the infections listed in section 2.2 (taking into account specific exposures mentioned by the patients and the local epidemiology) should be tested for, whenever possible. the results of the laboratory tests should then still be interpreted in the clinical context, i.e. the pre-test probability of the disease and the likelihood ratios of clinical predictors. indeed, for laboratory tests that have imperfect accuracy, these elements should be considered to calculate the post-test probability and be able to safely include or exclude certain diseases [27] . if this probability is above the "treatment threshold" (above which one treats the patients instead of continuing investigating them using tests), the appropriate antimicrobial should be administered [28] . if the probability is below the diagnostic threshold, no specific treatment is required, besides adjunctive treatment (e.g. i.e. acute respiratory infections, gastroenteritis, skin infections, meningitis and naso-pharyngeal infections (infections due to enterovirus, adenovirus or influenza, however presenting without any respiratory symptom). source: figure provided by v. d'acremont, adapted from [13] . classification of fever-causing pathogens in africa (parasitic, bacterial, viral, fungal systemic infections). "na": not applicable; "?": unknown; "naats": nucleic acid amplification technologies; "lft": lateral flow test. diseases not included because generally considered after more than one week of fever: sinusitis, visceral leishmaniasis, amoebic liver abscess. diseases not included because, although they are fairly prevalent, they rarely present with fever: pertussis, cryptosporidium. diseases not included because clinical diagnosis is generally sensitive enough and specific enough: measles. a medium sensitivity in hiv patients with low cd4 cell count (urinary test). k. mitsakakis et al. microelectronic engineering 201 (2018) intravenous rehydration in patients with severe diarrhea) and treatment for chronic conditions. if the probability falls between these two thresholds, further testing (when available) may be required and a second-line test should then be used. in practice, antimicrobials are often administered presumptively while waiting for the test results (except for malaria that can be excluded in < 15 min based on the results of a rapid test). in summary, evidence-based integrated management of fever is a complex process, both at hospital and primary care levels, that would clearly benefit from electronic clinical decision algorithms (ecda) based on mobile technology [23, 29, 30] to guide clinicians through the necessary steps, reach an accurate evaluation of the disease probability, and decide on the most appropriate treatment and management. electronic clinical decision algorithms are e-health tools pertaining to the broader category of "computerized decision support systems" (cdss). contrary to differential diagnosis generators (ddx) that provide a long list of possible diagnoses and have shown poor accuracy [31] , ecda provide structured decision pathways that align with the clinician's workflow and guide the clinician throughout the consultation, including on final treatments and advice (section 6). the diagnostic tools target in-and outpatients that visit urban and rural hospitals, peripheral health facilities and laboratories, and community level health posts. in particular, the following broad categories of target groups are identified: (i) patients living in resource-limited countries are clearly in need of new diagnostic tests for infectious diseases. these patients are very diverse in their immune status, the chronic conditions they may suffer from, and their ability and willingness to pay for a diagnostic test. (ii) people traveling from endemic to non-endemic areas. although it is difficult to determine the number of returning travelers and migrants to northern countries that are infected with an endemic disease, because of variation in reporting methods for different pathogens, an estimated 20,000-30,000 cases of travelers with malaria are, for example, reported in europe each year [32] . the number of people crossing borders between the southern countries is much higher and represents also an important target group. they can be tested at airports, harbors, cross-border controls, immigration control checkpoints, where reliability and short time-to-result tests are required. (iii) pre-and post-vaccination, elimination, and screening programs run by local governments, international organizations, and pharmaceutical industries. disease prevention and elimination programs should be supported by comprehensive evidence to demonstrate the underlying prevalence of the pathogen in question and the efficacy of the strategy proposed. using a multiplexingcapable diagnostic platform allows for effective modeling of the: (a) true prevalence and variation of multiple species, enabling the monitoring of impact of the intervention on disease dynamics; (b) local and regional differences in pathogen distribution. (iv) epidemics surveillance, particularly of interest in: (a) sentinel sites, where the diagnostic tools can be installed at strategic geographic locations, in order to cover several regional, cultural, environmental, and epidemiological settings; (b) ministries of health and regional alarm centers that should regularly receive reports from the sentinel sites for assessment of the frequency of patients and the diversity of diseases. the diagnostic systems should operate independently of patients' age, although there are some inherent difficulties to acquire proper quality sample matrix from an infant (e.g. sputum for alleged respiratory infection). furthermore, for some tests, such as blood culture or polymerase chain reaction (pcr) for bloodstream bacterial infections, the same diagnostic tool may have different performance in different age groups, due to different pathogen load [33] . not all diagnostic tools are, thus, suitable for all cases. in addition, the training level of the users, the facilities available at each level of care, and the target patient population are parameters that developers of novel diagnostic tools must take into consideration. there is an extensive debate regarding whether diagnostic tests with short time-to-result, but applicable only in laboratories, are true poc technologies or if the term "poc" should be used for tests that can be performed "under the tree". considering that diagnosis should be closely associated with treatment and that, at the peripheral level, there is often shortage of health staff and medicines, the "point-of-care" becomes by default the "point" where "proper care" can be provided, and this may well be a clinic, a peripheral lab, or a hospital, depending on how well-equipped these settings are, especially in terms of human resources [34] . the diversity of available diagnostic systems, but more importantly of target groups and local requirements, has posed the need to define generic requirements. to this scope, the "assured" criteria were developed [35] to summarize the characteristics of the ideal diagnostic test for the developing world, which should be affordable by the target groups; sensitive and specific; user-friendly by limited trained personnel; rapid and robust; equipment-free; and delivered to those in need. these criteria were initially developed for sexually transmitted infections but are generic for developing countries-targeted diagnostics. however, it has been evident that the users' requirements call for elaboration (or "fine-tuning") of these criteria. for example, the "equipment-free" criterion may be risky, because saving the test results in the device decreases the risk of mis-interpretation by inexperienced personnel. therefore, there is an increasing trend to use mobile technology to read results provided by lateral flow tests (lfts). moreover, there is need for guidance on how to select patients who should be tested, interpret test results in the clinical context, and triage patients who should be referred and admitted due to severe illness. in case of epidemics, where several patients arrive simultaneously at the checkpoint, the capacity of the tool to process several samples at the same time is an advantage. the assured criteria, together with additional specific requirements, are summarized in target product profiles (tpps), which technology developers should meet during their development stages. examples of tpps exist for tuberculosis, ebola, and differentiation between viral and bacterial infections, and are driven by the who, the médecins sans frontières (msf), the foundation for innovative new diagnostics (find), and other organizations [36, 37] . microscopy for screening of malaria parasites by examining a drop of blood smeared on a slide after proper staining remains a widely used diagnostic tool. giemsa-stained thick and thin blood films are used to detect the presence of plasmodium parasites, to quantify the parasite density, and to identify the species [38] . considering the progress in light technology and the use of light emitting diodes (leds), microscopy has become more easy to use. however, it still requires well-trained personnel and a degree of experience to prepare the samples and to interpret the results. poor slide/ blood film preparation may lead to artifacts which are mistaken for parasites, while in reality they are bacteria, fungi, dirt, or cell debris. as a consequence, poor specificity leads to poor diagnostic quality [39] . two studies in tanzania have shown that the low quality of malaria microscopy and the lack of trust by clinicians in microscopy outcomes resulted in higher mortality among patients with non-malaria febrile illnesses than patients with malaria [40, 41] . an additional major disadvantage of this method is the fact that microscopy cannot identify sources of fever other than malaria (except for borreliosis, which is a relatively rare cause of acute fever). thus, in the case of malaria-negative diagnosis, or in the case of co-infection of malaria with some viral/bacterial agent, the microscopy-only diagnosis is not sufficient for clinicians to decide on the suitable treatment. for bloodstream bacterial infections, including typhoid fever, the main test used in equipped laboratories is conventional bacterial culture. for pneumonia, culture is also based on respiratory secretions, including sputum (but it is then impossible to distinguish between chronic carriage and infection), bronchoalveolar lavage, or pleural fluid. detection of pneumococcal bacteremia can orient the diagnosis; however, this test results in low yields, due to the relative fragility of these bacteria. indeed, the main general drawback of blood culture is the inability, except for a few cases, to diagnose localized infections such as pneumonia or urinary tract infection. for typhoid, in addition to blood culture, stool culture and/or bone marrow culture are used but are rarely available. other limitations are that cultures take several days to provide results and that collection of high quality samples from pediatric patients is rather difficult and often leads to false results [42, 43] . in adults, sensitivity of blood culture is limited by the low bacterial load. specificity of blood culture is also hampered by contamination by cutaneous bacteria during blood withdrawal, because sample acquisition from two different sites to overcome this problem is rather challenging in practice, especially in children. lateral flow tests [44] consist of a plastic device containing a composite assay strip flanked by a reagent/sample pad at one end and an absorption pad at the other. the strip contains two or more lines striped into the nitrocellulose membrane: one or more test line(s) consist of a disease-specific antigen or antibody and one line is used as a control. thus, both antigen-or antibody-specific tests can be performed. the reagent pad holds the dried conjugate consisting of colloidal goldlabeled antibodies or antigens specific for each test. the reaction is performed by adding the sample (typically finger-prick whole blood, or urine) to the sample pad, followed by the addition of a few drops of buffer. the sample is mixed with the conjugate, forming antibody-gold complexes that flow through the nitrocellulose membrane. when specific antibodies/antigens are present in the sample, they bind the respective conjugate at the test zone, indicating a positive result through the captured red-colored gold nanoparticles carrying the antibody/antigen of interest (fig. 3) . multiplexing is possible with the lfts in two ways: either via multiple analytes on the same strip (one sample inlet for all), or via several strips on the same cassette (as many inlets as the number of strips). the former option is more challenging and less generic because it depends on the assays and the (compatibility of) reagents with all target proteins to be detected. the results are read visually after 10-20 min, depending on the test. a valid test result is obtained if staining of the control line is observed. the equipment-free readout, however, is not sufficiently subjective, while it becomes even more complicated when (semi)quantitative readout is desired, e.g. to assess the intensity of the strip line. in addition to the mis-interpretation and ambiguity of the test results, there is also the risk of data loss, which is not the case when a readout equipment (with data storage and transmission capacity) is used. especially in the case of multi-strip cassettes, the use of a readout unit may help to diminish the risk of confusion between results from different lines. thus, a current trend in lft diagnostics is towards an automated readout, such as the deki-reader that has been well validated in the field for malaria testing [46] . several technologies have been developed based on colorimetric (e.g. colloidal gold or colored latex) or fluorescence detection, by commercial suppliers: esequant™ lft strip reader by qiagen lake constance (germany); an imaging system by axxin inc. (australia); the handheld image capture lft reader by detekt biomedical, lcc (usa); the cpoc™ reader from lre medical (germany); the handheld dcn fluorescent test visualizer (usa) [47] . although these systems are well suited for developed countries, they are often not affordable for lmics. a more promising approach is the use of smartphones, which has several advantages over other systems to: (i) become increasingly accessible in africa; (ii) be used as an image processing device with the built-in camera and suitable software; (iii) be able to support an application (app) for processing the acquired data; and (iv) enable global positioning system (gps) tracking of patients during an epidemic (following the appropriate ethics rules and after related permissions). furthermore, data storage and transmission to external databases offers a high added value to lfts. the camera can also typically scan 1-or 2-d barcodes on the lft cassette in order to identify the type of test. to this direction, the non-profit global health organization "global solutions for infectious diseases (gsid)" [48] is developing a mobile tool to read, digitize, and transmit poc diagnostic results for important infectious diseases. in cooperation with the university of washington (seattle, usa) and dimagi (boston, usa), gsid developed an open-source software to capture the results of many different poc tests and report them immediately via wireless technology. a proof-of-concept study was conducted in zimbabwe in 2014 [49] . other examples of this approach are the mreader of mobile assay (usa), the assayquest™ from clearbridge bioloc (singapore), and the skansmart and skaneasy products from skannex (norway). extensive work on this field has been performed regarding software development, for example by novarum dx™ (uk), or the commcare mobile platform by dimagi (usa). due to the relatively low complexity in manufacturing lfts, there are > 100 manufacturers worldwide for clinical applications (which is why we are not able to provide a list of these manufacturers). the average price of an lft is approximately us$1. several studies assessed the performance of lfts [50] [51] [52] [53] and in a non-exhaustive list of infections, lfts currently exist for malaria, dengue, typhoid, hiv, hepatitis b virus (hbv), hepatitis c virus (hcv), and recently ebola and lassa [47] . however, the average quality of these tests is inversely proportional to the multitude of suppliers. it is often the case that their accuracy is inadequate [54] . the main drawback of lfts that detect antibodies is that, at the acute phase, antibodies are often not yet present and the tests lack sensitivity. also, the dynamics of igm/igg can be complex and hard to interpret, especially when re-infection is possible (e.g. dengue). another problem is that antibodies -even igm -persist for several weeks after acute infection, which decreases the specificity of the test (e.g. chikungunya). for this reason, the who has a standard operating procedure for pre-qualification, and publishes and regularly reviews recommendations for lfts for particular diseases, especially malaria and hiv [54, 55] . nucleic acid amplification technologies (naats) are based on sequence-specific recognition and amplification of unique target regions in the genome of pathogens to be detected. thus, naats are highly specific and sensitive, as single microorganisms can be detected. compared to microscopy (the reference in malaria diagnosis), naats are much more sensitive, while in contrast to culture methods (the reference in bacterial disease diagnosis), naat process times can be reduced from days to hours. the following sections present the most frequently used naats. polymerase chain reaction (pcr) represents the most commonly used naat-based diagnostic technology [56] . in this reaction, a thermostable polymerase (taq, from thermus aquaticus) is employed. two primers are used that flank the target region of the template dna to be amplified. a three-step process is then repeated~30-45 times: (i) a denaturation step at~95°c, where the double-stranded dna (dsdna) template is denatured resulting in two single-stranded dna (ssdna) molecules; (ii) a primer annealing step at~68°c, where the primers are hybridized to the ssdna target region to be amplified; and (iii) an elongation step at~72°c, where the polymerase generates a copy (amplicon) of the target region starting from the primer in 3′-to 5′-end direction. in an ideal reaction, the targeted region of the template dna is doubled after each cycle. the (endpoint) amplicons are detected via gel-electrophoresis (non-specific method) or by using different kinds of specific molecular probes that generate an amplicon-specific signal. here, the signal also depends on the template concentration. the need for cyclic heating and cooling to perform the previously described steps for pcr renders this method relatively time-consuming (several hours depending on the configuration and the thermocycler) and energy-consuming (a power supply is required). recently advanced methods in dna amplification use isothermal naats (inaats) [57, 58] , which overcome the need for thermal cycling. these methods are less energy consuming than pcr and can be potentially executed in battery or solar panel operated devices, which is an important advantage for their application at the point of care. furthermore, as a consequence of continuous amplification rather than cycling, the reaction will often generate detectable products faster than pcr. a disadvantage of inaats is in general (not referring to specific methods) that quantitation is difficult as they do not follow a strictly controllable reaction scheme. in all methods mentioned above and in the next sections, rna can also be amplified by the addition of a reverse transcriptase (rt), which transcribes rna into complementary dna (cdna) for further amplification. in the following sections some of the main isothermal amplification technologies are briefly described. nucleic acid sequence based amplification (nasba) [59] is used to detect rna and is performed at 41°c. due to the fact that it targets rna, nasba was first used for the rapid diagnosis and quantification of hiv-1 [60] . nasba reaction is facilitated by three enzymes (avian myeloblastosis virus reverse transcriptase, rnase h, and t7 rna polymerase) leading to the amplification product. in brief, during the amplification process, cdna is initially produced from rna templates through primer (primer 1) annealing to the template rna and reverse transcription forming a cdna from the latter. during cdna production, rnase h hydrolyzes the rna template. the resulting cdna with the promoter sequence is annealed to a second primer (primer 2) and double-stranded dna (dsdna) is produced with reverse transcription. following this step, t7 rna polymerase transcribes an rna molecule from the previously generated dsdna. the above procedure is cyclic and in each round the number of produced rna is increased. a unique advantage of this method is that it can selectively amplify rna in the presence of high dna background, while the process can also start with a dna template. in this case, the rnase h step in the non-cyclic phase is not necessary. with nasba, amplification of a nucleic acid sequence to > 10 9 copies can be accomplished in~90 min. loop-mediated isothermal amplification (lamp) was developed by eiken chemical [61] and is based on a bst polymerase with strand displacement activity. thermal denaturation of dsdna is not required, as the polymerase itself can denature dsdna prior to elongation. due to the reaction temperatures employed (60-65°c), dsdna is in a metastable condition and the structure may hybridize/dehybridize spontaneously. this enables primers to bind to the template dna in melted regions. six primers are used resulting in high specificity. the forward (f3) and backward (b3) primers, together with the forward inner primer (fip) and backward inner primer (bip), generate an artificial starting structure with single-stranded loop regions in each end. based on this "dumb-bell" structure, a cauliflower-like structure is generated with the fip and bip primers binding at the loop region. loop primers (lf, lb) may help to decrease the reaction time [62] . this process results in very fast generation of dsdna products that can be detected either with intercalating dyes or with specific probes [63] in potentially < 10 min. in short, lamp is advantageous due to: (i) high specificity, through the use of a minimum of four primers that define six binding sites on the target; (ii) sensitivity equal to, or higher than pcr; (iii) robustness against inhibiting compounds. recombinase polymerase amplification (rpa) [64] is typically performed at 37-42°c. recombinase/primer filaments scan the template dna for homologous sequences. if a match is found, recombinase mediates the hybridization of the primer to the complementary sequence. the denatured part of the template dna is stabilized by singlestrand binding (ssb) proteins and the polymerase can extend the primer complementary to the template sequence. due to the low reaction temperature, the reaction may start at will, with the addition of mg 2+ . this may be necessary, especially in applications where ambient temperature is the same as the reaction temperature. a recent international quality assessment of molecular detection of rift valley fever virus via rpa performed equally well as the best rt-pcr tests [65] . helicase-dependent amplification (hda) is typically performed at 63°c. two primers are used to initiate dna replication by a polymerase. in a first step, the dsdna unwinding activity of a helicase is exploited to achieve denaturation [66] . subsequently, the primers anneal to the single-stranded dna and then are extended by the dna polymerase. each dna duplex is doubled in one cycle. the produced dsdna molecules are separated by helicase and the chain reaction is repeated. similar to rpa, single-strand binding proteins are used to stabilize the denatured ssdna templates. the simplicity of the method, the uncomplicated cycle reaction, and its high speed (especially for circular hda (chda), speed can reach 100 bp/s) are the major advantages of hda [67] . hda has been demonstrated to be efficient with several different types of sample matrices, including urine, stool, blood, and plasma [57] . further promising dna isothermal amplification techniques, such as strand displacement amplification (sda) [68] , nicking enzyme amplification reaction (near) [69] , strand invasion based amplification (siba) [70] , or rolling circle amplification (rca) [71] are available and advantageous in various applications. despite the advantages of naats regarding sensitivity, a drawback is the relatively high cost of the reagents, compared to the cost of lfts. the main cost driver in naats is typically the amplification enzyme (the polymerase). cost further increases when viral rna should be amplified and a reverse transcriptase should be incorporated in the reaction mix. in the case of some inaats, the assay design can be very complicated; for example, primer design in lamp or the simultaneous use of three enzymes, as required in nasba. costs increase further, when enzymes are not pre-stored in liquid, but in dry format (e.g. lyophilized). such approaches may be required to help enzyme stability at the challenging environmental conditions of tropical regions. however, enzyme activity loss may be observed in lyophilized compared to liquid reagents. further problems may arise as naats inherently measure both living and dead pathogens (as both can provide template dna). this may lead to false positive diagnosis, for example during monitoring the progress of a patient over a treatment period. from the infrastructure point of view, specialized laboratory facilities are needed. in this case, well-trained personnel must also be employed to achieve proper assay set-up. in case of multiplexed assays, the risk of human error (e.g. pipetting error) increases, even with experienced personnel. to reach satisfying sensitivity, it is generally still necessary to purify nucleic acids from raw sample materials, such as blood, sputum, or urine. such purification processes may represent > 50% of the manual workload. the labor costs for manual workflows add substantially to the overall costs. this section summarizes poc and near-patient platforms for nucleic acid extraction and amplification, representing a big category of emerging and promising technologies that address the end-users' needs expressed in sections 2.3 and 2.4, and overcome some challenges outlined in section 4.2 related to lack of automation. in this context, naats mentioned in section 4 should be integrated into automated systems in order to overcome the need for specialized personnel as well as laboratory infrastructure. this includes reagent pre-storage in the system for full hands-off operation. the automated platforms must withstand the challenging environmental conditions (temperature, humidity) of the tropical regions. there are dozens of technologies for diagnostics, in various stages of development, used in various settings and detecting various targets. the list is long, and it would be impossible to include all in the present review, which does not aim to provide a list of all existing technologies, but to present those that apply in the context defined in the previous sections. within this framework, we applied a specific methodology and followed certain (inclusion/exclusion) criteria to select which technologies to present. in particular, we included those that: (i) focus on febrile illness and other syndromic infectious diseases with the potential to be expanded to febrile illness (thus, we excluded technologies for blood chemistry/clinical chemistry analyzers); (ii) include at least nucleic acid amplification testing, which can also be combined with immunoassays (thus, we excluded technologies for cd4 cell counting); (iii) are at least at late stage development, pre-commercial, or commercial stage (thus, we excluded academic, research, or laboratorylevel solutions). the description of each platform is divided in two sections: the performance as well as the technology and operating principle behind the platform. the former refers to issues such as portability, modularity, time-to-result, etc. the latter describes the core technical component(s) and main innovative features, e.g. the detection technology, the fluidics handling, and the manufacturing technology. in short: (i) in terms of detection technology, most platforms use optical methods (fluorescence in real-time signal monitoring or imaging mode; or light scattering as in verigene®), thus, it appears that progress on the label-free technologies is still required to reach commercial level. exceptions are the q-poc™ that uses silicon nanowires for detection and easynat™ that uses an integrated lft. (ii) regarding fluidics, it is noteworthy that not all described technologies implement microfluidics but also "macro"fluidics. in particular, enigma® ml and cobas® liat use cartridges that incorporate macrofluidic handling (e.g. syringes, plunges). fluid handling is mostly pressure-driven, although centrifugal microfluidics are increasingly used (e.g. labdisk, genepoc™, liaison® mdx). (iii) manufacturing-wise, the majority of the cartridges are plastic disposables, with some exceptions that are based on cmos (complementary metal-oxide-semiconductor) technologies such as the vereplex™ chip and the q-poc™ silicon nanowire array. (iv) in terms of amplification, all platforms allow for both dna and rna analysis. most of them use rt-pcr although isothermal amplification is also increasingly used (e.g. in alere™ i, easynat™, labdisk). notably, the assembly "assay-cartridge-instrument" is common in all cases, constituting a platform-based approach. regarding performance, some of the platforms aim at single or few pathogens (e.g. genexpert®, alere™ i, easynat™, cobas® liat, q-poc™, genepoc™, liaison® mdx), while others follow a syndromic approach, offering panels of several pathogens being simultaneously tested (e.g. filmarray®, vereplex™ biosystem, verigene®, diagcore®, labdisk). furthermore, it is important to emphasize that many of the technologies have a sample-to-answer time > 1 h (except alere™ i, cobas® liat, and q-poc™), while the cost is in the range of tens of us$ for the cartridge and several thousands of us$ for the instrument. therefore, naats seem to be outcompeted by lfts. however, the added value that multiplex naat platforms offer in comparison to the lfts should be taken into account. moreover, the cost should be considered in a broad perspective, taking into account "hidden" materials and labor costs when comparing with the standards of care. a comprehensive overview of the technologies and some of their technical features are shown in table 2 . performance features (e.g. time-to-result, portability) are mentioned in the platform description but not in the table, as the latter intends to focus mostly on technical aspects. furthermore, the information provided regarding the performance characteristics is based on the most up-to-date search on publicly available resources. the authors cannot exclude the possibility that these data may be updated in the future by the developers/manufacturers during the progress of the platform, or that manufacturers terminate or delay the development/manufacturing/market entry due to corporate or financial reasons. furthermore, table 3 summarizes some "customer" related information such as prices of instrument and test, pathogens/syndromes detected, and regulatory status. this information is provided for those platforms that public information is available, and it may vary between the european and us market. the genexpert® system ( fig. 4(a) ) from cepheid [72] is a widely used system in developing countries implementing molecular diagnostics to identify tuberculosis (tb) [73] [74] [75] . despite the complexity of its cartridge (assembly of more than five parts), the system was adopted by resource-limited countries due to the endorsement of the xpert® mtb/rif test for tb by the who in 2010 and the financial support by the global fund for aids, malaria, and tuberculosis. the system contains several additional versions to detect drug-resistant tb, hiv, mrsa, chlamydia trachomatis, neisseria gonorrhea [76] , while it included ebola testing during the 2014 outbreak. the time-to-result is estimated to be 60 min. the system of the xpert® series is developed in single-cartridge or multi-cartridge configurations for the end-users to decide which option is more suitable to their needs: low/medium throughput (1-, 2-, 4-, 16-cartridge system) for the point-of-care, or high throughput (80-cartridge station) for centralized laboratories. the most portable system of the xpert® series aims to truly decentralize tb diagnosis. the genexpert® omni* ( fig. 4(b) ) is a single-cartridge version, battery operated and wirelessly connected to the cloud (operable also via smartphone), which allows highly decentralized utility and real-time data transmission. the price of a test is us$40-60 in the us market, however there are special arrangements with ngos that subsidize this test for tb in developing countries, thus eventually costing slightly below us$10 (excluding shipping and distribution; this is the sales price ex works). the genexpert® iv device cost is approximately us$17,000, while the genexpert® omni* costs less than us$3,000. the genexpert® is a sample-to-answer system, where sample preparation and extraction of nucleic acids take place in the cartridge in an automated way as the necessary reagents are pre-stored. only a few minutes of hands-on steps are required for liquefaction of crude samples such as sputum. regarding fluidics, the cartridge contains a total of 11 chambers and reservoirs ( fig. 5(a) ). at its core, there is a cylindrical rotary valve, which is handled by a plunger rod that moves vertically, as soon as the cartridge is inserted in the processing device. the interface between the valve and the plunger activates the liquids (sample and pre-stored liquid reagents) movement between chambers, by positive or negative pressure through small openings at the base of the reservoirs. an important component of the cartridge is an integrated filter, which is located at the base of the valve body and captures the microorganisms. subsequently, a sonic horn approaches the filter area and mediates the mechanical lysis of the microorganisms and the release of nucleic acids. the latter are pumped through a chamber where beads with freezedried amplification reagents are stored. these are rehydrated upon sufficient filling of the corresponding chamber (the filling level acts also as internal fluidic quality control). the entire mixture is then directed to the pcr reaction tube, located at the back of the cartridge [79] . regarding detection, the pcr tube is surrounded by heating/cooling plates that enable fast thermal response, and by optical blocks that enable amplification and real-time fluorescence-based pcr product detection. in contrast to traditional thermal cycling systems, in which all samples are subjected to the same time/temperature/optical protocol, each sample in a genexpert® system can be subjected to a different protocol. one more innovative feature of the processing module is the intelligent cooling/heating optical reaction (i-core) module [80] , a sophisticated, solid-state, miniaturized microelectronic and micro-optical system capable of performing pcr reactions with multichannel fluorescence detection in < 30 min. each i-core includes a four-channel optics system capable of exciting and detecting multiple fluorescent dyes in the same reaction tube, thereby being able to accurately measure four dna targets simultaneously. the filmarray® (fig. 6 ) from biomérieux [83] is a benchtop diagnostic instrument that has three fda-cleared panels: the respiratory panel [84] , the blood culture identification panel [85] , and the gastrointestinal panel [86, 87] . filmarray® responded to the 2014 ebola outbreak and received emergency use authorization (eua) from the us fda, based on the filmarray® biothreat panel that specifically detects ebola zaire. a field study was conducted in sierra leone in patients and healthcare workers [88] . only a few minutes hands-on preparation time is required before the cartridge is inserted in the processing device, including the loading of the pouch into the loading block, the insertion of the hydration solution into the pouch, and the loading of the sample. the simplicity of use, the lack of refrigeration storage requirements, and the multiplexing capacity are positive features for use by limitedtrained personnel. up to 27 different targets can be analyzed per test, yet the throughput is still low, as only one patient can be screened per test run [89] . in addition, the manufacturer's list price for the reader (~us$40,000) and reagents (~us$130/cartridge) may not be affordable in resource-limited settings. the filmarray® is a sample-to-answer system, whose core technology is based on the freeze-dried vacuum reagent pouch, and on the nested multiplexed pcr that allows for the detection of multiple organisms. the microfluidic pouch ( fig. 6(b) ) consists of two areas, namely the fitment, where the reagents are pre-loaded and freeze-dried, and the main body. these two are bonded via heat welding. the fitment is fabricated with injection molding of polypropylene. the main body is fabricated with two sheets of a polyester/polypropylene film that contain a copolymer adhesive layer. these layers are welded together using heated plates. the fitment contains 12 reservoirs (a in fig. 6(b) ) that contain the biochemical reagents. during the entire manufacturing process, vacuum is maintained in each well through a set of plunges (b). it is very important to maintain the vacuum in the fitment and the blisters because: (i) it helps to maintain the freeze-dried nature of the components and, thus, their long-term storage; (ii) it allows for the injection of the sample and the hydration solutions in the pouch in the correct volume; (iii) it minimizes the formation of bubbles in the microfluidic system. the sample matrix is a naso-pharyngeal swab for the respiratory panel (different matrices are used for other panels). a total of 300 μl liquefied sample (100 μl sample and 200 μl lysis buffer) are used for the test. the user injects the hydration buffer (used for rehydrating the freeze-dried pre-stored reagents) and the sample in two differently designated inlets. the vacuum in the pouch automatically draws the correct volume, thus eliminating the need for precise measuring and pipetting by the user. liquid handling is applied through pneumatic pressure and "bladder" assembly. this contains a bladder plate on which several inflatable bladders are located, each corresponding to one chamber of the pouch. the assembly is subjected to different gas pressures and as it (and the bladders) is externally in contact with the pouch chambers, any change in the volume of the bladders causes liquid to move between the chambers of the pouch. the bladders are pressed through pneumatically-driven pistons (figs. 2, 3 in [90] ). the sample moves to the lysis chamber where the filmarray® performs mechanical lysis (bead beating) using ceramic (zirconium silicate) beads. the procedure follows the "boom chemistry" [91] . the released nucleic acids are bound on silica-coated magnetic beads, which are transported from the lysis chamber into the purification chamber, using a magnet integrated in the device. in this chamber, any remaining cellular or viral debris are removed through washing. an elution step releases the nucleic acids from the magnetic beads. the 1st stage pcr follows (15-20 cycles, including a reverse transcription step to convert rna into cdna), where dozens of primer pairs of all target molecules are mixed for multiplex pcr. a dilution step follows with the addition of a mixture of a fresh mastermix containing an intercalating fluorescent dna dye. subsequently, the mixture is aliquoted in a high-density array of 102 microfluidic wells where the 2nd stage (nested) pcr [92] takes place. the 2-step pcr is used to eliminate limitations associated with single-step multiplex pcr. at the end of the 2nd stage pcr, the temperature is slowly increased and the fluorescence in each well is measured to create a melting curve, the shape and position of which can be used for differentiation of amplification products with differences of < 2°c in melting temperature [93] . for the two stages of pcr, the instrument has two peltier elements. a blue led is used to illuminate the array at the 2nd stage pcr and the fluorescence emitted by the dyes is imaged by a ccd camera. alere inc. markets the molecular diagnostics point-of-care system alere™ i (fig. 7(a), [94] ) for the detection of influenza a and b [95] , human respiratory syncytial virus (rsv), as well as group a streptococcus (clia-waived, different cartridges for virus and bacterium detection). recent studies have shown that the alere™ i system exhibited at least equal or superior sensitivity over conventional rapid tests [96, 97] . the rapid isothermal amplification technology implemented by alere™ i (near, [69] ) enables the sample-to-answer analysis within 15 min. the instrument itself is portable and stand-alone (no need for a laptop), has a user-friendly interface, data storage capacity, and interface ability. the test price, as of 2014, is approximately us$70-85 while the device costs approximately us$8,500. the sample matrix is a naso-pharyngeal swab eluted in transport medium. the analysis procedure is from sample to answer with prestored reagents. the single use disposable test cartridge consists of three parts (fig. 7) . the test base is inserted into the first available device slot. the test base has two reaction tubes, each of which contains a lyophilized pellet with a pair of templates (similar to primers) for the specific amplification of the target nucleic acid, and a fluorescentlylabeled molecular beacon designed to specifically identify the amplified targets. the sample receiver is inserted in the second available device slot. this cartridge contains the elution buffer to elute the pathogens from the swab (10 s stirring of the swab in the cartridge). the transfer cartridge is pressed against the sample receiver to collect the necessary amount of material and then transferred and locked over the test base in order to initiate amplification. the amplified products are detected via dual-channel fluorescence, deriving from the fluorescent probes. indepth technical design or information regarding the cartridge are not publicly available in detail for this system. another system from the same manufacturer is the alere™ q hiv-1/2 detect (fig. 8, [98] ). it is a fully automated platform for hiv-1 and hiv-2 detection within 50 min based on real-time pcr with multiplexing capability [99, 100] . it is suitable for point-of-care use due to its portable nature, battery operation, no laptop requirement, easy to use touch screen, while the cartridge can be stored at room temperature without need for cold chain. the alere™ q platform has been launched in multiple developing countries, initially for qualitative hiv, and allows for finger-prick detection of hiv virus in whole blood collected directly into the cartridge (25 μl are sufficient) that is then inserted into an instrument for automated processing, extraction, amplification, and detection. technology and operating principle of the alere™ q alere™ q hiv-1/2 detect is a sample-to-answer system. lysis is based on chaotropic salts acting upon the sample and releasing the nucleic acids. biotinylated oligonucleotides that are specific to hiv-1 and hiv-2 genome hybridize with the post-lysis released viral rna, and are subsequently bound on streptavidin-sepharose particles. after washing, reverse transcription and pcr follow. detection of the pcr product is the core technological innovation of the system. it is based on competitive reporter monitored amplification (cma) technology utilizing an array of immobilized oligonucleotide probes and complementary fluorescently labeled reporter oligonucleotides in solution [101] . these reporters compete with the amplicons for hybridization on the surface probes. as in the beginning there are only few amplicons, labeled reporters hybridize with the surface-probes, thus, the initial signal is high. as the pcr reaction progresses, the labeled reporters preferably bind to the amplicons, therefore, less reporters are bound on the microarray. consequently, the progress of pcr is monitored via the decrease of fluorescence from the microarray spots until a plateau in the amplification reaction is reached. fluorescence imaging follows the progress of the reaction, as one image is collected during each annealing cycle. regarding fluidics (fig. 8(b) ), the sample is inserted in a capillary (position 1), the inlet being specially designed to receive finger/heel prick. a control window (5) helps the user to assess the filling level. the cap (6) ensures hermetic sealing and minimizes the risk of sample spilling or aerosols exiting the cartridge, thereby avoiding any contamination risk. dry reagents are pre-stored in internal compartments, along with a buffer reservoir (4) . a fluidic network of valves in the cartridge regulates the liquid/air movement. a septum, which is pricked by a needle connected to the pneumatic module of the cartridge, is used to apply air pressure and move the liquids into the different compartments. the compartment (3) is the reaction chamber where pcr takes place and the microarray is located. the enigma® ml (minilab™) (fig. 9) is a point-of-care platform commercialized by enigma® diagnostics, which, since mid-2017, is in the process of selling their intellectual property underlying its point-of-care molecular diagnostics platform [104] (nevertheless the platform is described here as one of the most representative ones). although the main application of the system has been on respiratory tract infections [105] , its functional and performance characteristics would enable potential use in febrile illness diagnosis. indicatively, the pipeline of infectious diseases includes influenza a and b, as well as rsv. the enigma® ml requires no laptop due to its internal controller and user-friendly touch screen. the instrument is available in a modular configuration, i.e. in a single-module up to a six-module option (for one to six cartridges, respectively). each module can operate independently via the central controller, therefore, different assays (requiring different protocols) from the same or different specimens can be performed at the same time. the overall analysis time for each module is 95 min. the analytical and diagnostic verification process of the fluab-rsv assay on the enigma® ml that led to the subsequent ce marking was completed in july 2013, at guy's and st. thomas' nhs foundation trust, london, uk [105] . the molecular analysis is based on real-time pcr. all required reagents are pre-stored in the cartridge allowing for fully automated analysis with a very short hands-on time of 2 min, while there is no need for cold chain as the reagents can be stored at ambient temperature. the biochemical analysis is initiated with a combination of thermal and chemical lysis to release nucleic acids, which are then captured by magnetic beads. magnetic beads are robotically transferred in situ to two washing buffers for purification and removal of any inhibitory agents. the last step before amplification is the elution of the nucleic acids from the beads, followed by mixing with the amplification reagents (freeze-dried). each test contains bacteriophage ms2 as an internal process control. the cartridge consists of three main blocks (fig. 10) : (i) the sample inlet block, where the swab is introduced in the collection tube that is then clamped onto the cartridge; (ii) the block with the pre-stored (liquid) reagents (indicated with yellow in fig. 10) ; and (iii) the block with specific tools (movable components such as pipettors, magnetic wand, and foil cutter). the core of the cartridge operation is based on robotic actuation of fluids between the vessels in order to facilitate the biochemical reactions, handling of beads, etc. in particular, a robotic arm picks any movable items and inserts them in the corresponding liquid tubes [107] . a highly innovative feature of the system is the pcr reaction vessel [108] . it consists of an electrically conducting polymer (ecp), which acts as a resistive heating element. at the same time it is arranged in such a way that the electrically conducting profile differs in different areas of the vessel (along its axis), thereby controlling thermal gradients that are necessary for thermocycling. the different electrical conducting profile is regulated during the manufacturing of the vessel by varying its radial thickness (key parameter), thus varying unevenly its resistance profile. the vessel consists of a combination of a highly conductive material (aluminum) and an ecp, the two being separated by an insulating layer. the aforementioned induced temperature gradients also assist in convection-based liquid mixing in the reaction vessel. the ecp fabrication technology is typically injection molding. detection is achieved via hybridization probes and fluorescent resonant energy transfer (fret) [109] . taking advantage of this effect, the enigma® ml system uses two probes to hybridize with the amplified dna/rna product, acting as energy donors and acceptors. when amplification occurs, the two probes hybridize and are located in close proximity, thus, the energy of the donor (induced by the excitation source) is transferred to the acceptor, which, in turn, emits this energy as fluorescence. the latter is then monitored in real time during the amplification process, as the amount of the acceptor fluorescence is proportional to the amount of the generated pcr product. roche diagnostics offers the cobas® liat (lab-in-a-tube) system [110] as a compact solution for point-of-care molecular diagnostics (fig. 11) . the system is currently used for detecting: (i) influenza a and b and rsv viral rna in naso-pharyngeal swab specimens from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors; (ii) streptococcus pyogenes (group a β-hemolytic streptococcus, strep a) in throat swab specimens from patients with signs and symptoms of pharyngitis. however, it is not a multiplexed system, as the aforementioned infections are detected in different cartridges. the instrument is compact and stand-alone (laptop-free) with small footprint operating in such way that minimizes possible interference by, and contamination of the user. with minimum training needed, the system can provide results in as fast as 20 min. the cost of the device and cartridge is approximately €25,000 and €55, respectively. although the system has not yet been used for febrile illness diagnosis in developing countries, its performance features make it a good candidate. technology and operating principle cobas® liat is a sample-to-answer platform thanks to the full integration of all necessary reagents in its cartridge. the in situ sample preparation starts with mixing the sample (200 μl) with chaotropic agents inducing pathogen lysis, followed by the "bind-wash-elute" protocol (boom-chemistry [91] ), which involves binding of nucleic acids in silica-shelled magnetic beads, subsequent washes and a final elution step. nucleic acids are then ready for real-time pcr with internal controls. the core technology of the system is the cartridge, which consists of a rigid frame and a transparent flexible tubule. the latter consists of several pouch segments, which are separated via breakable seals and are flexible enough to be compressed. the system uses peristaltic fluidic actuation for liquid handling. after the cartridge is loaded, the analyzer, which contains clamps, plunges, and actuators, controls the selective release of reagents from tube segments, leading to mixing and propulsion of liquids by precisely applying pressure to the pouches. the pressure needed to compress the tubule to unload the pouch is approximately 1 atm. the analyzer also provides control of reaction conditions at different temperatures, and alternating movement of the reaction mix between two different temperature zones for rapid pcr (fig. 12) . the outcome of the reaction appears as a real-time fluorescence signal, detected by the six-channel photometer module in the analyzer [112] . from the fabrication perspective, the tubule is based on polyolefins and is fabricated via injection molding or blow molding. in order to serve specific processing needs, some tubules are surface-processed via plasma treatment or introduction of additives. the wall material is multi-layered, with the inner layer being biocompatible, while the outer is resistant to gas permeability. the flexible tubule is supported by a rigid frame, which is made of polypropylene with lower melting temperature than the tubule to ensure uniform melting during sealing. according to the relevant patent [112] (although the patent does not ensure that these exact methods are used in the final production) sealing between these components is achieved via welding of the flexible tubule on the outer frame using thermal and/or ultrasonic sources. an alternative approach is to seal the two components with a hot-melt adhesive joint with ethylene vinyl acetate, or with uv cure epoxy. the dimensions of the frame, surrounding the tubule cartridge, are 150 mm (height) × 25 mm (width). the agency for science, technology and research (a*star), singapore, stmicroelectronics, italy, and veredus laboratories, singapore [114] have partnered to develop a lab-on-a-chip for diagnosis of infectious diseases (fig. 13) . the system is already in the market and was used by the brazilian authorities as the tool of choice during the 2014 world cup. a number of application-specific cartridges have been developed, some of which are: vereflu™ (for respiratory infections); veremtb™ (for tuberculosis); verethreat™ (for biothreat agents such as anthrax, small pox, plague and tularemia); verevet™ (for multiple avian pathogens); veremers™ (for the middle east respiratory syndrome coronavirus). in 2013 the partnership announced the launch of veretrop™ for multiple tropical diseases, providing the necessary multiplexity that febrile illness diagnosis requires. the cost per veretrop™ test is approximately us$100. sample preparation is performed ex situ, either via spin columns or an automated extraction kit, and the overall time-to-result (including extraction, amplification, optical readout) adds up to approximately 3.5 h. vereflu™ [115] and veretrop™ [116] assay chips and multi drug resistant tuberculosis [117] have been clinically validated. the core of the system is based on a lab-on-a-chip cartridge (the "in-check" platform, fig. 14) in the size of a standard microscope slide. it contains a silicon chip covered with a glass coverslip and bonded to a plastic support. an on-chip printed circuit board (pcb) provides the necessary electric contacts as an interface to the processing instrument (e.g. for temperature control). the silicon chip has distinct processing areas (fig. 14) : (i) the sample inlet microfluidic ports with buried microchannels to facilitate easy chip filling and fluid displacement. (ii) the pcr area (12 × 8.5 mm 2 ), which contains four pcr chambers, with 1 μl liquid volume capacity, each. four pairs of independently controlled aluminum resistors are used to heat the silicon substrates and fluidic reactors, thus providing the thermocycling environment. the temperature is regulated by the temperature control system (tcs) in the chip processing device. (iii) the outlet holes, which provide microfluidic contact to (iv) the post-pcr hybridization and detection area, which consists of a 3.5 × 9 mm 2 microarray with 126 spots, where oligonucleotide probes are spotted using a piezo-array system [119] . fabrication is performed with a semiconductor-based mems process technology, named simich at 8″ wafer diameter, which allows for the fabrication of micro-mechanical, sensor, and microfluidic components. more details on the process steps are available in [120] . fig. 14 shows some key microfabricated components of the chip and a cross-sectional view of the entire lab-on-a-chip device. post-fabrication processing includes surface chemical treatment in order to immobilize dna probes in the microarray area, and to achieve biocompatibility of the pcr area for dna amplification [121] . post-amplification detection is performed in a separate, dedicated instrument, through fluorescent microarray detection of chip-hybridized amplification products using an image acquisition camera. quantumdx [123] is a uk-based nanomedtech company that develops and commercializes affordable handheld sample-to-result diagnostic and dna sequencing technologies using nanowire biosensors and innovative microfluidic technologies. their collaboration with the institute of microelectronics (ime) of the agency for science, technology and research (a*star), singapore, is strategic for the fabrication of the key microelectronic components. the q-poc™ platform (fig. 15 ) of quantumdx is a handheld, battery-powered molecular diagnostic device that promises a sample-to-answer dna-based result in as few as 15 min. it contains integrated reagents with current stability targets of up to 40°c for up to 18 months. the system can test one patient per run and is a pioneering approach to the handheld sequencing market. in 2014, quantumdx partnered with find in order to develop a solution for the combined detection of tb and drug susceptibility determination for the causative agent of tb. the q-poc™ system seems to be especially promising for antimicrobial resistance profiling given that many different genes can be screened through a nanowire array. there are several other assays in the pipeline, for diagnosis of e.g. hiv, sexually transmitted infections, neglected diseases, as well as for tumor profiling [124] . the core of q-poc™ lies in its biosensor chip, which is equipped with a silicon nanowire (sinw) array based field-effect biosensor. sinws have large surface-to-volume ratio, tunable electric properties, and exhibit high potential as biosensors, thus enabling next-generation point-of-care diagnostics. the binding events are detected in a labelfree way through changes in the nanowires' resistance, effectively converting the genetic code into a binary code and providing the output in a digitized format. in particular, upon hybridization of the target dna on the sinw, the charge density (and subsequently the electric field) changes. this is reflected as a change in the resistance of the nanowire, which produces the biosensing signal (fig. 16) . the more negative charges present during hybridization, the higher the resistance of the n-type sinw sensor. this principle was shown by zhang et al., by varying the hybridization sites (thus varying the distance of the charge layer) but keeping the total number of charges unchanged [126] . functionalization of sinws is performed with peptide nucleic acid (pna), instead of dna oligonucleotides. the neutral nature of pna minimizes the formation of dense charge layer near the nanowire surface, subsequently minimizing the signal to noise ratio. the n-type sinws feature typical dimensions of (w × h × l) 50 nm × 60 nm × 100 μm and are fabricated using standard cmoscompatible top-down approaches, through the oxidation of polysilicon beams patterning using conventional photolithography on p-type test wafers (more details available in gao et al. [127] ). this method is compatible with mass-manufacturing, is reproducible and convenient, and allows for scaling the sensing area and multiplexing as required by the application. with a capacity of 1000 nanowires in each silicon biosensor chip, the multiplexing potential becomes huge [128] . the sample-to-answer operation is achieved by housing the biosensor chip in a microfluidic cartridge (fig. 17) . ustar biotechnologies (hangzhou) ltd. [130] commercializes its proprietary isothermal amplification technology into an equipment-free diagnostic system, the easynat™ (fig. 18) . therefore, the system is especially attractive for resource-limited settings and is intended for batch testing in microscopy centers. however, the system lacks multiplexity (1 target/cartridge) and requires hands-on extraction steps (centrifuge, vortex, syringe). the extraction is performed ex situ using an equipment-free extraction kit. after boiling lysis, a syringe is used to extract and elute the dna using appropriate buffers. initial tests with easynat™ kit have been performed in clinical studies in tanzania [131] and china [132] for tuberculosis. the manufacturers note that the reagents require refrigeration ("storage in −20°c"). the system is based on an isothermal amplification method, called cross priming amplification (cpa), which uses multiple (5-8) crosslinked primers to amplify dna or rna at a constant temperature of reprinted with permission from [126] . copyright © 2008 american chemical society. 63°c [134] . due to its isothermal nature, amplification does not require any complex temperature control system: a water bath or a heating block are used in order to maintain constant temperature and incubate the cpa assays. following the 60 min amplification step, the reaction tube is transferred to the cartridge, where a lateral flow immunochromatographic strip provides the visual readout after 15-30 min. the patented cross-contamination-proof (xcp) nucleic acid detection device (fig. 19 ) offers the advantage of minimizing the risk of amplicon cross-contamination [135] , through: (i) maintaining closed reaction tubes after amplification (thus avoiding release of amplicon aerosols); (ii) using an enclosed unit (the amplicon tube) to insert the reaction tube; (iii) placing the amplicon tube into an additional enclosed unit (the detection device). running buffer and reaction tubes are inserted in the amplicon cartridge. the amplicon cartridge is subsequently inserted into the detection chamber. as the sealing of the detection chamber is closed, it activates a blade that punctures simultaneously the two tubes. the running buffer drives the amplicon to the adjacent lateral flow strip and amplification results are visually readable (fig. 19(c) ). the detection principle in the lateral flow nucleic acid testing strip is based on a combination of the nucleic acid hybridization and immunoassay principles. a probe is used, which carries a colored particle, and can hybridize with the amplicon. the complex is then captured by the antibody on the nitrocellulose membrane of the test strip. in the absence of an amplification product, there is no interaction/hybridization with the probe, which then flows uncaptured through the membrane to the absorbent pad ( fig. 19(b) ). (i) meander-shaped microfluidic channel filled with reagents that are released at will to area (g)/(h). in particular, reagents may include various nucleotide mixtures and wash solutions, all separated by air bubble(s), in order to flow sequentially and in a well-controlled way. when nucleotides flow sequentially over a nanowire-immobilized single stranded oligonucleotide, then according to the hybridization sequence, the local charge changes, causing a resistance change in the sinw. image source: used from [129] . luminex corp. commercializes the verigene® rp flex system, (fig. 20) (developed and previously commercialized by nanosphere), which received an fda approval in 2015 [138] . the target application is respiratory tract infections and the system covers a broad panel including 13 viruses and 3 bacteria [139] . analysis is performed from naso-pharyngeal swabs. verigene® also offers other panels such as gastrointestinal, while it also identifies eight gram-negative organisms and resistance markers from blood culture [140] . the full-automation mode of the system (pre-stored reagents) requires minimum hands-on time (< 5 min), while the overall time-to-result is 2 h. the system is benchtop and consists of two components, the reader (as a controller) and the processor sp, performing the automated test processing steps. it is a modular system since one reader, which acts as the user interface and central control unit, can handle several processors sp. each verigene® test uses four single-use consumables: an extraction tray, a tip holder assembly, an amplification tray, and a test cartridge. these are all loaded in the processor sp for all automated processing the glass support of the test cartridge, which carries the microarray, is inserted in the reader of (a) for detection. image sources: (a) used from [138] ; (b,c) reprinted from [141] , copyright © 2009, with permission from elsevier. steps, specimen extraction, target amplification, and hybridization. fluidic handling is performed via an in-system servo actuator and positive displacement pump, e.g. syringe pump, in combination with pneumatic valves. a comprehensive sequence of steps is provided in one of the company's patents [142] and the system's technical bulletin [138] . after sample processing in the extraction and amplification trays, nucleic acids are transported to the test cartridge. at the end of the procedure, the substrate (which supports a glass slide with the microarray for capturing the target nucleic acids) must be removed from the cartridge and placed in the reader (readout takes a few seconds). all necessary reagents are contained in the cartridge. the amplification technology is based on multiplex rt-pcr between heating and cooling zones followed by hybridization to target specific capture oligonucleotides in a microarray format. end-point detection is achieved with gold nanoparticles (aunps), which constitute the core technology of the system (proprietary nanogrid technology, fig. 21 ). with dimensions of 13-15 nm, the advantage of aunps over conventional fluorophores is the increased sensitivity by several orders of magnitude (e.g. the light scattered from one nanoparticle is equivalent to the light emitted from 500,000 fluorophores [143] ). after amplification is completed, the workflow for detection is as follows: inside the test cartridge, the amplified targets are hybridized to capture oligonucleotides that are pre-immobilized on the glass slide microarray. after removal of the unbound dna, the hybridization products are labelled with aunps. subsequently, intermediate oligonucleotides are flushed into the chamber and bind specifically to the immobilized complex, leaving a poly-a tail sticking out and being available for subsequent attachment of poly-t-bound aunps, which act as probes. after removal of unbound aunps, captured aunps are covered by silver nps (during the analysis procedure) to enhance the scattered light. thus, the overall size of the nps becomes larger and the subsequent optical signal is enhanced. illumination begins with an led (λ max 630 nm) shining through the edge of the glass slide (acting as a waveguide to the led illumination). the evanescent wave that is generated through the glass slide and towards the np coated surface causes light scattering from the illuminated au/silvernps towards the camera, which captures images of the array multiple times and at various exposure times [144] . notably, the aunp-based detection technology may be adapted for protein analysis as well. the spanish molecular diagnostics company stat-dx [146], acquired by qiagen in january 2018, has been developing the diagcore® (fig. 22 ) platform operating as a near-patient testing solution for low test volumes or as a platform to cover mid-throughput requirements. the company announced in early 2018 the approval of ce-ivd clearance for the system and the respiratory panel. stat-dx expects full commercial launch of the diagcore® in the 2nd quarter of 2018. the the cartridges of diagcore® are pre-filled with dry and liquid reagents with a shelf-life stability of 12 months at room temperature. the cartridges can be inoculated either using swab directly (with the appropriate cartridge housing for sample elution [147] ), or using liquid samples of volumes from 100 μl to 1.5 ml. automated cell lysis is used for releasing nucleic acids from pathogens. nucleic acid purification follows using high quality extraction columns. the sample is then subjected to rapid thermocycling for amplification; eight real-time pcr reaction chambers with six-plex capability allow for the simultaneous detection of a total of 48 targets. regarding fluidics, the cartridge includes a transfer module that moves laterally along a guide that is located within the cartridge in order to align various fluid ports and to control the movement of fluids through the cartridge channels and chambers [148] . fig. 23(a) shows a typical cartridge from the back side, where the areas designated with the numbers #110, #114, and #116 represent the testing unit, the dilution/dosing unit, and the fluid guiding unit between various chambers, respectively. the testing unit is shown in more detail in fig. 23(b) . the fluidic testing arrangement is aligned with the gravity field in order to avoid formation of bubbles in the various chambers. the inlet port of the structure is indicated as #204, while the top chamber (#216) is unvented and acts as an air reservoir for the air trapped within the microchannels. by avoiding venting, the elimination of aerosol leakage out of the cartridge is achieved. channel openings (#206a, #206b) are used as fluidic controls, where optic sensing defines whether there is liquid or not. a mixing chamber is included (#208). its large crosssection causes turbulent liquid flow, which enhances mixing (passive mixing). the chamber #210 is the actual reaction chamber (with a volume of up to 50 μl) serving also for detection. chamber #212 contains dry stored reagents. by applying variable pressure in the inlet port #204, the liquid may be pushed to chamber #212, rehydrate the reagents, which are then pulled back to chamber #210 in order to participate in the reaction. the structure indicated in fig. 23 (b) may be repeated several times, with a common inlet port and different stored reagents, in order to serve in geometrically multiplexed reactions. the concept of centrifugal microfluidics (in contrast to pressuredriven microfluidics that all the aforementioned platforms use) has evolved to a mature and powerful technology for automation of diagnostic workflows in the fields of clinical chemistry, immunodiagnostics, nucleic acid analysis and others. this microfluidic approach benefits in particular from the straightforward mechanism for liquid actuation based on centrifugal forces that are a result of simple rotation of the cartridge with defined spinning frequencies [150] . no external pressure sources or complicated (micro)pumping systems are required, which reduces the risk of cross-contamination (due to openings in the cartridge) and allows for the building of simple, portable processing devices with small footprint and weight. these facts qualify centrifugal microfluidics as a technology for automation of diagnostics at the point of care. a main advantage of centrifugal microfluidics is the employment of microfluidic modules ("unit operations"), each one serving a particular fluidic functionality for: liquid handling (e.g. metering and aliquoting [151, 152] , valving and switching [153] [154] [155] , pumping [156, 157] or mixing [158, 159] ); assay integration (reagent pre-storage and supply [160, 161] ); separation [162, 163] ; detection [162, 164] ; blood plasma separation; nucleic acid purification and amplification, or washing and blocking. a comprehensive overview on unit operations and process chains is provided by strohmeier et al. [165] . combination of these unit operations and process chains allows for the integration of complex laboratory processes into one system for fully automated sample-to-answer analysis. challenges are mainly in the field of cartridge-integrated sample preparation required for completely integrated sample-to-answer solutions [165] [166] [167] . apart from the world-to-disk interface that defines how the sample can be added by the user to the cartridge, the preparation of complex samples such as sputum, a classical specimen in diagnostics e.g. for detection of tuberculosis [81] , is an unsolved issue yet, as samples can be diverse in their physical constitution, and harsh chemicals or strong forces are required for pretreatment. further challenges are the handling of increased sample volumes (> 1 ml) of blood or urine that are required when the pathogen load is low or when the required clinical sensitivity for diagnosis is high. the maximum sample volume is typically limited by the available footprint of the disk and it has to be considered that e.g. for nucleic acid extraction by classical bind-wash-elute chemistries, equal volumes of lysis and binding buffer are required for sample treatment, hence posing further challenges to cartridge integrated reagent pre-storage. from the research perspective, increased application of disk-based systems has been recently observed in resource-limited settings, especially in the field of immunodiagnostics. a disk-based test for detection of a four-parameter liver assay panel for defining liver function has been demonstrated to be completed within 20 min by nwankire et al. [168] using a portable, battery powered processing device and including field testing in a laboratory in nigeria. furthermore, hosseini et al. [169] have implemented a novel mixing method using microspheres on disk in order to detect dengue virus antigen ns1. the following sections provide a short overview of some centrifugal microfluidic platforms that are at late development or market stage. the inclusion criteria in this review were that the technologies focus on infectious diseases and have at least a few publications in the field. one representative platform of centrifugal microfluidics at late stage development is the labdisk (fig. 24) from hahn-schickard [170] and imtek, university of freiburg, germany. the technology has been applied in a variety of applications ranging from clinical chemistry [164, 171] , to immunoassay [172] and nucleic acid analysis, indicating universal use. in the latter field, two significant advances in fully automated sample-to-answer (nucleic acid extraction and amplification) analysis were announced, particularly for neonatal sepsis, detecting s. warneri, h. influenza, e. coli, and s. agalactiae [173] , as well as for h3n2 influenza virus [174] . the amplification was based on pcr and the time-to-result was 3-3.5 h. yet, when the labdisk was used to integrate rpa isothermal amplification for the detection of biological warfare pathogens (b. anthracis, f. tularensis, and y. pestis) the total time-toresult was < 45 min [175] . in addition, the processing device for isothermal amplification operates with a power consumption of < 75 w, which facilitates its application in remote environments [176] and its potential use with battery or solar panels. the application of the labdisk at the point of care in resourcelimited settings has been demonstrated with the detection of yellow fever virus in laboratory and field settings in senegal using rpa and 20 min amplification time [177] . recently, a labdisk was developed for the detection of malaria, dengue, chikungunya, pneumonia, and typhoid fever that used lamp providing the necessary means for nucleic acid based multiplex amplification with a time-to-result between 70 and 120 min [178] . currently, one disadvantage of labdisk is that, while several parameters can be analyzed per disk, only one disk, corresponding to one patient, can be processed per test run. the concept behind the labdisk is to translate laboratory processes and steps into microfluidic unit operations and interface them towards fully functional sample-to-answer solutions. sample volumes can range from (sub)μl to several hundreds µl. one of the labdisk innovations is the pre-storage of liquid reagents in stick-packs [160] . these pouches tightly seal the buffers and release them on demand upon centrifugation. the manufacturing technology is microthermoforming of polymer foils [179] , a technology that is routinely used in the macroscale for blister packages, but was adapted to the microscale for micro/nanostructures in the labdisk (fig. 25) . typical foil thicknesses are between 150 and 300 μm. a mold and a thin polymer foil placed above the mold are heated at a temperature near the glass transition temperature of the polymer foil. when the conditions are favorable, air is blown from the top so that the foil takes the shape of the mold. main process parameters are the applied air pressure and the temperatures of mold and foil. various materials can be used, with most preferable candidates being the cyclic olefin polymer/copolymer (cop, coc) and polycarbonate (pc), depending on the application requirements. sealing is performed either via a pressure sensitive adhesive foil (containing nanocapsules, which break and release adhesive upon application of pressure) or via thermal sealing using one more foil material (e.g. coc on coc). the elastomeric molds are suitable for prototyping, where fast iterations are required. however, each elastomeric mold may be worn out after repeated use for some tens of cartridges. thus, for large manufacturing series it is more efficient to use a metal mold. it may be more challenging to deform the foil (and methods such as coating on metal are used), however it is economically more sustainable and the durability of one metal mold can be sufficient for tens of thousands of disks. from the commercial point of view, the genepoc™ system (fig. 26 ) from genepoc inc. [181] is a simple and integrated portable instrument for the prevention and early detection of infectious diseases. the system received in 2017 fda clearance for its clostridium difficile assay and a ce mark for its group b streptococcus assay. in the future, genepoc inc. plans to develop cartridges for detection of respiratory, gastrointestinal, neonatal, and hospital-acquired infections, as well as sexually transmitted diseases. the system operates in a sample-to-answer mode, with minimum hands-on steps (a few minutes). the manufacturers declare a time-to-result of up to 1 h. importantly, the system operates in a stand-alone mode, without the need for laptop, which is replaced with a touch screen instead. its universal sample-to-cartridge interface (accepts liquefied matrix) allows compatibility with several types of swabs, urine, etc. [182] . the cartridge consists of only three pieces of plastic and has a wedge-shaped form (unlike an entire disk), which allows the device to process up to eight cartridges simultaneously, thus offering an increased throughput that is often necessary in cases of "extreme" poc testing [182] . the genepoc™ disposable is composed of three distinct operational sectors: (i) world-to-chip interface: 100 to 200 μl of clinical sample conditioned in genepoc™ sample buffer is inserted in the disposable with a transfer pipette. the developers mention that the use of blood is not feasible yet, as it would require off-chip microbial preconcentration. subsequently, the inlet chamber is sealed with a lid that prevents aerosols from leakage. a series of siphons and passive valves prime the liquid to the next chambers. (ii) universal sample preparation, starting with pathogen mechanical lysis: a metallic disk is magnetically actuated in the chamber where glass beads are present leading to efficient lysis and release of nucleic acids. the lysate is centrifugally driven to a dilution chamber, where pcr inhibitors are inactivated by heating up to 95°c and with proper ionic condition adjustment of the extracted material in order to optimize subsequent amplification. (iii) the amplification and detection sector, which is the only assay-specific area of the cartridge where rt-pcr is performed (reagents are drystored). this sector has three chambers available for amplification (thermocycling is achieved via air heating of the overall process chamber). the device includes four non-overlapping excitation and emission wavelengths for fluorescence detection of chemical dyes, therefore, up to 12 targets (4 in each chamber) can be detected simultaneously, and under rotation. further details regarding cartridge material and fabrication methods are not available by the manufacturer. the liaison® mdx (fig. 27 , formerly 3m cycler with simplexa™ integrated assays) is a centrifugal-based automated platform commercialized by diasorin inc. (formerly by focus diagnostics, which is currently owned by, and is part of diasorin group [184]). the system has acquired fda/ce-ivd approval and its target applications currently aim at respiratory tract infections for influenza a and b as well as rsv viruses, hsv 1 and 2, group a streptococcus, and c. difficile [185] . the system is classified as benchtop and is operated via a laptop. the cost per cartridge is approximately €30, while the instrument costs approximately €40,000. the device is compatible with two types of disks, targeting different types of end-user specifications (both are made of polypropylene with a nucleic acid compatible adhesive): (i) the direct amplification disk has eight individual slots/cartridges (all are connected into one disk, but one or more can be used simultaneously). the sample volume is 50 μl, which is mixed with buffer (50 μl) and the sample-to-answer time is 60 min based on high-speed thermocycling and rapid rt-pcr using high heating/cooling rates (> 5°c/s and > 4°c/s, respectively). samples require no pre-processing (dna/rna extraction) and direct amplification is applicable. (ii) the universal disk is a 96-well singleuse cartridge able to perform multiple assays per disk. the reaction volume is 10 μl. the device has four excitation and emission wavelengths for optical multiplexing in addition to the geometric (multichamber) multiplexing. when the universal disk is used, extraction should be performed ex situ and the eluate is inserted into the disk. a rather different approach from the typical benchtop-based or fully-automated systems has been suggested by the "diagnostics-in-a-suitcase (dias)", initially developed for the emerging avian influenza a (h7n9) virus [186] . in contrast to the aforementioned systems, where the core was a (micro)fluidic cartridge handled in a fully automated way, the dias system does not proclaim miniaturization or fully-integrated components and requires a few hands-on steps. yet, it smartly includes all the necessary biochemical components (extraction and amplification reagents), tools (gloves, pipettes, tube tracks, pipette boxes, waste container), and equipment (a tube scanner, vortex, microcentrifuge, even a mask in case of a highly infectious disease/epidemic), within a suitcase for a complete on-site analysis. it is an entire portable lab, which functions with solar panels or power packs (fig. 28) . the workflow includes a typical sample preparation starting from the lysis of the pathogens, extraction of nucleic acids by binding to magnetic beads, two washing steps, and elution. this process requires 30 min. the subsequent amplification step is conducted via rt-rpa and the time-to-positive is 15 min. in total, nine pipetting steps are required, which can be reduced by replacing some liquid components with lyophilized reagents. during the 2014 ebola outbreak, the system was applied in field trial in guinea in collaboration with the institut pasteur in dakar, the public health institute of guinea, the university of stirling, the robert koch institute, and twistdx ltd. [187] . this innovative approach tackled several problems including lack of (constant) electricity; lack of cold chain for material storage; risk and time loss from transferring infectious material from the field to central laboratories for analysis; and the need for fast time-to-result for healthcare providers who screened patients for ebola virus. a natural drawback of the dias system is the lack of connectivity with all the accompanied consequences (manual registration of data, risk of loss, ambiguous storage of data, etc.). furthermore, it requires a certain degree of user training for the handling of manual steps. along a similar concept, msf is developing a small-scale, autonomous diagnostic bacteriology laboratory based on appropriate and feasible, existing or adapted techniques, at affordable cost, with high accessibility and ease of use, which may respond to clinical needs in rt-pcr/isothermal . the feasibility of using robust blood culture bottles with visual readout, with a new type of media that bypasses the need for blood and, thus making it easy to use by non-experts, is being currently evaluated. a simple pathogen identification system, restricted to genus, as well as an antibiotic sensitivity testing system, restricted to the six antibiotics used frequently at the peripheral level, have also been developed. the target patient populations are children presenting with severe illness, those with risk factors for bloodstream infections, such as neonates, malnourished or hiv positive children, those who fail to respond to first-line antibiotic treatment, hospitalized war patients, and patients with trauma or prior surgery by providing organism-directed antibiotic therapy. with the developed autonomous diagnostic laboratory, it will be possible to acquire information regarding more general questions such as proportions and antibiotic resistance profiles of key bacterial pathogens, thereby providing evidence-based information for antibiotic policy (appropriate prescribing) and allowing for the improvement of epidemiological investigations as well as supporting healthcare infection control in emergencies and in neglected populations in resource-limited settings. novel diagnostic platforms, such as those mentioned in the previous sections, require validation in the target patient population, as well as in users. this should be a requirement for all new diagnostic tests, but it is even more important for point-of-care tests that are often used in nonselected patients (for example, any patient with fever attending any level of the health system) and by users with no technical background. before large-scale implementation in resource-limited countries is decided, the test should also preferably be endorsed by who and by international donors and governments that provide funding. unfortunately, many point-of-care tests have been launched in the market with only limited laboratory validation using convenient patient samples (for example that have a high pathogen load), or even cultured pathogens in relatively high concentration. field evaluation of the performance of the test in real patients, who often harbor low pathogen loads, is indeed often undertaken only after registration of the product. this is due to the lack of national and international regulatory mechanisms applicable to diagnostic tests, which, in contrast, exist for medicines [189] . the net result is that health workers, especially those working in private clinics, and even patients going to pharmacies, have access to poc tests that have in fact very low sensitivity and/or specificity. for example, lft for chikungunya is commercialized, although it has a sensitivity of maximum 50% in the field [190] [191] [192] . the gap between the performance measured in the laboratory and that in the field is due to several factors, depending on the type of pathogen and technology used. the sensitivity of naats to detect bacteria in the blood compared to blood cultures is often disappointing [33] . even though naats are able to detect additional cases compared to blood-culture, they are not positive in all blood culture-positive patients [193] because bacterial load can be very low, especially in adult patients with robust immunity. this explains the failure of numerous molecular-based multiplex platforms licensed in northern countries, aiming at diagnosing rapidly the cause of sepsis in admitted patients. fig. 29 . the whole diagnostic test development process, from innovation, to validation and application. this development process is in reality a reiterative process that is more circular than linear. source: v. d'acremont. these tests work well for blood cultured for a few days but not for native blood, which implies that the potential gain of time, essential in these patients, is lost [194] . tests based on the detection of antibodies, even of igm type, often lack sensitivity during the very first days of an acute febrile episode, but also specificity because of antibody persistence for several weeks. these challenges show how important it is to undertake clinical trials in real conditions to assess first the real performance of the test in the field (accuracy against the best available standard), and then the impact of using such tests on the management of patients (efficacy, measured through the impact on the health outcome of patients, cure rate after a certain number of days, secondary hospitalizations, or deaths and sequelae), done by routine health staff in their real working environment (effectiveness, measured generally through the level of compliance to test results and guidelines, and the appropriateness of antimicrobial prescriptions) (fig. 29 ). outcomes measured in these trials should be related to the health outcome of patients and the public health benefits, and not only to the diagnosis based on laboratory reference standards (which for some diseases are anyway suboptimal or even non-existent). "we have to treat the patient, not a test result". to follow this adage, it is even more important nowadays that a continually increasing number of diagnostic tools is becoming available at the point of care. that should, however, not be interpreted as it has been for years with malaria microscopy, as an invitation to disregard test results and treat diseases presumptively based on clinical elements with low sensitivity and/or specificity [195] . this adage rather suggests that, because no diagnostic test is 100% perfect, their results do not provide a black-orwhite answer, but rather an indication on the disease probability. this probability depends on two factors: (i) the prevalence of the disease in the corresponding patient population (pre-test probability), and (ii) the performance (positive and negative likelihood ratios) of the test used. when a disease is rare in the population (low pre-test probability) even if the test has a reasonable accuracy, the post-test probability after a positive result remains low. for example, if a typhoid rapid test with 80% sensitivity and 80% specificity (and thus, a likelihood ratio (lr) of 0.8/(1-0.8) = 4) is used in an area of asia where prevalence of typhoid fever among patients with febrile illness is 20%, the post-test probability will be 50% (it can be calculated using fagan's nomogram [196] ), which suggests that it is reasonable to implement an antibiotic treatment to that patient. if the same test is used in an area of africa where the prevalence is 2%, the post-test probability will only be 7.5%, which hardly justifies treating the patient for typhoid but rather to consider alternative diagnoses. based on these principles, decision charts can be built to precisely guide clinicians on how to use diagnostic tests and prescribe medicines in the most rational way. paper versions of such guidelines have been developed by who and unicef, and details on their strengths and weaknesses can be found in the literature [24, 25, 197] . these guidelines, however, were deemed difficult to be adopted and properly implemented, due to several reasons including the long duration of the training required and the conflicting recommendations provided in different guidelines due to the time required for harmonization and then update of all paper material at large scale. mobile electronic technologies are promising tools to overcome these challenges. an electronic algorithm is, indeed, able to guide clinicians through the specific branches that apply to their patients, to accelerate training by teaching health workers directly on the mobile support (and even integrating training modules into the device), and to help to disseminate quickly to all health workers updated versions of e-algorithms through 3g connection. fig. 30 provides a schematic representation of an integrated tool that aims at managing children with febrile disease at primary care level in resource-limited countries (derived from the existing e-poct tool [23] ). the tool integrates a few key clinical data, biosensors measuring respiratory rate, oxygen saturation, cardiac rate, glycemia, and hemoglobinemia, as well as rapid diagnostic tests detecting malaria antigen (other pathogens can be added according to local endemicities) and host biomarkers (to differentiate bacterial from viral infections). it then provides guidance to health workers on when to refer a sick child, and whether to prescribe or not an antimicrobial (and other type of medicines), while it also calculates drug dosage based on weight or age. it is beyond the scope of this review to elaborate on the content and performance of existing e-algorithms and platforms for the integrated management of children with febrile disease, however, the reader can refer to a recent review published by keitel et al. [30] . the consultation process data collected through the computerized decision-support tools used by clinicians and sent with 3g remote connection to a userfriendly data management system can be used for surveillance and outbreak detection. to document the cause of patient's fever, and at the same time the cause of the potential epidemic, a decision-support system should integrate the results of a pathogen poc test, through a reader directly connected to the tablet supporting the clinical e-algorithm, and if possible also remotely to the central server. the source of information would be patients seen at routine health facilities tested by poc tools that have a direct impact on case management (e.g. malaria or typhoid), but also from a restricted number of sentinel sites where additional poc test, aimed at detecting pathogens of particular interest (that cause large epidemics, even if non-treatable such as dengue, or rare such as ebola virus disease), would be used. the data accumulated on a daily or weekly basis (depending on the level of 3g connection availability) at the central level would allow health authorities to conduct real-time disease surveillance, through weekly/monthly reports, and would also enable immediate outbreak alarms when the preset incidence threshold for the surveyed disease has been reached. from the central server, automated feedback reports can also be created, sent to clinicians' tablets and discussed during supervision visits at the health facility. training e-tools, aimed at improving management of patients in general or in the particular situation of an outbreak, could also be sent to the tablet to guide clinicians. finally, a patient file supported by an electronic card owned by the patient, could be synchronized at each new consultation with the data collected by clinicians in the tablet (fig. 31) . such a fully integrated system seems to be overoptimistic; however, each of its components has already proven to be feasible and the challenge now is to find appropriate and sustainable diagnostic and information and communication technology (ict) platforms that can be deployed at large scale and in the long term. to address the clinically complex differential diagnosis of an episode of fever, that is a frequent presenting syndrome, this review outlined some point-of-care and near-patient diagnostic technologies that can assist to this scope. in the long, but non-exhaustive, list of described technologies, none seems to satisfy simultaneously all criteria of end-users' needs, target product profiles, and the assured criteria. regarding performance, some technologies may be fully automated and multiplexed, but they are prohibitively expensive; some are cost-effective, but compromise sensitivity or multiplexity. in contrast, some solutions offer multiplexity, yet they are benchtop, require laptop, electricity, and are barely portable (due to size and weight). regarding technology, most of the described platforms implement nucleic acid amplification (pcr-or isothermal-based) and almost none of them includes a parallel immunoassay analysis to measure the host response to an infection. a few of the suggested approaches are equipment-free but therefore lack connectivity and interfacing ability. however, one can observe a common rationale in the structure of the described technologies. the vast majority are based on the combination of three components: the assay, the cartridge, and the readout device. several variations may exist in the format of the assay, the cartridge, or the detection principle; however, this three-element model seems to be the backbone of all described diagnostic platforms. this modular nature should motivate the developers and manufacturers to consider more (semi)open and adaptable platforms to address end-users' needs in a dynamic way, being able to quickly modify their panels and adapt to endemic or epidemic needs. the review concludes with the need of electronic decision trees to support diagnostic tools, as interactive assistance to clinicians. such algorithms are derived from paper-based guidelines to computerized versions (which enforce the provision of output for any possible combination of clinical elements, which is not the case for traditional guidelines), operating on 3g mobile tools such as tablets. they receive, as input, data from several (poc and non-poc) diagnostic tests, which are processed via clinical algorithms, to provide clinicians with guidance regarding the handling of the patient (admission, treatment, etc.). such interoperability between ict components and interfaced physical systems (diagnostic tests) for decision-support can provide solutions of clinical and public health utility, therefore, this trend is expected to increase rapidly, offering high impact diagnosis in developing countries, and transforming the global healthcare landscape. this process, being rather complex and requiring know-how from a variety of disciplines and partners, requires multiple interactions between universities, industries, governmental and non-governmental organizations, as well as philanthropic institutions, that are not only based in wealthy countries but also in resource-limited countries. to increase the chances to make progress, most successful technologies should be supported and developers be motivated to partner with each other. it is only with such a spirit that the development pathway can be coherent and harmonized, and the new tools eventually may be implemented at large scale. looking into future perspectives, it seems that a combination of technological and non-technological factors will be critical to decide whether the diagnostic poc technologies can be used in the management of patients with febrile illness. from the technological perspective, it would be interesting to observe the landscape in the next 5-8 years and whether label-free detection technologies can be transferred from the laboratory to the clinics, and then to the market. magnetic field, electrochemical, gravimetric (e.g. surface acoustic waves), and micromechanical (e.g. cantilever based) detection technologies are some laboratory-mature fig. 31 . schematic representation of the potential integration of a mobile patient management system (efever) and a centralized electronic disease surveillance system (emergence). source: valérie d'acremont. candidates that could potentially substitute fluorescence detection and offer the advantage of cheaper read-out devices. regarding label-free technologies, it is important to consider how they can potentially be integrated into sample-to-answer systems, in view of the requirements outlined in sections 2.3 and 2.4. in addition, it is important to keep track of the progress on the lft-based nucleic acid analysis and detection, a concept that is rapidly catching up and combines the easiness of lfts with the molecular analysis. the transition from the laboratory to the market is crucial and depends on multiple parameters. a fundamental one is the sustainable and scalable manufacturability of the disposable cartridges (in terms of materials and processes). ideally, developers should have access to fabrication facilities that could perform the scale-up manufacturing in two phases: first, from prototyping to medium scale manufacturing (a few thousands or tens of thousands cartridges/chips/components per year) in order to serve clinical validation studies; and in a next level to aim for large scale manufacturing (tens of thousands to hundreds of thousands, that are fairly realistic quantities for infectious diseases of global impact). special attention should be given to the heterogeneous integration of components, i.e. when a system consists of not only plastic/silicon/glass core components, but integrates biological and biochemical agents, which are necessary for the sample-to-answer analysis. issues of stability and compatibility of (bio)chemical reagents with cartridge materials and manufacturing processes, adsorption (and loss) of reagents may prove critical for reaching the desired limit of detection. a future perspective is also that the diagnostic tools are no longer operating in a stand-alone mode, but in a broader "ecosystem". to this direction, integration of ict is of primary importance, as it is the means to connect a variety of different tools and generate networks (existing "traditional" approaches, reference methods, and new innovative tools), create databases, and process data (since we are in the era of big data). along these lines, the combination of nucleic acid with protein biomarker testing seems to be a path for more reliable diagnosis, especially in case of syndromes caused by several different pathogens of bacterial, viral, parasitic, or fungal nature. further elaborating on the ecosystem approach, and especially for diagnostics for resource-limited countries, special business model developments are required that would render marketization in the target areas attractive. large industries or small and medium enterprises (smes) may choose their own way to market, but a new path may be to try marketing in partnership with the pharmaceutical industry. it is obvious that diagnostics and therapeutics are the two sides of the same coin, and only through their combination and interplay can healthcare truly progress. especially in an era that antimicrobial resistance rises dramatically, this exact issue could and should become the bridge to unite the worlds of diagnostics and therapeutics. one parameter that should also be considered during the transition of the poc technologies from the laboratory to the market is the healthimpact modeling, including health economics and cost-impact analysis. one technology may be (comparatively) cheap but may not offer added clinical value; another may be (seemingly) expensive but it may prove overall cheaper than the existing test because the clinical impact is higher. the 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integrated management of childhood illness: caring for newborns and children in the community the authors k.m., s.h., f.v.s., and r.z. acknowledge the european commission for funding under the fp7 project "discognosis" (ga-318408), the h2020 project "diagoras" (ga-633780), and the h2020 project "dmc-malvec" (ga-688207).the author v.d.a. thanks blaise genton for critical reading of the manuscript. conflicts of interest: none. key: cord-030341-uora9qcb authors: ruland, sebastian; lochau, malte; jakobs, marie-christine title: hybridtiger: hybrid model checking and domination-based partitioning for efficient multi-goal test-suite generation (competition contribution) date: 2020-03-13 journal: fundamental approaches to software engineering doi: 10.1007/978-3-030-45234-6_26 sha: doc_id: 30341 cord_uid: uora9qcb in theory, software model checkers are well-suited for automated test-case generation. the idea is to perform (non-)reachability queries for the test goals and extract test cases from resulting counterexamples. however, in case of realistic programs, even simple coverage criteria (e.g., branch coverage) force model checkers to deal with several hundreds or even thousands of test goals. processing each of these test goals in isolation with model checking techniques does not scale. therefore, our tool hybridtiger builds on recent ideas on multi-property verification. however, since every additional property (i.e., test goal) reduces the model checker’s abstraction possibilities, we split the set of all test goals into different partitions. in test-comp 2019, we applied a random partitioning strategy and used predicate analysis as model checking technique. in test-comp 2020, we improved our technique in two ways. first, we exploit domination information among control-flow locations in our partitioning strategy to group test goals being located on (preferably) similar paths. second, we account to inherent weaknesses of the predicate analysis by applying a hybrid software model-checking approach that switches between explicit model checking and predicate-based model checking on-the-fly. our tool hybridtiger is integrated into the software analysis framework cpachecker. the hybridtiger algorithm is implemented within the software verification framework cpachecker [4] . cpachecker utilizes the eclipse cdt c-parser 3 . if ( n == 2) return 1; 5 return fib (n -1) 6 + fib (n -2) ; 7 } (a) c-program cpachecker allows developers to easily integrate new algorithms like hybrid-tiger, which may use other algorithms implemented in cpachecker, such as counterexample-guided abstraction refinement (cegar) [5] . additionally, new reachability analyses can be integrated as configurable program analyses (cpas) [2] . each cpa consist of an abstract domain with the operators post, merge, and stop. multiple cpas can also be combined into one cpa. hybridtiger uses the coveritest [3] algorithm to sequentially combine test-case generation runs utilizing different verification techniques. each test-case generation run applies the cpa/tiger-mgp 4 (tiger multi-goal-partitioning) algorithm, which utilizes the cegar algorithm. hybridtiger first extracts test goals from input programs and repeatedly executes reachability analyses provided by cpachecker until every reachable test goal is covered by at least one test case. to this end, test goals are encoded into (non-)reachability properties. if a test goal has been reached, cpachecker thus returns a counterexample and hybridtiger extracts a test case (i.e., a vector of input values), writes the test case to disk and marks the test goal as covered. hybrid test-case generation. hybridtiger receives as inputs a c program and a property specification (i.e., a set of test goals). next, hybridtiger transforms the c program into a control-flow automaton (cfa) [1] . figure 1 shows an example c program and the corresponding cfa. after cfa generation, the coveritest algorithm as configured in hybridtiger (see fig. 2 ) is executed. in every new iteration, each analysis of our configuration first (re-)partitions the set of uncovered test goals (e.g., partitions p1, p2, p3 and p4 for cpa/-tiger-mgp-value and p1 and p2 for cpa/tiger-mgp-predicate in fig. 2 ). in each iteration, cpa/tiger-mgp-value is performed first using explicit model checking and is stopped after 120s. after that, cpa/tiger-mgp-predicate is partitioning. hybridtiger utilizes domination information of test-goal locations according to the respective cfa paths. this meta-information is retrieved from the generated cfa: each cfa node (i.e., basic block of program locations) in fig. 1 is annotated with a post-order id such that a node will only be reached after all nodes on the same path with a larger id have been reached at least once. hence, we use the ids of predecessor nodes related to the cfa edges of test goals as sorting criterion for the overall set of test goals before splitting this set into partitions of predefined sizes. in this way, test goals sharing similar paths are more likely to be assigned to the same partition thus facilitating reuse potentials of reachability-information during reachability analysis. hybridtiger has three main strengths. first, the directed generation of test cases aiming at covering particular test goals significantly reduces the overall number of test cases. additionally, most test cases produced by hybridtiger effectively increase the overall coverage (i.e., hybridtiger produces mostly correct and nonredundant test cases). second, hybridtiger uses control-flow information to partition test goals which potentially enhances efficiency of test-case generation due to information reuse among similar test goals. lastly, hybridtiger uses combinations of different analysis strategies (i.e., value analysis and predicate analysis) to cope with structural diversity of input programs. one weakness of hybridtiger is that the partitioning approach does not improve performance of a goal-by-goal approach if being applied to programs with a small number of test goals (e.g., reaching one single error location as demanded in the cover-error category). results. in test-comp 2020, hybridtiger has participated in all categories and managed to reach the 4th rank in code coverage and the 6th rank in finding bugs, where hybridtiger performed better on tasks with many test goals. the version of hybridtiger submitted to test-comp 2020 is built from the tigerintegration2 5 branch revision 32283 of the cpachecker repository and is archived at https://gitlab.com/sosy-lab/test-comp/archives-2020. hybridtiger can be applied to a single file using the command where spec is the property file (e.g., coverage-error-call or coverage-branches) and file is the input c program. statistics of the analyses are printed to console and meta data on generated test cases as well as the test suite are written to files in the output folder. in order to run hybridtiger for the test-comp 2020 benchmarks a linux system with java 8, benchexec 6 and the sv-benchmarks 7 is required. finally, run benchexec with: the benchmark definition cpa-tiger.xml (archived at https://gitlab.com/sosylab/test-comp/bench-defs/tree/master/benchmark-defs), and the tool-info module cpachecker.py (archived at https://github.com/sosylab/benchexec/tree/master/benchexec/tools). cpachecker is maintained by the software systems lab at lmu munich as open-source project, contributed by an international group of researchers from lmu munich, university of passau, technical university of darmstadt and the institute for system programming of the russian academy of sciences.the branch tigerintegration2 from which hybridtiger is built is mainly developed at the technical university of darmstadt. additional information is available at https://cpachecker.sosy-lab.org/. software model checking via large-block encoding configurable software verification: concretizing the convergence of model checking and program analysis coveritest: cooperative verifier-based testing cpachecker: a tool for configurable software verification counterexample-guided abstraction refinement for symbolic model checking ), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use acknowledgement. this work was funded by the hessian loewe initiative within the software-factory 4.0 project. key: cord-311766-m9yv4qkm authors: demey, baptiste; daher, nagib; françois, catherine; lanoix, jean-philippe; duverlie, gilles; castelain, sandrine; brochot, etienne title: dynamic profile for the detection of anti-sars-cov-2 antibodies using four immunochromatographic assays date: 2020-05-07 journal: j infect doi: 10.1016/j.jinf.2020.04.033 sha: doc_id: 311766 cord_uid: m9yv4qkm in order to fight the sars-cov-2 pandemic infection, there is a growing need and demand for diagnostic tools that are complementary and different from the rt-pcr currently in use. multiple serological tests are or will be very soon available but need to be evaluated and validated. we have thus tested 4 immunochromatographic tests for the detection of antibodies to sars-cov-2. in addition, we assessed the kinetics of antibody appearance using these assays in 22 patients after they were tested positive by rt-pcr. we observed great heterogeneity in antiboy detection post-symptom onset. the median antibody detection time was between 8 and 10 days according to the manufacturers. all the tests showed a sensitivity of 60 to 80% on day 10 and 100% on day 15. in addition, a single cross-reaction was observed with other human coronavirus infections. thus, immunochromatographic tests for the detection of anti-sars-cov-2 antibodies may have their place for the diagnostic panel of covid-19. in order to fight the sars-cov-2 pandemic infection, there is a growing need and demand for diagnostic tools that are complementary and different from the rt-pcr currently in use. multiple serological tests are or will be very soon available but need to be evaluated and validated. we have thus tested 4 immunochromatographic tests for the detection of antibodies to sars-cov-2. in addition, we assessed the kinetics of antibody appearance using these assays in 22 patients after they were tested positive by rt-pcr. we observed great heterogeneity in antiboy detection postsymptom onset. the median antibody detection time was between 8 and 10 days according to the manufacturers. all the tests showed a sensitivity of 60 to 80% on day 10 and 100% on day 15. in addition, a single cross-reaction was observed with other human coronavirus infections. thus, immunochromatographic tests for the detection of anti-sars-cov-2 antibodies may have their place for the diagnostic panel of covid-19. keywords: sars-cov-2; covid-19, antibody, lateral flow assay since december 2019, the world has been facing a pandemic of covid-19, an infectious disease caused by sars-cov-2, a virus that emerged in china (wu and mcgoogan, 2020) . although rt-pcr testing of sars-cov-2 has become the standard method for direct diagnosis, these real-time pcr tests have some limitations, primarily dedicated infrastructure to avoid any biorisk, limited capacity and a long turnaround time (y. . there is increasing pressure from the medical community and society to screen the population on a large scale. serological tests in elisa format or as immunochromatographic lateral flow assay (lfa) have recently become available from many manufacturers (z. (haveri et al., 2020) . these serological tests will be complementary to pcr tests both for screening and diagnosis of the population, for the purpose of population exits from containment in different countries and finally for future epidemiological studies. however, it is necessary to evaluate the analytical performance of these assays and also their place in clinical practice. thus, the objective of our study was to evaluate four immunochromatographic assays for the detection of igm and igg antibodies to sars-cov-2 and to evaluate the kinetics of their detection by these lfa. twenty two patients diagnosed positive in amiens university hospital for sars-cov-2 on a nasopharyngeal swab using a rt-pcr technique (national reference center in pasteur institute, paris, france) were included in our study. the date of reporting of the first symptoms was retrieved from the medical records. the samples were tested regularly during the hospitalization until the tests were positive, with an evaluation at most on day 24 post-symptoms. in order to evaluate a possible crossreaction with the other human coronaviruses described to date (nl63, hku1, 229e and oc43), sera following such viral respiratory infection diagnosed in our lab were tested. this project was conducted in accordance with the reference methodology (mr-004 france) in accordance with article 30 of the gdpr. we evaluated 4 immunochromatographic tests for the detection of igm and igg directed against sars-cov-2 ( figure 1 ). these tests were kindely provided by asian manufacturers, namely biotime biotechnology co, autobio diagnostics co, isia bio-technology co and biolidics. for the biotime, autobio, and biolidics tests the detection of igm and igg is performed on the same diagnostic cassette. for isia 2 different cassettes are available. each test requires between 10 and 20 µl of serum, plasma or whole blood and is read 10 to 15 minutes after the sample and diluent have been deposited. for the biotime and biolidics assays, respectively 15 and 17 of the 22 patients could be tested for lack of immunochromatographic tests. longitudinal immunochromatographic testing in all patients shows heterogeneity in the time to detection of antibodies after symptom reporting (figure 2 ). the median antibody detection time was 8 days since onset of symptoms for autobio and biotime (igm or igg), 9 days for biolidics (igm or igg) and 9 and 10 days for isia igm and igg respectively (figure 2 and supplementary data). igg was detected in all patients on day 15 since onset of symptoms, while igm was not detected in 3 patients with autobio and isia. igm was detected before igg in 1, 1, 7 and 0 patients with the biotime, autobio, isia and biolidics assays respectively. in the other cases, igm was detected at the same time as igg. thus, the diagnostic interest of detecting igm directed against cov-2-sars appears limited. the clinical sensitivity of the different tests could be assessed longitudinally during follow-up and we observed an increasing sensitivity in the post-symptom period ( figure 3 and table 1 ). as described above, the clinical sensitivity of igm does not appear to be superior to igg for these immunochromatographic tests. with either igm or igg detection for a patient on days 5, 10 and 15 since onset of symptom, we calculated a clinical sensitivity between 9 and 24%, 67 and 82% and 100% respectively ( figure 3b and table 1 ). the autobio test appears to have better sensitivity at day 10 (81.82%) versus 70.59%, 68.18% and 66.67% for biotime, isia and biolidics respectively (not significant). in order to evaluate the specificity of these different immunochromatographic tests, particularly regarding previous infections to other viruses of the human coronavirus family, we evaluated 12 sera from patients who had a rt-pcr diagnosis of respiratory infection by different coronaviruses in 2019 (table 2) . of the 41 tests performed, only one (autobio) was positive from the serum of a patient with a respiratory diagnosis 39 days previously of hcov-229e. the same test for the other three samples with hcov-229e was negative each time. cross-reactions with these different coronaviruses therefore appear to be limited but may require further investigation. in this study we demonstrated the kinetics of detection of antibodies to sars-cov-2 using immunochromatographic lfa. these simple rapid unit tests are also easy to read (figure 1 ). profiling early humoral response were already observed with elisa assay (guo et al., 2020) (zhao et al., 2020) . we calculated increasing clinical sensitivities over time from the onset of symptoms in patients. moreover, we did not observe any real added value in igm staining from these immunochromatographic tests. with this kind of test, it is very difficult to distinguish the very recent infection from the older one because some patients present early with igg without igm and for some a detectable igm threshold appears later. serological elisa tests from research laboratories or portfolios of in vitro diagnostic manufacturers may allow a clearer distinction between igm and igg kinetics and their respective interest. nevertheless, the value of these point of care tests seems obvious in countries with limited resources but perhaps also as the epidemic progresses in individuals in the form of self-homemade assay from a drop of blood on the fingertip. this type of test could be easily delivered to individuals and thus limit contact. in addition, in countries with strong health systems, these rapid detection tests may also find their way as doctor tests in emergency departments. during this covid-19 epidemic there is a rebound in symptoms on days 7 to 10 leading to a visit to a doctor or an emergency department. in figure 3 and table 1, we have calculated a sensitivity of these tests between 22% and 81% during this post-symptom reporting period. even if symptom reporting remains very subjective and time-varying but if we add an average incubation period of 5 days (linton et al., 2020) , we can say that at 14-15 days post-infection these tests seem reliable. finally, regarding specificity that we evaluated with respect to sera of other common coronavirus infections, we observed a single cross-reaction. however, we were unable to test serum from people formerly infected with sars-cov (tian et al., 2020) . we would probably have many more cross reactions between these very close viruses as already report. also, due to the lack of available tests, we could not test for specificity with samples containing antibodies to other viruses (hiv, hcv, hbv and others pathogens). in conclusion, we described the kinetics of detection of post-symptom antibodies in 22 patients using immunochromatographic rapid tests and demonstrated the good performance of these tests for the detection of antibodies after sars-cov-2 infection. our results suggest that these rapid and simple tests should be seriously considered in this 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to sars-cov-2 in patients of novel coronavirus disease key: cord-179749-qdbmpi7j authors: sacks, daniel w.; menachemi, nir; embi, peter; wing, coady title: what can we learn about sars-cov-2 prevalence from testing and hospital data? date: 2020-08-01 journal: nan doi: nan sha: doc_id: 179749 cord_uid: qdbmpi7j measuring the prevalence of active sars-cov-2 infections is difficult because tests are conducted on a small and non-random segment of the population. but people admitted to the hospital for non-covid reasons are tested at very high rates, even though they do not appear to be at elevated risk of infection. this sub-population may provide valuable evidence on prevalence in the general population. we estimate upper and lower bounds on the prevalence of the virus in the general population and the population of non-covid hospital patients under weak assumptions on who gets tested, using indiana data on hospital inpatient records linked to sars-cov-2 virological tests. the non-covid hospital population is tested fifty times as often as the general population. by mid-june, we estimate that prevalence was between 0.01 and 4.1 percent in the general population and between 0.6 to 2.6 percent in the non-covid hospital population. we provide and test conditions under which this non-covid hospitalization bound is valid for the general population. the combination of clinical testing data and hospital records may contain much more information about the state of the epidemic than has been previously appreciated. the bounds we calculate for indiana could be constructed at relatively low cost in many other states. constructing credible estimates of the current prevalence of sars-cov-2 in the united states is challenging. despite growing since the start of the epidemic, testing rates remain low in most of the country: only a small fraction of the population is tested for sars-cov-2 on any given day. moreover, tests are often allocated to people exhibiting covid-19 symptoms or who are thought to have come into contact with the virus (abbott and lovett, 2020) . for example, new york state and texas both use a self-diagnostic tool to screen people for testing. 1 the low rate of testing in the general population means that the number of confirmed sars-cov-2 cases almost certainly understates the true number of infections in the population. at the same time, statistics like the fraction of tests that are positive likely overstate population prevalence because the tested population is more likely to be infected than the population as a whole. in this paper, we propose a new approach to measuring the point-in-time prevalence of active sars-cov-2 infections in the overall population using data on patients who are hospitalized for non-covid reasons. there is less uncertainty about sars-cov-2 prevalence for the non-covid hospital patient population because people in the hospital are tested at much higher rates than the general population, even if they are hospitalized for reasons unrelated to covid-19 (sutton et al., 2020) . we combine detailed testing data and hospital data from indiana with a family of weak monotonicity assumptions that seem to have high credibility. the combination of these assumptions with linked testinghospital data leads to relatively tight upper and lower bounds on the prevalence of active sars-cov-2 infections in the overall population in indiana in each week from mid-march to mid-june. the detailed indiana data allows us to conduct robustness checks that partially validate some of our assumptions. our basic method (without the validation checks) could be implemented using data that many states are already collecting and partially reporting. thus, our approach could help states extract timely prevalence information using existing surveillance data. importantly, our paper is focused on estimates of the fraction of the population that would test positive in each week. these estimates are distinct from recent efforts to estimate the share of the population ever infected with sars-cov-2 (manski and molinari, 2020) . the distinction between active prevalence and cumulative prevalence is important because the prevalence of active infections is a key determinant of the spread of the epidemic, given that the level of immunity in the population is thought to be quite low. estimates and forecasts of the prevalence of active sars-cov-2 infections are crucial for public and private responses to the disease. they have shaped decisions about disruptive non-pharmaceutical interventions such as school closures, non-essential business closures, gathering restrictions, stay-at-home mandates, and temporary increases in the generosity of the unemployment insurance system . reported estimates of prevalence likely also motivate individual precautionary behaviors ranging from wearing a mask to reducing demand for goods and services that require physical interaction allcott et al., 2020; philipson, 2000 philipson, , 1996 kremer, 1996) . finally, estimates of prevalence are a necessary input into efforts to measure other quantities of interest, like the infection fatality rate and the infection hospitalization rate. given its importance, researchers have developed several approaches to measuring sars-cov-2 prevalence in light of the challenge of non-representative testing. one of the most credible is to conduct a biometric survey in which tests are offered to a representative sample of the population (menachemi et al., 2020; richard m. fairbanks school of public health, 2020; gudbjartsson et al., 2020 ). other studies have tested a census of smaller populations such as cruise ships (e.g. russell et al. (2020) ). however, it is difficult and costly to regularly implement a survey with accurate coverage and high response rates, especially a new survey that has not been in the field for long. a second approach involves backcalculation methods, which use data on observed hospitalizations or deaths to infer disease prevalence at earlier dates using assumptions about the unobserved parameters that determine the progression of the disease, hospitalization rates, and case fatality rates (brookmeyer and gail, 1988; egan and hall, 2015; flaxman et al., 2020; salje et al., 2020) . backcalculation may work well if hospitalizations or deaths are well measured, and if previous research has already reached consensus about key parameters related to the disease. however, backcalculation may be less credible for a novel virus because the scientific knowledge base is smaller. and even when backcalculation is based on credible assumptions, it may be of limited value for public health decision making because both hospitalizations and deaths lag current infections (verity et al., 2020) . an alternative to biometric surveys and backcalculation is to combine non-random clinical testing data with weak distributional assumptions to construct bounds on population prevalence (e.g. manski (1999) ; wing (2010) ). in the covid-19 epidemic, stock et al. (2020) provide bounds on population prevalence using a testing encouragement design, which is not always available. manski and molinari (2020) bound the share of the population that has ever been infected under a "test monotonicity assumption" that the infection rate is weakly higher among the tested than among the untested. this assumption is appealingly credible, and the bounds can be calculated from widely available test data. however, even when the focus is cumulative prevalence under test monotonicity, the prevalence bounds are often wide because testing is so rare. we pursue a related strategy in this paper. our analysis is based on the insight that test rates are especially high among hospitalized patients, even patients hospitalized for reasons that are apparently unrelated to covid-19, such as labor and delivery or vehicle accidents. the upper and lower bounds on prevalence in these populations will tend to be much tighter than similar bounds in the general population. in addition, it is plausible that some types of hospitalizations occur for reasons that are independent of infection risk. for example, people who are hospitalized for injuries sustained in a traffic accident might be expected to have about the same risk of sars-cov-2 infection as the general population. we build on this insight to estimate more informative upper and lower bounds on weekly sars-cov-2 prevalence in the population. our paper makes some methodological contributions that may be relevant to the development of more informative public health surveillance systems. we describe the conditions under which upper and lower bounds on active prevalence among non-covid hospitalizations are valid estimates of the upper and lower bounds on prevalence in the general population. we maintain the test monotonicity assumption throughout, and we derive upper and lower bounds on prevalence in the population under two alternative assumptions about the representativeness of non-covid hospitalizations for the broader population. the first assumption is a relatively weak "hospital monotonicity" assumption that prevalence is at least as high among non-covid hospital patients as it is in the general population. this assumption would be satisfied even if hospitalized patients are at greater risk of covid because, for example, people who get into car accidents have more social interactions. the second assumption is a stronger "hospital independence" assumption that prevalence is the same among non-covid hospital patients as it is in the general population. the resulting bounds are informative for prevalence at a point in time, not just cumulative prevalence. this is important because the information required to estimate the bounds is closely related to the simple statistics that many states already report. it appears possible for many states to use our method to report upper and lower bounds on prevalence in near real time using data that they already collect and report. in particular, states already report the covid-hospitalization rate, as well as overall test rates and test positivity rates. to report a version of the upper and lower bounds we describe in this paper, states would simply have to compute testing and positivity rates among non-covid hospitalizations. our first empirical contribution uses this framework to estimate upper and lower bounds on weekly prevalence of sars-cov-2 in indiana. to operationalize the basic idea, we work with two definitions of non-covid hospitalizations. the first, which we call non-influenza-or covid-like-illness (non-icli) hospitalizations, simply excludes all hospitalizations with diagnosis for icli (center for disease control and prevention, 2020; armed forces health surveillance center, 2015) . this definition would be easy to implement in many different hospital data sets and yields a large population of hospital patients. however, we also work with a definition using a narrower set of patients who are hospitalized for six groups of clear non-covid causes: (i) cancer; (ii) appendicitis and vehicle accidents; (iii) labor and delivery; (iv) ami and stroke; (v) fractures, crushes, and open wounds; and (vi) other accidents. the clear-cause analysis is more intuitive and transparent, but it is based on smaller samples and might be harder to implement as part of a public health surveillance system. we show that, in indiana, test rates are much higher among non-covid hospital patients than in the general population. for example, in june, about 0.4 percent of the general population was tested in a given week, compared with 24 percent of non-covid hospital patients; test positivity rates are lower among non-covid hospital patients than in the general population. in the general population, 4.1 percent of tests were positive. in contrast, among non-covid hospital patients who were tested, only 2.6 percent of tests were positive. these testing and positivity rates can be combined to estimate upper and lower bounds on prevalence. under the test monotonicity assumption, between 0.01 and 4.1 percent of the general population was infected with sars-cov-2 on june 15. under the same test monotonicity assumption, the bounds are half as wide for non-covid hospital patients: prevalence was between 0.6 and 2.6 percent. under the hospital monotonicity assumption, the upper bound on prevalence in the non-covid hospital population is a valid upper bound on population prevalence. and under the stronger hospital independence assumption, the upper and lower bounds on prevalence in the non-covid hospital population are valid upper and lower bounds on population prevalence. the bounds on prevalence based on the non-icli definition are typically very similar to the bounds based on the clear-cause definition. we present bounds under alternative hospital representativeness assumptions (none, monotonicity, independence) to allow readers to make their own judgements about which assumptions are credible, and to better understand how much information about prevalence is derived from the data versus assumptions. we also calculate bounds among icli hospitalizations. we find, of course, much higher prevalence among this group, but our bounds still rule out very high prevalence. specifically, we find that sars-cov-2 prevalence among icli hospitalizations is no higher than 50 percent at its peak, and no higher than about 30 percent by mid-june. this shows that there is value to testing even highly symptomatic patients, as their sars-cov-2 rate is far from 100 percent, and testing outcomes would be informative for treatment and quarantine decisions. our second empirical contribution is to assess the credibility of key assumptions that would be difficult to study in other data sets. manski and molinari (2020) point out that the accuracy of sars-cov-2 virological tests is not well understood. incorporating information about testing errors alters the bounds on prevalence. we use data on people who were tested twice in a two day period to shed some light on the fraction of people who test negative but are actually infected. we tentatively conclude that test errors are have a negligible effect on the upper and lower bounds on prevalence reported in our paper. we also assess the credibility of the hospital representativeness assumptions at the core of the paper. the most restrictive hospital independence condition assumes that sars-cov-2 prevalence is the same in the non-covid and general populations, and the weaker hospital monotonicity condition assumes that prevalence is at least as high among the hospitalized. although we are not able to directly validate these assumptions, we probe their credibility in two ways. first, we compare the hospitalization bounds to estimates of population prevalence obtained from tests of a random sample of indiana residents in april and june (menachemi et al., 2020; richard m. fairbanks school of public health, 2020) . second, we examine the pre-hospitalization test rates of non-covid hospitalized patients. these validity checks are roughly consistent with the the hospital independence assumption, and highly consistent with hospital monotonicity. this suggests that these assumptions might be considered reasonable in other states where it would be easy to estimate the upper and lower bounds but harder to perform elaborate validation exercises. overall, our results indicate that combining testing data and information on non-covid hospitalizations may be a feasible and informative way of measuring sars-cov-2 prevalence. in the most recent weeks of our data (and under test monotonicity but without hospitalization data), we can only conclude that at most 4 percent of the overall indiana population was actively infected. with hospitalization data and the hospital monotonicity assumption, we can conclude that at most 2.6 percent of the indiana population was infected. despite this substantial improvement, the bounds remain wide enough that we cannot conclude whether prevalence has fallen since early april. similar bounds could be constructed in other states using aggregate data on non-covid hospitalizations and their testing and test positivity rates, potentially improving covid surveillance systems across the country. the empirical goal of our study is to bound the fraction of the indiana population that is infected with sars-cov-2 in each week. to fix ideas, we use i = 1...n to index the population of indiana. let c it = 1 indicate that person i is currently infected with sars-cov-2 on date t. the population prevalence of active sars-cov-2 infections in indiana at date t is p r(c it = 1) = 1 n n i=1 c it , where we leave conditioning on date t implicit to reduce clutter. we are also interested in prevalence among hospital inpatients with various covid-and non-covid-related diagnoses. this is simply the probability that a person is infected, conditional on being hospitalized for a specified condition or set of conditions. let h it be a binary indicator set to 1 if the person was hospitalized with a non-covid-related diagnosis. then p r(c it = 1|h it = 1) is the prevalence of active sars-cov-2 infections in the sub-population of people who were admitted with a non-covid-related diagnosis on date t. a key inferential challenge in estimating prevalence is that values of c it are unknown for most people on most days, because testing is rare. let d it = 1 if person i was tested on t and d it = 0 if the person was not tested. let p r(d it = 1) represent the proportion of the population tested on date t, where conditioning on t is implicit. continuing with the notation laid out above, p r(c it |d it = 1) and p r(c it |d it = 0) represent prevalence among people who are tested and not tested, respectively. the value of c it is observed for people where d it = 1, but unknown for people where d it = 0, which means that p r(c it |d it = 0) is not identified by the data on testing and test outcomes. 2 we define prevalence in the tested and untested hospital populations similarly, but with conditioning on both testing status and hospitalization status. in the absence of any distributional assumptions, the observed clinical tests partially identify prevalence overall, and in any sub-populations that can be defined by observable covariates. use the law of total probability to decompose population prevalence: the only unknown quantity on the right-hand side of the expression is p r(c it |d it = 0), which is prevalence among people who were not tested. without any additional assumptions or data, all that is known is that this value lies between 0 and 1. substituting 0 and 1 for the unknown prevalence yields worst-case lower and upper bounds l w and u w on population prevalence: these bounds define the set of values for unknown population prevalence that are compatible with the observed data and the logical definition of prevalence. the lower bound is the confirmed positive rate, and the upper bound is that rate plus the untested rate. the width of the worst-case bounds on a given day is decreasing in that day's testing rate. testing more people can only increase the confirmed positive rate and decrease the untested rate. however if few people are tested, the bounds can be very wide. to narrow the bounds, manski and molinari (2020) propose the "test monotonicity" condition. this condition requires that the prevalence of sars-cov-2 is at least as high in the tested population as it is in the untested population. formally, test monotonicity implies that p r(c it |d it = 1) â�¥ p r(c it |d it = 0). this is an appealing condition because virological tests are typically allocated to symptomatic individuals, who have a higher than average likelihood of infection. under test monotonicity, prevalence in the tested sub-population represents an upper bound on the unknown prevalence among the untested sub-population. the lower bound remains the worst-case lower bound. the lower and upper bounds under monotonicity, l m and u m , are: the new upper bound will be lower than the worst-case upper bound as long as prevalence in the tested sub-population is less than 1. in our data, test rates are less than 1 percent and positivity rates in the population are roughly 10 percent, so this assumption brings the upper bound down from 99 percent to 10 percent or less. a similar assumption could be made for the non-covid hospitalization population, yielding bounds l h m and u h m on prevalence among the non-covid hospitalized population. test monotonicity can be used to narrow the the bounds on prevalence in the population and among non-covid hospitalized patients. because testing rates are much higher in hospitals than in the general population, the bounds on prevalence in hospitalized subpopulations are much narrower. thus, assumptions that link hospital and population prevalence may be a powerful way to reduce uncertainty about population prevalence. we pursue two types of assumptions that enable extrapolation from non-covid hospital populations to the general population: (i) monotone selection into hospitalization and (ii) risk-independent hospitalization. these are both forms of hospital instrumental variable assumptions, and we refer to them collectively as hospital iv assumptions. the hospital monotonicity assumption requires that the prevalence of active sars-cov-2 infections among non-covid hospital patients is not lower than the prevalence of active infections in the general (non-hospitalized) population. formally, p r(c it |h it = 1) â�¥ p r(c it ). when prevalence is bounded in the hospitalized and general populations, the hospital monotonicity assumption may further reduce the width of both sets of bounds by ruling out values that would violate it. in particular, under hospital monotonicity, the upper bound on population prevalence cannot be larger than the upper bound on hospital prevalence. when both the hospital monotonicity assumption and the test monotonicity assumption are imposed, the upper bound on sars-cov-2 prevalence in the population is where u m is the upper bound on prevalence in the population under hospital monotonicity and u h m is the upper bound on prevalence among non-covid hospital patients. in our data, the hospital upper bound is typically lower than the population upper bound, so the hospital monotonicity condition in practice implies that the positivity rate among 8 non-covid hospitalizations is an upper bound population prevalence. 3 a stronger assumption that also facilitates extrapolation from a hospitalized sub-population to the general population is a "hospital independence" assumption, which means that hospitalization for non-covid-related health conditions is mean independent of infection with sars-cov-2. independence implies that people infected with sars-cov-2 have the same probability of being hospitalized for a non-covid condition as people who are not infected with sars-cov-2 so that p r(h it |c it = 1) = p r(h it |c it = 0). equivalently, the independence assumption implies that sars-cov-2 prevalence is the same among people who are hospitalized for non-covid conditions and the general population. that is, under the hospital independence assumption: p r(c it |h j it ) = p r(c it ). the independence assumption implies that non-covid hospitalizations are an instrumental variable for testing. this assumption would be satisfied if non-covid hospitalizations arose randomly in the population, for example because of health conditions (such as pregnancy or heart disease) determined prior to the epidemic. the hospital independence assumption would fail, however, if hospitalization risk was systematically related to covid-19 risk, for example because essential workers have more social interactions and greater likelihood of hospitalizations. under the hospital independence assumption, the bounds on the common prevalence parameter are defined by the intersection of the hospital and population bounds. the lower and upper bounds under test monotonicity and hospital independence, l m,ind and u m,ind , are: under hospital independence (as well as test monotonicity), the upper bound on prevalence is the same as under hospital monotonicity. what the hospital independence assumption buys us is a tighter lower bound, which is now the greater of the lower bounds on population and hospital prevalence under test monotonicity. in practice we find that the lower bound is always higher in the non-covid hospitalization sub-population than in the general population, so in practice this assumption implies that the lower bound on population prevalence is the confirmed positive rate among non-covid hospitalizations. virological tests for the presence of sars-cov-2 may not be perfectly accurate, and so far there are no detailed studies of the performance of the pcr tests that indiana is using to test people for sars-cov-2. to clarify how error-ridden tests complicate our prevalence estimates, we augment our notation to distinguish between test results and virological status. we continue to use c it to represent a person's true infection status, and we still use d it to indicate whether a person was tested at date t or not. but now we introduce r it , which is a binary measure set to 1 if the person tests positive and 0 if the person tests negative. using this notation, p r(c it = 1|d it = 1, r it = 1) is called the positive predictive value (ppv) of the test among people who are tested and who test positive. p r(c it = 0|d it = 1, r it = 0) is called the negative predictive value (npv) among people who are tested and who test negative. 1â��n p v = p r(c it = 1|d it = 1, r it = 0) is the fraction of people who test negative who are actually infected with sars-cov-2. our initial worst case bounds assumed no test errors. relaxing that assumption yields a different set of upper and lower bounds on prevalence. following manski and molinari (2020), we assume that (i) p p v = 1 so that none of the positive tests are false, but (ii) the second condition imposes a bound on 1â��n p v , which is the fraction of people who test negative who are actually infected. under these two restrictions, the new worst case bounds work out to: allowing for test errors in this way increases the worst case lower bound by the best-case fraction of missing positives, and increases the worst case upper bound by the worst-case fraction of missing positives. similar expressions hold for prevalence bounds under test monotonicity and other independence assumptions. the upshot of this analysis is that knowledge of test accuracy is important for efforts to learn about prevalence. in their study of the cumulative prevalence of sars-cov-2 infections, manski and molinari (2020) computed upper and lower bounds on prevalence under the assumption that î» l = .1 and î» u = .4, citing peci et al. (2014) . manski and molinari (2020) view this choice of .1 â�¤ 1 â�� n p v â�¤ .4] as an expression of scientific uncertainty about test errors, and they refer to the resulting prevalence bounds as "illustrative". however, the structure of the test error bounds makes it clear that assumptions about the numerical magnitude of test errors have inferential consequences. for example, setting î» u = .4 implies that, regardless of the outcome of the test, at least 40 percent of the people who are tested for sars-cov-2 are infected. although there is little published evidence on the properties of the sars-cov-2 pcr test, previous research suggests that pcr test errors are uncommon in other settings. for example, peci et al. (2014) study the performance of rapid influenza tests using pcr-based tests as a gold standard. pcr tests are used as a gold standard because they are expected to have very high ppv and npv. to shed more light on test errors, we constructed a sample of people who were (i) tested on day t, (ii) not tested on day tâ��1, and (iii) were tested again on day t+1. a total of 16,401 test pairs met this criteria. using r1 i and r2 i to represent the results of a person's first and second test, we found that p r(r1 i = 1, r2 i = 1) = .11 and p r(r1 i = 0, r2 i = 0) = .88 among the people in the twice-tested sample. the two tests were discordant for less than 1 percent of the twice-tested sample. in appendix c, we estimate prevalence and npv in the twice-tested sample under the strong assumptions that the tests have specificity equal to 1, sensitivity does not depend on initial test result, and retesting is random. using this method, we find that prevalence was 12 percent and n p v = 0.995 in the twice-tested sample. with 1 â�� n p v = 0.005, the estimates imply only about 0.5 percent of people who test negative are actually infected. the twice-tested sample is likely not representative of the population, of course. people who are re-tested after a negative test are probably highly symptomatic. 4 this suggests that 1 â�� n p v = p r(c it |d it , r it = 0) is probably higher in the twice-tested sample than in the population. accordingly, we think that a plausible value for î» l is nearly zero, and a plausible value for î» u is 0.005. accounting for test errors in this range would have almost no effect on the upper and lower bounds reported in the paper. our bounds turn out to be fairly simple objects. under test monotonicity, the lower bound on prevalence is the confirmed positive rate, the share of the population that tests positive. the corresponding upper bound under monotonicity is the test positivity rate, the share of tests that are positive. under hospital monotonicity, the upper bound becomes the test positivity rate among non-covid hospitalizations. and under hospital representativeness, the lower bound becomes the confirmed positive rate among non-covid hospitalizations. an appealing feature of these bounds is that they can be calculated with little additional data beyond what public health organizations already report. every state already reports the number of tests and the number of positive tests, and many states report the number of covid-related hospitalizations. 5 states would only have to report test and positivity rates for non-covid-related hospitalizations. this appears possible because many states already report "suspected" or "under investigation" covid hospitalizations, defined as hospitalized patients exhibiting covid-like illness. some states actually report both the number of hospitalizations of patients with covid-or influenza-like illness and, separately, the number of hospitalizations of patients with a positive sars-cov-2 test (e.g. arizona and illinois (arizona department of health services, 2020; illinois department of public health, 2020a,b)). 6 thus states have the capacity to identify icli-related hospitalizations and link hospitalization and testing data. our analysis is based on two main data sources managed by the regenstrief institute. first we obtain data on the near universe of clinical virological tests for sars-cov-2 conducted in indiana between january 1, 2020 and june 18, 2020. second we obtained data on all inpatient hospital admissions from hospitals that belong to the indiana network for patient care (inpc), which is a health information exchange that centralizes and stores data from health providers across the state of indiana, including all hospitals with emergency departments. 7 the hospital data are derived from the same database that the state uses for reporting hospitalizations on its dashboard (indiana state department of health, 2020). we link the testing and hospital data using an encrypted common identifier. of 5 see, e.g., the covid tracking project (2020). course, only a subset of hospital patients are tested and only a subset of tested people appear in the inpatient hospital data. for both data sets, we are also able to link the data with basic demographic data collected by the inpc; this information is available only for a subset of patients. the test data contain individual records for nearly all of the sars-cov-2 tests conducted in indiana during 2020. a small number of tests are excluded from our data because some institutions that conduct tests provide data to inpc but do not allow the data to be used for research purposes. the consequence of these exclusions is that we are missing some tests, which will result in a reduced lower bound in our framework. despite these exclusions, our data set tracks the state's official case counts fairly closely; see appendix figure a.1. as an aggregate summary, our data contain 39,472 total positive tests, and the state reports 41,541 positive tests as of june 18 (indiana state department of health, 2020). 8 each test record in our testing data includes information on the date the test was run, the outcome of the test (positive, negative, or inconclusive), and a patient identifier that we use to link the test data to demographic files and inpatient hospital files. the hospital inpatient data contain separate observations for each admission. we always observe admission time and a patient identifier that we use to link to the test data and inpatient files. we observe discharge time and diagnosis information only for a subset of admissions. (not all fields are available for all admissions because different institutions contribute different information to the inpc.) because the inpc data come from health care providers and payers, the same hospitalization can appear in the data set multiple times. to de-duplicate these records, we keep one observation per admission time (defined second-by-second), keeping the observation with the most diagnosis codes. 9 in-hospital testing, positivity rate, and confirmed positives the fraction of people who are tested in the hospital is an important quantity of interest in our analysis because hospital patients are tested at a higher rate than the general population, and hospital testing may be less correlated with covid symptoms. our data do not distinguish whether a person was tested in the hospital or whether the test was initiated independently of the hospital visit. we say that a hospitalized patient was tested in-hospital if she had at least one sars-cov-2 test dated between 2 days prior to admission to 4 days after admission. we chose to focus on this week-long period, rather than strictly between admission and discharge, for three reasons. first, some patients will be tested prior to admission, as part of their preparation for admission. thus it is valuable to look prior to admission. second, we observe the date the test is run, not the the date the sample is collected. backlogs in the testing system may mean that a patient is discharged before the test is run. third, for some admissions, we lack discharge dates, but we can still define in-hospital tests using this measure. we say that a patient tests positive in the hospital if she has at least one positive covid test between 2 days prior to admission and 4 days after admission. we define the positivity rate as the fraction of tests that are positive; the confirmed positive rate is the fraction of the population with a positive test. in some analyses we compare hospital testing and positivity to population testing and positivity. hospital testing and positivity are defined over a week-long span for a given hospitalization. to make the comparison with the general population clean, we examine test rates and positivity in a given week-long period. we say that a person was tested if she was tested at least once in a given week, and we say she was positive if she was positive at least once in that period. throughout, a patient is in the "test sample" if they are tested at least once. we say a patient is in the "inpatient sample" if they are hospitalized at least once. we limit our analysis to admissions with non-missing diagnostic information, and we say a patient is in the "inpatient diagnoses sample" if they meet this restriction. this limitation is important because diagnostic information is necessary for distinguishing covid-related admissions from non-covid-related admissions. we construct three analytic samples from the inpatient data. we start by defining hospitalizations for influenza-and covid-like illness (icli) using icd-10 codes. we identify admissions with any of a standard set of icd-10 codes for icli following armed forces health surveillance center (2015). then we identify admissions with any of the additional icd-10 codes that the cdc recommends using for coding covid hospitalizations (center for disease control and prevention, 2020). both the influenza-like and covid-like diagnoses include general symptoms such as cough or fever, as well as more specific diagnoses like acute pneumonia, viral influenza, or covid-19. we classify hospitalizations as icli-related if they have any influenzaor covid-like illness (icli) diagnoses, and we classify hospitalizations as non-icli if they are not icli-related. appendix b lists the icd-10 codes used to define the analytic samples. we view the non-icli sample as a useful starting point for our analysis for two reasons. first, our hospital iv assumptions are most plausible for hospitalizations that are not obviously covid-related, and this sample meets that criteria. second, as we have noted, many states already classify hospitalizations as icli-related; thus non-icli hospitalizations are identifiable and measurable in near-real time, so this sample can be studied more broadly. we acknowledge, however, that the non-icli sample may not satisfy the hospital iv assumptions for at least two reasons. first, it may condition on covid itself, since a patient with a reported covid diagnosis would be excluded from it. (in practice we observe many patients with positive covid tests but no covid diagnosis.) second, covid is a new disease with heterogeneous symptoms, so even if a patient is hospitalized because of covid, she may not have one of our flagged diagnoses, and we may incorrectly call her hospitalization non-icli . to avoid these problems, we study a third sample, which we call the "clear cause" sample. these are hospitalizations with a clear cause that is not obviously covid-related. we define clear-cause hospitalizations as hospitalizations with a diagnosis code for labor and delivery, ami, stroke, fractures, crushes, open wounds, appendicitis, vehicle accidents, other accidents, or cancer. for all of these conditions except cancer, we flag hospitalizations with a diagnosis at any priority. for cancer, we flag hospitalizations with a cancer diagnosis code as the admitting diagnosis, the primary final diagnosis, or any chemotherapy diagnosis. appendix b lists the icd-10 codes used for these classifications. we view the clear-cause sample as important for two reasons. first, we believe the hospital iv assumptions are most plausible for this sample, so we believe the bounds on prevalence are most likely to be valid. second, we view the clear-cause sample as offering a test of the validity of the non-icli sample. to the extent that the two samples generate similar bounds, we can be more confident that the non-icli sample is informative of broader population covid prevalence, despite the problems with the non-icli classification. this would be valuable because classifying hospitalizations as icli-related or not requires less information than ascertaining a clear cause of the hospitalization. we show summary statistics for all of our samples in table 1 , as well as for the state as a whole (from census fact finder and united states census bu-reau (2019)). the average tested and hospitalized patient is substantially older than the population as a whole, and also more likely to be female. because the tested and hospitalized samples are not age representative of the general population, we reweight all samples to match the population age distribution. the tested and hospitalized samples are fairly similar to the general population in terms of racial composition. limiting the inpatient sample to admissions with diagnoses reduces our sample size substantially, but it does not appear to change its demographic profile. although our main analysis looks at test rates at the admission level (rather than the person level), the summary statistics show that hospitalized patients are vastly more likely to have ever been tested than the population as a whole -about 21 percent of ever-hospitalized patients, compared to about 280,000 out of 6.64 million (4.2 percent) for the general population. icli-related hospitalizations are especially likely to have ever been tested. figure 1 shows the sars-cov-2 testing rate for each of the sub-populations in our analysis. we report the exact values of each of the test rates and the weekly number of admissions in appendix table a .1. because the tested and hospitalized populations have very different age distributions compared to the rest of the population, we reweight the hospital sub-populations to match the coarsened age distribution in the population. specifically, we calculate test rates in week-by-age-group cells, for age groups 0-17, 18-30, 30-50, 50-64, 65-74, and 75 and older. then we average these age-specific testing rates across the age groups, weighting each group by its population share. we report unweighted test rates in appendix table a .2. figure 1 shows vastly higher test rates in the hospitalized samples than in the general population. the testing rate in the general population grew from 0.2 percent in april to between 0.4 and 0.6 percent in may and june. so despite tripling, the weekly test in the indiana population rate remained below 1 percent. in contrast, people hospitalized for icli were tested at a very high rate, between 65 and 75 percent in most weeks. testing rates among non-icli hospital patients and among the clear-cause non-covid hospital patients were lower than the icli sample but much higher than the population overall, about 20 percent in april and 25-30 percent in may and june. in other words, testing rates among non-covid hospital inpatients are about 50 times higher than testing rates in the general population, but they are typically less than half as high as testing rates in the icli population. these high test rates imply tighter bounds on population prevalence under test monotonicity, as we show in figure 2 and report in appendix table a .3. we age-weight the bounds, analogously to our weighting of test rates, and we report unweighted bounds in appendix table a .4. several patterns are clear in the figure. first, the icli hospitalized population has higher upper and lower bounds on prevalence than the other groups. for the icli patients, the prevalence bounds are 5-22 percent in the first week of our sample, then increase to 30-40 percent in the last week of march, before declining steadily to 11-18 percent in the final week of the sample. these bounds rule out the possibility that all or nearly all symptomatic patients are infected with sars-cov-2. in most weeks the icli bounds lie outside the other groups' bounds, implying (unsurprisingly) unambiguously higher covid prevalence. this separation shows that the data are sensible and that the bounds are informative enough to tell apart these highly distinct populations. the second clear pattern in the figure is that the prevalence bounds are tighter for the non-icli and clear-cause hospitalization samples than for the all-test sample. in fact the bounds for both of these hospitalizations samples are always contained within the all-test bounds. even at their tightest, the bounds for the all-test sample are as wide as 0.02 to 3.6 percent in the last week of our data. in that week the bounds for non-icli hospitalizations are [0.6%, 2.4%] and for clear-cause hospitalizations they are [0.5%, 1.9%]. this tight bound implies that the hospitalization data could be informative for population prevalence (under hospital monotonicity or mean independence). the final pattern evident in figure 2 is that the bounds for the non-icli hospitalization sample and for the clear-cause hospitalization sample are nearly indistinguishable. the only noticeable difference is that the upper bound for non-icli hospitalization is perhaps slightly higher. this fact is important because non-icli hospitalizations are potentially easier to measure, but they may be negatively selected in the sense that by construction they may exclude covid-likely cases. the similarity of the non-icli bounds with the bounds for the clear-cause sample (which is not selected based on covid-likelihood) provides some evidence in support of using non-icli hospitalizations to measure general prevalence. our clear-cause hospitalization sample pools many distinct causes, including among others labor and delivery, vehicle accidents, and other accidents, including falls. in principle these hospitalizations may differ in their covid likelihood. one might worry, for example, that pregnant women are especially cautious and careful not to become infected, whereas people getting into vehicle accidents may be less cautious (either because they are not careful drivers, or because they are out of the house at all). we therefore report test rates and bounds for disaggregated causes as well as the overall clear-cause sample. we focus on six sets of causes: cancers, appendicitis and vehicle accidents, injuries (fractures/crush/wounds), non-vehicle accidents, and ami/stroke. these six groups have reasonable sample sizes throughout (they each have 1,000-1,500 admissions per month), and the demographic profiles within each group are roughly similar; see appendix table a .5 for age profiles of admitted patients by cause of admission. all ages are represented in the cancer sample: appendicitis and vehicle accidents both afflict young people; ami, stroke, and other accidents-primarily falls-afflict older people; and labor and delivery is limited, of course, to women of childbearing age. 10 because not all age groups are represented in every category, we do not age-weight these results. we report the monthly test rates for each of these groups in figure 3 . we also report sample sizes, exact tests rates, and bounds in appendix tables a.6 and a.7, by month and cause. all causes had relatively low test rates in march before rapid increases in april and may. by june there are some differences in the test rates, with higher rates for the injury, accident, and ami/stroke admissions, and less testing for cancer and labor and delivery. we report the bounds on prevalence by cause of admission in figure 4 . in march there is little testing; the bounds are wide and uninformative. the bounds tighten in april, may, and especially june. importantly, we do not see obvious, systematic differences across the groups. typically the bounds overlap, and there is no strong evidence that the upper or lower bounds differ by cause of admission. it is true that in june, cancer patients had zero positive tests, and the upper bound for appendicitis and vehicle accidents was below the lower bounds for injuries, other accidents, and ami/stroke. however in april and may the cancer and appendicitis/vehicle patients had similar or even higher upper bounds than did the injury and other accident patients. this evidence shows that patients admitted to the hospital for different reasons and with different demographic profiles are all nonetheless tested at a high rate and with similar bounds on prevalence. this is perhaps reassuring for the view that pooling many distinct causes of admissions can nonetheless generate meaningful bounds on prevalence. the results so far show that the bounds on prevalence are much tighter for the non-icli hospitalized population than for the population as a whole. this tighter bound is informative for general population prevalence only under assumptions about hospital representativeness, either a monotonicity assumption or an equal prevalence assumption. how valid are these assumptions? assessing them directly is of course impossible because we lack data on prevalence in the population as a whole or in the hospital sample. we have already provided one piece of indirect evidence in support of our hospital representativeness assumptions. the non-icli and clear-cause samples generate similar bounds, and, within the clear-cause sample, there are not large differences in bounds across different causes of admission. this suggests that prevalence does not vary with the exact set of hospitalizations studied, although of course this does not prove monotonicity or representativeness. in this section, we provide two additional pieces of evidence on the hospital iv assumptions. first we show that the hospital bounds are consistent with the estimates of population prevalence from the indiana covid-19 random sample study (menachemi et al., 2020; richard m. fairbanks school of public health, 2020) . 11 second, we compare the hospital sample to the general population in terms of their likelihood of prior testing (prior to the hospital data) and the test rate of their home counties. we take these to be proxies for their concern about covid, although other interpretations are possible. a valuable benchmark for the hospital-based prevalence bounds comes from a largescale study of sars-cov-2 prevalence in indiana. the study invited a representative sample of indiana residents (aged 12 and older) to obtain a sars-cov-2 test. the first wave of the study took place april 25-29, and the second wave took place june 3-7. the preliminary results are reported in menachemi et al. (2020) and richard m. fairbanks school of public health (2020). the response rate was roughly 25 percent, and no attempt was made to correct for non-random response. nonetheless this survey appears to be the best benchmark available. we report the point estimates for prevalence (assuming random nonresponse) and their confidence intervals in the top panel of table 2 . the first wave estimates 1.7 percent prevalence and the second 0.5 percent. 12 we compare our prevalence bound during the same time periods in the bottom panel of the table. we limit our sample to tests of people aged 12 and older, for comparison with the population study. using population testing we obtain very wide bounds that contain the random sample study estimates. this fact provides some support for the test monotonicity assumption. for both the non-covid hospitalization and clear-cause hospitalization samples, the bounds are much tighter. both sets of upper and lower bounds contain the april prevalence estimate, and both sets exclude the june estimate point estimates. however the confidence interval for the june 3-7 point estimates overlaps substantially with the non-icli and clear-cause bounds. thus for both dates the prevalence point estimates are consistent with the bounds obtained from the non-covid hospitalizations. as a comparison we also report the bounds from the icli-related hospitalizations, which always exclude the random sample estimates. a standard way of measuring representativeness is to compare the distribution of covariates in a study population to their distribution in the target population. in our case, this approach is most convincing if we have well-measured covariates that proxy for having covid-19. two candidate covariates are the community sars-cov-2 testing rate and the prior testing rate. the idea behind these proxies is that people who come from areas with high test rates, or who have been tested in the past, may themselves have a higher current likelihood of having covid-19. to operationalize these measures, we define the community testing rate for person i as the fraction of people in i's county who have ever been tested, as of the end of our sample period. we define the prior test rate of person i as of date t as the probability that i was tested at least once during the week-long period [t â�� 15, t â�� 9]. we focus on this window because it is the second week prior to our hospital testing window (which runs from t â�� 2 to t + 4 for a patient admitted at t). we allow for a week of time to elapse between the hospitalization and the "prior" testing because it is possible that some pre-hospital testing would occur in the window [t â�� 8, t â�� 3]. when studying prior tests, we limit the sample to each person's first hospitalization after march 1, 2020, to avoid picking up the higher testing that mechanically results from the fact that people hospitalized once are more likely than the general population to have been previously hospitalized. as with our bounds, here we weight the data to match the population age distribution. table 3 shows the community testing rate. the average county has a testing rate of 3.5%, with an interquartile range of 2.5% to 4.3%. the average person lives in a county with a test rate of 3.6%. the average non-icli hospitalized patient comes from a county with a test rate of 4.1%: for clear-cause hospitalizations it is 4.0%, and for icli hospitalizations it is 4.2%. these rates are all significantly different from the population average. figure 5 shows the prior testing rate as a function of admission date for the non-icli hospitalization sample, the clear-cause hospitalization sample, and the general population (for which the prior test rate on day t is defined as the fraction tested between t â�� 15 and t â�� 9). the rates in the hospitalization samples are initially close to the population rate (when testing is low in general), but the lines diverge. by the last week of the sample, the prior testing rate is about 1.5 percentage points in the hospitalization samples, relative to approximately .75 percentage points in the population. although the weekly differences are not individually statistically significant, overall the greater rate of prior testing is consistent with positive selection into hospitalization. however it is also consistent with the possibility that the hospitalization sample simply has more contact with the medical sector, resulting in greater testing at a given sars-cov-2 prevalence. we have calculated weekly bounds on the prevalence of sars-cov-2 for the indiana population as a whole and for three hospitalized populations: people hospitalized for influenza-and covid-like illness, people hospitalized for other reasons, and people with clear (and clearly not covid) causes of hospitalization. the bounds are valid under weak monotonicity assumptions. the bounds for the general population are wide but narrow over time. the bounds for the hospitalized population are much tighter, because the hospitalized populations are tested at a much higher rate than the general population. the hospitalized populations are informative for the general population only under additional, stronger assumptions. in particular, if the hospitalized population is representative of the general population in terms of sars-cov-2 prevalence, then both the upper and lower bounds are valid. if the hospitalized population has a higher prevalence than the general population, then only the upper bound is valid. we assess these assumptions in multiple ways. we find that the non-covid hospitalized population bounds contained the point estimate of prevalence from a random sample in late april. by early june the point estimate from the random sample was below our lower bound, although we cannot reject that it was inside the bound. hospitalized patients also appear to be tested for sars-cov-2 at somewhat higher rates than the general population, even outside the hospital. this last fact suggests that the representativeness assumption may be violated (although the magnitude may not be too severe), but both are consistent with a weaker monotonicity condition. even under this monotonicity condition, the hospital data are still useful, bringing down the upper bound on population prevalence by a third or more. we believe that the main promise of the non-covid hospitalization population is that it can provide near-real time information about population prevalence. only three numbers are necessary to calculate bounds from the non-covid hospitalizations: the count of non-covid admissions, the number of tests among this group, and the number of positive results. although these numbers are not currently reported, many states already report related numbers, including both the number of covid tests and the count of iclirelated hospitalizations. thus the infrastructure largely exists already to calculate these bounds. the results here help validate this approach for real-time surveillance. notes: column 1 reports characteristics for the set of people appearing in the test data, and columns 2-6 for people appearing the hospital data, ever (column 2), with at least one diagnosis (column 3), at least one non-icli hospitalization for icli (column 4), at least one clear cause hospitalization (column 5, see text for details), or at least one icli hospitalization with a diagnosis and not for icli (column 6). notes: the first two rows of the table report the estimated population prevalence and 95% confidence interval from the indiana covid-19 random sample study, conducted over the indicated dates, which assumes random nonresponse (menachemi et al., 2020; richard m. fairbanks school of public health, 2020) . the remaining rows report the (age-adjusted) bounds on prevalence from our different samples: population testing, non-icli hospitalizations, clear cause hospitalizations, and icli-related hospitalizations. we limit our sample to people aged 12 and older, for consistency with the random sample study. setup and identification here we show how to use data on multiple tests to simultaneously identify prevalence, test error rates, and how to use this information to obtain the negative predictive value, npv. assume in particular that people have been tested exactly twice, with r1 i the outcome of the first test and r2 i the outcome of the second test for person i, and c i person i's true infection status, which we assume is fixed between the tests. let p = p r(c i = 1) be the prevalence of active sars-cov-2 infections in this twice tested population. test outcomes may differ from true infection status because of test errors. in general, therefore, there are four possible sequences of test outcomes: (0, 0), (0, 1), (1, 0), (1, 1). we let p ab = p r(r1 i = a, r2 i = b) for (a, b) â�� {0, 1} 2 . we make three strong assumptions to simplify the analysis. 1. the specificity of the test is 1. that is, î² = p r(rj i = 0|c i = 0) = 1. 2. the sensitivity of the test, î± = p r(rj i = 1|c i = 1), does not depend on the initial test result. 3. retesting is random, i.e. independent of r1 i and c i . assumption 1 is the weakest of these assumptions. it implies that there are no false positives, which is consistent with typical practice (ucsf health hospital epidemiology and infection prevention, 2020). the remaining assumptions are stronger. assumption 2 says that the test errors are independent of the initial test result. it would be violated, for example, if false negatives are more common for patients with high levels of mucus, and mucus levels are correlated across test results. assumption 3 says that retesting rates do not depend on possible testing errors. we would expect this condition to fail if highly symptomatic people with negative tests are especially likely to test negative. we view this assumption as the most suspect. under these assumptions, the probabilities p ab simplify considerable. as the probabilities sum to one, and the assumptions imply that p 10 = p 01 , the only non-redundant probabilities are p 00 = (1 â�� p) + p(1 â�� î±) 2 p 11 = pî± 2 . we can observe p 00 and p 11 . solving for the unknowns p and î±, we have p = (p 00 â�� p 11 â�� 1) 2 4p 11 î± = 2p 11 1 â�� p 00 + p 11 this shows how to get p and î± from two tests and the assumption that specificity equals 1. our goal is to find the negative predictive value, but we can calculate it given knowledge of î±, î² and p. in general the npv of a single test is p r(c i = 0|r i = 0). applying bayes rule, n p v = 1 â�� p p(1 â�� î±) + (1 â�� p) to implement this approach, we construct a sample of all people who are tested on a given day, not tested the previous day, and then tested again in the next day. there are 16,401 such test pairs. we find p 00 = 0.88 and p 11 = 0.11. nearly all the mass is on the diagonals; test results switch less than 1% of the time. this fact, together with the assumption that specificity is equal to 1, implies very low false negative rates. plugging these values into our formula, we have p = 0.12 and î± = 0.96, which imply n p v = 0.995. using instead, all people who are retested once within a three day period, we find similar results: p = 0.13, î± = 0.91, n p v = 0.986. we emphasize that these estimates are valid for the twice-tested population and under assumptions 1-3, in particular, random retesting. the prevalence estimate is the prevalence among people tested twice, not the population prevalence. and it is only a valid estimate under assumptions 1-3. in reality, it is likely that retests are most common among suspected false negatives (i.e. when a highly symptomatic patient tests negative). we see some evidence for this: p 01 = 0.006 and p 10 = 0.003, inconsistent with the random retesting assumption. we therefore do not view our estimates of prevalence and sensitivity as definitive; rather we think of the sensitivity estimate as a lower bound on sensitivity, because we have selected a retest sample which has a disproportionate number of false negatives. as n p v is increasing in sensitivity, î±, our implied estimate of 1 â�� n p v is likely an upper bound on 1 â�� n p v . covid-19 test shortages prompt health authorities to narrow access polarization and public health: partisan differences in social distancing during the coronavirus pandemic data dashbaord influenzalike illness. afhsc standard case definitions a method for obtaining short-term projections and lower bounds on the size of the aids epidemic icd-10-cm official coding and reporting guidelines april 1 case data a review of back-calculation techniques and their potential to inform mitigation strategies with application to non-transmissible acute infectious diseases estimating the effects of non-pharmaceutical interventions on covid-19 in europe how disease surveillance systems can serve as practical building blocks for a health information infrastructure: the indiana experience spread of sars-cov-2 in the icelandic population tracking public and private response to the covid-19 epidemic: evidence from state and local government actions mandated and voluntary social distancing during the covid-19 epidemic covid-19 hospital resource utilization covid-19 syndromic surveillance gov, last accessed integrating behavioral choice into epidemiological models of aids identification problems in the social sciences estimating the covid-19 infection rate: anatomy of an inference problem population point prevalence of sars-cov-2 infection based on a statewide random sample indiana interactive chart: mississippi covid-19 hospitalizations performance of rapid influenza diagnostic testing in outbreak settings private vaccination and public health: an empirical examination for us measles economic epidemiology and infectious diseases isdh release findings from phase 2 of covid-19 testing in indiana estimating the infection and case fatality ratio for coronavirus disease (covid-19) using age-adjusted data from the outbreak on the diamond princess cruise ship estimating the burden of sars-cov-2 in france identification and estimation of undetected covid-19 cases using testing data from iceland universal screening for sars-cov-2 in women admitted for delivery current covid-19 hospitalizations the covid tracking project covid-19 diagnostic testing quick facts: indiana estimates of the severity of coronavirus disease 2019: a model-based analysis. the lancet infectious diseases current activity in vermont three essays on voluntary hiv testing and the hiv epidemic clinical characteristics of patients with coronavirus disease 2019 (covid-19) receiving emergency medical services in king county, washington we define icli-related hospitalizations as ones with at least one ili or cli diagnosis code. we define non-icli related hospitalizations as hospitalized with diagnosis codes, but no ili or cli code. we also define "clear cause" hospitalizations. these are hospitalizations for labor and delivery, ami, stroke, fractures and crushes, wounds, vehicle accidents, other accidents, appendicitis, or cancer. with the exception of cancer, we define a hospitalization as belonging to one of these groups if it has any diagnosis codes for that group i22. â�¢ appendicitis k35-k38 â�¢ other accidents w00-w99 â�¢ vehicle accident v01-v99 notes: the county test rate is the share of the county population tested at least once in our test data. table reports county-level statistics, as well as the average county test rates for the general population, the non-icli hospitalizations, clear cause hospitalizations, and icli hospitalizations, as well as t-statistic for the null hypothesis that the average person and the average hospitalization have the same county test rate. key: cord-286539-3sr4djft authors: mentus, cassidy; romeo, martin; dipaola, christian title: analysis and applications of adaptive group testing methods for covid-19 date: 2020-04-07 journal: nan doi: 10.1101/2020.04.05.20050245 sha: doc_id: 286539 cord_uid: 3sr4djft abstract testing strategies for covid-19 to maximize number of people tested is urgently needed. recently, it has been demonstrated that rt-pcr has the sensitivity to detect one positive case in a mixed sample 32 cases [9]. in this paper we propose non-adaptive and adaptive group testing strategies based on generalized binary splitting (gbs) [2] where we restrict the group test to the largest group that can be used. the method starts by choosing a group from the population to be tested, performing a test on the combined sample from the entire group and progressively splitting the group further into subgroups. compared to individual testing at 4% prevalence we save 74% at 1% we save 91% and at 1% we save 97% of tests. we analyze the number of times each sample is used and show the method is still efficient if we resort to testing a case individually if the sample is running low. abstract in addition we recommend clinical screening to filter out individuals with symptoms and show this leaves us with a population with lower prevalence. our approach is particularly applicable to vulnerable confined populations such as nursing homes, prisons, military ships and cruise ships. testing capacity for covid-19 is still too scarce to meet the needs to meet global health needs. conned are at particular risk for rapid contagion. they may include those who reside in facilities such as prisons, ships, military units and nursing homes. the goal of this paper is to increase the capacity to identify asymptomatic carriers of covid-19 by applying group testing methods. current practice typically involves a one-patient-one-test strategy. recently the potential to detect covid-19 rna in a mixture of samples from individuals has been validated [9] using rt-pcr. the method was rst devised in 1943 during world war ii to test large groups of us servicemen for syphillis prior to deployment [2] . now, just as in the wwii era, large scale testing is necessary. covid-19 is a highly contagious disease that can lead to pneumonia, acute respiratory distress syndrome (ards) and death. clinical symptoms and phyiscal exam features may include fever, cough, shortness of breath, malaise, lethargy, ageusia, anosmia and gastrointestinal (gi) symptoms. besides this diseases potential lethality, it is highly contagious. with the demonstrated success of group testing using rt-pcr for nding sars-cov-2 rna [9] , there is a renewed interest in the practical applications of mathematical group testing algorithms. in [5] a nonadaptive group testing method and practical applications are explored. we describe both non-adaptive and adaptive group testing based on generalized binary splitting (gsb) [3] . the adaptive approach optimizes group size according to the prevalence. we provide an algorithmic specication for subdividing groups and account for the limited test accuracy and the possibility that the an individual's sample size constrains the number of tests. we explain how prescreening symptomatic cases and separately testing them ca dramatically improves the performance. pre-screening reduces the prevalence in the test groups, this approach is relevant for large scale populations and particularly for conned groups. 1 figure 1 .1: (image credit: matthew heidemann) our group testing method consists of rst clinically screening out symptomatic individuals. this will lower the prevalence in the test population. our group testing is especially eective when we can assume the prevalence is uniform over the whole population. therefore, it is especially applicable to conned or cohesive populations. we split the asymptomatic individuals up into groups for which we mix the samples from each individual and test them. a negative result will conrm many negative cases. we subsequently divide groups and test the mixtures of samples for positive results. this we show, conserves the number of tests and, as a result, the time spent testing. using numerical experiments, we also show that our methods can be performed without running out of samples from passing through too many rounds. this application utilizes a primary clinical screening step to ensure that the tested population is composed of asymptomatic covid-19 (-) and (+) patients. this ensures the lowest prevalence of disease in the population and enhances the ecacy of the method. clinical screening should rst take place in the selected population to screen out as many potential positive patients as possible before administering the test. all patients with history of cough, shortness of breath, nausea, gastrointestinal symptoms, fever, malaise, lethargy, recent contact with positive covid-19 patients, have physical exam consistent with those ndings should be segregated out of the population. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint clinical screening will decrease the prevalence of covid-19 carriers in the test population if the probability of being asymptomatic given covid-19 (+) is less than the proportion of the population that is asymptomatic (regardless of covid-19 (+) or (-)). we demonstrate this by proving the following claim. claim. the sub-population not showing any symptoms will have a lower proportion of symptomatic carriers when the proportion of people not showing any symptoms in our group is less than the estimated proportion of covid-19 carriers who are asymptomatic. we can prove this through a couple of applications of bayes' theorem. we refer to the event that an individual does not show symptoms as no symptoms. the event that an individual has symptoms that are signs of covid-19 is referred to as symptoms regardless of whether they carry the disease. we denote the event of carrying the covid-19 virus as covid-19. then an asymptomatic carrier is referred to as no symptoms and covid-19. let p (a) denote the probability of an event a occuring. written in terms of probabilities, screening will help when p (covid-19|no symptoms) < p (covid-19). re-writing the left side of the inequality, we get p (covid-19|no symptoms) = p (covid-19 and no symptoms) p (no symptoms) = p (no symptoms|covid-19) p (no symptoms) p (covid-19). 3 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint therefore it is necessary and sucient for p (no p (no symptoms) < 1, which is the same as p (no symptoms|covid-19) < p (no symptoms). this is the probability of being asymptomatic given that they have covid-19 for the population we are testing. if a population has a low prevalence of covid-19 then it is likely for groups of individuals to not have any positive cases. therefore it is often the case that one test of the mixture of their samples is all that is needed to determine that they are all negative. in this example 32 cases are conrmed either (+) or (-) using 11 tests. some cases are left un-determined for future rounds of testing. otherwise, a positive result on the combined samples indicates that there are some positive cases. we are therefore able to design a strategy to use less tests to determine whether each individual is positive or negative for the disease by testing mixtures of samples from groups. to suciently identify negative cases and positve cases the groups must be large enough to balance nding many negative cases with one test, and locating some positive cases. step one of the group test method is: 1. a group size is chosen so that the frequency that a group has only (-) cases (gure 3.1) is roughly equal to the probability we nd at least one (+) case in the group. 4 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint table 3 .1: group size specied for the divide and test method. by dtg we mean to apply divide and test, using the minimum of g and the group size for divide and test (e.g. dt32 will use a group of size 32 if dt uses a group of size 64). we study limited group sizes to explore how many tests can be saved depending on the limitations of the instruments. if the test of the group's combined samples is positive, indicating that one of the individuals in the group is positive, apply a routine of repeatedly dividing the group into subgroups applying tests to the subgroups until a positive individual is identied. this is step two of the group test method. 2. if the rst test is (+), divide the group into two subgroups of equal size. test the combined sample from individuals in one subgroup to determine which halve contains some positive individuals. repeat this step on the subgroup that contains the positive cases. this routine is referred to as binary search. 5 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint when the test result of the group's samples is (+) we split the group into two equally sized subgroups testing one of them to decide if it contains some positive cases or if it consists entirely of negative cases. in the second situation, the second half must contain positive cases. continue the search on the subgroup with positive cases in the same way dividing it into two halves test-6 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint ing the mixed samples from one of the half-subgroups. repeating this step we are able to nd one positive case. along the way many groups of several individual are likely to be conrmed negative with a relatively small number of tests. any subgroup that was not tested retains its undetermined status and is returned to the general set of samples to be conrmed positive or negative. 3. repeat the procedure starting with 1. on the individuals in the population for which whether they carry the disease has yet to be determined. in this paper we consider group testing methods that follow two dierent interpretations of step 1 in the procedure above: methods with a xed group size and methods where the group size changes based on the results of previous tests. each type of method uses a slightly dierent denition of prevalence. prevalence is dened as the probability p an individual in the population has covid-19. for each level of prevalence we specify a xed number of individuals to test their combined samples, performing binary search if it is positive. the xed group size method we analyze in this paper is divide and test (dt). we also consider dorfman's method as a method to compare the methods against, as well as a simpler alternative that only ever uses an individuals samples for a maximum of two tets. prevalence is dened as the count of positive cases in the test population. as testing is carried out, the conrmed positive and negative cases are set aside are both removed from the undetermined test population. therefore, the population and the prevalence changes during the testing process. the group size depends on both the population and the prevalence. the adaptive group testing method presented in this paper is generalized binary splitting (gbs). in addition to studying these two classes of methods, we make modications to the methods to take into account limitations of the devices and techniques used to test samples for covid-19 rna. specically, we study methods with limited group sizes. these are dt and gbs with maximum group g (dtg and gbsg). they are dened so that any time either dt or gbs species a group size over the capped limit g we take the group size to be g. to cover a wide range of limits of detection we consider groups of sizes 4,8,16,32 and 64. 4 non-adaptive group testing method: divide and test (dt) this method is can be thought of as a xed group size version of the generalized binary splitting method [3] . 1. if p > .5 then test each member of the population individually. otherwise, let α = log 2 1 p − 1 . select a group of individuals g with |g| = 2 α and apply one test to g. if it is negative, then we conclude that the result of each case in g is negative. 2. if g is positive we use binary search to nd exactly one case that we can deduce is positive. 3. repeat the method starting with step 1. on the yet to be conrmed portion of the population. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint 5 adaptive testing method: hwang's generalized binary splitting (gbs) the group testing methods suggested in this paper are based on hwang's generalized binary splitting method. in [3] gsb is designed to nd up to d (+) cases in a population containing n individuals. it is described as follows: take g a group of cases such that |g| = 2 α and apply one test to g. if it is negative, then we conclude that the result of each case in g is negative. 2. if g is positive we use binary search to nd exactly one case that we can deduce is positive. 3. set the population to the set of individuals not determined (+) or (-). we set n to be the new population size. if we have found a positive case in the last two steps, we take d − 1 to be the new number of positives in the un-tested population. this method is extended to populations with a random number of (+) cases by setting the upper limit on positive cases according a chosen condence level (i.e. the probability that the number of (+) exceeds this is very small). in probability terms, if the number of positive cases d is generated acccording to a probability distribution, and we assume probability c that will identify every positive case, then let d c be such that p (d ≤ d c ) < c. generalized binary splitting is then applied to nd at most d c positive cases in the population. the setting of hwang's paper is group testing for general purposes, including identifying defective parts or products in addition to determining individuals with a disease. it is an ethical requirement to determine if each person is (+) or (-) if they are tested for covid-19. therefore gsb is appplied for an upper bound d c of positive cases at a xed level of condence c. if exactly d c positive cases are found, then there with non-zero probability there are some cases that have not been determined. otherwise if < d c positives are identied, then we are certain we have found them all and it will also happen that a positive or negative conrmation was given to each individual by the end of the testing process. in the case where there could be more positive cases, we repeat gsb with the same level of condence c on the remaining population. rather than considering the application of a level of condence as an idiosyncracy of the method, it is actually a fundamental property of identifying asymptomatic covid-19 carriers. asymptomatic positives cannot be identied without testing them, we can only pick an upper bound on the cases with a high level of certainty. it might seem preferable to then choose a method that instead prescribes a group-size for each level of prevalence, such as dorfman's method or divide and test, but for those methods the possibility that we have misjudged the prevalence still needs to be factored in! 6 condence bounds on group testing methods we will apply dt and gbs with groups capped at 4, 8, 16, 32 and 64 samples using two dierent levels of condence. • gbsg with d .5 (i.e. p (d ≤ d .5 ) = .5). we repeat gbsg at this level of condence if there are undetermined cases remaining. • we apply dtg with the group size 2 log( 1 p −1) . • gbsg with d .99 . for a population generated by a prevalence level p, this will perform worse on average than the application with d .5 , but level of eciency theoretically guarenteed for gsb [3] will hold with 99% condence. therefore, we can be more condent of the algorithms performance. • for populations of size n generated with prevalence p, we apply dtg at the actual prevalence level of the population with 99% condence. that is, we apply dtg with the group size (with ceiling g) corresponding to p .99 = d.99 n . 8 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint for small to medium sized populations, the 99% condence upper bound is very dierent from the mean. the dierent condence levels are very close for large n for a constant probability of having covid-19. how condent we are needs to be taken into account in this scenario as well because assuming a uniform probability of having the disease is likely to be too broad of an assumption. there are many dierent factors that lead to sub-populations having distinct frequencies of carrying covid-19 and the certainty about the prevalence varies as well. 7 dorfman's method [2] we compare the results from dt and gbs methods to dorfman's method. dorfman's method is as follows: 1. for a the optimal group size b (dened below), apply one test to a group of b cases. if the result is negative, then conclude that every case in the group is negative. if the test result is negative then one test was used for b cases. if it is positive, then b+1 tests were used. the quantity b is chosen to minimize the expected number of tests used to determine the result of one individual case, we will use this formulat to compute the expected proportion of tests saved for several dierent levels of prevalence to compare to the successor binary search based methods we choose study. to evaluate and analyze the performance we assume that the populations are sequences of randomly generated i.i.d. variables representing cases with the probability of having the disease p. the population is then modeled by probability distribution with the following parameters. • n -the size of the population/number of cases • pthe probability that a case is positive. we refer to this as the prevalence. • a case is then a bernoulli random variable that is positive with probability p and negative with probability 1 − p. • the number of positive cases is then a binomial random variable with n samples and probability p of a success, we write this as binomial(n, p). in the section 9 we analyze the performance of the group testing methods at a selection of prevalences for populations of size 100 and 1000. in section 10 we analyze the performance at the .99 condence upper bound. this can be thought of as overestimating the prevalence by a large enough amount so that we are 99% sure that the actual number of positive cases falls at or below this level. in section 11 we analyze the number of rounds of testing each sample goes through. our results suggest that not only do group testing methods save tests and time, but they can be done within the realistically expected amount of usage for each test. an analysis of the histograms of the number of tests per case from which we compute the average performances is in the numerical results section after the bibliography (section 14). 1 please visit https://github.com/cmentus/group-testing/ to nd the jupyter notebook for all numerical experiments. 9 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint 9 performance of group testing methods at dierent prevalences for our performance analysis, we sample populations of sizes n = 100, 1000 at prevelance levels p = .001, .01, .02, .03, .04, .05, .1, .15, .2, and .25. we present the mean of 1000 samples and present them in the table below. for dtg results, some of the entries are left empty. this is because the xed group size is less than the capped group size so it would be redundant to list it, and for the performance refer to the nearest entry to the left. we do not do this for gbs because the group size is adaptive and there is nothing restricting it from becoming small. in each table we list the percentage of tests saved from employing the group test methods. . we analyze the eect of picking the prescribed method for a conservatively large estimate on the prevalence. a larger estimate on the prevalence will still take advantage of the frequent occurence of many consecutive negative cases. the overestimate we use corresponds to a probability of having less positive cases of .99. we sample the populations at least 100 times and take the mean of the proportion of tests saved over all samples. specically for dtg applied to populations of 100 and 1000 we generated 100 samples. for gsbg we applied to populations of size 100 we sampled 1000 times except for gsb16. for gsbg applied to populations of size 1000 we sampled 100 times except for gsb64 we sampled 1000 times. the dierences in number of samples 11 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint are due to the overall computational expensiveness of simulating gsb and dt. the mean savings of each numerical experiment are presented in the following and various prevalences at the .99 condence level d .99 . note that d .99 is closer to d .5 for higher populations (that is d.99 d.5 is closer to 1). therefore the performance is closer to the results depicted in the previous section. although demonstrated to be more ecient in test usage, generalized binary splitting requires some samples to be used at least as many times as the logarithm of initial group size. for maximum group sizes of 32 and 64 this implies that positive cases will be tested at least 6 or 7 times. as is demonstrated by gures 11.1 and 11.2 it is possible for tests to be used more than twice as many times. 12 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint figure 11 .1: for gbs32 we sample a population of size 10,000 and record the number of times each case is tested. note that for prevalence of p = .1% a case at most 5 times in the population. in gure 11.2 where we perform the same experiment we nd cases that are tested up to 9 times. we apply gbs32 to nd at most d .99 (see section ??) we see in gure 11.3 that the proportion of cases that are tested many times (more than 6) is very low. this is evidence that testing the cases indivually when we are running low (if we are limited to 6 uses) adds up to less than 1% extra tests. if we are able to test a sample more than this many times, individually testing when we are about to run out eects the eciency negligibly. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint we plot the histograms of the number of times postive results and negative results are tested given that they are tested more than once. interestingly, sometimes the number of times negative cases are tested seems to have a larger maximum. gaining knowledge of covid positive status in asymptomatic carriers is of prime importance in the ght to contain and eliminate the disease. the group testing strategy will generate certainty and a margin of safety in conned populations and may be useful in detection of disease burden in geographically dispersed populations as a mode of surveillance. a key factor in our strategy is the two phase approach that we propose. clinical screening of symptomatic patients cuts down the prevalence of covid-19 in the chosen asymptomatic test populations. 14 . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint rapid, large scale testing is urgently needed for naval ships and military bases. a recent outbreak upon the uss roosevelt has brought this concern to the forefront of military proporities [4] . recently italian prisoners have rioted rioting due to lack of testing [6] . 300 prisoners have been released from nyc prisons due to concern for spread of coronavirus [7] . nursing homes have been sites of extensive outbreaks [8] . screening an asymptomatic population is challenging but important. sensitivity of tests is paramount but also utilization of resources must be ecient. this study proposes a statistically valid mathematical model to optimize number of tests performed on chosen populations. the future direction of our work will require clinical validation with real world application of this group testing strategy. the clinical screening of symptomatic patients out of our population is helpful but it will also make detecting virus in asymptomatic people potentially tougher because of the chosen viral load. current rt-pcr methods are standard but have a concerning false negative rate in symptomatic patients [1] . this may be further exacerbated in asymptomatic patients. future studies may explore or consider the use of more sensitive techniques including ddpcr and addition of other sampling techniques such as fecal specimens and perhaps advanced imaging such as chest ct scan. based on the above evidence it is clear that a comprehensive strategy is necessary to test all asymptomatic people. this strategy will uncover the hidden silent carriers of disease. group testing has the chance for saving tests, while giving an exit to revert to individual testing if there are more cases than estimated. these strategies are not necessarily the theoretical optimums. nevertheless, by our calculation and numerical simulations they have the power to cut down the number of tests used. the group testing method is applicable to conned populations from both a clinical and mathematical stand point. we can clinically screen conned groups to decrease the prevalence in a predictable way. this method allows each patient in the population to get a test result. we explore adaptive and non-adaptive strategies for group testing for covid-19, therefore we add adaptive and binary search based non-adaptive strategies. both perform well even when we restrict the size of the group based on the sensitivity of rt-pcr. we test the performance of two algorithms: divide and test and generalized binary splitting. for populations of size 100 and 1000 with each case being positive independent of one another with probability p, we nd that both methods save many tests. in fact, even when we restrict the size of the groups they make substantial savings. for example at prevalence .001 and group sizes capped at 16, both dt16 and gsb16 are capable of outperforming dorfman's method, that requires a group size of 33 for optimal performance. for a population of 1000 and condence level .99, the dt16 and gbs16 also have a similar performance to dorfman's method at .001 where dorfman's method is chosen as if we knew the exact prevalence. we nd that dt outperforms gsb on average even though gsb in theory uses close to optimal number of tests for a known xed number of (+) cases (bounded above by #tests − 1 + information lower bound). this indicates that non-adaptive binary search methods have the potential to save many tests. it is possible that gsb is more generally applicable to populations that cannot be thought of as being generated since samples can be divided frozen and stored for continuous use we mathematically analyze how many divisions we have to use for each sample. we nd that testing individually when the sample is about to run out does not subtract substantially from the savings. we demonstrate that for a group size of 32 the generalized binary search on 10000 cases uses the same sample more than 6 times for < 1% of all cases. therefore, applying a test individually to samples when they are running low is a viable way to save many tests while not depleting material from one person's sample. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint for both gbs and dt. the maximum group size is 64. for gsb we sampled 1000 populations, for dt we sampled 100 populations. note that the spread of the dt algorithm is much wider and has many cases that need a much lower number of tests than gsb. the small bars in gsb are caused by the 1% chance of not identifying all of the cases in one run and so repeating the process on the rest of the population. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.05.20050245 doi: medrxiv preprint correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases the detection of defective members of large populations a method for detecting all defective members in a population by group testing evaluation of group testing for sars-cov-2 rna. medrxiv italian coronavirus prison riot six dead coronavirus new york update: mayor announces immediate release of 300 prisoners convicted for non-violent oenses all 94 residents of new jersey nursing home presumed positive for coronavirus evaluation of covid-19 rt-qpcr test in multi-sample pools. medrxiv we thank martin douglas for interesting and useful discussions about group testing methods using binary search. we are also grateful to matthew heidemann, blake elias, kevin schallert, gary chizever, michael wells, olga buchel and patrick o'neill for contributing ideas and useful feedback.we are indebted to yaneer bar-yam for important recommendations that greatly improved the ideas, clarity and organization of the paper. we thank matthew heidemann for the diagram at the beginning of the paper that skillfully explains our process. thank you to david f. schaeer, marcel dvorak, julia naso and marthe kenny charles for discussions about the limit of detection for sars-cov-2 rna by rt-pcr and recommendations that lead us to simulate group tests with maximum sizes 16, 8 and 4.we thank endcoronavirus.org and the new england complexity institute for giving us the platform to meet and collaborate as a team and meet great people with diverse backgrounds and skillsets united in the cause to ght the current pandemic. key: cord-296588-q2716lda authors: hanson, kimberly e; caliendo, angela m; arias, cesar a; englund, janet a; lee, mark j; loeb, mark; patel, robin; el alayli, abdallah; kalot, mohamad a; falck-ytter, yngve; lavergne, valery; morgan, rebecca l; murad, m hassan; sultan, shahnaz; bhimraj, adarsh; mustafa, reem a title: infectious diseases society of america guidelines on the diagnosis of covid-19 date: 2020-06-16 journal: clin infect dis doi: 10.1093/cid/ciaa760 sha: doc_id: 296588 cord_uid: q2716lda background: accurate molecular diagnostic tests are necessary for confirming a diagnosis of coronavirus disease 2019 (covid-19). direct detection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) nucleic acids in respiratory tract specimens informs patient, healthcare institution and public health level decision-making. the numbers of available sars-cov-2 nucleic acid detection tests are rapidly increasing, as is the covid-19 diagnostic literature. thus, the infectious diseases society of america (idsa) recognized a significant need for frequently updated systematic reviews of the literature to inform evidence-based best practice guidance. objective: the idsa’s goal was to develop an evidence-based diagnostic guideline to assists clinicians, clinical laboratorians, patients and policymakers in decisions related to the optimal use of sars-cov-2 nucleic acid amplification tests. in addition, we provide a conceptual framework for understanding molecular diagnostic test performance, discuss the nuance of test result interpretation in a variety of practice settings, and highlight important unmet research needs in the covid-19 diagnostic testing space. methods: idsa convened a multidisciplinary panel of infectious diseases clinicians, clinical microbiologists, and experts in systematic literature review to identify and prioritize clinical questions and outcomes related to the use of sars-cov-2 molecular diagnostics. grading of recommendations assessment, development and evaluation (grade) methodology was used to assess the certainty of evidence and make testing recommendations. results: the panel agreed on 15 diagnostic recommendations. conclusions: universal access to accurate sars-cov-2 nucleic acid testing is critical for patient care, hospital infection prevention and the public response to the covid-19 pandemic. information on the clinical performance of available tests is rapidly emerging, but the quality of evidence of the current literature is considered low to very low. recognizing these limitations, the idsa panel weighed available diagnostic evidence and recommends nucleic acid testing for all symptomatic individuals suspected of having covid-19. in addition, testing is recommended for asymptomatic individuals with known or suspected contact with a covid-19 case. testing asymptomatic individuals without known exposure is suggested when the results will impact isolation/quarantine/personal protective equipment (ppe) usage decisions, dictate eligibility for surgery, or inform administration of immunosuppressive therapy. ultimately, prioritization of testing will depend on institutional-specific resources and the needs of different patient populations. a c c e p t e d m a n u s c r i p t background in late december 2019, an outbreak of pneumonia cases of unclear etiology was reported in wuhan city, hubei province, china [1] . unbiased next generation sequencing (ngs) using lower respiratory tract (lrt) specimens collected from affected patients subsequently identified a novel coronavirus as the cause of illness now known as coronavirus disease 2019 . the entire viral genome was shared online within days and phylogenetic analyses established close relationship to human severe acute respiratory syndrome coronavirus (sars-cov) as well as several other sars-like bat coronaviruses [1, 2] . based on genetic similarities, the novel coronavirus was officially named sars-cov-2 [3] . by march 11 th , 2020, the virus had spread to at least 114 countries and killed more than 4,000 people, prompting the world health organization (who) to officially declare a global pandemic [4] . public availability of the sars-cov-2 genome was an essential first step enabling development of accurate molecular diagnostic assays. nucleic acid amplification tests (naats) designed to detect one or more gene sequences specific to sars-cov-2 are essential for confirming covidto date, multiple commercial test manufacturers and clinical laboratories, including academic medical centers, have received eua for a sars-cov-2-specific molecular diagnostic test. the first home-based test collection kit was also recently granted an eua [5] . it is important to recognize, however, that eua guidance differs substantially from the standard fda approval process. in the setting of a public health emergency, the fda only requires test developers to establish acceptable analytical accuracy. clinical test performance (i.e., sensitivity and specificity) has yet to be determined or comprehensively compared across eua platforms. as a result, most of the naat performance data used to inform this guideline was derived from a c c e p t e d m a n u s c r i p t studies evaluating assays not widely used in the u.s. we assumed, therefore, that performance of standard naat methods to be comparable across countries (which may or may not be correct). given increasing test availability combined with a rapidly growing number of naat-focused studies published online or in academic journals, the infectious diseases society of america table 1 . at the time of this review, there was little evidence to inform use of serologic testing. therefore, the first version of the idsa diagnostic guideline focuses only on the use of targeted naat applied directly respiratory tract specimens. it is anticipated that these guidelines will be frequently updated as substantive new information becomes available; subsequent versions will also address sars-cov-2 serology due to the rapidly evolving information and uncertainty of the reliability of serological tests. m a n u s c r i p t this guideline was developed using the grading of recommendations assessment, development and evaluation (grade) approach for evidence assessment. in addition, given the need for rapid response to an urgent public health crisis, the methodological approach was modified according to the gin/mcmaster checklist for development of rapid recommendations [6] . the the conflict of interest (coi) review group included two representatives from idsa who were responsible for reviewing, evaluating and approving all disclosures. all members of the expert panel complied with the coi process for reviewing and managing conflicts of interest, which required disclosure of any financial, intellectual, or other interest that might be construed as constituting an actual, potential, or apparent conflict, regardless of relevancy to the guideline topic. the assessment of disclosed relationships for possible coi was based on the relative weight of the financial relationship (i.e., monetary amount) and the relevance of the relationship (i.e., the degree to which an association might reasonably be interpreted by an independent observer as related to the topic or recommendation of consideration). the coi review group ensured that the majority of the panel and chair was without potential relevant a c c e p t e d m a n u s c r i p t (related to the topic) conflicts. the chair and all members of the technical team were determined to be unconflicted. clinical questions were developed into a population, intervention, comparison, outcomes (pico) format [7] prior to the first panel meeting (table s1) . idsa panel members prioritized questions with available evidence that met the minimum acceptable criteria (i.e., the body of evidence reported on at least test accuracy results can be applied to the population of interest). panel members prioritized patient-oriented outcomes related to sars-cov-2 testing such as requirement for self-quarantine, eligibility for investigational covid-19 treatment, timing of elective surgery or procedures, and management of immunosuppressive therapy. we also considered the impact of sars-cov-2 results on infection prevention and public health practices, including the use of personal protective equipment (ppe) and contact tracing. the national institute of health and care excellence (nice) and the center of disease control (cdc) highly-sensitive search was reviewed by the methodologist in consultation with the technical team information specialist and was determined to have high sensitivity. an additional term, covid, was added to the search strategy used in addition to the terms identified in the pico questions (table s2) . ovid medline and embase were searched from 2019 through april 20, 2020. horizon scans were performed daily during the evidence assessment and recommendation process to locate additional grey literature and manuscript preprints from the following sources litcovid, medrxiv, ssrn, and trip database. reference lists and literature suggested by panelists were reviewed for inclusion. no restrictions were placed on language or study type. two reviewers independently screened titles and abstracts, as well as eligible full-text studies. we included studies reporting data on diagnostic test accuracy (cohort studies, cross sectional a c c e p t e d m a n u s c r i p t studies and case-control studies). when questions compared the performance of different tests (e.g., different testing or sampling methods) or testing strategies, we included studies that provided direct test accuracy data about both tests in the same population. when these direct studies where lacking, we included studies that assessed a single test and compared its results to a reference standard. we did not limit our inclusion to a specific reference standard due to sparsity of data. we also included studies that assessed the prevalence of covid-19 in different populations. reviewers extracted relevant information into a standardized data extraction form. two reviewers completed data extraction independently and in duplicate. disagreements were resolved by discussion to reach consensus and in consultation with expert clinician scientists. data extracted included general study characteristics (authors, publication year, country, study design), diagnostic index test and reference standard, prevalence of covid-19, and parameters to determine test accuracy (i.e., sensitivity and specificity of the index test). accuracy estimates from individual studies were combined quantitatively (pooled) for each test using openmetaanalyst (http://www.cebm.brown.edu/openmeta/). we had planned to conduct a bivariate analysis for pooling sensitivity and specificity for each of the test comparisons to account for variation within and between studies. however, this was not feasible due to the sparsity of available data and lack of information on specificity in most instances, so we either presented data as a range of the extreme sensitivity and specificity presented in the studies or pooled as proportions to facilitate decision making. we had also planned to use the breslow-day test to measure the percentage of total variation across studies due to heterogeneity (i 2 ) but were not able to do that due to the sparsity of data. forest plots were created for each comparison. to calculate the absolute differences in effects for different testing or sampling strategies, we applied the results of the sensitivity and specificity to a range of plausible prevalence in the population. we then calculated true positives, true negatives, false positives, and false a c c e p t e d m a n u s c r i p t negatives. to determine the prevalence for each question, we considered the published literature in consultation with the clinical experts. in general, for questions addressing symptomatic individuals we considered the following prevalence: 10% which is typically seen in symptomatic outpatients who have not reached a hospital facility [8] [9] [10] ; 40% which is typically seen in patients meeting clinical definition for covid-19 who were hospitalized [11, 12] ; and 80% which is typically seen in patients meeting clinical definition for covid-19 who were admitted to intensive care units. for questions addressing asymptomatic individuals who were exposed to covid-19, we considered that the prevalence may range from 10% to 50% based on household clusters, nursing home outbreak, active surveillance of passengers quarantined on a cruise ship or passengers of repatriation flights, hospital employees with close contact with covid-19 positive patients and customers and employees of a restaurant that had a covid-19 outbreak [13] [14] [15] [16] [17] [18] [19] . for questions addressing asymptomatic individuals, we considered that the prevalence may range from <1% in general population who are not in hotspots to 10% in asymptomatic patients in hotspots [8, 20, 21] . we conducted the risk of bias assessment for diagnostic test accuracy studies using the quality assessment of diagnostic accuracy studies (quadas)-2 revised tool (table s3 ) [22] . grade framework was used to assess overall certainty by evaluating the evidence for each outcome on the following domains: risk of bias, imprecision, inconsistency, indirectness, and publication bias [23, 24] . grade summary of findings tables were developed in gradepro guideline development tool [25] . the panel considered core elements of the grade evidence in the decision process, including a c c e p t e d m a n u s c r i p t as per grade methodology, recommendations are labeled as "strong" or "conditional". the words "we recommend" indicate strong recommendations and "we suggest" indicate conditional recommendations. figure 2 provides the suggested interpretation of strong and weak recommendations for patients, clinicians, and healthcare policymakers. rarely, low certainty evidence may lead to strong recommendations. in those instances, we followed generally recommended approaches by the grade working group, which are outlined in five paradigmatic situations (e.g., avoiding a catastrophic harm) [26] . for recommendations pertaining to good practice statements, appropriate identification and wording choices were followed according to the grade working group [27] . a "good practice statement" represents a message perceived by the guideline panel as necessary to health care practice, that is supported by a large body of indirect evidence difficult to summarize, and indicates that implementing this recommendation would clearly result in large net positive consequences. for recommendations where the comparators are not formally stated, the comparison of interest was implicitly referred to as "not using the test". some recommendations acknowledge the current "knowledge gap" and aim at avoiding premature favorable recommendations for test use and to avoid encouraging the rapid diffusion of potentially inaccurate tests. detailed suggestions about the specific research questions that should be addressed are found in table 2 . a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t committee reviewed and approved the guideline prior to dissemination. regular, frequent screening of the literature will take place to determine the need for revisions based on the likelihood that new data will have an impact on the recommendations. if necessary, the expert panel will be reconvened to discuss potential changes. in addition, future searches will include critical appraisal of the sars-cov-2 serology literature. systematic review and horizon scan of the literature identified 2,909 references of which 23 informed the evidence base for these recommendations (figure s1 ). characteristics of the included studies can be found in table s4 . figure 1 summarizes a testing algorithm for covid-19 diagnosis guidelines. however, testing is not widely available in some areas.  this recommendation does not address testing a combination of specimen types due to lack of evidence.  the panel considered symptomatic patients to have at least one of the most common symptoms compatible with covid-19 ( table 1) . thirteen studies informed this recommendation [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] and they provided varying descriptions of specimen type (supplement c). in an effort to maintain consistency in the analysis of evidence, reported specimen types were grouped into nasopharyngeal (np), mid-turbinate (mt), nasal, throat, or saliva. in studies that did not define collection techniques for "nasal", we assumed it to mean anterior nasal and not deep-nasal or a c c e p t e d m a n u s c r i p t nasopharyngeal. saliva collection methods were also inconsistent. saliva studies incorporating a "coughed-up" sample were excluded from the urti and ili analysis under the assumption that they likely included some mixture of pure saliva and sputum. analyses of "tongue" swabs were also excluded. it is important to note as well, that not all specimens were collected from the same patient at the same time, the time of collection from symptom onset was not provided in all studies and various approaches for establishing sars-cov-2 positivity were used to define positive results (i.e., clinical evaluation, detection different gene targets versus nucleic acid sequencing). a total of 11 reports presented data about test accuracy of a specific sample type(s); eight of these [35, 36, 38-41, 43, 44] provided comparative data for two or more sample collection sites; and three others [33, 37, 45] provided data for one site only. studies with comparative data showed a lower sensitivity for oral sampling in comparison to np, mt, or nasal sampling. summary statistics different specimen type are shown in table 3 . two studies [38, 39] a c c e p t e d m a n u s c r i p t types are false negative results, which could promote unchecked sars-cov-2 transmission. one potential benefit of the alternative methods are the less-invasive nature of nasal, mt and throat swabs or saliva as compared to np sampling. in addition, the ppe requirements for healthcare providers collecting non-cough inducing specimen types may be less. lastly, the non-np sampling is amendable to patient self-collection, which has the potential to further reduce healthcare worker exposure to infectious droplets and possible droplet nuclei. additional considerations: indirect evidence from influenza and respiratory syncytial virus studies suggest that alternative nasal cavity collection sampling methods such as anterior nasal and mt swabs provide comparable sensitivity to np swabs [46] . using np swab collection as the reference method will bias evaluation of the comparator method by definition. saliva is an easily obtained specimen and there is significant recent interest in its use for sars-cov-2 detection. at the time of this literature review we identified a single study assessing true saliva as a specimen type. this is a promising specimen type given the simplicity of collection. the panel anticipates multiple additional studies to follow, which will be included in future guideline updates. the panel considered indirect evidence for nasal swabs and mt swabs from other respiratory viruses in the decision to list these specimen types are preferred over saliva. in addition, saliva is complex matrix and clinical laboratories will need to carefully assess rna stability during specimen transport and the efficiency of nucleic acid extraction using their own specific methods. we did not identify any studies assessing combinations of specimen types. although oropharyngeal swabs or saliva can be utilized for the diagnosis of covid-19, the available evidence combined with indirect evidence from other respiratory viruses suggests that collection of anterior nares, mt, or np swabs has higher sensitivity. at the current time, there is little evidence to support use of oropharyngeal swabs or saliva alone. however, future studies of saliva as a specimen type for sars-co-2 detection are anticipated. a c c e p t e d m a n u s c r i p t evaluation of alternative collection devices and methods are critically needed as we are facing shortages in test collection supplies such as swabs, transport media and ppe. while np swab collection is widely used and the primary specimen type for commercial direct sars-cov-2 test platforms, based on current available evidence, clinical practice, and availability of testing resources, the panel believes there are comparable alternative methods for sampling the nasal passages. clinical laboratories will need to validate use of individual specimen types. future studies of saliva should clearly describe collection methods, specimen transport media and processing requirements. moving forward, it will be critical to standardize these processes. 9 (0 to 9) 0 (0 to 9) 0 (0 to 9) 0 (0 to 9) 18 (0 to 216) 0 (0 to 18) explanations: this table is based on applying the sensitivity and specificity estimates to calculate true and false positives and negatives in a hypothetical population of 1000 individuals. *no studies reported on the specificity of oral and np a. the case-control design leads to a serious study population bias. b. some studies compared two or more of the specimen types, but no studies compared all specimen types in the same patient population. studies reported test accuracy results but did not report on patient-important and population-important outcomes based on the results. c. there is serious unexplained heterogeneity.  appropriate specimen collection and transport to the laboratory is critical. general instructions for swab-based sars-cov2 testing are shown in table 4 . additional resources are available on the idsa website.  a clear, step-by-step protocol needs to be presented to patients attempting self-collection. this could be in the form of a short video or printed pamphlet with illustrations.  the majority of self-collection studies were performed in the presence of a healthcare worker.  the available evidence for nasal and mt swabs as alternatives to healthcare personnel collection is based on assessment of symptomatic patients. data on self-collection in asymptomatic individuals is currently unavailable. a c c e p t e d m a n u s c r i p t  the panel considered symptomatic patients to have at least one of the most common symptoms compatible with covid-19 ( table 1) . this recommendation is based on three cohort studies (supplement d). in the first study, test accuracy results were provided for self-collected non-invasive specimens compared to healthcare-collected np swabs as the standard [39] . for self-collection, participants were provided with instructions and asked to self-collect tongue, nasal, and mt swabs, in that order. tongue samples were collected with a nylon flocked swab. nasal samples were collected with a foam swab bilaterally. mid-turbinate samples were collected with a nylon flocked swab bilaterally. after patient sampling was completed, np samples were collected by a healthcare worker using a polyester tipped swab on a skinny wire. in the second study, patients attending dedicated covid-19 collection clinics were offered the option to first self-collect nasal and throat swabs followed by healthcare provider collection of nasal, throat or oropharyngeal swabs [44] ; concordance of results were presented. the third study compared positivity for supervised oral fluid sampling, supervised self-collected deep nasal swabs, unsupervised oral fluid sampling and provider collected np swabs [43] . in this analysis, any positive test, obtained from any of the reported sampling methods including the index test, was considered to be a true positive. although the study reported the results for "oral fluid", it is likely these samples were mixed with sputum. lastly, the panel considered unpublished data submitted to the fda on home collection, which demonstrated good stability of specimens stored in universal transport media (utm) during transport from homes to laboratories and comparable quantities of virus in self-collected compared to healthcare provider collected swabs. summary statistics for self-collected versus health-care worker collected nasal swabs are shown in table 5 . the studies used to inform the recommendation were small and heterogeneous. sources of heterogeneity included variable swab and transport media types as well as use of unilateral versus bilateral nares self-collection. the timing of collection relative to symptom onset is also important but was not well documented in available data. due to the mentioned concerns with a c c e p t e d m a n u s c r i p t the studies and the lack of direct comparisons between different specimen types in the same patient population, the panel agreed that overall certainty of evidence was low. benefits and harms: the panel placed a high value on avoiding the close exposure of healthcare providers to patient droplets and possible droplet nuclei generated during specimen collection. we assumed that self-collected specimens including anterior nasal swabs, mt swabs and saliva (without cough) would reduce provider exposure and could reduce mask or respirator use. the overall sensitivity of testing when samples were collected by patients was comparable to those collected by healthcare providers. other potential benefits of self-collection include increasing the availability of testing outside the healthcare system and increased patient satisfaction with selfcollection. concerns with self-collection include lack of experience or documentation for actual collection methods by patients; inappropriate sample collection and/or handling could then lead to inaccurate results. although data is limited, both healthcare provider collected, and self-collected nasal or mt swabs appear to result in similar rates of detection of sars-cov-2. self-collection of np swabs is unlikely to be an option as a self-collection method. there are advantages of having multiple strategies to collect clinical specimens, particularly in times of ppe shortages when limiting exposure to healthcare personnel or other patients is important, or when testing in specific populations without access to the healthcare system is required. further comparative studies of self-collected non-invasive specimens (i.e., nasal, mid-turbinate, and throat swabs, as well as saliva) compared with m a n u s c r i p t abbreviations: np = nasopharyngeal; op = oropharyngeal; mt = nasal mid-turbinate; ns = anterior nares swab. ^c autions: do not use calcium alginate swabs or swabs with wooden shafts, which may contain substances that interfere with nucleic acid amplification. rayon swabs may not be compatible with all molecular platforms. clinical laboratories should confirm compatibility of collection devices during assay validation. # pediatrics: swab insertion distance will differ for pediatric patients. swabs with stoppers make estimating distance easier for mt self-collection. two-sided mt sampling not always performed. we identified nine studies that performed both an upper respiratory tract (urt) swab and lower respiratory tract (lrt) sample collection consecutively on the same patient (supplement e). two reported on viral load and did not report on sensitivity [47, 48] . seven studies reported on sensitivity, of which three had a case control design [35, 49, 50] and one reported results per sample and not per patient [51] . the three cohort studies [43, 52, 53] were used to inform the panel's decision-making process. the sample type varied by study and included throat and nasal swabs for urt sampling and sputum and bronchoalveolar lavage (bal) fluid specimens for lrt sampling. summary statistics for urt versus lrt sampling in 3 cohort studies are shown in table 6 . the timing of specimen collection with regards to clinical course was not reported for all these studies and different diagnostic reference standards were a c c e p t e d m a n u s c r i p t m a n u s c r i p t a c c e p t e d m a n u s c r i p t 0 (0 to 9) 0 (0 to 9) 0 (0 to 6) 0 (0 to 6) explanations: this table is based on applying the sensitivity and specificity estimates to calculate true and false positives and negatives in a hypothetical population of 1000 individuals a. studies reported test accuracy results but did not report on patient-important and population-important outcomes based on the results. b. considering the lower vs upper limit of the sensitivity confidence interval may lead to different clinical decision, and the low number of patients lead to very serious imprecision c. typically seen in symptomatic outpatients who have not reached a hospital facility d. typically seen in patients meeting clinical definition for covid-19 who were hospitalized e. certainty of evidence (coe) m a n u s c r i p t missing data in the studies included timing of specimen collection in relationship to onset of clinical symptoms and specimen type used for testing. additionally, the performance and accuracy of different rapid tests was very inconsistent. given all these issues, the overall certainty of the effect of using rapid tests on patients was very low. the major benefit of a rapid result is the ability to make clinical decisions while the patient is present in a timely manner and implement interventions to protect others. a possible harm of rapid tests is the potential for increased numbers of false negative results, which could lead to missed diagnoses and patients not being isolated when they are indeed infected, if sensitivity is lower than non-rapid tests. diagnostic accuracy should be stratified by duration of symptoms and severity of disease. furthermore, the diagnostic reference standard must be clearly defined. performance characteristics of eua rapid tests, especially those that are clia-waived, should be collected in the field and performed by the non-laboratory staff running the test (which is how they are used in real life). ideally, studies should assess the impact of rapid results on clinical outcomes, such as time to appropriate treatment or therapeutic intervention. individuals who are either known or suspected to have been exposed to covid-19 (conditional recommendation, very low certainty of evidence).  known exposure was defined as direct contact with a laboratory confirmed case of covid suspected exposure was defined as working or residing in a congregate setting (e.g., longterm care, correctional facility, cruise ship, factory, among others) experiencing a covid-19 a c c e p t e d m a n u s c r i p t  the risk of contracting sars-cov-2 may vary under different exposure conditions.  this recommendation assumes the exposed individual was not wearing appropriate ppe.  the decision to test asymptomatic patients will be dependent on the availability of testing resources. summary of the evidence: we did not identify any studies that directly assessed a strategy of testing versus no testing of asymptomatic individuals exposed to sars-cov-2. therefore, the effect of testing on the pre-specified outcomes could not be directly assessed. we also did not identify test accuracy studies directly assessing the performance of sars-cov-2 naats in asymptomatic individuals. however, based on evidence that asymptomatic or pre-symptomatic patients may have similar viral loads and shedding compared to those who are symptomatic [15, 62, 63] , the panel agreed that it is reasonable to apply test accuracy data based on symptomatic patients to the asymptomatic populations. hence, it was essential to determine the pre-test probability or prevalence of covid-19 in the asymptomatic groups. we assessed studies that reported the prevalence of covid-19 among asymptomatic individuals in household clusters [15, 17, 19] , a nursing home outbreak [14] , active surveillance of passengers quarantined on a cruise ship or passengers of repatriation flights [18] , hospital employees with close contact to covid-19 positive patients [13] , and customers and employees of a restaurant that had a covid-19 outbreak [16] . overall, prevalence ranged from 10 to 50% in settings where substantial transmission was suspected prior to testing. summary statistics for single versus repeated testing are shown in table 8 and supplement h. we acknowledge that information on individual exposure was limited in the evidence base. all these limitations led to very low certainty in the evidence overall. testing asymptomatic individuals who have been exposed, or suspected to have been exposed, allows for isolation for those who are positive. whether in an institutional cluster or a wider community outbreak, isolation will help reduce further transmission. there is potential harm in a false negative naat result collected from an exposed individual who is a c c e p t e d m a n u s c r i p t actually infected; these individuals may incorrectly consider themselves non-infected, and unknowingly expose others to sars-cov-2 as a result. given the lack of evidence, a negative test post-exposure does not mean quarantine can be discontinued. some individuals may still be in the incubation phase, subsequently develop active viral shedding, and incorrectly consider themselves non-infected. as a result, a negative post-exposure test cannot necessarily be used to avoid quarantine. a positive result, however, would reinforce the importance of isolation as well as inform contact tracing, cohorting, or other mitigation strategies. additional considerations: diagnostic test performance in asymptomatic individuals has not been established. assuming an overall test sensitivity between 75%-95% [35, 36, [38] [39] [40] 44] , false negative test results are expected. there is also cost to testing asymptomatic exposed individuals; since quarantine may still be indicated regardless of test results, such testing may add cost without changing practice. data are limited to define definitions of close contact. risk stratification of a given exposure can be made in consultation with public health authorities. in addition, the cdc has published guidance on defining healthcare exposures and categorizing exposure risks [64] .the ideal time to test an asymptomatic contact of a known or suspected covid-19 case is also unknown. timing also becomes complicated for household contacts with ongoing exposure. the average incubation period for sars-cov2 has been determined to be five days [65] . thus, 5-7 days following exposure may be a reasonable time frame to consider post-exposure testing. in addition, data to inform the definition of a significant exposure or close contact are limited. considerations when assessing the risk of a known contact include the duration of exposure and the clinical symptoms (e.g., cough) of the person with covid-19. with known or suspected exposures should be coordinated with local public health officials. this indication for testing is especially important in situations where knowledge of asymptomatic or pre-symptomatic infection is essential for determining medical follow-up, defining risks for other vulnerable individuals in the household, congregate setting or hospital. special consideration should also be given to healthcare personnel exposed without a c c e p t e d m a n u s c r i p t appropriate ppe in healthcare settings. definitions of appropriate ppe can be found on the cdc website [66] . comparative studies (preferably randomized controlled trials) along with cost-effectiveness analyses of testing strategies in asymptomatic populations are needed. studies on the ideal time and collection method to test asymptomatic individuals who have been exposed to covid-19 should be performed. in addition, what constitutes an exposure that would justify testing requires further research. whether early diagnosis of covid-19 might provide an opportunity to intervene therapeutically and change the ultimate course of infection (i.e., prevent severe pneumonia) is unknown. if this is shown to be the case, the opportunity for therapeutic intervention might justify screening exposed individuals. this table is based on applying the sensitivity and specificity estimates to calculate true and false positives and negatives in a hypothetical population of 1000 individuals a. reference standard considered to be nasopharyngeal specimen rt-pcr. b. studies report test accuracy results but do not report on patient-important outcomes based on these results. c. a small number of patients included. d. we assessed studies that reported the prevalence of covid-19 among asymptomatic individuals who were exposed to covid-19 and determined that the prevalence may range from 10% to 50% based on household clusters, nursing home outbreak, active surveillance of passengers quarantined on a cruise ship or passengers of repatriation flights, hospital employees with close contact with covid-19 positive patients and customers and employees of a restaurant that had a covid-19 outbreak. e. certainty of evidence (coe)  asymptomatic individuals are defined as those with no symptoms or signs of covid-19.  a high prevalence of covid-19 in the community was considered communities with a prevalence of 10%. a c c e p t e d m a n u s c r i p t  the decision to test asymptomatic patients (including when the prevalence is between 2 and 9%) will be dependent on the availability of testing resources. we did not identify any studies that directly assessed a strategy of nucleic acid testing for sars-cov-2 versus no testing before hospitalization for non-covid-19 related reasons. we also did not identify test accuracy studies directly assessing the performance of sars-cov-2 viral rna tests in asymptomatic individuals. however, based on existing evidence suggesting that asymptomatic or pre-symptomatic patients may have similar virus loads and shedding as those who are symptomatic [62, 63] , the panel agreed to infer test accuracy for asymptomatic populations before being hospitalized. it was also essential to determine the pre-test probability or prevalence of the disease in asymptomatic patients admitted to the hospital. we assessed studies that reported prevalence  this recommendation defines immunosuppressive procedures as cytotoxic chemotherapy, solid organ or stem cell transplantation, long acting biologic therapy, cellular immunotherapy, or high-dose corticosteroids.  testing should ideally be performed as close to the planned treatment/procedure as possible (e.g. within 48-72 hours).  many of these patients require frequent, repeated or prolonged visits to receive treatment.  this recommendation does not address risks or strategies to deal with sars-cov-2 transmission in outpatient settings such as infusion centers. we did not identify any studies that directly assessed a strategy of testing for sars-cov-2 versus no testing of asymptomatic individuals before receiving chemotherapy or transplantation. in addition, we were unable to evaluate the risks of delaying necessary treatments or transplants if testing was not available and quarantine/delay of treatment was then required. we also did not identify any test accuracy studies directly assessing the performance of naat in asymptomatic individuals. based on existing evidence supporting that asymptomatic or pre-symptomatic patients may have similar virus loads and a c c e p t e d m a n u s c r i p t shedding as those who are symptomatic [62, 63] , the panel agreed that test accuracy data from symptomatic patients would apply to asymptomatic populations being hospitalized. it was essential to determine the pre-test probability or prevalence of covid-19 in asymptomatic patients who will be receiving chemotherapy. we assessed studies that evaluated prevalence of covid-19 among asymptomatic individuals and patients with cancer to estimate prevalence a between <1 to 10%. we identified three studies reporting data on the prevalence of cancer among covid-19 patients and the percentage of complications (e.g., icu admission, death) among these patients. liang et al [67] showed that the prevalence of cancer among covid-19 patients to be 1%, which was higher compared to their general population (0.2%). yu et al [21] showed the prevalence of covid-19 among patients admitted to the radiation and medical oncology floor to be 0.8%. lastly, a systematic review conducted by desai et al. (2020) [68] showed the pooled prevalence of cancer among covid-19 cases to be 2-3%. the overall certainty of the evidence about testing effects in immunocompromised individuals was very low due to extremely limited data in this population. the panel determined that a maximum threshold of <2-5 missed cases per 1000 would be acceptable. not testing individuals regardless of low versus high prevalence areas would lead to higher numbers of missed cases which the panel considered to exceed the acceptable threshold. the threshold was set very low due to concern about catastrophic outcomes in this population. although data is limited, there are reports documenting outbreaks of respiratory viruses in hospitalized immunocompromised hosts [69] . in addition, increased risks of severe adverse respiratory virus-related outcomes in this population are documented [70] . for false negative test results, so caution should be exercised by those who will be in close contact with/exposed to the upper respiratory tract (e.g., anesthesia personnel, ent procedures). the decision to test asymptomatic patients will be dependent on the availability of testing resources.  the panel defined time-sensitive procedures as medically necessary procedures that need to be done within three months.  procedures considered to be aerosol generating are listed in table 9 .  decisions about ppe will be dependent on test results because of limited availability of ppe. however, there is a risk for false negative test results, so caution should be exercised for those who will be in close contact with/exposed to the patient's airways.  procedures considered to be aerosol generating are listed in table 9 .  the decision to test asymptomatic patients will be dependent on the availability of testing resources.  this recommendation does not address the need for repeat testing if patients are required to undergo multiple procedures over time. the panel did not identify any studies that directly assessed a strategy of testing for sars-cov-2 versus no testing of asymptomatic individuals before undergoing major surgery or aerosol generating procedures (agps). the panel also did not identify test accuracy studies directly assessing the performance of sars-cov-2 naats in asymptomatic individuals. however, based on existing evidence supporting that asymptomatic or presymptomatic patients may have similar viral loads and shedding as those who are symptomatic, the panel agreed that test accuracy data from symptomatic patients could be applied to asymptomatic populations before surgery. it was essential to determine the pre-test probability or prevalence of disease in the asymptomatic patients who will undergo surgery. we assessed studies that evaluated the a c c e p t e d m a n u s c r i p t prevalence of covid-19 among asymptomatic individuals and determined that the range of prevalence would be between <1 to 10% based on assessing rates of infection in asymptomatic individuals in the general population in low prevalence and in "hotspot" areas [8, 20, 21] . the panel recommendation was based on emphasizing the importance of preventing infection in healthcare providers during major time-sensitive surgeries and agps. in addition, the limited data showing poor outcomes in covid-19 positive patients undergoing a major surgical procedure requiring intubation informed decisions to reduce this risk for asymptomatic patients [71] . there are no data that assess the outcome of agps in sars-cov-2 positive patients. the benefit of suggesting testing for sars-cov-2 in asymptomatic patients undergoing major time-sensitive surgery is that it allows for the identification of infected patients before the procedure; thus allowing surgery to delayed based on the limited data suggesting that patients testing positive may have poor outcomes [71] . this approach also has the potential to inform healthcare workers in terms of ppe use, particularly in areas where ppe is limited. of note, there is very low certainty evidence from retrospective case series suggesting poor outcomes of time-sensitive surgeries for those with covid-19. the surgeries included were variable in complexity and it was not clear if the poor outcomes came mostly from major or minor surgeries. however, it is plausible that poor outcomes were driven by the major surgeries. a potential harm of testing of immunocompetent, asymptomatic patients before a major surgery or agp is depletion of testing supplies and the diversion of all associated resources away from symptomatic patients. an additional harm of testing is related to the sensitivity of the naats for sars-cov-2, which will not detect all asymptomatic patients with covid-19 infection. therefore, some patients may be missed and healthcare workers at high risk could be exposed. thus, the panel suggests that healthcare workers at the highest risk during surgical procedures (e.g., those performing intubation or ent procedures) consider wearing ppe at all times, regardless of test results. this would be especially important in high prevalence areas a c c e p t e d m a n u s c r i p t (i.e., "hotspots"). an additional harm is that false positive tests for sars-cov-2 may unnecessarily delay a major time-sensitive surgery. there is no standard definition of what constitutes a major surgery. in general, the panel in consultation with surgical colleagues, agreed that major surgeries would be defined as more complicated and/or prolonged surgeries that require general anesthesia and intubation (which is an agp). additionally, time-sensitive surgeries/procedures were defined as those for which a delay greater than 3 months would negatively affect outcomes. in addition to the clinical questions addressed above, there is significant interest in the use of serologic sars-cov-2 tests both for diagnosis and public health surveillance. at the time of our literature review, however, additional data were needed to formulate recommendations. important areas that need to be addressed include assessment of the sensitivity and specificity of commercial antibody tests, determinations of protective immunity and measures of antibody responses over time. whether seroconversion can inform return to work or hospital staffing policies needs to be assessed. in the absence of evidence to guide the use of sars-cov-2 serologic testing, the idsa diagnostics committee published a serology primer for clinicians which highlights potential benefits, limitations and unmet research needs [73] . antigen detection tests may be on the horizon. how these will compare with naat needs to be defined. in addition, current naats detect viral rna but cannot distinguish infectious from noninfectious virus. this determination requires viral culture, which is not routinely performed in clinical laboratories for biosafety reasons and is likely less sensitive than naat. it will be important to define whether individuals who remain nucleic acid positive after symptom resolution, and potentially seroconversion, are infectious to others because this will have important ramifications for quarantine and reducing restrictions around social distancing. some "test of cure" algorithms require two sequential negative naats, yet some studies describe a c c e p t e d m a n u s c r i p t prolonged rna positivity. future studies are required to determine the significance of nucleic acid or antigen shedding after clinical recovery. molecular tests designed to detect sars-cov-2 nucleic acids are essential both for confirming covid-19 diagnosis and for public health responses aimed at curbing the pandemic. several countries have deployed naat on a massive scale as the cornerstone of a successful containment strategy. although the u.s. was hampered by limited test availability early in the outbreak, there are now more than 25 different commercially available sars-cov-2 assays and multiple clinical laboratories have developed their own laboratory-developed tests. aggressive efforts are underway to assure access to testing, but regional differences in availability persist. individual medical centers and clinics are likely to have different testing capacity as well. furthermore, which test a laboratory or facility chooses to perform will vary based on the resources of a given setting (e.g., near-patient versus high complexity laboratory) and turnaround-time to result requirements (i.e., rapid versus standard). the primary recommendations set forth in this guideline assume that sars-cov-2 testing is available to healthcare providers on the front lines. however, the panel also recognized that resources may vary, and contingency recommendations were developed for situations where naat supplies or ppe are limited. individual institutions will need to prioritize testing based on available resources and unique patient populations. testing for symptomatic patients should be prioritized. when testing capacity for symptomatic individuals is considered sufficiently robust, testing for asymptomatic individuals should be considered. there will undoubtedly be challenges prioritizing and implementing testing strategies for asymptomatic groups. the strongest recommendation for testing in asymptomatic individuals in this guideline pertains to immunocompromised patients being admitted to the hospital or in advance of immunosuppressive procedures. a c c e p t e d m a n u s c r i p t molecular tests have been central to our understanding of sars-cov-2. however, much about the biology of sars-cov-2 remains unknown. early experience suggests that sars-cov-2 is detectable in the upper respiratory tract, with peak levels typically measurable during the first week of symptoms [48, 62, 74] . rna detection rates, however, appear to vary from patient to patient and change over time. some patients with pneumonia, for example, have negative upper respiratory tract samples but positive lower airway samples [35, 75] . much less it known about the frequency of viral detection in asymptomatic individuals, although the concentration of detectable virus in some people with infection may be quite high [62, 63] . a better understanding of the spectrum of viral load kinetics over time at different anatomic sites is needed to inform decisions about the optimal testing strategies, including when and how to repeat if the first test is negative. like other respiratory viruses, shedding of viral rna in respiratory secretions may persist beyond resolution of symptoms and seroconversion [76] . whether such patients remain infectious to others is uncertain and this is an important area for future study. the clinical performance of commercially available sars-cov-2 molecular diagnostic tests has not yet been defined and will depend in large part on the biology of the virus. typically, when tests for the detection of viral respiratory pathogens are submitted to the fda, both analytical and clinical performance data are provided. under eua, however, only analytical data are required. diagnostic developers may test contrived specimens, by spiking viral rna or inactivated virus into the desired matrix, rather than using real clinical specimens collected from patients with covid-19. thirty contrived positive and 30 negative specimens tested, with 95% sensitivity and 100% specificity required for eua. therefore, while we have information regarding the limit of detection of the test and evidence (both in vitro and in silico studies) that the primer design is specific for sars-cov-2, there is no information on how each test performs clinically at the time the eua is issued. clinical laboratories using commercial eua tests must verify analytic test performance at some level in their own hands, including evaluation of different specimen types and collection methods (e.g., swab types and transport media). a c c e p t e d m a n u s c r i p t clinical performance metrics include sensitivity, which is the ability of the test to correctly identify those with infection, and specificity, the ability of the test to correctly identify those without the disease. in practice, the positive and negative predictive values of the test are also essential for interpreting test results. estimations of community prevalence and patient pre-test probability combined with knowledge of test sensitivity and specificity are essential for determining the likelihood that an individual has covid-19. in practice, however, the true prevalence of covid-19 in the community may not be well-defined and may be underestimated when test availability is limited. in addition, while sars-cov-2 rna tests are highly specific, their respective sensitivities are likely to vary. recognizing these complexities, estimates of prevalence/pre-test probability and assay sensitivity were varied in our analyses based on the available literature in an attempt to mirror what may be encountered in clinical practice. going forward, robust prevalence studies are needed. clinical test performance should also ideally be determined in prospective multicenter studies using a well-defined reference standard as the benchmark for test comparisons. table 2 outlines the type of clinical studies needed to address the most pressing covid-19 diagnostic knowledge gaps. one of the most important problems with current covid-19 diagnostic literature is the lack of a standard definition to define covid-19. the studies included in the systematic reviews that informed this guideline used variable case definitions and many classified disease based in part on the results of the index test under investigation. incorporation of the investigational index test into the diagnostic "gold" standard falsely inflates sensitivity and specificity estimates (i.e. incorporation bias). table 10 outlines options for defining a confirmed covid-19 case in diagnostic trials. it is recognized that not all individuals with covid-19 will have detectable sars-cov-2 nucleic acid. therefore, a "probable" case definition is also proposed. false negative naat results may be due to a variety of factors, including assay limit of detection, anatomic location and adequacy of specimen collection, timing of sampling relative to symptom onset, and underlying biology of disease. to fully understand sars-cov-2 viral dynamics, studies need to be designed to obtain specimens from multiple sites, ideally from the same a c c e p t e d m a n u s c r i p t patient at the same time. in addition, information on the duration of symptoms (if present), assessment of potential exposures and longitudinal follow-up of outcomes will be essential to define optimal diagnostic test strategies across a variety of patient populations. nucleic acid sequencing matches sars-cov-2 reference sequences positive results from at least 2 different naats (one of the two may be the index test) option 3 dual positive results from a single naat targeting 2 different genes (cannot be the index test) compatible clinical signs and symptoms in a setting with known community transmission, negative reference naat and documented sars-cov-2 seroconversion. compatible clinical signs and symptoms in a setting with known community transmission, negative reference naat and positive index test from two different anatomic sites. compatible clinical signs and symptoms in a setting with known community transmission, negative reference naat and positive sars-cov-2-specific serology. the guideline panel used a methodologically rigorous process to critically appraise the available diagnostic literature and formulate sars-cov-2 testing recommendations. the quality of existing evidence, however, was limited and not all of the data used to inform these recommendations had undergone peer-review. based on low certainty evidence, the idsa panel recommends nucleic acid testing for all symptomatic individuals suspected of having covid-19. in addition, testing selected asymptomatic individuals is suggested when the results will have significant impact on isolation/quarantine/ppe usage, dictate eligibility for surgery, or inform use of immunosuppressive therapy. ultimately, institutional resources will dictate test this project was funded by idsa. the following list displays what has been reported to the idsa. to provide thorough transparency, the idsa requires full disclosure of all relationships, regardless of relevancy to the guideline topic. evaluation of such relationships as potential conflicts of interest is determined by a review process which includes assessment by the board of directors liaison to the standards and practice guideline committee and, if necessary, the conflicts of interest (coi) and ethics committee. the assessment of disclosed relationships for possible coi is based on the relative weight of the financial relationship (i.e., monetary amount) and the relevance of the relationship (i.e., the degree to which an association might reasonably be interpreted by an independent observer as related to the topic or recommendation of consideration). the reader of these guidelines should be mindful of this when the list of disclosures is reviewed. k.h. serves as an advisor for biofire and quideland and receives centers for disease control and prevention. health care infection prevention and control faqs for covid-19 prevention strategies for seasonal influenza in health care settings advice on the use of masks in the context of covid-19 epidemic-and pandemic-prone acute respiratory diseases -infection prevention and control in health care day zero, visby, and chroma code; receives research funding from arcbio and hologic; and has served as an advisor for luminex. c.a. receives royalties from uptodate and receives research funding from merck entasis pharmaceuticals and the national institute of allergy and infectious diseases (niaid)/nih. j.e. serves as a consultant for sanofi pasteur; an advisor/consultant for meissa vaccines; and receives research funding from the centers for serves as an advisor for sanofi, seqirus, and medicago; has served as an advisor for pfizer, sunovion, and md brief; and receives research funding from the canadian institutes of health research and the medical research council (united kingdom). r.p. receives grants from shionogi the infectious diseases society of america (idsa), the national board of medical examiners, uptodate, and the infectious disease board review course; y.f.y. receives honoraria for evidence reviews and teaching from the evidence foundation, honoraria for evidence reviews for the american gastroenterological association, and serves as a director for the evidence foundation and for the u.s a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the species severe acute respiratory syndromerelated coronavirus: classifying 2019-ncov and naming it sars-cov-2 novel 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c c e p t e d m a n u s c r i p t key: cord-127025-9ubhd4vf authors: abraham, louis; b'ecigneul, gary; scholkopf, bernhard title: crackovid: optimizing group testing date: 2020-05-13 journal: nan doi: nan sha: doc_id: 127025 cord_uid: 9ubhd4vf we study the problem usually referred to as group testing in the context of covid-19. given $n$ samples taken from patients, how should we select mixtures of samples to be tested, so as to maximize information and minimize the number of tests? we consider both adaptive and non-adaptive strategies, and take a bayesian approach with a prior both for infection of patients and test errors. we start by proposing a mathematically principled objective, grounded in information theory. we then optimize non-adaptive optimization strategies using genetic algorithms, and leverage the mathematical framework of adaptive sub-modularity to obtain theoretical guarantees for the greedy-adaptive method. lacking effective treatments or vaccinations, the most effective way to save lives in an ongoing epidemic is to mitigate and control its spread. this can be done by testing and isolating positive cases early enough to prevent subsequent infections. if done sufficiently regularly and for a sufficiently large fraction of individuals at risk, this has the potential to prevent a large fraction of the infections a positive case would normally cause. however, a number of factors, such as limits on material resources as well as on work force, necessitate economical and efficient use of test resources. group testing 1 aims to improve properties of tests by testing groups of items simultaneously. we wish to leverage this framework to improve covid-19 testing. one needs to differentiate between two different settings: adaptive and non-adaptive. in the former, tests can be decided one at a time, taking into account previous test results. in the latter, one has to select all tests before seeing any lab result and could thus run them in parallel. one can also imagine a semi-adaptive setting in which tests are selected in small batches between each lab evaluation. a simple example of a semi-adaptive group test is to first split n samples into g groups of (roughly) equal size, pool the samples within the groups and perform g tests on the pooled samples. all samples in negatively tested pools are marked as negative, and all samples in positively tested pools are subsequently tested individually. this strategy has recently been validated for covid-19 pcr tests [schmidt et al., 2020] . non-adaptive group testing. most of the existing research on non-adaptive group testing is concerned with identifying at most k positive samples amongst n items, which is referred to as non-adaptive hypergeometric group testing [hwang and sós, 1987] . this assumption yields asymptotic bounds on the number of tests needed to recover the ground truth [knill et al., 1998 , indyk et al., 2010 , cheraghchi et al., 2012 , chan et al., 2014 . however, these are of limited practical relevance when constructive results on small numbers of samples are required. a different problem formulation. we formulate the problem differently: given n people and m testing kits, the characteristics of the test and prior probabilities for each person to be sick, we seek to optimize the way the tests are used by combining several samples. for simplicity, samples are assumed to be independent. [mazumdar, 2016] considers these assumptions as well, for the non-adaptive setting. however, his results are also asymptotic, i.e., valid for large n. adaptive group testing. in the adaptive setting, subsequent test designs may take into account earlier test results. by leveraging the framework of adaptive sub-modularity initially developed for sensor covering by [golovin and krause, 2011] , we prove near-optimality of the greedy-adaptive strategy. our contributions are as follows: • we provide a mathematically grounded objective function to optimize when designing a strategy, leveraging information theory. • we implement different strategies, both non-adaptive and adaptive, which can readily be used in a web browser to know (i) which tests to run and (ii) how to interpret the outcome. • we provide a guarantee of near-optimality of the greedy-adaptive strategy which is based on the mathematical objective we proposed. • software is available at https://louisabraham.github.io/crackovid/crackovid.html. intuition. our mathematical objective is designed such that the mixture tests it proposes to run in the lab will maximize the amount of information we gain on the ground truth once their lab results are revealed − in expectation, over the randomness of both imperfect tests and prior probabilities of infection per individual. notations are progressively introduced throughout, but also all gathered in appendix a. denote the number of patient 2 samples by n, and the number of tests to run by m. tests are assumed to be imperfect, with a true positive rate (or sensitivity) tpr 3 and true negative rate (or specificity) tnr. 4 patient sample i is infected with probability p i ∈ [0, 1] and we assume statistical independence of infection of patient samples. denoting by a '1' a positive result (infection), the unknown ground truth describing who is infected and who is not is a vector of size n made up of '0's and '1's: we call this the secret, denoted as s ∈ {0, 1} n . a design of a test d ∈ {0, 1} n to run in the lab is a subset of patient samples to mix together into the same sample, where d i = 1 if patient sample i is mixed into design d and d i = 0 otherwise. note that the outcome of a perfect design d for a given secret s can simply be obtained as , a test result is positive if there is at least one patient i for which d i = 1 (i is included in the sample) and s i = 1 (i is infected). recall that the secret s is unknown. however, since we assume initially that patient sample i is infected with probability p i and that patient samples are independent, this yields a prior probability distribution over the possible values of s. we hence represent the random value of s as a random variable (r.v.) , denoted by s, with probability distribution p s (s) := pr[s = s] over {0, 1} n . let us now recall the definition of the entropy of our random variable, representing the amount of uncertainty that we have on its outcome, measured in bits. it is maximized when s follows a uniform distribution, and minimized when s constantly outputs the same value. as we perform tests, we gain additional knowledge about s. for instance, if a test pools all samples and returns a negative result, then our posterior probability that all patients are healthy goes up, i.e., p s ((0, . . . , 0)) increases, governed by bayes' rule of probability theory. more generally, we may perform a sequence of tests of varying composition, updating our posterior after each test. our goal will be to select designs of tests so as to minimize entropy, resulting in the least amount of uncertainty about the test outcome for all individuals. note that since tests are imperfect, for a given pool design d ∈ {0, 1} n and a given secret s ∈ {0, 1} n , the boolean outcome t (s, d) of the test in the lab is not deterministic. if tests were perfect, we would have t (s, d) = 1 d,s >0 . to allow for imperfect tests, we model t (s, d) as a r.v. whose distribution is described by pr since the secret s is also unknown (and described by the r.v. s), the outcome t (s, d) has now two sources of randomness: imperfection of tests and unknown secret. 6 in practice, one will not run one test but multiple tests. we now suppose that m tests of pool designs are run and let their designs be represented as a multiset d ∈ ({0, 1} n this leads us to the following question: given an initial prior probability distribution p s over the secret, how should we select pool designs to test in the lab? we want to select it such that once we have its outcome, we have as much information as possible about s, i.e. the entropy (uncertainty) of s has been minimized. since we cannot know in advance the outcome of the tests, we have to minimize this quantity in expectation over the randomness coming from both the imperfects test and unknown secret. this requires the notion of conditional entropy. conditional entropy. given pool designs d, we consider two random variables s (secret) and t := t (s, d) (test results). the conditional entropy of s given t is given by: in this formula, the joint probability pr[s = s, t = t] has been computed with the conditional probability formula pr[s = s, t = t] = pr[s = s] pr[t = t|s = s], and the posterior distribution is computed using bayesian updating, i.e., where it represents the amount of information (measured in bits) needed to describe the outcome of s, given that the result of t is known. mutual information. equivalently, one can define the mutual information between s and t as: it quantifies the amount of information obtained about s by observing t . a well-motivated criterion for test selection. since h(s) does not depend on d, selecting the pool design d minimizing the conditional entropy of s given the outcome of d is equivalent to selecting the one maximizing the mutual information between s and t (s, d). we now have a clear criterion for selecting d: this criterion selects the pool designs d whose outcome will maximize our information about s. expected confidence. we report another evaluation metric of interest called the expected confidence. it is the mean average precision of the maximum likelihood outcome. the maximum likelihood outcome it defined by: which yields the following definition of expected confidence: ml is of particular practical interest: given test results t, a physician wants to make a prediction. in this case, it makes sense to use the maximum likelihood predictor. the interpretation of confidence is straightforward: the probability of the prediction to be true (across all possible secrets). updating the priors. both scoring functions described above compute the expectation relative to the test results of a score on the posterior distribution p s|t =t (s). after observing the test results, we are able to replace the prior distribution p s by the posterior. by the rules of bayesian computation, this update operation is commutative, i.e., the order in which designs d 1 and d 2 are tested does not matter, and compositional in the sense that we can test {d 1 , d 2 } simultaneously with the same results. thus, we can decompose those steps and make different choices as we run tests (see the adaptive method below). (a) compare two outcomes: (1) the posterior is concentrated on two points s ∈ {0, 1} n , taking the values 0.6 and 0.4. (2) it is concentrated on three points, taking the values 0.6, 0.2, 0.2. the entropy is higher, but maybe we still prefer (2) since the margin to the second best explanation is larger? foreword. non-adaptive methods have the benefit over their adaptive counterparts that they can be run in parallel. on the flip side, they describe a strictly more restrictive class of algorithms, since any non-adaptive method is an adaptive one ignoring the information obtained adaptively. moreover, non-asymptotic (i.e., for small values of n) performance guarantees are harder to obtain than for adaptive methods. given numbers n & m, test characteristics tpr & tnr as well as prior probabilities of sample infection p i , the best multiset d of m pool designs is the one maximizing some score, like i(s, t (s, d)) or confidence(s, t (s, d)). the tests are order insensitive, which gives a search space of cardinality 2 n +m m . evaluating the score of every multiset separately takes o (2 n+m ) operations. 8 hence, brute-forcing this search space is prohibitive even for small values of n and m. we resort to randomized algorithms to find a good enough solution. our approach is to use evolution strategies. we apply a variant of the (1 + λ) es with optimal restarts [luby et al., 1993] to optimize any objective function over individuals (multisets of tests). detailed description. we maintain a population of 1 individual between steps. at every step of the es, we mutate it in λ ∈ n + offsprings. in the standard (1 + λ) es, each offspring is mutated from the population, whereas our offsprings are iteratively mutated, each one being the mutation of the previous. these offsprings are added to the population, and the best element of the population is selected as the next generation of the population. we initialize our population with the "zero" individual that doesn't test anybody. our mutation step is straightforward: flipping one bit d i of one pool design d, both chosen uniformly at random. our iterative mutation scheme allows us to step out of local optima. after choosing a basis b proportional to n × m (which is approximately the logarithm of our search space), we apply restarts according to the luby sequence: this sequence of restarts is optimal for las vegas algorithms [luby et al., 1993] , and our es can be viewed as such under two conditions: (i) that the population never be stuck in a local optimum, which can be achieved in our algorithm using λ = n × m (note that much smaller constant values are used in practice); (ii) the second condition is purely conceptual and consists in defining a success as having a score larger than some (unknown) threshold. the fact that our algorithm does not use this threshold as an input yields the following result: theorem 1. under condition (i), the evolutionary strategy using the sequence of restarts illustrated in eq. (10) yields a las vegas algorithm that restarts optimally (as defined by [luby et al., 1993] ) to achieve any target score threshold. future improvements would consist in (i) initializing the population with random pool designs (following randomized testing methods from the literature), (ii) design new mutation rules and (iii) a dynamic restart strategy based on the detection of lack of progress. foreword. adaptive methods have the clear advantage over their non-adaptive counterparts to be more efficient in the number of tests, although they require waiting for the lab results after each sequential test run. although searching the space of all possible adaptive strategies would yield a prohibitive complexity of ω(2 2 m ), it turns out that a simple adaptive strategy can yield provably near-optimal results. detailed description. we describe our adaptive method as algorithm 1 which greedily optimizes the criterion defined in eq. (5). leveraging the framework of adaptive sub-modularity [golovin and krause, 2011] , and the fact that the criterion defined by eq. (5) is adaptive sub-modular and adaptive monotone, algorithm 1 has the guarantee below. we prove it in appendix b.1. algo, the expectation being taken over all 2 m outcomes of lab results. denote by 'optimal' the best (unknown) adaptive strategy. if we run algorithm 1 for m 1 tests and optimal for m 2 tests, we have: where α is defined as follows: assume that our priors p i are wrong, in the sense that there exist constants c, d with cp i ≤ p ′ i ≤ dp i for i ∈ {1, ..., n}, with c ≤ 1 and d ≥ 1, where p ′ i denotes the true prior: we set α := d/c. remarks. theorem 2 states that algorithm 1 is (i) robust to wrong priors and (ii) nearoptimal in the sense that the ratio of its performance with that of the optimal strategy goes to 1 exponentially fast in the ratio of the numbers of tests run in each algorithm. for α = 1 and m 1 = m 2 , this yields 1 − 1/e ≃ 0.63. there exists recent work applying group testing to covid-19: [seifried and ciesek, 2020] report using mini pools of patient samples of size 5 yielding no error in the prediction of healthy patients, over a set of 50 patient samples; [yelin et al., 2020] report the possibility of mixing up to 32 patient samples together, with a false positive rate of 10%; [jeffay, 2020] made a press release about the possibility of reliably testing mixtures including as many as 64 patient samples; [kadri, 2020] mentions simple mathematical methods to approach the problem; [deleforge, 2020] published a blogpost with appealing illustrations vulgarizing the mathematics of group testing. we have discussed some interesting special cases using relatively restrictive assumptions. while theoretical results often require such assumptions, our implementation can in principle be generalized into various directions... we have used the number of tests and samples as given, and then optimized a conditional entropy. however, from a practical point of view, other quantities are relevant and may need to be included in the objective, e.g. the expectation (over a population) of the waiting time before an individual is "cleared" as negative (and can then go to work, visit a nursing home, or perform other actions which may require a confirmation of non-infectiousness). semi-adaptive tests. instead of performing m consecutive tests, one could do them in k batches of respective sizes m 1 , ..., m k satisfying m 1 + ... + m k = m. adaptivity over the sequence of length k could be handled greedily as in algorithm 1, except that instead of selecting a single pool design d * , we would select m i designs at the i th step. we named this semi-adaptive algorithm the k-greedy strategy. pool size optimization. one could analyze the simple setting of [schmidt et al., 2020] , i.e., what is the best pool size given an average prior probability. "best" here could mean something different than in our setting. one might want to take into account the number of tests kits used as well as the number of people which are "cleared" as negative after a given time duration. pool allocation. how do we allocate people to the (first round) pool designs in this setting if prior probabilities are non-uniform? this will naturally arise if we apply group testing as per [schmidt et al., 2020] every day for part of the workforce of a company, in which case a negative result for a person on a given day would imply a low prior on the next day. one could also wonder how to allocate people to the pool designs in the presence of correlations between patient samples. one initial guess could be to place correlated people into the same pool in order to minimize the expected number of what one might call false pool alarms, i.e., cases where a person ends up in a positively tested pool even though they are personally negative. further practical considerations. a good practical strategy could be to perform one round of pooled tests to disjoint groups every morning as individuals arrive at work, being evaluated during work hours. those who are in a positive group (adaptively) get assigned to a second pool design tested later, which can consist of a non-adaptive combination of multiple designs, tested over night. they receive the result in the morning before they go to work, and if individually positive, the enter quarantine. if the test is so sensitive that it detects infections even before individuals become contagious (which may be the case for pcr tests), such a strategy could avoid most infections at work. dependencies between tpr, tnr and pool size. the reliability of tests may vary with pool size. in our notation, the outcome of the tests is a random variable that need not only depend on whether one person is sick (1 d,s >0 ) but it may also depend on the number of tested people |d| and the number of sick people d, s (cf. footnote 5); it could even assign different values of tpr and tnr to different people. the tpr may in practice be an increasing function of the proportion of sick people d, s /|d|. estimating prior infection probabilities. currently, we start with a factorized prior of infection that not only assumes independence between the tested patients but is also oblivious to the individual characteristics. we could, however, build a simple ml system that estimates the prior probabilities based on a set of features such as: job, number of people living in the same household, number of children, location of home, movement or contact data, etc. 9 those prior probabilities can then be readily used by our approach to optimize the pool designs, and the ml system can gradually be improved as we gather more test results. prevalence estimation. similar methods can be applied to the question of estimating prevalence. note that this is an easier problem in the sense that we need not necessarily estimate which individuals are positive, but only how many. ethical considerations. we identify two families of concern to address. the first family concerns the accuracy of the tests. indeed, when the number of tests and patients are equal, it is natural to compare the tpr/tnr of the individual test to the tpr/tnr of the individual results in our grouped test framework (obtained by marginalizing the posterior distribution). in some situations with unbalanced priors, the marginal tpr/tnr of some people in the group could be lower than the test tpr/tnr, even if the test will be more successful overall. however, reporting the marginal individual results gives doctors a tool to decide whether further testing should be needed; hence we cannot rule out that individuals might be worse off by being tested in a group. the second family of concerns, directly resulting from the first, is the responsibility of the doctor when assigning the people in batches and giving them prior probabilities (using another model). the assignment of people in batches should be dealt with in a future extension of our framework, while the sensitivity of our protocol to priors should be studied in more depth. in particular, the adaptive framework is more robust with respect to the choice of priors than the non-adaptive one. • h(p s|t =t ) : f (e(π, φ), φ); • h(s | t ) : f avg := e[f (e(π, φ), φ)]. this allows one to define, following definition 1 of [golovin and krause, 2011] , the conditional expected marginal benefit of a pool design d given results t as: it represents the marginal gain of information obtained, in expectation, by observing the outcome of d at a given stage (this stage being defined by p s , i.e. after having observed test results t). adaptive monotonicity holds if ∆(d) ≥ 0 for any d. adaptive sub-modularity holds if for any two sets of results t and t ′ such that t is a sub-realization 10 of t ′ , for any pool design d: the below lemma concludes the proof. lemma. with respect to ∆ defined in eq. (12), adaptive monotonicity and adaptive sub-modularity both hold. proof. adaptive monotonicity is a consequence of the "information-never-hurts" bound h(x | y ) ≤ h(x) [cover and thomas, 2012] . moreover, as mentioned in lemma 1 of [guestrin et al., 2005] , sub-modularity also follows directly from this bound. in this section, we show how our program (available at https://louisabraham.github.io/crackovid/cra can be used for practical applications. suppose a doctor already made some pooled tests. our program can compute the posterior distribution and report the most probable diagnosis, its confidence as well as the marginal probabilities for each person to be sick. in our numerical example, tpr=0.95, tnr=0.99, we test 3 persons with 3 tests. the i-th test is applied to everybody but the i-th person. we can then enter the results of each test. the input of our program is thus 10 i.e. there exist d and d ′ such that t (s, d) = t, t (s, d ′ ) = t ′ and d ⊂ d ′ . nonadaptive group testing: explicit bounds and novel algorithms graph-constrained group testing elements of information theory the maths of group testing: mixing samples to speed up covid-19 detection adaptive submodularity: theory and applications in active learning and stochastic optimization near-optimal sensor placements in gaussian processes non-adaptive hypergeometric group testing efficiently decodable nonadaptive group testing to ease global virus test bottleneck, israeli scientists suggest pooling samples enhancing the number of lab tests with a 'poisoned wine' approach non-adaptive group testing in the presence of errors optimal speedup of las vegas algorithms nonadaptive group testing with random set of defectives fact -frankfurt adjusted covid-19 testing -a novel method enables high-throughput sars-cov-2 screening without loss of sensitivity pool testing of sars-cov-2 samples increases worldwide test capacities many times over evaluation of covid-19 rt-qpcr test we use upper case letters exclusively for random variables (r.v.), except for mutual information i and entropy h. • n: number of patient samples • m: number of tests to run in the lab 1} n : the secret to unveil, with s i = 1 if and only if patient sample i is positive over possible values of s whose law describes the current information we have about s 1} n : a pool design, with d i = 1 if and only if patient sample i belongs to pool design d 1} n ) m : random multiset describing the pool designs output by the strategy 1} m : lab result of a list of m tests • t : r.v. over possible values of t describing lab results • tpr: true positive rate, sensitivity, hit rate, detection rate, recall • tnr: true negative rate, specificity, correct rejection rate, selectivity a]: probability of event a to happen ) is both adaptive monotone and adaptive sub-modular. direct respective correspondence between our notations and that of • set d of selected designs : set e(π, φ) of selected items by policy π gary bécigneul is funded by the max planck eth center for learning systems. 9 subject to privacy considerations with [results] a binary code representing the test observations. if the results are 000, the program indicates that with very high probability nobody is sick: 11 most probable diagnosis: 000 confidence: 0.999963 marginals: 1.23414e-05 1.23414e-05 1.23414e-05if the results are 011, the program predicts that the first person is sick and the other two are healthy: most probable diagnosis: 100 confidence: 0.973086 marginals: 0.975488 0.00292 0.00292interestingly, we observe error correction when the results are 001 (an impossible outcome with perfect tests) as the program still infers that nobody is sick: most probable diagnosis: 000 confidence: 0.955646 marginals: 0.0221854 0.0221854 6.64093e-05 this mode, given prior probabilities of people to be sick and a number of tests, finds a pooling scheme to optimize the expectation of some score on the posterior probabilities, eg minimize the entropy or maximize the confidence.in fact, the framework we applied above is optimal as suppose that instead of testing 3 people, we want to test 6 people. a possible return value of our randomized algorithm is: as 0.958704 2 = 0.919113 < 0.937214, grouping 6 people together gives much more accurate results than dividing then in two groups of 3 people. key: cord-326148-9wpxm5of authors: van walle, i.; leitmeyer, k.; broberg, e. k.; group, the european covid-19 microbiological laboratories title: meta-analysis of the clinical performance of commercial sars-cov-2 nucleic acid, antigen and antibody tests up to 22 august 2020 date: 2020-09-18 journal: nan doi: 10.1101/2020.09.16.20195917 sha: doc_id: 326148 cord_uid: 9wpxm5of we reviewed the clinical performance of sars-cov-2 nucleic acid, viral antigen and antibody tests based on 94739 test results from 157 published studies and 20205 new test results from 12 eu/eea member states. pooling the results and considering only results with 95% confidence interval width [≤]5%, we found 4 nucleic acid tests, among which 1 point of care test, and 3 antibody tests with a clinical sensitivity [≤]95% for at least one target population (hospitalised, mild or asymptomatic, or unknown). analogously, 9 nucleic acid tests and 25 antibody tests, among which 12 point of care tests, had a clinical specificity of [≤]98%. three antibody tests achieved both thresholds. evidence for nucleic acid and antigen point of care tests remains scarce at present, and sensitivity varied substantially. study heterogeneity was low for 8/14 (57.1%) sensitivity and 68/84 (81.0%) specificity results with confidence interval width [≤]5%, and lower for nucleic acid tests than antibody tests. manufacturer reported clinical performance was significantly higher than independently assessed in 11/32 (34.4%) and 4/34 (11.8%) cases for sensitivity and specificity respectively, indicating a need for improvement in this area. continuous monitoring of clinical performance within more clearly defined target populations is needed. testing is one of the central pillars of public health actions in epidemic and pandemic situations to allow timely identification, contact tracing, and isolation of infectious cases to reduce the spread of infectious diseases. in addition, it allows e.g. estimating disease incidence, disease prevalence, and prevalence and duration of humoral immunity. reliable testing for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and timely reporting of the data to public health authorities is therefore key for management of the coronavirus disease 2019 pandemic. this requires appropriate and accurate diagnostic tests, with sufficient availability, for both identifying individuals that are currently infected with sars-cov-2 as well as those that have been infected in the past. timely access to testing, sufficient supply of testing materials, availability of tests and related reagents and consumables as well as high-throughput testing are pivotal in this context. a large number of commercial tests for sars-cov-2 rna (nucleic acid tests or nats) or viral antigen detection are currently available, as well as serological tests for sars-cov-2 specific antibodies. the various types of tests can be used for different purposes and many of these tests have the ce-ivd certificate that indicates compliance with the european in vitro diagnostics directive (98/79/ec) and can thus be marketed within the eu/eea countries. in addition, the us food and drug administration has granted emergency use authorisations for use of many commercial tests in the us and the world health organisation maintains an emergency use listing of commercial tests. [1, 2] it is however important to note that the ce-marking is based on a self-declaration of the test manufacturer, including the claims on performance of the test. independent information on the clinical performance of these tests in terms of sensitivity and specificity is still limited, and yet this is critical for proper interpretation of results. for this reason, the european centre for disease prevention and control (ecdc) launched a continuous call to eu/eea member states and the uk on 1 april 2020 to provide any such clinical performance data for sharing with other member states. these data, provided by 12 member states, are presented in this paper. in addition, publicly available data were added. finally, minimal performance criteria for different intended uses were gathered from public sources and aided by a survey conducted among eu/eea member states and the uk from 20 may to 1 june 2020. studies containing potentially usable data on clinical performance of sars-cov-2 nucleic acid, antigen and antibody tests were first extracted from systematic reviews on this topic. these reviews were identified through an initial pubmed (medline) search for systematic reviews and meta-analysis for 'covid-19' and 'sars-cov-2', followed by snowballing using the 'find similar articles' feature. the selection was then extended with the studies listed in the foundation for innovative diagnostics (find) database and the european commission (ec) covid-19 in vitro diagnostic devices and test methods database. both databases attempt to exhaustively identify peer-reviewed as well as grey literature on clinical performance of covid-19 tests and are continuously updated. [3, 4] results from the latter were further filtered on those with a description indicating that they contain clinical performance results. results produced by united states food and drug administration were also included. [5] finally, pubmed was searched according to the query in supplementary figure s1 . the resulting studies were subsequently assessed for eligibility. at present there are virtually no clinical performance studies that can be judged as being at low risk of bias and low applicability concerns, and systematic reviews up to this point have not used risk of bias or applicability concerns as exclusion criteria. [6] [7] [8] [9] this was not done in this work either. instead, studies were excluded if they did not contain data on commercial tests, or if one or more of the authors were employed by the developer or manufacturer of the index test, to avoid possible conflicts of interest. studies with an ineligible design, such as blinded tests, analytical validation only, use of another threshold for positivity than in the instructions for use, comparisons between different specimen types or use of an antibody rather than nucleic acid test as reference test for any type of index test were subsequently excluded as well. further exclusions were done at sample level based on the reference test employed. samples classified as actual negatives, i.e. used for determining specificity, had to either (i) be taken before the covid-19 outbreak, in practice before 2020, (ii) be taken from an individual without covid-19 compatible symptoms, or (iii) be taken from an individual with covid-19 compatible symptoms but who was confirmed with another respiratory illness. samples classified as actual negatives that were taken during the outbreak and were negative according to a nucleic acid test were therefore excluded. this was done to maximally reduce misclassification as actual negatives due to known issues with sensitivity of nucleic acid tests. such misclassified samples would artificially lower index test specificity in particular when the index test is more sensitive than the reference test. [10] [11] [12] [13] [14] [15] [16] for the same reason the reported sensitivity of nucleic acid or antigen index tests, based on a nucleic acid reference test, was considered to be a positive agreement instead, calculated as part of a headto-head comparison between the two tests. for antibody index tests on the other hand, a nucleic acid test was considered to be a valid reference test to determine actual positive samples and sensitivity, in accordance with who interim guidelines. [17] manufacturer reported clinical sensitivity and specificity data were extracted from instructions for use where available, or otherwise from the manufacturer's website. sensitivity results derived from contrived samples spiked with purified viral rna were excluded. primary clinical performance data generated by the covid-19 microbiological laboratories group's co-authors were assessed by ecdc according to the same criteria as those of the literature review. meta-analysis of the included clinical sensitivity and specificity results was performed per test and per target, i.e. the genomic region for nucleic acid tests and the antibody isotype for antibody tests. antigen targets were not distinguished further. antibody test sensitivity results below the threshold days after onset were excluded. sensitivity and positive agreement results were further stratified by case population: hospitalised cases, mild or asymptomatic cases or unknown. pooled sensitivity and specificity values were calculated using fixed effects analysis, i.e. separately summing and dividing the number of correct predictions by the total number of samples in the group. wilson 95% confidence intervals were calculated for pooled results. study heterogeneity was assessed through the i 2 statistic, calculated through random effects analysis using r version 4.0.2 and the metafor package. [18] i 2 values <50.0% were considered low heterogeneity, 50.0-74.9% moderate and ≥75% high heterogeneity. as of 1 june 2020, minimum performance criteria for tests were publicly available from belgium, france, the netherlands and the united kingdom (supplementary table s1 ). all were applicable solely to antibody (ab) tests. the intended uses included diagnosis of covid-19, determination of exposure to sars-cov-2 and determination of the immune status against sars-cov-2. minimum clinical sensitivity for all of the specified intended uses ranged from 85 to 98%, with a median of 95%. these thresholds applied to samples collected at least >14 days post onset of symptoms (dpo), taking into account the time to seroconversion. minimum clinical specificity for all of the specified intended uses was 98% in six countries and 98.5% in one. for nucleic acid and antigen confirmatory tests, the draft who target product profiles for priority diagnostics to support response to the covid-19 pandemic state >95% ->98% sensitivity (acceptable/desired) and >99% specificity. [19] general thresholds of >95% sensitivity and >98% specificity were used for further analysis, together with a maximum 95% confidence interval (ci) width of ≤5%. for igm only results, an upper limit of ≤28 dpo, or the highest dpo category having a lower limit ≤28 dpo, was added as well since igm antibodies decrease fairly rapidly and such tests are not intended to be used long after exposure. [20] primary clinical performance data eight systematic reviews were identified, including one by health technology assessment bodies not listed as a peer-reviewed study, and the primary studies included in the analysis extracted. [6] [7] [8] [9] [21] [22] [23] [24] the full list of studies in the find and ec databases was retrieved on 22 august 2020. pubmed was searched on the same date. from the ec database, 268 out 385 studies were screened out since their description did not indicate that they would contain clinical performance data on commercial tests. analogously, 1520 out of 1738 studies were screened out from the pubmed results. from the combined list of 364 studies, 99 had no clinical performance data on commercial tests, 34 were excluded due to a potential conflict of interest and 74 were excluded due to an ineligible design, leaving a total 157 included studies. a complete overview of the study selection is given in figure 1 . after exclusion of antibody test sensitivity results ≤14 dpo and ineligible specificity results, a total of 38202 and 56537 index test results remained for calculation of sensitivity and specificity, respectively. after addition of original, previously unpublished results provided by the authors of this study, this increased to 48310 and 66634 index test results, respectively, for 201 tests (supplementary tables s2-s4) . pooled estimates for clinical sensitivity and specificity per test, target and, for sensitivity, case population were made as described in methods. for antibody tests, results were restricted to those estimates that had a 95% ci width of ≤5% and were derived from at least two studies, to be able to assess study heterogeneity. based on the minimum performance criteria analysis, results above 95% sensitivity and/or 98% specificity for a particular population are highlighted (table 1) . among these results, there were two clias, one elisa and no lfias/pocs, that had 95% sensitivity. analogously, there were nine clias, four elisas and twelve lfias/pocs, that had 98% specificity, among which the three with 95% sensitivity. study heterogeneity was low for 4/10 (40.0%) sensitivity and 53/69 (76.8%) specificity results with ci width of ≤5%. there were few sensitivity results for igg for mild or asymptomatic cases, iga and total antibody, none of which had a ci width of ≤5% (table 1) . compared to the same test used for hospitalised cases, drops in sensitivity were . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.16.20195917 doi: medrxiv preprint observed of 7.4%, 11.0%, 13.1% and 19.2% for igg, 28.8% for iga and -6.0% for total antibody. the latter negative value is likely due to low number of samples for both populations. for nucleic acid tests that were not point of care (poc) tests, results were restricted as for antibody tests (table 2) . three tests had 95% positive agreement with a ci width of ≤5%, and nine had 98% specificity. study heterogeneity was low for all 4 sensitivity and all 15 specificity results with ci width of ≤5%. limited data were available for five poc antigen tests and five poc nucleic acid tests (table 3) . large variability in positive agreement was observed. the best performing nucleic acid poc achieved a positive agreement of 98.9% (97.3-99.6%), i.e. with a ci width of ≤5%. virtually no data were available on specificity, but no false positives were observed among them for either antigen or nucleic acid pocs. the correlation between independently assessed clinical performance results and manufacturer reported results is shown in figure 2 . only independently assessed results with ci width of ≤5% are included. a total of 11/32 (34.4%) of sensitivity and 4/33 (12.1%) specificity results were significantly different (p<0.05). this review represents, to our knowledge, the most complete independent overview so far of clinical performance of commercially available covid-19 tests. a substantial amount of previously unpublished data from european countries are included as well. to date, there are numerous commercial tests for which sufficient performance data are available to allow calculation of clinical sensitivity or positive agreement, and specificity with narrow confidence interval ranges. reassuringly, the clinical performances of several nucleic acid and antibody tests exceeded the minimum performance criteria. as time progresses, the list of tests with sufficient available performance data is expected to increase. at the same time, the available evidence for point of care nucleic acid and antigen tests remains scarce, even though these tests can have substantial practical advantages for e.g. screening. we therefore recommend more emphasis on the validation of these tests, including as part of a testing algorithm, whereby the sensitivity and specificity of taking two tests with a number of days in between is assessed, and which can e.g. be useful to reduce the duration of a quarantine period. the comparison between the independently assessed clinical performance data and manufacturer reported clinical performance revealed that in particular sensitivity is frequently (40.7% of the cases in this study) significantly overestimated by the manufacturer. at a minimum, this emphasises that such independent assessments are clearly necessary. in the longer term, an explicit and proactive regulatory mechanism in europe to compare available independently generated evidence on these tests with manufacturer reported values, coupled with appropriate regulatory action, could be useful. this could also be rewarding towards those manufacturers that do provide robust estimates of their product's performance. limitations of this paper include that most of the included studies have a substantial risk of bias in the sample selection, especially for the sensitivity panel, as established also in the assessments performed in the systematic reviews that were used as a source. results were mainly based on hospitalised cases or poorly defined populations, whereas the population of interest consists often of symptomatic cases in general or even asymptomatic cases, and differences in performance may exist depending on disease severity. while this review addresses a pressing need for actionable . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . clinical performance data, ideally, the clinical performance should be assessed through prospective studies or clinical trials with a guaranteed unbiased sample selection for a clearly defined target population and intended use of the test. given the difficulty of assessing and extracting the data from individual studies in a coherent way, we recommend that the standard for reporting of diagnostic accuracy studies (stard) should also be followed when publishing the results. [25] in this context, the selection of the reference test is particularly important with respect to reference negative samples. as described in some of the assessed studies, it should be avoided that index test results are considered as false positives while the samples are from actual cases and for this reason we excluded nucleic acid negative samples from suspected covid-19 patients altogether. we expect therefore little bias in the specificity results, except potentially from under or overrepresentation of confounders. this is especially relevant for seroprevalence studies, where, in the current lowprevalence situation, in particular the specificity of the test needs to be well-defined and high. on the other hand, sensitivity results using a nucleic acid test as reference should be interpreted with caution, since the positive samples may exclude some actual cases. possibilities to improve the reference test can include testing -potentially only the false positiveswith a second reference nucleic acid test preferably targeting different genes, testing more than one sample from the same patient including for antibodies at a later time point, testing samples from both upper and lower respiratory tracts, and sequencing the sample. the handling of intermediate index test results is an issue that needs to be described in studies, and in general these should be considered as positive results rather than either negatives or being excluded from the validation, since they would normally require further follow-up to confirm the positivity of the sample. for the above reasons, the authors and organisations contributing to this study in no way recommend the use of the listed commercial tests over other not listed commercial or in-house tests. finally, it should be kept in mind that this study is a snapshot in time and that the clinical performance of tests may change over time as the virus population evolves. we therefore recommend a continuous monitoring of clinical performance both in europe and globally, which is key for reliable monitoring of the pandemic and which will also support vaccine and antiviral development. these results should be shared in a timely manner publically is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.16.20195917 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.16.20195917 doi: medrxiv preprint centre for infectious disease control, national institute for public health and the environment, the netherlands), maaike j.c. van den beld (centre for infectious disease control johan reimerink (centre for infectious diseases research, diagnostics and laboratory surveillance ,centre for infectious disease control, national institute for public health and the environment unit of medical microbiology, star-shl medical diagnostic center jeroen bosch hospital, 's-references 1. united states food and drug administration. emergency use authorizations for medical devices world health organisation. coronavirus disease (covid-19) pandemic -emergency use listing procedure (eul) open for in vitro diagnostics joint research centre of the european commission. covid-19 in vitro diagnostic devices and test methods database find evaluation update: sars-cov-2 molecular diagnostics united states food and drug administration openfda dataset on covid-19 serological testing evaluations antibody tests for 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in suspected human cases: interim guidance conducting meta-analyses in r with the metafor package covid-19 target product profiles for priority diagnostics to support response to the covid-19 pandemic v.0.1 of 31 interpreting diagnostic tests for sars-cov-2 testing for sars-cov-2 (covid-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays systematic review with meta-analysis of the accuracy of diagnostic tests for covid-19 meta-analysis of diagnostic performance of serological tests for sars-cov-2 antibodies up to 25 april 2020 and public health implications diagnostic characteristics of serological-based covid-19 testing: a systematic review and meta-analysis stard 2015 guidelines for reporting diagnostic accuracy studies: explanation and elaboration we would like to acknowledge the technicians in the european microbiology laboratories who work hard to support the control of covid-19 and supported the validations described in this manuscript.we would like to acknowledge the companies that made some of the kits available for evaluation to some of the laboratories. key: cord-271920-1dzkgt6w authors: carpenter, christopher r.; mudd, philip; west, colin p.; wilber, erin; wilber, scott t. title: diagnosing covid‐19 in the emergency department: a scoping review of clinical exam, labs, imaging accuracy and biases date: 2020-06-16 journal: acad emerg med doi: 10.1111/acem.14048 sha: doc_id: 271920 cord_uid: 1dzkgt6w in december 2019 a novel viral respiratory pathogen emerged in china, ultimately named severe acute respiratory syndrome coronavirus 2 (sars‐co‐v‐2) with the clinical illness dubbed coronavirus disease (covid‐19). covid‐19 became a global pandemic in early 2020 forcing governments worldwide to enact social isolation policies with dire economic ramifications. emergency departments (ed) encountered decreased patient volumes before some in seattle, new york city, new orleans, and detroit experienced waves of covid‐19 patients mixed with asymptomatic patients or those concerned about potential exposures. diagnosing covid‐19 was hampered by inadequate supplies of reagents and kits, which was compounded by clinical and radiographic features that overlap with numerous seasonal viral respiratory infections. dr. christopher r. carpenter (orcid id : 0000-0002-2603-7157) article type : evidence-based diagnostics in december 2019 a novel viral respiratory pathogen emerged in china, ultimately named severe acute respiratory syndrome coronavirus 2 (sars-co-v-2) with the clinical illness dubbed coronavirus disease . covid-19 became a global pandemic in early 2020 forcing governments worldwide to enact social isolation policies with dire economic ramifications. emergency departments (ed) encountered decreased patient volumes before some in seattle, new york city, new orleans, and detroit experienced waves of covid-19 patients mixed with asymptomatic patients or those concerned about potential exposures. diagnosing was hampered by inadequate supplies of reagents and kits, which was compounded by clinical and radiographic features that overlap with numerous seasonal viral respiratory infections. 1 the united states (us) food and drug administration (fda) issued an emergency use authorization on february 4, 2020 to enable centers for disease control (cdc)-qualified laboratories to perform covid-19 testing. as of june 3, the fda provided emergency use authorization for 85 commercial assays, including polymerase chain reaction tests and immunoglobulin assays. 2 early real-time reverse transcription polymerase chain reaction (rrt-pcr) tests had false-negative (1-sensitivity) rates as high as 40%. 3 as waves of covid-19 patients present to ed's in coming months with symptoms or potential exposures, understanding the diagnostic accuracy and reliability of history, physical exam, routine labs, advanced imaging, and an evolving array of covid-19 diagnostics will be essential knowledge to inform the timing of testing, optimal specimen and test selection, shared decision-making, and ultimately derivation of clinical instruments to guide disposition, follow-up, and shared this article is protected by copyright. all rights reserved decision-making choices ( figure 1 ). 4 this review provides a narrative overview of published research with the primary objective to describe the frequency, causes, and implications of falsenegative rrt-pcr for diagnosis and surveillance. secondary objectives include describing potential diagnostic biases in current rapid cycle covid-19 diagnostic research reports, while providing recommendations for clinicians for interpreting results with knowledge of these design and reporting limitations. a final objective is to add context to rrt-pcr ordering and interpretation by understanding the diagnostic accuracy and additive value of history, physical exam, routine hematology and chemistry tests, computed tomography (ct), and serology for covid-19 immunoglobulins. this is a scoping review that adheres to prisma-scr reporting recommendations. 5 the published literature was searched using strategies created by a medical librarian for covid -19 and diagnostic accuracy. the search was implemented in pubmed 1946-and embase 1947with a date limit from january 1, 2020 until present with an english language limit. the search strategy used a combination of standardized terms and key words, including but not limited to (covid-19 or novel coronavirus or sars-cov-2) and (diagnosis or polymerase chain reaction or serology or crispr-cas or sensitivity/specificity) (appendix). the testing search was based on cheng et al., 6 adding to this prior publication by incorporating clinical exam, imaging, and serology into the synthesis of current diagnostic research. the searches were run on april 23 and may 5, 2020 . one author (crc) reviewed the title and abstracts for all identified citations. other authors reviewed the manuscripts identified and added pertinent references. original research studies describing the frequency of history/physical exam findings or diagnostic accuracy (sensitivity, specificity, likelihood ratios) of history/physical exam, labs or imaging for covid-19 were included. exclusion criteria included non-english, animal research, study protocols, prevention, pathophysiology, lab processing, or policy manuscripts. two authors (crc, sw) abstracted this article is protected by copyright. all rights reserved diagnostic accuracy data and reported adherence to the standards for reporting of diagnostic accuracy (stard) guidelines. 7 compliance with stard was used as a measure of research quality. the same two authors synthesized the results into summary conclusions. a total of 1,907 citations were screened (figure 2) . none of the studies cite or adhere completely to either the standards for reporting of diagnostic accuracy (stard) 7 or the updated reporting framework for history and physical examination. 8 many of these early publications are letters or case reports with uncertain editorial rigor judging by the turnaround time from initial submission to publication. many studies rely upon rrt-pcr as the criterion standard for covid-19, but few contemplate the possibility or likelihood of false-negative or false-positive rrt-pcr results. none of the studies discuss the possibility of various diagnostic biases (spectrum, incorporation, partial verification, differential verification, or imperfect gold standard), nor the potential skew of these biases in observed estimates of sensitivity or specificity. 9 fever is the most commonly reported finding in 84%-87% of covid-19 cases, 10-12 but fever alone does not distinguish this virus from other infections. 13 therefore, absence of fever is inadequate for travel screening and likely for other decision thresholds such as whether ed staff can work shifts. 14 hyposmia (diminished sense of smell) and hypogeusia (diminished taste) have also emerged as covid-19 symptoms. both hyposmia (positive likelihood ratio [lr+] 5.3, negative likelihood ratio [lr-] 0.61) and hypogeusia (lr+ 7.1, lr-0.38) are better to rule-in than to rule-out covid-19, but neither may be fully adequate for either purpose. 15 although multiple covid-19 studies report acute smell or taste disorders as a distinguishing symptom, no other studies report diagnostic accuracy or sufficient details to compute likelihood ratios for hyposmia or hypogeusia. [16] [17] [18] [19] [20] [21] loss of smell is not necessarily associated with nasal obstruction or rhinorrhea. 22 in one case-control study, new onset smell and taste disorders are more common with covid-19 than with influenza (39% vs. 13%). 16 consequently, influenza decision this article is protected by copyright. all rights reserved aids or diagnostic algorithms do not incorporate hyposmia or hypogeusia. 23,24 anosmia, which may be the only complaint in some covid patients, 25 is noted by 47%-73% of covid-19 patients and is the initial symptom in 27%. [18] [19] [20] additionally, 71% recall an acute onset of symptoms associated with taste and smell. 16 anosmia is more common in women and may persist for two weeks. 17 predictive models incorporating a change in taste or smell to distinguish covid-19 from viral mimics appear most sensitive. 26 cough is only present in 58% patients. 10, 12 neither cough, dyspnea, sore throat, nor fatigue distinguish covid-19 from other illnesses, 13 but current studies do not quantify accuracy. 12,27 lymphopenia occurs in over 50% of covid-19 patients. 10, 11, 28 neutrophil to lymphocyte and platelet to lymphocyte ratios do not distinguish elevated ldh is also frequently described. 10, 28, 30, 31 none of these lab findings are commonly utilized in the diagnosis of influenza, but their prevalence and accuracy to distinguish covid-19 from other viral mimics merit further evaluation. 23,24 elevated prothrombin time (pt), ferritin, d-dimer or il-6 are associated with severe covid-19. 31-33 existing studies do not report sensitivity or specificity of these labs. most studies use rrt-pcr as the criterion standard for diagnosing covid-19. this test as used in current assays provides a qualitative detection of nucleic acid from the sars-cov-2 virus. this article is protected by copyright. all rights reserved capacity and time-to-diagnosis in many settings, 34,35 prompting laboratory researchers to explore the concept of specimen pooling in which multiple patients' samples are tested simultaneously with further individual testing only if the pooled specimen is positive. 36 the optimal pool specimen when covid-19 community prevalence is less than 10% is four patients, which improves testing efficiency by 69%. 37 there is limited information on the diagnostic accuracy of the rrt-pcr test. although an increasing number of studies provide head-to-head comparisons, 38-40 systematic reviews provide little quantitative accuracy data and no meta-analysis or assessment of individual study quality. 41 no rrt-pcr test is clearly superior to others in terms of diagnostic accuracy, but some provide faster results and commercial tests may be less sensitive than hospital-developed tests. 40,42 it is known, however, that false negatives are frequent, so current recommendations advise incorporating patient's exposure risk, clinical signs and symptoms, routine lab and imaging findings, serology, and (when available) ct results into real-time determination of covid-19 status. repeat or even serial rrt-pcr testing is required to confidently exclude covid-19. multiple studies report initially negative rrt-pcr results becoming positive with subsequent rrt-pcr tests in the following days or weeks. 43,44 others report hospitalized covid-19 patients with initially positive rrt-pcr tests becoming negative prior to discharge with subsequent readmission for positive tests in the ensuing days. 45 ren et al. noted rrt-pcr sensitivity with one test was 78% and increased to 86% with a second test. 46 a strategy of three negative rrt-pcr results is superior to two negative rrt-pcr followed by bronchoalveolar lavage. 47 repeating initially negative rrt-pcr up to five times increases sensitivity to 98%. 48 covid-19 patients identified on first rrt-pcr often have more severe disease associated with higher mortality, likely due to higher quantities of virus in those individuals. 48 older patients are more likely to remain rrt-pcr positive for an extended period, but whether this means they are contagious has yet to be determined. 44 potential reasons for false negative rrt-pcr results are summarized in table 1 . 49-51 emergency physicians will rely upon the rrt-pcr assay selected by their hospital laboratory, which may this article is protected by copyright. all rights reserved balance the limit of detection and sensitivity against turn-around-time, complexity, cost, workflow, availability of reagents and kits, specimen type, and lab personnel risk handling those specimens. 52 patients under investigation for covid-19 who ultimately rule-out are rarely reported in currently available studies, so specificity and false positives are generally not reported in the literature. however, false positives appear rare. 53 in cdc testing, there was no significant cross-reactivity with other common respiratory viruses or seasonal coronaviruses. 54 contamination of the specimen or reagents used in the rrt-pcr is therefore the main mechanism for false positives results. the cdc recommends protocols to prevent and detect potential contamination in order to minimize false positive results. 49, 54 nasopharyngeal (np) samples are most commonly obtained and studied, but oropharyngeal (op), saliva, sputum, stool, blood, and/or urine specimens can also be evaluated. obtaining np samples requires time and appropriate training, increases exposure to staff secondary to coughing and gagging, and is uncomfortable for patients. methodologically, few rrt-pcr accuracy studies describe how research or clinical staff were trained to collect np specimens, so fidelity and reproducibility remain in question. 55 wang et al provide videos describing np and op collection methods, note poor agreement between the two sampling methods (kappa = 0.31), and a higher yield with np. 56 saliva can be collected outside the hospital without training, perhaps as part of a telemedicine evaluation for covid-19. 57,58 one small italian study indicated that saliva specimens demonstrate detectable sars-co-v-2 virus and the limit of detection is not affected by patient age. 59 sputum samples exhibit higher viral load than op sites. 60-62 however, as already noted many patients under investigation lack a cough and fewer still have sputum production. expectoration of sputum may also expose health care workers collecting the sample to aerosols that would not have been generated without a sample collection attempt. furthermore, a bayesian analysis of prevalence-dependent positive and negative predictive values by ghosal and sinha demonstrates that even when the covid-19 prevalence is high (54%) the positive predictive value (ppv) of sputum rrt-pcr is only 95.7% and the negative predictive value (npv) is 52%. 63 ppv and npv vary with disease prevalence. specifically, ppv increases with higher disease prevalence and npv increases with lower disease accepted article prevalence making extrapolation to clinical populations challenging if the study prevalence does not match the patients to whom the test is applied. 64 for this reason, diagnosticians prefer likelihood ratios. 65 blood and urine are inadequate specimens for rrt-pcr as most patients do not exhibit virus in these body fluid compartments. 66 in addition to the cdc developed rrt-pcr test, manufacturers have developed molecular tests that target different portions of the sars-cov-2 viral genome and run on rapid testing platforms. for example, reverse transcription loop mediated isothermal amplification (rt-lamp) can detect sars-cov-2 within 30 minutes. [67] [68] [69] other laboratories are exploring highthroughput sequencing as for inconclusive fluorescence quantitative polymerase chain reaction specimens as a rapid mediator for the presence or absence of sars-cov-2. 70 the diagnostic accuracy of these tests is similarly not reported, but these tests have not shown cross-reactivity to other respiratory viruses and bacteria. on may 8, 2020, the us fda issued an eua for a sars-cov-2 antigen test. 71 this test detects sars-co-v or savs-cov-2 nucleocapsid protein antigens in np or nasal specimens using a lateral flow immunofluorescent sandwich assay. 71 this assay is run on a point-of-care device in laboratories that are able to perform high, moderate, or waived complexity tests, and can provide tests within minutes. 72 while the diagnostic accuracy of this test is not available at this time, the fda reports high specificity but sensitivity that is less than rrt-pcr. the fda and the manufacturer recommend negative results "be treated as presumptive and confirmed with a molecular assay, if necessary for patient management". 73 chest x-ray chest x-ray is essential to evaluate for covid-19 mimics such as pneumonia, pleural effusion, or pulmonary edema. typical covid-19 findings include hazy opacities that are often bilateral and peripheral. 74 with the exception of one outlier, the reported sensitivity of single view chest xray for covid-19 ranges from 33% to 60%. 75 this article is protected by copyright. all rights reserved accuracy of chest x-ray early in the covid-19 pandemic. 80 currently available covid-19 chest x-ray studies do not report specificity or reliability. 75 computed tomography (ct) findings suggesting covid-19 include ground glass opacity (often bilateral) and peripheral predominant lesions without mediastinal adenopathy or pleural effusions, though these findings represent non-specific manifestations of acute lung injury associated with numerous infectious and non-infectious etiologies. 81, 82 incidental findings consistent with covid-19 are observed on ct of the chest in patients without respiratory symptoms. 83 multiple studies report covid-19 identified by ct after one or more negative rrt-pcr tests. [84] [85] [86] [87] when the covid-19 epidemic erupted in china, clinicians lacked access to rrt-pcr kits and then as rrt-pcr became available, low rrt-pcr sensitivity reinforced belief in the additive value of ct for many. 88 these observations and scenarios prompted some to advocate for ct as a first-line supplement to the diagnostic evaluation for covid-19, combining rrt-pcr with ct. 89, 90 if ct alone or in combination with rrt-pcr reduced false-negative rates, the positive public health implications for case identification and control of disease transmission could be substantial. however, these benefits must be balanced against the cost of ct, medical radiation dangers, or practical limitations in busy hospitals with hourly trauma and stroke arrivals and potentially time-dependent emergencies juxtaposed against advised ct shutdowns for covid-19 cleaning requiring 30 or more minutes. 91, 92 this cleaning time would also delay access to ct for every patient in the ed, thereby prolonging potential exposure to those in the ed to other patients with covid-19. 92 some propose covid-19 patients wear n-95 masks and plastic bags over their heads to eliminate or reduce these cleaning times. 27 pragmatically, among those detected by ct no defined benefit, such as reduced mortality or faster resolution of covid-19 symptoms has been described. 93 in addition, radiologists' sensitivity for diagnosing covid-19 by ct findings ranges from 72% to 94% with specificity from 24% to 100%. 94 preliminary artificial intelligence studies report radiologists' sensitivity improves from 79% to 88% and specificity this article is protected by copyright. all rights reserved from 88% to 91% with this artificial intelligence image augmentation, 95 while others hypothesize that the most valuable role for this technology may be quantify the proportion of lung affected by covid-19. 96 despite these issues, multiple studies highlight that the sensitivity of ct is substantially higher than that of the first rrt-pcr, while combining ct and rrt-pcr provides maximal sensitivity (~97%). 89, 97, 98 theoretically, the sensitivity of ct would decline when testing populations outside of an epidemic (low prevalence rates), while specificity would be reduced when mimics like influenza are more common. 9, 27 the british society of thoracic imaging recommends against ct when rrt-pcr is positive, but to consider ct when the initial rrt-pcr is negative in order to identify co-existing disease or potential covid-19 complications such as pulmonary embolism. 99 tavare et al. developed a single-center protocol to prioritize inpatient ct decision-making for initial covid-19 negative rrt-pcr patients based upon initial clinical suspicion and chest x-ray findings. 100 the us fda has also issued an eua for the development of sars-cov-2 antibody tests. these antibody tests detect circulating igm, igg or both that are reactive against sars-cov-2 virus using lateral flow assays (lfa) or enzyme-linked immunosorbent assay (elisa). 2 however, unlike rrt-pcr tests, there is data regarding the diagnostic accuracy of these serologic tests. whitman and colleagues evaluated 10 lfa and 2 elisa tests for sars-cov2 antibodies. 101 they used plasma or serum samples from patients with symptomatic, rrt-pcr-confirmed positive patients as the gold standard for disease, and pre-covid-19 specimens from the american red cross as negative controls. sensitivity of both igm and igg varied by days since symptoms onset, with sensitivities for either igm or igg at >20 days ranging from 82% to 100%. specificity for either igm or igg also varied by test, ranging from 87% to 100%. 101 similarly, benavid and colleagues used a commercially available lfa test to perform a seroprevalence study in santa clara county, california and reported a sensitivity of 80% and a specificity of 99.5%. 102 true positive serologic tests for sars-cov-2 antibodies indicate prior infection with sars-cov2 and the development of an immune response. this may be helpful in identifying those who this article is protected by copyright. all rights reserved were asymptomatic or minimally symptomatic at the time of infection, as well as those who were unable to receive a molecular test when symptomatic. while some experts believe that the presence of igm or igg reactive against sars-cov-2 will confer immunity, 103 this has not yet been shown. 104 if the presence of antibodies on a true positive serologic test does confer immunity, the titer of antibodies required to confer immunity remains unknown, as does the duration of that immunity. false positive results may be due to cross-reactivity with other coronavirus strains which cause the common cold. the fda recommends the following information be included in the instructions for use and patient test reports: knowledge of the diagnostic characteristics, including sensitivity, specificity, and likelihood ratios of tests for sars-cov2, the virus that causes covid-19, is important to understand how to best apply these tests for patient care and disease surveillance. because this novel virus emerged as a significant pathogen in humans only a few months ago, diagnostic tests have been developed rapidly under fda euas in the us. consequently, we have less information about the diagnostic accuracy of these tests than we would under normal circumstances, but we do know that both false negatives and false positives may occur. an illustration of the false positive and false negative rate as a function of prevalence for two serologic tests for sars-cov-2 is provided in figure 3 . false negative tests commonly occur with rrt-pcr tests for several reasons (table 1 ). there are a number of potential implications of a false negative rrt-pcr test for sars-cov-2. from the this article is protected by copyright. all rights reserved patient's standpoint, a patient with a negative test may lead to an assumption that they are not infected and subsequently diminished adherence to instructions to isolate and take other infection control measures, increasing the risk of infecting others. in the hospital setting, precautions may be relaxed in the presence of a negative test, increasing the risk of transmission to healthcare workers and other patients. in patients with moderate or high pretest probability of disease, a negative test may not reduce the posttest probability of disease below a level where precautions to prevent spread of disease become less necessary. in patients with a low pretest probability of disease, the likelihood of disease given a negative test will be low. however, even low individual likelihoods of disease can cumulatively contribute to substantial risk of outbreaks across larger groups for more contagious infectious diseases, such as covid-19. false negative tests are also a consideration with serological testing. however, since these tests should generally not be used to assess an active infection, the risks of a false negative are less significant for disease transmission. a false negative serological test would incorrectly classify a person as not having an immune response to sars-cov-2. if "immunity passports" became a reality, this could incorrectly and adversely affect a person's ability to travel or work. 104 the increased sensitivity of ct for covid-19 might provide a net public health benefit if falsenegative rrt-pcr patients with higher clinical suspicion were accurately identified during the initial ed evaluation. mathematical models provide a theoretical basis for the concept that increasing diagnostic efficiencies (for example, by improving sensitivity with addition of ct) will decrease the risk of covid-19 transmission. 106 pending the availability of rapid, reliable, and sufficiently accurate covid-19 tests in ed settings, the identify-isolate-inform (3i) approach to decrease spread might be improved with more liberal ct use. 107 one italian hospital reported liberal ct screening for respiratory patients with possible false-negative chest x-ray results, but thus far has not reported on the positive or negative impact of that approach on individual patient care or public health. 108 in the early stages of covid-19, as many as 50% of patients this article is protected by copyright. all rights reserved with respiratory symptoms may have normal imaging. 96 radiologists have also noted that the quality of early ct accuracy studies is questionable because the rrt-pcr assay used as the criterion standard is either not described or the accuracy of that standard undefined. 82 in addition, ct findings are not pathognomonic for covid-19 as influenza, cytomegalovirus, and atypical pneumonia have similar findings. 96, 109 as a consequence of these ct limitations in addition to the costs, radiation exposure, and downstream effect on other patients in terms of diagnostic delays and cross-contamination, multiple groups, including the fleischner society and the british society of thoracic imaging discourage ct as a routine screening approach. 99, 110 nonetheless, ct likely plays a role when rrt-pcr tests are either too inaccurate, unavailable or suffer unacceptably slow turnaround times in patients with higher levels of covid-19 concern based on exposure history or other clinical findings. 74 the public health benefits of a more liberal ct screening approach from the ed merit additional research. false positive tests associated with rrt-pcr for sars-cov2 are believed to be rare and would most commonly occur due to contamination. false positive tests may occur more commonly with serological tests, which have reported specificities ranging from 87-100%. 101 the positive predictive value is a function of both test sensitivity and specificity as well as the pretest probability of disease. this implies that positive test results are more likely to represent false positive results when the pretest probability of disease is low. 111 the instability of positive predictive value is especially important as we apply imperfect diagnostic tests to low risk patient populations, such as asymptomatic patients in low prevalence communities. as an example, the antibody test used in a california community study has a reported sensitivity of this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved society with a false positive serologic test for sars-cov2 antibodies. patients may assume that they have developed immunity to covid-19, leading to a reduction in risk-mitigating activities such as physical distancing. healthcare workers with false positive tests may similarly reduce their vigilance and use of precautions due to an incorrect assumption that they have immunity. this could place these individuals and their close contacts at increased risk of contracting covid-19. multiple forms of diagnostic bias exist and each skew measured estimates of sensitivity and specificity in different directions. 112 incorporation bias is possible when the criterion standard includes the index test (for example, rrt-pcr) in ultimately determining whether the disease is present or absent. incorporation bias increases measured sensitivity and specificity. this is pertinent to covid-19 because most early studies incorporate rrt-pcr into the criterion standard. 9,113 differential verification bias is possible when patients with a positive or concerning index test (e.g., ct findings associated with covid-19) are more likely to receive an immediate invasive gold standard such as repeat rrt-pcr testing or bronchoalveolar lavage specimens. 82, 97 differential verification bias raises specificity in diseases that resolve spontaneously or lowers specificity for diseases that only become detectable during follow-up. 9 imperfect gold standard bias is possible when the standard used to classify the presence or absence of disease misclassifies some patients. imperfect gold standard bias raises observed specificity if errors on the index test and "copper standard" are correlated with true disease status and lowers observed specificity if errors on the index test and the copper standard are independent. 9 this is pertinent to covid-19 because no well-accepted criterion standard yet exists. a better criterion standard for covid-19 will certainly emerge and we propose some ideas in table 2 . 114, 115 temporal bias reflects variation in observed accuracy based on the period of time or stage of disease when index testing occurred. 116 in covid-19 viral shedding is highest in the early stages of disease with the highest positive rates noted within the first week. 117, 118 spectrum bias is possible when the spectrum of disease severity differs between the study and clinical application (example, critically ill covid-19 patients in the intensive care unit versus this article is protected by copyright. all rights reserved asymptomatic patients evaluated in ambulatory clinics). spectrum bias skews observed sensitivity upwards in sicker populations and skews specificity upward in healthier patients. 9, 119 spectrum bias is worth considering when applying diagnostic accuracy results from patients with varying severity of illness to dissimilar populations. for example, among covid-19 patients from cruise ships evaluated with ct, those with symptoms more commonly had covid-19 ct findings than those without symptoms (80% vs. 40%). 120 the rapidly expanding evidence base around covid-19 diagnostic accuracy for clinical exam, routine labs, imaging, and advanced testing provides important lessons moving forward for clinicians, researchers, and journal reviewers. covid-19 researchers need to contemplate myriad biases carefully in reporting observed diagnostic accuracy. if a bias is likely and the anticipated skew in observed sensitivity or specificity is upwards and the observed accuracy is already too low, further studies of that diagnostic test may not be warranted. the stard reporting guidelines provide manuscript protocols to ensure adequate description of methods and results so that diagnostic biases are easier to identify. 7, 8 unfortunately, none of the early covid-19 diagnostic research cites stard or adheres to these reporting standards, which is not uncommon in emergency medicine. 121, 122 clinical decision aids consist of three or more findings on history, physical exam, routine labs, or imaging that, in combination, more accurately identify patients at lower or higher risk of a disease or outcome. diagnostic and prognostic decision aids are commonly developed and employed in emergency medicine to reduce practice variability without compromising patient outcomes. 123 efforts to develop covid-19 decision aids might include something like the pulmonary embolism rule-out criteria (perc) rule to identify subsets of ed patients at lower risk of covid-19 pending definitive testing. 124 alternatively, a decision aid might serve prognostic purposes to identify covid-19 patients more likely to decompensate in response to the viral infection. [125] [126] [127] [128] when decision aid investigators attempt to derive and validate these this article is protected by copyright. all rights reserved instruments, higher quality emergency medicine research quantifying accuracy and reliability (or the elements of history, physical exam, labs, and imaging that become predictor variables of the decision aid) will be required as the basis for selecting variables likely to improve model performance. most laboratory tests are quantitative rather than qualitative, including covid-19 rrt-pcr and serological assays. when sensitivity and specificity are reported, the quantitative labs have been dichotomized at some level. another approach to evaluating diagnostic accuracy for quantitative data is interval likelihood ratios (ilr). 129 one advantage of ilr's is that indeterminant results are more readily interpreted. as covid-19 diagnosticians identify the rrt-pcr and serological tests that best balance availability, accuracy, reliability, and cost, reporting ilr's could provide added value for clinicians. ultimately, ed physicians' clinical impressions concerning the presence or absence of covid-19 are communicated to patients and families -usually without access to definitive testing. patient communication tools to convey the basics of covid-19 personal protection and infection prevention exist, 130,131 but shared decision-making instruments that communicate the uncertainties of clinical exam, imaging, and even rrt-pcr do not exist. figure 4 provides one example of a cates plot that could be used to communicate the accuracy limitations of rrt-pcr based on current evidence. actual shared decision-making instruments will require scientific investigation using accepted methodology before widespread implementation. 132 this scoping review has several limitations. the pace of publications around covid-19 and diagnostics in the first half of 2020 has been astonishing. at best, this review will serve as a snapshot in time, although hopefully illuminating issues that require higher methodological standards and peer-review attention moving forward. due to time constraints the search strategy was limited to english language and published research. more research undoubtedly exists in the gray literature. since earlier systematic reviews exploring aspects of covid-19 this article is protected by copyright. all rights reserved diagnostic testing did not identify or report additional measures of sensitivity, specificity, or likelihood ratios for hyposmia, hypogeusia, or rrt-pcr, we are confident that this search presents a complete scoping review of current knowledge. 10, 133 others have also noted the absence of diagnostic accuracy reporting amidst the flurry of covid-19 publications. 134, 135 additionally, this scoping review does not focus on special emergency medicine populations such as pediatrics, geriatrics, or obstetrics because other reviews already exist for these patients. 136, 137 most importantly, we report no formal assessment of study quality using accepted instruments such as the quadas-2, 138 although informal assessment of published research to date suggests limited adherence to the full set of recommended methodological standards for studies of diagnostic test performance. clinicians should be aware of the current limited knowledge around history, physical exam, labs, and imaging for covid-19. fever and acute onset disorders of taste and/or smell are the most common findings on history and physical exam associated with covid-19. lymphopenia is associated with covid-19 diagnosis, while elevated ldh and pt are associated with severe disease. rrt-pcr has emerged as the primary diagnostic test for suspected covid-19, but access has been limited, diagnostic accuracy is under-reported, and between-assay comparative accuracy is rarely evaluated. however, typical testing algorithms and diagnostic accuracy studies rely heavily on rrt-pcr results with frequent false negatives. chest ct is indicated for equivocal cases or when considering diagnoses like pulmonary embolism, but is not recommended as a general screening protocol. in cases with high clinical suspicions, repeat rrt-pcr testing with or without ct scanning may be beneficial to reduce community spread. antigen tests have only recently been approved, and diagnostic accuracy information is similarly limited. serology may identify past covid-19 exposure, but the role of antibody testing and implications for ed decision-making remain undefined. current clinical, imaging, and laboratory studies neglect diagnostic accuracy reporting standards and likely suffer from various biases. differential diagnosis for coronavirus disease (covid-19): beyond radiologic features. ajr american journal of roentgenology. 2020:w1. 2. fda: emergency use authorization (eua) information, and list of all current euas food and drug administration covid-19 testing: the threat of false-negative results the critical role of laboratory 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disease-2019: is fever an adequate screening for the returning travelers? tropical medicine and health utility of hyposmia and hypogeusia for the diagnosis of covid-19 acute-onset smell and taste disorders in the context of covid-19: a pilot multicenter pcr-based case-control study anosmia as a prominent symptom of covid-19 infection anosmia reporting tool: initial findings. otolaryngol head neck surg objective evaluation of anosmia and ageusia in covid-19 patients: single-center experience on 72 cases viral load of sars-cov-2 in clinical samples. the lancet infectious diseases quantitative detection and viral load analysis of sars-cov-2 in infected patients. clinical infectious diseases : an official publication of the infectious diseases society of america rapid sputum testing and not thermal screening alone should be the first-line screening test at airports: a bayesian analysis. diabetes and metabolic syndrome evidence-based emergency medicine/editorial. the problem with 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another advantage for the evidence-based diagnostician stopping the spread of masks and coronavirus disease state of the science: tools and measurement for shared decision making covid-19 diagnosis and management: a comprehensive review incorporating test characteristics into sars-cov-2 testing policy-sense and sensitivity. jama health forum web site all rights reserved 135. bachelet vc. do we know the diagnostic properties of the tests used in covid-19? a rapid review of recently published literature a systematic scoping review of covid-19 during pregnancy and childbirth covid-19 in older adults: key points for emergency department providers. geriatric emergency department collaborative quadas-2: a revised tool for the quality assessment of diagnostic accuracy studies accepted article covid-19 covid-19 diagnostic testing covid-19 serotherapy covid-19" or epidem*[tiab] or epidemic* or epidemy or new[tiab] or "novel"[tiab] or "outbreak" or pandem* or "sars-cov-2" or "shanghai" or "wuhan") and ("coronavirus infections sensitivity and specificity"[mesh] or "point-of-care testing or "next generation sequencing"[tiab] or "point-of-care test"[tiab] or "point of care tests 2019-ncov' or '2019ncov' or 'cov 2' or 'covid-19' or 'sars coronavirus 2' or 'sars cov 2' or 'sars-cov-2' or 'severe acute respiratory accepted article this article is protected by copyright. all rights reserved syndrome coronavirus 2' or 'coronavirus 2' or 'covid 19' or 'covid-19' or '2019 ncov' or '2019ncov' or 'corona virus disease 2019' or 'cov2' or 'covid-19' or 'covid19' or 'ncov 2019' or 'ncov' or 'new corona virus' or 'new coronaviruses' or 'novel corona virus' or 'novel coronaviruses' or 'sars coronavirus 2' or 'sars2' or 'sars-cov-2' or 'severe acute respiratory syndrome coronavirus 2'):ti,ab,kw or ((19 or 2019 or '2019-ncov' or 'beijing' or 'china' or 'covid-19' or epidem* or epidemic* or epidemy or new or 'novel' or 'outbreak' or pandem* or 'sars-cov-2' or 'shanghai' or 'wuhan'):ti,ab,kw and polymerase chain reaction'/exp or 'reverse transcription polymerase chain reaction'/exp or 'high throughput sequencing'/exp or 'sensitivity and specificity'/exp or 'point of care testing'/exp or 'antigen'/exp or 'serology'/exp or 'immunoglobulin g'/exp or 'immunoglobulin m'/exp or 'clustered regularly interspaced short palindromic repeat'/exp or 'crispr cas system'/exp or 'differential diagnosis'/exp or pcr:ti,ab,kw,de or 'digital droplet':ti,ab,kw,de or 'next generation sequencing':ti,ab,kw,de or 'point-of-care test':ti,ab,kw,de or 'point of care tests':ti,ab,kw,de or antigen:ti,ab,kw,de or analyte:ti,ab,kw,de or serology key: cord-034948-w59wxu8i authors: kuriyama, akira; jackson, jeffrey l.; kamei, jun title: performance of the cuff leak test in adults in predicting post-extubation airway complications: a systematic review and meta-analysis date: 2020-11-07 journal: crit care doi: 10.1186/s13054-020-03358-8 sha: doc_id: 34948 cord_uid: w59wxu8i background: clinical practice guidelines recommend performing a cuff leak test in mechanically ventilated adults who meet extubation criteria to screen those at high risk for post-extubation stridor. previous systematic reviews demonstrated excellent specificity of the cuff leak test but disagreed with respect to sensitivity. we conducted a systematic review and meta-analysis to assess the diagnostic accuracy of the cuff leak test for predicting post-extubation airway complications in intubated adult patients in critical care settings. methods: we searched medline, embase, scopus, isi web of science, the cochrane library for eligible studies from inception to march 16, 2020, without language restrictions. we included studies that examined the diagnostic accuracy of cuff leak test if post-extubation airway obstruction after extubation or reintubation was explicitly reported as the reference standard. two authors in duplicate and independently assessed the risk of bias using the quality assessment for diagnostic accuracy studies-2 tool. we pooled sensitivities and specificities using generalized linear mixed model approach to bivariate random-effects meta-analysis. our primary outcomes were post-extubation airway obstruction and reintubation. results: we included 28 studies involving 4493 extubations. three studies were at low risk for all quadas-2 risk of bias domains. the pooled sensitivity and specificity of cuff leak test for post-extubation airway obstruction were 0.62 (95% ci 0.49–0.73; i(2) = 81.6%) and 0.87 (95% ci 0.82–0.90; i(2) = 97.8%), respectively. the pooled sensitivity and specificity of the cuff leak test for reintubation were 0.66 (95% ci 0.46–0.81; i(2) = 48.9%) and 0.88 (95% ci 0.83–0.92; i(2) = 87.4%), respectively. subgroup analyses suggested that the type of cuff leak test and length of intubation might be the cause of statistical heterogeneity of sensitivity and specificity, respectively, for post-extubation airway obstruction. conclusions: the cuff leak test has excellent specificity but moderate sensitivity for post-extubation airway obstruction. the high specificity suggests that clinicians should consider intervening in patients with a positive test, but the low sensitivity suggests that patients still need to be closely monitored post-extubation. morbidity, duration of mechanical ventilation, and icu stay [2] [3] [4] [5] [6] [7] [8] . in select cases, systemic corticosteroids before extubation can be used to prevent post-extubation airway complications [9] . therefore, it is important to estimate the risk of laryngeal edema before extubation. since the endotracheal tube precludes direct visualization of the upper airway, the cuff leak test was proposed to predict the presence of laryngeal edema and post-extubation airway obstruction [10, 11] . theoretically, when there is no laryngeal edema, there is an air leak around the tube after deflating the balloon cuff of the endotracheal tube [12, 13] . in contrast, a failed cuff leak test suggests little or no air leak around the tube, suggesting potential airway obstruction from laryngeal edema [12, 13] . the clinical practice guideline published by the american thoracic society and american college of chest physicians in 2017 recommends performing a cuff leak test in mechanically ventilated adults who meet extubation criteria to screen those at high risk for post-extubation stridor [14] . this guideline referenced two systematic reviews on the diagnostic accuracy of cuff leak test [15, 16] , published in 2009 and 2011. both reviews demonstrated excellent specificity of the cuff leak test but disagreed with respect to sensitivity. in addition, there have been several studies of the diagnostic accuracy of cuff leak test published after these reviews were completed. consequently, we conducted a systematic review and meta-analysis to assess the diagnostic accuracy of the cuff leak test for predicting post-extubation airway obstruction and subsequent reintubation. the conduct and reporting of this systematic review followed the prisma-dta statement [17] . our review protocol was registered at prospero (crd42018084357). we searched medline, embase, scopus, isi web of science, and the cochrane library for eligible studies from inception to march 16, 2020 , without language restrictions [18] . our search strategy was developed with the help of a medical librarian (additional file 3: table s1 ). we hand-searched the references of included articles for potentially relevant studies. we examined the diagnostic accuracy of cuff leak test in intubated adult patients awaiting extubation in critical care settings. the index test was cuff leak test regardless of the type of cuff leak test (quantitative or qualitative) and threshold used. the reference standards included post-extubation airway obstruction determined by the original authors and subsequent reintubation. we included observational studies (cross-sectional and cohort studies) that examined the diagnostic accuracy of cuff leak test in critical care settings if: (1) the data were extractable into a 2 × 2 table from the reported data, (2) post-extubation airway obstruction after extubation or reintubation was explicitly reported as the reference standard. we considered both published studies and conference proceedings; however, we included the abstracts from conference proceedings only when they provided data in enough detail to be extractable. we considered interventional studies in critical care settings that examined the efficacy of systemic corticosteroids to prevent post-extubation airway complications; however, we excluded patients to whom systemic corticosteroids were administered after they were judged at high risk of postextubation airway complications. two authors (ak and jk) independently screened titles and abstracts obtained from the search and selected potentially relevant articles. disagreement was resolved through discussion. the first author (ak) and one of the other authors (jlj and jk) in duplicate and independently extracted the following data from each study: (1) patient demographics (age, sex); (2) study characteristics (country, study population; duration of mechanical ventilation; mode of ventilation; observation period after extubation); (3) the type of cuff leak test (quantitative or qualitative); (4) numbers of true-positive, false-positive, true-negative, and falsenegative; and (5) the reference standards used. quantitative cuff leak test measures the air leak volume with a cuff deflated and judges the post-extubation airway obstruction based on its absolute volume or proportion in comparison with the expiratory tidal volume against a certain threshold [19] . qualitative cuff leak test examines the presence or absence of audible expired air around an endotracheal tube, which indicates the pass or failure of the test [19] . in this study, a lack of a cuff leak, having a risk of post-extubation complications, was considered a positive test, while having a leak, suggesting low risk of post-extubation complications was a negative test [19] . two authors (ak and jlj) independently assessed the risk of bias using the quality assessment for diagnostic accuracy studies-2 (quadas-2) tool [20] . inconsistency was resolved through consensus. we had two primary outcomes: (1) post-extubation airway obstruction and (2) reintubation due to post-extubation airway obstruction. the reference standards for post-extubation airway obstruction included stridor (audible high-pitched inspiratory wheeze) [21] , or laryngeal edema defined by the study authors (including confirmation with bronchoscopy [22] or laryngoscope [23] ). we pooled the data using a generalized linear mixed model approach to bivariate random-effects meta-analysis to calculate summary estimates of sensitivity, specificity, and likelihood ratios as well as the associated 95% confidence intervals (cis) [24] . we pooled prevalence using a random-effects model, with exact binomial estimates of standard deviation and the freeman-tukey transformation for zero cells [25] . to examine the sources of heterogeneity, we examined the receiver operating characteristic (roc) curves, assessed the correlation between sensitivity and specificity, and analyzed whether sensitivity and specificity of cuff leak changed with the type of cuff leak test (qualitative or quantitative), the proportion of women, inclusion or exclusion of reintubated patients in a study, and the length of intubation, using subgroup or meta-regression analysis [14, 19] . we also calculated the sensitivity and specificity of cuff leak test using a cutoff of 110 ml, a value that is frequently used in clinical practice [21] . we tested for publication bias using deeks' method [26] . we created a fagan's nomogram, which determines the posttest probability according to the pretest probability and the calculated positive and negative likelihood ratios [27] . we followed standard diagnostic meta-analytic approaches in focusing on the sensitivity, specificity, and likelihood ratios instead of positive and negative predictive values because predictive values are dependent on the population prevalence of post-extubation complications, which can vary considerably. the threshold of statistical significance was set at p < 0.05. all analyses were performed with stata se, version 15.1 (stata corp; college station, tx). our literature search produced 2236 studies. after application of inclusion and exclusion criteria, 28 studies involving 4493 extubations were included in the analysis ( fig. 1) [12, 13, [21] [22] [23] . among the 28 included studies, 27 were published in english and one in korean [35] . twenty-seven studies (96%) were prospective [12, 13, 21-23, 28-37, 39-50] . nine studies and ten were conducted in medical [21, 22, 31, 32, 35-37, 43, 49] and mixed intensive care units [12, 13, 23, 28, 33, 34, 40, 41, 44, 45] , respectively ( table 1 ). the included studies were published between 1992 and 2019. all but one were published studies and the remaining one an abstract in a conference proceeding. eight studies were conducted in the usa, four each in france and taiwan, three in thailand, two in egypt, one each in australia, belgium, india, iran, italy, south korea, and turkey. the median sample size was 101, ranging 34-543 (interquartile range 51-236). the reported mean/median duration of mechanical ventilation ranged from 2 to 28.1 days. five studies used a qualitative cuff leak test (auscultation of airflow) [23, 28, 31, 39, 45] , and 21 used a quantitative measurement of the cuff leak [12, 13, 21, 22, 29, 30, 32-37, 40-44, 46, 47, 49, 50] (table 2 ). one study reported the results from both qualitative and quantitative cuff leak tests examined in a single cohort [48] . the remaining study reported on the data of patients who underwent either qualitative or quantitative cuff leak test [38] . the most frequent cutoff values for quantitative cuff leak tests ranged from 50 to 283 ml (median, 110 ml) in volume and from 10 to 57% in proportion. twenty studies used assist control ventilation [12, 13, 21, 22, 29, 30, 32-38, 41-44, 46, 47, 49] , and four used spontaneous breathing including pressure support ventilation [23, 31, 40, 45] . another study examined either of these two ventilation modes [28] . one study applied the ambu bag, while the cuff leak was tested [39] . one study performed a qualitative cuff leak test during spontaneous breathing via t-tube and during cough while still intubated [48] . the remaining one did not report the mode of ventilation used [50] . twenty-four studies used stridor [12, 13, 21, 28-39, 41-43, 45-50] and three used direct visualization of the airway following extubation as reference standards [22, 23, 40] for airway obstruction. one study used either of these reference standards [44] . three of the 28 studies were at low risk of bias for all quadas-2 risk of bias domains (table 3) . seventeen studies (60.7%) were deemed at low risk of bias for the domain of patient selection. eight out of 22 studies that assessed quantitative cuff leak prespecified the cutoff of cuff leak; fourteen studies (50%) were deemed to have adequately assessed the index test. a reference standard was adequately assessed in 18 studies (64.3%). study participants were adequately followed up in 19 studies (67.9%). the prevalence of post-extubation airway obstruction ranged from 4 to 37% (pooled estimate 9%; 95% ci 7-11; i 2 = 86.3%). the pooled sensitivity and specificity of cuff leak test for post-extubation airway obstruction were 0.62 (95% ci 0.49-0.73; i 2 = 81.6%) and 0.87 (95% ci 0.82-0.90; i 2 = 97.8%), respectively. the forest plots are shown in additional file 1: figure s1 . the pooled positive and negative likelihood ratios were 4.63 (95% ci 3.44-6.22) and 0.44 (95% ci 0.32-0.60), respectively. the area under the summary receiver operating characteristic (sroc) curve was 0.85 (95% ci 0.82-0.88) (fig. 2) , and the pooled diagnostic odds ratio (dor) was 10.54 (95% ci 6.26-17.76) (additional file 4: table s2 ). there was no evidence of publication bias (p = 0.189). subgroup analysis suggested that the specificity was similar between the qualitative and quantitative cuff leak tests ( a nomogram based on the pretest probability of 9% (the incidence of stridor in the studies included in our study) is provided (fig. 3) . the prevalence of reintubation varied from 0 to 11% (pooled estimate: 3%; 95% ci 1-5; i 2 = 79%). the pooled sensitivity and specificity of the cuff leak test for reintubation were 0.66 (95% ci 0.46-0.81; i 2 = 48.9%) and 0.88 (95% ci 0.83-0.92; i 2 = 87.4%), respectively. the forest plots of the sensitivity and specificity of the cuff leak table s2 ). there was no evidence of publication bias (p = 0.52). our study found that the cuff leak test has excellent specificity but moderate sensitivity for post-extubation airway obstruction. the cuff leak test thus works better to rule in than to rule out potential post-extubation airway obstruction. however, the false-negative rate of nr not reported 38% suggests that the cuff leak test may fail to identify some patients with post-extubation airway obstruction. our study found that the specificity of the cuff leak test for post-extubation airway obstruction was excellent, which is consistent with two previous systematic reviews [15, 16] . in contrast, ochoa et al. and zhou et al. concluded that the sensitivity of cuff leak testing for postextubation airway obstruction was 56% and 80%, respectively [15, 16] : our pooled sensitivity was 62%, which fell between those two findings. we included nearly double the number of studies that their reviews did. furthermore, the additional studies we included were higher quality, potentially making our findings more reliable. our analysis found that the qualitative cuff leak test had low sensitivity (35%) in predicting post-extubation airway obstruction. this has been consistently found in recent studies. the most likely explanation is the subjective nature of this test. in addition, since schnell et al. provided data from three different methods of qualitative cuff leak testing [48] , the sensitivity of which were all around 30%; this study may have been overweighed, although repeat analysis limiting schnell's study to a single data contribution did not change the sensitivity of the qualitative test. in contrast, the specificity of both qualitative and quantitative cuff leak tests was high, nearly 90%, while there was a statistically significant difference between two methods, clinically both performed equally well. a cutoff of 110 ml also had a low sensitivity (44%) and high specificity for predicting post-extubation airway obstruction. we thus conclude that the cuff leak test has high specificity and can be used to select patients to consider treating with systemic corticosteroids, but its low sensitivity suggests that the traditional practice of closely observing all patients in the immediate post-extubation period should be continued. consistent with these findings, the nomogram suggested that while a negative cuff leak test represents low possibility of post-extubation airway obstruction, a positive test still provides a relatively low posttest probability. the guideline by ats/accp provided a conditional recommendation regarding cuff leak test [14] , because failing the cuff leak test might lead a delay in extubation and an increase in complications such as barotrauma and ventilator-associated pneumonia. the guideline weakly recommended that the cuff leak test be reserved for highrisk patients, who experienced a traumatic intubation, were intubated more than 6 days, have a large endotracheal tube, are female, or were reintubated after an unplanned extubation [14] . our analysis found that the length of intubation had a small impact on the specificity of cuff leak test. female sex and reintubation had no impact of the accuracy of cuff leak test. since the original studies included in our review examined non-selected patients with respect to the risk of post-extubation airway obstruction and the sensitivity of cuff leak test is moderate, we support the idea of the ats/accp guideline to reserve cuff leak test for high-risk patients. our study suggested that the cuff leak test has moderate sensitivity and excellent specificity for reintubation. although the sensitivity in our study was similar to those of previous meta-analyses, the specificity in our study was slightly higher [15, 16] . the area under the sroc curve and dor were also greater than previously reported [16] . thus, a failed cuff leak test may serve as a good marker for those at risk of reintubation, if postextubation airway obstruction is not treated adequately. the limitation of cuff leak test has been repeatedly discussed. cuff leak test can be susceptible to relationship of tube size to laryngeal diameter [41] , respiratory system compliance and resistance, inspiratory flow, expiatory flow and time, and airway collapse [51] , and clinicians should bear in mind that the ability of cuff leak test may vary according to the condition or type of patients [52] . additionally, coughing during cuff deflation test hinders accurate measurement of the leak volume and lowers the reproducibility. a previous physiological study suggested that while patients were sedated and paralyzed, the cuff leak volume was reliably measurable [53] . an adequate amount of sedatives and opioids can suppress coughing during the airway suctioning before cuff leak test or cuff deflation during the test. further, cuff leak testing is recommended several hours before extubation, which allows the arousal of patients from sedation by the time of extubation. thus, we may be able to at least attempt to increase the reliability of cuff leak measurement. few tests are available to estimate the risk of postextubation airway complications. a case series of three patients suggested that video laryngoscopy enabled visualization of laryngeal edema prior to extubation [54] , but its clinical efficacy in estimating post-extubation airway complications is yet to be determined. several studies have examined the role of laryngeal ultrasonography in adult patients. laryngeal air column width difference is the difference between width of airway at the level of the vocal cord with cuff inflated and deflated. its reported sensitivity and specificity varied across studies, ranging from 50 to 91% and 54 to 72%, respectively [44, 46, 49] . laryngeal air column width ratio is the ratio of air column width before extubation over that after intubation. it has been examined in only one study [55] and needs further validations. thus, no single available options can correctly estimate the risk of post-extubation airway complications. clinicians should not overly rely on one single test in predicting the success or failure of extubation. our study had several strengths and limitations. strengths included a comprehensive search in five databases without language restrictions. this allowed us to conduct relevant subgroup analyses with a larger number of studies. further, inclusion of non-english studies facilitates the generalizability of our findings in various clinical settings [56] . our study had some limitations. first, the definition of post-extubation airway obstruction differed across studies. stridor was more frequently used as the reference standards than laryngeal edema (as assessed with endoscopy). laryngeal edema may be more frequent than stridor, because stridor and respiratory distress occur when laryngeal edema narrows the airway by ≥ 50% [57] . however, laryngeal edema is not always screened for in extubated patients, and the presence of stridor is an accepted sign of respiratory distress that triggers a concern for airway obstruction. thus, the finding of our study is generalizable to the clinical practice. second, although we attempted to include in the analysis the incidence of stridor due to post-extubation airway events, some patients might have had a concurrent clinical state that necessitated high minute ventilation or tachypnea through an edematous airway, which manifested as 'stridor. ' therefore, we might not have been able to completely separate stridor due to post-extubation airway events from stridor due to other etiologies, such as respiratory insufficiency. this limitation also applies to reintubation. third, whether to reintubate patients is subject to treating physicians' discretion and the effect of treatment to abort post-extubation stridor. therefore, the value of cuff leak test in predicting the need for reintubation in clinical practice may be limited along with the third limitation. however, prevention of post-extubation airway obstruction is more important than reintubation per se. once the cuff leak test identifies patients at high risk of postextubation airway obstruction, prophylactic systemic corticosteroids are indicated [9, 14, 58] . fourth, 15 out of 23 studies that assessed the quantitative cuff leak test determined the cutoff with the knowledge of the results of the reference standards. it is known that data-driven optimization of the cutoff can lead to overestimation of test performance [59] . thus, the pooled accuracy of quantitative cuff leak test in our study can be an overestimation; the optimal cutoff is still unknown. fifth, the quantitative cutoff for a positive test varied between the studies. since we had aggregate data from each included study, we failed to determine the optimal cutoff of cuff leak test. finally, there was substantial statistical heterogeneity in the pooled sensitivity and specificity for both outcomes. the presence of statistical heterogeneity is a common issue intrinsic to the the prediction region illustrates the extent of statistical heterogeneity by depicting a region within there is 95% confidence that the true sensitivity and specificity of a future study should lie meta-analysis of diagnostic accuracy of a test, given the clinical and methodological diversity in original studies as well as the possible relationship between sensitivity and specificity, as exemplified in roc curves in which more sensitive cut points have lower specificity (and vice versa). our subgroup analyses suggested that the type of cuff leak test (quantitative versus qualitative) and length of intubation might have been the cause of statistical heterogeneity of sensitivity and specificity, respectively, for post-extubation airway obstruction. the cuff leak test has excellent specificity but moderate sensitivity for post-extubation airway obstruction. the cuff leak test is a useful tool in the decision-making about extubation, but the low sensitivity suggests that a negative test cannot completely exclude post-extubation airway obstruction and that patients still need to be closely monitored post-extubation. the higher specificity suggests that clinicians should consider intervening in patients with systemic corticosteroids in response to a positive test. continued research to find better modalities to rule out post-extubation airway obstruction is needed. postextubation laryngeal edema and 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intravenous injection of methylprednisolone reduces the incidence of postextubation stridor in intensive care unit patients risk factors of extubation failure and analysis of cuff leak test as a predictor for postextubation stridor dexamethasone to prevent postextubation airway obstruction in adults: a prospective, randomized, double-blind, placebo-controlled study the role of the cuff leak test in predicting the effects of corticosteroid treatment on postextubation stridor the cuff leak test is not predictive of successful extubation risk factors evaluation and the cuff leak test as predictors for postextubation stridor cuff-leak test predicts the severity of postextubation acute laryngeal lesions: a preliminary study intra-individual variation of the cuff-leak test as a predictor of post-extubation stridor cuff leak volume as a clinical predictor for identifying post-extubation stridor cuff leak tests at the time of extubation correlate with voice quality assessment predicting laryngeal edema in intubated patients by portable intensive care unit ultrasound cuff leak test and laryngeal survey for predicting post-extubation stridor ultrasound-guided laryngeal air column width difference and the cuff leak volume in predicting the effectiveness of steroid therapy on postextubation stridor in adult. are they useful? laryngeal ultrasound versus cuff leak test in prediction of post-extubation stridor cuff leak test for the diagnosis of post-extubation stridor laryngeal ultrasonography versus cuff leak test in predicting postextubation stridor clinical risk, cuff leak test and laryngeal ultrasound based scoring system to predict postextubation stridor in intensive care unit patients determinants of the cuff-leak test: a physiological study the cuff-leak test: what are we measuring? is the leak test reproducible? use of video laryngoscopy and camera phones to communicate progression of laryngeal edema in assessing for extubation: a case series laryngeal air column width ratio in predicting post extubation stridor how often do systematic reviews exclude articles not published in english? tracheoesophageal compression associated with substernal goitre. correlation of symptoms with cross-sectional imaging findings adverse events associated with prophylactic corticosteroid use before extubation: a cohort study bias in sensitivity and specificity caused by data-driven selection of optimal cutoff values: mechanisms, magnitude, and solutions publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to sincerely thank ms. elizabeth suelzer, mlis, ahip for her help with literature search. supplementary information accompanies this paper at https ://doi. org/10.1186/s1305 4-020-03358 -8.additional file 1: figure s1 . forest plot of the sensitivity and specificity of the cuff leak test for predicting post-extubation upper airway obstruction.additional file 2: figure s2 . forest plot of the sensitivity and specificity of the cuff leak test for predicting reintubation.additional file 3: table s1 . search strategy. table s2 . the pooled diagnostic accuracy of cuff leak test for post-extubation airway obstruction and reintubation. authors' contributions ak and jlj substantially contributed to conception of the study design, data acquisition, data analysis, interpretation, and the writing and critical revision of the manuscript. jk substantially contributed to data acquisition, interpretation, and critical revision of the manuscript. all authors read and approved the submission of the final manuscript. there was no funding source for this study. all data generated or analyzed during this study are included in this published article and its supplementary information files. this study is a systematic review and an ethics approval was not necessary. not applicable. the authors declare that they have no competing interests. received: 27 july 2020 accepted: 23 october 2020 key: cord-202376-440zapcw authors: wilder, bryan; mina, michael j.; tambe, milind title: tracking disease outbreaks from sparse data with bayesian inference date: 2020-09-12 journal: nan doi: nan sha: doc_id: 202376 cord_uid: 440zapcw the covid-19 pandemic provides new motivation for a classic problem in epidemiology: estimating the empirical rate of transmission during an outbreak (formally, the time-varying reproduction number) from case counts. while standard methods exist, they work best at coarse-grained national or state scales with abundant data, and struggle to accommodate the partial observability and sparse data common at finer scales (e.g., individual schools or towns). for example, case counts may be sparse when only a small fraction of infections are caught by a testing program. or, whether an infected individual tests positive may depend on the kind of test and the point in time when they are tested. we propose a bayesian framework which accommodates partial observability in a principled manner. our model places a gaussian process prior over the unknown reproduction number at each time step and models observations sampled from the distribution of a specific testing program. for example, our framework can accommodate a variety of kinds of tests (viral rna, antibody, antigen, etc.) and sampling schemes (e.g., longitudinal or cross-sectional screening). inference in this framework is complicated by the presence of tens or hundreds of thousands of discrete latent variables. to address this challenge, we propose an efficient stochastic variational inference method which relies on a novel gradient estimator for the variational objective. experimental results for an example motivated by covid-19 show that our method produces an accurate and well-calibrated posterior, while standard methods for estimating the reproduction number can fail badly. a key goal for public health is effective surveillance and tracking of infectious disease outbreaks. this work is motivated in particular by the covid-19 pandemic but the methods we describe are applicable to other diseases as well. a central question is to estimate the empirical rate of transmission over time, often formalized via the reproduction number r t , t = 1...t . r t describes the expected number of secondary infections caused by someone infected at time t. accurate estimates of r t are critical to detect emerging outbreaks, forecast future cases, and measure the impact of interventions imposed to limit spread. r t is typically estimated using daily case counts, i.e., the number of new infections detected via testing each day. standard methods, including prominent dashboards devel-oped for covid-19, provide accurate estimates under idealized conditions for the observation of cases and have been successfully used at a national or state level where many observations are available and sampling variation averages out flaxman et al. 2020; systrom, vladek, and krieger 2020) . however, successful reopening will require programs which track spread at the level of particular colleges, workplaces, or towns, where partial observability poses several challenges. first, only a small number of infected people may be tested. it is estimated that only about 10% of sars-cov-2 infections in the us result in a confirmed test (havers et al. 2020 ) and we could expect even fewer in populations with a high prevalence of asymptomatic or mild infections (e.g., college students). second, the biological properties of the test play an important role. for example, a pcr test which detects viral rna will show positive results at different times than an antibody or antigen test. further, there can be substantial heterogeneity across individuals. third, testing programs may collect samples in a particular way which impacts the observations. for example, one suggestion for schools and workplaces to reopen is to institute regular surveillance testing of a fraction of the population in order to detect outbreaks and catch asymptomatic carriers (larremore et al. 2020) . the observations will depend on the fraction of the population enrolled in testing (potentially small due to budget constraints) along with the sampling design (e.g., cross-sectional vs longitudinal). this paper presents gprt, a novel bayesian approach to estimating r t which accounts for partial observability in a flexible and principled manner (illustrated in figure 1 ). this method yields well-calibrated probabilistic estimates (the posterior distribution). our model places a gaussian process (gp) prior over r t , allowing it to be an arbitrary smooth function. then, we explicitly model the sampling process which generates the observations from the true trajectory of infections. while this substantially improves accuracy (as we show experimentally) it creates a much more difficult inference problem than has been previously considered. specifically, our model contains tens or hundreds of thousands of discrete latent variables, preventing the application of out-of-the-box methods. moreover, the values of many variables are tightly correlated in the posterior distribution, further complicating inference. to make inference computationally tractable, we propose a novel stochastic figure 1 : illustration of our gprt method. top row: the generative model gprt posits for the observed data. bottom row: the inference process to recover a posterior over r t . variational inference method, enabled by a custom stochastic gradient estimator for the variational objective. extensive experiments show that our method recovers an accurate and well-calibrated posterior distribution in challenging situations where previous methods fail. there is a substantial body of work which attempts to infer unknowns in a disease outbreak. a frequent target for inference is the basic reproduction number r 0 (majumder and mandl 2020; riou and althaus 2020; wilder et al. 2020) ; by contrast, we attempt the more challenging task of estimating a reproduction number which can vary arbitrarily over time. there is a literature of both classic methods for estimating r t (wallinga and teunis 2004; cori et al. 2013; campbell et al. 2019 ) and newer methods developed for the covid-19 pandemic and implemented in popular dashboards systrom, vladek, and krieger 2020) . none of these methods incorporate partial observability and we empirically demonstrate that gprt improves substantially over methods in each category. another strand of literature develops maximum likelihood or particle filter estimates of the parameters of an epidemiological model (king et al. 2008; dureau, kalogeropoulos, and baguelin 2013; cazelles, champagne, and dureau 2018) . however, their work focuses on accommodating a complex model of the underlying disease dynamics; by contrast, we develop methods for probabilistically well-grounded inferences under a complex observation model. there is also a great deal of computational work more broadly concerned with disease control. examples include optimization problems related to vaccination or quarantine decisions (saha et al. 2015; zhang and prakash 2014; zhang et al. 2015) , machine learning methods for forecasting (without recovering a probabilistic view of r t ) (chakraborty et al. 2014; rekatsinas et al. 2015) , and agent-based simulations of disease dynamics (barrett, eubank, and marathe 2008) . our work complements this literature by allowing inference of a distribution over r t from noisy data, which can serve as the input that parameterizes an optimization problem or simulation. we now introduce a model for a disease process with a time-varying reproduction number. subsequently, we introduce example models for how the observations are generated from the disease process which our framework can support. we use a standard stochastic model of disease transmission similar to other r t estimation methods (wallinga and teunis 2004; cori et al. 2013; campbell et al. 2019 ); our contribution is a more powerful inference methodology which can accommodate complex observation models alongside a gp prior. let r = [r 1 ...r t ] be the vector with r t for each time. from r, the disease model defines a distribution over a vector n = [n 1 ...n t ] with the number of people newly infected each day. the main idea is that, over the course of a given individual's infection, they cause a poisson-distributed number of new infections with mean determined by r. specifically, if on day t individual i has been infected for h i days the expected number of new infections caused by i that day is here w h i gives the level of infectiousness of an individual h i days post-infection. w is normalized so that h w h = 1. for later convenience, we will define φ t = n i=1 w h i to be the total infectiousness in the population before scaling by r t (n is the total population size). each day, each infected individual i draws n i t ∼ poisson(λ i t ) other individuals to infect. we also incorporate infections from outside the population, with a mean of γ such infections per day. we assume the rate of external infection is constant with respect to our time but our model could be extended to a time-varying γ. we treat γ mostly as a nuisance parameter: our true objective is to infer r, but doing so requires accounting for the potential that some detected cases are due to infections from outside the population. we define n t = n i=1 n i t + poisson(γ) to be the total number of new infections. since φ is fixed given the time series n, we denote it as a function φ(n). we denote the probabilistic disease model induced by a specific choice of r and γ as m (r, γ) and the draw of a time series of infections from the model as n ∼ m (r, γ). we now depart from the standard disease model used in previous work and describe a wide-ranging set of examples for how our framework can accommodate models of the process which generates the observed data from the latent (unknown) true infections. previous work assumes either perfect observability or else the simplest of the three observation models we describe below (uniform undersampling). our focus is where observations are generated by a medical test which confirms the presence of the pathogen of interest. individuals have some probability of being tested at different times (depending on the testing policy adopted) and then test positive with a probability which depends on the biological characteristics of the disease and the test in question. the kind of test employed determines when an individual is likely to test positive during the course of infection. for example, for covid-19, pcr tests are commonly used to detect sars-cov-2 rna. they are highly sensitive and can detect early infections. most infected individuals become pcr-negative within the week or two following infection as viral rna is cleared (kucirka et al. 2020) . by contrast, serological tests detect the antibodies produced after infection. an individual is not likely to test serologically positive until a week or more postinfection, but may then continue to test serologically positive for months afterwards (iyer et al. 2020) . the observable data generated by a serological testing program is likely quite different than a pcr testing program since the time-frame and variance of when individuals test positive differs strongly. a range of other examples are possible (e.g., antigen tests) and can be easily incorporated into our framework. our model adopts a generic representation of a particular test as a distribution d over t convert , the number of days post-infection when an individual begins to test positive and t revert , the number of days post-infection when they cease to test positive. for an infected individual i, we write t i convert , t i revert ∼ d. our method only assumes the ability to sample from d, meaning that we can directly plug in the results of lab studies assessing the properties of a test. t i convert , t i revert are unobserved: we only get to see if an individual tests positive at a given point in time, not the full range of times that they would have tested positive. next, we describe a series of example models for how and when individuals are tested, which reveal observations depending on the status (t i convert , t i revert ) for each person who is tested. for convenience, we let t i convert = ∞ for an individual who is never infected. note however that we can model false negatives by having d sometimes set t i convert = ∞ for an infected individual, or false positives by returning finite t i convert for an uninfected individual. we denote the number of observed positive tests on day t as x t . an observation model is a distribution over x given n, denoted x ∼ obs(n). each setting below describes one such distribution. uniform undersampling in this setting, each individual who is infected is tested independently with some probability p test (e.g., if they individually decide whether to seek a test). to model this process, we introduce two new sets of latent random variables. first, a binary variable z i ∼ bernoulli(p test ), indicating whether individual i is tested. second, a delay c i , giving the number of days between t i convert and when individual i is tested. we can integrate out the z i and obtain the following conditional distribution for the observed number of positive tests x t : denotes the indicator function of an event. however, we cannot analytically integrate out t convert and c. cross-sectional testing here, a uniformly random sample of s t individuals are tested on each day t. this models a random screening program (e.g., testing random employees each day as they enter a workplace). in this case, we have this expression provides the likelihood of x after conditioning on the latent variables t i convert , t i revert , though there is no closed-form expression conditioning only on n. longitudinal testing in this setting, a single sample from the population is chosen up front and every individual in the sample is tested every d days. we again denote the total number of individual tested on day t as s t , but note that now the group of individuals who are tested repeats every d days. longitudinal testing offers different (and potentially more revealing) information than cross-sectional testing since when an individual first tests positive, we know that they did not test positive d days ago. however, it complicates inference by introducing correlations between the test results at different time steps. let a t denote the set of individuals who are tested at time t. we assume that the complete sample d t=1 a t is chosen uniformly at random from the population, with the chosen individuals then randomly partitioned between the d days. we have where t i convert > t−d captures that i was not positive on their previous test. this introduces correlations between x t and x t−d , so there is not a simple closed-form expression for the distribution of the time series x even after conditioning on t i convert and t i revert . (as there is in the cross-sectional case). we will instead build a flexible framework for inference which can just as well use a kind of sample of the log-likelihood. we will place a gaussian process (gp) prior over r, resulting in the following generative model: where gp(1, k) denotes a gaussian process with constant mean 1 and kernel k and exp(γ) is an exponential prior on γ with meanγ. given the observation x, our goal is to compute the resulting posterior distribution over r and γ. however, is complicated by the fact that x is determined by a large number of discrete latent variables, primarily n (the time series of infections) and {t i convert , t i revert } n i=1 , the times when each individual tests positive. a typical strategy for inference in complex bayesian models is markov chain monte carlo (mcmc). however, mcmc is difficult to apply because of tight correlations between the values of variables over time: due to the gp prior, we expect values of r to be closely correlated between timesteps, and successive values of n are highly correlated via the model m . formulating good proposal distributions for high-dimensional, tightly correlated random variables is notoriously difficult and has presented problems for gp inference via mcmc in other domains (titsias, lawrence, and rattray 2008) . the other main approach to bayesian inference is variational inference, where we attempt to find the best approximation to the posterior distribution within some restricted family. modern variational inference methods, typically intended for deep models such as variational autoencoders (kingma and welling 2013) , use a combination of autodifferentiation frameworks and the reparameterization trick to differentiate through the variational objective (kucukelbir et al. 2017 ). this process is highly effective for models with only continuous latent variables. however, our model has many thousands of discrete latent variables which cannot be reparameterized in a differentiable manner. typical solutions to this problem would be to either integrate out the discrete variables or to replace them with a continuous relaxation (jang, gu, and poole 2017; vahdat et al. 2018) . neither solution is attractive in our case -the structure of the model does not allow us to integrate out the discrete variables analytically, while a continuous relaxation is infeasible because our latent variables have a strict integer interpretation (every infection requires in a particular individual becoming testpositive at particular points in time). the last resort to differentiate through discrete probabilistic models is the score function estimator (paisley, blei, and jordan 2012) , which is often difficult to apply due to high variance. gprt uses a combination of techniques which exploit the structure of infectious disease models to develop an estimator with controlled variance. first, we develop a more tractable variational lower bound which is amenable to stochastic optimization. second, we hybridize the reparameterization and score function estimators across different parts of the generative model to take advantage of the properties of each component. third, we develop techniques to sample low-variance estimates of the log-likelihood for each of the observation models introduced earlier. these techniques are introduced in the next section. we now derive gprt, a novel variational inference method for r t estimation. gprt approximates the true (uncomputable) posterior over (r, γ) via a multivariate normal distribution with mean µ and covariance matrix σ. µ t is the posterior mean for r t while σ t,t gives the posterior covariance between r t and r t . µ γ is the mean for γ and σ γ,· gives its covariance with r. the diagonal σ t,t gives the variance of the posterior over r at each time, capturing the overall level of uncertainty. the aim is to find a µ and σ which closely approximate the true posterior. let q(r, γ|µ, σ) denote the variational distribution. let p be the true generative distribution, where p(r, γ, x) is the joint distribution over x and (r, γ), p(r, γ) is the prior over (r, γ), and p(r, γ|x) is the posterior over (r, γ) after conditioning on x. the aim of variational inference is to maximize a lower bound on the total log-probability of the evidence x: where the right-hand side is referred to as the evidence lower bound (elbo). our goal is to maximize the elbo via gradient ascent on the parameters µ and σ. this requires us to develop an estimator for the gradient of each term in the elbo. the first term is the negative cross-entropy between q and the prior p(r, γ). because both q and p have simple parametric forms, this can be easily computed and differentiated. the last term is the entropy, which is similarly tractable. the middle term is the expected log-likelihood. developing an estimator for the gradient of this term is substantially more complicated and will be our focus. in fact, for computational tractability we will actually develop an estimator for a lower bound on the expected log-likelihood; substituting this lower bound into the elbo still gives a valid lower bound on log p(x) and so is a sensible objective. the essential problem is that computing the log-likelihood of r requires integrating out the discrete latent variable n induced by the disease spread model, which is computationally intractable. the aim of this section is to develop the following stochastic estimator: theorem 1. let l be the cholesky factor of σ, ξ ∼ n (0, i), there exists a function g(µ, σ) with e r,γ∼q [log p(x|r, γ)] ≥ g(µ, σ) ∀µ, σ and e[∇] = ∇g(µ, σ). essentially, theorem 1 states that∇ is an unbiased estimator for a lower bound on the expected log-likelihood, exactly what we need to optimize a lower bound on log p(x) by stochastic gradient methods. moreoever, as we will highlight below, we can efficiently compute the terms of∇ via a combination of leveraging the structure of the disease model to apply autodifferentiation tools and novel sampling methods for the observation model. we now derive∇. proof. expanding the dependence of x on n we can rewrite the log-likelihood as it is not clear how to develop a well-behaved gradient estimator for this expression because we wish to differentiate with respect to the parameters governing two nested expectations, one within the log. however, via jensen's inequality, we can derive the lower bound pushing the log inside the expectation. we will substitute this bound into the elbo, obtaining a valid lower bound to maximize. the key advantage is that our new lower bound admits an efficient stochastic gradient estimator. we start with the inner expectation and attempt to compute a gradient with respect to r (which controls the distribution of the simulation results n). using score function estimator gives which expresses the gradient with respect to (r, γ) in terms of the gradient of the probability density of the disease model m with respect to (r, γ). it turns out that log m (n, φ|r, γ) can be easily computed. recall that n using the poisson superposition theorem, we have that n t ∼ poisson( n i=1 r t φ i t + γ) (while φ t is a deterministic function of n 1 ...n t−1 ). accordingly, we have that where the second line substitutes the poisson log-likelihood. this expression can be easily differentiated with respect to r and γ in closed form. accordingly, we obtain an unbiased estimate of the gradient of our lower bound by sampling n ∼ m (r, γ) and computing ∇ r,γ log m (n|r, γ) log p(x|n). this suffices to estimate the gradient with respect to (r, γ). however, our goal is to differentiate with respect to µ and σ, which control the distribution over (r, γ). fortunately, r and γ are continuous. so, we can exploit the reparameterization trick by writing (r, γ) as a function of a random variable whose distribution is fixed. specifically, since σ is positive semi-definite, it has a cholesky decomposition σ = ll t (for convenience, we actually optimize over l instead of σ). sampling a standard normal variable ξ ∼ n (0, i) and letting r γ = µ + lξ is equivalent to sampling r, γ ∼ n (µ, σ). we rewrite the lower bound as where µ and l appear only as parameters of the deterministic function expressing r and γ in terms of ξ, instead of in the distribution of a random variable. taking a sample from each of the expectations and substituting the score function estimator now gives the desired expression for∇. using theorem 1, our final gradient estimator will sample b values for ξ, run the model m once for each of the resulting values of r to sample n, and then compute 1 b b k=1 ∇ µ,l log m (n(k)|r(ξ(k)), γ(ξ(k))) log p(x|n(k)), easily accomplished with standard autograd tools given the closed-form expressions for r(ξ), γ(ξ), and log m (n|r). in practice, we also use the mean log p(x|n(k)) as a simple control variate to reduce variance (sutton and barto 1998) . we now turn to the task of computing the log-likelihood function log p(x|n), which measures the log-likelihood of observing the sequence of positive test results x given n new infections per day. unfortunately, the log-likelihood is not available in closed form for any of the settings that we consider because it depends on additional latent variables (e.g., t convert , t revert , c, or a). we will show that it suffices to develop an estimator which lower-bounds the log-likelihood and that such estimators can be efficiently implemented for each of the observation models we consider. specifically, denote the collection of latent variables used in a particular observation model as α. then, we have which presents a similar difficulty as in developing our earlier lower bound: sampling α to approximate the inner expectation does not result in an unbiased estimator due to the outer log. using jensen's inequality in the same way gives and so substituting the right-hand side into our variational objective preserves validity of the lower bound. the rhs has the crucial advantage that we can now develop an unbiased estimator by drawing a single sample of α, which can then be substituted into the stochastic gradient estimator of theorem 1. that is, for each of the simulation results n(1)...n(b) we sample a corresponding value for the latent variables, α(1)...α(b) and use the gradient estimator ∇µ,l log m (n(k)|r(ξ(k)), γ(ξ(k))) log p(x|n(k), α(k)) this works without issue for the uniform undersampling and cross-sectional models where we can obtain a closed form for the log likelihood after conditioning on the appropriate latent variables. however, the longitudinal testing model presents additional complications. in particular, after sampling the latent variables t convert , t revert , and a t , the number of positive tests becomes deterministic quantity. denote this simulated trajectory of positive testsx. if x = x, then p(x| t convert , t revert , a) = 1 and otherwise p(x| t convert , t revert , a) = 0. this renders the above gradient estimator useless because log p(x|n(k), α(k)) = −∞ unless the simulated trajectory exactly matches the observed data (a very low-probability event). while −∞ is technically a valid lower bound for the variational objective, it is not very useful for optimization. essentially, we need to develop a lowervariance estimator where the lower bound is more useful. we now present one such improved estimator. the intuition is that we can marginalize out a great deal of the randomness in the naive estimator by only revealing the results of random draws determining a t a single individual at a time. we start by sampling t convert and t revert . note that we can expand log p(x|n, t convert , t revert ) = t t=1 log p(x t |x 1 ...x t−1 , n, t convert , t revert ) and consider the likelihood at each day t after conditioning on the results observed on previous days. to compute an estimate for this table 1 : mean absolute error of each method averaged over instances and time points for each setting, along with standard deviation of the absolute error. "pcr" and "serological" denote settings where the observations are generated by the respective testing method. individual column headings give the percentage of the population enrolled in testing. sum, we introduce a new object, the series of matrices c t . at each time t, c t [t 1 , t 2 ] denotes the number of individuals who have t convert = t 1 , t revert = t 2 , and have not yet actually tested positive by time t. since a t is selected uniformly at random from the population, independent of the infection process, the x t individuals who test positive on day t are drawn uniformly at random from the set of all individuals who converted between days t − d and t, and who have not yet reverted. let n draws denote the number of individuals in a t who have not yet tested positive by time t and n conv = t t1=t−d t t2=t+1 c t [t 1 , t 2 ] denote the number of individuals who are "eligible" to test positive at time t. now x t |x 1 ...x t−1 , n, c t follows a binomial distribution with n draws draws and success probability nconv n − t−1 i=1 xi . accordingly, the log-likelihood log p(x t |x 1 ...x t−1 , n, c t ) can be computed in closed form. after this, we can sample c t |c t+1 by selecting a uniformly random individual to remove from c t+1 . we can view this as iteratively revealing the test-positive members of a t after conditioning on the se-quence of previous test results, instead of sampling the entire set up front as in the naive method. we test the performance gprt vs standard baselines on a wide variety of settings. we choose three baselines which have been recommended by leading epidemiologists as methods of choice for covid-19 (gostic et al. 2020) . first is the wallinga-teunis (wt) method (wallinga and teunis 2004) , which uses the distribution of the time between an infected person and their secondary infections to simulate possible who-infected-who scenarios, each of which induces a particular r t . wt assumes that cases are observed exactly and that there is no delay in observation. second is the method of figure 2 : observed x t and the distribution over r t returned by each method on an example in the outbreak setting with longitudinal sampling, d = 14, and a 1% sample. the green line gives the ground truth r t . we test each method in an array of settings, with different distributions for both the true value of r t and the observations. we include two different settings for the ground truth r t . first, the outbreak setting where r starts below 1 and rises above 1 at a random time. second, the random trend setting where r follows a linear trend which changes randomly at multiple points in time. details of the settings and other experimental parameters are in the appendix. we also include different observation models characterized by the test used, the sampling method, and the sample size. we include both pcr and serological tests, using previously estimated distributions for d (kucirka et al. 2020; iyer et al. 2020) . we also include three sampling models introduced earlier: uniform underreporting, cross-sectional, and longitudinal. finally, for each of the four combinations of tests and sampling method, we include four different sample sizes. many sizes model a challenging setting with sparse observations, representing highly limited testing capacity. note that the sample sizes evaluated are different for each method because they have different interpretations, e.g. 1% in the cross-sectional case means sampling 1% of the population each day while in the longitudinal case it would mean 1% every d days. for each setting, table 1 shows the mean absolute error between the posterior mean r produced by each method and the ground truth. each entry averages over 100 instances. for longitudinal testing we use d = 14; results for other values are very similar (see appendix). across almost all settings, our method has lower mae than any baseline, often by a substantial margin (reducing error by a factor of 2-10x). notably, gprt performs well even with extremely limited data (e.g., when testing 0.05% of the population per day or when 1% of infections are observed). performance improves with more data, but the gains limited (e.g., 0.02-0.04 mae), indicating that our method is able to make effective use of even very sparse data. figure 2 shows a representative example. the observed data is quite sparse, with 0-8 positive tests observed per day. our method recovers a posterior which closely tracks the ground truth. wt produces an estimate which is correlated with the ground truth but has many fluctuations and overly tight confident intervals. cori is not appropriate for data this sparse and produces a widely fluctuating posterior. epinow does not return an estimate for much of the time series, only estimating the part with denser observations (we gave the baselines an advantage by only evaluating their mae where they returned an estimate). moreoever, even in the higherobservation portion, it is less accurate than gprt. finally, figure 3 shows calibration, a metric which evaluates the entire posterior (not just the mean). intuitively, calibration reflects that, e.g., 90% of the data should fall into the 90%-credible interval of the posterior. calibration is critical for the posterior distribution to be interpretable as a valid probabilistic inference, and for it to be useful in downstream decision making. figure 3 shows the fraction of the data which is covered by the credible intervals of each method. this figure shows cross-sectional testing in the outbreak setting, but results for other settings are very similar (see appendix). gprt is close to perfectly calibrated (the dotted diagonal line) while the baseline methods are not well calibrated. the baselines suffer from two problems. first, as to be expected from their higher mae, they are biased and so their credible intervals often exclude the truth. second, they are over-confident: paradoxically, their calibration worsens with increased data since the larger sample size makes them more confident in their erroneous prediction. we conclude that gprt offers uniquely well-calibrated inferences. estimating the time-varying reproduction number of sars-cov-2 using national and subnational case counts an interaction-based approach to computational epidemiology bayesian inference of transmission chains using timing of symptoms, pathogen genomes and contact data accounting for non-stationarity in epidemiology by embedding time-varying parameters in stochastic models forecasting a moving target: ensemble models for ili case count predictions a new framework and software to estimate timevarying reproduction numbers during epidemics capturing the time-varying drivers of an epidemic using stochastic dynamical systems estimating the effects of nonpharmaceutical interventions on covid-19 in europe practical considerations for measuring the effective reproductive number seroprevalence of antibodies to sars-cov-2 in 10 sites in the united states dynamics and significance of the antibody response to sars-cov-2 infection categorical reparameterization with gumbel-softmax inapparent infections and cholera dynamics auto-encoding variational bayes variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure automatic differentiation variational inference test sensitivity is secondary to frequency and turnaround time for covid-19 surveillance early in the epidemic: impact of preprints on global discourse about covid-19 transmissibility variational bayesian inference with stochastic search sourceseer: forecasting rare disease outbreaks using multiple data sources pattern of early humanto-human transmission of wuhan approximation algorithms for reducing the spectral radius to control epidemic spread introduction to reinforcement learning improved inference of time-varying reproduction numbers during infectious disease outbreaks markov chain monte carlo algorithms for gaussian processes dvae++: discrete variational autoencoders with overlapping transformations different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures modeling between-population variation in covid-19 controlling propagation at group scale on networks dava: distributing vaccines over networks under prior information key: cord-288982-63ddlh20 authors: peeling, rosanna w. title: diagnostics in a digital age: an opportunity to strengthen health systems and improve health outcomes date: 2015-11-09 journal: int health doi: 10.1093/inthealth/ihv062 sha: doc_id: 288982 cord_uid: 63ddlh20 diagnostics play a critical role in clinical decision making, and in disease control and prevention. rapid point-of-care (poc) tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests vary, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. a new generation of poc molecular tests that are highly sensitive and specific, robust and easy to use are now available for deployment in low resource settings. decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. a feature of many of these poc molecular devices is that they are equipped with data transmission capacities. in a digital age, it is possible to link data from diagnostic laboratories and poc test readers and devices to provide data on testing coverage, disease trends and timely information for early warning of infectious disease outbreaks to inform design or optimisation of disease control and elimination programmes. data connectivity also allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes. 'without diagnostics, medicine is blind.' alain merieux. diagnostics and laboratory services have been labelled as the achilles heel of global health. 1 in the past, low quality diagnostics and ineffective laboratory services resulted in a mistrust of laboratory results, which in turn led to a decreasing demand for laboratory services and a low priority for funding. 2 the 2008 maputo declaration has called on governments, multilateral agencies, development partners, professional associations and academic institutions to address laboratory challenges that limit the scale-up of services for tb, malaria and hiv diagnosis and care. together with the 2010 us centers for disease control and prevention's call to end the neglect of laboratories, this has provided a major milestone on a pathway to place diagnostics and laboratory services as important pillars of a healthcare system. 3, 4 recent outbreaks of influenza, middle east respiratory syndrome coronavirus (mers-cov) and ebola virus disease illustrate the importance of diagnostics in outbreak investigations. [5] [6] [7] with rapid technological innovations in the last 10 years and donor investments in the development of improved diagnostics for infectious diseases of public health importance, it is time to re-examine the achilles heel and explore the promises and challenges of diagnostics in a digital age. accurate diagnostic tests play a key role in clinician trust and patient management. diagnostic testing is traditionally considered as a tool to rule in or rule out a condition or infection when clinical presentation in a patient is non-specific. for diseases such as hiv and other sexually transmitted infections, most infections are asymptomatic but can cause serious long-term consequences. diagnostics are used to screen for infection so that treatment can be given to prevent the development of long-term complications and to interrupt the chain of transmission to sexual partners or to the fetus in the case of pregnant women. a test for the human papillomavirus helps to determine risk of disease progression to cervical cancer. for pathogens that have developed antibiotic resistance, such as neisseria gonorrhoeae, diagnostic testing is used to determine antibiotic susceptibility to ensure treatment efficacy. for hiv patients, a cd4 test result is used to determine if the patient is eligible for antiretroviral treatment and, for those on treatment, a viral load assay is used to determine treatment compliance and drug efficacy. a test of cure is used to determine when treatment can be stopped ( figure 1 ). beyond patient management, diagnostics play a critical role in various aspects of public health through disease prevention and control. diagnostics are critical in surveillance, to monitor trends in disease and antimicrobial resistance and assess the impact of interventions. diagnostic tools 'of sufficient sensitivity and specificity to detect levels of infection that can lead to transmission' were identified as one of the three essential requirements for disease elimination or eradication. 8 who has set eradication or elimination targets for a number of neglected tropical diseases (ntds), including guinea worm, polio, yaws, trachoma, schistosomiasis, lymphatic filariasis, onchocerciasis, human african trypanosomiasis, chagas' disease and visceral leishmaniasis (vl) 9 and the elimination of mother to child transmission (emtct) of hiv and syphilis. mass drug administration (mda) is the main control strategy for many of these diseases, resulting in the need for highly sensitive tests to detect the few remaining cases in areas of low transmission intensity after multiple rounds of mda. highly specific tests are then needed to certify elimination and maintain post-elimination surveillance. there is often little commercial interest in the development of diagnostics that are used mainly for surveillance because of the low return on investment. for a ntd such as schistosomiasis, the chinese national control programme has defined stages for schistosomiasis control as morbidity control (prevalence over 5% as defined by stool examination), infection control (prevalence of 1-5%), transmission control (prevalence lower than 1%), transmission interruption (no case detected in 5 years successively) and elimination (no case detected in the 5e years after transmission was interrupted). diagnostics results are used to inform national and regional control policy, redirect resources and to transition to the next phase of the control and treatment strategy. 10 the who targets for emtct and who/unaids 90-90-90 targets for hiv by 2020 also include diagnostic test coverage as indicators for efficiency of service delivery that help countries with setting a course to have an aids free generation and to end the hiv epidemic by 2030. diagnostic testing is usually performed in laboratories. in countries where the laboratory infrastructure is limited, who advocates for the use of a syndromic approach for patient management whereby patients are treated for all the major causes of that syndrome. this often leads to overtreatment and increases risk for the development of antimicrobial resistance. in the last decade, rapid point-of-care (poc) diagnostic tests fulfilling the assured criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable) have become commercially available and are widely used for infectious diseases such as malaria, hiv and syphilis. [11] [12] [13] [14] [15] these tests have allowed control programmes to increase access to testing, identify those who need to be put on treatment, optimise disease control and save lives. however, the quality of these tests varies, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. a new generation of immunoassays, molecular and nanotechnology platforms has been developed in recent years that can improve patient management and disease surveillance. these platforms have superior performance and make use of optical readers, mobile phones and data transmission capabilities to improve reading of results and data transmission. such technologies provide real-time results to inform patient management decisions. data, linked to precise geographical locations using gps, can be transmitted to a central database to inform disease control programmes or to monitor progress towards elimination. many of these assays can also yield quantitative results to give estimates of pathogen copy number or, in the case of hiv, to monitor treatment compliance or efficacy. these technology platforms can also be used to detect multiple targets from a single specimen. for geographic areas with multiple diseases targeted for elimination, if surveillance is conducted in the same sentinel population, such as children under the age of 10 years, using the same specimen, such as a blood sample, exposure to multiple ntds can be detected on a multiplex platform. this may be a more cost-effective means of conducting post-mda surveillance than collecting specimens and testing for a single ntd at a time, 16, 17 although cost-effectiveness of using these platforms for multiple ntds remains to be demonstrated. increased sensitivity of detection using these advances has made it possible to have tests that detect antibodies from oral fluids. the use of these non-invasive specimens has made it possible to design hiv tests for self-testing or for home use. 18 the availability of self-testing at home has allowed testing to be de-stigmatised and should help countries with achieving the first 90 of the unaids/who 90-90-90 targets. 19 bead-based immunoassays bead-based technologies are more versatile and potentially offer higher sensitivity compared to traditional solid phase immunoassays. 20 instead of using enzymatic amplification of a colorimetric substrate, bead-based assays can use a range of labels, including laser or fluorescence to detect the binding of the secondary antibody to an antigen or antibody target. the beads are in solution, offering more sites for ligand binding compared to a solid phase support. fluorescent intensities can yield quantitative results. protein arrays offer the potential of quantification of the antigen target as a surrogate measure of bacterial, parasite or viral load to inform treatment strategies. detection of multiple targets from the same pathogen can lead to increased efficiency over single-target assays. detection of targets from multiple pathogens using a single specimen can offer substantial cost and sample savings over traditional elisa measurements. the detection platforms are often equipped with data transmission capacities and allows near real-time surveillance of disease trends and monitoring the effect of special interventions. microfluidic devices offer many advantages, such as high throughput, short analysis time, small volume and high sensitivity, which make them an ideal immunoassay format for clinical diagnoses. the current microfluidic immunoassays have limited multiplexing capability compared to flow cytometric assays but are improving. immunoassays developed on a microfluidic platform that reproduce all the steps of a traditional elisa in a miniaturised format are now available. 21 the microfluidic disc can fit into the palm of a hand and is rapid and inexpensive to manufacture. these platforms allow multiplexing, but have only been validated for antibody detection in blood samples. antigen detection from urine or stool samples will require multistep specimen processing and/or purification or concentration before being put into the antigen-antibody reaction within the microfluidic channels. a recent publication showed that it is possible to use the power of smart phones to power a microfluidic immunoassay reaction by plugging the reaction chamber into a smart phone using a dongle. 22 over 80 assays can be run before recharging is necessary. the results are interpreted and displayed on the phone and the data transmitted to a central database if required. a dual test to detect antibodies to hiv and syphilis has been designed on this platform and will have utility not only to improve access to prenatal screening but also to allow surveillance data for the dual elimination of mtct of hiv and syphilis to be available in real time. 23 nanosensor assays a growing number of promising diagnostic tools are based on nanotechnology. the application of nanomaterials to detect host or pathogen biomarkers has the potential to yield ultrasensitive assays. quantum dots are fluorescent semiconductor nanoparticles typically between 10 and 100 atoms in diameter and the technology has been applied to the development of ultrasensitive tests for hiv and other viral infections. 24 it is also possible to use these technologies to combine pathogen detection and speciation with genetic analysis, such as detection of single nucleotide polymorphisms. hence, they can be used for high throughput screening of drug mutation targets. quantum wires are being used in the development of a fully automated diagnostic test for malaria infection, speciation and drug resistance status in less than 20 minutes. 25 these nanosensors can ultimately be configured to a handheld pda-type device or a thin plate about the size of a small business card containing a tiny nanowire sensor. operationally, these tests are simple to use and give answers in less than 30 minutes. they can be used during a doctor's visit or at home by a patient, or early detection of bioterrorism in a community setting. linking mobile connectivity and gps will enable anonymised disease data to be sent to a data cloud for real-time surveillance. advocacy to apply these novel technologies to ntds and antimicrobial resistance monitoring is an urgent priority. this requires precise engineering of nanomaterial surfaces as the interface between the nanomaterial and the specimen is where the reaction occurs. molecular assays offer superior diagnostic performance compared to the limit of detection of immunoassays. in the last two decades, nucleic acid amplification tests (naats) have become the 'gold' or reference standard to which the performance of other diagnostic tests is compared. until recently, they have remained largely laboratory based and are expensive and inaccessible. a number of poc molecular assays, with or without amplification, are now on the horizon that may transform the delivery of health services. 26, 27 the first sample-in/answer-out real-time pcr platform that can be used wherever there is a source of electricity has been introduced for the diagnosis of tb and detection of rifampicin resistance. [28] [29] [30] the genexpert real-time pcr platform has a large menu of test targets; thus, allowing polyvalency to test for different pathogens. it requires minimal onsite expertise and provides a result in less than 2 hours. it has additional advantages of random access and remote quality control. however, the cartridges are expensive and not affordable or sustainable unless subsidised in most high burden countries. to maximise the impact of these novel technologies, it is critical that patient pathways be modified to take advantage of the speed to result and to find cost-efficient means of implementation compared to traditional laboratory testing. 31 isothermal amplification platforms such as helicase dependent amplification (hda), cross priming amplification (cpa), recombinase polymerase amplification (rpa) and loop-mediated amplification (lamp) assays can be engineered into poc naats as there is no requirement for sophisticated equipment to perform thermal cycling. the details of these technologies have been described elsewhere. 27, 32 poc naats based on these technologies have largely been applied to high burden diseases such as hiv and tb, but other technologies, such as lamp, have been developed and evaluated for the diagnosis of vl and hat. in the case of vl, the lamp assay for leishmania donovani is highly sensitive and specific for the diagnosis of vl (using a blood sample), and for the diagnosis of post-kala azar dermal leishmaniasis (pkdl) (using a skin biopsy). 33 diagnosis of pkdl will be important for the elimination agenda since patients with this condition are an important source of infection. in the case of hat, the lamp assay was shown to have a sensitivity of 87.3% (95% ci 80.9 to 91.8%) and a specificity of 92.8% (95% ci 86.4 to 96.3%) compared to a pcr reference standard. 34 advocacy and investments are needed to apply these technologies to the control and elimination of ntds. 35 the promise of diagnostics in a digital age in a digital age, data from social media and poc diagnostics in communities can be used to provide early warning for infectious disease outbreaks, and timely information to inform disease control and elimination programmes. the isense project, an epsrc irc funded project in early warning sensing systems in infectious diseases, aims to create low-cost latent sensing systems to analyse self-reported symptoms on the web, including social networks and micro-blogging sites (twitter) and searches (bing, google). isense will develop a new generation of early warning sensing systems to identify outbreaks of deadly infectious diseases, such as flu, methicillin-resistant staphylococcus aureus (mrsa) and hiv, by linking self-reported symptoms on the web to a new sensor-enabled disease surveillance infrastructure for an early warning sensing system for infectious diseases (http://www.i-sense.org.uk/research). the simplest poc diagnostic tests that are widely used today are rapid diagnostics tests (rdts) in a lateral flow format. these are read with the naked eye, which is subjective and prone to human error and may be further exacerbated by poor lighting in health posts. in addition, rdts lack on-board quality control and are often used in remote areas where health workers receive minimal training. as a result, accuracy of rdts performed in the field can be quite variable. this may adversely affect patient care and the accuracy of the data gathered for surveillance. digital imaging technology in electronic readers and smart phones can be used to capture and interpret rdt results and transmit data. given the large number of brands of rdts for hiv, malaria and syphilis, it would be ideal to have diagnostic test readers that could accommodate a variety of test technologies (e.g., lateral flow, immunofiltration) and formats (e.g., strips, cassettes of various sizes and shapes). a number of rdt readers are already on the market and others are in the pipeline. given the widespread adoption of smart phones in resource-limited settings, rdt readers using this technology have the potential to combine high-resolution test images with the computing capability required to run image analysis software and transmit data. the readers range in price depending on the technical complexity of the instrument and their compatibility with the type of rdts. data collection for the emtct is difficult when testing is highly decentralised and data quality is difficult to verify. to track global progress towards elimination of mtct of hiv and syphilis many challenges need to be addressed. currently, data from antenatal screening need to be manually accessed from individual testing sites. electronic readers for rdts are able to capture and automatically transmit data and may, therefore, serve a critical purpose for strengthening data collection and surveillance in the context of monitoring and evaluation of dual elimination. 36, 37 improving supply chain management real-time data monitoring via electronic readers could help to improve coordination of supply chain management by multiple partners. operational data on stocks, device usage and condition can be uploaded via wi-fi or cellular networks and transmitted to central databases. by linking the data to supply chain management software, stock-outs can potentially be avoided and health system efficiency improved. in recent years, rdts have been introduced to increase hiv and syphilis screening in antenatal clinics, enabling same day testing and treatment of infected mothers to prevent adverse outcomes of pregnancy and fetal loss. the emtct targets to be achieved are 95% of pregnant women access antenatal care, 95% of pregnant women at antenatal care tested for hiv and syphilis, and 95% of those testing positive receive treatment. to achieve these targets, countries will need to have a system to track progress the capacity for ongoing surveillance once the elimination target is achieved. studies have shown that the introduction of rapid rdts for syphilis for prenatal syphilis screening has strengthened health systems by improving access to diagnostics, health worker job satisfaction and reducing stillbirths and neonatal mortality. in peru, the introduction of rdts for prenatal screening has resulted in infected pregnant women being screened and treated in a single visit instead of 5-6 visits over 27 days. 38, 39 challenges of new technologies assuring quality of poc lateral flow tests and devices decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. when testing is decentralised, programme managers are often unable to monitor testing coverage and quality, making it difficult to identify problems of sub-standard testing and stock-outs in a timely manner. 40 international health for poc molecular devices, requirements for instrument calibration, ongoing maintenance and frequency of failure, power usage and environmental sustainability should also be considered. creation of a central database to collect surveillance data from village antenatal clinics all the way through to national and global databases is needed. this involves obtaining consensus on where to host the data, what data to collect, who has access to the data and finding affordable software. middleware solution that provides 'open access' will allow for rapid coordinated transmission of data to governments. technology is needed to ensure data is collected, transmitted and stored in a way that conforms to ethical and legal standards, maintaining patient privacy and confidentiality. these governance issues may be resolved by ensuring separate levels of access to operational data versus patient data. in addition, data storage should be in-line with country specific regulations. further discussion needs to take place around data ownership. an excellent example of a national database that informs critical programmatic decisions in real time is the kenyan national early infant diagnosis (eid) portal (www.nascop.org/eid), which has now been extended to cover hiv viral load and other diagnostics. this database is the result of a successful public-private partnership that involved the government of kenya ministry of health, usaid/president's emergency plan for aids relief (pepfar), the us cdc, the clinton health access initiative (chai), hewlett packard, safaricom and strathmore university. hewlett packard built a basic data centre at the national aids/std control programme (nascop) and at testing laboratories for early infant diagnosis. safaricom, the largest provider of mobile services in kenya, set up a short code service for sms. strathmore university students built the application. pepfar, cdc and chai rolled out the eid programme countrywide. the result is a national eid portal running on a government data centre-hosting all the testing and program data entered from computers in laboratories equipped with laboratory information systems (web-based applications). over 2000 health facilities that provide eid services have access to all the data and test results over sms, mobile web, web and email in near real time. data analytics include sample transport tracking, volume of testing at each facility, test results, including the number of rejected samples and indeterminate results. results are delivered via electronic and paper means (sms printer, email, courier, web) and real time eid/prevention of mtct programme analytics are available online by facility, and at regional and national levels. the new generation of diagnostics equipped with digital technologies are transforming the field of clinical decision-making and disease control and prevention. rapid poc tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests varies, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. electronic readers have the potential to provide fast, accurate, standardised rdt interpretation and real-time data reporting with a huge range of positive functions, including improving quality assurance, supply chain management and providing accurate timely data for surveillance. although countries need to consider data governance and confidentiality issues, data connectivity allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes. author's contribution: rp has undertaken all the duties of authorship and is guarantor of the paper. achilles heel" of global efforts to combat infectious diseases laboratory medicine in africa: a barrier to effective health care the maputo declaration on strengthening of laboratory systems. geneva: world health organization laboratory systems and services are critical in global health: time to end the neglect? severe respiratory disease concurrent with circulation of h1n1 influenza identification of the novel coronavirus in patients with severe acute respiratory syndrome the case for improved diagnostic tools to control ebola virus disease in west africa and how to get there the principles of disease elimination and eradication tools to support policy decisions related to treatment strategies and surveillance of schistosomiasis japonica towards elimination diagnostics for the developing world point-of-care tests for diagnosing infections in the developing world point-of-care diagnostics for global health diagnosing infections -current and anticipated technologies for point-of-care diagnostics and home-based testing point-of-care diagnosis of tuberculosis: past, present and future a diagnostics platform for the integrated mapping, monitoring, and surveillance of neglected tropical diseases: rationale and target product profiles development of a new platform for neglected tropical diseases surveillance hiv self-testing in resource-limited settings: regulatory and policy considerations unaids. 90-90-90 -an ambitious treatment target to help end the aids epidemic bead-based microfluidic immunoassays: the next generation microfluidics-based diagnostics of infectious diseases in the developing world smartphone dongle for simultaneous measurement of hemoglobin concentration and detection of hiv antibodies multisite laboratory evaluation of a dual human immunodeficiency virus (hiv)/syphilis point-of-care rapid test for simultaneous detection of hiv and syphilis infection integrated quantum dot barcode smartphone optical device for wireless multiplexed diagnosis of infected patients plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth emerging technologies in point-of-care molecular diagnostics for resource-limited settings point-of-care nucleic acid testing for infectious diseases feasibility, diagnostic accuracy, and effectiveness of decentralised use of the xpert mtb/rif test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study point-of-care system for detection of mycobacterium tuberculosis and rifampin resistance in sputum samples geneva: unitaid; geneva opportunities and challenges for cost-efficient implementation of new point-of-care diagnostics for hiv and tuberculosis application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis diagnostic accuracy of loopamp trypanosoma brucei detection kit for diagnosis of human african trypanosomiasis in clinical samples multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an openarray platform using electronic readers to monitor progress toward elimination of mother-to-child transmission of hiv and syphilis: an opinion piece elimination of elimination of mother-to-child transmission (emtct) of hiv and syphilis. global guidance on criteria and processes for validation. geneva: world health organization point-of-care tests to strengthen health systems and save newborn lives: the case of syphilis rapid syphilis tests as catalysts for health systems strengthening: a case study from peru the costs of accessible quality assured syphilis diagnostics: informing quality systems for rapid syphilis tests in a tanzanian setting the author would like to acknowledge david mabey and debi boeras in providing helpful comments in the preparation of this manuscript.funding: none. ethical approval: not required. key: cord-117424-mp6h9dyl authors: abraham, louis; becigneul, gary; coleman, benjamin; scholkopf, bernhard; shrivastava, anshumali; smola, alexander title: bloom origami assays: practical group testing date: 2020-07-21 journal: nan doi: nan sha: doc_id: 117424 cord_uid: mp6h9dyl we study the problem usually referred to as group testing in the context of covid-19. given n samples collected from patients, how should we select and test mixtures of samples to maximize information and minimize the number of tests? group testing is a well-studied problem with several appealing solutions, but recent biological studies impose practical constraints for covid-19 that are incompatible with traditional methods. furthermore, existing methods use unnecessarily restrictive solutions, which were devised for settings with more memory and compute constraints than the problem at hand. this results in poor utility. in the new setting, we obtain strong solutions for small values of n using evolutionary strategies. we then develop a new method combining bloom filters with belief propagation to scale to larger values of n (more than 100) with good empirical results. we also present a more accurate decoding algorithm that is tailored for specific covid-19 settings. this work demonstrates the practical gap between dedicated algorithms and well-known generic solutions. our efforts results in a new and practical multiplex method yielding strong empirical performance without mixing more than a chosen number of patients into the same probe. finally, we briefly discuss adaptive methods, casting them into the framework of adaptive sub-modularity. lacking effective treatments or vaccinations, the most effective way to save lives in an ongoing epidemic is to mitigate and control its spread. this can be done by testing and isolating positive cases early enough to prevent subsequent infections. if done regularly and for a sufficiently large fraction of susceptible individuals, mass testing has the potential to prevent many of the infections a positive case would normally cause. however, a number of factors, such as limits on material and human resources, necessitate economical and efficient use of test resources. group testing aims to improve test quality by testing groups of samples simultaneously. we wish to leverage this framework to design practical and efficient covid-19 tests with limited testing resources. group testing can be adaptive or non-adaptive. in the former, tests can be decided one at a time, taking into account previous test results. in the latter, one can run tests in parallel, but also has to select all tests before seeing any lab results. a popular example of a semi-adaptive group test is to first split n samples into g groups of (roughly) equal size, pool the samples within the groups and perform g tests on the pooled samples. all samples in negatively tested pools are marked as negative, and all samples in positively tested pools are subsequently tested individually. figure 1 : we formulate the group testing problem as a constrained information maximization problem. samples are grouped into testing pools so that the information gain is maximized while obeying practical constraints (i.e. no more than 64 samples in one group). here, positive samples are shown in black and positive tests are shown in blue. the tests are decoded with error correcting probabilistic methods. practical constraints for covid-19. although group testing is a well-studied problem, the recent covid-19 pandemic introduces specific constraints. in contrast to seroprevalence antibody tests, pcr tests aim to detect active cases, and only successfully do so during part of the disease course [8] ). this results in a small prevalence (prior probability of population infection; we will assume a default value of 10 −3 ), assuming we screen the general population rather than only symptomatic individuals. group testing has recently been validated for covid-19 pcr tests [22, 26] . it is facilitated by the fact that pcr is an amplification technique that can detect small virus concentrations. nevertheless, there are limitations on the number of samples l that can be placed in a group ([26] considers up to 64), and constraints on the number of times a particular sample can be used ( [22] uses serial incubation of the same respiratory sample in up to k = 10 tubes). besides, there are practical issues: adaptive testing is time consuming and hard to manage. complex multiplex designs are prone to human error. existing research on non-adaptive group testing is generally concerned with identifying at most k positive samples amongst n total samples, which is referred to as non-adaptive hypergeometric group testing [9] . this assumption yields asymptotic bounds on the number of tests needed to recover the ground truth [13, 10, 4, 3] . however, these are of limited practical relevance when constructive results on small numbers of samples are required. the specific constraints for covid-19 force us to revisit the general framework of group testing. novel formulation. we formulate the problem based on the principle of information gain: given n people and m testing kits, the characteristics of the test and prior probabilities for each person to be sick, we seek to optimize the way the tests are used by combining several samples. for simplicity, samples are assumed to be independent analogous to [18] . however, we focus on implementable tests, unlike [18] which focuses on asymptotic results that are valid for large n. figure 1 summarizes our approach. optimal characterization: by leveraging the framework of adaptive sub-modularity initially developed for sensor covering by [6] , we prove near-optimality of a simple greedy-strategy for adaptive testing. despite the simplicity, it turns out that this greedy strategy has exponential running time and becomes infeasible for n ≥ 16. fortunately, the near optimally of the greedy-adaptive method points toward a simple and scalable non-adaptive solution leveraging randomization akin to the bloom filter structure [20] . bloom origami assays: 1 [19, 2] recently showed that pooling using random hash functions, similar to a bloom filter structure, can lead to an efficient and straightforward group testing protocol. we will show that such a protocol, based on random hash functions, is unnecessarily restrictive. bloom filters were designed for streaming data, where there is no choice but to use universal hash functions for pooling. for covid-19, the computational situation is much simpler. leveraging our information gain framework, we propose superior but straightforward hashing strategies. a bigger problem with bloom filters is the (necessarily) simple decoder. the decoder trades accuracy for efficiency, as it was designed for internet-scale problems where linear time decoding is prohibitive. for covid-19, we instead propose a message-passing decoder, similar to counter braids [16] , which is more accurate. our proposal of connecting probabilistic graphical model (pgm) inference with bloom filters could be broadly applicable to situations beyond covid-19 group testing. since the graphical model is a bipartite graph for which no narrow junction tree can be found, message passing does not necessarily converge to the global optimum. therefore, we propose a new method for graphical model inference leveraging probabilistic concentration and meet-in-the-middle (mitm) techniques, which may be of independent interest. our mitm method is particularly useful for the low prevalence scenario. this paper illustrates the power of algorithmic re-design to target practical constraints. we obtain significant gains even on the relatively well-studied topic of group testing. notations are progressively introduced throughout but are gathered in the appendix, which also contains the proofs. denote the number of patient 2 samples by n. as previously mentioned, we consider the group testing task in the particular context of the covid-19 pandemic. this choice of problem setting naturally introduces new mathematical constraints of a practical nature: impracticality of adaptivity. adaptive methods require several hours in between each lab result of the adaptive sequence. this inspires us to only consider either non-adaptive methods or semi-adaptive methods with no more than two phases of testing. low concentration and test accuracy. excessive mixing of patient swabs may result in prohibitively low viral concentration with negative consequences for testing. a recent study reports that one can safely mix a patient swab up to 10 times [22] ; another relays that mixing up to 32 patient samples into the same probe yields a false negative rate below 10% [26] . there is clearly ambiguity in the limitations of the experimental protocol. for instance, [15] validate double-digit numbers of patients per sample for pcr tests. while dilution effects are relevant for such large pools, they can be partly addressed by incubating respiratory swabs multiple times [22] . also note that we are only concerned with the accuracy of the tests per se rather than the biological sampling protocol (i.e. whether swabs are taken when viral load is detectable in patients). in what follows we consider group sizes of n = 100 as a sensible upper limit. notations and reminders denote the number of tests to run by m. tests are assumed to be imperfect, with a true positive rate (or sensitivity) tpr 3 and true negative rate (or specificity) tnr. 4 as simple default values, we will use tpr = 99% [21] and tnr = 90% [26] . 5 patient sample i is infected with probability p i ∈ [0, 1] and we assume statistical independence of infection of patient samples. denoting by a '1' a positive result (infection), the unknown ground truth is a vector of size n made up of '0's and '1's. this vector describes who is infected and who is not. we call this the secret, denoted as s ∈ {0, 1} n . a design of a test d ∈ {0, 1} n to run in the lab is a subset of patient samples to mix together into the same sample, where d i = 1 if patient sample i is mixed into design d and d i = 0 otherwise. note that the outcome of a perfect design d for a given secret s can simply be obtained as 1 d,s >0 where d, s := n i=1 d i s i . that is, a test result is positive if there is at least one patient i for which d i = 1 (patient i is included in the sample) and s i = 1 (patient i is infected). figure 1 illustrates the problem setting. recall that the secret s is unknown. however, since we assume that patient sample i is infected with probability p i and that patient samples are independent, we have a prior probability distribution over the possible values of s. we hence represent the random value of s as a random variable (r.v.), denoted by s, with probability distribution p s (s) := pr[s = s] over {0, 1} n . let us now recall the definition of the entropy of our random variable, the entropy represents the amount of uncertainty that we have on its outcome, measured in bits. it is maximized when s follows a uniform distribution, and minimized when s constantly outputs the same value. as we perform tests, we gain additional knowledge about s. for instance, if we group all samples into the same pool and have a negative result, then our posterior probability that all patients are healthy goes up. that is, p s ((0, . . . , 0)) increases according to bayes' rule of probability theory. more generally, we may perform a sequence of tests of varying composition, updating our posterior after each test. our goal will be to select designs of tests so as to minimize entropy, resulting in the least amount of uncertainty about the test outcome for all individuals. given n people, test characteristics tpr & tnr and a set of prior probabilities of sample infection (p i ) 1≤i≤n , the best multiset d of m pool designs is the one maximizing the information gain. the tests are order insensitive, which gives a search space of cardinality 2 n +m m . evaluating the information gain of every multiset separately takes o (2 n+m ) operations. 6 hence, brute-forcing this search space is prohibitive even for small values of n and m. we resort to randomized algorithms to find a good enough solution. our approach is to use evolutionary strategies (es). we apply a variant of the (1 + λ) es with optimal restarts [17] to optimize any objective function over individuals (multisets of tests). detailed description. we maintain a population of 1 individual between steps. at every step of the es, we mutate it in λ ∈ n + offsprings. in the standard (1 + λ) es, each offspring is mutated from the population, whereas our offsprings are iteratively mutated, each one being the mutation of the previous. these offsprings are added to the population, and the best element of the population is selected as the next generation of the population. we initialize our population with the "zero" design that doesn't test anyone. our mutation step is straightforward: flipping one bit d i of one pool design d, both chosen uniformly at random. we also restrict our search space if needed: the number of 1's in a column must be less than the number of times a given swab can be mixed with others, the number of 1's in a line is constrained not to put too many swabs into the same pool. our iterative mutation scheme allows us to step out of local optima. after choosing a basis b proportional to n × m (which is approximately the logarithm of our search space), we apply restarts according to the luby sequence: , ...). this sequence of restarts is optimal for las vegas algorithms [17] , and our es can be viewed as such under two conditions: (i) that the population never be stuck in a local optimum, which can be achieved in our algorithm using λ = n × m (note that much smaller constant values are used in practice); (ii) the second condition is purely conceptual and consists in defining a success as having a score larger than some threshold. the fact that our algorithm does not use this threshold as an input yields the following result, proved in appendix c.1: theorem 1. under condition (i), the evolutionary strategy using the luby sequence for restarts yields a las vegas algorithm that restarts optimally [17] to achieve any target score threshold. note that since tests are imperfect, for a given pool design d ∈ {0, 1} n and a given secret s ∈ {0, 1} n , the boolean outcome t (s, d) of the test in the lab is not deterministic. if tests were perfect, we would have t (s, d) = 1 d,s >0 . to allow for imperfect tests, we model t (s, d) as a r.v. whose distribution is described by pr[t (s, d) = 1 | d, s > 0] = tpr and pr[t (s, d) = 0 | d, s = 0] = tnr. 7 since the secret s is also unknown (and described by the r.v. s), the outcome t (s, d) has now two sources of randomness: imperfection of tests and unknown secret. 8 in practice, one will not run one test but multiple tests. we now suppose that m tests of pool designs are run and let their designs be represented as a multiset d ∈ ({0, 1} n ) m . this leads us to the following question: given an initial prior probability distribution p s over the secret, how should we select pool designs to test in the lab? we want to select it such that once we have its outcome, we have as much information as possible about s, i.e. the entropy (uncertainty) of s has been minimized. since we cannot know in advance the outcome of the tests, we have to minimize this quantity in expectation over the randomness coming from both the imperfect test and unknown secret. this requires the notion of conditional entropy. conditional entropy. given pool designs d, we consider two random variables s (secret) and t := t (s, d) (test results). the conditional entropy of s given t is given by: in where it represents the amount of information (measured in bits) needed to describe the outcome of s, given that the result of t is known. the mutual information between s and t can equivalently be defined as i this criterion selects the pool designs d whose outcome will maximize our information about s. expected confidence. we report another evaluation metric of interest called the expected confidence. it is the mean average precision of the maximum likelihood outcome. the maximum likelihood outcome it defined by: m l is of particular practical interest: given test results t, a physician wants to make a prediction. in this case, it makes sense to use the maximum likelihood predictor. the interpretation of confidence is straightforward: it is the probability that the prediction is true (across all possible secrets). updating the priors. both scoring functions described above compute the expectation relative to the test results of a score on the posterior distribution p s|t =t (s). after observing the test results, we are able to replace the prior distribution p s by the posterior. by the rules of bayesian computation, this update operation is commutative, i.e., the order in which designs d 1 and d 2 are tested does not matter, and compositional in the sense that we can test {d 1 , d 2 } simultaneously with the same results. thus, we can decompose those steps and make different choices as we run tests (see the adaptive method below). although searching the space of all possible adaptive strategies would yield a prohibitive complexity of ω(2 2 m ), it turns out that a simple adaptive strategy can yield provably near-optimal results. we describe an adaptive scheme in algorithm 1 which greedily optimizes the criterion defined in eq. (4). update p s accordingly (see eq. (3)) to the realization of d * in the lab ; 9 decrease the number of remaining tests k by 1; 10 end leveraging the framework of adaptive sub-modularity [6] , and assuming that the criterion defined by eq. (4) is adaptive sub-modular 9 , algorithm 1 has the guarantee below. theorem 2. denote by 'algo' an adaptive strategy. let i(algo) be the expected mutual information obtained at the end of all m tests by running algo, the expectation being taken over all 2 m outcomes of lab results. denote by 'optimal' the best (unknown) adaptive strategy. if we run algorithm 1 for m 1 tests and optimal for m 2 tests, we have: where α is defined as follows: assume that our priors p i are wrong, in the sense that there exist constants c, d with cp i ≤ p i ≤ dp i for i ∈ {1, ..., n}, with c ≤ 1 and d ≥ 1, where p i denotes the true prior: we set α := d/c. remarks. accordingly, algorithm 1 is (i) robust to wrong priors and (ii) near-optimal in the sense that the ratio of its performance with that of the optimal strategy goes to 1 exponentially in the ratio of the numbers of tests run in each algorithm. for α = 1 and m 1 = m 2 , this yields 1 − e −1 0.63. our previous methods are effective, but they are prohibitively expensive for n > 30 patients. to address this, we present a randomized approach to selecting d by grouping patients into pools using bloom filters [1] . randomized test pooling may be attractive to practitioners because it is straightforward to understand and implement in the laboratory. the simplest method partitions n patients into random groups of equal size. patients are either re-tested or reported positive if their group tests positive (single pooling). in [2] , the authors propose an extension to this idea that inserts patients into two sets of pools, named double pooling, which offers impressive advantages at the same cost. we present a generalization of this idea that uses an array of bloom filters to improve the error characteristics of the test. while bloom filters have been considered for the low-prevalence covid-19 testing problem [19, 12] , current methods are based on a simple randomized encoding and decoding process that was designed for internet-scale applications where even linear time was prohibitive and where the keys are not known beforehand. this sacrifices accuracy. we now design an improved algorithm. 9 empirical validation in appendix f. encoding. bloom filters use universal random hash functions for load balancing because the streaming algorithm setting does not allow us to control the number of items in each group. here, we can improve the filter with perfect load balancing. we divide the m tests into g groups of b pools. in each group, we assign the n patient samples to the b pools so that each pool contains n/b patients. 10 . this procedure constrains the multiset d of possible test designs. with uniform prior probabilities, we implement a perfectly load balanced hash by assigning each patient a number based on a permutation π j of the integers {1, ...n} thus patient i is assigned to pool h j (i) := π j (i) mod b in group j. for non-uniform priors, we can resort to a variable load hash to balance total weights into pools. due to the concavity of the entropy, the information gain is maximized if all pools have the same probability of testing positive. this is maximized for 1/2, the mode of the binary entropy. load balancing implies information gain: load balancing, as exhibited by our encoding, maximizes the information gain for a practical subset of constrained bloom filter group test problems. theorem 3 motivates bloom filters in the context of our information theoretic framework. with a constraint on the number of samples in each pool, our load balancing hash allocation is the optimal pooling strategy provided that pr[t b = 1] is sufficiently small (∼ 20%). we defer a detailed discussion to the appendix. decoding for perfect tests. assuming perfect tests, one can easily decode the pooled test results t ∈ {0, 1} b×g because all patients in negative pools are healthy. we can then identify positive (and ambiguous) samples by eliminating healthy samples from positive pools, as described in the appendix. in the case where g = 1 and g = 2, we have the widely-used single pooling method and the recently-proposed double pooling method [2] . assuming there are no false negative pool results, one can use the decoder to identify all positive samples and derive optimal dimensions b × g that minimize the number of tests, as shown in the below theorem: the analysis borrows tools from regular bloom filters and the results shown in [20] . note that the problem with no test error and 1/2 prevalence is a #p-complete restriction of #sat, called monotone cnf [24] . realistic tests with nontrivial fnr and fpr are technically more interesting. a natural idea is an algorithm dating back to [11] when decoding diseases from the qmr database. decoding via message passing. indeed, false negative rates are often as high as 10%. the decoder fails for imperfect tests because even negative pools might contain positive samples. a small number of healthy pools might even test positive for some protocols (e.g. due to spurious contamination). when viewed as a probabilistic graphical model we can interpret t gb as a corrupted version of the true state y gb . it is our goal to infer the secret s that produced t gb . belief propagation is a common technique to estimate the posterior distribution p s|t =t for a graphical model. since our graphical model cannot be rewritten as a junction tree with narrow tree width there are no efficient exact algorithms. instead, we resort to loopy belief propagation [14] . while inexact (loopy-bp isn't guaranteed to converge to the minimum) the resulting solution can classify samples as positive or negative with reasonable performance. while the degree of each pool figure 3 : intuition behind probabilistic decoding. in each group, we suspect that patients in positive pools are positive. if a patient falls within multiple positive pools, the likelihood that their test status is positive increases. even if a false positive or negative occurs, we may still report the correct diagnosis thanks to information from other groups. this process is known as "error correction" and can be implemented with message passing or our mitm algorithm. figure 5 : intuition behind the mitm approach. if the prevalence is low, then we do not need to consider inputs with many positives. this restricts the set of possible secrets and the set of ways we can encode those secrets. the figure shows the inputs for at most 3 positives. given a (potentially corrupted) output, there are only a small set of true encodings that could have produced that output -it is highly unlikely that every test had a false result. the two conditions "meet in the middle" to produce a small set of states. our mitm algorithm efficiently approximates the posterior probabilities by summing over this restricted state space. node is so high that the clique potential would naively involve an intractable number of states, the clique potentials have a simple form that permits an efficient implementation (detail in the appendix). decoding for imperfect tests: meet-in-the-middle (mitm). the structure of the problem also enables an efficient approximation to the exact solution in the (realistic) setting where the tests are fairly accurate and the disease prevalence is low. low prevalence implies that there are relatively few "likely secrets" s ∈ {0, 1} n , because most s i are 0 with high probability. thus, we only need to consider secrets with a small number of positive patients. since the secrets concentrate in a small subset of {0, 1} n , we expect to see relatively few bloom encodings y ∈ {0, 1} t for low-prevalence problems. furthermore, the output space is likely to be corrupted in relatively few ways. the true state y gb is likely to be the same as the observed output t gb , so we only need to consider states that are similar to the observed output. by restricting our attention to "likely secrets" and "likely outputs", we can reduce the o(2 n ) complexity of the naive brute-force algorithm. this process constitutes a "meet in the middle" approach where we only need to consider illustrative values for mitm decoding. using stirling's formula n! ∼ √ 2πn(n/e) n , one can easily show that for a fraction x ∈ [0, 1], we have f (xn) = o((2p x ) n ) when n → +∞. if k := xn is such that 2p x < 1, i.e. x > log(2)/ log(1/p), then f (k) will be exponentially small w.r.t. n. with our default p = 0.1% we only need to consider secrets with a fraction smaller than x * = log 2 (2)/ log 2 (1/10 −3 ) ≈ 10.03% of infected people to yield negligible error. for n = 60, choosing x = 13% reduces 12 the search space of secrets from 2 60 ≈ 10 18 to 60 * 0.13 −1 i=0 60 i < 6 · 10 7 with an error ε < (2p 0.13 ) 60 < 5 · 10 −6 . we ran simulations to compare test designs for a large variety of group testing parameters (n, tpr/tnr, b × g, prevalence) in the appendix. in this section we present results for a practical scenario where tnr = 0.9, tpr = 0.99, and 0.1% prevalence. in figure 6 , we compare the entropy-minimizing solution found by genetic algorithms with several bloom filter designs. the bloom filter performance closely resembles the optimal solution, albeit with higher variance. this validates our claim that the load balancing permutation hash implies a good information gain. we also apply our graphical model framework to 3 × 5 arrays of bloom filters, single pooling and double pooling designs. we use the mitm technique to compute posteriors for all designs and we compare performance. while mitm provides the best results, computational constraints may demand belief propagation for situations where there are many positive group tests. in the high-prevalence scenario, belief propagation will still provide sufficient error correction for good diagnostic results (figure 4) . the vanilla bloom decoding (single and double pooling) is unnecessarily inaccurate, clearly implying the need for specific tailored algorithms. we have presented a framework for group testing taking into account specifics of the current covid-19 pandemic. it applies methods of probability and information theory to construct and decode multiplex codes spanning the relevant range of group sizes, establishing an interesting connection to bloom filters and graphical models inference along the way. our empirical results, more of which are included in the appendix, show that our methods lead to better codes than randomized pooling and popular approaches such as single pooling and double pooling. furthermore, we provide an approximate inference algorithm through theorem 5 that outperforms the message passing approach for realistic parameter values by pruning the exponential search space. we also prove compute-time bounds on its error, highly useful in practice because they are strict. we believe that the test multiplexing problem is an ideal opportunity for our community to make a contribution towards addressing the current global crisis. by firmly rooting this problem in learning and inference methods, we provide fertile ground for further development. as more information about test characteristics becomes available, we could take into account dependencies of tpr, tnr on pool size. the framework could be adapted to different objective functions, or linked to decision theory using suitable risk functionals, e.g., taking into account the downstream risk of misdiagnosing an individual with particular characteristics (comorbidities, probability of spreading the disease, etc.). it can be combined with the output of other methods providing individualized estimated of infection probabilities, to optimize pool allocation for non-uniform priors/prevalence. statistical dependencies (e.g., for family members) could be taken into account. finally, similar methods also permit addressing the problem of prevalence estimation. further details as well as some concrete design recommendations derived from our methods are available in the appendix. the motivation for this work was to help address the worldwide shortage of testing capacity for cov-sars-2. testing plays a major role in breaking infection chains, monitoring the pandemic, and informing public policy. countries successful at containing covid-19 tend to be those that test a lot. 13 on an individual level, availability of tests allows early and targeted care for high-risk patients. while treatment options are limited, it is believed that antiviral drugs are most effective if administered early on, since medical complications in later stages of the disease are substantially driven by inflammatory processes, rather than by the virus itself [23] . finally, large-scale testing as enabled by pooling and multiplexing strategies may be a crucial component for opening up our societies and economies. people want to visit their family members in nursing homes, send their children to school, and the economy needs to function in order to secure supply chains and allow people to earn their livelihoods. 14 however, the present work also poses some ethical challenges, of which we would like to list the below. the first family concerns the accuracy of the tests. indeed, when the number of tests and patients are equal, it is natural to compare the tpr/tnr of the individual test to the tpr/tnr of the individual results in our grouped test framework (obtained by marginalizing the posterior distribution). in some situations with unbalanced priors, the marginal tpr/tnr of some people in the group could be lower than the test tpr/tnr, even if the test will be more successful overall. however, reporting the marginal individual results gives doctors a tool to decide whether further testing should be needed; hence we cannot rule out that individuals might be worse off by being tested in a group. we furthermore show in the appendix that some designs are more fair than others, in that the individual performances are more equally distributed. the second family of concerns, directly resulting from the first, is the responsibility of the doctor when assigning the people to batches and giving them prior probabilities (using another model). the assignment of people in batches should be dealt with in a future extension of our framework, while the sensitivity of our protocols to priors should be studied in more depth. the adaptive framework may be more robust with respect to the choice of priors than the non-adaptive one. finally, the possibility of truly large scale testing may allow countries with sufficient financial resources to perform daily testing of large populations, with significant advantages for economic activity. this, in turn, could exacerbate economic imbalances. we use upper case letters exclusively for random variables (r.v.), except for mutual information i and entropy h. different objective functions. we have used the number of tests and samples as given, and then optimized a conditional entropy. however, from a practical point of view, other quantities are relevant and may need to be included in the objective, e.g. the expectation (over a population) of the waiting time before an individual is "cleared" as negative (and can then go to work, visit a nursing home, or perform other actions which may require a confirmation of non-infectiousness). semi-adaptive tests. instead of performing m consecutive tests, one could do them in k batches of respective sizes m 1 , ..., m k satisfying m 1 + ... + m k = m. adaptivity over the sequence of length k could be handled greedily as in algorithm 1, except that instead of selecting a single pool design d * , we would select m i designs at the i th step. we named this semi-adaptive algorithm the k-greedy strategy. further practical considerations. a good practical strategy could be to perform one round of pooled tests to disjoint groups every morning as individuals arrive at work, being evaluated during work hours. those who are in a positive group (adaptively) get assigned to a second pool design tested later, which can consist of a non-adaptive combination of multiple designs, tested over night. they receive the result in the morning before they go to work, and if individually positive, they enter quarantine. if the test is so sensitive that it detects infections even before individuals become contagious (which may be the case for pcr tests), such a strategy could avoid most infections at work. "bloom" denotes the use of the bloom encoding described in section 5 while "entropy" denotes the use of the conditional entropy / mutual information encoder described in section 4. in both comparisons, we observe that the entropy encoder yields more similar pr curves across patients, compared to the bloom encoder. dependencies between tpr, tnr and pool size. the reliability of tests may vary with pool size. in our notation, the outcome of the tests is a random variable that need not only depend on whether one person is sick (1 d,s >0 ) but it may also depend on the number of tested people |d| and the number of sick people d, s (cf. footnote 7); it could even assign different values of tpr and tnr to different people. the tpr may in practice be an increasing function of the proportion of sick people d, s /|d|. estimating prior infection probabilities. currently, we start with a factorized prior of infection that not only assumes independence between the tested patients but is also oblivious to the individual characteristics. we could, however, build a simple ml system that estimates the prior probabilities based on a set of features such as: job, number of people living in the same household, number of children, location of home, movement or contact data, etc. 15 those prior probabilities can then be readily used by our approach to optimize the pool designs, and the ml system can gradually be improved as we gather more test results. prevalence estimation. similar methods can be applied to the question of estimating prevalence. note that this is an easier problem in the sense that we need not necessarily estimate which individuals are positive, but only how many. c.1 theorem 1 statement: under the condition that the population never be stuck in a local optimum, the evolutionary strategy using the luby sequence (b, b, 2b, b, b, 2b, 4b, b, b, 2b, b, b, 2b, 4b, 8b , ...) for restarts yields a las vegas algorithm that restarts optimally [17] to achieve any target score threshold. proof. let us remind the main result we use on optimal restarts [17] : the simple luby sequence of times of restart given by (b, b, 2b, b, b, 2b, 4b, b, b, 2b, b, b, 2b, 4b, 8b , ...) is optimal (up to a log factor) for las vegas algorithms (i.e. randomized algorithms that always provide the correct answer when they stop, but may have non-deterministic running time). our theorem is a direct consequence of conceptually casting our problem as a las vegas algorithm: indeed, we seek to optimize a fitness function f . for a given threshold a > 0, we can replace the maximization of f by the condition f > a. applying the result of [17] for an exhaustive family of thresholds yields the desired result. we wish to invoke theorem 1 of [6] . in order to do so, we need to prove that the conditional entropy which we introduced in eq. (2) is adaptive monotone. concerning adaptive sub-modularity, we make it an assumption upon which our results is conditioned, and validate it numerically with high precision for small values of n (see appendix f). direct respective correspondence between our notations and that of [6] is given by: • pool designs d : items e; • test results t : realizations φ; • set d of selected designs : set e(π, φ) of selected items by policy π; this allows one to define, following definition 1 of [6] , the conditional expected marginal benefit of a pool design d given results t as: it represents the marginal gain of information obtained, in expectation, by observing the outcome of d at a given stage (this stage being defined by p s , i.e. after having observed test results t). adaptive monotonicity holds if ∆(d) ≥ 0 for any d. adaptive sub-modularity holds if for any two sets of results t and t such that t is a sub-realization 16 of t , for any pool design d: the below lemma concludes the proof. lemma. with respect to ∆ defined in eq. (9), adaptive monotonicity holds. proof. adaptive monotonicity is a consequence of the "information-never-hurts" bound h(x | y ) ≤ h(x) [5] . we are interested in the information i(s, t ) for a single bloom filter row with b cells. because each test in the row contains a disjoint set of patients, i(s, t ) is the sum of the information for each test (i.e. the t b random variables are independent and there are no cross terms). using the basic definition of h(t b ), we have that we use the fact that since the relationship between the ideal test results y b and the patient statuses s i is deterministic, conditioning on s i is the same as conditioning on y b . in particular, one can write pr(y b = 0) = (1 − p i ). observe that tpr = pr(t b = 0|y b = 0) and tnr = pr(t b = 1|y b = 1) and let ρ b = pr(y b = 1). this gives us a simple expression for pr[t b = t] and thus we approach the second term h(t b |s patients ∈b ) the same way. = − t∈{0,1} y∈{0,1} put β = (tnr log 2 (tnr)+(1−tnr) log 2 (1−tnr)) and α = (tpr log 2 (tpr)+(1−tpr) log 2 (1−tpr)). then, the information i(s, t ) is equal to information is concave in ρ: to show that there is a single, constant, and optimal probability for each group test to be positive, we prove that i(s, t ) is concave in ρ. it is sufficient to show that each term i(s, t b ) in the sum is concave in ρ b . taking derivatives, we have the second derivative is we wish to show that d 2 dρ 2 b i(s, t b ) ≤ 0, which we will do by proving that the two fractions are both positive. the squared terms in the numerators are positive, as is the expression (2tpr − 1)ρ b + 1 − tnr because tnr > 0.5. this leaves the (1 − 2tnr)ρ b + tnr term in the denominator. this term is linear in ρ b ∈ [0, 1], with a minimum of 1 -tnr. thus, i(s, t ) is concave. optimal value of ρ b : since the information is concave, there is an optimal value of ρ b that maximizes the information gain from each grouped test. since h(t b ) depends only on tpr, tnr and ρ b , it is easy to see that this value is constant and the same for all groups b. this proves the theorem. however, it is of practical importance to find or approximate the optimal value of ρ b . if one wanted to load balance a variety of (possibly different) priors into groups that have the optimal probability of testing positive, one needs to know the desired value of ρ b . we obtain the following equation by setting the derivative to zero: where c = (1 − 2tnr) + (2tpr − 1) + log(2)(β − α). one can obtain the optimal ρ b by numerically solving this equation. when tpr = tnr, we have c = 0 and the optimal value of ρ b = 0.5. we prove the theorem using an analysis that is similar to the one for standard bloom filters. the bloom filter decoder identifies a sample as positive if all of the pools containing the sample are positive. it is easy to see that the decoder cannot produce false negatives under perfect tests, because each positive sample will always generate a positive pool result. we now analyze the systemic false positives introduced by the pooling operation. each pool contains either n b or n b − 1 patients, where the latter situation occurs when b does not perfectly divide n and there are a few "leftover" elements. thus, any given sample will share a bin with up to n b − 1 other elements, each of which has independent probability ρ of testing positive. to correctly identify a sample as negative, we require that all of these n b − 1 samples also test negative. hence the probability that our sample will not collide with a positive sample is at least the -1 arises from the fact that the sample cannot collide with itself. this analysis holds for a single bloom filter row, but we have g independent opportunities to land in a negative pool. the rows are independent because independent random hash functions are used to form the groupings. the probability that we collide with a positive in all g groups is at most this expression gives the probability that we fail to identify the sample correctly. we want to bound the failure probability p f and choose parameters that minimize the bound. note that we replaced n b − 1 with n b -the inequality still holds because (1 − ρ) < 1. the optimal dimensions for the bloom filter come from minimizing the upper bound. we use the relation m = b × g to put p f in terms of m and g. we find that the optimal g = m n ρ log 2. notations. we use tildax to denote the estimation of a quantity x. error bounds and confidence levels. given a test result t ∈ {0, 1} m , and a patient i ∈ {1, ..., n}, we seek to estimate p [s i |t], i.e. the probability of patient sample s i being positive. we can rewrite: where we defined λ := p [s i , t] and µ := p [s i , t]. hence, we seek to estimate λ, resp. µ. we use the term "code space" to refer to the space {0, 1} m of encodings of secrets s ∈ {0, 1} n . we write λ and µ in terms of the joint distribution of secrets s, encodings c, and results t. summing across the code space yields: suppose that we have (under-)estimatesã andb such that 0 ≤ max c (a(c) −ã(c)) ≤ ε and 0 ≤ c (b(c) −b(c)) ≤ ε. later, we will describe how to obtain these estimates. for now, observe that we can (under-)estimate λ = c a(c)b(c) withλ := cã (c)b(c), with the following error bound 17 : and similarly 0 ≤ µ −μ ≤ 2ε, which would imply 0 ≤ (λ + µ) − ( which concludes the proof that we can estimate p [s i |t] with error less than 5ε/p [t], wherep then, similarly, letã this concludes our presentation of the estimatorsã(c) andb(c). note that we presented a confidence interval together with a bound on its size, i.e. we showed that the true value p [s i |t] is within an interval that depends on the observed quantityp [s i |t]. however, we can also provide an interval for the observed quantity as a function of the true value: whose size can be bounded by: where we assumed ε < λ/4 to justify that 3λε 2 (λ+µ) 3 1 1− ε λ+µ < ε λ+µ . one might also be interested in the error rather than a confidence interval. we will now present an algorithm that efficiently computes these estimators. in our algorithm, a( ) is the number of secrets with at most k nonzeros, c( ) is number of codes produced by this restricted set of k-sparse secrets, and b( ) is a set of probable ideal codes for the potentially-corrupted output t that we observe. 1 preprocessing: (independent of results t) 2 compute k such that f (k) < ε and initialize c = ∅; 3 enumerate all the codes c := enc(s) for s with less than k positives a ; 4 use these codes to approximatep [s i , c] andp [s i , c] using the formula forb(c) in eq. (42). store the results in c; 5 query: (dependent upon results t) 6 compute p := i t i , n := m − p and a(c) (see eq. (48)) for f p ≤ p , f n ≤ n ; complexity analysis. since the outcome of a test t is conditionally independent to s w.r.t. c, we can pre-compute all encodings c := enc(s) for s belonging to the reduced search space of size a(ε) := k−1 i=0 n i . saving all these resulting encodings with a hashmap or a set structure gives a space of complexity proportional to c(ε) ≤ a(ε), since the output function image of an input set is always smaller than (or equal to) the size of the input set. finally, at query time, we seek to estimate p [s i |t]. note that we have pruned two search spaces: the space of encodings of a(ε) many secrets, reduced from 2 m to c(ε), and the space of codes c such that for our given t, prob[f p ][f n ] > ε, reduced from 2 m to b(ε). given a test result t, we can compute n, p in o(m) operations, which then allows us to compute b(ε) for this t. also note that we approximate p [s i , t] via cã (c)b(c). sinceã(c) = 0 for c such that t doesn't belong to the reduced test results space of size b(ε), we can choose to perform this sum on either this set, or the reduced code space of size c(ε): whichever is the smallest. this is where the denomination "meet-in-the-middle" comes from. the c++ code can be used in the browser through an interactive webassembly demo: https://bloom-origami.github.io/ the following features are implemented: • bloom assay generation • greedy adaptive strategy simulation • design optimization using genetic algorithms • posterior decoding using mitm our designs assume that the prevalence ρ is known, at least approximately. however, we can also use our bloom filter design to estimate the prevalence in the overall infected population. when we randomly and independently sample an individual from the population, they have probability ρ of being infected. the prevalence estimation problem is to determine ρ using as few tests as possible. here, we assume perfect tests to simplify the analysis. of course, one could individually test a large number of people from the population and report the fraction of positive test results. the challenge is that if we screen individuals, we end up with a random variable for which the mean to variance ratio is unfavorable. consider a random variable x ∈ {0, 1} with e[x] = ρ and variance var[x] = ρ − ρ 2 = ρ(1 − ρ). the error of the empirical average of m individual tests is the relative error is 1−ρ ρ . clearly this is minimized for ρ = 1. unfortunately, this value is entirely useless since it corresponds to the situation where every test returns positive. in practice, we encounter the unfortunate situation of ρ << 1 where the relative error diverges. under a naive random sampling approach to prevalence estimation, a very large number of tests are required. to amend this situation, it is beneficial to increase the probability of a positive test by testing multiple candidates at once. our pooled tests are no longer positive with probability ρ but with probability q = 1 − (1 − ρ) k , where k is the number of samples combined in a single pool. knowing q, we can solve for ρ via we will use the central limit theorem and the delta method to show that we need fewer bloom filter pooled tests than random individual tests to estimate the prevalence. the central limit theorem states that suppose we combine k samples into each bin. now x is the test status of the bin and it is positive with probability q = 1 − (1 − ρ) k . hence µ = q and σ 2 = q(1 − q). use the delta method with space/time trade-offs in hash coding with allowable errors a note on double pooling tests non-adaptive group testing: explicit bounds and novel algorithms graphconstrained group testing elements of information theory adaptive submodularity: theory and applications in active learning and stochastic optimization near-optimal sensor placements in gaussian processes temporal dynamics in viral shedding and transmissibility of covid-19 non-adaptive hypergeometric group testing efficiently decodable non-adaptive group testing variational probabilistic inference and the qmr-dt network non-adaptive group testing in the presence of errors probabilistic graphical models: principles and techniques pooling of samples for testing for sars-cov-2 in asymptomatic people. the lancet infectious diseases counter braids: a novel counter architecture for per-flow measurement optimal speedup of las vegas algorithms nonadaptive group testing with random set of defectives bloom-filter inspired testing of pooled samples (and splitting of swabs!) probability and computing: randomization and probabilistic techniques in algorithms and data analysis reconstructed diagnostic sensitivity and specificity of the rt-pcr test for covid-19. medrxiv fact -frankfurt adjusted covid-19 testing -a novel method enables high-throughput sars-cov-2 screening without loss of sensitivity. medrxiv the trinity of covid-19: immunity, inflammation and intervention model counting of monotone cnf formulas with spectra paper-origami-based multiplexed malaria diagnostics from whole blood evaluation of covid-19 rt-qpcr test in multi-sample pools << aux ( { } ) << ' ' << aux ( { t e s t 1 } ) << ' ' 131 << aux ( { t e s t 2 } ) << ' ' << aux gary bécigneul is funded by the max planck eth center for learning systems. benjamin coleman and anshumali shrivastava are supported by nsf-1652131, nsf-bigdata 1838177, afosr-yipfa9550-18-1-0152, amazon research award, and onr brc grant for randomized numerical linear algebra. key: cord-002626-jzwwses4 authors: kaul, karen l.; sabatini, linda m.; tsongalis, gregory j.; caliendo, angela m.; olsen, randall j.; ashwood, edward r.; bale, sherri; benirschke, robert; carlow, dean; funke, birgit h.; grody, wayne w.; hayden, randall t.; hegde, madhuri; lyon, elaine; murata, kazunori; pessin, melissa; press, richard d.; thomson, richard b. title: the case for laboratory developed procedures: quality and positive impact on patient care date: 2017-07-16 journal: acad pathol doi: 10.1177/2374289517708309 sha: doc_id: 2626 cord_uid: jzwwses4 an explosion of knowledge and technology is revolutionizing medicine and patient care. novel testing must be brought to the clinic with safety and accuracy, but also in a timely and cost-effective manner, so that patients can benefit and laboratories can offer testing consistent with current guidelines. under the oversight provided by the clinical laboratory improvement amendments, laboratories have been able to develop and optimize laboratory procedures for use in-house. quality improvement programs, interlaboratory comparisons, and the ability of laboratories to adjust assays as needed to improve results, utilize new sample types, or incorporate new mutations, information, or technologies are positive aspects of clinical laboratory improvement amendments oversight of laboratory-developed procedures. laboratories have a long history of successful service to patients operating under clinical laboratory improvement amendments. a series of detailed clinical examples illustrating the quality and positive impact of laboratory-developed procedures on patient care is provided. these examples also demonstrate how clinical laboratory improvement amendments oversight ensures accurate, reliable, and reproducible testing in clinical laboratories. the field of pathology offers the opportunity to better understand the science behind the mechanisms of disease, to lead innovation of new diagnostic technologies, to provide quality oversight of these developments, and to have enormous impact on the lives of patients every day. patients benefit from laboratory medicine testing throughout their lives as every medical decision can be impacted by the result of a laboratory test. laboratory results constitute the majority of data in a patient's electronic medical record, and our procedures dictate the majority of downstream medical decisions for patients. 1, 2 clinical laboratory medical professionals have the unique responsibility to patients for assessing the performance of technologies in providing the most accurate information to ensure the most appropriate and efficient course of care. it is an understatement to say that the medical field is rapidly changing. technology and new genomic data developed as a result of the human genome project are leading to an explosion of knowledge applicable to individual patient care; this is the promise of precision medicine. we must continue to innovate and integrate novel diagnostic tools, genomic information, and new treatments into clinical practice. considerable technologic advances allow clinical laboratory professionals to offer new testing that provides more information than ever before and often within a time frame that allows rapid patient care and better outcomes. next-generation sequencing (ngs) in genetics and oncology, maldi-tof in the clinical microbiology laboratory, and a variety of mass spectroscopy-based methods in clinical chemistry now allow precise and rapid testing with demonstrated improvements in patient care. it is often thought that when "laboratory tests" are done to reach a diagnosis, they are done with a kit or on a machine, but in fact, most are procedures done with the direct involvement of a laboratory professional or physician. laboratory tests are generally not fully encompassed by a "test kit" but often start with the pathologist examining the tissue section, bone marrow aspirate, or gram stain and determining what additional information is needed to provide the clinician with the best scenario so that the patient can be treated most effectively. often, clinical laboratory professionals lead the development and optimization of these approaches to improve care and fill a clinical need, and their involvement in the process helps ensure that the highest quality standards are maintained. ongoing development of these novel methods is critical to medicine and must be done with the highest level of safety and accuracy, yet simultaneously addressing growing demands to lower the cost of medical care in the united states. the regulatory oversight of laboratory-developed testing procedures (ldps) has a critical impact on patients' access to testing and diagnosis. currently, there is national discussion regarding the optimal regulatory oversight of laboratory tests and procedures that will balance the needed accuracy and safety with ensuring that new tests are made available to patients safely and expeditiously. oversight provided by the clinical laboratory improvement amendments (clia) and the food and drug administration (fda) currently exists in the clinical laboratory. it must be noted that a spectrum of testing activities take place in clinical laboratories. these activities range from complex procedures, such as evaluation of a tissue biopsy, classification of a tumor, or culture of a microbe, to more automated or standardized tests for which an fdaapproved kit is utilized. all of these activities take place under the direction of a laboratory professional and in accordance with the detailed requirements of clia. the fda review process is well suited for diagnostic assays that are commercially marketed as kits designed to operate across a spectrum of laboratory settings, in laboratories with a range of expertise. currently, fda approval or clearance requires prospective clinical trial data and a lengthy review process, and thus, fda approval takes considerable effort and time. the investment needed for this process impacts the types of tests, and also sample types, that are submitted for approval, as manufacturers must recover their investment afterward. additionally, fda approval for an in vitro diagnostic (ivd) specifies the sample type, clinical purpose, and other aspects of performance of the assay. when a new clinical need for the ivd arises, the need to use a new sample type arises, or any needed modification that arises in response to the ongoing and sometimes rapid advancements seen in medicine, incorporation of these improvements renders the test an ldp, with the validation needs of an ldp under clia. laboratories can be caught between the regulations and the needs to best serve their patients. the clia provide for oversight of clinical laboratories by defining all aspects of laboratory operation, including the quality programs required for clinical tests, personnel requirements, and the validation requirements for ldps. the clia certification of laboratories can be accomplished through several deemed agencies such as the college of american pathologists (cap) or the new york state department of health (nys-doh). these organizations provide operational guidelines and perform on-site inspections based upon checklists developed via consensus of laboratory experts, which include hundreds of pages of requirements and data points. compliance with clia has been built into clinical laboratory operations, mechanisms for data collection, training, proficiency testing (pt), and test implementation. laboratories are subject to unannounced inspections and must demonstrate satisfactory performance characteristics for any test offered to ensure that results are accurate. for testing not reviewed by the fda, such as ldps, laboratories must go through a rigorous validation process before offering the test for clinical use. under clia, the validation data collected by these laboratories are subject to ongoing peer review. in particular, organizations with deemed status overseeing laboratories (such as cap and nysdoh) conduct rigorous peer-inspection using detailed criteria developed specifically for molecular pathology. laboratories also participate in required pt to demonstrate assay quality, with interlaboratory data sharing and assessment (most often led by the cap) that leads to ongoing broad improvement in ldps. recent public documents have presented examples of specific ldps in which the clinical validity of the test, the interpretation or use by clinicians, or the reproducibility of the laboratory data was questioned, raising concern about the safety and efficacy of this category of tests. 3, 4 enhanced regulatory oversight has been proposed to help ensure laboratories are delivering meaningful, high-quality results to patients. however, additional regulations have the potential to slow innovation and to limit and delay patient access to novel testing that impacts their clinical care, as well as add redundant reporting efforts and significant cost. expanded oversight of laboratories through a revised clia process has also been proposed (http://www.cap.org/showproperty?nodepath=/ucmcon/con tribution%20folders/webcontent/pdf/2015-cap-ldt-legisla tive-proposal.pdf. accessed april 27, 2017). 5 in an effort to illustrate the need for and positive impact of laboratorydeveloped procedures on patient care, the following series of vignettes have been assembled. in each case, timely and highquality laboratory-developed procedures filled a key clinical need. the impact on patient care has been summarized. as possible, data describing interlaboratory comparisons are provided to demonstrate the quality of these ldps, as validated and performed in accordance with clia. these examples also serve to highlight the quality efforts and interlaboratory comparisons that take place before ldps are offered by laboratories for clinical use; these activities and data are often unknown to the clinicians who utilize these testing services. table 1 provides a guide to the examples included and summarizes the impact of each assay along with key points about its utility. encephalitis is the most serious complication of herpes simplex virus (hsv) infection. 6 while rare, hsv is commonly included among causes of viral encephalitis. since hsv infection can be treated effectively with acyclovir, it is critical to rapidly and accurately establish the diagnosis and initiate treatment to reduce morbidity and mortality. culture of cerebrospinal fluid (csf) is insensitive for the diagnosis of hsv encephalitis, so brain biopsy, a very invasive procedure with significant morbidity, was historically required to make a diagnosis, with results not available for days afterward. several studies demonstrated that detection of viral dna by polymerase chain reaction (pcr) using csf samples performed equivalently to brain biopsy, including a landmark study in 1995. [7] [8] [9] given the ease of collecting csf samples, the lower risk of complication, the lower cost, and speed of results, pcr became the standard of care for diagnosing hsv cns infections in the mid-1990s, with ldps used for nearly 20 years. it was not until 2014 that the first fda-cleared pcr test for the diagnosis of hsv encephalitis became available, with a second test cleared in 2015. laboratory-developed testing procedures dramatically improved the quality of care for many patients and continue to be used successfully today, as the 2 cleared tests have limitations, requiring specific instrumentation not standard in many laboratories. additionally, one of the tests is highly multiplexed, which may not be appropriate in all clinical situations. herpes simplex virus can also cause infections in neonates, with a frequency of 1:3500 to 1:5000 deliveries in the united states. the infection is acquired by exposure to maternal genital secretions. the presentation of neonatal hsv varies and includes skin, eye, and mouth infection, with encephalitis and disseminated disease in over 50% of cases. again, rapid diagnosis is needed to prevent morbidity and sequelae from encephalitis. the diagnosis of encephalitis is made via pcr of csf samples, while testing of plasma or serum is critical to diagnose disseminated disease. the ability to use plasma or serum is very important as it may be difficult to obtain enough csf from a newborn to assess all diagnoses in the differential. although there are 2 fdacleared assays to test csf for hsv, there are no assays cleared for testing serum and plasma. laboratory-developed testing procedures continue to play a critical role in the diagnosis and management of disseminated hsv infection in newborns. additionally, ldps have performed with a high degree of accuracy and interlaboratory agreement. blinded pt samples were distributed to 383 laboratories as part of a cap pt survey for analysis of hsv; 91.6% correctly identified hsv-2, and 7.8% identified hsv, but not noting the subtype; an overall accuracy of 99.4% was obtained. previous proficiency surveys showed similar results for samples containing hsv1. over 90% of the laboratories used ldps. 10 bk virus infection is very common, with a seroprevalence in the adult population of *90%. after primary infection, the virus colonizes the renal and urinary tracts; healthy individuals will occasionally shed virus in their urine without consequence. however, in renal transplant recipients, bk virus is the major cause of polyomavirus-associated nephropathy (pvan), putting 1% to 15% of kidney transplant patients at risk of premature allograft failure. 11 given the lack of effective antiviral therapy for bk virus, the key to preventing allograft loss is to identify at-risk renal transplant recipients early and reduce immunosuppressive therapy before pvan develops. reduction of immunosuppressive therapy helps control viral replication and in most cases prevents the development of pvan. 12 there is a critical balance between too much immunosuppressive therapy, which can lead to pvan, and too little immunosuppression causing rejection. the ability to monitor bk virus levels in the blood allows for informed clinical decisions. studies have shown that monitoring patients with viral load testing during the first 2 years after transplant can dramatically reduce the development of pvan. consensus guidelines recommend that screening for bk replication be performed at least every 3 months during the first 2 years posttransplant and then annually until the fifth year posttransplant. 13 when the plasma viral load value rises above a threshold (10 000 to 50 000 copies/ml), a renal biopsy may be performed to assess for pvan, and immunosuppressive therapy is reduced based on the results of the biopsy. viral load testing is also done if there is an increase in serum creatinine, as the level of bk virus aids in distinguishing rejection from pvan. although bk viral load testing has been the standard of care for several years and is used in transplant centers across the country, there is no fda-cleared or fda-approved test available. all testing is performed using ldps. if these ldps were not available, most cases of pvan would not be identified promptly, leading to negative patient outcomes such as allograft loss or rejection. cytomegalovirus (cmv) can cause a wide range of complications among both solid organ transplant and hematopoietic stem cell transplant recipients, as well as in other immunocompromised patients. cytomegalovirus has a high seroprevalence, and large numbers of transplant patients experience reactivation or primary infection, with probability increasing based on pre-transplant serostatus, severity of pretransplant conditioning, and allograft relatedness. 14, 15 although infection can be subclinical, high or increasing viral load signals increased the risk of symptomatic disease, which can range from relatively mild constitutional symptoms to severe end-organ infection or potentially fatal disseminated disease. preemptive therapy of high-risk patients based on the detection of increasing cmv load in peripheral blood was shown to be effective in the 1990s 16, 17 ; however, the first fda-approved ivd test did not appear on the market until 2012. 18 in the intervening years, laboratory-developed assays played a critical role in bridging the gap. the presence of such methods and their adoption across the country enabled the routine use of cmv screening for asymptomatic patients in transplant centers soon after data supported its utility. the availability of such methods has likely saved many lives over the years, supporting early diagnosis, preemptive treatment strategies, and the assessment of therapeutic treatment efficacy. 14, [18] [19] [20] the widespread use of ldp cmv quantitative methods produced a generation of transplant physicians comfortable with the use of such methods and changed the epidemiology of posttransplant cmv disease, markedly reducing the incidence of early disease in such patients. over the years of ldp use, numerous studies focused on continuous improvement and optimization of methods, 21 including the development of international quantitative standards in 2010. 22 the ability to rapidly incorporate advances in technology (including the advent of real-time quantitative methods) has been demonstrated by the improvement in sensitivity and other performance characteristics of such tests over time. the data accumulated throughout these experiences informed the development of the first commercially available cmv ivd assays. in fact, the absence of ldps and the vast clinical and laboratory experience that they provided likely would have delayed the availability of commercial methods, and their performance characteristics taken longer to optimize to today's levels. human papilloma virus (hpv), the causative agent of genital warts, has been implicated in the development of *99% of cervical cancers. 23 more than 200 hpv genotypes have been described, 24 and approximately 40 are capable of infecting the human genital tract. 25 of these, a relative few are known to cause cervical cancer and other malignancies; these can be identified by genotyping. today, 4 assays are fda approved for detecting high-risk hpv genotype strains in cervical specimens. [26] [27] [28] [29] these tests are routinely used to confirm the presence of an hpv infection, screen for cervical cancer, and refer cases with an indeterminate cytology examination. 30 during the past decade, a rise in oropharyngeal squamous cell cancer, primarily in 40 to 55-year-old caucasian males with limited alcohol and tobacco exposure, has been described. 31 unexpectedly, investigators discovered the frequent presence of high-risk hpv genotype strains in these lesions. human papilloma virus-positive head and neck cancer is biologically distinct from hpv-negative disease. patients with hpv-driven tumors have a significantly better prognosis, including response to chemoradiation therapy and overall survival, compared to hpv-negative patients. 32 therefore, testing head and neck cancer specimens for high-risk hpv genotypes has become standard of care and is used to guide treatment. 33, 34 however, the hpv tests that are fda approved for cervical specimens have not been approved for head and neck cancer, leaving a critical gap for patient care. clinical laboratories have thus had to develop new assays or modified the existing fda-approved ones to detect high-risk hpv genotypes in head and neck cancer specimens. inclusion of patients in several clinical trials is based on these ldps (www.clinicaltrials.gov. accessed april 27, 2017). without laboratory-developed tests, patients with head and neck cancer cannot benefit from personalized treatment strategies. clinical laboratories must be highly vigilant for infectious disease outbreaks, since they are likely the first to recover the pathogen and recognize the potential for an outbreak. recent examples of emerging or reemerging pathogens causing substantial human morbidity and mortality include avian influenza virus ("bird flu"), chikungunya virus, ebola virus, middle eastern respiratory syndrome virus, severe acute respiratory syndrome virus, and zika virus. 35, 36 at the time of their emergence, no fda-approved tests were available to detect any of these pathogens. in response, many clinical laboratories developed, validated, and implemented the ldps needed to care for patients at their institutions. 37, 38 these assays were based on extensive data sets and reported in peer-reviewed journals, supporting their quality claims. since performance characteristics such as analytic sensitivity (limit of detection) may vary between different assay designs, 39 as was recently discussed for several different zika virus tests, 40 these sorts of collaborative efforts are essential. in many cases, laboratories leading these efforts collaborated with one another to share validation materials, perform interlaboratory comparisons, and exchange blinded testing samples (http://www.usatoday.com/story/ news/2016/02/23/texas-hospitals-develop-rapid-zika-test/ 80776382/. accessed april 27, 2017). while perhaps not ideal, there is an urgent need for a more integrated and coordinated mechanism for rapid diagnosis of novel infectious agents that incorporates both the public health and hospital laboratories. the recent emergence of zika virus, which has been linked to severe birth defects, 41 underscores the need for clinical laboratories to have rapid access to diagnostic tools. when the secretary of health and human services declared zika virus to be a public health emergency, the first fda emergency use authorization for zika virus testing was granted to a test available only to the public health laboratory system, not to hospital laboratories on the front lines of patient care (https://www.fe deralregister.gov/documents/2016/03/28/2016-06888/authori zation-of-emergency-use-of-an-in-vitro-diagnostic-device-fordiagnosis-of-zika-virus. accessed april 27, 2017). as a result, the public health system quickly became overwhelmed, as it has been during past outbreaks, such as influenza. 42 in many areas, turnaround times for zika virus testing exceeded 4 to 8 weeks (http://www.nytimes.com/2016/09/13/us/zika-testdelays-florida-pregnant.html?_râ¼0. accessed april 27, 2017, http://medicalxpress.com/news/2016-10-lab-constraints-zikaresults.html. accessed april 27, 2017, http://www.miamiher ald.com/news/health-care/article104856631.html. accessed april 27, 2017). these delays critically impact patient care, particularly for pregnant women with possibly affected fetuses. in comparison, many hospital-based and national reference laboratories are able to report results within hours (http:// www.usatoday.com/story/news/2016/02/23/texas-hospitalsdevelop-rapid-zika-test/80776382/. accessed april 27, 2017, http://www.nytimes.com/2016/09/13/us/zika-test-delays-flor ida-pregnant.html?_râ¼0. accessed april 27, 2017). for example, in miami-dade florida, where more than 1000 people have been confirmed zika positive and the virus is circulating in the local mosquito population, the public health laboratory system encountered a backlog of nearly 1000 untested specimens (http://www.miamiherald.com/news/health-care/arti cle104856631.html. accessed april 27, 2017). diagnostic assays for zika and other emerging pathogens will continue to evolve, 43 but it is clear that rapid identification of pathogens during other outbreaks facilitates rapid treatment and appropriate isolation of patients, leading to improved patient outcomes and potentially slowing the spread of infections such as influenza (http://www.cdc.gov/flu/professionals/diagnosis/molecu lar-assays.htm. accessed april 27, 2017). implementation of the first rapid diagnostic tests for influenza was directly associated with reduced length of hospital stay, decreased mortality, and reduced costs. 44 access to diagnostic tests, early in the course of an outbreak and in hospital laboratories, has a positive public health impact. the kras gene encodes a gtpase critical in signal transduction that is known to be mutated in a wide range of tumor types. 45 a landmark study presented at the american society of clinical oncology (asco) meeting in 2007 demonstrated that patients with metastatic colorectal cancer harboring a mutated kras failed to respond to targeted therapy with cetuximab. 46, 47 at the time, there were no clinical tests for kras mutations available. molecular pathology laboratories worked quickly to fill this need, to define the best analytic approaches, and to ensure that test results done in one laboratory matched those done in another. [48] [49] [50] within a few months, laboratories were able to offer fully validated kras assays that worked reliably and were safe for patient care. under clia, the validation data collected by these laboratories were subject to ongoing peer review, and laboratories participate in ongoing pt to demonstrate consistent assay quality. in 2009, the asco and the national comprehensive cancer network (nccn) recommended mutational profiling of kras exons 12 and13 before institution of anti-epidermal growth factor receptor (egfr) therapy for patients with metastatic colorectal cancer; it became standard of care to assess formalin-fixed paraffin-embedded tumor tissues from patients with metastatic colon cancer for kras mutation status. 51, 52 it was not until 5 years later that the fda cleared qiagen's therascreen kras test, designed to detect the presence of 7 mutations in the kras gene in colorectal cancer. 53 by this time, new data demonstrated that kras analysis alone was not enough; mutation analysis of other genes was necessary, 54, 55 and the fda-approved assay was already inadequate for patient testing compliant with national treatment guidelines. without ldps, an estimated 10% of patients with non-exon 2 kras mutations would be overtreated with expensive anti-egfr therapy. 56 the use of ldps has thus persisted in clinical practice to provide patients with the most complete information to guide treatment. in the cap kras-b-2015 mailing, 204 laboratories reported results from testing 3 blinded proficiency-testing specimens. the specimens contained recurring somatic mutations in kras exons 12 or 13 (nm_004985.3), c.35g>t (p.g12 v), and c.38g>a (p.g13d). an acceptable response was reported by over 96% of the laboratories for both mutations (197/204 for p.g12v and 195/202 for p.g13d). the vast majority of reporting laboratories utilized ldps. 57 kras and ras family gene mutation analysis is also critical in the management of patients with non-small-cell lung cancer (nsclc) and other tumors, 58 for which fda approval of kits has not occurred; ldps or off-label use of kits is required. braf belongs to a family of serine-threonine protein kinases that participate in signal transduction cascades involving ras, raf mek, and erk family members. this pathway is important in the regulation of normal cell proliferation and differentiation. 59 activating mutations in braf can lead to increased proliferation and prolonged cell survival in a variety of tumor types. 60 laboratories will typically test for braf mutations in low-grade gliomas (for diagnosis), colorectal cancer (to establish the sporadic origin of msi-h tumors and response to anti-egfr therapy), hairy cell leukemia (for diagnosis), lung adenocarcinomas (to predict response to therapy), thyroid cancer (for preoperative detection of thyroid cancer in fna samples and prognosis in papillary thyroid carcinoma), or melanoma (to predict response to braf kinase inhibitors). for one of these indications, malignant melanoma, fdaapproved companion diagnostics are available. vemurafenib (zelboraf) is a kinase inhibitor indicated for the treatment of patients with unresectable or metastatic melanoma with braf v600e mutation. dabrafenib (tafinlar) is a kinase inhibitor indicated as a single agent for the treatment of patients with unresectable or metastatic melanoma with braf v600e mutation or in combination with trametinib (mekinist) for the treatment of patients with unresectable or metastatic melanoma with braf v600e or v600k mutations (www.fda.gov. accessed april 27, 2017). the cobas 4800 braf v600 mutation test (roche molecular systems, pleasanton, ca, usa) sporadically cross reacts with braf v600k and braf v600d mutations 61,62 and thus is neither sensitive for braf v600 mutations nor specific for braf v600e mutations, confounding accurate outcome evaluations and preventing its usefulness in selecting patient for tafinlar therapy. the thxid braf kit (biomerieux, boston, ma, usa) does detect both braf v600e and braf v600k mutations (but not other braf v600 mutations) and is necessary to distinguish between alternate therapeutic options (single agent vs combination therapy). it is important to note that increased cell proliferation has been seen in tumors treated with braf inhibitors with normal braf. 61 it is therefore important to identify all braf activating mutations to assist in the selection of appropriate therapy. the fda-approved assays are therefore not adequate for current clinical needs. in a 2015 european multicenter study, 62 420 consecutive tumor samples of histologically proven melanoma tumor tissue were assessed for braf mutation status by the cobas system and a variety of laboratory developed procedures. testing was concordant for 392 (93.3%) of 420 samples but discordant for 28 (6.7%). among the discordant cases, 11 had invalid results (8 samples with the cobas and 3 with ldps). of 10 samples with braf v600 mutations detected by the ldps (but not by the cobas mutation test), 5 were v600k, d, or r mutations, and 2 contained only 20% tumor cells. for the 7 samples with braf v600 mutations detected by the cobas but not by the ldps, 4 were confirmed with retesting, 1 was not mutated, and 2 were considered invalid results. this study documents similar results between the performance of braf ldps and ivds. further data can be gathered from the cap pt program. in the braf-b-2015 cap proficiency survey, 173 laboratories reported on the detection of v600e and other braf mutations by a variety of analytic methods. two of the well-characterized specimens in the proficiency test contained braf v600e alleles, and 98.8% and 99.4% of laboratories correctly reported the mutation. the majority of these laboratories used ldps. 63 there are no currently available braf mutation tests approved for use in other tumor types such as those mentioned above, and use of the existing ivd tests would constitute offlabel use and hence ldps. microsatellite instability is the presence of hypermutability in repetitive dna sequences resulting from impaired dna mismatch repair. microsatellite instability can be an inherited or acquired feature of tumors. microsatellite instability occurs in approximately 15% of all colorectal carcinomas and is a consistent feature of colorectal and other tumors in patients with lynch syndrome. 64 tumors are classified as showing high levels of msi (msi-h phenotype) if 2 or more of 5 microsatellite markers (or !30%) exhibit instability, a microsatellite-stable phenotype if none of the markers show instability, and an msilow phenotype if only 1 of 5 or less than 30% of the markers show instability. 65 studies have confirmed that the appropriate cutoff for determining an msi-h phenotype is the finding of instability in 30% or more of the markers tested. the finding of an msi-h phenotype is consistent with the presence of defective dna mmr in the tumor. 66 the finding of an msi-h phenotype in a crc increases the likelihood that the patient has ls but is not specific for ls. the definitive establishment of a diagnosis of ls requires the finding of a pathogenic germline mutation in one of the dna mmr genes. additional testing that can be offered to determine whether a patient with an msi-h crc is likely to have ls includes testing the tumor for dna mmr protein expression using ihc, braf v600e point mutation analysis (since braf-mutated msi-h colorectal carcinomas are known to have sporadic mmr gene mutations), and mlh1 promoter hypermethylation. studies have shown that an msi-h phenotype is a favorable independent prognostic indicator in patients with crc. 67 in addition, some reports indicate that msi-h tumors may not be responsive to 5-fluorouracil-based therapies. 68 recent draft guidelines developed collaboratively by 4 professional societies recommend that deficient mismatch repair/microsatellite instability testing must be performed in all colorectal cancers for prognostic stratification and identification of patients with lynch syndrome. 69 although numerous laboratories offer msi testing using ldps, there are currently no fda-approved tests for the evaluation of microsatellite instability. a summary of cap pt results 70 demonstrates excellent performance of laboratories participating in the msi proficiency surveys. this good performance of laboratories over the years may be partly due to the educational nature of the cap pt, which provides laboratories with an external mechanism to monitor the quality status of their testing. epidermal growth factor receptor is a membrane-bound tyrosine kinase which activates several signaling pathways known to be altered in human cancer, including nsclcs. [71] [72] [73] nonsmall cell lung cancer tumors with egfr-activating mutations are responsive to gefitinib and erlotinib, small molecule tyrosine kinase inhibitors of egfr. 74, 75 the fda approval of anti-egfr therapies based on clinical trial outcomes data resulted in the need for clinical laboratories to test tumor tissue for the egfr-sensitizing mutations in order for patients to be eligible for treatment. with no fda-approved "companion" diagnostic test on the market, clia-licensed laboratories developed and validated ldp tests for the 2 most common egfr mutations as early as 2004. 73 the fda followed with approval of the roche cobas egfr mutation test in 2013 along with the qiagen therascreen egfr rgq kit. both assays tested for the exon 19 deletions and the exon 20 l858r point mutation. of note was that each test was approved for specific therapeutic indications and specimen types. as new drugs became available, approval for new claims was needed. laboratory-developed procedures continue to be the method of choice due to the limitations of claims made for fda-approved assays and performance characteristics, including types of mutations being detected. 76 clinical laboratories participate in twice-yearly proficiency test challenges of unknown samples that must be analyzed and reported, with results graded and compared to other laboratories performing the testing. in the cap egfr-b-2015 proficiency test, 192 laboratories reported results from testing 3 unknown proficiency-testing specimens in late 2015. the specimens connote: some laboratories do not test for certain mutations, hence, the denominator is often less than 192. 77 as patients being treated with these new targeted anti-egfr therapies began to relapse, further studies revealed that egfr harbors both sensitizing and resistance mutations. the clia laboratories have demonstrated the ability to detect all mutations in the egfr gene as well as in other genes using ngs assays to sequence panels of cancer-related genes. 78 this panel approach allows the laboratory to provide the oncologist with a more comprehensive profile of the tumor, using a costeffective technology that makes maximal use of small tissue samples and thus makes treatment strategies more effective. in addition, the time saved by testing a broader panel of gene targets can result in better outcomes for the patient as well as fewer adverse drug reactions. for the payer, the cost savings of such an approach versus algorithm-based testing with single gene assays is significant. currently, there are no fdaapproved sequencing assays for egfr mutation status, and ldps are solely used for the detection of these mutations that define therapy selection. the complexity of cancer biology and the ever-evolving therapeutic approach to management of the patient with cancer more often requires expanded knowledge of the tumor beyond single-gene mutation status. over the past several years, the laboratory's ability to multiplex testing for several genes or genetic variants has been limited by the available technologies. next-generation sequencing or massively parallel sequencing has allowed the laboratory to provide a more comprehensive genomic profile of tumor cells in a single assay than previous methods. the ability to detect numerous mutations in multiple genes results in information that can allow the oncologist to develop a more accurate treatment strategy including therapies selected based on a "responsive" tumor profile and those not selected based on the presence of resistance mutations. [79] [80] [81] instrumentation from thermo fisher (thermo fisher life-technologies, carlsbad, ca, usa) and illumina (illumina, san diego, ca, usa), the personal genome machine (pgm), and miseq, have made ngs suitable for routine clinical laboratory testing. [82] [83] [84] in 2015, the miseq dx obtained fda approval. despite this approval and the routine use of ngs in ldps setting, there are no currently fda-approved ngs tests for application in oncology. although many drug package inserts require or allude to the use of a companion diagnostic for eligibility, very few companion diagnostic tests are fda approved and none using ngs technology. as an ldp, clinical laboratories are required to demonstrate rigorous performance criteria for wet-bench testing and analysis pipelines to ensure the test is functioning properly for its intended clinical purpose. 73, [85] [86] [87] most ngs testing for therapeutic selection in cancer consists of panels of genes that range from 10 to 400 or more genes. each test can be designed to detect hotspots of known mutations in those genes, to sequence the entire coding region of the genes, or to sequence the entire gene. the end result is a comprehensive profile of the tumor genome that can then be used to tailor therapy for the individual patient. although ngs assays cost more than single-gene assays, the cost per gene sequence is dramatically reduced and results in cost savings over using multiple single-gene tests. furthermore, ngs panels can be applied to very small specimen samples, using as little as 10 to 250 ng of input dna depending on the analytic platform utilized. since evaluation of therapeutic biomarkers is usually needed in the setting of advanced disease and such patients often have only limited tissue samples available for testing, ngs assays allow for much more extensive genomic information to be obtained compared to single-gene assays, each of which can require dna input comparable to that of an entire ngs panel. a total of 111 laboratories recently participated in a capsponsored proficiency assessment of ngs cancer panel testing (ngsst-a-2016), with data collection on 10 gene mutations (akt1, alk, braf, egfr, fbxw7, idh1, kit, kras, nras, and pik3ca). of 1010 genotyping calls across the spectrum of mutations tests, 993 (98.3%) were called concordantly (unpublished data). 88 no other ldp in the field of oncology has had a greater impact on patient care than has the quantitation of rna in chronic myelogenous leukemia (cml). one of the first (and arguably most successful) molecularly targeted cancer therapies is the bcr-abl1-targeted tyrosine kinase inhibitor, imatinib, which was fdaapproved in 2001. in those very early days of targeted therapy, long before the advent of fda-approved "companion diagnostics," the ubiquitous and obvious method to determine the efficacy of novel leukemia treatments was to directly quantitate the target of the inhibitor drug, namely, the cancer cellspecific bcr-abl1 fusion gene. a reduction in posttreatment bcr-abl1 rna levels, as measured by sensitive laboratorydeveloped pcr-based methods, was shown to be the best available test for predicting therapeutic response and long-term progression-free survival in tki-treated patients with cml. 89 consensus oncology practice guidelines in both the united states (nccn) 90 and europe (eln), 91 going back at least a decade, have universally recommended that tki-treated patients with cml should be serially monitored with a (laboratory-developed) bcr-abl1 rt-pcr blood test at least every 3 to 6 months during their lifelong course of tki therapy. the nccn and eln guidelines have also long recommended serial bcr-abl1 rna testing to directly inform not only the appropriate dose of tki (to overcome developing resistance) but also the therapeutic switch from one tki to another (depending on the drug's known resistance profile). to directly support optimal therapeutic decision-making in the routine care of patients with cml, clinical laboratories have been offering accurate and sensitive pcr-based laboratory-developed procedures for bcr-abl1 for at least the last 15 years. recognizing that standardization of these ldp's was necessary to promote uniform therapeutic decisionmaking, the laboratory community undertook an extensive multiyear project to create a standardized "international scale" (is) of measurement for bcr-abl1 messenger rna. 92 follow-up efforts resulted in the creation of a world health organization-recognized panel of reference materials directly linked to the bcr-abl1 is 93 and the subsequent creation of secondary is-calibrated reference materials that could be used for routine daily qc in clinical laboratories. 94, 95 recognizing the additional need for pt, the cap has been offering semiannual bcr-abl1 pt surveys for at least 10 years, with a progressive increase in the number of participating laboratories (from *100 in 2009 to *190 in 2016). as proof of the nearuniversal recognition of assay standardization, approximately 90% of these accredited laboratories now report their pcr results using the standardized is. a 2016 cap survey confirmed excellent interlaboratory precision, with over 90% of laboratories reporting a bcr-abl1 is result within internationally acknowledged acceptable tolerance limits (0.5 logs) for is reporting of a sample with an approximate 1000-to 10 000-fold reduction in pretreatment bcr-abl1 levels 96 the primary driving force behind the remarkable increase in longevity and quality of life for patients with cml over the past 15 years has no doubt been the availability of noveltargeted tki therapies. this has become the paradigm for personalized/precision cancer medicine programs, coupled with parallel effort of the laboratory community toward building, improving, and standardizing accurate, precise, and sensitive laboratory-developed tests for bcr-abl1. of note, this 15year targeted therapy program for cml occurred entirely without the availability of fda-approved bcr-abl diagnostic reagents, which have only become available in 2016. these fda-approved assays were based upon those developed in clinical laboratories; they are not approved for diagnosis of cml nor do they cover the spectrum of breakpoints that occur in the disease. during those ground-breaking first 15 years of the targeted cancer therapy era, if the laboratory community had been prohibited from providing high-quality, standardized ldp-based testing under existing clia guidelines, the negative consequences to patient care in the past and the future would have been substantial. fragile x (fx) syndrome is one of the most common inherited causes of intellectual disability. the causative molecular mechanism is an expansion of a cgg repeat region of the 5 0 regulatory region of the fmrp gene. when the cgg repeat expands beyond approximately 200 cgg repeats, the gene is methylated and silenced. laboratory testing for fx includes sizing the number of repeats as well as methylation analysis and has been available for clinical diagnostic and carrier status testing for over 20 years. the american college of medical genetics and genomics (acmg) has published practice guidelines for appropriate test ordering. 97 it is a first tier test for individuals and families in which an x-linked inheritance pattern of intellectual disability is suspected. once an expanded fx allele has been identified, other family members can be tested to identify premutation carriers at risk of having affected offspring. prenatal (fetal) or preimplantation genetic diagnostic testing is available for known fx carriers. according the genetic test registry, over 50 laboratories in the united states offer testing for fx (https://www.ncbi.nlm. nih.gov/gtr/; accessed 11-01-2016). currently, all fx testing is performed as ldps: no fda-cleared assay is available. pcr primers and southern blot reagents are available commercially as analyte-specific reagents (asr) or as investigational use only. clinical laboratories use these commercial reagents, or design primers or probes, combine them internally to develop the assay and establish performance characteristics. the acmg has published standards and guidelines for clinical laboratories that perform this test. 98 reference materials to standardize sizing were developed through the genetic testing reference material program 99 sponsored by the centers for disease control and prevention (cdc), the national institute of standards and technology (https://www.nist.gov/node/ 608501, accessed november 1, 2016), and the world health organization. 100 proficiency testing through the cap has demonstrated the excellent performance of clinical laboratories of this high-complexity ldp. 101 as the genetic basis for many human diseases became apparent, sequence-based diagnostic testing was implemented as a way to provide definitive diagnoses for patients and their families. many laboratories began offering sequence-based testing for heritable disorders in the 1990s, using a variety of mutationscanning techniques, such as single-strand conformation polymorphism, denaturing hplc, mlpa, and others. 102 sanger-based sequencing was the gold standard, despite being slow and expensive. the multigenic nature of some of these disorders made these sequencing approaches challenging due to the sheer number of genes and size of the sequence requiring analysis. in recent years, however, most of this testing has been converted to ngs, which offers significant advantages in terms of analytic capabilities, quality, speed, and cost. 103 the repetitive sequence reads in a single region ensure an enhanced level of quality, and ngs assays can be designed to interrogate anything from small to large gene panels, whole exomes, and beyond, depending on the clinical need being addressed. consensus guidelines for ngs assays have been developed by multiple professional societies and address the development, validation, and quality control of these assays. 104, 105 the cap has developed inspection checklists for laboratories performing ngs for detection of somatic mutations in cancers as well as germline mutations that cause heritable diseases. progress has also been made in the planning and production of reference materials and proficiency samples. 106 together, these practices and resources have yielded laboratory-developed assays that can be demonstrated to meet the quality levels needed for patient care. in addition to continuous quality assessment of the "wet laboratory" procedures, ongoing national and international efforts to share data, and construct and maintain up-to-date curated databases for variant interpretation, are critical for quality care. as new mutations and variants are detected, interpretation and assessment of clinical impact, including determination of the clinical importance of variants of undetermined significance, is critical. rapid generation of genomic data, disorganized data sharing, and a lack standardization have created challenges, and a more consistent approach to clinical sequence interpretation is needed, along with centralized and openly accessible databases for sequence and clinical information. in recognition of the urgent need for up-to-date variant classification resources, the acmg and association for molecular pathology released a landmark guidance document in 2015, 105 which has been implemented by us and international laboratories. additional resources are outlined and include ncbi's clinvar database (https://www.ncbi.nlm.nih.gov/clinvar/), which has quickly become a valuable centralized resource for clinically classified variants, and the clinical genome resource (clingen, www.clinicalgenome.org), which serves as a centralized site for managing genomic knowledge surrounding genes and variants. with exome and genome sequencing being increasingly implemented, guidance has been issued by the acmgg on how to deal with the incidental identification of variants in the so-called "actionable" genes in patients tested for unrelated conditions. 107 additionally, quality assessment focusing on the informatics pipeline and variant interpretation could effectively utilize sequence data sets, as has been recently outlined. 108 the genetics and pathology communities are increasingly embracing data sharing, which will lead to needed improvements in divergent interpretation of gene sequence variants. these interpretative tasks would be beyond the scope of an fda approval for a kit, or the clia oversight of an ldp, but have a critical impact on patient care based on genomic information. next-generation sequencing analysis of a variety of gene panels has become routine in clinical care and has had a positive impact on the diagnosis and treatment of patients and families with complex syndromes and disorders. examples of 3 of the most common clinical settings in which gene panels are tested for potential germline mutations are provided below, along with information on the clinical setting, potential benefits, and also challenges in utilizing these approaches. inherited cardiomyopathies are common disorders and include hypertrophic cardiomyopathy (hcm), dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and restrictive cardiomyopathy. 109, 110 several characteristics of all inherited cardiomyopathies provide compelling reasons for genetic testing and include (1) a substantial genetic component with detection rates currently ranging between 30% and 50%, 111, 112 (2) long presymptomatic phases with acute disease onset typically not before adolescence, and (3) a predisposition to sudden cardiac death (scd), which can be the first presenting sign. frequent and heartbreaking publicity following cases of sudden death of competitive athletes has brought these diseases to public attention, 113 and a recent study shows that 16% of scd cases are due to an underlying unrecognized inherited cardiomyopathy. 114 the importance of these heritable abnormalities is underscored by the fact that nearly a third of the genes for which the acmg recommends return of results, regardless of the test indication, are made up of these cardiomyopathy genes. these disorders are heterogeneous, which can lead to clinical diagnostic uncertainty or error; hence, large multigene panels are particularly useful to cover the spectrum of genes that may cause a clinical disorder. importantly, the identification of pathogenic variants in affected individuals can inform medical management of family members and can identify those at risk early and also release negative individuals from clinical screening. 111 genetic testing can also identify phenocopies such as fabry disease, which can masquerade as isolated hcm. while fabry disease is rare, disease-modifying treatment is available, and therefore, genetic testing provides essential clinical information for these patients. epilepsy is a common disorder of the central nervous system characterized by periodic loss of consciousness with or without convulsions associated with abnormal electrical activity in the brain. this can significantly affect the quality of life and has major psychological and socioeconomic consequences. it is estimated that there are more than 50 million people with epilepsy worldwide, and an estimated 1 in 26 people in the united states will develop epilepsy at some point in their lifetime. a significant proportion of cases show a familial distribution, and there is an increased risk of epilepsy with a family history. the prime requirements for successful management of epilepsy are a complete diagnosis and selection of an optimal treatment to benefit the patient. development of epilepsy may involve multiple gene abnormalities or a gene abnormality in concert with an environmental trigger. more than 100 genes have been shown to be associated with epilepsy, and a precise genetic diagnosis can help in deciding the accurate treatment and follow-up. 115, 116 evaluation of all genes implicated in epilepsy is most efficiently accomplished using an ngs panel to provide accurate information to the physician for treatment planning. nextgeneration sequencing assays are performed as ldps under clia. an example of such one gene is scn1a, which causes dravet syndrome and can be successfully treated. 117 it is important to avoid treatment with sodium channel blockers as these can worsen seizures in dravet syndrome. these include phenytoin (dilantin), fosphenytoin (cerebyx, prodilantin), carbamazepine (tegretol), and other medications. as ongoing research reveals new genes and mutations relevant to these diseases, it is important to classify newly found variant quickly and accurately. open access to new information and consensus efforts to define standards for classification and reporting of variants and vuss will be critical in ensuring that patients get the most upto-date and complete information from genomic testing. 104, 105 another example is the recently discovered gene tbckrelated epilepsy. 118 tbck-related intellectual disability syndrome is rare with developmental delay, hypotonia, and seizures. children with lower levels of the tbck protein have slower cell mtor signaling, which can be improved by the addition of leucine, which may provide future therapeutic options. it is important that clinical laboratories adapt their ngs panels to incorporate new targets as clinical utility is established. neuromuscular diseases (nmds) refer collectively to the many disorders that affect the peripheral nervous system, either by impairing the proper development or functioning of muscles or by damaging the associated nerves or neuromuscular junctions. muscular dystrophies that form the majority of inherited nmds share clinical, genetic, and pathological characteristics, including muscle degeneration and wasting, progressive muscle weakness, hypotonia, and variably elevated serum creatine kinase levels. cardiac involvement is often present, accounting for high morbidity and mortality. there are 80 different genetically defined types of muscular dystrophies categorized based on the age of onset, the specific muscles involved, and common characteristic clinical features. [119] [120] [121] [122] congenital muscular dystrophies and limb-girdle muscular dystrophies are the 2 major subgroups; these are genetically heterogenous, with many new genes being implicated in recent years. lack of pathognomonic signs or specific biochemical markers and the presence of high phenotypic overlap with other forms of nmds make diagnosis difficult. molecular assessment is critical not only to establish a diagnosis but also to allow participation in clinical trials of therapeutic treatments that are designed for a specific set of variants or variant types. an extensive diagnostic workup involving protein studies on muscle biopsy may be used to narrow the number of single genes to be tested, but many patients never are specifically diagnosed. comprehensive approaches to expedite molecular diagnosis now include ngs-based panel testing for sequence analysis of all disease-associated genes in a single analysis. [123] [124] [125] heritable cancer panel genomic testing for familial cancer syndromes has become routine over the past 2 decades. approximately 50 heritable cancer syndromes are recognized and are causative of 5% to 10% of all cancers. although familial breast cancer has perhaps become the most publicly known example of testing, genes for other inherited cancer syndromes may be included in ngs multigene panels. patients who carry a germline mutation of brca1 or 2 have a lifetime risk of breast cancer of 60% to 70%. the us preventative task force recommends brca1 and 2 testing for women who have family members with breast, ovarian, fallopian tube, or peritoneal cancer or meet other criteria. 126 numerous studies have demonstrated the psychosocial benefits of genetic counseling and testing. 126, 127 for patients who carry a germline mutation of brca1 or 2, surgical interventions may significantly reduce the risk of cancer or death. contralateral mastectomy has been shown to reduce the risk of death in carriers by 48%. 128 prophylactic salpingooophorectomy has been shown to dramatically reduce the mortality due to ovarian cancer or breast cancer in brca1 mutation carriers. 129 similarly, identification of lynch syndrome mutations can permit surveillance leading to earlier detection and marked improvement in survival in patients developing colorectal, endometrial, or ovarian cancer. 130 although more than 2 decades have passed since sequencebased analysis of high-penetrance cancer genes has been performed, only laboratory-developed procedures have been available. countless patients and families have benefited from the availability of these ldps. epidermolysis bullosa (eb) is an inherited skin and connective tissue disease that causes skin and oral blistering with only mild trauma (http://www.niams.nih.gov/health_info/epidermolysis_ bullosa/epidermolysis_bullosa_ff. accessed november 4, 2016) . the severity of the disorder depends on the layer of skin where the tissue separation occurs. 131 approximately 99% of patients with biopsy-proven eb will have mutation(s) in 1 of 18 genes known to cause the disorder (http://www.niams.nih.gov/health_info/epider molysis_bullosa/epidermolysis_bullosa_ff. accessed november 4, 2016). knowledge of the specific gene can direct therapy and provide reproductive options for the family. for many years, it was possible only to sequence the suspect genes one by one, taking months to years, and only on a research basis. recently, with the advent of ngs technology, a small number of clinical laboratories have stepped in to develop a rapid multigene ngs approach that provides answers quickly in time to make treatment decisions as well as to provide carrier and prenatal testing in at-risk families. 131, 132 as new candidate genes have been identified, ngs has been validated and offered for clinical testing. under clia, the validation data collected by the laboratory was subject to ongoing peer review, and the laboratory participates in ongoing pt to demonstrate assay quality. as the disease is rare (200 children born with eb annually), 133 the cost of bringing such a test through fda for approval is prohibitive. without an available ldp, patients and families affected by this disease would go without specific diagnoses, be unable to enroll in gene/mutation specific therapies (currently in development by at least 4 pharmaceutical companies and academic centers) (http://www.debra.org/research-trials. accessed november 4, 2016), or have control over their reproductive lives. whole-exome sequencing (wes) involves evaluating the coding regions of all *20 000 human genes at once, to search for the underlying molecular cause of an undiagnosed but presumed genetic disorder. these tests are used for patients who already have undergone extensive genetic diagnostic testing and exhausted the (limited) fda and ldp singlegene tests or in cases where it is more cost-and timeeffective to start with wes. whole-exome sequencing is used for the so-called "diagnostic odyssey" patient and has a high diagnostic yield for these patients, with 25% to 30% of studies yielding a diagnosis. 134, 135 the tests are highly complex and involve capture of the relevant dna segments, sequencing of those segments, bioinformatics approaches to sequence alignment and identification of variants (differences between patient dna and reference), interpretation of the identified variants, and report generation. under clia and nysdoh, the technical validation data collected by the laboratory prior to offering wes are required, and the laboratory participates in ongoing pt (through cap and sample exchanges) to demonstrate assay quality. as this is cutting-edge science and medical practice, the capture and sequencing technology, bioinformatics, and interpretation tools are evolving at a very fast pace. to provide the best service to patients, laboratories must frequently update, revalidate, and offer new services. whole-exome sequencing is offered as an ldp by laboratories which have extensive experience in genetic testing. the time delay involved in bringing this test and its frequent modifications to the fda is prohibitive. as these tests serve the rare disease community, and reimbursement is limited by the lack of pricing for a specific cpt code, the cost of bringing wes through fda approval would be a major deterrent. innovation would be slowed, and likely several laboratories would remove the test from test menus. with 30 000 affected individuals in the united states, 136, 137 huntington disease (hd) falls under the category of a rare (or orphan) disorder, and given its limited market, no commercial genetic testing platform has been developed or submitted to the fda for review. the relatively small number of laboratories offering diagnostic or predictive (presymptomatic) testing for this disorder must therefore rely entirely on ldps, without which patients with hd and their at-risk relatives would have no access to testing and diagnosis. despite the characteristic clinical features (the movement disorder [chorea] along with intellectual decline), and lack of preventive or curative treatment for hd, genetic testing is widely relied upon by hd families and their physicians. onset of symptoms is often insidious and nonspecific, and definitive early diagnosis can only be accomplished at the dna level. 132 presymptomatic testing, which is offered to the adult offspring of patients with hd (who are at 50% risk of inheriting this autosomal dominant disease), can only be accomplished using ldps and allows crucial life-planning decisions, such as educational pursuits and career choices, marriage, and whether to have children (and if so, affording the ability to pursue prenatal diagnosis) and whether to begin planning for inevitable disability. without this test, all of these at-risk relatives (of which there are an estimated 200 000 in the united states) 137 would lead their lives anxiously waiting for the symptoms to begin, when half of them are actually at no risk because they did not inherit the mutant gene from their affected parent. although there are at-risk relatives who choose not to avail themselves of the predictive test, the many who do opt to be tested credit it with freeing them of years of obsessive uncertainty. the results can afford the affected patients the opportunity to enroll in clinical trials (involving drugs or neuronal stem cells), with the aim of preventing or delaying the onset of symptoms. 138 although these studies are still in an early phase with no outcome data yet available, they give patients some hope for the future, perhaps for themselves but also for future patients. given the mechanism of gradual neuronal cell death in the basal ganglia, it stands to reason that the earlier such intervention is initiated-ideally in the presymptomatic stage-the higher the chances of success. huntington disease is one of the trinucleotide repeat disorders, caused by expansion of a tandem repeat of cag in the first intron of the huntingtin (htt or it15) gene. in contrast to fx syndrome and some of the other trinucleotide repeat disorders, the difference between the mutated and nonmutated repeat length can be as little as a single repeat (ie, 3 nucleotides). thus, extreme care is required in the sizing of the repeat, especially when it falls near the cutoff length of 40 40 repeats or higher which is diagnostic or predictive of hd, with 100% penetrance. fortunately, the ldps in current use, relying on capillary electrophoresis, are very accurate in determining repeat length, as attested by the excellent performance in cap proficiency surveys. busulfan is a bifunctional dna alkylating agent typically given to patients as a conditioning agent prior to hematopoietic cell transplantation (hct) for the treatment of hematologic malignancies. therapeutic drug monitoring (tdm) is crucial for the safe and efficacious use of busulfan due to a narrow therapeutic index based on area under the curve (auc) calculations. too low a dose places the patient at risk of either graft failure or early relapse. 139, 140 on the other hand, too high a dose increases the risk of neurotoxicity 141 as well as a severe and life-threatening complication termed hepatic sinusoidal obstruction syndrome (sos). 139, 142 hepatic sos, previously termed hepatic veno-occlusive disease (vod), refers to the occlusion of terminal hepatic venules and hepatic sinusoids. severe cases of vod can lead to hepatorenal syndrome, causing multi-organ failure, hepatic encephalopathy, and death. veno-occlusive disease typically occurs in the context of hct, particularly after administration of conditioning regimens prior to hct. it is one of the most feared complications of hct and accounts for a significant fraction of hct-related mortality. 143 severe cases, which account for approximately 25% to 30% of sos, are almost always fatal. 142, 144, 145 despite a clear need for busulfan tdm, there are currently no fda-approved assays available for the quantitation of busulfan in blood. for this reason, various bioanalytical methods have been developed 146 and are currently in use by multiple laboratories. data from a busulfan proficiency program organized by the university of washington/seattle cancer care alliance show a total of 24 participating laboratories at the present time. of these, 6 laboratories use gas chromatography (gc) methods, 3 use hplc methods, and 14 use liquid chromatography-tandem mass spectrometry (ms) methods. 147 all of these methods are non-fda-approved tests independently developed and validated for clinical use by their respective clinical laboratories. the use of these methods is also driven by the need for high precision and accuracy. current criteria for acceptable laboratory performance in the analysis of busulfan is +10% of the known concentrations for medium and high concentrations and within +15% of the known concentration for low concentrations. 147 this is due to the fact that dosing change decisions are based on auc calculations, which depend on blood concentrations of busulfan measured from multiple timed blood draws. multiple small analytical errors can easily add up to big differences in calculated auc values. busulfan testing is currently available from reference laboratories. however, many busulfan regimens call for intravenous infusions every 6 or 24 hours for 3 to 4 days. bone marrow transplant teams need quicker turnaround times than can be reasonably provided by sendout testing in order to be able to make dosing adjustments within this limited timespan. guidelines have also been recently published by the american society for blood and marrow transplantation guidelines committee advocating for personalized busulfan dosing using busulfan tdm in certain busulfan regimens. 148 for these reasons, laboratory-developed methods for busulfan will continue to play key roles in the management of hematologic malignancies at cancer centers throughout the world. very sensitive measurements of serum androgens are important in adult and pediatric endocrinology and oncology. very-low-level testosterone (te) measurements are needed for adult women, whose values are routinely <50 ng/dl, in children, and men undergoing antiandrogen therapy whose values are usually <10 ng/dl. 149 the most commonly used methods for steroid analysis are fda-approved immunoassays because they are rapid and sensitive enough for most routine applications involving healthy adult males. however, te immunoassays lack the sensitivity requirements for chemically castrated males, women, and children. many immunoassays also lack specificity and accuracy as immunoassays may show cross-reactivity with structurally similar compounds. [150] [151] [152] in addition, most immunoassays are not standardized against internationally recognized standards. for these reasons, a number of sensitive and specific assays using ms have been described for te. [153] [154] [155] the lack of sufficient accuracy and standardization of te assays is a major concern for the clinical and public health communities. 156 several years ago, the endocrine society, in partnership with the cdc, convened a meeting with various relevant professional societies and industrial partners to create the partnership for the accurate testing of hormones (path) whose mission is to improve the accuracy and standardization of a variety of steroid hormone tests. [156] [157] [158] the path has worked with the cdc and begun to address this concern through its host program, which provides laboratories with specimens spanning their analytical measurement range that have been previously analyzed using the cdc reference method. 155 many assays using ms have been approved by the cdc host program, 159 however not a single fda-approved immunoassay has met the performance requirements. testosterone is a perfect example of an ldp that is indispensable for patient care and allows for accurate measurements to be made on children, women, and male patients with cancer receiving antiandrogen medication. ethylene glycol is a colorless, sweet-tasting liquid commonly encountered in automobile antifreeze. because of this widespread availability, it is also a commonly encountered toxicological agent in both accidental and self-inflicted poisonings with 6078 exposures in 2014. 160 ethylene glycol poisoning classically presents with a metabolic acidosis caused by the production of toxic metabolites, primarily glycolic acid and oxalic acid. this is also often accompanied by an anion gap and osmolal gap. untreated ethylene glycol poisoning can also progress to acute renal failure when high levels of oxalate anions combine with calcium to develop crystals in the kidneys and urinary tract. 161 ethylene glycol poisoning is an urgent, toxicological emergency. once ethylene glycol is identified, the drug fomepizole is typically administered. fomepizole inhibits alcohol dehydrogenase, the enzyme that metabolizes ethylene glycol, to slow the accumulation of toxic metabolites. fomepizole and ethanol dramatically lengthen the half-life of ethylene glycol, and therefore, hemodialysis is often required to clear the poison. 162 both the diagnosis and treatment of ethylene glycol poisoning are heavily dependent on laboratory measurements. no fda-approved assays for ethylene glycol are currently available, and all testing is performed by laboratory-developed procedures. the 3 most common methods for the analysis of ethylene glycol are gc with flame ionization detector, gc with ms, and enzymatic assays. 163, 164 gas chromatography with mass spectrometry is considered the gold standard for the analysis of ethylene glycol, as it can differentiate it from interferences that plague the other 2 methods. 163, 165 in addition to initial detection needed for diagnosis, the ethylene glycol blood concentration is used to determine when hemodialysis has cleared ethylene glycol to undetectable levels. measurement of thyroglobulin (tg) in serum has proven useful for detecting recurrence of treated differentiated thyroid carcinoma (dtc). according to the american cancer society, the united states has over 60 000 new thyroid cancer cases each year. the death rate is almost 2000 per year. differentiated thyroid cancer accounts for over 90% of cases. differentiated thyroid cancer produces tg, making its measurement useful as a tumor marker for detecting recurrence. the nccn and american thyroid association (ata) guidelines recommend tg testing following total thyroidectomy and radioiodine ablation treatment, including tests at baseline, 6 to 12 weeks after treatment, 6 months, 12 months, and annually thereafter. patients free of disease have undetectable tg. older competitive tg-ria methods are available but produce falsely high tg results in the presence of tg autoantibodies (tg-ab). 166 newer fda-cleared tg assays are immunometric immunoassays (tg-ia) and can detect tg at concentrations down to approximately 0.1 ng/ml. generally, tg is captured with a solid-phase antibody, then quantitated using a detection anti-tg reagent. the signal is directly proportional to the amount of tg. the assay design is susceptible to interference from endogenous anti-tg-ab. 166 the intended use of these tg-ia are tg measurement in tg-ab-negative (tg-ab ã� ) patients. the fda requires tg-ab testing whenever tg is measured using these cleared methods. depending on the method used, up to 36% of treated patients with dtc are tg-ab positive (tg-ab ã¾ ). thus, many of these patients have falsely low or even falsely negative tg results when measured by ia. four tg-ab assays are in common use and are not harmonized, detecting tg-ab in widely divergent numbers of treated patients with dtc. 167 in addition, the degree of tg interference cannot be predicted from the magnitude of the tg-ab result. thus, up to 20% of patients with recurrence are missed by tg-ia testing. to circumvent the tg-ab interference, hoofnagle and wener developed and validated a ms tg method (tg-ms) using tryptic digestion and immunocapture of tg-specific peptides followed by ms focused on those peptides. 167 they demonstrated the tg-ms method accurately measures tg in the presence of tg-ab. 162 the tryptic digestion destroys the tg-ab, eliminating their interference with the assay. four national reference laboratories have adopted versions of this method, and harmonization efforts are underway. a recent clinical outcome study compared tg-ia, tg-ria, and tg-ms in both tg-ab ã� and tg-ab ã¾ patients. as predicted from the assay designs, all methods were equivalent for tg-ab ã� cases, but the tg-ms was more accurate for tg-ab ã¾ cases. tg-ia methods had more false negatives and tg-ria had more false positives. 168 although tg-ms has not yet been incorporated into the current guidelines, the ata guideline mentions it as a promising new technique. without tg-ms, thyroid cancer recurrence in tg-ab ã¾ patients can be missed, thus delaying follow-up and creating patient harm. antimicrobial susceptibility testing, used to determine whether antibiotic treatment will be successful, is an essential component of the microbiology culture report. emerging resistance among pathogenic bacteria and new antimicrobial agents require frequent updates to both testing methods and interpretation of the results. most laboratories use automated instruments to perform minimal inhibitory concentration (mic) testing to determine whether a patient's isolated bacteria are susceptible, susceptible dose dependent, intermediate, or resistant to a panel of antibiotics. these interpretations are based on fda breakpoint criteria published at the time of drug approval and periodically updated to respond to the appearance of new resistance mechanisms. 169 the fda also clears automated instrumentation used to determine mic values (via the 510k process). 170 although the mic testing process may not be changed, new breakpoints added to the instrument software require a revised 510k application. because the fda does not have the authority to require manufacturers to submit data for revised breakpoints within a specified time frame, manufacturers may elect to use outdated breakpoints rather than face the expense of a 510k resubmission. a recent example was the 3year delay between the release of updated breakpoints for diagnosing carbapenem-resistant enterobacteriaceae in 2010 and the ability to use these breakpoints in the clinical laboratory. this delay was used to calculate the potential for additional carriers of multidrug-resistant enterobacteriaceae in southern california health-care systems. as many as 1821 additional carriers of mdro enterobacteriaceae were estimated to have occurred in orange county, california because of this delay. 171 the disastrous spread of mdro enterobacteriaceae can be mitigated by laboratories validating testing methods that enable use of updated antimicrobial breakpoint interpretations before fda-cleared testing is available. specifically, modifying a manufacturer's instructions, including interpreting mic results using a revised breakpoint other than that listed in the product insert, is a change that renders the procedure an ldp. without the option of using an ldp, one is left reporting outdated interpretations that miss resistant strains leading to unacceptable patient care. as illustrated, ldps are an integral part of the spectrum of tests and procedures performed by clinical laboratories which fulfill a critical need for patient care, particularly in rapidly evolving areas such as testing for personalized medicine. laboratory testing should be consistent with national/international consensus treatment guidelines, which may require development of procedures earlier or for new clinical purposes not fulfilled by fda-approved kits. in such cases where clinical testing needs exist beyond the original fda purpose, laboratories must be able to perform additional validation of new sample types or develop additional assays to include new mutations or analytes that are needed. as illustrated by these case examples, laboratories and professional organizations often work together to broadly compare and optimize assay performance, creating consensus standards that raise the quality of testing overall. in contrast, the current structure for fda approval requires review of assay kits individually, or in comparison with the predicate method, rather than assessing and improving performance across the spectrum of assay options available. the science of laboratory medicine has advanced dramatically in the almost 3 decades since clia was enacted, and updates and expansions to clia regulations could be useful. additional resources, such as reference materials, and consensus practice guidelines would extend the quality framework that all laboratories and manufacturers utilize. for example, consensus guidelines that include such details as the target for percent allele frequency detectable, requirements for percent tumor cell content, what mutations and variants should be included, and sample types to be tested would be useful as a guide for assay validation as well as in standardization of the practice. professional expert groups are already generating assay and practice guidelines. 69, 73, [172] [173] [174] [175] ideally, clinical laboratories and kit manufacturers would utilize appropriate reference materials to help standardize the results obtained for any particular analyte regardless of technology platform or laboratory setting. to address the needs for reference materials to facilitate assay result standardization, a multistakeholder initiative, the diagnostic quality assurance pilot, has been launched to design, develop, and evaluate traceable reference sample materials (referred to as reference materials) to better provide molecular pathology laboratories with the means to demonstrate equivalent performance of ldps and companion diagnostic ivds for targeted cancer therapy. this quality pilot emerged from the sustainable predictive oncology therapeutics and diagnostics working group, launched in 2013 by tapestry networks (waltham, massachusetts), which was composed of diverse stakeholders (oncologists, pathologists, patient advocates, third party payers, and regulators) for the purpose of designing a quality pilot to advance these goals (http://www.ta pestrynetworks.com/initiatives/healthcare/oncology-therapeu tics-and-diagnostics/diagnostic-quality-assurance-pilot.cfm. accessed april 27, 2017). 176 the tapestry pilot proposes that laboratories would be allowed to utilize assays that best serve the needs of their patients based upon performance, quality, clinical needs, and the test menus and volumes of that particular laboratory. it is not critical that laboratories all use the identical assay or test platform, provided that all are able to get the correct answer. to close, the overarching goal is the efficacy and safety of our clinical laboratory tests and procedures for patients. pathologists and laboratory professionals need the best and most upto-date tools to do their jobs and optimize patient care. some of these will be fda approved or cleared kits, and others will be laboratory-developed procedures performed under clia; both have their place. laboratories have a long history of success performing ldps, as illustrated by these case studies. as much as possible, these capabilities need to be performed on-site to insure that the results can be integrated with other clinical and laboratory findings, interpreted as a whole and completed in a timely fashion. also important is the handson training of the next generation of physicians, for whom, we hope, maximal use of genomic and other laboratory information will be a way of life as they treat human disease. that is the promise of personalized medicine! the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. the '70% claim': what is the evidence base? evidence-based laboratory medicine the public health evidence for fda oversight of laboratory developed tests: 20 case studies the public health evidence for fda oversight of laboratory developed tests: 20 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document m100-s27 but no tests! the challenge of antimicrobial susceptibility testing in the current us regulatory landscape impact of delays between clinical and laboratory standards institute and food and drug administration revisions of interpretive criteria for carbapenem-resistant enterobacteriaceae the spectrum of clinical utilities in molecular pathology testing procedures for inherited conditions and cancer next-generation sequencing for infectious disease diagnosis and management: a report of the association for molecular pathology laboratory practice guidelines for detecting and reporting jak2 and mpl mutations in myeloproliferative neoplasms the role of mgmt testing in clinical practice diagnostic quality assurance pilot: a model to demonstrate comparative laboratory test performance with an oncology companion device assay the authors would like to thank the college of american pathologists, dr jason merker and ms patty vasalos, for facilitating access and allowing use of data from cap interlaboratory proficiency testing programs. ms mary williams, dr barbara zehnbauer, dr peter hulick, and dr david klimstra provided valuable comments on the manuscript. ms laura metz provided clerical support. key: cord-298086-pbfi5c8e authors: lyngse, f. p.; kirkeby, c. t.; halasa, t.; andreasen, v.; skov, r. l.; moller, f. t.; krause, t. g.; molbak, k. title: covid-19 transmission within danish households: a nationwide study from lockdown to reopening date: 2020-09-09 journal: nan doi: 10.1101/2020.09.09.20191239 sha: doc_id: 298086 cord_uid: pbfi5c8e background the covid-19 pandemic is one of the most serious global public health threats in recent times. understanding transmission of sars-cov-2 is of utmost importance to be able to respond to outbreaks and take action against spread of the disease. transmission within the household is a concern, especially because infection control is difficult to apply within the household domain. methods we used comprehensive administrative register data from denmark, comprising the full population and all covid-19 tests, to estimate household transmission risk and attack rate. results we studied the testing dynamics for covid-19 and found that the day after receiving a positive test result within the household, 35% of potential secondary cases were tested and 13% of these were positive. after a primary case in 6,782 households, 82% of potential secondary cases were tested within 14 days, of which 17% tested positive as secondary cases, implying an attack rate of 17%. among primary cases, those aged 0-24 were underrepresented when compared with the total population. we found an approximately linearly increasing relationship between attack rate and age. we investigated the transmission risk from primary cases by age, and found an increasing risk with age of primary cases for adults, while the risk seems to decrease with age for children. conclusions although there is an increasing attack rate and transmission risk of sars-cov-2 with age, children are also able to transmit sars-cov-2 within the household. in late 2019, increased numbers of severe respiratory infections were reported in the wuhan region in china caused by the sars-cov-2 virus (coronaviridae study group of the international committee on taxonomy of viruses, 2020). since its emergence, the virus has spread rapidly throughout all five continents affecting millions of people (world health organization, 2020) and causing massive economic losses (fernandes, 2020) . the effective reproduction number was estimated in different studies with different methods to range from 2.1 to 4.7 (boldog et al. (2020) ), revealing a high transmission potential. person-to-person transmission is a major transmission mode of sars-cov-2, including transmission through aerosols or droplets on surfaces (chan et al., 2020; leclerc et al., 2020) . quantifying the transmission risk in different settings is essential for improving our understanding of the viral transmission dynamics, to implement effective preventive measures, to minimize economic damage, and to avoid overloading the healthcare system. close person-to-person contact is one of the main risk factors and transmission within the household is thus a major setting for virus transmission (prem et al., 2017; davies et al., 2020; cauchemez et al., 2009; leclerc et al., 2020) . furthermore, infection control and isolation are challenging in the (often crowded) household domain. quantifying the extent of transmission within the household can help improve our understanding of the effects of implementing quarantine for household members, physical distancing and improved hygiene. furthermore, these estimates are useful in constructing reliable prediction models for the spread of sars-cov-2 from households to the community. data from contact tracing and monitoring of individuals have been used to investigate household transmission of sars-cov-2 (he et al., 2020; jing et al., 2020; liu et al., 2020; li et al., 2020; madewell et al., 2020) . contact tracing is laborious and requires large resources when there are many new cases, as in the case of the sars-cov-2 pandemic. thus, these studies have been limited to include a maximum of a few hundred primary cases, selected mainly due to history of hospitalization or clinical disease (see a systematic review by madewell et al. (2020) ). the relatively small sample size as well as the selection and recall bias caused by contact tracing may limit the generalizability of these studies. in this study, we exploit national register data from denmark to investigate transmission patterns within households. to our knowledge, this is the first nationwide study that uses estimates of household attack rates and transmission risks that exploit sars-cov-2 test data from an entire population. the epidemic in denmark on february 27, 2020, the first case of sars-cov-2 was diagnosed in denmark (reilev et al., 2020) . shortly thereafter, the number of cases began to rise with an estimated effective reproduction rate (re) of about 2.5 (statens serum institut (2020)). on march 11, a comprehensive lockdown of the public sector was implemented by the government. moreover, the private sector was encouraged to work from home as much as possible. in figure a.1 (panel a) , key indicators of the epidemic are shown: number of tests, positive test results, hospitalized cases, and deaths for the early stage of the epidemic, the lockdown period and the two following stages of reopening. the figure shows a clear reduction in the number of positive test results, hospitalized cases and deaths over time. panel b shows a summary of the main measures for controlling the epidemic over time, including test and contact tracing strategy, and restrictions on public and private workplaces, education and childcare institutions, as well as in the community in general. all danish residents have access to tax-paid universal health insurance, and a test for sars-cov-2 is free of charge. furthermore, denmark has comprehensive social welfare insurance, and sars-cov-2 sick leave is fully reimbursed by the state. thus, financial reasons are not a major obstacle to obtaining a test. the test capacity has increased throughout the epidemic, and in the study period, the number of tests has been fairly stable since late april 2020 (figure a.1) . in the beginning of the epidemic, all suspected cases of covid-19 were tested and their contacts traced. however, soon after, due to capacity constraints on testing, only cases with severe symptoms (i.e., admitted to hospital) were tested and the overstretched contact tracing was halted. during reopening testing again became generally accessible, so that all residents could obtain a test without a referral (panel b of figure a.1) . in the current study, we used danish administrative register data. all residents in denmark have a unique personal identification number that allows a completely accurate linkage of information across different registers at the individual level. all microbiological data in denmark is registered in the danish microbiology database, from where we obtained individual level data on all national tests for sars-cov-2 for the period february 27, 2020 (the first positive test in denmark) to july 24, 2020. information on the reason for being tested (e.g., symptoms, potential contact with infected persons etc.) was not available. from the danish civil registry system, we obtained information about the sex, age, and home address for all individuals living in denmark. all data management and analyses were carried out on the danish health data authority's restricted research servers with project number fseid-00004942. we constructed households by linking all individuals living at the same address, and only considered households with six or fewer members, in order to exclude e.g., institutions. the data set captured 98.3% percent of the danish population. person level data, which included information on the test result and date and time of sampling as well as time of the result, were linked to individuals within households. for each household, we identified the first positive test for sars-cov-2; this case was defined as the primary case and referred to as such throughout this paper. we considered all subsequent tests from other members in the same household as tests taken in response to the primary case. we defined secondary cases as those who had a positive test within 14 days of the primary case being tested positive. primary examination of the data revealed that this cutoff provided a stable proportion of potential secondary cases (see figure 1b ). in addition, we assumed that the secondary household members were infected by the household primary case, although some of these secondary cases could represent co-primary cases. a longer cutoff time period could result in misclassification of cases among household members with somewhere else being the source of secondary infections. in order to investigate potential bias in our results in relation to time, due to changing test strategy and capacity over the epidemic period (see figure a .1 for an overview of test strategy and capacity), we separated the data into three data sets representing the three time periods based on the test date of the primary case, and analyses were performed separately on these data sets. the defined periods were: lockdown: march 12 -april 14; early reopening: april 15 -may 17; and late reopening may 18 -july 5 (see figure a .1). during lockdown, educational institutions were kept closed and the public sector with non-essential functions stayed at home. children's daycare was limited to children of employees in essential functions, such as doctors, nurses and police. employees in the private sector were encouraged to stay home if possible, and international travel was minimized by closing the borders except for essential activities. in the early reopening phase, children's daycare became available again, and schools were re-opened for classes up to 5th grade. in the late reopening, systematic contact tracing was resumed, schools (6-10th grade) and higher educational institutions reopened, along with restaurants and smaller bars (with physical distancing). . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint to determine the probability that an additional household member would test positive after the primary case in the household was tested positive, we used all records for potential secondary cases within the household. we defined the attack rate as the proportion of additional household members that tested positive, whereas the transmission risk was the proportion of secondary cases per primary case. we did not exclude any potential co-primary cases in the main analysis. we further conducted a sensitivity analysis for the robustness of the time cutoff of transmission from the primary case to potential secondary cases. we used sas 9.4 to manage and analyze the data (sas, 2015) . in order to reach sufficient sample sizes, we separated all records into age groups of five-year intervals. to investigate the testing dynamics, we took an event study approach (ball & brown, 1968) . following this method, we used the date of diagnosis of the primary case in each household as an event and observed all other household members for five days before until 14 days after the event. in the case of two or more primary cases detected on the same date, we randomly assigned one of them as the primary case. we estimated the probability of being tested (β τ ) for each day relative to the first positive test result within the household, using the following equation: where y i,t is a binary variable for individual i being tested at time t. τ is days relative to the date of the primary case's positive test result. i τ =t denotes indicators for time since the primary case's positive test result. β τ represents parameters estimating the probability of being tested on day τ relative to receiving the primary case's test result in the household. ε i,t denotes the error term, clustered on the household (event) level. we used the same equation to estimate the probability of y testing positive conditional on being tested. furthermore, to estimate the proportion of potential secondary cases that had been tested on day τ or previously, we estimated the absorbing probability, using the following equation: where i τ ≥t = 1 if individual i was tested on day τ or previously, and zero otherwise. we also used the same equation to estimate the probability of y ever being tested positive. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint to investigate the proportion on positive tests originating from households, we defined new cases that live in a household with another case that tested positive within the preceding 14 days as a case originated from the household domain. we used a seven day rolling average in order to take account of variation in testing rates across the weekdays. to estimate the attack rate, we estimated the proportion of potential secondary household members who received a positive test within 14 days after the test date of the primary case. we estimated attack rates using the following equation: where y i,t = 1 if the individual had a positive test within the 14 days after the primary case, and zero otherwise. γ denotes a vector of fixed effects for the three periods. female is a binary variable for sex. β 0 measures the 14 day attack rate. ε i,t denotes the error term, clustered on the household (event) level. to investigate the age structure of the attack rate for potential secondary cases and transmission risk from primary cases, we used a non-parametric approach. we separated the data into five-year age groups. we estimated the attack rate using the following equation: where y i,t = 1 if the potential secondary case i had a positive test within the 14 days after the test date of the primary case, and zero otherwise. agegroup s is five-year age groups of the potential secondary cases. α is a vector that measures the age structured attack rate. ε i,t denotes the error term, clustered on the household (event) level. we estimated the transmission risk using the following equation: where agegroup p is five-year age groups of the primary cases. β is a vector that measures the age structured transmission risk. we estimated the age structured interaction between the attack rate and transmission risk using the following equation: 6 . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10. 1101 where γ is a vector that measures the age structured interaction between attack rate and transmission risk. to quantify the effect of age on attack rate, we estimated the approximately linear relationships between attack rate and age using equation: where β measures the probability of being infected as a linear function of age. φ is a vector of fixed effects for each time period (see "robustness of estimates over time" in section 2.1). δ measures the differential age gradient for each period. π measures the effect of sex for each period. in order to investigate the potential difference between male and female cases, we explored the age dependent attack rate separately for each sex. we also separated the data into households where the primary cases were children (below 15 years of age) and adults (above 25 years of age). these thresholds were chosen to ensure the primary cases were either children or adults. we estimated the attack rate stratified by the number of household members for households with one to six members. furthermore, we estimated the proportion of households with n number of positive cases, conditional on the size of the household being greater than or equal to n (with a maximum household size of six). in total, we obtained positive test results from 6,782 household primary cases and 1,904 positive secondary cases, see table 1 . further summary statistics are presented in appendix b. 7 . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint appendix figure a.2 presents the probability of being tested and testing positive over the epidemic for five-year age groups. we found that all age groups are being tested, though children generally had a lower probability. the increase in test capacity over time was approximately equally distributed across ages. in late june, we saw an increase in the probability of children being tested. appendix figure b.1 shows summary statistics of the primary cases compared to the overall danish population. panel (a) shows the age distribution and panel (b) shows the household size distribution. there were proportionally fewer children as primary cases than in the total population. the household sizes of the primary cases generally matched the household sizes of the population. in this section, we focus only on the testing dynamics during the late reopening, where test capacity was stable. (in appendix d, we illustrate changes over all three periods.) figure 1 panel (a) shows that after receiving a positive test result in the household (t = 0), 36% of potential secondary cases were tested (blue) the day after the positive test result (t = 1) of the primary case was available and 13% of these 36% were positive (red). on the day preceding the test result (t = −1), 12% were tested and 24% of these tests were positive. panel (b) shows the proportion of individuals that were tested (blue) and those that tested positive (red), daily up to 14 days after the primary case was tested (t = 0). 18% of potential secondary cases were tested on the same day as the primary case and 4% of potential secondary cases tested positive on that day. within 14 days after the primary case was tested, 82% of the potential secondary cases were tested and 17% tested positive. there were 4% who tested positive on the same day (t = 0) as the primary case. these may represent co-primary cases, and therefore do not represent household cases. under this assumption, in order to estimate the attack rate, one should exclude these cases (by subtracting 4 percentage points (pp) from 17%), thereby resulting in an attack 8 . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint rate of 13%. similarly, cases detected within one day of the primary case may represent co-primary cases (t ≤ 1). this leaves an attack rate of 11% (by subtracting 6 pp from 17%). (appendix f provides robustness analysis on the definition of co-primary cases.) on the day preceding the result (t = −1) 12% were tested and 25% of these tested positive. the day after the households received their first positive test result (t = 1), 35% were tested and 13% of these tested positive. in panel b, t = 0 denotes the time the first positive test was taken (not when the test result was given). 18% of all potential secondary cases were tested on the same day as the primary case (t = 0) and 4% of all potential secondary cases tested positive. this implies a 22% probability of testing positive conditional on being tested on the same day as the primary case. 14 days after the day of the first positive test, 82% had been tested and 17% tested positive. shaded areas show the 95% confidence bands clustered on household level. figure 2 shows the proportion of positive tests originating from households within the 14 days after the primary case tested positive. after a rapid decrease immediately after lockdown, there was an increasing proportion of new cases originating from households during lockdown, and a decreasing proportion after reopening. the last day of school was june 26, and many families start their vacation at that time. after this date, the proportion started to increase again. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint we found an approximately linearly increasing relationship between attack rate and age. panel a of figure 3 shows the probability of having a test (blue) and the probability of having a positive test (red) (unconditional on being tested) across 5-year age groups. the shaded areas show the 95% confidence bands. panel b shows the transmission risk from primary cases by age group, i.e., the probability of infecting others. the figure shows an increase with age for adults, while the risk seems to decrease with age for children. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint notes: panel a illustrates the probability of having a test (blue) and the probability of having a positive test (red) (unconditional on being tested) across 5-year age groups. panel b illustrates the transmission risk from primary cases by age group, i.e. the probability of infecting others. the shaded areas show the 95% confidence bands clustered on the household level. as figure 3 shows an approximately linear relationship between attack rate and age, we estimated the linear relationship using equation 7 (see table e .2, column i). the results show that individuals had a baseline risk of 9.4% of testing positive. the risk increased by 0.24 percentage points for each year of age. thus, a 10-year-old had a risk of 11.8%, a 30-year-old had 19.0%, and a 60-year-old had 33.4%. the estimates were robust to different specifications, including period and sex covariates, table e .2, column ii-iv. the attack rates conditional on the primary case being a child or an adult are presented in figure c.1. when the primary case is a child (under 15 years old), the attack rate seemed uniform, regardless of the age of the potential secondary case. when the primary case is an adult (over 25 years old), the attack rate increased (linearly) with age. the attack rates by sex are presented in appendix c.1. the results indicated no difference in attack rates between males and females. the interaction between the attack rate and transmission risk for each combination of age groups is presented in figure c .2. the age range of the primary case is depicted on the horizontal axis and the age of potential secondary cases on the vertical axis. panel a shows the probability of being tested (for each age group combination); panel b shows the probability of obtaining a positive test result. the probability for potential secondary cases of being tested was generally highest when the primary case was a child (below 15 years old). the probability of being tested was also high when the age difference between the primary case and the potential secondary case was small. the attack rate was highest when the primary and potential 11 . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint secondary cases were above 60 years of age. when children were the primary case, an increased attack rate was observed for all age groups. figure 4 illustrates the proportion of cases by the number of household members for households with one to six members. for instance, in a household with two members (red), 79% of the households had one positive case, while 21% had two positive cases. similarly, in a household with three members (green) 79% of the households had one case, 16% had two cases, and 5% had three cases. (table e .4.) this implies that in a three-person household, 79% have an attack rate of 0, 16% have an attack rate of 50%, and 5% have an attack rate of 100%. this amounts to an overall attack rate of 13% (79% × 0% + 16% × 50% + 5% × 100% = 13%). once a primary case was found within a household, the probability of at least one secondary case was 23%, regardless of household size, and consequently 77% of the primary cases do not generate any additional cases. in particular, the primary cases had a 17% probability of generating one additional case, 6% of generating two additional cases, 3% of generating three additional cases, and 2% of generating four additional cases. this pattern was consistent regardless of the number of persons in the household, showing a transmission pattern that was exponentially decreasing with the number members within the household. the pattern was consistent over the three phases of the epidemic examined here, indicating that it is not a result of the change in the testing strategy (figure d is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint the epidemic in denmark changed over time, both due to changes in policy response (e.g., lockdown), and changes in test capacity and strategy (see appendix a for an overview). in this section, we investigate the robustness of the previously described results over the three defined periods of the epidemic (see appendix d). the analyses show that despite the substantial changes in probability of being tested, the estimated attack rates were consistent over the epidemic. in appendix f, we investigate the sensitivity of the estimated attacks to the definition of co-primary cases. the analysis showed that the estimated age structured attack rates did not change noticeably by excluding secondary cases found within the same day, one day or two days of the primary case. we estimated an overall attack rate within households of 17% (table e .1), ranging from 11% during lockdown, 16% during early reopening, and 17% during late reopening (figure d.1) . this suggests that attack rates estimated early in the danish epidemic underestimated the true attack rate. this bias likely comes from the limited testing capacity 13 . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint early on. these estimates are in line with the estimates from the literature; madewell et al. (2020) conducted a meta-analysis of household transmission of sars-cov-2 from 40 studies and found an overall attack rate of 18.8%, which is very close to our estimate. we studied the testing dynamics for covid-19 and found that the probability of obtaining a test relative to a primary case positive test result within the household peaked on the day after the primary case received the result, where 35% of all potential secondary cases were tested and 13% of these were positive (figure 1a) . interestingly, 12% of the potential secondary household cases were tested one day preceding the test result of the primary case, and 25% of these were positive. this could indicate that these individuals were tested for a reason other than the primary case result, e.g., for having symptoms themselves. the probability of being tested after a primary case in the same household increased from 18% on the same day as the primary case until it flattened out at around 80% on day six (figure 1b) . 76% of the secondary cases were found during the first three days after the primary case (13% / 17% = 76%). this highlights the importance of fast contact tracing, as most secondary cases are found in the first couple of days after the primary case, which is also concluded by moghadas et al. (2020) . the proportion of positive cases originating from households ( figure 2 ) increased during the lockdown until the late reopening period when the borders reopened. thereafter, it increased again after the school holidays started. in other words, the school holidays (which also include three-weeks of annual leave for most parents) essentially functioned as another lockdown period, because families tend to stay together during the holidays. we suggest that this may have contributed to a low incidence of community transmission over the summer. when the testing capacity was relatively stable (from late april 2020), the proportion varies between 20% and 45% of total cases. this indicates that many cases originate within the household domain, and this should be taken into account when monitoring the epidemic as well as in national guidelines for covid-19 prevention. evidence from other countries also finds a substantial proportion of cases originating from households, as for instance, in israel, where 67% of all infections were found to have been originated at home (jaffe-hoffman, 2020). we estimated the age structured attack rate and transmission (figure 3 ) and found a (linear) relationship between age and both the attack rate and transmission risk from primary cases. this suggests that susceptibility to infection increases with the age of the susceptible person. however, children (younger than 15 year of age) also have an elevated transmission risk, likely due to closer contact with parents ( figure c. 2), indicating that children may represent an overlooked risk. our findings correspond well with findings in the literature: madewell et al. (2020) found that susceptibility to infection increased with 14 . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint age, and a large seroprevalence study in spain also found an increasing linear relationship with age (pollán et al., 2020) . similar findings were found by li et al. (2020) and bi et al. (2020) . we further estimated the attack rates conditional on the primary case being a child or an adult (figure c.1) . when the primary case is a child (under 15 years old), the attack rate seemed to be uniform, regardless of the age of the potential secondary case. when the primary case is an adult (over 25 years old), the attack rate increased (linearly) with the age of the potential secondary case. this suggests that transmission from children is fairly constant, and depends on close contact with susceptible cases, whereas transmission from adults is more effective the older the potential secondary case is. one could think that if a child is sick, caregivers are likely to have more even close contact with the child -and more so the younger the child is. the opposite may be true for adult cases, indicating that the susceptibility to covid-19 increases with the age of a person, reflecting immunological properties. although there is agreement that transmission from and between children is not the main driver in this epidemic (ludvigsson, 2020) , transmission from sick children to parents in the household domain may represent a hitherto overlooked risk factor. we also estimated the age structured attack rates by sex and found no difference in attack rates between males and females, corresponding to the findings of jing et al. (2020). we used a large administrative data set to investigate the attack rate and transmission risk from lockdown to reopening, comprising 6,782 primary cases and 14,233 potential secondary cases. exploiting this rich nationwide data, we were able to address several concerns regarding the sensitivity of our results. as the testing capacity and strategy (and hence access to obtaining a test) has changed remarkably over the epidemic, robustness of results are a primary concern when comparing positive test cases over the covid-19 epidemic. we addressed this by dividing our sample into three periods with different testing capacities (appendix d) and found that the probability of obtaining a test did increase remarkably across the periods. our results were, however, relatively consistent over time, suggesting that the findings are not due to changing testing strategies. there are no formal guidelines for defining co-primary cases. madewell et al. (2020) provided a review on household transmission of sars-cov-2: most of the studies included in madewell et al. (2020) did not describe how co-primary cases were handled, several studies stated they assumed all secondary cases were infected by the primary case, one study excluded secondary cases if they developed symptoms before exposure to the primary case, 15 . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint and another study randomly selected one primary case as the source of infection. we addressed this important question by showing the sensitivity for different specifications. we found that the attack rate is strongly dependent on the definition. for instance, in figure 1b we show that if one defines co-primary cases as individuals tested on the same day as the primary case, the secondary attack rate is reduced by 24% (4% / 17% = 24%). increasing the period for co-primary cases further caused the estimated attack rate to decrease even more. therefore, it is important to further investigate the dynamics of sars-cov-2 in homes, schools, workplaces and major places in the community in order to quantify their impacts on virus transmission and develop effective control measures. mathematical modeling is a widely used tool for researchers in order to understand and predict the spread of disease; and policy relies on proper results from these models when making decisions such as choosing between keeping some parts of society open and other parts closed. closing of childcare and schools has been widespread in most countries. the results from this study can be used as direct input in parameterizing such mathematical models in terms of virus transmission at home. furthermore, we estimated the age structured attack rate and transmission risk, as well as the interaction of these, which are important inputs in mathematical models, for instance, for contact matrices between age groups (davies et al., 2020; zhang et al., 2020) . when modeling the spread of covid-19, many researchers assume that each contact has the same probability of transmission (conditional on time and distance of contact), i.e., a binomial process (e.g., jing et al. (2020); kucharski et al. (2020) ). our results, however, suggest that this should be modelled as a two-step procedure when simulating contacts between individuals: first, it should be determined whether a case is infectious or not (i.e., a bernoulli process), and second, conditional on being an infectious case, a binomial process should be used to represent actual transmission. this allows replicating the transmission dynamics of the virus more realistically and hence allows more realistic predictions from these models. in conclusion, to the best of our knowledge, this study presents the results of the first nationwide register-based study on household transmission of covid-19. these results are important as they show differences in transmission pattern with the age of both the primary case and the potential secondary cases within households. a large proportion of transmission was found to occur within households, highlighting the severity of covid-19 transmission, and that preventative measures within households are urgently needed in order to prevent transmission. moreover, monitoring the proportion of positive cases originating from households may be an important tool for public health authorities to measure community transmission. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https: //doi.org/10.1101 //doi.org/10. /2020 appendix a is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint -44 61 38 23 122 45 -49 77 53 27 157 50 -54 87 57 36 180 55 -59 74 52 18 144 60 -64 65 35 17 117 65 -69 39 22 11 72 70 -74 34 14 13 61 75 -79 23 5 9 37 80 -84 13 7 7 27 85 -89 6 <5 -7 90 -94 -<5 -<5 95 -99 ----100 -104 ---1 ----2 276 189 103 568 3 161 156 64 381 4 209 190 119 518 5 118 143 57 318 6 43 48 28 119 notes: this table provides summary statistics on the potential secondary cases included in the study. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint appendix c age structured attack rate notes: panel a shows the probability of being tested and panel b the probability of having a positive test. the age of the primary case is depicted on the x-axis and the age of potential secondary cases on the y-axis. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020 . . https://doi.org/10.1101 appendix c.1 age structured attack rate by sex is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020 . . https://doi.org/10.1101 appendix d dynamics over the epidemic appendix d.1 testing dynamics over the epidemic figure d .1 panel a-c presents the testing dynamics for the three periods of the epidemic. panel a shows that during lockdown only few percent of the other household members were tested and that there was a high positive rate of the ones tested. panel b shows that during the early reopening, more household members were being tested and the positive rate declined accordingly. lastly, panel c shows that in the late reopening even more household members were being tested and the positive rate declined further. panel d-f shows the proportion of household members ever being tested and ever being positive. during lockdown 25% of secondary household members were tested after 14 days, during early reopening 65% were tested, and during late reopening 82% were tested. similarly, 11% had tested positive after 14 days during lockdown, 16% during early reopening and 17% during late reopening. another aspect of testing is how long a person has to wait for the result after obtaining the test. table a .1 shows that 75% of all cases received the result within 1 day during lockdown, within 2 days during early reopening, and within 1 day during late reopening. notes: shaded areas are 95% confidence bands clustered on the household level. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint appendix d.2 age structured attack rate over the epidemic figure d .2 illustrates the age structured attack rate separately for the three periods of the epidemic. the testing rate clearly increases during the epidemic, while the rate of positive test results is relatively constant. both rates increase with age of the case. notes: this figure illustrates the age structured attack rate separately for the three periods of the epidemic. figure 3 shows the estimates for the pooled analysis. the testing rate clearly increases during the epidemic, while the positive rate is fairly constant. shaded areas are 95% confidence bands clustered on the household level. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint notes: the estimates are calculated from households with at least one potential secondary case, i.e., households with two to six members. the estimate for two secondary cases is calculated from households with at least two potential secondary cases, i.e., households with three to six members. estimates are in percentages. standard errors in parenthesis, clustered on the household level. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint notes: this table provides estimates on the robustness of the estimates for the attack rate depending on the definition of co-primary cases. column (i) corresponds to column (ii) in table e .1. in column (ii) we exclude secondary cases that test positive on the same day (t = 0) as the primary case. in column (iii) we exclude secondary cases that test positive within one day (t ≤ 1) of the primary case. in column (v) we exclude secondary cases that test positive within three days (t ≤ 5) of the primary case. standard errors in parenthesis, clustered on the household level. *p<0.05, **p<0.01. . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint notes: this figure illustrates robustness for the definition of co-primary cases with respect to the age structured attack rate. panel a has no restrictions and is the same as figure 3. panel b excludes secondary cases testing positive the same day as the primary case (t = 0). panel c excludes secondary cases testing positive within 1 day of the primary case (t ≤1). panel d excludes secondary cases testing positive within 2 days of the primary case (t ≤2). . cc-by 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.09.20191239 doi: medrxiv preprint an empirical evaluation of accounting income numbers epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study. the lancet infectious diseases risk assessment of novel coronavirus covid-19 outbreaks outside household transmission of 2009 pandemic influenza a (h1n1) virus in the united states a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 age-dependent effects in the transmission and control of covid-19 epidemics economic effects of coronavirus outbreak (covid-19) on the world economy temporal dynamics in viral shedding and transmissibility of covid-19 early dynamics of transmission and control of covid-19: a mathematical modelling study. the lancet infectious diseases what settings have been linked to sars-cov-2 transmission clusters? the characteristics of household transmission of covid-19 secondary attack rate and superspreading events for sars-cov-2. the lancet children are unlikely to be the main drivers of the covid-19 pandemic-a systematic review household transmission of sars-cov-2: a systematic review and meta-analysis of secondary attack rate the implications of silent transmission for the control of covid-19 outbreaks prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study projecting social contact matrices in 152 countries using contact surveys and demographic data coronavirus disease (covid-19): situation report changes in contact patterns shape the dynamics of the covid-19 outbreak in china 1: robustness for definition of co-primary cases key: cord-337458-dc90ecfe authors: markwalter, christine f.; kantor, andrew g.; moore, carson p.; richardson, kelly a.; wright, david w. title: inorganic complexes and metal-based nanomaterials for infectious disease diagnostics date: 2018-12-04 journal: chem rev doi: 10.1021/acs.chemrev.8b00136 sha: doc_id: 337458 cord_uid: dc90ecfe [image: see text] infectious diseases claim millions of lives each year. robust and accurate diagnostics are essential tools for identifying those who are at risk and in need of treatment in low-resource settings. inorganic complexes and metal-based nanomaterials continue to drive the development of diagnostic platforms and strategies that enable infectious disease detection in low-resource settings. in this review, we highlight works from the past 20 years in which inorganic chemistry and nanotechnology were implemented in each of the core components that make up a diagnostic test. first, we present how inorganic biomarkers and their properties are leveraged for infectious disease detection. in the following section, we detail metal-based technologies that have been employed for sample preparation and biomarker isolation from sample matrices. we then describe how inorganicand nanomaterial-based probes have been utilized in point-of-care diagnostics for signal generation. the following section discusses instrumentation for signal readout in resource-limited settings. next, we highlight the detection of nucleic acids at the point of care as an emerging application of inorganic chemistry. lastly, we consider the challenges that remain for translation of the aforementioned diagnostic platforms to low-resource settings. in 1906, william osler, one of the founders of modern american medicine, stated, "diagnosis, not drugging, is our chief weapon of offence." 1 despite the medical advances of the 20th and 21st centuries, infectious diseases remain major global health issues and continue to claim millions of lives each year in low-and middle-income countries (lmics). 2−4 as the global community moves toward control and elimination of infectious disease, it has become evident that there is a pressing need for diagnostic strategies that can be applied in primary health care settings. 5−7 this review shines a spotlight on how the applied uses of inorganic chemistry advance the concepts of metals-in-medicine beyond therapeutics and vaccines and into the realm of diagnostics, enabling new tools to meet these global challenges. the world health organization depicts lmic health care accessibility and infrastructure as a tiered pyramid structure, in which the best-equipped facilities are the least accessible and facilities with the least amount of resources are most common ( figure 1 ). 8 in this system, a level 1 facility is a primary care setting with little laboratory infrastructure, trained personnel, or advanced diagnostic technology. in lmics, both urban and rural level 1 facilities frequently lack essential resources such as consistent electricity and clean running water. district hospitals (level 2), regional laboratories (level 3), and national reference laboratories (level 4) have more advanced diagnostic technology available with increasing infrastructure; however, these facilities are often inaccessible to most patients in need due to geography, cost, and lack of transportation. 8 consider the case of early human immunodeficiency virus (hiv) diagnosis in exposed infants. coulibaly et al. highlight the challenges faced by this population in their 2011 burkina faso study. 9 hiv-positive mothers were advised to attend a 6week postnatal appointment at their nearest primary healthcare facility for collection of dried blood spot samples from their exposed infants. qualified sample collection technicians were often only available once each month, so mothers would have to return to the clinic on that day for testing. once collected, the dried blood spot samples were sent to district-level hospitals (level 2), which then sent the samples to a reference laboratory (level 3). the tertiary laboratory then determined viral load by performing polymerase chain reaction (pcr) to detect hiv genetic material. test results followed the same circuitous path back to the patients, a process that could take as long as four months. additionally, in the case of a positive dried blood spot result, the process had to be repeated in order to validate the original results. 9 in contrast, the same high-risk newborns in the united states are screened for hiv at birth with additional tests at 2−3 weeks, 1−2 months, and 4−6 months. 10 for these patients, results are usually available within 1 or 2 days of sample collection, allowing immediate antiretroviral treatment for hiv-positive children. 11 the consequences of diagnostic efficiency versus inefficiency could not be more striking; in the burkina faso study, 10% of the hiv-positive infants died before antiretroviral therapy could even be started, while the early treatment initiated in the u.s. improved infant survival by a remarkable 76%. 9, 12 the stark disparity in response time between these two settings shows just how devastating outcomes can be for patients relying on level 1 facilities for their healthcare needs. disease diagnosis in a level 1 setting represents a challenge that often requires simple tools that can be used without skilled personnel or significant laboratory or physical infrastructure. the world health organization has developed a set of criteria ("assured") that defines the ideal characteristics for pointof-care (poc) tests in low-resource settings. 13 in accordance with these criteria, an ideal test should be affordable to those who are at risk of infection, result in few false-negatives (sensitive) and false-positives (specific), and be user-friendly, rapid and robust, equipment-free, and deliverable to the populations in need of the test. additionally, the required performance parameters of a test depend on its intended usecase scenario. for individual patient case management, sensitive multiplexed tests are advantageous for determining the source(s) of nonspecific symptoms and selection of appropriate treatment. from an epidemiological standpoint, if the goal is simply to reduce the prevalence of high-intensity infections within a geographic region, the need to quantify disease burden outweighs the need for high analytical sensitivity. if the goal is disease elimination, high analytical sensitivity is one of the most important parameters, since the interruption of local transmission requires the detection of every infection, including extremely low-intensity infections. the most sensible approach to designing and developing a poc diagnostic is to examine and optimize each individual component before integrating into a single diagnostic device. in this review, we define the components of a diagnostic to include: (1) the target biomarker, an endogenous indicator of a disease state, which is most often a pathogen or host protein, carbohydrate, or nucleic acid sequence, (2) sample preparation, which allows for biomarker isolation, purification, and/or concentration from complex biological matrices, (3) molecular recognition elements, which specifically capture and detect the target biomarker, (4) signal generation and amplification, and (5) instrumentation for signal read-out. simple components requiring only a single user step are generally preferable in lowresource level 1 settings. however, there are few diagnostic technologies that currently can be deployed in these settings, and the lack of appropriate diagnostics in resource-limited settings can lead to tragic outcomes. fortunately, emerging technologies based on inorganic chemistry and nanomaterials can be exploited as potential solutions. in this review, we focus on how inorganic chemistry and nanomaterials have been utilized in each component of a poc diagnostic for infectious disease detection. section 2 describes inorganic biomarkers that are associated with infectious diseases and how their properties have been exploited in diagnostic applications. in section 3, we discuss how coordination chemistry has been harnessed for sample preparation and protein biomarker enrichment for poc tests. section 4 details the critical advancements in inorganic chemistry and nanomaterials for signal generation probes that have allowed the field of poc diagnostics to rapidly progress. in section 5, we provide an overview of field-deployable instrumentation that incorporates the fundamental inorganic chemistries discussed in sections 2−4 for poc diagnosis of infectious disease. finally, section 6 discusses the emerging trend of using inorganic chemistry and nanomaterials for nucleic acid detection at the point of care. biomarkers are quantifiable characteristics of a disease state that can be measured accurately and reproducibly. the vast majority of biomarker targets for infectious diseases fall into three main categories: (1) pathogen genetic material, (2) protein and carbohydrate antigens produced by a pathogen, or (3) human host responses to the presence of infection, such as pathogen-specific antibodies. while inorganic markers of infectious disease are far less common than their bioorganic counterparts, metal-based biomarkers have unique properties that can be leveraged for innovative diagnostic strategies, as illustrated in the following two examples. hemozoin, often called malaria pigment, is a biomineral produced by some parasites that rely on hematophagy as their primary source of nutrients. 14 hemozoin was first described for the malaria parasite plasmodium falciparum over a century ago and is produced by all plasmodium species. 15−17 in the erythrocytic life stages, malaria parasites digest host hemoglobin in the acidic digestive food vacuole as a source of amino acids. the breakdown of one hemoglobin molecule releases four molecules of heme (ferriprotoporphyrin ix [fe(iii)-ppix]), which are highly toxic to the parasite. 17 as plasmodium spp. lack functional heme oxygenase activity, the parasites remove the free heme by crystallizing it into insoluble, inert hemozoin crystals. 17, 18 these crystals accumulate in the parasite over the course of its erythrocytic life cycle. after the mature schizont ruptures, hemozoin is released into circulation and rapidly taken up by phagocytes. 19 the heme detoxification pathway that produces hemozoin is crucial for malaria parasite survival; therefore, hemozoin indicates the presence of plasmodia parasites, making it an attractive biomarker for malaria detection. 19 structurally, hemozoin crystals consist of reciprocating fe(iii)ppix dimers linked through coordination between the central ferric ions and a carboxylate group of one of the propionate moieties on protoporphyrin iv. 17, 20, 21 dimers assemble into chains through hydrogen bonding of the second propionate porphyrin side chain, and these networks of crosslinked dimers are held together via π−π interactions ( figure 2 ), resulting in an anisotropic rectangular crystal with unique magnetic, optical, and photoacoustic properties. 22 one such property of hemozoin is its paramagnetism, which is derived from the unpaired electrons on the central fe (iii) ions. 23 like all paramagnetic substances, hemozoin has a magnetic moment and is thus attracted up a magnetic gradient in the presence of an externally applied magnetic field. the paramagnetism of hemozoin has been leveraged for malaria diagnosis in several ways. first, the magnetic properties have been exploited for separation, purification, and concentration of infected red blood cells (irbcs) from blood samples. with the use of a high-gradient external magnetic field, irbc separation was first performed by heidelberger et al. 24 in 1946 for the purpose of vaccine development, though at the time the method was described as "applicable only to small blood volumes, [and requiring] apparatus difficult of access." 24 since then, the availability of high-field permanent magnets and electromagnets has substantially improved, and the time required for magnetic separation of irbcs has been reduced. both commercially available systems as well as custom-made microfluidic devices have been used to enrich irbcs from whole blood samples for analysis by microscopy. 25−32 three groups have explored detection of hemozoin based on the fact that paramagnetic materials decrease nuclear magnetic resonance (nmr) transverse relaxation time (t 2 ) of water, the same phenomenon leveraged for magnetic resonance imaging (mri) contrast agents. karl et al. 33 first demonstrated that hemozoin could be detected based on decreased t 2 , though only at parasitemias greater than 10000 parasites/microliter, which is 2 orders of magnitude greater than the detection limits of commercial rapid diagnostic tests. several years later, peng et al. 34 and kong et al. 35 developed a low-cost (<$2000), homemade micromagnetic resonance device for hemozoinbased malaria detection. they reported detection limits less than 10 parasites/microliter using their method and attributed this drastic improvement to the use of ultrashort echo times and a premeasurement centrifugation step that pelleted red blood cells from whole blood. 34 these discordant conclusions led to a reaction by karl et al. 36 and a response by peng et al. 37 recently, gossuin et al. 38 confirmed the results of karl et al., indicating that conventional relaxometry alone is insufficient for sensitive malaria detection. these findings led to two hypotheses related to the superior sensitivity measured by peng et al.: (1) the premeasurement centrifugation of higherdensity irbcs (compared to uninfected rbcs) significantly enriched hemozoin in the portion of the pellet measured, and (2) ultrashort echo times may have allowed for probing of other protons, such as macromolecular protons, which may be more susceptible to the presence of hemozoin. 38 a simple, lowresource relaxometry-based detection device remains to be developed, and such a device would ultimately require resources typically not available in primary healthcare settings in lmics (i.e., consistent availability of electricity and clean water). however, these studies highlight the importance and advantages of sample preparation and enrichment steps in the development of sensitive infectious disease diagnostics. in addition to its paramagnetic properties, the unique optical properties of hemozoin enable the development of new diagnostic tools for malaria detection. in particular, hemozoin strongly scatters and absorbs light. 39 the latter characteristic led to the remarkable development of the microvascular microscope (mvm) for needle-free, in vivo detection of hemozoin. 40, 41 the mvm employed cross-polarized epiillumination to optically access vessels below the surface of the skin, with green light (λ = 532 nm) illumination for locating vessels and red light (λ = 655 nm) illumination for detecting hemozoin. this method was able to detect parasitemias as low as 0.03% in mouse models, corresponding to approximately 1000 parasites/microliter in humans. unfortunately, lower parasite density infections were not explored. 40 the mvm was able to differentiate between irbcs and hemozoin-containing white blood cells, which can persist in circulation for weeks after an infection is cleared, based on the diameter and relative intensity of the detected hemozoin. 41 additionally, hemozoin particle velocity could be measured in vivo (figure 3 ), potentially allowing for identification of cytoadhesion and sequestration, two phenomena related to disease severity. 41, 42 though further sensitivity analysis and field testing is required, the mvm represents a promising, noninvasive tool for malaria detection, particularly if a portable and battery-powered device is developed. photoacoustic spectroscopy has also demonstrated potential for transdermal malaria detection. 43−48 photoacoustic (pa) hemozoin detection requires excitation with intense light and measurement of the resulting acoustic signals generated by local thermal expansion of the surrounding medium. the utility of in vitro pa detection of hemozoin was first demonstrated by balasubramanian et al. in 1984 . 43 since then, it has been shown that short, intense laser excitation of hemozoin in the parasite can localize heat around the crystal, evaporating the surrounding medium and creating transient, nanosized vapor bubbles. the expansion and collapse of these nanobubbles enhance acoustic detection. 44 this technique has allowed for transdermal malaria detection with exceptional sensitivity in mice. this method was also applied to one human patient with high parasite density (8900−65000 parasites/microliter). 45−48 further studies with increased patient sample size will be necessary to determine the true limits of the technology. magneto-optical detection exploits both the paramagnetic and optical properties of malarial hemozoin crystals, which have a rectangular shape that affords a high level of both magnetic anisotropy and optical dichroism. in this technique, whole blood (or lysed whole blood) is inserted into a magnetic field, orienting the paramagnetic crystals along the applied field direction. this phenomenon, known as the cotton-mouton effect, induces an optical dichroism across the dispersion of crystals, resulting in a detectable change in transmittance of modulated polarized light (λ = 660 nm) ( figure 4 ). 49, 50 when applied to patient whole blood samples, the diagnostic sensitivity and specificity of magneto-optical detection were not competitive with rapid diagnostic tests and pcr, though the ease of use is encouraging for poc applications. 49, 50 in particular, model simulations have suggested that magnetooptic detection of hemozoin could be exploited for the development of a noninvasive probe capable of measuring transmittance of polarized light through a fingertip. 51 a similar magneto-optic strategy that employs a fluctuating magnetic field and measures the change in amplitude of transmittance has shown potential in mouse models, but further evaluation of the diagnostic sensitivity and specificity in human samples is needed. 52−55 recently, a commercial magneto-optic device for malaria detection based on hemozoin has demonstrated promise in field evaluations, detecting clinical parasite densities as low as 5 parasites/microliter (hemex health). 56 additional techniques used to detect malarial hemozoin include flow cytometry, 57−60 laser-desorption mass spectrometry, 61−64 and raman spectroscopy. 65−69 while these strategies are highly sensitive, they require expensive and resourceintensive instrumentation that typically prevent their use in a district hospital setting, local health outpost, or field setting. however, as instruments become more compact, robust, and affordable, similar to some of the technologies detailed in this section, their implementation in field settings could become more feasible. importantly, hemozoin is synthesized by other hematophagous parasites that infect humans, including the schistosoma, echinostoma, and opisthorchis trematodes, as well as blood feeding insects, such as rhodnius and boophilus. 17, 70, 71 despite being produced by these additional parasites, hemozoin has not yet been used as a diagnostic biomarker for nonmalarial pathogens. further, it is important to note that in all of the aforementioned hemozoin-based malarial detection strategies, hemozoin originating from trematodes would produce a positive signal. this is particularly important to consider regarding false positive tests since the geographic distribution of malaria overlaps with schistosoma. 72, 73 in addition to confounding signal originating from other hematophagous parasites, hemozoin as a biomarker presents some unique challenges to malaria diagnosis. first, the biocrystal is present at much lower concentrations in the ring stage when compared to the later trophozoite and schizont stages of the erythrocytic cycle. 32 thus, magnetic enrichment or malaria diagnosis strategies based on hemozoin could miss early stages of infection, potentially resulting in false-negatives. additionally, phagocytic cells ingest irbcs and hemozoin released into circulation after schizont rupture, and hemozoincontaining white blood cells have been shown to persist in circulation up to 17 days after parasite clearance. 74 this persistence can lead to false-positive results in hemozoin-based malarial diagnostics unless an algorithm for distinguishing pigment-containing white blood cells and irbcs is devised, such as the one developed in the aforementioned work by burnett et al. 41 despite these challenges, researchers continue to develop innovative techniques for malaria detection using hemozoin as a biomarker. implementation of these methods will require careful design with cost and equipment reduction in mind in addition to rigorous field evaluation. schistosome eggs are the most common biomarker for schistosomiasis detection. they are produced by adult schistosoma worm pairs that reside in the intestine and bladder and deposited into the vessels of the gut wall. 75 many eggs become permanently lodged in the intestine, liver, bladder, or urogenital system, leading to chronic inflammation and systemic tissue damage; 76 however, some mature eggs are excreted in urine or feces to allow the parasites to continue the transmission cycle. 75 eggs from distinct species are distinguishable by their morphologies. differences include the overall egg shape (round to oval), spine position, and extent of filamentous coating. 77 all of these features are observable by microscopy, which is the gold standard for schistosomiasis diagnosis. voided eggs from all schistosoma species contain mature miracidia within a relatively impermeable eggshell. 75 while the dense biopolymer composition of schistosoma eggshells has been studied since the 1960s, 78 the inorganic components of the eggshells were not investigated until 2007, when jones et al. 79 demonstrated the presence of iron in schistosoma japonicum eggshells ( figure 5a ). the group used inductively coupled plasma mass spectrometry (icp-ms) and energy dispersive spectroscopy (eds) to provide evidence that iron localized to the eggshell. although these elemental characterization techniques did not provide specific information about the oxidation state or organization of iron present, the group found that the eggshells contained 121 mg/kg (dry weight) fe, making up nearly 89% of the iron found in intact eggs. 79 in the same year as the jones study, teixeira et al. 80 set out to increase the sensitivity of microscopy for schistosoma detection using magnetic microparticles to enrich eggs from fecal samples. to accomplish this, stool samples were suspended in water and repeatedly sieved and washed before the sediment was incubated with magnetic particles that bound to the schistosoma eggs. this technique for egg concentration was dubbed the helmintex method 80 and was demonstrated to be more sensitive than kato-katz, the current who recommended sample preparation method for schistosome diagnosis in a field setting. 81 the initial hypotheses of the helmintex method were that biotinylated lectins would bind to carbohydrates on the surface of schistosoma mansoni eggs and that streptavidin-coated magnetic beads could bind the biotinlectin-egg complexes. using an external magnet, the eggs could then be isolated and concentrated before detection by microscopy. however, control experiments demonstrated the following: (1) lectins were not necessary for egg binding to magnetic particles, (2) nonmagnetic latex particles did not bind to s. mansoni eggs, and (3) the eggs alone did not migrate to an external magnet ( figure 5b ). 80 these observations, coupled with the discovery of iron localized to eggshells, led some to hypothesize that, in the presence of an external magnetic field, the paramagnetic beads act as weak bar magnets, enabling attraction of the iron in the shells of the eggs. 82 in 2013, karl et al. 83 further probed the iron distribution and magnetic properties of schistosome eggshells to elucidate the mechanism of the helmintex method. the study found that iron was localized to pores in eggshells, and schistosome eggs demonstrated moderate paramagnetic behavior. however, the eggs did not move in a magnetic field, and the paramagnetic particles bound to parasite eggs in the absence of a magnetic field, suggesting that the mechanism of binding was not magnetic in origin. 83 the authors also found that s. japonicum eggs bound more microparticles than s. mansoni, leading to the hypothesis that the filamentous outer structure of the eggs, more prominent in s. japonicum compared to s. mansoni eggs, provided strong, nonmagnetic adhesion based on many relatively weak van der waals interactions. 83 in a separate study, candido et al. 84 also observed that s. japonicum eggs bound more microparticles than s. mansoni. the group used poisson analysis and found two distinct populations within each species: a high "affinity" group and a low "affinity" group, where affinity was determined by the number of microparticles bound to the visible edge of a single egg. interestingly, the magnitudes of the high affinity values were similar between the two species, but the fraction of eggs in the high affinity group for s. japonicum was greater than for s. mansoni. 84 collectively, these results suggest that differences in filamentous microspines between the two species do not fully explain the mechanism of egg-microparticle binding. additionally, the work by candido et al. opens up many questions, the answers to which could greatly inform further optimization of the helmintex method. for instance, what are the physiochemical differences between high and low affinity egg groups? is there a biological difference between the two groups, such as egg maturity or surface antigen expression? both the karl et al. and candido et al. studies were performed on eggs isolated from livers of infected mice, but do these two egg populations appear in the urine or feces of infected humans? if so, should two types of magnetic microspheres be used to ensure the helmintex method captures all egg types present in a sample? these questions should be answered carefully and systematically. given the greater number of microspheres bound to eggs in the high affinity group, the populations should be separable using external magnetic fields of differing strengths. egg-microparticle binding mechanisms for each population could be interrogated by determining solution properties (e.g., ph, salt, surfactant, chelators, blocking proteins, etc.) required to interrupt the interactions. additionally, functionalizing microspheres in a way that systematically varies their zeta potential and hydrophobicity could determine the role these physiochemical properties play in binding. these studies will be crucial if the helmintex method aims to determine infection intensity using quantitative egg enrichment. this is particularly important for schistosomiasis surveillance and morbidity control campaigns, which aim to quantify and reduce the prevalence of high-intensity infections. the helmintex method originated with the idea that iron in the eggshells of schistosomes could serve as a handle for magnetically enriching schistosoma eggs for more sensitive detection in the field setting. while the helmintex method has already been shown to improve the sensitivity of kato-katz, elucidation and optimization of the true binding mechanisms could lead to an even more impactful method for diagnosing low-intensity schistosomiasis infections. metal-based sample preparation techniques that enrich biomarker concentration improve the sensitivity of diagnostics by delivering more biomarker to the test. these strategies can be applied to existing assured diagnostic tests, such as lateral flow assays (lfas). while the easiest way to deliver more biomarker to the tests would be to add larger sample volumes (100−500 μl), many lfas cannot accommodate large sample volumes for several reasons: 85 first, limited bed volumes in porous paper substrates physically limit the amount of liquid a test can hold. second, the increase in the number of interfering molecules that results from an increase in sample volume could cause nonspecific cross reaction with the test's molecular recognition elements (e.g., antibodies). third, colored biological matrices, such as whole blood, can increase the background signal and decrease the user's ability to distinguish between positive and negative results. metal-based sample preparation techniques mitigate these shortcomings, allowing for the concentration of a target from a large volume sample into a smaller, test-compatible volume. moreover, metal-based sample preparation also removes the target from its original complex biological matrix, decreasing the potential for nonspecific cross-reactivity. 85 these low-cost, simple, and robust methods represent a powerful tool for use-case scenarios in which improved sensitivity is needed, such as elimination campaigns. this section will discuss how metal coordination chemistry has been leveraged for simple sample preparation methods applicable to poc diagnostics. coordination chemistry, which is defined as the chemistry of metal atoms or ions that form complexes with one or more ligands, 86 can be utilized to coordinate biomarkers with a particular affinity for metal ions. the most significant illustration of this capacity is the isolation of proteins via immobilized metal affinity chromatography (imac). 87,88 this technique involves the conjugation of various ligands to a solid support matrix such as agarose, cellulose, or silica. these ligands act as lewis bases, chelating metal ions with a high affinity. the fixed metal ions serve as lewis acids, coordinating to both the immobilized ligands and specific amino acid residues in a protein biomarker. 89 while amino acid affinities toward metal ions vary, histidine demonstrates a particularly high affinity toward metal ions in this format. these interactions can be leveraged to isolate a biomarker from a complex biological matrix. once separated, the target can be competitively eluted and concentrated into a smaller volume suitable for a poc diagnostic test. 87, 88 the choice of chelating ligand is critical to imac protein separation efficiency. ligands both fix the metal ion to the solid support and modulate metal-protein binding based on the number and conformation of chelation sites. 88 ,90−92 a multitude of ligands with varying coordination numbers have been employed. most imac platforms contain tridentate or tetradentate chelators, although some bidentate and pentadentate options exist ( figure 6 ). tetradentate nitrilotriacetic acid (nta) 93 and tridentate iminodiacetic acid (ida) 94 are two of the most common commercial ligands, but many companies have also developed their own proprietary structures such as talon 95 or ni-penta. 96 the variety of chelating ligands, many of which possess different coordination numbers, make imac tunable, which is beneficial when tailoring the chemistry to different diagnostic applications. 87,88,97−100 guided by pearson's hard−soft acid−base theory, numerous metal ion and ligand combinations have been exploited in imac. borderline acids such as co 2+ , ni 2+ , cu 2+ , and zn 2+ have predominated, 86−88 displaying varying levels of affinity and specificity. the determination of which ion will be optimal in a given isolation often depends on the protein in question. ni(ii)-nta has been one of the most widely utilized metal− ligand combinations in imac applications. until recently, a thorough investigation that compared divalent metals and their molecular recognition capability was lacking. bauer et al. 99 filled this void by conducting a systematic investigation of the affinity of malarial biomarker histidine-rich protein 2 (hrp2) to m 2+ -functionalized biosensors and solid phase resins (m 2+ = co 2+ , ni 2+ , cu 2+ , or zn 2+ ). hrp2 is the most frequently detected target in malarial rapid diagnostic tests and possesses a unique primary structure containing 35% histidine, making it an ideal histidine-rich target. 101 the authors used biolayer interferometry (bli), an optical biosensor technique, to determine kinetic constants (e.g., k on , k d ) for the binding of the various metals to hrp2. the authors found that co 2+ and zn 2+ exhibited the fastest binding kinetics (highest k on ) and the highest hrp2 binding affinity (lowest k d ) on bli biosensors. 99 when the same imac complexes were incorporated into a loose solid phase resin, zn(ii)-nta resulted in the fastest complete binding of hrp2 per mole of divalent metal while also allowing for the most efficient elution of hrp2 upon the addition of imidazole. once a metal had been selected, hrp2's binding and elution behavior was evaluated against a variety of ligands and solid supports. the selected zn(ii)-ida resin was then integrated into a pipet tip format, enabling direct hrp2 capture from spiked lysed blood samples. this field-deployable sample isolation and concentration tool displayed up to a 4-fold signal enhancement in commercial rapid diagnostic tests (figure 7 ). this study 99 demonstrated that optimizing the imac metalligand combination for a particular application is critical for efficient biomarker capture and elution; the initial binding cannot be too tight so as to prevent biomarker elution with a competing ligand (e.g., imidazole) nor too weak so as to attain suboptimal biomarker enrichment. 88, 99 imac has been adapted to a variety of solid support platforms, from agarose gels to rigid silica particles. 102, 103 although many of these platforms have become wellestablished research tools, the emergence of novel materifigure 6 . coordination of imac ligands (ida, nta, and ted) to a divalent metal for binding histidine residues in proteins. co 2+ , ni 2+ , cu 2+ , and zn 2+ have predominantly been utilized in this application. ida (iminodiacetic acid) is a tridentate ligand (coordination number = 3), with the remaining three coordination sites in the metal available to bind two histidine residues and a water molecule. nta (nitrilotriacetic acid) is a tetradentate ligand (coordination number = 4) with two coordination sites for histidine residue binding. ted (tris(carboxymethyl)ethylene diamine) is a pentadentate ligand (coordination number = 5) with one remaining coordination site for protein binding. als 104−108 has enabled imac chemistry in new applications. for example, wang et al. 109 polymerized polyhedral oligomeric silsesquioxanes (posss) into micron-sized particles with nanosized pores, which were subsequently functionalized with ida and charged with cu 2+ . this material was then used for specific capture of hemoglobin, which is rich in surface-exposed histidine residues. incubation of the test solution with the cu(ii)-ida functionalized material enabled selective capture of hemoglobin versus other proteins without these accessible histidine residues. 109 the development of this novel material using the principles of imac demonstrates that nanomaterials can be modified for molecular recognition of histidine-rich proteins. these modified nanomaterials have the potential to be incorporated into poc diagnostic frameworks for biomarker capture. imac-functionalized cellulose membranes could provide another excellent option for sample preparation in lowresource settings. given that these membranes already hold an established place as a laboratory research tool, they could readily be incorporated into field-friendly paper fluidic devices. opitz et al. 110 utilized commercial ida-functionalized cellulose charged with zn 2+ as a platform for separating influenza virus particles from culture lysate. after an initial metal screening, they saw that zn 2+ -charged membranes separated particles with significantly higher affinity the other metals tested. the authors postulated that the influenza hemagglutinin glycoprotein on the particular strain studied (influenza virus a/ puerto rico/8/34) contained histidine-rich zn 2+ -coordinating sequences on each subunit and that the repeated configuration of these subunits as part of the capsid structure contributed to a higher overall binding avidity. 110 although this imac-based method for separating virus particles from cell culture was developed to improve the efficiency of laboratory-based research, it is not difficult to imagine extending this concept to a simple sample preparation method for increasing the sensitivity of an influenza rapid test. applying the developed protocol to a diagnostic test would require feasibility testing on clinical patient samples, ideally, resulting in the design of an integrated device for both sample preparation and virus detection. imac-functionalized cellulose membranes present a unique advantage in relation to other materials because of the ease in which they can be incorporated into paper-based diagnostic formats that are usable in low-resource poc settings. magnetic particles hold a number of advantages over other solid imac supports. the increased surface area of the particles provides increased availability for chemical reactivity and functionalization. 111, 112 the independent response of paramagnetic particles to an externally applied magnetic field enables active mixing, which has proven especially useful in microfluidic applications. this magnetic susceptibility also allows for easy manipulation of the particles once a targeted biomarker is bound, permitting biomarker isolation and concentration before downstream detection. 111, 112 multistep protocols that would otherwise rely heavily on laboratory techniques such as filtration, centrifugation, and column chromatography can easily be performed using magnetic beads and a hand-held external magnet. aside from chromatographic capabilities, magnetic particles have been employed as signal generation labels that are detected by inductive or magnetoresistive sensors. this application is discussed in detail in sections 4.2.6 and 5.4. magnetic particles thus represent a promising vehicle for imac chemistries in low-resource settings. the wright research group recently demonstrated that imac-functionalized magnetic beads have the potential to improve commercial poc diagnostic sensitivity for malaria. 113 hrp2 was captured from large-volume (100 μl whole blood samples diluted and lysed to 200 μl) p. falciparum-spiked samples via chelation to ni(ii)-nta on the surface of . flow-through device for capture of malarial biomarker hrp2 via zn(ii)-ida chelation to histidine residues. the device incorporated a silica resin solid phase functionalized with zn(ii)-ida into a pipet tip, which enabled rapid capture of hrp2 from whole blood samples using the principles of imac. subsequent addition of imidazole at a high concentration competitively eluted the hrp2 from the zn(ii)-ida resin into a smaller volume. this small volume sample was then deposited onto a commercially available rapid diagnostic test which enhanced sensitivity for hrp2 detection in a poc setting. adapted with permission from ref 99. copyright 2017 american institute of physics. commercially available magnetic particles. then, using a specially designed extraction cassette and a hand-held magnet, the hrp2-bound particles were guided through stationary liquid chambers separated by surface-tension valves to wash the sample and remove background. once purified, the magnetic beads with bound hrp2 were transferred to a final 10 μl elution chamber preloaded with imidazole to release the biomarker. this resulted in a 10-fold theoretical concentration factor for hrp2 due to the transfer from a 100 μl blood sample to a 10 μl elution chamber. after extraction using this self-contained system, the authors added the 10 μl elution volume to a variety of commercial hrp2-based malaria rapid diagnostic tests and found significant signal enhancement across all brands. 113, 114 in subsequent studies, the group developed and optimized a hand-held, easy-to-use device 85, 115 (figure 8a ) in which hrp2-bound, imac-functionalized magnetic beads were directly transferred to the sample pad of commercial malaria lateral flow assays. the biomarker was eluted with an imidazole-spiked running buffer. as shown in figure 8b , this device enabled highly sensitive hrp2 detection with few added user steps, improving the detection limits of commercial malaria tests by over an order of magnitude with minimal added cost per test. 85, 115 this enhanced sensitivity could make a substantial difference in malaria elimination campaigns, which rely on detecting and treating all infected individuals, including low-density carriers. one of the primary limitations of imac chemistry is its ability to recognize only those proteins with affinities for metal ions. for purification of recombinant proteins from cell culture, this drawback has been overcome via incorporation of short hexahistidine tags (his 6 -tags) into expressed protein sequences. 116,117 using a similar principle, bauer et al. 117 developed a platform in which target-specific antibodies modified with his 6 -tags were combined with imac magnetic beads to isolate and concentrate biomarkers from biological samples. thus, the advantages of imac for biomarker enrichment were no longer limited to histidine-rich proteins but rather could be extended to any protein biomarker target. in the context of malaria diagnosis, this expanded capability allows for the detection of the nonhistidine-rich biomarker plasmodium lactate dehydrogenase (pldh) alongside hrp2, which is beneficial for several reasons. plasmodium ldh is essential to parasite survival and is conserved across all species of malaria known to infect humans, whereas hrp2 is produced only by p. falciparum. 118 in addition, hrp2-only tests fail to account for parasitic infections containing pf hrp2 gene deletions, which are increasing in prevalence worldwide. 72 finally, hrp2 has been shown to persist in host circulation up to 52 days after successful treatment, but pldh clears within 24 h after parasite clearance, so a dual assay can distinguish between resolved and active p. falciparum infections. 119−122 for these reasons, bauer et al. 117 employed their imacbased his 6 -tagged antibody approach to concentrate both pldh and hrp2 from large volume samples (200 μl parasitespiked whole blood) utilizing commercially available imac magnetic beads. 117 in this dual capture system, hrp2 coordinated directly to the co(ii)-nta-functionalized surface of the magnetic bead, while pldh was bound using his 6tagged anti-pldh antibodies ( figure 9 ). following isolation of both biomarkers, hrp2 and the his 6 -tagged-antibody/pldh complex were simultaneously eluted with imidazole. when applied to whole blood samples spiked with the p. falciparum parasite, this strategy resulted in a nearly 20-fold improvement in the limit of detection of commercial lfas. 117 this unique platform demonstrates that imac-based biomarker enrichment is not limited to histidine-rich proteins and could be expanded to virtually any biomarker for which there is a commercially available antibody. one of the challenges associated with employing a his 6tagged antibody/imac concentration strategy is that the biomarker remains bound to the antibody during the detection step. the antibody used for sample preparation has the potential to block target binding (via overlapping epitopes or steric effects) to the molecular recognition elements employed in the downstream assay, leading to false-negative results. this challenge can be addressed by designing the diagnostic such that the his 6 -tagged antibody and downstream detection elements bind to orthogonal epitopes of the target. furthermore, other his 6 -tagged molecular recognition elements such as aptamers could be used in place of his 6 -tagged antibodies. aptamers are short, single-stranded nucleic acid sequences with secondary structures that afford high (∼nanomolar−picomolar) binding affinities. compared to antibodies, aptamers are smaller and may target unique epitopes, potentially allowing for this innovative sample preparation strategy to be more universally applied. 123−126 coordination chemistry-based sample preparation technologies have enabled significant improvements in the sensitivities of point-of-care diagnostics. the enhanced sensitivities, which in several examples were improved by a whole order of magnitude, make a strong case for the use of these technologies in elimination campaigns. 85, 115, 117 previously, biomarker enrichment strategies were not incorporated into low-resource diagnostics because of the restrictive format of the tests. the imac-based chemistries described in this section represent innovative yet simple solutions that open the door for the development of poc diagnostics that incorporate sample preparation. while some of the sample preparation methods presented in this section increased the total number of assay steps, these methods were not designed in tandem with the diagnostic devices that were utilized for detection. moving forward, human-centered device design that integrates sample preparation with detection elements will permit the next generation of poc diagnostics to be developed with minimized user steps and improved sensitivities. inorganic metal complexes and nanoparticles have been instrumental in the development of targeted reagents for disease-associated dna, proteins, or cells. their biomedical applications include the development of metallodrugs, 127, 128 enzyme inhibitors, 129−131 photothermal 132,133 and photodynamic 133 therapy agents, and mri contrast, 127, 132 in vivo bioimaging, 133, 134 and protein/cell imaging agents. 135, 136 their unique optical, electrochemical, magnetic, and catalytic properties have also enabled their use as signaling modalities in diagnostics. this section focuses on the application of metalbased signal generation probes for the detection of protein biomarkers at the point of care (summarized in table 1 by biomarker and in table 2 by signal generation method). (the detection of pathogenic nucleic acids via metal-based signal generation probes is discussed in detail in section 6). first, we describe how inorganic complexes have been utilized in photoluminescent, electrochemical, and electrochemiluminescent applications, respectively. subsequently, we detail the essential bioconjugation chemistries that permit nanoparticles to be functionalized with molecular recognition elements to generate useful diagnostic reagents. we then discuss how various classes of these functionalized nanoparticles have been used in poc diagnostic assays. lastly, we consider the catalytic properties of metalloenzymes and metal nanoparticles that have been utilized for signal amplification in poc diagnostics for infectious diseases. the broad array of properties that result when a ligand coordinates to a metal has been leveraged for signal production in a variety of diagnostic formats. inorganic coordination compounds are valuable as signal generation probes due to their low molecular weight, tunability, and unique catalytic and photophysical properties. 130 these compounds have been used in photoluminescent (i.e., fluorescent and phosphorescent), 137−142 electrochemical, 143−148 and electrochemiluminescent (ecl) 149−157 sensing platforms. each signal generation method has trade-offs with regard to ease-of-use and interpretation, as well as complexity of instrumentation required to measure signal output. when designing a lowresource diagnostic, these trade-offs must be evaluated based on the use-case scenario and testing environment. collectively, these considerations better inform the developer as to which inorganic complexes are suitable for a particular signal generation method. 4.1.1. luminescence. photoluminescent inorganic complexes possess unique properties and offer several advantages over organic fluorophores as signaling molecules for diagnostics. these advantages include long-lived phosphorescence, significant stokes shifts, and emission tuneability via ligand composition. 137, 158, 159 long-lived phosphorescence is beneficial because it is spectrally distinct from the autofluorfigure 9 . a his 6 -tag was coupled to an anti-pldh antibody via sulfo-smcc to allow for chelation of the his 6 -tagged antibody to co(ii)nta-functionalized magnetic bead. remaining sites on the bead surface (unoccupied by antibody) permitted coordination of hrp2 directly to the bead surface. subsequent to biomarker capture, imidazole was added to elute hrp2 and the his-tagged antibody/ pldh complex. adapted from ref 117 158 the large stokes shifts of metal complexes also result in improved sensitivity because self-quenching of signaling molecules is minimized. 158 additionally, the tunable emissions of metal complexes enable their use as signal transducers, whereby ligand exchange reactions that induce changes in emission spectra can be correlated to biomarker concentration. 159 to date, cyclometalated d 6 complexes of ir (iii) are the most commonly utilized photoluminescent signal generation probes. [137] [138] [139] 141, 142, 160 ligands in these cyclometalated ir (iii) complexes (e.g., deprotonated 2-phenylpyridine) demonstrate strong σ-donor effects, which significantly increase the energy of the 1 mc (metal-centered) excited state (i.e., the ligand field stabilization energy) that gives rise to nonradiative decay pathways. as a result, excited electrons in these complexes preferentially populate the 1 mlct (metal-toligand charge transfer) excited state (as opposed to 1 mc). the excited electrons then undergo intersystem crossing and subsequently phosphoresce from a mixture of the 3 lc (ligand-centered) and 3 mlct excited states ( figure 10 ). the high phosphorescent quantum yields with large stokes shifts make these complexes ideal for signal generation. 160, 161 davis et al. 138 designed a "switch-on" ir(iii) detection probe that selectively produced phosphorescent signal in the presence of malarial biomarker hrp2. the cyclometalated ir(iii) complex, [ir(ppy) 2 (h 2 o) 2 ] + , was poorly luminescent in the absence of hrp2; however, when aqua ligands were displaced by hrp2's histidine residues, a biomarker-dependent 3 mlct/ 3 lc phosphorescent signal was produced ( figure 11 ). similar to the principles of imac chemistry discussed in section 3, this approach capitalized on the chelation of hrp2's histidine repeats to the inorganic coordination compound. the assay design aptly showed how the tunable luminescence of ir(iii) complexes could be harnessed for signal transduction, demonstrating that a ligand exchange reaction's phosphorescent signal could be correlated to biomarker concentration. in addition, the ir(iii) complex displayed a significant stokes shift (δλ = 145 nm), 138 making it less prone to self-quenching effects and decreases in sensitivity associated with its organic fluorophore counterparts. 162 this study 138 also incorporated the ir(iii) probe into an imac magnetic bead-based biomarker isolation and detection assay for recombinant hrp2 which had a detection limit of 14.5 nm. although less sensitive than an elisa (enzymelinked immunosorbent assay), this hrp2 concentration was within the relevant range for clinical diagnosis of malaria. moreover, the bead-based assay could be completed in less than 2 h, whereas traditional elisas require 6−8 h. while the current assay format would only permit its application in better-equipped laboratories, the developed cyclometalated ir(iii) probe could be an exciting detection element in an lfa with hrp2-specific antibodies at the test line. however, this photoluminescence detection mechanism is dependent on existence of a histidine-rich biomarker, so further applications of this particular strategy are limited. a more generalizable platform using photoluminescent ir (iii) probes was developed by lin et al. 139 for the detection of interferon-gamma (ifn-γ), an inflammatory cytokine biomarker for immunological diseases such as hiv. for molecular recognition and detection of ifn-γ, the assay used two aptamers: one was highly specific for ifn-γ, and the second was a guanine-rich nucleic acid sequence. the two aptamers were designed to be partially complementary and were prehybridized prior to the introduction of a sample containing ifn-γ. in the presence of ifn-γ, the ifn-γ-specific aptamer bound to its target and liberated the guanine-rich sequence. introduction of k + ions induced the formation of guanine quadruplexes, which subsequently bound an ir (iii) probe for "switch-on" luminescence that correlated biomarker concentration with phosphorescent signal (figure 12 ). this ir(iii) probe possessed a long phosphorescence lifetime (>2.9 μs) and significant stokes shift (δλ = 215 nm), thus exhibiting the canonical advantages of inorganic probes over organic fluorophores. the assay demonstrated adequate specificity for ifn-γ versus other proteins commonly found in biological matrices (e.g., human serum albumin and immunoglobulins) and had a limit of detection of 0.12 nm and a dynamic range of 1−300 nm. however, the assay suffered a reduction in sensitivity when conducted in a 0.5% cell extract, and no attempts were made in the complex biological matrices that are pertinent to hiv diagnosis (e.g., blood, urine, saliva, etc.). nonetheless, the assay established a proof-of-principle for using a photoluminescent cyclometalated ir(iii) complex as a detection probe for an hiv biomarker. 139 moreover, the assay format is generalizable such that it could be applied to virtually any biomarker for which there is a high-affinity aptamer. if combined with one of the sample preparation strategies discussed previously (section 3) or integrated with paper or another field-ready substrate, this ir(iii)-based detection strategy could produce a robust and sensitive assay that is applicable in low-resource diagnostic settings. 4.1.2. electrochemistry and electrochemiluminescence (ecl). by applying a potential (or range of potentials) to a sample containing an electroactive inorganic probe, current or luminescence can be measured as an output for electrochemical or ecl detection, respectively. electrochemical or ecl-based assays that combine molecular recognition elements, such as antibodies or aptamers, with electroactive inorganic probes have shown promise for infectious disease detection. 149,163−165 in particular, inorganic low-spin d 6 complexes of ir (iii) and ru(ii), as well as hemin (ferriprotoporphyrin ix), have been utilized in developing electrochemical-and ecl-based probes. 149 both electrochemistry and ecl are signal generation strategies that are well-suited to the development of poc diagnostics, largely due to (1) capability for miniaturization, (2) low cost, (3) simplicity, (4) rapid time-to-result, and (5) high sensitivity. 149, 163 this has been aptly demonstrated with the development of paper-based electrochemical diagnostic platforms, 166 often termed microfluidic paper-based electrochemical devices (μpeds) 167−169 or electrochemical paper analytical devices (epads). 170 these tests utilize paper as the assay substrate and have been combined with instrumentation amenable to low-resource settings, such as cell phones, for detection. incorporation of existing electrochemical-and eclbased assays into these formats could result in robust and highly sensitive infectious disease diagnostics. this would be impactful for disease surveillance or case management, where robust and sensitive diagnostics are needed. electrochemistry. diagnostic assays with electrochemical detection commonly consist of a three-electrode system: a working electrode (often gold or carbon modified with a molecular recognition element for biomarker capture), a counter electrode (typically platinum), and a reference electrode (commonly a saturated calomel electrode or ag/ agcl electrode). 171 in this format, incubation of a biological sample in an electrochemical cell initially results in capture of the biomarker at the working electrode. introduction of a biomarker-specific inorganic probe and a supporting electrolyte and subsequent application of an electrochemical method (e.g., cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, etc.) results in significant current production at the working electrode. this current can then be correlated to biomarker concentration. with regard to infectious disease biomarker detection, hemin, ir(iii) complexes and ru(ii) complexes have been utilized as electrochemical probes. 144−146 hemin (ferriprotoporphyrin ix) is a fe 3+ -containing porphyrin derivative that mimics the enzymatic activity of horseradish peroxidase in catalyzing the reduction of h 2 o 2 (see section 4.3) . this enzymatic activity is further enhanced when hemin is incorporated into a g-quadruplex aptamer. 143, 144, 172 zhang et al. 144 developed an electrochemical assay for the detection of hiv-associated ifn-γ that utilized hemin as a key component of the signal generation mechanism. a 3′-thiolated dna construct was conjugated to a gold working electrode, where the dna construct contained a gquadruplex sequence that was specific for hemin and an ifn-γspecific aptamer. both the g-quadruplex and the ifn-γ-specific aptamer were prehybridized by an 8-nucleotide hairpin. the authors utilized this functionalized working electrode to the assay utilized two aptamers, the first being an ifn-γ-specific aptamer (green) that was prehybridized to a g-quadruplex aptamer (blue). addition of a sample resulted in the capture of ifn-γ by the first aptamer and liberation of the g-quadruplex aptamer, which underwent a structural switch to form guanine tetrads upon introduction of k + ions. the gquadruplexes were specific for a cyclometalated ir (iii) perform cyclic voltammetry on samples containing ifn-γ to monitor the reduction of h 2 o 2 , which could be correlated to ifn-γ concentration ( figure 13 ). in samples containing ifn-γ, the ifn-γ-specific aptamer bound to ifn-γ, opening the hairpin and subsequently allowing for hemin to bind to the g-quadruplex. the potential sweep catalyzed the reduction of h 2 o 2 by the hemin/gquadruplex dnazyme, producing a cathodic current that was proportional to the concentration of ifn-γ in the sample. the assay yielded a detection limit of 0.1 nm and a dynamic range of 0.1−120 nm 144 and thus performed similarly to the luminescent assay developed by lin et al. 139 (section 4.1.1). though the authors demonstrated the specificity of the assay for ifn-γ versus nontarget proteins bsa and igg, they did not report whether the assay could be performed in more complex matrices, which can significantly compromise assay performance. more rigorous testing of the assay in biological matrices is therefore needed to demonstrate the robustness of the assay. miao and colleagues 145 synthesized and implemented an ir(iii) complex-based electrochemical probe for detection of ifn-γ ( figure 14 ). first, a gold working electrode was functionalized with a molecular beacon (mb) that contained an ifn-γ-specific aptamer as well as a sequence that was partially complementary to a second oligonucleotide strand (h 1 ). in the absence of ifn-γ, the portion of the mb complementary to h 1 was hybridized into a hairpin structure. when ifn-γ bound to the aptamer, the h 1 sequence was freed and available to hybridize with its complement, h 2 . the developed ir (iii) probe then bound to the major and minor grooves of the h 1 −h 2 double helix. oxidation and reduction of the 1,10-phenanthroline-5,6-dione ligand via differential pulse voltammetry (dpv) resulted in ifn-γ concentrationdependent current, thus correlating an electrochemical signal to the presence of target biomarker. the electrochemical assay had a detection limit of 16.3 fm and a dynamic range of 50 fm−3.0 pm and could be conducted in samples that were 5.0% human serum. importantly, this assay yielded an ∼7000-fold improvement in limit of detection compared to the two previously discussed assays for detection of ifn-γ. 145 this is largely due to the signal amplification that resulted from the groove-binding of numerous ir (iii) probes to the h 1 −h 2 double helix, which provided multiple signal-generating elements for every one ifn-γ biomarker bound. in its present form, this assay could not be applied in a primary healthcare setting because it utilizes an electrochemical workstation that requires trained personnel and significant laboratory resources. however, a portable electrochemical detection system such as the one described in section 5.5 173 could allow for this highly sensitive assay to be applied in a poc setting. electrochemiluminescence. similar to most electrochemical assays, ecl systems employ a working electrode that is functionalized with a target-specific molecular recognition element. the potential manipulation is similar to voltammetric measurements; however, in ecl, the applied potential also generates an excited state in the ecl probe, which then produces luminescence upon relaxation. 149, 165 the canonical probe for ecl-based detection is [ru(bpy) 3 ] 2+ , a complex (or derivative thereof) which has been used extensively in the development of ecl-based sensors for infectious disease detection. [150] [151] [152] [153] [154] 156 in diagnostic assays, ecl with [ru-(bpy) 3 ] 2+ occurs primarily through a coreactant mechanism, where the coreactant is most commonly n-tripropylamine (tpra) (figure 15 ). in coreactant ecl, simultaneous figure 13 . hemin dnazyme-based catalytic assay for electrochemical detection of ifn-γ. a thiolated dna construct was conjugated to a au working electrode. the dna construct consisted of a gquadruplex aptamer specific for hemin (purple) and an ifn-γ-specific aptamer (blue). both aptamers in the dna construct were prehybridized by an 8-nucleotide hairpin. in the presence of ifn-γ, the ifn-γ-specific aptamer bound to ifn-γ and opened the hairpin. hemin was then added, which bound to the g-quadruplex, forming a dnazyme that could catalyze the reduction of h 2 o 2 . this reduction produced a current that was proportional to ifn-γ concentration in the sample. 3 ]• 2+ that is analogous to the 3 mlct state that is populated through absorption of a photon. the triplet excited state then undergoes phosphorescent decay to the ground state, allowing for luminescent detection of target biomarkers. 149 of the various mechanisms for electrogeneration of a luminescent excited state, coreactant ecl has proven to be the most advantageous for the development of diagnostic assays because it permits measurements that can be obtained in aqueous solvents and matrices. one platform for detection of infectious diseases, pioneered by yoon et al., 150 used immunoliposomes to encapsulate ecl signal generation probes. liposomes equipped with a maleimide handle were synthesized by the freeze−thaw method and loaded with [ru(bpy) 3 ] 2+ . the maleimidefunctionalized liposomes were then conjugated to thiolactivated antibodies specific for the target antigen. the resulting immunoliposomes then served as a target-specific reagent containing an ecl probe that could be released with the application of detergents such as sds or triton. the authors 150 incorporated the [ru(bpy) 3 ] 2+ -loaded immunoliposome into a membrane strip test for detection of the legionella antigen. the strip test contained the following components: (1) a nitrocellulose membrane impregnated with legionella antigen, (2) a glass fiber pad presoaked in sds, and (3) screen-printed electrodes that could interface with an ecl analyzer. the immunoliposomes were incubated with a buffer solution containing the legionella antigen, and then the sample solution was allowed to wick up the nitrocellulose strip. both the antigen in the sample and the antigen embedded in the nitrocellulose competed for binding with the immunoliposome. in positive samples, the immunoliposome was bound by the legionella antigen in solution and migrated past the nitrocellulose strip to the glass fiber pad. the sds in the glass fiber pad lysed the immunoliposome, releasing [ru(bpy) 3 ] 2+ , which was detected by coreactant ecl and correlated to the antigen concentration ( figure 16 ). the assay could be completed in 20 min and had a limit of detection of 2 ng/ml. 150 compared to a traditional lateral flow assay, the test required additional user manipulation steps (preincubation of the sample and immunoliposome, addition of coreactant, etc.). the assay was also dependent on a benchtop ecl analyzer and, therefore, ill-suited to a level 1 healthcare setting in its current format. however, this novel platform demonstrated the capabilities of ecl for detecting a target antigen and showed how a [ru(bpy) 3 ] 2+ -loaded immunoliposome could be utilized to amplify signal. variations of this loaded immunoliposome platform have since been applied to the detection of the influenza virus biomarker hemagglutinin. 151, 152 recently, zhou et al. 153 developed an ecl-based immunosensor for p24, a hiv biomarker associated with early stage infection and detection of which promotes earlier intervention in hiv cases. the detection probe in this assay was a nanocomposite consisting of gold nanoparticle-decorated graphene (p-rgo@au) and [ru(bpy) 3 ] 2+ -doped silica nanoparticles (ru-sio 2 ) ( figure 17 ). a gold nanoparticle-modified glassy carbon electrode (gce) was functionalized with an anti-p24 antibody (ab1) for molecular recognition of p24. a second anti-p24 antibody (ab2) conjugated to the p-rgo@ au@ru-sio 2 nanocomposite was used as the detection element. once the ab1-p24-ab2 sandwich was formed on the electrode, coreactant n-tripropylamine (tpa) was added to produce the ecl signal through the mechanism previously discussed. 149 the p-rgo@au@ru-sio 2 nanocomposite offered several benefits to the system, namely an increased surface area for the ecl reaction and an increased rate of electron transfer. the sio 2 nanoparticles permitted greater [ru(bpy) 3 ] 2+ loading, and the gold nanoparticle-graphene composite increased loading of the [ru(bpy) 3 ] 2+ -doped sio 2 nanoparticles. the authors contended that these aspects led to an increased concentration of ecl-probe at the working electrode surface, which ultimately improved the assay's review detection limit (lod = 1.0 pg/ml) and linear range. additionally, the authors demonstrated that the p24 antigen could be detected in diluted human serum. 153 a remaining challenge for developing luminescent, electrochemical, and ecl assays that incorporate inorganic complexes is thorough validation in complex biological matrices and clinical samples. the aforementioned luminescent assays 138, 139 were not rigorously evaluated in biological samples. this is an important consideration in developing luminescent assays because of the high background that can result from the presence of interferents in biological samples. biofouling of electrodes is a known challenge in developing electrochemical sensors as well. 174, 175 although several of the previously mentioned electrochemical and ecl assays were tested in dilute biological matrices, 145,153 a more thorough evaluation of how the biological matrix affects the limit of detection is still needed. however, the issue of interference and biofouling could potentially be addressed by integrating these detection platforms with the metal-based sample preparation strategies discussed in section 3, resulting in more robust and sensitive assays. inorganic complexes have demonstrated promise for detection of infectious disease biomarkers. the luminescent, electrochemical, and ecl detection platforms discussed in this section all capitalized on the unique properties that result from the interplay between ligands and metal centers. however, inorganic complexes are rarely used as stand-alone probes for biomarker detection. instead, inorganic nanoparticles have emerged as the predominant probes in infectious disease diagnostics. 176, 177 integration of inorganic complexes with nanomaterials represents a potential avenue to developing the next generation of detection probes. one interesting nanomaterial that incorporates inorganic complexes is the metal− organic framework (mof), which is a nanosized network of organic ligands and metal ion/complex connecting points. 178, 179 the ligands provide functional handles for the conjugation of molecular recognition elements, while signalgenerating metal complexes are installed at the connecting points or encapsulated within the network. recently, electrochemical aptasensors have been developed that contain aptamer-conjugated mofs for specific recognition and detection of various protein or small molecule targets. 180−182 moreover, two of the complexes discussed in this section, ir(ppy) 3 and [ru(bpy) 3 ] 2+ , have already been incorporated into mofs for sensing and imaging applications. 183, 184 by integrating molecular recognition elements into the mof architecture, the applications of these ir(iii)-and ru(ii)-based mofs could be further expanded to include luminescent, electrochemical, or ecl detection of infectious disease biomarkers. the recent expansion of nanochemistry has drastically influenced many areas of science. in diagnostics, inorganic nanoparticles have become ubiquitous as detection elements in numerous platforms (e.g., colorimetric, luminescent, electrochemical, thermal, etc.), and each distinct class of nanoparticles possesses unique advantages and caveats. 176,177,185−187 the diagnostic setting and use-case scenario determine the applicable detection methods which, in turn, direct which nanoparticles to use. in order to apply inorganic nanoparticles as diagnostic reagents, target-specific molecular recognition elements (i.e., antibodies, aptamers, and oligonucleotide probes) must be functionalized to the nanoparticle surface. this section will first review bioconjugation reactions and nanoparticle characterization methods (section 4.2.1). the list of reactions is not meant to be exhaustive but, rather, to highlight the most essential covalent and affinity-based coupling strategies for nanoparticle bioconjugation. then, we survey the various types of nanoparticles that have been applied to the detection of infectious diseases with potential applications at the point of care (sections 4.2.2−4.2.6). 4.2.1. functionalization and characterization of nanoparticles. developing a nanoparticle detection reagent that can be targeted toward a biomarker requires conjugation of molecular recognition elements to its surface. these types of conjugations often require linkers that facilitate attachment to the nanoparticle surface. the chemistries employed for coupling molecular recognition elements to nanoparticle surfaces for diagnostic applications generally rely on a few key bioconjugation reactions and are similar to those used for planar solid supports. however, the smaller size regime of nanoparticles significantly influences the reaction kinetics on nanoparticle surfaces; therefore, bioconjugation reactions must be optimized for a given class and size of nanoparticle. 188, 189 covalent coupling. given the prevalence of carboxylates and primary amines on nanoparticle surfaces and molecular recognition elements, one of the most common bioconjugation strategies for generating functionalized nanoparticle detection probes is the formation of amide bonds. the conventional reagent for forming amide bonds on nanoparticle surfaces is 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (edc), which produces an activated ester that can subsequently react with a primary amine. however, alone, the edcactivated ester reacts slowly with amines, often leading to hydrolysis products that inhibit amide coupling. for this reason, edc is usually paired with sulfo-nhs (n-hydroxysuccinimide) esters to improve the stability of the activated ester intermediate. the sulfo-nhs ester-activated intermediate readily reacts with primary amine nucleophiles to form stable amide linkages ( figure 18a ). 190, 191 edc/sulfo-nhs coupling is useful in generating both antibody-and nucleic acid-nanoparticle conjugates as diagnostic detection probes. due to the intrinsic abundance of primary amines in antibodies, they are readily conjugated to carboxylate nanoparticles without the need for initial derivatization. 188, 192 edc coupling with imidazole enables conjugation of ethylene diamine to the 5′ phosphate group of nucleic acids, yielding 5′-amine-functionalized oligonucleotides that can be coupled to carboxylated particles. 193 quantum dots, magnetic nanoparticles, and polymeric (latex) particles have found applications in poc diagnostics and are commonly equipped with carboxylates or amines on the surface to enable conjugation via edc coupling. quantum dots are conventionally stabilized by dihydrolipoic acid (dhla) derivatives or various copolymers that install a carboxylate on the surface, and magnetic nanoparticles are coated with carboxylatecontaining polymers to enable further functionalization with primary amine-containing biomolecules. 188,194−196 alternatively, both quantum dots and magnetic nanoparticles can be coated with a silica shell derivatized with carboxylate or amine functional groups. silica nanoshells are formed via the condensation of silica oxide monomers, such as tetraethyl orthosilicate (teos) or 3-aminopropyl-triethoxysilane (aptes), yielding surface aminopropyl-silanols ( figure 18b ). the surface of the silica can subsequently be coupled to molecular recognition elements via edc. 188 in addition to supplying functional groups for further bioconjugation, the silica encapsulation of other nanoparticles provides biocompatibility and protects the core material from degradation, making it a suitable strategy for the preparation of diagnostic nanoparticle detection probes. 197 the other significant reaction class for generating nanoparticle detection probes is the bioconjugation of thiols present either in molecular recognition elements or on nanoparticle surfaces. thiolated nucleic acids are prepared by the same method as amine-functionalized nucleic acids, except cystamine is used in place of ethylene diamine for the addition of a disulfide that can be reduced with dithiothreitol (dtt) to give a 5′-thiolated oligonucleotide probe. 193, 198 on the other hand, antibodies generally do not contain free thiols and, therefore, either have to be thiolated (often with traut's reagent) or cleaved at the interchain disulfides (via dtt or papain) to generate thiols for subsequent bioconjugation to a nanoparticle surface. 192, 198 coupling of thiolated biomolecules to noble metal nanoparticles (referring to gold and silver nanoparticles) is conventionally done via coordinate covalent (dative) bonds, where the lone pairs of the sulfur atoms form stable linkages directly to the nanoparticle surface ( figure 18c ). 188, 191 alternatively, thiol-containing affinity reagents or nanoparticles can be coupled via a maleimide-derived heterobifunctional cross-linking agent, commonly sulfo-smcc (succinimidyl-4-(n-maleimidomethyl)cyclohexane-1-carboxylate). thiols react chemical reviews with the maleimide functionality of sulfo-smcc to form stable thioethers, whereas the nhs-ester component of sulfo-smcc reacts with primary amines in molecular recognition elements or on nanoparticle surfaces ( figure 18d ). 191, 194, 195, 197 photochemical cross-linking and "click chemistry" have also been utilized to produce nanoparticle-based detection probes. photosensitive functional groups, including phenyl azides and diazirines, generate reactive nitrenes and carbenes, respectively, upon exposure to uv light and allow for insertion into carbon−hydrogen and nitrogen−hydrogen bonds ( figure 18e ). photochemical agents can be integrated into heterobifunctional cross-linkers, such as sulfo-nhs-lc-diazirine, for conjugation of nanoparticles to molecular recognition elements. 191, 199, 200 "click" chemistry generically refers to bioorthogonal reactions with high yields, minimal side products, and mild conditions. while the most common "click" reaction is the conjugation of an azide to an alkyne in the presence of a cu(i) catalyst ( figure 18f ), there are many classes of "click" reactions that couple nanoparticles and molecular recognition elements. 191,201−205 biotin−streptavidin. the predominant affinity-based conjugation strategy for nanoparticle functionalization is the biotin−streptavidin system. biotin is a small molecule that binds with an extraordinarily high affinity (k d = 1.3 × 10 −15 m) to the bacterial protein streptavidin. 206 streptavidin is an approximately 60 kda tetrameric protein, permitting a 4:1 stoichiometry of biotin to streptavidin. 206 in general, streptavidin is added to the nanoparticle surface via passive adsorption or a heterobifunctional cross-linker such as sulfo-smcc. the most common method for biotinylating a molecular recognition element is the coupling of sulfo-nhsbiotin or nhs-(peg) n -biotin to a primary amine. although streptavidin−biotin bioconjugation is generally easy, a (peg) n spacer may be included to optimize for the depth of the streptavidin binding pocket (9 å) as well as the low relative solubility of biotin. 206 nanoparticle characterization. characterization of nanoparticle conjugates is essential for determining the success and extent of particle functionalization, which can drastically affect particle performance in a diagnostic format. there are several characterization techniques applicable to the development of detection probes for diagnostics. uv−visible spectroscopy (uv−vis) is one of the most commonly used methods for biomolecule-functionalized nanoparticles given its simplicity and applicability to the detection of nucleic acids and proteins. 207, 208 particle size and distribution can be assessed using dynamic light scattering (dls) and transmission electron microscopy (tem), 209−211 while functional group characterization on the nanoparticle surface can be evaluated with nmr and/or infrared (ir) spectroscopy. 212−215 all of these analytical methods have trade-offs with respect to cost, sensitivity, time of analysis, and complexity of sample preparation. however, because many of the techniques offer complementary information, they can be used in combination to ensure an extensive characterization of inorganic nanoparticle detection probes. the principal concern associated with antibody modification is decreased antigen-binding activity as a result of overfunctionalization of the variable region of the antibody. optimization of the bioconjugation reaction minimizes this effect by achieving a balance between sufficient nanoparticle coupling efficiency and retention of antigen binding. the extent of antibody modification can be monitored with functional group-specific chromophores that are detectable by uv−vis, such as ellman's reagent for free-thiol detection or haba (4′-hydroxyazobenzene-2-carboxylic acid) for biotin detection. 192, 198 also, biosensor techniques, such as surface plasmon resonance (spr), quartz crystal microbalance (qcm), and bli, can be employed to assess the impact of modification on the binding affinity for the antigen. 216−218 decreased target affinity can also originate from suboptimal loading of antibody or nucleic acid on the nanoparticle surface. insufficient coverage may lead to the nonspecific binding of interferents, whereas overloading the nanoparticle with molecular recognition elements can result in steric effects, decreasing target binding. simple absorbance-based supernatant assays that measure antibody or nucleic acid concentration both before and after nanoparticle functionalization reactions give an estimation of the element's surface density and aid in determining the nanoparticle surface saturation. 207, 208 the straightforward nature of these procedures make them popular strategies for characterizing the extent of functionalization of nanoparticle conjugates for use in infectious disease diagnostics. the bioconjugation chemistry for attaching a molecular recognition element to a nanoparticle surface is the key component for transforming a nanoparticle with interesting signaling properties into a functional reagent that can be employed in diagnostic assays. the characterization of the nanoparticle postfunctionalization is critical to ensuring optimal signal in downstream diagnostic application. several classes of functionalized nanoparticles can be utilized in developing poc diagnostics for infectious disease, the choice of which depends on the use-case scenario and desired signal output. sections 4.2.2−4.2.6 describe the fundamental principles and poc applications for these classes of nanoparticles. 4.2.2. noble metal nanoparticles. both gold nanoparticles (aunps) and silver nanoparticles (agnps), collectively referred to as noble metal nanoparticles, possess many unique properties that make them advantageous probes for signal generation in infectious disease diagnostics. noble metal nanoparticles display a vivid color that is observable with the naked eye due to surface plasmon resonance (spr). the phenomenon of spr, illustrated in figure 19a , occurs when incoming photons strike the nanoparticle surface and generate a dipole that causes electron oscillations (i.e., surface plasmons) with a frequency that resonates with the frequency of the incoming light. visible light fulfills this resonance condition for noble metal nanoparticles, giving rise to large molar extinction coefficients (on the order of ∼10 9 m −1 cm −1 ) at visible wavelengths. 219 moreover, a nanoparticle's color and λ max can be manipulated by controlling its size ( figure 19b ). 220, 221 noble metal nanoparticles are also easily functionalized with antibodies, aptamers, or oligonucleotide probes to afford specificity to a target biomolecule. 177, 188 altogether, these qualities have made noble metal nanoparticles a frequent choice for colorimetric labels in diagnostic assays designed to be interpreted by the naked eye. it is important to note that we have limited further discussion of aunps since their use in diagnostics and sensing applications has been extensively reviewed. 177,219,222−226 given the vast literature that exists for aunp applications, we have elected to focus on the applications of agnps as signal generation probes for infectious disease diagnosis. similar to aunps, localized spr of agnps has been capitalized upon to chemical reviews review develop colorimetric assays for infectious disease protein and nucleic acid biomarkers. 227−231 additionally, the conjugation of fluorescent probes (e.g., fam) to agnp surfaces, as well as the intrinsic luminescence of some agnps, has permitted the development of luminescence-based assays. 232, 233 the high agnp conductivity has also been exploited for electrochemical detection of nucleic acids. 234−236 lastly, surface-enhanced raman spectroscopy (sers) of agnps has been employed for the direct detection of malaria-infected red blood cells because of the significant light scattering of agnps and the high specificity of raman signatures. 69,237−240 colorimetric detection using nanoparticles in paper-based assays has been a widely implemented method for signal readout in primary healthcare settings. in fact, the success of lateral flow assays can be credited in part to the nanoparticlebased detection systems that allow for visual interpretation of results by the end user. the use of functionalized agnps as colorimetric detection probes in paper-based diagnostics for infectious disease has been reported for both protein and nucleic acid biomarkers. 227, 241 for example, yen et al. 229 employed agnps in a multiplexed lateral flow assay for detection of dengue, yellow fever, and ebola virus. the authors synthesized 30-, 41-, and 47 nm agnps, which were visually detectable as orange, red, and green, respectively, due to sizedependent spr shifts. each nanoparticle of a particular size was specifically functionalized with a distinct target-specific antibody associated with one of the viruses, allowing for detection of all three diseases in a single assay ( figure 20) . the detection limit for each biomarker was found to be 150 ng/ml, which is within clinically relevant ranges for each disease but is higher than other published methods. 242, 243 such multiplexed diagnostic assays are critical for discriminating between febrile illnesses at the point of care to allow for selection of the appropriate treatment course. colorimetric agnp aggregation assays also rely on the sizedependence of spr. when agnps aggregate, the decrease in distance between nanoparticles results in interparticle spr, producing a large red-shift in absorption and distinct color change that is visible to the unaided eye. 222, 227 these reaction mechanisms are classified as either "cross-linking" or "noncross-linking." in cross-linking assays, agnps are functionalized with both molecular recognition elements and capping ligands that stabilize the nanoparticles in solution. addition of a sample of target biomarker results in cross-linking of the functionalized nanoparticles, drastically decreasing the nano review particle−nanoparticle distance and producing in a spectral shift. 244, 245 agnp aggregation assays in this format have been implemented for detection of both infectious disease protein 228 and nucleic acid 230 biomarkers; however, to the best of our knowledge, cross-linking-based agnp aggregation has yet to be employed in paper diagnostics or other lowresource platforms. in non-cross-linking aggregation assays, nanoparticles are either aggregated or stabilized with molecular recognition elements that electrostatically adsorb to the nanoparticle surface. subsequent addition of a sample containing the diagnostic target then either disrupts or promotes nanoparticle stabilization, producing an observable color change. 222 for example, teengam et al. 231 developed a non-cross-linking aggregation assay using agnps for multiplexed visual detection of middle east respiratory syndrome coronavirus, mycobacterium tuberculosis, and human papillomavirus oligonucleotides in a microfluidic paper analytical device (μpad). the assay lacked the sensitivity required for detection of pathogenic nucleic acids; however, most sensitive nucleic acid diagnostics incorporate amplification techniques (e.g., pcr) that require extensive resources and infrastructure only available in betterequipped laboratories. 246 nonetheless, the use of a agnp aggregation-assay in a μpad enabled multiplexed visual detection of three different nucleic acid biomarkers in a format that could be applicable to a primary healthcare setting. taking advantage of the intrinsic fluorescence of some agnps, kurdekar et al. 233 developed a fluorescent immunoassay for detection of p24 that utilized a streptavidin-coated agnp as the detection probe. using a standard well-plate immunoassay format, adsorbed anti-p24 antibodies captured p24 from samples. a biotinylated anti-p24 detection antibody and the streptavidin-conjugated fluorescent agnp were subsequently added to generate a fluorescent signal that correlated with p24 concentration. the assay successfully detected p24 standard spiked into plasma as well as the p24 present in hiv-positive patient plasma samples. moreover, no cross reactivity was observed in samples that were hivnegative but hbv-or hcv-positive. the limit of detection of the assay was 10 pg/ml, which is an order of magnitude higher than a previously discussed assay for p24 (section 4.1.2). 153 this discrepancy could be explained by the lack of signal amplification in the fluorescent agnp-based immunoassay. in its current format, the fluorescent agnp immunoassay is not amenable to a resource-limited primary healthcare setting; however, fluorescent agnps could be employed in a lateral flow assay format and combined with a portable fluorescent reader, allowing for implementation at the point of care. 247 4.2.3. quantum dots (qds). quantum dots (qds) are semiconductor nanocrystals with luminescent and conductive properties that make them advantageous as detection probes in infectious diseases diagnostics. canonically, qds consist of combinations of elements from groups ii and vi or groups iii and v and range in size from 1 to 100 nm. 248, 249 at these small sizes, qds absorb photons, generating excited electrons in the conduction band and positively charged holes in the valence band of the semiconductor ( figure 21a ). the distance that separates the electron−hole pair is confined to less than the diameter of the nanocrystal, resulting in "particle in a box" behavior known as quantum confinement. 249−251 as a result of quantum confinement, qd emission wavelength increases as nanoparticle size increases; thus, qd emission can be tuned via size-controlled synthesis ( figure 21b ). 252 because qds exhibit absorption across a broad spectrum yet have emission spectra with very narrow and tunable bandwidths, they are particularly useful for multiplexed detection of disease biomarkers. 194, 253 additionally, qds demonstrate increased biocompatibility, photostability, quantum yield, and semiconducting properties compared to traditional organic fluorophores, further rendering qds advantageous as signal generation probes for diagnostics. 254 265 escherichia coli, 266 and cholera 267, 268 in fluorescent assays for laboratory and resource-limited settings. functionalized qds or qd-loaded microparticles have been utilized as fluorescent probes for both protein and nucleic acid biomarkers. 194, 256, 269 the surface of qds can be functionalized with antibodies or antigens for protein detection or oligonucleotide probes for target dna detection using the bioconjugation strategies discussed in section 4.2.1. for a thorough evaluation of the bioconjugation of qds, the reader is directed to a number of reviews. 194, 196, 270 goldman et al. 271, 272 pioneered the earliest uses of antibody-functionalized qds as fluorescent detection probes. the authors utilized affinity-based coupling chemistry (i.e., biotin−avidin) as well as electrostatic interactions between charged moieties to functionalize antibodies to the metal surfaces of cdse/zns core−shell quantum dots. the biotin−avidin system was found to be the more simple, reproducible, and robust method for qd bioconjugation of the two, and these functionalized qds were successfully utilized in a multiplexed fluorescent assay for cholera toxin and staphylococcal enterotoxin b. 272 these early works exploited the narrow emission profile and enhanced quantum yield of qds for antigen detection and laid the groundwork for the development of enhanced, fluorescencebased diagnostics for a variety of targets. building upon this previous work, klostranec et al. 253 constructed a multiplexed fluorescence-based microfluidic device for detecting hiv, hepatitis b, and hepatitis c antibodies utilizing antigen-functionalized qd barcode particles. two types of cdse/zns qds were synthesized and loaded into polystyrene beads at varying ratios, resulting in three distinct qd barcode particles. each qd barcode particle was covalently bound to a disease-specific antigen via edc coupling. the assay employed a "sandwich" format where disease-specific antibodies were captured by the antigenfunctionalized qd barcode particles, and a fluorophoreconjugated (alexafluor 488) antihuman antibody was used to detect the presence of a target antibody. the narrow peak width of qd emission enabled detection of both the antibodyconjugated fluorophore at ∼520 nm to indicate the presence of target antibody as well as both of the qds at 570 nm (yellow) or 615 nm (red). the ratio of the red:yellow signal intensity allowed for barcode identification and determination of which of the three target antibodies was present ( figure 22 ). 253 the detection limits for antibodies correlating to all three diseases were in the picomolar range, which is a 50-fold improvement compared to commercial elisas. 253 while the assay enabled multiplexed and sensitive detection of infectious disease biomarkers, the microfluidic platform employed by the authors required extensive user manipulations and complex instrumentation and analysis that would only be viable in higher level healthcare settings. given the improvements in sensitivity of the assay, adaptation of the technique to a handheld fluorimeter or cell phone-adapted fluorescence device could potentially allow for deployment in a primary healthcare low-resource setting. the benefit of this strategy is that the biomarker signal and barcode signal are both fluorescencebased, resulting in a multiplexed detection platform that is simplified compared to assays with different detection schemes for biomarkers and barcodes. the authors hypothesized that up to 10 6 barcode combinations are theoretically possible using this approach due to the narrow and distinct fluorescent peak widths generated by different qd particles, allowing for even greater multiplexing. 253 however, such hypotheses should be approached with caution, as highly multiplexed assays can be more susceptible to cross-reactivity, thereby reducing the diagnostic specificity of the individual assays. qd-loaded particles have also been functionalized with antibodies for the detection of protein biomarkers for infectious disease. recently, hu et al. 257 developed a lateral flow assay that utilized antibody-coated polymeric particles (260.9 nm) loaded with both qds (3−6 nm) and aunps (20 nm) for detection of ebola virus (ebov) glycoprotein. the reporter probes, which the authors dubbed "dual signal readout nanospheres" (rns), enabled detection of the ebov glycoprotein either visually via aunps or fluorescently via qd emission. characterization of the rns revealed that there were dozens of aunps adsorbed and hundreds of qds embedded per every one antibody-coated polymeric particle ( figure 23 , panels a and b); thus, for every one biomarker arrested at the test line, there were significantly more signaling review moieties to permit more sensitive detection. additionally, two sets of antibody-functionalized rns were fabricated and used for each lfa: (1) streptavidin-conjugated and (2) biotinconjugated rns. application of the sample and streptavidinconjugated rns to the sample pad resulted in migration of the sample and rns to the test line and formation of an antibody "sandwich" at the test line. subsequently, the biotin-conjugated rns were added and bound to the streptavidin-coated rns, enhancing the signal based on the high affinity of the biotin− streptavidin interaction. the authors 257 utilized the lfa to detect either the ebov glycoprotein or the whole virion spiked into samples. the assay demonstrated a broad dynamic range that spanned 3 orders of magnitude for detection of aunps with a limit of detection by visual inspection of 2 ng/ml. when the qd fluorescence signal was used, the detection limit was improved to 0.18 ng/ml (figure 23 , panels c and d). the fluorescent detection of this assay was 1−2 orders of magnitude more sensitive than 2 commercial brands of rapid tests for ebov glycoprotein. moreover, the fluorescent signal could be quantitated by image processing software that is readily available on a smartphone. though the authors utilized a benchtop fluorimeter for qd excitation and emission, the use of a smartphone adapted hand-held fluorimeter or dedicated fluorescent lateral flow reader could enable measurements at the point of care. 257 the sensitive dual signal readout provides versatility to this platform such that it could be employed in a number of use-case scenarios. if morbidity control were the primary concern, visual detection of aunps would suffice. if more sensitive and quantitative measurements were needed for surveillance, intervention management, or an elimination campaign, the fluorescent capabilities of the detection probes could be exploited. however, for a given use-case scenario, likely only one signal readout modality is needed. as a consequence, the additional cost of implementing a dual signal rn, when using a single signal (visual or fluorescent) rn would suffice, needs to be considered. nonetheless, the assay demonstrated that quantum dots could be incorporated into tests that are readily deployable in primary healthcare settings, though more rigorous field evaluation and validation of the assay would be necessary for clinical use. there have been significant advances in the use of qd-based detection methods for infectious disease biomarkers. while the complexity of instrumentation required for detecting quantum dots is often still a barrier to their implementation at the point of care, developments in smartphone-based devices are currently enabling the detection of fluorescent labels such as qds at the point of care. 273−275 fluorescent assays have demonstrated a capacity for multiplexing. because of the narrow emission profiles demonstrated by qds, the potential for continued expansion in multiplexing likely lies with qdbased fluorescent assays. given the importance of detecting comorbidities and coinfections or discriminating between febrile illnesses, 276,277 the qd-based technologies represent a platform that has the potential to make a significant impact at the point of care. 4.2.4. lanthanide chelate-doped nanoparticles. nanoparticles doped with lanthanide chelates are advantageous as detection labels owing to their large stokes shifts, sharp emission peaks, stable luminescence, long fluorescence lifetime, and biocompatibility. 278, 279 in a lateral flow assay format, these particles are often functionalized with targetspecific antibodies and then embedded in the conjugate pad of the test. like qds, detection of lanthanide chelate-doped nanoparticles requires additional instrumentation for particle excitation and measurement of emission. 280, 281 despite this need for additional equipment, these particles are attractive labels for poc diagnostics because they enable highly sensitive biomolecule detection. in the context of elimination or surveillance of a particular infectious disease, the added sensitivity provided by lanthanide chelate-doped nanoparticles could allow for detection of low-intensity infections that would otherwise be missed. except lanthanum, all lanthanides are f-block elements; the 4f orbital is gradually filled as the atomic number increases. because the 4f orbital is largely shielded from the chemical environment by the filled 5s and 5p shells, the lanthanides are very similar in their chemical properties. for example, the most common oxidation state for aqueous lanthanide ions is +3. 282, 283 most of these ions are luminescent, though some lanthanides have greater quantum yields than others due to the large energy gaps between the lowest emissive energy level and the highest sublevel of the ground state. as the size of the energy gap increases, lanthanide ions become less prone to lower energy nonradiative decay processes, resulting in greater quantum yields. 278 eu 3+ is the most commonly utilized lanthanide for labels in lateral flow assays because of its (1) ideal energy gap between the nondegenerate emissive state of 5 d 0 and the ground state of 7 f j and (2) convenient emission in the visible region. consequently, eu 3+ will be the focus of this section. 279 europium(iii) itself is not strongly luminescent because most excitations involve forbidden electric dipole f-f transitions, which have low absorption cross sections. 278 however, this can be overcome when the ion is coordinated to a sensitizing structure, such as an organic ligand. a simplified jablonski diagram of the luminescence process for a eu 3+ chelate is depicted in figure 24 . the ligand absorbs the excitation energy and transfers it to a triplet state (t 1 ) via intersystem crossing (isc). next, this energy is transferred to one of the 5 d j levels of eu 3+ , and, after internal conversion (iet), emission occurs from the 5 d 0 energy level to any of the 7 f j levels. 278, 279 in addition to sensitization, chelating ligands also promote luminescence by shielding eu 3+ from aqueous quenching effects and allow for coupling to biomolecules or polymeric materials. 278 chelating ligands typically absorb in the uv (330−370 nm), and the subsequent eu 3+ emission occurs at 580−690 nm. 279 ligands usually contain a chromophore portion (e.g., pyridine, salicylate, phenanthroline, coumarin, pyrazole, triphenylene, and quinolone) and a chelating structure based on multidentate polyacids or macrocycles (e.g., edta). 278 tetracycline has also been employed as a ligand for eu 3+ . 284 these chelate dyes are incorporated into nanoparticles functionalized with target-specific molecular recognition elements for use in lateral flow assays. most lateral flow assays that employ eu 3+ chelates use commercially available particles, which are often superior due to their monodispersity and quality control standards. the majority of commercially available eu 3+ chelate lateral flow labels are 100−300 nm carboxyl-or streptavidin-decorated polystyrene particles and are readily available from large vendors such as thermo-fisher 285 and expedeon ltd. 286 eu 3+ chelates are easily adsorbed into polymeric (e.g., polystyrene) nanoparticles based on hydrophobic interactions between the polymers and the organic chelating ligands of the complexes. for example, huhtinen et al. 287 found that simply incubating 50 nm polystyrene particles with eu 3+ chelated with dipicolinic acid derivative ligands resulted in 1000−2000 chelates per nanoparticle. this represents a density of approximately 0.02 chelates/nm 3 . harmäet al. 288 found a similarly high density of approximately 0.05 chelates/nm 3 (31000 chelates/particle) in 107 nm commercially available particles. thus, for each biomolecule bound in a lateral flow format, thousands of associated eu 3+ chelates produce a signal, resulting in a highly sensitive measurement. the advantages of eu 3+ chelate-loaded polystyrene nanoparticles as labels for lateral flow assays were demonstrated by yeo et al., 289 who developed a sensitive lateral flow assay for the detection of avian influenza h7 subtype virus. these viruses can be highly pathogenic; for a 2013 h7n9 outbreak in china, a mortality rate of roughly 36% was estimated for hospital patients with laboratory-confirmed infections. 290 currently, laboratory-based molecular testing is required to differentiate between h7 subtype avian influenza and other viral infections. thus, there is a need for rapid tests that can be performed at the point of care. the authors approached this problem by developing and evaluating a panel of 10 h7 subtype-specific monoclonal antibodies against hemagglutinin 1 of h7n9 influenza. after the best antibody pair was determined, h7 subtype-specific lateral flow assays were constructed with both europium-chelate and gold nanoparticle (aunp) conjugates. using a benchtop time-resolved fluorescence reader, the authors found that the detection limits for the eu 3+ chelate-based tests were 25-fold better than those of aunp-based tests. additionally, the developed eu 3+ -based tests performed better than commercially available tests detecting influenza a nucleoprotein. 289 these enhancements over gold particles are impressive, although given the high density of chelates per nanoparticle, it is surprising that larger enhancements were not achieved. one possible explanation could be the efficiency of molecular recognition element adsorption for the two types of nanoparticles. in addition to avian influenza, 289, 291 eu 3+ chelate-embedded polymeric nanoparticles have been employed for the detection of e. coli, 292 hepatitis b, 293 and procalcitonin, 281 a host marker for bacterial infections. several commercial platforms based on these particles have been developed as well, including the sofia influenza a+b tests (quidel, inc.) 294−299 and the aqcare chlamydia trf test (medisensor, inc.), 300, 301 which have been extensively evaluated in the literature. the high sensitivity of these assays is derived not only from the high number of eu 3+ chelates embedded in the polymeric nanoparticles but also from the use of time-resolved fluorescence measurements. one of the primary disadvantages of using luminescent probes as labels in lateral flow assays is the high autofluorescence of biological samples and assay components occurring at excitation and emission wavelengths similar to those of the probes. 302 this fluorescence can interfere with lateral flow readings, causing high background that leads to low-sensitivity measurements. however, lanthanide chelates overcome these effects because their fluorescence lifetimes are several orders of magnitude longer than conventional fluorescent materials (microseconds or milliseconds compared to nanoseconds). 278, 279 this allows for the use of time-resolved measurements in which samples are excited with a pulse of uv light, and emission signal is collected after a time delay on the order of 100 ns for a given time period. 284 by delaying the collection of emitted signal, the short-lived autofluorescence from biological and assay components is excluded from measurements. while the intensity of the delayed luminescence is slightly decayed from the initial signal output of the chelates, these measurements can be cycled to improve signalto-noise. 303 of course, the unique instrumentation required for time-resolved measurements makes field-deployment technically challenging, though currently available benchtop devices could allow for measurements in district clinics and laboratories. recently, paterson et al. 304 developed a promising smartphone-based platform for time-resolved luminescent measurements, which could allow for point-of-care measurements based on eu 3+ chelate particles. however, the lengthy 100 ms time-delay employed in their device is only compatible with the most persistent luminescent nanophosphors such as crystalline sral 2 o 4 :eu 2+ ,dy 3+ particles. while polymeric eu 3+ chelate nanoparticles are the primary materials employed by lanthanide-labeled lateral flow assays, these particles present some disadvantages. their large size and tendency to swell can cause aggregation in aqueous solutions. additionally, since they are not covalently bound to the polymers, eu 3+ chelates can leach from the particles. 284 silica particles avoid some of these problems. in one example, xia et al. 305 developed a lateral flow assay for hepatitis b detection using eu 3+ chelate-loaded silica nanoparticles. to synthesize these particles, the authors relied on an iterative method involving silica condensation from teos, surface-amination using aptms, covalent functionalization of chelating ligands to the particles, and loading of eu 3+ . this process was repeated five times to achieve a high density of eu 3+ chelates. the resulting 55 nm nanoparticles contained a remarkable 686000 chelates/particle, corresponding to a density of roughly 7.9 eu 3+ chelates/nm 3 , more than 100 times greater than commercially available polymeric particles. 306 these particles were then functionalized with oxidized dextran to serve as a hydrophilic linker, and free amine moieties on antihepatitis b surface antigen (hbsag)-specific antibodies were coupled to the surface via reductive amination of the aldehyde groups. after synthesis and characterization, the particles were used as labels in a traditionally formatted lateral flow assay for hbsag. to measure signal, the strips were illuminated with a uv lamp and imaged using a digital camera. signal intensity was determined using image analysis in adobe photoshop. unlike the previous example presented, this imaging technique does not take advantage of the long fluorescence lifetime of eu 3+ chelates. however, even without time-resolved measurements, the detection limit of the lfa was found to be 30 pg/ ml hbsag, 100-fold better than a similarly formatted goldbased lateral flow assay. 305 the study was then validated by analyzing 286 clinical serum samples using the developed eu 3+ chelate-based lateral flow assay as well as a quantitative elisa. the two methods were concordant for all samples analyzed and had similar sensitivities. 305 thus, the developed method represents an innovative tool for diagnosis of hepatitis b and provides sufficient sensitivity when compared to clinical methods. in order to implement this assay directly at the point of care, two modifications are necessary: (1) the assay should be optimized for field-friendly whole-blood specimens, and (2) a mobile phone-based detection strategy should be implemented. lanthanide chelate-based nanoparticle probes are a promising avenue for developing highly sensitive lateral flow assays. similar to other fluorescent probes, instrumentation remains the primary barrier to implementation of eu 3+ chelate nanoparticles at the point of care. however, as optical devices are miniaturized and become more affordable, these probes could become commonplace among rapid tests for infectious diseases. 4.2.5. up-converting phosphor nanoparticles. upconverting phosphor (ucp) nanoparticles represent a unique and exciting reporter technology for bioassays. unlike typical fluorescent labels, these ceramic nanoparticles doped with rare earth elements absorb low-energy ir radiation and emit higher-energy visible light. 307−309 ucps are often superior to conventional colorimetric and fluorescent reporters for a number of reasons. first, the high anti-stokes shift from ir to visible does not occur in nature. thus, the inherent autofluorescence typically associated with biological samples and assay components does not interfere with ucp measurements. second, ucp signal is highly stable and does not photobleach, allowing for indefinite storage and repetitive analysis of a single test. this is especially useful for confirming field results in a central laboratory. 309 third, assays are easily multiplexed; distinct particles absorb at the same ir excitation energy and emit wavelengths defined by the dopant. finally, optical detection of ucp particles offers an inherent boost in analytical sensitivity over visual detection of gold; as few as 10−100 ucp particles can be detected at the test line of a lateral flow strip, while approximately 40000 gold particles are needed for visual detection. 310 this increase in sensitivity makes ucp particles attractive labels for diagnostic tests employed in surveillance or elimination campaigns in resourcelimited settings, since they would enable the detection of lowintensity or asymptomatic infections. however, similar to qds and lanthanide-chelate particles discussed in the preceding sections, these benefits of ucp particles must be weighed against the additional cost and resource requirements needed for detection instrumentation. the primary mechanisms for up-conversion include excited state absorption, energy transfer, and cooperative sensitization. excited state absorption is the absorption of light by electrons already in an excited state. this requires two photons and equal separation between the ground state, first excited state, and second excited state of a single ion. energy transfer upconversion requires two ions, a sensitizer and an activator. in this process, the sensitizer ion is excited and sequentially transfers its energy to the ground state and first excited state of the activator ion. 307 cooperative sensitization is similar to energy transfer, though it involves excitation of two sensitizer ions which simultaneously transfer their energy to the activator ion. other up-conversion mechanisms include photon avalanche and cross relaxation. 311 energy transfer up-conversion is the most common upconversion mechanism for bioassay reporter particles, which typically involve a yb 3+ sensitizer (excitation 980 nm) and a rare earth (er 3+ , tm 3+ , pr 3+ , ho 3+ , and gd 3+ ) activator codoped in yttrium fluoride (yf 3 ), yttrium oxide (y 2 o 3 ), yttrium oxysulfide (y 2 o 2 s), or sodium yttrium fluoride (nayf 4 ) crystals. 307, 309 an illustration of the up-conversion processes for two of these materials are shown in figure 25 . ucps have been utilized as labels for both protein and nucleic acid targets in lateral flow assays for a variety of infectious diseases. recently, corstjens et al. 312−317 developed an ultrasensitive, ucp-based lateral flow assay for schistosomiasis that detects schistosoma circulating anodic antigen (caa), a proteoglycan biomarker waste product produced by the parasite. caa is present in the serum and urine of patients with schistosoma infections of all known species and has been found to correspond well with worm burden, clearing soon after successful treatment. 312, 317, 318 the developed lateral flow assay employed 400 nm y 2 o 2 s:yb 3+ , er 3+ ucps, which are excited at 980 nm and emit at 550 nm (green) and can detect caa for a single schistosoma worm pair in serum. 314, 317 this assay has been applied to patient samples from endemic areas, and due to its superior sensitivity, it has demonstrated much higher schistosomiasis prevalence than microscopy, serology, and nucleic acid-based tests. [315] [316] [317] 319 the caa lateral flow assay format is similar to conventional lateral flow assays. caa-specific antibodies are printed on the test line, and antimouse igg is printed on the control line. the workflow of the assay in its current form, however, differs from a typical field-ready test. first, a trichloroacetic acid (tca) extraction is performed on a urine or serum sample, requiring a centrifugation step. the extract supernatant is then combined with running buffer and anti-caa-functionalized ucp particles and incubated for 1 h on an orbital shaker at 37°c before the lateral flow strip is added to the solution. the test is allowed to develop and must dry completely (at least 3 h) before scanning and analyzing the strip. 312, 313 in order to simplify the preparation procedure and decrease the number of steps, a dry reagent kit was later developed in which all buffers and particles were lyophilized. 313 this kit was shipped to a tertiary lab in south africa, where nearly 2000 samples were evaluated by local technicians using both the kit and a caa elisa. the researchers found that the ucp-based lfa successfully identified more low-level infections than the caa elisa. 320 to increase the analytical sensitivity of the assay, corstjens et al. 317 added a spin-filter concentration step to the sample preparation method. this allowed for caa in urine sample volumes of 0.5−7.5 ml to be concentrated into 20 μl before addition to the lateral flow strip. the resulting detection limits improved as sample volume increased, reaching as low as 0.03 pg/ml for the 7.5 ml assay. to demonstrate clinical applicability, the concentration step was successfully performed on 2 ml patient urine samples from kenya (high-endemic, s. mansoni) and china (low-endemic, s. japonicum) 316, 317 it is important to note that, though this sample concentration step improved the sensitivity of the assay, it required laboratory equipment to carry out; all patient samples in these studies were processed in well-equipped laboratories. further, the additional concentration step increased the cost of this ultrasensitive caa assay, though sample pooling could make this test more cost-effective and allow for monitoring of worm burdens at the subpopulation level for large-scale surveillance. 320 for this ucp-based ultrasensitive assay to be utilized in a field setting, a more robust, field-ready sample preparation method is needed. lateral flow assays with ucp reporters have also been developed for detection of protein markers of other infectious diseases, including neurocysticercosis, 321 334 and hiv. 335 additionally, ucp-based multiplexed lateral flow assays have been developed for detection of protein biomarkers for multiple infectious diseases. 336, 337 these multiplexed diagnostics typically consist of one strip with multiple test lines, each capturing a distinct protein biomarker. in this format, the ucp crystals are the same for each biomarker, though they are functionalized with target-specific antibodies. one disadvantage of highly multiplexed assays on a single strip is the potential increase in cross-reactivity and nonspecific binding that could lead to false-positive results. to mitigate this risk, hong et al. 330 developed a unique, circular cassette with 10 channels for 10 different singleplex lateral flow assays using ucp particles as the reporter labels. one unexplored application of multiplexed detection on a lateral flow assay is the use of ucp particles with identical excitation but differing emission profiles. this could be particularly advantageous for large biomarkers with multiple accessible epitopes and could provide additional clinical information. for example, an assay that captures and detects whole organisms could include a second ucp probe for detecting surface proteins that confer drug resistance, providing both detection and susceptibility results in one assay. ucp nanoparticles have also been used in nonlateral flow diagnostic formats, including immunohistochemistry, 338 microarrays, 339 magnetic bead assays, 340, 341 and plate immunoassays, 309 though these platforms are not readily amenable to low-resource, point-of-care settings. even in the lateral flow format, ucp nanoparticles present interesting challenges for field deployment. clearly, sample preparation methods, including purification, concentration, and amplification, must be adapted for environments lacking controlled laboratory conditions and should rely on as little electricity as possible. while hand-powered centrifuges 342, 343 and battery-powered mixers 115, 344 have been developed, the ideal poc assay will be optimized to preclude these additional resources. another issue, often unaddressed, is that the lateral flow strips must be completely dry before optical measurements and analysis. this is because water also absorbs in the near-ir, decreasing excitation efficiency of the sensitizer. 345 typically, protocols require at least 3 h of drying time after the lateral flow assay has developed, a long time-to-result for a point-of-care test. finally, detection of ucp particles relies on optical instrumentation. although the most sensitive of these devices are benchtop instruments, portable battery-powered devices have been developed, such as the ucp-modified version of qiagen's esequant lateral flow reader ("ucp-quant"). 313, 314, 346 many of the sample preparation methods described in section 3 can be applied to ucp lateral flow formats. additionally, innovations in hand-held optical readers discussed in section 5 require only simple adaptations to detect the anti-stokes shift of ucp particles. there are clear advantages of using ucp nanoparticles as labels in lateral flow assays, including their inherently low background interference and high analytical sensitivity. innovations in sample preparation methods and the development of portable optical readers will allow for these advantages to be exploited in low-resource, poc settings. 4.2.6. magnetic nanoparticles. while most frequently used for sample preparation and biomarker enrichment, magnetic nanoparticles are emerging as promising labels in poc assays. similar to noble metal nanoparticles, magnetic nanoparticles can be easily functionalized and possess strong optical absorbance, which has led to their use as visual labels on lateral flow assays. 347 however, their magnetic properties can also be leveraged for detection, which has advantages over traditional optical detection in a lateral flow format. first, when visual or optical detection of absorbent or fluorescent particles is employed, only signal from the surface (∼10 μm) of the membrane is measurable due to the opacity of the membrane. typically, nitrocellulose membranes used in lfas are 100− 200 μm thick, so optical detection only allows for signal from the top 10% of the membrane to be measured. in contrast, magnetization measurements can be performed regardless of the opacity of the substrate, taking advantage of the entire volume of the test line on a lateral flow assay. 348 in addition, metal-oxide nanoparticles are highly stable, and drying and aggregation do not influence the intensity of magnetic measurements. 349 finally, there are very few biological or assay components that interfere with magnetic measurements. 350 the properties of magnetic particles are size-and temperature-dependent. 195, 351 iron oxide (typically magnetite or maghemite) particles are the most common magnetic nanoparticles employed in diagnostic applications. in the bulk, these materials are ferri-or ferromagnetic and thus retain magnetization after an external field has been applied. this hysteresis reaches a maximum when particle size is decreased to the point that the material becomes single-domain. as particle size is even further reduced, the hysteresis effect decreases until the particles reach a critical diameter, below which brownian forces are strong enough to overcome magnetic forces. thus, at these small sizes, the particles are superparamagnetic; the magnetic moments of the particles are aligned in the presence of an external magnetic field, but they revert back to a nonmagnetic state when the field is removed. 351 in the lateral flow format, magnetic nanoparticles are labeled with target-specific molecular recognition elements and employed as conjugates for analyte detection. 352 superparamagnetic behavior is ideal for particles applied as labels review for lateral flow assays because the lack of magnetization in the absence of an external field allows for the particles to wick along the strip without aggregating due to magnetic effects. 353 then, the magnetic properties of the particles can be leveraged after labels have bound to the test and control lines. magnetic nanoparticles are typically detected on lateral flow assays based on induction (figure 26 ) or using magnetoresistive sensors. 350 in the inductive format, which employs the principles of faraday's law, a magnetic particle-based test is placed in an oscillating magnetic field above a set of induction coils. in the absence of magnetic particles, the net current induced in the coils is zero. however, when particles are present, the direction of their magnetic moments oscillates with the external magnetic field, resulting in a measurable net voltage induced across the coils that is proportional to the total number of particles at the test or control line. 354, 355 in contrast, magnetoresistive sensors are based on the principle that the electrical resistance of certain materials, which are incorporated in the sensors, can change upon application of an external magnetic field. 350 in this format, a magnetic field is applied to para-or superparamagnetic detection elements in order to align their magnetic moments and produce a fringe field. magnetoresistive sensors are placed close enough to the particles to detect the fringe field they produce based on the change in resistance of the materials within the sensor. 356, 357 a detailed description of the instrumentation required for these detection methods is provided in section 5.4. magnetic nanoparticles have been employed as detection labels in lateral flow assays for protein and whole-cell targets. for example, handali et al. 349 developed two magnetic particle-based lateral flow assays for detection of taenia solium, a cestode that is prevalent around the world for which swine are intermediate hosts. when contaminated, undercooked pork is ingested, the parasites develop into tapeworms in the human gut (taeniasis). however, if eggs are ingested, the larval stage can infect the human nervous system, potentially forming cysts in the brain. this severe form of the disease, called neurocysticercosis, is a leading cause of epilepsy worldwide. 358 the gold standard for diagnosis of taeniasis is observation of eggs in stool by microscopy, though this method is insensitive and labor-intensive. diagnosis of neurocysticercosis currently requires ct scans of the brain, a technology that is unavailable in low-resource settings. 359 as such, there is a pressing need for t. solium rapid diagnostic tests. handali et al. 349 developed two serological magnetic beadbased lateral flow assays that detected the immune response (i.e., host antibodies) against taeniasis-specific (es33) and cysticercosis-specific (t24) antigens. for each test, one batch of the recombinant antigen was printed at the test line, and a separate batch was coupled to commercially available carboxylated superparamagnetic particles via edc/nhs chemistry. the recombinant antigen at the test line and the antigen conjugated to magnetic particles were able to simultaneously bind to host antibodies in the sample, taking advantage of the multivalent nature of anti-es33 and anti-t24 igm and igg antibodies. the lateral flow assays were evaluated using an inductionbased reader and visual reading to detect antibodies in serum samples from endemic areas and nonendemic control regions ( figure 27 ). 349 it was found that the taeniasis-specific es33 assay had a diagnostic sensitivity and specificity of 95% and 96%, respectively, when evaluated with the magnetic reader. the sensitivity and specificity of the neurocysticercosis t24 test using the magnetic reader were 94% and 99%, respectively, for patients with two or more viable cysts as determined by ct scan or mri of the brain. for patients with only one viable cyst, the t24 assay only identified 5/15 cases of neurocysticercosis, indicating that the burden of infection was not great enough to elicit an immune response. 349 despite this decrease in sensitivity for single-cyst infections, the developed lateral flow assays are promising alternatives to the current gold standards. further, because the assays were not speciesspecific, they could be used to detect porcine cysticercosis as a marker of disease control and transmission. one disadvantage of utilizing superparamagnetic labels is that many magnetic readers are benchtop devices requiring electricity and a laboratory setting. however, in over 70% of the cases, the magnetic nanoparticles could be detected visually. 349 while this sensitivity is not ideal, it is possible that initial visual results could be applied in the field with follow-up laboratory-based measurements assessed for the visually negative assays. as hand-held magnetic readers become more commonplace, these assays will become truly impactful. in addition to t. solium, magnetic particles have been employed in protein-detecting lateral flow assays for hiv 360, 361 and h1n1 influenza. 362 whole cell-detecting magnetic particle-based assays have also been developed for a number of targets, including vibrio parahemolyticus, 363,364 listeria monocytogenes, 365 and bacillus anthracis. 366, 367 the demonstrated sensitivity of these magnetic particle-based lateral flow assays makes them promising alternatives to traditional assays with colorimetric or fluorescent labels. however, current instrumentation, which will be discussed further in section 5.4, limits their utility at the point of care. one exciting avenue that has yet to be explored for infectious disease detection is the employment of the same superparamagnetic nanoparticles for dual purposes: immunomagnetic biomarker enrichment from large-volume samples and visual labeling for lateral flow assays. ideally, such a diagnostic would integrate sample preparation and assay into one device. once developed, the combined effects of target enrichment and magnetic detection could lead to a highly sensitive test capable of detecting low-density infections. one of the most widely utilized techniques for improving the sensitivity of diagnostics is signal amplification, where thousands of signaling molecules are generated for every one biomarker molecule. though there are now numerous methods for amplifying signal in diagnostic assays, the use of metalloenzymes was one of the first approaches. 368, 369 metalloenzymes are metal-containing catalytic proteins in which the presence of particular metals or metal complexes in the tertiary structure is critical to the catalytic turnover of the substrate. 370 the predominant metalloenzymes employed in diagnostics are alkaline phosphatase (alp), horseradish peroxidase (hrp), and catalase. the high catalytic efficiencies of these enzymes enable the rapid conversion of substrate to detectable products. 371 using cross-linking chemistry discussed in section 4.2.1, these metalloenzymes can be conjugated to antibodies or nucleic acids that afford specificity for a target biomarker. 192, 193 the three primary enzymes used in metalloenzyme detection conjugates rely on different metal ions for catalytic activity. alp possesses two zn(ii) ions and one mg(ii) ion per enzyme monomer and hydrolyzes phosphate monoesters. 372, 373 the conventional substrates for alp in point-of-care diagnostic applications are bcip (5-bromo-4-chloro-3-indolyl phosphate) and nbt (nitro blue tetrazolium), where dephosphorylation and oxidation of bcip allows for reduction of yellow nbt dye to a blue/purple formazan precipitate. 371 both hrp and catalase are heme-containing monooxygenases that catalyze reactions with h 2 o 2 . 374 4.3.1. elisas (enzyme-linked immunosorbent assays). metalloenzyme-antibody conjugates have been widely implemented in elisas for the detection of protein biomarkers for disease. this is due to the sensitivity afforded by the use of enzymes for signal amplification as well as the specificity of the antibody−antigen interactions used for molecular recognition. 371, 377, 378 despite the high sensitivities of elisas, their application in poc settings is limited by several factors: 378−381 signal readout for elisas typically requires a spectrophotometer unavailable in a primary healthcare setting, though there are a growing number of portable alternatives 382 (e.g., cellphones or hand-held scanners; see section 5.1.3). elisas are time-consuming (∼6−8 h), demand extensive sample handling and solution manipulation, and often require specialized training. lastly, the lack of thermal and long-term stability of metalloenzymes in elisas can lead to suboptimal assay performance, as elisas are intrinsically dependent on these metalloenzymes for signal amplification and readout. thus, elisas are usually limited to well-equipped tertiary laboratories. to mitigate these issues, investigators have begun to use paper as the elisa solid support, increasing the likelihood that these sensitive assays could be used in resource-limited settings. paper-based elisas have been developed for detection of diagnostic markers of hiv and malaria. 381, 383, 384 long-term stabilization conditions of metalloenzymes on paper have also been investigated to maximize catalytic activity in poc settings. 385−387 paper-based elisas seek to address many of the issues that inhibit the use of elisas in more resource-limited poc settings. the time to result for paper-based elisas is ∼1 h due to the comparatively smaller volumes required when contrasted with conventional benchtop elisas. these smaller reagent volumes also significantly reduce the overall cost of elisas. cheng et al. 381 first pioneered this method for the detection of serum antibodies against hiv-1 envelope antigen gp41, where an alp-antibody conjugate was utilized to turn over bcip/nbt substrates to purple precipitate products. the authors used hydrophilic paper that was patterned with hydrophobic photoresist to fabricate 96 test "wells" that were spatially separated just as in a polystyrene 96-well plate. samples containing biomarker were added to the paper elisa card, which was placed on top of a blotting pad to enable wicking of the reagents through the test wells. though the assay boasted a faster time to result and a lower cost than the analogous conventional elisa, the sensitivity of the paperbased elisa was decreased by an order of magnitude compared to the conventional elisa format. lathwal and sikes 383 conducted a systematic investigation of several signal amplification methods for paper-based colorimetric detection of malarial biomarker hrp2. to investigate multiple metalloenzyme−substrate combinations, the same capture antibody was immobilized on aldehyde-modified cellulose for all experiments, and the following detection systems were evaluated: (1) hrp with substrates dab and h 2 o 2 , (2) hrp with substrates tmb and h 2 o 2 , (3) alp with substrates bcip and nbt, (4) ag deposition onto aunps, and (5) polymerization-based amplification. because all factors (i.e., capture antibody, capture antibody loading, biomarker, detection antibody) were held constant, the differences review observed in each assay were presumed to be the direct result of the signal amplification system used. positive and negative controls were evaluated at various time points for each signal amplification method to determine an optimal window for visual readout of the signal ( figure 28 ). 383 all of the methods had an optimal time point for visual signal readout with the exception of hrp-tmb/h 2 o 2 , which produced significant false-positive signals and a color change from blue to brown that was difficult to interpret. of the 4 remaining methods, the metalloenzyme−substrate combinations (hrp-dab/h 2 o 2 and alp-bcip/nbt) had detection limits an order of magnitude better than ag deposition and polymerization-based amplification methods when quantified by rgb color intensity. 383 any portable colorimetric reader, even a cell phone, could enable the use of these highly sensitive metalloenzyme conjugates in a primary healthcare setting. the advantages of paper-based elisas are potentially compromised by the instability of metalloenzyme conjugates, since denaturation of metalloenzymes leads to poor turnover of substrate and lower sensitivities. 388 for this reason, notable research efforts have been devoted to optimizing long-term storage of metalloenzymes, particularly hrp, for use in microfluidic paper analytical devices (μpads) or two-dimensional paper networks (2dpns). 385−387 one method involves vacuum drying or freeze-drying enzymes and substrates in the presence of trehalose, a disaccharide often used as a cryoprotectant because of its capacity to stabilize the protein's interaction with solvents, forming a protective "glass" as it dries. 389, 390 ramachandran et al. used a combination of trehalose, bovine serum albumin (bsa), and feso 4 -edta for vacuum drying of hrp-conjugated antibodies against malarial biomarker hrp2 into glass fiber pads. 387 the stabilized hrp could be stored for 5 months at 45°c under these conditions without a significant loss in catalytic activity. a glass fiber pad containing all of these reagents was incorporated into a 2dpn for detection of hrp2 with a lod of 6.5 ng/ml, a value well within clinically relevant concentrations. this assay also demonstrated the utility of metalloenzymes in paper-based poc diagnostics, providing a signal amplification step that is rarely present in conventional paper diagnostics (e.g., lateral flow assays). these advances in enzyme stabilization when combined with simplified paperbased assay formats could potentially allow for elisa sensitivity to be translated for direct use at the point of care. 4.3.2. nanoparticle-assisted enzymatic signal amplification. the integration of noble metal nanoparticles has further augmented the signal amplification capabilities of metalloenzymes in infectious disease diagnostics. nanoparticles have served as surfaces for the coupling of antibody-metalloenzyme conjugates and have been implemented in electrochemical sensors for detection of infectious disease-associated protein biomarkers. 391−393 zheng et al. 392 developed an amperometric immunosensor for detection of hiv antigen p24 that employed a standard "sandwich" format. a glassy carbon electrode was modified with gold nanoparticles to allow for immobilization of anti-p24 capture antibodies. hrp-conjugated anti-p24 antibodies were used for detection, catalyzing the oxidation of substrate hydroquinone in the presence of h 2 o 2 . the reaction generated a reductive current at the electrode surface proportional to p24 concentration. the detection limit of the assay was 8 pg/ml, similar to the p24 assays 153, 233 previously discussed in sections 4.1.2 and 4.2.2. while the assay was robust to human serum samples spiked with p24, the serum samples tested contained a p24 concentration 3 orders of magnitude higher than the lod. 392 the currently required electrochemical workstation would limit the assay's application to higher level laboratories. nonetheless, this amperometric method demonstrated the value of integrating noble metal nanoparticles with metalloenzyme conjugates for signal amplification in diagnosis of infectious diseases. noble metal nanoparticles have also been utilized as colorimetric signaling probes in elisas for detection of protein biomarkers for disease. 394−398 so-called "plasmonic" elisas utilize the standard "sandwich" assay format with an immobilized capture antibody and a detection antibody that is conjugated to a metalloenzyme. however, as opposed to using conventional metalloenzyme substrates for colorimetric detection, plasmonic elisas employ the enzyme as a kinetic tool for nanoparticle nucleation. this causes drastic shifts in spr and in the absorbance spectra of the nanoparticles that are detectable with the naked eye. 399 the plasmonic elisa pioneered by de la rica and stevens 396 utilized a catalase-conjugated detection antibody to control the growth of aunps in such a manner that was proportional to the concentration of hiv biomarker p24. in the absence of biomarker, bulk au(iii) was reduced by h 2 o 2 to form stable, spherical aunps that appeared red in color. when p24 was present in a sample, the catalase-conjugated (figure 29) . the red-shift in the spr was highly sensitive toward h 2 o 2 concentration; thus, depletion of h 2 o 2 with catalase that was only present in p24-positive samples allowed for very sensitive detection of p24 with the naked eye. the absorbance was also quantified using a simple spectrophotometric readout, yielding a detection limit of 1.0 ag/ml, 396 nearly 6 orders of magnitude more sensitive than the previously discussed detection methods for p24 antigen. 153, 233, 392 the authors also demonstrated that the plasmonic elisa could detect p24 in hivpositive serum samples, even identifying p24 in samples from hiv-positive patients with viral loads less than 50 copies/ ml. 396 the remarkable sensitivity of this assay for naked-eye detection of p24 provides the necessary improvement required for elimination campaigns and active case management. it would permit earlier detection of p24, leading to improved outcomes for hiv patients. the assay could be particularly impactful for the challenges associated with early infant hiv diagnosis in low-resource settings. the plasmonic elisa presented by the authors still calls for extensive sample handling and user manipulation and is currently unsuitable to a primary healthcare setting. adaptation of the previously discussed enzyme stabilization measures and integration of the assay to a field-ready format (e.g., paper-based elisa or 2dpn) could enable its translation to an ultrasensitive fieldready diagnostic for hiv detection. metalloenzymes play a critical role for signal amplification in a number of assays, enabling the sensitive detection of infectious diseases. several research efforts have been aimed at translating the sensitivity of metalloenzyme-based assays to formats such as paper-based diagnostics that are amenable to resource-limited settings. although the issue of enzyme storage in paper has been addressed to certain extent, 385−387 the longterm stability of enzymes remains a concern for poc diagnostics, especially in climates with elevated temperatures and prevalent infectious disease. there are numerous methods that utilize nonenzymatic means for signal amplification that eliminate the issue of long-term enzyme storage altogether, including nanoparticles that act as enzyme mimics and other metal-based methods. these will be covered in the following section. while the majority of signal amplification in bioassays is enzyme-based, several interesting metal-based amplification strategies have been developed. these strategies include metal nanoparticle dissolution, nanocrystal ion exchange, enzyme mimics, and reductive nanoparticle enlargement. 394,400−403 compared to enzymes, metal-based signal-amplification has the distinct advantage of long-term storage and thermal stability. however, many of these strategies suffer from the following drawbacks: (1) amplification often requires additional steps to be integrated into point-of-care test workflow, and (2) incorporation into a paper-based format can be technically challenging. however, innovative assay design can overcome these challenges. integration of signal amplification into point-of-care tests can drastically improve analytical and diagnostic sensitivity, resulting in earlier diagnoses and detection of low-density infections. therefore, diagnostics which incorporate novel and user-friendly signal amplification steps could fill a need for elimination campaigns and surveillance programs. this section reviews metal-based signal amplification strategies that show promise for application to low-resource infectious disease diagnostics. 4.4.1. nanoparticle dissolution and cation exchange. at the heart of signal amplification is the principle that, for each biomarker target captured in an assay, many signalgenerating elements (particles, molecules, atoms, electrons, photons, etc.) are produced. in a typical nanoparticle-based assay, a single nanoparticle often indicates the presence of one biomolecule. however, a single nanoparticle is a densely packed lattice of thousands to millions of metal atoms, each of which, after nanoparticle dissolution or cation exchange, can be leveraged for signal generation (figure 30 ). 199,404−406 metal ion chelating reagents that result in chromogenic or fluorescent signal have long been used for detection of trace metals for a myriad of applications. 407, 408 recently, tong et al. 405 exploited ferrous ion-chelating ferrozine in an iron oxide nanoparticle-linked immunosorbent assay (ilisa). antibodyfunctionalized wustite (feo) particles were used as detection elements in direct, competitive, indirect, and sandwich wellplate assays. after functionalized iron-oxide particles bound to the target, the nanocrystals were treated with acid and reducing agents, resulting in stoichiometric conversion to ferrous ions. thus, thousands of fe 2+ ions were released for each biomarker present. when excess ferrozine was added, solutions changed from transparent to purple upon chelation, with intensities directly proportional to the iron concentration in solution. proof-of-concept assays detecting mouse igg had low picomolar detection limits, similar to assays that depend on enzymatic signal amplification. to extend the applicability of their amplification technology, the group also demonstrated its use in a western blot. by switching the chelating reagent to potassium ferrocyanide (prussian blue), the reagent could easily be precipitated onto a cellulose membrane when iron oxide particles were used as detection elements. 405 while wellplate assays are far from applicable to field settings, the ilisa system could be translated to an aptly designed paper diagnostic format. another avenue for signal amplification similar to nanoparticle dissolution is nanocrystal cation exchange, in which the cations within a nanocrystal are place-exchanged with different cations. 409 while bulk, solid-phase cation exchange often requires elevated temperatures and several weeks, complete nanoscale exchange can occur within seconds due to increased surface area and lower energy barriers to diffusing ions. 410 for cation exchange to be employed in a bioassay, nanocrystals (znse, zns, cdse, or cus) are first functionalized with targetspecific molecular recognition elements, such as antibodies or aptamers, which are utilized as detection probes. using rapid silver ion exchange, each bound nanocrystal releases thousands of zn 2+ , cd 2+ , or cu 2+ ions. zinc and cadmium ions are then detectable using fluorogenic chelating reagents such as fluozin-3 or rhod-5n, respectively, and cu 2+ can be detected in a chemiluminescent reaction with luminol and h 2 o 2 . these reactions produce an amplified, stoichiometric signal and have been employed in well-plate 403, 411 and magnetic beadbased 412−415 immunoassays for protein 403, 411, 415 and cell 412 detection as well as rolling circle amplification for dna/ mirna 413, 414 detection. despite this versatility, the cation exchange method has yet to be applied in paper-based formats. similar to the ilisa example discussed previously, signal amplification using nanocrystal cation exchange would require a precipitating reagent for metal ion detection as well as clever design of a paper microfluidic device. enzymatic signal amplification results in high analytical sensitivity and is easily performed in a controlled laboratory environment. however, as discussed in section 4.3, elevated temperatures often lead to decreased catalytic turnover, making it difficult to incorporate enzymatic signal amplification into low-resource infectious disease diagnostics. in recent years, some inorganic nanoparticles, including noble metal, rare earth, and magnetic nanoparticles, have been found to display surprising enzyme-like catalytic activity. 416−418 these nanomaterial-based artificial enzymes, originally dubbed "nanozymes" by manea et al., 419 are highly stable toward a range of temperatures and phs and cannot be degraded by proteases. further, catalytic activity of inorganic nanoparticles can be tuned with particle size, shape, coating, modification, and composition. for these reasons, nanozymes have been incorporated into a myriad of sensors for various applications. in 2007, gao et al. 420 made the surprising discovery that magnetite (fe 3 o 4 ) nanoparticles have intrinsic peroxidase-like activity, displaying michaelis−menten kinetics consistent with a ping-pong mechanism. fe 3 o 4 magnetic nanoparticles were found to catalytically turn over several common horseradish peroxidase substrates, including tmb, dab, and o-phenylenediamine (opd), with catalytic turnover numbers equal to or improved over horseradish peroxidase. the group demonstrated that the particles could be used in place of horseradish peroxidase in a traditional immunoassay and that the magnetic properties of fe 3 o 4 could be leveraged further to enrich biomarkers before detection. 420 as a result of this seminal work, magnetic nanoparticles have been employed as signal amplification elements in protein and dna bioassays for a variety of infectious diseases, including ebola, 242 rotavirus, 402 mycoplasma pneumonia, 421 vibrio cholerae, 422 chlamydia trachomatis, 401 l. monocytogenes, 423 enterobacter sakazakii, 424 and salmonella typhimurium. 425 beyond magnetite, several other types of nanoparticles, including gold, 426 platinum, 427 hybrid particles, 428−432 and mofs, 433 have been utilized as enzyme mimics in bioassays for targets such as respiratory syncytial virus, 432 avian influenza a, 426 salmonella, and e. coli, 431 hepatitis c and hiv, 428 and staphylococcus aureus. 433 many of the assays that utilize review nanozymes as catalytic turnover reagents for detection are formatted similarly to immunoassays on the surface of well plates. 402,421−423 several groups have translated this technology to the lateral flow format and found that the nanozyme detection element resulted in at least 100-fold improvement in limits of detection when compared to traditional gold nanoparticle-based lateral flow strips. 242, 424, 427, 431, 434 for example, loynachan et al. 435 developed an ultrasensitive lateral flow assay for the detection of hiv p24 antigen using porous platinum au@pt core−shell nanozymes that catalyzed the precipitation of cn/dab (4-chloro-1-naphthol/3,3′-diaminobenzidine tetrahydrochloride) in the presence of h 2 o 2 ( figure 31 ). incorporation of this detection scheme, as well as careful and systematic design of the affinity reagents for p24 capture and detection, resulted in a lateral flow assay with femtomolar detection limits, more sensitive than laboratory-based elisa methods and nearly 2 orders of magnitude more sensitive than commercially available rapid tests. 435 because visual signal varied linearly with concentration before and after catalytic signal amplification, the test also had an ultrabroad dynamic range. the advantages of the nanoparticle enzyme mimics were directly demonstrated in a stability test in which the activities of lyophilized porous pt core−shell nanocatalyst conjugate and lyophilized hrp conjugates were measured over time. while the enzyme conjugates lost nearly all activity after 15 days of storage at room temperature, the nanocatalysts maintained 100% of their initial activity while stored at room temperature and nearly 80% of their initial activity while stored at 44°c for up to 42 days. 435 however, it should be noted that the optimal hrp lyophilization and storage conditions 387,389 discussed in section 4.3.1 were not employed in this study. to achieve such significant improvements in sensitivity using nanozymes, a wash step, a substrate addition step, and a reaction quenching step were inserted into the lateral flow assay workflow after the initial signal development. 435 these additional steps make this assay and other similar diagnostics more difficult to perform in the field. redesigned paper devices or automated lateral flow cassettes could simplify this workflow review and allow for application of nanozymes in field settings, providing much-needed signal amplification and improved sensitivity for low-resource infectious disease diagnostics. 4.4.3. reductive nanoparticle enlargement. the use of gold nanoparticles as detection elements in commercially available lateral flow assays is ubiquitous. 436 particles used in these assays are typically 15−50 nm and can be detected either visually (without optical equipment) or using simple hand-held readers. 177, 219, 437 often, the detection limits of these assays are not low enough to diagnose low-density infections. one simple solution for improving sensitivity is chemically enlarging the particles, making them more visually intense. in this process, a lateral flow assay is run in the typical manner such that, if the antigen is present, a gold nanoparticle-containing sandwich complex is formed at the test line. next, an enhancement solution consisting of a molecular precursor and reducing agent is added to the test. the gold nanoparticles at the test and control lines serve as nucleation sites for solid metal deposition. the most common format for particle enlargement, silver enhancement, is based on 19th-century photographic techniques and involves the reduction of silver ions (e.g., silver nitrate) to silver metal on the surface of gold particles in an acidic buffered solution containing hydroquinone. 438 this process is similar to the seeded-growth nanoparticle synthesis method and can result in particles from hundreds of nanometers to several micrometers in diameter ( figure 32 ). 439 because silver enhancement found its first biological application in tissue staining in 1935, 440 there are many commercially available silver enhancement solutions designated for microscopy applications (sigma, thermo, ted pella, nano, etc.). the first application of silver enhancement to paper diagnostics was in 1991 when horton et al. 441 developed a proof-of-concept lateral flow assay for the detection of mouse igg and demonstrated that silver treatment resulted in a 100fold improvement in detection limits. since then, silver enhancement has successfully improved the detection of many infectious diseases by several orders of magnitude, including influenza, 439, 442 hiv, 443 malaria, 444−449 y. pestis, 450 and e. coli. 451 one of the drawbacks to silver enhancement on lateral flow assays is that it must be performed after the test has completed its initial development. increasing the total number of steps required reduces the likelihood that the test could be applied in the field. for example, one group 450 found that silver enhancement improved the detection limits of their y. pestis test 1000-fold. in order to take advantage of this impressive enhancement, the authors allowed the gold test to develop as usual, washed the strip three times, submerged it into enhancing reagents for 5 min, and then stopped the reaction with sodium thiosulfate. this procedure is feasible in a district hospital or even a local health outpost with minimal resources, and the enhanced assay would be a true asset in these settings should a plague pandemic occur. however, the additional steps required for signal amplification make poc application in a low-resource field setting unlikely. for this reason, some groups who employ a silver enhancement step in their paper diagnostic assays have moved away from the traditional lateral flow assay format and toward alternative formats that allow for automated delivery of silver enhancement reagents to the test line. fujifilm 439, 452 applied their expertise in photo technology to develop a benchtop workstation that automated silver enhancement for influenza rapid diagnostic tests. in this format, a user added the sample to a lateral flow cartridge that contained all reagents necessary for test washing and silver enhancement. the cartridge was inserted into the benchtop workstation, which released the solutions in a timed, automated manner and provided a quantitative readout of the strip after test development was completed. despite the fact that the instrument greatly simplified the assay protocol for the user, the requirement of a reliable source of electricity greatly inhibits its application to a field setting. recently, the yager and fu groups have designed 2dpns that rely on unique paper geometry or embedded paper-fluidic valves to enable programmed, electricity-free delivery of silver enhancement reagents to the assay test line. [444] [445] [446] [447] [448] 453, 454 these 2dpns simply require the addition of sample and buffer to pads that have been preloaded with assay reagents, mirroring the same number of steps required to run a traditional lateral flow assay. the unique physical structures of 2dpns allow for precisely timed delivery of sample, then gold conjugate, and finally silver enhancement reagents to the test line. similarly, cho et al. 455 developed a cross-flow chromatography system, in which silver enhancement reagents were embedded into paper and then rehydrated after the lateral flow assay developed. the enhancement reagents flowed perpendicular to the initial immunochromatographic test over the test and control lines. these creative approaches to redesigning the lateral flow assay make silver enhancement the most field-deployable metal-based signal amplification strategy to date, although large-scale manufacturing requirements must be considered to determine the scalability of such test designs. finally, it should be noted that while silver enhancement on gold particles is most common, other metals have been used for reductive nanoparticle enlargement for lateral flow enhancement. gold has been deposited onto gold and silver nanoparticles, 443,456−458 and silver onto silver and platinum nanoparticles. 459 while there are many other examples of solution and microfluidic assays employing reductive nanoparticle enlargement for visual and electrochemical signal amplification, these formats are less conducive to low-resource disease diagnosis. 451, 457, 460 4.4.4. bio-barcodes. the amplification methods discussed thus far increase the number of signaling elements after the initial assay has developed, requiring additional steps post hoc. another amplification strategy is to generate a greater number of detectable molecules midassay, thereby increasing the downstream signal output of the assay. in 2002, the mirkin group 461 demonstrated that protein biomarker targets could be detected indirectly in a highly sensitive manner by employing gold nanoparticles functionalized with dna tags. in this strategy, magnetic microparticles functionalized with targetspecific antibodies were added to a biological sample. after biomarker capture, the supernatant was removed and intermediate detection elements were added to the particles. these detection elements consisted of gold nanoparticles functionalized with a low density of target-specific antibodies and a very high density of double-stranded barcode dna. using an external magnet, the gold nanoparticles that bound the target were separated, and then the barcode dna was dehybridized and detected downstream ( figure 33 ). 462 because the number of barcode tags was much higher than the number of captured biomarkers, the resulting signal was amplified when compared to a direct detection method. one powerful advantage of this modular assay format is it could be easily multiplexed, with distinct barcode tags for each target of interest. 463 further, while the biobarcode method was initially applied to the detection of proteins, it has also been applied to nucleic acid detection. simply replacing the biomarker-specific antibodies with dna complementary to the nucleic acid target allows for detection of dna or rna targets with pcr-like sensitivity. 464 since its development, the biobarcode assay format has been utilized for the detection of infectious diseases such as hepatitis b, 463 hepatitis c, 465, 466 variola virus, 463 ebola, 463 hiv, 463, 467, 468 b. anthracis, 464 and salmonella, 469 as well as c-reactive protein 470 and a variety of cytokines. 471, 472 despite its promise for sensitive detection, the biobarcode assay format suffers from two major drawbacks: (1) the high number of user steps required before the barcode tags are released for detection makes it extremely difficult to adapt to a lowresource format, and (2) low-resource detection of the nucleic acid tags remains a challenge. initial detection strategies for the biobarcode assay were scanometric; the barcode tags were detected in a sandwich hybridization assay on the surface of a glass slide, with gold nanoparticles as the detection elements. signal was further enhanced using reductive silver deposition before the slides were evaluated using image analysis. [461] [462] [463] [464] 467, 473 methods such as pcr, 466,468 mass spectrometry, 474 and electrical detection 465 have been employed to detect the barcode tags as well. nam et al. 461 ,472 developed a colorimetric assay in which the released gold nanoparticle biobarcode tags were introduced to a second solution of gold nanoparticles functionalized with single-stranded dna complementary to the tags. the hybridization of the biobarcode tags with complementary dna resulted in aggregation of the two gold nanoparticle-dna conjugates, yielding a detectable color change. none of these detection methods can be employed in a field setting; however, it is not difficult to imagine developing a hybridization-based lateral flow assay for barcode tag detection or functionalizing the tags themselves with moieties such as biotin or a flag peptide that could make them readily detectable in a paper diagnostic format. as is evidenced by sections 4.3 and 4.4, significant advances have been made in developing metalloenzyme-and metal nanoparticle-based signal amplification strategies for infectious disease diagnosis. however, even with the advancements in metalloenzyme stabilization and highly stable nanoparticlebased approaches, poc assays utilizing signal amplification are still scarce. most of the assays discussed, even those that employ paper substrates or lateral flow formats, require too many user steps before the signal is generated. future work that minimizes user steps, for instance, through use of 2dpns or vertical flow assay formats, will be critical in translating these signal amplification strategies from laboratory tests to assays that can be run at a primary healthcare facility. moreover, most of the assays discussed in sections 4.3 and 4.4 were also heavily reliant on benchtop, laboratory-based instrumentation for signal readout. the next section will discuss the various types of field-deployable instrumentation that have been developed in an effort to bring quantitative and sensitive assays, such as those discussed in section 4, to low-resource settings. traditionally, point-of-care diagnostics such as lateral flow assays have delivered qualitative visual results. although this simplicity is advantageous in the field, the diagnostic sensitivity and specificity can suffer from subjective interpretation and user bias errors. quantitative measurements mitigate these factors, avoiding the data loss associated with qualitative tests and providing insight into important variables such as infection intensities and biomarker expression patterns. in an elimination setting, these parameters allow for robust epidemiological studies into transmission dynamics and intervention efficacy. novel detection strategies that offer highly sensitive and quantitative results, including many of those outlined in section 4, require instrumentation for signal readout. as a result, the development of appropriate and simple instrumentation will likely become an integral part of the application of diagnostic technologies in low-resource settings. incorporation of detection devices into point-of-care diagnostics effectively eliminates the possibility of "equipment-free" tests (e in assured criteria) and reduces the "affordable" nature of simple tests (a in assured criteria). 13 however, the potential benefits of instrumentation, including improved sensitivity and reduced bias, will outweigh these disadvantages in many use-case scenarios, and the overall cost of instrumentation per test can be reduced when a device is used to evaluate many tests over time. additionally, many instruments incorporate common consumer electronic devices, such as mobile phones and smart phones, thereby reducing the equipment burden. this section outlines some of the devices developed for point-of-care infectious disease diagnosis. table 3 is included at the beginning of section 5 to highlight selected examples of point-of-care instrumentation. a vast majority of the metal-based probes and nanoparticles highlighted in section 4 produce optical signals such as absorbance, fluorescence, or phosphorescence. as a result, a diverse array of instrumentation has been developed to facilitate assay readout for imaging and quantitative purposes. this section provides an overview of efforts to develop portable, affordable, and sensitive instruments for optical detection in low-resource settings. 5.1.1. microscopy. conventional microscopy remains the gold standard diagnostic technique for many infectious diseases, including malaria, tuberculosis, schistosomiasis, and intestinal protozoa. 475 to detect these diseases, a variety of microscopic techniques, such as bright field, dark field, and fluorescence microscopy, are used for direct inspection of stained or unstained smears (blood, sputum, urine, etc.). while microscopy can be a useful diagnostic tool, results depend strongly on infection intensities, sample preparation methods, and the training level of the microscopist. 476 these challenges lead to variability that reduces the utility of microscopy-based diagnosis in field settings, especially in low-transmission areas. additionally, microscope instrumentation can be expensive and bulky. to address these drawbacks, many groups are developing portable, affordable, and easy-to-use microscopy tools suitable for low-resource settings. one approach to bringing laboratory-based microscopy tools to low-resource settings involves building simple, portable, and/or miniature microscopes from low-cost materials. while this solution does not address some of the primary disadvantages of microscopy as a poc diagnostic (i.e., the need for skilled microscopists and sample preparation requirements), it does allow for conventional microscope platforms to be deployed in difficult settings at low cost. such devices typically replace high-energy, expensive light sources with leds, reducing the expense and power required for illumination in addition to increasing the light source lifetime. 477 structural components of these microscopes are often three-dimensionally (3d) printed, 477−480 and affordable plastic or hybrid lenses and fiber optics can replace expensive and delicate glass lenses. 477 tuberculosis. when samples were evaluated as positive or negative for m. tuberculosis, results from the global focus microscope and a laboratory-grade fluorescence microscope were in agreement for 98.4% of cases. 485 many similarly portable and lightweight microscopes have been developed, including tomographic, 482 holographic, 483 wide-field, 484 and fluorescence 486 microscopes, some impressively weighing in at less than 200 g. 479,482−484 these microscopes have been applied not only to the detection of tuberculosis 480, 485, 486 but also to malaria, 481 soil-transmitted helminths, 487 schistosomiasis, 487 and hymenolepis nana. 482 the foldscope is an example of a low-resource microscope that most embodies the assured criteria. 488 assembled using the principles of origami, the foldscope is constructed from flat paper, copper tape, a button-cell battery, an led, polymer filters, and a ball lens ( figure 34b ). the entire device costs less than $1, can achieve magnification up to 2180×, and was shown to be capable of detecting giardia lamblia, leishmania donovani, trypanosoma cruzi, e. coli, and schistosoma hematobium eggs. 488 despite these accomplishments, when the foldscope was applied to s. hematobium detection in a field setting, the diagnostic sensitivity was just 55.9% when compared to conventional light microscopy. 489 this limited sensitivity was likely a result of challenges with physical manipulation of the foldscope when integrated with a smartphone display. in other words, the foldscope failed to satisfy the "u" for user-friendly in the assured criteria for this particular use-case. this example demonstrates the importance of rigorous field evaluation of novel diagnostic devices and indicates that improvements to the foldscope must be made before implementation for case management. recently, mobile phone-based microscopy has emerged as a potential alternative to conventional microscopes for infectious disease diagnostics. phone-based bright-field, fluorescence, and dark-field microscopy devices have been developed, often employing compact, pocket-sized attachments ( figure 34c ). 490 these devices have been applied to the detection of a myriad of infectious diseases, including malaria, 491, 492 tuberculosis, 492 schistosomiasis, 493, 494 human papillomavirus (hpv), 495 soil-transmitted helminths, 496 filarial diseases, 497, 498 and intestinal protozoa. 494, 499 repurposing mobile phones is advantageous due to their widespread adoption and growing connectivity in low-resource settings. additionally, the acquisition of digital images could allow for image-processing (locally or remotely), automated diagnoses, and near real-time data transmission. these advantages, coupled with diseasespecific software applications, could potentially decrease the user variability typically associated with microscopy, improving case management and epidemiological studies. one example that leverages these advantages is the loascope, developed by the fletcher group ( figure 34d ). 500, 497, 498 the loascope is a smartphone-based device that combines video microscopy and automated software with onboard quantitative detection based on the movements of filarial parasite loa loa in whole blood. an application downloaded to the smartphone is used for stage control, video capture, image analysis, and data reporting of quantitative l. loa microfilaria densities. detection of l. loa is important because high levels of microfilariae are associated with potentially fatal adverse events after treatment with ivermectin, a drug frequently used in mass drug administration efforts against onchocerciasis and lymphatic filariasis (lf). these adverse events have led to the suspension of mass drug administration campaigns and subsequent major setbacks in onchocerciasis and lf elimination efforts. rapid, point-of-care measurement of l. loa infection intensity could allow for these elimination campaigns to resume. this device was validated in a field setting and yielded results similar to those of thick smears. 497 it was then applied in a large-scale (16,259 participants) test-and-not-treat approach for onchocerciasis in an area with high prevalence that had not participated in mass drug administration campaigns since 1999 due to the prevalence of l. loa. 498 each patient was screened for l. loa burden using the loascope, and those with high counts were not given ivermectin but were treated for l. loa with albendazole. this is a striking example of mobile phone-based microscopy in the field; all loascope measurements were performed by operators with only one hour of training, and the application-specific software significantly decreased the probability of user error by eliminating the need for subjective interpretation of results. in total, the loascope enabled ivermectin delivery to 15522 participants that otherwise would not have received treatment for onchocerciasis, demonstrating the incredible impact mobile phone microscopy can have when applied to detection of infectious diseases. 498 despite the fact that many mobile phone-based microscopes lack the analytical and diagnostic sensitivity of conventional microscopes, the loascope is an excellent demonstration that mobile phone microscopy can be highly impactful if applied in an appropriate use-case scenario. because this particular study focused on identifying patients with very high l. loa burdens, it is unclear to what extent this device could detect low-burden infections. generalization of this technology to other filarial diseases will also require further study. additionally, potential interference from other filarial parasites needs to be studied. as work continues in the field of low-resource microscopy, it will be important to develop new tools with specific use-case scenarios in mind. one potential avenue for application-based innovation involves development of low-resource sample preparation methods coupled with novel probes, such as the inorganic complexes and nanoparticles discussed in section 4. 5.1.2. lateral flow analysis. while much progress has been made in the development of rugged, field-applicable microscopy devices, lateral flow assays currently remain the most field-friendly format for infectious disease diagnosis. however, the simple, direct visual readout of lateral flow assays, often cited as one of the primary advantages of this format, is also considered its achilles heel; difficulties in interpretation resulting from visual impairment and operator bias can result in user-to-user variation. after tests are interpreted, maintaining accurate records and communicating test results to local and national health organizations remain substantial challenges. to overcome these challenges, a number of optical readers have been developed and commercialized to provide rapid, objective, and quantitative measurements of signal on lateral flow assays. the underlying instrumentation within these readers can generally be grouped into two categories: (1) optomechanical scanning and (2) imaging. for scanning readers, the primary optical components include a light source (led or laser) and photodiode. 501 the specifications of these components are tailored to the specific detection element used in a particular assay. light source power and wavelength are determined by the absorbance or excitation wavelength of the labels employed in each specific test. the light source is rastered along the lateral flow strip, and a photodiode then detects reflectance or emission, depending on whether colorimetric or fluorescent labels are used. measurements are typically recorded with millivolt (mv) units, though biomarker concentration can be obtained if a calibration curve is established. when colored particles are present at the control and test lines, they absorb incident light, resulting in a quantifiable decrease in reflectance. for fluorescent labels, the intensity of emitted light increases as the light source passes over the control and test lines. optics are configured in either a specular or confocal arrangement. 501 in the specular arrangement, which is primarily used for colorimetric labels, the photodiode is positioned such that it collects light that is reflected from the test at the same angle as the incident light from the source. confocal scanning systems can be used to measure reflectance or fluorescence. in this case, incident light is focused to a point on the lateral flow strip, and the photodiode is placed above this focal point. light returning from the strip, whether reflected or emitted, passes through an aperture that excludes rays that were not directly from the focal point, eliminating background scattering. 502 in the case of fluorescent labels, a dichroic mirror or filter is often placed before the aperture. both specular and confocal scanning configurations are sensitive to the z position of the lateral flow strip. in the specular arrangement, any small deviation along the z axis can result in the reflected light completely missing the photodiode due to misalignment. confocal systems are slightly more tolerant to small variations along the z axis, depending on the size of the pinhole aperture. however, even slight variations off the focal plane can drastically decrease the number of photons reaching the photodiode, altering the quantitative output. another challenge associated with confocal and some specular opto-mechanical scanning strip readers is that a point of incident light only covers 10−20% of the test and control lines. 503 this is especially problematic if the concentration of particles varies along the test or control line due to improper flow or manufacturing variations. despite these challenges, scanning systems are advantageous due to their low cost and relative simplicity. one of the most widely used commercial systems is the esequant line of lateral flow readers manufactured by qiagen, inc. ( figure 35a ). 346 qiagen offers customization of optics for colorimetric, fluorescent, chemiluminescent, and upconverting phosphor labels. the user should take into account that these systems often vary in sensitivity. as a general rule, smaller portable systems rarely perform as well as their benchtop counterparts. for example, van dam et al. 313 found that the portable commercial reader, the ucp-quant (qiagen) , was at least an order of magnitude less sensitive than a custom-modified benchtop reader for evaluating upconverting phosphor-based schistosomiasis lateral flow assays. consequently, selecting a scanning lateral flow strip reader for a particular application requires consideration of both portability and sensitivity. imaging-based lateral flow readers greatly reduce the optical equipment required for quantifying lateral flow assays. these systems produce a picture record of a lateral flow assay using a charge coupled device (ccd) or complementary metal-oxidesemiconductor (cmos) detector and rely on image processing software to detect and/or quantify the intensity of the test line. 503 benefits of this format include the ability to store test images alongside patient records and a lack of moving parts, making imaging-based readers more robust to field conditions. further, image analysis software can identify and correct for nonuniform signal, allowing for analysis of the full test line. imaging-based readers can be used for detection of colorimetric and fluorescent labels. in the latter case, hardware for excitation is required, and filters can be added to improve image quality. since test position and illumination can affect image analysis, many camera-based lateral flow readers include housing units for run-to-run consistency. commercial lateral flow imaging devices include the deki reader (fio), ax-2x (axxin inc.), skansmart (skannex), and the rds-2500 pro (detekt biomedical llc) ( figure 35b ). 504−506 these devices have been used for quantification of malaria, 507−510 dengue, 509 burkholderia pseudomallei, 509 and y. pestis 509 lateral flow assays, and some, such as the deki reader, have been tested extensively in the field. 507−510 image-based lateral flow quantification translates well to mobile phone technology, especially as smartphone camera quality improves. mobile phones have been used to quantify lateral flow assays for numerous diseases, including malaria, 511−514 hiv, 511 and tuberculosis. 511 similar to commercially available imaging-based readers, many of these studies required external devices to ensure consistent illumination and distance from the camera in order to maintain consistent image analysis. recently, scherr et al. developed a lateral flow imaging platform using an unmodified mobile phone as the only hardware. 512 relying only on software for image analysis, lateral flow images were captured under ambient lighting conditions. the images were sent to a secure web database (redcap) and analyzed using a matlab-based algorithm. quantitative results were sent back to redcap and delivered to the phone in real time. this software-based imaging platform was tolerant to moderate variations in test orientation, distance, and illumination, making data collection as easy as taking a cell phone picture. when compared to the scanning esequant lateral flow reader, this processing platform was found to have improved detection limits. additionally, the algorithm reliably worked for camera resolutions between 0.5 and 8 megapixels, suggesting that any modern mobile phone camera could capture images with sufficient quality for the image processing software to work. 512 as mobile phones are incorporated into low-resource diagnostic workflows, it is important to ask whether the conventional lateral flow format can be redesigned to take full advantage of potential benefits smartphones can offer. in other words, what changes can be made to the diagnostic test format that maintain the simplicity of lateral flow assays but will enable improved quantification, communication, and aggregation of test results? one option is to embed quick response (qr) codes into the lateral flow test. this idea has been approached in a number of ways, including placing stickers on lateral flow tests and adding a transparent overlay in which the test and control lines become part of the qr code. 514, 515 in these formats, the embedded barcode is either static, providing useful information about the brand, lot, and patient id, or dynamic, producing a positive/negative read-out. scherr et al. developed a novel platform in which the control line of the lateral flow assay was replaced with a control grid patterned in the shape of a qr code ( figure 35c ). 513 the qr code functioned as a flow control, appearing on both positive and negative tests, but not invalid tests. the qr codes also triggered data analysis and enabled corrections for lighting effects. when scanned, manufacturing details were quickly and easily accessed at the same time as the test line was quantified. 513 it is not difficult to imagine the impact such a test format could have when combined with the data communication and aggregation abilities of smartphones. epidemiological and surveillance studies could account for variables that are not even currently considered in most studies, such as manufacturing batch-to-batch variation. geographic data could also be coupled to specific rapid tests. as tools for automated reading of lateral flow assays become more prevalent, establishing standards for consistent reporting and data management will be important. just like with the lateral flow assays themselves, test-reading instrumentation will need to be validated to ensure proper and consistent biomarker quantification across different devices and test brands. although optical lateral flow readers may detect test lines invisible to the human eye, readers alone will not replace the need for ultrasensitive lateral flow tests. 5.1.3. other smartphone-enabled optical measurements. mobile phones have been adapted for optical measurements beyond lateral flow quantification. one example is the mobile phone-based, hand-held microplate reader developed by berg et al. 382 this device consists of a 3dprinted attachment in which an led array illuminates each well, and the transmitted light is collected via 96 individual optical fibers. results are visualized and delivered to the user in one minute within a custom mobile phone app. the device was shown to be more than 98% accurate for immunoassays detecting mumps igg, measles igg, and herpes simplex virus igg (hsv-1 and hsv-2) when compared to an fda-approved clinical spectrophotometer. 382 while microplate-based immunoassays are not feasible in a field setting, this device could make a true impact in resource-limited clinics as an excellent alternative to traditional bulky and expensive spectrophotometers. additionally, because the device is attached to a mobile phone, it offers the potential to rapidly communicate and aggregate results, further enabling large-scale epidemiological studies. mobile phone attachments for other optical detection modalities have been developed as well. in one exciting example, long et al. 516 developed the tri-analyzer, a trimodal smartphone attachment capable of measuring transmission, reflection, and emission intensities. this multifunctional device leveraged white light from the smartphone's rear-facing flash led or an integrated green laser diode for sample illumination via optical fibers. the optical fibers collected light transmitted through or emitted from the sample or light reflected from a photonic crystal biosensor. this collected light was then collimated and focused before being dispersed by the diffraction grating onto the smartphone cmos sensor. the intensity at each wavelength was determined by the integration of pixel intensity at the corresponding position along the sensor. this clever detection strategy allowed for full absorbance and emission spectra to be collected with a simple smartphone-based device. in two proof-of-concept assays, the developed device could detect analytes at lower concentrations than those detected using conventional laboratory instrumentation, indicating that the miniaturization of the technology did not result in a trade-off of sensitivity. 516 thus, this trimodal smartphone attachment enables quantitative laboratory-quality optical measurements at one's fingertips. similarly formatted mobile phone-based spectrophotometers, 517,518 surface plasmon resonance sensors, 519−521 and flow cytometers 522, 523 have also been developed. the miniaturization and decrease in cost associated with these smartphone-enabled tools will make chemical reviews laboratory techniques possible that would otherwise be inaccessible in low-resource settings. noble metal nanoparticles have extremely high molar absorptivities, making them ideal visual labels for lateral flow assays. this intense absorbance occurs when particles are excited with light that matches the resonant oscillation of their free electrons. as these electrons relax down to the ground state, they release energy in the form of heat to the surroundings. 524 the amount of heat generated is equal to the product of the number of noble metal nanoparticles, the cross-sectional area of the nanoparticles, and the intensity of the incident light. 524 recently, bischof's group took advantage of these unique thermophysical properties to develop a novel method for lateral flow quantification based on the thermal contrast of gold nanoparticles. 524−526 in this method, a highpowered green laser was used to excite the gold nanoparticles, and an infrared-detecting camera was employed to measure the resulting thermal contrast. an early benchtop prototype of the device demonstrated a 32-fold improvement in detection limits of an fda approved lateral flow assay for cryptococcal capsular polysaccharide glucuronoxylomannan antigen (crag). 525 crag is a biomarker for cryptococcal infections, which are opportunistic fungal infections that are among the leading causes of death for patients with aids and a common cause of adult meningitis in sub-saharan africa. 525 the thermal contrast-coupled crag lateral flow assay was further validated in a second study, displaying 92% accuracy in quantifying crag levels in patient samples. 527 after successful validation of their prototype device, the group developed a reader that incorporated a green laser, infrared camera, and automated control system into a benchtop box device ( figure 36 ). 526 the study demonstrated the utility and versatility of thermal contrast as a method for quantification of lateral flow assays from a variety of vendors and for multiple diseases. an 8-fold improvement in detection threshold compared to visual inspection was found for lateral flow assays for influenza (bd veritor), malaria (first response, sd bioline, and parahit), and clostridium difficile (ridaquick). 526 in a separate study, the group found that this enhancement factor was greater as nanoparticle size increased, suggesting that redesign of lateral flow assays could further improve test sensitivity using a thermal contrast reader. 528 while thermal contrast represents a unique and versatile method for lateral flow quantification, several issues must be addressed before large-scale implementation. these include cost, portability, and background signal reduction from biological interferences such as blood. additionally, nitrocellulose membranes are highly flammable; while it does not appear that full strip ignition has occurred in the currently published studies, visible burn marks along the nitrocellulose membrane 526 suggest that safety studies to determine the limits of the thermal contrast system should be undertaken before widespread implementation. nevertheless, thermal contrast represents a highly sensitive and promising tool for lateral flow quantification. surface-enhanced raman spectroscopy (sers) is a highly sensitive vibrational spectroscopy technique for structurebased analyte detection. in this technique, excited localized surface plasmons, typically from noble metal (e.g., gold and silver) substrates with nanoscale features, generate amplified electromagnetic fields, enhancing the inelastic raman scattering signals of analytes of interest up to 10 14 times. 529 instrumentation for sers detection requires a high-powered laser excitation source, which is typically tuned according to the resonant frequency of the plasmonic substrate. after excitation, filters are used to exclude elastic rayleigh scattering, allowing only raman-scattered light to reach the detector. 530 the high sensitivity of sers makes it an attractive detection platform for infectious disease diagnostics, particularly for the detection of low-density infections. many laboratory-based bioassays for protein, nucleic acid, and whole-cell biomarkers that leverage sers detection have been developed for a myriad of infectious diseases. 68,531−536 these assays are generally either performed on the surface of particles in solution or on a bulk solid substrate. detection elements typically consist of nanoparticles functionalized with raman reporter molecules and target-specific molecular recognition elements. 529 once a complex is formed, the nanoparticles provide the plasmonic surface for raman signal enhancement of the reporter molecule. nanoparticles are typically gold, silver, or au@ag particles and come in a variety of shapes that provide optimized signal enhancement. 537 small-molecule raman reporters, such as 4-mercaptobenzoic acid, malachite green, and 4-aminothiophenol, are often employed because biomolecule targets typically have complex vibrational signatures and are frequently too far away from the plasmonic surface for sensitive detection and identification. 529 the bulky nature and resource requirements of sers detection remain its primary barrier to implementation in point-of-care settings. several hand-held raman spectrometers have been developed and commercialized, including the firstdefender (thermofisher scientific, inc.), 538, 539 nano-ram (b&w tek), 540, 541 and progeny (rigaku corp.). these devices are advertised for identification of unknown substances in forensic, environmental, military, and first-response contexts and for quality control in industrial settings. while many studies that utilize sers-based detection for infectious disease biomarkers suggest that these commercially available portable devices could be utilized for analysis of a sers-based lateral flow assay, none of the studies have actually implemented a hand-held device. 542−547 accordingly, it is unknown whether such devices have sufficient sensitivity or spatial resolution to use effectively with low-resource diagnostics. these parameters, along with cost analysis, should be studied to determine whether current commercial devices could be implemented. magnetic nanoparticles possess advantages over optical lateral flow labels because their signal is rarely hindered by test opacity or biological interferents. a detailed discussion of these qualities as well as examples of specific assays that employ magnetic nanoparticles are provided in section 4.2.6. here, we discuss instrumentation required for magnetic particle detection. there are a variety of methods for detecting paramagnetic and superparamagnetic nanoparticles on lateral flow assays based on either the inductive effect or magnetoresistance. for example, magnabiosciences (usa) has developed an induction-based device that has been used extensively in the literature for detection of infectious diseases such as anthrax, 366, 367 t. solium, 349 hiv, 360,361 v. parahemolyticus, 364 and l. monocytogenes. 365 in this format, the lateral flow assay is placed inside an oscillating magnetic field on a set of induction coils, and the instrument measures the induced electromotive force (v) on the coils. assays quantified by this technology generally had a greater sensitivity compared to visual detection of iron oxide particles. 548 while the current iteration of the magnabiosciences reader is a relatively large benchtop device that is unsuitable for use in low-resource settings, a more portable version is being developed. 549, 550 other companies, such as magnasense (finland) and magnisense (france), have developed portable, hand-held devices based on inductive detection of paramagnetic particles, though these devices have not been as extensively tested in the literature. several groups have developed lateral flow assay readers using magnetoresistive sensors. these devices are based on the principle that the electrical resistance of certain materials, which are incorporated in the sensors, can change upon application of an external magnetic field. 350 there are several types of sensors based on magnetoresistance, but the most common are giant magnetoresistive (gmr) sensors, which were once used in the reading heads for hard disk drives. 551 when applied to lateral flow assays, gmr sensors are placed in close proximity to the test and control lines, and an external magnetic field is applied to the test. this external field aligns the magnetic moments of paramagnetic or superparamagnetic lateral flow detection reporters. this alignment then produces a fringe field that is detectable by a change in resistance when a constant current is applied across the gmr sensor. 551 magnetoresistive sensors have been applied extensively by researchers to the detection of magnetic particle labels on lateral flow assays; however, most have been proof-of-principle studies using imitative lateral flow assays or tests for biomarkers such as hcg, cardiac troponin 1, and ifnγ. 357,552−555 the high sensitivity of magnetic detection on lateral flow assays makes this technique attractive for infectious disease diagnosis. while magnetic detection necessitates instrumentation for result readout, iron-containing magnetic nanoparticles also have the added advantage of being highly absorbent visual labels. 347 thus, magnetic nanoparticles can potentially be applied to different use-case scenarios with differing sensitivity requirements. in a use-case scenario such as morbidity control, where it is most important to identify high-intensity infections in the field, the visual detection of magnetic particles would be sufficient. when improved sensitivity is desired to detect lowintensity infections for epidemiological or surveillance purposes, analysis using the current instrumentation for reading magnetic nanoparticle labels could be performed in a district hospital or laboratory. however, further work on miniaturization of instrumentation and validation of hand-held readers will be required for field application of magnetic detection on lateral flow assays. as discussed in previous sections, electrochemical detection offers several benefits for poc infectious disease detection, namely that it is inexpensive, sensitive, rapid, and quantitative. the electrochemical detection systems previously discussed were largely proof-of-concept diagnostics for detection of protein or nucleic acid biomarkers; the infrastructure required to carry out these assays would limit their utility in a primary healthcare setting in low-and middle-income countries. integration of validated and field-ready electrochemical detection systems with developed electrochemical assays represents a viable strategy for implementing the next generation of poc diagnostics. the blood glucose biosensor is a remarkably successful electrochemical device that is ubiquitously applied at the point of care. generally, electrochemical glucose meter kits consist of three components: a lancet for sample collection, plastic test strips for wicking of the glucose-containing sample and generation of current, and a device for digital readout of the generated current. 556 the test strips house an electrochemical cell that contains a counter-reference electrode, fill-detection electrode, enzyme-coated working electrode, and mediators ( figure 37a ). in these devices, the working electrode is coated with the enzyme glucose oxidase (gox) or glucose dehydrogenase (gdh), and the enzyme oxidizes the glucose present in a blood sample. ferrocene derivatives or other group viii metal complexes act as mediators to facilitate electron transfer from the enzyme redox center to the electrode surface, thereby producing a measurable current. 556 since the development of the first commercial blood glucose biosensor in 1987, the use of this technology has drastically expanded with the current market now offering numerous blood glucose meters that are portable, rapid, sensitive, and quantitative. 557 the blood glucose biosensor has had an enormous impact on management of types i and ii diabetes where constant monitoring and a rapid time to result are critical to preventing incidences of hyper-and hypoglycemia. successful adaptation of this technology for field detection of infectious diseases would have a profound impact in disease surveillance and elimination campaigns, where sensitive and quantitative results are desired to best inform healthcare professionals about disease distribution. 556, 557 several groups have repurposed commercial blood glucose meters for detection of a variety of targets. 167,558−562 xiang and lu 558 developed a platform that utilized a personal glucose meter for signal output to detect protein (ifn-γ), nucleic acid (adenosine), small molecule (cocaine), or toxic ion (uranium) targets. their strategy employed a magnetic bead-conjugated aptamer specific for the biomarker of interest. the aptamer was initially prehybridized to a partially complementary dna strand that was linked to invertase, an enzyme that catalyzes the hydrolysis of sucrose into its fructose and glucose subunits. in biomarker-containing samples, the aptamer bound to the biomarker, releasing the invertase-conjugated complementary dna into solution. the magnetic beads were then removed from solution using an external magnet, and the addition of sucrose to the supernatant resulted in glucose production that was readily detectable by a personal glucose meter. while this creative application of an extant platform expanded its functionality, it also increased the number of assay steps, potentially limiting its use directly at the point of care. to address this concern, the lu group 562 has since integrated this solution-based assay into a lateral flow strip with dried reagents, eliminating several user steps and further simplifying the platform such that it could be applied at the point of care. this innovative approach is an excellent example of how metalbased sample preparation using magnetic beads can be combined with an existing electrochemical platform for rapid detection of a variety of disease targets. as the blood glucose meter clearly demonstrates, portable electrochemical instrumentation has enabled minimally trained users to measure analytes at the point of care and rapidly obtain information regarding their health status. mobile health technologies (i.e., mobile phones and telemedicine) augment these advances in instrumentation, connecting individual users to an entire healthcare infrastructure. mobile health technologies are useful for tracking patient information and facilitating communication between the patient and the care provider. as these technologies mature, they are becoming increasingly important for modeling diseases and disease trends throughout various populations. 563 integration of portable electrochemical diagnostics with mobile health technologies in so-called "connected" diagnostics has been pursued by interfacing electrochemical devices with mobile phones. 173, 564 nemiroski and colleagues 173 developed a universal mobile electrochemical detector (umed) that combined a potentiostat with virtually any mobile phone using audio-based data transmission ( figure 37b ). the device could accommodate several electrochemical techniques (amperometry, coulometry, voltammetry, and potentiometry), and the voice system-based data ensured broad compatibility with a variety of mobile phones and networks. the authors employed the umed to detect blood glucose, heavy metals in water, electrolytes, and malarial biomarker hrp2. following sample collection and measurement using the potentiostat, the user placed a phone call to a skype number in a remote location to vocally report the value of the analyte measured. the remote application then extracted the value from the audio data and sent an sms message back to the user with additional diagnostic information (e.g., "low" if the reported blood glucose level was low). although the umed was applied to detect an infectious disease biomarker (hrp2), it is limited by the availability of existing compatible testing technology. for instance, hrp2 detection required an offline 96-well plate elisa to be conducted prior to analysis on the umed and transmission over a mobile network. as more progress is made in electrochemical diagnostic design, the umed and other similar devices will be able to reach their full potential as "connected" diagnostics. as battery-operated and portable devices become more robust, electrochemical detection devices are becoming increasingly viable as poc platforms. the success of the personal glucose meter is a testament to the effective and reliable diagnostic performance that is possible with electrochemical measurements. with the concurrent expansion of information technologies, "connected" electrochemical diagnostics could serve as the link between the chemistries utilized at the point of care and the remote higher level healthcare systems that map disease trends and inform treatment decisions. in high-income countries, advances in portable electronics and instrumentation have enabled live health tracking at consumers' fingertips. wearable commercial health diagnostics such as apple watch, garmin, and fitbit are simple to use and provide real-time updates of heart rate, activities completed, or calories burned. in a similar vein, there are several emerging healthcentered tools, such as press-on tattoos, 565,566 skin patches, 567 and face masks, 568 capable of detecting changes in human physiology at the point of use. in addition, the advent of technologies such as google glass have allowed for "connected" interpretation of rapid diagnostic tests. 515 overall, wearable instruments combine sample collection, sample preparation, analysis and readout, and data transmission into one device, representing significant progress in diagnosis and health management at the point of care. small electrochemical instrumentation has become a focus of innovation, and research in this field has led to a number of skin-based wearable diagnostics. jia et al. 565 566 detected glucose in the interstitial fluid of the skin ( figure 38a ). these technologies are extraordinarily promising for future diagnostic devices, particularly as new biomarkers of infectious diseases are discovered in surface biofluids such as sweat. 569 recent advances in microprojection 570,571 and microneedle 572,573 technology have allowed researchers to expand the use of skin-based wearables beneath the surface of skin. in several applications, patches consisting of many microprojections were easily applied to the skin with a gentle press and could draw hundreds of microliters of blood with little-to-no pain. 567,574−576 in 2014, lee et al. 567 outlined the design of a multiplexed patch that captured both igg and malarial biomarker hrp2 from the skin of inoculated mice. the patch consisted of microprojection arrays (mpas) that were subsequently functionalized with anti-hrp2 antibodies, fc-specific antimouse igg antibodies, and antidengue antibodies. the anti-hrp2-functionalized mpas detected the biomarker, while the anti-igg-functionalized mpas acted as a positive control for skin penetration, and the antidenguefunctionalized mpa served as a negative control. though sample collection and preparation were applicable at the point of use, the assay required an elisa for colorimetric readout of the captured antigen. further research into integration of this technology with a diagnostic such as a stacked paper-based assay would allow for real-time skin-based monitoring of infectious diseases. another forthcoming application for wearable instrumentation is in breath analysis via paper masks. guder et al. 568 developed an electrical sensor to monitor respiration. the sensor consisted of a paper surgical mask printed with graphite electrodes that were attached to a battery and signal amplifier. as a user breathed, the humidity changes from the inhalation and exhalation generated a measurable current across the electrode, which was then detected using a bluetoothconnected mobile device ( figure 38b ). as this technology evolves, it may be possible to adapt it for analysis of breathbased biomarkers, particularly for respiratory infectious diseases such as tuberculosis and influenza. in addition, incorporating the external battery/amplifier pack into the material of the mask may open avenues for broader distribution, particularly to low-resource areas. likewise, personal computer displays such as google glass have enabled users to harness the power of a technological workstation in a hands-free glasses format. this capacity could significantly improve diagnostic capabilities and effectiveness. in 2014, feng et al. 515 developed a method of lateral flow assay analysis which combined visual examination of a lateral flow test with computerized image analysis and wireless storage capability ( figure 38c ). this wearable imaging technique allowed users to collect images of tests and store them in a cloud-based system or locally on the google glass headset if internet access was unavailable. once the google glass headset was connected to a wireless network, the test images could be sent to a secure server to be analyzed, and then the test's results and analysis were sent back to the viewer's eyepiece. this method has been demonstrated using rapid diagnostic tests for both hiv antibodies and prostate specific antigen (psa), 515 though it has the potential to be adapted for any lateral flow assay with visual labels. while the use of computer-based imaging may remove some of the inaccuracies that arise from subjective human-based interpretation of results, it must also be noted that there are a number of technological hurdles that should first be addressed. the google glass is not capable of autofocusing, and the images were taken under good lighting against white and universally solid, simple backdrops. thus, this technology is limited in its field application. in addition, test recognition was dependent on preprinted qr codes that were prepared and attached to the tests by researchers before use. in order to more efficiently utilize this technology, the google glass application could be adapted to identify test features without the need for a usersupplied qr code and web-based test input. this would simplify the workflow and allow for more high-throughput analysis of diagnostic tests. overall, there is significant room for growth and development in the field of wearable instrumentation. wearable diagnostics, which may have previously seemed more at home in the world of science fiction, are becoming commonplace in health-related research efforts. the development of wearable diagnostic technologies could drastically improve the simplicity of diagnostic measurements and interpretation in regions with little or no laboratory infrastructure. as these technologies continue to be developed for applications in high-resource areas of the world, the scientific community would be remiss not to consider the impact they could have on infectious disease diagnosis in resource-limited settings. inorganic chemistry for nucleic acid detection thus far, we have highlighted innovative examples of metalbased strategies for improving point-of-care diagnosis of infectious diseases based primarily on protein and inorganic disease biomarkers. in most cases, leveraging inorganic chemistry for sample preparation methods and detection probes would require minimal modification for application at the point of care. it is clear that, despite the potential trade-offs in complexity of workflow and instrumentation, the increased sensitivity offered by these metal-based strategies for biomarker detection could significantly improve morbidity control and elimination efforts. in many instances, though, sensitive and quantitative detection of the presence or absence of a disease does not paint the full picture needed to implement successful control or elimination campaigns. in some cases, the pathogen's own genetic material must be detected and extensively studied on a population scale in order to inform strategy. this research provides valuable information on pathogen species and strain distribution and drug resistance, which can inform treatment and diagnostic strategies in a control or elimination campaign. 579 the information-rich and highly specific nature of nucleic acid detection makes these biomarkers attractive targets for point-of-care diagnostics. in fact, several groups have begun to apply some of the metal-based detection strategies for proteins and inorganic biomarkers discussed in the preceding sections t o t h e d e t e c t i o n o f p a t h o g e n i c d n a o r rna. 231,234,235,273,580−587 for instance, many metal-containing nanoparticle probes have been functionalized to detect pathogen-specific nucleic acid sequences in a variety of formats. the size-dependence of spr for noble-metal nanoparticles has been leveraged in an aggregation assay for multiplexed detection of middle east respiratory syndrome coronavirus, m. tuberculosis, and hpv in a μpad format. 231 the high conductivity of agnps has been exploited for electrochemical detection of m. tuberculosis and hiv. 234, 235 luminescent particles (i.e., quantum dots, europium chelatedoped particles, and upconverting phosphors) have been functionalized for the detection of a myriad of diseases, including hiv, 273,580−583 hbv, 273 hcv, 273 hpv, 584 p. falciparum malaria, 585 bacillus cereus, 581, 582, 586, 587 and streptococcus pyogenes. 581 moreover, these nanoparticle-based methods occur in a variety of formats, including lateral flow assays. because specific nucleic acid sequences are typically present at much lower concentrations than protein biomarkers in patient samples, a target amplification step is often required before the detection method can be applied. the most common target amplification technique is pcr, a timeconsuming enzyme-based method that requires technical expertise, expensive equipment, and significant laboratory infrastructure. these requirements are the primary barriers to sensitive detection of pathogen genetic material in the field. fortunately, alternative target amplification techniques with potential point-of-care applications have emerged. for example, the biobarcode signal amplification strategy discussed in section 4.4.4 has been adapted for amplification of nucleic acid targets. 464 however, in general, isothermal enzymatic amplification techniques have demonstrated the most sensitivity and promise for point-of-care diagnostic applications. isothermal amplification techniques do not require any thermal cycling, are simpler to operate, and require fewer resources when compared to traditional pcr. this class of enzymatic amplification techniques includes loop-mediated isothermal amplification (lamp), helicase-dependent amplification (hda), rolling circle amplification (rca), multiple displacement amplification (mda), recombinase polymerase amplification (rpa), and nucleic acid sequence-based amplification (nasba). 588 among these strategies, lamp is one of the most widely validated, having been employed for amplification of nucleic acid targets for a variety of diseases and infections. these include targets for malaria, 589−592 hiv, 593 hpv, 594 bordetella pertussis, 595 c. trachomatis, 595,596 neisserie gonorrheae, 595 h1n1 influenza, 595,597 e. coli, 598 and several filarial parasites. 599 in addition to malaria diagnosis, lamp has also been used to detect artemisinin resistance in p. falciparum malaria. 600 this technique consists of iterative elongation and recycling of stem-loop dna structures, usually requiring a constant temperature between 60 and 65°c. 588 the relatively simple workflow of lamp (when compared to other enzymatic amplification techniques) and its potential for colorimetric visual readout have led to the development of several low-resource devices for nucleic acid detection. one example is the noninstrumented nucleic acid amplification device (nina), which relies on an exothermal chemical reaction (mgfe powder, saline, and oxygen) coupled to a phase change material for prolonged and precise heating inside a thermos cup. 589, 601 despite its streamlined workflow, the nina device does not account for sample preparation. the preamplification nucleic acid extraction step still requires laboratory equipment such as a heated water bath and a centrifuge. 589 although the amplification step itself has been modified for use at the point of care, the complete protocol relegates it to the laboratory environment. in an effort to mitigate these limitations, the klapperich group 594 has developed an inventive method in which nucleic acid extraction, amplification, and detection are fully integrated into a single paper fluidic device. to accomplish this, dna from clinical samples was precipitated from solution and applied to a paper membrane, which was washed with ethanol to remove impurities. lamp reaction reagents, which included fitc-conjugated forward and biotinylated reverse loop primers, were then applied to the sample port, which was sealed for a 30 min heating step on a hot plate. the amplified solution was then wicked onto a lateral flow assay equipped with anti-fitc antibodies on the test line and streptavidincoated aunps for detection ( figure 39 ). in the presence of the target, both primers hybridized to the amplified genetic material, forming a "sandwich" on the lateral flow assay and resulting in a visible test line signal. 594 this all-in-one paper-based nucleic acid test is a perfect example of how target extraction and amplification can be innovatively combined with metal-based detection elements. this integration will allow for complex nucleic acid detection protocols to be performed at the point of care that previously were only possible in well-equipped laboratories. moving forward, several considerations must be addressed, including integration of laboratory-free heating elements, reducing the number of user steps, and large-scale manufacturing feasibility. nonetheless, combination of the paper-based devices developed by the klapperich group with simple, integrated heating elements similar to the nina device could allow for sensitive and information-rich nucleic acid detection in low resource settings. this would enable case management and elimination strategies to be precisely carried out in nearly any healthcare setting. the application of inorganic chemistry and nanomaterials to point-of-care infectious disease diagnosis has enhanced the sensitivity and specificity of diagnostic tests, leading to improved outcomes and reduced transmission. the sample preparation and detection methods presented herein are promising examples of how metal-based strategies can expand the use-case scenarios of diagnostic tests, resulting in access to care that would otherwise be unattainable. however, we have also seen that these same metal-based strategies that improve review diagnostic sensitivity and specificity can lead to more complex tests, potentially requiring more user steps, expertise, and time to complete. these trade-offs of complexity, cost, and speed must be weighed against the potential impact that improved sensitivity and specificity could have in a given use-case scenario. for example, in the case of morbidity control, where the goal is to reduce the prevalence of high-intensity infections, the demand for simple, affordable, and rapid tests likely outweighs the need for highly sensitive tests. once in the elimination phase, however, where the goal is to completely interrupt local transmission, high sensitivity tests are imperative for detecting and treating low-density infections. consequently, the need for more sensitive diagnostics likely outweighs small increases in test complexity or cost for elimination campaigns. in our evaluation of various inorganic complexes and nanomaterials, advantages and caveats became apparent for different classes of materials. the properties of these materials directly affect how they can be applied in diagnostics. noble metal nanoparticles have frequently been incorporated into paper-based assays, such as lateral flow assays, with visual signal readout because of their stability and superior molar absorptivities. 219, 221, 229, 231 despite the strong optical signal that noble metal nanoparticles afford, these colorimetric assays are often still limited by poor sensitivity. this potentially limits their impact in a use-case scenario such as an elimination campaign. if better sensitivity is needed, there are essentially three metal-based strategies that we have discussed in this review for further improvement: (1) sample preparation for biomarker enrichment, (2) luminescent detection probes, and (3) signal amplification techniques. the imac-functionalized materials used for sample preparation collectively represent one approach for improving assay sensitivity by enriching the biomarker concentration. while some of the sample preparation methods demonstrated increased diagnostic sensitivity, these methods did add to the number of user steps. 85, 99, [113] [114] [115] 117 metal-based sample preparation is perhaps the simplest of the three metal-based approaches to increasing sensitivity; however, for it to be truly impactful, it will need to be incorporated into diagnostics that are developed with the end user in mind such that the overall workflow is considered and the assay steps are minimized. alternatively, noble metal nanoparticles can be replaced with luminescent detection probes to afford greater sensitivity. quantum dots, lanthanide chelate-doped nanoparticles, and up-converting phosphor nanoparticles all potentially offer improvements in sensitivity over colorimetric labels, albeit through different mechanisms. 257, 289, 305, 309 while luminescent inorganic complexes were incorporated into proof-of-principle assays for the detection of infectious disease biomarkers, they have yet to be utilized in a field-deployable format and lack the sensitivity of the aforementioned nanomaterials. 138, 139 quantum dots and up-converting phosphor nanoparticles also have the capability for multiplexing that could be beneficial for case management. 252, 253, 310 although the various luminescent nanoparticles offer improvements in sensitivity, they also require more instrumentation for signal readout. the increased cost and complexity of signal readout for luminescent nanoparticles must therefore be considered when developing diagnostic strategies to combat infectious diseases. lastly, metal-based signal amplification techniques could be incorporated to improve sensitivity. almost all of the signal amplification techniques discussed in this review (metalloenzyme-based amplification, nanoparticle dissolution, nanoparticle enzyme mimics, reductive nanoparticle enlargement, and biobarcodes) offer colorimetric readouts with improved sensitivity. however, these improvements come at the cost of an increased number of assay steps and thus longer total assay times. while some efforts, such as the p24 lfas developed by loynachan et al., 435 demonstrated progress toward more fieldfriendly signal amplification methods, further improvements in the ease of use and time to result must be made to translate these methods to primary healthcare facilities. for these metal-based strategies that improve sensitivity, it is clear that simplifying the user interface is a significant challenge that remains. aside from the user interface, there are several other challenges to translating novel diagnostic strategies that must be considered before implementation. these include: (1) the need for clinical validation, (2) the potential for large-scale manufacturability, (3) the availability of distribution channels, figure 39 . workflow for detecting nucleic acids on a paper fluidic device. different dyes were used to illustrate the various components of the assay. (a) the dna-containing sample (blue dye) is added to the sample port, where it wicks down the test by capillary action (b). the test is then washed twice with ethanol (yellow), which also flows down the test (c−f). (g) the absorbent pad is then removed and discarded. (h) the lamp reaction mix is then added to the sample port, and the test is folded to protect the sample/lamp mixture from evaporation. (i) once the test has been heated on a hot plate for 30 min, the hydrophobic barrier is removed so that the sample port is exposed on top and bottom, allowing it to come into contact with the lfd strip for dna elution. (j) the sample, once eluted, can then wick down the length of the test strip to generate a signal. adapted with permission from ref 594. copyright 2016 royal society of chemistry. review and (4) integration into current health systems. most of the metal-based strategies for improved infectious disease diagnostics presented in this review have demonstrated some level of feasibility in a small set of patient or mock clinical samples. however, new technology must be clinically validated on a large set of patient samples from endemic regions before implementation. such studies provide insight into the diagnostic performance and tolerance of day-to-day and interpatient variation in the appropriate clinical context. this rigor can draw attention to challenges and weaknesses that would otherwise go unnoticed in a research laboratory setting. production scalability of novel diagnostic technology is another important consideration for successful translation. even in the research phase of diagnostic development, material choice, device design, and fabrication should be based, in part, on manufacturing scalability in order to streamline implementation. many of the metal-based strategies presented in this review, particularly the diverse inorganic nanoparticle detection probes used in lateral flow assays, are advantageous in this context because they are easily integrated into existing lfa manufacturing infrastructure. beyond the laboratory, it is important to consider distribution channels and how novel tools will be integrated into existing health systems. region-specific health delivery systems and their respective regulatory approvals are ultimately what define a novel diagnostic device's implementation. for example, in one region, most rapid testing for infectious diseases may be performed at local health clinics run by a national ministry of health, whereas in another area, privately run dispensaries may be the most common source of primary health care. understanding the structural differences between such health delivery systems as well as the respective availability of resources could impact device design in the research phase. although there are many considerations and barriers to translating novel technologies to the clinical setting, we are optimistic that the continued development of metal-based point-of-care diagnostic strategies will have a profound impact on global health. returning to the example presented in the introduction, if some of the ultrasensitive p24 detection methods presented in this review 396, 435 were adapted for use at primary healthcare facilities, the time to result for infant hiv diagnosis at the point of need would be significantly reduced. in areas like burkina faso, this would enable earlier initiation of very effective antiretroviral therapies and drastically reduce infant mortality stemming from opportunistic infections. finally, the continually increasing connectivity in low-and middle-income countries coupled to sensitive diagnostics will allow for real-time disease surveillance, intervention analysis, and epidemiological studies. "connected" diagnostics have the potential to accelerate communications between field workers, local health outposts, district hospitals, and national health ministries. the ability to rapidly convey diagnostic results could not only enable active case detection strategies but also would provide faster responses to epidemics such as the 2014− 2016 ebola and 2015−2016 zika outbreaks. these faster responses could potentially reduce mortality and improve clinical outcomes. metal-based strategies for sample preparation and signal generation show great promise for use in the development and improvement of sensitive diagnostic devices for low-resource settings. these devices, coupled with connected, fielddeployable instrumentation, will empower rapid response, effective control, and successful elimination of infectious diseases worldwide, ushering in an era of sustainable and improved global health. (2011) and an american association for the advancement of science fellow (2014). for the past decade, he has unraveled the mechanism of heme detoxification within the parasite plasmodium falciparum, the causative agent of malaria. additionally, he has made important contributions in understanding hemozoin's immunomodulatory activity in parasite-host interactions, the mechanism of action for antimalarial compounds, and the development of biomarkers for low-resource diagnostics. more recent efforts have focused on the challenges of innovation in low-resource diagnostics. he has focused primarily on malaria biomarkers and new approaches to formatting and improving performance of diagnostics for the detection of asymptomatic patients, drug-resistant infections, and drug-sensitive patients. his lab works in the field in macha, zambia; cape town, south africa; and san marcos, peru. this work is supported by the national institutes of health (r01 ai135937; r21 tw010635-01), nih/fogarty international center (d43 tw009348), the bill and melinda gates foundation (opp1123092), and vanderbilt university through the laboratories for innovations in global health technologies. c.f.m. and k.a.r. acknowledge support from the national science foundation graduate research fellowship program (dge-1145197). we gratefully acknowledge shellie richards for her tireless proofreading efforts that sharpened the manuscript. aequanimitas, with other addresses to medical students trends in infectious disease mortality in the united states during the 20th century convergence of non-communicable and infectious diseases in lowand middle-income countries uneven progress in preventing and treating hiv infections worldwide disease eradication, elimination and control: the need for accurate and consistent usage the principles of disease elimination and eradication lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. a literature survey no. e111240. 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electrodes and nanoparticle-based dna amplification an improved gold nanoparticle probe-based assay for hcv core antigen ultrasensitive detection nanoparticle-based biobarcode amplification assay (bca) for sensitive and early detection of human immunodeficiency type 1 acquired immune defic fluorescent bio-barcode dna assay for the detection of salmonella enterica serovar enteritidis sandwich np-based biobarcode assay for quantification c-reactive protein in plasma samples colorimetric bio-barcode amplification assay for cytokines detection of proteins using a colorimetric bio-barcode assay multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes barcode dna-mediated signal amplifying strategy for ultrasensitive biomolecular detection on matrix-assisted laser desorption ionization time of flight (maldi-tof) mass spectrometry professional/infectious-diseases/laboratory-diagnosis-of-infectiousdisease/introduction-to-laboratory-diagnosis-of-infectious-disease 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influenza a (h1n1) from clinical specimens paper machine" for molecular diagnostics colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (nina-lamp) a novel snp-lamp assay for detection of artemisinin-resistant plasmodium falciparum malaria noninstrumented nucleic acid amplification assay. in microfluidics, biomems, and medical microsystems vi key: cord-256568-mbkrg98v authors: jantzen, r.; noisel, n.; camilleri-broet, s.; labbe, c.; de malliard, t.; payette, y.; broet, p. title: epidemiological and socio-economic characteristics of the covid-19 spring outbreak in quebec, canada: a population-based study date: 2020-09-01 journal: nan doi: 10.1101/2020.08.26.20182675 sha: doc_id: 256568 cord_uid: mbkrg98v background: by mid-july 2020, more than 108,000 covid-19 cases had been diagnosed in canada with more than half in the province of quebec. to be prepared for a potential second wave of covid-19 in the fall, it seems of utmost importance to analyze the epidemiological and socio-economic characteristics of the spring outbreak in the population. method: we conducted an online survey of the participants of the cartagene population-based cohort, composed of middle-aged and older adults. we collected information on socio-demographic, lifestyle, health condition, covid-related symptoms and covid-19 testing. we studied the association between these factors and two outcomes: the status of having been tested for sars-cov-2 and the status of having received a positive test when having been tested. these associations were evaluated with univariate and multivariate analyzes using a hybrid tree-based regression model. results: among the 8,129 respondents from the cartagene cohort, 649 were tested for covid-19 and 41 were positive. medical workers and individuals having a contact with a covid-19 patient had the highest probabilities of being tested (32% and 42.4%, respectively) and of being positive (17.2% and 13.0%, respectively) among those tested. 7.6% of the participants declared that they have experienced at least one of the four covid-related symptoms chosen by the public health authorities (fever, cough, dyspnea, anosmia) but were not tested. results from the tree-based model analyzes adjusted on exposure factors show that the combination of dyspnea, dry cough and fever was highly associated with being tested whereas anosmia, fever, and headache were the most discriminant factors for having a positive test among those tested. during the spring outbreak, more than one third of the participants have experienced a decrease in access to health services. there were sex and age differences in the socio-economic and emotional impacts of the pandemic. conclusion: we have shown some discrepancies between the symptoms associated with being tested and being positive. in particular, the anosmia is a major discriminant symptom for positivity whereas ear-nose-throat symptoms seem not to be covid-related. the results also emphasize the need of increasing the accessibility of testing for the general population. epidemiological and socio-economic characteristics of the covid-19 spring outbreak in quebec, canada: a population-based study background by mid-july 2020, more than 108,000 covid-19 cases had been diagnosed in canada with more than half in the province of quebec [1] . the province of quebec confirmed its first case of covid-19 on february 27. with only seventeen confirmed cases, the state of emergency was declared by the quebec government on march the 13th with a shutdown of daycare facilities, primary and secondary schools, cegeps and universities followed by a more extensive shutdown of restaurants and bars, indoor sport facilities and non-essential economic activities. in early march, most cases could be traced to infected travelers returning to canada or to close contact with those travelers. by march 23, according to the public health agency of canada, nearly half of canadian covid-19 cases have been acquired through community spread [2] . in mid-july, more than 56,000 covid-19 postive cases have been confirmed in the province of quebec with nearly half in the montreal metropolitan area and around one-third of the confirmed cases have been diagnosed among middle-aged and older adults. as in many countries over the world, the covid-19 testing strategy in canada has evolved over the first wave, taking into account each province's specific situation. in order to be prepared for a potential second wave of covid-19 in the fall, it seems of utmost importance to analyze the epidemiological and socio-economic characteristics of the spring outbreak in the population, together with the results of the public health policies that have been implemented. such information can provide useful knowledge for planning public health strategies for the next coming months. despite the impressive number of publications about covid-19 infection, most of them are hospital-based series, [3, 4, 5, 6] to name a few, while population-based studies are scarce [7, 8, 9] . however, population-based cohorts that have been established before the pandemic may yield unbiased estimates of the characteristics and consequences of the pandemic in the general population. it may also provide useful information about the effectiveness of public health interventions such as testing strategies. in this context, the existing population-based cohort cartagene (cag), composed of middle-aged and older adults, which was established before the covid-19 outbreak, offers a unique opportunity to analyze the characteristics and consequences of the outbreak among a population that seems to be at greatest risk. a large survey (hereinafter referred to as cag covid-19) was launched in early june. an online questionnaire was sent to the participants of the cag cohort for collecting information about covid-19-related symptoms, diagnosis, comorbidities and social impacts of the spring outbreak. the aim of this survey was to analyze the demographic, clinical and socio-economic characteristics associated with the covid-19 pandemic in quebec in spring 2020. we also investigated the risk exposure, pre-existing medical conditions and clinical features that led to being tested and to have a positive result among those who have been tested. cartagene is a population-based cohort composed of more than 43,000 quebec residents aged between 40 and 69 years at recruitment [10] . original survey design . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint was defined by gender, age groups and forward sortation area (fsa -defined by the first 3-digit postal codes). participants have been recruited during two phases (phase a: 2009-2010 (n = 23,000) and phase b: 2013-2014 (n = 20,000)) in metropolitan areas where nearly 70% of quebecers live and prospective follow-ups are conducted on a regular basis. with a rich collection of data including phenotyping and biological data, cag is the largest ongoing prospective population cohort and biobank in quebec, canada. the collected data includes a self-administered socio-demographic and lifestyle questionnaire (phase a and b), an interviewer-administered health questionnaire (phase a and b); non-invasive physical measurements (phase a) and biospecimen collection (blood (phase a and b) and urine (phase a)). more than 30,000 individuals have provided blood samples. the lifestyle questionnaires cover topics such as socio-demographic factors, lifestyle, mental state, psychosocial environment, personal and family history of disease, health care utilization, medication use, reproductive health and history and declared health conditions. a cohort-wide follow-up has been carried out in 2018 aiming at updating lifestyle and health information 5 to 10 years after the baseline data. cag is part of the canadian partnership for tomorrow's health (canpath, former called cptp) which is the canada's largest population health research platform [11] . cag covid-19 online questionnaire for this study, we have developed a specific online questionnaire [12] for collecting updated information on basic demographic variables (age, sex, household composition and postal code), covid-19 testing, test dates and results, whether participants suspect they had an undiagnosed covid-19 infection based on symptoms (cough, shortness of breath, fever, etc.). we also collected information on exposure status: health care or essential workers, contact with someone who has been diagnosed with covid-19, international travel. those with confirmed covid-19 infection were asked if they were hospitalized along with details related to their care, treatment and outcome of the hospitalization. in addition, participants were asked to report existing chronic health conditions and medication use. finally, they were asked about the psychosocial and socio-economic impacts of the pandemic on their lives. this online questionnaire was unfolded in close collaboration with the canpath consortium for allowing future inter-provincial comparisons as well as other national and international research initiatives. a weblink to the consent and questionnaire was sent by email to all the cartagene participants with a valid email address, that is 33,019. initial invitation was followed by up to 3 emails reminders. the digital invitations were sent early in june 2020 and were valid for 4 weeks. survey closed early july 2020. a positive exposure status was defined as an individual being either medical or essential workers, having been in contact with a covid-19 positive patient or coming back from international travel. a medical worker was defined as either a physician, nurse, hospital/aged-care facility employee, first responder, pharmacist with patient exposure. an essential worker was defined as either a grocery store attendant, public transit, police, fireman. a contact with a covid-19 positive . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint individual was defined as being in the same room as a person who was told by a health professional that he or she has covid-19. international travel was considered as a travel outside canada returning after the 1st of february. pre-existing conditions were defined as medical condition currently treated among the following list: cardiovascular diseases (hbp, coronary artery disease,...), autoimmune diseases, diabetes, infectious diseases, gastro-intestinal and liver diseases, cancers, renal diseases. the following list of symptoms were considered in the questionnaire: dry cough, wet cough, shortness of breath (dyspnea), fever (≥ 38 • c), shivering, fatigue, runny nose, sinus pain (sinusitis), ear pain (otitis), sore throat, hoarseness, loss of taste (ageusia), loss of smell (anosmia), diarrhea, nausea, vomiting, loss of appetite, headache, general muscle aches and pains. the possible responses were: none, mild, moderate or severe. for the association studies, the following symptoms were considered as present only when being severe: fatigue, loss of appetite, sinus pain, ear pain, sore throat, hoarseness, headache, muscle pain, diarrhea. hereafter, we will refer to the symptom-based case definition chosen by the quebec public health authorities with at least one of the four major symptoms: fever (≥ 38 • c), a new cough or a cough that gets worse, difficulty of breathing, anosmia with or without ageusia [13] . two outcomes were studied. the first was the status of having been tested for sars-cov-2 (rt-pcr). the second was the status of having received a positive test when having been tested. confirmed covid-19 infection was defined as at least one positive result test. if multiple tests were performed, we considered the date of the first positive one. the participants' baseline characteristics have been reported and tested : demographic (age, sex, geographical location, level of education, financial resources, household income, dwelling type), health history (high blood pressure, cardio-vascular disease, pulmonary disease (chronic obstructive pulmonary disease (copd), asthma, ...), cancer, diabetes, autoimmune diseases, mental health, body mass index (bmi)), lifestyle behaviors (smoking status, alcohol intake,...). mean (or median) with standard deviations (or interquartile ranges) were reported for continuous outcomes. frequencies with 95% confidence intervals were reported for qualitative variables. for comparing means between groups, we used an anova test. for categorical variables, we used chi-squared test or exact test if needed. for the univariate association analyzes (between factors and studied outcomes), we used a chi-square test (qualitative) or a logistic regression model (quantitative). for each hypothesis test, we reported the p-values. moreover, in order to address the multiple testing problem, we also indicated those that are still significant for a fdr (the expected proportion of false discoveries among all discoveries) controlled at a level of 1% using the benjamini-hochberg procedure [14] . . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint for the multivariate analyzes of exposure and risk factors, we used a multiple logistic regression model. for the multivariate analyzes of symptoms associated with the outcomes, we had to cope with complex interplay between clinical symptoms. thus, we considered a generalized partially linear tree-based regression (gpltr) model. gpltr models represent a class of semi-parametric regression models that integrate the advantages of generalized linear regression and tree-structure models [15] . the linear part is used to model the main effects of confounding variables (e.g. exposure) while the nonparametric tree part is used to address potential collinearity and interactions between explanatory variables (e.g. clinical symptoms). this tree-based model provides a classification of individuals in homogeneous groups in terms of risk for the event of interest and identifies relevant combination of explanatory variables. the optimal gpltr tree is selected using a penalized maximum likelihood method with the bayesian information criterion (bic) [15] . regression trees are prone to instability, especially when dealing with a low number of outcomes, making variable selection somewhat precarious. thus, for the analyzis of the positive status outcome, we constructed multiple trees using a bagging approach, which provides a way to assess the relevance of each variable across the set of trees using variables' importance measures. these later results provide us some arguments regarding the reliability of the selected optimal gpltr tree. we reported the depth deviance importance score (ddis) of each symptom that is computed for each gpltr model as the sum of the values of the deviance at each split based on this variable, weighted by the location of the split in the tree. these scores are summed across the set of trees, and normalized to take values between 0 and 1, with the sum of all scores equal to 1. a set of 300 gpltr models was done and we reported the symptoms as ranked by the ddis. we also evaluated in our dataset the discriminative power of the menni et al. logistic-based predictive model [9] which is based on age, sex, loss of smell and taste, fatigue, persistent cough and loss of appetite. we reported the area under the roc curve (auc) together with the sensitivity and specificity applying the 50% probability threshold (as proposed by menni et al. and corresponding to the bayes rule). statistical analyzes were performed using r software [16] . regression tree analyzes were performed with the 'gpltr' r package [15] . estimation of the probability of being positive when experiencing a symptom since the tested participants are selected based on the symptoms being hypothesized to be associated with a positive test, the tested participants constitute a non-random set (ascertainment bias). thus, it is not possible to estimate directly the probability of being infected given that the person has experienced a particular symptom since non-selected (untested) individuals are unobserved. we can only estimate the probability of being positive given that the person has experienced a particular symptom and has been tested. instead, we propose to report the minimum, mean and maximum values that these probabilities can take using the law of total probability. more precisely, we . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint know that the probability of being positive given that the person has experienced a particular symptom (p (+|s)) can be expressed as: where p (+|s ∩ t ) (resp. p (+|s ∩t )) are the probabilities of being positive when the symptom is present and he/she has been tested (resp. not tested) and p (t |s) is the probability of being tested when having the symptom. from our data, p (+|s ∩t ) and p (t |s) could be directly estimated while p (+|s ∩ t ) could not. however, we know that this probability ranges from zero (complete dependence) to p (+|s ∩ t ) (independence between positivity and test). thus, we calculated the minimum, mean and maximum values that p (+|s) can attain for each symptom. survey respondents among the 43,038 participants of the cartagene cohort, we sent the questionnaire to 33,019 individuals. as compared to the non-respondents (n=24,882), the respondents (n=8,137) were slightly older (median age of 62.9 vs. 60.6, p < 0.001), composed of more women (58.8% vs. 53.9%, p < 0.001), had a higher education level (58.3% vs. 44.8%, p < 0.001) and were more often living in montreal (68% vs. 65.9%, p < 0.001). the same results were observed when comparing the individuals who answered the questionnaire to the whole cartagene cohort. of the 8,137 respondents, 8,129 had responded to the questions regarding covid-19 testing ("have you been tested for covid-19?" and "what was the result of your 1st covid-19 test?"). hereafter in this article, we analyzed this set of 8,129 respondents. both the proportion of tested participants for covid-19 and positive individuals among those being tested are consistent with those reported in quebec at the closure of our survey (tested: cag 8.0% among the 4,855 individuals who experienced at least one covid-related symptom (as defined above), 1,022 declared that their first action was to call or consult their family doctor (39.3%), to call the 811 or a dedicated coronavirus hotline (24.6%), to go to the pharmacy (24.6%), to the hospital emergency room (6.4%) or to a covid-19 screening clinic (5.1%). among the 1,522 individuals who tried to contact a healthcare professional, 31.3% (476) were not able to reach someone on the phone or to see someone because of too much waiting. among the 288 participants who experienced a covid-19 contact, the majority was at work (123), in a health care facility (69) or at home (54). in the later case, 13 covid-19 contacts were the spouse or partner. 56.2% (162/288) were not tested. among the respondents, 649 (8.0%) have been tested for covid-19 and 41 (6.3%) were declared positive. among these tested individuals, 548 had one test, 55 had . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. when looking to the number of tests performed per day, we can see a low number of tested participants during april with a maximum at the end of may. this trend reflects the shortage faced by the province in early april followed by a rebound by end of may. in our series, the highest number of tests per day was may 22, consistent with the entire province [17] (supplementary figure s1 ). among the 649 tested individuals, 134 were medical workers, 123 were in contact with a covid-19 positive individual and 69 had both risk exposures. 47 individuals were essential workers including 28 without other risk exposure. 161 individuals declared an international travel, including 117 having no other risk exposure. among the 309 tested individuals with no declared risk exposure, 74 declared no covidrelated symptom with 41 patients having a chronic pre-existing medical condition (mainly cardio-vascular or respiratory disease). the proportion of positive cases varied widely across risk exposure status: 17.2% (23/134) for the medical workers, 4.2% (2/47) for essential workers, 13.0% (7/54) for people in contact with a covid-19 positive individual without professional exposure, 4.3% (5/117) for people having declared an international travel without other exposure risk and 1.9% (4/214) for people having one of the major symptoms but without known exposure risk. none of the 74 patients without risk exposure or covid-related symptoms were positive. there were 619 individuals who declared that they have experienced at least one of the four covid-related symptoms chosen by the quebec public health authorities but were not tested. among them 3.4% were medical worker, 3.1% had a contact with a covid-19 patient. among those having none of these later exposure factors, 20.3% declared an anosmia. moreover, the percentage decreased with the number of covid-related symptoms from 38.1% (one symptom) to 8.4% (all the four symptoms). among the 41 individuals with at least one positive test, 10 (24.4%) participants had a first negative test followed by a positive test. twenty-three (56%) were working as a medical worker and 2 (4.9%) as an essential worker. seven (17.0%) were in contact with a covid-19 positive individual without professional exposure and five (12.2%) with no other risk exposure traveled outside of canada. three individuals have been hospitalized for covid-19 infection but none of them was hospitalized in icu. five were treated with an experimental therapy prescribed by a clinician for covid-19. the colchicine was the only one prescribed treatment. none of them had chloroquine/hydroxychloroquine, remdesivir, lopinavir-ritonavir or tocilizumab. among the positive cases, 35 (89.7%) felt completely or mostly back to normal. eight (23%) were sick for seven days or less, 12 (34%) between eight and fourteen days, 10 (29%) between fifteen and thirty days, and 5 (14%) more than thirty days. the two individuals who felt not really back to normal were at 30 and 43 days of symptoms. they had 7 and 15 different symptoms, respectively. among the positive individuals who responded to mental and emotional questions, 35/39 (89.7%) had someone to help meeting their immediate needs. 26/38 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint (68.4%) received help, aid or support (including from friends, family, community or government). socio-economic, lifestyle and health early impacts of the covid-19 spring 2020 outbreak general and socio-economic impacts while the household income had substantially decreased for 447 (5.8%) of all the participants, there was a moderate or major impact on the ability to meet financial obligations or essential needs (e.g. rent or mortgage payments, utilities and groceries) for 609 (8%). among the 1,302 individuals who regularly took public transit before march 2020, 919 (70.6%) did not continue to take public transit after march, while 297 (22.8%) took public transit less frequently. individuals changed their transport habits because of the lockdown (923), being afraid of covid-19 exposure (522), quarantine/in self-isolation (206) or covid-19 symptoms (11) . regarding the relationship with the intimate partner during the lockdown, 4,443 (76.9%) of the participants experienced no change, 954 (16.5%) declared that they became closer than before the pandemic, while 380 (6.6%) felt more distant or strained than before the pandemic. same trends were observed with friends and colleagues who became closer in 1,266 (16.4%) and 921 (15.9%), respectively, while more distant in 742 (9.6%) and 421 (7.3%), respectively. the family relationship became more distant or strained than before the pandemic for 1,552 (20.2%) of the participants while 641 (8.3%) became closer. during the spring outbreak, 2,982 (37.7%) of the participants have experienced a change in access to health services. among them, 225 (4.5%) had a surgery canceled or deferred, 411 (8.3%) a medical procedure canceled or deferred, 289 (5.8%) a treatment canceled or deferred and 711 (14.4%) experienced a delay for seeing a healthcare professional about an preexisting problem and 344 (6.9%) about a new problem. however, 2,157 (43.7%) have used virtual appointments with their health care provider. among the 100 individuals who have initiated new use of mental health services, 48 did for anxiety, 35 for depression and 39 for stress. overall, the mental/emotional health at the time of completing the questionnaire was good/excellent for 5,370 (69.2%) participants. over the last 2 weeks, some individuals have been bothered more than half of the days or nearly everyday by the following problems: trouble falling/staying asleep or sleeping too much (8.9%), feeling tired or having little energy (8.8%), feeling nervous (8.2%), trouble relaxing (6.4%), excessive worrying (5.7%) or constant worrying (4.6%). since march, the alcohol consumption increased for 1499 (20.8%) of the participants. among the 544 current smokers (6.9% of the participants), 175 (32.6%) increased their consumption, 60 (11.1%) decreased their consumption whereas 302 (56.3%) did . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint not change. among the 605 individuals who used cannabis in the past 12 months (7.6% of the participants), 84 (14.2%) increased their consumption, 125 (21.0%) decreased their consumption whereas 385 (64.8%) did not change. the physical activity decreased for 1,757 (22.2%) of the participants. sleep duration and quality did not change for 7,497 (94.8%) and 7,433 (94.0%), respectively. the food intake increased for 131 (1.7%), while the food quality decreased for 95 (1.2%) of the participants. because of the pandemic, 1,355 of the participants (18.6%) looked for support from family, 937 (13.4%) from general media (tv, internet, social media), 890 (12.6%) from the government, 870 (12.4%) from friends, 706 (10.3%) from provincial or federal health authorities, 485 (7.09%) from professional and 278 (4.14%) from colleagues. the covid-19 outbreak had different impact between men and women. the alcohol consumption increased more among women (22.4% versus 18.5%, p < 0.001). physical activity and quality of sleep decreased more for the women (23.4% versus 20.5%, p < 0.001; 5.6% versus 3.3%, p < 0.001, respectively). the quantity of food intake increased (2.1% versus 1%, p < 0.001) more for the women. since march, women accessed more mental health services for anxiety (2.6% versus 1.1%, p < 0.001), stress (2% versus 0.7%, p < 0.001) and depression (1.6% versus 0.8%, p = 0.0033). nevertheless, the current mental/emotional health was very good or excellent for 74.5% of men but only 65.4% of women (p < 0.001). women have looked more for support from family (22.5% versus 12.9%, p < 0.001), general media (16.2% versus 9.6%, p < 0.001), friends (15.4% versus 8.1%, p < 0.001), provincial/federal health authorities (11.7% versus 8.3%, p < 0.001), professional (8.7% versus 4.9%, p < 0.001), colleagues (4.9% versus 3.1%, p < 0.001). looking for support from the government (financial support, financial relief, resources) was not different between women and men. over the last 2 weeks, women were more bothered by the following problems, more than half of the days or nearly every day: trouble falling/staying asleep or sleeping too much (10.7% versus 6.2%, p < 0.001), feeling tired or having little energy (10.6% versus 6.2%, p < 0.001), feeling nervous (10.9% versus 4.4%, p < 0.001), trouble relaxing (8.2% versus 3.8%, p < 0.001), excessive worrying (7.3% versus 3.2%, p < 0.001) or constant worrying (6.1% versus 2.3%, p < 0.001). when looking to the covid-19 outbreak impact among individuals under and over 65 years, the income substantially decreased for less elderly (7.8% versus 2.6%, p < 0.001). the alcohol consumption increased more for the younger (24% versus 15.9%, p < 0.001). the quality of sleep and food decreased more for the younger (5.7% versus 3.1%, p < 0.001; 1.6% versus 0.6%, p < 0.001, respectively). the quantity of intake increased more for the younger (2% versus 1.1%, p = 0.0021). the younger accessed more mental health services for anxiety (2.5% versus 1.2%, p < 0.001), stress (1.9% versus 0.8%, p < 0.001) and depression (1.7% versus 0.6%, p < 0.001). nevertheless, the current mental/emotional health was very good or excellent for 74.3% of elderly but only 65.8% of younger (p < 0.001). . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint since march, elderly have looked more for support from family (26.2% versus 13.6%, p < 0.001), friends (17.4% versus 9.1%, p < 0.001), general media (15.8% versus 11.9%, p < 0.001), provincial/federal health authorities (11.6% versus 9.4%, p = 0.0037), professional (8.7% versus 6.1%, p < 0.001) or community/volunteer organization (4.1% versus 1.8%, p < 0.001). looking for support from colleagues was less frequent among the elderly (2.2% versus 5.3%, p < 0.001). over the last 2 weeks, the younger were bothered more by the following problems, more than half of the days or nearly every day: trouble falling/staying asleep or sleeping too much (10.4% versus 6.5%, p < 0.001), feeling tired or having little energy (10.4% versus 6.3%, p < 0.001), feeling nervous (9.8% versus 5.7%, p < 0.001), trouble relaxing (7.9% versus 4%, p < 0.001), excessive worrying (6.9% versus 3.8%, p < 0.001) or constant worrying (5.7% versus 2.8%, p < 0.001). association between being tested and demographic, risk exposure, pre-existing medical conditions and covid-related symptoms we compared the demographic, exposure and clinical characteristics of the participants who have been tested to those who have not. participants below the median age were most frequently tested (p < 0.001). there was no difference between men and women. participants living in the montreal area were more likely to be tested than those living outside of montreal (9.3% versus 5.2%, p < 0.001). participants living in a house or duplex were less likely to be tested than those living in apartment or condominium (7.3% versus 9.4%, p = 0.002). this relationship is still significant when adjusted for living in montreal or outside. participants coming back from international travel were most frequently tested (9.8% versus 7.4%, p = 0.001). medical workers and people having contact with a covid-19 positive individual were most frequently tested (32.0% versus 6.5%, p < 0.001 and 42.4% versus 6.1%, p < 0.001, respectively). participants having at least one pre-existing medical condition (as defined above) were more frequently tested (p = 0.002). in particular, individuals having a preexisting condition such as asthma (p < 0.001), copd (p < 0.001), chronic bronchitis (p < 0.001), nonalcoholic fatty liver disease (nafld) (p < 0.001) were more frequently tested (supplementary table 1) . a multiple logistic regression analyzis showed that place of residence, dweling, risk exposure (medical worker, contact with a covid-19 positive patient, international travel), having at least one pre-existing condition were independent factors associated with the outcome ( table 1) . all the covid-related symptoms were more frequent among tested participants (supplementary table 1) . taking into account socio-demographic, medical and exposure factors (place of residence, dweling, medical worker, contact with a covid-19 positive patient, international travel, pre-existing condition) as confounding factors and covid-related symptoms as explanatory variables, we performed a gpltr analyzis for identifying the combinations of symptoms leading to the most homogeneous sub-groups with respect to being tested. the procedure with the bic criterion led to a final tree having five leaves built with three selected covid-related symptoms: dyspnea, dry cough and fever ( figure 1a) . . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint we identified: (i) a leaf with a very low rate for being tested (4.8%, 251/5206) with none of the selected covid-related symptoms; (ii) a leaf with the highest rate 19.2% (150/780) of tested individuals having both dyspnea and dry cough; (iii) three leaves with at least one of the symptoms with a rate of being tested: dyspnea (11.0%, 55/500), fever (15.5%, 71/459), dry cough (9.6%, 94/978). table 2 displays odd-ratio estimates and p-values for the confounding variables and terminal leaves corresponding to the optimal gpltr tree for being tested. association between being covid-19 positive and demographic, risk exposure, medical pre-existing conditions and covid-related symptoms participants below the median age were most frequently positive (p < 0.001). there were more women among positives (8.2% versus 3.4%, p = 0.02) but it was no longer significant after multiple testing adjustment. there was no difference between geographical area of residence or international travelers. medical workers (p < 0.001) and people having contact with a covid-19 positive individual (p < 0.001) were most frequent among positives (supplementary table 2 ). participants having at least one medical pre-existing condition (as defined above) were less frequently positive (4.3% versus 8.7% p = 0.04) but it was no longer significant after multiple testing adjustment. other variables such as blood group, treatments, pre-existing conditions, vaccination (influenza and bcg), smoking, bmi, presence in the household of children or pets were not significantly associated with positive status. a multiple logistic regression analyzis showed that being a medical worker or having contact with a covid-19 patient were the only independent factors associated with the outcome (table 3) . patients with loss of taste and smell, fever, dyspnea, headache, pain, fatigue, shivering, loss of appetite, dry cough and nausea symptoms were more frequent among positive individuals. wet cough and other symptoms were not significantly associated (see supplementary table 2) . when estimating the probability of being positive given that the person has experienced a particular symptom (see statistical section), we showed ( figure 2 , left panel) that symptoms with the highest probabilities are ageusia, anosmia, loss of appetite, headache, fatigue. in contrast, ear, nose and throat (ent) symptoms (running nose, sore throat, hoarseness, sinus pain) and wet cough showed the lowest probabilities. taking into account exposure status (contact with covid-19 infected patient, medical worker) as confounding factors and covid-related symptoms as explanatory variables, we performed a gpltr analyzis for identifying the combinations of symptoms leading to the most homogeneous sub-groups with respect to being positive. our procedure with the bic criterion led to a tree with four leaves built with three different covid-related symptoms selected: anosmia, headache and fever. we identified: (i) a leaf with a very low rate of being positive (1.8%, 8/446) with no anosmia and no fever; (ii) a leaf with a low rate (6.8%, 8/118) of being positive with fever but without anosmia; (iii) a leaf with a moderate rate (23.5%, 12/51) of being positive with anosmia but no headache; (iv) a leaf with a very high positive rate with both anosmia and headache (61.9%, 13/21). results from the bagging . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint procedure associated with the tree-based model provided a ranking of the variables based on depth deviance importance scores (ddis). anosmia, fever and headache selected in the optimal gpltr tree showed high importance scores (figure 2 , right panel). table 4 displays odd-ratio estimates and p-values for the confounding variables and terminal leaves corresponding to the optimal gpltr tree for positivity. among the participants tested, the menni et al. predictive model was available for 634 individuals. the model led to an auc of 83.6% (supplementary figure 2) . the sensitivity was of 53.7% and the specificity was of 91.4%. we report the results of an online survey of population-based cohort participants whose main objective was to analyze the characteristics and consequences of the first pandemic wave of covid-19 spring outbreak in quebec, canada. we also report the exposure risk factors and covid-related symptoms associated with the status of having been tested for sars-cov-2 and having been declared positive. as seen from our analyzis, the demographic characteristics of the respondents are broadly representative of the middle-aged and older population of quebec. montrealers, individuals living in an apartment/condominium, having a pre-existing medical condition and having risk exposure (medical worker, contact with a covid-19 patient, international travel) were more frequently tested. as expected, medical workers and individuals with known or suspected contact with a covid-positive individual were the most tested. these later findings are in accordance with public health notices giving priority to health care workers and individuals in contact with people tested positive for sars-cov-2, whether they had symptoms or not. results from the extended tree-based model analyzis, adjusted on exposure factors, show that the combination of dyspnea, dry cough and fever are highly associated with being tested. these results are consistent with the case definition chosen by the public health authorities in early spring. the fact that anosmia is not selected reflects its paucity in the general population (4.2% as compared to 11.7% to 25% for the other three symptoms) and that it has been added later in the official list of the main symptoms. among the covid-related symptoms associated with testing, ear, nose and throat (ent) symptoms (running nose, sore throat, hoarseness, sinus pain) and wet cough were not related to a positive test in univariate analyzis. results from the extended tree-based model analyzis, adjusted on exposure factors, show that a combination of anosmia, fever and headache are the most discriminant factors for a positive test in our series. individuals with both anosmia and headache had the highest chance (almost two third) of being positive while those with anosmia alone had almost one-fourth chance of being positive. individuals without anosmia and fever had less than two percent chance of being positive. these results underline the importance of neurological symptoms such as anosmia and headache, as compared to ent and gastro-intestinal symptoms. results obtained from the bagging procedure confirm the importance of the selected symptoms and suggest that the final tree-based model is sufficiently reliable. they also show the importance of fatigue and loss of appetite that are, with anosmia, the main factors of the menni et al. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint predictive model. in our series, the operating characteristics of the menni et al. predictive model are close to those published with a weak sensitivity and a high specificity. however, it is difficult to disentangle the effect of the testing criteria applied in our population from those of the menni et al. algorithm since the positive cases are only those being tested. it is worth noting that among the 41 positive individuals, five did not meet the four criteria chosen by the public health authorities, two of them reported few symptoms such as fatigue, shivering without fever and/or loss of appetite and three individuals did not experience covid-related symptoms. interestingly, all these five patients were tested for having a contact with people being tested positive for sars-cov-2. this highlights that a non-negligible fraction of infected people could be asymptomatic or pauci-symptomatic, even in this age group. it underlines the importance of contact tracing as an essential component of the toolbox for containing the covid-19 outbreak. interestingly, we observe some discrepancies between being tested and being positive among the studied factors. some of them are linked to both outcomes such as being a medical worker, having a contact with a covid-19 patient and fever. anosmia increases the discriminative power for being positive as compared to being tested, reflecting its value for positivity. in contrast, dyspnea, that was the main factor for being tested, has a lower discriminative power for positivity. some factors associated with testing were not related to a positive test such as international travel, pre-existing conditions and ent symptoms. while the first covid-19 cases were linked to international travelers, the public health measure (e.g. quarantine, border closure) mitigated this risk. the lack of relevance for the ent symptoms should lead to withdraw these symptoms from the list of covidrelated symptoms. for the pre-existing conditions, this discrepancy reflects the testing policies that have focused on group of patients that might be of high risk for severe disease if exposed to the virus. it is worth to note that this over-representation of people having a pre-existing condition led to an inverse relationship with a positive test that is however no longer significant after multiple testing adjustment. such spurious association could be related to an ascertainment bias caused by testing primarily individuals reporting specific conditions and thought to be positive. as reported by the participants, the economic consequences in this early stage of the pandemic seem still moderate but is more important for younger participants. however, the real economic impact of the pandemic may appear in the coming months. the alcohol and food consumption slightly increased during the lockdown, especially among women and younger participants. the current emotional health is considered good or excellent for the majority of the participants, but lower for women. as feared, one third of the participants have experienced a decreased access to health services with surgery or medical procedures canceled or deferred for more than ten percent of them. however, it is interesting to note that virtual consultations were widely used. one of the main strengths of this study is its embedding within the cartagene cohort. it provides a rich body of information collected before the pandemic that is representative of the middle-aged and older population, which seems to be the most commonly affected group. thus, it offers a unique opportunity to appreciate the . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint impact of covid-19 infection and public health measures in the quebec population. in particular, we highlight the selection process for testing at the population level. we show that the most exposed people (medical worker and people having a contact with a covid-19 positive patient) were more widely tested than the rest of the population. in this later group, only the individuals having all the covid-related symptoms criteria had a high probability of being tested. this emphasizes the need of increasing the accessibility of testing for the general population who meet the testing criteria through easy to access point-of-care or drive-thru testing centers. there are however some limitations to this study. firstly, we experienced a low response rate that might be explained by the online survey in a population that was previously used to paper surveys and by the short time allowed to respond. nevertheless, our series is broadly representative of the entire cohort. secondly, it relies on self-reported data, which could be subjected to biases in respondents' recall and to potential effect from mainstream media coverage. thirdly, when analyzing the factors related to a positive test, there is an ascertainment bias caused by testing primarily individuals reporting specific exposures or symptoms. this may blur some relationship between factors and a positive outcome. this highlights the interest of seroepidemiological studies at the population level. fourthly, our population is limited to middle and older adults and has less than one percent of individuals living in senior's house that were severely affected by the covid-19 outbreak in the province. finally, this study is a first step toward a follow-up study intended to understand the course of the pandemic and its consequences in the population. it will also allow us to compare the public health policies between provinces and countries. moreover, it will be enriched with a serological study in order to analyze more thoroughly the non-tested symptomatic and asymptomatic cases. to our best knowledge, this is one of the first report about the consequences of covid-19 outbreak at the population level. we have shown some discrepancies between the symptoms and risk factors associated with being tested and being positive. in particular, the anosmia is a major discriminant symptom for positivity whereas ent symptoms should be withdrawn from the list of covid-related symptoms. it also emphasizes the need of increasing the accessibility of testing for the general population. the authors declare that they have no competing interests. author's contributions rj: conceptualization, data curation, formal analyzis, investigation, methodology, writing -original draft. nn: conceptualization, resources, writing -original draf. scb: writing -original draft. cl: resources. tm: data curation, software. yp: data curation, software. pb: conceptualization, formal analyzis, methodology, project administration, supervision, validation, writing -original draft. ethics approval and consent to participate this project has been approved by the research ethics board of the chu sainte-justine on 28th may 2020 under the reference mp-21-2011-345, 3297. all participants have given their consent. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 1, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint additional file 2 fig s1. tif figure s1 : number of tested participants and cumulative number of tested participants over time. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 1, 2020. . https://doi.org/10.1101/2020.08.26.20182675 doi: medrxiv preprint situation of the coronavirus (covid-19) in québec public health agency of canada presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area features of 20 133 uk patients in hospital with covid-19 using the isaric who clinical characterisation protocol: prospective observational cohort study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet covid-19: a retrospective cohort study with focus on the over-80s and hospital-onset disease ethnic and socioeconomic differences in sars-cov-2 infection: prospective cohort study using uk biobank serocov-pop): a population-based study. the lancet real-time tracking of self-reported symptoms to predict potential covid-19 cohort profile of the cartagene study: quebec's population-based biobank for public health and personalized genomics the canadian partnership for tomorrow project: a pan-canadian platform for research on chronic disease prevention questionnaire study on coronavirus and covid-19 définition de cas de covid-19 -québec controlling the false discovery rate: a practical and powerful approach to multiple testing a novel tree-based procedure for deciphering the genomic spectrum of clinical disease entities r: a language and environment for statistical computing we would like to thank all the cartagene participants for their generous investments in health research. we would also like to thank the ramq and the commission d'accèsà l'information (cai) for their support in obtaining the data. not applicable. the data that support the findings of this study are available from cartagene but restrictions apply to the availability of these data. data are available directly from cartagene (http://cartagene.qc.ca; access@cartagene.qc.ca; +1 514-345-2156). key: cord-252959-ktet18wl authors: lim, jong-min; do, eunju; park, dong-chan; jung, go-woon; cho, hyung-rae; lee, seo-young; shin, jae wook; baek, kyung min; choi, jae-suk title: ingestion of exopolymers from aureobasidium pullulans reduces the duration of cold and flu symptoms: a randomized, placebo-controlled intervention study date: 2018-05-30 journal: evid based complement alternat med doi: 10.1155/2018/9024295 sha: doc_id: 252959 cord_uid: ktet18wl aim: the objective of the study was to assess the efficacy of exopolymers from aureobasidium pullulans (eap) on the incidence of colds and flu in healthy adults. methods: we conducted a randomized, double-blind, placebo-controlled study at the onset of the influenza season. a total of 76 subjects (30–70 years of age) were recruited from the general population. the subjects were instructed to take one capsule per day of either eap or a placebo for a period of 8 weeks. the duration of cold and flu symptoms, a primary variable in assessing effectiveness, and serum cytokine levels as well as wbc counts as secondary variables were also evaluated. results: eap was associated with a statistically significant decrease in the duration of cold and flu symptoms, a primary variable in assessing effectiveness. although cold and flu symptom levels were not significantly different at a significance level of 5%, the cold and flu symptom levels of the eap group were less severe compared to the placebo group. no statistically significant changes of serum cytokine levels as well as wbc counts were observed. conclusion: the results showed that eap is a useful pharmaceutical and functional food material for preventing and treating colds and flu. the immune system has evolved to protect multicellular organisms, including humans, from pathogens. immunostimulatory effects are regulatory actions that counteract reduced immune responses caused by immunodeficiency diseases, viral infections, malnutrition, tumors, and aging. two strategies have been developed to stimulate the immune system. one method is to increase antigen-specific or nonspecific immune responses, and the other method involves the use of appropriate immunosupplements for antigen administration [1] . respiratory viruses are among the most infectious pathogens in humans, and many differences occur in the kinds of pathogens, their antigenicities, and their worldwide infection patterns. recently, respiratory diseases such as middle east respiratory syndrome, severe acute respiratory syndrome, and the flu caused by novel swine-origin influenza a strains have emerged and begun to spread rapidly [2] [3] [4] . as the associated respiratory viruses show very strong tendencies to spread, there is a growing interest in foods that promote immune functions as well as a global monitoring system for influenza [5] . the results of several researches have revealed a positive correlation between food nutrition and the control of human immune functions, and various foods have been recognized as functional foods that can improve immune functions. these food materials may be derived from vegetables [6] , 2 evidence-based complementary and alternative medicine seafood [7] , mushrooms [8] , microbes such as lactic acid bacteria [9] , chitosan [10] , and peptides [11] . -1,3/1,6-glucan is derived from yeast cell walls and modulates many in vivo and in vitro activities [12, 13] . its main immunopharmacological activities [14] [15] [16] are associated with antitumor effects [17] , increased host resistance to viral, bacterial, and parasitic infections [18] , and adjuvant effects [19] . proximate composition of extracellular polysaccharides of aureobasidium pullulans is carbohydrate 79.5%, sugar 3.7%, crude protein 4.2%, and moisture 3.2%. namely, above 80% of the proximate composition is polysaccharides and there is a small amount of protein and fat (personal comm. dr. dong-chan park). it has been reported that the extracellular polysaccharides isolated from aureobasidium pullulans are pullulan (poly--1,6-maltotriose-based exopolysaccharide, eps) [20] , aubasidan ( -1,3-d-glucan with -1,6-branches to -1,4 side chains) [21] , and -glucan ( -1,3/1,6-d-glucan) [22] . however, the physiological activity of polysaccharides produced by aureobasidium pullulans has been mainly studied for -1,3/1,6-d-glucan [12] . exopolymers purified from aureobasidium pullulans sm-2001 (eap; its solubility is about 10% in water), the test food material used in this study, containing 13% -1,3/1,6-glucan [23] , are used in foods and have shown potent immunomodulatory activities in a mouse model [16] . in addition, eap showed no adverse events in clinical studies designed to evaluate its effectiveness on bone metabolism improvement [24, 25] and on atopic dermatitis [26] . therefore, -glucan is considered safe for human ingestion. many reports have described the immunomodulatory activities of -glucan against immune-related diseases [12] [13] [14] [15] [16] [17] [18] [19] , but only a few reports have described its effects against colds and flu. for this reason, a clinical study is now being performed to evaluate the efficacy and safety of -glucan from aureobasidium pullulans against colds and flu in patients. in this study, we selected adult subjects who provided written, informed consent to participate, who satisfied our selection criteria, and who did not meet exclusion criteria. we randomly administered eap or a placebo, where subjects took a daily dosage of 150 mg for 8 weeks. we evaluated immunological improvements of the test food by observing functional changes in the immune system before and after administering the test food materials in the eap and placebo groups. we also observed the duration and total levels of cold and flu symptoms during the test period. the eap and placebo products used in this clinical research study were packaged inside boxes provided by glucan co., ltd. (busan, korea) and were supplied and distributed by aribio co., ltd. (seongnam-si, gyeonggi-do, korea). some eap samples were deposited in the herbarium of silla university (busan, korea; encoded as eap2015choi01). sixty-five capsules were provided to each subject, taking into consideration an 8-week dosage (56 days, 56 capsules), a visitation window of 5 days (5 capsules), and storage products (4 capsules). the test food was manufactured as maroon-colored hard capsules, and each capsule of test food contained 150 mg of eap and 100.0 mg of microcrystalline cellulose, whereas the placebo food contained 220.0 mg of microcrystalline cellulose. the test food was supplied to a testing agency in sealed white plastic bottles, each containing 65 capsules intended for 1 person. the test foods used in this study were identical in appearance and description and would be difficult to distinguish with the naked eye. the experiment was conducted in doubleblind format for the subjects and researchers by attaching identical labels to the test foods. the main ingredients, generic names, and lot number were not recorded on the test food labels to avoid exposing the differences between the test groups. subjects took 1 eap hard capsule once daily for 8 weeks on an empty stomach, with a sufficient amount of water. the subjects were healthy adults who did not have any disease that required proactive treatment. the number of subjects assigned to each group was 38 (76 overall). in consideration of a potential 20% dropout rate, the number of subjects in each group would decrease to 30, the minimum number of subjects for an effectiveness analysis ( figure 1 ). subjects included in this study had to meet the following criteria: adults between 30 and 70 years of age; individuals with a white blood cell (wbc) count of 4-7 × 10 3 / l; individuals who could be monitored throughout the test period; individuals who listened to a thorough explanation of the study's purpose and contents and voluntarily signed an informed consent form to participate in the experiment. subjects for whom the following criteria were applicable were excluded from the experiment: individuals with a body mass index (bmi) under 18 or over 35; individuals who exceeded the normal maximum alanine transaminase and aspartate transaminase levels by 2-fold; females who were pregnant or were breast-feeding; females of childbearing age who did not agree to use contraceptives via medically proven methods (e.g., condoms, lubricant, and femidom) during the test period; individuals with a fasting plasma dextrose concentration over 126 mg/dl; individuals with high blood pressure (systolic blood pressure of 160 mm hg or diastolic blood pressure of 100 mm hg); individuals continuously using medicine that could affect the effectiveness assessment (hyperlipidemia medicine, steroid medicines, hormone medicines, immunosuppressants, and antibiotics); individuals who require continuous treatment for psychiatric disorders such as anorexia, depression, and manic depression; individuals with systemic diseases such as immunity-related diseases, serious hepatic and renal insufficiencies, malignant tumors, pulmonary disease, collagenosis, multiple sclerosis, allergic skin conditions, and other autoimmune diseases; individuals with a medical history of drugs and clinically significant allergic reactions; individuals with a history of gastrointestinal disorders that could affect the absorption of the test foods or a history of gastrointestinal surgery (excluding a simple appendectomy or hernia operation); individuals who consumed medicine or herbal medicines within a month of participation in the experiment which could affect immunity; individuals who participated in a different human study or clinical test and took experimental products within 3 months of participation in this experiment (excluding human studies with cosmetics); and individuals whom the researchers otherwise determined might have difficulty completing the experiment. dosages of the experimental products were temporarily suspended at instances where it was appropriate to stop the experiment for the welfare of the subjects. events that may have occurred. in the following cases, administration of the test foods was temporarily suspended: (a) a temporary interruption on the occurrence of an adverse reaction or for the treatment of an adverse reaction; (b) when showing adverse events related to the safety of the test foods; and (c) in case of showing acute reactions (allergies and hypersensitivity) to the test foods. however, in the case of a temporary interruption for the occurrence of an adverse reaction or for the treatment of an adverse reaction, if there was no problem in its compliance, the study was continued. cases that were processed as dropouts included those in which test foods were allocated and the subject or a legal representative withdrew consent from participation in the experiment, cases where results for a final inspection could not be obtained, cases where subjects did not adhere to the test protocol resulting in a significant effect on the test results, cases where there was an interruption in contact with the subject (i.e., when the subject could not be traced), cases where the subject failed to make visits, subjects for whom a disease was discovered which went unnoticed during the screening inspection, cases where compliance with usage of the test food fell below 80%, as assessed with the usage log, cases in which, without the direction of the researcher, the subject used a medicine or product while they were taking the test food which could affect the research results or interpretation of data, cases in which the subject had an average weekly drinking quantity as ethanol of 490 g/week throughout the test period, or cases where the lead researcher determined that the research proceedings were inappropriate or that there could be a significant effect on the safety of the subjects or the experiment results. subjects stopped taking test foods and were processed as dropouts for the following reasons, in which it was not possible to continue the human study: cases in which a serious adverse event occurred with the subject, cases in which an adverse event made participation in the experiment impossible, cases in which the researcher determined that the usage and observation of the test foods would be impertinent, or cases in which clinical tests were not possible due to the death of a subject or emergence of a disease. subjects who took the test foods and later dropped out of the study, where a portion or the entire estimate value of the final assessment (visit 3) was omitted, were included in the itt group's analysis data. missing values were processed after a dropout occurred using missing value processing method for effective assessments. the results of all subjects who dropped out were excluded from assessment of the per protocol (pp) group ( figure 1 and table 1 ). 2.2.6. drinking, diet, and exercise among the subjects. during the experiment, subjects maintained regular levels of drinking, diet, and exercise which were similar to levels prior to participation in the experiment. to verify this, on the day of base evaluations, we examined the dietary contents 4 evidence-based complementary and alternative medicine for 3 contiguous days prior to the visitation day and the weekly exercise types and times prior to the visitation day. the number of alcoholic drinks and the amount of ethanol consumed during the week prior to the visitation day were also examined. subjects kept a daily record of whether they consumed the test foods and recorded 3 contiguous days of their diets, including saturday or sunday. exercise, alcoholic drinks, and test food consumption were recorded in a chart provided by the testing agency, a comparison was made against these quantities prior to participation in the experiment, and observations were made as to whether there were any changes that could have significantyl affected the experiment results. subjects were assigned to the eap group (the group taking eap) and the placebo group (the group taking the placebo) based on a randomized list produced with a random sampling table generated using microsoft excel 2010 on microsoft windows ( figure 1 ). the exclusion criteria used to select subjects during the screening visit (visit 1) were verified, and individuals who met selection criteria and had met no exclusion criteria were selected as subjects for this study. food materials were allocated to the subjects during visit 2. the subjects selected for the experiment were issued a subject number in the order of visits made during visit 2 and were provided with the test foods, which were randomly allocated. the foods used in the human test were provided only to the participating subjects, based on the process described in the human test protocol ( table 1) the visitation dates allowed for the set base visitation days + 5 additional days. those who were selected as test subjects received the test foods within 14 days from the base evaluation date (selection inspection date) and began taking the test foods by the next morning. demographic data including the initials, age, gender, birthdate, weight, and height of the subjects and basic information such as medical history, medicinal intake history, combined treatment, concomitant drugs, smoking, drinking, and exercise habits were recorded in detail. the contents during the last 3 years were recorded in the medical histories, and contents during the last 4 weeks were recorded in the medicinal intake history. the types of exercise and the exercise times were examined for 1 week prior to the time of the visitation day. diet and exercise were compared during the base evaluation period and experimental period and observed to determine whether any important changes occurred which could affect the test results. the number and quantity of alcoholic drinks consumed during the week preceding the visitation day were examined, and that information was converted into the number of grams of ethanol consumed and recorded. levels. blood pressure, body temperature, and pulse were measured and recorded as vital signs. obesity levels were measured using a body fat analyzer (inbody 3.0, biospace, korea), and the bmi (kg/m 2 ), percent evidence-based complementary and alternative medicine 5 my daily life was very uncomfortable due to symptoms, and proactive treatment was needed. in the case of severe discomfort, i may stop working or studying or be admitted to the hospital body fat (%), waist-hip ratio, and visceral fat area (cm 2 ) were recorded. hematological examination of wbcs, red blood cells, hemoglobin levels, hematocrits, and platelets was performed using an automated analyzer (sysmex xp-300; sysmex, kobe, japan). the levels of blood urea nitrogen, creatinine, uric acid, total bilirubin, albumin, total protein, alkaline phosphatase, glucose, alanine transaminase, aspartate transaminase, gamma-glutamyl transferase, triglyceride, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were measured using a clinical chemistry analyzer (dimension xpand plus; siemens, munich, germany). all urine samples were stored in the dark at 4 ∘ c and were analyzed within two hours of collection. the ph, specific gravity, and protein and glucose levels were measured using a semiautomated analyzer (uriscan pro ii; yd diagnostics, seoul, korea). pregnancy testing was performed using a kit detecting human chorionic gonadotropin. serum concentration of cytokines was analyzed by using hmaglxsa (6 plex) multiplex elisa kit (r&d systems, minneapolis, mn). cytokine levels were determined using luminex 200 (luminex, austin, tx, usa) and the data were reported as median fluorescent intensities. comparisons. if immune functions improve, the disease duration and symptom levels should decrease due to increased resistance to viral diseases such as colds and flu. accordingly, we examined the duration and levels of cold and flu symptoms after consuming the test food products as a functional index of immunological improvements. for the survey following the administration of test foods, a form was provided in which subjects made a daily record of their health conditions, with a daily record of their test food dosages. nine symptoms were monitored to inspect the health conditions of the subjects in the survey: fever, chills, runny nose, nasal congestion, coughing, sneezing, sore throat, headache, and gastrointestinal symptoms (nausea, vomiting, diarrhea, and abdominal pain). subjects marked i for "symptoms exist" and × for "no symptoms." to record the symptom levels, subjects used a 4-step scoring point system, where they marked (0) for no symptoms, (1) for minor symptoms, (2) for moderate symptoms, and (3) for serious symptoms [27] and marked whether they took early leave or were absent from work or school due to symptoms. table 2 shows a detailed explanation of the criteria used for reporting symptom levels. to assess the effectiveness of the experimental food products, subjects kept daily records in their dosage logs of cold and flu symptom durations and symptom levels during the testing period. compliance with ingesting test foods was evaluated based on the food-usage logs. during the final visit, the number of remaining foods collected was compared with the usage log records. in cases where there were differences between the recorded and actual amounts of food taken, compliance was verified when the differences were small. primary effectiveness assessment variables included the duration and total score of symptom levels for cold and influenza. secondary effectiveness assessment variables included serum il-1 , il-6, il-8, il-10, tnf-, and inf-levels, as well as wbc counts and differential blood counts. evidence-based complementary and alternative medicine on significance comparisons between the eap and placebo groups. the methodologies described below were followed for detailed analysis. analysis sets obtained from the test subjects were composed largely of an itt analysis group and a pp analysis group. the itt group comprised the primary data set used for effectiveness assessments, and the pp group was additionally analyzed to assess the effectiveness. subjects in the itt group included those who had taken the test food materials and whose assessment variables were measured at least once during visit 1. subjects in the pp group included those in the itt group who completed the test according to the study protocol. it has been predicted that fewer subjects will contract viral diseases such as cold and flu if their immune functions are enhanced and that the duration and levels of symptoms will decrease if one of the diseases is contracted. the activation state of principal cells related to immunity can be determined by measuring changes in the serum contents of cytokine il-1 , il-6, il-8, il-10, tnf-, and inf-. to assess the effectiveness of the test food materials, the significance of the mean difference (visits 1-3) was tested for each variable included in the effectiveness test. in other words, a statistical comparison was made between the average variation before and after food ingestions in the eap and placebo groups. to this end, normality was tested for the evaluation data. the parametric method of a twosample t-test was chosen if normality was satisfied, whereas the nonparametric method of a wilcoxon's rank-sum test was chosen if normality was not satisfied. in addition, the above significance tests were performed on mean differences between the eap and placebo groups between each visit after test food ingestions. to analyze the prevalence of cold symptoms, subjects recorded the duration and levels of symptoms when they contracted a cold or the flu prior to ingesting the test food. during visit 3, subjects recorded the duration and total symptom scores of cold and influenza symptoms during the test period. accordingly, it was not meaningful to compare the mean difference between visits, considering that the rating scales used in visits 1 and 3 were different. in other words, we confirmed homogeneity between the test groups in visit 1 and evaluated the effectiveness by comparing the results between test groups in visit 3. forward (locf) method (a single-imputation method) for data missing in the final effectiveness assessment. typically, the locf method is easy to use in clinical trials, despite the restricting assumption where missing data are treated as a constant. the locf method is commonly used because it provides conservative results. however, there are no commonly recommended methodologies for processing missing values [29] . in this study, the statistical analytic data collected was comprised of only the baseline assessment prior to the ingestion of test food materials and the final assessment after ingestion of test food materials. the missing values during the final assessment were not replaced by the base assessment values but were substituted by the average of each group and assessed. in this study, we screened 117 individuals who voluntarily agreed to participate in this human study, and 76 subjects who met the selection criteria and did not meet any exclusionary criteria were enrolled. among the 76 subjects, 38 were assigned to the eap group and 38 were assigned to the placebo group ( figure 1 ). four instances were verified in which subjects violated the study protocol while consuming the test foods. these 4 subjects were processed as dropouts. among the 76 subjects who were selected, 2 individuals from the eap group dropped out due to violations in the visitation schedule and shortfalls in compliance, and 2 individuals from the placebo group dropped out due to withdrawal of informed consent and shortfalls in compliance. the remaining 72 subjects adhered to the test protocol and completed the test. the current states of dropouts are separated according to the test group ( figure 1) . compliance with ingesting test food materials was assessed in visit 3. the contents recorded in the daily record filled out by the subjects as well as the quantity of remaining products returned during the final visit were confirmed and assessed. compliance was calculated using the following formula: compliance (%) = (number of food materials ingested/number of food materials that should have been ingested) × 100, where the number of food materials ingested = number of food materials provided − number of food materials returned. subjects whose compliance was below 80% were set as dropouts, and there were 2 dropouts in this study due to compliance shortfalls. the compliance of test food material ingestion was 93.82 ± 6.73% in the eap group and 94.81 ± 6.09% in the placebo group. a significant difference in compliance between the test groups was not observed (table 3) . a homogeneity test was conducted between test groups for the following: measurement values prior to food material ingestion regarding demographic information of the subjects including age, gender, medical history, treatment history, medication history, and concomitant medication and measurement values before and after ingestion of food materials regarding items that could affect the test results based on changes in lifestyle habits during the test period including regular exercise, smoking, and drinking. the results of a preliminary investigation on the age, gender, medical history, treatment history, medication history, concomitant medication, exercise, smoking, and drinking among the subjects are summarized in tables 3 and 4 . no significant differences were found between the test groups regarding any of the items examined. missing values were replaced with averages and used in the itt group data analysis. (a) (b) figure 2 : foundational statistics and comparisons of cold symptom durations (days; (a)) and levels (b) between test groups: eap group (black columns) and placebo group (gray columns). variables in the itt group. the results of a -test performed to evaluate significant differences between 2 groups before and after ingesting the test food materials verified that the duration of cold symptoms ( value = 0.5748) and cold symptom levels ( value = 0.2462) at visit 1 were not significant at a significance level of 5%. statistical analysis also showed that no difference occurred between test groups prior to the ingestion of food materials. during visit 3, the duration of cold symptoms in the experiment group (3.1579 ± 4.4814 days) was lower than that of the placebo group (6.6842 ± 9.3581 days). the difference between the test groups was statistically significant ( value = 0.0410). the eap group also had a lower score for cold symptom levels than the placebo group (8.7105 ± 13.7269 versus 18.3421 ± 30.1566, resp.). this difference was significant at a level of 10% ( value = 0.0790) but not 5% (figure 2 ). were verified with respect to the mean difference of each measured item between the visitation times to assess the effectiveness of the test food materials on the blood test results. the results were not significantly different between the eap and placebo groups (table 5 ) at a significance level of 5%. for wbc, basophil, and lymphocyte counts that wilcoxon's rank-sum test was used to analyze variables that did not follow normality. missing values were replaced with averages and used in the itt group data analysis. did not follow normality, a nonparametric wilcoxon's ranksum test was conducted. the results were not significantly different between the eap and placebo groups (table 6 ) at a significance level of 5%. to assess the effectiveness of the test food materials, a -test was conducted to evaluate significance differences in the mean cytokine concentrations between visits 1-3. the results were not significantly different between the eap and placebo groups (table 7 ) at a significance level of 5%. il-10, il-6, il-8, and tnf-values did not satisfy normality; a nonparametric statistics wilcoxon's ranksum test was conducted, revealing no significant differences in any of the items at a significance level of 5% (table 8) . adverse events were observed in 3 individuals from the placebo group and in 1 individual from the eap group. in the placebo group, nausea and pruritus were confirmed in subject e11, asthenopia and rash in subject e49, and a sore throat in subject e68. no other adverse events were observed aside from these. in the eap group, headache was confirmed in subject e59, but no other adverse events were confirmed aside from this. all these adverse events subsided within a few days. in the case of subject e59, who ingested the test food materials, the headache was completely alleviated after 2 days, and the subject was judged as having no cause-and-effect relationship with ingesting the test food materials (data not shown). while the sgot or sgpt exceeds normal upper limit by 3fold, the finding is not considered to represent an abnormal phenomenon. however, the -gtp value was found to be abnormally high during visit 3 for subject e57, but examination of the results showed that the abnormal values were not related to the ingestion of test food materials. instead, the results were due to excessive drinking, where the subject consumed alcoholic drinks frequently (5 days/week) and a high quantity of ethanol (average = g/week) (data not shown). with the occurrence of adverse events and abnormal changes in clinical test results, no items were found to correlate with the use of test food materials or were suspected to have a correlation. thus, the test foods were found to be safe in the context of this study. the various biological activities of -glucan were verified through in vitro testing, animal testing, and clinical trials [12] . recently, the effectiveness of -glucan on respiratory diseases has been studied in rodent [30] and human [31] [32] [33] because their prevalence has increased rapidly. eap, exopolymers purified from aureobasidium pullulans sm-2001, containing 13% -1,3/1,6-glucan [20] , are used in test foods and have shown potent immunomodulatory activities in a mouse model [16] . however, this study is the first to evaluate the use of eap against respiratory diseases. to assess the ability of eap to improve human immunological responses and to alleviate cold or flu symptoms, 117 adults aged 30-70 years with a white blood cell count of 4-7 x 10 3 / l were screened. 76 individuals were enrolled as subjects who satisfied the selection criteria and did not fall under any exclusion criteria (data not shown). in an aspect of subject's respiratory morbidity, none of the subjects had a respiratory disease at the time of enrollment. within 4 weeks prior to the screening date of the subjects, there were 2 subjects with light respiratory disease. after confirming no symptoms at the enrolment, the 2 subjects were proceeded to obtain informed consent. the 76 subjects (administrated with the test foods for a period of 8 weeks) were randomly assigned to either eap or a placebo group. blood tests and surveys of cold or flu symptoms were conducted before and after ingesting test food materials, and the duration and symptom level of colds or flu, as well as the results of the blood test and cytokine analysis, were compared. the eap and placebo groups were composed of individuals that exhibited homogeneity and showed no statistically significant differences in their compliance of ingesting test foods or in gender, age, compliance, regular exercise, smoking, drinking, medical history, medicinal history, or concomitant drug administration. clinical data have shown that immunity-promoting functions can prevent acute respiratory infections from cold or influenza viruses and reduce the frequency of contracting colds or flu, symptom levels, and symptom durations based on effectiveness criteria [34] . in this study, statistical analysis of the duration and level of cold symptoms, a primary variable for assessing effectiveness, showed that there were no differences in assessment values between the eap and placebo groups before ingesting test food materials. however, the duration of cold and flu symptoms after ingesting test food materials was significantly lower in the eap group than in the placebo group at a 5% significance level. the eap group also showed lower levels of cold and flu symptoms, which were statistically different at the 10% (but not 5%) significance level than placebo group. it is predicted that individuals whose immune functions are enhanced will contract fewer viral diseases such as colds and flu, with reduced durations and levels of symptoms. the activation state of primary cells related to immunity can be determined by measuring the serum levels of the cytokines il-1 , il-6, il-8, il-10, tnf-, and inf[1] . according to the "guideline for safety and efficacy assessments of functional food on immune function improvement" from the ministry of food and drug safety of korea, changes in cytokine levels, wbc counts, t cell counts, and nk cell activity in the peripheral blood should be measured when determining the functionality of immunity reinforcement in humans [35] . in this study, secondary variables including serum cytokines (il-1 , il-6, il-8, il-10, tnf-, and inf-) and wbcs (neutrophil, eosinophil, basophil, lymphocyte, and monocyte) for assessing effectiveness did not exhibit statistically significant differences before and after ingestion of test food materials at a significance level of 5%. these results are consistent with those of choi et al. (2009) [14] , where the daily intake level of eap (polycan tm ) or placebo food materials was set at 400 mg, and changes in serum cytokines were studied (with the intention of observing effectiveness in immunity improvements of eap), but no statistically significant changes were found. although eap has shown potent immunomodulatory activities in a mouse model [16] , no immunity improvement of eap on the human was observed in this study. the cytokine is a factor that needs to change suddenly according to the individual health condition and then return to the normal range [1] . on the normal range of blood cytokine concentration, its individual variation is very large. thus, a large number of subjects are required to measure the normal range of cytokine concentrations [36] . indeed, in a study of normal korean cytokine levels, 110 subjects were studied [37] , and in a study of normal american cytokine levels, 144 subjects were examined [38] to estimate the normal range of blood cytokine concentration. serval studies have shown that no changes in blood cytokine levels have been observed following ingestion of multivitamin and mineral supplement [39] , probiotic supplement [40] , fish oil [41] , and cordyceps militaris [42] , which are known to help improve immunity. this may be because the cytokine level increased during the test period and then returned to normal, or the number of subjects was small, indicating that the individual deviation range was not statistically significant. in this study, the total number of subjects was 72: 36 in the test group and 36 in the control group, respectively. therefore, the results of this study may not statistically overcome the individual variation range of the subject's cytokine level due to the small number of subjects. further research is needed on extensive clinical studies with clear statistical numberings to confirm the effects of eap on plasma cytokines. no significant adverse events occurred during the period of test food material intake, and adverse events (excluding colds) were observed in 3 individuals from the placebo group (nausea, pruritus, asthenopia, rash, and sore throat) and in 1 individual from the experiment group (headache). all these adverse events subsided within just a few days and were determined to have no cause-and-effect relationship with the ingestion of test food materials. changes in blood test values were also observed in the eap group with an increase in sgpt, tg, and -gtp values, but this was difficult to determine as being clinically abnormal. in addition, in an aspect of the respiratory morbidity, the subjects who suffered from cold during the test period appeared and then fully cured after the treatment, and no respiratory morbidity was shown in all subjects. therefore, eap was verified to be harmless to the human body. collectively, our data revealed that eap was associated with a statistically significant decrease in the duration of cold and flu symptoms, a primary variable in assessing effectiveness. although cold symptom levels were not different at a significance level of 5%, the cold and flu symptom levels of the eap group were less severe compared to the placebo group. no statistically significant changes were observed in the serum cytokine concentrations, wbc, and differential blood counts (secondary variables for assessing effectiveness). therefore, our data showed that eap is a useful medicine and functional food material for preventing and treating colds and flu. the data used to support the findings of this study are available from the corresponding author upon request. the authors declare that there are no conflicts of interest. human infection with mers coronavirus after exposure to infected camels from sars to avian influenza preparedness in hong kong whole-genome characterization of a novel human influenza a(h1n2) virus variant, brazil epidemiological characterization of respiratory viruses detected from acute respiratory patients in seoul a review on immunostimulatory plants bioactive compounds from marine processing byproducts-a review mushroom immunomodulators: unique molecules with unlimited applications probiotic immunomodulation in health and disease application of chitin and chitosan derivatives in the pharmaceutical field bioactive peptides: production and functionality clinical and physiological perspectives of -glucans: the past, present and future functional food for immune regulation-beta-glucan a 4 week randomized, double-blind human trial to compare the efficacy and safety of aureobasidium pullulans cultured solution and placebo on improvement of immune in subjects immunomodulatory activities of oat -glucan in vitro and in vivo immunomodulatory effects of aureobasidium pullulans sm-2001 exopolymers on the cyclophosphamide-treated mice effect of glucan on natural killer (nk) cells: further comparison between nk cell and bone marrow effector cell activities purification of solubleglucan with immune-enhancing activity from the cell wall of yeast glucan as an adjuvant for a murine babesia microti immunization trial characterization and enzymatic hydrolysis of hydrothermally treated -1,3-1,6-glucan from aureobasidium pullulans an aubasidan-like -glucan produced by aureobasidium pullulans in thailand the structure of the carbohydrate moiety of an acidic polysaccharide produced by aureobasidium sp. k-1 production of ß1, 3/1, 6glucan by aureobasidium pullulanssm2001 safety and efficacy of polycalcium for improving biomarkers of bone metabolism: a 4-week open-label clinical study randomized, doubleblind, placebo-controlled trial of the effects of polycan, ß-glucan originating from aureobasidium pullulans, on bone biomarkers in healthy women a clinical study for the efficacy and safety of ß-glucan from aureobasidium pullulans in mild to moderate atopic dermatitis patients a placebo-controlled trial of korean red ginseng extract for preventing influenza-like illness in healthy adults korea food and drug administration (kfda, guidelines for clinical trial statistics sample size calculations with dropouts in clinical trials β-glucan derived from aureobasidium pullulans is effective for the prevention of influenza in mice immunomodulatory effect of pleuran ( -glucan from pleurotus ostreatus) in children with recurrent respiratory tract infections preventive effect of pleuran ( -glucan from pleurotus ostreatus) in children with recurrent respiratory tract infections -open-label prospective study placebo-driven clinical trials of yeast-derived ß-(1,3) glucan in children with chronic respiratory problems efficacy of cold-fx in the prevention of respiratory symptoms in community-dwelling adults: a randomized, doubleblinded, placebo controlled trial guidelines for functional evaluation of health functional foods, 'can help improve immune function conceptual and methodological issues relevant to cytokine and inflammatory marker measurements in clinical research serum cytokine profiles in healthy young and elderly population assessed using multiplexed bead-based immunoassays baseline levels and temporal stability of 27 multiplexed serum cytokine concentrations in healthy subjects the effects of a multivitamin/mineral supplement on micronutrient status, antioxidant capacity and cytokine production in healthy older adults consuming a fortified diet probiotic supplement consumption alters cytokine production from peripheral blood mononuclear cells: a preliminary study using healthy individuals proand anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year regulation of human cytokines by cordyceps militaris this research was performed with the support of the industry core technology development project (no. 10049026), ministry of trade, industry, and energy, republic of korea. jong-min lim and eunju do contributed equally to this work. key: cord-273324-xhpv783y authors: land, kevin j.; boeras, debrah i.; chen, xiang-sheng; ramsay, andrew r.; peeling, rosanna w. title: reassured diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes date: 2018-12-13 journal: nat microbiol doi: 10.1038/s41564-018-0295-3 sha: doc_id: 273324 cord_uid: xhpv783y lack of access to quality diagnostics remains a major contributor to health burden in resource-limited settings. it has been more than 10 years since assured (affordable, sensitive, specific, user-friendly, rapid, equipment-free, delivered) was coined to describe the ideal test to meet the needs of the developing world. since its initial publication, technological innovations have led to the development of diagnostics that address the assured criteria, but challenges remain. from this perspective, we assess factors contributing to the success and failure of assured diagnostics, lessons learnt in the implementation of assured tests over the past decade, and highlight additional conditions that should be considered in addressing point-of-care needs. with rapid advances in digital technology and mobile health (m-health), future diagnostics should incorporate these elements to give us reassured diagnostic systems that can inform disease control strategies in real-time, strengthen the efficiency of health care systems and improve patient outcomes. or in sequence. in case of discrepancies, a third test is used as a tiebreaker. an example of this approach is for hiv case detection. specificity. diagnostics should have low false positive rates. the ideal scenario is where the sensitivity and specificity achieved from the diagnostics used at the poc approach those of laboratory-based assays wherever possible. however, a lower specificity can be tolerated if the harm of overtreatment is much less critical than missing the diagnosis of an infection. screening syphilis during pregnancy is an example of this approach, as missing maternal syphilis can lead to stillbirths, preterm birth and congenital syphilis in a third of pregnant women compared to the harm of overtreatment with a single dose of penicillin 5 . user friendliness. tests should be easy to perform in 2-3 steps and require minimal user training with no prior knowledge of diagnostic testing. typically, results should be available in 15-60 minutes after sample collection and enable patient management and treatment during the same visit. the benefits of a rapid test versus a more accurate test that requires patients to return for results have been previously demonstrated 6 . robustness refers to the ability of the test to withstand the supply chain (temperature, humidity, time delays, mechanical stresses) without requiring additional (and often costly) transport and storage conditions (for example, refrigeration). ideally the test does not require any special equipment or can be operated in small portable devices that use solar or battery power. deliverable to end-users. delivery refers to the organizational structures and relationships established with the purpose of coordinating and steering the logistics of selecting, procuring, shipping, storing, distributing and delivering a new health technology to ensure it reaches the end-users in resource-constrained settings 7 . compared to when assured was proposed, access to laboratories in resource-limited settings has improved dramatically both in numbers and in quality 8, 9 , especially in areas such as hiv and malaria testing, cd4 counting and tb. however, there remains a critical need for a wider range of diagnostics that can be performed at the poc. from this perspective, we undertake a review of the assured benchmark over the last 14 years, assess the factors contributing to the success and failure of the assured diagnostics over the past decade, identify areas that have remained difficult to address as well as lessons learned from implementation in resourcelimited settings, and highlight additional conditions that should be considered in addressing poc needs. since 2003, several diagnostic tests that satisfy (or nearly satisfy) the assured criteria have been developed to identify major human pathogens. this includes tests for hiv, malaria, syphilis and tb ( table 1) . the hiv rapid test is the first test to fulfil the assured criteria, followed closely by the malaria and syphilis rapid tests and, recently, a near-poc test for tb. much of this development was the result of advocacy by donors such as the bill & melinda gates hiv rdts. early detection of hiv infection has been a critical component of hiv control programmes worldwide since the early days of the epidemic. since most infected individuals do not show specific symptoms and the period of viraemia is short, screening for hiv antibodies in blood as a marker of exposure has been the most cost-effective means of identifying infected individuals. the who developed a process and criteria for validating the performance of hiv antibody detection assays more than a decade ago and showed that hiv rdts using finger-pricked whole-blood specimens has acceptable performance compared to laboratory-based immunoassays 12, 13 . many donors -for example, global fund and implementation partners, including the who, unaids and pepfar -have helped affected countries with the procurement and introduction of rapid hiv tests and enable early detection and prevent onward transmission. as a result of funding from donors, increased accessibility of rdts, and the 'architecture' provided by national hiv/ aids control programmes, hiv tests successfully reached as many as 150 million children and adults in 129 low-and middle-income countries in 2014 (ref. 14 ) . two major challenges remain: training and quality assurance. with thousands of poc testing sites across a country, even the most well-organized control programmes would find it difficult to provide adequate training on an ongoing basis. this problem is most acute in health facilities where there is high turnover of staff. the us centres for disease control and prevention (cdc) has developed a convenient means of external quality assurance by working with countries to generate thermally stable proficiency panels that can be shipped to every poc testing site to ensure competence of testing personnel. however, a study in south africa found that only 3% of hiv rdts were performed correctly 15 . although not every error will result in an incorrect diagnosis, the alarming reality is that with 150 million tests being performed annually worldwide, assuming a 99% accuracy rate, as many as 1.5 million incorrect results per year could potentially be generated. ongoing quality assurance efforts include developing key policy and quality documents for the implementation of hiv-related poc testing 16 . for example, as poc technologies for hiv viral load and early infant diagnosis were being developed, there was tremendous emphasis on quality, given the complexity of the test and lessons learned from hiv rdts. malaria is estimated to be the cause of at least a million deaths a year worldwide, most of which are in sub-saharan africa. while microscopic identification of parasites in blood smears has been the traditional means of diagnosing malaria in patients presenting with fever, microscopy requires equipment, a source of electricity and trained laboratory technicians. malaria rdts were developed as many rural communities lack these resources, and to date there are over 120 brands of malaria rdts made by approximately 60 companies 17 , which vary widely in performance, manufacturing quality and price. the who has set up a pre-qualification programme with the cdc and the foundation for innovative new diagnostics to evaluate these tests to inform test selection and procurement for national malaria control programmes 17 . in countries that permit the sale of malaria rdts and medicines over the counter, the quality of these commodities cannot be guaranteed. the price of most rdt brands is between us$0.65 and us$2.50 per test 7, 18 , and as with all diagnostics, it is important to caution that price pressure will ultimately affect quality. as with hiv, with the support of donors, selection of high-quality rdts and the architecture provided by national malaria control programmes, malaria rdts have reached millions of patients every year. this promising trend, coupled with the effectiveness of bednets for preventing transmission, has led to the call for malaria elimination in many parts of the world 19 . however, challenges remain for the future of malaria poc testing and the global elimination of malaria. first, highly accurate tests are required for all malaria species affecting humans, but these panspecific tests are usually about 40% more expensive than rdts that only detect plasmodium falciparum. most p. falciparum rdts detect a malarial antigen, histidine-rich protein (hrp) 20 , and the discovery of parasites that have a deletion in this gene has raised concerns about false negative results and ongoing transmission. also, the problems of providing adequate training and quality assurance of malaria tests and testing at remote poc sites are similar to those described for hiv rdts 21, 22 . in the near future, as countries progress towards malaria elimination, funding for rdts may become an issue. as the intensity of transmission decreases due to lower parasite density in infected individuals, more sensitive tests will be needed, which can only be achieved with costlier amplification steps or with ultrasensitive platforms for antigen detection. also, decreasing numbers of cases mean that malaria tests are no longer cost-effective, and thus it is more difficult to justify funding in light of multiple competing priorities for limited health budgets. syphilis. syphilis, caused by the spirochete treponema pallidum, has a long latent period during which patients have no symptoms, but can remain infectious. syphilis in pregnancy can lead to adverse outcomes of stillbirths and miscarriage, and babies born with congenital syphilis in the developing world only have a 50% chance of survival during the first 2 years of life 23, 24 . despite the availability of simple diagnostic tests for antenatal screening and the effectiveness of treatment with a single dose of long-acting penicillin, syphilis is re-emerging as a global public health problem 23 . it is estimated that 500,000 babies die each year as a result of syphilis-associated stillbirths and congenital syphilis, largely because of lack of access to antenatal screening 25, 26 . rdts exist for detecting syphilis and are reported to have acceptable performance 27, 28 and operational characteristics. the introduction of rdts was also acceptable to patients and health care providers and was shown to contribute to the improvement of antenatal care in low-resource settings 29 . despite all this, syphilis rdts have not had the same success as hiv and malaria rdts. the main reasons for this are the lack of advocacy and political will to translate the evidence to national policy, lack of funding to make the tests affordable to those in need and the lack of a national control programmes to provide the architecture needed to coordinate all the different aspects of testing. ensuring adequate training for health care workers and supplies of commodities were cited as key implementation barriers in africa 30, 31 . dual hiv and syphilis rdt testing in countries was prioritized by the who for the elimination of mother-to-child transmission (mtct) of hiv and syphilis by 2020 32,33 , but a recent review showed that while prevention of mother to child transmission (pmtct) programmes for hiv have resulted in a dramatic decrease in the number of hiv-positive infants in sub-saharan africa, the rate of syphilis screening of pregnant women in the same countries has remained at unacceptably low levels of approximately 30% (ref. 34 ). this is due largely to disparity in funding and lack of political will despite the global fund allowing countries to purchase syphilis rdts with hiv rdts since 2007 (ref. 35 ). countries need to harness the architecture provided by hiv pmtct programmes to screen women for both infections using a single drop of blood in a single visit to a health care facility. dual rapid hiv-syphilis rdts with acceptable performance are now available 36 and the who, as well as many countries, has adopted these hiv and/or syphilis rdts into their national guidelines 31, 37 . tuberculosis. tb causes 1.7 million deaths a year, 95% of which occur in low-and middle-income countries. ending the tb epidemic by 2030 is among the health targets of the sustainable development goals 38 . the tb lipoarabinomannan (lam) antigen test provides poc screening for active tb in hiv positive patients. this novel rapid test detects lam in urine samples, providing results in just minutes, enabling earlier treatment for patients 39 . the lam test does not assess tb drug susceptibility, but multidrug-resistant tb is a threat to global health security with an estimated 600,000 new cases a year showing resistance to rifampicin (rif) -the most effective first-line drug 40 . nucleic acid tests (nats) for tb are available and provide highly sensitive and specific means of diagnosis. the recent development of a sample-in-answerout automated testing device that allows for simultaneous detection of mycobacterium tuberculosis (mtb) and rif resistance in 1 h 45 min has improved case detection and could decrease transmission, though nats still require patients to make a return visit for test results and treatment. this test system is available in 1-80 modules, allowing for flexibility in throughput. the mtb/rif near-poc assay can improve time to diagnosis and treatment and increase the efficiency of the health system if introduced appropriately 41-44 , and as of june 2012, two-thirds of high-burden mtb countries and half of countries with a high multidrug-resistant mtb had adopted this assay into their national tb programme guidelines. a wider adoption of such high-performing assays would allow countries to increase their case detection rates and potentially reach the 2020 milestones of the end tb strategy 45 . although national tb programmes provide a robust architecture for the implementation of new technologies, challenges associated with the near-poc nat assay remain as barriers -affordability (molecular assays are device-based and costly, even with subsidy), expertise (more technically demanding than lateral flow rdts) and sustainability 46 , in addition to power and per-test time. we expect that addressing these barriers would improve patient outcomes, but a true poc test is still needed to overcome remaining challenges such as sample preparation and demands on human resources 47 . while culture has been a long-standing microbiological gold-standard and is highly specific, it is also the most technically demanding, costly and slowest of diagnostic options, requiring patients to return for another clinic visit to obtain their test results and treatment if necessary. thus, it does not conform well to the development of assured diagnostics. successful poc tests achieve high sensitivity, which is needed as screening tools to ensure that all true and suspect cases are brought to the attention of control programmes and appropriately managed. in particular, antibody-based detection tests, such as those for hiv and hepatitis c virus, are highly sensitive as antibodies are present in large quantities in blood, and blood samples can be collected with a finger prick and put directly into the test without any prior processing. also, antibody detection can be rapid and easy to perform with minimal training required (table 2) . however, the choice of the antibody target affects a test's effectiveness -unless the diagnostic target is specific to the intended infection, there is the potential for false positive results due to cross-reactivity, which is the case with dengue and zika immunoassays 48, 49 . therefore, antibody detection assays should be interpreted within the appropriate epidemiological context and findings from physical examination. the other major disadvantage of antibody-based tests is that antibodies are usually a marker of exposure to a pathogen and cannot be used to distinguish between those with active and past infection, which is the case of the rk39 tests for visceral leishmaniasis (vl) 50 . a presumptive diagnosis of active vl can only be made using a combination of a positive rk39 test and more than 2 weeks of fever and splenomegaly. the same is true of most syphilis rdts, which detect long-lived antibodies to treponemal antigens and thus do not diagnose active syphilis. antibodies to a non-treponemal antigen, which appears with infection and declines in the months after successful treatment, are used in a flocculation assay called the rapid plasma reagin assay, which can yield a result in 8 minutes but requires electricity to operate, a centrifuge for processing serum from whole blood, a shaker and cardiolipin as an antigen. a combination treponemal and non-treponemal rapid test has been developed in an immunochromatographic test in a lateral flow format with acceptable performance characteristics 51 . antigen detection poc tests, such as those for chlamydia and gonorrhoea, suffer from three major drawbacks (table 2) . first, the specimen used for sexually transmitted infections are usually from urethral, cervical or vaginal swabs that require multiple steps to solubilize the bacteria and free antigen before reaction with the capture antibody. this sometimes requires a heating step and adds to the complexity and cost of the poc test. urine is the preferred specimen in men with such infections, and current poc tests require a urine centrifugation step to concentrate the bacteria before processing and reaction. another common drawback of antigen detection tests is the potential for false positive results due to cross-reactivity with antigens from closely related bacterial or viral species. this is especially true if polyclonal antibodies are used as capture antibodies. finally, a common disadvantage of current antigen detection poc assays is low sensitivity, often requiring 10 4 to 10 5 bacteria for the rdt to become positive. malaria rdts are an exception to these drawbacks in that the parasites are present in large quantities in blood and no pre-processing or concentration steps are required. for most antigen detection poc tests, the low sensitivity may be due to low concentration of target antigens, inefficiency of extraction or limited optimization of reagents, which has hampered chlamydia and gonorrhoea tests 52, 53 . however, gift et al. showed that rapid chlamydia tests with a sensitivity of 65% can lead to more infected patients being treated compared to nats, which require patients to return for their test results 54 . they called this the rapid test paradox, which was also recently seen with a p24 assay for early infant diagnosis of hiv, where a sensitivity of 72% can still lead to a higher 'diagnostic yield' than nats performed on dried blood spots sent from remote antenatal clinics 55 . nats offer a more sensitive and specific alternative to antigen detection assays as nucleic acid targets can be selected from a genome sequence with high specificity, and the amplification process to increase sensitivity can be fairly rapid (table 2) . for example, the near-poc tb diagnostic involves primer-based amplification of mtb dna specifically, including regions associated with rif drug resistance. thus, nat tests are generally highly sensitive and more specific than antigen-or antibody-based tests, and may help inform on drug susceptibilities. while instruments are available for highthroughput screening of patients and subsidies can bring test costs down to us$10, the equipment (capable of four tests at a time) costs more than us$10,000, which may be prohibitive in high-incidence regions. thus, a truly poc diagnostic based on nats without needing any equipment is still a promise and not a reality as yet 56, 57 . of all the criteria originally accepted, the requirement of equipment-free is perhaps the only one which is not as critical as originally defined. a wide range of near-poc nats have been developed for use outside of laboratory settings 58 and most of these assays are automated, requiring only 2-3 minutes of hands-on time and minimal training. these near-poc devices come in sealed units with internal self-calibration making it easier to ensure quality of testing and most are also equipped with data transmission capabilities, which make these platforms very attractive in the context of disease surveillance and epidemic preparedness. most of these near-poc devices have a broad menu of diagnostic targets which makes the capital cost of purchasing the device less of an issue. reagents are pre-measured and dried ready for hydration with the introduction of the specimen. however, near-poc nat cartridges are expensive to manufacture and not affordable to most developing countries without subsidies. rapid advances in miniaturization, material science, electronics and data transmission in recent years have made minimal instrumentation a reality for new diagnostics, including the use of smart phones, which are widely available, custom developed and low cost. the use of a dongle to connect a smart phone to a microfluidic disc to detect hiv and syphilis antibodies in finger-pricked blood allows the phone to power the reaction, interpret the results and transmit the data to a central database. this smart phone-based poc assay is currently undergoing clinical trials 59 . use of a smart phone to power nats is in development and will play an important role in future poc applications 60,61 . in particular, there has been considerable interest shown in utilizing mobile phones as readers and connectivity for rdts using rfid (radio frequency identification) to prevent errors in subjective interpretation and transcription [62] [63] [64] [65] . while phone-based diagnostics are attractive as an option, regulatory approval and rapid updates of phone software that can affect test performance are important challenges. defining and quantifying risks and benefits. while successes need to be celebrated, significant challenges remain. first, as no diagnostic is perfect, countries continue to struggle with defining acceptable risks of false positive or negative results when a novel diagnostic technology may provide incremental clinical benefits. in the developing world where over 60% of the population reside in rural areas, and the sustainable development goals urge countries to provide universal health coverage and leave no one behind, the trade-off between accuracy (sensitive and specific) and accessibility (user friendly, rapid and deliverable) becomes paramount 66 . while it impossible to give absolute values across different tests, both sensitivity and specificity remain critical. in selecting an ideal test, the positive and negative predictive values, which are dependent on the prevalence of the disease as well as the characteristics of the diagnostic test, should be taken into account, but few studies have been performed to determine quantitatively what tradeoffs are acceptable. an analysis for syphilis screening compared the use of a laboratory-based immunoassay with treponemal rdts in tanzania 67 . it showed that a test that has 100% sensitivity but can only be used in sophisticated laboratories can realistically only be accessed by 30-40% of the population, while a rapid lateral flowbased antibody test that has only 90% sensitivity, but can be used at all levels of the health care system effectively gives a correct diagnosis to as many as 81% of the population, highlighting that tests should not be evaluated purely on technical performance, but rather on diagnostic performance and clinical impact. training and quality assurance. assuring the quality of tests and testing is the biggest challenge when testing is decentralized at poc. there are many rapid tests from various manufacturers available, and test quality may impact accurate and inconsistent diagnosis across different sites, with studies showing that high rates of errors were observed even in performance of simple rdts for hiv and malaria 15, 68 . quality of testing requires proficiency panels be sent to all the poc testing sites by a national reference laboratory. including positive and negative controls with each box of tests will allow all tests to be approved once they arrive at their destination and before first use. recently, nine african countries developed a national system for assuring the quality of poc testing for hiv 69, 70 . with data connectivity from each testing site, quality assurance results can be linked to test results at each poc testing site at a centralized database to trigger alerts for corrective action. supply chain software can also be linked through these connectivity solutions, avoiding stock-outs. demonstrating the value of a novel diagnostic technology. the value of a diagnostic goes beyond the technology, as each test needs to be matched to its 'testing environment' , which includes population characteristics, prevalence of target diseases, comorbidities/coinfections and health system characteristics. national programmes should take advantage of the rapid turnaround time of assured tests to streamline patient pathways and increase the efficiency of the health care system. the roll-out of poc diagnostics requires the use of implementation science to ensure success 71 , taking into account the cultural, behavioural, socioeconomic and health systems contexts. this includes a careful assessment of acceptability and feasibility linked to possible increased stresses on the health system/provider when testing is introduced into settings where thus far no or limited testing was performed. studies using the genexpert mtb/rif tb test showed that new diagnostic tests would not have the expected impact on outcomes if test introduction is not accompanied by changes in patient pathways or practices 72, 73 . these studies highlight the importance of programmatic monitoring of the impact of novel technologies beyond studies that are usually conducted under controlled conditions. likewise, same-day testing and treatment using a syphilis rdt strengthened health systems in the amazon forest and rural china when policy makers were involved and testing was introduced within the appropriate social and cultural contexts 71, 74 . for example, in peru, women normally need to present six times to the largest maternal hospital in lima for their prenatal syphilis screening to be completed and treatment provided, but the introduction of the rapid syphilis test streamlined testing and treatment to one visit, reducing patient out-of-pocket expenses and increasing the efficiency of a busy health care facility 75 . there are few detailed analyses which have been conducted to accurately determine the economic impact of many of the available tests, including the diagnostic cost versus the overall economic impact. while these benefits are intuitively important, they are often difficult to quantify 76 . it would, therefore, be extremely useful to have economic analysis tools for each of the major diseases or conditions, so that developers have an understanding of cost implications and what cost structures would be acceptable to health service implementers. as an example, it may be necessary to accept cost trade-offs when addressing global health threats (for example, zika, ebola, and so on), where speed of intervention is critical. this having been stated, low-cost diagnostic tests remain critical in resource-limited settings. in the past two decades, the biggest drivers of diagnostic development were well-resourced diseases such as hiv, malaria and tb. in recent years, the increasing frequency and severity of global health emergencies caused by infectious diseases of epidemic potential, such as severe acute respiratory syndrome (sars) coronavirus, middle east respiratory syndrome coronavirus (mers) co-v, ebola and zika virus, have made the global community realize the need to accelerate assured test development, validation, manufacturing and deployment. there is a need for innovation in rapid detection technologies that are assured but allow multiplex detection of a panel of pathogens such as major causes of respiratory illness, haemorrhagic fevers or enteric infections in a single specimen. these tests should not only identify the cause of outbreak but also be used to process multiple specimens with high throughput throughout the outbreak. another major driver of test development is the need for cheaper, better and faster tests that can be used at poc to reduce inappropriate antimicrobial use. an estimated 700,000 people die from untreatable conditions due to antimicrobial resistance (amr) every year worldwide 77 , and if amr continues to spread, by 2050, 10 million people may die from resistant infections annually at an economic cost estimated at $100 trillion. at the primary care level, a simple rapid test that can be used to distinguish bacterial from nonbacterial causes of fever, respiratory and enteric infections would allow health care providers to reduce antibiotic use and preserve them for future generations. the o'neill report 77 also pointed out that a test that can identify a pathogen and its antibiotic susceptibility at the poc would allow the safe use of first-line drugs or drug combinations with savings to the health care system. to stimulate interest in innovation in diagnostic tests that can be used at poc, several countries have set up challenge prizes. in 2015, the united kingdom announced the longitude prize of â£10 million, which seeks an affordable, accurate, fast and easy-to-use test for bacterial infections that will allow health professionals worldwide to administer the right antibiotics at the right time. the challenge is currently ongoing, with final submission by 2020. more information available at: https://longitudeprize.org. in september 2016, the us department of health and human services announced a challenge prize competition in which up to $20 million will be awarded for one or more novel and innovative poc diagnostics that would have clinical and public health value in combating the development and spread of antibiotic resistant bacteria. more information is available at: https://dpcpsi.nih.gov/amrchallenge. these prizes may catalyse new technologies, but there are still no guarantees to the sustainability of these tests in resource-limited countries given that cost is a critical factor woven into every step needed to bring about and sustain testing. donors and funders of diagnostics, such as the the bill & melinda gates foundation, global fund, unitaid and aid agencies from national governments such as pepfar and dfid, play a critical role in incentivizing diagnostic innovation and addressing market dynamics. technology advances to support innovation. technology 78 , markets and medical devices have matured to enable connected diagnostics to become a useful tool for epidemiology, patient care and tracking, research, and amr and outbreak surveillance. the ability to digitize data from laboratory and poc platforms, including lateral flow rdt results, can standardize the interpretation of results and allows data to be linked to proficiency testing to ensure testing quality, reducing interpretation and transcription errors. remote monitoring of poc instrument functionality and utilization through connectivity allows programmes to optimize instrument placement, algorithm adoption and supply management. alerts can be built into the system to raise alarm at unusual trends such as outbreaks. the application of mobile devices and related technologies to health care is improving patients' access to health information and treatment, offering possibilities to diagnose, track and assess the impact of infectious disease interventions across the world 79, 80 . smart devices can also be used to automatically add data to a central database and allow systems to assess likelihood of a diagnosis or predict disease outbreaks through machine learning, providing a route to smart diagnostics that incorporates historical or epidemiologic data to make diagnoses more accurate. finally, new engineering fields such as the internet of things (iot), industry 4.0 and printed electronics [81] [82] [83] [84] promise to add significantly to the technologies that can be chosen and incorporated into new diagnostic devices. many of these technologies are aimed at high-volume and low-cost distributable manufacturing, thus fitting in well with the goal of poc diagnostics. new detectors and light sources also suggest that incorporating these into tests could allow for more accurate result reading and the possibility of multiplexing. while it is clear that the original assured criteria remain relevant, opportunities exist to improve future diagnostics by incorporating new technological elements to provide real-time quality control for testing and treatment and overcoming the difficulties in specimen collection and/or processing 85 , which currently limits scaling-up of diagnostics in resource-limited areas. we therefore propose two additional criteria of r (real-time connectivity) and e (ease of specimen collection and environmental friendliness) into the original assured, to create a new acronym of reassured (table 3) . to use testing to effectively survey or treat patients, it is critical to obtain and analyse results at the poc 47 . however, one of the main challenges to poc testing is ensuring that results are rapidly provided to the patient once testing has been completed 86 ; this is formidable challenge when testing is decentralized at hundreds or thousands of different sites by health care workers who are not consistently skilled in reading test results, leading to risk that incorrect data is being recorded and/or transmitted. one solution would be to allow the analysis of test results at a centralized level for consistent diagnosis and epidemiological surveillance. many manufacturers are now embedding connectivity into poc and near-poc instruments (for example, the alere pima poc cd4 device 87 ). other manufacturers have developed innovative connectivity solutions that can use mobile phones to read the results from lateral flow assays and provide electronic result exchange, while a third option would be to include connectivity directly onto the test 88 . ideally, connectivity should not only include collection and transmission of test data, but also analysis to provide feedback for immediate patient treatment or for surveillance. with the addition of barcodes, two-dimensional codes or electronic storage into tests, a number of other data points can be collected, including manufacturing information (for example, lot numbers), dates, expiry dates, stock availability and possibly even environmental conditions such as temperature and humidity under which the tests have been manufactured, transported, stored and used. for instrumented tests, maintenance and other machine data can be collected. thus, connectivity solutions can increase quality assurance for poc tests and would allow for centralized and real-time decision-making, even across tiered laboratory systems and during outbreak investigations and global health emergencies. moving forward, it is critically important to make provisions for poor or non-existent internet availability to support sustainability standards and that systems are developed with compatibility so that they can be linked into central databases. ease of specimen collection. specimen collection and processing may need further in-depth development. first, it is ideal to have noninvasive specimen collection given that more tests become available as self or home tests. second, samples come in many different formats (blood, urine, sputum, stool, swab, breath, and so on) and require different preparation depending on the test to be performed. this may include concentrating the sample (or possibly titration), purification, lysing of cells, target amplification, and so on. while most of these processes can be easily performed in the laboratory, there remain few solutions for collecting and performing these tasks at the poc 89 . oral tests for hiv and hepatitis c virus are good examples of advances in non-invasive sampling, although the collection device is more expensive than blood collection via disposable capillary tubes. related, given that rapid lateral flow antigen detection tests often have lower sensitivity, extra processing steps such as heating or other forms of extraction are required when using specimens other than blood. environmental friendliness. advances in technology have made the assured requirement for equipment somewhat less daunting. however, in light of more diagnostic tests now being performed in both urban and rural areas, there could be more future effort devoted to the environmental impact of these tests. the cumulative effect of many tens of thousands of tests performed at remote sites away from laboratory infrastructure may pose health and environmental risks and put a strain on limited resources. some examples include the plastics used in current rapid tests that cannot be recycled and give out toxic fumes when burned, and testing cartridges that may even contain harmful chemicals and need to be disposed of properly. recyclable materials should be used, when possible, for the housing, substrate matrix materials, and reagents used in tests. paper, an obvious choice of substrate material that has many inherent advantages over standard plastic materials, will see increased usage, not only in lateral flow formats but other paper based formats as well such as in micro paperbased analytical devices (î¼ pads) [90] [91] [92] . and recently, cell-free synthetic gene networks for in vitro applications at poc, have been achieved by freeze-drying cell-free systems onto paper, which increases stability at room temperature and can be easily transported and stored, and simply re-activated by adding water 93 . other factors also need to be considered, such as disposal of test reagents, clinical samples and materials used for active components such as electronic tracks and electrodes. the assured criteria have been a valuable framework for developing devices and methods to detect major human diseases in challenging poc contexts. however, over the last decade, new technologies, not envisaged or available at the time of the first assured criteria definition, have given rise to the possibility of including new considerations into the next generation of devices and tests. we propose the acronym reassured for the design of future diagnostic tests to address important priorities such as global health emergencies and amr. an ideal test would be one that combines the best of both existing diagnostic worlds (instrumented laboratory based tests with lateral flow tests) and technologies currently being developed (fig. 2) , which would provide an important extension to diagnostic laboratory systems and fulfil the sustainable development goals of 'no one left behind' in terms of effective health care service delivery. such tests can only be created by forming strong collaborative partnerships across many disciplinary boundaries, and we look toward a future when data connectivity linking cost-effective assured diagnostics to laboratory systems will form the backbone of health care systems and provide real-time data for evidence-based disease control and prevention strategies, more 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frequency identification tags on flexible substrates, facilitating low-cost and integrated point-of-care diagnostics a low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami sensing approaches on paper-based devices: a review diagnostics for the developing world: microfluidic paper-based analytical devices recent developments in paper-based microfluidic devices paper-based synthetic gene networks printed microfluidic channels the authors declare no competing interests. reprints and permissions information is available at www.nature.com/reprints.correspondence should be addressed to x.-s.c. or r.w.p.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-150218-javbnjrg authors: gupta, prateek; maharaj, tegan; weiss, martin; rahaman, nasim; alsdurf, hannah; sharma, abhinav; minoyan, nanor; harnois-leblanc, soren; schmidt, victor; charles, pierre-luc st.; deleu, tristan; williams, andrew; patel, akshay; qu, meng; bilaniuk, olexa; caron, ga'etan marceau; carrier, pierre luc; ortiz-gagn'e, satya; rousseau, marc-andre; buckeridge, david; ghosn, joumana; zhang, yang; scholkopf, bernhard; tang, jian; rish, irina; pal, christopher; merckx, joanna; muller, eilif b.; bengio, yoshua title: covi-agentsim: an agent-based model for evaluating methods of digital contact tracing date: 2020-10-30 journal: nan doi: nan sha: doc_id: 150218 cord_uid: javbnjrg the rapid global spread of covid-19 has led to an unprecedented demand for effective methods to mitigate the spread of the disease, and various digital contact tracing (dct) methods have emerged as a component of the solution. in order to make informed public health choices, there is a need for tools which allow evaluation and comparison of dct methods. we introduce an agent-based compartmental simulator we call covi-agentsim, integrating detailed consideration of virology, disease progression, social contact networks, and mobility patterns, based on parameters derived from empirical research. we verify by comparing to real data that covi-agentsim is able to reproduce realistic covid-19 spread dynamics, and perform a sensitivity analysis to verify that the relative performance of contact tracing methods are consistent across a range of settings. we use covi-agentsim to perform cost-benefit analyses comparing no dct to: 1) standard binary contact tracing (bct) that assigns binary recommendations based on binary test results; and 2) a rule-based method for feature-based contact tracing (fct) that assigns a graded level of recommendation based on diverse individual features. we find all dct methods consistently reduce the spread of the disease, and that the advantage of fct over bct is maintained over a wide range of adoption rates. feature-based methods of contact tracing avert more disability-adjusted life years (dalys) per socioeconomic cost (measured by productive hours lost). our results suggest any dct method can help save lives, support re-opening of economies, and prevent second-wave outbreaks, and that fct methods are a promising direction for enriching bct using self-reported symptoms, yielding earlier warning signals and a significantly reduced spread of the virus per socioeconomic cost. dct, however, is not free of disadvantages. due to a wide range of privacy concerns about smartphone communications, dct suffers from poor adoption by the public [10] . additionally, most countries using dct have adopted a simple form which informs and recommends quarantine to all digitally-recorded contacts of cases confirmed through testing. we call these systems binary contact tracing (bct) because they recommend users either to quarantine or not (binary decisions) based on whether a past contact took place with a confirmed index case (binary input feature). covid-19 is a challenging disease to mitigate with bct for two primary reasons (i) bct currently relies on reverse transcriptase pcr (rt-pcr) tests which have high disease phase-dependent false negative rates. to make it worse, these tests are expensive, and may require a long time to obtain results [11, 12] (ii) the majority of transmissions of sars-cov-2 take place before the infector shows any symptoms, thereby reducing the likelihood that a potential infector would have been tested before transmission [13] . we observe that there are a wide variety of clues potentially available to a contact tracing app that would allow for non-binary, individualized recommendations, thereby offering significant improvements to bct. we call these methods feature-based contact tracing (fct), and hypothesize they could provide an important and effective means of reducing the spread of the disease, perhaps even more effectively than bct at lower adoption rates. recognizing this potential, we propose covi-agentsim -a software testbed 2 to design, evaluate and benchmark dct methods using cost-benefit analysis in terms of lives saved, reduction in effective reproductive number (r t ) of the virus, disability-adjusted life years (dalys) averted, and productive hours lost. by using an agent-based model (abm) as the foundation of this testbed, we are able to simulate a rich set of individual-level input features. covi-agentsim can be adapted to a region of interest by providing appropriate demographics and contact pattern information for that region. it can then be calibrated to match published data for that region of interest. we calibrate covi-agentsim to reproduce covid-19 case and hospitalization data for the region of montreal, canada. in order to ensure the simulator is a fair and reliable testbed, we also check that the relative ordering of methods is preserved across wide ranges of simulator parameters and over several metrics. we propose a simple rule-based fct method which leverages individual-level features to make non-binary recommendations, and compare this approach to bct and compare both to no-dct via cost-benefit analyses. we find that both bct and fct methods are able to reduce spread of the disease, and our results echo those of recent research [14] suggesting that dct methods can still save lives even at low adoption rates. we find evidence that fct approaches, which leverage rich individual-level features to make graded recommendations, are promising for improving dct even further. additionally, by stratifying dalys over age groups, we observe the most dalys averted per person for those over 80 years of age, even with low app adoption rates in that age group, thus showing the protective effects of younger people using dct. these results are conservative in estimating the benefits for the most vulnerable populations, since we randomly assign dct app usage proportional to smartphone usage, yet more vulnerable people (or those close to more vulnerable people) may be more likely to use dct. our results thus strongly support the usage of dct methods as a component of effective public health strategies, and we hope covi-agentsim will be a useful resource for development, benchmarking, evaluation, and improvement of dct methods. agent-based models (abms) are frequently used to study geospatial and other patterns of disease which vary at an individual level (e.g. [15, 16] ). they are thus often useful for studying differential effects of policy decisions and interventions on different subgroups of the population; for instance [17] use an abm to study which post-lockdown measures most effectively protect the most vulnerable, in terms of disease incidence, mortality, and icu occupancy, and [18] and [19] study patterns of covid-19 spread in different representative locations (university, workplace, and highschools), and the impact of different intervention scenarios in each of these locations. one of the interventions studied by these works is dct, and both find evidence that it can reduce icu admissions and help curb the spread of the disease. abms are also useful for modeling the impact of outlier individuals or events such as super-spreading, e.g. [20] , which are not easily captured by mathematical models of population-level spread. several works have studied the use of smartphone apps in epidemic management, e.g. [21, 22] , and some work has begun specifically on covid-19. for instance, ferretti et al [23] propose a mathematical model of infectiousness based on early epidemic data in china and compare binary contact tracing to manual tracing. they quantify the contribution of different transmission patterns (infection through symptomatic individuals, presymptomatic individuals, and from the environment) and the requirements for effective contact tracing. assuming a 3-day delay in notification (and thereby quarantine of the individual), the authors demonstrate mct could not bring r t below 1 and hence, could not control the epidemic. instantaneous contact tracing by a digital tracing application on smartphones could do so (r t <1). shamil et al. [24] follow a similar approach, but with an abm taking into account realistic contact patterns, studying the potential efficacy of bct in controlling the spread of the disease. they find strong dependence of the efficacy of digital contact tracing on app adoption, suggesting that bct alone is insufficient to control a pandemic unless over 60% of the population is using the app. [14] find similar results, emphasizing however that even at very low adoption rates dct is able to save lives. this suggests that dct should be considered an important component of public health strategy for mitigating covid-19. we find similarly that bct and fct are unlikely to control a pandemic on their own at low adoption rates (see section 8) , showing that even at 60% adoption rate these methods must be combined with other strategies to contain the disease. perhaps most similar to our work is hinch et al. [25] , who also propose an open-source abm which allows manual and digital contact tracing methods to be compared, with benefits stratified across age. developed concurrently to our simulator, similarities between the two approaches highlight the importance of several design decisions made independently but converging to the same solutions, e.g. the use of abms, a python interface, and the need for empirical testbeds of this nature. a key difference in our simulator is the rich set of individual-level features (including e.g. pre-existing medical conditions), which allow us to benchmark feature-based contact tracing methods, and also allow for stratification over a larger variety of subgroups. the cost of this level of detail is computation; our simulator models smaller populations at higher fidelity for the same computational budget. we perform a scaling analysis (appendix k) in order to ensure the dynamics we produce on these smaller populations are representative of larger populations. however, the simulator of [25] may be preferable for studying binary-only contact tracing methods, or when faster computation is needed relative to individual-level detail. the simulator is an agent-based compartmental model [26] implemented in python [27] and c [28] , using simpy [29] , a process-based discrete-event simulation framework. for each agent the simulator tracks transitions through susceptible, exposed, infectious, and recovered (seir) states, as well as a variety of individual characteristics, including pre-existing medical conditions, self-reported symptoms, and test results. this rich set of individual features enable the simulation of contact tracing apps which make use of such features. at the same time, we parameterize our simulator using real-world data when available, and when no data is available we make weak assumptions and investigate the sensitivity of our results with respect to these assumptions (see section 5.3). covi-agentsim simulates the spread of the sars-cov-2 virus in a city through contagion events between agents. simulator is initialized with a synthetic population along with the mobility and contact patterns informed by census and empirically derived data. it can be configured easily for any region of interest (see appendix f). each agent i in the simulation has individual characteristics (e.g. age, sex, pre-existing medical conditions) denoted x i . dwelling characteristics, workplace association, and contact patterns are derived from age-stratified surveys and empirical studies (see appendix e.2). at start of a simulation, a fraction α of the agent population is randomly exposed to covid-19. infection spreads through communities via contagion events at households, workplaces, schools and other random locations. agents move around the city transitioning between locations like households, workplaces and other locations. the pattern of each individual's mobility (i.e., which locations they visit, how often they visit them, and how they interact with other individuals at these locations) is set according to [30, 31] . while at a location, agents sample contacts according to age-stratified contact matrices derived from [32] . a detailed discussion of agents' mobility patterns and location dependent contact pattern is provided in appendix e. figure 1 compares simulated age-stratified contact patterns with surveyed matrices. virus transmission takes place anytime an infectious and a susceptible individual are within 2 meters of each other for at least 15 minutes, thereby possibly transmitting viral load to a newly infected agent. we model the probability of covid-19 transmission p according to [23] . borrowing notation from the authors, briefly, this probability is proportional to age-dependent susceptibility s a of the susceptible agent with age a, location-dependent multiplicative factor b n (for location n), symptom status (asymptomatic, mild, severe) dependent ratio a s of the infectious agent, and a surrogate for cumulative viral load (evl) transmitted from the infectious agent for duration δt. we discuss evl in the next section. a proportionality factor r is used to calibrate the reproductive number of the disease spread. for the sake of completeness, a mathematical form of this transmission model is presented in eq 1 and eq 2. where p (δt, s a , a s , n) is the probability of the contagion event. we use the same values for constants as used by authors in their open-sourced code 3 . after such a contagion event, we sample for the infected agent the variables controlling the course of the disease, including symptoms and severity. additionally, we sample a time-series of a quantity proportional to viral load as measured by [33, 34] , which we call effective viral load ev l; this quantity represents the interaction of the virus with the host's immune system. we further discuss (ev l), its dependence on age and preexisting conditions, and how it is sampled in appendix b. a model for sampling symptoms for each agent conditional on whether the agent has cold, flu, or covid-19 is discussed in details in appendix j. in covi-agentsim, we model rt-pcr testing and its relation to contact tracing applications. as discussed earlier, the disease trajectory for each agent depends on their age and preexisting conditions. thus, there are agents who experience symptoms on a spectrum from none (asymptomatic) to severe: agents who experience more severe symptoms are more likely to seek an rt-pcr test. given the limited testing capacity at the onset of the pandemic, we model a testing facility with a fixed maximum capacity. for an infectious agent, the outcome of a test is modeled according to disease phase dependent false negative rates as per [35] . as an example, 4 days after a sars-cov-2 infection, the infected agent will have a false negative rate of 67%. upon receiving a positive test result, the agent is put to self-isolation for d max days with a probability of not following such interaction modeled via dropout parameters. we discuss details about testing in appendix c and hospitalization in appendix d. unlike existing covid-19 abms, our agents are designed to follow varying levels of contact patterns. for example, number of contacts sampled by an agent in level k at location l corresponds to a fraction (γ l k ) reduction in contacts with respect to pre-pandemic number of contacts for that location. these pre-pandemic number of contacts are available through surveyed studies [32] which we discuss in appendix e. thus, if there are n + 1 such levels ranging from 0, 1, ..., n, an agent at location l in level 0 will draw contacts using pre-pandemic number of contacts (γ l k = 0), and an agent at location l in level n is in quarantine i.e. samples no contacts (γ l k = 1). fraction γ l k for intermediate levels k ∈ {1, ..., n − 1} are obtained by interpolation scheme (e.g. linear) between γ l 0 and γ l n . our choice is motivated by the desire to have a simple model grouping together the effect of choices individuals can make to reduce their likelihood of becoming infected like washing hands, wearing a mask, and physical distancing. each of these levels are further associated with a dropout parameter that represents the fraction of time an agent in level k will drop to level 0, returning to a higher level of activity (specifically, pre-pandemic numbers of contacts). covi-agentsim can be configured to quarantine individuals due to any of the following triggers (a) confirmed positive test (b) self-reporting of symptoms, (c) recommended by the app, and (d) household member of any of the above cases. additionally, in order to model population-wide mobility restrictions we use a bernoulli distribution with parameter β ∈ [0, 1] to sample contacts. thus, an agent that usually draws 12 contacts will now draw β * 12 contacts on average. this modeling choice is a simplification of the varying degrees of government imposed mobility restrictions, controlled by a population-wide β. dct methods rely on capturing high-risk contacts and communicating risk information. the population-level app adoption rate is a key parameter because the fraction of contacts captured by this system will be proportional to the square of the app-using fraction of the population. to distribute apps throughout our simulated population, we use an age-dependent distribution across smartphone owners. we use an age-based breakdown of smartphone users as in [23] , and use an u p t ake parameter to vary the population-level adoption rate. if an agent is assigned an app, there is a provision to report individual characteristics like age and preexisting conditions as well as daily symptoms. we further model dropout rates for reporting symptoms as well as a "drop-in" rate for falsely reporting symptoms to account for malicious users and other confounding factors that produce symptom inputs, such as colds or flu. when app users i and j have a significant encounter on day d (which can lead to contagion) the ct method can exchange messages between them, on the same day or later. we have considered two kinds of messages: a contact message exchanged on day d allows i and j to keep track of the encounter event (such as when it happened) as well as a handle allowing them to send each other messages later (potentially via some server), while minimizing breaches in privacy. a contact message could also contain information about the current degree of contagiousness risk they pose to each other on day d. later, if i observes new clues suggesting their contagiousness on day d was actually higher, i can send an update message to j, with information which can help j update their own evaluation of contagiousness. we denote m d i,j (t) the risk message sent on day t by i to j regarding their encounter on day d. if t = d this is the initial (contact) message, whereas for t > d this is used to update the information j will have at day t about their encounter with i on day d. note that i may send multiple update messages to j, as they acquire more information about having been infectious on day d. additionally, we restrict the frequency of communication: if t is the time of the last risk message sent by i about some encounter, the next message is sent the clues which agent i may use to come up with its risk messages include symptoms, test results, pre-existing conditions and received risk messages. the way to come up with these risk messages, as well as how to adjust behavior based on estimated risk, is specified by the ct method, such as those described below. in digital contact tracing we have two goals: to reduce the individual's spread of disease (by recommending a reduction in mobility to risky individuals), and to inform contacts of additional risk. agent's adherence to a recommendation level k entails sampling location specific contact patterns according to that level (see section 3.6). we denote agent i's behavior level on day d by ζ i d such that for a total of n + 1 behavior levels, ζ i d ∈ {0, 1, ..., n}. determination of this recommendation level is done via a risk estimator that uses a rich suite of features to evaluate agent i's risk history on day t. that risk history is denoted r i t , where we constrain risk levels to be non-negative integers with a maximum value of r max . we use r i t,d to denote estimated risk of agent i for day d such that t − d max < d < t. if an agent i had an encounter with an agent j on day d such that t − d < d max , r i t,d is sent to the past contacts as a risk message m d i,j (t) = r i t,d if j was a contact of i on day d (see section 3.7) . this determines what information is available to agent's contacts such that they may modify their own behavior or to propagate risk further. useful notations we use n (i) to denote a set of agent indices that had at least one digitally recorded contact with agent i in the past d max days. further, we use the colon symbol (:) to iterate through all possibilities for the variable at that position. for example, m d i,: (:) represents risk messages from agent i to all agents j ∈ n (i) for encounter on day d, if there was one. similarly, m : i,: (:) represents risk messages sent by agent i to agent n (i) within the past d max days. we the most common class of digital tracing methods, binary contact tracing (bct), can be viewed as a binary classifier with final decision (behavior recommendation) being whether the agent should quarantine or not. most often, the decision boundary is simple: did the individual had a recent contact with someone who received a positive rt-pcr test? we refer to these methods as test-based bct, which we describe formally next. in test-based bct method, for an agent i with test d i = +1, agents j ∈ n (i) are notified and recommended to quarantine themselves as discussed in section 3. we call this particular method bct1 because it only affects the immediate contacts (and their household members) of individuals with positive test results; in section 5.3 we show results for this baseline, bct2, where second order contacts may also be quarantined. we describe a class of methods we call feature-based contact tracing (fct), which leverage the potentially rich set of features available on a smartphone to make graded recommendations. as discussed earlier, we make use of following available information on an app to update an agent i's estimated risk history r i d on day d: i,: (:), and (e) previous estimated risk history r i d−1 . the agent's estimated risk for the past d max days is then propagated as discussed in section 3.7. in addition to risk estimation, the agent's behavior is set to a level ζ i d based on the estimated risk for that day i.e. r d,d . in this section, we describe one such rule-based implementation of fct -heuristic-fct which forms the basis of our experiments. with the help of available information about how covid-19 spreads and manifests itself in an individual in the form of symptoms, we designed a rule-based fct method. specifically, for every available aforementioned feature type, we determine the agent's risk history for the past d max days. the agent's risk history on each day d is taken as the maximum across these per-feature estimated risks. broadly, heuristic-fct uses the following rules: • test results, t i d : the agent's risk is set to r max = r max if there is a positive test result in the past d max days. this rule takes priority over any other rules (assuming that a positive test gives us maximum certainty about being in the top risk level). • symptoms, s i d : we identify three categories of symptoms based on how indicative they are of covid-19. the presence of a highly informative symptom in s i d results in a high risk level r high ; a moderately informative symptom results in a moderate risk level r moderate ; and a mildly information symptom results in mild risk level r mild . we assign these risk level values for the past d max /2 days in r i d , similarly to [25]. • risk messages, m : i,: (:): the risk of an agent receiving a risk message r max is estimated to be ρ = r high , while one receiving r high is estimated to be at ρ = r moderate , and so on. the rationale is that the level of risk decreases rapidly as we move away from one agent to its contacts, to the contacts of these contacts, etc. we then compute the duration of time when agent i could have been infectious if this contact had caused their infection as d + 1 < d < d. thus, we set the agent's risk to this value ρ until d days in the past. • other rules: there are some rules that are designed to override the above rules. for example, an agent with a negative test on day d is assigned a risk of 0 from that day onwards. further, an agent is estimated to be at risk level 0 if there had been no positive test in d max days, no symptoms in the last d max /2 days, and no high, moderate of medium risk messages in a certain past time horizon. we present a formal description of this risk estimator in appendix i. in this section we seek to provide following evidence: (1) our simulator is producing a reasonable approximation of real-world covid-19 dynamics, and (2) it is a reliable testbed for comparing contact tracing methods. we address (1) by checking the output epidemiological characteristics match published literature (see figure 2 and table 1 ), and by checking that the hospitalization and mortality statistics are well-aligned with those found in real world data ( figure 3 ). we address (2) by performing a sensitivity analysis of the simulator to a wide range of parameters, and checking that the ranking between contact-tracing methods is preserved over these settings, for different metrics. we calibrate the simulator so that the observed statistics in the simulator are similar to what is observed for covid-19, plotting seir curves in 2 and comparing statistics to published data in 1. simulator reported numbers are average µ in days and corresponding 1 standard deviation σ, computed over 10 random seeds on a population size of 3000. it is important to note that these statistics are a result of the many processes happening within the abm; there are no parameters that specifically encode these values. we run 10 simulations using random seeds and plot the mean and standard deviation of the hospitalization and mortality statistics over 100 days. we compare these results to the real data reported in montreal from the same time period. our simulations use 30,000 people and we report results as a proportion of population. we see that under these settings the proportion of the population that is hospitalized or deceased aligns with the data from montreal (see figure 3 ). a primary contribution of this work is the creation of an abm which can act as a testbed for comparing covid-19 contact tracing methods. while the majority of the parameters in our abm are chosen according to published literature, much about covid-19 is still unknown or uncertain. for this reason, we conduct a sensitivity analysis of some key parameters which exhibit high variance across different studies. specifically, we study the impact of the asymptomatic proportion of the population (see figure 4 ), which is a difficult to measure without widespread serological testing, and the initially infected proportion of the population ( figure 5 ). we observe that the relative efficacy of different methods (i.e. the ranking of methods) holds across a wide range of settings, a desirable characteristic for a comparison testbed. we compare against the real hospitalization and mortality data (as a percentage of population) during the first wave of covid-19 in quebec. it is important to note that we only report a post-lockdown scenario. we report results with our simulator from 10 runs with a population of 30,000 people. figure 4 : sensitivity to asymptomaticity: these figures show a comparison between 4 contact tracing methods (including the no tracing setting) and varying the proportion of the asymptomatic population between 0 and 100%. we plot two views of the same data with different y-axes: fraction infected andrt. the relative performance between the methods are consistent across rates of asymptomaticity and choice of y-axis. we report the mean and standard deviation across 5 seeds with a population of 3000 people. on the right, we see a counter-intuitive result: thert at the end of the simulation is lower for simulations which started with a larger initially infected population. this can be explained by noting thatrt in some sense reports the instantaneous change in rate of spread of the disease while the fraction infected reports the absolute extent of the spread. crucially, the ranking between the methods remains similar across choice of visualization and initially infected population. we report the mean and standard deviation for 5 runs under each condition with a population of 3000 people. we focus our analysis on the region of montreal for the period between march and june. our agents are initialized with dwelling and workplace characteristics informed from publicly available census data [38] . we use n + 1 = 4 recommendation levels γ i , with γ 0 = 0 (full pre-pandemic mobility), γ 4 = 1 (full quarantine), γ 3 as per post-lockdown contact patterns reported in [39] , and rest of the levels as γ k = γ k+1 /2. given a memory intensive infrastructure required for managing risk messages, we run simulations on a smaller population size of 3000 people. we initialize each simulation with 0.2% of the population as infected (6 infections), and run 10 different seeds for each value of β ∈ {0.25, 0.275, 0.30, ..., 0.85} resulting in a total of 420 runs. these values are chosen so that we can get estimates ∆r of the change in the reproduction number of the virus, as discussed in section 6.3. we compare the heuristic-fct method proposed in section 4.2 with the test-based bct method discussed in section 4.1, and a no tracing scenario. the scenario of no tracing corresponds to agents initialized at level 1 (post-lockdown contacts with some restriction advised) instead of level 0 (pre-pandemic contacts), in order to compare our methods in the context of the scenario where economies are gradually reopening. we use test-based bct1 and test-based bct2 to distinguish between the two variants when necessary, otherwise we use test-based bct to imply test-based bct1 which is the method being considered by most of the countries. we compute the following metrics to evaluate various scenarios: • average number of contacts per day per human (c): we empirically compute the average number of daily contacts per agent in simulation. • proxy r (r): we use an empirical calculation to estimate the reproductive number r, we call this estimater. at any timepoint in the simulation we may approximate r t byr t , by computing the infection tree and taking the ratio of number of children number of parents , where parents are recovered agents. this ratio gives an approximate rate of growth of the tree. we user t at the end of the simulation as ourr. • daily cases (%): percentage of population exposed, i.e., new cases on any given day of a simulation. • cumulative cases (%): percentage of agents in exposed, infectious, or recovered state up until a particular day of a simulation. • prevalence (%): percentage of population in infectious or exposed state on any given day of a simulation • incidence (%): number of agents exposed per 1000 susceptible agents on any given day of a simulation. it is also referred to as attack rate. as the contact tracing methods proposed in this paper change agents' behaviour to varying degrees, it is crucial to compare different methods across similar social mobility restrictions. for example, bct can only use levels 1 and n while fct can use all levels from 1 to n. therefore, at the same value of β, bct will likely under perform as it can not reduce infections by using intermediate levels. thus, for a fair comparison of bct with fct, we run simulations with varying values of β ∈ (0, 1) and match them for average number of contacts. to compare the performance of different methods for the same mobility restriction we empirically compute pairwise difference in meanr for a fixed number of contacts c. this is achieved by obtaining the performance in terms ofr of a method across a spectrum of values of c (by varying β), and fitting a gaussian process (gp) regression [40] to obtain a functional dependence for each method between c andr. we denote this fitted regression for method a byr gp a . to compute the advantage of method a over b, we find note that r = 1 is the threshold between exponential growth and exponential decay of the virus and serves as a good point of interest when comparing methods since the objective is to choose a method which brings r below 1, all else being equal (e.g., general restrictions on social mobility). figure 6 shows a comparison of the gp regression fits between aforementioned dct methods at 60% adoption rate, and a no tracing scenario, across a range of values of c. we observe that heuristic-fct significantly improves over test-based bct by reducingr by 6.7%. of note is the region around c = 5.61 ± 0.5 wherer = 1.2 for no tracing. we compare the performance of dct methods in this region to set our comparisons in the context of current scenarios (partial lockdown) where government-imposed restrictions keep r under control. to further investigate the reason for the improvement of heuristic-fct over test-based bct, we peek into the simulations in the concerned region of c. figure 7 shows that on average, heuristic-fct exhibits lower incidence as well as prevalence on any given simulation day. this is expected as heuristic-fct makes use of far richer features as compared to binary test results used by test-based bct to evaluate individual's risk of infecting others. finally, due to network effects, it is likely that adoption rate plays an important role in the performance of dct methods. thus, we evaluate dct methods at various adoption rates. to do this, we run simulations of dct methods at different adoption rates in a similar way as explained in section 6.3, and concern our analysis on simulations chosen according to the selection criterion described above. figure 8 shows the performance of the considered dct methods at different adoption rates. as expected, the performance diminishes with lower adoption rates, for all the methods. however, we observe that heuristic-fct retains its advantage over bct methods even at the lower adoption rates. at the same time, we also note that poor adoption brings both the dct methods close to no tracing, figure 6 : pareto front. we compare dct methods at 60% adoption rate, and a no tracing scenario. for each method, a gp regression is fit as discussed in section 6.3, approximating a trade-off between mobility and spread of disease. we plot the mean fitted function along with 95% confidence intervals. relative to the no tracing scenario, we observe a statistically significant 17.4% reduction inr by heuristic-fct as compared to 10.7% by test-based bct method. figure 7 : case curves. we investigate the dynamics of the simulations chosen as per the criterion mentioned in section 6.4 to compare the performance of dct methods at 60% adoption rate in the context of partial-lockdown. shown are the mean with one standard error band of the metrics described in section 6.3. simulations under heuristic-fct exhibit lower attack rates (incidence) as compared to test-based bct method, thereby explaining the advantage visible in figure 6 . thereby making it more difficult to measure significant advantages over the partial lockdown scenario. thus, we argue that by continuity while at any adoption rate dct methods can save lives, adoption rate is crucial for their efficacy. figure 8 : effect of adoption rates. performance of dct methods is heavily dependent on adoption rates. however, heuristic-fct retains its advantage over test-based bct across this spectrum. covid-19 has been a challenge for health agencies and economic policy decision makers alike. countries around the world have taken unprecedented measures to prevent the collapse of health and economic welfare of people. yet, at times, these two objectives have seemed to be at odds with each other in efforts to contain the pandemic. therefore, we acknowledge that the evaluation of dct methods should also stand on sound assumptions of utility maximization in economic theory. in this section, we attempt to relate simulation dynamics to economic outcomes that policy makers can use for decision making. to assess the socio-economic burden of covid-19, we examine the following metrics: (a) disability-adjusted life years (dalys) averted [41] , a measure of lost years of healthy life, and (b) temporary productivity loss (tpl), a measure of economic cost to society of restrictive measures. of particular interest is the trade-off between these two measures. we use tpl per daly averted to compare the cost-effectiveness of dct methods with respect to the no tracing baseline. this can be thought of as an incremental cost-effectiveness ratio (icer), which answers the following question: how much more does each unit of additional benefit (in averted dalys) cost with respect to the no tracing baseline? a breakdown of the methodology used to calculate dalys and tpl can be found in appendix l. one assumption made in computing tpl is that total foregone work hours due to quarantine are scaled by a factor of 0.49 [42] to account for the proportion of agents able to work from home. to assess the impact of the aforementioned ct methods on dalys and tpl, we run 10 simulations of 3000 agents for each method. for a reliable economic analysis, it is necessary to have data on the full trajectory of simulations reaching a post-epidemic steady state comprised of agents that are either susceptible or recovered. such a trajectory would help assess whether the dct methods actually avert the loss of life years, or simply delay them. since ct methods are used to stop individuals from spreading the disease, it is important to know whether the ct methods have successfully reduced the overall proportion of individuals, or if they have simply delayed these infections. if the simulations are only run for 60 days with 0.2% infections seeded at the start, then the epidemic has still not reached a post-pandemic steady state, resulting in a possible overestimation of the benefits of the aforementioned ct methods. thus, we run longer simulations of 90 days with 5% infections seeded at the start of simulations. this particular choice lets us draw preliminary insights into the cost-effectiveness of ct methods without having to scale up the simulations which are extremely compute intensive. as a first step towards understanding simulation dynamics in terms of healthcare and economic costs, we compute dalys averted and tpl for each scenario considered in section 6 under the conditions described above. we use no tracing as a reference scenario to independently evaluate differential benefit of dct methods over doing nothing. icer for no tracingunder the assumptions discussed at the onset of this section, our experiments (results in table 2) suggest that heuristic-fct saves almost six times more dalys than test-based bct, a significant number of life years saved. however, we note that the tpl of implementing test-based bct is 30% that of heuristic-fct: higher proportions of agents are quarantined under heuristic-fct. finally, we note that the cost per daly averted ofheuristic-fct is 58% of that of test-based bct. thus, the cost per healthy year of life saved by heuristic-fct is lower than the test-based bct method. icer for test-based bct to quantify the cost-effectiveness advantage that heuristic-fct provides over test-based bct, we also calculate the icer of heuristic-fct over test-based bct. on average, heuristic-fct saves (58.12 − 10.01) = 48.11 more dalys than test-based bct. however, it costs ($755k − $223k) = $532k more as well. therefore, the icer of heuristic-fct over test-based bct is 532k 48.11 = $11k per daly averted. dalys per age group since there is evidence in the literature that covid-19 disproportionately affects the elderly [43] , we stratify the dalys per person by age in figure 9 . it is highest among people aged 80-89 years for both ct methods, which is consistent with the literature. it is worth noting that the mean of heuristic-fct performs at least as well as test-based bct across all age ranges, and notably better for agents aged 60 and over. figure 9 : impact of different ct methods on dalys, binned by age group. dalys per person are total dalys per age group over the size of each group. heuristic-fct consistently outperforms test-based bct across older age groups, most vulnerable to covid-19. we attempted to create an easily configurable platform to assist in evaluation of dct methods and variants thereof. further, we proposed an fct method that has a potential to improve upon bct methods, that is widely used in practice. however, we would like to point out some of the limitations our work. first, although most of the parameters in covi-agentsim are informed from published literature, there are assumptions we had to take in the absence of available data. in this paper, we have enlisted most of these parameters and corresponding references that informs them. it is important to note that as more about covid-19 will be known these parameters are subjected to change, thereby affecting results in section 6. additionally, we introduced intermediate levels of user behavior to contrast fct with bct. this is done with the help of introducing a factor γ n for level n that represents fraction reduction in number of contacts relative to pre-pandemic number of contacts (γ 0 ), empirical estimates of which have been widely used in epidemiological modeling. to obtain intermediate levels, we used interpolation such that γ k = γ k+1 /2. although it is trivial to experiment with various interpolation schemes, in the absence of user-behavior research, there is not enough that can be done in this regard other than providing the sensitivity analysis of the parameters involved therein. in addition to this, we foresee the use of such assumptions to be translated into government policies such that resulting number of contacts can approximate these assumptions. second, our modeling framework of fct relies on certain assumptions of technology which might not hold in practice. for example, our assumptions of proximity detection using bluetooth signals (appendix g) might be unrealistic. however, [44] 's work on improving the reliability of bluetooth signal can be a way to address this. further, we assume the app to be active (in foreground) to be able to communicate with nearby phones all the time, an assumption that might not hold depending on the app design. there are a variety of such technological and ethical considerations which are discussed in [45] to design a successful peer-to-peer fct app. covi-agentsim incorporates most of the technological assumptions in [45] , however, with additional effort, other assumptions can also be incorporated in covi-agentsim to evaluate dct methods in different settings. third, we designed the heuristic-fct method using rules that were informed by domain knowledge about covid-19's spread characteristics. at this point, we acknowledge that there should be room for improvement in these rules which can be brought upon by amalgamation of ideas in disciplines such as epidemiology, user behavior research, computer science, and statistics to name a few. alternatively, machine learning methods could be used to learn such rules. thus, we think that a unified mathematical framework to analyze fct methods might help further in development of better fct methods. this work presents covi-agentsim, a simulation testbed for evaluation of dct methods. covi-agentsim is an agent-based compartmental model that is initialized with a synthetic population sampled from the census data. daily activities as well as interactions for each agent are sampled according to the empirically derived contact matrices. we calibrate covi-agentsim to approximate the spread of covid-19 virus to the region of montreal, however, the simulator can be easily configured for other regions via change of an appropriate configuration file. finally, we propose the fct class of contact tracing methods that utilize a richer set of input features as compared to bct methods (which rely on binary signals like presence or absence of a positive test result). in doing this, we aim to provide infected individuals with a warning signal earlier than bct methods. to put fct in practice, we designed heuristic-fct which uses hand-designed rules to inform an individual's risk of infection and infectiousness to others. our empirical results show that heuristic-fct results in 6.5% improvement in r t over bct methods, and both the methods themselves provide a significant improvement over a partial lockdown scenario. experiments with varying adoption rates suggest that the efficacy of dct methods is heavily dependent upon adoption rates. it is, however, observed that heuristic-fct retains its benefit over test-based bct method across the adoption rate spectrum, but this advantage was not statistically significant in the face of very low adoption rates, at the scale of our simulations. using an agent-based compartmental model as the foundation of this testbed allows us to simulate a rich set of individual-level features, which we show can potentially be leveraged by dct methods to improve over the existing bct methods. we hope that the baselines established in this work will encourage and enable the informed development of dct methods as a first step in their responsible deployment as an epidemic intervention tool, potentially saving lives at lower economic cost during deconfinement and/or second-wave prevention. finally, this work joins a growing body of work in considering novel methodologies for rigorous evaluation of interdisciplinary technologies. epidemiology is fundamentally an intersectional science, touching sociology, biology, behavioural psychology, geography, political science, ecology, mathematical and computational modeling, and many other fields, in a society which is increasingly digitized and globalized. by working together across fields, with careful empirical study, we have hope in dealing with the important issues we face. a major direction for future work is to benchmark a wider variety of ct methods, including probabilistic and machine-learning based methods which could make even better use of the features our simulator provides than the heuristic-fct proposed here. the data generated by our simulator are potentially of interest for training such models to estimate individual-level characteristics which are predictive of the spread of the disease. our cost-benefit analysis using dalys and tpl is a first step towards an integrated framework to help policy makers in their decision process. we see imbuing economically sound decisions in our simulated agents as a step in creating an integrated framework for richer evaluation of dct methods. although covi-agentsim is designed to evaluate dct methods, we foresee directions for the simulator to investigate the impact of a gamut of non-pharmaceutical interventions on containing covid-19. for example, with some work and expertise in manual ct methods, one can compare and evaluate various variants thereof. another example is to analyse various covid-19 testing strategies in conjunction with dct methods. studies of this nature could potentially help public health agencies as well as policy makers in their decision process. a far cry, though worth mentioning, is the fact that covi-agentsim has essential components for simulating an outbreak, thereby enabling adaptation to other infectious diseases like influenza or tuberculosis. [19] giulia cencetti, gabriele santin, antonio longa, emanuele pigani, alain barrat, ciro cattuto, sune lehmann, and bruno lepri. using real-world contact networks to quantify the effectiveness of digital contact tracing and isolation strategies for covid-19 pandemic. medrxiv, 2020. [20] yunhwan kim, hohyung ryu, and sunmi lee. agent-based modeling for super-spreading events: a case study of mers-cov transmission dynamics in the republic of korea. a test, this agent is added to the queue. for different symptom severities, we assign a different probability of seeking a test. at some points during the pandemic, montreal experienced a restricted testing capacity. in order to model that setting, we limit the daily testing capacity to a proportion of the population (generally set to 0.1%). to determine which people get tested under such a restriction, we allocate tests to people with more severe symptoms and app-based recommendations. after a test has been conducted, there is a delay before results are returned (2 days in our experiments). during this period, the individual who is being tested is isolated, and if the test returns positive then an additional 12 days of isolation is recommended. any quarantine which is applied to the person receiving the test is also applied to other members of their household. we adopt a simple model of hospitalization and interaction within them. to simulate post-lockdown scenarios, we assumed no infections in hospitals. we adopt a probabilistic model of hospitalization where likelihood of being admitted to the hospital or icu depends on symptom severity and age. mortality rates are conditioned on age-group following data from quebec public health (date?) [59] . the duration of a hospitalization, likelihood of requiring critical care, and mortality rate given critical care by age follow nationally conducted surveys available publicly 5 . the number of hospitals is defined in relation to the population, using the same ratio of hospitals to people as are found in quebec (1.99 hospitals per 100,000 people). the number of hospital beds per capita, and occupancy ratios are taken from 6 , and the number of icu beds per capita and occupancy ratios are taken from 7 . hospitals are staffed by doctors and nurses, who are modelled as people with a profession that requires they work at the hospital and have protected interactions with patients. literature on the link between underlying medical conditions and covid-19 encompasses risk of being hospitalized due to covid-19, risk of severe complications (e.g. mechanical ventilation) and risk of mortality. preexisting medical conditions were used to model: 1) hospitalizations and deaths outcomes and 2) conditional probability of symptoms. to model hospitalizations and deaths in the population, we used risk ratio estimates from studies focusing on risk of hospitalisation and risk of death as outcomes. risk ratios adjusted for other individual characteristics were preferred over crude estimates. to inform the hospitalisation and death outcome model from the simulator, we selected the following risks ratios: diagnosis of heart disease, 1.17 [48] , stroke history, 2.16 [48] , asthma with recent oral corticosteroid use, 1.13 [48] , copd, 1.08 [47] , cancer (excluding haematological malignancy), 1.72 [48] , diabetes, 2.24 [47] , obesity stages 1 and 2 (body mass index (bmi) = 30-39.9 kg/m 2 ), 1.8, obesity stage 3 (bmi >40 kg/m 2 ), 2.45 [47] , ckd, 2.60 [47] , immuno-suppressed because of asplenia, 1.34 and because of immunosuppressive conditions (excluding asplenia and haematological malignancy), 1.70 [48] . given the uncertainty on the association between smoking and covid-19 prognosis [56] , we did not consider this risk factor in the simulator. in this section we describe contact patterns in pre-pandemic situation. scenarios of lockdown and contact patterns in intermediate behavior levels are a modification by a factor γ n as discussed in the section 3. we use empirically derived matrices in 2017 from [32] for canada that we further project on to montreal's demographical structure. projection of country-wide matrix to a regional matrix is done via method described in [60] . however, abm can be configured to bypass the step of regional projection of contact matrices. given a discrepancy between population wide mean daily contacts inferred from projected matrices and montreal's number of contacts reported in a 2020 survey [39] , we scale the projected matrices appropriately. we ran 12 simulations with no infected agent i.e. α = 0 to observe the pre-pandemic contact patterns to yield simulated contact patterns in this section. additionally, the simulated contact patterns shown in this section are descaled with the same multiplicative factor that is used to scale the projected matrices. as discussed in the main section, agents are grouped into houses according to census data [61] . thus, we simulate dwelling characteristics of the city of montreal. however, it can be configured easily for any other city by using appropriate parameter values. we consider house of sizes ranging from 1 to 5. age distribution of agents living solo also follows census data. further, house sizes ranging from 2 to 5 consider three broad categories of dwelling characteristics -(a) couple with x kids, (b) single parent with x kids, (c) random allocation, where x represents number of kids required to complete the house size. for example, for a house with single parent and of size 5, x = 4. the distribution of these characteristics also follow from census data. finally, we also consider senior residencies where a proportion of agents above age 65 live. we inform this proportion from the census data as well 8 . we explicitly model older adults living in assisted care resulting in oversampling of contacts in that age group. we discuss it further in the appendix e.2 as a result of housing allocation discussed above, we yield a contact pattern as shown in figure e .12. we make two observations (a) there is an oversampling of contacts towards the older age groups: it is because older agents grouped in collectives like senior residencies are modelled explicitly. this choice was motivated from [62] which suggests inclusion of collectives in proper response to the covid-19 pandemic. (b) a slight discrepancy we observe in the intensity of the main diagonal is due to insufficient social gatherings at households. we consider an age-dependent workplace allocation such that agents in each age group have a probability of attending a school or a workplace. we consider schools for the following age groups (a * ) 2-4 years old (y.o) (b) 4-5 y.o (c) 5-12 y.o (d) 12-17 y.o (e * ) 17-19 y.o: (f * ) 19-24 y.o (g * ) 25-29 y.o, where * marks the age group in which only a fraction (informed by census data) of agent population was allocated schools. further, we assume 100% employment so that all agents older than 17 y.o and younger than 65 y.o. were allocated a workplace. agents in senior residencies are allocated a common room as their workplace where they get together during working hours. such allocation of younger agents to schools give a contact pattern as shown in figure e to model infections at locations other than workplace and house, we consider locations where agents remain for a relatively shorter duration as compared to house and workplace. specifically, we model interactions at locations like restaurants, grocery stores, and parks. note that this category of locations is also termed as "other" in [32] . further, as the mean number of contacts at house are greater than the number of residents, it was important to consider socializing activities organized at houses. to do this, we maintain a pool of agents that an agent interacts with, and bring them together for a social activity at either a restaurant or at the agent's house. we discuss scheduling of these activities next. a contact pattern resulting at these random locations is shown in figure e. 15. we consider adult supervision for agents below 14 y.o i.e. except for when agent goes to school, at least one adult agent (older than 14 y.o) has to be present all the time. thus, we pre-plan the schedule of agents older than 14 y.o at the start of the simulation, and plan the schedule of agents younger than 14 y.o during the simulation. planning the schedule takes into account workplace opening hours as well as regularly scheduled activities like social gatherings 9 , exercising, and grocery shopping 10 . thus, an agent who has gone to a grocery store or a restaurant on one day will be less likely to go again during that same week, and so on. the schedule additionally depends on the day of the week. for instance, agents with school as workplaces are scheduled to be at school on weekdays, whereas most of their time will be spent at home on weekends. on the day of activity, however, these activities might stand cancel due to sickness, quarantining requirements, or hospitalization. in these situations, location of activity is appropriately changed for a required duration. at the same time, if an agent requiring adult supervision is sick, has to quarantine, or is hospitalized, an adult from the same house has to cancel the activity to stay with the agent. of note, agent's mobility i.e. presence in locations other than house is reduced when they are experiencing symptoms (to a degree proportional to symptom severity). note that we do not change the schedule unless the agent is quarantining i.e. normal mobility is maintained all the time unless the agent is put in the level n. figure e.16 show a breakdown of contacts at each location on weekdays and weekends. we implement age-stratified contact sampling as informed from empirically derived contact matrices as described in appendix e.1. specifically, for each agent we draw number of contacts as per the location-specific agestratified number of contacts obtained from the contact matrices. we use a negative binomial distribution [64] to draw number of contacts. further, we use these matrices to infer probability of interaction with other agents in each age group, thereby, implementing location dependent assortativity in interactions. we call these interactions as encounter. finally, we also figure e.16: simulated mean daily contacts on weekdays and weekends broken down by age groups. agent activities are scheduled such that the mean number of contacts on work and non-work days follow surveyed data as reported in [63, 37] draw amount of time spent in each encounter as per the survey conducted in california [65] standardized to the demographics of montreal. thus, we obtain an aggregated contact pattern as shown in figure e.17 . we briefly describe the required steps for customization of the simulator. location-specific demographic and contact data may be modified simply by adding a new configuration file to the configs folder. configuration files are written using yaml, a human friendly data serialization standard. essentially, these files contain key-value pairs. the values for a new region must be specified and contained in the new configuration file. examples of modifications required to model a new region include: population-level distribution of age, housing distribution i.e. number of houses from size 1 to 5, occupation characteristics including age for kids to go to schools, retirement age, etc. and, finally, how often people go out to stores, socialize, we provide details of the messaging protocol that uses bluetooth signals to exchange tokens. the privacy protocol which provides anonymous message exchange between phones which have exchanged these tokens is covered in more depth in another paper 11 . in the context of the current work, we focus on the description of how we model the bluetooth communication range of phones and on the description of the clustering algorithm we use to prepare received messages for input to a risk prediction model. given a ground-truth distance and duration for our encounter, we want to determine if two app-users should exchange encounter messages. we only exchange encounter messages if the perceived distance is below 2 meters and the duration greater than 15 minutes. to compute this, we use a naive model of the bluetooth noise which uses a per-person noise and a per-encounter noise. each smartphone has a different noise sampled uniformly between 0 and 1. each encounter takes the mean of this value across both users. we then apply a relative offset to the real encounter distance by multiplying the combined user noise and a uniform random variable centered at 0 with range [0.5, −0.5]. the magnitude of the distance offset is up to 2 metres when the real distance is 2 metres, and up to 0.5 metres when the ground truth distance is 1 metre. an encounter message m enc is composed of the day the encounter occurred d and the sender's quantized risk at the time of the encounter r d . the formal definition of a risk message is: an app user (say "alice") receives new encounter messages four times per day in batches; we call the set of new encounter messages on day d, m new . the risk messages which alice has already clustered are noted m enc (which is an empty set when alice first installs the app). it is useful to think of encounter messages as database records. alice inserts records into her database whenever she encounters other app users ("bobs") with their current risk estimate. update messages are like database update statements, and are used to change the risk values in old encounter messages. 11 full citation to be provided in the camera-ready version if a user (say "bob") had many encounters with alice, and bob subsequently receives a positive test result, becomes sick (reporting symptoms), or otherwise re-evaluates his risk, then bob will send risk update messages to alice. similar to encounter messages, these risk update messages may be sent through the server up to four times per day. more specifically, if bob updates his estimate of his risk on some previous day d old , then bob will construct a risk update message for every encounter message that he had on d old and these messages are sent to the users with whom bob had a contact. if this update message is sent on day d, it is composed of the current day d, his new risk r new , his previous risk estimate for that day r old and the day of the encounter d old . therefore, the formal definition of a risk update message is: m update = (r new , d, r old , d old ) ∈ m updates by virtue of the strong privacy protocol in place, we are not able to create message clusters that correspond the true chains of contacts for the encountered users. the only information we have to create clusters is the day and risk level of the encounters. our hypothesis is that a user's contact patterns can provide a rough idea on the number of individual people they encounter on each day. all encounter messages received on a given day with a similar risk level are put into the same cluster. given that there are 16 risk levels, it is only possible to create 16 new clusters on a given day, unless the user receives update messages. every update messages is created for exactly one encounter message. if there are fewer update messages for a given day / risk combination than there are encounter messages for that day / risk, then we split the existing cluster for that day / risk into two, with one cluster containing newly updated encounter messages, and another containing the rest of the messages. we do not claim that this clustering algorithm is optimal, that the input format to the neural network is optimal, or that this messaging protocol is optimal. there exists an interesting trade-off between privacy and risk precision which we hope will be explored in future work. agent-agent transmission all encounters are assumed to be at a distance equal or less than two meters. transmission events can only take place between infectious and susceptible individuals. although the abm models all encounters taking place between individuals, a susceptible individual is only considered exposed (i.e. the model considers that an effective contact has taken place) when the encounter lasts a minimum of 15 minutes and bernoulli distribution with probability determined via equation 2 gives 1. the likelihood of viral transmission during a given effective contact is proportional to the time duration of the encounter, and depends broadly on characteristics of both the infected and susceptible individual. we use the transmission model as explained in [23] . thus, encounters taking place in certain locations (i.e. senior residencies and households) are also inherently more prone to result in a transmission event, all other factors being equal. this is modeled via b n . at the same time, characteristics affecting infectiousness (of the infected individual) include progression of the disease (i.e. effective viral load) (ev l), whether (and to what extent) they are symptomatic (a s ). susceptibility of the exposed individual depends notably on age (s a ). environment-agent transmission please note, that the probability of environmental transmission is not supported by any published study, and we consider 0 environmental transmissions in the experiments in this paper. however, abm can be configured to simulate environmental transmissions. empirical estimates of such transmissions stands at 10% as per [23], therefore, we consider a transmission model that models environmental infections in pre-pandemic scenario. given the lack of such estimation in post-lockdown scenario, we do not consider any such transmissions. we model these transmissions by considering a linearly time-decaying probability of location being contaminated which is triggered by the presence of an infectious individual. initial magnitude of contamination is dependent on the agent's current phase of the disease. further, the duration of such contamination is informed from experimental study [66] , which lists surfaces and the duration for which virus survives on them. we consider an experimental environment transmission model that estimates probability of infecting agent as proportional to (a) contamination strength of the location, and (b) susceptibility of an agent. the proportionality factor being the environmental transmission control knob which lets us model the disease spread as per the observed data. we denote s high as the row indices in s i d ∈ {0, 1} nsymptoms×dmax such that the symptom at those indices are highly informative symptoms (see sec. j.8). similarly, we obtain sets of indices s moderate and s mild . we used n + 1 = 4 recommendation levels, thereby allowing an agent behavior level to be either of {1, 2, 3, 4}. we also denote the set of days {d 1 , d 2 , ...d max } as d. the heuristic-fct algorithm is implemented as algorithm 1. we next describe each function of the heuristic-fct. in each of the following sections we detail the probability of a given symptom in each of 5 phases of the disease. the probability of a person having fatigue as a symptom is given in the first column, while the probabilities of having the other symptoms given that this person has fatigue are given in the columns following the fatigue column. in order to have unusual symptoms, the person must also be over 75 years of age; that is, the probabilities in the table given for unusual symptoms are the probabilities of having unusual symptoms given this person is over 75 years of age and is experiencing fatigue. the probability of having trouble breathing as a symptom is given in the tb column for the corresponding covid-19 phases. for an individual to experience severe chest pain (scp), the individual must also be extremely sick; that is, the probabilities given for having scp as a symtpom are the probabilities of having scp given an individual has trouble breathing and is extremely sick. the probability of having light/moderate/heavy trouble breathing is given by the probability of an individual having light/moderate/severe symptoms of covid-19 and the probability of having trouble breathing as a symptom. the probability of having loss of taste as a symptom of covid-19 is 25% during the onset phase, 35% during the plateau phase, and 0% for all other phases of covid-19. aches are not caused by covid-19 in this simulator, but are caused by the flu. the probabilities of having aches on the first and last days of the flu are 30% and 80% respectively, while the probability of having aches for all other days with the flu is 50%. we wish to verify that the dynamics of the simulator at the population sizes we model are representative of larger populations. because of the computational demands of an agent-based model, particularly with messaging between agents, it is more efficient to model smaller populations, as long as the dynamics remain reprsentative. as shown in figure k .19, we find that population sizes above 2k to be representative of the dynamics of larger populations across a range of metrics. we thus ensure all experiments are run with populations of 2k and over. disability-adjusted life years (dalys) are a summary measure of the public health burden associated to a specific cause's premature mortality and morbidity. to calculate dalys, we individually compute the years of life lost due to premature mortality (ylls) [67] for agents that died during the simulation, as well as the years of life lost due to disability (ylds) [68] for agents that were infected and symptomatic. disability weights (dw) are taken from the 2017 global burden of disease study [69] : they represent health preferences such that a dw of 0 is perfect health and a dw of 1 is equivalent to death. hence, the higher the amount of dalys, the worse health outcomes are. dalys are calculated without discounting or age-weighting, following the who methodology [70] . for each agent i, tpl temporary productivity loss (tpl) is the loss in productivity due to absenteeism from work. to calculate tpl, we extract from the simulator the number of work hours that agents aged 25 to 65 years had to forego due to quarantine, taking care of a dependent (supervision), or illness to the point of being unable to work. we then multiply this quantity of foregone work hours by the 2019 average hourly wage in montreal [71] to obtain total tpl. we follow the methodology for calculating tpl presented in [43] . disability weights covid-19 is a novel disease, and there are no published disability weights for different levels of severity of the disease. therefore, we use similar conditions as proxies for different health states of the agents. agent hospitalization status is used as a proxy for actual health status, and can be divided into three categories: agents that are symptomatic and not hospitalized, agents that are hospitalized but not in critical are, and agents that are in critical care. the disability weights, as well as their equivalent causes in the gbd 2017 study can be found in table l. 3. full trajectory due to the computational strain of the message-passing within the simulations, observing the full trajectory under binary contact tracing and feature-based contact tracing is currently unfeasible. future work will consider longer simulations that reach a post-pandemic steady state.. ppl in addition to temporary productivity loss (tpl) due to absenteeism from work, cost-benefit analyses following the human capital model (hca) [43] typically include a permanent productivity loss (ppl) due to premature mortality component. when longer simulations become possible, future work will take into account ppl. . to properly evaluate the impact of different tracing strategies on the socio-economic burden of a disease, it is important to evaluate whether the proposed strategies avert dalys, or simply delay them [72] . such evaluations require a longer trajectory to arrive at a postpandemic steady state, which we will consider in future work. . the focus of this paper is not the cost-benefit analysis of the outcomes of the simulator, but rather the simulator itself, a sensitivity analysis of the cost-benefit results has been relegated to future work.. table l .5: yll, yld and dalys ×1000 for different ct methods. in all three cases, the bulk of dalys is due to years of life lost due to premature mortality (yll), rather than years of life disabled (yld). women are also disproportionately affected in all three scenarios. as can be seen in figure l .20, under no tracing, the total dalys is 129.42, and the tpl is $1.370m. test-based bct slightly affects the health and economic outcomes: the tpl is $1.591m and the total dalys is 119.42. however, heuristic-fct has a comparatively large effect on the total dalys, which drops to 71.31. however, this is contrasted by a rise in the tpl to $2.122m: health outcomes are drastically improved at the cost of a greater drop in productivity. of note is the difference in impact of both tracing methods: whereas test-based bct has very little effect on the health and economic outcomes, heuristic-fct reduces total dalys by 44.90% at the expense of an increase in tpl by 54.89%. bootstrapping dalys and tpl are computed for each seed separately before being aggregated to obtain a mean and standard error. when measures are calculated across subgroups of the simulation's population, such as across age groups, bootstrapping is used to capture todo figure l.20: impact of different ct methods on dalys and total foregone work hours. no tracing foregoes the least amount of work, but results in a high amount of dalys. test-based bct foregoes more work, but still results in a large loss of health. heuristic-fct foregoes even more work than no tracing does, but results in a large decrease of dalys, alleviating the health burden. total foregone hours due to quarantine, aggregated across individuals, are scaled by a factor of 0.49 [42] to account for the proportion of agents able to work from home. standard errors are computed by bootstrapping 100 samples of 6 runs over 10 seeds. lockdown timing and efficacy in controlling covid-19 using mobile phone tracking lockdown-type measures look effective against covid-19 covid-19 pandemic and lockdown measures impact on mental health among the general population in italy economic and social consequences of human mobility restrictions under covid-19 mitigate the effects of home confinement on children during the covid-19 outbreak the economic cost of covid lockdowns: an out-of-equilibrium analysis. economics of disasters and climate change a national plan to enable comprehensive covid-19 case finding and contact tracing in the us temporal dynamics in viral shedding and transmissibility of covid-19 modes of contact and risk of transmission in covid-19 among close contacts. medrxiv rapid review of contact tracing methods for covid-19 variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure fast coronavirus tests: what they can and can't do covid-19 pandemic planning scenarios modeling the combined effect of digital exposure notification and non-pharmaceutical interventions on the covid-19 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polymerase chain reaction for severe acute respiratory syndrome coronavirus 2: role of deep-learning-based ct diagnosis and insights from two cases estimating effective reproduction number using generation time versus serial interval, with application to covid-19 in the greater toronto area, canada. medrxiv social contacts and mixing patterns relevant to the spread of infectious diseases census profile, 2016 census -montreal, quebec and canada epidémiologie et modélisation de l global, regional, and national disability-adjusted life-years (dalys) for 359 diseases and injuries and healthy life expectancy (hale) for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study 2017 remote work and employment dynamics under covid-19: evidence from canada impact of the burden of covid-19 in italy: results of disability-adjusted life years (dalys) and productivity loss risk scoring calculation for the current nhsx contact tracing app report from the canadian chronic disease surveillance system: heart disease in canada factors associated with hospital admission and critical illness among 5279 people with coronavirus disease 2019 in new york city: prospective cohort study factors associated with covid-19-related death using opensafely characteristics associated with hospitalization among patients with covid-19-metropolitan atlanta, georgia determinants of covid-19 disease severity in patients with cancer public health agency of canada. diabetes in canada predicting mortality due to sars-cov-2: a mechanistic score relating obesity and diabetes to covid-19 outcomes in mexico overweight and obesity based on measured body mass index, by age group and sex prevalence estimates of chronic kidney disease in canada: results of a nationally representative survey world health organization. smoking and covid-19 age-dependent effects in the transmission and control of covid-19 epidemics temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study. the lancet infectious diseases quebec public health projecting social contact matrices to different demographic structures the need to include assisted living in responding to the covid-19 pandemic contacts in context: largescale setting-specific social mixing matrices from the bbc pandemic project. medrxiv wikipedia contributors. negative binomial distribution using time-use data to parameterize models for the spread of close-contact infectious diseases sustainability of coronavirus on different surfaces global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the global burden of disease study global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study global burden of disease collaborative network. global burden of disease study 2017 (gbd 2017) disability weights world health organization. who methods and data sources for global burden of disease estimates weekly and hourly earnings of employees by sex, montréal and all of québec covid-19 and the health policy recession: whatever it takes, grandma or the economy or what makes sense? information on the montreal population regarding age, sex and occupation distribution was retrieved from canadian census data [38] . prevalences of selected medical conditions considered as covid-19 infection and prognosis risk factors were determined based on prevalence estimates from nationally representative surveys or medical surveillance programs in canada: heart disease [46, 47, 48, 49] , stroke [48] , asthma [47, 48] , chronic obstructive pulmonary disease (copd) [47, 48, 49] , cancer [47, 48, 50] , diabetes [47, 48, 49, 51] , obesity [47, 48, 49, 52, 53] , chronic kidney disease (ckd) [47, 48, 49, 54] , immuno-suppressed conditions [48] and smoking [55, 56] . national prevalence estimates were extracted based on age group (<10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80 and >80 years of age) and sex.we determined conditional probability of developing covid-19 in abm based on symptoms and risk factors associated with covid-19 in published literature. a mathematical modelling study of the epidemic with canadianspecific estimates [57] was used to model covid-19 susceptibility in the pediatric population of the simulator. we model inoculum, the amount of virus transmitted during an exposure event, as a random variable uniformly distributed between 0. and 1.. the magnitude of inoculum is used to determine the type and severity of symptoms.we sample parameters for a piece-wise linear model of what we call effective viral load (evl) 4 . we think of evl as a piecewise linear function, attributes of which are sampled for each individual separately. this approximation follows empirical studies on viral load progression [58, 34] . figure b .11 is the mean of sampled effective viral load curve. we model rt-pcr test allocation as a priority queue. when an agent experiences symptoms, or is advised by a contact tracing application to seek key: cord-022659-chwk2bs4 authors: nan title: abstracts: poster session date: 2004-10-08 journal: ann neurol doi: 10.1002/ana.410320224 sha: doc_id: 22659 cord_uid: chwk2bs4 nan an immune etiology has been postulated for acute cerebellar ataxia of childhood (acac) since it frequently follows viral infections. we analyzed serum and cerebrospinal fluid (csf) from 6 acac patients for antibody cross-reacting with cerebellar neurons. serum and csf were obtained within 7 days of onset of pancerebellar ataxia from subjects aged 3.5 to 11 years. varicella infection preceded 4 cases. results of enhanced cranial ct scans were normal; csf demonstrated 2-122 cells/mm3 with sterile cultures. serial dilutions from 1 : 20 of serum and undiluted csf were screened for antineuronal antibody by indirect immunofluorescence (iif) using frozen, unfixed normal human cerebellum. serum (1 : 400) was examined further for antineural antibody by western immunoblotting using purified cerebellar neuronal extracts as antigen. serum from age-matched, neurologically normal pediatric inpatients served as the control group for iif and immunoblot experiments. in acac patients, no antineuronal immunoreactivity was observed by iif. immunoblots demonstrated no consistent pattern of immunoreaction when comparing acac to controls, though 1 patient exhibited distinct bands at 200 kd (neurofilament protein) and 54 kd. although antecedent infection suggests an immune etiology for acac, our preliminary results do not support a humoral mechanism for this disorder. ingrid taff; joseph zito, robert gould, and steven pavlakis, great neck and manhasset, ny in a 20-month period we studied 69 patients between the ages of 7 weeks and 2 1 years with magnetic resonance angiography (mra). studies were performed on a 1.5t magnet (siemens magnetom sp) with a circular polarized head coil. a three-dimensional time-of-flight technique was utilized. occasionally, images were obtained after gadopenetate dimeglumine infusion. two-dimensional projection images were calculated using a maximum intensity projection algorithm and recorded on laser film. sixty-seven patients also had routine mri. a sampling of vascular lesions was demonstrated. nineteen patients had clinical and mri evidence of stroke. mra revealed intracranial vascular occlusion in 2 patients, diminished focal cerebral flow in the affected area in 4, and generalized ipsilateral underdeveloped cerebral circulation in 4. a moya-moya vascular pattern was found in 4 and sickle-cell vasculopathy was found in 1 patient. seven mras were normal. seventeen vascular hamartomas were demonstrated including 2 vene of galen malformations, 7 arteriovascular malformations, and 8 venous angiomas. three aneurysms were found. thirty-one mras were normal. we find m u to be a valuable adjunct to routine mr imaging in the evaluation of pediatric patients with potential cerebrovascular disease. it demonstrates a spectrum of pathology, is noninvasive, and allows for serial follow-up examinations. angiography in pediatric cerebrovascular disease p4. thalamic change in acute encephalopathy of adult rats. twelve weeks after grafting, clinical and histological studies were performed. we developed a protocol for evaluating functional deficits that follow spinal cord injury in the rat. the survival, growth, differentiation, and parenchymal integration of the graft were documented histologically on semi-thin section. animals that received the transplants demonstrated qualitative and quantitative improvements in several parameters of locomotion. donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. we suggest embryonic rat spinal cord transplantation may be a useful treatment of spinal cord injury and a possible therapeutic strategy in human spinal cord injury and amyotrophic lateral sclerosis. the basic neuropathophysiology of hemineglect after unilateral cerebral lesions is still not clear. one theory holds that degraded perceptual processing occurs in the damaged hemisphere due to intrahemispheric deficits. another holds interhemispheric interaction at fault, with the intact hemisphere actively inhibiting spatial cognitive processes in the damaged one. we tested 11 adult macaca fascicuhris with acute neglect on a task in which the whole visual surround was restricted to 15 degrees from central fixation, and a second in which an opaque lens occluded the eye either ipsilateral (ipsi) or contralateral (contra) to the lesion. using paired t tests, in the first task there were no differences in reaction time to the ipsi and contralesional hemifields. in the second, there was no change in extent of the ipsilesional field (obtained with the contralesional eye occluded), as compared to its extent without occlusion. the contralesional field, however, improved significantly ( p < .03) with the ipsilesional eye occluded. since reducing sensory input to both hemispheres leads to no worsening of hemineglect, but reducing sensory input to the intact hemisphere alone leads to improvement of hemineglect, we conclude that adverse interhemispheric interactions play a major role in the pathophysiology of hemineglect. we assessed the sensitivity and applicability of a new, cornbined cognitive and mood screening battery for multiple sclerosis (ms). sixty consecutive, untreated clinically active ms patients, 30 each relapsing-remitting and chronic progressive, underwent the battery and head mri upon entering 2 concurrent treatment trials. the battery combines the faust-fogel brief cognitive screen and visual analogue dysphoria scale, both previously validated in other neurological diseases. cognitive domains tested were immediate and delayed sentence and word-pair recall, verbal fluency, and conflicting response suppression. patients marked ''usual mood" along a "happy-sad" cartoon continuum. relapsing patients were program and abstracts, american neurological association 235 younger (32.8 vs 41.2 yr mean), with shorter ms durations (6.4 vs 11.3 yr), and had lower kurtzke disability scores (1.7 vs 5.8). modified qualitative mri grading (lesion burden, confluence, localization) was compared. half as many relapsing patients (43% vs 83%) scored ''abnormal'' cognitively, despite similar "sadness" rates (20% vs 23%). subjective dys-~ phoria in both groups correlated with denser periventricular lesion burdens. the battery was well tolerated and easily administered within 10 minutes without special equipment. this combined cognitive and mood screening battery is sensitive and convenient for clinically active ms. alternate forms of the battery are needed for repeatability. questionnaires may be reliable and valid supplements to laboratory tests for brain-damaged patients, as they can be applied to situations for which laboratory testing is not possible. we investigated the usefulness of informant-based data in alzheimer's disease (ad) by comparing caregivers' subjective evaluations of 83 probable a d patients' performance on an abbreviated version of the memory self-report questionnaire to objective evaluations derived from an extensive battery of neuropsychological tests and to clinicians' evaluations. similar information was obtained from 39 healthy agematched controls. caregivers' subjective appraisals of patients' memory correlated significantly with objective measures of secondary memory, with all cognitive variables, measures of activities of daily living, and clinicians' evaluations of dementia staging. scores were independent of clinical indicators of depression. the abbreviated memory questionnaire showed good reliability, internal consistency, and external validity. its positive predictive value is 63.5 and its negative predictive value is close to 100%. results suggest that (1) informant-based questionnaires may be useful for obtaining valid information on cognitive ability outside of laboratory settings; (2) the scale reflected more than just memory functions; and (3) the scale may be promising for screening cognitive difficulties in epidemiological or clinical settings. although neglect along the horizontal dimensions of extrapersonal space is well recognized, there are only a limited number of observations documenting neglect along the vertical and radial spatial dimensions. we report an investigation of neglect along the 3 principal dimensions of extrapersonal space in a patient with bilateral mesial temporo-occipital infarctions. neglect was assessed by asking the patient and controls to bisect lines of 4 lengths oriented in 3 directions with respect to the body: horizontal, vertical, and radial. our patient showed significant neglect of upper vertical and far radial space, as well as neglect of left hemispace. his line bisection errors were consistently in a direction opposite the slight directional biases shown by controls for all 3 line orientations ( p < .05). the magnitude of the patient's bisection errors increased by moving the lines toward the neglected sectors of 3-dimensional space. neglect of upper vertical and far radial space was also evident on line cancellation tasks. our results suggest that following focal brain injury, neglect may be observed along all 3 dimensions of extrapersonal space. these findings provide further empirical support for functional specialization within inferior and mesial temporooccipital regions for attending to upper vertical and far visual space (previc, 1990) . p12. posterior cortical atrophy: degenerative disease with primary visuospatial and visuosemantic deficits a. kertesz, m . polk, and a. kirk, london, ontario, and saskatoon, saskatchewan, canada posterior cortical atrophy is a recent, and heidenhahn's disease is an old, label for a miscellaneous group of patients with imaging or pathological and clinical evidence of visuocognitive deficits and cortical atrophy localized to the posterior cortex. the extent of this cortical localization and the nature of the pathological findings are not fully agreed upon, but spongiform degeneration and alzheimer pathology have been described. detailed examination of patients who are representative of the problem and have uniquely specific deficits is presented. one patient had visual associative agnosia, prosopagnosia, and transcortical sensory aphasia. lexicosemantic experiments of categorization, word retrieval, and comprehension of auditory and visual stimuli showed a specific impairment of visuoverbal semantics. a striking preservation of phonological, orthographic and visual structural input, and intercategory dissociations was demonstrated. consistency of errors argued for specific loss of semantic knowledge. another patient with apraxia, primary visuospatial deficit, agraphia, and amnesia at the beginning had predominantly right-sided posterior cortical atrophy, demonstrating further fractionation of the entity and the striking specificity of visuospatial function. the behavioral specification of degenerative disease is clinically and theoretically important. permanent neurological deficits after ischemic stroke are mainly determined by the location and size of the infarct. clinical recovery also depends on the functional state of adjacent brain tissue, where both neuronal loss and deactivation without gross morphological damage may affect flow and metabolism to a varying degree (g. mies et al, stroke 1983; 1422-27) , and where the ability to respond to stimulation by appropriate neuronal recruitment may be impaired. therefore, degree of resting hypometabolism and of responsiveness to functional activation may provide a measure of prognosis. in 26 patients (age 60.7 10.9 yr) with aphasia consequent to ischemic stroke of the dominant hemisphere, regional cerebral metabolic rate of glucose (rcmrgi) was measured at rest and in 17 of them also during spontaneous speech, using positron emission tomography (pet) of 2-(f18)-fluoro-2-deoxy-~-glucose (fdg). the pet study and a standardized neuropsychological test battery to assess the main aspects of language were performed around the fourteenth day after the stroke, and the language functions were assessed again 3 to 5 months later. performances in various dimensions of language 2 weeks and 3 to 5 months after stroke were related to rcmrgl in topographically meaningful areas at rest and during activation using wilcoxon-rank program and abstracts, american neurological association 237 sum test and multiple regression analysis. severity of aphasia was assessed by the token test, which showed a bimodal distribution to slight and severe, and a lower representation of moderate cases. global and all regional cmrgl at rest and during activation were significantly correlated to scores in token test at first and second examination, with the highest correlation coefficients ( -0.7 to -0.61) for broca's, wernicke's, and left temporoparietal regions. for performance after 3 to 5 months, the relationships were still significant with lower coefficients. verbal fluency also was correlated to kmrgi, but with lower coefficients that slightly increased for the recovery state. language performance at different stages in the course after ischemic stroke was significantly related (r = 0.84 for token test, r = 0.93 for verbal fluency). however, there exists a high variability in recovery that may be explained by stepwise regression of metabolic values. significant effects were observed only for cmrgl of the left hemisphere outside the infarct (partial r' = 0.21) at rest and for cmrgl within the infarct (0.27), the contralat-era1 mirror region (0.16), and broca's region (0.17) during activation, with a sum of all partial weight factors of 0.46 at rest and 0.72 during activation. our results furnish 2 indicators for recovery of aphasia: the resting metabolism of the left hemisphere outside the infarct, and the activated metabolism in residual tissue within the infarct and in languagerelated areas. although the hemispheric metabolism at rest might be related to neuronal loss and thereby to the brain's reserve capacity, the extent of metabolic activation indicates neuronal recruitment and the capability of neuronal networks for functional recovery. heparin therapy for acute myocardial infarction: the timi-i1 pilot and randomized trial combined experience m . a. sloan, t . r. price, m . l. tevrin, and s. forman for the timi investigators, baltimore, m d of 3,924 myocardial infarction (mi) patients treated with rt-pa and heparin, 29 (0.7%) developed ischemic cerebral infarcts (ci). all ci patients had detailed neurological evaluations and 27 (939%) had c t scans. age range was 40 to 74 years (mean 60 yr), 25 were male, and 22 were caucasian. electrocardiographic location of mi was anterior in 22 (76%) and nonanterior in 7 (249%). six cis occurred within 6 hours; 1 between 6 and 12 hours; 2 between 12 and 24 hours; 4 between 24 and 48 hours; 13 during the second week; and 3 others distributed over the 4 weeks after study entry. six of 29 cis did not involve cerebral cortex; 9 (319%) had multiple cis. of 24 cis thought to be embolic in origin, 17 had at least 1 cardiac abnormality (mural clot, wall motion abnormality, aneurysm, or transient atrial fibrillation) known to be associated more specifically with embolism than just the diagnosis of myocardial infarction. eight of 27 (30%) with ct scans had hemorrhagic conversion of varying degrees. the time of occurrence and sites of ci after rt-pa and heparin therapy for acute mi are similar to those reported in the prethrombolytic area. nancy futrell andjeanne m. riddle, detroit, m i photochemical irradiation of the carotid artery of rats has been used to induce endothelial damage, producing a nonocc h i v e thrombus (that apparently embolizes spontaneously) thrombi and emboli and multiple cerebral infarcts. evidence for embolism generally has a presumptive component. to document further that cerebral infarcts in this model are indeed due to embolism, we studied the ultrastructure of the carotid thrombi and the presumed cerebral emboli using scanning and transmission electron microscopy (sem, tem). the right carotid artery of 9 wistar rats was irradiated with a laser (632 nm, 200 mw/cm2, 15 min) following the injection of the photosensitizing dye photofrin 11, 12.5 mgikg. rats were sacrificed from 1 to 24 hours later. endothelial damage with formation of a fragmenting thrombus, composed mainly of platelets and erythrocytes (with no fibrin in most areas), was present in the carotid arteries of all rats by sem. sem was done on 36 cerebral vessels, 3 1 containing peripheral blood elements, with single (1) and aggregated (6) platelets (causing occlusion in 3), single (10) and aggregated (10) erythrocytes (without occlusion), and single (1) and aggregated (3) leukocytes (without occlusion). tem demonstrated that the platelet aggregates did not adhere to the cerebral endothelium. the endothelial surface of all cerebral vessels was normal, which provided additional evidence that the mechanism of cerebral infarction in this model is embolism. model of repetitive ischemia: this effect is significantly enhanced when combined with mild hypothermia ashfaq shuaib and elisabeth sechocka, saskatoon, saskatchewan. canada there is considerable evidence that glutamate release resulting in activation of postsynaptic receptors (especially nmethyhaspartate) is a major mechanism of ischemic neuronal injury. in vivo experiments have shown that a more severe release of glutamate may be responsible for the excessive damage seen with repeated ischemic insults. we have shown that in cell cultures the effect of brief repeated insults is more severe than a single insult of similar duration. in the present study, we tested the protective effects of cgs-19755 in a cell culture model of single ischemic and multiple-insult paradigm. in the multiple-insult paradigm, in some cultures cgs-19755 was combined with mild hypothermia to see if this would offer additional protection. cgs-19755 offered a dose-dependent protection in cell cultures exposed to a single ischemic insult. cgs-19755 was protective to cultures exposed to repeated ischemic insults. the protective effects were enhanced significantly when they were combined with hypothermia, resulting in almost complete protection of the cultures. the combination of therapies appears to be a valuable strategy in neuronal protection during cerebral ischemia. toby i. gropen, i . prohovnik, t . k. tatemirhi, z. sharif; and m. hirano, new york, n y although a rare syndrome, mitochondria1 encephalomyopathy, lactic acidosis, and stroke (melas) may offer a unique insight into stroke mechanisms. we report novel observations in a patient with melas studied with serial and quantitative cerebral perfusion after stroke using """tc-ceretec spect and '3ixe rcbf. a 24-year-old man with melas presented with left-sided headache, generalized seizures, fluent aphasia, and right hemianopia. serial ct and mri showed infarction of the posterior left hemisphere in a multiterritorial distribution. spect performed 15 days after stroke showed 20 to 30% greater flow in the infarct than in normal brain, which reversed 109 days after stroke. quantitative rcbf (m2 isi, reflecting mostly gray matter), when corpatients died. we recommend ct evaluation in all patients who have a seizure or lose consciousness during the peripar-tum period. despite intensive management, mortality is high. seizures should not be attributed to eclampsia without careful neurological assessment. when prenatal care is sought, women should be counseled about the dangers of cocaine to themselves as well as to their babies. changes in circulating blood volume following stephan a. mayer, matthew e. fink, laura lenniban, louise m. klebanoff; auis beckford, lsak prohounik, william young, and robert a. solomon, new york, n y reduction of blood volume (bv) has been implicated as a risk factor for delayed cerebral ischemia (dci) due to vasospasm after aneurysmal subarachnoid hemorrhage (sah). volume expansion guided by target filling pressures has gained popularity as a means of preventing or reversing dci; however, the adequacy of central venous pressure (cvp) as a reflection of bv in this setting remains unclear. we measured bv and cvp concurrently in 10 patients (5 males, 5 females; mean age 57 yr) 1 day after craniotomy (mean 3.5 days after sah) and an average of 5.5 days later. the mean bv (mllkg) measured using chromium5'-labeled red blood cells (rbcs) fell from 69.7 to 55.3 (normal range 55-80), a reduction of 21% ( p = .05, paired student's t test). despite this, mean cvp (mm hg) remained unchanged (7.1 vs 7.9). similar reductions of plasma volume (2 1%) and rbc volume (24%) accounted for no change in mean hematocrit (33.2 vs 33.5). bv fell 25.7% among grade iiiliv patients (n = 5 ) compared to 14.7% among grade 1/11 patients (n = 5). a moderate correlation between bv and cvp ( r = .48, p = .16) was found only with the first set of measurements. time-related alterations in venous capacitance, myocardial contractility, or systemic vascular resistance may explain our findings. axel rosengart, louis r. caplan, michael s. pessin, atherostenosis of the extracranial vertebral artery (ec-va) has rarely been studied systematically in series of patients with acute vertebrobasilar strokes or transient ischemic attacks. we identified by conventional angiography and neuroimaging (ct, mri, ultrasound, mr angiography) 45 patients with ec-va disease among 154 patients with posterior circulation ischemia. patients with cardiac sources of emboli were excluded. the probable etiologic mechanisms were: group a: vertebral artery origin (vao) atherostenosis with embolism-19 patients; group b: vao atherostenosis with hemodynamic spells-7 patients; group c: 1 patient with vao atherostenosis and both intra-arterial embolism and hernodynamic spells; group d: ec-va dissection-8 patients (6 unilateral, 2 bilateral); 1 patient had perioperative compromise of the ec-va with presumed intra-arterial embolism; group e: vao disease in addition to other distal vascular lesions-9 patients (2 with intracranial va and 7 with basilar artery [baf occlusive disease). ec-va disease is not always benign. vao atherostenosis and dissection of the ec-va are sources of intra-arterial emboli. hemodynamicrelated ischemia occurs with bilateral and unilateral va disease but is often transient. vao atherostenosis is often accompanied by severe occlusive disease of the intracranial va and ba. program and abstracts, american neurological association 239 sebastian e. ameriso, vicky l. y. wong, andvas gruber, hidemi ishii, and mark fisher, los angeles and la jolla, c a , and kanagawa, japan hemostasis abnormalities are associated with ischemic stroke. these changes typically are demonstrated in antecubital venous blood samples and may not necessarily represent changes within the vasculature of the brain. the purpose of this study was to identify potential differences in hemostatic profile from samples of cranial versus noncranial venous sites in patients with acute ischemic stroke. eight patients were studied within 7 days of acute brain infarction. some patients were studied on 2 separate days. blood was drawn from the external jugular vein and immediately thereafter from an antecubital vein without the use of tourniquet. we measured hematocrit, leukocyte count, platelet count, fibrin d-dimer (cross-linked fibrin fragment), plasminogen activator inhibitor-1 (pai-1, an important antifibrinolytic protein), and anticoagulant proteins thrombomodulin and activated protein c. a jugular-to-antecubital ratio was calculated for each paired blood sampling. thirteen paired samples were obtained from the eight patients. external jugular-to-antecubital ratios (mean to-antecubitat ratio for pal 1 was significantly different from 1 ( p < 0.05), with higher concentrations in jugular samples. in conclusion, levels of hemostatic proteins measured from cranial venous blood may differ from antecubital samples in patients with acute ischemic stroke. in animal models of transient cerebral ischemia, the effects of repetitive insults are more severe than a single ischemic episode of similar duration. we used the cell culture model of ischemia to determine if the effects of repetitive ischemia are similarly more severe in this model of ischemia. for cell culture, we used fetal mice cortical astrocytic and postnatal cerebellar (glutamatergic) granular neurons and cerebral gamma-aminobutyric acid (gaba)ergic cells. lactic dehydrogenase (ldh) (activity per gram protein) release in the medium was used as a measure of cellular damage. compared to a single insult, there was a large increase in ldh release during repetitive ischemia in astrocytes (233 vs 7 5 , p < 0.02) and granular cells (129 vs 41, p < 0.001) (highly significant) and a modest (but significant) increase in the cortical neurons (11.6 vs 7.5 p = 0.05). the demonstration that repetitive ischemia produces more severe damage in cell culture would suggest that the mechanisms are not predominantly vascular. cell culture could prove useful to study the mechanisms of neuronal damage with repetitive ischemia. we studied the spontaneous recovery of neurological function after acute ischemic stroke using a standardized stroke nih stroke scale scale ( n i h stroke scale) to assess the extent of improvement, differences in stroke types, and early predictors of later outcome. we performed serial neurological assessments on admission; 24, 48, and 72 hours after admission; and 7 to 10 days and >30 days after admission. twenty-six patients had presumed embolic occlusion of the middle cerebral artery (mca) and 14 had a clinical diagnosis of lacune. admission score was better in the lacune group compared to the mca group. the mean scores for all patients improved by the 7to 10-day and the >30-day examination, but the degree of improvement was greater in the mca group than in the lacune group at >30 days ( p < 0.004). the degree of change at 7 to 10 days correlated with the change in score at 24 hours ( r = 0.45, p < 0.05) and 48 hours ( r = 0.86, p < 0.05). most patients improve after acute ischemic stroke, but to variable degrees and at different rates. david w. desmond, thomas k. tatemichi, miguel figueroa, dew'itt t . cross, and yaakov stern, new yovk, n y to investigate the effects of lacunar infarction (li) on cognitive function, we examined 57 li patients 3 months after stroke (age = 71.6 ? 9.0 yr; education = 9.2 2 4.7 yr) and 241 stroke-free nondemented control subjects (age = 70.6 k 6.5 yr; education = 12.4 k 4.5 yr) with a battery of neuropsychological tests. li was defined as a presenting infarct of 5 2 cc and a mean volume of any additional subcortical infarctions of 5 2 cc on ct scan. using multiple regression analyses, with significance set at p < .01 to minimize the risk of type i error, we considered the role of li as a correlate of performance in multiple cognitive domains. controlling for the effects of demographic factors, vascular risk factors, alcohol use, and depression within the multivariate models, li was a significant independent correlate of deficits in memory ((3 = -.40, p = .0002), verbal (p = -.28, p = .0025), visuospatial (p = -.51, p < .oool), abstract reasoning (p = -.31, p = .0029), and attentional skills (p = -.50, p < .0001). we further investigated the effects of infarct number, volume, and location, as well as atrophy, on global cognitive function within the li group. the only significant independent correlate of global cognitive performance was a preponderance of left-hemisphere infarctions (p = -.07, p = ,0454). these results suggest that li may produce dysfunction in multiple cognitive domains, particularly when the left hemisphere is differentially involved. p30. increased intracranial atherosclerotic stroke in hispanics and blacks from northern manhattan ralph l. sacco, christina zamanillo, t . shi, andj. p. mobr, new york, ny intracranial atherosclerosis has been found to be more frequent in blacks compared to whites, whereas hispanics have rarely been characterized. among 2 10 consecutive patients from northern manhattan over age 39 hospitalized at the presbyterian hospital from 1990 to 1991, cerebral infarction occurred in 32 whites, 84 blacks, and 74 hispanics. all patients had at least one c t scan, 96% had duplex doppler, 94% transcranial doppler, and 12% angiography. strokes were classified as atherosclerotic (ath), cardioembolism, lacunar, and as infarcts of undetermined cause. ath was further subdivided into extracranial (eath) or intracranial (iath) . overall, the frequency of ath was similar in the three racelethnic groups (white 2096, black 16'96, hispanic 2096) . the distribution of the atherosclerosis, however, was different in whites compared to blacks and hispanics. whites had more eath stroke than blacks and hispanics (white 17%, black 7 % , hispanic lo%), while iath was similar in blacks and hispanics and greater than in whites (white 394, black 895, hispanic 10%). nonwhites have more iath stroke than whites. the similarity in the distribution of atherosclerosis between blacks and hispanics argues for shared environmental risk factors, rather than genetic differences. ethnic differences in stroke risk factors may help explain differences in infarct subtype. we studied 10 mild to moderate alzheimer's disease (ad) patients with a series of "0 water bolus positron emission tomographic (pet) activation studies, and compared them to similar studies in 10 age-matched normal controls. for each group, pet images were mapped onto the subjects' mri scan, and results of a particular activation condition were averaged across the group. naming a series of pictures (line drawings of animals) minus counting abstract designs as a baseline produced strong activation of the anterior cingulate gyrus only in the ad group. silent reading of words minus viewing a baseline series of "xs" similarly showed strong activation of the anterior cingulate gyms in the ad subjects but not the normals. naming 1 block (activation condition) of 80% unnamed pictures, minus a second block (baseline) of easily named pictures, demonstrated much greater cingulate activation in the ad patients, for naming of the more difficult pictures. we conclude that this cingulate activation may reflect the greater involvement of an attentional network (of which the anterior cingulate is a part) in tasks requiring a higher degree of "mental work" on the part of ad patients. dementia in alzheimer's disease j. w. pettegrew, k. panchalingam, w. e. klunk, and r. j alzheimer's disease (ad) predominantly affects the brain, resulting in the loss of multiple cognitive abilities. some studies suggest the membranes of peripheral cells are involved in the disease. to investigate erythrocyte membrane molecular dynamics in ad patients and age-matched controls, we investigated erythrocyte membrane molecular motion at the surface (fluorescamine), aqueous-hydrocarbon interface (dppe-ans), and hydrocarbon core (1219j-as; ppc-dph) by steady-state fluorescence anisotropy measurements of 16 probable ad patients (5 males; 11 females) and 20 (1 1 males; 9 females) age-matched controls. cognitive function was assessed by the mini-mental, mattis, and blessed scales. we found that intergroup comparisons revealed decreased motion at the surface ( p = 0.001) and aqueous-hydrocarbon interface ( p = 0.03) and increased motion in the hydrocarbon core ( p = 0.01) of the moderately to severely impaired ad patients compared to the controls. in the ad patients, there were significant correlations between decreasing membrane surface motion and worsening blessed scores (males p = 0.05; r = 0.7; femalesp = 0.04; r = 0.5). these findings suggest that molecules are being produced in the brain of ad patients that gain access to the circulation. these molecules insert into the erythrocyte membrane and secondarily alter erythrocyte membrane molecular motion. the production of these molecules correlates with the dementia and could contribute to the molecular pathophysiology of the disease. parkinson's disease (pd) and alzheimer's disease (ad) are 2 common disorders of old age and may therefore coexist. the prognosis in demented pd patients is poor and early recognition of such cases is therefore desirable. the objective of this study was to identify characteristics that distinguish pd + ad from pd patients during early stage. all patients were clinically evaluated over a 22-year period . clinical diagnosis of dementia was made only when unequivocal clinical evidence of progressive decline in memory and cognitive function was documented, and pathological diagnosis of ad and pd was made using standard criteria. twentysix patients who had only pd or pd + ad were identified; 20 had no dementia and at autopsy had pd. six patients had clinical evidence of parkinsonism and dementia and at autopsy had 2 distinct pathological findings-pd and ad. these 6 cases could be classified as having simultaneous or sequential evolution of pd + ad. those with sequential onset had pd before age 65 years but were inexplicably functionally disabled early on, whereas those with simultaneous onset manifested pd after age 65 years. pd + ad patients had rapid disease progression, shorter survival, poorer drug response, and more side effects of levodopa than pd patients. to study the prevalence ofwhite matter lesions in the general elderly population, and to investigate whether white matter lesions were relatively frequent in subjects with classic vascular risk factors and with hemostatic risk factors, magnetic resonance scans were obtained of 11 1 participants, aged 65 to 85 years, of the rotterdam elderly study. the subjects for the imaging study were a random sample from the general population, stratified by age and gender. t2-weighted images were obtained in the axial plane. white matter lesions were considered present when moderate or severe periventricular hyperintensities or when more than 5 small focal lesions or focal confluent lesions were found. overall, 27% of subjects had white matter lesions. the prevalence and severity of le-program and abstracts, american neurological association 241 sions increased with age. history of stroke or myocardial infarction, presence of peripheral arterial disease, factor viic activity, and fibrinogen level were each significantly and independently associated with the presence of white matter lesions. significant relations with actual systolic as well as diastolic blood pressure, with a history of hypertension, and with plasma cholesterol were observed only for subjects between 65 and 74 years. this study suggests that white matter lesions in the elderly may be related not only to the classic cardiovascular risk factors. but also to hemostatic factors. joan m. swearer, paula nelligan, hanno muelher, beatrice woodward, and david drachman, worcester, ma although behavioral disturbances occur frequently in alzheimer's disease and other dementing disorders, little is known about the factors that predict their development or predispose to their occurrence. in the present study we examined 2 sets of possible predictive/predisposing factors retrospectively for behavioral disturbances in 35 mildly to severely demented, community-dwelling patients. the factors examined included: individual distinguishing features (age, gender, age of onset, premorbid personality traits, prior psychiatric history) and dementia severity (dependence in activities of daily living [adls) and self-care, duration of dementia, global disease severity). spearman correlations and t tests were used to assess the relative influence of these factors on the occurrence of 3 types of aberrant behaviors: aggressive behaviors, disordered ideation, and motor abnormalities. forty percent of the patients exhibited aggressive behaviors, 77% exhibited disordered ideation, and 63% had motor abnormalities. neither a prior history of psychiatric disorders nor premorbid personality traits were associated with the occurrence of the target behaviors. dependence in adls and self-care and greater global severity were associated ( p < .01) with the frequency and severity of aggressive behaviors, disordered ideation, and motor abnormalities. these results suggest that severity of dementia is a consistent and reliable factor in the development of aberrant behaviors, whereas preexisting personality traits are not. dementia of the alzheimer t y p e w. j. burke, a. ranno, w. h . roccafrte, s. p. wengel. b. l. bayer, and n . k. willcockson, omaha, ne l-deprenyl is an irreversible inhibitor of mao-b that has been reported to cause modest improvements in short-term memory and behavioral symptoms in persons with dementia of the alzheimer type (dat). thirty-eight subjects meeting research criteria for mild d a t were enrolled in a placebo-controlled, double-blind trial of r-deprenyl at a dose of 5 mg twice a day. subjects underwent extensive clinical and neuropsychological assessments at entry, and at 2 and 8 months. after 8 months, subjects taking both l-deprenyl and placebo showed a significant decline in their scores on the mini-mental state examination, the clinical dementia rating (cdr) scale, and the sum-of-boxes score derived from the cdr. when the change in scores on these clinical measures was examined across the 2 groups, there was no significant difference. there were no significant differences within or between groups on several behavioral measures including the brief psychiatric rating scale and the cornell rating scale for depression in dementia. neuropsychological testing demonstrated no significant differences berween groups based on mean score change. l-deprenyl did not affect cognition or behavioral symptoms of dat in this 8-month study. k. marder, m-x. tang, r. ottman, l. cote, y. stern, and r. mayeux, new york, n y the etiology of dementia in parkinson's disease (pd) is probably multifactorial but there may be a shared susceptibility for p d and alzheimer's disease (ad). reliable risk factor interviews were conducted with informants of 15 1 nondemented p d patients (pd-d) and 65 demented p d patients (pd + d) enrolled in a longitudinal community study of pd. p d + d were older (78.9 yr) than pd-d (71.4 yr) and had later age at onset of motor signs (71.1 yr) than pd-d (64.8 yr) ( p < ,001). the frequency of smoking, alcohol use, head injury, and family history (fh) of pd did not differ but fh of ad was significantly more frequent in the pd + d group (or 2.25, ci 1.01-5.01). using stepwise logistic regression, only age of onset of motor signs 2 6 5 (or 2.86), education <8 years (or 2.53), and the interaction of age of onset of motor signs and fh of a d (or 3.49) were independent predictors of dementia in pd. to address variable years at risk for development of dementia, life table analysis revealed the cumulative risk of a d to age 90 in first-degree relatives of p d + d was .312, and .119 in pd-d relatives ( p < .05). cox proportional hazards analysis controlling for the differences in ages of the relatives of both groups yielded a rate ratio of 2.06 (ci 1.2-3.7) for the development of a d among p d + d compared to pd-d relatives. we conclude that a genetic susceptibility to a d may raise the risk for dementia in patients with pd. differentiated from alzheimer's disease? john c. mowis, elizabeth grant, rita canfield, eugene rubin, and daniel mckeel, jr, st louis. mo vascular dementia (vd) is believed to account for 20 to 30% of all us cases of dementia; however, pathologically confirmed cases are quite rare. this discrepancy suggests that current diagnostic criteria lead to the clinical overdiagnosis of vd. twenty v d subjects (mean age 78.5 yr; 9 men, 11 women) were diagnosed solely on the basis of the presence of dementia, a history of stroke(s), and a documented relationship of stroke to onset andlor course of dementia; ischemic scores (is) and neuroradiographic findings were not used for diagnosis. compared with 89 subjects (mean age 75.2 yr; 34 men, 55 women) with dementia of the alzheimer type (dat), there were no significant group differences for comparable clinical dementia rating stages of dementia for measures of language, activities of daily living, or general cognition. the vd group scored significantly higher than the dat group on the modified is (f [91, 64] = 138.2, p < ,0001). all 27 autopsied d a t subjects had verified alzheimer's disease (ad); 8 also had cerebral infarctions. the 3 autopsied v d subjects had 168, 160, and 55 cc of brain tissue affected by stroke; 1 (168 cc) also satisfied histological criteria for ad. we conclude that (1) the clinical features of vd and a d overlap considerably; (2) diagnostic criteria based on the temporal association of stroke with dementia may have predictive value for vd; and (3) the frequent coexistence of a d and strokes indicates that refinement of criteria is needed to distinguish "mixed" and "pure" vd. clinicopathological correlation remains essential for any study of putative vd. left-handedness has been proposed as a marker for decreased survival in the general population, but possible effects of handedness on longevity in alzheimer's disease (ad) have not been examined. we hypothesized that left-handed ad patients would evince more rapid deterioration and therefore die at an earlier age than right-handed patients. subjects were 103 demented patients consecutively confirmed at autopsy to meet nincds-adrda criteria for "definite" ad. handedness was determined from structured interviews with primary caregivers and validated for most subjects with the edinburgh inventory of handedness. age at onset of dementia symptoms retrospectively determined by caregivers was used to calculate the duration of illness at the time of death. because of reported gender differences with regard to longevity, we first partialled out effects of gender before using hierarchical regression procedures to test the hypothesis. four of 46 men and 2 of 57 women with definite ad were left-handed. the mean age at onset did not differ significantly between handedness groups (f [ l,loo] = .82), but the mean duration of symptoms ( alterations in the optical properties of brain can be used to detect pathological changes in patients with alzheimer's disease (ad). using time-resolved spectroscopy (trs) and phase-modulation spectroscopy (pms), we measured the absorption (ua) coefficient, scattering (us) coefficient, and mean photon pathlength (pl) of red light directed through the base of the frontal lobes of 28 patients with ad and 11 age-matched control subjects. the measured values and the asymmetry index (ai) (an indication of the symmetry of the measurements between the left and right side of the brain) were correlated with the severity of disease as determined by mini-mental state score. there were significant differences between the ad and control group for ua, us, pl, and the standard deviation of ai. there was no correlation between the mms score and ua, us, or pl. however, the highest asymmetry index values were seen in moderately impaired patients , which suggests that the asymmetrical nature of the pathological process detected by optical spectroscopy is most marked during this stage of the illness. this noninvasive technique may provide a convenient method to detect and monitor the pathological changes that occur in the brain of patients with ad. disease p41. memory impairment in very mild alzheimer's a memory impairment is often the earliest indication of alzheimer's disease (ad). we investigated 3 components of disease learning and recall to determine which aspect of memory function is impaired the earliest in incipient ad. using the mayo clinic alzheimer's disease patient registry, which is a longitudinal prospective project on ad and normal aging, we identified 34 patients with very mild ad (i.e., with a mini-mental state score of 24 or greater) and 34 age-and sex-matched controls. we assessed performance on 2 memory measures: the rey auditory verbal learning test and the buschke free and cued selective reminding test (fcsrt). the 3 parameters evaluated included a measure of acquisition, total learning over trials (tl), and delayed recall (dr). on the fcsrt, an index of facilitation of performance with semantic cues (sc) was assessed. results indicated that all 3 indices, tl., dr, and sc, were capable of separating the mild ad group from the controls ( p < 0,001). using a linear discriminant analysis with stepwise variable entry, the measure that assessed the patient's ability to use semantic cues (sc) was the most sensitive parameter for separating the 2 groups ( f = 15.38, p < 0.0002), and the acquisition parameter (tl) was also useful at adding some additional predictive power (f = 8.48, p < 0.005). the delayed recall measure, however, did not add anything to the previous 2 measures. it appears that very early ad can be detected using appropriately structured memory tasks, and these procedures can be helpful in identifying at-risk individuals. alzheimer's disease (ad) has an insidious onset that is difficult to date reliably. we developed a standardized interview to provide objective criteria for dating the onset of 7 different symptoms (memory complainr, performance problems, language deficits, disorientation, depression, behavior problems, and psychosis), yielding an estimated disease onset date. inrerrater reliability (icc = 0.99;p < ,001) and interinformant reliability (icc = 3 5 ; p < .001) for the onset of first symptom was high. interrater agreement for the order in which symptoms appeared was high (icc = 0.72-0.98) as was interinformant reliability for all symptoms except memory complaint. the interview was administered to 2 16 patients with ad. mean estimate duration of illness was 4.26 years k 3.47 years and correlated significanrly with problems in instrumental activities of daily living. sixty-six percent had memory complaint and 45% had performance problems as their initial symptom. this technique provides a reliable characterization of disease onset. longitudinal studies will determine if particular onset symptoms differentially predict disease progression. the purpose of this study was to determine whether there is an excess of white matter disease (wmd) in alzheimer's disease (ad). brun and englund (1986) reported an excess of wmd in brains of patients with ad vs age-matched controls. there have been reports both confirming (bowen et al, 1990; fazekas et al, 1990) and refuting (leys, 1990) these findings using ct and mri in patients with clinically diagnosed ad. postmortem t2-weighted mri scans and program and abstracts, american neurological association 243 neuropathology were graded on 9 brains of pathologically confirmed ad subjects and 6 brains of age-matched neuropathologically normal controls. white matter lesions were scored on a 0 to 3 scale (none, mild, moderate, severe) separately for periventricular (pvl) and deep white matter (dwm) areas in mri scans and lux01 fast blue (lfb)-stained brain sections. correlations between mri and neuropathology were good ( r = 0.61 for pvl, r = 0.66 for dwm). pvl scores were higher in ad than in normal subjects on mri (ad: 2.2 l 0.9 vs controls: 0.2 rfr 0.3; p < .005). on pathology the difference in scores did not reach significance (ad: 1.3 2 0.7 vs controls: 0.5 * 0.4; p = 0.2). similarly, dwm scores were higher in ad subjects than normals on mri, but not neuropathology. in conclusion, ad brains have a significant excess of wmd on mri compared to controls. although the pvl and dwm scores for pathological sections are not different in the 2 groups, mri is much more sensitive than lfb-stained sections for wmd. thirteen autopsy cases of progressive supranuclear palsy (psp) were investigated for clinical-neuropathological correlations and heterogeneity. we reviewed clinical records of 11 men and 2 women aged 61 to 89 years (mean age 72 yr) with disease duration ranging from 4 to 12 years. most patients had classic features of psp including ophthalmoplegia, postural instability, and extrapyramidal signs. dementia was eventually observed in 9 of the 13 patients (67%). six of 9 patients (67 %) on whom adequate initial documentation was available presented with memory loss or behavior change. five of the 13 patients (395%), including 4 with an initial presentation of memory loss, were diagnosed clinically as having alzheimer's disease (ad) rather than psp; neuropathological diagnoses in these cases varied: i had combined ad-psp; 1 had ad-psp combined with parkinson's disease (pd) changes; 1 had psp-pd; and 2 had "pure" psp. the 2 patients with concomitant pd changes showed lewy bodies in the substantia nigra, locus coeruleus, nucleus basalis, and neocortex. the remaining 8 patients were clinically diagnosed as having psp; neuropathological diagnoses in these cases included 4 with "pure" psp and 4 with psp that also met neuropathological criteria for ad (psp-ad). these findings emphasize the clinical and neuropathological heterogeneity in psp. the neuropsychological battery developed for the consortium to establish a registry for alzheimer's disease (cerad) is currently used in many research studies to index the cognitive impairments of alzheimer's disease. in spite of its widespread use, normative informarion on the battery, important for interpretation of performance, has not been available. we report norms for the cerad battery based on a large sample of elderly control subjects (n = 398; white men and women; ages 50-89 yr) enrolled in the national study of cerad. performance on the neuropsychological measures was examined separately for subjects with high (2 12 yr) and low (< 12 yr) education. distribution of scores and basic descriptive information (means and sd) for each measure were determined. significant age and sex effects were observed on most cognitive measures in the highly educated group. in contrast, no significant age effects were observed in the low education group. effect of sex was not explored in this group due to the limited sample size (n = 48). further exploration of cerad performance in normal controls from underrepresented groups including minorities, residents of rural communities, and individuals with low education is in progress. intraneuronal inclusions of cytoskeletal proteins appear in several neurological diseases; for example, the neurofibrillary tangles of alzheimer's disease contain a cytoskeletal protein, tau. because the previously described slowing of axonal transport in aged animals might lead to accumulation of cytoskeletal proteins in nerve-cell bodies and axons, we assessed the abundance of 2 major cytoskeletal proteins in brain tracts of rats at age 4 months or 25 months. immunoassay was performed with monoclonal antibodies to alpha and beta tubulin and to nf-l (the core neurofilament protein) by published methods. samples were dissected in a standardized fashion and 2-mrn pieces of the following tracts were assayed: optic nerve, corticospinal tract (medulla), superior cerebellar peduncle, l5 dorsal root, and l5 ventral root. between 4 months and 25 months, the nf-l content approximately doubled in each brain site. tubulin substantially increased at of aged rats all sites except the fimbria-fornix. in contrast, tubulin did not change in the spinal roots. nf-l increased slightly in the ventral but not the dorsal root. this tendency of senescent brain neurons to accumulate cytoskeletal proteins in their axoplasm may predispose them to formation of intraneuronal inclusions in various degenerative diseases. we performed a prospective study of preoperative magnetic resonance imaging (mri) in 13 consecutive patients with intractable partial epilepsy who underwent a stereotactic resection of an extrahippocampal temporal lobe foreign-tissue lesion, "lesionectomy," between june 1986 and january 199 1. interpretation of the mri studies was performed by an investigator blinded to the presurgical evaluation, surgical outcome, and pathology. hippocampal formation (hf) atrophy was assessed using mri-based volumetry (n = 10) and visual grading of the h f (n = 13). mri-detected hf atrophy has been shown to be a reliable marker of moderate to severe mesial temporal sclerosis (mts) (cascino gd, et al, ann neurol 1991; 30:31-36 evidence from experimental animals indicates that endogenously produced platelet-derived growth factor (pdgf) is an important regulator of glial proliferation and differentiation. because of the striking degree of glial proliferation in chronic epilepsy, we sought to determine whether cultured glia from human epilepsy tissue would be responsive to pdgf. the effects of pdgf on dna synthesis, proliferation, and relative distribution of a2b5(+) glia were studied in a cell culture derived from temporal lobe white matter of adult epilepsy lobectomy tissue. by immunocytochemistry, glial fibrillary acidic protein (gfap) was detectable in 90% of cells in untreated or 4-day pdgf-treated (10 ng/ml) cultures, which confirmed their astrocytic nature. in contrast, a2b5( +) cells increased from 10 to 30% in untreated cultures to 75% after pdgf treatment, which suggested that type 2 astrocytes (a2b5[ + 1, gfap[ +]) had been elicited. dna synthesis of cells resembling oligodendrocyte-type 2 astrocyte (0-2a) progenitors occurred within 24 hours after program and abstracts, american neurological association 245 pdgf treatment as evidenced by nuclear incorporation of brdu in bipolar a2b5( +) cells. these studies demonstrate that expansion of adult human gfap( +) astrocyte populations is sensitive to regulation by pdgf. further, these data imply the existence of pdgf-responsive 0-2a progenitor cells in astrocyte-rich cultures derived from human epilepsy tissue. ( we have recorded vagal and esophageal-evoked potentials after electrical (e) and balloon (b) stimulation in 10 epileptic patients who had vagal stimulators for the control of intractable epilepsy and the results were compared with esophagealevoked potentials in 10 healthy controls and 3 diabetic patients. the vagal and esophageal-evoked potentials showed similar configurations with 3 major positive and negative potentials. the amplitudes of the responses habituated rapidly over 30 trials at 2 per second up to 1 per 6 seconds. latencies were shorter from the upper esophagus (20 cm above lower esophageal sphinctereles]) cf. lower esophagus ( 5 cm above les) yielding conduction velocities of 5 to 7 meters per second but conduction was significantly slower in the diabetics. the vagal-evoked potentials have validated the use of esophageal-evoked potentials as a practical method of assessment of the integrity and speed of conduction in vagal afferent pathways in man. ronald e. kramer and neil l. rosenberg, englewood. co seizure disorders were analyzed in patients in whom toluene was the sole or major drug of abuse. toluene abuse is increasing; therefore, physicians should gain experience with its neuropathological and ciinical sequelae. a retrospective chart review found 15 patients meeting criteria. the average patient age was 28 years; abuse onset averaged 16.7 years; abuse averaged 10.7 years; and 11 patients were male. ten were daily, 3 were weekly, and 2 were intermittent users. seizures occurred in 2. one suffered a single generalized tonic-clonic seizure without recurrence and without treatment. his we characterized the clinical dose-response curves for relief of parkinsonism and production of dyskinesias as a function of plasma levodopa and 3-0-methyldopa levels in 6 patients with parkinson's disease (pd) and fluctuating responses to oral levodopa/carbidopa. dose response to graded intravenous levodopa was measured after overnight drug withdrawal on 2 occasions, first after chronic, intermittent oral levodopa/ carbidopa and second after 3 to 5 days of continuous intravenous levodopa. continuous intravenous levodopa shifted the dyskinesia dose-response curve to the right, and reduced maximum dyskinesia activity, but did not significantly alter dose response for relief of parkinsonism. improvement in dyskinesia was apparent by the second day of continuous levodopa, during which ratios of plasma dopa/3-0-methyldopa remained constant. our results support the hypothesis that relief of parkinsonism and production of dyskinesias occur by separate mechanisms. continuous dopamine-mimetic therapy should be sought as a therapeutic goal for advanced pd. dopa-responsive dystonia (drd) is a distinct subset of idiopathic dystonia with diurnal fl uctuation and a dramatically beneficial response to l-dopa. it has hitherto been considered an autosomal-dominant disease with reduced penetration (mckusick no. 12823) . we studied an arabic family of 67 members with d r d spanning 6 generations. we examined 43 members and 6 of their spouses. l-dopa was withheld for 24 hours from patients in treatment. five family members had generalized dystonia with diurnal auctuation ( i male, 4 in an arabic family females). dystonia started between the age of 2 and 7 years with gait difficulty and involvement of the legs. mri, eeg, evoked potentials, and screening for wilson's disease were negative. an excellent response to l-dopa was noted in all 5 patients with continued long-term clinical stability for as long as 17 years. the 5 patients were the products of 2 consanguineous marriages, and their 7 siblings were normal. the patients were descendants of the same great-great-grandparents. this pedigree suggests an autosomal-recessive type of inheritance. we believe this is the first report of d r d with an autosomal-recessive type of inheritance. a. achiron, m . gornish, h . goldberg, i . ziv, r. djaldetti, y. zoldan, h. smka, and e. mekzmed, petah tiqva, israel freezing gait is an incapacitating symptom that occurs often in advanced parkinson's disease and also in other neurological disorders, eg., multiinfarct state, multisystem atrophies, and normotensive hydrocephalus. we evaluated, videotaped, and rated 18 patients (15 men, age 74 k 6,60-82 yr) who developed pure progressive freezing gait during 2.5 2 1.9, 0.5-6 years. severity was mild in 4 with sudden motor blocks mainly when confronted with obstacles; moderate in 9 with gait arrests upon any attempt to initiate walking and changing direction, requiring a walking stick or partial external assistance; and severe in 5 with total inability to start walking, requiring a walker, massive assistance, or a wheelchair. in all, freezing was associated with postural instability. they could mimic normal gait when seated or lying prone and could overcome arrests by the "walking over lines" maneuver. neurological examination was otherwise normal with no signs of dementia, parkinsonism, or pseudobulbar palsy. ischemic risk factors including ischemic heart disease, hypertension, and diabetes occurred in 7 and previous strokes in 6. brain c t and mri were normal or showed mild cortical atrophy in 12 and putative lacunae in only 6 patients. none responded to levodopa or dopamine agonists. progressive pure freezing gait should be recognized as a separate nonparkinsonian neurological entity. it may be due to degenerative or ischemic non-nigral brainstem lesions. we describe 19 patients with causalgia and dystonia, triggered by peripheral injuries in 15 patients and occurring spontaneously in 4 patients. the injury was often trivial. the mean age at presentation was 28.6 years. the legs were affected in 12 patients, and the arm was affected in the remaining 7 patients. all had burning pain, allodynia, and hyperpathia, along with vasomotor, sudomotor, and trophic changes. all developed typical dystonic muscle spasms in the affected part. the spasms typically were sustained, producing a fixed dystonic posture, in contrast to the mobile spasms characteristic of idiopathic torsion dystonia. dystonia always followed the causalgia and was painful. there was spread of the causalgia and of the dystonia from its initial site both in the affected limb and to other extremities, the latter in a hemiplegic, transverse, and triplegic distribution. all forms of conventional treatment failed to relieve either the pain or the dystonia. we suggest that functional changes in the corticobasal ganglia-thalamic system are responsible for this painful dystonic syndrome. program and abstracts, american neurological association 247 holiday for parkinson's disease: a controlled clinical trial r. kurlan, c . m . tanner, c. g. goetz, j . sutton, p. carvey, c. deeley, l. cui, c. itvine, and m . mcdemzott, rochester, ny, chicago, il, and san jose, c a the efficacy and mechanisms of levodopa (ld) drug holiday for parkinson's disease (pd) remain controversial. we performed a double-blind, randomized study with 11 advanced pd patients (5 men, 6 women; aged 52-79 yr) with entry criteria of inadequate response to ld plus dose-limiting ld-induced side effects (dyskinesias, hallucinations, and confusion). subjects were assigned to: (1) 100% placebo for ld (complete drug holiday) or (2) 50% ld and 50% placebo for ld (50% drug reduction) for 6 days. after subsequent open-label ld dose optimization, subjects were followed to end point (defined as the time when entry criteria were again satisfied or a maximum of 1 year). median survival time to end point was not significantly different for the complete drug holiday (182 days) and 509% drug reduction (100 days) groups ( p = 3 5 ) . aspiration pneumonia occurred in 2 complete drug holiday patients and no significant morbidity occurred with drug reduction. after a 100-mg dose of ld, clinical and pharmacological responses were no different before and after drug holiday ( p = .75) or reduction ( p = 254). subject to the limitations of our small sample size, we conclude that complete drug holiday is associated with greater morbidity and confers no major advantage over 50% drug reduction. we found no evidence of significant alterations of pharmacokinetic or pharmacodynamic properties of ld after drug holiday or reduction. ( (pc) of the substantia nigra on t2-weighted images. the narrowing of the pc signal has been attributed either to atrophy of the pc or to increased deposition of iron in this region. we have studied details of iron distribution in the midbrain of 8 formalin-fixed human brains by scanning pixe analysis. three p d brains, 1 juvenile pd brain, and 4 control (amyotrophic lateral sclerosis) brains were studied. in the controls, the iron content of the pars reticulata (pr) was almost equal to that of the red nucleus (rn), but that of the pc was less than 50% of the pr. in parkinsonian brains, the iron content in the pc was significantly higher than in the controls. the iron content ratios of pc/pr and pc/rn in parkinsonian brains were significantly higher than in the controls. these findings suggest that iron deposition increases in the pc of parkinsonian brains. the difference in the pattern of iron content corresponds to the intensity profile pattern noted on mri. mri findings in parkinsonian patients may reflect a change in the iron distribution pattern. (jankovic and brin, nejm, 1991) . such resistance within 2 exposures is not likely due to toxin antibody formation. of 61 cd patients evaluated per protocol receiving 2 or more botox tx under multichannel emg monitoring, 7 (11.5%) showed no benefit after the first and second tx, despite mild neck weakness on static muscle testing and, in some instances, emg signs of denervation. the 54 responders (16 men, 38 women) had younger age onset cd (mean 45 yr vs 60 yr), but similar duration (mean 10 yr vs 9 yr) compared to the 1 male and 6 female nonresponders (in contrast to jankovic and schwartz, arch neurol, 1991) . both groups had similar degrees of severity and tx dose (mean 200 iu, range 112.5-300). although disparate group size prohibits statistical analysis, some interesting comparisons include: nonresponders were more apt to have extranuchal dystonic sites (72% vs 33%), antecollis (29% vs 4%), dark eyes (57% vs 19%), women with elevated antinuclear antibody titer (67% vs 24%), history of intracranial operation (29% vs o%), significant for age focal mri abnormality (5 of 6 vs 3 of 19). history of cervical operation (6 patients) did not limit responsiveness. rates of prior remission, perinatal stress, antecedent trauma, left-handedness, and family history of movement disorder were similar for both groups. antecollis presents problems for optimizing tx to affected muscles, but central mechanisms may play a role in why some patients with focal dystonia do not improve with botox tx from the outset. enrico fazzini, new york, n y with parkinson's disease does deprenyl have a symptomatic effect on patients with untreated and l-dopa-treated parkinson's disease (pd)? once deprenyl is started, how long is it before another medication is needed to control symptoms of continued disease progression? there has been controversy over whether deprenyl has effects on delaying disease progression (nejm 1989; 321:1364) and/or in alleviating the symptoms of pd. one hundred seventy-five patients already taking l-dopa (group 1) and 33 patients who had never taken l-dopa (group 2) were treated with deprenyl 10 mg/day. unified pd rating scale (updrs) scores were measured before and after deprenyl. patients were followed until pd symptoms progressed to the point of requiring additional medication. one hundred eighteen of 175 (67%) patients in group 1 (reduced updrs mean activities of daily living [adl) 11 to 9, motor [mtr] 30 to 22) and 29/33 (88%) patients in group 2 (reduced updrs mean adl 11 to 6, mtr 26 to 20) reported symptomatic benefit. an average of 12 months' duration was found in both groups before further medication adjustments were needed. deprenyl provides symptomatic benefit for an average of 12 months in the majority of patients with p d regardless of whether or not they are being treated with l-dopa. yasuo iwasaki, masao kinoshita, toshiya shojima, and ken ikeda, tokyo, japan, and cleveland, oh in parkinsonian patients we measured fasting plasma amino acids in 30 parkinson's disease patients and 30 controls matched for age and sex. all patients were receiving l-dopa and they were free of any medications other than l-dopa. normal controls were free of any medication. there were no differences in diets between patients and controls. fasting blood specimens were collected in heparinized tubes and immediately were centrifuged at 20,000 g for 10 minutes. analysis of plasma amino acids was performed by automated ion-exchange chromatography with lithium-based buffer and an amino-acid analyzer. parkinsonian patients had significant elevations of aspartate, glutamate, and glycine. the other amino acids were not significantly different from those in controls. n o correlation between severity or activity and degree of abnormality in plasma level of amino acids in patients was established. we conclude that excitatory amino-acid metabolism is altered in patients with parkinson's disease. bonnie e. levin, rachel tomer, and william weiner, miami, fl disease there is evidence linking obsessive-compulsive symptoms (ocs) to basal ganglia dysfunction. we investigated the presence and severity of ocs in a sample of 44 patients with an unequivocal diagnosis of idiopathic parkinson's disease (pd) using the leyton obsessional inventory. ocs was found in the majority of the patients, with 24 (55%) scoring above the normative cutoff for the symptom score and 32 (73%) scoring above the normative cutoff for the trait score. when severity of oc symptoms was correlated with a battery of neuropsychological measures, significant relationships were observed between ocs and a preponderance of tests associated with right-hemisphere functions. these findings were observed especially on those tests with a strong frontal lobe component (block design: r = -.50; embedded figures: r = -.534; set shifting: r = -.42; and perseverative responses: r = .58; p < .005 for all measures). in all cases, the more severe oc symptoms, the poorer the performance. a similar trend was observed between the leyton trait scores and the cognitive measures. these findings suggest that ocs is present in a subgroup of pd patients, which may reflect greater compromise of right-hemisphere basal ganglia-frontal lobe pathways. intraclass correlations (iccs) were calculated for the total motor score and for each individual sign. results indicated excellent agreement (icc > .7) for the total motor score, resting tremor, gait, arising from a chair, and speeded, repetitive movements; good agreement (icc > .5) for rigidity, action tremor, posture, postural stability, and bradykinesia; and poor agreement (icc < .2) for speech and facial mobility. a factor analysis was then performed on updrs motor scores for 146 pd patients from a community-dwelling cohort. three factors were extracted by principal components analysis with subsequent varimax rotation, accounting for 63.5% of the total variance: factor 1-balance and stability (posture, postural stability, gait, arising from a chair, and bradykinesia); factor 2-rigidity and motor speed (rigidity, speech, facial mobility, rapid alternating movements, leg agility, hand movements, and finger tapping); factor 3-tremor (resting and action). these results indicate that the updrs motor examination is reliable between raters and measures the cardinal signs of pd. an open pilot study was performed to evaluate the efficacy of botulinum a toxin (botox) injections for disabling hand tremors. a previous report on the use of botox for hand tremors suggested that it was helpful, but relied on subjective clinical rating scales. the extent of normal clinical fluctuations or a placebo response could not be determined. to investigate these issues more objectively, 7 patients with parkinson's disease and 1 with essential tremor with refractory hand tremors underwent electromyographically guided intramuscular injections of botox into wrist flexors and extensors. patients without great medication-related tremor fluctuations were selected. results before and after botox were determined by comparing (1) patient perceptions of functional improvement, (2) clinical assessments using the unified pd rating scale for tremor and the webster rating scales, and (3) physiological measurements using accelerometric analysis of hand tremors of tremor frequency, amplitude, and waveform characteristics. all patients reported some improvement, ranging from mild to marked with a mean of 2.6 on a 0 to 4 (4 = marked) global rating scale. however, only 3/8 patients showed a significant improvement in the clinical rating scales, confirmed by >50% reduction in tremor amplitudes. these findings show that most patients reported improvement not confirmed by the clinical or physiological measures. efficacy of botox injections for tremors is implied, but controlled trials are needed before this procedure can be generally recommended. christopher g. goetz and glenn t . stebbins, chicago, i l we tested whether hallucinations, motor disability, and cognitive decline were risk factors for nursing home placement in advanced parkinson's disease (pd) and whether these effects were independent or synergistic. between 1987 and 1991, we identified 11 patients admitted to long-term nursing homes. using case control methodology, we matched each for age, pd duration, and sex with 2 control pd patients remaining at home. parkinsonism was assessed by the motor and activities of daily living subscales of the unified p d rating scale (updrs); hallucinations and dementia were determined by scores 2 2 on the thought disorder and intellectual impairment items of the updrs. tests of synergy were based on a mantel-hentel model. hallucinations were a significant risk factor with odds ratio = 18.0, x2 = 17.0, p < ,001. motor impairment alone and cognitive impairment alone were not significant risk factors for nursing home placement (x2 for motor severity = ,00084, p > .05, and x2 for cognitive impairment = .0023, p > .05). furthermore, combined odds ratios for hallucinationslmotor severity and hallucinations/cognitive impairment showed no synergy of effect (x2 <1.0 for both,p > .05). of the 3 variables studied, hallucinatory behavior is the most prominent and independent risk factor for nursing home placement in these patients; the data suggest that aggressive control of hallucinations may be warranted to prevent nursing home admission. temperature-sensitive paramyotonia congenita phenotype* louis j . ptacek, philip mcmanis, hzrbert kwiecinski, alfred george, robert barchi, launce gouw. and mark leppert, salt lake city, u t , rochester, mn, warsaw, poland, and philadelphia, pa the periodic paralyses are a group of autosomal-dominant muscle diseases sharing a common feature of episodic paralysis. in one form, paramyotonia congenita (pc), the paralysis is temperature-sensitive, usually occurring with muscle cooling. electrophysiological studies of muscle from patients with pc have revealed temperature-dependent alterations in sodium channel (nach) function. this observation led to the identification of 2 distinct mutations in an s4 segment of a skeletal muscle nach in 3 unrelated pc families. we describe the use of the single-strand conformation polymorphism (sscp) technique to define a third allele specific to pc patients in an additional family. this aberrant pattern, though distinct from the first 2, occurs in the same exon of this nach gene. sequencing is currently underway to define the molecular alteration causing this aberrant pattern. two additional families with the pc phenotype have been sampled and do not demonstrate these 3 sscp variants. we are currently searching for new mutations in these 2 families to define further the molecular heterogeneity of this temperature-sensitive pc phenotype. parag mehta and roger w. kukz, brooklyn, n y abnormal accumulation of calcium (ca) in myofibers is thought to play a role in pathogenic myonecrosis. attempts at reducing intracellular ca content with ca channel blockers in duchenne muscular dystrophy (dmd) have been clinically unsuccessful. dantrolene, however, which acts at the sarcoplasmic reticulum to inhibit ca release from intracellular stores, has produced dramatic reductions in serum creatine kinase (ck) in dystrophic mice and more recently in dmd. we investigated the effect of low-dose dantrolene in a group of 20 patients with limb girdle dystrophy (lgd), dmd, and other myopathic disorders. all subjects received dantrolene in incrementing doses from 25 to 100 mg daily over a 6to 12-week period. mean baseline ck was compared to ck with dantrolene treatment. dramatic reductions in serum ck levels averaging 50% were seen at 50to 100-mg doses in lgd patients (8). dmd patients (3) and patients with other myopathies (9) showed a similar but less dramatic reduction in ck. three of the weakest patients complained of increased fatigue while taking 100 mg, which suggests that higher-dose dantrolene may confound longer-term trials assessing clinical muscle strength and function. dantrolene in dosages well below conventional antispastic doses has a dramatic effect on serum ck and possibly myofiber necrosis in lgd, other dystrophies, and other muscle disorders. p77. antigen-specific therapy in myasthenia gravis: myasthenia gravis (mg) is mediated by anti-acetylcholine receptor (achr) antibodies, believed to be t-cell dependent, and antigen-specific therapy would be preferable to current nonspecific immunosuppression. exposing mouse t-cell clones to mhc class i1 molecules complexed with relevant antigen on planar membranes induced proliferative unresponsiveness (quill and schwartz, 1987) , and soluble mhc class i1 molecules complexed with myelin basic protein (mbp) peptide resulted in unresponsiveness of specific tlymphocyte clones in vitro (sharma et al, 1991) . we have used our well-defined dr4-restricted t-cell clone (ong et a [, 1991 ) isolated from an mg patient and specific for p138-167 of the achr alpha subunit. overnight incubation of these t cells with a soluble p138-167 : dr4 complex substantially inhibited the subsequent response to challenge with soluble antigen and presenting cells. in contrast, antigen response after preincubation with dr4 complexed to an irrelevant peptide (mbp 1-14), soluble dr4 alone, or an experimental approach studied in vitro p138-167 alone (at equimolar concentrations) did not differ appreciably from that in untreated cells. the p138-167:dr4 complex had no effect on other non-achrspecific cell lines/clones. these results suggest that the use of soluble mhc-peptide complexes may be an approach to selective immunotherapy in mg patients. p78. high-dose intravenous immunoglobulin in the shawke a. soueidan and marinos c. dalakas, bethesda, md inclusion body myositis (ibm) is a severe disabling inflammatory myopathy with characteristic clinical and histological features. it is commonly suspected when a patient with presumed polymyositis does not respond to available immunotherapies. the need for an effective treatment in patients with ibm prompted the present pilot study using high-dose intravenous immunoglobulin (hd-ivig), an apparently effective immunomodulating agent in several autoimmune neuromuscular disorders. we treated 4 patients with muscle biopsy-proven ibm with up to 2 monthly infusions of 2 gml kg ivig. after the first infusion, 3 of the 4 patients showed definite functional improvement consisting of independent ambulation, fewer falls, and increased ability to lift weights. the muscle strength of the proximal and less atrophic muscle groups improved by one grade mrc scale (from 4 to 5), whereas the distal and atrophic muscles remained unchanged. the improvement, sustained up to 7 months, was greater in patients with the most severe endomysial inflammation. we conclude that hd-ivig may be the first promising agent that can improve the strength of certain muscle groups in patients with ibm. because ivig is prohibitively expensive, the present encouraging results warrant a large-scale controlled therapeutic study. hays, and n . lutov, new york, n y , and milan, italy anti-myelin-associated glycoprotein (mag) antibodies from patients with neuropathy cross-react with the glycolipid 3-sulfated glucuronyl paragloboside (sgpg). among 24 patients tested by enzyme-linked immunosorbent assay and western blot, 15 had highly elevated antibody titers (56,400) to both mag and sgpg, 2 had highly elevated titers to mag alone, and 7 had highly elevated titers to only sgpg. immunostaining of normal nerve myelin by the antibodies correlated better with anti-mag than anti-sgpg activity. twenty-one of the patients, including patients in all 3 groups, had predominantly sensory or sensorimotor neuropathy, and biopsy specimens revealed deposits of igm and complement on affected myelin sheaths. three patients presented with motor syndromes, all with antibodies specific for sgpg; 1 had a predominantly motor demyelinating neuropathy, 1 had upper and lower motor neuron signs and peripheral neuropathy, and 1 had amyotrophic lateral sclerosis confirmed post mortem. all 3 had deposits of complement on peripheral nerve myelin sheaths. these studies suggest the following: (1) that anti-mag or sgpg antibodies may differ in their fine specificities and biological activities, (2) that anti-sgpg antibodies also may occur in motor neuron diseases, complicating the clinical presentation, and (3) that both mag and sgpg should be used as antigens in testing for autoantibody activity in peripheral neuropathy. david b. williams, john steele, ulla-katrina craig, sandra bryant, peter o'brien, and leonard kurland, newcastle, new south wales, australia, mangilao, guam, and rochester, m n continuing surveillance of neurodegenerative diseases in the mariana islands reveals changes in frequency and clinical characteristics since the 1950s that resemble those in other known western pacific foci (kii peninsula, japan, and irian jaya, new guinea). recent surveys of patients 50 years and older were conducted on rota, tinian, and yigo, guam. possible cases of dementia, parkinsonism, and amyotrophic lateral sclerosis (als) were identified by local trained personnel using a questionnaire, world health organization neurology test, and cognitive screening. those who failed the screening were examined by a neurologist. in the small populations of rota and tinian, there were no definite cases of als compared to i to 3 cases present in previous surveys. the high prevalence of parkinsonism-dementia complex (pdc) was unchanged and dementia was increased compared to earlier surveys. in yigo, als and pdc continue to be prevalent; however, the als patients are predominantly long-term survivors (> 10-year disease duration). in areas of previous high prevalence of als/pdc, dementia (as pdc) was associated with extrapyramidal signs, whereas in areas of previously low prevalence of als/pdc, dementia alone, possibly of alzheimer type, predominated. these observations help to confirm previous reports of changing clinical patterns, but suggest that the geographic distribution of the (presumed) environmental etiological agent for als/pdc remains stable after almost 40 years. p. v . fragokz, 0. frongillo, m. michisanti, g. antonini. derangements of the cardiac conducting system are the most common features of heart involvement in myotonic dystrophy (md). in view of chis 65 patients with various grades of md (43 males and 22 females, mean age 37 yr) underwent 12-lead ecg and holter monitoring. in 31 patients (48%), almost all with a severe grade of md, 1 or more conduction defects were found: first-degree atrioventricular block (i-avb) in 21 cases, second-degree avb in 1 case, right bundle branch block in 3 cases, left anterior hemiblock in 2 cases, left bundle branch block in 2 cases, and trifascicular block in 1 case (pacemaker implanted). an 18-year-old boy had a chronic atrial fibrillation with slow ventricular rate; he died suddenly while awaiting electrophysiological study. thirty-seven patients were followed over a mean period of 40 months (range 11-75 mo). a 54-year-old woman experienced a myocardial infarction and was excluded from subsequent considerations. conduction defects de novo appeared in 6 patients: i-avb in 5 and i-avb plus 11-avb (mobitz i and i1 type) in 1. nine patients, all with i-avb at initial evaluation, showed deterioration of their defects' conduction: a bifascicular block was observed in 6 cases and a trifascicular block in 3 cases (pacemaker implanted). conduction defects may run a malignant course in md, mainly in patients with more severe grades of the neuromuscular disease; thus, a close cardiological evaluation is mandatory for a proper therapeutic approach in single cases. hiroshi mitsumoto, surest5 kumar, kevy h. levin, robert w. shields, jr, michelle secic, asa j . wilbourn, and rajendra g . desai, cleveland, oh, and santa ana, ca twenty patients with amyotrophic lateral sclerosis (als) entered a pretreatment study with monthly quantitative isometric muscle strength tests (4 muscles in each extremity) and quantitative tufts scales including vital capacity, bulbar diadochokinetic rate, timed water drinking, timed rising from a chair, and timed walking 5 meters. after 4 to 6 months of pretreatment observation, the patients received 400 mg/kg intravenous immunoglobulin (ivig) (gamimune-n, miles) every month for up to 12 months. during the study period, 5 patients died and 2 patients withdrew from the study. the slope of each variable's changes over time before and after the ivig treatment were compared statistically. none of the quantitative scales showed significant change with ivig. however, the slope of the upper extremity muscle strength revealed improvement with ivig treatment ( p = .002 and .013, right and left, respectively). when all extremities were combined, the slope was also significantly improved with ivig ( p = .035). lower extremity muscle strength alone showed similar trends but no statistical significance. electrophysiological and immunological data were also analyzed. our results warrant a double-blind, controlled study with ivig for the treatment of als. leber's hereditary optic neuropathy (lhon) is a mitochondrial disorder with predominantly optic nerve abnormality. it can be associated with dystonia, ataxia, encephalopathy, cardiac abnormalities, and other less well-characterized neurological syndromes. we describe 2 members of a family with lhon with a slowly progressive motor polyneuropathy. a 22-year-old man and his 11-year-old sister have had mild motor impairment since early childhood. a gait disorder and distal muscle weakness became evident at puberty. the girl, but not the man, also has blindness, distal numbness, type i diabetes mellitus, and short stature. physical examination showed in both: bilateral foot drop, limb hyperreflexia but absent ankle reflexes, distal sensory loss, and a slight brownish scaly skin discoloration over the forearms. only the girl had clonus and optic nerve atrophy. the man had peripapillary telangiectasias. the jaw jerk was normal in both. motor nerve conductions in both showed absent tibia1 and peroneal responses, whereas other motor and sensory nerve conductions were normal. emg revealed denervation, more so distally. muscle biopsy findings showed recent denervation and previous denervation followed by reinnervation. n o raggedred fibers were observed with the modified trichrome and sdh stains. brain mri was normal in both. cerebrospinal fluid in the girl was normal. blood samples from both patients and maternally related family members revealed a mitochondrial dna point mutation at position 11778 (d. c. wallace). in this family, only 1 male had lhon; 3 females had lhon and 2 others, including the patients' mother, were asymptomatic carriers. this association of chronic motor neuropathy and hyperreflexia with lhon appears to be a distinct syndrome. the pathogenic mechanism by which the mitochondrial dna defect causes the neuropathy (and other neurological deficits) requires analysis. duchennelbecker muscular dystrophy henry j . kaminski, mazen al-hakim, r. john leigh, bashar katirji, and robert l. ruff; cleveland, oh fast-twitch extremity muscle fibers are preferentially affected in duchenne/becker muscular dystrophy (dbmd). since saccades are thought to be mediated by fast-twitch fibers, saccadic velocities would be expected to be decreased among these patients. to investigate involvement of extraocular muscle (eom) by dbmd, we studied with infrared oculography 3 patients who were wheelchair-bound and able to perform only minimal activities of daily living. saccades were slightly slowed but were within 95% confidence limits of normal. all 3 patients showed square wave jerk movements (swj). in 2 patients, the frequency of the swj exceeded that of normal subjects, which suggested central nervous system dysfunction. clinical neuroophrhalmological examination of 7 other dbmd patients was normal. this investigation is the first study of ocular motility in dbmd and demonstrates that eom function is relatively preserved even in far advanced patients. eom is composed of a heterogenous mix of fiber types that differ in anatomical and physiological characteristics from extremity muscle. study of eom in dbmd may prove to be useful in understanding why some muscles are resistant to dbmd and in characterizing properties that limit muscle degeneration. (supported by nih grants ey09186, ey067 17, the department of veterans affairs, and the evenor armington fund.) since patients with myotonic dystrophy (mtd) exhibit a marked resistance to insulin effect on glucose uptake and an impaired handling of the insulin-sensitive amino acids, it is possible that muscle wasting in mtd may reflect a derangement of insulin action on muscle protein metabolism. increased muscle protein breakdown in mtd would be expected if the normal inhibitory effect of insulin on protein catabolism is impaired. the forearm perfusion technique combined with measurements of 3-methylhistidine (3-mh) arteriovenous (a-v) differences by high-performance liquid chromatography provides a unique method to investigate skeletal muscle myofibrillar protein degradation in vivo. we studied 3-mh (a-v) and efflux from the forearm muscles in 8 men moderately affected with mtd and 10 normal men. efflux values (q) were calculated as the product of 3-mh (a-v) times forearm plasma flow measured by the indicator dilution technique. forearm 3-mh release (estimated as {a-v} or q) of mtd patients did not differ significantly from normal controls. we conclude that myofibrillar degradation is not increased in mtd even when measured in a muscle compartment selectively affected by wasting. the possibility of an impaired anabolic action of insulin in mtd has yet to be determined. to study the relationship between the ragged red fibers (rrf) and age, we have reviewed 500 muscle biopsy specimens prospectively. patients with well-established mitochondrial myopathy syndrome and with myopathies known to produce secondary rrf were excluded. the number of ragged red fibers (rrf) was counted under 40 x (lpf) magnification. rrf were identified by the modified trichrome and sdh stain. for the final analysis, the sdh staining was used. the frequency of rrf was analyzed in relation to patients' ages. the frequency of cases with more than 1 rrf increased with aging: 0% in the first decade, 16% in the fourth decade, and 55% in the eighth decade. the frequency of cases with more than 10 rrf also increased with aging: 0% in the first three decades, 4% in the fourth decade, and 19% in the eighth decade. of 25 patients with well-established mitochondrial myopathy syndrome, 24 had more than 10 rrf under lpf. the number of rrf in muscle increases with aging, indicating that mitochondrial activity in muscle is affected by aging. this finding may complicate the diagnostic criteria of mitochondrial myopathy in older individuals. ten rrf under lpf seems to be a reasonable cutoff point for the diagnosis of mitochondrial myopathy. schwartz-jampel, a rare autosomal-recessive syndrome characterized by short stature, myotonia, skeletal abnormalities, and peculiar facies, was reported by aberfeld in 1965. the same sibship was earlier reported by schwartz and jampel in 1962 with emphasis on blepharophimosis. as of this writing about 30 cases have been reported in the literature. most of the features of this syndrome are believed to be secondary to primary muscle disease. several peripheral electrophysiological studies showing features of myotonia have been reported. we describe 4 patients with schwartz-jampel syndrome showing evidence of central conduction disturbance documented by somatosensory-evoked potentials (seps). median nerve seps showed normal latencies to erb's point and n-13 in all. interpeak latencies between n13 and n19 were prolonged in 3 with complete block in 1. emg showed typical myotonic discharges in all. motor nerve conduction velocities, visual and brainstem auditory-evoked potentials, ct, and mri were normal in all. seps in the parents were normal. we believe this is the first report documenting evidence of central nervous system (cns) involvement in schwartz-jampel syndrome. schwartz-jampel syndrome and myotonic dystrophy may have similar cns ''lesion'' as sep abnormalities also have been shown in myotonic dystrophy. epidemiological studies have associated consumption of certain batches of l-tryptophan (lt) with development of the eosinophilia-myalgia syndrome (ems). 1,1 '-ethyledenebisltryptophan) (ebt or peak e), a derivative of lt, is a trace contaminant associated with implicated batches of lt. three female lewis rats received ebt, 4 mg per 100 gm daily, by intraperitoneal injection. four control rats received unimplicated lt. n o peripheral eosinophilia, rash, or weakness were observed in either group. one rat from each group died during the experiment (control-bowel infarct; ebt-death under anesthetic). after 132 days, forelimb and hindlimb muscles of the remaining animals were frozen and fixed for program and abstracts, american neurological association 253 ultrastructural and histological studies. two ebt rats had a myopathy involving soleus with a perimysial infiltrate containing lymphocytes, macrophages and sparse eosinophils, and necrotic fibers; the other showed few necrotic fibers in gastrocnemius. occasional eosinophils were seen in fascia in both animals given ebt but not in controls. fiber-type specific quantitative analysis of the microvasculature showed no decrease in the capillary index. ultrastructural examination revealed an increase in the size of microvessels in ebt animals. no denervation or reinnervation were demonstrated. the perimysial inflammation replicates an important feature of human ems and supports the epidemiological evidence that ebt is the causative agent of the disease. britta ostermeyer-shoaib, bernard m. patten, and tetsuo ashizawa, houston, t x five women (patients 1-5) developed motor neuron disease (mnd) 10 years (range 2-23 yr) after receiving silicone gel-filled breast implants. at explant in 4 patients, 2 had both and 2 had the left implant ruptured with silicone spilled into tissue. one woman (patient 6) developed amyotrophic lateral sclerosis (als) 5 years after numerous injections of free silicone into her face. biceps muscle biopsy specimens in all 6 showed neurogenic atrophy. patient 1 developed als with bulbar involvement and died 7 years later of respiratory failure. she had anti-gm1 antibodies and autopsy findings confirmed the diagnosis of typical als. patient 2 developed als, but also fatigue, myalgia, arthralgia, and skin rash. she had anti-gm 1 antibodies, antisilicone antibodies, positive antinuclear antibodies (ana), and decreased serum igg, iga, and c3, but increased igm and creatine phosphokinase (cpk) and chronic inflammation was revealed in muscle biopsy specimens. patient 3 developed als, but also had hair loss, skin rash, fatigue, headache, sjogren's syndrome, and positive ana. patient 4 developed als, but also had myalgia and arthralgia. patient 5 developed lower mnd, but also fevers, arthralgia, and joint stiffness. she had anti-gm1 antibodies, positive ana, antimyelin antibodies, and decreased serum igg and iga with chronic inflammation shown in nerve biopsy findings. patient 6 developed a steroidresponsive and steroid-dependent als with bulbar involvement. she had a monoclonal gammopathy in the cerebrospinal fluid and increased cpk. we suggest that silicone acts as an adjuvant that damages motor neurons via an indirect autoimmune mechanism. implants and silicone injections into the face p90. silicone adjuvant breast disease: more forty-five women developed mixed sensory-motor neuropathy (35), motor neuron disease (5), multiple sclerosis (2), multiple sclerosis-like syndrome (2), or myasthenia gravis (1) 7 years (range 1 mo-23 yr) after receiving silicone-gel breast implants (38), saline-filled silicone-covered breast implants (6), or direct injections of silicone into the breast (1). most patients had, in addition, severe fatigability, myalgia, arthralgia, morning stiffness, skin rash, lymphadenopathy, sjogren's syndrome, and short-term memory problems. laboratory results revealed in most of the women decreased or increased serum immunoglobulins, autoantibodies, a serum monoclonal gammopathy, or oligoclonal bands in cerebrospinal fluid. at explantation in 34, 14 had both and 7 had 1 implant neurological cases ruptured. biopsy of the fibrous implant capsule in most patients showed foreign-body giant cells containing refractile material consistent with silicone whether or not the elastomer shell was ruptured, indicating silicone bleed. the major finding on surd nerve biopsy was loss of myelinated fibers, on biceps muscle biopsy was neurogenic atrophy, and on pectoralis muscle biopsy was myositis with vasculitis and free silicone in some. we suggest that silicone may provoke damage to nerve and muscle, probably indirectly promoting autoimmunity. james f. howard, jr, m . kathleen donovan. and m. susan tucker, chapel hill, nc urinary symptoms of urgency and incontinence have been reported only rarely in patients with myasthenia gravis (mg) and then most often in association with myasthenic crisis. we report the case of a 31-year-old woman who in december 1987 had the onset of chest pain and was found to have a lymphocytic thymoma. in june 1989 she developed urinary incontinence, was found to have an open bladder neck. and underwent a suspension procedure for stress incontinence in january 1990. eight months later she developed exertional fatigue and a diagnosis of m g was made. in july 1991 there was a recurrence of urinary incontinence. these symptoms clustered toward the end of the day and at trough mestinon dose. neuro-urophysiological studies demonstrated her previous open bladder neck, the inability to sustain a pelvic floor contraction, and increased bladder wall contraction. singlefiber electromyography (sfemg) recordings from the anal sphincter demonstrated a mean consecutive difference (mcd) of 88 ysec, and 11% of fiber pairs had impulse blocking while recordings in the extensor digitorium communis muscle were normal. following a course of plasma exchange, there was significant clinical improvement with a reduction in the frequency of urinary incontinence, and improvement in anal sphincter sfemg studies (mcd, 60 ysec with no blocking). this case demonstrates that in those myasthenic patients with predisposing bladder outlet dysfunction, urinary incontinence may be a manifestation of worsening mg. diana m. escolar, mohamed eldaly, and jaime rich, boston, m a debate still exists as to the role of antibody versus celmediated factors in the pathogenesis of guillain-barre syndrome (gbs). we describe a patient with increased proportion of circulating t cells and a t-cell lymphoma who developed gbs and responded to intravenous immunoglobulin (ivig). a 57-year-old man with t-cell lymphoma drveloped gbs by clinical, nerve conduction, and cerebrospinal fluid criteria. he had an elevated proportion of t cells and markedly reduced b cells with a normal cd4/cd8 ratio. he responded rapidly to ivig, with return of nearly normal motor function in 1 week. three weeks later, he relapsed and his vital capacity dropped. ivig was again administered and within 24 hours he nearly recovered. a third relapse, 2 1 days later, again responded to ivig. he has remained asymptomatic with ivig maintenance. the role of t-cell lymphocytes in initiating experimental autoimmune neuritis has been shown by adoptive transfer experiments. this patient with t-cell neoplasia may represent an analogous model in hu-guillain-barre syndrome mans supporting the role of t-cell (cell-mediated) autoimmunity in the pathogenesis of gbs. ivig therapy may act primarily by inhibiting the t-cell-mediated attack on myelin. p93. sympathetic skin response: age effect it is frequently stated that the sympathetic skin response (ssr) can be elicited in all normal subjects, but the age of the investigated population usually is not considered to be a significant factor. we have examined the ssr in the upper and lower limbs of 100 normal subjects, aged 20 to 88 years (5 5 of them males). the ssr was elicitable in the lower limbs in all subjects under the age of 60 years and in the upper limbs in all subjects younger than 70 years. in contrast, it could be elicited in the lower limbs in only 345% and in the upper limbs in 655% of octogenarians. the amplitude of the response, though highly variable, showed a remarkable decline with age, both in the upper ( p < 0.0001, r = 0.51) and in the lower ( p < 0.0001, r = 0.65) limbs. these results indicate that age affects both the elicitability and the amplitude of the ssr. this has to be taken into consideration when evaluating the autonomic function in the elderly. we reviewed records of patients appearing to have motor neuron disease (mnd) to whom we recommended immunosuppression over 4 years (17 of 2 10 m n d patients). atypical findings engendered hope rhat they might have treatable neuropathy. electrophysiological studies were mainly consistent with mnd, but also showed conduction block or other evidence of relatively mild peripheral nerve disease in 12. sensory symptoms were present in 7 ; 1 or more reduced or absent deep tendon reflexes in 6; elevated cerebrospinal fluid protein in 8; and nonspecific abnormalities on sural nerve biopsies in 6 of 7. anti-gm1 levels were measured in 6 (including 1 who improved), but none was significantly elevated. immunosuppression included cyclophosphamide (12 patients); prednisone (10); plasma exchange (4); intravenous gammaglobulin (1); cyclosporine (1); and total lymphoid irradiation (1). seven treated patients died, 6 worsened, 1 remained stable for 2 years, and 1 improved. two patients declined treatment. one died and the other did not worsen in 3 years. we conclude that immunosuppression by our methods is, at best, rarely effective in atypical mnd. we studied serum antiglycolipid antibodies by enzymelinked immunosorbent assay in 11 patients with typical miller fisher syndrome (mfs), 6 patients with atypical mfs who were lacking in some of the 3 cardinal signs, 4 patients with guillain-barrc syndrome (gbs) with ophthalmoplegia, 20 patients with gbs without ophthalmoplegia, 20 patients with multiple sclerosis (ms), and 17 patients with other immunological disorders (oid) including systemic lupus erythematosus, polymyositis, and mixed connective tissue disorder. all patients with typical mfs had increased activity of igg antibody against ganglioside g q l b in the early phase, and it reduced with time. such anti-gqlb igg activity also was detected in 5 of the 6 patients with atypical mfs and in 3 of the 4 patients with gbs with ophthalmoplegia. in atypical mfs, the only patient without increased anti-gq1 b igg activity demonstrated normal eye movement with ptosis, whereas eye movement was impaired in the other 5 patients. no patients with gbs without ophthalmoplegia, ms, or oid had increased anti-gqlb igg activity. these findings suggest the close association between increased anti-gql b igg activity and impaired eye movement in mfs and gbs. serum anti-gqlb igg activity possibly plays a role in impaired eye movement in mfs. our goal is to develop an assay that can be used to monitor a relevant immune effect of interferon p (ifnp) in multiple sclerosis (ms) patients during the course of ifnp immuno-' therapy, since recombinant ifnp is being tested in multicenter clinical trials. this report extends our prior studies of the inhibitory effect of ifnp on t-cell activation. peripheral blood mononuclear cells (pbls) from 11 healthy donors and 10 clinically stable ms patients were studied. pbl cultures were stimulated with cona, mab to cd3, or with the phorbol ester pma in the presence of the calcium ionophore ionomycin. parallel cultures were studied in the presence of ifnp, 100 uiml. t-cell activation was monitored by determining the percent cells positive for il-2 receptor (il-2r) using facs analysis, or with a sensitive enzyme-linked immunosorbent assay for ifny. ifnp markedly inhibited il-2r expression induced by cona, by mab to cd3, or by pma and ionomycin, which activate t cells via different pathways. the results suggest that ifnp inhibits t-cell activation by actions independent of membrane receptors. we observed significant inhibition of cona-induced t-cell il-2r expression in both ms patients (20.6% inhibition, p < 0.05) and controls (26.8% inhibition, p < 0.05). there was no significant difference in percent inhibition between ms and controls, but there was more variance among the ms patients. variability of biological effects of ifnp on t cells may relate to differential therapeutic responses to exogenously administered ifnp in ms patients. preliminary experiments suggested that ifnp inhibited ifn gamma secretion by pbl stimulated with cona. ifnp inhibits a number of events associated with t-cell activation, in both normal and ms t cells. response to ifnp appears more variable in the ms cases. immunological monitoring of t-cell activation in patients receiving ifnp may assist in understanding the observed therapeutic responses and planning clinical protocols. myelin destruction is associated with many central nervous system disorders, such as multiple sclerosis, head trauma, and ischemic injury. inflammatory cells, monocytes/macrophages, and polymorphonuclear leukocytes (pmn) may me-program and abstracts, american neurological association 255 diate myelin injury, and lipid peroxidation may be an important mechanism. inhibiting myelin oxidation could have substantial benefit, so we evaluated the ability of a 21-minosteroid, u74500a, to inhibit myelin oxidation by monocytes and pmn. fresh rat brain myelin was harvested by multiple sucrose gradient, ultracentrifugation steps, and the final product was confirmed to be pure myelin by sds-page electrophoresis. human monocytes or pmn were obtained from 5 healthy, unmedicated volunteers by gradient separation techniques. monocytes (1.47 x lo6 cells/ml) and pmn (3.40 x lo6 cells/ml), myelin (460 pg protein/ml), and lipopolysaccharide (100 pg/ml) were incubated for 16 hours with or without 100 wm u74500a in 1-ml wells. myelin oxidation was evaluated by a thiobarbituric acid reactive substance assay for production of malondialdehyde (nmol/ ml). myelin oxidation by monocytes was 1.80 +-0.35 (mean 2 standard error of mean) without u74500a and was reduced to 0.76 ? 0.15 by 100 pm u74500a ( p < .005). pmn-mediated myelin oxidation was 1.89 _t 0.44 without drug and 0.64 ? 0.10 with drug ( p < 0.02). these results demonstrate that u74500a markedly inhibits monocyte-and pmn-mediated myelin oxidation and suggest that the 21aminosteroids may help disorders associated with inflammatory cell-induced myelin injury. microglia cells participate in the pathological reactions of the cns to multiple insults including trauma, inflammation, and neuronal degeneration. functional roles for these cells could include mediating tissue in jury, promoting repair, or modulating immune responses. with regard to the latter, we have observed that the majority of adult human-derived microglia express major histocompatibility complex (mhc) class 11 molecules under basal culture conditions, in contrast to astrocytes derived from the same surgical biopsy specimens. all morphological subtypes of the microglia (ameboid, bipolar, and ramified) expressed mhc class i1 molecules, indicating a discordance between morphology and mhc antigen expression as markers of microglia activation. the microglia actively ingest myelin constituents, as assessed using fluorescein-labeled myelin basic protein and laser confocal microscopy. autologous t cells (e') freshly isolated from the systemic blood and cocultured with candida antigen underwent active proliferation in the presence of 1 to 10% microglia, indicating the functional capacity of the microglia to serve as antigen-presenting cells. y-interferon augmented both mhc class 11 expression and functional antigen-presenting capacity. these results indicate the potential of the adult human microglia to promote immune reactivity within the cns. protein (mbp) neutralize anti-mbp purified from multiple sclerosis cerebrospinal fluid active phases of multiple sclerosis (ms) are associated with increased titers of intrathecally produced antimyelin basic protein (anti-mbp). anti-mbp can be purified by antigenspecific affinity chromatography from csf igg of patients with acute relapses of ms. eighteen synthetic peptides of human myelin basic protein (h-mbp) containing between 8 and 25 amino-acid residues and covering the entire length of the molecule were synthesized by the fmoc method. purified anti-mbp was reacted with increasing amounts of h-mbp as well as each of the 18 peptides in an initial liquid phase assay, and subsequently titers of f anti-mbp in all resulting mixtures were measured by a solid-phase radioimmunoassay . purified anti-mbp was neutralized by h-mbp and 6 of the 18 synthetic peptides containing overall residues corresponding to 61 to 106 of h-mbp. the remaining 12 synthetic peptides covering both the amino and carboxyl terminals of h-mbp did not significantly react with purified anti-mbp from these patients. in conclusion, anti-mbp purified from csf of ms patients has affinity for epitopes located between residues 61 and 106 of h-mbp. in a double-blind study involving 70 patients, we recently demonstrated that 4-aminopyridine (4-ap) is superior to placebo in the treatment of multiple sclerosis (ms) (ann neurol, in press). the related agent 3,4-diaminopyridine (dap) also appears to be effective. to enable a preliminary comparison, 14 patients, who in our previous study had not benefitted from 4-ap, were now treated (4 wk) with dap (up to 1.0 mg/kg/day) for 4 weeks in an open-label fashion. instruments for assessment and registration of side effects were the same as in the previous trial. the optimal dose of dap was 56.4 mg/day compared to 30.7 mg/day for 4-ap. significant changes in the edss (1.0 point or more) were not found, whereas significant improvements in neurophysiological parameters were found (no difference between 4-ap and dap, allp > 0.05). subjective side effects during 4-ap (8 patients) mainly suggested cns-function disturbance (dizziness and gait disturbance) and during dap (10 patients) mainly suggested peripheral nervous system-function disturbance (paresthesias). systemic tolerability clearly was diminished for dap compared to 4-ap, with 2 patients withdrawing because of severe gastric complaints and 1 developing liver function abnormalities. these data suggest that 4-ap is more valuable than dap in the treatment of ms. cy clophosphamide/methylprednisolone therapy in multiple sclerosis multiple sclerosis (ms) is a presumed autoimmune disease in which various forms of immunotherapy have been attempted. mri studies show the disease to be more chronically active than is clinically evident, thus a single treatment is unlikely to provide lasting benefit. recently, the northeast cooperative treatment group found that pulse cyclophosphamide (700 mg/m2 every other month for 2 years) slows progressive ms. we initiated a pilot study to determine the effect of a more intensive and prolonged pulse therapy regimen in both progressive and earlier stages of the disease. pulse therapy was given after induction with either iv cyclophosphamide/corticotropin (600 mg/m2 x 5 over 8 days) or iv methylprednisolone (1 gm x 7 over 7 days). patients received a single iv dose of cyclophosphamide (800-1,400 mg/m2) adjusted to produce leukopenia plus 1 gram of iv methylprednisolone monthly for a year, every 6 weeks for the next year, and every 2 months in the third year. another group received pulse methylprednisolone without cyclophosphamide. as of this writing, 128 patients have been treated, of which 24 have completed 3 years. interim analysis shows that patients treated with pulse methylprednisolone were more likely to become treatment failures than those treated with pulse cyclophosphamide/methylprednisolone (78% vs 45%) independent of induction therapy. withdrawal due to toxicity, however, was higher in the pulse cyclophospharnide group. current regimens involve methylprednisolone induction alone followed by pulse cyclophosphamide/methylprednisolone, analogous to lupus nephritis pulse therapy. this regimen can be given solely on an outpatient basis, does not cause alopecia, and is more amenable for use in earlier stages of the disease. to determine the incidence of pathologically confirmed malignancy in multiple sclerosis (ms) patients in funded clinical trials of cyclophosphamide (ctx) and of azathioprine (aza), data were collected longitudinally using telephone interviews, written questionnaires, physical examinations, and medical records for ctx and aza patients from a community in-hospital and out-patient ms clinic in fargo, nd. in the ctx study (goodkin et al, arch neurol 1987; 44: 823-4327) , 5 1 clinically definite (cd), chronic progressive ms patients were enrolled. twenty-four were controls and 27 received a mean induction dose of 6.08 grams. fourteen of the induced patients then received boosters every other month for 24 months resulting in a mean total dose of 15.92 grams. no malignancies were detected. in the aza study (goodkin et al, neurology 1991; 41:20-25) , 54 c d relapsing ms patients participated. twenty-five were controls and 29 received 3 mgtkg of aza daily by mouth adjusted to maintain a white blood count of greater than 3,50o/cmm for 2 years (mean dose 2.6 mg/kg). two of 29 aza patients developed resectable skin cancers: 1 basal cell (bcc) at 18 months and i squarnous cell (scc) at 43 months. the incidence of developing a bcc or scc was not significantly increased after initiating therapies as compared to the untreated ms controls (aza: fisher exact testp value = 0.49). no other malignancy has been detected during 46 to 100 months of clinical follow-up. allergic encephalomyelitis paula dore-dufly, ruth washington, and robert h. swanborg, detroit, m i postcapillary endothelium at sites of inflammation undergoes many changes referred to as activation. activated endothelial cells (ec) exhibit increased surface expression of immunorelevant proteins (icam-1; ncam, elam, and mhc class i and class i1 antigens [ags)). the sequence of events that characterizes ec activation may be important in susceptibility, induction, and perpetuation of experimental allergic encephalomyelitis (eae). in this study we examine expression of ec activation antigens in central nervous system (cns) microvessels in response to interferon gamma (ifn-y). cns microvessels from sjl and bio.s mice were incubated for 18 hours in ifn-y (500 u/ml), fixed, permeabilized, and then stained with an antibody that recognizes class i, class i1 mhc antigens, icam-1, and factor viii. relative fluorescence intensity was determined using a laser cytometer. results indicate that microvessels from all strains tested expressed no detectable icam-1 and class i1 ags. little class i antigen and transferrin receptors were expressed. upon stimulation with ifn-y, sjl microvessels exhibited increased surface expression of all ec activation ags. b1o.s microvessels exhibited icam-1 and class i mhc but mhc class i1 ags were not upregulated. results indicate that there are strain differences in the ec response to ifn-y. resistance of b1o.s mouse ec to activation by ifn may be a factor in decreased susceptibility or induction of eae, or both. protein-t-cell line-mediated experimental to investigate a possible pathogenic role of interferongamma (ifn-y) in experimental allergic encephalomyelitis (eae), an immunocytochemical study was undertaken to localize this cytokine in the spinal cord of lewis rats in which eae was produced by adoptive transfer of myelin basic protein-specific t cells. one pm-thick cryosections of spinal cord were labeled with monoclonal antibodies (mab) db-1 and db-12 recognizing different epitopes of rat ifn-y. in the spinal cord of naive rats, mab db-i, but not db-12, stained processes of astrocytes, suggesting that astrocytes contain a protein with an epitope cross-reacting with ifn-y. in rats with at-eae, numerous ifn-y-positive cells stained with both mab db-1-and db-12-positive cells were present from days 3 to 7 after cell transfer and had disappeared on day 10. at day 2 they entered the spinal cords predominantly through subpial vessels. ifn-y-positive cells could be identified as w3/13+ leukocytes as well as ed1-positive macrophages. as in naive rats, astrocytes in at-eae were labeled only with mab db-1, but not db-12. we never observed labeling of motor neurons with these mab. the transient presence of ifn-y in the rat spinal cord at the onset of at-eae suggests a pathogenic role of this cytokine in acute immune-mediated demyelination of the cns probably as a local stimulus for expression of mhc class i1 antigens and adhesion molecules, as well as for the release of tnf-a and toxic oxygen radicals from macrophages and microglia. immune responses to stress or heat shock proteins are implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis (ms). we examined the hypothesis that antigens in myelin cross-reacted with stress protein antigens. two techniques were used: immunocytochemistry and western blotting. frozen histological sections were prepared from normal human central and peripheral nervous system tissues. sections were incubated with 4 murine monoclonal antibodies to different mycobacterial stress proteins. antibody binding was determined using avidin-biotin complexed antimurine antibody linked to alkaline phosphatase. program and abstracts, american neurological association 257 a monoclonal antibody to the stress protein sp65 from m . leprae strongly stained both central and peripheral nervous system myelin. no myelin staining was noted with antibodies to sp70 or sp17. proteins from purified central and peripheral nervous system myelin were separated by sds-page. western blots were prepared using a monoclonal antibody to sp65 and a polyvalent rabbit antibody to myelin basic protein (mbp) as primary antibodies. antibody binding was determined using antimurine or antirabbit igg antibody coupled to alkaline phosphatase. strong staining of central but not peripheral mbp by the anti-sp65 antibody was observed. the rabbit anti-mbp antibody stained both central and peripheral nervous system mbp. the presence of antigenic epitopes shared by a stress protein and the potential autoantigen, mbp, supports the hypothesis that immune responses to stress proteins may be involved in the pathogenesis of presumed autoimmune diseases such as ms. (1). of 5 patients with borderline or positive csf lyme titer, 3 received parenteral ceftriaxone. all had later relapses consistent with ms. one patient received a month of oral doxycycline. the fifth patient had a negative western blot and was not treated. we conclude that an incidental borderline or positive lyme serology in an ms patient is unlikely to indicate neurological lyme disease. borderline serologies should be documented to rise on later testing; positive serologies should be confirmed by retest in a different laboratory or by western blot. csf abnormalities suggestive of neurological lyme disease (pleocytosis, protein elevation, intrathecal lyme antibodies) are distinct from those suggestive of ms (ogb, elevated igg index, mbp). in such patients antibiotic treatment may be appropriate, but will not alter disease course. when designing and analyzing therapeutic trials for multiple sclerosis (ms), investigators commonly compare the proportion of patients in the experimental and control groups who worsen one or more steps on the disability status scale (dss) or expanded dss during the study (typically 2 years' duration). however, the intervals between the scores in the dss may not be equal. it may be easier to change by one or more steps in the lower end of the scale (e.g., dss = 1-3) than in the midportion of the scale (e.g., dss = 4-6). to evaluate this possibility, we compared the proportion of patients who worsened by one or more steps in the 2 years after entering our program with dss = 3 or with dss = 6. fifty-one percent (29157) natural history data may be useful for designing therapeutic trials for multiple sclerosis (ms). since 1971, we have collected such data in a standard format on patients in the ucla multiple sclerosis research and treatment program. t o describe the course in our group, we have performed survival analysis (kaplan-meier) of patients with 3 or more assessments who entered the clinic with disability status scale (dss) scores of 1 to 7 (n = 395). an increase of one or more steps in the dss score persisting for more than 3 months defines worsening. median times to worsening for a dds at entry of 1 to 5 were approximately 2 years (range 1.7-2.2); but were 4.6 years for dss 6 and 3.6 years for dss 7. for those starting at dss 6 (n = 129), only 13% worsened by 1 year, 26% by 2 years, and 39% by 3 years. the percent worsening when starting at dss 3 (n = 61) were 41%, 48%, and 54%, respectively. these variable rates of worsening (i.e., time spent at each starting level) influence therapeutic trial design. including patients with dss 6 or 7 will increase the sample size and study duration. for testing nontoxic agents, we recommend enrolling patients with dss 1 to 5. for more toxic treatments, we suggest dss 3 to 5. (partially supported by usphs grant ns 087 1 1, the conrad n. hilton foundation, and various donors.) pi 12. cholinergic antagonists and p-adrenergic agonists inhibit experimental allergic encephalomyelitis in an additive manner mark a. jensen, avertano noronha, and bawy g. w. amason, chicago, il lymphoid organs receive a sympathetic (sns) and possibly a parasympathetic innervation. lymphocytes express padrenergic and cholinergic receptors and thus are sensitive to regulation by these neurotransmitters. the severity of experimental allergic encephalomyelitis (eae) is increased in sns-ablated animals. local parasympathectomy decreases plaque-forming responses in submandibular nodes. p,-adrenergic receptors are upregulated on cds t cells in progressive multiple sclerosis, as are m,-muscarinic acetylcholine receptors on cd4 t cells. we examined the effect of isoproterenol, a p-adrenergic agonist, and scopolamine, a cholinergic antagonist, on the course of eae in lewis rats. eae was induced by injection of 0.1 ml of incomplete freund's ad juvant containing guinea pig spinal cord (25% wlv) and m. tubercdosis (3 mgiml) in one hind footpad. scopolamine (6.6 mglkg, twice daily) and/or isoproterenol(o.15 mglkg, twice daily) or saline were injected subcutaneously starting on the day of immunization. scopolamine or isoproterenol alone reduced severity ( p < 0.05, t test) and duration ( p < 0.05, t test) of disease compared to controls. the combination of scopolamine and isoproterenol further reduced disease severity compared to either agent alone ( p < 0.05, x2 test), suggesting an additive protective effect of cholinergic antagonists and p-adrenergic agonists in eae. antigen-presenting cells a . conrad, v. sanders, p. schmid, and w. w . tourtellotte, los angeles, c a tissue is cryopreserved by the method of tourtellotte; this procedure minimizes or eliminates ice artifacts and preserves surface protein markers. the dissected plaques are lightly fixed in 5% paraformaldehyde and then suspended in 30% sucrose. blocks are mounted in oct, cryosectioned at 20 p,m, and picked up on gelatinized slides. the activity of plaques is determined by the presence or absence of myelin debris (antimyelin basic protein stain or lux01 fast blue) or presence or absence of neutral lipids indicating myelin digestion as seen by oil red 0 (oro) staining and the presence or absence of a class i1 major histocompatibility complex antigen (evidence for an antigen-presenting cell) on macrophages or microglia as seen by immunocytochemically staining for hla-dr (hb104 atcc). the following is our classification of the activity of multiple sclerosis (ms) plaques. type i, the most active, is defined as an area of hypercellularity, positive for hla-dr with no o r 0 staining or staining for myelin debris. type 11, or active, is defined as an area of hla-dr-positive cells that stain mildly with o r 0 at the plaque edge but positive for myelin debris; evidence for demyelinating activity <72 hours (prineas; raine). there is an inner area of plump cells that are positive for hla-dr and oro. type 111, or modestly active, is defined as a "shelf" of plump hla-dr-positive cells at the edge loaded with program and abstracts, american neurological association 259 oro-stained neutral lipid but a paucity of or no myelin debris. a region of hypocellularity is observed at the center of plaque. type iv, least active or inactive, is defined as scattered hla-dr-positive cells at plaque edge with little or no o r 0 staining and no evidence of myelin debris. the frequency of plaque types was determined from a random sample of 97 ms tissue blocks from 23 patients who died of ms. ten percent of the plaques were type i; 20%: were type 11; 209% were type 111. the majority of plaques (505%) were inactive (type iv). accordingly, it is necessary to classify the demyelinating activity of ms plaques for protocols designed to investigate etiopathogenesis. do these results suggest that whatever causes ms can be eradicated in half of the demyelinating areas by the time of death? p114. localization of g d l b ganglioside antigen in the human peripheral nervous system susumu kusunoki, atsuro chiba, tadashi tai, and lchiro kanazawa, tokyo, japan serum antibodies against ganglioside gm 1 and/or g d l b frequently are detected in autoimmune neuropathies such as rnultifocal motor neuropathy, igm paraproteinemic neuropathy, and guillain-barre syndrome. some of them bind to gm1 or g d l b monospecifically but the others cross-react with both of the antigens. to investigate respective localizations of gm1 and g d l b antigens in the human peripheral nervous system (pns), immunohistochemical study of dorsal root ganglia (drg), dorsal roots, ventral roots, and sympathetic ganglia (sg), which were obtained from human autopsy specimens, was performed by using 2 mouse monoclonal antibodies, each monospecific to gm1 and gdlb. ggr12, monospecific to gd1 b, immunostained nerve cell somas and axons of drg, sg, and dorsal and ventral roots. ggrl2 also recognized some myelin, including paranodal areas. however, gmb16, monospecific to gm1, did not bind to either neuron or myelin. thus, serum anti-gdlb antibody can bind to neurons and some myelin in the human pns. further study is necessary to identify the localization of gm 1 antigen, because previous biochemical studies have shown that gm1 is also present in the human pns. the purpose of this study was to compare t4 and t8 lymphocytes in migraine patients versus controls, and a review of the literature was done. fifty-six migraine patients, aged 18 to 53, were tested, as were 19 controls. the t4 :t8 ratio in migraine patients was 2 : 1 1, compared to 1 : 60 for controls. suppressors (t8) were reduced from 918 in controls to 482 in migraine patients. helpers (t4) decreased from 1,436 in controls to 985 in migraine patients, and total ?' lymphocytes decreased from 2,513 to 1,698. previous studies have revealed similar differences in t lymphocytes, with higher t4 : t8 ratios being found in both migraine and tension-type headache patients. in food-induced migraine, an increase in circulating immune complexes was noted, with increased t4 levels. differences in serotonin binding to mononuclear cells have been noted in migraine patients. a decreased sensitivity of the lymphocyte beta-adrenergic receptor in migraine patients was suggested by one study. after lymphocyte incubation with il-2, the natural cytotoxic response is augmented in cluster patients. the percentage of lymphocytes expressing receptors for il-2 was decreased in cluster patients in a fur-review of the literature ther study. this il-2 receptor defect was independent of whether the clusters were present. there is a loss of highaffinity binding sites for serotonin on lymphocytes in both episodic tension and chronic tension headaches. we used positron emission tomography (pet) to measure local cerebral blood flow in 6 volunteer subjects while they performed tasks of memory-guided saccades and a visual fixation control. tasks were performed continuously for 70 seconds during emission scans, after bolus injection of h2lso. eye movements were verified with electrooculography. areas of significant increase in regional blood flow between tasks were matched to three-dimensional reconstructions of brain magnetic resonance images of each subject. compared to the visual fixation control, saccade tasks evoked bilateral activation in the posterior superior parietal lobule with extension into the inferior parietal lobule. activation was also seen bilaterally in an area of frontal cortex immediately rostra1 to the precentral gyrus extending from the superior frontal gyrus to the inferior frontal sulcus. the increased blood flow in the posterior parietal cortex most likely corresponded to enhancement of visual attention required during saccades, while that in the frontal lobe, which included the frontal eye fields, indicated activation for saccade motor output. pamela blake, alexander s. mark, martin kolsky, and jorge kattah, washington, dc fifty patients with third cranial nerve (cn) palsy underwent precontrast and postcontrast mri to assess the utility of this study in this clinical context. mri demonstrated an appropriate lesion in 32 cases. six patients had brainstem lesions (2 infarcts, 1 mass lesion, 1 cryptic vascular malformation, 1 hemorrhagic shearing injury, 1 with compound q toxicity). lesions of the cisternal segment of the nerve were present in 12 patients (4 aneurysms, 4 lymphomas, 1 ophthalmoplegic migraine, 1 viral meningitis, 1 coccidiodomycosis, 1 nerve avulsion), with enhancement of this segment in 7 patients. fourteen patients had cavernous sinus lesions (2 lymphomas, 2 nasopharyngeal carcinoma, 4 tolosa-hunt syndrome, 2 cavernous carotid aneurysms, 3 pituitary apoplexy, 1 aspergillosis). eighteen patients, all with history of diabetes or vascular disease, had normal mri results, suggesting microvascular infarction of c n 111. in patients with cn i11 palsy, mri can detect the presence of brainstem or cavernous sinus lesions and often can suggest their cause. mri with contrast enhancement can demonstrate involvement of the cisternal segment of cn i11 in patients with inflammatory or infiltrative processes that previously could not be radiographically demonstrated. our study suggests microvascular infarction does not cause nerve enhancement on contrast-enhanced mri. we describe 2 patients with acute hyperglycemic, hyperosmolal, nonketotic stupor who had ocular flutter or opsoclonus clinically. these are the fourth and fifth adult patients reported with the acute onset of stupor and opsoclonus; all patients had nonketotic hyperglycemia and hyperosmolality (rapid eye movement sleep also causes stupor and saccadic eye movements). three of the 5 patients had myoclonic jerks in addition to opsoclonus. in the 5, opsoclonus began when glucose and osmolality acutely increased, and completely resolved when glucose and osmolality became normal, showing that opsoclonus is a specific and reversible effect of the metabolic disorder and implying that either acute hyperglycemia or hyperosmolality directly causes opsoclonus. since acute hyperosmolality caused by nacl or sucrose can cause a similar syndrome in experimental animals (trans am neurol assoc 1962; 87:33-36) and infants, hyperosmolality is probably more important. opsoclonus is thought to be due to abnormal activity of saccadic "burst" cells in the pons. acute hyperosmolality may cause spontaneous saccades by disinhibiting burst cells from normal "pause" cell inhibition or by directly activating burst cells. the combination of acute deterioration in mental status and either ocular flutter or opsoclonus should suggest acute hyperosmolality, in particular, nonketotic hyperglycemia. neural cell adhesion molecule (n-cam) and the related cell adhesion molecule l1 play significant roles in axon outgrowth and mediation of cell-cell contact in development, and after peripheral nervous system injury. to understand the role of these molecules after injury in the adult cns, we studied alterations in n-cam and l1 in the rat brain's response to entorhinal cortex (erc) lesion. post lesion, reactive synaptogenesis and axonal sprouting follow a well-defined temporal course in restoring the synaptic density of the dederented outer two-thirds of the hippocampal dentate gyrus molecular layer (ml) to near prelesion levels. we found striking regionand lamina-specific staining of n-cam and l1 in the normal hippocampus and marked alterations in these molecules after injury. embryonic n-cam, present in high amounts during development but expressed at very low levels in the adult hippocampus, was massively re-expressed in the denervated zone; the embryonic form was still heavily expressed 60 days later, when synapse number returned to >so% of prelesion levels. l1 staining, normally evenly distributed through the ml of the dentate, was completely lost in the outer ml, which is denervated by the erc lesion. this staining had not returned by 60 days after lesion. neural cell adhesion molecules play a role in specificity of neural connectivity both in development and after injury. in reactive synaptogenesis and axonal sprouting after injury, both ontogenetic (the reexpression of embryonic epitopes) as well as uniquely adult sequences of repair are utilized. following hemispherectom y" a. pascual-leone, h . t. chugani, l. g. cohen, j . p . brasil-neto, e. m . wassermann, j . and m. hallett, betbesh, md, and los angeles, ca we studied 7 subjects, aged 18 months to 32 years, who underwent hemispherectomy 6 months to 25 years earlier for intractable epilepsy. all had a spastic hemiparesis contralateral to the resected hemisphere, which was present presurgically. we used focal transcranial magnetic stimulation to map the areas of the preserved hemisphere targeting the abductor pollicis brevis (apb), the biceps, and the deltoid ipsilaterally and contralaterally. in 2 subjects who had hemispherectomy after age 10, the same area targeted ipsilateral and contralateral muscles. in the remaining 5 subjects, who were more functional, 2 areas targeted ipsilateral muscles. one area coincided with the contralateral representation, but stimulation induced motor-evoked potentials (meps) of lower amplitude and longer latency in the ipsilateral muscles. the other area, 2 to 4 cm anterolaterally, targeted exclusively ipsilateral muscles and stimulation induced meps of normal amplitude and latency. this separate ipsilateral representation was more distinct in subjects studied a long time after the hemispherectomy and in those who were younger at the time of the operation. these results show evidence of motor reorganization after hemispherectomy. better motor function is associated with topographically differentiated ipsilat-era1 and contralateral representations, which may depend on age at the time of hemispherectomy and the time since then. cynthia l. comella, glenn t . stebbins, nancy brown-toms, and christopher g. goetz, chicago, il in a single-blind, crossover study, we evaluated the effect of an intensive outpatient physical rehabilitation program (re-hab) on the severity of parkinson's disease (pd). the re-hab program consisted of 3 2-hour sessions per week for 4 weeks. sixteen patients completed 2 phases, a rehab phase and a control phase, separated by 6 months. the order of participation in each phase was randomized. all patients were evaluated using the unified pd rating scale with subscales for mentation (ment), activities of daily living (adl), and motor function (mot) by an investigator blinded to the rehab phase of the patient. all patients were evaluated immediately before and after each phase. pd medications were not changed during any phase. following the re-hab phase, there was significant improvement in adl score (pre-rehab 12, post-rehab 8, p = .005 wilcoxon) and in mot score (pre-rehab 26, post-rehab 2 1, p = ,008 wilcoxon), but no change in ment score. after the con-trol phase, there was no significant change in any outcome measure. this is the first controlled, crossover study of an intensive rehab program in pd. it demonstrates that re-hab improves objective motor function and adl scores. sensitive and quantitative measurements of leg weakness, one of the most common deficits in multiple sclerosis (ms) patients, can be made using specialized extremity testing equipment. to determine whether such determinations could be used in clinical trials, 6 ms patients with leg weakness that had been stable for at least 2 months were evaluated every 60 days over a 4-month period. each testing session was carried out at the same time of day and medication dosages and schedules were kept constant (only 1 patient was taking baclofen and his dosing remained constant). quadriceps and hamstrings strengths were measured in isometric contraction in a mechanical testing apparatus (kincom). variability for each series of determinations was expressed as the standard deviation of the mean as a percent of the mean. the overall variability then was expressed as the mean of the individual variabilities. the mean variability for the strength determinations over 4 months was 5.74 k 5.3896, and only one series of determinations out of 28 had greater than 20% variability. these results suggest that quantitative strength determinations might be useful in clinical trials, and early experience in trials of aminopyridines will be discussed. polymyositis (pm) is an inflammatory myopathy of unknown cause, but the accumulating data strongly suggest an autoim-mune pathogenesis. the histological picture is of muscle fiber necrosis and inflammation, whereas in postviral fatigue syndrome (pfs), a disorder characterized by severe fatigue with myalgia and psychiatric symptoms, the histological picture of muscle is essentially normal. enteroviruses have been implicated on epidemiological and serological studies in both. we have used the polymerase chain reaction (pcr) and an enteroviral-specific probe and found persistent enteroviral genomic material in both pm and pfs muscle biopsy specimens. furthermore, we used a radiolabeled full-length cdna probe derived from coxsackie b1 in an in situ technique to look for viral d n a in pcr-positive cases. coxsackie genome was clearly identifiable in the muscle biopsy specimens of patients with pm but negative in pcr enteroviral-positive cases of pfs. the virus excites an inflammatory reaction only in pm. a murine animal model for pfs developed in our laboratory showed positive muscle pcr using enterovirus probes and a conspicuous increase in interleukin 6 within the brain. these results provide major clues in the search for the etiology of these two puzzling disorders. human t-lymphotropic virus type i (htlv-i) is a cause of adult t-cell leukemia and tropical spastic paraparesis. in a specific population of iranian jews originating from the city of mashad, there is a high incidence of htlv-i infection (1 1.5%) and associated t-cell leukemia. we evaluated the incidence of possible correlation between htlv-i infection and spastic paraparesis in israeli mashadi-born jews. we have examined 41 mashadi-born immigrants in a mashadi community center (16 men, 25 women, mean age 66 t_ 13.7 yr) and 40 non-mashadi iranian-born jews. blood samples were tested for htlv-i antibodies by particle agglutination test. the polymerase chain reaction (pcr) was used to amplify htlv-i sequences of d n a from peripheral blood mononuclear cells. twelve mashadi-born immigrants (295%) were seropositive for htlv-i. in 10 of those serologically positive for htlv-i (83%), neurological examination revealed spastic paraparesis of varying severity. none of the non-mashadi iranian jews were seropositive for htlv-i or had clinical signs of spastic paraparesis. these results support other studies of htlv-i-associated myelopathy. the high incidence of htlv-i-associated spastic paraparesis in the mashadi community might be related to their unique history of a high rate of intermarriage among members of this ethnically segregated group. further epidemiological studies are underway to evaluate the incidence of htlv-i in mashadi families as well as in mashadi-originating jews born in israel, to identify whether infection might be genetically transmitted. lymphotropic virus t y p e i-associated acute t-cell leukemia/lymphoma william j . harrington, jr, william a. sheremata, susan snodgrass, and mark raven, miami, fl human t-cell lymphotropic virus type i (htlv-i)-associated acute t-cell leukernia/lymphoma (atl) is thought to produce important c n s disease infrequently. we wish to correct this impression by presenting neurological findings in 15 patients seen between 1989 and the present. all had positive western blots to htlv with polymerase chain reaction confirmation of htlv-i infection. only 2 had a concomitant human immunodeficiency virus infection. all were black, aged 31 to 86 years, 7 were men and 8 women. all but 3 americans came from the caribbean nations of haiti (51, dominican republic (l), jamaica (6), and trinidad (1). sexual transmission was the risk factor for htlv-i for all except for 1 intravenous drug user. all patients were systemically ill. three had preceding neurological abnormality (1, 8 months, and 14 yr) and 8 had major neurological disease concomitantly. two of these had tumor masses demonstrated in brain and 1 in the spinal canal. one had a minor facial sensory abnormality; only 3 had no deficits. we conclude that cns disease is commonly associated with atl, and that in the us atl occurs principally in caribbean natives. a. nath, v . hartloper, and m . furer, winni$eg, manitoba, canada microglia and astrocyte cultures were established from human fetal brain. microglia were infected with human immunodeficiency virus (hiv) strains, hiv,, or hiv,,,, resulting in a rising titer of p24 antigen in the supernatants. multinucleated giant-cell formation, vacuolar changes, and rising levels of lactate dehydrogenase in the supernatants were seen, indicating a cytopathic infection of microglia. astrocytes were infected with free virus or cocultivated with an hiv-infected lymphocyte cell line (hut-78). after a productive phase (rising titers of p24 antigen and detection of hiv antigens by immunocytochemistry), the cells went into a latent phase where hiv could be detected only by dna polymerase chain reaction. a 10-fold increase in astrocytes staining for hiv antigens was seen after cocultivation with hut-78 cells. lymphocytes adhered to astrocytes by 3 hours of cocultivation. no adhesion was seen to microglia. fusion of plasma membranes was seen on electron microscopy. infected astrocytes did not show cytopathic or morphological changes. cell-to-cell contact may be important in viral transmission to astrocytes. hszao-huei chen, steven b. stein, wing kong, and raymond p. roos, chicago, i l one of our goals is tq delineate molecular determinants for disease phenotypes produced by theiler's virus (tv), a mouse picornavirus. the identification of these genes and gene products may clarify viral pathogenesis and also lead to the identification of genes that are important in normal cns function and nonviral cns disease. members of the gdvii subgroup of tv cause an acute, fatal neuronal infection, whereas members of the to subgroup are less neurovirulent and produce a demyelinating persistent infection. our studies of infectious tv cdna clones have demonstrated that the gdvii 1 b(vp2)-2c segment is critical for neurovirulence. several other areas of the genome, including the 5' untranslated region (s'utr), also affect tmev-induced disease. the 5'utr of poliovirus, another picornavirus, has a critical role in paralysis; this effect on neurovirulence is believed to result from an altered translational efficiency related to bind-region ing of neural cell proteins. tv 5'utr has an unusual predicted secondary structure, even for picornaviruses. our studies demonstrate that the tv 5'utr affects translational efficiency and has a distinctive protein-binding pattern. investigations of the tv 5'utr may clarify features of translational regulation of cns genes in general. p129. coronaviruses infect primate brain from g a y f. cabirac, ronald s. murray, galen cai, kristen hoel, and kenneth soike, englewood, co, and covington, la recently, we described finding coronavirus (cv) rna and antigen in active demyelinating plaques of multiple sclerosis (ms) brain tissue (murray et al, ann neurol, in press). molecular analysis showed the cv rna to be more closely related to murine cvs than to human cvs. we then demonstrated that, following intracerebral inoculation, the murine cv jhm and the putative ms isolate cv-sd could infect and cause demyelination in primate brain (murray et al, virology, in press). we now have data showing that murine cv can infect primate brain following intranasal or intravenous routes of inoculation. standard virology, histopathology, and the molecular analysis of viral cytotropism will be presented. we conclude that cvs related to murine cvs can infect primate cns from peripheral routes and warrant consideration as potential human pathogens. probes (gag, pol, or enw). eleven were white and 9 were black. a history of transfusion was obtained in 8, and sexual risk of transmission was present in 8 but not in 4 others. fulminant disease occurred in 4 transfused men 3 months to 4 years later but such disease was only seen in 1 woman (in 1 month). in contrast, 2 of 3 women with multiple sexual partners had rapid progressive disease. the male-to-female ratio was 4 : 4 for transfusion association and 5:3 for those at sexual risk but 0:4 for unknown risk, transfusion is an important risk for tsp/ham and the diagnosis must be considered in all gait problems, regardless of the "diagnosis."transfusion association should decrease with regular testing of blood donors, but this will not affect the risk of sexual transmission. spastic paraparedhtlv-i-associated myelopathy (tsp/ ham). it is unclear why htlv-i infection causes atl in some individuals and tsplham in others. differences in the genome of viral isolates or immunological and host factors have been hypothesized to play a role in disease expression. at present there are no animal models of tsplham and no suitable animal models of htlv-i infection that can address these issues. we transplanted severe combined immunodeficient (scid) mice with peripheral blood mononuclear cells (pbm) from tsplham patients in an attempt to produce htlv-i disease. two weeks after transplantation of pbm from 2 tsplham patients, we detected anti-htlv-i igg in serum samples of 7 of 8 scid mice by enzyme immunoassay using sonicated whole virus. five weeks after transplantation, we detected anti-htlv-i igg to p19, p24, gp46, or gp61168 in serum samples of 5 of 7 scid mice by immunoblot of disrupted virus. these findings suggest that scid mice may be valuable in the study of molecular and pathological determinants of htlv-i-induced disease. theiler's virus produces an encephalomyelitis in susceptible mice. during the course of the disease, specific regions in the cns become infected. compared to the immunocompetent mouse, the nude mouse provides a useful model where viral dissemination can be studied in the absence of functional t lymphocytes and antibodies. we investigated the distribution and spread of the da strain of theiler's virus in the cns of nude mice. by immunohistochemistry, the hippocampus, arnygdaloid nuclei, entorhinal cortex, cingulate cortex, thalamus (anteroventral nuclei), midbrain, and spinal cord all contained viral antigens by 2 weeks after infection. in addition, the olfactory nuclei, mamillary body, hypothalamus, basal ganglia, and nucleus raphe dorsalis often were involved. in the brain, the limbic system was the site commonly infected by theiler's virus. the time course of virus dissemination varied depending on the site of initial virus infection, though the final distribution of virus was the same. olfactory bulb injection, which is a direct inoculation into the olfactory pathway, resulted in more rapid spread than did cortex injection. we demonstrated the constant presence of viral antigen in the limbic system and a different kinetics of viral dissemination between the two different routes of intracerebral inoculations. these results suggest that limbic structures and their connections are important to the dissemination of theiler's virus. 1-associated myelopathy in a northwest native indian d. foti, d. werke, g. dekaban, g. p. a . rice, andj. oger, vancouver, bc, and london, ontario, canada a 59-year-old indian of the oweekeno tribe was admitted for investigation of a myelopathy. initially symptomatic with midthoracic radicular pain, she developed progressive spastic paraparesis over 1 year with mild sensory symptoms and urinary retention. mri of the cord and brain were normal. csf showed elevated protein (930 mgll), 13 lymphocytes, and weak oligoclonal banding with evidence of intrathecal igg synthesis. antibodies to human t-lymphotropic virus type i (htlv-i) were positive by enzyme-linked immunosorbent assay and western blot on both serum and csf. presence of htlv-i was demonstrated by polymerase chain reaction of blood lymphocytes. htlv-i-associated myelopathy, or tropical spastic paraparesis, is endemic in southern japan, the caribbean basin, and several tropical islands but has not been reported in natives of northwest canada. this patient's only risk factors for htlv-i infection were 3 blood transfusions 30 years previously. ongoing familial and epidemiological studies as well as virus sequencing should indicate if this case represents an indigenous or an imported infection. caused by herpes simplex virus type 1 lawy blankensbz) and herbert 0. newton, columbus, oh myeloradiculitis occasionally occurs secondary to herpes simplex virus type 2 (hsv2) infection, but rarely has been reported after herpes simplex virus type 1 (hsv1) infection without encephalitis. we describe a 30-year-old man who developed cervical myelopathy and radiculitis, never developed symptoms of encephalitis, and had positive hsvl spinal fluid cultures. h e initially developed extremity weakness and incoordination, numbness, paresthesias, and neck pain. evaluation was negative except for mri results, which showed a high-signal lesion centrally within the cervical cord. the weakness, sensory loss, and radicular pain progressed over several months. subsequent mri showed extension of the high-signal abnormality and mild enlargement of the cervical cord. symptoms stabilized briefly with dexamethasone but soon worsened, and were accompanied by paroxysmal kinesiogenic dystonic episodes of his arms and right leg. repeat evaluation was unrevealing except for the spinal fluid, which grew out hsv1. the patient was treated with dilantin and a i-month course of intravenous acyclovir, with slow improvement of neurological status and resolution of the dystonic episodes. this case illustrates that hsvl can cause a myelopathy with a subacute and protracted course, requiring serial was more frequent in the middle-aged group ( p < 0.0001, p = 0.001). history of hypertension, previous strokes, diabetes mellitus, and antithrombotic treatment was similar, as were sex ratio, qualifying event type (transient ischemic attack or nondisabling stroke), and angiographic features; stenosis severity in either side and presence of ulceration were all similar. in elderly patients, ecad is associated with symptomatic cardiac disease (scad or af), whereas in middleaged patients it is associated with precursors of generalized atherosclerosis (smoking and hyperlipidemia). to identify the anatomic factors correlating with dementia in patients with lacunar infarctions, we examined digitized ct data on 58 elderly patients (mean age = 71.6 yr; education = 7 yr) who presented with acute lacunar infarction. dementia was diagnosed in 13 patients (22.4%) based on neuropsychological tests given 3 months after stroke onset. the following ct variables were assessed: infarct location and number, total infarct volume, brain parenchymal and csf areas at 3 levels, and width of the frontal horn, third ventricle (tvw), and lateral ventricle plus their ratio to the intracranial width. atrophy and leukoaraiosis were rated semiquantitatively using a standard scoring method. in the group overall, mean infarct volume was 0.64 cc and mean infarct number was 3.8. in univariate analyses, dementia was significantly related to infarct volume, number, tvw, bilaterality, and infarct predominance on the left side, but not to leukoaraiosis or atrophy. in a regression model adjusting for demographic factors, the ct variables correlating independently with dementia status were infarct number (p = 0.37, p = .01) and the presence of left cerebral infarcts (p = 14.17, p = .002). we conclude that the most important ct variables related to dementia in lacunar stroke are lesion multiplicity and the presence of lesions on the left side. brain ischemia results in potassium (k+)-induced voltageregulated presynaptic calcium (ca") accumulation, which may contribute directly to neuronal in jury presynaptically, and also promote excessive release of excitatory neurotransmitters leading to cell damage postsynaptically. k+-induced depolarization of brain synaptosomes may be used as an in vitro model to study the therapeutic potential of pharmacological agents to alter ischemia-induced presynaptic ca2 + accumulation. we preincubated gerbil cerebral cortical synaptosomes in r56865 (janssen pharmaceutical) at concentrations of to lo-' m, subsequently depolarized the synaptosomes with k + , at concentrations of 5 to 6 0 mm, and measured intrasynaptosomal ca2' ([ca2+]) with the fluorescent indicator fura 2. r56865 had no effect on ecaz'i in nondepolarized synaptosomes, but significantly (<.01, student t ) depressed depolarization-induced {ca2+] at to lo-' m in a dose-dependent fashion. the greater the degree of depolarization employed, the greater degree of depression in [ca2+] was seen at each dose of r56865. since r56865 had no effect on [ca2+] when utilizing ca2+-free incubation media, it was demonstrated that r56865 prevents depolaritation-induced [ca2 ' ] increase by blocking voltage-regulated influx. because r56865 appears to block voltage-regulated presynaptic ca2' accumulation, it should be further evaluated as a potential therapeutic agent after cerebral ischemia. sneddon's syndrome (ss) is a focal and diffuse arthropathy affecting mainly the vascular wall of the skin and cerebral arteries. etiology is not determined. we studied 37 patients with ss (27 females, 12 males), aged 15 to 56 years. clinical, radiological, and immunological studies were done. all patients developed cerebrovascular disorders: ischemic stroke in 82%, transient ischemic attack (tia) in 64%, and ischemic stroke and tia in 46%. cerebral scan showed small and medium (less than 3 cm) ischemic lesions in 77%. these lesions were superficial in the cerebral cortex (57%); deeper in the centrum semiovalis, internal capsule, and basal ganglia (18%); and both superficial and deeper (25%). ultrasound and angiogram studies revealed obstruction of intracranial arteries in 30%, and of extracranial arteries in 3%. partial or complete improvement of cerebrovascular symptoms was observed in 81%. immunological studies showed increased content of b-cell lymphocytes ( p < 0.05), increased levels of igm ( p < o.oool), and circulating immunocomplexes ( p < 0.0001). anticardiolipin antibodies were increased (58%) and lupus anticoagulant detected (5 1%). cerebrovascular disorders in ss that lead to small ischemic cortical lesions have a good prognosis. pathogenesis of this syndrome may be related to antiphospholipid antibodies. the cheiro-oral syndrome is characterized by pure sensory deficit limited to the hand and mouth. this syndrome has been described in diverse lesions of the parietal operculum and brainstem, but occurs most commonly due to lesions of the contralateral thalamus, and suggests a humuncular representation of sensation in the ventral posterior lateral (vpl) and ventral posterior medial (vpm) nuclei. we describe a 54-year-old hypertensive woman who presented with sudden onset of numbness of the left side of her body. examination revealed a left hemisensory deficit to all modalities that spared the hand and peri-oral regions. cat scan and mri demonstrated an acute infarction of the posterolateral right thalamus, with a rim of preservation adjacent to the internal capsule. pure hemisensory loss with sparing of the hand and mouth ("inverse cheiro-oral syndrome") has not been reported previously, and complements previously published studies of the cheiro-oral syndrome in demonstrating somatotopic sensory representation in the thalamus. to examine the relationship between depression and dementia after stroke, we administered the 17-item hamilton depression rating scale (hdrs) and neuropsychological tests to 237 elderly patients 3 months after ischemic stroke. using dsm-111-r criteria, we found dementia in 57 (24.1%). hdrs score was 5.0 -t 4.6 overall, and higher in demented compared to nondemented patients (6.2 2 5.0 vs 4.6 t 4.5, p = 0.025). the frequency of depression (total hdrs score >11) was also higher with dementia (17.5% vs 7.8%, p = 0.03). however, demented patients did not differ from nondemented patients on ratings of depressed mood. instead, hdrs items for psychomotor retardation, reduced work activities, and impaired insight best distinguished the 2 groups. in multiple regression analysis, stroke severity (p = 0.23, p = 0.0003) was the most important correlate of hdrs score; dementia status was not correlated independently. mean scores on mini-mental state examination and neuropsychological tests assessing memory, orientation, verbal, spatial, attentional, and abstract reasoning skills did not differ by depression status. although weakly related to intellectual impairment, hdrs score in our stroke sample was most importantly associated with stroke severity. higher scores on hdrs in demented stroke patients may be explained by physical and cognitive symptoms that are expected with dementia. these findings do not support a causal link between depression and dementia, and argue against the importance of "depressive pseudodementia" as an explanation for intellectual decline after stroke. to investigate the pathophysiological mechanism of the white matter lesions in progressive subcortical vascular encephalopathy (psve) of binswanger type, we measured regional water partition coefficient (pc) (reflecting water content) and effective p h (pht) (weighted average of intra-and extracellular ph) with dynamic positron emission tomographic technique using 0-15 co,, o,, and c-11 co,. si-multaneously, regional cerebral blood flow (rcbf) and regional cerebral metabolic rate of oxygen (rcmro,) were evaluated. eight subjects (3 normal, 3 psve, 2 multiple infarction) were examined. in the white matter lesions in psve, corresponding to high-intensity areas in t2-weighted images of mr, the pc increased and pht was unchanged or decreased, whereas both rcbf and rcmroz declined. the ratio of white matter pc to gray matter pc was 0.92 in psve and 0.87 in normals. in the frontal gray matter, the pc decreased in psve, whereas rcbf and rcmroz also decreased. these results suggest that tissue water content increases in the white matter lesions in psve reflecting the edematous status of the damaged regions. elevated pht with high pc may reflect the increase of extracellular water of the tissue. measurement of pc and pht provides useful information about the pathogenesis of psve. stroke has been the second most common cause of death in korea but its risk factors (rfs) have not been studied intensively, so the rfs or causes were investigated prospectively in 1,507 consecutive stroke patients who were admitted to the chungnam national university hospital, taejon, korea, between 1989 and 1991. they included 775 cases of cerebral ischemia (ci) (51.4%), 467 cases of intracerebral hemorrhage (ich) (31.0%), and 265 cases of subarachnoid hemorrhage (sah) (17.6%). control data were obtained from 209 healthy spouses of the patients. multivariate analyses showed that hypertension (ht) was the strongest rf for all stroke types and was followed by old age, diabetes mellitus (dm), smoking, high-density lipoprotein cholesterol, and fibrinogen. the rfs for atherothrombotic ci included old age, ht, dm, smoking, fibrinogen, and cholesterol. for lacunar infarcts, ht, dm, old age, fibrinogen, alcohol abuse, smoking, and female sex were the significant rfs. ht was the cause of ich in 347 (74.3%) and alcohol abuse (44, 9.4%) and vascular anomalies (32, 6.9%) followed. most alcoholassociated ichs occurred characteristically in the posterior fossa. frequencies of hospital admission of ich patients correlated positively with diurnal variation of temperature ( r = 0.5229, p < 0.001). aneurysmal rupture was the cause of sah in 222 patients (83.8%). three major sites of 247 aneurysms identified on cerebral angiograms were the anterior communicating artery (83, 33.6%), the middle cerebral artery (70, 28.3%), and the posterior communicating artery (56,22.6%). thirty-nine patients (14.7%) had no identifiable cause of sah. in korea curvilinear subinsular lesions have been noted on ct but the underlying pathology is unknown. we reviewed the cranial mr scans (1.5 tesla ge machine) of 49 serial patients over the age of 60 who had no cause for mr hyperintensities (hi) other than age or vascular risk factors. seven (14%) had linear subinsular h i (sihi). four of 7 (57%) patients with sihi and 4 of 42 (12%) without sihi had marked periventricular h i compatible with subcortical arteriosclerotic encephalopathy (pvhi) ( p < 0.05). patients with sihi were older (75.3 yr) than patients without sihi (69.1 yr) ( p < 0.05). four of 7 (57%) patients with sihi had hypertension program and abstracts, american neurological association 267 or vascular risk factors. a further patient with diabetes, hypertension, and the opercular syndrome (bilateral facial, pharyngeal, and lingual weakness) had subinsular lesions on ct. the brain arteries were injected postmortem with a lead/ gelatin suspension, and mr of the whole brain and x-ray films of the brain slices were taken. bilateral sihi were seen on mr and corresponded with linear cavitary infarction pathologically, which on microangiograms was found to lie in the border-zone between cortical and basal penetrating arteries. we conclude that sihi are associated with older age and pvhi, and can be due to infarction in a deep watershed territory and can be associated with clinical deficits. hemodilution-treatment results in 12 consecutive cases james l. frey, phoenix, az because precipitous neurological deterioration occurred during blood pressure reduction in a seminal case of lacunar infarction, 11 subsequent patients with partial or evolving lacunar deficits were treated with hemodilution and blood pressure nonintervention to test the hypothesis that lacunar strokes represent perfusion failure. isovolemic hemodilution was performed using hetastarch with target hematocrit of 30 to 33. results of pretreatment ct brain scans, carotid ultrasound, and echocardiograms were normal. nine patients recovered normal neurological function, and 2 regained complete functional independence in close temporal correlation with hemodilution. mri brain scans demonstrated appropriate single white matter lesions in 9 cases. no specific risk factor combination could be identified. n o patient has had recurrent stroke in follow-up from 12 to 30 months. response to hemodilution suggests a hemodynamic pathophysiology. successful treatment requires (1 } blood pressure nonintervention and (2) hemodilution prior to severe clinical deterioration. the hemodynamic classification for the carotid-cavernous sinus fistula (ccf) is important for the implication of prognosis and therapy, but satisfactory objective criteria for such differentiation is still lacking. retrospectively, we studied the application of extracranial duplex sonography in 9 cases of ccf with emphasis on the hemodynamic parameters of resistivity index and flow volume. a correlation was made with the angiographic findings in an attempt to evolve an objective hemodynamic classification by this noninvasive method. the alterations in membrane metabolism and structure could be the primary etiological event in alzheimer's disease (ad) that results in the clinical and neuropathological findings. to investigate in vivo brain membrane phospholipid and highenergy phosphate metabolism in probable ad patients and control subjects, the building blocks (pme) and breakdown products (pde) of membranes and the high-energy phosphates pcr and atp were measured noninvasively by in vivo brain 31p mrs in 11 probable ad patients ( 5 males; 6 females) and 18 controls (9 males; 9 females). all subjects were assessed by mini-mental, mattis, and blessed scales. we found that, at clinical onset, ad females had elevated pme ( p = 0.004), decreased pcr ( p = 0.001), and decreased atp ( p = 0.03). similar changes were not seen in ad males at clinical onset, but the severely demented ad males had increased atp ( p = 0.05). correlation analysis for the ad patients revealed that increasing dementia was associated with decreasing pme ( p = 0.05; r = 0.4), increasing pde ( p = 0.08; r = 0.4), increasing pcr ( p = 0.05; r = 0.4), and increasing atp ( p = 0.02; y = 0.5). similar metaboliccognitive correlations were not seen in the controls. these results demonstrate alterations in membrane and energy metabolism at the earliest clinical stages of ad. increasing dementia correlates with markers of membrane degeneration and decreased utilization of high-energy phosphates. both of these findings suggest the dementia in ad is secondary to membrane changes resulting in synapse loss. alzheimer's disease: postmortem mri and histological correlates fen-lei f. chang, j. e. purisi, c. r. jack+ jr, and r. c. petersen, rochester, mn hippocampal atrophy, defined by mri-derived volumetric measurement, has been useful in differentiating alzheimer's disease (ad) patients from normals. since several studies have demonstrated anatomical and functional gradients along the rostral-caudal (r-c) axis of the hippocampus, it is tempting to speculate on the existence of a differential distribution of morphometric changes along this axis. the hippocampi from 19 patients with clinically and pathologically confirmed ad were studied by postmortem mri and by reconstruction of serial histological sections. there was good correspondence between these two methods. the r-c length of the hippocampus in ad was preserved compared to normal. this length was longer on the left, both in normals and ad. the adassociated atrophy was due primarily to the reduction of the coronal cross-sectional area of the hippocampus. with increasing hippocampal atrophy, volume reduction was more prominent in the rostral area (pes hippocampus). this nonuniform reduction in volume may be associated with different connectivity patterns between rostral and caudal hippocampus. the purpose of this study was to determine the neuropathological validity of nincds-adrda criteria (nac) for probable and possible ad. mckhann et al (1985) provided clinical criteria for the categories probable ad and possible ad. the validity of these categories has not yet been reported. a retrospective, blinded evaluation of the complete neurological history, examination, neuroimaging, laboratory, and psychometric data was done for 68 subjects from the state of florida brain bank. pathological classification, which was blind to clinical diagnoses, was in 3 categories: pure ad, ad + , and other dementias. ninety-three percent of probable ad (n = 42), whereas only 75% of possible ad (n = 26) patients, had ad or a d + ( p = 0.1). pure ad was found in 62% of probable ad and 38% of possible ad patients ( p = 0.1). pathological evidence of coexisting parkinson's disease was present in 14% of all ad brains. these results suggest differential predictive power of nac for probable and possible ad, as suggested by the labels. nac are, therefore, usefui for research and clinical purposes. our prior study of falls in the elderly had shown significantly more white matter low attenuation (wmla) on ct scan among fallers than nonfallers, but no association between wmla and cognitive impairment among nondemented subjects. as these subjects were followed over time, it appeared that fallers were becoming demented at a more rapid rate than nonfallers. as a result, we analyzed the relationship between wmla and rate of change on the blessed test of information, memory, and concentration (bimc) in a combined cohort of 144 subjects from the falls study and from a longitudinal study of dementia and normal aging. we selected 61 of these 144 subjects whose initial bimc score was less than 25 (i.e., not already severely impaired) and who had at least 4 yearly bimc evaluations. ct scans were scored on an 8-point ordinal scale for hemispheric wmla. linear regression was used to summarize the rate of change for each subjects' test scores. the rate of change on bimc was 2.3 points per year with standard error (se) of 0.2 among subjects who eventually became demented; nondemented subjects declined at 0.4 points per year with se of 0.2. multiple regression analysis was performed with initial bimc score and wmla as the independent variables and rate of change of bimc as the dependent variable. wmla accounted for 16% of the variance (partial r = 0.396, t = 3.26, p < .05). these data suggest that elderly subjects with wmla may be at increased risk for rapid cognitive decline. such as g proteins and adenylate cyclase. the activities of adenylate cyclase, and of the g protein-associated enzyme activity, low-km glutamyl transpeptidase (gtpase), were assayed in membranes prepared from the postmortem brains of 8 alzheimer-diseased and 8 age-matched control subjects. both basal and fluoroaluminate-stimulated adenylate cyclase activities were significantly reduced in ad frontal cortex compared to control subjects ( p < 0.01; two-tailed student's t test). in addition, a significant, though smaller, reduction in basal gtpase activity also was detected in ad frontal cortex ( p < 0.05). in contrast, no significant change in the activity of either enzyme was detected in the hippocampus. the stimulation of gtpase activity by muscarinic and gababreceptor agonists was not altered significantly by the presence of alzheimer's disease. however, the degree of stimulation was much lower in human tissue compared to that observed using fresh rat brain, suggesting that the ability of receptors to activate g proteins declines post mortem. these results suggest that alzheimer's disease causes alterations in some key components involved in signal transduction. the association of lobar hemorrhage (lh) with cerebral amyloid angiopathy (caa) and that of caa with alzheimer's disease (ad) are well known. to determine how frequently lh and caa occur in ad, we reviewed 13 patients with cerebral or cerebellar hemorrhage and caa. five patients were treated surgically, none was demented, 2 died, and 1 came to autopsy. eight patients died of lh and came to autopsy. of the 9 autopsied patients, 3 had ad, both clinically and neuropathologically. they comprised 1.2% of 258 cases of autopsy-confirmed ad in our laboratory. none of the other patients were known to be demented. five had senile plaques, with or without neurofibrillary tangles, in the hippocampus, and occasional senile plaques in the cortex, but none met the consortium for establishing a registry for alzheimer's disease neuropathological criteria for ad. ad patients were 70, 85, and 85 years old and the ages of the 10 nondemented patients ranged from 63 to 87 (mean = 73.1 yr). seven were younger than 75 years of age. despite the frequent occurrence of caa in ad, we found that lh was uncommon. in addition, most of our patients with lh and caa were not demented and tended to be younger than ad patients with lh. these results indicate that the hf is involved primarily in acquisition or leaning processes and less so in retrieval of previously learned information. these findings relate to memory and structural brain changes found in normal aging. alzheimer's disease y . stern, l. stricks, g. alexander, 1. prohovnik, and there is an inverse relationship between parietotemporal cerebral blood flow and years of education in alzheimer's disease (ad) patients matched for clinical severity, which suggests delayed clinical manifestation of ad in patients with higher education (stern et al, soc neurosci abs 1771). we classified the lifetime primary occupations of 5 1 ad patients using the dictionary of occupational titles of the us department of labor and derived 6 factor scores describing intellectual, interpersonal, and physical job demands. after controlling for age, education, age at onset, illness duration, and dementia severity (mental status and activities of daily living), relative perfusion in the parietotemporal region (assessed using 133-xenon inhalation) showed significant correlations with job complexity (pl: r = -.36, p < .008) and interpersonal (p3: r = -.44, p < .001) factor scores. in a stepwise multiple regression, job complexity and interpersonal skills increased explained parietotemporal flow variance by 7.5% ( f = 4.7, p < .05) over that explained by demo-graphic and severity indices; physical demands then accounted for another 11.5% of the variance ( f = 8.4, p < .01). we conclude that occupational demands, similar to but independent of education, may provide a reserve that delays the clinical expression of ad. richard mayeux and ming-xin tang, new york, n y risk factors for alzheimer's disease (ad) were collected from 223 patients with ad and 278 healthy elderly controls in an urban community population consisting of 3 ethnic groups: black, hispanic, and white. advanced age (>80 yr) (or = 10.7; 75% ci 6.2-19.1) and head injury with loss of consciousness (or = 2.70; 1.2-7.3) were associated with ad, controlling for all known putative risk factors. factors such as low education (<8 yr) (or = 1.5; 0.8-2.6) and family history of ad (or = 1.7; 0.7-3.0) were not found to be significantly related to ad. head injury occurred in 15.7% of the patients and 6.?% of controls. most (70%) head injuries in the patients with ad occurred after age 50, prior to disease onset. in controls with head injury, 5592 had experienced a head injury before age 50. the duration of unconsciousness was consistently longer in patients with ad than in the controls. the overall effect in each ethnic group was similar (or,, 2.5; 1.3-4.8). these results confirm and strengthen the previously described putative relationship between head injury and ad. we also conclude that both the severity and the timing of the head injury as well as the frequency of head injury in the population at risk may be important factors in understanding the causal relationship between head injury and ad. the purpose of this study was to determine how native language affects the cutoff scores in screening tests for dementia. there is a paucity of data o n this issue at present. screening tests used in the study were: folstein mini-mental state (mms); clockdrawing (clock); preparing a letter for mailing (mail); 4-item grocery list (list); and hamilton depression scale (ham). the subjects included 175 (74 demented) native english speakers (eng) and 72 (22 demented) native spanish speakers (spa) with memory complaints. diagnosis of dementia was determined by neurological, neuropsychological, and psychiatric evaluation. age and gender were unrelated to mms. in spa only, education was positively related to mms. ham scores were higher in deand 0.77 (spa); c d and list did not add discriminative power to mms for eng but did so for list in spa. mms discriminates between demented and nondemented far better in english than in spanish speakers. only mail adds discriminative power to mms. a. heyman, g. fillenbaum, s. mirra, and participating cerad neuropathologists, durham, nc, and atlanta, ga the clinical diagnosis of alzheimer's disease (ad) has become more accurate in recent years due to the application of specific clinical criteria, wider use of neuroimaging procedures, and greater expertise among physicians. we report the frequency of clinical misdiagnosis of ad among 52 patients who at autopsy were found to meet the rigorous clinical diagnostic criteria imposed by the consortium to establish a registry for alzheimer's disease (cerad) study. these patients included 31 men and 21 women (mean ages 78 and 76 yr, respectively) who were among the group of 95 patients who died in 13 cerad medical centers in the us between 1987 and 1991. the clinical diagnosis of ad was neuropathologically confirmed in 47 (88.5%) of the 52 cases. of these 47 cases, varying degrees of concomitant cerebrovascular disease were present in 26% and coexisting parkinson's disease changes were found in 19%. in 4 of the 5 patients without neuropathological evidence of ad, the diagnoses were: lobar atrophy, diffuse lewy body disease, nonspecific neurodegenerative changes, and mesocorticolimbic dementia, respectively. the fifth patient showed no morphological abnormalities. on the basis of these results, it would appear that application of strict diagnostic criteria, as well as the use of brain scans and detailed clinical and neuropsychological tests by experienced clinicians, cannot yet distinguish some types of primary degenerative dementias from alzheimer's disease. we designed a scale that measures an underexplored facet of functional decline in alzheimer's disease (ad): the patient's dependence on others for supervising or performing activities. two hundred twenty-three informants for patients with mild ad (clinical dementia rating [cdr] = 1 for 200; cdr = 2 for 23) were interviewed and dependence was staged from 0 to 5. interrater reliability was assessed by separate interviews of 20 informants; agreement was 100% for dependence stage. dependence stage differed significantly at the 2 cdr levels (chi square = 41.0, p < .01) and correlated significantly with modified mini-mental state (mmms) ( r = -.30, p < .001) and blessed dementia rating scale-part 2 (bdrs) ( y = .41, p < .001). seventy-eight percent of patients in a health-related facility were at stage 3 or higher vs 25% of patients living at home. in a multiple regression model, both the bdrs and dependence scale accounted for unique portions of the variance in mmms, suggesting that they assess unique aspects of functional ability. dependence increased significantly in 118 patients retested at 1 year. we conclude that the dependence scale is reliable and relates to both disease severity and progression. formal assessment of dependence should prove useful for studies of the natural history of ad as well as for clinical trials. cortical-basal ganglionic degeneration (cbgd) is a disorder characterized by an asymmetrical akinetic-rigid syndrome and cortical signs such as apraxia, alien limb phenomena, and cortical sensory loss. dementia has been present in many cases, but always as a late manifestation. we report 2 cases pathologically consistent with cbgd, presenting as primary degenerative dementia, fulfilling nincds-adrda criteria for probable alzheimer's disease (ad). the first patient presented with changes in memory and personality, language dysfunction, decreased verbal output, and shuffling gait. follow-up examinations over 3 years showed progressive dementia, wide-based gait, and frequent falls. the second patient presented with complaints of memory loss. neuropsychological examinations showed progressive deficits in memory, attention, calculations, and visuospatial functioning. no movement disorder developed over 6 years of follow-up. at autopsy, both patients had typical changes of cbgd and lacked pathological features of ad. definitive diagnosis of cbgd rests on both clinical and pathological criteria. cbgd should be considered in the differential diagnosis of patients with dementia resembling that found in ad, especially if extrapyramidal signs are present. a quick, easily administered and scored test for praxis is desirable in evaluation of neurological patients. during 2 months in 1991, each patient seen in the outpatient setting by the principal investigator (e. k.) received 1 of 2 praxis screening batteries. thirty-six patients were tested with a short battery. eleven had normal cognition based on neurological history, examination, and a short test of mental status. twenty-five had cognitive decline (cd). handedness, male/ female ratio, education, and mean age were similar in both groups. mean time of completion of the test was 102 2 10 seconds in normals and 136 * 41 seconds in patients with cd. total scores (maximum 50) were 47.6 * 1.8 in the normals and 41.2 2 9 in the cognitively declined patients. twenty-five other patients, 12 with cd and 13 with normal cognition, were tested with a longer battery containing oral/ facial, upper and lower limb, axial, sequential, and imitation subtests (maximum score 110). normals completed the battery in 297 i 56, and cognitively declined patients completed the battery in 359 ? 53 seconds. of the various subtests, tests of sequential praxis were performed most poorly by patients with cd: 8.3 * 1.8 in normals vs 4.9 * 3.0 in patients with cd. oral/facial praxis was least affected by cd: olivopontocerebellar atrophy (opca) is generally understood to be a nondementing neurodegenerative disorder affecting the cerebellum, lower brainstem, and spinal cord. one of us (s. k.) recently reported that postmortem cerebral cortex from patients with dominantly inherited opca shows a widespread reduction of cholinergic markers similar to that observed in alzheimer's disease (ad). we were interested to determine the status of other neurotransmitter systems in opca postmortem cerebral cortex. samples of frontal, parietal, temporal, and occipital cortex were dissected from 10 confirmed cases of opca and 11 age-matched controls. after processing, neuropeptide levels were measured by radioimmunoassay. concentrations of somatostatin were significantly reduced by 42 to 58% in 3 of the 4 cortical areas of opca brain that were examined. the area that was spared was the inferior temporal gyrus, a region in which somatostatin levels are markedly reduced in ad. levels of neuropeptide y were normal in all 4 areas, while concentrations of cholecystokinin, vasoactive intestinal polypeptide, and substance p were significantly increased in 2 of the 4 areas. these data show widespread neuropeptide changes in the cerebral cortex of opca postmortem brain. in contrast to cholinergic markers, the pattern of neuropeptide changes is different from what is observed in ad. it has been postulated that demise of the corticomotoneuron is the initial event in amyotrophic lateral sclerosis (als) and that the anterior horn cell dies as the result of antegrade glutamatergic excitotoxicity (muscle nerve 1992;15:219). excitability of the corticomotoneuronal system can be tested by measuring threshold-to-cortical magnetic stimulation and the motor-evoked potential (mep)/compound muscle action potential (cmap) ratio, which estimates the number of corticornotoneurons stimulated. cortical threshold and mep/ cmap ratio were measured in 39 patients early in the course of als. the mean time interval from onset of first symptoms was 8.5 months. mean threshold and mepicmap ratio measured 64.2 f 16.6% and 22.0 rfr 21.2%, respectively. in 12 (30.8%) patients, threshold was paradoxically low (<55%, mean 49.1 +. 4.3%) and in 7 (17.9%) patients there was no response. there was a significant ( r z = 0.698) inverse power relationship between cortical threshold and mep/cmap ratio given by 84.1 x mep/cmap-'j. six months later, 7/24 (29.2%) patients still had low thresholds but the mean mep/ cmap ratio had dropped to 24.9 ? 24.5% and in 33.3% there was no response. we conclude that early in als the corticomotoneuronal pathways are abnormally excitable. this may explain early cramping and fasciculation, which characteristically diminishes as als progresses. one of the distinct clinical features in patients with amyotrophic lateral sclerosis (als) is loss of elasticity of skin. however, little is known concerning the biochemical nature of skin elastin in als. in our study, 2 cross-links unique to elastin, desmosine and isodesmosine, were measured and compared in skin tissue (left upper arm) from 10 patients with als and from 7 age-matched controls. the contents of desmosine and isodesmosine were decreased significantly ( p < 0.01 and p < 0.01, respectively) in patients with als (mean * sd, 0.94 ? 0.33 and 0.74 * 0.30 nmol/mg dry weight; range 0.36-1.51 and 0.27-1.33 nmol/mg dry weight, respectively) as compared with those of controls (mean 2 sd, 1.43 * 0.30and 1.17 2 0.21;range 1.11-2.03 and 0.92-1.52, respectively), and were negatively and significantly associated with duration of illness in patients with with amyotrophic lateral sclerosis als ( r = -0.77, p < 0.01, and r = -0.65, p < 0.05, respectively). the ratio of desmosine and isodesmosine was constant (1 : 3) in all samples analyzed. the decline in skin desmosine and isodesmosine is more rapid in als than in normal aging. thus, cross-linking of skin elastin is affected in als. (supported in part by n i h grants de 18522, de 11233, de 08611, ar 19969, ar 30587, and nasa grant nag-2-181.) pl69. natural history of amyotrophic (1) collection of large numbers of twin pairs in disease of low prevalence is difficult. to circumvent this, we devised a new approach termed the "death discordant twin pair" method. eleven thousand deaths from motor neuron disease (mnd) were extracted from the office of population censuses and surveys during 1979 to 1789. birth indexes from 1900 onward were searched for possible twins. for each twin so identified (13 1 pairs), the national health service central registry located the relevant family practitioner committee and thence the co-twin's general practitioner. the search produced: (1) 53 living co-twins; (2) 5 embarked; (3) 60 dying as adults or infants; (4) 3 not mnd; and (5) validity of the accuracy, sensitivity, and specificity of the world federation of neurology (wfn) subcommittee on motor neuron disease working group criteria for the clinical diagnosis of amyotrophic lateral sclerosis (als) has been tested against neuropathological criteria in 36 autopsied patients (neurology, in press). integration of clinical and electrodiagnostic data to meet wfn criteria for possible, probable, and definite als was studied in this samegroup. patients received 1.7 ifr 1.1 (mean * standard deviation) electromyograms (emgs) per patient and included 1.7 2 0.9 emg levels (bulbar, cervical, thoracic, lumbar) per patient. proportionately fewer emgs were performed as the level of diagnostic certainty at presentation increased: suspected (34.3%), possible (31.4%), probable (25.7%), definite (8.6%). in only 4.8% of all patients studied did the first emg alone change the level of diagnostic certainty of the diagnosis of als. only 16.7% of patients presenting with suspected als alone or with possible or probable als were associated with a change in level of diagnostic certainty following 1 or more emgs. however, although increasing the number of emgs performed per patient may be associated with an increasing chance of increasing the level of diagnostic certainty (22 emgdpatient = 33.3%; 2 3 emgdpatient = 60.0%), selection of the level of emg analysis was more crucial. the "el escorial" criteria emphasize the importance of emg evidence of lower motor neuron involvement in a limb with clinical upper motor neuron signs. our analysis of emg studies in autopsy-confirmed als patients suggests that complete evaluation of bulbar and thoracic levels for lower motor neuron changes and complete evaluation of motor unit recruitment patterns are important for the integration of emg data with clinical data in the application of the "el escorial" criteria for the diagnosis of possible, probable, and definite als. sibships on guam annette grefe, john steele, linda flares, and stephen waving, birmingham, al, and umatac and mangikao, guam reports in the 1950s indicated that 40% of guamanian chamorro patients with amyotrophic lateral sclerosis (als) gave october 20 a positive family history. subsequent investigators have inferred that purely genetic factors are not responsible for guamanian als or its clinical variant, parkinsonism-dementia complex (pdc). we report the first 4 chamorro sibships selected in an ongoing study of 17 1 familial aggregations. the basis for the initial selection was that the youngest patient in the sibship had als. age of onset was 30 to 46 years (mean 35 yr). fourteen of 23 persons in these sibships were affected. others in the sibship developed pdc, progressive supranuclear palsy, or pure dementia later in life (mean 61 yr, range 50-74 yr). these cases developed 14 to 47 years (mean 25 yr) after onset of disease in the first sibling. the age at onset of the first case and the intervals between earliest and latest cases in such sibships may help to determine minimal and maximal latency (i.e., interval between exposure to an exogenous agent and onset of symptoms). our observations suggest that exposure may have occurred early (before age 30) with varying and often long latency (up to 47 yr). we also find that variability of clinical expression may be correlated with the age at onset. concentrating on the study of familial cases on guam may enhance the identification of the etiologic agent(s) of this prevalent and tragic disorder. the spinocerebellar ataxias are an uncommon group of genetic disorders that have been well characterized in north america and europe. information concerning these conditions in africa and other parts is scant. to address this problem, a large-scale survey has been undertaken in the cape province of south africa. in this investigation, more than 500 persons in 18 affected families have been appraised and investigated and phenotypic features have been analyzed in detail. linkage studies have been undertaken in 4 families with similar phenotypes in which the condition was transmitted as an autosomal-dominant trait. human lymphocyte antigen (hla) typing was carried out on 79 members of the 4 families and linkage analysis was undertaken using the liped program to analyze the data with a correction factor for age of onset. maximum lod scores were: family a: 4.13 (0 = 0.005); family b: 0.6 (0 = 0.001); family c: 0.50 (0 = 0.30); family d: 0.0 (0 = 0.50). these results indicate linkage to hla in 1 out of 4 families. these findings provide support for the concept of genetic heterogeneity in these phenotypically homogeneous families. pcr typing with the reportedly more closely linked d6s89 locus is now being undertaken in these south african families. h.-p. hartung, g. f. hoffmann, and g. becker, wunburg, heidelberg, germany recently, l-2-hydroxyglutaric acidemia has been described as a novel metabolic disorder in 6 children. we report the occurrence of this disease in adults. one of 2 brothers developed at the age of 10 an abnormal gait and dysarthria; in the other 1, clumsiness and walking delay were noted at age 3. symptoms progressed and at the time of admission, when the patients were 33 and 39 years, neurological examination revealed a spastic ataxic gait, limb ataxia, dysmetria, dysarthria, dystonic posturing, and mental retardation. ct and mr imaging revealed subcortical white matter changes with loss of arcuate fibers, folial atrophy, and leakage in the cerebellar vermis, as well as atrophic changes in the cerebellar hemi-acidemia program and abstracts, american neurological association 273 spheres. on biochemical screening, highly elevated concentrations of l-2-hydroxyglutaric acid were found in csf, plasma, and urine. the pathological accumulation of l-2hydroxyglutaric acid in these 2 adults, along with the clinical picture characterized by cerebellar, extrapyramidal, and pyramidal symptoms and oligophrenia, and the neuroradiological findings of severe loss of myelinated arcuate fibers in subcortical white matter, conform with what previously has been described in the few neuropediatric cases. the biochemical abnormality underlying accumulation of this organic acid remains elusive. this study was undertaken to differentiate primarily affected areas from functionally suppressed areas due to remote effect in aphasic patients with focal brain degeneration, using an activation method with 0-15 water, in comparison with [sf]2-fluoro-2-deoxy-~-g~ucose (fdg) positron emission tomographic examination at rest. the subjects were 2 patients with slowly progressive aphasia showing contrasted clinical symptoms (nonfluent type vs fluent type). regional cerebral metabolic rate of glucose (cmrglu) was measured with intravenous injection of 130mbq of f-18 fdg at rest. regional cerebral blood flow (cbf) was measured with intravenous bolus injection of 1.5gbq of 0-15 water in 3 different conditions: at rest, under repetition tasks, and under naming tasks. changes of cbf were evaluated between a resting condition and task-performing conditions. regional cmrglu was decreased focally in both broca's and wernicke's areas similarly in these cases in spite of the difference in clinical symptoms, whereas the patterns of regional cbf changes were different. the fluent patient showed prominent activation in the bilateral frontal areas on repetition tasks. the nonfluent patient showed, whereas the fluent patient did not show, focal activation in the left occipitoparietal area on a naming task. evaluation with an activation method can provide detailed information about the pathophysiological process and the location of primary lesion. defective complex i activity has been linked to huntington's disease (hd) and parkinson's disease (pd). intrastriatal injection of inhibitors of complex i reproduces the pathological features of hd, and the neurotoxin mpp+ kills dopaminergic neurons by inhibiting complex i. defects in complex i have been reported in hd and pd, but the distribution of this enzyme in the brain is unknown. to map complex i in brain quantitatively, we developed an assay using e3h)dihydrorotenone to label the enzyme in tissue sections. this high-affinity binding is saturable and is displaceable by rote-in brain none and mpp+. using 100 pm rotenone to define nonspecific binding, more than 95% of binding is specific. highest levels of binding are found in kidney, followed by myocardium. moderate levels of complex i are seen in striated muscle and some brain regions. within the brain, binding varies more than 20-fold and is heaviest in the cerebellar molecular layer and dentate gyrus. lower levels of binding are found in cortex and striatum and very low levels are located in substantia nigra. this assay may help to clarify the role of complex i in neurodegenerative disorders. ( in 1988, 4 patients with medically intractable parkinson's disease underwent autologous adrenal medullary-to-caudate transplants at the university of california-los angeles (ucla). these persons had been followed for several years before operation at 3-or 4-month intervals. at each visit, their disability had been rated on the quantified ucla scale and the hoehn and yahr stage of disease. these provided a longitudinal assessment of the progression of disease in each patient, which could be compared to the rate of progression in the cohort of cases followed at ucla for 15 years. in addition, the unified parkinson's disease rating scale provided supplementary data for the immediate preoperative and subsequent postoperative evaluations. the hours "off" also were recorded for the preoperative and postoperative periods. after operation, the same evaluations were performed by the neurologist who previously had cared for the patients. the longitudinal postoperative data revealed that at 1 to 4 months after operation, all 4 patients improved. after 4 years, 3 continue to be less disabled than their preoperative baselines. the progression of their disease, while still evident, is nonetheless proceeding at a slower rate than before transplant. the fourth patient had brief improvement shortly after operation, but then rapidly worsened to his previous level. his disease has continued to progress at a rapid pace, unchanged from progression before operation. individuals at risk for huntington's disease h . p. h . kremer, w. shtybel, b. snow, c. clark, j . theilmann, m . r. hayden, and w. in huntington's disease (hd), caudate hypometabolism as demonstrated by positron emission tomography (pet) is a well-established feature in symptomatic patients. in individuals at risk, however, conflicting findings are reported. since 1984 we have performed 61 pet scans in 44 asymptomatic persons at risk for hd (age 27-76 yr) . linkage analysis with 9 independent dna probes or subsequent evolution to clinical hd (in 5 patients) allowed a risk estimate for 35 subjects. twenty were considered to be at increased risk (?85%), 14 at decreased risk (<15%), and in 1 individual no modification could be given. nine subjects were not tested. a pet scan was considered abnormal if either the caudate/thalamus ratio or the caudate/whole-brain ratio of rcmrglu was more than these criteria, 8 scans in 6 individuals were abnormal. none had received a decreased risk. comparison of the ratios of increased risk, decreased risk, unmodified risk, and control subjects failed to show statistically significant differences (analysis of variance). follow-up of 3 persons with an abnormal scan showed conversion to symptomatic status within 5 years after the first abnormal scan. this result suggests that an abnormal pet scan in a person at risk for h d heralds the onset of choreic movements. robitaille, m . el-awar, b. clark, l. scbut, m. ball, l. young, r. currier, and k. sbannak, toronto, ontario, and montreal, quebec, canada; pittsburgh, pa, minneapolis, mn, portkmd, or, and jackson, ms we measured the levels of dopamine in striatum of 14 patients with end-stage dominantly inherited olivopontocerebellar atrophy (opca). on average, dopamine levels were reduced in putamen ( -5396, as compared with controls), caudate ( -35%), and nucleus accumbens ( -31%). however, individual patient values showed a wide variation (normal to -9996), indicating that striatal dopamine loss is a common, but not constant feature of opca. seven patients had marked putamen dopamine loss (-62 to -8195) but without corresponding severe substantia nigra cell damage; this suggests a "dying-back'' phenomenon in which nerve terminal loss precedes cell-body degeneration. in this regard, opca may offer the possibility of examining nigrostriatal dopamine neuronal degeneration at an early stage. although 2 patients were found to have severe nigral cell loss with near total ( -95 to -99%) striatal dopamine loss, none had depression in parkinson's disease (pd) has been correlated with low cerebrospinal fluid (csf) levels of 5-hydroxyindoleacetic acid (5-hiaa). l-dopa may precipitate or exacerbate depression in 30% of pd patients. to determine if l-dopa affects 5-hydroxytryptamine (5ht) metabolism, 8 patients were studied. four had pd. of these, 2 were taking l-dopa and both were depressed. the diagnoses in the remaining 4 were progressive supranuclear palsy (psp), striatonigral degeneration (snd), normal pressure hydrocephalus, and pseudoseizures with depression. neuropsychological examinations and lps of all 8 patients were done. three patients were started on l-dopa (the 2 previously untreated pd patients and the psp patient) and retested 3 days later. one pd patient started on l-dopa became depressed. mood in the others was unchanged. csf was analyzed for 5ht and 5-hiaa. 5ht could not be detected in the csf of patients who were not taking l-dopa, but was easily detectable in all of the patients who were taking l-dopa. 5-hiaa levels were low in the untreated pd patients, and also in the patients with psp, snd, and pseudoseizures with depression. 5-hiaa levels were even lower in the l-dopa-treated patients. the ratio of 5-hiaa:5ht (an index of 5ht turnover) was lowest in the l-dopa-treated pd patients who were depressed. low csf 5-hiaa in untreated pd may reflect depletion of brain 5ht. l-dopa may induce depression by inhibiting 5ht turnover in 5ht-depleted brain. p18 1. ventroposterolateral medial pallidotomy in the e. fazzani, m. dogali, a. ben;, d. eidelberg, j. gianutsos, t. kay, b. newman, s. loftus, d. samehon, and l. laitinen, new york, n y , and stockholm, sweden in patients with parkinson's disease (pd), as a consequence of low dopamine there exists an increase in inhibitory output from the globus pallidus. ten patients (5 men and 5 women) with pd received unilateral (6 right, 4 left) ventroposterolateral medial globus pallidotomies (vplmp). the average patient age was 61 years (range 55-73 yr), and the average duration of disease was 14 years (range 6-24 yr). patients fluctuated between "on" chorea and "off" parkinsonism. hoehn and yahr stage "on" was i1 in 7 , 111 in 3; and "off" was i11 in 5, iv in 2, and v in 3 patients. unified pd rating scale (updrs) score averages 12 hours off medicines (12hom) were activities of daily living (adl): 20 and motor (mtr): 40 preoperatively (preop). capit score averages preop 12hom were pronation-supination (ps): 3 1 seconds (s), finger tap (ft): 31s, board (b): 39s for the most affected contralateral side, and gait: 57s (2 patients could not walk). re-examination was done 2 to 5 days after pallidotomy. updrs scores 12hom decreased an average of 60%. three patients had major bilateral improvement in bradykinesia. rest tremor, prominent in 3 patients, also was diminished. capit scores 12hom decreased to ps: 19s, ft: 20s, b: 22s; the average gait of the 8 patients who could walk preop improved to 36s. there were no side effects. vplmp leads to an immediate overall significant improvement in patients with pd. s. kish, y . there is a growing interest in the genetic aspects of parkinson's disease and other basal ganglia disorders. we have studied 3 families whose ancestors immigrated to north america from contiguous regions of northern germany and southern denmark. the pedigrees contain 77, 206, and 376 individuals spanning 6, 7, and 8 generations with 7,6, and 9 affected members, respectively. autosomal-dominant inheritance is clearly present in 2 families and probable in the third. typical levodopa-responsive parkinsonism with bradykinesia, rigidity, resting tremor, and impaired postural reflexes uniformly develops in affected individuals from all 3 families. n o downgaze impairment, pyramidal signs, sensory disturbances, cerebellar dysfunction, or orthostatic blood pressure changes have been observed. dementia, however, has developed in a few elderly individuals, especially in 1 family. laboratory studies are normal. mri shows moderately enlarged ventricles and cortical atrophy. 6-fd positron emission tomography demonstrated reduced striatal uptake in 1 examined patient and normal uptake in l individual at risk. autopsy of only 1 subject has been performed (in 1975). brain weight was 1,380 grams and there were no obvious gross abnormaliprogram and abstracts, american neurological association 275 tuesday, october 20 ties, but microscopic examination was limited. further research on these 3 families is planned. electrophysiological study s. maurri, m . cincotta, a. ragazzoni, g. descisciolo, and f. barontini, florence, ltah many neurophysiological examinations were conducted of a 32-year-old woman with familial mirror movements. n o other neurological abnormalities were detected. examination included voluntary electromyographic (emg) activity from various muscles, f-wave as well as short-and long-latency reflex responses of the thenar muscles from electrical stimulation of the median nerve, mapping of motor-evoked potentials (meps) to transcranial magnetic stimulation, movement-related cortical potentials (mrcps), and somatosensory-evoked potentials (seps). emg documented mirror activity in the upper limbs, most marked in the muscles of both hands. onset latency of emg activity in response to an auditory stimulus was identical in active and mirror muscle. long-latency responses from median nerve stimulation were recorded on contralateral as well as ipsilateral thenar muscles. short-latency reflexes and f wave were strictly ipsilateral. unilateral scalp magnetic stimulation evoked bilateral responses at similar latencies in the thenar muscles; midline scalp stimulation activated no responses. scalp distribution of median nerve seps and of mrcps associated with self-paced thumb abduction were normal. our findings suggest that congenital inherited mirror movements in otherwise normal subjects can be generated by corticospinal fibers pro jecting to ipsilateral motoneurons of the spinal cord. we have previously identified dysphagia and constipation (both slow transit and defecatory dysfunction types) as common gastrointestinal (gi) problems in parkinson's disease (pd). since apomorphine has been shown to be capable of terminating off-periods when injected subcutaneously, we have evaluated the effects of apomorphine injection on objective parameters of dysphagia and bowel dysfunction in pd patients. nine subjects underwent the following battery of studies to characterize their pd features, swallowing, and bowel function: gi assessment survey, unified pd rating scale, videoesophagram, colon transit study, defecography, and anorectal manometry. specific abnormalities on the studies were noted and the most abnormal study was repeated after subcutaneous administration of 6 mg apomorphine. all individuals were pretreated with domperidone 20 mg four times daily for 3 days prior to apomorphine administration. improvement in both esophageal motility and deglutition was noted in the 1 individual in whom videoesophagram was repeated. for the other 8 patients, defecography or anorectal manometry was performed. significant improvement in specific parameters was demonstrated after apomorphine administration, but 2 individuals experienced syncope during radiographic procedures. we conclude that subcutaneous apomorphine administration holds promise as a potential therapeutic approach to dysphagia and, especially, bowel dysfunction in pd, but that further investigation and refinement are necessary. asymmetrical effect of unilateral thalamotomy or subthalamotomy on tremor in parkinson's disease nico diedericb, christopber g. goetz, glenn t . stebbins, harold l. klawans, k. nittner, a. kozrlosakis, p. sanker, and v . strum, cologne, germany, and chicago, il in the past, stereotactic operation was a regular treatment for unilateral tremor in parkinson's disease (pd). however, follow-up studies were usually short term and always unblinded. we examined 17 pd patients in long-term follow-up (mean 10.9 yr after operation) who underwent unilateral thalamotomy for parkinsonian tremor. we used videotapes and the unified parkinson's disease rating scale to blindly compare tremor ipsilateral and contralateral to the side of operation. since the patients were specifically selected for stereotactic operation because of asymmetric tremor, we reasoned that a sign of long-term efficacy would be current postoperative reversal of tremor side predominance. upper extremity tremor was significantly better contralateral to the side of operation compared to the ipsilateral side ( z = 3.29; p < 0.023). for the lower extremities the difference was not statistically significant. in chronic follow-up, stereotactic operation improved the absolute magnitude of arm tremor or ameliorated its rate of progression. since asymmetric bradykinesia and dyskinesia were not prerequisites for the choice of surgical side, we cannot make any conclusion about longterm impact of operation on these features. subtle extrapyramidal signs resembles that of patients with parkinson's disease m . richards, k. marder, l. cote, y . stern, and r. mayeux, new york, n y to investigate the relationship between extrapyramidal signs (eps) and cognition, eps severity and neuropsychological function were assessed in 307 normal elderly individuals and 130 nondemented patients with idiopathic parkinson's disease (pd) from a community-dwelling cohort in new york city. multivariate analysis of variance (manova) indicated poorer neuropsychological performance ( p = .001) in pd patients on verbal memory, orientation, verbal fluency, visuomotor construction, and psychomotor speed, but not naming, abstract reasoning, or matching. controlling for eps severity abolished these differences. one hundred fourteen (37%) of the normal individuals had subtle eps (mostly postural abnormality, bradykinesia, or rigidity) but no identifiable neurological disorder. manova indicated poorer neuropsychological test performance ( j = ,001) in these individuals than in normals without eps on verbal memory, orientation, abstract reasoning, naming, verbal fluency, matching, and psychomotor speed but not visuomotor construction. we conclude that: (1) cognitive impairment in pd is specifically associated with eps, and (2) a similar association occurs in individuals with subtle eps but no neurological disorder. whether this represents a preclinical stage of pd or ad is yet to be determined. disease in olmsted county, minnesota emre kokmen, fatma sibel ozekmekci, c. mary beard, and peter c. o'brien, rochester, m n , and istanbd, turkey there have been many studies of prevalence of huntington's disease (hd) in diverse populations around the world. to study the incidence, we took advantage of the availability of detailed health care records for the population of olmsted county, mn, from mayo clinic, its affiliated hospitals, olmsted medical group, county hospital, state hospital, records of solo practitioners, nursing homes, death certificates, and autopsy records. we reviewed all records with a diagnosis of hd, huntington's chorea, chorea major, and chorea otherwise unidentified, and sought evidence for progressive chorea, progressive cognitive and/or behavioral dysfunction, and family history compatible with autosomal-dominant inheritance with onset of symptoms in the period between january 1, 1950, and december 31, 1989 , while the patient lived in the geographic boundaries of olmsted county. we found 3 males and 6 females who met these criteria. average annual incidence rate (age/sex adjusted to 1960 us white population) for hd for this 40-year period was 0.3 cases/ year/ 100,000 population. we also estimated prevalence by taking account of in-migration, out-migration, and deaths. the agelsex adjusted (1960) prevalence for 1-1-60 was 7.51 100,000 and for 1-1-90 it was 2.4/100,000. the small number of cases caused the instability of the prevalence rates, but our rates are similar to rates reported in other populations. alton e . btyant, 111, l. breeden hollis, john a. hamjian, john d. wooten, ill, and francis 0. walker, nc we described 4 patients with unusual episodic movement disorders and normal diagnostic work-ups: a 34-year-old woman who had recurrent episodes of tonic jaw deviation and forced right-eye closure; a 30-year-old woman who developed unexplained pain and subsequent spells of tonic inversion of the left leg; a 61-year-old man who presented with a bizarre episodic right-arm tremor; and a 15-year-old woman who experienced intermittent abdominal undulations. examination of the affected body part provoked or enhanced symptoms in all patients. using suggestion and placebo activation in the form of a medicated patch, intravenous saline, or cervical massage, we first induced and then aborted typical episodes of their abnormal movements. postinduction discussions of the procedure led to a marked reduction in the frequency of attacks in 2 patients. activation procedures are useful in diagnosing psychogenic disorders because they demonstrate that situational, not medical, factors govern the expression of the abnormal behavior. we speculate that patients who are refractory to simple suggestion may respond to induction because it offers the potential of validating their symptoms. as in the case of psychogenic respiratory distress or pseudoseizures, positive induction can assist in counseling and symptom control. had alzheimer's disease with parkinsonism (adp), 1 had essential tremor (et), 1 had cerebellar tremor in multiple sclerosis (ms), and 1 had tardive dyskinesia (td). clozapine was used either to treat psychosis (20 pd, 2 adp, 3 dys, 1 td) or tremor (1 5 pd, 1 et, 1 ms). two pd patients were retrospective analysis of 38 patients counted twice, 1 who was treated for psychosis and then tremor and 1 who was treated on 2 separate occasions for psychosis with different responses. all 3 dys patients improved, 2 with complete resolution of their dystonia on changing antipsychotic drugs. the patients with et (75 mg) and ms (25 mg) improved mildly but sedation and clumsiness caused drug discontinuation in the ms patient. one adp patient (112.5 mg) responded well and the other became sedated and confused (25 mg). the p d responses for psychosis at a dose range of 6.25 to 400 mg daily were good (5), very good (2), and excellent (8), whereas 4 were intolerant. pd tremor responses were good (51, very good (4), excellent (2), and poor (3) at doses of 12.5 to 100 mg daily. one patient died of unrelated causes shortly after initiation of the drug. adverse effects included sedation, weight gain, hypersalivation, fainting, clumsiness, transient granulocytopenia, and "spasms" necessitating discontinuation in 9 patients (7 pd, l td, and l ms). tourette's syndrome and attention-deficit disorder patients s. m. silverstein, p. g. coma, d. palumbo, l. west, and r. kurlan, rochester, n y impaired attention is a common comorbid behavioral feature of tourette's syndrome (ts) and a key clinical feature of attention-deficit hyperactivity disorder (adhd). however, the pattern of attentional impairments reported in adhd has not been observed in ts. we therefore compared 17 ts patients (9 male, 8 female; mean age 32 ? 11 yr), 17 adhd patients (8 male, 9 female; 36 * 12 yr), and 17 normal controls (8 male, 9 female; 3 1 ? 9 yr) on specific neuropsychological (np) and computer-administered tasks of attentional ability. adhd, but not ts, subjects performed significantly worse than controls on the n p tasks (digit symbol, perceptual speed) and had a trend toward poorer performance on a computerized measure of attention. however, both the adhd and ts groups had significantly greater test performance variability on some, but not all, tasks and had more subjects with deviant scores. among ts patients, higher scores on an obsessive-compulsive disorder (ocd) inventory and a greater number of adhd symptoms correlated significantly with poorer performance on the attentional tasks. moreover, ts patients with observed tics during testing had greater attentional impairment than those without tics. these results suggest that: (1) many adult ts patients do not have impaired attention; (2) attentional impairment in ts differs from that observed in adhd; and (3) attentional impairment in ts is associated with the full neurobehavioral spectrum of ts (i.e., tics, ocd, and adhd). frank r. sharp, cathleen miller, thomas rando, and steven greenberg, san francisco and palo alto, c a six patients are described with choreoathetoid movements and marked proprioceptive sensory loss. one patient had a traumatic injury to the right parietal cortex that produced severe proprioceptive sensory loss and choreoathetosis in the left arm. another patient had a left thalamic infarction that resulted in profound proprioceptive sensory loss and chorea on the right side of the body. two patients had cervical spinal cord disease, proprioceptive sensory loss, and diffuse choreoathetosis. another patient had dorsal root ganglionitis associ-a hypothesis program and abstracts, american neurological association 277 ated with small-cell lung carcinoma that produced diffuse loss of all sensory modalities and chorea. the last patient had an ulnar sensory neuropathy and choreic movements of the fifth finger. lesions anywhere along the pathway that transmits limb proprioception may cause pseudochoreoathetosis. furthermore, choreoathetosis without sensory loss caused by focal lesions of striatum may occur because of disruption of cortical proprioceptive inputs to striatum-perhaps explaining why most focal lesions of striatum do not produce chorea. sporadic inclusion-body myositis (s-ibm) and autosomalrecessive hereditary inclusion-bod y myositis (h-ibm) are of unknown cause and pathogenesis. in both there are muscle fibers with rimmed vacuoles containing 15to 21-nm cytoplasmic tubulofilaments (ctfs) and denervation atrophy; in s-ibm, but not h-ibm, there is a varying degree of inflammation. vacuolated fibers contain ubiquitinated inclusions (askanas 1991) and congo-red positivity indicating amyloid (mendell 199 1). because immunoreactive p-amyloid precursor protein (app) and p-amyloid protein (p-ap) are constituents of ubiquitinated senile plaques in alzheimer's disease (ad) brain, we studied immunolocalization of app and p-ap fibers in ibm muscle using antibodies against: (1) non-p-ap fragments of app, viz. (a) c-terminus (residue 676-695 courtesy d. selkoe) and (b) n-terminus (residue 45-62 courtesy b. frangione and d. levartovsky); (2) p-ap (sequence 8-17, courtesy g. glenner, and sequence 1-40 courtesy d. selkoe); (3) ub (chemicon). in 13 of 13 ibm patients, including one h-ibm, 100% of the vacuolated muscle fibers contained large or several small app and p-ap immunoreactive (ir) inclusions, which by double-labeling fluorescence were closely colocalized with each other and with ub-ir. none of 18 control muscle biopsy specimens (including 7 polymyositis) contained app-ir, p-ap-ir, or ub-ir inclusions characteristic of ibm. control experiments utilizing omitted, replaced, or absorbed primary antisera were negative. p-ap, a product of proteolytic cleavage of app, is receiving attention regarding the pathogenesis of ad. our study provides (1) the first demonstration of app and p-ap accumulations in abnormal human muscle, and (2) raises the possibility that in ibm muscle and ad brain rhey may form from similar cellular events. jeffrrey d. rothstein, lin jin, and ralph kuncl, baltimore, m d the pathogenesis of motor neuron death in amyotrophic lateral sclerosis (als) is unknown. accumulating evidence suggests that the disease is characterized neurochemically by a derangement in the control of neurotransmitter glutamate metabolism: csf levels of glutamate and aspartate are ele-of motor neuron degeneration vated and their high-affinity transporter is defective in brain and spinal cord. inefficient glutamate transport, and subsequent chronic increase in extracellular glutamate, could be responsible for selective motor neuron death. to test the hypothesis that chronic defects in glutamate uptake can produce motor neuron toxicity, we developed a tissue culture model employing organotypic rat spinal cord maintained under conditions of chronic glutamate uptake inhibition. slices (300 pm) of lumbar spinal cord from 8-to 14-day-old rat pups were cultured on millicell membranes. chronic uptake inhibition was produced by culturing tissue in the presence of threohydroxyaspartate (tha) or pyrrolidine-dicarboxylic acid, both known to be specific inhibitors of glutamate transport. tha produced chronic elevation of glutamate in the medium and produced motor neuron toxicity after 25 to 30 days in culture using 100 pm tha, and after 18 days using 500 pm tha, as determined by assay of tissue choline acetyltransferase (chat) activity and by histological analysis of 1-micron plastic sections. motor neuron toxicity was completely blocked by the non-n-methybaspartate (nmda) antagonists cnqx or nbqx, but not by the nmda antagonist mk-801. this model demonstrates that the chronic loss of glutamate transport in als can produce motor neuron degeneration and that motor neurons appear to be susceptible to non-nmda-mediated glutamate toxicity. guillain last year, we described a distinct acute paralytic syndrome in children and young adults from northern china and differentiated it from guillain-barre syndrome (gbs) by epidemiological, clinical, and nerve conduction (nc) features. to distinguish chinese paralytic syndrome (cps) from gbs more clearly, we measured nc in 21 cps patients (mean 8 yr, range 1.5-35 yr) and in 21 gbs patients from johns hopkins (mean 14 yr, range 5-24 yr). sensory nc was normal in all (n = 62) but 1 nerve of cps patients, whereas sensory nc was frequently abnormal in gbs patients: median nerve, 63%; ulnar nerve, 87%; and sural nerve, 21%. motor n c also differed between the groups. in all nerves, distal latency (dl) was significantly longer in gbs than in cps. for example, in the median nerve, mean dl was 8.6 ms (se 1) in gbs and 2.8 ms (0.2) in cps ( p < 0.005). motor conduction velocity was significantly reduced in gbs median and ulnar nerves compared with cps nerves. f-wave latency was significantly longer in gbs median nerves than in cps nerves. these data support the distinction both clinically and electrodiagnostically between cps and north american gbs. the use of n c may be especially important in field epidemiological studies in separating the 2 disorders. clinical manifestations and gene analysis of the first japanese kindred yoshihide sunada, teruo shimizu, lchiro kanazawa, and toru mannen, tokyo, japan familial amyloidotic polyneuropathy type iv (fap iv) has been clustered in the finnish population and only a few cases have been reported from the netherlands. denmark, and united states. we describe the first japanese family with fap iv. the family originates from nagano prefecture, a mountainous district in the middle part of japan, and has no relationship to the finnish population. this family has 42 members in 3 generations, and 14 individuals are affected with slowly progressive cranial neuropathy and corneal lattice dystrophy. the genetic trait is autosomal-dominant. polarizing microscopy and immunohistochemistry show abundant amyloid deposits reactive to an anti-gelsolin monoclonal antibody. direct sequence analysis of a dna fragment spanning codon 187 of the plasmagelsolin cdna from the propositus, and restriction analysis using a modified pcr from other family members demonstrate a single base substitution, g to a at the first base of codon 187, which is identical to the mutation of finnish fap iv. this suggests that the mutation causes the fap iv phenotype regardless of ethnic background. gene expression focal puncture injury has been used as a model to study degenerative and regenerative responses of skeletal muscle. previous studies have demonstrated the ultrastructural and metabolic effects of muscle injury. however, the early genomic response to focal injury is presently unknown. we asked whether the immediate early genes (iegs) or early response genes-~$268, c-jun, nur77, and junb-are responsive to muscle injury. these iegs encode transcription factors and are expressed rapidly after cell-surface stimulation. we have previously shown that surgical denervation and neural stimulation of muscle induced differential patterns of ieg expression. in this study, we produced injury of mouse gastrocnemius muscle by injection of 20 c1.1 of normal saline. we used the contralateral (uninjected) muscle as a control and examined the milna levels of each of these 4 iegs. we found that ~$ 2 6 8 and junb levels were increased at 1 and 4 hours and returned to basal levels by 24 hours. in contrast, mrna levels of nur77 and cjun remained unchanged. this pattern of ieg response is distinct from that seen after muscle stimulation or denervation. the selectivity of this pattern suggests that ieg expression may play a role in the response of muscle to injury. amyotrophic lateral sclerosis (als) is a degenerative disease that leads to the restricted loss of motor neurons (mn). the reason for the selective death of mn remains unknown. we hypothesize that mn-enriched or mn-specific genes are important for normal m n function and that their disturbance may play a role in the pathogenesis of als. we have produced clonal hybrid cells derived from embryonic and neonatal spinal cord m n for the study of m n gene properties. some of these hybrid mn clones express traits typical of mn, such as high levels of choline acetyltransferase enzyme activity and message, glycine receptor message, and neurofilament and neural cell adhesion molecule proteins. we are using molecular techniques to identify novel mn-enriched or mn-specific genes in these cells. with this strategy, we have identified several cdna clones preferentially expressed in mn hybrid cells but not in the parental neuroblastoma cells by differential hybridization of an embryonic mn hybrid cdna phage library. we are extending these observations by performing subtraction hybridization experiments. these results suggest that mn-enriched or mn-specific genes can be identified, and may lead to a greater understanding of the etiology of als. there is a syndrome of slowly progressive, mid-adult-onset fasciculating progressive muscular atrophy (pma) affecting upper more than lower limbs, without bulbar or corticospinal signs, more often in males, associated with igm monoclonal gammopathy, and no nerve conduction block. two such men, ages (a) 59 years and (b) 64 years, duration 11 and 3 and one-half years, csf protein 28 and 60, had failed to achieve sustained improvement with: prednisone, cyclophosphamide, total-body irradiation, and multiple lymphoplasmaphereses in a; and interferon alpha 2a in b. intravenous immunoglobulin (ivig), 0.4 gm/kg/day, has provided dramatic benefit, sustained and increasing for >5 and >4 months to date. (there is a continuing base of depotestosterone, 200 mg weekly, which initially alone provided very minimal improvement.) strength increase was evident at 1 and 3 days after the first course of 5 daily ivig infusions. it further increased for 2 to 2 and one-half weeks after treatment, and then began to diminish. repeat 5-day treatment 3 weeks after the first course resulted in summated improvement, now sustained and enhanced by an average of 1 treatment per week. quantitated strength testing by a blinded observer has shown a 2-fold to >loo-fold gradually increasing muscle function in all limbs. patient a regained the ability to feed himself, get out of a chair, walk unaided, and go up steps; quantitated hip flexors increased 2-and 20-fold. patient b regained the ability to feed himself, take care of personal toilet needs, walk securely, and drive 500 miles; quantitated hip flexors increased 5-fold, and biceps flexions increased from 0 with no weight to > 130 reps while holding 10-pound weights. bukhara jews: a new cluster with typical oculopharyngeal muscular dystrophy (opmd) is a rare, late-onset myopathy with autosomal-dominant inheritance. its ultrastructural hallmark is the finding in muscle fibers of intranuclear tubular filaments of 8.5-nm outer diameter. most opmd cases were described among french canadians; in france, the homeland of their ancestors, the prevalence is 1/200,000 (brunet et al, 1990) . in israel's central area live approximately 40,000 jews who have immigrated from the bukhara and samarkand regions in uzbekistan. they represent a homogeneous ethnic group with its own language and community life. among them we have identified opmd in 15 families (55 affected individuals). the inheritance, clinical, electrophysiological, and histological features of these pa-tients are similar to those described in other pdts of the world, with typical intranuclear inclusions seen on electron microscopy. the minimal estimated prevalence of opmd in this population is approximately 1 : 7 5 0 . this cluster of opmd among bukhara jews is the second largest in the world. because many bukharian families are large, they may be suitable for linkage genetic studies. human muscle during ontogenesis e . scarpini, g. conti, p. l. baron, and g. myoblast transfer has been proposed recently as a possible therapy for duchenne muscular dystrophy patients. because immune rejection can represent a major problem in myoblast implantation, immunological characteristics of human muscle should be investigated. previous studies showed that human muscle cells cultured in vitro can constitutively express human lymphocyte antigen (hla) class i, but not hla class 11. furthermore, human y-interferon induces the surface expression of hla class i1 on mononuclear myoblasts, but not on multinucleated myotubes. however, whether the cells produce and present the antigen by themselves or take this material from the environment, where it could be released by infiltrative cells, is not yet clear. in this study, we analyzed hla molecules at the protein level by immunocytochemistry with monoclonal antibodies against different hla-dr epitopes and hla-abc molecules on frozen serial sections of human muscle during development and at the adult stage. human muscle infiltration by macrophages and monocytesmacrophages also were studied with m718and leum3specific monoclonal antibodies at the same stages of development. our results show that during muscle development and maturation, hla-dr and hla-abc antibodies do not label muscle fibers but some m718-and leum3-positive cells within the muscle. these data can be useful to understand the role of infiltrating monocytes-macrophages in the muscle immune response. mounting evidence suggests that excitotoxicity, mediated via the glutamate receptor, is involved in the pathogenesis of amyotrophic lateral sclerosis (als), as well as in other neurological diseases. we therefore initiated an open label, phasen i trial of highdose dextromethorphan (dm), a noncompetitive, selective n-methybaspartate antagonist, in als. patients began with 2 mg/kg/day, divided into 4 doses, and incrementally escalated their medication to 10 mg/kg/day or their maximum tolerable dose. thirteen patients, all extensive metabolizers of dm, were enrolled. total daily doses ranged from 4.75 to 10 mg/kg. major side effects were lightheadedness (8), slurred speech (7), and fatigue (6). no biochemical, hematological, or neuropsychiatric abnormalities occurred after up to 6 months of maximal therapy, except for depression in 1 patient. plasma kinetics of dextrorphan (dt) (the major metabolite of dm) were studied after an acute oral dose of 2.5 mg/kg dm. median elimination halflife was 2.5 hours. plasma dt concentration peaked at a median of 2 hours, with a median cmax of 14.4 pm. median amyotrophic lateral sclerosis cerebrospinal fluid/plasma dt ratio was 9.796. this study demonstrates the feasibility of long-term, high-dose dm therapy. we are now conducting a phase i1 study of highdose dm in als, designed to assess its efficacy. polyneuropathy: a chronic inflammatory demyelinating polyradiculoneuropathy variant? d. cros, k. h. chiappa, s. patel, and s. gominak, boston, ma, we describe 4 patients (3 men, 1 woman) with a pure, adultonset sensory neuropathy. the course was chronic in all cases. three patients had a relapsing-remitting course over 2 to 22 years with several attacks every year; the onset was gradual and followed by a plateau in the fourth patient. all patients had positive and negative sensory symptoms, and 2 had positive motor symptoms (fasciculations). in all patients, muscle power was normal at the time of peak deficit. all were areflexic and had large fiber sensory deficits, and 3 patients had sensory ataxia. three patients had elevated csf protein, whereas the csf was normal in 1 patient. mri demonstrated marked thickening of the lumbosacral spinal roots in 1 patient. motor conduction studies were normal in all patients, and mild f-response abnormalities were noted in 2. neurophysiological investigations of the sensory pathways were abnormal in all. three patients had several studies over a 2-year period. sensory nerve action potentials were unobtainable in 2 patients, and normal in the others. median and tibial somatosensory-evoked potentials showed conduction slowing consistent with demyelinating lesions affecting the peripheral sensory pathways, either globally or focally in the proximal segments. two patients appeared to respond to plasma exchange or intravenous immunoglobulin therapy, or both. glial fibrillary acidic protein cells in experimental motoneuron disease raul n . mandler, pam c. allgood, and james a. wallace, albuquerque, nm neuronal degeneration in human and animal motoneuron disease has been emphasized, but glial phenotype alterations have not been studied as extensively. we carried out a developmental and topographic study of astrocyte expression in the wobbler mouse model of motoneuron disease. wobbler mice and normal littermates were studied at 3, 4 , 5 , and 6 weeks of postnatal development. anesthetized animals were perfused intracardially with paraformaldehyde. spinal cords were dissected and landmarks were identified carefully for systematic study. sections were stained with monoclonal antibodies against glial fibrillary acidic protein (gfap) neurofilament and neuron-specific enolase. cell quantitation was done with video-enhancing microscopy. in symptomatic animals, marked increases in gfap staining were found in rostral and caudal spinal cord areas. quantitation studies revealed a 15to 20-fold increase in gfap+ cells in the wobbler. we conclude that gfap+ cells are markedly increased in the wobbler mouse at cervical, thoracic, and lumbar areas. this cell may also be relevant in motoneuron disease pathogenesis. ( indirect evidence suggests that polio virus may persist in the human cns years after initial infection and may be a cause for the post-polio syndrome. to evaluate whether the polio virus genome can be detected in the cns of patients with previous polio infection, we identified 11 patients who had died with autopsy findings and clinical history consistent with poliomyelitis. rna was extracted from paraffin-embedded sections of brain or spinal cord and subjected to reverse transcription followed by dna amplification by polymerase chain reaction (rt-pcr) using primers specific for heat shock protein 70 (hsp70) and a conserved region of the polio viruses. hsp70 mrna could be detected in all specimens, indicating that amplifiable rna had been isolated. in no specimens could polio virus rna be detected. this study suggests that polio virus does not persist in the human cns in quantities detectable by the sensitive pcr method. with cmt type 11, seen at mayo clinic rochester between 1981 and 1991, for the frequency of selective calf weakness in cmt type 11, the form of cmt most similar clinically to distal sma. anterior compartment weakness exceeded calf weakness in 49 patients (74%); anterior and posterior involvement was equal in 16 (24%). calfweakness exceeded anterior compartment weakness in 1 patient (2%). selective calf weakness in distal sma thus helps distinguish this disorder from cmt type 11, and similarly from distal sma with weakness resembling cmt, in that we are unaware of the 2 distributions in distal sma occurring in the same family. given the possibility of genetic heterogeneity, linkage studies of distal sma probably should include patient selection criteria such that the distribution of leg muscle weakness is homogeneous. p206. conjugal amyotrophic lateral sclerosis: amyotrophic lateral sclerosis (als) is a sporadic neurodegenerative disorder of unknown cause. unusual cases may provide etiologic clues. we report a married couple, both of clue to etiology? whom developed als in 1 year. the couple grew up in southeastern pennsylvania and attended the same schools. they married after high school and have 2 healthy children. in september 1990, a 38-year-old woman noted right-hand weakness and associated fasciculations that progressed to the entire right upper extremity. by january 1991, the lower extremities were asymmetrically weak and fasciculating. she then developed left-arm weakness, d ysarthria, dysphagia, and emotional incontinence. she had hyperreflexia and bilateral extensor plantar responses. then, in may 1991, her husband, aged 38, noted difficulty whistling, which progressed to frank dysarthria. later, he developed dysphagia, emotional incontinence, and weakness, wasting, and fasciculations in the upper extremities. hyperactive gag, jaw, and limb reflexes were present. in both, electrodiagnostic testing revealed widespread evidence of lower motor neuron degeneration. numerous laboratory tests were normal. although these cases may represent a chance association, the development of als in a young husband and wife suggests a possible environmental cause. the authors welcome suggestions about these cases from the neurological community. conduction block in demyelinating neuropathies usually is assessed from differences in the sizes of surface-recorded maximum m-potentials evoked by supramaximal stimulation at successively more proximal sites along the course of motor nerves. as the maximum m-potential is comprised of many bitriphasic surface-recorded motor unit action potentials (muaps), differences in the relative latencies between muaps may lead to phase cancellations, reducing the m-potential size and rendering any quantitative assessment of the extent of conduction block relative to phase cancellation difficult. cooling a muscle (not the nerve), however, by as much as 15â°c increases the negative peak durations of muaps by as much as 2 to 3 times and moves the point at which maximum phase cancellation might occur to some theoretical point well proximal to the spinal roots. in 5 cases of guillain-barre syndrome (gbs) studied to date, cooling produced little change in percent reductions in m-potential negative peak areas between successively more proximal sites of stimulation. this finding suggests that "true" conduction block rather than interpotential phase cancellation best explains reductions in m-potential size at successively more proximal sites of stimulation in gbs. associated with trimethoprim-sul famethoxazole rare cases of primarily motor polyneuropathy have been associated with the use of sulfonamides. the incidence of polyneuropathy has diminished substantially with the abandonment of earlier methylated compounds. we describe 2 patients who developed allergic phenomena, including a skin rash and debilitating, painful sensory and autonomic polyneuropathy within days of receiving trimethoprimsulfamethoxazole. in 1 patient, examination revealed resting tachycardia, marked blood pressure orthostasis and near-program and abstracts, american neurological association 281 syncope, hyporeffexia, urinary incontinence, and reduced sensation distally in the lower extremities. his cerebrospinal fluid was acellular with a protein of 141 mg/dl. the other patient showed a resting tachycardia, sluggish pupils, reduced distal vibration perception, and hyperpathia of hands and feet. conventional nerve conduction studies demonstrated normal motor results in both patients, absent or reduced sensory amplitudes in the first patient, and normal sensory results in the second. autonomic studies identified profound abnormalities in testing of sympathetic skin potentials, sinus arrhythmia, and valsalva's ratio. in both cases, nerve biopsy was not performed for fear of exacerbating the patient's hyperpathia. subsequent hemodynamic and electrophysiological testing showed improvement in autonomic function, paralleling the patients' clinical amelioration. although uncommon, a painful, sensory and autonomic, partially reversible polyneuropathy may develop after the use of trimethoprim-sulfamethoxazole. the remote effects of botulinum a toxin injections into vocalis muscles for treatment of focal laryngeal dystonia were investigated using single-fiber electromyography (sfemg). botulinum a toxin injections have been proven effective therapy for various dystonic disorders including focal laryngeal dystonia, blepharospasm, and torticollis. previous sfemg studies have demonstrated remote effects of the toxin in noninjected muscles after treatment for both blepharospasm and torticollis. these effects include an increase in fiber density, mean jitter (mcd), and percentage of fiber pairs with increased jitter. other researchers have postulated that the distant effects of this toxin may be related in part to the dose of botulinum toxin injected. to investigate this hypothesis we have studied patients treated for focal laryngeal dystonia because the amounts of toxin required are 1/25th to 1/1ooth of the doses used to treat other dystonias. using electromyographic (emg) guidance, bilateral injections of 2.5 or 5.0 mouse units of botulinum a toxin were injected into each vocalis muscle of 11 patients. each patient had significant improvement in phonotory function within 48 hours after injections and have been followed serially (usually within 3 weeks and again at 2 mo) after injections, with sfemg recordings of the left extensor digitorum communis and sternocleidomastoid muscles. five patients have had more than 1 series of injections over the 10 months since we began this study. sfemg studies have revealed no significant change in the fiber density, mean mcd, or percent of fiber pairs with normal jitter in either muscle. in conclusion, our studies support the hypothesis that the presence of remote effects of botulinum toxin may be related, in part, to the amount of toxin used. the c57bl/6/01a mouse exhibits the remarkable characteristic of prolonged survival of axons separated from their cell bodies (slow wallerian degeneration). previous work has demonstrated that the axon itself is responsible for the phenotype of prolonged survival. we investigated whether the lack of rapid axonal degeneration after axotomy in this substrain is due to an inability to break down cytoskeletal components, a process that is normally accomplished by activation of intrinsic calcium proteases. segments of desheathed sciatic nerves from normal and ola mice were incubated for 2 hours under conditions that disrupt the axolemma (freeze/ thaw or in 1 % triton x-loo), allowing external calcium free access to axoplasm. nerves were analyzed by western blot for neurofilament (nf) proteins and by electron microscopy. in high-calcium media (1 mm caci,), nf immunoreactivity was lost and axoplasm was reduced to watery debris in both substrains, whereas in egta-buffered media, axoplasm was preserved. these results demonstrate that calcium-activated proteases are present and can be activated in ola nerves. the defect in these mice that allows for prolonged survival of transected axons is likely in the mechanism for calcium entry into the distal stump. the mechanism by which the analogue of adrenocorticotrophic hormone, acta4-9, prevents cisplatinum (cp) neurotoxicity is unknown. murine n1e. 115 neuroblastoma cells and neural crest-derived, squirrel fish erythrophore cells tuesday, have similar vesicular transport mechanisms to human neural cells. they were used to study the effects of cp and acth4, on cellular transport. differentiated n 1e. 1 15 cells were treated 1 hour prior to observation with serum-free media (sfm, control); sfm/cp 5 pg/ml; or sfm/cp 5 pg/ml and 100 ng/ml acth4-,. organelle transport was studied (7 neurites and 100-140 organelles per condition) using computer-enhanced video microscopy. mean fast anterograde (1.00 k 0.07 pmsec-' vs 1.65 * 0.07 pmsec-') and retrograde (0.67 2 0.04 pmsec-' vs 1.19 +-0.05 pmsec-') transport were decreased in cp-treated compared to control cells ( p < 0.0001). in cp/acth4,-treated cells, mean anterograde (1.46 +_ 0.07 pmsec-') and retrograde (1.13 2 0.04 pmsec-') velocities were greater than in cp cells ( p < 0.0001). velocities in control and cp/acth4, cells were not statistically different. erythrophore pigment granule transport was observed in a blinded study, using similar techniques. mean aggregation velocity was greater in control (1.99 k 0.09 msec-') and cp/acth4, (1.97 f 0.07 msec-')-treated cells compared to cp (1.43 * 0.07 msec-') cells ( p < 0.0001). incubation with cp for 1 or 72 hours affected velocities equally, but acute exposure was more easily reversed by control or acth,, containing media. there is striking inhibition by cp in cross-species models of organelle transport. this can be prevented by acth4,. erythrophores allow future study of individual transport components. neurotrophin to investigate signal transduction pathways involved in neurite growth, the cytoplasmic regions of p7sngfr, the common neurotrophin receptor monomer, were searched for a motif analogous to the predicted secondary structure of the tetradecapeptide mastoparan. potential sequences were modeled using a semi-empirical molecular mechanical force field approach. the sequence rat ~7 5~~~ 367-379 represents a highly conserved amphiphilic domain predicted to be involved in neurotrophin signal transduction via g-protein mechanisms. to test this prediction, peptides containing sequences homologous to p7 5ngfr 367-3 79 were examined for effects on trophic factor-induced survival/differentiation responses of rat pc12 pheochromocytoma cells, chick embryo drg neurons, and chick embryo ciliary neurons. a peptide identical to ~7 5~~~ 367-379 accelerated the neurite growth response to nerve growth factor (ngf) of pc12 cells and drg neurons in a time frame that paralleled uptake into cells, but mastoparan did not influence ngf-mediated neurite growth. millimolar mg+ + and benzalkonium chloride, known to block the actions of mastoparan, blocked the effect of the peptide on ngf-mediated neurite growth by pc12 cells. peptides mutated to alter cationic amino acid relationships or amphiphilicity were less effective than the peptide in accelerating ngf-mediated neurite growth. these observations complement and extend evidence suggesting a pivotal role of ~7 5~~~ in ngf-mediated signal transduction. these studies complement and extend evidence suggesting a pivotal role of p7sngfr in neurotrophin signal transduction and evidence that activation of intracellular signalling processes involving specific g-protein mechanisms are involved in neurotrophin-mediated neurite growth. crushing the hypoglossal nerve causes hypoglossal motor neurons to decrease expression of choline acetyltransferase (chat) and begin expressing p75ngfr, the low-affinity ngf receptor. these changes are evident within 3 days after the injury and continue for several weeks. inhibition of axonal transport by vincristine applied to uninjured nerves causes loss of chat expression without induction of ~7 5~~" . we sought to determine if topical vincristine would alter p7sngf' expression after nerve injury. hypoglossal nerves were surgically exposed unilaterally in anesthetized rats and crushed. one week later, rats were reanesthetized and the same nerves were re-exposed. vincristine or saline was applied at the crush sites by soaking a strip of cotronoid wrapped around the nerves. one week later, rats were anesthetized and perfused with aldehydes. frozen sections from the brainstems were stained by indirect immunoperoxidase to demonstrate chat and ~7 5~~~' . saline-treated controls showed decreased chat and abundant ~7 5~~" in hypoglossal motor neurons ipsilateral to the crush injury. vincristine-treated animals showed no chat and no p7tngf'. we interpret these results as indicating that a signal originating from the injury site maintains ~7 5~~" expression after nerve injury. catecholamines have been reported to be toxic to embryonic-derived rat neurons and glia via the formation of reactive oxygen species (rosenberg, 1988) . we tried to determine whether oligodendrocytes (ol) from adult 6-month-old rat brain are similarly susceptible. toxicity to ol was examined using light microscopy and galactocerebroside immunohistochemistry where the relative number of surviving ol and their extent of process formation were graded. five days of exposure to norepinephrine (ne) and epinephrine (epi) at 10 and 100 fm produced significant toxicity ( p < 0.05, analysis of variance [anova)) to adult rat ol; this toxicity was evident by 24 hours of exposure. treatment with catalase (50 p,g/ml), a free-radical scavenger enzyme, completely prevented the toxicity of catecholamines. to ascertain whether astrocytes, which have free-radical scavenging capacity, could prevent the catecholamine-induced injury to ol, rat ol were seeded on neonatal rat astrocytes. under such conditions, the toxicity of n e and epi was reduced significantly ( p < 0.05, anova). these findings suggest that impairment of this protective function of astrocytes may render ol and its myelin membrane susceptible to free-radical-mediated damage. lyme neuroborreliosis is an increasingly prevalent disorder, but the diagnosis generally has been indirect. thus, the pres-ence of other manifestations of the infection, consistent findings on neurological exam or lumbar puncture, or presence of csf antibody have been used rather than direct isolation or identification of the organism from the csf. we have previously developed polymerase chain reaction with hybridization (pcr/h) to identify borrelia burgdorferi in the blood and organs of infected mice, and found that the assay was equivalent or, in some cases, preferable to culture (ann neurol 1991; 30:302) . the assay used for primers oligos derived from a sequence of genomic b. burgdovfri d n a expressed on a plasmid by rosa and schwan. a two-stage nested pcr was performed on csf samples in which the d n a was isolated in a variety of ways. pcr products were subsequently hybridized with a digoxigenin-labeled internal probe by slotblot hybridization. the sensitivity of the assay was excellent, being <o. 1 fg of purified b. burgdoorfevi dnaiml of csf, and 1 to 10 spirochetes per ml of csf when "spiked" csf was tested. the assay then was applied to a battery of csfs stored over the past 15 years in patients with definite, probable, and possible lyme neuroborreliosis. the lack of pcr/h positivity in some patients with definite and probable disease signifies that the spirochete does not reside in the lumbar csf in all patients with lyme neuroborreliosis, and may be concentrated in the meninges or parenchyma. progenitor cell line on transplantation into the adult murine brain t . kitagwcbi, d.-h. chui, and t . tabira, tokyo, japan several multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into primary culture cells of the septum of embryonic murine brains (the retroviral vector, kindly provided by d r c. l. cepko, boston, ma). two of those cell lines, when transplanted back into 6to 8-week-old mice by stereotaxic operation, integrated into the septum in a nontumorigenic manner. the implanted cells, which had been labeled with pkh26 dye, could be identified in animals up to at least 10 weeks after transplantation. immunocytochemical analysis demonstrated that the transplanted cells were seen assuming a round morphology and no processes. they did not stain with any glial or neuronal markers except for a2b5 and hnk-i, in the same way as in vitro. interestingly, after 10 weeks of implantation, the cells were located discretely in the transplanted site and displayed a small and round morphology with fine processes. most of the transplanted cells showed immunoreactivity with monoclonal antibodies directed against neuronal markers such as neurofilament and map2. these data indicate that the immortalized cell lines might be useful in characterizing factors that regulate cell type differentiation in the mammalian nervous system. (supported by a grant from the science and technology agency of japan.) related to serum response factor in human brain and muscle dana leifer, dimitri krainc, racbael neve, roger bveitbavt, and stuart a. lipton, boston, m a we have identified cdna clones for a novel transcription factor that is expressed at high levels in human fetal brain and skeletal muscle, but not in a variety of other tissues, as demonstrated by northern blotting. sequence analysis indicates that the clones appear to have 2 alternatively spliced forms and have extensive homology with human serum re-sponse factor (srf) and a family of related transcription factors that has representatives throughout eukaryotic evolution in species from yeast to man. srf is thought to have a role in regulation of immediate early genes and of muscle-specific genes. in gel mobility-shift assays, the srf-like proteins coded for by our clones bind specifically to the myocytespecific enhancer binding factor-2 (mef-2) regulatory element, a dna sequence that has so far been found to be functionally important in the promoter and enhancer regions of a variety of muscle-specific genes. moreover, our srf-like proteins specifically activate transcription of reporter genes containing the mef-2 element. given the abundant expression of our clones in human brain as well as in muscle, our results suggest that these proteins may have a significant role in development not only of muscle but also of brain. a host of quantitative eeg studies, most of which employed variations of the fast-fourier transform, have been made on isolated clinical entities such as the epilepsies, metabolic syndromes, and sleep with varying degrees of satisfactory correlations. using a time domain wave-by-wave computer program that simulated the logical approach of the clinical electroencephalographer, we have studied more than 300 unselected routine neurological clinic and ward patients. the presenting problems ranged from headache to major cerebral involvement. employing the dichotomy of normal and abnormal, the computer-read eegs agreed with the routine eeg, the clinical manifestations (including imaging techniques), and neuropathological findings in 90% of all cases. this high level of correlation, which approached that achieved by welltrained eegers reading the same records, was obtained by solely utilizing the background eeg activity. such results became possible only when the computer program was so organized that the "raw" computed and original oscillograph records were made to match closely. the diagnosis, with topographically diagramed localization, was printed automatically as the final step of the program. our data strongly suggest that routine computer reading of the human eeg is feasible, reliable, and potentially useful. protein accumulation in damaged neurites and microglial cells abnormal accumulation or abnormal processing of amyloid precursor protein (app), or both, may be critical to the development of p-amyloid protein (bap) deposits. however, alzheimer's disease (ad) has another pathological hallmark, which is the appearance of neurofibrillary tangles. the question is, therefore, to clarify the possible relationship between altered app metabolism and neurofibrillary degeneration. one classic method for inducing neurofibrillary tangles is the administration of aluminum salts. although these tangles are morphologically different than those seen in ad brain, this phenomenon has given rise to the hypothesis that aluminum might be an environmental factor in the causation of ad. this theory is controversial, but the animal aluminum model remains one of the few ways that long-lasting neuroskeletal changes can be induced. to explore the effects of aluminum salts on app metabolism, we examined app immunodistribution in rat brain injected intraventricularly or intrastriatally with aici,. we found a long-lasting accumulation of app in affected neurites, as well as in activated microglia/macrophages. abnormal neurites also showed argentophilic changes, neurofilament accumulation, and alz-50 immunoreactivity. the results support the hypothesis that interruption of axoplasmic flow by aluminum salts, as by other means, can lead to both app accumulation and neuroskeletal alterations. p223. nondemyelinating conduction slowing of myelinated fibers due to inactivation of n a channel takanori yokota, yukinobu saito, and tadashi miyatake, tokyo, japan in 4 patients with acute or chronic inflammatory demyelinating polyradiculoneuropathy, the marked improvement of motor-nerve conduction velocity without change of amplitude or configuration of the compound muscle action potentials (cmaps) was observed in 3 days. to investigate the mechanism of the rapid improvement of nerve conduction, the influence of na channel blockade on nerve conduction was studied. in 4 healthy volunteers, the recovery of the cmap and sensory nerve action potential (snap) of median nerve stimulation were recorded after the intravenous infusion of 100 mg of lidocaine. after the loading, cmap and snap were reduced rapidly in amplitude and were slowed in latency. after the recovery of amplitude of cmap and snap, the nerve conduction velocities were improved gradually in 2 hours with the amplitudes and configurations of cmap and snap unchanged. this indicated that myelinated nerve conduction velocity could be slowed by the inactivation of na channels without demyelination or conduction block. the prolongation of rising time in depolarization of axonal membrane potential should be one of the mechanisms of this conduction slowing due to inactivation of na channels. paraneoplastic cerebellar degeneration (pcd) in gynecological and breast cancers frequently is accompanied by an autoantibody response (type i or "anti-yo") reacting with 34and 62-kd cytoplasmic antigens of cerebellar purkinje cells. sakai et al, using a cerebellar library (stratagene), recently have isolated a cdna clone expressing a 52-kd protein recognized by igg from a patient with pcd and uterine carcinoma (sakai et al, ann neurol 1990; 28:692) . this clone is believed to share some homology with the message encoding the 62-kd protein recognized by type i sera. because pcd17 was isolated using serum from a single patient, however, it is not known whether the expressed protein of pcd17 is recognized uniformly by all patients with type i antibody response. e. coli strain xl1-blue transformed with pwr590-2 containing the snabiihindiii fragment of pcd17, pwrsbo-pcdl?sn, was induced using isopropylthiogalactoside. bacterial lysates were electrophoresed on 10% sds-page gels and analyzed by western immunoblot program and abstracts, american neurological association 285 methods using sera and csf from patients with pcd associated with gynecological and breast malignancies who exhibited type i antibody response. the 52-kd expression product was labeled by 818 sera and 414 csf samples tested from antibody-positive patients but was not labeled by sera or csfs from controls. our studies demonstrate that the 52-kd protein expressed by pcd17 contains epitopes that are consistently recognized by both systemically and intrathecally produced antibodies from patients with pcd accompanying gynecological and breast malignancies. tax01 is a novel antitumor drug that has demonstrated impressive clinical activity in breast, ovarian, and lung cancers. although sensory neuropath y has been described secondary to both taxol alone and taxol-cisplatin combination, detailed neurophysiological and pathological studies have not been described. nineteen patients with solid tumors were treated with combinations of taxol (135-350 mg/m2), cisplatin (75-100 mg/m2), and granulocyte colony-stimulating factor (g-csf) (5 pglkglday). sequential neurological examinations, nerve conduction studies (ncs), and quantitative vibration testing (qst) were performed during and after treatment. eighteen of 19 patients developed sensorimotor neuropathy and 3 developed myopathy. thirteen were symptomatic with numbness or weakness and 17 had abnormal results on neuromuscular examination. ncs showed absent or prolonged h-reflexes (14), reduced peroneal motor amplitude (14), reduced sural sensory amplitude (13), and myopathic motor unit potentials in proximal muscles (3). qst results were abnormal in 7 of 15 tested patients. muscle biopsy specimen in a patient with myopathy showed features of a toxic myopathy. the neuropathy was worse with higher cumulative dosages of taxol (>600 mg), with higher single doses of taxol(>300 mg/m2), or with a preexisting neuropathy. we conclude that sensorimotor neuropathy and myopathy are dose-limiting neurotoxicities of combined cisplatin and taxol use, now that neutropenia can be controlled with neuropathy and myopathy g-csf. central nervous system lymphoma casilda balmacedz and lisa deangelis, new york, ny ally periventricular and may seed the csf by direct growth through the ependyma. we reviewed the csf profile of 83 non-acquired immunodeficiency syndrome (aids) patients (pts) with pcnsl. all pts had lumbar puncture (lp) and 46 had multiple samples from an ommaya reservoir. definite lm involvement was identified with a positive csf cytology, lymphomatous lm infiltration on a surgical specimen, or mri with gadolinium showing lm tumor. probable lm lymphoma was diagnosed in pts with suspicious or atypical csf cytology. there were 41 women and 42 men with a median age of 57 (range 20-81 yr). at diagnosis, mean white blood cell count was 49/mm3 (range 0-5,5 10); mean lumbar csf protein was 103 mgldl (range 3-1,893); and mean ventricular csf protein was 32 mgldl (range 4-265). glucose was always normal. nineteen of 57 pts sampled had oligoclonal bands, and 42/65 had elevated pz microglobulin. at diagnosis 43 (52%) had an abnormal csf cytology: 22 positive, 16 suspicious, and 5 atypical. one pt had pathological infiltration of the lm and 1 had lm tumor on spine mri for a total of 45 (54%) pts with definite or probable lm lymphoma. in 43 pts with abnormal cytology, the abnormality was found in 30143 (70%) lps and 32/55 (58%) of the ommaya and ventricular specimens. in 8 pts the lumbar cytology was the only abnormal specimen despite multiple ventricular samples, and in 10 only the ventricular csf was abnormal. thirty-six of 83 (43%) pts developed recurrent tumor afrer treatment. forty-two percent (15136) of all patients with relapse had lm recurrence. lm recurrence was accompanied by brain recurrence in 9 pts, systemic in 1, ocular in 1, both systemic and ocular in 1, and isolated in 3. at diagnosis 59/83 patients received treatment directed against the lm. of these, 8 (14%) had meningeal recurrence whereas 7 of the 24 (29%) patients who did not receive this treatment had lm recurrence. lm involvement by pcnsl is frequent, may be missed on a single csf sample, and requires specific therapy at diagnosis. sixty-three solid cancer patients with a single brain metastasis were prospectively randomized for neurosurgery and radiotherapy combined (arm 1) or radiotherapy alone (arm 2). they were stratified for lung or nonlung cancer and for active versus stable or absent extracranial disease. world health organization performance status was 9. age, sex, performance status, and location of brain metastasis were divided evenly over both groups. one-month mortality was 7% in arm 1 and 6% in arm 2. median survival of 11 months after combination therapy was significantly better compared to 6 months after irradiation alone ( p < .04). it made no difference whether they had lung or nonlung cancer. the largest difference between both treatment arms was observed in patients with stable or absent extracranial disease (12 vs 7 mo, p < .02). when systemic disease activity was present, median survival was 5 months irrespective of treatment arm. functional independent survival was 1 to 2 months shorter than overall survival and was significantly better for patients with stable extracranial disease after combined therapy. multivariate analysis showed that age was also an independent prognostic factor. patients older than 60 years had a hazard ratio for dying of 2.8 (p. 004). we will detail the type and pattern of neurological complications in t-cell non-hodgkin's lymphoma (nhl), and review how they differ from those associated with b-cell nhl, and the lymphomas in general. this study is the first step in a process to characterize these tumors to determine if special staging or cns prophylaxis are indicated in any of the subtypes of t-cell lymphoma. we recently have encountered 5 women with breast cancer and an unusual sensorimotor neuropathy. the neuropathy was the major clinical problem. in 4 women the initial symptom was severe itching, 3 generalized and 1 first localized to the involved breast and then generalized. all developed distal extremity numbness and burning that very slowly progressed proximally, and in 2 became generalized. four complained of painful muscle cramps in the extremities ( 4 ) and jaw (1). all had mild extremity weakness, distal (5) and proximal ( 1 ) . three women developed symptoms up to 21 months prior to cancer diagnosis, 1 shortly after diagnosis, and 1 5 years after diagnosis. four women had disease confined to the breast and regional lymph nodes, and 1 had metastatic disease in remission. although annoying, symptoms were generally not disabling. three women stabilized or had slight improve-associated with breast cancer ment with cancer treatment, and 2 continue to gradually progress while in cancer remission; 1 required a cane to ambulate after 9 years due to sensory ataxia. one who developed cancer relapse had concurrent neurological relapse. one woman treated with high-dose immunoglobulin did not improve. none had significant weight loss. laboratory abnormalities included elevated erythrocyte sedimentation rate (28-60) in 3, antinuclear antibody 1:40 in 1, csf with lymphocytic pleocytosis (7-14 white blood cellslmm) in 3/ 4 and elevated protein (5 1-97 mg/dl) in 414 available. emgi ncv showed mild sensory-to-motor polyneuropathy in 314 available. none had detectable antibodies against peripheral nerve or dorsal root ganglia. the etiology of sensorimotor neuropathy in these patients is unknown, but it may represent a distinct paraneoplastic syndrome that can herald the onset of, and parallel the course of breast cancer. our objective was to determine whether ceramide induces differentiation of anaplastic glioma cells. sphingomyelin hydrolysis resulting in ceramide production has been linked to differentiation of leukemia cells. t9 rat anaplastic glioma cells, seeded at 2 x lo4 cells per well, were grown in serum-a glioma cell line program and abstracts, american neurological association 287 free media. on day 4, cells were photographed, scored for processes longer than one cell body, and counted. c2 ceramide changed plump cells to flattened cells with many long processes: ceramide treatment increased the percentage of cells with processes from 22 2 4% (sd) to 49 * 12% (n = 4, p < 0.01). control cells grew to 6.3 x lo5 cells/well 2 0.6 x lo5 cells; ceramide-treated cells grew to 1.6 x lo5 ? 0.6 x los (n = 4, p < 0.0005). although one ceramide analog reproduced the c2 ceramide-associated changes, the optical isomer of this analog did not, demonstrating stereospecificity. cerarnide did not decrease cell viability by trypan blue. ceramide inhibits proliferation and induces process formation in a glioma cell line, causing it to assume a more differentiated phenotype. cerarnide or its analogs represent possible future therapeutic agents that would inhibit the growth and affect differentiation of anaplastic gliomas. [bashir, mod pathol 1990; 3(4) :429-434)). this is similar to the pattern seen in ebv-infected human b cells and unlike the uniform latent infection seen in burkitt's lymphoma. we tested the hypothesis that long-term passaging of ebv-immortalized human b cells in immunodeficient mice leads to emergence of a uniform nonlytic pattern of ebv infection associated with appearance of the malignant profile. ebv-infected normal human b cells were serially passaged intracerebrally in severe combined immunodeficient cb17 mice (5 x 10' cells per mouse, 5 mice per passage for a total of 10 passages). frozen mouse brain sections from each passage were stained with vca antibody (ebv lytic cycle) and hybridized with biotinylated bamh1-w sequence of ebv. all injected animals developed tumors as previously described (bashir, lab invest 1991; 65(6):702-709) . tumor cells continued to express vca and showed latent and lytic hybridization patterns with bamh,-w after 10 passages despite exhibiting monoclonality (surface immunoglobulins) and random chromosomal changes. lytic infection of immortalized b cells with ebv is stable, resembling brain lymphomas in aids, and unlike the latent infection seen in burkitt's lymphoma. a previously healthy 73-year-old man developed diplopia and incapacitating, diffuse weakness over a period of 6 weeks. examination showed a "one-and-a-half'' syndrome of horizontal gaze paresis, patchy severe weakness with atrophy and fasciculations, absent tendon reflexes in the legs and right biceps, and decreased vibration sense in the feet. csf contained a mild pleocytosis, elevated protein, and oligoclonal igg bands. electrophysiological testing indicated a generalized sensorimotor axonal neuropathy with diffuse denervation. small-cell lung carcinoma was diagnosed by bronchoscopy. prednisone produced mild subjective improvement. chemotherapy was begun but the patient developed fatal septicemia. serum was negative for anti-hu or anti-gm1 with paraneoplastic encephalomyeloneuritis antibodies. serum and csf contained high titers of igg antibodies reacting specifically with a protein antigen of approximately 130 kd in immunoblots of human cerebral cortical neuronal nuclei or of human purkinje cells. this pattern of autoantibody reactivity was not present in sera from any of 37 other patients with small-cell lung carcinoma, 17 of whom had paraneoplastic encephalomyeloneuritis and anti-hu antibodies, nor was it present in many patients with other neurological disorders. the patient's serum has been used to probe a human cerebellum expression library and to isolate a cdna clone that is being characterized. acute encephalopathy is the problem in 17% of neurology consultations reported at mskcc. we studied 94 patients (5 1 prospectively and 43 retrospectively) to determine clinical findings, causes, and outcome. fifty-five were women and 39 were men, and the average age was 63 years. all patients had cancer: lung (20%), gastrointestinal tract (19%), breast (12%), and others (49%), forty-two patients (45%) were delirious on admission and delirium developed an average of 12 days later in 55%. encephalopathy occurred postoperatively in 24%. symptoms included confusion (92%), lethargy (59%), agitation (5 1 %), hallucinations (29%), and seizures (10%). signs included deficits in attention (44%), memory (385%), language (15%), lateralizing signs (50%), and asterixis (33%). the average mini-mental status test (mms) score was 12 (30 = normal). a single cause for delirium was found in only 4% of patients with an average of 4 etiologies per patient. metabolic abnormalities were found in 89% of patients, and were a primary cause in 44%; disseminated intravascular coagulation contributed to delirium in 11%. cns metastases were found in 32% and were a major cause of delirium in all. fifty percent of the patients had fever/ systemic infection, but sepsis was present in only 4%; only 1 patient had cns infection. medication contributed to delirium in 97% but was a primary cause in only 29%. the 30-day mortality rate was 3 1 % and delirium improved in 68% (average mms = 23). patients with cancer have multiple, potentially treatable causes of delirium. delirium is associated with a high death rate, though patients generally improve. radiation therapy for brain tumors d. w. dodick, b. mokri, k. k. unni, g. m. miller, and e. g. shaw, rochester, m n osteosarcoma in a previously normal bone is a rare but recognized remote effect of radiation therapy. any bone in the field of radiation can be affected. involvement of cranial bones is exceedingly rare. we could identify only 4 patients (2 men and 2 women) with postirradiation osteosarcoma of the calvarium seen at the mayo clinic over a 50-year period, from 1931 to 1991. all had received radiation for brain tumor, osteosarcoma had appeared in the field of radiation in all, the interval from radiation therapy to the appearance of sarcoma ranged from 7 to 23 years, and diagnosis of sarcoma was confirmed histologically in all cases. the patients' age at the diagnosis of the brain tumor ranged from 18 to 41 years. the nature of the brain tumor was unverified in 2 cases, was a low-grade ependymoma in the third case, and a pilocystic astrocytoma in the fourth case. one patient is still alive 12 months after the diagnosis of the sarcoma. she received chemotherapy and subsequently underwent resection of the osteosarcoma. one patient died postoperatively after partial resection of the sarcoma. the other 2 patients died 7 months and 15 months after the diagnosis of the osteosarcoma despite additional radiation therapy in the former and aggressive chemotherapy in the latter. element-la2 fusion gene as a potential marker of neural tumor differentiation lawrence recht, chiffon wu, and louis j. degennam, worcester, ma inducing cancers to differentiate into more benign differentiated tumors represents a novel oncological strategy. to establish a model that would permit assessment of this phenomenon at a molecular level, we created and have partially characterized a murine neuroblastoma line that has been stably transfected with a synthetic fusion gene containing the promoter element of the rodent synapsin i gene (synapsin i regulatory element isre)). in vivo, this promoter directs the neuron-specific expression of the synapsin i gene in normal adults. the gene also is expressed in varying amounts in neuronal tumors including neuroblastoma. in the synthetic fusion gene, the sre has been linked to the lacz gene that encodes bacterial p-galactosidase. a simple histochemical assay for p-galactosidase therefore provides a specific marker of the expression of the fusion gene. our preliminary experiments as of this writing have shown that it is possible to detect p-galactosidase activity in the transfected neuroblastoma cells both in vitro and in transplanted tumors. it appears possible therefore that this transfected neuroblastoma cell line can provide a useful model system with which to assess the effects of differentiation therapies. larger lesions (>18 cm2), and larger midline shifts (>4 mm). twenty-eight of 36 (78%) patients with prior has had bt has compared to 25 of 75 (33%) patients without prior has. in 8 patients, their bt h a was similar to their previous ha but was more frequent or severe. we conclude that has in bt patients are common but usually not severe. nausea, vomiting, an abnormal neurological examination, or a change in prior headaches warrant further investigation. cairncross and macdonald showed that procarbazine, lomustine, and vincristine (pcv) are effective for recurrent anaplastic oligodendrogliomas (ao) (ann neurol 1988; 23:360-364) . pcv now has a major role in management of all forms of oligodendrogliomas (0), but the biological basis for this response is unknown. to evaluate one subset of possibilities, we studied 14 patients (6 ao, 8 0) with a ct method that permits measurement of blood-to-tissue transport (kj, tumor-to-blood transport (k2), and vascular volume (v,) (ann neurol 1991; 30:581-587) . pcv was used to treat 11 of the 14 patients. k, (~1 grr-' min-') values were highly variable for whole tumor, ranging from 1.49 to 14.37 (mean 4.87 k 4.17) with no difference between a 0 and 0. k2 and v, were also highly variable. k, of tumor-free brain was 1.40 to 2.69 (mean 1.89 ? 0.50). in comparison to malignant astrocytomas, which have a mean k, in the range of 21.0 with some as high as 33.9, a 0 and 0 appear to be much less permeable. this suggests that the efficacy of pcv may be due to factors other than capillary transport, such as tumor-cell sensitivity. frameless stereotactic localizer gene h. barnett, donald w. komos, and charles p . steiner, cleveland, oh extent of tumor resection has been shown to correlate with prognosis in malignant gliomas. although frame-based stereotactic techniques can provide information regarding tumor margin, they are often unwieldy and require expensive and elaborate computing systems. a frameless stereotactic neurosurgical localizing system was designed that overcomes these liabilities. this armless, frameless, stereotactic pointing device provides real-time three-dimensional localization information during operation. in addition to assisting in placement of a trephine craniotomy, it allows volumetric resection of the tumor with virtually complete excision of even large irregularly shaped tumors. mean error on localizing a point in space using this system has proven to be less than 2 mm. a technical description of the system as well as surgical results are presented. to investigate the effect of age on response rate to chemotherapy and time to progression (ttp) and death ('itd) in patients with recurrent astrocytomas and malignant astrocytomas, we reviewed case records and scans of 143 patients who received chemotherapy at the university of michigan with bischloroethylnitrosourea or procarbazine. three age groups were studied: (1) <39 yr (n = 64); (2) 40-60 yr (n = 56); (3) >60 yr (n = 23). tumors were grouped as grade 2r+ 3 (recurrent grade 2 plus grade 3, n = 72) or grade 4 (n = 7 1). serial computed tomographic or magnetic resonance scans were analyzed in a blinded fashion and graded as progressive disease (pd), stable disease (sd), or partial response (pr, >25% decrease in size). the pr rates for the 3 age groups were 58%/32%/0% for grade 2r+ 3 tumors ( p = 0.023) and 40%/10%/5% for grade 4 tumors ( p = 0.011). median 'itp was 3112316 weeks for grade 2 r + 3 and 221 16/10 weeks for grade 4. median ltd was 53/48/19 weeks for grade 2r+ 3 and 36/31/24 weeks for grade 4. we conclude that age is an important prognostic factor with respect to likelihood of response to chemotherapy, duration of response, and survival irrespective of grade. cheryl p. harris and kurt a. jaeckle, salt lake city, ut intravascular malignant lymphomatosis (iml), a b-cell lymphoma confined to small venules and capillaries, often presents with neurological symptoms. this disease is uniformly fatal (5-month mean survival); no successful treatment has been identified. we observed marked reproducible neurological improvement after plasmapheresis in a 43-year-old woman with iml. presenting with a cauda equina syndrome, she progressed over 1 year with neurological, hepatic, and hematological disease. persistent laboratory abnormalities included a high sedimentation rate (140 mm/hr), coagulopathy, hemolytic anemia, and elevated liver enzymes. extensive evaluations for infectious, autoimmune, and neoplastic processes, including bone marrow examination, were inconclusive. because of neurological progression, empiric therapy with high-dose steroids followed by cyclophosphamide was initiated without response. plasmapheresis (250 ml/kg in 6 exchanges) effected resolution of encephalopathy and normalization of the coagulopathy and sedimentation rate. neurological progression recurred within 2 weeks of pheresis; 6 repetitive courses reproduced neurological response. finally, progressive dementia ensued, and a decision was made to cease pheresis; the patient died 5 days later, 16 months after presentation. autopsy disclosed diffuse intravascular cd-20 positive malignant lymphoma cells in small vessels of all organs. although the mechanism is unknown, the serendipitous discovery of response to plasmapheresis in this patient warrants further consideration. morphine is an effective analgesic in the rat after injection into a number of discrete brainstem regions, including the periaqueductal gray (pag), the locus coeruleus (lc), and the nucleus raphe magnus (nrm). early work with morphine gray and the nucleus raphe magnus established the existence of synergy between the brainstem and the spinal cord in rats. more recently, studies from our laboratory revealed synergy between two brainstem structures, the pag and the lc. in the current study, we explored the analgesic interactions between the pag and the nrm using indwelling cannulae. first, we established morphine dose-response curves and calculated the ed,, independently in the pag (1.9 pg) and the nrm (2.5 pg). we then simultaneously injected various morphine doses into both regions. injecting morphine at 1 pg into either the pag or the nrm did not elevate tailflick latencies above baseline values. however, administered into both regions simultaneously, the 2 1-pg doses produced an 80% maximal response, corresponding to more than a threefold increase of baseline latencies. a fixed morphine dose of 1 pg in the pag shifted the morphine dose-response curve fivefold in the nrm (ed,, 0.5 pg), whereas a fixed nrm dose of 1 fg shifted morphine's dose-response in the pag approximately twofold. together, these results clearly show synergistic interactions for morphine between the pag and the nrm. the presence of synergistic interactions between brainstem nuclei as well as between the brainstem and the spinal cord underscores the complexity of opioid analgesic systems. p244. the glutamate uptake inhibitor ~-trans-2,4-pyrrolidine dicarboxylate is neurotoxic in neonatal rat brain john d. e. barks and faye s. siloerstein, ann arbor, m i important evidence of the neurotoxicity of endogenous glutamate (glu) in mammalian brain was provided by the observation that dl-threo-3-hydroxyaspartate, a high-affinity glutamate uptake (hagu) inhibitor, was neurotoxic in adult rodent striatum (j neurochem 1985; 44:247) ; however, the absence of neurotoxicity in neonatal brain was interpreted as evidence that immaturity of glutamatergic innervation limited the potential role of endogenous glu as a neurotoxin in the immature brain. yet, considerable data provide indirect support for the hypothesis that glu can be neurotoxic at this stage. to resolve this issue, we assessed the neurotoxicity of a novel, selective hagu inhibitor, ~-trans-2,4-pyrrolidine dicarboxylate (l-pdc) (j med chem 1991;34:717), in postnatal day (pnd) 7 rats (n = 8). l-pdc (ph 7.4) was stereotaxically injected into right anterior striatum (str) (568 nrnol, n = 2) or through dorsal hippocampus into posterior str (568 nmol, n = 4; 150 nmol, n = 2). animals were killed 5 days later, and neuropathology was assessed in cresyl violet-stained sections. after anterior injections, focal neuronal necrosis was evident in dorsal str; high-dose posterior injections caused prominent hippocampal lesions with pyramidal layer thinning and focal necrosis in dorsal thalamus, while 150 nmol produced small foci of pyramidal cell loss. in both groups, focal cortical necrosis and callosal cysts were apparent adjacent to the injection track. l-pdc-induced brain injury provides direct support for the hypothesis that endogenous glu may be neurotoxic in the developing brain. (sax et al, ann neurol 1991; 30:311a) . the signs and symptoms of 7 of these patients lessened for 12 to 22 months. furthermore, a patient with a severe oral-lingual biting dyskinesia improved when taking doses up to 300 milligrams per day for 4 months. we noted no-significant adverse reactions, although 4 patients had mild but labile elevations in sgop and sgpt. one patient receiving opioid analgesics for pain inadvertently failed to discontinue the naltrexone but noted no reduction in the pain-alleviating effects of the analgesic. although hyperkinesia, especially of midline functions, as well as quality of life improved for the hd patients, their cognitive deficits remained unaffected. these observations suggest that chronic naltrexone is a safe and effective agent to treat chorea, dysphagia, and oral dyskinesia in hd for periods longer than a year. furthermore, they indicate that naltrexone can be effective in ameliorating oral-lingual biting tardive dyskinesia. these findings support our previous hypothesis that endogenous opioids play a role in rhe modulation of the dopamine system in hyperkinetic stereotypic movement disorders. a. jon stoessl, elizabeth szczutkmuski, and hanna fydvszak, london, ontario, canada we previously have demonstrated (psychopharmacology 1989; 98:372-379 ) that intraperitoneally (ip) administered cholecystokinin (cck)-8s suppresses vacuous chewing mouth movements (vcms, a putative mode1 of tardive dyskinesia) in rats exposed to chronic neuroleptics. as cck is not thought to cross the blood-brain barrier in significant amounts, its site of action in this paradigm is unclear. other behavioral and neurochemical effects of ip cck are blocked by vagotomy. male sprague-dawley rats were administered fluphenazine decanoate (flu; 25 mg/kg im) or its vehicle every 3 weeks for approximately 20 weeks. cck (10, 20, or 50 ng intracerebroventricularly) had no effect on neuroleptic-induced vcms. another group of neuroleptic-treated rats was subjected to bilateral subdiaphragmatic vagotomy or a sham procedure. cck-8s (10, 30, or 50 pg/kg intraperitoneally) suppressed neuroleptic-induced vcms in shamoperated animals, which confirmed our previous results. in vagotomited animals, chronic flu failed to induce vcms and cck was without effect in vehicle-or neuroleptictreated animals. these data suggest that the effects of cck on flu-induced vcms may be mediated peripherally, and that vagal pathways may be important for generating this response. (supported by the ontario mental health foundation and the ontario ministry of health.) p247. excitotoxic amino acids are not involved in dopaminergic neurotoxicity of m p t p eldad mehmed, jutta rosenthal, and avinoam reches, petah tiqva, tel aviv, and jerusalem, israel the dopaminergic (da) neurotoxicity of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp) is mediated via its oxidation in cns to mpp + , which enters da neurons and poisons mitochondrial complex i. da neuronal damage induced by direct nigral mpp+ injection is prevented by pretreatment with n-methyl-d-aspartate receptor antagonists, which suggests that excitatory amino acids are involved in mptp toxicity. since local mpp + application may produce nonselective nigral damage, we examined whether excitotoxins have a role in toxicity of systemically administered mptp. c57 black mice were injected intraperitoneally, once, with mptp.hci(40 mg/kg) and decapitated 5 days later. groups of animals underwent the following pretreatments: (i) decortication 1 week prior to mftp; (2) intracerebroventricular injections of the excitatory amino acid receptor antagonists 2-amino-phosphonoheptanoate and d-glutamyl-glycine; and (3) intraperitoneal injections of the calcium channel antagonists nimodipine, diltiazem, and flunarizine 30 minutes prior to mptp. mptp produced marked striatal da depletions. decortication, destroying glutamatergic corticostriatal projections, intracerebral amino acid receptor antagonists, and systemic calcium channel antagonists did not protect mice against mptp toxicity; mptp-induced striatal da decreases were similar to those given the neurotoxin alone. this study suggests that excitotoxins are not involved in the mechanism of mptp toxicity. scopolamine-induced cognitive deficits k . j . meador, m . e. allen, p. franke, e. e. moore, and d. w. loring, augusta, ga a unique neurophysiological role for thiamine in cholinergic systems has been suggested. total thiamine content in cholinergic nerve terminals is comparable to that of acetylcholine, and the phosphorylation state of thiamine changes with release of acetylcholine. thiamine binds to nicotinic receptors and may exhibit anticholinesterase activity. based on these observations, we investigated the effects of pharmacological doses of thiamine on the cognitive deficits induced by the anticholinergic scopolamine in 13 healthy young adults using a randomized, double-blind, placebo-conrrolled, double crossover design. cognitive tests included the p3 eventrelated potential and free recall memory for a verbal paragraph. conditions included baseline (bl), thiamine 5 gm by mouth and scopolamine .007 mg/kg intramuscularly (b1 + scop), and lactose by mouth and scopolamine (plac + scop). testing was performed 3 hours post thiamine or placebo, and 1.5 hours post scopolamine. thiamine significantly reduced the adverse effects of scopolamine on p3 latency (f [i, 121 = 11.84, p 5 .005) and percent recall memory ( f el, 12) = 10.62, p 5 .007). means (+sd) and p3 latency (ms) were bl = 335 (24), b1 + scop = 360 (29), and plac neurol 1990; 27: 186-192) . we previously reported that cff improved in 11 of 17 ms patients (65%) given el-970 orally in divided daily doses ranging from 7.5 to 52.5 mg (stefoski et al, neurology 1991; 41:1344 -1348 . subsequent analysis revealed that in 14 of the 17 patients cff improvements and reversals followed in a phase-locked trend the rising and falling serum concentrations of el-970, including 3 patients whose changes remained below the 15% increase needed to qualify for the improved category. these results resemble the phase-locked effects of temperature on neurological function in ms. the cff changes, because they so closely reflect variations in serum concentration, suggest that el-970 tissue levels closely follow those in serum and that el-970 rapidly crosses the blood-brain barrier. efficacy of el-970 in ms is also predicted to have a close relationship to serum levels. p250. development of a n internal standard detectable by proton and phosphorus-3 1 nmr and hplc w. e. klunk, k. panchalingam, r. j . mcclure, and j . w. pettegrnu, pittsburgh, pa comparison of quantitative results from different analytical techniques can prove difficult due to the peculiarities of the particular techniques and the lack of a common standard applicable to all of the techniques. recently, both in vivo and in vitro nuclear magnetic resonance (nmr) have been applied to the quantification of a large variety of metabolites. although nmr can be applied to the study of living tissue, the question arises of how this technique compares with more traditional techniques such as high-pressure liquid chromatography (hplc). although this question can be addressed partly by studying perchloric acid (pca) extracts, it is difficult to directly compare this in vitro nmr data with results from hplc. to address this question, we have developed an internal standard that can be quantified directly by in vitro phosphorus-3 1 nmr, proton nmr, and by 9-fluorenylmethyl chloroformate (fmoc) derivatization followed by separation by hplc. a variety of aminophosphonic acids were studied by 3'p nmr, 'h nmr, and hplc. promising compounds were added to pca extracts of human brain. the optimal compound was found to be 3-aminopropylphosphonic acid (app, ho3p-ch2-ch,-ch2-nh2). app is easily detectable by all 3 techniques, falls well outside of the region of interest in phosphorus-31 nmr, and is well resolved by proton nmr at 500 mhz. it is easily separable from the phosphomonoesters and phosphodiesters observable by hplc and from the amino acids that occur in brain in significant concentrations. app appears to be a useful internal standard in the study of phosphorus and amino acid metabolites by in vitro 31p nmr, 'h nmr, and hplc. theories of sentence comprehension hypothesize at least a grammatical component that establishes the relationship among words in a sentence, and a semantic component that determines the meanings of these words. we used positron emission tomography (pet) to quantify regional cerebral blood flow (rcbf) in 12 neurologically intact subjects during their detection of a letter target, a grammatical target, or a semantic target in the same written sentences. a mixedmodel analysis of variance (anova) revealed significant main effects for region (f e30, 210) = 19.32; p < .001), condition (f 12, 143 = 3 . 9 6 ;~ < .05), and a significant region times condition interaction ( f {go, 4203 = 1.46; p < .05), but there were no differences between individual subjects. subsequent anovas revealed increased rcbf in a unique set of brain regions during the subjects' response to a grammatical probe when compared to their response to a letter probe of the same sentences. a unique distribution of rcbf also distinguished response to the semantic probe from response to the grammatical probe and the letter probe. other brain regions apparently contributed to performance for several activation conditions. these findings support the hypothesized dissociation of specific linguistic components based on their unique cerebral topographical representation, and that a distributed network of brain regions subserves sentence comprehension. cerebral music processing-a comparison study of musically trained and naive individuals louis s. russo, jr, jachonville, fl we performed topographical mapping of brain electrical activity in 6 right-handed symphony musicians and 5 righthanded, musically naive individuals during various musical tasks; namely, listening to solo piano music, silent singing of familiar music, and silent reading of unfamiliar music. fast-fourier transform ( f r ) of electrocortical activity was carried out during task performance and the eyes-open resting state. data were analyzed using a computer-assisted model. increases in regional beta activity of greater than 3 standard deviations from the resting state were considered significant of activation. during audition, the musicians showed activation in the right posterior parietotemporal region; the naive showed no change from the resting state. during silent singing, the musicians showed bitemporal activation, r > l; the naive showed activation in the right mid-and posteriortemporal regions alone. during silent sight reading, the musicians showed a major activation in both temporal regions, l > > r, the naive showed only a marginal change in the posterior temporal-occipital regions. these data suggest that music processing is primarily a right cerebral function in untrained individuals and a bilateral function in musicians. musicians, in contrast to the naive, show progressively more left brain activation as task complexity increases. the present study analyzes language profiles in 10 patients who presented with primary progressive aphasia (ppa) without global dementia for at least 2 years. language and cognitive impairment were evaluated using the western aphasia battery (wab) and the mattis dementia rating scale (drs). expressive language disability with reduced speech fluency and anomia, but preserved language comprehension and nonverbal cognition, were typical features in early stages. spontaneous speech was significantly more impaired in ppa than in anomic aphasia after left-hemisphere stroke and in language impairment in probable alzheimer's disease (ad) ( p = 0.0004). the profile of aphasia suggests that ppa tends to affect anterior parts of the language-dominant cortex first. neuroimaging generally showed mild to moderate brain atrophy. in 2 patients atrophy involved especially the left frontal progressive aphasia cortex. follow-up examinations that were done in 7 patients 1 or several years after the first assessment revealed continuous, most often rapid deterioration of language impairment. two patients died 3 and 4 years after the onset of ppa. neuropathological examination showed ad in 1 patient and pick's disease in the other patient. beverly clarke, adrian upton, markad kamath, and helene griff;., hamilton, ontario, canada eight patients implanted with a cyberonics neurocybernetic prosthesis model 100 to stimulate the vagus nerve were assessed for changes in cognitive performance. the patients had complex partial seizures for more than 10 years, with more than 6 per month. patients were 34 years * 7.8 sd old. cognitive evaluation included response time to a randomized light signal appearing on a switch box (test a); test b, in which the signal appeared bilaterally; and test c, in which a response to the signal was required while the patient simultaneously ignored a second signal. data were collected and analyzed using an apple i1 e computer and switch pad. all patients were taking therapeutic levels of 3 anticonvulsant medications and dosages were constant. testing occurred 10 times during a day preoperatively (day l), 2 weeks postoperatively with the stimulator on (day 2), and 3 months after turn-on (day 3). patients were randomized into high-and low-frequency stimulation groups (hfg and lfg). hfg parameters were 30 hz, so0 msec pulse width (pw), and lfg 1 hz, 130 msec pw. examiners were blinded as to group. student's t-test analyses of mean differences between groups and individual measurements showed a significant difference between hfg and lfg for test c ( p < 0.05). lfg showed a significant improvement for tests a, b, c between day 1 and 2, and for test c between day 2 and 3. no group effect was seen between day 1 and 3 in the lfg. individual measurements showed improvement for test b ( p < 0.05) for the hfg between day 1 and 2, test b ( p < 0.0s) between day 2 and 3, and tests a and b ( p < 0.01) and ( p < 0.05) day 1 vs 1. the lfg group improved between days 1 and 2 and 2 and 3. between day 1 and day 3, the lfg showed improvement only for test b ( p < 0.001). chronic stimulation of the vagus nerve improves cognitive function in epileptic patients and this improvement is more marked with low-frequency stimulation. a. guidotti, and j. d. rothstein, bologna, itah, washington, dc, and baltimore, m d we reported idiopathic recurring stupor (irs) in a patient with stuporous episodes without known causes and reversed by flumazenil, a specific benzodiazepine (bz) antagonist. ictal plasmdcerebrospinal fluid (csf) showed increased bz-like activity (ann neurol, in press). recently, an endogenous bzreceptor ligand (endozepine [ez]) has been purified from mammalian brain with properties similar to diazepam. it acts like diazepam to potentiate gamma-aminobutyric acidmediated postsynaptic inhibition. we hypothesized that irs might be due to an excess of this substance. irs was diagnosed in 2 patients, 41 and 58 years old, who had recurring stupor or coma episodes lasting hours to days. ictal brain ct/mri, kidney, liver, heart, blood glucose, ammonia, and osmolality were normal. eeg showed fast 13-hz background activity while the patients were unreactive to stimuli, reversed by flumazenil. ictal serum or csf revealed an enormous increase of the ez in both patients, with levels as high as 400 nm, compared to 5 to 10 nm in control serum/csf. interictal csf or serum in irs contained ez levels similar to control csf and serum. irs may be due to excess ez. the cause for increased ez is unknown. parkinson's disease: evidence from word learning murray grossman, jenger mickanin, barbara schaefr, kris onishi, matthew b. stern, steven gollomp, and howard hurtig, philadelphia, pa several reports have suggested that patients with parkinson's disease (pd) have intellectual impairments in several domains such as memory, but few studies have explored difficulties in language processing. we investigated the ability of 20 nondemented pd patients with mild motor impairments to learn about the grammatical and semantic information represented in a new verb. the new verb was presented to patients in a sentence-picture matching context, and we probed their recall of the verb 10 minutes later. a sentence judgment task assessed grammatical knowledge by asking patients to judge the new verb, known verbs, and pseudowords used appropriately or incorrectly in a sentence. we found that 65% of pd patients were significantly impaired in their grammatical appreciation of the new verb (f [i, 331 = 17.03;p < 0.003). this was not related to their motor disorder or neuropsychological performance. a picture classification task used pictures illustrating specific aspects of the new word's meaning to evaluate semantic knowledge. pd patients were as accurate as controls at deciding whether a picture illustrated the meaning of the new verb (f (1, 331 = 0.lo;p > 0.10). only 1 pd patient (5%) had difficulty sorting pictures. selective difficulty recalling only grammatical aspects of a new word suggests that the word learning impairment in pd cannot be entirely explained by poor memory. instead, in agreement with other recent findings, pd patients may be impaired in some aspect of grammatical processing in language. we discuss the hypothesis that defects in the frontocaudate axis in pd underlie this impairment. neuropsychological performance on both verbal and nonverbal tasks is reported to differ between healthy men and women. some of these cognitive differences are postulated to reflect differences in interhemispheric and intrahemispheric cerebral organization. our preliminary study indicated that women with alzheimer's disease (ad) performed worse than men on a composite neuropsychological battery, even after effects of potentially confounding variables were considered (buckwalter et al, j clin exp neuropsychol 1992; 14:23) . to explore further the nature of gender-associated differences in ad, we analyzed data from a verbal and a nonverbal task (the boston naming test and drawings from the spatial quantitative battery supplement to the boston diagnostic aphasia examination) for 22 men and 23 women who met nincds-adrda criteria for "probable" ad. prior to ana-grip strength 9 kg [range 1.5-20 kg]). the electrophysiological examination showed improvement with reversal of conduction block in 3 patients and was unchanged in 4. an apparent response to the placebo was seen in 3 patients. improvement after ivig therapy was maintained for variable durations (3-20 wk) and reoccurred with subsequent infusions. an equally effective response was documented after infusion of a single ivig dose of 1 gm/kg. we conclude that ivig therapy is effective in some patients with cidp, even after long duration of illness. the best responses were observed in patients with recent relapse. a single high-dose treatment may be equally effective. the object of this study was to examine whether borrelia burgdorjeri antigens could be detected in csf in the absence of detectable antibodies to b. burgdorjeri (the etiological agent of lyme disease). osp a is a 31-kd antigen that is specific for 8. duvgdorferi osp a was probed using western (immuno) blot and specific mouse monoclonal antibodies. polyclonal lyme antibodies were detected in csf using standard micro enzyme-linked immunosorbent assay. seven patients had osp a in csf without detectable lyme antibodies. there were 3 men and 4 women aged 20 to 58 years (mean 38 yr). disease duration ranged from 2 weeks to 8 years. neurological syndromes included confusion with acute flulike illness, optic neuritis, hemiparesis with inflammatory brain lesion, encephalitis, headache with erythema migrans, bilateral facial nerve palsies, and encephalomyeloradiculitis. three patients had csf abnormalities. in 4 patients csf parameters were otherwise completely normal. possible explanations for undetectable csf lyme antibodies included early infection (3 patients), prior antibiotics (2 patients), and prior steroids plus antibiotics (1 patient). in 1 patient there was no obvious explanation. we conclude that osp a, a specific antigen of b. burgdorferi, may be present in csf without a detectable humoral response. the diagnosis of neurological infection with b. bwgdorjeri should not require a positive csf serology. mollaret's meningitis: demonstration by polymerase chain reaction a 29-year-old man was seen in september 1991 for his fourth episode of aseptic meningitis over a 4-year period. the episodes conformed to criteria for mollaret's meningitis as published by bruyn, straathof, and raymakers, and subsequently by others; they lasted about 1 week, and were characterized by fever, headache, meningismus, lymphocytic pleocytosis, elevated protein in cerebrospinal fluid (csf), and spontaneous resolution without residua. extensive prior evaluations had failed to uncover a cause. the patient was otherwise well and neither he nor his wife had any history of sexually transmitted diseases. suspicion of herpes simplex virus (hsv) arose due to a single transient, raised skin rash several weeks earlier that failed to yield virus on culture. the patient was treated with acyclovir, which resulted in rapid resolution of symptoms. though culture and immunological studies of csf and blood again were unrevealing, polymerase chain reaction (pcr) studies of csf confirmed the presence of hsv type 2. we suspect that herpes simplex virus is a more common cause of recurrent aseptic meningitis than current culture and immunological techniques would suggest. pcr offers increased diagnostic sensitivity for neurotropic viruses and should be considered in patients with recurrent meningitis of cryptic etiology. differentiation by inorganic lead: a role for protein kinase c j. lutewa, j. p. bressler, r. r. indurti, l. belloni-olivi, and g. w . goldstein, baltimore, m d microvascular endothelial function in developing brain is altered by inorganic lead. this may result from changes in protein kinase c (pkc) modulation. we examined the effects of inorganic lead on an in vitro model of neural endothelial differentiation. astroglial-induced endothelial differentiation into capillary-like structures was inhibited by lead acetate with 50% maximal inhibition occurring at 0.5 pm lead. inhibition was independent of effects on cell viability or growth. we examined the effects of lead on cellular pkc pools under conditions that inhibited capillary-like structure formation. membranous pkc increased in c6 astroglial and neural endothelial cells after exposure to lead acetate. exposing c6 cells to 5 p,m lead for 16 hours increased membranous pkc by 150% as determined by immunoblotting. membranous pkc increased in response to as little as 50 nm lead and saturated at 1 pm. phorbol esters were used to determine if pkc modulation was mechanistically related to lead's inhibition of capillary-like structure formation. 12-myristate 13-acetate (10 nm) inhibited endothelial differentiation by 65 * 5%, whereas 4-alpha-phorbol 12,13didecanoate was without effect. these findings demonstrate that inorganic lead may induce cerebral microvessel dysfunction by interfering with pkc modulation in microvascular endothelial or perivascular astroglial cells. w. f. brown, b. v . watson, j . garland, g . c. ebers, and n . desai, london, ontario, canada gait difficulties in multiple sclerosis (ms) are commonly accompanied by fatigue and dyspnea. possible explanations for the latter include weakness andlor dyssynergia of the respiratory muscles, including possible abnormalities in central pathways regulating respiration. this study examined central and peripheral motor conduction to the diaphragm in 15 ms patients whose gait was notably labored and accompanied by breathlessness. peripheral conduction was assessed by measuring the latency and size of the surface-recorded diaphragmatic maximum m-potential responses to supramaximal stimulation of the phrenic nerve in the neck, and central motor conduction by comparable measurements in response to magnetoelectrical stimulation over the vertex. peripheral motor conduction was normal. the most striking abnormalities were in central motor conduction. cortical stimulus-evoked diaphragmatic responses were absent on both sides in 3 patients, and unilaterally in l patient, whereas in 5 others the latency of the cortical stimulus-evoked response was increased and the size clearly reduced and entirely normal in only 3 patients. these studies show that central conduction program and abstracts, american neurological association 295 to the diaphragm is commonly abnormal and may play a role in the fatigue and dyspnea experienced by ms patients. six men (40-53 yr) developed myelopathy that progressed slowly over several months and was characterized by asymmetrical, incomplete spinal cord syndrome manifested at the sensory level at the trunk, mild spastic paraparesis, and urinary incontinence. the spinal cord lesions at appropriate levels were recognized by mri as enhancing lesions in 4 of the men. coxiella burnetii infection was confirmed in the blood of all patients by immunofluorescence microscopic assay (ifa) and transmission electron microscopy (tem). in 2 patients, we detected c. burnetti by tem and ifa using csf of the patients inoculated onto fresh peripheral blood lymphocytes. four patients who were treated with appropriate antibiotics responded with either partial resolution of symptoms or arrest of further neurological progression. in 3 patients the lesion was shown on mri to have decreased in size. in summary, we report 6 cases of transverse myelopathy associated with c. barnetti infection. this is the first report, to our knowledge, of coxiella-related chronic myelopathy. we present a series of patients with different labyrinthine lesions diagnosed by mri. twelve patients with sensorineural hearing loss were studied by gadolinium-enhanced mri, including 3-mm contiguous t1-weighted images through the labyrinth. ten patients had enhancement of the cochlea or vestibule, or both. all patients with cochlear enhancement had severe neural sensory hearing loss. all patients with vestibular enhancement had severe vestibular symptoms. the patients' final diagnosis included viral labyrinthitis (3 patients), syphilitic labyrinthitis (2 patients), bacterial labyrinthitis (1 patient), and vestibular neuromas (3 patients). one patient had an acoustic neuroma extending in the basal turn of the cochlea. the enhancement in patients with vestibular neuromas was brighter and there was slight mass effect in comparison with the patients with inflammatory labyrinthine lesions. one patient had hemorrhage within the vestibule from an adjacent temporal bone hemangioma. one patient with ct-proven cochlear otosclerosis had pericochlear areas of enhancement on gadolinium mri. mri can diagnose a variety of labyrinthine lesions that correlate very well with the patient's clinical symptoms. gadolinium should be used routinely in patients with suspected labyrinthine disease. the diagnosis of meniere's disease and endolymphatic hydrops remains a diagnosis of exclusion. few radiographic findings have been correlated with the clinical symptoms of this entity. we describe 9 patients with symptoms of hearing loss or vertigo, or both, who demonstrated enhancement of the endolymphatic sac on gadolinium-enhanced mri. no enhancement was noted in a series of 20 controls with no symptoms of hearing loss and vertigo. enhancement in the brain correlates with inflammatory or neoplastic conditions. we thus can speculate that enhancement of the endolymphatic sac reflects an inflammatory process in this location that may interfere with the normal resorption of indulin and secondary hydrops. in addition to excluding an acoustic neuroma and a labyrinthine schwannoma (which clinically may be confused with meniere's disease), contrast-enhanced mri may provide objective evidence in favor of labyrinthine hydrops. it was intermittent (62%) but progressive. the ha was mild to moderate in severity; it was the worst symptom in only 24 (459%) and the first symptom in 32 (60%) patients. has were worse in the morning in 19 (36%) and interfered with sleep in 17 (32%) patients. unlike true tension-type has, bt has were worse with bending over in 17 (32%), with valsalva's maneuver in 12 (23%), and nausea or vomiting were present in 21 (40%) patients. an abnormal neurological exam was found in 38 (72%) patients with has and 43 (74%) patients without has lyzing the effects of gender, we used a hierarchical regression procedure to control for possible effects of subject age, education, age at onset of dementia symptoms, dementia duration, and family history of dementia. significant gender effects were found for the verbal task ( p < 0.005) (mean boston naming test score of 2 1.4 for women and 34.4 for men), but not for the drawing task. we conclude that verbal abilities are more severely affected in women than in men with ad, a difference that may in part reflect premorbid gender-associated differences in cerebral hemispheric organization. hemispatial placement is known to affect line bisection in patients with neglect. whereas placing stimuli in neglected space increases bisection error, placing stimuli in nonneglected space attenuates error. the effects of hemispatial placement on line bisection were examined in 4 patients with chronic neglect (over 5 months after stroke). all patients had large (frontotemporoparietal), unilateral, right-hemisphere lesions. each patient bisected lines of different lengths (24, 26, 28 , and 30 cm) in 3 hemispatial conditions (30 cm left of midline, midline, and 30 cm right of midline). like previous reports, when patients bisected lines in left hemispace, a consistent (20/24 trials) left-sided neglect was observed (2.6 cm). however, when lines were bisected in center space, misbisections occurred on either side of the midline; and, unlike previous studies, when lines were bisected in right hemispace, a consistent (20/24 trials) right-sided neglect was observed (2.0 cm). the magnitude and directional consistency of line bisection errors were significant. neither visual field defects nor limitations in reaching accounted for the results. recovery in chronic neglect may involve a realignment of limited attentional resources favoring the body's midline. consequently, performance in both hemispatial fields can be biased toward midline, resulting in neglect of opposite directions. despite agreement that depression is the most common neuropsychiatric symptom associated with multiple sclerosis (ms), many aspects of this emotional change are unclear. one of the more controversial issues concerns the relationship between severity of ms and depression. this relationship is used to evaluate whether depression is an integral or reactive symptom of ms. examination of this relationship is complicated by the presumed overlap between somatic features of depressive and neurological symptoms in ms. to clarify this situation, we examined the relationship between severity of ms and 4 categories of depressive symptoms using the beck depression inventory (bdi). eighty-nine patients and 47 normal controls were examined. for certain comparisons, patients were classified as mild (extended disability status scale of 0-2) or moderatelsevere (3) (4) (5) (6) (7) (8) . results indicated that total bdi scores and the depressive symptom categories (mood, self-reproach, vegetative, and somatic features) were elevated in patients with ms, but the extent of these elevations was not related to severity of disease. these results suggest that depression in ms is not a simple reaction to physical disability. furthermore, clinical examination of depressive symptoms is straightforward and not confounded by severity of ms. neurological involvement in wegener's granulomatosis was studied in 324 consecutive patients diagnosed at the mayo clinic. one hundred and nine patients (34%) had neurological involvement. peripheral neuropathy was seen in 53 (16.4%), cranial neuropathy in 2 1, external ophthalmoplegia in 16, cerebrovascular events in 13, seizures in 10, and miscellaneous involvement in 25. the mean age and sex ratio did not differ in those with or without neurological involvement. among the patients with peripheral neuropathy, 42 had multiple mononeuropathy, 6 had distal symmetric polyneuropathy, and 5 had unclassified peripheral neuropathy. multiple mononeuropathy was one of the major presenting symptoms in 8 patients. kidney involvement was significantly higher in the patients with peripheral neuropathy compared to those without it (p < 0.001). among the cranial nerves, the second, sixth, and seventh nerves were affected most frequently. multiple cranial nerves were affected in 8 patients. unusual neurological manifestations among the miscellaneous group included spastic paraparesis, temporal arteritis, homer's syndrome, and papilledema. this is the first comprehensive study on the frequency and distribution of neurological involvement in wegener's granulomatosis. chronic inflammatory demyelinating polyneuropathy: a double-blind placebo-controlled crossover study treatment with high-dose intravenous human immunoglobulin (ivig) has been reported to be beneficial in some patients with chronic inflammatory demyelinating polyneuropathy (cidp), yet most observations have been nonblinded. we examined the effect of ivig therapy in 10 patients (6 men, 4 women) with cidp in a double-blind, placebo-controlled crossover study. disease was chronic progressive (n = 5 ) or chronic relapsing (n = 5) and of variable duration (4 mo to 2 1 yr). the diagnosis was confirmed by electrophysiological (10) and nerve biopsy (7) examinations. the trial consisted of two 28-day periods each. patients were randomly treated with ivig (0.4 mg/kg/day) or placebo on 5 consecutive days and followed. function was assessed by a quantitative neurological disability score, functional grade, grip strength measurement, and electrophysiological examinations at the beginning and end of each treatment period. with ivig therapy, significant improvement was documented in 7 / 10 patients (improvement in neurological disability score mean key: cord-022653-qa1uph35 authors: nan title: poster discussion session pds date: 2017-08-30 journal: allergy doi: 10.1111/all.13251 sha: doc_id: 22653 cord_uid: qa1uph35 nan objectives: since bradykinin is a short-lived, low-abundance mediator in the systemic circulation, the discovery of additional biochemical biomarkers correlating hae disease activity with contact system dysregulation may be useful for further elucidation of hae pathophysiology and pharmacodynamic therapeutic modulation of the contact system. results: activated pkal cleaves single chain high molecular weight kininogen to generate bradykinin and cleaved 2chain hmwk. cleaved 2-chain hmwk was measured in human plasma using both a semi-quantitative western blot assay with fluorescent detection (licor) and a novel elisa with a capture antibody that specifically binds 2-chain hmwk. the western blot assay was previously used to monitor pharmacodynamic activity in hae patients treated with lanadelumab, a fully human antibody inhibitor of pkal that is in clinical development for hae attack prophylaxis. lanadelumab inhibited 2-chain hmwk generation following contact system activation in vitro, confirming that 2-chain hmwk is a product of pkal activity. plasma 2-chain hmwk levels from hae patients during or between attacks were compared to that from healthy volunteers using both the western blot assay and elisa. roc curve analyses with both methods suggest that 2-chain hmwk is a trait-specific biomarker capable of differentiating hae patients from healthy volunteers. the elisa was able to differentiate samples from hae patients collected during an attack from those collected between attacks with moderate sensitivity and specificity (c-statis-tic=0.8176). a comparison of 2-chain hmwk levels in citrated plasma versus plasma that contains protease inhibitors (scat169 plasma) provides estimates of endogenous versus ex vivo activation. the measurement of 2-chain hmwk using the specific assays described may find use in further dissecting the role of the contact system in disease pathology, to identify additional indications for modulators of this pathway, and to investigate therapeutics targeting the contact system. 0206 | g protein coupled receptor kinase 2 (grk2) regulates endothelial permeability induced by bradykinin 0208 | pharmacokinetics (pk) and pharmacodynamics (pd) of c1 esterase inhibitor of chronic urticaria challenges most commonly identified were the following: time of onset of disease; frequency/duration of and provoking factors for wheals; diurnal variation; occurrence in relation to weekends, holidays, and foreign travel; shape, size, and distribution of wheals; associated angioedema; associated subjective symptoms of lesions; family and personal history regarding urticaria, atopy; previous or current allergies, infections, internal diseases, or other possible causes; psychosomatic and psychiatric diseases; surgical implantations and events during surgery; gastric/ intestinal problems; induction by physical agents or exercise; use of drugs; food allergies; relationship to the menstrual cycle; smoking habits; type of work, hobbies; stress; quality of life and emotional impact; previous therapy and response to therapy, and previous diagnostic procedures/results. we included all of these aspects in our guide and as a result we developed a chronic urticaria check list. conclusions: our guide of clinical history for chronic urticaria (gur) contributes to have an easy tool in order to achieve a better diagnosis and evaluation of chronic urticaria. 0213 | clinical and diagnostic features in acquired cold urticaria patients in a coruña sanitary area, spain physicians and dermatologists/allergists; c/sa patients were more likely to visit dermatologists/allergists (51% vs. 47%) and less likely to visit general physicians (32% vs. 57%) than european patients. emergency room visits due to cu were more common in c/sa (40%, mean [sd] number=23.2 [124.3] ) than europe (29%, mean [sd] number=3.7 [11.4] ). conversely, hospital admissions due to cu were more likely to occur in europe (22%) than c/sa (8%), but the average (sd) number of admissions among those hospitalised was greater in c/sa (3.3 [4.7] vs. 2.0 [3.1]). variations were seen in subregion comparisons. mean (sd) overall wpai scores were 7.0 (18.9), 25. 1 (26.8), 27.3 (28.5), and 33.3 (30.8) for absenteeism, presenteeism, work productivity loss, and activity impairment, respectively; patients in c/sa reported a higher rate of impairment (range, 13%-36%) on all domains compared with patients in europe. conclusions: cu is associated with substantial hru and work and activity impairment in both europe and c/sa. general physicians should be considered key members of the treatment team in the care of patients with cu in these regions. objectives: cu patients (n=15) received monthly subcutaneous injections of omalizumab for up to six months. urticaria-related symptoms were assessed by both the urticaria control test (uct) and the chronic urticaria quality of life score (cu-q 2 ol). peripheral blood was drawn prior to each injection for determining the concentration-dependent reactivity of patients' basophils to specific anti-fceri and unspecific fmlp stimulation by basophil activation test. furthermore, the impact of anti-ige treatment on ige-bearing cell populations was characterized by flow cytometry analyzing the surface expression of both fceri (e.g. on monocytes, dendritic cells, basophils) and the low-affinity receptor for ige, fcerii (e.g. on b cells, eosinophils). results: anti-ige treatment of cu patients significantly improved clinical symptoms of cu already after the first injection as evaluated by cu-q 2 ol and uct, the latter of which correlated with an increase of basophil numbers and a decrease of basophil surface bound ige. of note, cell-bound ige on fcerii-expressing cells was not altered. furthermore, while clinical amelioration also was accompanied by reduced fceri expression on basophils, the basophil responsiveness to anti-fceri stimulation increased in 73% (11/15) of treated patients. in contrast, ige-independent activation of basophils by fmlp was unchanged. conclusions: clinical improvement of cu patients treated with omalizumab is associated with characteristic immune alterations in basophils, like rapid, cell-specific reduction of surface bound ige and fceri-expression as well as normalization of basophil responsiveness to fceri-stimulation. while our findings might help to better understand the mode of action of anti-ige therapy in cu, they also can shed new light on the pathomechanisms underlying cu. 0216 | omalizumab in patients with severe active chronic spontaneous urticaria (csu) heavily treated with corticosteroids and cyclosporine introduction: increased levels of blood d-dimers (d-d), the byproducts of fibrin degradation, is linked to the severity of chronic spontaneous urticaria (csu) and to poor response to antihistamines h1 (ah1). omalizumab (oma) is a human monoclonal anti-immunoglobulin-e antibody registered as an add-on treatment of csu in adults and adolescent (≥ 12 years old) and with insufficient response to ah1. the sunrise study assessed the efficacy of omalizumab on csu symptoms and the correlation between d-d levels and response over time to treatment with oma to explore its potential predictive value. objectives: sunrise was a french prospective non comparative phase 4 study. included patients had to have been diagnosed with csu for at least 6 months, be resistant to ah1 treatment, and have a uct score (assessed by patient over the 4 last weeks, values from 0 (maximal disease)to 16 (full control)) <8, indicative of a poorly controlled disease. the widely used uas score (assessed by patients over 1 week which captures intensity of pruritus and number of hives, values ranging from 0 (no disease) to 42 (severe disease)) was further used to evaluate the proportion of patients achieving a well controlled disease(uas≤6). all patients received 300 mg oma by sub-cutaneous injections at day 0, weeks (w) 4 and 8. blood levels of d-d were assessed (turbidimetric immunoassay) at d0, w4 and w8. response to treatment was evaluated at w12 by means of the uas7. results: median level of d-d assessed at d0 in 112 patients was increased at baseline (618 ng/ml, extremes 108-5170) and normalized as early as w4 reaching 286 ng/ml (108-481) at w8. correlation between d-d concentration and uas7 score at w12 was weakly positive (spearman coefficient 0.108). among the 10 patients with a very high baseline de d-d level (>3000 ng/ml) 8 were responder (uas7≤6) at w12. conclusions: baseline d-d levels were increased in more than half of patients of this study in line with relevant literature. a fast normalization was observed with oma as early as w4 of treatment. d-d levels at w8 were weakly correlated with uas7. subgroup analyses may help to better understand the link between d-d and clinical response, as these preliminary results do not yet allow the predictive use as a biomarker. the sunrise study explored for the first time in a prospective way the impact of oma on dd and found weak correlations were measured between dd level and response to oma. further studies will be needed to evaluate its predictive value for response. (crp, esr, il-6, il-10, il-33, ccl2/mcp-1), and the disease severity in patients with chronic spontaneous urticaria introduction: pru p 3 is the primary sensitizer of some fruits and responsible for severe reactions in the mediterranean area. sublingual immunotherapy (slit) using peach extract enriched in pru p 3 (prup3-enriched-slit) brings a new perspective to treat patients with reactions to peach considering that currently the treatment of the allergy to peach is based on avoidable ingestion of fruit. we performed a pilot study to examine the immune modulation by slit in patients with peach allergy over a 1-year treatment period. objectives: we aimed to evaluate the effect of the slit during one year in patients with peach allergy. we analysed the capacity pru p 3enriched-slit to modulate immune response, from a th2 to th1 response with increases of treg cells. we studied three groups of subjects: peach allergic patients who received prup3-enriched-slit for 1 year, peach allergic patients non treated, and healthy controls who tolerated peach. monocyte-derived dendritic cells (dcs) maturation, peripheral blood mononuclear cell phenotype and lymphocyte proliferation after prup3 stimulation were assessed by flow cytometry from samples obtained before, and 1, 6 and 12 months during slit. results: we found statistically significant differences in dcs activation and maturation between allergic patients and controls at the basal state. when we analyzed the effect of prup3-enriched-slit over time, we found a significant reduction of activation and maturation markers (ccr7, cd40, cd80, cd83 and cd86) at the first month of treatment that was maintained after 1 year. concerning lymphocytes, we observed a significant decrease of effector cells immune cells. recent studies showed that fructo-oligosaccharides (fos) increase the efficacy of oral immunotherapy (oit) in a mouse model for cow's milk allergy, however, the mechanism is unknown. objectives: investigating the effect of oit+fos on the effector response and the process of tolerance induction. methods: female c3h/heouj mice (5-6 weeks old, n=8/group) were sensitized to the cow's milk protein whey (20 mg in pbs, intragastrically (i.g.)) with cholera toxin (15 lg) once a week for 5 weeks (d0-d28). the mice received a diet with 1% fos or a control diet from d35-d70. oit (10 mg in pbs or pbs) was provided 5 days a week for 3 weeks (d41-d59). intradermal (i.d.) and i.g. challenges were performed to measure the acute allergic response. serum, bone marrow, caecum content, small intestines and mesenteric lymph nodes (mln) were collected at d50, d63 and d70. spleen-derived t cell fractions (whole spleen, cd4+cd25-and cd4+cd25+, using macs) were transferred to na€ ıve recipient mice at d70. the recipients were sensitized and challenged as described for the donor mice. conclusions: this study shows that oit+fos results in an early induction of functional tregs and a reduction of mast cell degranulation upon challenge. the latter may be caused by inhibition of mast cell activation by galectin-9 and/or butyric acid. moreover, the effect of fos on bone marrow suggests possible epigenetic changes reducing development of mast cells. further research is needed to investigate if this approach may be of potential value to treat food allergies. 0348 | safety and feasibility of slow low-dose oral immunotherapy sugiura s; kitamura k; tajima n; takasato y; kato t; tajima i; ono m; tagami k; sakai k; nakagawa t; ito k aichi children's health and medical center, obu, japan introduction: slow low-dose oral immunotherapy (sloit) is an ongoing clinical trial conducted in our institute (umin registry number 000017416). this is a type of oral immunotherapy with a low dose antigen increased very slowly. objectives: to evaluate the safety and feasibility of the protocol. results: sloit enrolled the patients who were diagnosed as severe egg, milk or wheat allergies, with a threshold dose of 5 g (or ml) or less in the oral food challenge (ofc, 20-minutes boiled egg white, whole milk, udon noodle). subjects were divided into two groups, intervention (sloit) group or elimination (control) group, based on their preference. sloit group began to ingest 1/2 to 1/10 amount of the final dose of the ofc based on the severity of provoked symptoms. intake was continued everyday with an increasing dose less than 1.5 times/ month, expecting a 10 times increase from the starting dose after 12 months. feasibility was evaluated based on the proportion of patients who complied the protocol. safety was evaluated based on the frequency of immediate allergic reactions observed in the programmed intakes. fifty-nine patients were enrolled from april to december 2015, and 36 of them (egg: 23, milk: 4, wheat: 9) preferred the sloit group. among them, 11 patients (30.6%) dropped out from the study protocol because of provoked allergic symptoms (n=2) or the other personal reasons (n=9). among a total of 7090 ingestions by 25 patients who continued the protocol over 10 months, mild symptoms were observed 25 times (0.35%), to which rescue medicine such as oral antihistamines was used in 9 cases (0.13%). no one needed an emergency visit or an adrenalin auto-injector. low threshold dose at the initial ofc (1.0 g or less, n=16) was associated with higher proportion of provoked allergic symptoms in the protocol (low threshold: 0.50%, non-low threshold: 0.12%, p=.019). the level of specific ige titer was not associated with the safety. conclusions: sloit protocol has sufficient safety and feasibility, but low threshold patients should be monitored closely. the efficacy of this protocol is being evaluated by an ofc after 12-15 months of the treatment, and the overall data will be presented after march 2018. objectives: the aim of this phase i, randomized, non-controlled, multicenter, opened, with parallel groups clinical trial, is to evaluate the safety and tolerability of subcutaneous immunotherapy (scit), in a polymerized mixture (100/100) depot presentation. patients with rhino-conjunctivitis polysensitized to olea europaea/ phleum pratense received a schedule consisting of two weeks of initiation with three weekly injections; or a program comprising two administrations in the same day separated by 30 minutes. both treatments continued with a maintenance period of three months with a monthly administration. the primary outcomes are the number, percentage, and severity of adverse reactions. secondary endpoint included subrogate efficacy parameters evaluation: changes in immunoglobulin titers (specific ige, igg and igg4) and changes in cutaneous reactivity at different concentrations. 2 systemic reactions were registered, representing 4.3% of the included patients: one grade 0, described as general discomfort plus dizziness and one grade i, such as rhinoconjunctivitis. there were no local reactions. all were classified as of mild intensity and took place with the cluster schedule. symptomatic treatment was not required. conclusions: both schedules with polymerized mixture of phleum pratense/olea europaea, (100/100), presented a good safety and tolerability. a statistically significant decrease in cutaneous reactivity to olive and grass allergens was observed after immunotherapy. 0351a | design of the pivotal phase iii study to assess the efficacy and safety of subcutaneous hdm allergoid immunotherapy in patients with hdm induced allergic rhinitis/ rhinoconjunctivitis introduction: in order to comply with ema guidelines on development of allergen immunotherapy products, a clinical development program was started to obtain full marketing authorization for a allergoid subcutaneous immunotherapy (scit) product for the treatment of house dust mite (hdm) allergy. previously, the safety and tolerability of increasing doses of this allergoid scit product was evaluated in patients with hdm-induced allergic rhinitis/rhinoconjunctivitis (arc) [eudract 2008-006261-81] . no safety or tolerability issues were identified for doses up to 40 000 aueq. subsequently, a dose-finding study to identify the optimal, i.e. effective and safe dose in hdm arc with or without concomitant asthma was performed [eudract 2011-000393-61; pfaar, allergy 2016] . this study demonstrated a dose response relationship with doses of 10 000 aueq (0.5 ml of 20 000 aueq/ml) up to 50 000 aueq (0.5 ml of 100 000 aueq/ml) showing significant improvements compared to placebo. the current pivotal phase iii study [eudract 2016-000051-27] aims to confirm safety and efficacy of this hdm allergoid scit product at a dose level of 50 000 aueq/ml (0.5 ml) compared to placebo after one year of treatment in patients with hdm-induced arc. objectives: the current study is a multi-center (80 clinical study centers in 7 european countries) randomized, double-blind, placebocontrolled, parallel-group study in 730 adult patients, with moderate to severe hdm induced arc with or without mild to moderate persistent asthma. the primary outcome of the study is the difference in mean combined symptom and medication score (nasal symptoms only) (csms (n)) between 50 000 aueq/ml allergoid scit and placebo treatment, assessed during the last 8 weeks of the approximately 1 year treatment period. results: 211 patients from 14 centers, (mean age 32.9 years) have been included and analyzed. the 49.8% are men, 65.9% presented associated asthma. a large majority of patients have received subcutaneous sit (98.6%), and 58.8% of them containing a single allergenic source. 55.8% in polymerized formulation and 40.9% in depot. accelerated schedule has been the most prescribed (56.9%), followed by clustered one (32.2%). from the 81 patients who have completed the study, 45.7% of them improved from persistent to intermittent rhinoconjunctivitis (p<.01) and 49.4% from moderate/severe to mild intensity (aria) (p<.01). moreover, 21% of asthmatic patients at baseline, did not have any bronchial symptoms after 1-year treatment. the improvement in quality of life was possible to be analyzed in 53 patients. mean values in rqlq questionnaire (total score) decreased from 3.1 to 1.5 points (51.6% of score reduction) in final visit, reflecting a statistically significant improvement (p<.01). mean value of treatment satisfaction was 7.3 (sd=1.7) and 7.2 (sd=1.9) for physician and patients respectively. for safety assessment, out of 199 analyzed patients, only 8 systemic reactions were reported in 8 patients (4.02%). seven of them were classified as grade i, and one as grade ii according to eaaci grading system. strasbourg is a 147 m 3 chamber, located into the university hospital of strasbourg at less than to 5 minutes to the intensive care unit. one of the characteristics of this unit is that the maximum parameters are controlled: temperature, relative humidity, ventilation rate, particles number and particles size, concentration of airborne of der p 1 and airborne voc. mite allergens extract were nebulized through a nebulizer. airborne der p1 concentrations were sampled using 5 glass fiber filters and measured with an elisa assay. particles number and particles size were monitored continuously during nebulization, using 10 particles counters distributed inside the exposure room. the cleaning process was also controlled and validated. objectives: to validate the technical parameters of the environmental exposure chamber (eec) of strasbourg with mite allergens. results: the reproducibility was excellent for the indoor temperature, relative humidity and airflow (5 cv interassay <20%). three concentrations of der p1 were measured: 63, 76, 105 ng/m 3 (n=45). for all 3 concentrations, the cv intra-assay of airborne der p1 was 22ae1.3%, the interassay was less than 30%. for the particle size 0.5-5 and 5-10 lm, the cv interassay was 8 and 13%, respectively (n=19). the cv of the mmad was 2.2% (n=10). no measurable airborne der p1 was detected in the toilets and the medical supervision room (n=6) neither in the exposure room 10 minutes after the end of the allergen exposure (n=9). no airborne voc was measured in the chamber and the other rooms of the clinical unit (n=10). there was no significant change in particles size and number when 2 to 6 persons entered the room (n=4). introduction: pathogenesis of systemic sclerosis (ssc) includes vasculopathy with endothelial dysfunction which is considered to be one of the earliest changes in the pathogenesis of ssc. several biological molecules, including e-selectin (e-sel), inter-cellular adhesion molecule 1 (icam-1), endothelin 1 (et-1), von willebrand factor (vwf) and interleukin 6 (il-6) have been associated with endothelial activation. objectives: we aimed to determine if these vascular biomarkers are associated with distinct capillaroscopic ssc patterns and/or more severe disease in ssc patients. results: correlations between serum levels of all 5 vascular biomarkers were good to moderate and statistically significant, with r indices varying between 0.660 and 0.332, the only exception being et-1 which did not correlate with e-sel. good correlations (r 0.465 to 0.727) were also found between all 5 biomarkers and crp. patients with severe vasculopathy, as reflected by the nfc "late" pattern, had higher levels of il-6 (median 12.06 vs 3.08 pg/ml, p=.001), et-1 (median 2.06 vs 1.59 pg/ml, p=.029), vwf (median 3284 vs 2730 iu/ml, p=.013) and e-sel (median 52.6 vs 42.3 ng/ml, p>.05), compared to patients with nfc "early" or "active" patterns. there was a significant, negative correlation between lung transfer for carbon monoxide (dlco) and e-sel, icam-1 (both p<.001) and vwf (p=.013). et-1 was higher in patients with more severe disease (dcssc, patients positive for anti-topoisomerase antibodies and patients with a history of digital ulcers-all p<.05). conclusions: serum endothelial activation biomarkers are elevated in patients with more severe ssc-associated vasculopathy and correlate with serum crp. together with nfc data they may be used for assessing vasculopathy severity in ssc. objectives: here we present the imagination findings of eye involvement in a family whose 11 members have mws. method: clinical data was collected during the course of ongoing patient care. results: we evaluated the clinical features of 11 patients who were referred to our center. the median age of the patients was 25 years (range: 9-65 years). the ratio of females /males was 1.2 (6/5). all patients had arthritis with exacerbation on exposure to cold and ocular involvement, mostly in the form of conjunctivitis and far less other forms. the median age of onset of ocular involvement was 8 years (2-45 y). chronic eye damage were detected in three patients. corneal involvement and clouding was detected in four patient. two conclusions: in this study, it has been shown that eye findings related to mws can vary from conjunctivitis to severe uveitis. we want to emphasize that ocular involvement in mws should be carefully assessed, since it can lead to visual impairment. 0485 | antiretroviral activity of the conjugates 3'-azido-3'deoxythymidine and derivatives of 1,3-diacylglycerides case report: aids epidemics remain the critical problem due to both their emergent and long development. treatment of aids with azt reduces p24 antigenemia, increases cd4 + lymphocyte counts, reduces the frequency and severity of opportunistic infections and prolongs life. however, zidovudine and other dideoxynucleotides do not decrease the ability to isolate hiv from pbmc, and in addition, these drugs are very toxic. this phenomenon is caused by insufficient inhibition of hiv due to low levels of nucleoside kinases in ccr5-positive cells, including in macrophages, which are a major reservoir of hiv. potential advantages of these liponucleotide prodrugs include: greater in vivo efficacy and lower toxicity due to a greater delivery to monocytes/macrophages, ability to bypass the initial anabolic phosphorylation due to the presence of the phosphorous center in the structure, and the prospect of improved pharmacokinetics and prolonged intracellular persistence. the aim of this work is the study of cytotoxicity and anti-hiv activity of glycerolipids derivatives of azt. evaluation of the cytotoxicity of azt and test compounds was per0488 | bone mineralisation defect in patients with hax-1 deficiency 4 and osteopenia (z score <à1) in 3 patients. bone mineralisation defect was found in all female patients while only one male had osteopenia (table 1) . conclusions: in this study, a significant decrease (87.5%) of bone mineralization was observed in patients with hax-1 deficiency. female patients were found more prone to bone mineralisation defect. to conclude on this subject, more studies are needed with large number of patients having not only hax-1 deficiency but also ela-2 mutations. gene mutation age ( introduction: bronchopulmonary diseases are kept as one of the actual problems of the pediatricians. nevertheless fulfilment of several scientific works on study of these diseases, presently we meet the complication of the respiratory diseases, recurrency, changing to the chronical type. objectives: the main purpose of our work to study the cytokine status and substance p, mutual connection they in frequently ill chilresults: in fic with respiratory diseases in the acute period of the disease increase of levels proinflammatory cytokines il-1beta, il-6, il-8, tnf-alpha and substance p,decrease of levels il-2 and ifngamma was marked. clinical remission in these children is not accompanied by normalization of cytokine status and substance p. the high level of proinflammatory cytokines and substance p testifies to proceeding of inflammatory process that is possible connected with persistence of the infections agent. acute decrease in level of cytokines il-2 and ifn-gamma in these children,most likely, is caused by the presence of a immunodeficiency of cellular type. conclusions: in this connection it is necessary to carry out an adequate therapy of fic with arvi. introduction: atopic diseases are known to be characterized by a t helper (th) 2-skewed immunity. th2-skewed immunity at birth, th2-associated cc chemokine ligand (ccl)-22, appears to be associated with high total ige levels but not of allergic outcomes later in life. the prevalence of asthma increases with a rapid upward trend after age 2; however, there are few studies addressing the changes of ccl22 chemokine levels during infancy related to the development of atopic diseases in early childhood. objectives: we investigated 182 children followed up regularly at the clinic for 5 years in a birth cohort study. the levels of th2 related chemokine ccl22 were quantified in cord blood and age 1.5 by multiplex luminex kits. specific immunoglobulin e antibodies against food (egg white, milk, and wheat) and inhalant allergens (d. pteronyssinus, d. farina, and c. herbarum) were measured at 6 months as well as 1.5, 3, 4 and 5 years of age. results: a total of 125 pairs of ccl22 chemokine levels from birth to age 1.5 were recruited in this study. k-means clustering was performed using r software and package mfuzz and this resulted in 3 groups of ccl22 chemokine levels that declined from around 2000 to 500 pg/ml (cluster a, n=51), from around 1200 to 400 pg/ml (cluster b, n=46) , and raised from around 400 to 600 pg/ml (cluster c, n=28) . in children with raised ccl22 chemokine levels appeared to be associated with a higher prevalence of house dust mite sensitization at age 1.5. furthermore, raised ccl22 levels during infancy were significantly associated with higher risk of asthma at age 3 (p=.026). conclusions: raised ccl22 chemokine levels during infancy appear not only to be associated with an increase in the prevalence of house dust mite sensitization but also the risk of asthma in early childhood. 0520 | serum periostin is "not" a biomarker for pediatric asthma suzuki n 1 ; hirayama j 1 ; nagao m 1 ; kameda k 1 ; kuwabara y 1 ; kainuma k 1 ; ono j 2 ; ohta s 3 ; izuhara k 3 ; fujisawa t 1 1 mie national hospital, tsu, japan; 2 shino-test corporation, kanagawa, japan; 3 saga medical school, saga, japan introduction: periostin is a matricellular protein induced by type 2 helper t-cell cytokines, expressed by airway structural cells and is thought to contribute to airway remodeling and progressive lung function decline in severe asthma in adults. we sought a possible clinical utility of serum periostin in children with asthma. objectives: we recruited volunteer children and adolescents (age range, 3 to 17 years) who were otherwise healthy except for allergic diseases including bronchial asthma. allergic diseases were classified with isaac questionnaire and serum levels of periostin were measured with elisa. abstracts | 167 results: a total of 661 volunteers were enrolled. among them 161 had no allergic diseases (na), 215 had only allergic rhinitis (ar) and the rest of 285 had bronchial asthma (ba) and/or atopic dermatitis (ad) . serum levels of periostin in na younger than 15 were significantly higher than the older counterpart and the levels in children <15 years old were similar across each age. data distribution of serum periostin in ar were very similar with that in na and we defined na and ar groups as reference population. reference values, geometric mean (+2 geometric sd range), for serum periostin were 101 (179) and 82 (151) ng/ml in children/adolescents <15 and ≥15, respectively. there were no gender differences in serum periostin in the reference population. we then compared the serum levels of periostin in ad and/or ba with the reference group and found that serum periostin in ad and ad+ba, not ba, in <15 years old were significantly higher than the reference group. in addition, severity of asthma had no association with serum periostin levels in age group of <15. on the other hand, the levels in ba aged ≥15 were slightly higher than non-ba (statistically significant). conclusions: serum periostin is physiologically high in children and adolescents, possibly in growing age, and the levels are elevated in those with ad, not ba. serum periostin is "not" a useful biomarker for pediatric asthma. 0521 | clinical, biochemical and radiological factors for response to aspirin desensitization in patients with aspirin exacerbated respiratory disease-pilot study introduction: aspirin desensitization is regarded as an effective and well-tolerated therapy for patients with aspirin exacerbated respiratory disease (aerd). despite many studies investigating the pathophysiology of aerd, the underlying mechanism responsible for the beneficial effects of aspirin therapy has not yet been clarified. the aim of the study was to evaluate the influence of aspirin desensitization on clinical, biochemical and radiological changes in aerd individuals. objectives: this is a prospective study of twenty-one aerd patients subjected to one-year aspirin desensitization. all participants were hospitalized three times over the period of one year. at baseline and during each follow-up visit (2 nd and 12 th month) blood, urine, induced sputum (is) and nasal lavage (nl) were collected from all participants. the acquired material was processed in order to evaluate 1) is and nl cell count 2) concentrations of is and nl eicosanoids 3) leukotriene e4 (lte4) in urine and 4) periostin in blood. additionally, participants underwent a ct scan of the paranasal sinuses at baseline and after 12 months of aspirin therapy. the lund-mackay score values were compared. for statistical analysis, summary statistics and repeated measures anova with post-hoc test were applied. results: twenty participants completed a one-year aspirin therapy. there was a statistically significant decrease in the number (p=.007) and percentage (p=.02) of eosinophils in is between baseline and after aspirin desensitisation. significant increase of urine lte4 in the course of aspirin therapy was observed (p=.02). the levels of prostaglandins and leukotrienes in is and nl as well as blood periostin level and the differential cell count in nl did not change during aspirin desensitization. in ct scan images the regression of the lesions in paranasal sinuses was observed in 32%, the worsening in 42% and in 26% of patients no changes were noted. in is count of eosinophils in aspirin-sensitive individuals, which may potentially be used in disease monitoring and tailoring asthma therapy. aspirin desensitization did not lead to significant changes in local eicosanoid levels in is and nl. blood periostin level is not a good marker for patient's response to aspirin desensitization. introduction: one of the main severe asthma phenotype is the "severe eosinophilic" or "eosinophilic refractory" asthma, for which novel biologic agents are emerging as therapeutical options. in this context, blood eosinophils count are one of the most reliable biomarker. objectives: the aim of our study is to evaluate the performance of a point-of-care peripheral blood counter in a clinical setting of severe asthmatics. seventy-six patients with severe asthma were evaluated, for blood cells count, by both a point-of-care and a standard analyser. results: the inter-and intra-assay variation was acceptable for leukocytes, neutrophils, lymphocytes and eosinophils. this was not the case of monocytes and basophils. a significant correlation between blood eosinophils assessed by the two devices was found (r 2 = 0.854, p<.0001); similar correlations were found also for white blood cells, neutrophils and lymphocytes. the point-of-care device showed ability to predict blood eosinophils cutoffs used to select patients for biologic treatments for severe eosinophilic asthma, and the elen index, a composite score useful to predict sputum eosinophilia. introduction: over 10 years ago there was revealed periostin should play an important role in pathogenesis of allergic inflammation including asthma and processes of tissue remodeling and fibrosis. its expression has been observed in the thickened basement membrane as well as in serum of asthmatic patients. thus, measuring of periostin serum levels may shed some light on these elusive asthma features. periostin has already demonstrated a convenient value in clinical studies as a companion diagnostics for lebrikizumab, tralokinumab or omalizumab treatment. however, to date, the changes of periostin serum levels following asthma therapy except for inhaled corticosteroids remain unclear. objectives: to emphasize this issue we have collected clinical and laboratory data including serum periostin of 48 asthma patients (19 males/29 females) in a cross-sectional study. all patients were treated either by conventional therapy comprising inhaled corticosteroids (n=38) or by inhaled corticosteroids with additional biological therapy (omalizumab) (n=10). asthma phenotype, control, complications, comorbidities and other available biomarkers have been assessed and statistically analysed. results: we have observed a weak but statistically significant correlation between serum periostin and total ige levels (p=.039) and absolute eosinophil count (p=.045). association between periostin and total ige had nonlinear character (p=.007), and was expressed particularly in non-severe asthma patients without omalizumab treatment (p=.018). despite mutual correlation between serum periostin a total ige levels, both biomarkers showed different reaction on asthma treatment. multivariate analysis demonstrated, that only periostin levels, in contrast to all other assessed biomarkers, were significantly decreased in severe asthma patients treated by omalizumab (p=.013). this relationship was independent of asthma control (assessed by asthma control test -act), exacerbation rate, hrct or spirometry measurement results or comorbidities, except chronic rhinosinusitis with nasal polyps (crswnp) (p<.001). conclusions: we demonstrate, serum periostin levels are dependent on therapy, thus it may contribute not only to asthma phenotype stratification, prediction of treatment responsiveness, complications such as remodeling but probably more precise monitoring of therapy effect too. 0527 | usefulness of serum pteridines as a biomarker for childhood asthma kasuga s 1 ; hamazaki t 1 ; fujitani h 1 ; fujikawa s 1 ; niihira s 2 ; shintaku h 1 1 department of pediatrics, osaka city university graduate school of medicine, osaka, japan; 2 the national institute of special education, tokyo, japan introduction: reliable and stable biomarker of airway inflammation is essential to determine intensity of asthma treatment. exhaled nitric oxide (feno) has been introduced to assess useful marker of airway inflammation but it fluctuates depending on steroid inhalation. nitric oxide is produced by nitric oxide synthase which requires tetrahydrobiopterin (one of pteridines) as a cofactor. objectives: to assess pteridines as a biomarker of childhood asthma control. results: asthmatic children were recruited for periodical asthma checkup program in japan to assess asthma control status by using objective questionnaire, respiratory function tests, airway resistance, feno, and serum pteridine levels. serum pteridine levels were measured by high performance liquid chromatography. total 131 japanese children (4-17 years) were participated in this program. 36 children who have no asthma attack over three years were divided as remission group to evaluate the long-term asthma control. the other 95 children were divided as asthma group. furthermore, we divided asthma group for three groups by childhood asthma control test (c-act) scores to evaluate the short-term asthma control. and we had 61 age matched children as control group. pteridine levels tended to decrease in patients who showed higher feno in asthma group. asthmatic children showed lower pteridine levels than other groups. there are significant differences between control group and remission groups and asthma groups (p<.001, tukey's honestly significant difference test). but there are no significant differences between the three groups in asthma group (well-controlled group, partly-controlled groups and uncontrolled groups) which were divided by c-act scores. the low concentration of serum pteridines in children with stable asthma may indicate poor long-term control of asthma. but that not indicate poor short-term control of asthma. since pteridine biosynthesis pathways were suppressed by th2 mediated immune response, these results suggest that th2 mediated immune response was dominating the th1 response in asthmatic children. therefore, serum pteridines could be a novel biomarker of stable asthmatic children. introduction: asthma is a syndrome with chronic airway inflammation. the goal in the treatment of asthma is to control the inflammation. however, there has been a scarcity of useful noninvasive tests for monitoring the airway inflammation in clinical setting. metabolites of the eicosanoids pathways in induced sputum of the patients with asthma could be valuable biomarkers that can reflect the airway inflammation. objectives: to investigate eicosanoid metabolites and to find out their phenotypic differences, induced sputum supernatants from 37 patients with refractory asthma, 40 patients with controlled asthma, and 6 normal control subjects were analyzed by using liquid chromatography tandem mass spectrometry. in addition, we evaluated the relationship between asthma exacerbation and eicosanoid metabolites. results: among 43 metabolites which were measured, 26 metabolites were detected in the induced sputum. we found that normal control subjects had higher concentrations of prostaglandin (pg) d2 (normal subjects vs controlled asthma vs refractory asthma, median conclusions: eicosanoid profiles in induced sputum supernatant were different between patients with refractory asthma, those with controlled asthma, and normal control subjects. our findings suggest that they could be biomarkers to differentiate refractory asthma from controllable asthma or non-asthma. this work was supported by the research program funded by the korea centers for disease control and prevention (2016-er7402-00) . 0529 | serum periostin in asthma is related to disease severity, eosinophilia and il-33 introduction: introduction: asthma is a chronic inflammatory disease where more than 100 powerful inflammatory mediators are associated with airway hyperresponsiveness, mucus hypersecretion, activation of fibroblasts and hyperplasia and hypertrophy of smooth muscles of the airways and if they do not use preventive antiasthma treatment can cause irreversible airway remodeling. objectives: the aim of this study was to determine the effect of adding montelukast to combined therapy icss/labas in patients with uncontrolled asthma by analyzing of serum level of il-13, il-5, eosinophils and symptom score. methods: in study we included 29 patients, they were treated with icss/labas (500/50 mcg-twice daily) plus montelukast (10 mgdaily). in each of them were measured serum levels of il-13 and il-5 by the elisa method, value of eosinophils were obtained with visual examination of peripheral blood smear, and assessing symptom scores with 5-point likert scale of breathlessness at the beginning and after 6 months of therapy. objectives: this study has been focussed on a detailed comparison of two samplers-cyclone and chemvol-and on the parameters that could influence their efficiency. results: airborne concentrations of two key olive and grass allergens, ole e 1 and phl p 5, respectively, were monitored over two years with different weather patterns, 2012 and 2014, in c ordoba, located in south-western spain. allergenic particles were quantified by elisa assay and results were compared with pollen concentrations monitored using a hirst-type volumetric spore trap over the same study periods. the influence of weather-related parameters on local airborne pollen and allergen concentrations was also analysed. inter-year differences were observed with regard to pollen season timing and intensity. for both olive and grass, the pollen season was longer and the pollen index (pi) higher in 2014; that year the peak value was higher and it was recorded earlier. although a positive correlation was detected between results obtained using the two samplers during the pollen season, results for the cumulative annual allergen index varied considerably. the two samplers revealed a positive correlation between pollen concentrations and both minimum temperature during the warmer year (2012) and maximum temperature during the cooler year (2014); a negative significant correlation was observed in both cases with rainfall and relative humidity. conclusions: in summary, although differences were observed between the two samplers studied, both samplers may be suitable for allergen detection. objectives: the scientific council of rnsa was asked to update the allergy potency (ap) of plant species that can be established in urban areas. to update the allergy potency of plant species, the rnsa used scientific work on the subject, and also the opinions of allergists and botanists. the pollen grains of anemophilous species are transported by wind; they produce very large quantities of pollen grains so that the fertilization of female flowers has a greater chance of being effective. the majority of allergenic species are anemophilous. results: the pollen allergy potency of a plant species is the ability of its pollen to cause an allergy to a significant part of the population. the allergenic potential can be: low or negligible: no problem to plant them in urban garden moderate: only a few species can be planted in the same garden high: this species cannot be planted in urban places. the table presented on the poster will permit to know the level of allergy potency of more than 30 species. conclusions: species or genus with a strong ap should be labeled as "not to be planted in habitation or residence area ", those with moderate ap should be labeled as "not to be planted in big quantities in habitation or residence area". other species with low or negligible ap may not be affected by public information. gadermaier g 1 ; metz-favre c 2 ; stemeseder t 1 ; de blay f 2 ; pauli g 2 1 universit e de salzbourg, salzbourg, austria; 2 chru strasbourg-allergologie, strasbourg, france introduction: the purpose is to study the profile of cutaneous and molecular sensitization of patients sensitized to plantain pollen living in the north-east of france. objectives: the sera of 28 patients with seasonal pollinosis and cutaneous polysensitization including a positive test to plantain, are investigated through immunoblot (ib), elisa and immunocap. the plantain immunoblot is inhibited with plantain, grass, ash and birch pollen extracts as well as with pla l 1. the specific iges against pla l 1, phl p 1-5 and profilin are measured. results: pla l 1, the major plantain allergen is detected by ib in 10 cases out of 28, either in glycosylated or non-glycosylated form. an allergen of 35 kda is detected in 21 out of 28 cases, always and exclusively inhibited by the grass extract. the intensity of the spot is proportional to the anti-phl p 1 / phl p 5 ige levels, and corresponds to an allergen which is cross-reacting with grass. other cross-reacting allergens with grasses are detected at 30 kda (14/28), and at 50-54 kda (5/28). at the molecular weights of 15 to 17 kda, we detected in addition to pla l 1, cross-reacting allergens with grass, ash and birch pollen which predominantly corresponded to profilin which was confirmed by specific ige measurements in 20 cases. conclusions: among the patients included in this study, only 28% had genuine sensitization to plantago, which never corresponded to a monosensitization in our cohort. most often cutaneous sensitizations to plantain pollen were based on ige cross-reactivity with grass pollen allergens mainly through a 35 kda allergen and/or profilin. the strong environmental grass pollen pressure, compared to the low plantain pollen exposure, seems to be at the origin of this profile of molecular sensitization. these results reinforce the superiority of molecular diagnosis in patients with pollinosis, who are polysensitized. 0536 | sensitisation to peach tree pollen in a highly exposed population results: the 14% of subjects were sensitized to pp, which was the most prevalent after olive tree, grass and cypress pollens, respectively. sensitization to peach fruit was 8%. pru p 3 spt was tested in 30/78 pp positive cases, being 46% positive (14/30). specific ige to pru p 3 was detected in 4/10 pp sensitized individuals. immunoblot showed specific ige to different components in the pp extract, being the most frequent band recognised a 20-25 kda protein. conclusions: pp is a prevalent inhalant allergen in highly exposure areas. in our population pru p 3 was not identified as the major allergen. other pp molecular components need to be identified and their clinical relevance should be further investigated. introduction: allergic respiratory diseases increase after an exposure to airborne pollen, as asthma and allergic rhinitis, are deeply increasing and nowadays, they represent one of the major public health problems. olive pollen is one of the main causes of allergic disease in the mediterranean area, especially in northwest and west of the turkey. olive pollen has been characterized 20 proteins with allergic activity and ole e 1 is the major allergen of olea pollen. objectives: the aim of this study was to estimate the correlation between daily airborne olive pollen and ole e 1 in the atmosphere. aeroallergen load of ole e 1 detected by cascade impactor (chemvol) using prewashed polyurethane foam and pollen counts recorded by hirst trap. chemvol sampler collects particles at 800 l/minutes and it contains 2 impaction stages pm>10 micron and 10>pm>2.5 lm. this sampler is being tested in the frame of the project hialine. results: generally, similar behaviour of pollen count and total allergenic load of ole e1 was observed during the main pollen season. nevertheless, in some occasions, before and later of main period, airborne ole e 1 activity was recorded and some differences between pollen grain/m3 and allergen concentration/m3 were detected. pollen from different days released 11-fold different amounts of ole e1 per pollen. average allergen release from pollen was much higher in 2011 (1.11 pg ole e 1/pollen, r 2 =.44) than in 2012 (0.65 pg ole e 1/pollen, r 2 =.3148). indeed, yearly olive pollen counts in 2011 were 3.8 times higher than in 2012, but ole e1 concentrations were 2.2 times higher. these results have shown that ole e 1 is mostly associated with olive pollen grains but aeroallergen load was not always directly proportional to airborne pollen counts. this suggests that ole e 1 quantification is a better marker for olive allergen exposure. in conclusion, aeroallergen monitoring may contribute to a better understanding of the ole e 1 exposure from airborne pollen. objectives: to describe the clinical profile of the patients sensitized to alt at 6 and to assess the sensitivity of the prick test with the alternaria extract in comparison to the patients sensitized to both allergens alt a 6 and alt a 1. out of the 726 patients, 94 (12.9%) were sensitized to alternaria with specific ige of ≥0.3 isu to alt at 1 or alt a 6. twenty patients (11 females and 9 males, age 6-57 years old) who were sensitized to alt a 6 were selected. we analyzed the clinical profile, total ige values, other co sensitizations as well as the clinical relevance of alternaria sensitization in these patients. : of the 94 patients sensitized to alternaria, 20 (21.3%) were sensitized to alt a 6, of which 9 (9.6%) were monosensitized to alt at 6. the mean age was lower in monosensitized (ẋ 16.2 vs 25.2). among the 9 patients monosensitized to alt a 6, 4 (44%) had prick test negative with the alternaria extract. eight out of 9 patients were polysensitized to more than three different aeroallergens and were asthmatics (6 persistent). the median total ige was higher in monosensitized (859 ku/l vs 479 ku/l). of these 8 patients, alternaria sensitization has clinical relevance in 5 (62%), of which 3 have positive prick test to alternaria. all the patients (12/ 12) sensitized to both allergens have positive prick test to alternaria. ten of them were polysensitized, 8 were asthmatic (3 persistent) and 5 sensitized patients showed clinical relevance. introduction: the role of vitamin d as a potential immune-modulator has been recently elucidated. dendritic cell, a key regulator driving towards th2 immune response in allergic diseases is known to be affected by vitamin d. however, the role of vitamin d in the pathogenesis of allergic rhinitis is unclear and its anti-allergic effect has not been established yet, especially in the mouse model. objectives: the aims of this study are to evaluate 1) the anti-allergic effect of topically applied vitamin d in the allergic rhinitis mouse model, and 2) the effect of vitamin d on dendritic cell activation. results: balb/c mice were intraperitoneally sensitized with ovalbumin (ova) and alum, and they were intranasally challenged with ova. intranasal 1, 25-dihydroxyvitamin d3 was given to treatment group and solvent was given intranasally to sham treatment group. allergic symptom scores, eosinophil infiltration, cytokine mrna levels (il-4, il-5, il-10, il-13, ifn-c) in the nasal mucosa, serum total and ova-specific ige, igg1, and igg2a were analyzed and compared with negative and positive controls. cervical lymph nodes were harvested for flow cytometry analysis. in the treatment group, allergic symptom scores, eosinophil infiltration, and the mrna levels of il-4 and il-13 were significantly reduced compared to positive control. il-5 mrna level, serum total ige, and ova-specific ige and igg1 levels showed a tendency to decrease in the treatment group, but did not reach to a significant level. il-10 did not show a significant difference between groups. cd11c + , mhcii hi , cd86 + activated dendritic cells were significantly reduced in the treatment group. cd4 + , cd25 + , foxp3 + treg cells tended to increase in the treatment group, however it was not significant. the intranasal instillation of 1, 25-dihydroxyvitamin d3 has an anti-allergic effect in the allergic rhinitis mouse model. we believe that the anti-allergic effect of vitamin d is mediated by the inhibition of dendritic cell activation, and therefore decreased objectives: we wanted to assess whether treatment with cyclosporine and tacrolimus allows children with vkc to improve the level of vitamin d due to the higher summer sun exposure for good control of the ocular symptoms. objectives: our objective was to assess the expression of smad2 and smad5 proteins in patients with asthma in correlation with clinical parameters and the expression's changes in response to allergen and methacholine challenge test. the study included 151 patients with asthma and 77 healthy volunteers. spirometry, skin prick tests (spt), allergen and methacholine provocation tests were performed in compliance with standards. personalized clinic surveys including act tm were collected. venous blood was collected before and after provocation. the expression levels of il-5 and il-15 and smads were evaluated by qrt-pcr using isoform-specific primers. results: we showed correlation between the mrna expression of smad2 and the asthma control according to act tm (p<.001). the expression of smad2 is higher in the group of uncontrolled (2 -δct =0.52, p<.001) and in the group of partially controlled patients (2 -δct =0.50, p<.05) in comparison to the group of patients with controlled asthma (2 -δct =0.40). we proved that expression of mrna of smad5 correlates significantly with expression of il-5 and il-15 in patients with asthma (il-5 r=0.34; il-15 r=0.36) and in the control group (il-5 r=0.36; il-15 r=0.27) (p<.05). expression of smad5 elevates more after methacholine provocation test (median 2 -δct =0.69) rather than after allergen provocation test (median 2 -δct =0.51; p=.04). we showed also that skin prick test results correlate positively with smad5 level after the provocation (p<.001). conclusions: the loss of asthma control is connected with expression of smad2, which is part of tgf-ßrii related pathway. il-5 and il-15 correlates with smad5 expression, which can indicate the participation of these cytokines in the regulation of this pathway. the expression of smad5 elevates after methacholine and allergen provocation as well as it correlates positively with skin prick test results, which can be useful clinically. to sum up, tgf-ß-tgfßrii-smad proteins are an important mediators of inflammation in asthma. 0545 | deletion of nfatc1 in t lymphocytes affects th2 and th17 cell differentiation as well as il-9-mediated mast cell activation in allergic asthma objectives: analysing the role of nfatc1 in the allergic trait of human asthma. additionally, we investigated the influence of nfatc1 on t cell differentiation and immunoglobulin class switch and explored its impact on mast cell differentiation in experimental asthma. with the children 0 s hospital in erlangen, we studied nfatc1 mrna expression in freshly isolated pbmcs from pre-school children with and without asthma. in asthmatic children, we found increased nfatc1 mrna expression, especially in those with a positive skin test. these results were confirmed in the asthma bio-repository for integrative genomic exploration (asthma bridge) study, where the isoform a and d of nfatc1 were found significantly increased in asthmatic adults with a positive skin test. moreover, il-9 was also found increased in the supernatants of pbmcs of asthmatic children with a positive skin test. furthermore, mice with a deficiency of nfatc1 in cd4 + t cells display significantly lower levels of il-9. additionally, these mice show reduced numbers of lung th2 and th17 cells. moreover, basic leucine zipper atf like (batf), which is known as an important transcription factor for t cell differentiation as well as immunoglobulin class switch, was found decreased in these mice. consequently, ova-specific ige and total igg1 levels were found significantly decreased after allergen exposure and in the absence of nfatc1. furthermore, nfatc1 deletion also resulted in decreased mast cell numbers. we then analyzed the effect of il-9 on mast cell differentiation and histamine release. we observed that bone marrow differentiated mast cells incubated with ova and il-9 had an induced histamine release. conclusions: thus, nfatc1 deficiency in t cells results in defective ige production affecting ige-orchestrated mast cell activation mediated through il-9. therefore, nfatc1 emerges as a novel target for anti-allergy intervention. 0546 | efficiency of the use of nitric oxide donors for the treatment of bronchial asthma bazarova s; djambekova g center of therapy, tashkent, uzbekistan introduction: to study the influence of nitric oxide donor-l-arginine-on indicators of endothelial system in patients with bronchial asthma. objectives: 58 patients aged 27-55 years (41 ae 4.12 years) with moderate persistent bronchial asthma were examined. ratio of men to women was 30/28. two age-and sex-matched groups were randomly selected. main group (32 patients) received nitric oxide donor -l-arginine in addition to standard background therapy (gina, 2012) . the medication (100 ml of 4.2% solution, tivortin, "yuria-farm", ukraine) was administered intravenously once daily for 10 days. the control group (26 patients) only received the background therapy. the efficiency was assessed with the use of conventional methods of study (clinical laboratory methods, instrumental methods: spirography, peak flow monitoring, bronchomotor tests). concentration of nitric oxide stable metabolites in exhaled breath condensate (ebc) and in blood was studied. their ratio was also assessed. results: baseline data in patients of both groups demonstrated the increase of level of nitric oxide stable metabolites in blood (0.78 ae 0.02 mmol/l) and in ebc (0.43 ae 0.01 mmol/l). after the treatment the positive clinical efficiency of inclusion of l-arginine was much higher than that in the control group (p<.05). in the main group, reduction of requirement for beta2-agonists, decrease of frequency of night-time symptoms, decrease of frequency of lowest pef rate in the morning and improvement of respiratory function were observed on average on the 3rd/4th day compared to the control group were such improvements were evident on the 7th/8th day. in the main group the concentration of nitric oxide stable metabolites significantly increased in blood (0.59 ae 0.02 mmol /l, p<.05) and in ebc (0.21 ae 0.03 mmol/l, p<.05). in the control group the concentration of nitric oxide stable metabolites changed in blood (0.69 ae 0.06 mmol/l) and in ebc (0.32 ae 0.05 mmol/l), but not significantly. introduction: abpa is currently believed to be an exaggerated form of aspergillus sensitization, and is probably the first step in its development. objectives: the aim of this study was to investigate the clinical and immunologic characteristics of fungal-sensitive asthma (fa), nonfungal-sensitive asthma(nfa) and abpa. conclusions: there were different clinical and immunological features among nfa, fa and abpa. the abpa had worse function as well as higher percentage of bronchiectasis, and higher dose of oral corticosteroid. besides, the sensitivity to aspergillus was more severe in abpa. the level of sige-a.f was associated with the damage of lung function. 0548 | self-reported allergic rhinitis and/or allergic conjunctivitis associate with il13 rs20541 genotypes in finnish adult asthma patients introduction: the increased prevalence of asthma and allergic diseases is a major public health problem worldwide. atopy, family history, inhaled irritants, and upper airway inflammation are known risk factors of asthma. a population-based sample of finnish adult asthma patients (n=1156) and matched controls (n=1792) filled a questionnaire. asthma was diagnosed based on a typical history of asthma symptoms and lung function tests. skin prick tests (spt) with 17 aeroallergens and blood tests including analysis of interleukin 13 (il13) rs20541 (g/a) genotypes were performed for a subsample (n=193). objectives: our aim was to observe in adult asthmatics with and without allergic co-morbidities e.g. subject-reported allergic rhinitis and/or allergic conjunctivitis (ar/ac) association with il13 rs20541 genotypes and other factors. results: the proportion of asthmatics reporting ar was 61.9% and ac was 40.7%. after adjustments, the presence of il13 rs20541aallele (or=3.06 ci=1.42-6.58, p=.004) or multi-sensitization (adjusted or=4.59, ci=1.48-14.26, p=.008) associated with ar/ac-asthma. nasal polyps and aspirin-exacerbated respiratory disease associated also with ar/ac-asthma. conclusions: adult ar/ac-asthma could putatively be a phenotype, characterized by the presence of atopic and/or eosinophilic factors and a high prevalence of the il13 rs20541a-allele. studies on the mechanisms behind this and in other populations are needed. 0549 | immunoregulatory role of nfatinteracting protein (nip) 45 in adaptive and innate immune responses in allergic asthma introduction: nfat-interacting protein (nip) 45 is a th2 associated transcription factor. after t cell receptor stimulation, the arginine methylation domain of nip45 supports the interaction with nfat, thereby enhancing the production of the th2 cytokine il-4. moreover, nip45 deficient mice have been shown to be deficient in il-4 and ifn-gamma production indicating that nip45 controls both th1 and th2 cytokine production and might therefore play a protective role in allergic asthma . objectives: we wanted to analyze the importance of nip45 in allergic asthma in pre-school children as well as adults. furthermore, we investigated the role of nip45 in a murine model of allergic asthma to find out more about its contribution to adaptive as well as innate immune responses in the disease. results: in the european study predicta, in collaboration with the children 0 s hospital in erlangen, we analyzed nip45 mrna expression by using quantitative real time pcr in rna extracted from pbmcs isolated from pre-school children with and without asthma. in the pbmcs of the asthmatic children, nip45 mrna expression was found significantly increased compared to healthy control children. furthermore, nip45 mrna expression was also found induced in cd4 + t cells in adult asthmatics from the asthma bio-repository for integrative genomic exploration (asthma bridge) study. we further analyzed the importance of nip45 in a murine model of allergic asthma. after allergen sensitization and challenge nip45 (-/-) mice showed decreased airway hyperresponsiveness, inflammation and mucus production, three of the main patho-physiological features of asthma. additionally, we discovered that nip45 (-/-) mice released decreased th2 type cytokines and also expressed less st2, which is the receptor for il-33, after allergen challenge. furthermore, a defect in innate lymphoid cell type 2 (ilc2) differentiation was observed in the absence of nip45 in allergic asthma and in bone marrow differentiated ilc2s indicating a crucial role for nip45 in mediating asthma via ilc2s. conclusions: in summary, we found that the lack of nip45 influences not only immune responses of the adaptive immune system but also influences components of innate immunity resulting in a abstracts | 181 protective phenotype to allergic diseases such as asthma. objectives: in the study were included 40 ap and 10 healthy controls (hc). fraction of exhaled no (feno), standard lung function parameters, complete blood count and absolute count of cells, serum ige, crp, il-6, il-17a and periostin were measured. results: four clusters were identified by cluster analysis: cluster 1 (c1)(n=14)-late-onset, non-atopic, eosinophilic asthma with impaired lung function, cluster 2 (c2)(n=13)-late-onset, atopic asthma, cluster 3 (c3)(n=6)-late-onset, aspirin sensitivity, eosinophilic asthma and cluster 4 (c4)(n=7)-early-onset, atopic asthma. we have found higher levels of il-6 in all clusters ap as compared to hc (c1: p=.001, c2: p=.017, c3: p=.012 and c4: p=.003). tendency for higher levels of serum il-6 in c1 compared with c2 or c4 (p=.089 or p=.062, respectively) was observed. periostin levels were significantly higher in c1 (p<.001), c2 (p<.001) and c4 (p<.01) as compared to hc. there were no differences in periostin levels between all clusters (anova, p=.389). il-17a levels were significantly higher only in c4 as compared to hc or to c1 and c2 (p<.05, for each comparison). we have found correlation between il-6 and crp (r=.640; p=.014) in c1, il-17a and periostin in c1(r=.631; p=.016) and in c2 (r=.641; p=.018). interestingly, we have observed negative correlation between the duration of asthma and il-17a (r=à.886; p=.019), but positive between the duration of asthma and crp (r=.886, p=.019) in c3. our results have shown higher levels of il-6 in all clusters as compared to hc that are associated with marker for systemic inflammation. periostin levels were significantly elevated in c1, c2 and c4 as compared to hc with no differences between the clusters. a positive correlation between periostin and il-17a in c1 and c2 was observed, that rising the question about their interrelationship in the pathogenesis of late-onset asthma. serum il-17a was significantly higher in c4 in comparison with hc or c1 and c2 suggesting that th17 mediated immunity may be involved in early-onset, atopic asthma. these data support the concept of heterogeneity of the bronchial asthma. 0551 | eosinophil activation with autophagy and extracellular dna traps is involved in severe asthma results: il-5+lps treatment significantly increased autophagy and eet levels from pbes of the study subjects (p<.05 for all), which were in a positive correlation (r=.802, p<.001). compared to nsa patients, both untreated and il-5+lps-treated pbes from sa patients had significantly higher autophagy levels (p=.007 and .002, respectively), while only il-5+lps-treated pbes from sa patients had higher eet level (p=.036). eet level from untreated pbes was correlated with serum eosinophil cationic protein level (r=0.608, p=0.036) and fev1% predicted value (r=à.620, p=.056). co-culture of aec with pbes slightly increased il-8 production, which was significantly enhanced by il-5+lps treatment (p<.001). the il-8 production in co-culture system was reduced by pretreatments with dexamethasone (1 mm, p=.001), antibodies against major basic protein (200 ng/ml), il-5 receptor (1 mm) and il-4 receptor (1 mm, p=.05 for all), but increased by cotreatment with micrococcal nuclease (10 iu/ml, p=.001). conclusions: pbes from sa patients are highly susceptible to be activated to produce high levels of autophagy and eet, which could enhance and maintain airway inflammation. we suggest that steroid and anti-il-5/il-4 receptor antibodies may be beneficial to control airway inflammation in severe eosinophilic asthma via inhibition of eet production, while dna digesting reagents may increase airway inflammation. introduction: it has been demonstrated that exposure to stress induce hyporeactivity of the hpa system by modifying the secretion of cortisol and also that psychosocial stressors such as poor social status are associated with an increased risk of childhood asthma, decreased serum cortisol and high ige response. thus, from this concept it could be postulated that a blunted hpa axis response may increase the risk for allergic inflammatory reaction. objectives: aim of this study was to evaluate the association of serum cortisol in pediatric allergic bronchial asthma and its influence on the ige immune response in a poor children community with psychosocial chronic stress. results: this was a pilot analytical case control study(50 ipa positive subjects and 50 healthy control paired by age and gender, both from poor areas of barranquilla objectives: in order to characterize bp14 an immunoproteomics analysis was conducted, i.e. electrophoretic separation of cypress pollen extracted proteins, ige western blotting using cypress pollen allergic patient's sera and mass spectrometry (lc/ms/ms) for identification of ige-binding proteins. results: ms analysis using chymotrypsin identified in bp14 a peptide also found in the family of protein snakin/gibberellin-regulated protein (grp). the snakin-1, an anti-microbial peptide of potato, produced as a 63 amino-acid recombinant protein (homologous to the c-terminal part of grp), is recognized by ige from cypress pollen allergic patients with ige to bp14. this ige reactivity is abolished after reduction of disulfide bridges and is inhibited by a cypress pollen extract. ige reactivity to bp14 is however barely inhibited by the recombinant potato snakin-1. conclusions: bp14 exhibits a molecular mass closer to grp than to snakin and the absence or very low inhibition of the ige reactivity to bp14 with snakin may be explained by peptidic ige epitopes on the n-terminal part of bp14. the potato snakin-1 share 83% sequence identity with peamaclein, the peach allergen pru p 7, also shown in citrus. these results might explain the peach/cypress and citrus/cypress syndromes described and point out bp14 as the cross reactive allergen. the proteins of snakin/grp family present in many fruits and vegetables might include allergens involved in pollen/food associated syndromes. introduction: exposure to high levels of grass pollen may lead to a high degree of allergic inflammation, sensitization to minor allergens, such as profilin, and development of severe profilin mediated food reactions, similar to those described due to sublingual immunotherapy (slit). objectives: our objective here was to identify genetic biomarkers in order to generate a model that can improve the classification and treatment of patients with a higher probability of developing severe adverse reactions. results: 6 healthy subjects (group 1, control) and 17 patients with mild (group 2) or severe (group 3) profilin mediated food reactions were studied. rna extraction was performed on ficoll-isolated pbmcs using the rneasy ® mini kit (qiagen) and its integrity was analyzed with experion rna stdsens analysis kit (bio-rad). the gene expression profile of all the samples was analyzed using the gene-chip ® wt plus reagent kit (affymetrix) and two specific software: affymetrix ® expression console ™ and affymetrix ® transcriptome analysis console (tac). finally, the microarray data was validated by quantitative real-time pcr (qpcr). the hierarchical clustering of the samples shows the separation of the patients into three clusters coincident with the three established clinical groups (control, mild and severe). genetic profile of patients with mild reactions is similar to healthy patients while severe subjects were significantly different from the other two groups. genes regulated in the severe group were related to histone modification pathways, human complement system, cell adhesion and tgf-b and its receptor. these changes may be associated with the different degree of inflammation between patients in each experimental group. in the course of our study we found out that severe patients had different rna expression patterns compared to mild and non-allergic patients. this lead to the identification of genetic biomarkers useful for the generation of a model able to predict severe reactions and/or adverse reaction during immunotherapy, thus improving the diagnosis and treatment of this type of allergic results: his-tagged recombinant allergens, namely parvalbumin, aldolase, enolase and tropomyosin from c. idella and l. crocea, as well as cod parvalbumin, were synthesized in e. coli and purified using immobilized metal-chelate chromatography. 14 children with history of immediate-type fish allergy were included in this study and their serological ige reactivity to the fish allergen components were measured by elisa. 11 children were positive to at least one allergen component, with nine of them being positive to parvalbumin. despite the high similarity between l. crocea, c. idella and cod parvalbumins (70.6-81.6%), three children only reacted to l. crocea and c. idella parvalbumin but not to cod parvalbumin, while the other six children were positive to all three parvalbumins. competitive inhibition elisa revealed that c. idella parvalbumin inhibited >85% of the binding of specific ige to both l. crocea and cod parvalbumin, while reciprocally only inhibition of 60% and 50% could be achieved respectively. two children were reactive to aldolase and enolase but not parvalbumin. one of them had positive sige to both enolase and aldolase from l. crocea, while the other reacted to aldolase from both species. these two children exhibited relatively mild subjective allergy symptoms (itchy skin and throat). notably, three children were non-reactive to all components tested, and two of them were outgrowing fish allergy clinically. introduction: anti-a-gal antibodies are naturally produced in response to the gastrointestinal flora. in red meat allergy, patients develop ige antibodies towards the a-gal epitope, which itself are structurally closely related to the blood group b antigen. objectives: this study aimed to explore the relationship between a-galand b-antigen-specific antibodies in red meat allergic patients compared to healthy individuals with blood group b or a/o. sera from 39 red meat allergic patients and 84 healthy blood donors of whom 52 belonged to blood group b and 32 to blood group a or o were included. ige reactivity against a-gal and the b-antigen were determined using immunocap. allergen-specific igg, igg1, igg2, igg3, igg4 and ige were assessed by indirect elisa assay. statistical analysis was performed using spearman rank correlation and unpaired t-tests. results: all red meat allergic patients, 12 of the healthy a/o and 3 of the healthy b donors were ige positive to a-gal. however, the ige levels to a-gal were significantly higher in the allergic group compared to the healthy a/o and b individuals. the majority of the meat allergic patients, but none of the healthy individuals had ige antibodies against the b-antigen. a moderate correlation between a-gal and b-antigen specific ige was noted (r 2 =.48). the red meat allergic patients had significantly higher igg levels against a-gal with igg1 and igg4 antibodies as the predominant difference compared to the healthy individuals. the 12 healthy a/o ige positive individuals had significantly higher igg1, igg3 and igg4 compared to the ige negative individuals. the igg response to the b-antigen followed the same pattern as to a-gal. there was a low correlation between the igg levels against a-gal and the b-antigen in both meat allergic patients and healthy a/o individuals (r 2 =.30 and .33). introduction: fx5, a food mixture of milk, egg white, fish, peanut, wheat and soybean, is largely used for food allergy detection. besides the fact that it is questionable if this is the better approach to identify a food allergy, the information that the test provides may represent a pitfall because a positive or negative result does not mean food allergy presence or absence, respectively. objectives: the goal of our study was to access the reason for fx5 request in a pediatric hospital and to analyze its suitability. methods: the fx5 requests, performed in a pediatric population of d. estefânia hospital over a five months' period, were analyzed, concerning demographic data, reason for request and attitude taken due to the result. this test was requested due to respiratory symptoms in 37 patients, gastrointestinal symptoms in 23 patients, cutaneous symptoms in 20 patients and nonspecific complaints in 48 patients. all of these symptoms were not directly related with food intake. a positive result was obtained in 25 patients; of those, only 15 were referenced to our immunoallergology department. in all of them a detailed clinical history was obtained and diagnostic tests (skin prick tests and specific ige) were performed in the ones considered suitable. food allergy was diagnosed in only one patient. conclusions: in the vast majority of patients, fx5 was asked for nonspecific complaints and often without a clinical history suggestive of food allergy. moreover, a positive fx5 test does not mean clinical reactivity or food allergy. on the other hand, the clinical history allows us to identify a suspect trigger in most of the children with food allergy. in such cases, it is preferable to request the specific ige towards the allergen, which gives a more precise and accurate result, instead of the fx5. as our results showed, in the majority of the cases, the fx5 request was often made without complying with a reasonable criterion, implying unnecessary costs. the high number of requests verified may be explained because it is an easily accessible analysis but this attitude should be discouraged. tuppo l 1 ; alessandri c 2 ; pasquariello s 3 ; petriccione m 3 ; giangrieco i 1 ; tamburrini m 1 ; rafaiani c 2 ; ciancamerla m 2 ; mari a 2 ; ciardiello ma 1 1 istituto di bioscienze e biorisorse -ibbr-cnr, naples, italy; 2 caam -centri associati di allergologia molecolare, rome, italy; 3 cra, research unit on fruit trees, caserta, italy introduction: pomegranate, punica granatum l., is one of the oldest cultivated fruit trees. the fruit contains the arils, which are seeds covered by a red pulp, that is a juice sac. the arils are surrounded by the white and fleshy mesocarp. pomegranate can trigger allergic reactions, but the allergenic pattern of this fruit is still poorly characterized and only one allergen, the ltp pun g 1, was reported. objectives: the aim of this study was the investigation of the allergen pattern in pomegranate tissues and cultivars. reported symptoms were food impaction (68%), dysphagia (65%), heartburn (33%) and vomiting (30%). the first 2 symptoms were more frequent in adolescents and adults (96%). children's main complaint was vomiting (73%) and 4 cases presented failure to thrive. most patients had associated allergic diseases (88%), 23% had previous food allergy and 74% were sensitized to aeroallergens. endoscopic evaluation revealed esophageal stricture in 5 patients. at least half of diagnostic esophageal biopsies had >20 eosinophils per highpower field and 33% showed microabscesses. food sensitization was found in 61% of the patients, mainly to cow's milk (46%), nuts and peanut (33%), cereals (30%) and egg (21%). considering therapeutic approach, 88% were treated with swallowed fluticasone and dietary elimination was recommended in 54%. oral corticosteroids were prescribed in 8 patients. at present time 42 patients had done endoscopic reevaluation, 30 (71%) showed histologic resolution. five patients had clinical and histologic relapse during follow-up. conclusions: eoe has a balanced distribution but a distinctly clinical presentation accordingly to age group: children may present unspecific symptoms like vomiting, whereas adolescents and adults complain of food impaction and dysphagia. other atopic diseases and food sensitization is very common. introduction: air pollution, particularly ambient air particulate matter (pm), is considered as one of the most important environmental risks for human health. pm could potentially disrupt immune regulatory mechanisms and predispose exposed individuals to asthma. in contrast, children exposed to traditional farm environment seem to have a natural resistance to asthma. this phenomenon links with exposure to stable dust and subsequent immune regulatory mechanisms initiated in the airways. the underlying exposure agents and definitive pathways determining the risk of asthma are still to be identified. objectives: our aim was to investigate the effect of urban air pm (high risk environment) and farm dust (protective environment) on immune responses in finnish children. briefly, peripheral blood mononuclear cells (pbmcs) of 4-year-old children (n=18) were stimulated with farm dust extract (40 lg/ml, stable in northern savonia, finland) and pm samples (75 lg/ml, pm 2.5-1 , pm 1-0.2 or pm <0.2 , nanjing, china) for 18 hours. expression of immunogenic cd80 and tolerogenic ilt4 in circulating myeloid dendritic cells (mdcs) and plasmacytoid dcs (pdcs) and monocytes were analyzed by flow cytometry. pm samples were analyzed for polycyclic aromatic hydrocarbons (pahs), inorganic ions and elements. farm dust sample will be analyzed for microbial content. results: pm stimulation decreased the percentage of cd80+ monocytes and dcs among children's pbmcs, whereas farm dust stimulation increased the percentage of cells positive for this marker. the percentage of tolerogenic ilt4+ was decreased in all cell types after stimulation with pm. farm dust also decreased the percentage of ilt4+ monocytes, but not dcs. specific metals and pahs in pm associated with the studied immunological parameters. conclusions: samples from high risk and protective environments have differing capacities to influence immunogenic and tolerogenic properties in children's immune cells. the importance of these findings in relation to the risk of asthma in exposed populations will be studied further. introduction: alterations in cell surface glycosylation pattern is a common feature of tumor cells that might be related to immune evasion and malignancy. objectives: to study the capacity of the carbohydrate a10 (ca10) located in the surface of murine ehrlich tumor (et) cells and also in certain human adenocarcinomas to condition the phenotype and function of human dendritic cells (dcs) and the capacity to polarize t cell responses. results: nf-jb/ap-1 are not activated in thp1 cells by ca10, however, this carbohydrate partially impairs the activation of these transcription factors induced by the tlr2 ligand pam3csk. ca10 induces the expression of the tolerogenic marker pdl1 in human monocyte-derived dcs (hmodcs) as well as the production of il-6 and il-10, analyzed by flow cytometry and elisa assays respectively. ca10-activated hmodcs generate functional il-10-producing cd4 + cd25 high cd127 -foxp3 + regulatory t (treg) cells that were able to inhibit the proliferation of cd4 + t cells from pbmcs in a dose-dependent manner. supporting the role of ca10 in the induction of treg cells, our in vivo data showed that the ca10 is present in the sera of tumor bearing mice and the percentage of foxp3 + treg cells is increased in the regional (inguinal) lymph nodes from tumor bearers. our results showed that ca10-activated hmodcs induce the generation of foxp3 + treg cells both in vitro and in vivo, which might well condition the immune response against the tumor and promote the tumor escape. 0567 | assessment of changes in expression of immune system biomarkers to assist the differential diagnosis of acute bacterial infections introduction: biomarkers for acute infections include c-reactive protein, mmp-9, sicam-1, procalcitonin, and neutrophil band counts for bacterial infection. a rapid means of assessment of acute bacterial infections via biomarker assessment was sought. objectives: the expression of toll-like receptors (cd282 and cd284), complement receptors (cd35 and cd88), integrins (cd11b and cd11c), fc-receptors (cd32 and cd64) and l-selectin (cd62l) on the surface of blood neutrophils and monocytes stimulated with inactivated e. coli, l. acidophilus, e. coli derived bacterial ghosts, e. coli lps and l. acidophilus cell walls was assayed using flow cytometry. both the percent of expression and mean fluorescence intensity (mfi) were analyzed for each molecule. results: all the bacterial components used exerted similar activation capabilities even in low concentrations. while the expression of cd11b, cd11c, cd32, cd35 and cd88 was enhanced by both neutrophils and monocytes under activation, the expression of cd64 significantly increased only in neutrophils. the expression of tlr2 and tlr4 was slightly increased by neutrophils and monocytes. the expression of cd62l by monocytes and neutrophils (the percent of activated cells as well as the mfi) was decreased during activation. there was a negative correlation between cd62l expression and integrins (cd11b and cd11b). the activation index was calculated for each molecule as a ratio of expression of molecule by activated cells vs cells used as a negative control (resting). the highest values for the activation index was seen with cd11b, cd11c, cd32, cd35, cd62l and cd88 mfi by neutrophils and monocytes, and the percent of cd64 expression by neutrophils. conclusions: e. coli and bacterial ghosts significantly increased the expression of cd11b, cd11c, cd32, cd35, cd62l and cd88 by neutrophils and monocytes even in very low concentrations, suggesting use as potential biomarkers in the differential diagnosis of the etiology of acute infections. objectives: the aim of this study was to evaluate changes in peripheral blood monocyte expression of cd16, cd163, cd206, cd209, hla-dr, and cd47 in kidney allograft recipients. in total, 88 patients who underwent renal transplantation from a deceased donor were enrolled in the study. the phenotype was evaluated by a multicolor flow cytometry in defined time points and in the case of complications requiring fine needle aspiration biopsy procedure. the results confirmed our pilot data, proportions of peripheral cd14+cd16+ monocytes were downregulated during the first week after the kidney transplantation while the percentage of cd14+cd163+ monocytes dramatically increased early after the kidney transplantation and remained high for at least four months in most patients. the expression of cd206 (marker of m2 macrophages) was limited only to a small population of monocytes (less than 5% in most patients) but the receiver operating characteristic (roc) curve analysis showed its potential importance by significant correlation with acute rejection with a sensitivity of 70% and specificity of 80.33% (area under the roc curve 0.7787, p-value: 0.004973). no correlation between two different m2 markers cd163 and cd206 has been found. the expression of cd209 (dc-sign) was low and did not show any changes in time or association with acute rejection. hla-dr (mhc ii) and cd47 (integrin associated protein) were constitutively expressed without any significant changes in patients with acute rejection of the allograft. we assume from our data that kidney allograft transplantation is associated with early reciprocal modulation of monocyte subpopulations (cd14+cd16+ and cd14+cd163+). a decreased proportion of cd206 positive blood monocytes seems to be associated with an increased risk of acute rejection of kidney allograft. introduction: thioredoxin (trx), a 12-kda oxidoreductase enzyme, is well known to be a redox-active protein that regulates reactive oxidative metabolism. in addition to its anti-oxidative activ0570 | progranulin-dependent regulation of th2 airway inflammation by house dust mite allergen introduction: progranulin is a growth factor that consists of 593 amino acids including 71/2 repeats of cysteine-rich motifs, and produced by variety kinds of cell. progranulin is involved in the regulation and maintenance of inflammatory response, and its role is wellstudied in neuronal and metabolic diseases such as neurodegeneration and type 2 diabetes. however, the role of progranulin during the development of airway inflammation induced by inhaled allergen is still obscure. objectives: in this study, we evaluated the role of progranulin in the development of th2 airway inflammation induced by house dust mite allergen. results: to find the main source of progranulin, we stimulated each cell line with various doses of house dust mite allergens. the production of each cytokine, including progranulin, was estimated in culture supernatant by elisa. to investigate the role of progranulin in airway inflammation, we intranasally administrated house dust mite allergens to 6-week-old female progranulin knock-out mice (macrophage-specific) or littermate mice. lung inflammation and immunological parameters were evaluated at 15 h after first sensitization with allergen or 24 h after final allergen challenge. the production of progranulin was significantly elevated by house dust mite allergen stimulation in innate immune cells, especially in alveolar macrophages over other cells. in the house dust mite allergeninduced airway inflammation model, we found that the level of progranulin increased earlier than other pro-inflammatory cytokines. in addition, in macrophage-specific progranulin knock-out mice, airway inflammation was down-regulated in the earlier phase after exposure to house dust mite allergen. moreover, we stimulated mice with house dust mite allergen for a longer period to observe the changes in the adaptive immune response of th2 airway inflammation, which was found to be decreased in conditional knock-out mice. conclusions: these findings indicate that th2 airway inflammation induced by house dust mite allergen is dependent on progranulin. objectives: the aim of the present study was therefore to investigate the immunomodulatory effect of epinephrine on m2a macrophages and its consequence on cross talk to mast cells in a human model of allergic inflammation. results: primary monocytes from healthy pbmcs were first differentiated into m2a macrophages using m-csf in the presence of il-4 and il-13 cytokines. after overnight incubation with epinephrine, supernatants were collected and analyzed by elisa for il-10, tnf, il-6, ccl1, il-12 and ifn-c, whereas cell surface markers including cd206, cd163 and cd86 were evaluated using flow cytometry. subsequently, both m2a and epinephrine-treated m2a supernatants were transferred onto cord blood-derived mast cells (cbmcs) for further overnight incubation, after which ige-mediated degranulation was assessed by the ß-hexosaminidase release assay. after overnight epinephrine treatment, m2a macrophages showed an increase in il-10, ccl1, tnf and il-6 production, but no ifn-c and il-12 expression was observed. epinephrine treatment also downregulated surface markers cd206 and cd163 and upregulated cd86. when supernatants from epinephrine-treated m2a macrophages were added to cbmc cultures, ige-mediated degranulation was impaired compared to cbmcs treated with supernatants of unstimulated m2a macrophages. conclusions: taken together, epinephrine promoted a phenotypic shift of m2a polarized human macrophages toward an m2b-like regulatory phenotype that was able to reduce the ige-mediated degranulation of cbmcs. we conclude that prolonged acute stress exposure in allergic patients may attenuate symptoms of acute allergy by directing macrophages towards an immunosuppressive phenotype, which can further dampen mast cell degranulation. objectives: a murine local lymph node assay was used to investigate the effect of oa on the immune response to the known skin sensitizer hexyl cinnamic aldehyde (hca, 25% w/v). the ear lobes tape stripped prior to immunization. test solutions (25 ll) were applied on the dorsal side of each ear on three consecutive days. female balb/c mice (8 groups a 6 mice), were exposed to the vehicle acetone:olive oil (4:1) alone, or in combination with hca, with or without oa in the concentrations 5, 10 and 20%, or oa alone (5, 10 and 20%). on day 5, the animals were weighed and exsanguinated by cardiac puncture. the auricular lymph nodes were harvested for single cell preparation, stimulation with cona and cytokine release of il-2 and il-17. the earlobes were excised and fixed for immunohistochemistry. results: no group differences were found for bodyweights or bodyweight change, number of lymph node cells or il-17 secretion. il-2 showed a tendency of dose-related increase, but a significant difference were only found between hca and hca+10% oa (p=.034) in one out of the two experiments. in he stained sections, the epidermal thickness was significantly increased in groups given hca + 10 and 20% oa (p≤0.001). sections immunostained with anti-ly6g showed significant increase in neutrophil influx for the same groups as above (p≤0.001). oa alone showed no effects or effects significantly lower than hca + oa. objectives: we hypothesized that plasma s1p levels in cf patients might be associated with cftr mutations and could influence disease presentation. results: plasma was collected with a defined standard operation procedure to impede unspecific s1p release from blood cells from 20 double lung transplanted adult cf patients as well as 20 sex-and age-matched, non-allergic healthy controls all being fasted overnight. total plasma s1p was measured by mass spectrometry and unbound plasma s1p by elisa. levels were correlated with cftr mutation status, routine laboratory parameters and clinical symptoms. we observed higher total and unbound plasma s1p levels in healthy controls compared to cf patients with the latter value reaching statistical significance (p=.044) after exclusion of two statistical outliers. unbound plasma s1p levels were significantly higher in df508 homozygous cf patients compared to patients with df508 heterozygosity (p=.029). 2 patients with other mutations were excluded. levels of unbound s1p were positively correlated with hemoglobin and negatively correlated with triglyceride levels. additionally, we observed a positive correlation of total plasma s1p levels in cf patients with hba1c. gastrointestinal symptoms were more common in df508 heterozygous (6/8) compared to df508 homozygous cf patients (4/10). fecal calprotectin levels were found to be significantly higher in df508 homozygous compared to heterozygous cf patients (p=.047). differences in unbound s1p levels were not correlated with immunosuppressive treatment after transplantation. conclusions: to the best of our knowledge this is the first clinical study directly correlating plasma s1p levels with cf genotypes and clinical presentation in cf patients. we emphasize to evaluate s1p as a potential novel disease biomarker as well as a therapeutic target for cf in future studies. supported by the austria science fund grant kli 284 (to eu). ochoa-grull on j 1 ; tejera-alhambra m 2 ; guevara k 1 ; guzm an-fulgencio m 2 ; benavente cm 3 ; mart ınez r 3 ; p erez c 3 ; peña a 3 ; rodr ıguez de la peña a 1 ; llano hern andez k 1 ; rodr ıguez-fr ıas e 1 ; s anchez-ram on s 1 objectives: we show preliminary data of one study aimed to evaluate the use of this strategy in the prevention of rrti in infants and preschool children. results: patients: 121 children (70 male and 51 female, age range 6-35 months) were included in a randomized double blind and placebo-controlled study (eudract 2012-002450-24) . all of them showed negative in vivo and in vitro allergy tests. active treatment consisted in a suspension of a mixture of selected strains, grown and inactivated in optimal conditions, of s. aureus (15%), s. epidermidis (15%), s. pneumoniae (60%), k. pneumoniae (4%), m. catarrhalis (3%) and h. influenzae (3%) in physiologic saline solution with 50% glycerol at a concentration of 300 formazin turbidity units (ftu)/ml (equivalent to 10 9 bacteria/ml). placebo did not contain any bacteria. patient were treated for a period of 6 months, receiving 2 daily sublingual puffs of active or placebo and with a follow-up of other 6 months (total period of evaluation was 1 year). symptom (cough, dyspnea, wheeze, mucus, fever, discomfort) and medication (inhaled corticosteroids, beta adrenergics, montelukast, antibiotics) scores were evaluated since the first day of treatment to the end of the study (1 year) . any adverse event was recorded to assess safety. for the comparison between both groups, t test was used. patients who received active treatment experienced an improvement of 39% over placebo in overall symptoms and 38% in medications scores (p<.01). in the combination of symptoms and medication scores the improvement was 38% (p<.0001). no adverse events related to treatment were recorded. conclusions: immunostimulation with these selected strains of bacteria is safe and can be successfully used in infants and preschool children in order to prevent rrti. introduction: to reduce the duration and the risk of the allergen specific immunotherapy using commonly used allergen extracts, new highly immunogenic and non reactogenic vaccines are needed. objectives: the goal of the present study was to employ the ap205 spytag/catcher system to develop a virus-like particle (vlp) vaccine based on the major house dust mite (hdm) allergen der p 2 and to evaluate its reactogenicity in vitro. spycatcher-ap205 vlps and recombinant der p 2, fused at the c-terminus to the 13 amino acid spytag binding-partner, were expressed in e. coli. purified spytagged der p 2 was mixed with spycatcher-vlps, which resulted in covalent conjugation of der p 2 to the surface of spycatcher-vlps. excess unbound der p 2 was removed by dialysis. dynamic lightscattering (dls) was used to analyse the size and aggregation state of vlp-der p 2. the ige reactivity of vlp-der p 2 was assayed by direct elisa and by rat basophil degranulation assays. conclusions: our results demonstrate that coupling of spy-tagged der p 2 to ap205 spycatcher-vlps dramatically reduces the reactogenicity of the allergen, suggesting that vaccination with ap205 vlp-der p 2 may be a safe and effective treatment for hdm allergy. objectives: the qm1s hybrid protein is a qm1 variant where cysteine amino acids have been replaced by serine. the expression of qm1s hybrid protein was performed in e. coli bl21(de3) after iptg induction. the purification of qm1s protein was performed from inclusion bodies by a three-step chromatography process. the stability of qm1s was analyzed by sds-page and total protein assay. qm1s ige-binding capacity was compared with natural der p 1 and der p 2 by elisa-inhibition and allergenicity was studied by mediator release from rbl cells. immunogenicity was evaluated in mice by analysis of the specific igg response to der p 1 and der p 2. results: qm1s was expressed in complex media as inclusion bodies that were solubilized in urea. soluble protein was purified by anionic ion exchange, hydrophobic interaction, and size exclusion chromatography in the presence of a detergent. qm1s purification process was shorter and more efficient than that of qm1. the purity obtained was >95%. elisa inhibition assay showed that qm1s hybrid protein was almost unable to inhibit ige-binding to the hdm extract, less than 10% in all the range of concentrations tested (0.1-10 000 ng/ml) and representing a 40-fold reduction as compared to qm1. qm1s showed a great reduction of the b-hexosaminidase release in rbl cells, compared to der p 1 and der p 2. qm1s was able to induce der p 1-and der p 2-specific lgg antibodies responses comparable with those induced by the mixture of wildtype allergens. mouse igg antibodies induced by the hybrid proteins qm1s and qm1 showed similar ige-blocking antibodies properties to mixture of der p 1 and der p 2. the stability of qm1s was studied in solution at 25°c, 4°c, and -20°c and lyophilized at 4°c, being the frozen and lyophilized forms the best conservation conditions. the qm1s hybrid exhibited less ige-binding activity than qm1 and the natural der p 1 and der p 2 while retained the immunogenicity. these properties together with the improved manufacturability made qm1s a good candidate for sit to hdm allergy. objectives: slit tablets of cockroach, slit tablets of parthenium, slit tablets containing both allergens together (mix) and slit bilayer tablets containing one layer with parthenium allergen and other layer with cockroach allergen, compress to single tablet were prepared. punches and dies of 11 mm were used for compression. slit drops containing cockroach, slit drops of parthenium and slit drops containing both allergens together, were prepared and filled in 10 ml amber colored dropper vials. results: tablet formulations were evaluated for thickness (3.5-3.6 mm), weight variation (357-402 mg), hardness (2.0-2.5 kg/cm 2 ), disintegration time (not more than 2 min.), and biologically active content (90%-110% of the stated label claim). in-vitro dissolution test was performed as per usp using distilled water as the medium and the release was shown between 70 to 80% in 5 minutes. the liquid formulations were analyzed for ph (7.0 -7.5), the biological content (90% -110% of the label claim), specific gravity ( introduction: virtually all patients suffering from the common birch pollen allergy exhibit ige against the bet v 1 relevant allergen. as such, an elisa method for the quantification of bet v 1 was selected and validated as part of the bsp090 project, aiming to establish reference methods for the european pharmacopoeia. herein, we report the mapping of the epitopes recognized on recombinant bet v 1 allergen by the two specific murine monoclonal antibodies (mabs) used for the accurate and precise quantification of bet v 1 within birch pollen extracts. objectives: in order to investigate the ability of mabs 5b4 and 6h4 to recognize various bet v 1 isoallergens, we first carried out immunochromatography combined with electrophoresis. epitope mapping was performed by hydrogen/deuterium exchange (hdx) coupled with mass spectrometry (ms) analysis, using a gmp-grade purified rbet v 1 molecule. results: immunochromatography unveiled that both mab 5b4 and mab 6h4 capture most of bet v 1 isoallergens present within birch pollen natural extracts. those two mabs cross-react with the aln g 1 allergen from alder pollen but do not react with the cas s 1 chestnut pollen allergen. hdx-ms experiments combined with site-directed mutagenesis evidenced that mabs 5b4 and 6h4 target two distinct epitopes. the hdx-defined 5b4 epitope is discontinuous and contains a dominating sequential element (i.e., loop ile56-lys68). the hdx-defined 6h4 epitope is also discontinuous and mainly composed of regions ile44-lys55 and arg70-phe79. conclusions: overall, this study provides a precise molecular characterization of epitopes within bet v 1 recognized by mabs 5b4 and 6h4, confirming that these two antibodies target distinct non-overlapping epitopes and recognize the vast majority of currently introduction: short or common ragweed (ambrosia artemisiifolia), belonging to the aster plant family, sheds enormous amounts of highly allergenic pollen late in the summer. due to its high allergenic potential ambrosia artemisiifolia is becoming a health threat in north america and europe. hal allergy is developing a subcutaneous immunotherapy for patients suffering from ragweed pollen allergy. a standardized ragweed pollen extract, chemically modified and adsorbed to aluminium hydroxide (al(oh) 3 ), is being investigated for its potential use in immunotherapy. objectives: ragweed extract (re) was modified by glutaraldehyde followed by adsorption to al(oh 3 ). in vitro, a mediator release assay (mra) using hurbl (humanized rat basophil leukemia) cells was performed. hurbl cells were pre-sensitized using individual sera of ragweed-allergic patients and challenged with serial dilutions of re and modified re starting at 10 lg/ml followed by eight 1/10 dilutions (0-10.000 ng/ml). antigen-specific release of ß-hexosaminidase was measured and calculated in relation to 100% release values. in vivo, the immunogenic potency of modified re was evaluated by measuring the induction of re-specific igg in mice. female balb/c mice (n=10 per group) were subcutaneously (s.c.) injected with 20.000 aueq/ml re or modified re adsorbed to al(oh) 3 (0.8 mg/ml) per mouse on days 0, 7, 14 and 21. control mice (n=6) were injected with matrix only. specific igg titres were determined in serum obtained at days -1, 13 and 28. results: the potency of modified re in mra was drastically reduced in all patients, with a mean reduction of 1000 fold or more. chemical modification resulted in a later onset of activation as well as a lowering of the maximum release of ß-hexosaminidase. in vivo, both re and modified re show comparable levels of re-specific igg antibodies in mice at day 28 of the immunogenicity model. shown that chemical modification impairs the capacity of re to activate basophils while retaining its capability to be immunogenic. therefore, chemical modification of re may be a promising approach for the development of a safe and effective immunotherapy for ragweed allergy. objectives: the children who had taken subcutaneous conventional venom immunotherapy in our pediatric allergy outpatient clinic between 2002 and 2015 were evaluated with respect to the side effects. in addition, each child was called to ask if the patient was exposed to bee sting and the result of a sting during immunotherapy. introduction: the major unmet needs for allergen immunotherapy (ait) are improved efficacy with good tolerability, and high adherence. to achieve these, allergoids, peptides and recombinant proteins are potentially the answer but their low rate of systemic aes make selection of the optimal dose difficult. to select the dose for an ultra-short course subcutaneous birch ait, the company has adopted the use of a conjunctival provocation test (cpt), a wide range of doses and the multiple comparison procedure -modelling (mcp-mod) statistical analysis to test for a dose response and to determine the shape of the dose response curve. objectives: a range of dose regimens of 5100 su, 15300, 20100 and 27300 su were compared with placebo with respect to reduction of total symptom score elicited by cpt after treatment. patients were administered 6 weekly injections outside the pollen season. cpt was performed at screening, at baseline and 4 weeks after completion of treatment. the study was undertaken in 28 sites in germany and austria with 370 patients. the primary efficacy analysis was performed on a modified full analysis set (fas). the mcp-mod methodology was used to test for a dose-response using the placebo and above doses to describe the shape of the dose response curve. three candidate models were pre-specified: a maximum possible effect for the agonist (emax) model, a logistic model, and a linear in log-dose model. a statistically significant dose-response (p<.01) was shown for the range of cumulative doses, which approached a plateau with the 27300 su dose. the median effective dose (ed-50) was 2600 su. only minor differences were observed between the six active treatment groups in prevalence of treatment-emergent adverse events (between 70.9% and 83.9% of patients with overlapping 95% two-sided confidence intervals), majority of which were local reactions, short-lived and mild. teaes classified as systemic reactions were seen in 2.0% (20100 su group) and in up to 15.1% (5000 su group) of patients in the active treatment groups, and in 11.3% of patients in the placebo group. no treatment related saes were observed. adherence was >90% in all treatment arms. the ed-50 was 2600 su, demonstrating that the currently marketed dose (5100 su) is effective. the highest 27300 su dose will be further investigated in a pivotal phase iii trial having achieved an increase in efficacy by 50% without differences in the onset of aes between the treatment arms. introduction: in order for allergen immunotherapy (ait) to induce long-term immunological and clinical effects prolonged administration is required. therefore adherence to treatment is crucial for its efficacy. there is currently limited data available on ait adherence beyond clinical trials i.e. in real-life clinical practice. objectives: this eaaci immunotherapy interest group endorsed survey aimed to prospectively evaluate adherence to sublingual and subcutaneous immunotherapy in adults with allergic respiratory diseases and hymenoptera venom allergy in real life practice across different european countries. in addition, the reasons for lack of adherence and discontinuation of treatment were explored. this was a prospective, multi-centre, observational survey which took place in eight countries: czech republic, georgia, germany, greece, italy, poland, portugal and spain. data collection involved an online survey that followed participants four-monthly for a period of 36 months from the start date of ait. results: a total of 1336 participants were included in the analysis. introduction: allergic rhinitis is a multiple gene-regulated disease involved in many immune cells such as mast cells and eosinophils, and various inflammatory mediators, and mirna probably plays a critical regulatory role in its pathogenesis. therefore, studies on the functions of critical mirna and its regulatory mechanisms in activated mast cells will lay an important theoretical foundation for our understanding of ar pathogenesis and the development of therapeutic strategies. objectives: to investigate the effect of mir-125a-3p on mast cell activation in an ar mouse model. the number of sneezes and the frequency of nasal rubbing in ar+mir-125a-3p group were significantly reduced compared to those for ar+mir-nc group (p<.05). histological examination showed that inflammation in the nasal mucosa from ar+mir-125a-3p group was slighter than that in ar+mir-nc group. the number of mast cells in ar+mir-125a-3p group was increased compared to ar+mir-nc group (p<.05). the levels of histamine and il-13 in nasal lavage fluid supernatants, histamine in plasmas and il-13 in sera were significantly decreased in ar+mir-125a-3p group compared to ar+mir-nc group (p<.05). conclusions: upregulation of mir-125a-3p can reduce allergic inflammation in the nasal mucosa of ar and alleviate ar symptoms through inhibiting mast cell activation in vivo. mir-125a-3p probably becomes a new target for gene therapy of ar. 0222 | correlation between chronic cough and chronic rhinosinusitis in adults: nationwide, population-based, and cross-sectional study the second hospital of shandong university, ji nan, china; 2 national university of singapore, singapore, singapore introduction: nasal polyp implies a refractory clinical course in case of chronic rhinosinusitis (crs). although hypoxia is believed to be associated with nasal polyposis, little is known about the mechanism underlying polypogenesis. objectives: the aim of this study was to assess mrna and protein introduction: nasal polyp is a multi-factorial disease commonly associated with inactive ciliary beating frequency (cbf), a condition partly attributed to the mis-localization of dnah5, a component of the outer dynein arm in ciliary axoneme. so far however, there have yet to be a systematic histopathological investigation directly linking dnah5 localization pattern in nasal polyps. therefore, we sought to examine the localization of dnah5 in cilia structure of both nasal polyps and inferior turbinates from healthy individuals, and assess whether there are any localization changes that can account for the extensive inactive cbf observed in nasal polyps. objectives: the focus of this work is to compare the localization of dnah5 from the nasal polyps biopsies (n=80) and normal inferior turbinates (n=19) by immunofluorescence. the characterization of each sample was obtained from an average of 10 fields at 4009 magnification. results: from the samples, we observed three distinct localization patterns of dnah5 in the nasal cilia. the three patterns were as follows: 1) the localization of dnah5 in normal cilia is present throughout the entire axoneme (pattern a); 2) the localization of dnah5 is within the axoneme except at their proximal regions (pattern b); 3) the localization of dnah5 is restricted exclusively at the ciliary base and not present in the entire axoneme (pattern c). approximately 96% of the samples exhibited more than one distinct localization patterns for dnah5 within the observed fields. the percentage of pattern a, pattern b and pattern c were observed in 36.5%, 24.7%, and 38.8% fields for samples from nasal polyps. correspondingly, 75.1%, 19.3%, and 5.6% were observed for samples from healthy controls. the results indicated that the predominance of "pattern c" in nasal polyps countered by "pattern a" in inferior turbinates from healthy individuals. conclusions: our study indicated that there is a significant increase in the mis-localization of dnah5 among the cilia in nasal polyps as compared to controls. this mis-localization may account for the inactive cbf, a hallmark characteristic, observed in nasal polyps. zhao l 1 ; zhi l 2 ; jin p 1 ; zi x 1 ; tu y 1 ; li a 1 ; li t 1 ; shi l 1 (3.64, 2.28-5.01, p<.05) and +gc np patients (10.39, 6.00-14.77, p<.05 the results showed that the number of th17 + cells were correlated with eosinophil cells and macrophage (r=.3210, p<.05, r=.3269, p <.05), but no correlation was found between th17 + cells and neutrophil cells. the significantly correlation were found between ilobjectives: this study aimed to reveal if some features of the sinus wall and content (as homogeneity and density of the sinus content, or the continuity, thickness and density of the sinus wall), differ between the afrs and other forms of crs. we tried also to establish early diagnostic parameters for recognition of fungal rhinosinusitis on ct. results: the study included 36 adult patients (mean age: 45.58ae14.29 years, m:f ratio=2.1:1) with clinically diagnosed crs. out of all maxillary sinuses (n=72) from study patients, 62 (86.11%) were opacified, and only these sinuses were included in further analyses. we found out that: (1) positive fungal finding had 33% (12/36) crs patients and 86% (31/36) of these patients had severe forms of crs, (2) patients with positive fungal finding had more often positive specific ige ab than those without fungi in sinuses (43.9% vs. 21.3%, p=.027), (3) foci of non-homogeneity, mean and maximum densities and wall density were more common found in maxillary sinuses with present fungi than those without fungi (p=.037, p=.05, p=.05; respectively) and (4) patients with crs lasting more than 10 years had more often foci of non-homogeneity and presence of hyperattenuation centres, than patients with shorter length of crs. results: neutrophil-related gene mpo and eosinophil recruitment genes ccl13 and ccl18 showed higher expression level in acp than in controls, which were in line with the significantly elevated neutrophils and eosinophils infiltration in acp compared to control. increased cd8 + t cells, macrophages and cd4 + t cells infiltration in acp were also observed. the expression level of t-reg transcription factor foxp3 was significantly higher in patients with acp than in controls, but the expression of th1/th2/th17 transcription factor tbet, gata3 and rorc were significantly decreased in acp vs controls. we further investigated the relationships between these t-cellassociated genes in acp. the expression of foxp3 was positively correlated with t-bet, gata3, il17r and il12a, while no significant correlation with rorc was evident. il6 was observed positively correlated with t-bet, gata3, foxp3, and il17r. il10 had significant correlation with t-bet and il12a. objectives: the aim of this study was to find the olfactory change pattern of crs after ess in short-term and the differences between crswnps and crssnps, secondary aim were to identify the relationship among olfactory dysfunction, ct scores and quality of life(qol). in this study, 48 crs patients who underwent ess were evaluated preoperative by t&t recognition threshold tests, snot-20 score and lund-mackay ct score. patient outcomes were re-evaluated at clinical follow-up 1 month, 3 months and 6 months postoperative. analysis of variance was performed and correlation was calculated, with results analysed separately for crswnp and crssnp subgroups. 1. subgroups of crs differed in the degree of olfactory dysfunction reported before and after the ess. a significant difference in the changes of olfactory dysfunction between the two groups was found at 6 month postoperative. 2. the mean t&t and snot-20 scores showed significant improvement within 6 months after ess in both crswnp and crssnp subgroups, however, no significantly recovery of olfactory dysfunction was observed at 3 months compared to 1 month postoperative. there is a plateau of olfactory recovery at 3 months postoperative. 3. in crswnps, the mean t&t scores preoperative were correlated with lund-mackay ct score significantly(r=.649, p<.001; r=.625, p<.001). however, no relations were found in crssnps and the changes of olfactory dysfunction at the 6 months postoperative with lund-mackay ct score. 4. olfactory scores, before and after the ess, and their changes did not correlate with sont-20 scores. conclusions: olfactory dysfunction was more severe in crswnps. olfaction and qol of crs patients were significantly improved after ess in both groups, but there was a plateau of olfactory recovery at 3 months postoperative. ct scan may predict olfactory disorder, but the olfactory scores were not related with the qol. objectives: in this study, we investigated the effect of hgf, tgf-b1, and pge 2 as effective components for allergic rhinitis treatment using in vitro and in vivo mouse model studies. results: pge 2 decreased infiltration of eosinophil in nasal mucosa. tgf-b1 decreased the infiltration of eosinophil in nasal tissue and increased the number of treg in spleen. however, there was no antiallergic effect of hgf in this experiment condition. in case of the combination treatment group (tgf-b1+pge 2 +hgf), eosinophil infiltrations and the expression of eotaxin-2 were reduced in the nasal tissue, and treg was increased in the spleen. in all treatment group, serum ige and systemic cytokine levels were not decreased due to intranasal administration rather than systemic administration. in vitro study showed that phosphorylation of map kinases such as erk, jnk, and p38 and translocation of p65 were inhibited after treatment of hgf, tgf-b1 and pge 2 , suggesting their anti-allergic mechanism. conclusions: we found that tgf-b1, and pge 2 decreased allergic inflammation and these effects might be derived from changes in the frequency of treg and the activation of map kinase and p65 in the t cell receptor signaling pathway. furthermore, we hypothesized that tgf-b1, and pge 2 would be effective components for allergic rhinitis therapy. introduction: interleukin (il)-10 is implicated in suppression of allergic inflammation. the role of il-10 in the early-phase reaction in type 1 hypersensitivity has been unclear, however. we investigated the contribution of il-10 in a mouse model of the ige-mediated early-phase reaction in allergic conjunctivitis. objectives: ige-mediated allergic conjunctivitis was induced in c57bl/6-kit(+/+) wild-type mice, kit(+/+) il-10-deficient mice, and kit(w-sh/w-sh) mast cell-deficient mice by means of passive conjunctival anaphylaxis. the mice were thus subjected to subconjunctival injection with anti-dinitrophenol ige (dnp-ige) followed after 24 h by intravenous injection with dnp antigen. kit(w-sh/w-sh) mice that had received a subconjunctival graft of cultured bone marrowderived mast cells from kit(+/+) wild-type mice or kit(+/+) il-10deficient mice were similarly treated. vascular permeability of the conjunctiva was examined 30 min after antigen injection by colorimetric evaluation of the extravasation of evans blue dye. results: passive transfer of dnp-ige followed by intravenous antigen injection increased vascular permeability in the conjunctiva of kit(+/+) wild-type mice but not in that of kit(w-sh/w-sh) mice, suggesting that this effect was dependent on mast cells. vascular permeability was increased to a significantly greater extent in kit(+/+) il-10-deficient mice than in kit(+/+) wild-type mice. reconstitution of kit(w-sh/w-sh) mice with kit(+/+) wild-type or kit(+/+) il-10deficient mast cells restored the dnp-ige-and dnp-induced increase in vascular permeability to similar extents. our results suggest that il-10 produced by cells other that mast cells suppresses the mast cell-mediated early-phase reaction in ocular allergy. objectives: our aim was to evaluate the effectiveness and the safety of ccl treatment for keratoconus in children with vkc. forty-two boys (mean age 13.5ae3.3 years) and 17 girls (mean age 11.8ae4 years) with vkc were included in the study. tarsal, limbal and mixed vkc were detected in 55.9%, 8.5% and 35.6% of the subjects, respectively. evaporative dry eye was detected in 23 children out of 43 (53.5%), schirmer test results were <10 mm/ 5 minutes in 20.5% and <5 mm (severe dry eye) in 4 out of 44 children (9.1%) and 39% of the subjects (n=23) had confirmed keratoconus/forme fruste keratoconus with corneal topography (sirius, cso, italy). allergic symptoms were controlled with topical steroids, cyclosporine, dual action antihistaminic/mast cell stabilizers and lubricant agents before the procedure. ccl surgery was performed under topical anesthesia. the children were followed-up at least 1 year and preoperative and postoperative corneal topographic parameters were compared using paired sample t test. results: the visual acuity was between 0.4 and 0.6 (moderate visual loss) in 16.9% of the subjects and less than 0.3 (severe visual loss) in 6.8% of the children. ccl procedure was performed to 19 eyes of 15 children. at the end of one year, the disease was stable in all children with no differences in k1 and k2 corneal parameters before and after cxl (p>.05). there was a statistically significant improvement in maximum keratometry value after the procedure (before 56.57ae4d, after 55.37ae3.5d, p=.005). in one subject, a corneal infiltrate was detected 3 days after ccl, which was treated successfully with topical moxifloxacin. otherwise, no complications were observed in the postoperative period. conclusions: as keratoconus is common in vkc, these children should be referred to ophthalmologists for an eye examination and corneal topography. ccl seems to be a safe and effective option to halt the progression of keratoconus, which might be very aggressive in children with vkc. results: surprisingly, we found among them 11 cases of celiac disease, 7 cases of thyroid dysfunction (thyroiditis), 3 cases of crohn's disease and 1 case of ulcerative colitis, 1 case of anterior uveitis and 1 case of pemphigus respectively. we realized that an average percentage of 7% of the total of vkc cases are affected by an "autoimmune" systemic disease. limbal form of vkc was prevalent and more than 70% of children showed it. we can suppose that a racial and genetic predisposition to systemic diseases can coexist with vkc or that there is a group of vkc affected subjects in which the immune disorder is predominant on the allergic disease. introduction: nasal polyposis (np) is a heterogeneous inflammatory disease of nasal mucosa affecting 1-4% of the population with a high rate of recidivism. polyps arise from nasal sinuses to nasal cavity and are often associated with a strong local eosinophilia. the pathophysiology of np remains controversial, as it seems to be a phenotypic manifestation of multiple possible immunologic processes, such as respiratory allergy, despite a lack of correlated systemic response. here we propose a multiparametric assessment of np patients, in order to shed light on the underlying mechanisms of the disease in allergic and non-allergic patients and aiming to find new predictive biomarkers. objectives: our main aim was to unravel the link between systemic and local allergic inflammation and polyp development, as well as the nasal epithelium condition in polyps and surrounding healthy tissue. methods: four groups of patients were included in the study: healthy donors with or without allergy and np patients with or without allergy. in this regard, several different approaches were followed: a metabolomics serum study, a polyp and nonpolypous nasal epithelium histology and transcriptomic study. results: as for the histological study, luna staining revealed differences in eosinophilia between allergic and non-allergic patients, especially when patients were polysensitized, including perennial allergens; and between nonpolypus tissue and polyps being higher in polyps. pas staining showed differences in epithelium integrity and submucous and goblet cell (pas positive) distribution. immunohistochemistry for cd4+ and cd11c+ reveal a significant inflammatory infiltration in polyps. this inflammatory response was also asses by abstracts | 209 gene expression quantitation. no differences were seen in the metabolic profile in patient sera between groups. for the first time, nasal epithelium from polyps and neighboring tissue were studied. histology techniques and image analysis revealed differences in eosinophil concentration in both mucosa and submucosa areas, as well as different features in epithelium and submucous tissue structure. some of these findings were confirmed by gene expression quantitation. conclusions: our data show an increased eosinophilia and inflammatory infiltration in polyp tissue suggesting a role for allergic inflammation in the progression of np. additionally, we provide clues for the role of inflammation in the damage on nasal mucosa and the following progression of the disease. 0235 | is specific immunotherapy effective in subjects suffering from vkc? a tertiary referral center ten years experience. objectives: our work shows the results of sit additional to usual treatments, in children suffering from vkc and followed in our tertiary referral center (lavagna hospital, genova, italy). we retrospectively analyzed the clinical data of 37 subjects (25 males and 12 females); their mean age at the beginning of treatment was 8 ae 1.2 years. the patients were treated both with scit (sub-cutaneous immune-therapy-56.7%) and slit (sub-lingual immune-therapy -43.3%) depending on patient's wishes. they had to be mono-sensitized to one of the usually more frequent allergens (dust-mites, grass pollen, and pellitory) which was detected by means of recombinant rast, prick test and conjunctival provocation test (cpt); these tests were performed after a complete ophthalmological and allergological history and examination. children selected for sit needed to be positive to all performed allergy tests. systemic involvement included 25 cases of asthma, 5 cases of atopic dermatitis and 1 case of rhinitis; the remaining 6 cases were asymptomatic. local involvement included only vkc cases, the 62% of which were of the limbal type and only 14 subjects were suffering from the tarsal papillary type. mean follow-up was 7.2 years. all the patients included into the study completed their treatment and followed the therapeutic protocol. after one year of sit, no variation in clinical course and treatment was recorded. after the third year of sit, an average improvement in symptoms and signs score (43%) and an average decreased need for allergy systemic medications (63%.) (i.e. antihistamines and corticosteroids) was registered. also topical therapy (including steroid and cyclosporine eye-drops) was discontinued in 43% of children, in this group, short courses of steroid drops were necessary in less than 30% of children (as rescue treatment in the acute phases of the disease). these positive results after sit treatment were stable for the following 5 years. few (only local sub-cutaneous) side effects were recorded and the treatment was generally well-tolerated. conclusions: our experience shows positive results with sit in vkc which can have sensitive-to sit-treatment subtypes. results: 39 deaths occurred in children (21 boys, 54%). median age at death was 11 years (iqr 2-15). pamr of any cause was 0.08 (95%ci, 0.05-0.10) per 10 6 children per year, with a decreasing rate over time (annual change: -2.5%; 95%ci, -5.6 to 0.9). triggers were iatrogenic causes (n=18, 46%), insect venom (n=3, 8%) and food (n=2, 5%). unspecified causes were frequently reported (n=16, 41%). there was no difference in overall pamr between boys and girls (p=.74). there was no age group related differences in fatalities: preschool children (<7 years) (n=14, 36%), school children (7-12 years) (n=12, 31%), adolescent and toddlers (>12 years) (n=13, 39%). the number of fatal cases was similar comparing the southern (n=16, 41%) and the northern regions of france (n=23, 59%) (p=.15). the first episode of anaphylaxis for each patient was captured to calculate incidence. we estimated incidence rate ratios using poisson regression models. results: between 2001 and 2015 there were 523 anaphylaxis episodes in 481 patients younger than 18 years in hong kong. the incidence of admission for anaphylaxis increased markedly from 2.46 to 6.63 per 100 000 person-years during the study period (p<.001). the incidence of food-related anaphylaxis increased significantly from 0.21 to 1.88 (p<.001). increases in anaphylaxis and food-related anaphylaxis were seen in all age groups, with the largest increase in those aged 0 to 4 years. at the beginning of the study period (2001), medication was a more common trigger for anaphylaxis than food (1.61 vs 0.21 per 100 000 person-years). by 2015, food had become the predominant trigger (1.88 vs 0.54 per 100 000 personyears for medication). the incidence of medication-related anaphylaxis decreased significantly (p<.05). the incidence rate of anaphylaxis was significantly higher in boys than girls in the 5-14 and 15-18 year age groups, while there was no significant gender difference in the 0-4 year age group. the most common food triggers of anaphylaxis were peanuts, seafood, eggs, milk products, tree nuts & seeds (in descending order). conclusions: even though the incidence of anaphylaxis among children in hong kong is lower compared with other western countries, it has recently increased significantly, with food-related anaphylaxis predominant. 0238 | prevalence of anaphylaxis and prescription rates of epinephrine self-injector in korea based on national health insurance data results: the prevalence of anaphylaxis over the 5 years were 0.02%. the annual prevalence of anaphylaxis increased over the 5 years. anaphylaxis was more prevalent in male than female (56% vs. 44%) and in population aged 50-59 years old. for the regional prevalence of anaphylaxis in korea, gangwon province showed the highest prevalence of anaphylaxis (41.5 per 100 000 individuals) and relatively low prescription rates (5.8%) of epinephrine self-injector for the patients with anaphylaxis. on the contrary, seoul showed relatively low prevalence of anaphylaxis (14.3 per 100 000 individuals) and the highest prescription rates (26.6%) of epinephrine self-injector for patients with anaphylaxis conclusions: the prevalence of anaphylaxis has increased annually in korea. the prevalence of anaphylaxis and prescription rates of epinephrine self-injector showed regional difference in korea. objectives: the aim of the study was to analyze the prevalence of allergic symptoms and anaphylaxis in mastocytosis patients analyzed in the registry of the ecnm. results: methods: a total of 1513 patient with mastocytosis were enrolled. in these patients, the prevalence of allergy, anaphylaxis, triggers of allergic reaction, and disease subtypes were analyzed. results: symptoms of allergy were observed in 28% of all patients. the most affected group were patients with bone marrow mastocytosis (bmm: 64.52%) and indolent systemic mastocytosis (ism: 32.78%). insect venom allergy (iva) was reported in 14.14% of all subjects. in ism/bmm patients iva affected 22.30% of the cases, while in other patient groups, only 4.3% of the cases were affected (p<.00001). most patients (64%) had wasp allergy, 18% had bee allergy, 2% polistes allergy, and 6.8% allergy to more than one venom. in 9.2% the culprit insect was not identified. food allergy was reported in 3.56%, drug intolerance/allergy in 4.7%, inhalant allergy in 4.43%, and physical triggers in 0.8% of patients. in mastocytosis patients iva is the most prevalent cause of anaphylactic reactions exceeding the prevalence of iva in the general population by far. iva affects mainly bmm and ism patients (22.3% of cases). but 267 (66.1%) subjects didn't know whether adrenaline was administered. only 6 within 44 patients who had adrenaline autoinjectors used their autoinjectors during an anaphylaxis attack. most common symptoms were skin (n=363, 89.9%) and respiratory symptoms (n=327, 80.9%). syncope, hypotension or hypoxemia were present in 273 cases(63.9%), at least three organ dysfunctions in 258 (63.9%) cases; 56 patients (13.9%) had to be hospitalized (f:36, m:20).nearly a third (26.2%) of the patients had stage 1-2 anaphylaxis and 298 patients(73.8%) had stage 3-4 reactions. in 184 cases (45.6%), basal tryptase levels were examined and the average value was correlated with the severity. concomitant drugs being used by the patients were antihypertensives (20.5%),oral antidiabetics (6.2%); angiotensin converting enzyme inhibitors or angiotensin receptor blockers(11.1%), beta blockers(8.4%), diuretics(4.7%) and nsaid's (7.2%). conclusions: male sex was noted as a risk factor for severe reactions and recurrent anaphylaxis. anaphylaxis requiring hospitalization was more frequent in the patients using oral antidiabetics or diuretics. baseline tryptase levels were higher in patients with neurological and gastrointestinal symptoms. cardiovascular symptoms were found to be higher if a cofactor was present. skin symptoms were seen more frequently and higher rate of hospitalization occurred in anaphylaxis in the presence of infections or nsaid use. this study is important to elucidate the factors affecting anaphylaxis severity. 0241 | serum levels of 9a,11ß-pgf2 in combination with apolipoprotein a1 or cysleukotrienes are reliable biomarkers of anaphylaxis objectives: we analyzed mast cell mediators in sera derived from patients with acute anaphylactic symptoms (n=18) versus patients with acute cardiovascular or febrile reactions (n=12) and patients with a history of anaphylaxis but without displaying any symptoms when sera were taken (n=27). in addition, we identified proteins with substantial changes during anaphylaxis. matched serum samples were used to compare basal mediator levels with corresponding levels during acute anaphylaxis in the same patient (n=9). roc curve analysis was performed to determine the sensitivity/specificity of each mediator. results: serum levels of histamine and tryptase were not increased upon anaphylaxis and showed no relation to anaphylaxis severity. however, serum 9a,11ß-pgf 2 , a metabolite of pgd 2 , was significantly increased in acute anaphylactic patients (~8-fold) and abstracts | 213 correlated well with anaphylaxis severity. 9a,11ß-pgf 2 distinguished anaphylaxis from cardiovascular or febrile reactions and showed the highest diagnostic power observed by roc curve analysis. cys-leukotrienes (cys-lts=ltc 4 , ltd 4 , lte 4 ) were increased upon anaphylaxis while apolipoprotein (apo) a1 was significantly decreased. the highest diagnostic power was observed with the combination of 9a,11ß-pgf 2 and apoa1. conclusions: in conclusion, histamine might only be used to detect anaphylaxis when assessed shortly after onset of an anaphylactic response because of its short half-life, whereas tryptase is a useful biomarker if the baseline level of the same patient is known. 9a,11ß-pgf 2 seems to be the most reliable marker as demonstrated by the distinct increase upon anaphylaxis and could be supported by apoa1 or cys-lts. further investigations are needed to prove the suitability of these markers. objectives: objective of this study was to estimate the long-term bv of tryptase using certain chronic disease models and to compare it with those in food and drug allergy. results: serial determinations of tryptase concentrations (n≥5 data points per patient) obtained from patients diagnosed with mastocytosis (n=10) or chronic urticaria (n=1) during a period of sometimes several years were measured by the immunocap assay and evaluated using sigmaplot software. polynomial curve fitting was performed and data points outside the 95% confidence interval of the curve were appointed as outliers and excluded. because the data points were not normally distributed due to long-term fluctuations in homeostatic set-point, a non-linear fitting was applied and used to compute the standard error of the estimate. these standard errors of the fit divided by the estimates themselves were used to calculate the total coefficient of variation within a subject (cv t ). the analytical cv (cv a ) was calculated based on quality control samples (3 levels, n=149 data points) in a conventional way, while the within-subject bv (cv i ) was defined as cv l =(cv t 2 à cv a 2 ) ½ . eleven patients with a chronic disease were selected, of which 1 patient was treated as a potential outlier and 2 patients had to be excluded because of cv t <cv a . the between-subject bv (cv g ) was without the outlier 3.68ae1.92% (n=85 data points during a period 5.6ae2.3 years) and with the outlier 5.75ae6.46% (n=92 data points during a period of 5.3ae2.4 years). as a food and drug allergy control group 2 additional patients were selected with an uncomprehended food (n=1) or drug allergy (n=1). this cv g was calculated under the same conditions and proved to be 9.61ae1.64% (n=16 data points during a period of 4.1ae1.2 years). the long-term between-subject biological variation of tryptase concentrations of patients in chronic disease models was smaller than that of patients with an uncomprehended food or drug allergy. chronic disease models may be of interest to determine the long-term "physiological" biological variation of tryptase. vilà-nadal g; rodr ıguez-sanz a; s anchez-villanueva r; fiandor-roman a; dom ınguez-ortega j; bell on-heredia t introduction: hypersensitivity reactions during hemodialysis sessions have been reported. our aim was to study the mechanisms of the allergic or "pseudoallergic" reactions to synthetic polysulfone (ps) dialysis membranes in "ps-allergic" patients who tolerated cellulose triacetate (cta) membranes. objectives: ten patients with adverse reactions to ps and 8 control non-allergic patients in hemodialysis were studied. 50 ml of blood were collected and an ex-vivo hd were performed on experimental external circuits with low or high priming volumes and pediatric membranes (fx-paed helixone 0.2 m 2 and sureflux). serum tryptase levels, basophil degranulation (hla-dr à cd123 + cd63 + leukocytes), and t cell activation (cd69 expression on cd4 + and cd8 + subpopulations) were analysed in pre-and post-dialysis samples. objectives: the aim was to investigate the influence of asa and alcohol on egg allergy. donor sera from 4 egg allergic patients (i-iv) with s-ige to ovalbumin at (i) 0.1, (ii) 8.87, (iii) 19.5, and (iv) 170 ku/l were injected intracutaneously to the forearm of 11 healthy/nonallergic volunteers. they were then orally challenged separately with: 1) egg white 2) egg white + asa and 3) egg white + alcohol. 'time to wheal' and 'wheal size' were compared between the three experiments. results: two sera (i-ii) with the lowest s-ige to ovalbumin did not react sufficiently and where excluded from further analysis. the 2 remaining sera (iii-iv) significantly decreased 'time to wheal' with both asa (p=.001) and alcohol (p=.019) added as co-factor compared to baseline. increase in 'wheal size' was only found with co-factors added for the less reactive serum (iii) and not for the serum with the highest s-ige (iv). introduction: eaaci have published numerous guidance documents providing evidence-based recommendations for the recognition, risk factor assessment and management of anaphylaxis. these guidelines clearly advocate intra-muscular adrenaline firstline for the treatment of anaphylaxis, and list absolute indications when an adrenaline auto-injector (aai) must always be prescribed; eg for those who have previously experienced an anaphylactic event, those who have a mast cell disorder and for those with co-existing unstable or moderate/severe asthma and a food allergy. furthermore, eaaci recommends that an aai should be prescribed to those at risk of anaphylaxis (termed relative indications), and that 2 aais should be prescribed and carried by patients at all times. despite widespread dissemination of these guidelines, anaphylaxis hospitalizations continue to rise in many european countries. objectives: in this environment eaaci developed the anaphylaxis survey (axis) as part of its allergy awareness initiative to gain insight into guideline awareness. this survey was sent by email to 8720 eaaci members. 873 members from 89 countries responded. data were anonymous and analysed descriptively. results: 81.4% (n=711) reported familiarly with the eaaci anaphylaxis management guidelines, and of these 96.8% (n=688) reported following them. 97.0% (n=847) responded that they would use adrenaline firstline for the treatment of anaphylaxis (although 9.6% (n=84) would still administer it subcutaneously). however, when confronted with a list of indications, only 41.8% (n=365) of eaaci members would prescribe an aai for all absolute indications. furthermore, although 90.1% (n=787) of respondents considered they would prescribe an aai for those at risk, when provided with a list of relative indications, 0% would prescribe an aai for all of them. there was also a lack of consensus about when an aai should actually be used, with 23.8% of respondents (n=208) considering aai use when individuals experienced breathing difficulties and felt that they were going to collapse. conclusions: these results reflect the latest findings that aais are under-prescribed in daily practice. there appears to be a mismatch between perception of guideline compliance and aai prescription behaviour. continued training, further simplification and wider dissemination of guideline messages are required. objectives: the aim of this study was to analyse the clinical practice of emergency physicians concerning the management of anaphylaxis in alsace and lorraine, a region of 3.5 million inhabitants, in france via an electronic survey. results: the response rate was 18.3% (44). fifty percent of physicians were female. 84.1% (37) (1). in case of grade 2 reactions, intramuscular adrenaline injection was performed by 11.2% (5) of physicians as first-line treatment, intravenous adrenaline injection by 8.4% (4), antihistamines (oral route or iv) by 16% (7) and corticosteroids (oral route or iv) by 17.8% (8). in case of grade 3 reactions, intramuscular adrenaline injection was performed by 13.6% (6) of physicians as first-line treatment, intravenous adrenaline injection by 64.3% (28), iv antihistamines by 6.8% (3) and corticosteroids (oral route or iv) by 16% (7). following an acute anaphylaxis episode, only 2.3% (1) of physicians systematically recommended an allergy assessment, 11.2% (5) sometimes, and 86.4% (38) never. conclusions: this study highlights the discrepancies between current guidelines for the management of anaphylaxis in emergency departments and common clinical practice. our results emphasised the need for specific programs to improve clinical management of anaphylaxis in ed. conclusions: a significant number of individuals among those surveyed reported a severe allergic reaction requiring an eai, yet less than a quarter used an eai. the study also identifies most common challenges faced by at-risk individuals including eai availability, cost, and concerns over short expiry dates. objectives: to evaluate the implementation of a stock epinephrine (stock epi) program in a mall setting. stock epinephrine devices are not prescribed for a particular person and can be used in anaphylactic emergencies. methods: a mixed methods approach was used to evaluate the implementation of the stock epinephrine program regarding its acceptability and adoption by diners, food service, mall administration and security personnel; and the appropriateness, costs (included costs for personnel, epinephrine auto-injector devices, and training materials), fidelity, reach, and sustainability (measured by the national health service (nhs) sustainability model, united kingdom) of the program in this setting (hamilton, ontario, canada). the study revealed that individuals with food allergy and food service establishments were in favour of a stock epi program. a total of 1580 individuals with food allergy completed our national online survey. approximately 42% dine out once or twice per month, and one-third had experienced an allergic reaction. we also completed in-person surveys with 120 diners at the mall food court. of these, 24% had a food allergy and 35% reported carrying their epinephrine auto-injector when dining out. the mean nhs sustainability score was 87 among survey participants, well above the mean considered a sustainable program (>55). total cost of the stock epi program over a one-year period varied by ontarian stakeholders: $2155 for the mall, $987 for fast-food restaurants, and $715 for sitdown restaurants. conclusions: this is the first study to evaluate the implementation of a stock epi program. the stock epi program was well received abstracts | 217 and sustainable. implementing a stock epi program provides enhanced access to emergency medication, however it does not replace the responsibility of individuals with food allergy to self-manage. objectives: to identify the optimal needle length for epinephrine prefilled syringe. results: three hundred seventy-two children aged 1 month to 18 years were enrolled. skin to muscle depth (stmd) and skin to bone depth (stbd), which can represent the minimum and maximum needle length respectively, were measured using an ultrasonography at the mid-anterolateral thigh. number of children who had stbd less than needle length (too long needle) and stmd greater than needle length (too short needle) were calculated. one hundred thirty-seven children weight <15 kg, 80 children weight >15-30 kg, and 155 children weight >30 kg were enrolled: 196 (52.7%) children were male. one inch needle was too long in 38 (27.7%) children weight <15 kg, 1 (1.3%) children weight >15-30 kg. it was too short in 8 (5.2%) children weight >30 kg. age≥3 months, weight≥6 kg, height≥59 cm, bmi≥13 kg/m 2 and thigh circumfer-ence≥23 cm, provided the sensitivity of 97-99% in predicting the appropriateness for using 1 inch needle for children weight <15 kg. in children weight >30 kg, thigh circumference≥48.75 cm provided the sensitivity of 86% and specificity of 75% for predicting the inappropriateness for using 1 inch needle. objectives: we present a patient with a probable mdh syndrome to unusual drugs, including ah and cct. results: we report a case of a 23-years-old female, with history of moderate-persistent asthma and chronic urticaria, who experienced angioedema and exacerbation of urticaria hours after the administration of multiple ah (desloratadine, loratadine, cetirizine), systemic cct (hydrocortisone, methylprednisolone, deflazacort) and nonsteroidal anti-inflammatories (nsaids) (paracetamol, ibuprofen, flurbiprofen). patch tests (pt) with excipients (bial arestegui ® ) and drug provocation test (dpt) with placebo were negative. skin prick tests (spt) and intradermal tests (id) with hydroxyzine, hydrocortisone, methylprednisolone and prednisolone were positive to hydroxyzine (5mg/ml) and pt with corticosteroids (bial arestegui ® ) and hydroxyzine were negative. dpts with desloratadine and an alternative ah, dimetindene, were positive with facial angioedema and generalized urticaria within 5 hours. lymphocytic transformation test (ltt) was positive to desloratadine, ebastine and clemastine. dpt with dexamethasone was negative, however, when administered as treatment, a reproducible reaction occurred. since dpt with montelukast was also positive, omalizumab 300mg was initiated to control angioedema and chronic urticaria. after 1 year of treatment, dpt with nimesulide was negative. omalizumab dose was reduced to half after the patient found out she was pregnant. there were no further episodes after anti-ige therapy introduction and pregnancy went uneventfully. conclusions: mdh syndrome is rare, more so when the drugs reported are ah and cct. hr to ah was confirmed, but diagnostic workup remains incomplete, postponed due to the patient's pregnancy. this case is as challenging in terms of diagnosis as it is in terms of therapeutic, so much so that omalizumab was initiated as an off label therapy, maintained during pregnancy based on the premise that risk was lower than benefit. objectives: in this study, we aimed to present our patients who were admitted with oral iron hypersensitivity. conclusions: according to our clinical experience, we think that oral iron salts with different conjugates are safe and acceptable option in patients with suspected oral iron hypersensitivity. introduction: antineoplastic agents are consider nowadays an essential treatment for many kinds of cancer. the increased use of these drugs in recent years is in parallel with a high rise of hypersensitivity reactions to them. these reactions range from mild to severe and as other allergic reactions, are not predictable. a nursing protocol in the desensitization schedules with these drugs is essential in allergy daily hospitals. objectives: the aim of this study are to describe a nursing protocol during desensitization schedules with antineoplastic agents carried out in our allergy unit in order to detect symptoms suggestive an allergic reaction during drug administration and to assess a correct intervention in case of reaction. conclusions: an appropriate nursing protocol in desensitization schedules with antineoplastic agents is essential in order to achieve the correct administration of the treatment in safety conditions. 0255 | long term clinical effects of aspirin desensitization in patients with nerd: comparison of maintenance doses of 300 mg vs 600 mg aspirin çelik ge 1 ; karakaya g 2 ; erkekol f € o 3 ; dursun ba 4 ; gelincik a 5 ; celebioglu e 6 ; y€ ucel t 7 ; yorulmaz i 8 ; dursun e 9 ; tezcaner ç 8 ; s€ ozener zç 10 ; b€ uy€ uk€ ozt€ urk s 11 ; kalyoncu f 12 ; aydin ö 1 introduction: aspirin desensitization (ad) treatment has been shown to be effective in relieving the respiratory symptoms as well in reducing recurrency of nasal polyps in patients with nsaids exacerbated respiratory diseases (nerd). however, a conflict occurs about effective maintenance doses of aspirin on clinical parameters. objectives: in this study, our aim was to compare the effects of two different maintenance doses of aspirin on clinical outcomes for 3 years of ad. this was a multicenter study which involved 4 tertiary centers. patients who completed at least one year of ad treatment were included to analysis. study outcomes were number of nasal surgery, sinus infections, asthma morbidity (number of severe asthma attacks, hospitalization) as well as medication uses for both clinical conditions. the study included 114 subjects, 33 of whom were under 300 mg aspirin daily as maintenance treatment whereas remaining 81 on 600 mg aspirin for a mean of 48.2ae32 months of ad duration. regardless of maintenance doses, number of nasal polyp surgery gradually decreased at 1 (0.04ae0.03/year) and 3 years (0.02ae0.01/year) compared to that of before ad (0.41ae0.06/year) (p<.0001) in all subjects and were comparable in 300 and 600 mg. considering asthma outcomes, decrease in asthma attacks were observed only in 600 mg aspirin group (p<.01) at 1 and 3 years whereas hospitalization due to asthma and systemic corticosteroid use decreased in both groups at 1 and 3 years. conclusions: ad has a reducing effect on nasal polyp recurrence for at least 3 years in patients with nerd. this effect was similar for both 300 and 600 mg maintenance doses of aspirin. considering both treatment arms provided decreased hospitalization due to asthma and systemic corticosteroid use, we think that at 3 years evaluation both 300 and 600 mg/day aspirin has comparable effects on asthma as well. however, reducing effect of ad on asthma attacks was only existed in patients taking 600 mg. aspirin. objectives: pregnant women with syphilis and history of immediate hypersensitivity reaction (hsr) to penicillin were enrolled. according to the risk stratification, which was based on the initial hsr, serum specific ige and skin tests, patients were re-exposed to penicillin either through desensitization or provocation. patients with a clinical history suggestive of penicillin-anaphylaxis and/or positive serum specific ige to penicillin and/or positive immediate skin test were considered at high risk and were desensitized. the remaining patients underwent penicillin provocation test. results: we evaluated 21 pregnant women with latent syphilis and history of penicillin allergy. clinical history was suggestive of immediate hsr in 13 out of these 21 (62%) patients, who were desensitized. all of them had negative serum specific ige to penicillin. intradermal tests were positive in 4/13 (30%). three out of those four were desensitized with an oral protocol and reacted during the procedure. one patient had a severe breakthrough reaction with uterine contraction and did not finish the procedure. the only patient with positive intradermal test that didn't react during the rdd underwent an intravenous protocol. the remaining 9/13 (70%) patients had negative skin tests and an uneventfully rdd. there was a statistically significant association between positive intradermal tests and breakthrough reactions during the rdd (p=.02). the other 8/21 (38%) patients with inconclusive history and negative skin test were submitted to penicillin provocation, which were negative in all of them. conclusions: risk stratification based on the initial clinical reaction and skin testing to guide penicillin re-introduction was safe and effective, as well as rdd. skin testing identified allergic patients to penicillin with increased risk of reactions during rdd. 0257 | utility of basophil activation test for monitoring the acquisition of clinical tolerance after subcutaneous desensitization to brentuximab-vedotin in two patients many hypersensitivity reactions (hsrs) produced by biologic agents have been seen and their true incidence is unknown. desensitization is a method to counter hsrs from monoclonal antibodies in patients with no other adequate alternative options. objectives: we describe a successful rapid desensitization to bv in two patients with scleronodular type hl, refractory to several lines of chemotherapy and asct. this clinical tolerance to bv is easily observed through the basophil activation test (bat) as a decrease in activated basophils after desensitization was done as a treatment of hsrs. because there was no therapeutic alternative in the two patients, we planned to pursue bv administration using a rapid desensitization 12-step protocol. a total dose of 125 mg of bv was given through increasing rate and concentration. the patients completed their infusion without difficulty. after desensitization to bv, bat was done in both patients. the percentage of activated stimulated basophils with bv descended in both patients. both values are similar to their corresponding negative controls. conclusions: the bat continues to be a useful in vitro tool for the study of drug allergic disease. also, the bat in flow cytometry is able to monitor an acquired tolerance induced by a desensitization treatment in hsrs to bv. however, studies involving a larger number of patients will be required to assess the safety and efficacy of this approach to bat as a method to validate rapid desensitization in patients with hsr to bv. objectives: we retrospectively reviewed 166 desensitizations in patients with a history of ihsrs to chemotherapy agents performed in our center from january 2014 to december 2016. the protocol consists of increases in infusion rate every 10 to 15 minutes, in a 10 to 12 steps depending on the drug. in all cases the protocol was performed without premedication (only using regular medication according to instructions for every drug). results: a total of 61 patients with a history of (ihsrs) received 166 desensitization protocol without premedication to chemotherapy agents. the most common involved drug was carboplatin in 24 (39.3%) patients (of these 66% presented positive skin test (st)), followed by paclitaxel in 15 (24.9%) patients (of these 46.6% presented a positive st) and oxaliplatin in 9 (14.75%) patients (of these 11.11% o the st were positive). other chemotherapy agents involved were cetuximab, rituximab, irinotecan, epirrubicin, etoposide, cisplatin and cyclophosphamide. all patients were able to successfully complete the desensitization protocol without premedication and none of them need to withdraw the drug. conclusions: this protocol for rapid desensitization to chemotherapy without premedication is safe and effective. in addition, minimizes secondary effects of the premedication in these patients that are polymedicated. 0259 | rapid desensitization for the management of hypersensitivity reaction to biologicals-infliximab and adalimumab in inflammatory bowel disease patients objectives: to identify barriers to best practice with regards to drug allergy history taking and documentation, and to elaborate the potential strategies to overcome them. results: a total 164 prescribers responded to the survey: doctors in training 47.6% consultants 44.5% non-medical prescribers 7.9% most respondents 56.1% (95%ci 47.9-64.3%) were not aware of the availability of penicillin allergy testing in our trust. among those that were aware of it, 63.9% (95%ci 51.9-76%) had not referred any penicillin-allergic patient to immunology during the past year. barriers to accurate allergy history collection: 55.2% (95%ci 47-63.4%) concurred that often it is not possible to draw a firm conclusion based on history alone. 59.4% (95%ci 51.4-67.5%) agreed with the statement saying that, regardless of the details of the allergy history, it is always better to "play it safe" and not to use alternative beta-lactams in patients labelled as being penicillin-allergic (figure will be attached in the poster). among the interventions proposed; practical educational sessions, an interactive questionnaire to guide allergy history taking and classification and a modified antibiotic policy to guide prescribing based on the allergy classification, were all rated as useful (average score >7 on a scale from 1-not useful at all-to 10-very useful). 0262 | the regulatory role of germinal center maturation during the early b cell response to inhalant allergens investigated in the piama cohort using the medall allergen microarray introduction: in contrast to common belief, igg to airborne allergens is higher in allergic subjects, even before immunotherapy. one of the confusing aspects of the allergic immune response is that not only the igg response, but also the ige response can follow more than a single trajectory (with or without gc maturation). for ige we assume that the direct isotype-switching pathway (igm to ige) is the most relevant for the initial, mature-gc independent phase of sensitization to low-dose airborne allergens (such a pollen, mite). in later phases and for higher exposure situations as well as for other immunization routes, indirect switching is assumed to be the more relevant pathway. methods: ige, igg1 and igg4 antibodies were measured using the medall allergen microarray in 105 children from the piama cohort at age 1 and 4. these results were analyzed in relation to the ige levels at age 4 and 12. objectives: to find support for the hypothesis that the ige/igg ratio reflects not only exposure, but also details on immunological processes during sensitization, such as germinal center maturation. results: sigg1 and sigg4 levels to the major inhalant allergens were low at age 1 and remained in general low at age 4. however, children who at that time were positive for ige an allergen had a significant increased sigg1 to the allergen in question. sigg4 also appeared, but this response was low. the sigg level at age 4 in sigenegative children was not consistently predictive for sige at age 12. conclusions: the initial igg1 response to inhalant allergens is synchronized with the ige response. this result fits with the hypothesis abstracts | 225 that in the initial phase of sensitization to inhalant allergens the allergen initiates a weak and incomplete germinal center response that allows parallel ige-and igg1 production. one of the consequences of the multiplicity of b cell developmental pathways is that the igg/ige ratio is potentially diagnostic: if a subject has sigg levels in the microgram range, and thus a high sigg/sige ratio, this indicates involvement of mature gcs and the indirect class-switching pathway for some or all of the sige in this subject. 0263 | synthetic allergoid consisted of plga nanoparticles covered with synthetic peptides from bet v1 objectives: the aim of this study was to test a hypothesis that the ahr signaling is critical in controlling sl homeostasis through the regulation of key sphingolipid enzymes involved in the s1p synthesis. results: we found that an ahr ligand and a tryptophan photoproduct, 6-formylindolo (3, 2-b) carbazole (ficz; 1 nm), induced a increase in s1p level in a ros and ca 2+ -dependent manner, leading to the degranulation as well as il-13 secretion in mast cells, when compared to those seen in vehicle-treated cells. this was concomitant with an increased level of sphk1 phosphorylation and with a reduction in the enzymatic activity of s1p lyase, which could be reversed by the addition of an anti-oxidant, nac. moreover, s1pl was found to be directly oxidized by ros in vitro and in vivo. conclusions: our findings suggested that ahr-mediated ros and ca 2+ signals are critical for controlling sl homeostasis through regulating sphk1 and s1pl metabolic pathways, providing a new regulatory pathway in mast cells. methods: peptide cytokine mimetics were selected by phage display technology. flow cytometry, elisa, elispot, t cell proliferation, reporter gene, mediator release, intravital microscopy and peritonitis assays were conducted to evaluate the capacity of the mimetic peptides to modulate the immune response. results: the synthetic tgf-b1-like peptide was able to down-regulate the production of tnf-a, il-4, ifn-c and il-8, up-regulate il-10, decrease basophil degranulation and induce t reg cell differentiation. furthermore, this peptide was able to decrease leukocyte rolling and neutrophil migration during an inflammatory condition in vivo. the synthetic il-10-like peptide was able to decrease basophil degranulation and to inhibit the proliferation of allergen-specific t cell lines established from the peripheral blood of birch pollen-allergic patients. conclusions: the peptide cytokine mimetics tested herein, were able to modulate the immune response in the tested conditions. they, thus, represent promising novel candidates for therapeutic approaches. nonetheless, most studies focus on changes occurring early in life and there are rare data on differences in responses between allergic and non allergic subjects. objectives: we aimed to evaluate i) the maturation trajectory of the tlr3 antiviral pathway ii) if this trajectory varies between atopics and non atopics. peripheral blood mononuclear cells (pbmcs) were isolated from otherwise healthy atopic and non atopic subjects. atopy was assessed by medical history and skin prick testing to 8 common aeroallergens and egg white. selected cytokines involved in the antiviral response were measured by luminex multiplexing technology in 24 hour culture supernatants of poly:ic-stimulated pbmcs. data were analyzed by estimating the non-parametric correlation between age and cytokine expression in atopics and non atopics and comparison of regression curves for each cytokine between the 2 groups was performed. results: the analysis comprised data from 39 atopic and 39 non atopic patients (mean age 10.8 years, age range 0-45 and mean 10.3 years, range 0-43.3, respectively). significant age-related increases in the production of ifn-a2, ifn-c, il-1b, il-17a, tnf-a, and mip-1b were found only in non atopics and of il-9 and il-10 in both groups. significant differences in the trajectories (slopes) of cytokine responses over time between atopics and non atopics were observed for ifn-a2, ifn-c, il-10, il-1b, tnf-a and mip-1b, with suboptimal production in atopics. conclusions: age-related increases in cytokines implicated in innate antiviral responses were observed mostly for non atopics. atopy was associated with suboptimal trl-3 induced cytokine responses. differences in the developmental pattern of those cytokines between atopics and non atopics may account for the reported increased susceptibility of atopics to infections. 0271 | a systems-immunology approach identifies a set of micrornas in shaping the th2 phenotype in allergic airway inflammation introduction: mouse allergy is a common disease in inner city households, affecting up to 18% of children who are exposed as determined by house dust analysis. it is associated with allergic rhinitis, atopic dermatitis and asthma and it has been reported that exposure and sensitization to mouse allergens is a strong predictor for asthmatic disease. despite a strong link between mouse exposure and asthmatic disease, the allergic immune response to mouse has been significantly understudied. to date, only one major allergen in mouse, mus m 1, has been identified and very little is known about the targets of the allergic immune response against mouse. objectives: using a proteomic/transcriptomic approach, we sought to identify t cell targets in 24 mouse allergic and asthmatic patients. results: mouse urine and epithelial extracts were analyzed by 2d-ige/igg immunoblots using pooled sera from mouse-sensitized donors. mass spectrometry of selected protein spots identified 30 novel antibody reactive proteins. predicted mhc binding peptides from these novel proteins and mouse homologs to mammalian allergens were screened for t cell reactivity in pbmcs from mouse allergic patients. overlapping peptides from the major mouse allergen mus m 1 and its major urinary protein isoforms were screened in parallel. our screen for t cell responses in pbmc from mouse allergic donors demonstrated that major urinary proteins account for >75% of the total t cell response but they are not the only target of mouse-specific t cell responses. reactivity to mouse peptides homologous to other mammalian allergens, specifically guinea pig, was also detected. conclusions: in summary, our data demonstrates that the cellular and serological targets of the allergic response overlap, with mus m 1 being the major target for both t cells and antibodies. to the best of our knowledge, this is one of the first comprehensive studies of t cell epitope targets in mouse allergy, which provides important insights into cellular and serological targets. this data may form the basis for the development of a mouse-specific immunotherapeutic approach. introduction: food allergy has a complex etiology with many potential underlying factors proposed to contribute to and modify its development and progression. the use of a databases to collect and analyse all relevant data related to incidents of food allergy is essential to fully understand causal factors and improve treatment. objectives: we developed a database using sql, hibernate and java server pages (jsp) that was designed to allow allergy professionals to easily add and modify patient data, including medical history, reaction details and in vitro/in vivo test results. we then filled this database with clinical data relating to reactions to plant-based foods for patients who visited the allergy service of the regional university hospital of malaga between 2012-2016. these data were then analysed in various ways. cluster analysis of skin test results was used to search for relationships between different allergens based on similarities between patient sensitisation profiles; descriptive statistics and graphical analysis were performed to search for relationships between food type, age of first reaction, number of reactions and reaction severity. results: cluster analysis placed the different skin test allergens in distinct groups, which generally correlated with the type of allergen. for example, nuts, rosacea plant-food, mites, trees and weeds formed distinct clusters. analysis of patient history data showed that the first reaction occurred most frequently between ages 10-20, with a right skewed distribution. a relationship was also found between age at first reaction and reaction severity, being urticaria and angioedema more common when the initial reaction occurs at a younger age, and anaphylaxis when the initial reaction occurs later in life. oas remained relatively prevalent at all ranges (around a quarter of all reactions in all age groups). we found fruits to be the most frequent triggers, followed by nuts; within fruits peach and banana were the most frequent. conclusions: this preliminary study show the importance and utility of recording patient allergy information in a well-structured and easily managed database. future work is currently underway to collect a new set of patients from the same geographical area and to analyse similar data from a different area in order to identify what results are replicable within our population and which results are generalizable to other areas. introduction: cow's milk allergy is very common in children and its correct diagnosis is important to prevent possible dangerous allergic reactions. the aim of the present work was to evaluate the prevalence of allergic sensitization to both cow's milk and to its main proteins. with medcalc 9 ® . normality distribution of data was evaluated through the kolmogorov-smirnov test. patients were divided into three age groups (0-2 years, 3-6 years-7-16 years). chi-squared test was performed to verify a statistical difference between sensitization to whole milk and to its main proteins and patients' age. introduction: the order fagales represents an important cause of tree pollen allergy, which is ubiquitous in the northern hemisphere. a high degree of allergenic cross-reactivity has been observed among allergens from these plants, mainly represented by pathogenesis-related protein class 10 (pr-10) pr-10s, including inhalant and food allergens. conclusions: testing ige reactivity to a panel of pr-10s unveils important associations between sensitization profiles and clinical presentation, and allows the identification of novel cluster patterns potentially useful to predict disease severity in patients with pr-10 allergy. results: 11 patients were included, seven boys and four girls, with a median age at diagnosis of six months. the most common offending foods were cow's milk protein (cmp, n=5) and rice (n=2). other foods were fish, egg, chicken, wheat, carrot and potato. average time of symptom onset was 2.5 hours. the most frequent symptoms were vomiting (n=10) and diarrhea (n=4). six patients had a history of hospital admission related to this problem. seven patients had concomitant atopic diseases, being the most frequent allergic comobility atopic eczema (n=5). skin prick tests and/or specific ige to culprit foods were negative at diagnosis, except for one patient with low specific ige to cmp. another patient become sensitized to cmp during follow-up. open food challenges were performed in 10 patients starting from six months of age. resolution was achieved in 6 patients, at a medium age of 36 months. results: a total of 2901 cases of immediate-type fa among 2056 children were reported, with 92.5% involving patients younger than 7 years of age. the major 7 causative foods were hen's egg (27.4%), cow's milk (26.6%), walnut (7.2%), wheat (6.2%), peanut (5.5%), soybean (2.4%), and shrimp (2.2%). the most common causative food in each age group was cow's milk (0-2 years), walnut (3-6 years), walnut and hen's egg (7-12 years), and buckwheat (13-18 years), respectively. the symptom onset time was less than 30 minutes in 65%. food-induced anaphylaxis was reported in 732 (25.2%) out of 2901 cases, and the major 7 causes were cow's milk (26.9%), hen's egg (20.1%), walnut (12.0%), wheat (9.6%), peanut (7.0%), buckwheat (4.0%), and shrimp (2.5%). the proportion of anaphylaxis was highest in buckwheat (60.4%), followed by walnut and pine nut (42.1% each). korean children were hen's egg, cow's milk, walnut, wheat, and peanut, with distinctive distributions according to different age groups. anaphylaxis was reported in 25.2% among immediate-type fa. results: a total of 263 children with a median (inter-quartile) age of 6.13 years (4.27-8.43) were enrolled to the study; (male 66.5%). their ages at diagnosis were 0.50 years (0.40-0.67); follow-up times were 5.63 years (3.57-7.69) and milk specific ige levels at diagnosis were 7.6 ku/l (1.9-27.2). in 30.8% of the children there was only cma; the other children were polyallergic to different foods having most frequently egg white allergy. concomitant diseases were 56.7% atopic dermatitis, 35.4% were asthma, 12.2% were allergic rhinitis. during the follow-up milk tolerance was developed in 43.1%, 65.2% and 68.1% at the ages of 3, 5 and 6 years respectively. the specific ige level at the beginning of the disease was found to be a risk factor for the persistence of the disease (p<.001). conclusions: cma is frequently present with other food allergies. nearly half of the patients develop tolerance to cm up to the age of 3 years; whereas 2/3 becomes tolerant when they are at the age of 5 years. most frequent concomitant diseases are atopic dermatitis and asthma. objectives: two survey tools were used; a questionnaire based on similar surveys done overseas, and the validated food allergy quality of life questionnaire (faqlq). this was distributed throughout paediatric allergy clinics at two metropolitan centres. children and adolescents aged 10-19 completed the questionnaire independently, whilst parents assisted with children aged 5-9 years. results: 102 surveys have been collected at the time of writing of which 64 were answered by parents for young children. overall, 44/ 97 (45%) reported bullying, with a higher portion in older children and adolescents (22/37; 59%). of this group, 10/20 (50%) reported being bullied or teased because of their food allergies. from parental reports, 11/19 (57%) stated that their child had experienced bullying or teasing because of food allergies. for those not bullied, parents mentioned that this may be due to their child having friends at school, being too young for bullying or because other children at school had a good understanding of the severity of allergies and were educated by teachers. the most common location for bullying was "in the playground or sportsground" (36/39). the most common form of bullying involved being "teased, called names or someone has said mean things to me" (31/39). whilst food allergens were involved in bullying in many cases (24/39), there were no reports of children being forced to eat food to which they are allergic. of concern however, two adolescents reported experiencing an allergic reaction as a result of the bullying. the majority reported experiences of sadness from bullying (30/39) while seven stated that it had no effect. conclusions: our current research shows that 45% of children and adolescents with food allergies experience bullying, and that 22% (21/97) experience bullying specifically because of their food allergies. this indicates a significant social problem that requires addressing to positively assist those children living with food allergies. introduction: recently we demonstrated that intake of specific foods, types of fat and micronutrients was associated with inflammation and mucosal integrity in adults with eosinophilic oesophagitis (eoe). the current study aimed to compare dietary intake of these patients with dietary guidelines and intakes of the general dutch population to further investigate our hypotheses on the protective or allergy-provoking role of specific nutrients in eoe. results: the total faqlq score was low when assessed by teenagers and children (4.0 and 3.9, respectively) and moderately low when assessed by parents (2.7). experience of anaphylaxis and having multiple food allergies impaired hrql according to faqlq parent form (p<.05). gender, having prescribed an adrenaline-auto injector, experience of food provocation test, peanut allergy and faim did not contribute to different hrql. hrql in kindergarten and schools were moderately diminished (sum score 2.6 in schools and 2.2 in kindergartens) (p>.05). perceived disease severity was moderately present with total faim scores being 3.4, 3.6 and 3.9, when assessed by children, teenagers and parents, respectively (p>.05). 68% of participants' reported at least some possibility of dying if child/teenagers would accidently eat a food allergen. after fulfilling faqlq and faim, all participants expressed content, ten children/teenagers decided to approach food provocation tests de novo, employees of children's schools/kindergartens were encouraged in written invitations to assess anaphylaxis training programs, and four families accepted additional psychological support. conclusions: food allergies impair hrql in children and teenagers. allergies to multiple foods and experience of anaphylaxis were associated with more severe impairment of hrql. hrqlq and faim are useful, additional tools to assess and discuss child's/teenager's/parent's fears and obstacles because of food allergy and identify further needs of support. introduction: fruit allergy is the most common cause of food allergy in children older than 5 years and adults. regional variations have been observed in europe but there are few studies in pediatric population. objectives: in the context of a prospective and multicentric study on pollen and vegetable food allergy in spain, we enrolled 45 patients (median age 12, range 2-18, female 64%), who had suffered at least two episodes of immediate symptoms after ingestion of fruits and had positive skin test to the implicated fruits. immunocap isac was analyzed in all patients. our aim was to describe the clinical characteristics with the fruits involved and the usefulness of the allergens included in the immunocap isac to improve its characterization. symptoms were categorized into oral allergy syndrome (oas), systemic symptoms (ss) and anaphylaxis. results: a total of 45 patients were included. all of them had symptoms with more than one fruit. 42 patients had pollen sensitization. the main offending food associated with allergic reactions were peach (49%), kiwi (27%), melon (24%), apple (22%) and banana (15%). 5 allergic patients to peach had oas, 13 ss and 4 anaphylaxis, were recognized prup3 in all patients with anaphylaxis, in 4 patients witch oas and in 10 with ss, also prup3 associated with abstracts | 235 prup1 in 2 patients with ss. for allergy to kiwi, 9 patients had oas and 3 ss, were recognized actd2 in 1 patient with sao and 3 patients with ss. actd1 in 1 patient with ss. conclusions: in our population, the most prevalent fruit allergy was the peach, as in spanish adults. patients allergic to peach were the ones that presented the most ss and anaphylaxis, followed by allergic to apple. as previously have been reported most of them had sige to its components in isac; being prup3 the most prevalent (9% had prup1). only patients with ss with kiwi were sensitized to kiwi allergens .the majority of patients allergic to apple, melon and banana were not diagnosed by immunocap isac. introduction: studies have shown that asthma and allergy are prevalent among production workers processing seafood, particularly in workers processing crustaceans. a major ige-reactive proteins is the muscle protein tropomyosin. specific ige to tropomyosin is suggested as a central marker for crustacean allergy, however it is not the only protein characterised as an allergen in crab processing. objectives: the aim of our study was to characterise tropomyosin exposure and prevalence of sensitisation to allergens in workers processing king crab (paralithodes camtschaticus) and edible crab (cancer pagurus) in land based processing plants in norway. results: personal air samplers collected air from the workers' breathing zone during crab processing. workers' blood was collected for ige testing. extracts of both king crab and edible crab raw meat, cooked meat, intestines and shell were made in our lab and used for skin prick testing and immunoblotting. while processing cooked crab yielded highest tropomyosin levels in the edible crab plant, processing raw crab yielded highest levels in king crab plants. ten (12.8%) edible crab and 11 (9.7%) king crab workers had positive ige test (>0.35 ku/l blood, immunocap systems) to crab. four (10%) of skin prick tested king crab workers and 15 (19%) of skin prick tested edible crab workers had one or more positive reactions to edible crab extracts. more workers reacted to cooked crabmeat extracts than to raw crabmeat extracts. immunoblotting showed ige binding to a large number of proteins in all four extracts of both king and edible crab. binding was found to high molecular weight proteins in all four extracts of the crab tested, and the ige-reactive proteins differed between king crab and edible crab. conclusions: workers are exposed to tropomyosin in their breathing zone during crab processing. both king crab and edible crab workers are sensitised to crab, shown with immunocap specific ige test to crab, as well as positive skin prick tests and immunoblots to four different crab extracts made in our lab. workers processing crab in norwegian processing plants have an increased risk for developing sensitisation to crab. objectives: the aim of the study was to assess frequency of skin symptoms in surgery clinic employees, to evaluate burnout as a predictor of the frequency of skin symptoms, and to determine latexspecific ige in surgery nurses with skin symptoms. results: skin symptoms were significantly more frequent in surgery nurses (25%) than in surgeons (2.5%), other physicians (0), and other nurses (6.7%) (v 2 =18.16; p=.001). skin symptoms were also significantly more frequent in workers with high/medium than in workers with low emotional exhaustion (14.3% vs 5.2%; v 2 =4.32; p=.038), as well as in participants with burnout than in subjects without burnout introduction: baker's asthma sensitization pattern is changing due to the introduction of different types of grains and seeds. objectives: a 43 year-old ecuadorian man showing ocular, nasal and pulmonary symptoms when handling grain flours (with or without seeds), while baking for the last 8 years. he tolerated grain flours oral intake, but had oropharyngeal symptoms, rhinoconjunctivitis and dyspnea when eating sunflower and sesame seeds, mustard, and beer with alcohol. he tolerated alcohol-free beer. we performed an allergy study: prick-test with commercial extracts of pollens, dust and storage mite, fungus, animal danders, cereals, yeasts and mustard. prick by prick with patient's products: wheat, multicereals and seeded flour, sunflower seeds, regular and alcoholfree beer spirometry and niox. serial peak flow measurement at and away from work, handling tests with wheat flour and sunflower seeds. laboratory studies: specific ig e to cereals and seeds (cap-isac-microarrays). immunoblot with regular beer (at room temperature and boiled), wheat flour and sunflower seeds, and sequential chromatography. results: skin tests were positive with commercial mustard and all provided products except for the alcohol-free beer. spirometry was normal. niox: 116 ppb. peak-flow monitoring showed a 27% variability during working period, remaining stable during holidays. handling tests with wheat flour and sunflower seeds were positive. specific ig e was positive for grains, malt, gluten, mustard and sunflower seed. the specific determinants were positive to 7s-viciline, 11-s globulin, several prolamins (2s-albumine, alfa-amylase inhibitors and gliadin) and ltp. immunoblot detected a band lower than 14 kda in both regular beer extracts (not detected in alcohol-free beer) identified as barley's ltp, a band of 18 kda in the sunflower seed extract (2salbumine), and two bands lower than 14 kda in wheat flour extract (two kinds of alfa-amylase inhibitors). we present a non-atopic baker with occupational rhinoconjunctivitis and asthma due to prolamins (alfa-amylase inhibitor and gliadin of wheat flour together with 2s-albumin sunflower seed), and anaphylaxis when eating seeds (2s-albumins, 7s-vicilin and 11s-globulin) or drinking beer (sensitization to barley's ltp). it is interesting that the manufacturing process of non-alcoholic beer (high temperature and high pressure) seems to degrade barley's ltp, as suggested by both tolerance to its ingestion and loss of immunoblot band. objectives: the main objective of this study is to evaluate longitudinal change of lung function in workers employed in food preparation and distribution potentially exposed to food allergens. spirometries performed between 2012 and 2015 as part of medical surveillance of 58 food-handlers workers were evaluated. data about occupational task, work years, smoking habits and diagnosis of atopy, asthma and copd were collected from a clinical database. differences in prevalence were calculated by chi-square test, differences in means were calculated using spirola software referred to european predicted values. results: the majority of workers were females (n=49; 84.5%) and caucasians (n=57; 98.3%). 25 (43.1%) subjects were current smokers, 6 (10.3%) were ex smokers, 9 (15.5%) were atopic and no one reported a diagnosis of asthma or chronic obstructive pulmonary disease. 69% workers were canteen service employees and 31% were cookery employees. mean yearly values of the pair-wise within person variation of fev1 and fvc were respectively à248 ml and à266 ml. 6.9% of last observations had a fev1 below lln and 8.6% of last observations had a fvc below lln. conclusions: this study may help in planning preventive programs and in facilitating early recognition and diagnosis of work-related respiratory diseases. wheat, foods and latex allergens may determine decline in fev1 and fvc; furthermore, in our study, a significant proportion of workers reported exposure to tobacco smoke. excessive loss in fev1 over time should be evaluated using a percentage decline (15% plus loss expected due to aging) that we will make afterwards adding more years of follow up spirometries. intervention of smoke avoidance are needed. 0288 | prevalence of wood dust sensitization in occupationally exposed workers in germany-what can be tested? objectives: in 373 serum samples from patients with suspiciously allergic symptoms to wood dust overall 2797 specific (s)ige-tests with standardized wood-dust extracts coupled to streptavidin-immu-nocaps were conducted. additionally, ccd as known source of non-protein ige-target was evaluated. sensitization rates were calculated for 21 different wood species. most frequently requested wood dust allergens were obeche (n=198), beech (n=186), oak (n=173), spruce (n=175) and pine wood (n=147) followed by 80-100 requested sige-tests to mahogany, ash, larch and maple wood. results: overall wood dust sensitization rate was about 11% (range: 0-29%) with obeche, box wood and kambala as most prominent sensitization sources obtaining each more than 20% sensitization. no sensitizations were detectable to red cedar and meranti wood in more than 50 requested tests, respectively. in 81 patients at least one sige-sensitization to any wood was measured. there from 77 were additionally tested with ccd resulting in 47% positive and 53% negative ige responses to ccd. some wood species were exclusively recognized by ccd-positive subjects: ash, maple, alder, mahogany, teak, mansonia and palisander. whereas other woods were recognized by sige of subjects with / or without sige to ccd: obeche, box wood, spruce, oak, beech, limba, pine and kambala. relevance of wood sensitization next to ccd was investigated in eight double positive subjects (wood + / ccd +). specific ige-binding to wood allergens was completely inhibited by ccd in three samples. these subjects were supposed to have no clinically relevant wood sensitization. whereas in five samples sige-binding to selected wood species was not significantly reduced by ccd. in four of these patients skin prick tests (spt) and challenge tests (bronchial and/or nasal) with corresponding wood allergens were performed. three of four challenge tests were positive with the respective wood extract and all spts with wood extracts whose sige-binding was not affected by ccd inhibition showed positive reactions. here clinical relevance of sige-mediated wood sensitization could be demonstrated. conclusions: in summary, our data demonstrate that standardized wood extracts and ccd tools are necessary for valid in-vitro diagnosis of wood dust sensitization. introduction: respiratory symptoms have been reported frequently among seafood processing workers. since seafood processing workers handle the raw material and participate in processing activities during work, they are exposed to inhalable bioaerosols. this put them at risk of developing respiratory symptoms, asthma and allergy. there is little knowledge about the respiratory health status among fish production workers on board fishing trawlers. objectives: the aim of this study was to assess the respiratory health status among norwegian fish production workers, processing fish on board fishing trawlers. the study population consisted of 92 fish production workers, 21 machinists, 22 support crew members and 38 non exposed controls, all were males. written informed consent was the fish production workers had a significantly decreased fev 1% predicted compared to the non-exposed control group, b=à5.9, 95% ci [à11.2, à0.6], when controlling for age, pack-years and family history of asthma/allergy/eczema. the effect did not change when controlling for doctor diagnosed asthma or after dividing fish production workers by doctor diagnosed asthma. machinists and support crew members showed a similar decrease in fev 1% predicted, but the difference from the non-exposed control group was not statistically significant. furthermore fish production workers, reported a non-significantly increased prevalence of wheezing and daily morning cough compared to the non-exposed control group. conclusions: fish production workers, processing fish on board fishing trawlers, showed reduced lung function values compared to a non-exposed control group, and this finding is in accordance with previous findings in seafood industry worker populations from our research group. results: study group comprised 264 patients with work-related respiratory symptoms suggesting wra. the research completion with sic allowed to recognise wra in 164 persons (108 oa and 56 wea) and to exclude asthma in 100 cases qualified to reference group (gr). workers with wea occupationally exposed do lmw-a manifested the highest level of baseline non-specific bronchial hyperresponsiveness (nsbhr) in comparison to the other groups (table 1) . patients with oa exposed to lmw-a more frequently than exposed to hmw-a revealed nsbhr before sic (p=.036) with lower level of median (me) provocative methacholine concentration value causing 20% fall in fev 1 (pc 20 induced sputum (is) was obtained before and 24h after sic from 159 patients (38 gr and 121 wra). in all gr samples and samples possessed before sic from wea subjects exposed to hmw-a, intermediate profile of is (neutrophils (ne)<61% and eosinophils (eo)<2%) dominated ( results: eg: in "granulation" exposure is relatively high and the number of different enzymes handled is low; here the risk of sensitization is highest. in contrast in the pilot plants the exposure compared to granulation in general is lower but the number of enzymes handled concurrently is higher. still the sensitization risk is lower than the range for granulation. conclusions: even though this approach may seem crude and not free from bias and potential misclassification, data does not support the hypothesis that the number of enzymes increases risk of sensitization, whereas increasing exposure level seems to be a risk factor. this suggests that each enzyme exposure acts as a risk in its own right and that the "cocktail effect" seems to be of minor relevance. phospholipids. bioinformatic studies of sequence homology conducted prior to this study showed no similarity between the mpla1s and known allergens including ves v 1. however, the common enzymatic activity in ves v 1 and the mpla1s might still lead to crossreactivity. the goal of this project was therefore to test for possible cross-reactions between sige towards ves v 1 and three different mpla1s. methods: serum from 10 known wasp allergic persons with sige towards ves v 1 spanning from 1.57 ku/l to 1734 ku/l were used for inhibition studies. from each, 125 ll serum was incubated with 125ll of either saline solution (negative control), 50 lg/ml alk802 soluprick solution (positive control) or one of three mpla1s, each in three concentrations (either 0.5 lg/ml, 5 lg/ml and 50 lg/ml (n=3) or 5 lg/ml, 50 lg/ml and 500 lg/ml (n=7)). the level of sige towards ves v 1 was measured using the i211 immunocap, and a decrease in this level was calculated as %inhibition compared to the sige measured from serum incubated with the negative control. inhibition by mpla1s would indicate cross-reactivity. results: the positive control caused 62.5ae28.6% (n=10) inhibition of sige towards ves v 1. this was lower than expected but was found to be caused by a few sera where the fraction of sige towards ves v 1 was <10% of all sige towards wasp venom. in the remaining sera, %inhibition was 78.1ae14.9% (n=7). for all three mpla1s, no inhibition was found for any serum tested (n=10) at the highest concentration tested with %inhibition being 3.5ae2.5%, 3.3ae2.9% and 1.8ae6.0% respectively. conclusions: no inhibition of sige to ves v 1 was found to any of the three microbial phospholipases tested. this indicates that no cross-reaction is found between the phospholipase a1 in wasp, ves v 1, and phospholipase a1 from microorganisms despite the common enzymatic activity. objectives: the aim of study was to evaluate the prevalence and the impact of polyvalent ige-mediated allergy on the course of ad and the occurrence of allergic symptoms from other organs and systems in infants and young children. conclusions: polyvalent ige-mediated allergy is common in young children with ad and seems to be a risk factor for the severe course of the disease. introduction: non-steroid anti-inflammatory drugs (nsaid) are the second most common cause of drug allergies in childhood. objectives: the aim of the study is to determine the frequency of nsaid hypersensitivity in asthmatic children. results: 976 patients who were being followed up for asthma were included in this study. the mean age of the patients was 10.61ae4.21 years, while 59.5% (575) were male. 1% (n=10) of the patients had a reaction history to nsaid (ibuprofen in 4, flurbiprofen in 2, diclofenac potassium in 1, metamizole+acetylsalicylic acid in 1, paracetamol+acetylsalicylic acid in 1 and ibuprofen+acetylsalicylic acid in 1). nsaid sensitivity was confirmed in 9 (0.9%; 9/976) patients who were tested with suspected drugs, while the provocation test was found negative in one patient who described reaction with ibuprofen. of the 2000 children who were assessed as a control group, only 1 had a reaction history to acetylsalicylic acid and no reaction developed in the provocation test. conclusions: nsaid hypersensitivity is more common in patients with asthma. thus, these patients should be evaluated for nsaid hypersensitivity. results: from jan 2012 to dec 2013, 8840 pediatric cases were received by kaers. of 8840 pediatric patients, 56.4% were male, 42.0% were female and 1.6% were unknown. these 8840 pediatric cases included a total of 17 319 adr events with an approximate average of 1.9 adr events per report. of those 8840 cases, 21.2% were in infants (age 0-1 years), 27.8% were in young children (age 2-7 years), 24.3% were in old children (age 8-13 years), 22.7% were in adolescent.(age 14-18 years) and unknown were 4.0%. male to female ratio was 1:0.8 and the mean age was 8.3ae6.3 years. regarding categorical ranking of reported adr agent groups, the most common group were antibiotics (24.4%) followed by antineoplastic agents (14.8%), vaccine (11.6%), antipyretics (7.1%), opioids (5.4%), sedatives (3.1%), antiepileptic drugs (2.9%), contrast media (2.3%), steroids (2.0%). the most common adr symptoms were gastrointestinal system disorder in 33.2%, skin-appendage disorder in 19.5%, abstracts | 249 body as a whole-general disorder in 9.5%, central-peripheral nervous system disorder in 7.5%. regarding seriousness of adrs, 610 events (6.9%) had episodes requiring hospitalization and were considered life threatening. of these, 126 cases had anaphylaxis or anaphylactoid reactions. introduction: multiple drug allergy is an adverse reaction to two or more structurally unrelated drugs that appears to occur by immune mediated mechanism. patients with a history of reaction to two or more drugs often apply to allergy clinics. objectives: the aim of this study is to evaluate the test results of the patients who have a history of multiple drug allergies and underwent drug provocation tests. results: during the study period, drug provocation tests were performed in 889 patients (1014 drug provocation tests). of these patients, 106 (11.9%) had a history of drug reactions to 2 or more drugs. the mean age of the patients who had a history of reactions to two or more drugs was 8.48ae3.94 years, and 58.5% (n=62) toms, and autoimmune manifestation in comparison to igm/iga responders (respectively, pneumonia: 64%, 31% and 0%; chronic diarrhea: 25%, 14% and 0%; autoimmunity 41%, 29% and 0%; autoimmune cytopenias: 17%, 8% and 0%). malignancies were found more frequently in the non-responders and igm-only responders groups in comparison to igm/iga responders (respectively, 23%, 14% and 0%). eleven (15%) patients died during the study time. survival analysis according to the igm/iga responder status showed that the 6-years estimated survival for non-responders vs igm-only vs igm/iga responders was respectively after one year 98%, 87% and 100%; after two year: 93%, 87% and 100%; after three years: 91%, 80% and 100%%; after 4 years: 87%, 80% and 100%; after 5 years: 87%, 80% and 100%; after 6 years: 83%, 80% and 100%. interesting, in our series only two deaths were due to infective complications: five were consequent to malignancies, one to autoimmune cytopenias and three to not-cvid related conditions. between-infusions intervals (10-14 days) than pid patients (6-7 days). finally, a small number of patients with anti-cd20-related sid was able to discontinue scig replacement therapy after recovery of spontaneous igg production. conclusions: this is, to our knowledge, the biggest single center cohort of scig-treated patients ever described. results suggest that safety and effectiveness of scig is similar in pid and sid, irrespective of the mechanisms underlying igg depletion. moreover, in sid a lower igg dosage is required and ig replacement does not always need to be lifelong, with obvious pharmacoeconomic implications. 0685a | should we screen children with bronchial asthma for primary immune deficiencies? miteva d 1 ; perenovska p 1 ; papochieva v 1 ; georgieva b 1 ; lazova s 1 ; naumova e 2 ; petrova g 1 the patient became febrile and cultures were repeated, being positive to campylobacter jejuni, resistant only to ciprofloxacin (blood) and to campylobacter coli, susceptible only to gentamicin and amoxicillin/ clavulanic acid (stools). treatment was thus switched to amoxicillin/ clavulanic acid (1875/125mg 8/8h). the patient became apiretic at day 2 and improvement of local inflammatory signs was noticed, treatment was prolonged for six weeks. one week after cessation, skin lesion worsened again in the same location, in association with fever. blood and stool cultures were repeated and gentamicin (240mg/day; iv) and cefixime (200mg 12/12h) were started, in agreement with previous cultures results. curiously, there was no history of diarrhea, but the patient referred a period of recurrent abdominal colicly pain, before cutaneous lesions appear. conclusion: bacteremia with campylobacter species requires specific laboratory workup. 7diagnosis of campylobacter jejuni bacteremia should be considered in hypogammaglobulinemic patients with recurrent fever, particularly when typical copper color erysipela-like skin lesions occur. campylobacter eradication can only be achieved with prolonged antibiotic therapy guided by antibiogram in cultures. conclusions: in this study, genetic defects of five higm patients have been identified, for other patients further genetic investigation such as next generation sequence (ngs) is required. the study results can help diagnose of the disease more definitively and also can provide valuable information for genetic counseling especially for those who have a history of immunodeficiency in their families and also for prenatal testing. conclusions: mutation analysis of unc13d gene can help the families with hlh patients give genetics counseling for carrier detection and prenatal diagnosis. woelke s 1 ; valesky e 2 ; bakhtiar s 3 ; bader p 3 ; schubert r 1 ; zielen s 1 1 department for children and adolescents, division of allergology, pulmonology and cystic fibrosis, goethe university hospital, frankfurt, germany; 2 department of dermatology, venereology and allergology, goethe university hospital, frankfurt, germany; 3 department for children and adolescents, division for stem cell transplantation and immunology, goethe university hospital, frankfurt, germany introduction: ataxia telangiectasia (a-t) is a devastating multi-system disorder characterized by progressive cerebellar ataxia, growth retardation, immunodeficiency, chronic pulmonary disease and genetic instability with an increased risk for malignoma. as described in other primary immunodeficiencies cutaneous granulomas are a known phenomenon also in a-t. still treatment indication and strategies remain controversial. objectives: from our cohort of 60 classical a-t patients, eight patients in the aged 2 to 11 years presented with granulomas. histopathology of the lesions confirmed the presence of granulomatous inflammation without detection of any microbiological agent in all patients. five patients suffered from cutaneous manifestation, in two patients we detected a bone and in one a joint involvement. both patients with bone involvement (patients 1 and 2) as well as one patient with massive skin manifestation (patient 3) were treated with tnf inhibitors (infliximab). the patient with granulomas in his finger joint (patient 4) was bone marrow transplant (bmt) for other reasons. year led to a total remission for three years now. in patients 2 and 3 treatment with tnf inhibitors led to a partial regression of granulomas. treatment interruptions caused deterioration again. in the course of treatment the effects of infliximab diminished most likely caused by drug antibodies. after changing treatment to subcutaneous adalimumab a further regression could be detected. in patient 4 granulomas totally disappeared with immune reconstitution after successful bmt. partially successful in treatment of granulomas. due to the known immunodeficiency in a-t patients, indication for immunosuppressive therapies as tnfa inhibitors should be held strictly. woelke s 1 ; hess u 1 ; knop v 2 ; krausskopf d 3 ; kieslich m 3 ; schubert r 1 ; zielen s 1 of 1 to 38 years regarding c-reactive protein (crp), liver enzymes, abdominal ultrasound and neurological status (ataxia score). we divided the patients into two age groups of 27 a-t patients aged 1 to 11 years and 26 a-t patients aged 12 years to 38 years. ataxia score (r=0.34), although the underlying pathomechanism is unclear. ultrasound revealed nonalcoholic fatty liver disease in only one young patient (3.7%) compared to 11 older patients (42.3%). one female patient aged 37 years died due to a hcc. conclusions: liver disease is present in almost all older a-t patients. structural changes, nonalcoholic fatty liver disease and fibrosis are frequent findings. there is a considerable risk for hcc. prospective studies are necessary using noninvasive techniques for the assessment of liver fibrosis (eg transient elastography) and to establish the risk of hcc in a-t patients. objectives: in this study, it was aimed to determine the frequency of pollen-food syndrome in children who have sensitization to pollens. results: 672 pollen-sensitized patients were included in this study. the mean age of the patients was 11.82ae3.90 years, while 63.1% (n=424) were male. in 45.2% (n=304) of the patients, allergic rhinitis was concomitant with asthma. 4.3% (n=29) of the patients described symptoms related to pfs. 31% (9/29) of them had a history of anaphylaxis with suspected food. the mean age of the patients describing pfs was 12.22ae4.26 years and 55% (n=16) of them were female. in 17 (58.6%) of these 29 patients, skin tests performed with suspected food was positive, but in one patient the skin test was negative while specific ige was positive. suspected food was fruit/ vegetables in 21 patients. in patients with pollen allergy, oropharyngeal symptoms related to fresh fruit, vegetables, dried fruits and nuts should be enquired. it should be noted that these patients might experience serious systemic reactions including anaphylaxis. patel nb 1 ; vazquez-ortiz m 1 ; lindsley s 1 ; abrantes g 1 ; bartra j 1 ; dunn galvin a 2 ; turner pj 1 conclusions: there is no evidence that the occurrence of anaphylaxis at fc, and self-treatment with an adrenaline auto-injector device, result in adverse impact on hrql measures. the impact of a reaction at food challenge appears to confer greater benefit on the parent than the child. the relationship between confidence in management and hrql needs further assessment, since it is likely that these outcomes will be affected in different ways following therapeutic interventions. results: fifty-one patients (23 females, 28 males) (median age: 6.5 years, range 0-18) with cma were studied. spt to cm was 7.3ae3.3 mm as mean diameter. forty-eight out of fifty-one (94.1%) patients underwent dm-opt (3 patients refused to underwent opt due to positive spt or s-ige to dm introduction: bovine milk is the most common food allergen in children under 3 years of age. milk allergy is treated by eliminating milk from the diet. the milk elimination diet endangers the child's energy intake and also exposes the child to shortage of multiple nutrients. this study was needed because there are no previous systematic reviews about this subject. objectives: the aim of the present study was to examine if the milk allergy or the other factors associated with milk elimination diet have an influence on child's growth. the present study was conducted according to international guidelines for systematic reviews. results: a total of 646 studies were initially identified, of which three fulfilled our criteria. these three studies included 186 children with cow's milk allergy and 122 control children. in all these three studies, children with cow's milk allergy were lighter than controls. in addition, in two studies, the growth of the children with cow's milk allergy was stunted. in two studies the milk elimination was substituted with special infant formula. also in one study where both the growth and the weight were stunted, no major differences in energy or nutrient intake between cow's milk allergic cases and controls were reported. conclusions: current evidence suggests that milk allergy is associated with stunted growth in childhood, but reasons for this are unclear. in order to clarify the effect of cow's milk elimination diet on growth in childhood and the underpinning mechanisms, more studies need to be conducted. in addition, special attention to the diet and growth of children with cow's milk allergy is needed. results: a total of 158 817 children were surveyed in this 6-year period (annual average: 26 470). in 2011, 704 children (2.6%) were diagnosed with food allergy, including 159 cases of pfas, 376 cases of egg allergy, and 235 cases of dairy-product allergy. in 2016, 939 children (3.6%) were diagnosed with food allergy, including 344 cases of pfas, 385 cases of egg allergy, and 249 cases of dairy-product allergy. over this 6-year period, the prevalence of pfas increased 2.2fold (from 0.59% to 1.31%). among the pfas cases, the prevalence of rosacea and apple allergy increased 2.9-and 3.3-fold, respectively. the most prevalent was apple allergy (0.76%), followed by peach (0.59%), loquat (0.53%), plum (0.44%), pear (0.40%), and strawberry (0.18%). the prevalence increased significantly for pfas but not for other types of food allergy (egg and dairy-product allergy). in the future, nationwide studies are needed to further elucidate the relationship between pfas and allergic rhinitis. 0327 | immune profile after oit in children with cow 0 s milk allergy patients with elimination diet, group 3: patients with natural tolerance, group 4: healthy control). in all groups specified laboratory tests were performed at onset and also at 6 month to treatment groups. in desensitization group at 6 month of treatment we evaluated increase in total ige level, sp iga and igg4 antibody responses, decrease in cow's milk spige levels and an increase in il-2, il-10, tgf-b cytokine responses without a difference in cd4+cd25+fox-p3% levels. il-13 levels were similar with pre-treatment levels whereas foxp3 mrna expression was similar with tolerance group. in elimination group at 6 month treatment, there was a decrease in cow's milk spige whereas there was no change in sp iga levels and a minimal increase in igg4 levels. there was no change in il-2 and il-10 cytokine levels. and an increase in tgf-b cytokine response less than group 1, decreased il13 response, a foxp3 mrna expression differed from tolerance group was identified. objectives: although hyperuricemia has a significant prevalence in the general population, and has been related to exercise-induced asthma, could be related to bronchial asthma and nasal polyposis. hitherto, its possible association with hypersensitivity to nsaids and its convenience as biomarker have not been acquainted. conclusions: in our population, hyperuricemia has not demonstrated to be a reliable biomarker related to nsaids allergy and could not be used as a risk factor for assessing the triad nsaids allergy, asthma and nasal polyps. to study the vas of the total score was seen significant variance in: nsaid-cu (3.38pt) and nsaid-pa (6.57pt) (p=.046), nsaid-cu conclusions: data support that cutaneous manifestations have a common response with aerd to cox inhibitors. conclusions: this study showed that in a positive drug oral provocation test, respiratory symptoms were accompanied with nitric oxide changes in both nasal and exhaled way. barrionuevo e 1 ; doña i 1 ; salas m 1 ; bogas g 1 ; guerrero ma 1 ; sanchez mi 1 ; cornejo-garcia ja 2 ; torres mj 1 1 allergy unit, regional university hospital of malaga-ibima, malaga, spain; 2 research laboratory, ibima-regional university hospital of malaga-uma, malaga, spain introduction: non-steroidal anti-inflammatory drugs (nsaids) are the most frequent triggers of drug hypersensitivity reactions, being cross-hypersensitivity reactions (chr) the most frequent. the categories included in chr are nsaids-exacerbated respiratory disease (nerd); nsaids-exacerbated cutaneous disease (necd) and nsaids induced urticaria/angioedema (niua). however, it has been reported patients with chr to nsaids who developed a combination of skin and respiratory symptomatology (blended reactions). objectives: our aim was to analyse the characteristics of patients with blended reactions and compare with those developing symptoms exclusively respiratory (nerd) or cutaneous (niua). episodes of cutaneous and/or respiratory symptoms after the intake abstracts | 265 of ≥3 different nsaids included strong cox-1 inhibitor (acetylsalicylic acid (asa) and/or indomethacin); ii) if they had <3 episodes of cutaneous and/or respiratory symptoms induced by <3 different nsaids, a positive drug provocation test (dpt) with asa was required; iii) if patients had respiratory symptoms accompanied or not by cutaneous involvement, a positive nasal provocation test with lysine aspirin (npt-lasa) was required. atopy was assessed by skin prick test using a panel of inhalant and food allergens. objectives: subjects with nsaids hypersensitivity were divided into two groups: a) those from 2 to 14 years and b) those from 15-20 years. diagnosis was established by a clinical history and controlled challenge with asa. atopic status was verified with a detailed allergological study including skin testing to inhalant allergens. clinical entities were classified in three categories: urticaria/angioedema, anaphylaxis and respiratory (asthma and/or rhinitis). cases. no differences were observed in the atopic status between both groups. there were significant differences between males/ females (p<.05). when we compared the clinical entities there were more cutaneous manifestations, mainly angioedema, in the group a) and more anaphylaxis in group b) although there were no significant differences due to sample size. conclusions: significant sex differences between hypersensitivity reactions to nsaid occurs with predominance of males in the first group (a). although there are also a predominance of clinical entities, an increase number of cases is needed to establish significance. studies on this direction are in progress. show an increase in all age groups. etiologic analysis was limited as the study was carried out using the icd-10 code (nhic records) and database of self reporting systems (kaers). so, further study was needed. introduction: genetic variants from the 17q21 locus are the strongest known genetic determinant for early-onset childhood asthma, and have also been associated with uncontrolled asthma despite asthma treatment. objectives: the aim of the study was to assess whether there is an association between a single nucleotide polymorphism (snp) in the 17q21 locus (rs7216389) and asthma exacerbations despite the objectives: our hypothesis is that the arg16 allele is associated with increased use of prescribed asthma medication, over a 9-year period. to explore this hypothesis, we have undertaken a secondary analysis of breathe, a study of gene-environment associations with asthma severity. breathe data were collected on participants with asthma, aged 3-22 years, between 2003 and 2005, in tayside conclusions: in children and adults, the homozygous arg/arg status is associated with long-term increased prescribing for asthma medication compared to those carrying at least one gly allele. defining subgroups of individuals requiring more medicines could help predict treatment costs and develop targeted management strategies. objectives: considering the role of ptgdr in allergy, the goal of this study was to analyze the effect of ptgdr expression on cytokine levels in the a549 cell line. analyze ptgdr expression in a549 cell cultures. cytokine production assays in the culture supernatant were measured by cytometric bead assay using the bioplexpro tm human cytokine standard 27-plex, group i. the assays were conducted with bioplex high-throughput fluidics system, powered by the luminex technology. every sample was run at least in triplicate. the ptgdr expression in the transfected cells with the haplotypic variants differed by 5 orders of magnitude relative to control cells (p<.001). we found significant differences in ptgdr expression between ctct and cccc haplotypic variants. the cccc (à613 c, à549 c, à441 c, and à197 c) introduction: c159t polymorphism in the cd14 gene has been suggested in susceptibility to asthma. cd14 is a multifunctional receptor endotoxin, which is expressed on the surface of macrophages, monocytes and neutrophils. it is likely to play a role in the inflammation pathway. though data is available regarding association of cd14 gene with asthma but independent studies are in conflict. objectives: the present study was conducted to examine the association of promoter c159t single nucleotide polymorphism (snp) in the cd14 gene for indian children with atopic asthma. we characterize the c159t polymorphism in children with asthma (50), cohort group (20) and healthy control group (50) by pcr rflp. association analysis was performed using v2 tests. we also analyzed the association of cd14 (c159t) with total ige levels by elisa and foxp3 expression using flow cytometry. in this snp a 497 base pair (bp) pcr product was generated using the standard primers. after restriction products showed that homozygous c allele was appeared as a single 497bp band, the homozygous t allele as bands of 144 bp and 353 bp, and heterozygous exhibits all three bands (144, 353 and 497 bp). the or of cc genotype frequency abstracts | 271 was 0.84 in study group and 0.78 in cohort group and the or of c allele frequency was 2.38 in study group and 2.52 in cohort group. total ige level were found to be significantly higher in cc genotype compared to ct and tt genotype. foxp3 level is significantly higher in control group in all genotypes compared to cohort and study group. conclusions: the present study concludes that in cd14 gene polymorphism cc genotype was not significantly associated with asthma but other factors ie total ige and foxp3 showed significant association of cc genotype with asthma. on the other hand, there was significant association of c allele with asthma. 0362 | the disbalance of tlr2, tlr4 gene expression and cytokines production in children with bronchial asthma svitich oa 1 ; gankovskaya lv 2 ; namazova-baranova ls 3 ; bragvadze bg 2 ; alekseeva aa 3 ; gankovskii va 3 objectives: examined were 38 patients with bronchial asthma aged from 3 to 12 years and 10 healthy children of the same age. cytokines were determined by elisa. determination of mrna expression in scrapings from the mucous membranes of the respiratory tract and in peripheral leukocytes was carried out polymerase chain reaction in real time. results: in scrapings from the mucous in patients with moderate to severe asthma found a significant increase in the gene expression of tlr2, 3 times, the gene tlr4 in 10 times in comparison with the control group. in children with severe asthma also found a significant increase in the gene expression of tlr2 4.8 times compared to healthy children, p≤.05). indicators of tlr4 gene expression in this group of patients have a tendency to increase, but not statistically significant. when comparing the indicators of innate immunity in samples with a leukocyte mass in the group of children with severe asthma showed a decreasing expression of tlr2 compared with the index in healthy children. also decreased the expression of tlr4. in children with moderate ba similarly, a significant decrease of tlr4 gene expression in 18 times. the trend towards reduced expression of tlr2 remains in this group, but is not reliable. in washings from the nasopharynx shows that patients with bronchial asthma il-1 is increased 4.5 times compared to the norm, to 45 pcg/ml, tnf increased in 6 times and 18 pcg/mg in severe asthma, and 7.7 pkg/ mg-for mild, il-17 increased in 3 times (16 pcg/mg), pkg of 7.5/ mild blood, il-10 also increased 4.5-fold and equal to 22.5 pkg/mgwith heavier with easy-7.5 pkg/mg, normal à5 pkg/mg. ie is an increase in proinflammatory and anti-inflammatory cytokines. conclusions: the overexpression of tlr2, tlr4 accompanied by increasing, the production of cytokines. this is a violation of mechanisms of innate immunity at the level of the mucous membrane of the nasal cavity on the role of inflammation in the pathogenesis of tlr. 0364 | sustained reduction in risk of experiencing asthma symptoms and using asthma medication in years following grass slittablet treatment-results from the paediatric gap trial were: wheezing, cough (for more than 10 consecutive days), shortness of breath, chest tightness. the odds ratio for having asthma symptoms, using asthma medication, or having asthma symptoms and using asthma medication was significantly lower in the grass slit-tablet group during the 2 followup years. for asthma symptoms, the results were: or=0.52, p=.016 days with csms<2 were defined as "no or minimal symptoms" and days with csms >6 were defined as severe symptoms. csms score was significant lower in the ilit group than in the placebo group for 2014, 2015 and 2016. in 2014 mean csms was 4.05 roth-walter f 1 ; schmutz r 2 ; mothes-luksch n 2 ; zieglmayreer p 2 ; zieglmayer r 2 ; jensen-jarolim e 3 objectives: here, we investigated whether the immune molecule lipocalin 2 (lcn2) may discriminate between allergic and sensitized individuals, and between responding and non-responding patients. results: lcn2-concentrations were assessed in sera of healthy and allergic subjects (n=160) as well as of house dust mite (hdm) allergies that underwent hdm-sublingual immunotherapy (slit) in a randomized, double-blind, placebo controlled trial for 24 weeks. sera pre -, post-slit and at least 3 months after slit were assessed for lcn2 and correlated with total nasal symptom score (tnss) obtained during chamber challenge at week 24 in patients receiving hdm-(n=30) or placebo-slit (n=10). allergic individuals had significant lower lcn2-levels than healthy controls, with women having lower lcn2-levels than men in the patient cohort. hdm-allergic patients who received hdm-slit had a significant increase in hlcn2 6 months after termination of hdm-slit, whereas in subjects receiving placebo no increase in hlcn2 was observed. within the hdm-slit treated group, lcn2-levels were significantly higher in patients whose symptoms improved during slit in contrast to those in which symptoms became more severe. hence, time-course of lcn2 in an allergic individual was predictive to assess clinical reactivity to hdm. objectives: here, we present the benefits of treatment in terms of nnt to prevent one additional child from having asthma symptoms and asthma medication use. children treated with grass slit-tablet had a reduced risk of asthma symptoms and asthma medication use during the 2-year follow-up period compared with placebo (or=0.28 [0.14, 0.57] for sq grass slit-tablet (n=377) vs placebo (n=398), p<.001, relative risk reduc-tion=71%). the risk reduction was independent of age at treatmentstart. younger children had a higher predicted probability of developing asthma symptoms and asthma medication use than older children. thus, the younger the children were at treatment-start, the greater the percentage was prevented from having asthma symptoms and asthma medication use during follow-up off treatment. for children aged 5 at treatment-start, the risk was reduced from 40% to 24% and for those aged 12 it was reduced from 10% to 5%. consequently, the nnt to prevent one additional child from having asthma symptoms and asthma medication use during the 2-year follow-up increased with age, with nnt=6 for children aged 5 and nnt=20 for children aged 12. conclusions: the grass slit-tablet reduced the risk of asthma symptoms and asthma medication use during the 2-year follow-up period; the risk reduction was independent of age. however, the nnt increased with age as younger children had a higher risk of developing asthma symptoms and asthma medication use, emphasising the importance of treatment-start early in life. results: 226 participants were screened for birch and grass allergy, of whom 93 ultimately met randomization criteria and were treated with either slit-t or placebo for 4 months. treatment was preceded by a successful baseline birch pollen challenge in the eeu where a minimum tnss of 6 was achieved in the first 2 of 5 hours of pollen exposure. 87 participants attended the post treatment challenge in the eeu, also 5 hours in duration. no significant differences were noted in the reduction of birch-induced tnss compared to baseline between the slit-t and the placebo treated participants (the primary outcome measure). adverse events with a minimum 5% frequency occurred in 50% of participants in the placebo arm and 70% of participants in the active arm, with upper respiratory tract infection (32% in active arm and 24% in placebo arm) being the most common. oropharyngeal itch was the most common adverse event with causality at least possibly related to study medication (23% in active arm and 2% in placebo). no serious adverse events occurred including no anaphylaxis. objectives: here, we present a pooled subgroup analysis in adolescents from 2 phase ii/iii-iii trials with the hdm slit-tablet (12 sq-hdm dose), p001 in north america and to-203-3-2 in japan. results: to-203-3-2 was a randomised, dbpc phase ii/iii trial investigating the efficacy and safety of the hdm slit-tablet in japanese adolescents and adults (12 to 64 years) with moderate-tosevere hdm ar (n=946, of which 302 were adolescents). subjects were randomised to treatment with the hdm slit-tablet in doses of 6 sq-hdm, 12 sq-hdm or placebo for 1 year. p001 was a randomised dbpc phase iii trial investigating the efficacy and safety of the hdm slit-tablet in north american adolescents and adults (≥12 years) with moderate-severe hdm ar with or without asthma (n=1482, of which 189 were adolescents). subjects were randomised to treatment with 12 sq-hdm or placebo for up to 1 year. in both trials, the primary endpoint was the average total combined rhinitis score (tcrs) during the last 8 weeks of treatment. the pooled analysis was performed on mean values using a linear mixedeffects model. treatment with 12 sq-hdm of the hdm slit-tablet resulted in a statistically significant reduction in the average tcrs of 1.04 (22%, p=.002) compared to placebo in the last 8 weeks of treatment. statistically significant differences were seen for both components of the tcrs, the rhinitis medication score (difference=0.06, p=.038) and rhinitis symptom score (difference=0.87, p=.004). furthermore, treatment with 12 sq-hdm resulted in a statistically significant reduction of the conjunctivitis symptom score compared to placebo (differ-ence=1.10, p=.026). treatment was well tolerated. the most frequent adverse events were mild-to-moderate local allergic reactions. the pooled subgroup analysis showed that the hdm slit-tablet (12 sq-hdm) was effective in treating hdm allergic rhinitis in adolescents (12-17 years old). the reduction in the primary endpoint tcrs was statistically significant and comparable to what has been observed in adults. results: at the time of the data-cut for this analysis, a total of 179 european patients were screened (58% male). 56% of the subjects were aged 4-11, 28% were aged 12-17, and 16% were adults. the mean age of the sample was 11.4 [sd 6.9] years, with a minimum of 4 and a maximum of 49 years. in europe were children. many were allergic to foods other than peanut, and most had at least one other atopic condition. even if allergic to multiple foods, patients and their families were nonetheless keen to participate in the trial. the majority of the screened patients were highly sensitive to peanut, reacting to 144mg (cumulative) or less of peanut protein but a significant proportion of screening failures were due to lack of reactivity to this dose. 0372 | efficacy of 300ir 5-grass pollen sublingual tablet: improvement in subjects with grass pollen-induced allergic rhinoconjunctivitis based on well days and severe days evaluation objectives: four phase ii/iii dbpc studies, 3 in adults and one in children/adolescents, were conducted worldwide. participants were abstracts | 277 randomised to receive the 300ir tablet or placebo starting 4 months (4m) prior to the pollen season and continuing for its duration. data from the first season of the 3 trials in adults were pooled. the well days were defined as those with not more than one moderate symptom or 2 mild symptoms of the 6 evaluated symptoms (sneezing, rhinorrhoea, nasal pruritus, nasal congestion, ocular pruritus, watery eyes) and no use of rescue medication. the severe days were defined as either at least one moderate symptom in subjects using rescue medication, or at least 2 moderate symptoms or one severe symptom whether rescue medication was taken or not. for each subject, the proportions of well days and those of severe days were evaluated during the pollen period while on treatment. treatment groups were compared using a wilcoxon 2-sample test. introduction: the safety of subcutaneous immunotherapy (scit) has been proven in several studies, but the occurrence of side effects (se) remains a concern in daily clinical practice and understanding of their risk factors and avoidance strategies remains limited. objectives: the aim of presented study was to assess the incidence and risk factors of early and late side effects in patients undergoing scit. we conducted a 2 year-long observation of over 1300 patients undergoing scit in our outpatient clinic. we recorded detailed information for each administration and screened subjects for both immediate and late reactions. we compiled the records with medical histories to build a database, which was analyzed using multiple logistic regression. casein is a major cow 0 s milk allergen and very resistant to high temperatures. higher levels of ige towards caseins have been reported to associate with persistent cow 0 s milk allergy and higher risk of adverse reactions before and during the milk oit. a follow-up study on oit to milk started in 2005 with a six-month built-up phase. seventy-six children (mean age 9.7 years) with challenge-verified cma participated the milk oit and their immunological changes were analyzed employing crd. during the year 2016, long-term follow-up report was collected by questionnaires (73%, n=49) and blood samples (46%, n=35). specific antibody responses were characterized before oit and on long term follow up and compared to long-term questionnaire results: milk consumption (yes/no) and side-effects from milk from past 12 months (yes/no). objectives: to define the utility of crd in long-term follow-up after milk oit. results: mean follow-up time was 8 (5-11) years. ten patients (20%) avoided milk completely and 39 patients (80%) reported routine consumption of dairy products. side effects after milk consumption from last 12 months were reported by 39% of the patients. increased specific ige levels towards caseins were seen among patients who did not consume milk at the long-term (p=.03*). there was no significant association between the long-term milk consumption and ige levels to whole milk (p=.08), caseins (p=.06), alpha-lactalbumin (p=.25) and beta-lactoglobulin (p=.08) measured before the oit in this small sample. patients who consumed milk at the longterm follow-up and reported side effects had higher specific ige levels towards caseins before oit (p=.02*). conclusions: there was a high incidence of side effects even after eight years of milk consumption, which may indicate desensitization instead of true tolerance. component-resolved diagnostics may corroborate in predicting prognosis, as suggested by higher incidence of side effects among patients with high levels of ige towards caseins also in the long-term follow-up. objectives: retrospective study of 346 patients who underwent subcutaneous ait for allergic rhinitis (20% had concomitant asthma). patients with less than 4 months of treatment were excluded. large local reactions (wheal≥25 mm) and systemic reactions were defined according to who grading system. patient's satisfaction was defined by vas score. results: the mean age of patients (54% males) was 28ae13 (range 5-72) years (11%<16 years).the most prevalent immunizing allergens were house dust mite (92%), olive (26%) mix of grasses (23%) and pets (17%). 47% of patients were immunized against a single allergen whereas 12% were immunized with≥4 allergens. patients were followed for 24ae18 months. following ait the vas score decreased from 7.2ae2.3 to 3.6ae2.5 (p<.0005). 92% of the patients declared that they will recommend immunotherapy to their relatives. systemic adverse reactions (37% class i, 62% class ii) were observed in 120 introduction: metabolomics, one of the core disciplines of system biology, is a high-dimensional biology method that may allow hypothesis-free profiling of biomarkers, rather than a traditional hypothesis-driven approach. objectives: this study aimed to apply the metabolomic approach to serum to longitudinal alterations during allergy immunotherapy (ait) in dermatophagoides pteronyssinus (der p) sensitized asthmatic children. results: a robust hydroxyeicosatetraenoic acids (hetes) of inflammatory responses was found for discriminating during ait, including 5-hete, 12-hete and 15-hete. 12-hete and 15-hete were significantly decreased continuously from 6 through 12 months of ait. moreover, compared with baseline of ait 5-hete was detected in very low level during 6 and 12 months. the metabolomic profiling could clearly longitudinal alterations biochemical-metabolic profiles in der p-sensitized children during ait. these markers might be involved in asthma pathophysiology but also represent the therapeutic target for ait. background: metabolomics, one of the core disciplines of system biology, is a high-dimensional biology method that may allow hypothesis-free profiling of biomarkers, rather than a traditional hypothesis-driven approach. this study aimed to apply the metabolomic approach to serum to longitudinal alterations during allergy immunotherapy (ait) in dermatophagoides pteronyssinus (der p) sensitized asthmatic children. methods: in this longitudinal study, we recruited 58 der p-sensitized asthmatic children with ait for 12 months. serum samples were analyzed using a metabolomic approach based on mass spectrometry. results: a robust hydroxyeicosatetraenoic acids (hetes) of inflammatory responses was found for discriminating during ait, including 5-hete, 12-hete and 15-hete. 12-hete and 15-hete were significantly decreased continuously from 6 through 12 months of ait. moreover, compared with baseline of ait 5-hete was detected in very low level during 6 and 12 months. the metabolomic profiling could clearly longitudinal alterations biochemical-metabolic profiles in der p-sensitized children during ait. these markers might be involved in asthma pathophysiology but also represent the therapeutic target for ait. we performed desensitization with asa according to the wong protocol (doses of 0. 1, 0.3, 1, 3, 10, 30, 40, 81, 162, 325 mg of asa at intervals of 10-20 min). we pre-medicate with cetirizine 10 mg. our patient successfully reached the necessary dose: 164 mg of asa (0. 1, 0.3, 1, 3, 10, 30, 40, and 80 mg) conclusions: approximately 10% of patients with asthma undergo respiratory symptoms due to exposure to aspirin, meanwhile 0.07% and 0.2% of the population shall undergo hives when exposed to it. desensitization with aspirin is an efficient and safe treatment in patients with hypersensitivity type i, iii and iv. due to the benefits and low toxicity of the wong protocol, we recommend this protocol in order to desensitize patients with allergy to aspirin who undergo rheumatologic or cardiovascular disorders and need antiplatelet treatment. we finally recommend our patients to take 150 mg of asa premedicated with 10 mg of cetirizine every day. we warn that if you interrupt the administration of asa for more than 48 hours, it shall have to be administered again under medical supervision. semedo fm 1 ; cruz c 2 ; reis r 2 ; tomaz e 2 ; in acio f 2 horiuchi t 1 ; takazawa t 2 ; saito s 1 1 department of anesthesiology, gunma university hospital, maebashi, japan; 2 intensive care unit, gunma university hospital, maebashi, japan introduction: sugammadex is a synthetic c-dextrin derivative that is designed to selectively bind to steroidal neuromuscular blocking agent molecules. although sugammadex has been used in many cases of general anesthesia, there are several reports of anaphylaxis following its use. skin testing is the gold standard for detecting the causative agent of anaphylaxis. however, the test itself sometimes precipitates serious complications, including recurrence of anaphylaxis. hence, development of a novel test that can be performed in vitro without causing such complications is desired. recently, the basophil activation test (bat) has been established as a tool to detect the causative agent of anaphylaxis with high sensitivity and specificity. yet, there are few studies examining the utility of the bat in diagnosing sugammadex-induced anaphylaxis, besides our previous report. although both cd63 and cd203c are currently used as the major markers for activated basophils, which one of them is more suitable for the bat depends on the targeted drugs. objectives: the aim of this study was to investigate whether bat could be utilized to diagnose sugammadex-induced anaphylaxis. in addition, we compared the capability of cd203c and cd63 as markers for activated basophils. seven patients with perioperative anaphylaxis demonstrating a positive skin test for sugammadex were included. furthermore, 21 individuals who tolerated sugammadex and had a negative skin test for allergy to this drug were enrolled as controls. results: the ratios of activated basophils in the patients were much higher than those in controls (mann-whitney u test, p<.005). cd203c up-regulation. this was also true for cd63. in the case of cd203c, the sensitivity of bat for sugammadex was 83% and specificity was 100%, while sensitivity and specificity for cd63 were 71% and 100%, respectively. there were no significant differences between cd203c and cd63 in the areas under the roc curve. conclusions: this study showed that bat is a reliable instrument to diagnose sugammadex-induced anaphylaxis. we did not find any difference between cd203c and cd63 as markers for activated basophils in the bat for sugammadex. objectives: we report a retrospective study of 10 patients who were referred to our allergology departments between 2012 and 2016 with a suggestive history of immediate hypersensitivity to ppis. our purpose was the analysis of the clinical presentations and the allergological investigation performed in order to confirm the diagnosis of drug hypersensitivity to ppis and to study the cross-reactivity among ppis. results: the culprit drugs were pantoprazole (n=5), omeprazole (n=3) and lansoprazole (n=2). the allergological investigation confirmed the diagnosis of hypersensitivity to ppis in 5 patients (4 by skin tests and 1 oral challenge). we observed cross-reactions between omeprazole, pantoprazole and esomeprazole (n=1), omeprazole and pantoprazole (n=2), respectively omeprazole and esomeprazole (n=1). in 5 patients the severity and the recurrence of the reaction did not allowed the provocation test with the culprit drug. two oral challenges allowed the use of an alternative ppi (based on the pattern of less cross-reactivity depending on the chemical structure: pantoprazole for a patient who had a grade iii anaphylaxis to lansoprazole and lansoprazole for a patient who had a grade ii anaphylaxis to pantoprazole). conclusions: a complete allergological investigation (skin tests and cautiously, oral challenge) is needed in order to confirm the diagnosis of drug hypersensitivity to ppis and to take a therapeutic decision. the diagnostic approach is limited by the low sensitivity of skin tests and the patient's background (comorbidities and severity of the reaction) which do not always allow the oral provocation test. an ige dependent mechanism may be involved in hypersensitivity reactions to ppis or their metabolites. herrero-lifona l 1 ; muñoz-rom an c 2 1 hospital quironsalud campo de gibraltar, los barrios, spain; 2 hospital regional universitario de m alaga, m alaga, spain introduction: hypersensitivity reactions to beta-lactams are an important problem to study, especially in children, given that these antibiotics are the gold standard for the treatment of many infectious diseases in the infancy. most hypersensitivity reactions to betalactams in children are due to non-immediate response and to diagnose them is essential to perform a drug challenge test. although hypersensitivity is usually ruled out in children by drug challenge test, it is positive test up to 5%-10%. objectives: a 6 year-old boy is suspected to have experienced two drug reactions after the intake of amoxicillin with and without clavulanic acid for pharyngitis treatment. in the first one, he presented a maculopapular eruption in the back after one day treatment with amoxicillin-clavulanic acid. in the second reaction, two hours after the intake of amoxicillin, it appeared an itching maculopapular eruption in the back that was resolved with symptomatic treatment. in both cases, the patient tolerated treatment with cefuroxime afterwards. results: intradermal test with amoxicillin was negative in immediate and delayed reading. oral drug challenge test with amoxicillin (50 mg/kg/day, total cumuoral drug challenge test with penicillin v (total cumulative dose 250 mg): it was well tolerated and the patient continued taking it for epicutaneous patch testing with amoxicillin in the lesion area and in an unaffected area: in the lesion area, it appeared mild erythema, but it was considered a negative result in both areas. conclusions: we report a case of an amoxicillin-induced multiple fixed exanthema: an unusual non-immediate reaction in childhood. most cases of non-immediate hypersensibility need to be confirmed by drug challenge test, given that skin testing lacks sensitivity both intradermal and patch tests. the patient presents a selective hypersensitivity to amoxicillin, being tolerant to penicillin and cephalosporins. jimenez-rodriguez t 1 ; soriano-gomis v 1 ; gonzalez-delgado p 1 ; cueva-oliver b 1 ; venegas-diaz ij 1 ; fernandez j 2 results: data from 62 patients were analyzed, of which 64.5% (40) were women. the overall mean age was 46.1ae16.9 years, with no statistical difference between sexes. the 69.4% (43) of the patients presented symptoms with a single group of nsaids, and 30.7% (19) with 2 or more different groups. the 90% (56) of patients presented a history of atopy. in patients with rhinitis 58% had symptoms with only one nsaid and 42% with two or more groups of nsaid, while patients with asthma, 83% reacted to one nsaid and 17% to two or more groups. the most frequent clinical manifestations were: urticaria/angioedema in 71% (44), pruritus in 35.5% (22), bronchospasm in 19.4% (12), other respiratory symptoms in 16% (10) and anaphylaxis in 13% (7) patients. the most frequently involved nsaids were: metamizole (46.8%) and ibuprofen (37.1%). skin tests were performed in only 24 patients, of whom 13 (54%) were positive to metamizol. dpt was performed in 66.1% (41) objectives: fifty patients diagnosed with "dress" (2012-2016), in a tertiary center were retrospectively analyzed, with 31 cases meeting the regiscar criteria. we collected demographic, clinical, laboratory and therapeutic data from the electronic medical records. results: all cases occurred during hospitalization and in several hospital areas. the mean age was 49.16 years (7-94) with 58.06% women and 95% of caucasian origin. reported clinical manifestations were skin rash (97%), fever (48.39%), digestive symptoms (22.58%), respiratory (12.9%), neurological (9.68%), head and neck (6.45%), urological (6.45%), ophthalmologic (3.23%) and musculoskeletal (3.23%). the most relevant laboratory findings were eosinophilia (77%), elevated transaminase levels (77%), lymphocytosis (53%), altered coagulation (34%) and altered renal function (32%). virus serology was positive in 4 cases (12.9%) involving hhv-6, ebv, cmv and hiv. antinuclear antibodies were positive in 2/13 cases (15.38%). skin biopsy, performed in 12 cases (38.7%) revealed findings suggestive of drug induced skin reaction. suspected culprit drugs were antibiotics (45%), nsaids (26%), antiepileptic drugs (13%), and antiparasitic agents (10%). one case was related to the administration of heparins and other to a monoclonal antibody. in 10% of cases, the episode was not related to a particular drug. treatment was accomplished by the administration of corticosteroids (97%), antihistamines (96%), and immunosuppressant drugs in 2 cases. fluids and other medications were administered attending to symptoms. death occurred in 3 patients, although only in 1 case it was related to dress syndrome. the average time from reaction to death was 4.33 months. allergy evaluation, performed in 11 cases addressed the potential role of drugs. skin tests were positive in 3/11 patients (28.6%) and basophil activation test was negative (2/2 cases). results: from 17 patients admitted with possible diagnosis of scar, only 8 met the inclusion criteria (6f/2m, age 15-81, average 49.1 years-old). they were all admitted to a medicine ward. five patients had diagnosis criteria of dress, 2 of sjs and 1 of ten. as for the drugs related with the reaction, antiepileptic drugs were the most frequent (4 patients); allopurinol (1), betahistine (1), ciprofloxacin+nimesulide (1) were the causative drugs in the remaining patients. average time to the beginning of symptoms was 30.7 days (2 patients unknown). in 1 patient the cause was unknown (he had criteria of ten and also diagnosis of malaria and was transferred to another hospital). there were no deaths. objectives: the objectives of the present study were to determine the efficacy of nfeno to: 1. establish the diagnosis of rhinitis; and 2. discriminate allergic rhinitis (ar) from non-allergic rhinitis (nar). material and methods: prospective and controlled study with healthy subjects, which included patients with rhinitis. ar and nar were phenotypically defined according to positive or negative prick test results, respectively; the control group was collected from people without upper or lower respiratory tract disease and the prick test was negative. in all sample included the following measurements were performed: feno and nfeno (novario analyzer) were performed; spirometry; eosinophil counts in blood and nose (by nasal brushing); bilateral nasal endoscopy; and acoustic rhinometry. results: we included 147 cases (ar=61, nar=53 and controls=33), with a mean age of 37 (sd 11.2) years, 66% men. the table below shows the results of the variables analyzed by group. patients with rhinitis compared to controls had significantly greater feno production, as well as a higher proportion of eosinophils in blood and nose, but not in nfeno production. however, when the two subgroups of rhinitis were compared, ar patients had significantly higher feno and nfeno than nar, in addition to blood and nose eosinophils. there were no differences in acoustic rhinometry values between groups. conclusions: although nfeno does not appear to identify patients with rhinitis, it may be useful in discriminating ar from nar. in routine clinical practice, it could help guide to identify an ar in cases with inconclusive results of the complementary tests commonly used in its diagnosis. introduction: fractionated exhaled nitric oxide (feno) is used as a marker of eosinophilic airway inflammation in asthma, whereas clinical presentation of nasal no (nno) is unknown. objectives: the objective of this study was to evaluate the factors influencing nno levels. in patients with chronic nasal symptoms, total-nasal-symptom-scores (tnss) were calculated; skin-prick-test conclusions: in conclusion nasal no is useful in allergic inflammation in the absence of sinus obstruction in nasal cavity. however, its value is limited with paranasal sinus ostium occlusion. were recruited across australia and the uk via a patient panel. the aim was to assess which ar treatment attribute(s) drove patient preference. results: table 1 shows the willingness to pay (wtp) for each attribute. all attributes were significant predictors of treatment choice, except administration method. however, patients in both countries showed a considerable preference for treatments that were more efficacious, fast acting and affordable. conclusions: although treatment relief remains of primary concern, time to treatment benefit and cost could dictate treatment preference. these data may be of value in optimizing the acceptability of future ar treatments and informing trial endpoint selection. australia ( izquierdo l; chiriac am; molinari n; demoly p introduction: allergic rhinitis is a frequent disease with an important impact on quality of life. self-administrated control assessment tests can help achieve a better management of this disease. however, none of these tools has been validated in teenagers. test in its original version, following the same protocol as the original study in adults. we designed a multicentre, observational, crossconclusions: while the number of included patients is low, the first results are promising. indeed, a significant improvement of the rhinitis control score was found between d1 and d15. this led us to the hypothesis that this questionnaire could be used as is to estimate the control of the allergic rhinitis in teenagers. analyses concerning the validation of the questionnaire itself do not reach at the moment the threshold of significance, the recruitment is on-going, to increase its power. objectives: the objective of this study was to evaluate the role of serum vitamin d in patients with symptomatic allergic rhinitis and active asthma during the allergy season and observe the effect of montelukast 10 mg daily as treatment. results: this study included 130 asthmatic and seasonal allergic rhinitis patients following a single-blind, placebo run-in period of 3-5 days, patients were randomized to oral montelukast 10 mg (n=70) or placebo (n=60) daily during the 2-week, double-blind, active-treatment period. the serum vitamin d was also evaluated in both the groups. the serum vitamin d levels were found to be higher in patients taking montelukast compared to placebo after 2 weeks (p<.001). montelukast reduced the daily rhinitis symptoms score: difference between montelukast and placebo (p<.001). similar improvements were seen in daytime nasal symptoms (p<.001) and nighttime symptoms (p<.001). conclusions: montelukast provides significant relief from symptoms of seasonal allergic rhinitis, while also conferring a benefit for asthma, in patients with both allergic rhinitis and asthma. further, it has a beneficial role in improving vitamin d levels. venegas-diaz ij 1 ; jimenez-rodriguez t 1 ; canto-reig vj 1 ; lindo-gutarra m 1 ; soriano-gomis v 1 ; gonzalez-delgado p 1 ; cueva-oliver b 1 ; fernández j 2 1 allergy section. general hospital alicante. isabial, alicante, spain; 2 allergy section. general hospital alicante. isabial-umh, alicante, spain introduction: local allergic rhinitis is an accepted entity, which only can be recognized by nasal provocation test (npt) in patients with clinical rhinitis with negative skin tests or specific ige (sige). our aim was study this entity in our patients in alicante, spain. objectives: 100 patients with symptoms of rhinitis with skin tests, and sige negative for common aeroallergens were selected, since 2015. half of them (47) most were young adults, 50% belonging to the range of age between 18-30 years; more than 43% had family history of atopy; the mean age of onset of symptoms was 25 years and up to 18.7% had had symptoms before the age of 14 years; 56.2% were nonsmokers. regarding comorbidity, 62.5% of the patients presented with concomitant symptoms of conjunctivitis and 6.2% of asthma. symptoms in 93.7% of the patients were persistent and most of moderate (50%) to severe (43.7%) intensity. the majority of patients (87.5%) presented perennial symptoms and a tpn positive to mites in 37.5%, to salsola pollen in 25% and to cypress pollen in 12.5%. but 3 patients (18.75%) were simultaneously positive to 2 allergens. conclusions: local allergic rhinitis represents an important proportion of patients with clinical rhinitis with negative skin tests and sige. we have to pay attention to identify clinical and demographic factors of ral and to use npt to demonstrate it. objectives: we report the extent of sinus involvement at baseline (bl) in pts with bilateral np refractory to intranasal corticosteroids from a dupilumab phase 2a study (nct01920893). ct scans from enrolled pts, 59 adults <65 years with nasal polyp score of ≥5/8, were pooled and analyzed at bl. for opacification, standard lund-mackay (lmk) scoring (0=normal, 1=partial opacification, 2=total opacification) in maxillary, anterior/posterior ethmoid, sphenoid, frontal sinuses was used. ten sinus scores plus bilateral ostiomeatal complex (omc) score (0=not occluded, 2=occluded) were summed to a bilateral total lmk (0-24). zinreich modified lmk score (zlmk), providing more granularity on opacification degree, was evaluated post-hoc; each sinus was given a 0-5 score based on opacification % from mucosal thickening (0=0%, 1=1%-25%, 2=26%-50%, 3=51%-75%, 4=76%-99%, 5=100%); omc results: the prevalence of asthma at the age of 12 years was 6.4%. 15% of them had an act tm value below 20, ie uncontrolled asthma, median 22.0 (range 12-25). independent risk factors for uncontrolled asthma at the age of 12 were current doctor diagnosed rhinitis (adjusted or, aor 2.8; 95% ci 1.1-7.0), wheeze triggered by exercise (aor 5.6; 1.9-16.6) and cat at home (aor 3.5; 1.2-10.0). if at least one of the parents had higher education the risk of uncontrolled asthma was decreased (aor 0.3; 95% ci 0.1-0.8). six children reported hospitalisation due to asthma during the last 12 months (ie 2.6%) at 12 years of age. hospitalisation was more common in individuals with uncontrolled asthma (or 12.3; 2.2-70.5) or when mites (or 9.8; 1.9-51.5) or pollen (or 5.8; 1.0-32.5) was reported as trigger factors. also, oral corticosteroids (betamethasone), in the last 12 months increased the risk for hospitalisation (or 8.2; 1.4-48.7). to have a parent with asthma (17% compared to 37%, or 0.3; 0.04-2.9) or higher education (40% compared to 62%, or 0.3; 0.07-2.5) was numerically less common for children who were hospitalised for asthma but did not reach statistical significance. conclusions: uncontrolled asthma was associated with current allergic rhinitis, having a cat and exercise as trigger factor. at least one parent with higher education reduced the risk. hospitalisation due to asthma was more common in individuals with uncontrolled asthma. abstracts 0404 | adolescent asthma in relation to severity and gender-data from a prospective population based cohort study introduction: asthma often debuts early in life, but recent studies show that the development of asthma is a dynamic process with disease turnover throughout child-and young adulthood. objectives: to describe adolescents' asthma with focus on severity and gender. the study population consisted of 3115 adolescents participating in the cohort, followed since birth up to 16 years of age with questionnaires and clinical investigations. blood samples from 2452 adolescents (78.7%), sera analyzed for specific ige against common inhalant allergens. asthma at 16 years was defined as fulfilling at least 2 of the following 3 criteria: symptoms of wheeze and/or breathing difficulties in the last 12 months, ever doctor's diagnosis of asthma and/or asthma medicine occasionally or regularly last 12 months. uncontrolled asthma was based on parental information on symptoms of asthma in the last 12 months prior the 16-year follow-up, including 3 or 4 features: nocturnal asthma, activity limitation, wheeze 4 times and hospitalization due to acute asthma. we looked into 2 phenotypes; late-onset, defined as asthma at 8, 12 or 16, but not at 1, 2 and 4 years of age and persistent, defined as asthma at 1, 2 or 4 and 8, 12 or 16 years of age. severe asthma, defined as asthma as above combined with prescribed and dispensed corticosteroids used within the last 18 months plus uncontrolled asthma and/or lung function (fev 1 <80% of predicted). results: at 16 years of age, 14.2% (n=437) fulfilled the study definition of asthma, 49.3% late onset and 50.7% persistent asthma. persistent asthma was more frequent among boys than girls 59.1% vs 43.3% (p=.001). overall ige sensitization was common among adolescents with asthma, 70.1% were sensitised to common inhalant allergens, and equally common in late-onset and persistent asthma (71.7% vs 68.8%, p=.54). however, more boys than girls were sensitised (79.9% vs 62.6%, p<.001). in total 8.1% (n=25) adolescents had severe asthma, 11.2% (n=15) boys and 5.8% (n=10) girls (p=.08). the overall prevalence of severe asthma in the cohort was estimated to 1.0%. severe asthma did not significantly differ among adolescents with late-onset compared to persistent asthma (17/25, 8/25, p=.09). conclusions: among adolescents with asthma, late-onset (age ≥8 years) and persistent asthma were equally common. persistent asthma were more common among boys. there were no difference in asthma severity, or proportion of ige sensitization among adolescents with late-onset or persistent asthma. however, data investigating associations with wheeze and asthma in later childhood are scarce. objectives: our aim was to explore the association of maternal milk fatty acid composition with childhood wheezing phenotypes and asthma up to age 13 years. breast milk was collected 6 weeks and 6 months post-delivery in the ulm birth cohort study (n=720 and n=454, respectively). concentrations of 10-24 carbon atom chain length fatty acids were measured by high-resolution capillary gas-liquid chromatography. to control for constant sum constraint, concentration data were transformed using the centered log ratio method. compositional biplots and correlation matrices were used to group fatty acids based on within sample correlation. adjusted risk ratios with parent-reported wheezing phenotypes and doctor-diagnosed asthma were computed using a modified poisson regression. results: we observed no straightforward evidence of associations between overall breast milk fatty acid composition and specific wheeze phenotypes or doctor-diagnosed asthma. conclusions: despite our use of sophisticated statistical methodology, our results may have been biased toward the null by several cohort-specific factors associated with breast milk collection and fatty acid composition. to overcome potential selection bias by maternal lifestyle, further research should investigate fatty acid intake during the first year of life including sources other than breast milk among children who were never or not exclusively breastfed. introduction: fall is the most common season for asthma exacerbation. previous studies found that exposure and sensitization to house dust mites (hdms) can exacerbate asthma. the seasonal variation in indoor hdm concentration is highest in the fall, correlating with the incidence of asthma exacerbation. most of the studies conducted to date have focused on the effect of hdm exposure on the incidence of acute asthma exacerbation, but reports about the effect of hdm sensitization are few. thus, we aimed to determine whether sensitization to hdms acts as a risk factor of asthma exacerbation in the fall. in addition, we investigated whether asthma exacerbation in the fall had any distinctive features by comparing levels of various cytokines and chemokines with those in other seasons. objectives: we enrolled 55 children aged 3-14 who visited the emergency department because of acute asthma exacerbation from january 2008 to december 2013 (63.6%, males; mean age, 6.0ae2.8 years). they were treated in accordance with the standardized treatment protocol. blood samples were collected from the children during the course of treatment. by using residual sera from the blood samples, we measured the levels of total immunoglobulin e (ige), hdm-specific ige (sige), eosinophil cationic protein (ecp), and various cytokines and chemokines, and classified them according to season. we compared the date divided into fall group (from september to november, n=31) and other season group (from december to august, n=24). ci, confidence interval; or, odds ratio; der f-sige, dermatophagoides farinae -specific immunoglobulin e; der p-sige, dermatophagoides pteronyssinus-specific immunoglobulin e; ecp, eosinophil cationic protein; ige, immunoglobulin e. data are presented as n (%) or as meansaesems and median (range). p value refers to the difference between the fall and "other season" groups and was calculate by the chi-square statistics, fisher's exact test, student's t-test, or wilcoxon rank-sum test. a adjusted for total ige. results: this retrospective study obtained archived nasopharyngeal aspirate (npa) samples from patients aged below 18 years who were hospitalized for acute respiratory illnesses in a university-affiliated hospital during the periods september-november 2014 and january-april 2015. their clinical information was retrieved from computerised record. hrv was detected by rt-pcr, and isolates were sequenced to determine the genogroups and serotypes. ninety patients whose npa was positive for hrv and 160 patients being negative for an extended panel of respiratory viruses by multiplex pcr method were identified. mean age of these groups was 3.6 years and 3.5 years respectively. hrv infection was significantly associated with asthma exacerbation (or 16.54, results: a total of 203 children were included: 73.4% were boys; 26.6% aged 6 months to 2 years, 51.0% aged 3-6 years, 20.1% aged 7-13 years and 2.4% aged 14-18 years; 44.0% were hospitalized, results: the long-term remission (≥5 years without treatment) rate 16 years after initiating early anti-inflammatory therapy was 88.1% (intermittent asthma, 100%; mild persistent asthma, 72.2%; moderate persistent asthma, 90.0%; severe persistent asthma, 66.7%). longterm remission rates improved compared with past asthmatic convaobjectives: the aim of this study is to explore the potential value of feno level for diagnosing chronic cough in children. objectives: in this study, we aimed to compare the efficacy of classical spirometry and impulse oscillometry (ios) in evaluation of late reversibility in children who received treatment with the diagnosis of atopic asthma. we enrolled 83 patients aged 7-17 years who were diagnosed with atopic asthma. exclusion criteria were having received asthma treatment during the previous two months, having acute asthma exacerbation findings, having any other respiratory disease or cardiac disease that may affects the lung function test results. allergic sensitization was determined by skin prick test performed according to eeaci guidelines. lung function test measurements were performed at enrollment and after two months of inhaled steroid treatment. conclusions: classical spirometry is more valuable compared to ios in evaluation of late airway hyperreactivity in children with atopic asthma in children older than seven years age. 0413 | computer bronchophonographyfrequency analysis of the respiratory cycle. objectives: we performed mct in 144 children with symptoms suggestive of asthma and 30 without respiratory symptoms. after each inhalation step oscillometry and spirometry was performed. parameters analysed for ios were z5, r5, r20, x5, x15 and ax (the integrated impedance reactance at r5 and above) and fev1 for spirometry. a fall of 20% in fev1 from baseline after mtc was considered as a positive challenge. pc20-fev1 and pc40 r5, x5 and ax were calculated. results: a total of 174 patients, 79 female, with a mean age of 9.5 (ae3.03) years were enrolled. 132 had a ≥ 20% fall in fev1 after mtc. the mean variation in fev1 was 7% (-12.2%/ +45.8%), the mean variation in z5, r5, r20, x5, x15, ax and fres were 21.88% objectives: the objective of our study was to synthesize cationic peptides and study their antiviral activity (aa) in vitro. results: 16 peptides were synthesized by solid phase method with different structures (linear, helical and dendrimeric). cytotoxicity of the peptides was studied by mtt assay using hela cells. objectives: the aim of the study is to evaluate the changes of ilresults: 48 subjects aged 18-65 years were enrolled in this study, which were divided into 4 groups: 23 patients with diagnosed moderate to severe ba (1st group), 6 ba patients with rvi (2nd group), 8 subjects with rvi only (3rd group) and 11 healthy volunteers (4th group). all patients with ba from group 1 and 2 received inhaled corticosteroids. clinical blood test revealed increase of eosinophils in patients with ba and ba accompanied with rvi up to 7% and 4%, respectively. fev1 was decreased in 1st and 2nd groups to 77% and 79% compared 98% and 97% for 3rd and 4th groups, respectively. conclusions: this study is alarming for asthmatic nosocomial hazards in this govt. hospital. identification of ige specific reactive components of predominant fungal allergens and cross-reactivity among each other, delined in this study could minimize the hazard of therapeutic and diagnostic use of these cross-reactive components, in fungal allergen-specific immunotherapy. objectives: we conducted a longitudinal prospective study to examine the development, composition and diversity of the gut microbiota in healthy and allergic children. we followed 93 children from 4 months to 8 years of age with clinical evaluation; specific ige levels and skin prick testing. fecal samples were collected at 4, 6, 13 months and 8 years. 16s rrna sequencing was used to profile the gut microbiome using illumina miseq. the composition and diversity of the gut microbiome were assessed using quantitative insights into microbial ecology (qiime). comparisons between groups were made using the lefse pipeline; non-parametric factorial kruskal-wallis test, unpaired wilcoxon rank test and linear discriminant analyses (lda) with score >2.0. to assess the interaction effect, the likelihood ratio test (lrt) was applied using r statistical program. p<.05 was considered significant after correction for multiple testing. results: to achieve our aim we divided balb/c mice into 4 groups: mice with viba (1st group), mice with viba treated with nonspecific sirna against gfp (sigfp) (2nd group) and against il-33 (siil-33) (3d group). 4th group was intact mice. groups 1-3 were i.p. sensitized on days 1, 14, 28 with ovalbumin (ova) mixed with aluminum hydroxide and i.n. challenged with ova on days 40-42. the same mice were i.n. infected with 5910 6 tcid 50 /mouse rsv strain a2 on day 39. mice from group 2 and 3 were i.n. treated by sirnas on days 38-42 in dose 120 lg/mouse. on day 43 hyperresponsiveness (ahr) to methacholine was measured. on day 44 and lungs were removed for histological analysis. viral rna (vrna) load and il-33 gene expression were evaluated by qpcr in lungs. bronchoalveolar lavage (bal) was collected for differential cell count by light microscopy. so i.n. administration of siil-33 suppressed il-33 gene expression in lungs by 50% compared to sigfp treated mice. there were no significant changes in body weight and lung vrna amounts between mice received sigfp and siil-33. mice treated with siil-33 demonstrated the tendency to improve lung function compared to mice of group 1-2, that expressed in 13% reduction of specific resistance of airways and 15% increase of peak expiratory flow. bal cell count revealed decrease of total cell number, eosinophils and lymphocytes in mice received siil-33 by 55%, 90% and 63% compared to sigfp treated mice, that indicate reduction of inflammation, that was confirmed by histopathological studies showed 50% leukocyte reduction. downregulation of il-33 resulted in 2-and 1.7-fold decrease of bronchial epithelium metaplasia and hyperplasia. and femur length measurements were collected from routine antenatal screenings. these and derived head to abdominal circumference ratio and estimated fetal weight were converted into z-scores adjusted for gestational age and gender and categorized as "low" (≤1 sd below mean), "normal," or "high" (≥1 sd above mean). ad cases were children with parent-or pediatrician-report of physician ad diagnosis assessed yearly up to age 3 years and supplemented by clinical diagnoses during dermatological exams at 0.5, 1, and 2 years. modified poisson regression models were used to compute risk ratios (rr) adjusted for potential confounders. conclusions: these results provide further evidence for a role of fetal growth as an influence on atopic disease outcomes. unlike previous studies, our data suggests several patterns of fetal growth beginning as early as the 1st trimester may influence ad outcomes. objectives: we aimed to examine the role of eosinophil cationic protein (ecp), eosinophil derived neurotoxin (edn) and total immunoglobulin (ig) e as a bio-marker of disease severity. we examined the difference in level of total ige, ecp and edn between the two groups and whether any correlation existed between disease severity and ecp or edn. objectives: we aimed to identify the subgroup of ad patients with a good clinical response to probiotic treatment. we recruited children who suffered from moderate to severe ad with the scoring ad (scorad) index of 20 or higher. after 2 weeks of washout period, all patients were given lactobacillus plantarum cjlp133 at a dosage of 1910 10 colony-forming units once a day for 12 weeks. we measured eosinophil counts in the peripheral blood, the proportion of cd4 + cd25 + foxp3 + regulatory t (treg) cells in cd4 + t cells, serum total ige levels, and specific ige to common allergens before the start of the treatment (t1) and at discontinuation (t2). logistic regression models were used for the statistical analysis. seventy-six patients (48 boys and 28 girls) with a mean age of 8.7ae4.7 years completed the study. there were 36 responders and 40 non-responders after probiotic treatment. the median scorad was reduced from 29.5 (range 20.6-46.3) at t1 to 16.4 (range 6.3-30.8) at t2 in the responder group (p<.001). in multivariable logistic regression analysis, a good clinical response was significantly associated with high total ige levels (aor 5.1, 95% ci 1.1-23.6), increased expression of , and high proportion of cd4 + cd25 + foxp3 + cells in cd4 + t cells (aor 3.7, 95% ci 1.1-12.7). in responder group, the proportion of cd4 + cd25 + foxp3 + cells of cd4 + t cells were significantly increased after 12 weeks of treatment (p=.004), while the levels of tgf-b mrna expression were decreased (p=.017). there were no differences in total ige levels between t1 and t2 (p=.414) conclusions: the therapeutic effect of l. plantarum cjlp133 on ad is more pronounced in children with high total ige levels, objectives: the objective of the study was to examine the effect of a specific synbiotic mixture of short-chain galacto-, long-chain fructo-oligosaccharides (scgos/lcfos, ratio 9:1) and bifidobacterium breve m-16v on the severity of ad and correlation to serum chemokines in infants with moderate to severe ad and elevated ige. in an exploratory randomized, double-blind, placebo-controlled trial the effect of extensively-hydrolyzed whey-based formula without intervention. serum obtained prior to start and at the end of intervention were analysed using luminex. six chemokines and nine ratio's thereof were correlated to ad severity (sample size=24). introduction: dyshidrotic eczema is one of the most common skin conditions. contact allergy is often associated with dyshidrotic eczema although the exact impact and the influence of contact allergens in different forms of dyshidrotic eczema remain unknown. hypersensitization to nickel is one of the most common contact allergies associated with pompholyx. the standard of care protocol is to use a medical treatment with topical corticosteroids and calcineurin inhibitors to treat the symptoms, together with occlusive barrier creams to avoid skin exposure to the allergens. after the symptoms have been cleared with the topical treatment, the recommendation is to use occlusive barrier creams to prevent recurrence of the symptoms. objectives: a new emollient with specific metal-scavenging agents and no occlusive ingredients has recently been developed and made commercially available. the aim of this study was to evaluate the effect of such cream to provide relief for patients with dyshidrotic eczema associated with nickel allergy. results: thirty-two subjects with dyshidrotic eczema and a positive patch test ppt (contact sensitized) reaction to nickel were selected. these were divided into two randomized groups, group-a was given nickel-scavenging cream (skintifique creamtm, paris) after medical treatment, (n=9) and group-b followed the standard protocol for pompholyx, (n=23). hand eczema was scored according to the dyshidrotic eczema area and severity index (dasi). dasi scores were evaluated at the beginning of the study (day-0), after the medical treatment (day-15) and two months after the end of medical treatment (day-75). results show a significant difference in the efficacy of treatment between the two groups at day-75. a higher percentage of at least 75% reduction of initial dasi score (77.8%) and a higher percentage of total clearance (56%) in patients using nickel-scavenging nonocclusive moisturizing cream was observed as compared to standard-of-care occlusive creams (26.1% and 22%, respectively). objectives: the goal of this study was to investigate whether long-term emollient therapy is associated with alterations of skin barrier function and shifts of the skin microbiome in infants at high risk for developing ad. we prospectively enrolled newborns with a family history of ad to be randomized to either emollient treatment group or control group. at 6 months of age, we tested the skin barrier (transepidermal water loss/tewl, water capacitance/cap, ph) and skin microbiome (16s rdna sequencing of skin swabs from cheek, dorsal and volar forearm). results: the emollient group (n=10) had significantly lower skin ph compared to controls (n=9) (p=.02), but without a statistically significant difference in tewl or cap. the emollient group had higher numbers of different bacterial taxa (chao richness) at cheeks (p=.003), dorsal forearms (p=.008), and volar forearms (p=.003) as compared to controls. both streptococcus pneumoniae and s. salivarius statistically significantly contributed to the observed skin microbiome differences between patient groups. s. salivarius was significantly more abundant in emollient subjects at all sampling sites (p=.02). we then analyzed our previous larger cohort of older children with ad and also observed higher s. salivarius proportions in ad patients with treated and less severe disease (p=.01). objectives: to evaluate the effect of overnight treatment with a temperature-controlled laminar airflow (tla) device in children/adolescents with severe eczema over a 12-month period. in an open-label study, 15 subjects aged 2-16 years (median 10 years) with longstanding severe eczema attended 3 visits during the run-in period lasting 6-10 weeks (median 7.14 weeks) to optimize eczema management. the run-in was followed by a 12month treatment period using overnight tla device (airsonett ® , sweden), which included 8 study visits. we used scorad-index results: the median duration of eczema was 116.5 months (interquartile range 82-145.5). all subjects were sensitised to ≥1 perennial allergen, and had multiple comorbidities (15/15 rhino-conjunctivitis, 14/15 food allergy, 11/15 asthma). there were no significant changes during the run-in period in any of the outcome measures. we observed a significant improvement in scorad after the 12-month tla-treatment period, from 34.9 [28.75-45.15 ] to 17.2 [12.95-32.3] , p=.015. iga improved significantly from a median of 4 [3-4] to 2 [1] [2] [3] , p=.001. improvement in symptoms was paralleled by a significant reduction in medication usage. by 12 months, there was a significant improvement in .0] to 12 [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] , p=.032), and an improvement in cdqli (marginal, p=.084). however, we observed no changes in poem (p=.196) . post-hoc cluster analysis of the patterns of changes in scorad over the 12month treatment period identified two clusters, with 9 participants classified as responders and 6 as non-responders. introduction: intestinal degradation has been shown to determine allergenicity of food allergens. however, it is unclear how allergens are degraded inside pivotal immune cells such as dendritic cells (dc), and how this affects the subsequent immune response to these allergens. in our studies, we determined whether we are able to measure uptake and degradation of allergens inside dc and whether differences in above mentioned factors exist between allergens. objectives: using mouse bone marrow-derived dc and fluorescent-labeled proteins, we studied the cellular uptake of the peanut proteins ara h 1, 2, 3, and 6. results: first, we observed that dc uptake of ara h1 was much higher than ara h2, 3 and 6. using blocking reagents and receptor binding assays for various uptake routes in dc, we observed that one of the principal routes of uptake for ara h 1 was the mannose receptor. other uptake routes, and the routes of uptake for ara h2, 3 and 6 are currently under investigation. second, using proteins coupled to beads we observed that intracellular protein degradation was higher for ara h1 and ara h3 than for ara h2 and 6. finally, cd4+t cells from ara h 1, 2, 3 or 6 sensitized mice were added to matching allergen-pulsed dc. we observed that while ara h1, 3 and to lesser extend ara h6 elicited strong th2 type responses, ara h2 did not elicit any t cell responses. conclusions: together, we show that allergenicity of (peanut) proteins may be, at least partly, determined at the level of allergen uptake and breakdown inside the antigen presenting cell. these findings may be relevant to risk evaluation of existing allergens but also of novel or modified proteins. this also illustrates the usefulness of in vitro, cell based assays to examine initial processes of allergenic sensitization to proteins. 0445 | sensitising capacity of unmodified and acid hydrolysed gluten through the skin-a comparative study in na€ ıve vs tolerant brown norway rats ballegaard ar; madsen cb; bøgh kl national food institute, technical university of denmark, søborg, denmark introduction: allergic sensitisation to foods may occur in infancy without prior oral exposure to the offending food. this has led to the assumption that food allergy sensitisation may occur through alternative routes, such as the skin, supported by the observed correlation between skin barrier disruption and food allergy. recently, concerns have been raised regarding the safety of use of cosmetic and personal care products containing hydrolysed wheat proteins, since these products have been shown to induce allergy towards acid hydrolysed wheat through the skin, and even to cause an abrogation of the already established oral tolerance against unmodified wheat. objectives: the aim of the study was to compare the sensitising capacity of an unmodified and an acid hydrolysed wheat product via slightly damaged skin, in order to evaluate differences in conditions necessary for skin sensitisation in na€ ıve vs tolerant individuals. brown norway rats were raised and bred on either (1) a diet free from wheat, resembling individuals with a na€ ıve immune system, or (2) a conventional wheat containing rat chow, resembling individuals tolerant to wheat. results: in the na€ ıve rats both products were able to induce a statistically significant specific antibody response after application of the products on the slightly damaged skin, whereas in the wheat tolerant rats, only the acid hydrolysed wheat product was able to induce a statistically significant antibody response. for both the na€ ıve and the wheat tolerant rats the response was dose-dependent. in the na€ ıve rats both products were able to sensitise through the skin, inducing a specific ige response, whereas in the tolerant rats only the acid hydrolysed product were able to induce a specific ige response, though this ige response was much lower than in the na€ ıve rats. results from competitive elisas demonstrated that new epitopes had developed as a result of acid hydrolysis, though original epitopes were maintained at the same time. this may explain why only the acid hydrolysed wheat could induce specific antibody responses in the tolerant animals. conclusions: this study showed that the sensitising capacity through the skin of two different wheat products is heavily influenced by the tolerance status of the immune system and the degree of modification of the wheat products. results: using a germ-free c3h/hen mouse model of food allergy, we examined the presence of a major peanut allergen, ara h 2, in the blood after intraperitoneal injection in previously sensitized and control (non-sensitized) mice using an untargeted, quantitative proteomic approach. previously sensitized mice underwent physiological (core temperature decrease, clinical symptoms) and biochemical (mast cell protease increase, ara h 2 specific ige positivity) changes associated with severe allergic reactions. we were able to confidently detect multiple peptides derived from the ara h 2 protein after intraperitoneal injection in both control and ara h 2 sensitized mice. however, the ara h 2 protein was present at 15-40 fold higher levels if mice were previously sensitized, suggesting increased transit across the peritoneal mesothelium with sensitization. an untargeted proteomic approach also allowed changes in the blood proteome of mice undergoing a severe allergic response to be examined. we identified proteins with significantly altered quantity in serum between control and sensitized mice and were therefore apparently associated with the allergic response. we demonstrate the applicability of untargeted proteomics to the study of the allergen transport and proteomic changes which co-occur with a resultant allergic reaction. the transit of allergens into the bloodstream is heavily dependent upon previous sensitization. we are continuing this work to examine the specificity of observed increases in trans-mesothelial transport and to address transport of allergens after intragastric challenge. 0447 | iga to cow's milk differs between breast milk and serum for its epitope specificity objectives: we sought to assess whether the profile of epitopespecific iga differs between mother's serum and bm. we also examined how infants' food epitope-specific iga develops in early infancy and the relationship of iga epitope recognition with development of cow's milk allergy (cma). results: . we measured iga specific to an array of overlapping peptides in major cow's milk allergens (alpha s1-, alpha s2 -, betaand kappa-caseins and beta-lactoglobulin). diversity of peptide-specific iga (ie epitope diversity) was determined in paired maternal and infant sera as well as breast milk samples in 31 mother-infant dyads within the first 15 postpartum months utilizing peptide microarray. microarray data was converted to z-scores and filtered for noise. peptide epitopes were determined based on jaccard distance between neighboring peptides, and intra individual correlation between sample types was estimated using phi coefficient. comparisons between groups and individuals was done using non-parametric tests. we noted marked discordance in epitope recognition in paired breast milk and maternal serum samples. at least one shared epitope was recognized by both milk and serum samples ranging from 13% of mothers for alpha-s1 casein to 73% for kappa-casein. epitopespecific iga was detectable in infants' sera starting at less than 3 months of age. sera of mothers with a cma infant had increased binding of epitope-specific iga to cow's milk proteins compared to those with a non-cma infant (p<.05 for all five proteins). conclusions: these findings support the concept that mothers' milk represents a product of the mucosal immune system that has an antibody repertoire distinct from that of peripheral blood. results: multiple ige-binding proteins were detected in the different wn preparations and many of which were dissemblance in 2dblotting. profiles of detectable proteins (peptides) of raw, roasted and boiled wn extracts were profoundly different in emi analysis. introduction: consumption of tree nuts is on the rise due to their beneficial health effects. however, tree nuts can led to severe allergic reactions in sensitized patients. thermal processing can modify the structure and function of food proteins and may alter (increase or decrease) their allergenic properties. knowledge about the effects of thermal processing on tree nuts such as cashew or pistachio is rare and based on traditional in vitro immunoassays. objectives: to elucidate the influence of thermal treatments (boiling and heat / pressure) on the ige reactivity of cashew and pistachio proteins, by means of traditional in vitro immunoassays and mediator release assays (mra). results: the allergenicity of untreated and treated cashew and pistachio nuts was evaluated by ige-elisa, ige-immunoblot and elisa inhibition assays using sera from spanish patients with clinical allergy to cashew and pistachio. rat basophilic leukaemia cell line transfected with the a-chain from the human high-affinity ige receptor fceri, was sensitized with a pooled sera and used for mra. sensitized cells were stimulated with untreated and treated protein extracts, in order to investigate the capability of untreated and treated cashew and pistachio proteins to cross-link ige on effector cells. the results showed that heat and pressure treatment at the harshest conditions considered in this study produced a higher decrease of the ige-binding capacity of cashew and pistachio proteins than boiling without pressure or soft conditions of heat and pressure. interestingly, although the treatments of heat and pressure seemed to affect cashew allergens to a greater extent than pistachio allergens (evaluated by ige-elisa and elisa inhibition), the results of mra using the cell line rbl-48 indicated that cashew proteins treated with heat and pressure, still retained some capacity to cross-link ige. introduction: previous research has indicated an important role for dietary non-digestible oligosaccharides in decreasing the incidence of atopic dermatitis in children at risk of allergy. it is assumed that these prebiotics promote colonization of beneficial bacteria in the gut. children with atopic dermatitis receiving a diet of galacto-and long chain fructo-oligosaccharides (scgos/lcfos) with bifidobacterium breve m-16v had enhanced serum galectin-9 levels. galectin-9 has ige-binding capacities and can hereby suppress degranulation of mast cells and basophils. next to their function in the gut, oligosaccharides may also affect other immune cells directly, since they were found in plasma and urine. objectives: we investigated whether non-digestible oligosaccharides or galectin-9 can have a direct effect on immune cells, by determining the effect on basophil degranulation in peanut-allergic patients. whole heparinized blood samples were collected from 12 peanut-allergic adult patients and incubated for 24 hours with either a mixture of 0.05% 9:1 scgos/lcfos or scfos/lcfos, or galectin-9 (1 or 5 lg/ml) at 37°c in the presence of il-3 (0.75 ng/ml). after 24 hours, a basophil activation test (bat) was performed. basophils were stimulated for 30 minutes at 37°c with increasing concentrations of whole roasted peanut extract or human anti-ige. degranulating basophils were determined as cd63+ cells and calculated as percentage positive cells. results: in each patient, the concentration of anti-ige or peanut extract that induced maximal degranulation in the untreated control sample was used as reference value to compare degranulation of the samples pretreated with scgos/lcfos, scfos/lcfos, or galectin-9. pre-treatment of whole blood with scgos/lcfos resulted in an average decrease in degranulation of approximately 8%, while a significant reduction of 19% was observed after pre-treatment with scfos/lcfos (p<.05). pre-treatment with 1 lg/ml galectin-9 decreased basophil degranulation with 12%, whereas 5 lg/ml galectin-9 caused a significant decrease of 25% (p<.05). no differences were observed in the ec50, indicating that the basophils are not becoming less sensitive to the peanut extract or anti-ige. the prebiotic mixture scfos/lcfos and galectin-9 can contribute to decreased degranulation of basophils in a igemediated bat assay using whole blood. further analysis is warranted to define the exact working mechanism of these oligosaccharides. introduction: the intestinal mucosa plays a key role in the development of food allergies. we studied the interaction between intestinal epithelial cells (iecs) and pbmcs of peanut-allergic patients in a transwell co-culture model. exposure of iecs to a mixture of galacto-and/or long chain fructo-oligosaccharides (scgos/lcfos) in combination with synthetic cpg dna (tlr9 ligand),modulated the cytokine response of activated pbmcs, driving away from the allergic phenotype. objectives: our aim was to compare the efficacy of scgos/lcfos with a scfos/lcfos mixture and to evaluate these effects in an allergen-specific co-culture model. iecs (ht-29, human colon adenocarcinoma) were grown on transwell filters until confluence. iecs were apically exposed to 0.5% 9:1 scgos/lcfos or scfos/lcfos either or not in combination with 2.5 lmol l à1 cpg dna to mimic presence of dna of beneficial bacterial in the gut. these iecs were co-cultured with basolateral pbmcs from peanut-allergic patients, either activated with anti-cd3/cd28 (24 hours) or peanut-extract (pe, 6 days).cytokines ifn-c, il-10, il-13 and tnf-a were measured in the basolateral supernatant and t-cell polarization of the pbmcs was determined (th2, th1, treg and tfh). results: apical exposure of iecs to cpg dna increased basolateral ifn-c and il-10 production by anti-cd3/cd28 activated pbmcs (p<.01), and was enhanced by both oligosaccharide mixtures (p<.01). cpg exposure in transwells with pe stimulated pbmcs also increased ifn-c and il-10 production, which was further enhanced by scfos/lcfos (p<.05). in this pe-specific model, percentages of th1 and treg cells increased upon cpg exposure of iecs, and th1 frequency was increased by scfos/lcfos (p<.05). cpg dna exposed iecs suppressed il-13 and tnf-a production by anti-cd3/ cd28 activated pbmcs. both scgos/lcfos and scfos/lcfos reduced tnf-a production in combination with cpg dna (p<.01). tfh cells can produce il-21, inhibiting class-switching to ige2. upon cpg exposure of iecs, we observed an increase in tfh cells (p<.01) and similar tendency for cd4 + il-21 + cell frequency in anti-cd3/ cd28 activated pbmcs. conclusions: epithelial exposure to both scgos/lcfos and scfos/lcfos enhances the cpg dna induced th1 and regulatory il-10 response in an anti-cd3/cd28 co-culture model, whereas only scfos/lcfos was effective in an peanut-specific co-culture model. introduction: in areas of spain with high level of grass pollen exposure, 60% of grass allergic patients were sensitized to profilin, and most of them developed severe profilin mediated food reactions. this specific population of patients constitute an ideal model to study the relation among respiratory and food allergy objectives: our aim here was to analyze the links between epithelial barrier integrity and inflammation in oral mucosa. methods: 30 allergic patients and 6 controls were included in the study. allergic patients were stratified into mild or severe according to their clinical history and response to profilin oral challenge test. immunohistochemistry (ihc) for cd11c, cd3, cd4, claudin-1; and dapi nuclear staining were performed in formalin-fixed, paraffin embedded sections of oral mucosa biopsies. rt-pcr was performed to analyze il1b and il33 gene expression and the number of circulating cd4+ cells in pbmc was measured by flow cytometry. additionally, basophil activation test was carried out in whole blood samples upon profilin stimulation and the ec50 was calculated. results: regarding epithelial barrier integrity, claudin-1 expression resulted inversely proportional to pollen-associated food allergy severity. furthermore, by dapi staining, we noticed a lower number of epithelial cells in allergic patients than in non-allergic, and the gene expression of il1b and il33 resulted significantly increased in oral mucosa from severe group. as for immune response in oral mucosa, the number of cd11c and cd3+ cells resulted significantly higher in severe group. in allergic patients, double ihc for cd11c-cd4 showed an increase colocalization of t cells and apcs in the interface between epithelium and connective tissue. a decrease in blood circulating cd4 + cells was detected in allergic patients compared to non-allergic. as for the basophil sensitivity, ec50 in severe patients was 10-times lower than in mild. our results show that damage in oral mucosa epithelium, probably induced by high grass pollen exposure, might allow profilin to penetrate inside oral mucosa and induce inflammation with local recruitment of immune cells. furthermore, analyzing the immune response developed by effector cells, basophils from severe group result more sensitive to profilin as they react to a lower allergen concentration. these data explain the differences in food allergy severity between mild and severe group and propose oral mucosa as a new sensitization route. introduction: th2 cells producing the hallmark cytokines il-4, il-5 and il-13 have been found to constitute the majority of the allergen-specific th cell responses in allergic diseases. subpopulations of the th2 responses have been described with an early primed th2 subtype characterized by production of il-4 and il-13, and a highly differentiated th2 subtype, which in addition produce at least il-5. objectives: we aimed to investigate the polarity of tree nut and peanut allergen-specific th cells in subjects with confirmed tolerance or allergy to multiple nuts, and hereby detect differences in allergenspecific th cell responses within the same study population. we also wanted to stress the question whether a th2 phenotype dominates in asymptomatic sensitization. methods: pbmcs from 35 donors all assessed for clinical reactivity to hazelnut, walnut, cashew nut, pistachio nut and peanut was stained with cfse and stimulated (2910 6 cells/ml) with and without whole nut extracts (50-200 lg/ml) for 7 days. allergen-specific cd4+ t cell phenotypes and cytokine release was analyzed with flow cytometry and luminex, respectively. the allergen-specific th cells of the allergic donors showed a trend of more highly differentiated il-4+il-5+ th2 cells and higher abstracts | 319 release of il-5 than in the tolerant donors. unexpected, except in the cashew nut stimulated cells, no difference was found in the relative percentage of the less differentiated th2 cells (single il-4+ allergen-specific cd4+ t cells) when comparing allergic with tolerant donors. when subdividing the tolerant donors into asymptomatically sensitized or ige-negative (<0.35 kua/l), increased frequency of highly differentiated il-4+il-5+ allergen-specific th2 cells were found in asymptomatically sensitized compared to tolerant-ige-negative donors. interestingly, a positive association of the allergen-specific ige level and the frequencies of allergen-specific il-4+il-5+ and il-4+ th2 cells were found in hazelnut, pistachio nut and cashew nut but not in walnut and peanut allergic subjects. conclusions: an overrepresentation of allergen-specific highly differentiated il-4+il-5+ th2 cells and an elevated il-5 production were observed in allergic subjects compared to subjects that tolerated the nuts. we furthermore found a trend that subjects asymptomatically sensitized to nuts differed from tolerant-ige-negative subjects by having relatively more highly differentiated il-4+il-5+ allergen-specific th2 cells. introduction: it remains largely unknown which features of food proteins that render them allergenic vs tolerogenic. however, it has been suggested that the protein-chemical features affects protein uptake in the intestine, and that protein uptake route may impact on the risk of sensitisation. objectives: the aim of this study was to investigate the interplay between protein-chemical characteristics, the allergenic vs tolerogenic properties and the intestinal uptake of two protein products. the allergenic vs tolerogenic capacity of a heat-treated whey product, consisting of partly denatured and aggregated proteins, was compared to an unmodified whey product in: (1) results: though this study showed that both unmodified and heattreated whey had immunogenic, sensitising and eliciting capacities as well as tolerance inducing capacity, significant differences between the two products were observed. the heat-treated product was found to have a lower allergenicity combined with high tolerogenicity compared to the unmodified product. competitive igg1 elisas indicated that heat-treatment of whey induced de novo epitopes while the original epitopes were maintained. newly established methods to study in vivo intestinal uptake were successfully applied to compare the uptake kinetics of the two products in different small intestinal tissues and serum. collectively the in vivo and in vitro uptake experiments suggested that uptake kinetics and the major intestinal uptake route differed between the heattreated and unmodified product. conclusions: this study showed that heat-treatment, which induces partly protein denaturation and aggregation, changes the immunological properties and intestinal uptake of a whey protein product. the heat-treated product was found to have a lower allergenicity combined with high tolerogenicity compared to the unmodified product, which highlights this products promising potential for induction of cow's milk tolerance. objectives: in this study, we enumerated tregs in esophageal tissue of patient with eoe, gerd and normal controls. ten patients with eoe, 10 patients with gastroesophageal reflux disease (gerd), and 8 patients with normal endoscopy and normal esophageal tissue were included. tregs were enumerated in paraffin embedded esophageal biopsy blocks, using immunohistochemistry assay. tregs were identified as foxp3+, cd3+ cells. results: tregs were counted in 3 high power fields (hpf, 9400) for 28 patients and the average of 3 hpf was recorded. the number of tregs in esophageal tissue of patient with eoe (mean 10.9 cells/ hpf) was significantly more than gerd(mean 2.77 cells/hpf) and control groups(mean 0.37 cells/hpf) (p value <.001). conclusions: there is an increase in number of regulatory t cells in esophagus of patients with eoe in comparison to gerd and control groups. the presence of these cells might be due to eosinophilic inflammation and help controlling the inflammation. objectives: to evaluate whether anti-cd38 (daratumumab) treatment results in a reduction of total and specific ige levels. results: samples from patients with relapsed/refractory multiple myeloma, treated with daratumumab monotherapy or daratumumab plus lenalidomide-dexamethasone in the umc utrecht between april and august 2014, were tested for total ige levels as well as presence of specific ige against common inhalant allergens. in patients with detectable ige at baseline, the total and specific ige levels were evaluated during treatment up to 20 weeks. of eight patients receiving treatment, four had detectable ige levels at baseline. one patient demonstrated sensitization to common inhalant allergens. for this patient, levels of total ige gradually decreased during 20 weeks of treatment (from 356 to 44 ku/l; 88%), as well as specific ige against timothy grass (9.8-1.7 ku/l; 83%) and house dust mite (3.2-0.4 ku/l; 88%). a second patient, not sensitized to common inhalant allergens but with ige levels of 41 ku/l at baseline, also demonstrated a decrease in ige levels, to 5 ku/l (88%). the last two patients had total ige levels <10 ku/l at baseline, which dropped below detection limit after eight weeks of treatment. conclusions: this proof of concept demonstrates that (specific) ige depletion occurred during treatment with anti-cd38 (daratumumab). anti-cd38 could potentially play a role in the management of severe ige-mediated diseases. objectives: following individual and assessment, patients established and stable on omalizumab have been commenced on home therapy. training and education in self-administration has been provided. a service assessment has been carried out to review the ongoing safety, quality and patient experience of the service. results: to date, over 90 doses of omalizumab have been self administered in the community with no adverse events or incidents related to home therapy. conclusions: self-administration of omalizumab at home by patients offers the potential to improve quality of life while also providing efficiency savings. introduction: chronic idiopathic/spontaneous urticaria (csu) is a chronic urticarial subtype, defined as itchy hives that last for at least 6 weeks, with or without angioedema, and that have no apparent external trigger. although csu is more frequent in adult populationup to 0.5%-1%-, it can affect children and generally has a prolonged duration and has a detrimental effect on patients' quality of life. before the fda and ema approval of the anti-ige monoclonal therapy omalizumab for adults and children 12 years and above, nonsedating h1-antihistamines were the only agents licensed for use in patients with csu. however, a majority of patients did not respond to these drugs, even when they were administered at three to four times their licensed dose. objectives: we present 3 pediatric patients (≥12 years old) with csu non-responding to antihistamines, treated with omalizumab. results: at the diagnosis of csu, the patients were 11, 13, 14 years old, respectively. a trial with non-sedating h1-antihistamines, administered up to three to four times their licensed dose, were performed for all patients, without clinical efficacy. omalizumab was started at 12, 14, 14 years, respectively. before anti-ige therapy, the urticaria activity score (uas) was 5, 4, 5 respectively, indicating poor symptom control. omalizumab therapy was administered every 4 weeks for 6 months at the dosage of 300 mg s.c. after 1 month of therapy, all 3 patients were symptom free with uas 0; the patients remained asymptomatic for all the 6 months of duration of monoclonal therapy and antihistamines were discontinued. by now, after 2, 5, 6 months respectively from discontinuation of omalizumab, all 3 patients are asymptomatic with uas 0. introduction: mepolizumab is a humanized monoclonal antibody directed against il-5, and is licensed for the treatment of severe asthma in patients aged >12 years (eu only adults) with an eosinophilic phenotype as an add-on treatment. we were interested to know whether the substance has an antiallergic effect, too. objectives: here, we report the case of a 60 year old man (nonresults: in march 2016 he was switched from omalizumab to mepolizumab (100 mg/month) 4 weeks after the last omalizumab because of two asthma exacerbations under omalizumab in the last 6 months and 380 eosinophils/ll blood under 10 mg prednisolone/day. after two injections of mepolizumab his fev1 increased from 55% to 64% pred., the act from 14 to 21 points, feno decreased from 48 to 32 ppb and the amount of prednisolone from 10 mg to 5 mg/day. but -the patient reported new nasal symptoms after the 2nd injection of mepolizumab, mainly blocked nose which has not been observed under omalizumab. he agreed to be challenged in the mobile gae²len exposure chamber* with house dust mite allergen for 40 min. the total symptom score increased from 2 to 7 points after 20 min exposure time remaining stable till the end, the positive nasal inspiratory flow decreased from 90 to 50 l after 30 min and 40 l after 40 min, the fev1 decreased from 62% to 51% pred. following the inhalation of salbutamol the fev1 reversed. conclusions: mepolizumab has an anti-asthmatic effect but the anti-allergic efficacy seems to be small or absent. methods: all patients treated with omalizumab for severe allergic asthma or chronic spontaneous urticaria at the department of respiratory medicine and allergy at aarhus university hospital (n=64) were grouped after their home municipality. the number of omalizumab treated patients/inhabitant in these municipalities was correlated to the distance to the treatment centre at aarhus university hospital. results: mean distance to the treatment centre was 53.5 km for all inhabitants in the central region, while patients treated with omalizumab lived in average 38.7 km away. we found a negative linear correlation between the number of patients treated with omalizumab/inhabitant and the distance from the municipality to the treatment centre (slope: à0.038 95%ci: à0.072 to à0.003), p=.034, n=64). conclusions: patients living at long distance from the treatment centre are less likely to be offered omalizumab treatment for severe allergic asthma or chronic spontaneous urticaria. objectives: demographics, clinical characteristics, and response to mepolizumab, were evaluated for atopic and non-atopic subgroups. sensitization to any one of the following; house dust mite, dog dander, cat dander, alternaria, or cockroach was considered as atopic, as assessed by specific serum ige of ≥0.35 ku/l. mensa (nct01691521) was a gsk sponsored study. results: of the 576 severe asthma patients, 275 (48%) were considered atopic, 272 (47%) were considered non-atopic and atopic status was missing for 29 (5%) patients. the majority of atopic patients (n=147) were sensitized to ≥3 antigens. compared to the non-atopic sub-group the atopic sub-group was younger with a mean age of 46 vs 54 years of age, had a longer mean duration of asthma (21.6 vs 18.4 years), and a higher total baseline ige level (293 u/ml vs 85 u/ml). mean number of exacerbations in the 12 months prior to the study was similar in the atopic and non-atopic subgroups (3.6 vs 3.8) as was the mean baseline peripheral blood eosinophil level (300 cells/ll vs 300 cells/ll). with mepolizumab an 80% reduction in eosinophils was achieved by week 4 irrespective of atopic status and maintained throughout the treatment period. mepolizumab reduced the rate of exacerbations relative to placebo by 48% in the atopic subgroup and by 54% in the non-atopic subgroup. conclusions: the mensa study recruited an equivalent portion of atopic and non-atopic severe asthma patients. the atopic population was younger and had a longer duration of asthma than the non-atopic subgroup. while the baseline total ige level was >3 times greater in the atopic subgroup, the pharmacodynamic and efficacy response to mepolizumab treatment was similar. introduction: a treatment goal in the management of oral corticosteroid (ocs) dependent severe asthma is to reduce daily ocs use due to the side effects associated with long term use. anti ige treatment is used in atopic patients to reduce daily ocs. this analysis characterizes ocs reduction and asthma control in the sub-set of atopic and non-atopic ocs-dependent severe eosinophilic asthma (sea) patients from the 24-week sirius ocs reduction study. objectives: sirius study participants (n=135) were sub-grouped by atopic status in a post-hoc analysis; demographics, clinical characteristics, and response to mepolizumab were evaluated. sensitization to any one of the following; house dust mite, dog dander, cat dander, alternaria, or cockroach was considered as atopic, as assessed by specific serum ige of ≥0.35 ku/l. sirius (nct01691508) was a gsk sponsored study. results: of the 135 ocs-dependent sea patients, 49 (36%) were considered atopic (61% female), 77 (57%) were considered non-atopic (51% female), atopic status was missing for 9 patients (7%). compared to the non-atopic sub-group, the atopic sub-group was younger; mean age of 46 vs 52 years of age, while the mean duration of asthma was the same irrespective of atopic status (19 years). the mean ocs daily dose and acq-5 score at baseline were similar between subgroups (12.6 mg vs 13.2 mg and 2.16 vs 2.04, in the atopic and non-atopic subgroups respectively). the mean baseline peripheral blood eosinophil level was comparable in both subgroups (250 cells/ll vs 220 cells/ll) while the total ige level was higher in the atopic subgroup (182 u/ml vs 86 u/ml). mepolizumab lead to a ≥ 80% reduction in eosinophils by week 4 irrespective of atopic status and eosinophil reduction was maintained throughout the study. mepolizumab reduced the ocs dose in the atopic group and non-atopic subgroups (odds ratio of a greater ocs reduction category of 5.6 (95% ci 1.71, 18.49) and 1.6 (95% ci 0.66, 3.93), respectively) and led to improvement in asthma control with a change in acq-5 of à0.60 (95% ci à1.20, 0.00) in the atopic subgroup and à0.45 (95% ci à0.94, 0.04) in the non-atopic subgroup. in an ocs dependent sea population mepolizumab reduced eosinophils independent of ige level and atopic status. in this limited post-hoc analysis, mepolizumab was an effective ocs sparing agent while improving asthma control in both atopic and non-atopic patients. patients who responded to retreatment had a similar mean time to response between the 1st dosing period (3.5 weeks) and 2nd dosing period (3.1 weeks). of all patients who were retreated (n=56), symptom control (uas7 ≤ 6) after two doses was achieved in 80% (1st period) and 85% (2nd period) of patients; complete response (uas7=0) occurred in 63% (1st period) and 56% (2nd period) of these patients. omalizumab was well-tolerated during both dosing periods. conclusions: omalizumab retreatment is safe and effective in patients with ciu/csu who respond to initial treatment and relapse after withdrawal, with most patients regaining symptom control after a 2nd course of omalizumab. : total ige reactivity of the grass allergoid formulation was diminished compared with native unmodified extracts. a difference in igg profiles was observed and indicated enhancement of accessible reactive igg epitopes across size distribution profiles of the grass allergoid formulation. detailed analysis of the epitope specificity showed retention of six lol p 1 igg binding epitopes in the grass modified extract. all epitopes were mapped on the solvent exposed area of lol p 1 homology model accessible for igg binding. one of the epitopes was identified as an "immunodominant" lol p 1 igg binding epitope (62-ifkdgrgcgscfeik-76) and classified as a novel epitope. lastly, lol p 1 igg antibodies showed functional capacity to block up to 50% of ige binding sites which provides evidence in protective function of immunotherapy induced antibodies against native allergens. the structural and immunological changes which take place following the grass allergen modification process were further unravelled revealing distinct igg immunological profiles. the results from this study support the concept that modification not only enhances the safety profile of scit but allows shorter-course objectives: design targeted sirna delivery system into liver cells. results: liposome surface was modified by lactose derivatives with different structures: lac(c 16 ) 2 and (lac) 7 lac(c 16 ) 2 . the basic physicochemical and biological properties of the carriers have been examined. it is shown that glycoconjugate introduction has no effect on the physico-chemical characteristics. however the carbohydrate modification of the liposomal surface leads to increasing in transfection efficiency on a specific cell line hepg2. moreover, the introduction of mono-carbohydrate derivative increases the transfection efficiency better than using a branched derivative of lactose. in the pharmacokinetic study target effect also was shown. unmodified liposomes start to be detected in the liver at 10 minutes after administration, whereas modified liposomes were detected at 2 minutes after administration. similarly, the excretion of modified liposomes from the liver was more slowly. also worth noting the high concentration of modified liposomes in the liver. furthermore liposomes modified by carbohydrates reduces the intensity of its accumulation in the lung. conclusions: it was shown that increasing attraction of drugs to the liver cells can be achieved by using liposome modification with lactose derivative. 0472 | sialylated fetuin-a is a candidate predictive biomarker for successful grass pollen allergen immunotherapy objectives: to identify new biomarkers of ait efficacy, pre-treatment sera obtained from 82 grass pollen allergic patients responding or not to sublingual ait were differentially assessed by 2d-dige or label-free mass spectrometry. the role of fetuin-a in allergy physiopathology was studied by using gene silencing in a mouse asthma model, human dendritic cell stimulation assays and surface plasmon resonance. results: using comparative proteomics, we provide evidence for an increased o-linked sialylation of fetuin-a in sera collected before treatment from patients exhibiting clinical responses, when compared with low responders. whereas feta may either inhibit or promote chronic inflammation, no specific role in allergy had been ascribed to it until now. in ovalbumin-sensitized mice, silencing of the fetuin-a gene increased airway hyperresponsiveness and th2 responses. fetuin-a, but not its non sialytated counterpart, synergizes with lps and grass pollen or mite allergens in a tlr4-dependent pathway to enhance the proallergic profile of human monocyte-derived dendritic cells. conclusions: quantification of sialylated fetuin-a glycoforms in the blood before treatment allows to identify patients more likely to benefit from grass pollen immunotherapy. validation of the hypothesis that this marker associated with "an inflammation status" can be used to predict ait efficacy is ongoing in larger cohorts of patients. introduction: kawasaki disease (kd) is a vasculitis that mainly affects small to medium-sized vessels, particularly the coronary arteries, and is a leading cause of acquired heart disease in children. previously, we found that the development of kd is associated with an elevation of both th1 and th2 immunity. gene hypomethylation is abstracts | 329 an important form of epigenetic regulation, which results in increased gene expression. because m1 macrophages are associated with inflammation and th1 immunity while m2 macrophages are associated with immune regulation and th2 immunity, we hypothesize that kd is associated with hypomethylation of both m1 and m2 macrophage related genes. objectives: our objective was to investigate the methylation profile of m1 and m2 related genes in patients with kawasaki disease. twenty-four patients with kd and 12 age-matched healthy controls (hc) were included in this study. in patients with kd, blood was sampled 24 hours before ivig therapy (kd1) and 21 days after ivig therapy (kd2). after dna extraction, samples were analyzed using human methylation bead chip 450 (illumina) to examine the methylation ratios of genes related to m1 macrophages (64 genes, 1536 cpg sites) and m2 macrophages (46 genes, 1329 cpg sites). cpg sites with more than 10% difference in methylation levels and a pvalue less than 0.05 between groups were considered significant. objectives: primary aim of the study is the identification of differentially expressed genes (deg) associated with allergic rhinitis (ar) by using genome-wide transcriptomics data from blood immune cells. this set of candidate genes will then be used to define gene networks relevant for onset, severity and potential treatment outcome of house dust mite associated ar. with different ethnicity or populations exposed to a different environment is currently in preparation. introduction: allergic patients display abnormal immune responses to harmless antigens, leading to various symptoms from hay fever to life-threatening conditions. these responses include abnormal type 2 immunity polarization and induction of allergen-specific memory t and b cells, resulting in development of allergy instead of immune tolerance. allergen-specific immunotherapy (ait) is currently the only causative treatment of allergic disorders. yet, in depth understanding of the underlying differences in allergen-specific cd4+ t cell and treg responses between allergy and tolerance and their changes during immunotherapy is lacking. objectives: we investigated whole-genome transcriptomics of circulating birch (bet v 1) and grass (phl p 5a)-specific cd4+ t cells in allergic patients before and at 3, 6, and 12 months of ait, as well in non-allergic healthy controls in corresponding seasonal time points. detailed immunophenotyping with flow cytometry and cd4+ mhc class ii tetramer staining with low rna/single cell next generation sequencing were performed. results: at baseline, out of the pollen season, there were more allergen-specific cd4+ t cells in allergic patients than in controls. at this time, over 1500 genes were significantly changed in allergenspecific as compared to total cd4+t cells in patients, yet we found substantial differences in signal transduction and inflammatory response gene expression when compared to healthy controls. during ait we noted significant increase of allergen-specific cd4+ cells in patients with subsequent gene expression changes in the immune tolerance pathways. finally, we found increase in allergen-specific treg cells in patients upon ait, but not in tolerant controls in corresponding seasons. of interest yet, at early ait time points, allergenspecific treg cells displayed gene profiles suggesting their insufficient suppressive functions. conclusions: in summary, in vivo allergen exposure causes profound changes in the transcriptomic profiles of allergen-specific t cells. these gene profiles seem to be deficient at baseline in allergic patients, but ait is skewing them into the tolerant controls levels. introduction: the variability of the pharmacological response to beta 2 (b2)-agonists may be due to the polymorphism of the gene of results: singled out statistically significant differences of the genotype distribution. the gly/gly16 homozygous allele was discovered twice as often in the group with an insufficient response to b2-agonists than in the group with a good response (66 vs 38%, p<.001), while the distribution of heterozygous allele was detected the opposite pattern (55 vs 28%, p<.001). in the arg/arg16 genotype distribution, there were no considerable differences in both groups (6% in each group). in the subgroups of children, receiving the high doses of the inhaled glucocorticoids, there was a trend for the prevalence of gly/gly16 homozygous allele prevalence. we have discovered the association of the gly/ gly16 genotype of the adrb2 gene with an insufficient effect of broncholytic therapy by means of short-term b2-agonists; we also revealed the participation of the gly16 allele in the phenotype formation with the severe run of bronchial asthma and tolerance towards the therapy both by b2-agonists and inhaled glucocorticoids. mckenna oe 1 ; posselt g 1 ; lackner p 1 ; schmitt a 2 ; h€ ollbacher b 1 ; briza p 1 ; wessler s 1 ; gadermaier g 1 ; ferreira f 1 1 universit€ at salzburg, salzburg, austria; 2 georg august-universit€ at g€ ottingen, g€ ottingen, germany introduction: an excess of 100 million people worldwide have a reported allergy to birch pollen. proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. until now the protease content of betula pendula, a species endemic to the northern hemisphere, has not been studied in detail. hence, we aim to identify and characterise pollen and bacterial derived proteases found within betula pendula pollen. objectives: birch pollen transcriptome was constructed via de novo transcriptome sequencing. reads were assembled using the trinity software suite.. analysis of the birch pollen proteome was achieved via mass spectrometry and use of zymographic gels with the embedded substrates gelatinase and casein, which enabled visualisation of proteolytic activity. further to this, protease activity was quantified using a fluorescently labelled casein substrate protease assay. results: by using mass spectrometry, we were able to identify up to 26 proteases within birch pollen. we could cluster the proteases into specific families based on their distinct proteolytic activities. further comparative analysis of the proteome and transcriptome revealed the relationship between transcript levels and the proteins they encode. zymographic methods enabled distinct visualisation of proteolytic activity for both casein and gelatin substrates. using fluorescently labelled casein, the birch pollen protease activity was quantified as 0.68 lg/ml when compared to a trypsin standard curve. additionally, 26 bacterial isolates of the birch pollen were identified, and the proteolytic activity analysed. we report successful discovery of pollen and bacterial derived proteases endogenous to betula pendula. whilst none of the known birch pollen allergens have been recognised as a protease, we aim to investigate the role of tight junction degradation within epithelial cells and further enhance understanding of proteolytic activity on immune-polarization. objectives: to investigate the frequency and reasons of shortand long-term reintroduction failure in adults after a negative fc. method:: these are preliminary results of an ongoing prospective study. after a negative fc, patients receive standardized aftercare consisting of an introduction scheme to introduce the food at home and consultation by phone for advice and support 24 hours, 1-3 weeks and 6 months after the fc. short-term data about the frequency that patients failed introduction, defined as not completing the introduction scheme or patient-reported allergic complaints repeatedly during introduction, was obtained using a telephone interview. long-term data (5-12 months after negative fc) about frequency and reasons of reintroduction failure in daily diet, defined as not eating the food or only eating products with traces of the food, was obtained using a patient-reported questionnaire. results: from 2014 until now 38 patients were included (mean age: 34 years, male: 26%), who underwent a total of 110 fcs with: hazelnut (23%), other nuts (17%), fruit (7%), composite meals (7%), fish and crustaceans (7%), cow's milk (6%), hen's egg (5%) and other (28%). in 96 (87%) of the negative fcs, introduction using an introduction scheme was advised. in 13%, patients did not receive standardized aftercare for different reasons, eg negative fc with a composite meal. in 19%, introduction with an introduction scheme failed. long-term information was available for 40 fcs. introduction failed in 12 fcs (30%), including 1 patient that even avoided traces of the food. patient-reported reasons for introduction failure were (n=11): symptoms after ingestion of the food (n=8), fear for allergic reactions (n=2) and not like the taste of the food (n=1). conclusions: short term introduction with an introduction scheme failed in 19%. however on the long-term in almost one third of the negative fcs, patients failed to reintroduce the food in daily diet, despite careful aftercare. it is recommended to give these patient even more intensive and tailored support. introduction: in order to help allergic patients manage often severe symptoms, food manufacturers are required to list allergens on labels and take precautions to avoid inadvertent contamination of foods with allergens. existing tools using generic immunoassays do not provide precise identification or quantification of specific food allergens. furthermore, existing elisa immunoassays are not able to be run simultaneously and are often unreliable. objectives: our goal was to develop and validate an accurate, sensitive and high throughput immunoassay that would enable simultaneous quantification of multiple allergens in foods. fluorescent beads coupled to allergen specific monoclonal antibodies were used to develop a multiplex array for simultaneous quantification of major allergens from peanut (ara h 6), milk (bos d 5), egg (gal d 2), and shrimp tropomyosin (pen a 1). target allergens were detected using biotinylated antibodies and streptavidin conjugated fluorochrome. the array was quantified using highly purified natural allergens as standards. allergen content was measured in various samples including samples spiked with purified allergen and allergen incurred food matrices. the results were compared to elisa. a multi laboratory validation was performed to validate the performance characteristics of the assay. results: there was a high correlation between the multiplex array and allergen specific elisa. the limit of detection of the array was as low as 20 pg/ml. no significant cross reactivity was observed between the various food allergen assays. the recovery of allergen from spiked samples was generally between 70 and 130%. inter and intra assay variability was observed to be less than 15%. in conclusion, an accurate, sensitive and reliable multiplex array for major food allergens was developed and validated. the flexibility of the microsphere technology allows for further expansion to produce a comprehensive array for the most important food allergens. this quantitative multiplex array may help to reduce the risk of inadvertent contamination of food. objectives: our aim was to create a food allergy web-based educational program for both schools and restaurants professionals. results: an interactive platform that hosts a free learning program, conclusions: the program and the toolkit are currently available online and are being implemented in schools at the north of portugal. we expect that fac program will give us an important insight on the professionals' knowledge about food allergy. additionally, acting as free integrated service and awareness effort, this program could be an educational tool easily adapted and disseminated, which may improve professionals 'commitment and skills to deal with food allergy in the community. 0495 | development of parallel reaction monitoring (prm) methods for soy and milk detection: consideration of allergen-derived ingredients chen s 1 ; krawitzky m 1 ; yang ct 2 ; downs m 1 1 university of nebraska-lincoln, lincoln, united states; 2 thermo fisher scientific, san jose, united states introduction: the presence of undeclared food allergens poses both regulatory and health risks. in order to assess the magnitude of these risks, accurate quantitative detection methods are required. for some allergenic foods, the food industry uses not only the allergenic source but also a number of source-derived ingredients. some detection methods (both immunoassays and mass spectrometry methods) may fail to accurately detect and quantify these allergen-derived ingredients if they are not taken into consideration during development. objectives: the objective of this work was to select peptide targets for parallel reaction monitoring (prm) mass spectrometry methods that are suitable for detecting a variety of soy-and milk-derived ingredients. six soy-derived and six milk-derived ingredients were obtained for this study. the ingredients were extracted (50 mmol l à1 tris-hcl, ph 8.6, with 6 mol l à1 urea, 20 mmol l à1 dtt, and 1% pvpp) and subjected to in solution trypsin digestion. discovery proteomics analysis was conducted by lc-ms/ms using a q exactive tm plus orbitrap tm running in top 10 data-dependent acquisition mode. peptides were identified using proteome discoverer 2.1 and relative, labelfree quantification was conducted on high-confidence (fdr<0.01) peptides. selected peptides were subsequently analyzed in a targeted prm mode. results: the relative abundance of identified peptides varied among both the soy-derived and milk-derived ingredients. in the case of soy ingredients, for example, there were particularly marked decreases in the abundances of numerous peptides, across different proteins, in a hydrolyzed soy protein isolate. in the milk ingredients, variation in peptide abundance could be more directly attributed to differences in product protein fractionation (eg a decrease in abundance of whey proteins in a sodium caseinate product), although some peptides and proteins maintained consistent abundance across ingredients. after applying specificity and performance criteria, 57 peptides from 14 soy proteins and 64 peptides from 10 milk proteins were selected for further analysis in a targeted prm method. conclusions: peptide abundances vary widely among ingredients derived from allergenic foods. consideration of these peptide differences during ms method development by incorporating discovery proteomics may lead to more versatile and widely-applicable quantitative detection methods for food allergens. introduction: milk and its derivates are usual ingredients in many food products, which must be excluded by cow's milk allergic (cma) patients. oit protocols in patients with cma appear to be safe and effective in inducing desensitization and milk introduction in the diet. objectives: we conducted a survey in cma patients after successful completion of an oit programme, to assess their dietetic profile after introduction of fresh milk and dairy products (unrestricted diet) we considered 42 cma patients (pts), 32 males and 10 females, age 5-29 (median 12.5), who successfully completed the oit protocol. these patients were on an unrestricted diet for milk and derivates, and they had been recommended to assume at least 100 ml of fresh milk or yogurt every day. patients were given an ad hoc questionnaire to assess the milk/dairy products intake at least 24 months after oit completion. results: from our investigation all of the pts have been assuming milk protein everyday without significant reactions after oit. 71% of the interviewee (30 pts) referred to be drinking fresh milk at least three times a week, in a quantity of 100 ml or higher. of note, 85% have to add cocoa powder (14 pts) or coffee (11 pts) to mask milk taste. dislike of milk taste was the cause of the refuse to take fresh milk in 10 pts (over 12 patients that didn't take milk at all); 5 pts who didn't drink milk had at least 125 ml yogurt instead, almost every day. fresh cheese was eaten at least once a week by 50% of all the pts; hard cheese was eaten as main course almost once a week by 62%. 86% of patients consumed biscuits and sweet baked products containing milk/derivates at least once a week, 62% every day. pizza was also present in the diet of the majority of pts (86%), once a week/month. ice cream was appreciated by all the patients and regularly assumed especially during the summer time. conclusions: taste seems to be the main factor that directs the daily choice of milk derivates or milk containing products. all pts easily consume baked products containing milk/derivates, and the majority of them accept fresh milk as advised in order to maintain unresponsiveness. our survey confirms that oit is effective in expanding food choice, but for some patients the change of dietetic habits is hampered due to taste reasons. a more detailed, qualitative and quantitative analysis of the milkunrestricted diet will derive from the 7-days food diary given to these patients. 0497 | frozen-defrosted dried skimmed milk is a suitable product for sublingual immunotherapy for cow's milk allergy introduction: sublingual immunotherapy (slit) is a promising treatment for cow's milk allergy (cma) due to its favourable safety profile. however, its efficacy is limited-probably due to the small volumes and doses that can be delivered sublingually, especially in children. milk products with preserved allergenicity that allow higher protein concentrations in smaller volumes might potentially improve the efficacy of slit for cma. dried skimmed milk (dsm) could fulfill these characteristics. in addition, once dsm is reconstituted, freezing individual vials from the same batch for further administrations could increase dose-consistency throughout the treatment, which would help ensure safety. however, little is known about whether processing to produce dsm, and further freezing-defrosting, may alter its allergenicity. objectives: to evaluate the allergenic protein composition of dsm, including once frozen-defrosted, in comparison to fully-allergenic usually consumed fresh milk. methods: sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by silver staining was performed following the laemmli method to determine the soluble protein composition of the following products: fresh pasteurized milk (friesland campina, amersfoort, the netherlands), dsm (marvel, the premier foods group ltd, london, uk) and frozen-defrosted dsm to identify all protein bands present. western blot with anti-whey and anti-casein antibodies was performed to identify the specific allergenic proteins. results: no significant differences were found amongst the milk products tested, with sds-page displaying all the bands corresponding to the major allergenic proteins in milk (alpha-lactalbumin, beta-lactoglobulin and alpha-, beta-and kappa-caseins) and the western blot showing recognition to all proteins with similar intensity. the major allergenic proteins present in unprocessed milk are well-preserved in dsm, even after freezing-defrosting, making it a convenient product for sublingual immunotherapy. results: nineteen children were randomized into the low dose group (n=11) and the elimination group (n=8). there were no significant differences in background between these groups. after 24 weeks of therapy, the rate of passing the oral food challenge test with 1/8 th of a whole egg was significantly higher in the low dose group (11/11 (100%) vs 4/8 (50%), p=.018). ovomucoid-specific ige levels in the low dose group after 24 weeks were significantly lower than those at baseline. adverse events associated with both therapies did not occur during the study period. conclusions: these results show that low dose ingestion of egg is safe and effective for tolerance or desensitization induction in children with egg allergy, without the need for dose elevation. 0499 | another brick in the wall: toward a national food allergy strategy for canada introduction: the world is experiencing an apparent epidemic of food allergies. with rising prevalence and increasing global spread, basic scientists continue to search for causes while clinical and social scientists continue to study the consequences. these include life-long chronic illness, fear and anxiety, social exclusion and stigma, all of which are underscored by inequalities across vulnerable groups (eg, low income families, immigrants, indigenous peoples). concomitantly, consumer organizations and patient support groups attempt to influence policy in order to maximize choice and minimize risk for individuals and families affected by food allergy, both directly and indirectly. this includes food labeling legislation, food safety regulations, and policies and practices in public places such as schools, restaurants and transportation modes (eg, airplanes). objectives: in canada, with the support of 10+ years of science undertaken by the allergy genes and environment (allergen) networks of centre of excellence in allergic disease, we are leading a national team that aims to establish the building blocks for a national food allergy strategy. conclusions: the greatest challenges to building a national food allergy strategy are not those related to the basic or clinical science, but rather the activities and commitment of individual stakeholders. while gaps remain in the science, the challenges of transdisciplinary integrated knowledge translation hinder progress. we present lesions learned regarding culture shifts that will facilitate the progress we need to maximize choice and minimize risk for canadians affected by food allergy. 0500 | component resolved diagnostics reduces diet restrictions by half among finnish school children savolainen j 1 ; mascialino b 2 ; pensamo e 3 ; hermansson l 4 ; silvan m 3 ; borres mp 4 ; korhonen k 5 1 university of turku, turku, finland; 2 department of allergology, uppsala, sweden; 3 thermo fisher scientific-immunodiagnostics, turku, finland; 4 university of turku, uppsala, sweden; 5 thermo fisher scientific-immunodiagnostics, lieto, finland introduction: there are 50 000-100 000 special diets because of food allergy in finnish schools. the finnish allergy program 2008-2018 was launched to reduce problems and costs induced by allergies. one of the special aims was to reduce diets caused by food allergy will by 50%. the h€ ark€ atie social and health care service region schools have required doctor's certificates for diets for over abstracts | 337 10 years. all diets are listed in a register and checked on a regular basis. objectives: the purposes of this study were to evaluate whether it is possible reduce the special diets when the special diet system appears to be working well and to assess the added value of immu-nocap ® isac allergen component diagnostics in the intervention. results: there were 2885 children attending the schools in the h€ ark€ atie region. there were 205 children with diet due to food allergy. children were recruited to study from the diet register and contacted by a nurse's phone interview. there were 48 children who refused the study leaving 157 children for the assessment of the diet. 23 children were not following any diet and in 44 children the diet was not regarded necessary by the study nurse. 90 children agreed to take part in the study and were referred to laboratory tests for food specific ige and isac. 7 children dropped out leaving 83 children in the study. after the food specific ige results were available, children were contacted by physician. based on the results and/or interview a free diet was allowed for 3 children. 15 children were eating small amounts of the food and were encouraged to increase the use of the foods. 48 children were advised to avoid the foods only if they caused symptoms. only 17 children were advised to continue diet. after the results of isac were available, children conclusions: the study confirmed that it is possible to have diets decreased over 50% in accordance to the finnish allergy program. additionally, the study showed that food allergic children can benefit from the use of component resolved diagnostics. 0501 | omalizumab treatment for severe food allergy caused by lipid transfer protein: a preliminary case series mari a; zennaro d; ferrara r; bernardi ml; alessandri c caam-centri associati di allergologia molecolare, rome, italy introduction: lipid transfer proteins (ltp) are allergens from plantderived foods causing symptoms ranging from oas to anaphylaxis. ltp are present in almost all kind of plant species used for human feeding. ltp allergic patients suffer of reactions to a broad variety of foods, with a very personal reactivity profile which can involve few or many foods. in addition, patients who start to record severe reaction to several foods begin to be afraid of eating any other related or non-related food, leading to a poor diet. omalizumab (ozm) has been documented to be effective in treating food allergies with sensitization to other allergens. objectives: to document the approach for recruiting, studying, monitoring ltp allergic patients, and do a preliminary evaluation of the clinical impact of ozm therapy. results: six adult patients with documented reactions to plantderived foods in the last 12 months and diagnosed to be ige positive to multiple ltp by using multiplex testing, namely isac112 at the beginning and faber244 during the follow up, were recruited. ozm was offered as an off label treatment adopting an open label study design. a questionnaire was submitted to each patient before, during and after the treatment. foods listed as "no more eaten" where considered for reintroduction, starting from those excluded because of fear, going for those with an increasing risk of reactions. re-introduction was done at home after receiving detailed instructions, having all rescue medication at hand. the allergist was connected real time with the patients using one or more ict tools. one patient dropped out because was not complying with the scheduled reintroduction. three patients completed the treatment with a successful re-introduction of all or almost all excluded foods, in two cases peach was reintroduced. the last two patients reintroduced 50%-60% of the excluded foods, including some of those previously causing reactions. the attempt of re-introducing the most risky foods failed causing local allergic reactions promptly controlled by the therapy. all foods re-introduced were tolerated once the treatment was stopped. conclusions: ozm seems to be a promising treatment for severe ltp allergics, improving their qol. starting from the recruitment phase to the re-introduction, the overall procedure looks quite complicated and deserve much resources. ige sensitization to ltp, carefully defined before treatment, can be monitored during the ozm course as the drug doesn't interfere with faber test ige detection. 0502 | oral immunotherapy with peach-juice in lipid transfer protein (ltp) allergy: is it possible to reach tolerance? introduction: food allergy to rosaceae fruits and nuts, due to sensitization to lipid-transfer protein (ltp), is frequent in the mediterranean area countries. based on milk allergy oral immunotherapy (oit) protocols, a study is proposed to obtain an oit regimen in patients allergic to ltp. objectives: we included patients with anaphylactic reactions due to ltp-food allergy, from january/2015 to october/2016. skin prick test (spt) was performed with a food and inhalant battery, ltp bial-aristegui ® (0.1 mg/ml), prick by prick (pbp) with a specific commercial peach-juice (dilutions to endpoint), and determination of pru p3 ige levels (immunocap). the presence of pru p 3 protein in the peach-juice, was confirmed by sds-page and elisa method, and quantified with anti-pru p 3. we elaborated an oit guideline with progressive administration of peach-juice, 1-2-4-5 drops (5drops=0.13 ml), sublingually, starting concentration chosen according to the endpoint spt results, up to 5 ml daily dose. the dose reached at the allergy service, was continued at home, daily, for 1-2 weeks. all the increasing doses were performed at the allergy service, after pbp with peach-juice and ltp control spt. after a time, an oral challenge up to 250 ml peach-juice was performed. patients were instructed on the importance of maintaining regular intakes and avoiding cofactors. results: 16 patients started this protocol (6 male/10 female), aged 7-38 (media 19.67). peach-juice analysis in agarose gel electrophoresis, showed a 10 kda band quantified as 21.16 lg/ml of ltp. there was no difference between wheal diameter on spt for ltp bial-aristegui ® and pbp with commercial peach-juice along all the study. average concentration on pbp to endpoint: 1/1000 (4), 1/100 (8), 1/10 (4). pru p 3 ige level average: 30.22 ku/l. none anaphylactic or serious reactions during the oit protocol appeared. only five patients (31%) reported mild occasional symptoms (oral pruritus and mild located urticaria). 11 patients reached 5 ml daily dose (68.75%), and 4 of them (25%) completed oral food challenge with peach-juice up to 250 ml, with good tolerance. this pattern also allowed us to reintroduce withdrawn foods from the diet due to previous reactions, with good tolerance. results: 60 children (age 10-18 years) sensitized to dog dander extract (median 11.5 ku a /l), with or without known dog induced allergic airway disease, were included. nasal provocation testing with dog dander extract was performed. measurement of ige to dog dander extract and to can f 1-can f 6 was performed with immunocap. an ige level ≥0.1 ku a /l was considered positive. among all 60 children sensitization to can f 1 (65%) and can f5 (62%) was most frequent. corresponding numbers for can f 4, can f 2, can f 6 and can f 3 were 48%, 47%, 47% and 27% respectively. based on the results from the nasal challenges three groups were identified; no (n=21), mild (n=14) and strong reaction (n=25). the median ige levels to dog dander and to all 6 allergen molecules were higher in the strong reaction group than in the mild and the no reaction group. among children with a strong reaction, ige to can f 4 was found in 68% (17/25) compared to 24% (5/21) among children with no reaction (p=.003). ige to can f 6 showed a similar pattern (p=.006). 40% (10/25) of the children with a strong reaction were sensitized to can f 3 compared to 9.5% (2/21) of the patients with no reaction (p=.01). no associations were found between the ige levels to can f 1, can f 2 and can f 5 and the no reaction or strong reaction groups. using multiple regression analysis, with all 6 allergens in the model, sensitization to can f 4 showed a statistically significant difference between the groups. conclusions: molecular allergology may improve diagnostics of dog allergy in children. sensitization to the lipocalin can f 4 was significantly associated to a strong positive nasal reaction implying its usefulness as a marker of clinically relevant dog allergy. tsolakis n 1 ; malinovschi a 2 ; nordvall l 1 ; janson c 2 ; mattson l 3 ; lidholm j 3 ; borres m 3 ; alving k 1 1 uppsala university, women's and children's health, uppsala, sweden; 2 uppsala university, medical sciences, uppsala, sweden; 3 thermo fisher scientific, uppsala, sweden introduction: cat allergy is common worldwide and a major trigger of asthma in many countries. molecular patterns of cat sensitisation vary between individuals but their relationship with allergic inflammation has not been extensively studied. objectives: the aim was to investigate the prevalence of ige to different cat allergen components and their relationship to type-2 inflammation and bronchial responsiveness in young asthmatics. conclusions: ige sensitisation to cat serum albumin (fel d 2) and cat lipocalins (fel d 4, fel d 7) , but not to secretoglobin (fel d 1) or cat dander extract, were independently associated with feno and b-eos count. we suggest that cat serum albumin and cat lipocalins can be used as markers for increased risk of type-2 inflammation in young asthmatics, as shown in multiple regression analyses. 0505 | complete sequence and recombinant production of horse dander allergen equ c 2 lidholm j; lundgren t; larsson h; mattsson l thermo fisher scientific, uppsala, sweden introduction: horse dander is an increasingly important cause of respiratory allergy. equ c 2 was one of the first horse allergens to be recognised but only a small part of its amino acid sequence has been reported. objectives: the aim of this study was to determine the complete sequence of equ c 2 and express it as an immunoreactive recombinant protein. methods: equ c 2 was purified by size exclusion, hydrophobic interaction, anion exchange and reversed phase chromatography. recombinant equ c 2 was expressed as a hexahistidine tagged protein in e. coli and purified by immobilized metal ion affinity and ion exchange chromatography. ige antibody reactivity to natural and recombinant equ c 2 and other horse dander allergens was determined in sera of 25 subjects allergic to horse. results: a putative equ c 2 sequence predicted from genomic data revealed a lipocalin protein of 159 amino acids with a highest sequence identity among known lipocalin allergens of 33% to can f 4. n-terminal sequencing and mass spectrometric analysis of purified natural equ c 2 confirmed 97.5% (155/159 residues) of the predicted sequence. recombinant equ c 2 displayed ige antibody binding activity comparable to that of purified natural equ c 2 (r=.98). of the 25 horse allergic subjects studied, 19 (76%) showed ige antibody binding to equ c 1, 13 (52%) to equ c 2, 5 (20%) to equ c 3 and 7 (28%) to equ c 4. the complete sequence of equ c 2 was established. as a fully immunoreactive recombinant protein, it represents an important addition to the panel of allergens useful in the diagnosis of allergy to horse. 0506 | component resolved diagnosis using guinea-pig allergens elucidates allergen sensitization profiles in allergy to furry animals objectives: to identify major guinea-pig allergens and to evaluate their potential as reliable markers for a specific ige-diagnosis in comparison to dander extracts. results: forty-three patients with a clinical history of allergy to guinea-pig and 45 patients allergic to cat and/or dog were recruited for the study. major guinea-pig allergens were identified by ige-immunoblot and n-terminal sequencing of ige-reactive proteins. corresponding cdnas were cloned and allergens were expressed as recombinant proteins in e. coli. specific ige to animal dander, fel d 2 and can f 3 were determined, specific ige to fel d 4, can f 6 and to guinea-pig allergens were quantified by elisa. two new guinea-pig lipocalin allergens, cav p 1 and cav p 6, were identified in guineapig dander. the combination of 4 guinea-pig allergens, the 2 new allergens and the previously isolated lipocalins cav p 2 and cav p 3, enabled the identification of 39 out of 43 patients sensitized to guinea-pig. the vast majority of the patients had specific ige to cav p 1 (84%). cav p 6 shares 53% sequence identity with fel d 4 and can f 6 and was found to be cross-reactive with these cat and dog allergens. in the group of cat and/or dog allergic patients, 87% had also specific ige to guinea-pig dander. nearly half of those (47%) had ige to cat serum albumin fel d 2 or to fel d 4 (58%) and to can f 6 (56%), explaining the high degree of cross-reactivity to guinea-pig dander. only 25% of the cat/dog allergic patients with a positive isle test to guinea-pig dander had specific ige to any of the non crossreactive guinea-pig allergens cav p 1, cav p 2 or cav p 3. however, none allergen has been characterized from mongolian oak. objectives: in this study, we tried to characterize a major allergen from mongolian oak. results: a molecule homologous to pathogenesis-related protein 10, a putative que m 1, was cloned by rt-pcr and its recombinant protein along with que a 1, an allergen from white oak (q. alba) was produced. cloned que m 1 sequence shares 57.5%-96.2% amino acid sequence identity (96.2%) with pr-10-like allergens from various plants. allergenicity and diagnostic value of recombinant que m abstracts | 341 1, que a 1 and bet v 1 proteins were compared by elisa using sera from oak sensitized subjects. specific ige to recombinant que m 1, que a 1, and bet v 1 were detected in 90.0%, 78.0%, and 94.0% of 50 serum samples from korean tree pollinosis patients. recombinant que m 1 was able to inhibit ige reactivity to que a 1 and bet v 1, indicating its strong cross-reactivity. activation pattern of basophils from five patients was similar in terms of cd203c expression and protein concentration of challenged bet v 1 and que m 1. objectives: over the course of one year, all patients who were seen at our immunoallergology clinic for the first time underwent spt with our standard series, to which four olive tree cultivar extracts were added (arbequina, blanqueta, hojiblanca e picual). we then recorded wheal size diameters, considering a wheal >3 mm to correspond to a positive test. we recorded 832 individual sessions of spts, in which 613 presented at least one positive result. two hundred and thirtysix of these patients (38.5%) had a positive spt for olive tree pollen, only one of them being monosensitized. when looking only at pollen-sensitized patients, twenty-two patients (5.9%) tested positive solely for olive tree pollen. in all allergic patients, the most frequent cultivar sensitization was to the cultivars hojiblanca (31.5%) and arbequina (31.3%), followed by the cultivars picual (29.2%) and blanqueta (27%). twenty-six patients (11% of all olive pollen sensitizations) had a positive spt for the conventional extract and negative spt for the cultivars; 192 patients (81.4%) had a positive spt for both. we noted that 7.6% (n=18) of patients sensitized to olive tree pollen had a negative spt with the conventional extract and positive with one of the cultivar extracts. the cultivar hojiblanca was the most frequent in this group (66.7%, n=12), followed by the arbequina cultivar (55.6%, n=10). in the group as a whole, there was a positive correlation between the results of the spts with the conventional extract and each of the cultivar extracts. this correlation was weaker with the cultivar hojiblanca (r=.785). conclusions: some patients, sensitized only to the pollen of certain olive tree cultivars, are not identified with the conventional extract. spt with the hojiblanca cultivar could identify most of these patients in our country, and therefore should be considered in patients with a history of pollinosis and a negative spt for the common extract of olive tree pollen. 0509 | structural and immunological comparison of heat treated pru p 3 and art v 3, the non-specific lipid transfer proteins of peach and mugwort pollen wildner s 1 ; stock l 1 ; regl c 2 ; alessandri c 3 ; mari a 3 ; huber c 2 ; stutz h 2 ; gadermaier g 4 objectives: recombinant art v 3.0201 and pru p 3.0102 were expressed in e. coli rosetta-gamib plyss and purified using cation exchange chromatography. proteins were analyzed in gel electrophoresis and mass spectrometry. proteins were incubated at 95°c in time intervals up to 120 min using buffers at ph 3.4 and 7.3. physico-chemical properties of native and heated allergens were analyzed by circular dichroism spectroscopy, dynamic light scattering and size exclusion chromatography (sec). using sera from italian patients sensitized to pru p 3 and art v 3 (n=26), ige binding to native and heat-denatured allergens was investigated in elisa. results: highly pure recombinant pru p 3 and art v 3 were obtained as non-tagged proteins from e. coli. identity and formation of disulfide bonds was verified by mass spectrometry. circular dichroism showed high thermal stability of both proteins at acidic ph. the alpha-helical fold of art v 3 was lost upon heating for 15 min at ph 7.3 while pru p 3 was already altered after 5 min. purified pru p 3 and art v 3 are monomeric molecules with a hydrodynamic radius of 1.6 and 1.8 nm, respectively. structural relaxation is observed upon heat treatment which is not attributed to protein aggregation as determined by sec. the ige reactivity to both allergens was largely unaffected upon heating at ph 3.4. notably, ige reactivity to art v 3 was already significantly decreased upon 15 min heating and was completely abrogated to both proteins after 120 min denaturation at neutral conditions. conclusions: even though the fold of pru p 3 is more compact compared to art v 3, susceptibility to structural changes upon thermal treatment at neutral conditions are more pronounced which do however not directly translate to lower ige binding capacities. particularly the buffer environment needs to be considered when formulating ltp-containing products which undergo heat treatment. objectives: we sought to determine the ige binding capacity and potential diagnostic value of a recombinant hybrid molecule. results: the codon-optimized nucleotide sequences of a hybrid molecule comprising the full sequences of blo t 5 and der p 2 at the amino and carboxyl ends respectively, here named bp-2, was cloned into a plasmid vector and expressed in escherichia coli as a 6xhis tag protein. two hundred and thirty three sera from colombian (n=118) and cuban (n=115) allergic patients were tested by elisa for ige reactivity. thirty seven sera from non-allergic subjects and negative skin test (spt) to mites were used as controls. all subjects provided written informed consent to their participation in this study. hdm allergy was diagnosed on the basis of clinical symptoms in combination with mite extract spt. potential diagnostic value of bp-2 specific ige was determined by receiver operating characteristic (roc) analysis and area under roc curve (auc) calculated. positive serum ige values to hybrid molecule were defined as optical density (od) higher than 0.13 (mean od plus 3 standard deviations in 15 nonallergic subjects). in respiratory allergic patients, the overall frequency of positive ige reactivity to bp-2 was 70.9%, in non-allergic subjects the frequency was 21%. serum ige levels to bp-2 were positively correlated to spt to b. tropicalis (spearman r=.54, p=.001), and to d. pteronyssinus (spearman r=.4, p=.001). using spt to mite extracts as gold standard, the sensitivity and specificity of serum ige levels to bp-2 were 71.4% and 94.1% respectively, with an auc of 0.82 (95% confidence interval 0.74-0.89). conclusions: these data suggest that bp-2 could be a useful reagent for identifying allergic patients sensitized to b. tropicalis and/or d. pteronyssinus. 0511 | igg, ige and igg4 specific antibodies to molecular allergens of aspergillus fumigatus introduction: in clinical allergy, alongside with skin prick tests, in vitro determination of specific ige for a particular patient contributes to the diagnosis and helps to estimate the risk associated with different food allergens. however, with commercial methods of specific ige antibodies detection (component-resolved diagnosis, crd), the clinician is typically limited by the list of the available allergens. objectives: to overcome this limitation, we developed two component-resolved diagnostic tests for food allergy in which natural extracts can be used. in the first developed method, the crd is performed using immunoaffinity capillary electrophoresis (iace) coupled with matrixassisted laser desorption/ionization mass spectrometry. meanwhile, the second method is based on in-tube immunomagnetic separation (ims) with mass spectrometry identification (mass spectrometry or peptide mass fingerprinting). in both techniques, magnetic beads coated with antihuman ige antibodies are used to extract the ige antibodies from the blood serum of the allergic patient. then, the immunocomplex, obtained on the magnetic beads, is used to quantify the total ige level or to probe the ige binding with standard allergens or natural allergenic extracts. afterwards, the identification of the extracted proteins, ie potential allergens, is performed by mass spectrometry with or without ce separation. after optimisation, the proposed methods have been successfully applied to a commercial blood sample of a patient with a known allergy to cow's milk, with results confirmed by standard tests. as a proof-of-concept, the sensitization profile of a patient suffering from protein contact dermatitis to the cow's whey fraction has been determined. we confirmed the presence of circulating ige antibodies binding lactoferrin and bovine serum albumin. cross-reactivity tests were also performed using goat and sheep milk and revealed the patient sensitivity to serum albumins from these two milks. such approaches open the possibility for direct identification of ige-bound allergens molecular mass and structure. these methods allow the discovery of yet unknown allergens and could be useful for precise personalized allergy diagnosis, allergens epitope mapping, and cross-reactivity studies. objectives: the objective of this study was to investigate the validity of cord blood ige for predicting atopy at 6 years of age. methods: a total of 385 children born in 2010 participated in the longitudinal investigation of global health in taiwanese schoolchildren (lights) cohort. total ige was measured in umbilical cord blood at birth. perinatal history was collected from medical records in the chang gung memorial hospital, taiwan. total and specific serum ige and questionnaires were carried out at 6 years of age. receiver-operating characteristic (roc) curves were used to determine the validity of cord blood ige for predicting atopy at 6 years of age. logistic regression models were applied to assess the association between cord blood ige and atopy at 6 years of age. results: cord blood ige levels was significantly associated with total serum ige level at 6 years of age (r=.314, p<.001). the cord blood ige levels in atopic children aged 6 years (meanaesd, 1.61ae6.2 ku/l) were significantly higher than those in nonatopic children (0.36ae0.94 ku/l) (p<.001). the area under the receiveroperating characteristic (roc) curve of cord blood ige for predicting atopy at 6 years of age was 0.702. the sensitivity, specificity, and positive and negative predictive values of cord blood at the optimal cutoff of 0.34 ku/l on the roc curve for predicting atopy were 50.9%, 88.1%, 92.1%, and 39.8%, respectively. higher cord blood ige levels (≥0.34 ku/l) was associated with a higher likelihood of atopy at 6 years of age (or=7.66; 95% ci: 2.81-20.90; p<.001). our results indicate that cord blood ige is a potential predictor of atopy at school age, with an optimal cutoff of 0.34 ku/l. bogomolov a vinnitsa national pirogov memorial medical university, vinnitsa, ukraine introduction: allergen sensitization is being diagnosed by commonly available methods in clinical practice-skin prick tests (spts) and specific immunoglobulin e test (sige). recently, a new thermographic (th) method for the assessment of spt was developed, and it was demonstrated that the th measurements of forearm temperature distribution during spt, supported by a mathematical model, offer a new quantification method of allergen-induced skin reactions. objectives: the aim of this study is a comprehensive comparison of the th method with spt and sige techniques. the group of patients who were participated in this study consist of 45 patients. among them were 24 patients (53.3%) with allergic rhinitis and 21 patient (46.7%) with asthma, 35.5% of them were men and 64.5% were women aged 18-53 years (mean age 38.3ae6.5 years). spt and sige testings were performed by the standard techniques. for th analyses, set of thermograms of both forearms were acquired after prick and analyzed with the use of developed software. all results were converted into categorized scale for comparison. after counting patients who were true positive (tp), true negative (tn), false positive (fp), and false negative (fn), the sensitivity, specificity, and accuracy were calculated according to the following formula using the results of spt as the standard; sensitivity=tp/(tp+fn), specificity=tn/(tn+fp), and efficacy=(tp+tn)/(tp+tn+fp+fn). the results showed high correlation coefficients between the methods equal to 0.73-0.97. the sensitivity and accuracy of the th assessment in respect of both the classical methods is at a good level (0.70-0.94). the acceptable level of specificity 0.60-0.88 was also achieved for the majority of allergic reactions. the best accordance was observed between th and sige results (r=.92), while th-spt was the most divergent pair r=.85. in case of particular allergens, the biggest correlation was 0.99, while the smallest value amounted to 0.76. the results of diagnostic indicators of thermographic measurement of skin reactivity in comparison with the classical methods of determining the sensitivity to allergens show the prospect of using the method in routine practice. the main advantages of this method are its higher measuring ability and objectivity, by which the possibility of making error in diagnosis is significantly reduced. introduction: chronic urticaria symptoms may be worsened by factors such as temperature, exercise, hormones and stress. a salicylate rich diet has been reported to worsen symptoms in these patients. the mechanism by which natural salicylates do this is unclear but, like aspirin, is thought to be due to their ability to interfere with the arachidonic pathway via cyclooxygenase inhibition. studies have shown that low dose aspirin increases serum il-3 levels in patients with antiphospholipid syndrome. il-3 is important in basophil and mast cell function, inducing mediator release and cd203c upregulation in the absence of ige stimuli. objectives: the gold standard for diagnosing salicylate exacerbated chronic urticaria is by challenge testing. there is no in vitro laboratory test approved for routine diagnosis. this study investigated whether il-3 levels are raised in patients with chronic urticaria and if these levels were affected by salicylate intake. we aimed to find an optimum method of measuring il-3 levels by comparing levels in serum and salivary samples. the quantikine human il-3 enzyme linked immunosorbent assay (elisa) was validated and used on both saliva and serum of patients with chronic urticaria and normal controls. this was a case control study of 19 medicated patients with chronic urticaria and 26 controls at university hospital southampton, to see whether there were any correlations with il-3, and degree of salicylate intake (based on a questionnaire). introduction: since the introduction of molecular components in allergy, a big challenge of allergy diagnostics is to connect clinical symptoms with optimal test use and correct interpretation of results. objectives: the aim of the study was to (1) develop an algorithm that would meet that need, and (2) to evaluate the effect of introduction of algorithm to clinical practice. the algorithm was developed which groups clinical symptoms into six categories: rhinoconjunctivitis/ asthma, oral allergy syndrome (oas), urticaria/angioedema, eczema, anaphylaxis, and a combination of symptoms and combines them with knowledge of possible allergen specificity. this information is combined with two basic allergen mixtures (panels), reflex testing of selected food molecular components and accompanied by interpretative comments. the introduction of our algorithm led to less inhalation screenings, more food screenings and an increase in the requested molecular components. the oas was seldom recognized or used as a symptom by specialists. the reduction in costs, by using the possibility that the disease presentation may be a consequence of an relatively not dangerous oas, was therefore not achieved. all pr-10 positive proteins in various allergen sources showed also positivity for birch antigen. the screening based on this algorithm has potential to enable clinicians/general practitioners with a tool to increase the pre-test probability of allergy for the most frequently occurring allergens. allergy diagnostics may be more efficient if pr-10 component of birch (r bet v 1) is included in early screening and can help in early recognition of oas. helbling a 2 department of otorhinolaryngology-head and neck surgery 0492 | clinical and immunological evolution of patients who failed milk-oral immunotherapy there is lack of evidence on evolution among failures. objectives: to analyze clinical and immunological evolution of patients who failed milk-oral immunotherapy. data were obtained from medical records of a cohort of 20 patients who failed moit in the past 10 years in hospital infantil universitario niño jesus 14(70%) patients failed during build-up phase [13(92%) due to adverse events (ae) and 1(8%) to family decision. 6 failed during maintenance phase: 3(50%) due to eosinophilic esophagitis, 1(16%) to family decision, 1 to psychiatric disorder and (range 5-1023). the most frequent aes were cutaneous and gastrointestinal symptoms. 10/20 (50%) patients underwent a second moit and was successful in 5. the second moit was performed between 2 and 9 years after the first one. there was no statistical differences between specific ige(ku/l) levels at baseline and 12 months after the moit end portugal introduction: fish allergy is common in countries where consumption is high. parvalbumins present in fish muscle are the major allergens. allergy to multiple fish species is caused by parvalbuminspecific cross-reactive ige. cross-reactivity with parvalbumin from baltic cod retrospective study of patients with fish allergy followed in our immunoallergology department. fish tolerance acquisition was evaluated by oral food challenge. statistical analysis was performed using spss version 23 (descriptive statistics, student test results: 81 pts were included (55 male, 26 female), 62 children and 19 adults (age 14ae9 years). 63(78%) had previous history of rhinitis, 35(43%) of asthma and 54(67%) of eczema age of first contact with fish averaged 8.9ae4.0 months (min4, max36) and the possible types of contact were: oral in 78(96%), cutaneous in 22(27%) and inhalation of cooking fish vapours age of first clinical manifestation (excluding the 4 pts who developed allergy in adulthood) was at 22ae25 months (min4, max132) the clinical manifestation of the reaction was: angioedema/urticaria 58(72%), gastrointestinal symptoms 28(35%), eczema 27(33%), respiratory symptoms 19(23%), oral allergy syndrome 10 (12%), cardiovascular symptoms 2 (2%) age at the first immunoallergology out-patient clinic consult averaged 7ae9 years (min 0.4, max 48) and time from first symptom to first thermo-fisher) was evaluated before and after acquisition of tolerance to at least 1 fish. before tolerance, sige (ku/l) averaged 21 roc curve (area under curve 0.854) showed that, for gad c 1 105 ku/l, pts had a sensitivity of 90.2% and specificity of 62.5% that they would have a negative oral food challenge to a fish an sige<0.635 ku/l had sensitivity of 98% of a negative challenge 34 ku/l had specificity of 92.9% of a positive challenge. conclusions: fish allergy is a common allergy in early childhood however, acquisition of tolerance is possible. rgad c 1 appears to be a good marker for fish tolerance and could help allergologists as to when start testing for fish tolerance key: cord-019490-m1cuuehi authors: nan title: abstracts cont. date: 2015-12-28 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2005.clm_1134_02.x sha: doc_id: 19490 cord_uid: m1cuuehi nan addition of a biocatalytic oxygen-reducing agent may be required in the absence of fresh media (<12 hours old) when testing tigecycline using broth microdilution t. stevens, b. johnson, s. bouchillon, j. johnson, d. hoban, m. dowzicky, p. bradford (schaumburg, pearl river, usa) objectives: tigecycline (tgc), a new glycylcycline antimicrobial in development has demonstrated excellent in vitro activity against gram-positive and -negative pathogens. recent reports suggest that tgc mic values, against some organisms, may be elevated if broth used in microdilution panels is (>12 hours old (aged). the nccls has tentatively recommended that testing of tgc be performed in broth that is (<12 hours old (fresh). this study looks at the difference between panels using fresh broth, aged broth and broth with a biocatalytic oxygen-reducing agent (bora) added to compensate for any potential broth differences. materials and methods: testing was performed on approximately 120 organisms including: e. coli; k. pneumoniae; m. catarrhalis; s. epidermidis; and s. pneumoniae. the bora used in this study is oxyrase ò for broth at 2% concentration. tig microdilution panels evaluated in this study include: panels and aged broth without oxyrase (aged); panels and aged broth with oxyrase (ao); panels and fresh broth without oxyrase (fresh); and panels and fresh broth with oxyrase (fo). panels and aged broth were prepared by microscan. fresh broth was prepared internally. each organism was tested on all four panel types. quality controls were performed using nccls approved atcc strains. results: combined test results showed an mic correlation (with in 1 log2 dilution) as follows: 97.5% between fresh/ao; 85.6% between fresh/aged; 94.3% between fo/ao; and 87.9% between fo/aged. quality controls ranges for fo, fresh and ao were all in compliance, but aged panels were out of range 29.3% of the time. conclusion: the addition of a bora to aged broth produced results equivalent to fresh broth without a bora. objectives: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many grampositive pathogens including methicillin-resistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillin-tazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from multinational evaluation centres in the test program. methods: a total of 1245 clinical isolates were identified to the species level at each of 15 sites in 8 countries and confirmed by the central laboratory. isolates were collected between january 2004 and november 2004. mics were determined by each participating laboratory using supplied broth microdilution panels from dade microscan. all testing was performed according to nccls guidelines and manufacturer's instructions. results: the mics of tigecycline ranged from 0.06 to 1 mcg/ml for all isolates of s. aureus. tigecycline's mic50/mic90 of 0.12/ 0.25 mcg/ml, respectively, against mssa was similar to imipenem and minocycline and 4/8 fold lower than the remaining comparative agents. tigecycline's mic50/mic90 of 0.25/0.5 mcg/ml, respectively, against mrsa was 8/4 fold lower than vancomycin, 2/4 fold lower than minocycline and 4/8 fold lower than linezolid. all isolates of s. aureus were inhibited by tigecycline at an mic of 1 mcg/ml regardless of methicillin phenotype. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. background: rapid increasing resistance in nosocomial pathogens has always been a challenge for clinicians and hospital infection control. tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of enterobacteriaceae (mainly e. coli, klebsiella spp., enterobacter spp., and serratia spp.) collected from hospitals in north america, europe and asia. methods: a total of 3204 clinical isolates of enterobacteriaceae were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity was equivalent to imipenem presenting a mic50/mic90 of 0.25/1 mcg/ml against all strains of enterobacteriaceae. in comparison to other antimicrobials tested, the mic90 of 1 mcg/ml for tigecycline was also the lowest being 8 fold lower than commonly prescribed broad spectrum antimicrobials such as ceftriaxone, levofloxacin, and minocycline and 16 fold lower than ceftazidime and piperacillin/tazobactam. the frequency of esbl production among k. pneumoniae and e. coli was found to be 10.3% and 2.2%, respectively. tigecycline inhibited >98% of all e. coli and k. pneumoniae esbl producers at an mic of 2 mcg/ml. approximately 20% of enterobacter spp. and serratia spp. presented resistance to third generation cephalosporins (ceftazidime and ceftriaxone) suggestive of ampc-type resistance. tigecycline also inhibited a majority of these isolates with an mic90 of 2 mcg/ml. conclusion: tigecyclines in vitro activity was comparable to the activity of a broad spectrum antimicrobial, carbapenem (imipenem) , and greater than other commonly prescribed broad spectrum agents tested in this study. the presented data suggest that tigecycline may be an effective therapeutic option against both susceptible strains of enterobacteriaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have a potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram-positive, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against isolates of enterobacteriaceae collected from hospitals across the usa. methods: a total of 2446 clinical isolates, collected in 2004, were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: the efficacy of all broad spectrum antimicrobial agents still remain highly active against enterobacteriaceae in the united states. the susceptibility rates for amikacin, cefepime, ceftazidime, ceftriaxone, imipenem, levofloxacin, minocycline and piperacillin/tazabactam are 99.3%, 97.5%, 89%, 92.2%, 98.8%, 85.5%, 85.7% and 91.8%, respectively. tigecycline's activity was similar to imipenem presenting a mic50/mic90 of 0.25/1 mcg/ ml against all strains of enterobacteriaceae. the frequency of esbl production among k. pneumoniae and e. coli was found to be 8.5% and 2.2%, respectively. tigecycline successfully inhibited >98% of all e. coli and k. pneumoniae esbl producers at a mic of 2 mcg/ml. it was also noticed unusual resistance to imipenem in 29 of these isolates. while still under more detailed analysis, preliminary data have shown that tigecycline presented a mic50/ mic90 of 1/2 mcg/ml against these multi-resistant isolates. conclusion: most of broad spectrum antimicrobial agents still remain active against enterobacteriaceae from the us. tigecycline's activity was comparable to the activities of broad spectrum antimicrobials and with greater activity against most esbl and ampc producing isolates. tigecycline also showed in vitro activity against isolates that were intermediate or resistant to imipenem, which in many instances is considered as a last therapeutic option. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible strains of enterobacterieaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered gram-positive and gram-negative species, including anaerobic pathogens responsible for community and hospital infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/ tazobactam against gram negative rods in addition to linezolid, penicillin and vancomycin for the gram positive species. isolates were collected from hospitals in the united states throughout 2004. methods: a total of 4100 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with a mic equal or lesser than 2 mcg/ml. tigecycline also showed in vitro activity with a mic90 2 mcg/ml against 29 imipenem resistant enterobacteriaceae strains. although similar to other classes of broad spectrum antimicrobial agents against non-fermenters, tigecycline was especially active against acinetobacter spp. with the lowest mic90 of 2 mcg/ml. tigecycline inhibited s. aureus with mic90 of 0.25 mcg/ml for both mssa and mrsa isolates. against enterococci, tigecycline's mic90 was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable or greater than most commonly prescribed broad spectrum antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible common gram-positive and gram-negative pathogens, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, and piperacillin/tazobactam against gram negative rods in addition to linezolid, penicillin, and vancomycin for the gram positive species. isolates were collected from hospitals located in germany, italy, spain, and united kingdom throughout 2004. methods: a total of 1064 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with mics equal or lesser than 2 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against glucose non-fermenters, tigecycline was especially active against acinetobacter spp. presenting the lowest mic90 of 1 mcg/ml. tigecycline inhibited s. aureus with a mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. the same results were noticed against enterococci where tigecycline's mic90 of 0.25 mcg/ml was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered spe-cies responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/ tazobactam against gram negative rods in addition to linezolid, penicillin and vancomycin for the gram positive species. isolates were collected from hospitals located in asia throughout 2004. methods: a total of 424 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity was similar to imipenem against enterobacteriaceae with mic50/mic90 of 0.25/1 mcg/ml. resistance to third generation cephalosporin was found in 63.2% of e. coli and 77.8% of k. pneumoniae consistent with esbl phenotype. tigecycline inhibited esbl and ampc producers with mics equal or lesser than 1 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against glucose non-fermenters, tigecycline was especially active against acinetobacter spp. presenting the lowest mic90 of 1 mcg/ml. tigecycline successfully inhibited s. aureus with mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. same phenomenon was noticed against enterococci where tigecycline's mic90 of 0.12 mcg/ml was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram-positive, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against isolates of enterobacteriaceae collected from hospitals in germany, italy, spain and united kingdom. methods: a total of 627 clinical isolates, collected in 2004, were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory abstracts using supplied broth microdilution panels and interpreted according to nccls guidelines. results: it was observed that the in vitro activity of all broad spectrum antimicrobial agents still remains highly active against enterobacteriaceae in europe. the susceptibility rates for amikacin, cefepime, ceftazidime, ceftriaxone, imipenem, levofloxacin, minocycline,andpiperacillin/tazobactamare99.8%,94.6%,84.8%, 87.6%, 100%, 86.1%, 84.5%, and 92.2%, respectively. tigecycline's activity was similar to the most effective antimicrobial agent, imipenem (100% susceptibility) presenting a mic50/mic90 of 0.25/1 mcg/ml against all strains of enterobacteriaceae. the frequency of esbl production among k. pneumoniae and e. coli was found to be 21.7% and 2.4%, respectively. tigecycline inhibited 100% of all esbl producing e. coli at a mic of 0.5 mcg/ml and at a mic of 4 mcg/ml for esbl producing k. pneumoniae. approximately 30% of enterobacter spp. and 6% of serratia marcescens presented resistance to third generation cephalosporins (ceftazidime and ceftriaxone) suggestive of ampc-type resistance. conclusion: most of the broad spectrum antimicrobial agents still remain active against european representatives of enterobacteriaceae. tigecycline's in vitro activity was comparable to the activities of all broad spectrum antimicrobials with greater activity against esbl and ampc producing isolates with a mic 90 of 2 mcg/ml. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible strains of enterobacterieaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, and piperacillin/tazobactam against gram negative rods in addition to linezolid, penicillin, and vancomycin for the gram positive species. isolates were collected from hospitals in north america, europe and asia throughout 2004. methods: a total of 6383 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with a mic equal or lesser than 2 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against non-fermenters, tigecycline was especially active against acinetobacter spp. demonstrating the lowest mic90 of 2 mcg/ml. tigecycline successfully inhibited s. aureus with mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. the same results were noticed against enterococci. tigecycline's mic90 was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: resistance to glycopeptides in enterococci was first recognized in the late 1980s, and since then has been a major challenge to clinicians and infection control. tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to vancomycin, linezolid, ampicillin, imipenem, ceftriaxone, levofloxacin, minocycline, penicillin and piperacillin/tazobactam against members of enterococcus spp. collected from hospitals in north america, europe and asia. methods: a total of 685 clinical isolates of enterococcus spp. were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: of 437 e. faecalis evaluated, resistance to vancomycin in 15 (3.4%) isolates was observed. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.06/0.12 mcg/ml) among all antimicrobial agents evaluated. among 248 e. faecium, 50 (20.2%) were resistant to vancomycin, of which three isolates were also resistant to linezolid. tigecycline also presented the lowest mic50/mic90 of 0.03/0.06 mcg/ml. five isolates of vancomycin susceptible e. faecalis and one vancomycin susceptible e. faecium were nonsusceptible to linezolid. no abnormal resistance phenotype was observed in other enterococcus species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multidrug resistant strains regardless of degree or type of resistance. tigecycline evaluation surveillance trial (test) -global in vitro antibacterial activity against selected species of glucose non-fermenting organisms objective: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many gram-positive pathogens including methicillinresistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillintazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from 23 us centres in the test program. methods: a total of 794 clinical isolates were identified to the species level at each of participating sites and confirmed by the central laboratory. isolates were collected throughout 2004. mics were determined by each participating laboratory using broth microdilution panels from dade microscan. all testing was performed and interpreted according to nccls guidelines and manufacturer's instructions. results: among the 794 isolates, 389 (49.0%) were found to be resistant to methicillin (mrsa). besides the cross resistance of mrsa isolates to imipenem, ceftriaxone, penicillin, ampicillin, and piperacillin/tazobactam, it was observed a high rate of nonsusceptibility to levofloxacin (74.9%). no resistance was observed against vancomycin and linezolid. the mics of tigecycline ranged from 0.03 to 1 mcg/ml for all isolates of s. aureus, and tigecycline presented the lowest mic50/mic90 of 0.12/0.25 mcg/ml against mrsa isolates, being several folds lower than all the comparator agents. the mssa isolates showed the expected profile of high resistance to ampicillin and penicillin. the only unusual pattern was the 20.4% nonsusceptibility to levofloxacin. tigecycline's mic50/mic90 of 0.12/0.12 was also the lowest among all mssa isolates. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. tigecycline evaluation surveillance trial (test) -united states in vitro antibacterial activity against selected species of enterococcus spp. results: of 276 e. faecalis evaluated, it was observed resistance to vancomycin in 8 (4.5%) isolates. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.06/0.12 mcg/ml) among all antimicrobial agents evaluated. as a typical profile of e. faecalis, fluoroquinolone (levofloxacin) and tetracycline (minocycline) had limited activities against this species. among 144 e. faecium, 35 (24.3%) were resistant to vancomycin, of which three isolates were also resistant to linezolid. tigecycline also presented the lowest mic50/mic90 of 0.06/0.12 mcg/ml. five isolates of vancomycin susceptible e. faecalis and one vancomycin susceptible e. faecium were non-susceptible to linezolid. no abnormal resistance phenotype was observed in other enterococcus species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multi-drug resistant strains regardless of degree or type of resistance. background: glucose non-fermenting gram negative rods are known to be highly resistant in hospital settings and have always been a challenge for clinicians and hospital infection control. the degree or type of resistance may be due to several sophisticated mechanisms such as production of broad spectrum beta-lactamases, efflux pumps and altered membrane permeability, inactivating most classes of antimicrobials that are available for treatment (cephalosporins, carbapenems, aminoglycosides, fluoruquinolones). tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae and selected species of non-fermenters, as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of acinetobacter spp. and pseudomonas aeruginosa collected from hospitals in the united states. methods: a total of 1687 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity against p. aeruginosa showed a mic90 of (16 mcg/ml. towards a. baumannii (n = 849), which cephalosporins were ineffective, tigecycline showed the lowest mic50/mic90 of 0.5/2 mcg/ml outperforming amikacin mic50/mic90 4/32, imipenem mic50/mic90 0.5/16 and minocycline mic50/mic90 1/8. similar findings were found in other species of acinetobacter genus. conclusion: the presented data suggest that tigecycline may be an effective and reliable therapeutic option against strains of acinetobacter spp., including multi-drug resistant strains regardless of degree or type of resistance. objective: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many gram-positive pathogens including methicillinresistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillintazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from 5 european centres in the test program. methods: a total of 216 clinical isolates were identified to the species level at each of participating sites and confirmed by the central laboratory. isolates were collected throughout 2004. mics were determined by each participating laboratory using broth microdilution panels from dade microscan. all testing was performed and interpreted according to nccls guidelines and manufacturer's instructions. results: among the 216 isolates, 76 (35.2%) were found to be resistant to methicillin (mrsa). besides the cross resistance of mrsa isolates to imipenem, ceftriaxone, penicillin, ampicillin, and piperacillin/tazobactam, it was observed that all of mrsa isolates were also non-susceptible to levofloxacin. no resistance was observed against vancomycin and linezolid. the mics of tigecycline ranged from 0.06 to 0.5 mcg/ml for all isolates of s. aureus, and tigecycline presented the lowest mic50/mic90 of 0.25/0.25 mcg/ml against mrsa isolates, being several folds lower than all the comparator agents. the mssa isolates showed the expected profile of high resistance to ampicillin and penicillin. opposite to mrsa isolates, mssa presented very little resistance to levofloxacin (2.5%). tigecycline's mic50/mic90 of 0.12/0.25 was also the lowest among all mssa isolates. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. tigecycline evaluation surveillance trial (test) -european in vitro antibacterial activity against selected species of enterococcus spp. been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to vancomycin, linezolid, ampicillin, imipenem, ceftriaxone, levofloxacin, minocycline, penicillin and piperacillin/tazobactam against members of enterococcus spp. collected from hospitals in germany, italy, spain and united kingdom. methods: a total of 146 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: of 82 e. faecalis evaluated, resistance to vancomycin in 3 (3.6%) isolates was observed. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.12/0.25 mcg/ml) among all antimicrobial agents evaluated. among 40 e. faecium, 1 (5.0%) was resistant to vancomycin. tigecycline also presented the lowest mic50/ mic90 of 0.03/0.06 mcg/ml. no abnormal resistance phenotype was observed in other enterococci species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multi-drug resistant strains regardless of degree or type of resistance. tigecycline evaluation surveillance trial (test) -european in vitro antibacterial activity against selected species of glucose non-fermenting organisms background: glucose non-fermenting gram negative rods are known to be highly resistant in hospital settings and have always been a challenge for clinicians and hospital infection control. the degree or type of resistance may be due to several sophisticated mechanisms such as production of broad spectrum beta-lactamases, efflux pumps and altered membrane permeability, inactivating most classes of antimicrobials that are available for treatment (cephalosporins, carbapenems, aminoglycosides, fluoruquinolones). tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae and selected species of non-fermenters, as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of acinetobacter spp. and pseudomonas aeruginosa collected from hospitals in germany, italy, spain, and united kingdom. methods: a total of 826 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity against p. aeruginosa showed a mic90 of (16 mcg/ml. the cephalosporins were ineffective towards a. baumannii (n = 552). tigecycline showed the lowest mics against a. baumannii mic50/mic90 of 0.25/1 mcg/ml, outperforming amikacin mic50/mic90 4/>64, imipenem mic50/mic90 0.5/16 and minocycline mic50/mic90 1/8. similar findings were found in other species of acinetobacter genus. conclusion: the presented data suggest that tigecycline may be an effective and reliable therapeutic option against strains of acinetobacter spp., including multi-drug resistant strains regardless of degree or type of resistance. p818 antimicrobial activity of tigecycline tested against bacterial pathogens from intensive care units h. sader, t. fritsche, r. jones (north liberty, usa) objectives: to evaluate the antimicrobial activity of tigecycline (tig) and selected antimicrobials against bacterial pathogens isolated from patients hospitalized in intensive care units (icus) worldwide. methods: a total of 7129 were consecutively collected in >70 medical centres located in north america (3164), south america (1465), europe (2428) and the asia-australia region (72). the isolates were collected from (no. of isolates/%): bloodstream (5349/75%), respiratory tract (746/10%), skin/soft tissue (323/ 5%), and urinary tract (182/3%) infections in the 2000-2004 period, and susceptibility tested by nccls broth microdilution methods. results: the antimicrobial activity of tig and the frequency of occurrence of bacterial pathogens are summarized in the table: all gram-positive pathogens (4817) were inhibited at <1 mg/l of tig. resistance (r) to oxacillin was detected in 43% of sa and 84% of cons, and r to vancomycin was detected in 19% of enterococci. tig was very active against enterobacteriaceae (ent; 1468) with a mic90 <1 mg/l, except for serratia spp. 10% of e. coli and 30% of klebsiella spp. showed an esbl phenotype while 28% of enterobacter spp. were r to ceftazidime. 14% of ent showed r to ciprofloxacin. tig and trimethoprim/sulfamethoxazole were the most active compounds against s. maltophilia (mic90, 2 and 1 mg/l respectively). tig was also highly active against asp (mic90, 1 mg/l), but psa showed decreased s to tig (mic90, 16 mg/l). non-s to imipenem (mic, >8 mg/l) was observed in 16% of asp and 31% of psa isolates. conclusions: isolates from icu patients showed high rates of antimicrobial r. the most alarming problems detected were vancomycin r among enterococci, esbl mediated beta-lactam r and fluoroquinolone r among ent, and carbapenem r among psa and asp. tig exhibited potent in vitro activity against the vast majority of clinically important pathogenic bacteria (except psa) isolated from icu patients and may represent an excellent option for the treatment of infections in this clinical environment. potency and spectrum of tigecycline tested against an international collection (1999) (2000) (2001) (2002) (2003) of bacterial pathogens producing skin and soft tissue infections t. fritsche, h. sader, m. stilwell, r. jones (north liberty, usa) objective: tigecycline (tig) is the sentinel representative of the glycylcycline class to be developed as a parenteral agent targeting bacterial pathogens responsible for pneumonia, intraabdominal sepsis and ssti. the aim of this study was to evaluate the activity and potency of tig when tested against a large collection of bacterial pathogens causing ssti. methods: consecutive, non-duplicate bacterial isolates (3,421 strains) were collected from 1999 to 2003 from patients with documented community-acquired or nosocomial ssti in >70 medical centres participating in the tig surveillance program in north america (38.6%), europe (54.0%), latin america (3.7%) and the asia-pacific region (3.7%). all isolates were tested using nccls broth microdilution methods against tig and representative comparator agents used for empiric and directed therapy of ssti. results: ssti pathogen rank order (top ten), potency and cumulative inhibition rates for tig are in the table: all sa, streptococci, enterococci, cons, ksp and ec were inhibited by £2 mg/l of tig, along with 98% of asp and 96% of ent. the broad-spectrum of activity exhibited by tig included tetracycline resistant subsets as well as mrsa, vre, and esbl-producing strains. only pm and psa isolates were less susceptible (mic90 values at 8 and 16 mg/l, respectively). conclusions: among the top ten-ranked pathogens producing ssti, 94% of isolates were inhibited by £2 mg/l of tig and 98% were inhibited by £4 mg/l (the current nccls breakpoint for tetracyclines). tig may represent a welcome choice among newer parenteral agents for the common gram-positive and negative pathogens producing serious ssti given in vitro testing results, thus warranting continued investigation for this indication. objective: to assess the activity of tigecycline (formerly gar936), a novel glycylcycline, against recent bloodstream infection (bsi) pathogen isolates from six continents. frequency of clinical occurrence of these pathogens was determined and their antibiograms assessed using nccls reference broth microdilution methods. methods: a total of 22,950 strains were tested by the m7-a6 (2003) method with interpretations from m100-s14 (2004) . a tigecycline susceptible (s) breakpoint was defined as £2 mg/l for comparison purposes only, although £4 mg/l has been used for tetracyclines. the rank order of pathogens was: s. aureus (sa; 34.2%), coagulase-negative staphylococci (cons; 14.1%), e. coli (ec; 13.2%), enterococci (ent; 12.7%), klebsiella spp. (ksp; 5.3%), p. aeruginosa (psa; 4.0%), enterobacter spp. (ebs; 2.9), beta-haemolyticstreptococci (bst; 2.8%); s. pneumoniae (spn; 2.4%), and viridans group streptococci (vgs; 1.6%). more than 20 comparison agents were tested including tetracycline (tc) and ciprofloxacin (cip) . results: bsi pathogens (gram-positive and enterobacteriaceae) tested against tigecycline are shown in the table. tigecycline was consistently active against tc-resistant (r) strains (89-100% s versus 45-90%). mic50:% s results for other bsi species were: p. mirabilis (4 mg/l:10), acinetobacter spp. (0.5 mg/l:77), serratia spp. (1 mg/l:81), s. maltophilia (1 mg/l:77) and indole-positive proteae (1 mg/l:54). tigecycline exhibited a broader spectrum of activity against bsi isolates when compared to cip, tc, older aminoglycosides and imipenem. tigecycline was not active against psa (mic50, 8 mg/l; 4.0% of bsi isolates). conclusions: tigecycline exhibited a wide spectrum of antimicrobial potency versus bsi isolates collected worldwide. serious infections in nosocomial environments should benefit from tigecycline among the investigational phase 3 agents focused on r strains. evaluation of the in vitro activity of tigecycline against gram-negative anaerobic bacteria objectives: the evaluation of the in vitro activity of tigecycline in comparison to tetracycline, penicillin, piperacillin ( tazobactam, cefoxitin, clindamycin, metronidazole and imipenem against recently isolated gram-negative anaerobic bacteria. materials and methods: a total of 185 gram-negative anaerobic clinical strains (116 bacteroides fragilis group, 20 other bacteroides spp. non-fragilis, 34 prevotella spp. and 15 miscellaneous) isolated during the period 11/2002-11/2004 were tested using the e-test and the agar dilution methods on brucella agar plates supplemented with 5% horse blood, vitamin k1 and hemin. incubation in a chellab anaerobic chamber was performed for 48 hours. interpretation of the results was according to nccls guidelines. for quality control the strains b. fragilis atcc25285 and b. thetaiotaomicron atcc29741 were used. results: overall mic90 to tigecycline, tetracycline, penicillin, piperacillin + tazobactam, cefoxitin, clindamycin, metronidazole and imipenem were 8, 128, 256, 2, 64, 256, 1 and 0.5 mg/l, respectively, whereas mic50 were 0.5, 16, 32, 0.125, 8, 1, 0.5 and 0. 064 mg/l, respectively. bacteroides fragilis group mic90 were 8, 256, 256, 4, 128, 256 , 1 and 0.5 mg/l, respectively, whereas mic50 were 0.5, 16, 64, 0.25, 16, 1, 0.5 and 0. 064 mg/l, respectively. at the tigecycline mic90 concentration of 8 mg/ l the compound inhibited a higher percentage of isolates than clindamycin, cefoxitin or tetracycline (which inhibited 76, 56 and 45% of the isolates, respectively). only one strain was resistant to imipenem (mic > 32 mg/l), having a tigecycline mic of 8 mg/l. in addition, six of the eight strains with a metronidazole mic > 16 mg/l had a tigecycline mic < 1 mg/l. conclusions: in general, tigecycline showed better in vitro activity than clindamycin, cefoxitin, tetracycline or penicillin against gram-negative anaerobic bacteria and somewhat lower clinical microbiology and infection, volume 11, supplement 2, 2005 activity than imipenem, metronidazole or piperacillin + tazobactam. nevertheless, among most metronidazole and imipenem resistant isolates tigecycline displayed good activity. the results of this in-vitro evaluation show that tigecycline should be considered as a possible alternative for the treatment of mixed aerobicanaerobic infections. objectives: to ascertain the variation between mics of tigecycline determined in broth and agar, and by nccls and bsac methods. to assess the effect of adding tigecycline to the medium one day before use and of using media that had been aged for one week before addition of tigecycline and inoculation. method: mics for 96 non-fastidious bacteria and 20 streptococci were determined in mueller-hinton broth (mhb), mueller-hinton agar (mha), iso-sensitest broth (isb) or iso-sensitest agar (isa). fresh antibiotic stock solutions (tigecycline, tetracycline and minocycline) were incorporated into bulk media by rolling, to minimise aeration. preparation conditions otherwise were as fol-lows: (a) freshly-prepared media, antibiotic added upon cooling, then inoculated; (b) freshly-prepared media, antibiotic added upon cooling thenstored for 1 day before inoculationand (c) media stored for 7 days before addition of the antibiotic and inoculation. results: mics in fresh mhb (the nccls reference method) were taken as a standard. for all the tetracyclines, these mics were 0-2 doubling dilutions higher than on mha or isa (bsac reference method), or in isb. media with tetracyclines added a day before use gave raised mics, though rarely by more than one dilution. tigecycline mics were increased in 7-day-old mhb or isb. this effect was greatest (2-3 dilutions) for the most susceptible strains (mic £ 0.5 mg/l) and was absent for the resistant organisms (mic ‡ 8 mg/l); it did not occur in agar. minocycline mics were not raised in aged media, and tetracycline mics were only marginally affected. the addition of blood to the mhb largely abrogated the effect on tigecycline mic, as did boiling the broth prior to adding the antibiotic. conclusion: the raised mics of tigecycline in aged broth probably reflect inactivation by dissolved oxygen. this accords with lack of any mic increase in newly boiled (i.e. degassed) mhb or on aged agar (which is boiled to melt before use). the effect of blood may be to add to the reducing capacity, protecting the tigecycline. at a practical level, broth mic methods for tigecycline (e.g. the nccls reference method) require, freshly prepared or steamed medium; this is not a concern if agar dilution methods are used, such as that of the bsac. sexually transmitted diseases p823 prevalence of chlamydia trachomatis and neisseria gonorrhoeae in native and immigrant population (ambits study) in barcelona (spain) testing of all danish gc strains isolated in 2002-2004 (n = 953) . clinical and epidemiological data were obtained when possible. results: the 25 pip-negative gc isolates were all designated as serovar ib-4 and similar mic values were identified in the antibiograms of these isolates fingerprints were identified using spei and three using bglii among the 25 ib-4 isolates. four distinguishable pfge . however, all these isolates differed by less than six bands in both fingerprints. the phylogenetic tree analysis of the porb1b sequences, suggested that these minor differences represented the ongoing evolution of the same strain. during the period jan. 2002 to nov. 2004 the proportions of pip-neg gc per year in denmark were 15/332, 47/261 and 27/360, respectively, and the pip-neg gc infected mostly men (only 5 women). the antibiograms of all danish isolates 2002-2004 indicate a spread of at least one pip-neg gc strain. the results of the present study indicate a circulation of at least one n. gonorrhoeae prolyliminopeptidasenegative strain in the danish homosexual community. an increased awareness for pip-negative n. gonorrhoeae, which create large problems in diagnosis using e.g. api nh, rapid nh, gonocheck ii or neisseria pet, is essential. a new recombinant antigen haemagglutination test for the serological diagnosis of syphilis n. chepurchenko, t. ulanova, v. puzyrev, l. loginova, a. burkov, a. obriadina (nizhniy novgorod, rus) introduction: passive treponema pallidum hemagglutination (ha) -the routine treponemal test has equal sensitivity with the fluorescent treponemal antibody absorption (fta-abs) test and with the enzyme immunoassay (eia) in later stages of syphilis, but some studies have indicated that it is less sensitive in the primary stage of the disease. the use of recombinant t. pallidum antigens instead of a poorly defined mixture of antigens from wild-type t. pallidum has the potential for improving the sensitivity of hemagglutination test. objectives: the purpose of this study was to evaluate the diagnostic relevance of 4 recombinant proteins that efficiently model the antigenic epitope(s) of the treponema pallidum proteins. methods: four full-length recombinant proteins (tmpa, 47, 17, 15 kda) were expressed in e. coli and then used individually to develop the hemagglutination test (ht) for the detection of anti-treponema pallidum (anti-tp) activity in serum specimens. serum samples (n = 267) from patients with clinically proven syphilis in various stages of the disease (primary (n = 36), secondary (n = 70), latent (n = 161)) and from normal blood donors (n = 505) were tested. 24 samples from patients with primary syphilis, 21 samples from patients with secondary and 17 samples from patients with latent stage were tested initially with commercially available hemagglutination test based on mixture of treponema pallidum antigens (http). all specimens were additionally tested for specific antibodies by commercially available enzyme immunoassay (eia). results: the sensitivity of the ht for the detection of anti-tp activity in human serum specimens varied from 38.9% to 97.2% with primary syphilis sera, from 87.1% to 100% with secondary, and from 96.9% to 98.1% with latent stage sera for each protein. the tmpa was found as the most immunoreactive when serum samples from patients with untreated syphilis were tested. the sensitivity of tmpa ht was identical to those of the http in cases of untreated secondary and latent syphilis and significantly higher with specimens from primary stage of disease. the overall specificity and sensitivity of the tmpa ht were comparable to those of eia 99% and 99.6%; 98% and 99.6% respectively. the results of this study indicate that the tmpa ht may be a reasonable alternative to the routine hemagglutination test and the elisa as a confirmatory test for syphilis. use of treponema + vdrl virablot test as a useful ôall in oneõ confirmatory assay for treponema pallidum antibodies a. kostoula, g. vrioni, c. bobogianni, c. stergiopoulou, e. papantoniou, s. levidiotou (ioannina, gr) objectives: as detection of treponema pallidum subsp. pallidum by dark-field microscopy or direct immunofluorescence is possible only in the early stages of the disease, serology has become the method of choice for syphilis diagnosis. except for conventional treponemal tests, the t. pallidum western immunoblot assay has also been used as an alternative confirmatory assay for t. pallidum antibodies. the aim of the present study was the use of a multiparameter immunoassay, which simultaneously detects treponemal specific and lipoid antibodies, for the confirmation of syphilis antibodies. methods: a total of 86 serum specimens were tested including, 56 reactive sera from patients with documented treponemal infection; a specificity panel of 10 sera from normal subjects, 10 sera with rapid plasma reagin (rpr) reactivity (fta-abs and tpha negative), 5 leptospira igm serology-positive sera and 5 borrelia burgdorferi igg or igm serology-positive sera. all serum samples were tested with conventional syphilis tests according to the manufacturersõ instructions: rpr, venereal disease research laboratory (vdrl), t. pallidum haemagglutination (tpha) at a serum dilution of 1:80, fluorescent t. pallidum absorption (fta-abs) and igm capture enzyme immunoassay (eia). subsequently, the results were compared to those of the treponema + vdrl virablot igg, igm test (viramed, labor -diagnostika), a immunoassay for the qualitative detection of specific igg or igm antibodies to t. pallidum using the specific treponemal proteins p47, p44.5, p17 and p15, and the quantitative determination of vdrl reaction on one nitrocellulose strip. results: treponema + vdrl virablot igg and igm tests confirmed the results for 56 serum specimens positive for t. pallidum antibodies: reactive vdrl band and at least two clear bands from p47, p44.5, p17 or p15 for igg treponemal antibodies or at least one clear band p47, p17 or p15 for igm treponemal antibodies. the 10 rpr reactive sera were vdrl virablot reactive, but treponema virablot specific antibodies negative. none of the remaining sera reacted in tests with the treponema + vdrl virablot assay. conclusions: the treponema + vdrl virablot assay combining the necessary serologic markers in one test strip (i.e. specific and non-specific treponemal antigens) is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques. the great imitator: syphilis with predominant acute severe hepatic involvement the ground of a significant increase of serum transaminiases (up to 2200 and 900 u/l of gpt, respectively), frank jaundice, elevated bilirubin (up to 11.7 and 9.6 mg/dl, respectively), and evident rise of serum alkaline phosphatase and gamma-gt. clinical history did not show risk factors for viral hepatitis, and all laboratory examinations for major and minor hepatotropic viruses proved negative, as well as autoimmmune, dysmetabolic, or drug-induced liver damage. repeated ultrasonography detected increased liver and spleen dimensions, in absence of other abnormalities. in the second p, histopathologic study performed after liver biopsy, showed an aspecific cholestatic hepatopathy, a moderate granulocyte and lympho-mononocyte intralobular infiltrate, appreciable necrotic foci, and a mild portal fibrosis (knodell score 5). only syphilis serology, performed despite the absence of significant history or clinical clues, allowed to recognize an igm positive serology associated with tpha titres ranging from 1:1250 and 1:680, respectively. specific chemotherapy (i.v. penicillin g at 24 mu daily for 7 days, followed by 12 mu/day in the second p),led to a complete resolution of hepatic involvement, associated with an improvement of serology after 1 month. discussion: while in the majority of episodes of syphilis liver involvement remains missed or subclinical, an early diagnosis of syphilis with predominant hepatic disease remains an unresolved problem of differential diagnosis, due to the rare occurrence of isolated organ involvement compared with typical syphilis signs and symptoms, and the aspecific clinical, histopathological, and imaging picture (jozsa l,acta hepatogastroenterol 1977; 24:344-7; young mf, j clin gastroenterol 1992; 15:174-6) . during the recent recrudescence of syphilis in italy, a diagnostic suspicion should be maintained by clinicians who face an apparent acute icteric hepatitis, and syphilis serology should be included among initial laboratory workout. the great imitator: syphilis with predominant meningoencephalitic features r. manfredi, s. sabbatani, d. pocaterra, f. chiodo (bologna, i) introduction: a significant recrudescence of syphilis was observed in recent years, not contained by prophylactic measures against hiv and other stds. meningeal and meningoencephalitic involvement may occur also in early stages of syphilis, thus posing problems of differential diagnosis. case reports: a young woman and an hiv-infected male developed a syphilis with predominant meningoencephalitis expression. in the first p, based on an isolated positive borrelia burgdorferi serology (later deemed as a cross-reaction), early ceftriaxone was started, due to a suspected neurological lyme disease. also after the diagnosis of neurosyphilis (on the ground of positive csf serology), antimicrobial therapy was left unchanged for 3 weeks at our day-hospital facilities, until complete clinical-microbiological cure. a second, hiv-infected p was hospitalized owing to mild fever lasting 2 weeks, associated with cephalalgia and anxiety.the favorable virological-immunological status (cd4 + count 439 cells/ll and undetectable hiv viraemia, under an effective haart regimen), did not exclude all searchs for possible hiv-related disorders.although serum examination detected a potential latent syphilis (isolated positive serum treponema pallidum igg, tpha 1:640,igg-positive rpr, and mute history for syphilis), only csf examination disclosed an increased cell content (50/ll), altered brain-blood barrier indexes with increased intrathecal ig synthesis, and frank vdrl,tpha (1:1520), and borderline t. pallidum igm serology.penicillin g at 24 mu/day for 14 days led to a slow resolution of neurological signs and symptoms, and a tendency to improvement of specific csf-serum syphilis serology during subsequent controls. discussion: focusing on differential diagnosis, a luetic etiology should not be underestimated, when facing young p suffering from a meningoencephalitis of unclear origin.our cases were characterized by a young age (34 and 44 years, respectively), when compared with usual mean age of tertiary neurosyphilis. in absence of suggestive history and other syphilis signs, the diagnosis was achieved only after the retrieval of elevated syphilis serology positivity on both csf and serum, together with some clinical signs, such as seizures, altered mentation, cognitive abnormalities, and anisochoria in the first p, and persisting headache and anxiety in the second p. our experience with ceftriaxone (started in the first p when neuroborreliosis was suspected), was favorable like that with high-dose i.v. penicillin g. comparison of the genomes of pathogenic treponemes, treponema pallidum ssp. pallidum, ssp. pertenue and treponema paraluiscuniculi objectives: the cervical human papillomavirus (hpv) infection is common among young sexually active women. because the cytology tests have some limitations, including up to 30% false-negative results, molecular techniques are increasingly used for identification of hpv-induced cell changes in the cervix. the polymerase chain reaction (pcr) for hpv diagnostics was successfully introduced recently in bulgaria. however limited data on specific prevalence and determinants of subclinical hpv infections are available. in this study we designed a multiplex primers pcr system for simultaneous identification of the most common low-and high-risk hpvs in women with cytologically normal cervical smears. materials & methods: dna from 102 cervical cell samples from women aged between 18 and 40 years with normal pap smear tests was extracted by standard procedure and studied for hpv presence. six type-specific primer sets for e6 hpv gene were used in a single-tube reaction for detection and identification of . respective controls and statistic analysis were included. results: the overall hpv dna prevalence was 23.5% (24/102). ten of the hpv positive women (41.6%) were infected with high-risk (hpv-16 or hpv-18) virus types; eight (33.3%) with low-risk types (hpv-6 or hpv-11), and six (25%) were infected with both high-and low-risk types (3 hpv-6/-16; 2 hpv-6/-18 or 1 hpv-6/-33). hpv prevalence among women younger than 23 years was 58.3% (14/24). for low-risk types the peak prevalence was observed in women between 35-40 years. besides age, there was a positive association between the detection rates of hpv infection and number of sexual partners and oral contraceptive use. conclusion: the used technique is simple, economic, rapid and reliable for screening studies of hpv infections. the prevalence of hpv in cytologically normal bulgarian women is similar to the reported in other countries. our findings suggest that molecular techniques for hpv detection might be useful as an adjunct to pap smear screening. were examined. mycoplasmas were determined by use of a commercially available system (liofilchem s.r.l, italy) and c. trachomatis antigen by dif (cellabs pty ltd, australia). results: of 1284 specimens, 1121 were positive. the isolated species in each age group are summarized in table 1 . in 818 cases of lower genital tract (lgt) infection, only one species was isolated. in all three age groups, mycoplasmas were the most commonly identified. in particular, the species of the isolated pathogens were: (1) group a (n = 43): mycoplasmas 52%, streptococcus spp 24%, candida spp 8%, c. trachomatis 8% and anaerobes 8%, (2) in 39 women of this group a mixed infection with three species was found. from 818 women with positive cultures, 562 (68.70%) were symptomatic, while in 256 asymptomatic women (31.20%), sexually transmitted pathogens were identified. noticeably 40.58% and 37.5% of the infected women with candida spp and c. trachomatis respectively, presented no symptoms. conclusions: (1) the rate of mixed lgt infections was 27.02% (2) asymptomatic infections were 31.20% (3) all teenagers in group a were symptomatic. ureaplasma mba size variants in woman with spontaneous preterm delivery j. spergser, a. witt, l. petricevic, p. husslein, a.m. hirschl, r. rosengarten (vienna, a) objectives: despite colonization of the lower urogenital tract, ureaplasmas reach the upper genital tract only in a subpopulation of women, and only in some of these individuals symptoms like preterm delivery ensue. while some studies indicated that certain serovars are more frequently implicated with particular diseases, others have suggested that size variability of the multiple banded antigen (mba) may contribute to ureaplasmal pathogenesis. to address these hypotheses, vaginal and placental samples from women with spontaneous preterm delivery were tested for ureaplasma species, serovars and mba size variants. methods: placenta/amnion specimens and corresponding vaginal samples from women that delivered via caesarean section less than 34 weeks of pregnancy were collected. samples were examined for ureaplasma species and serovars by cultivation and pcr assays. to determine mba size variants, pcr with primers amplifying the non-repetitive and the c-terminal repeat region of the mba gene were performed. results: out of 100 cases examined so far, 32 placenta/amnion specimens and 40 corresponding vaginal samples were positive for ureaplasmas by cultivation and pcr. while ureaplasma positive samples from the lower genital tract were carrying u. parvum (100%) and u. urealyticum (50%), colonization of the placenta/amnion was restricted to u. parvum which was only recovered from women with preterm labor or preterm prom. in contrast, ureaplasmas were not detected in samples from women with hellp syndrome, iugr and preeclampsia. as opposed to some previous studies, particular serovars were not implicated in adverse pregnancy outcome. however, significant differences between number and lengths of mba pcr products among placental and vaginal ureaplasma isolates were obvious. ureaplasma isolates from placenta/amnion membranes gave shorter pcr products than did corresponding vaginal isolates and while a majority of vaginal ureaplasma isolates were composed of multiple size variants, placental isolates were predominantly restricted to a singular mba size variant. conclusion: only u. parvum was found to be frequently present in the placenta/amnion of women with preterm delivery and certain serovars were not more frequently implicated with particular diseases. in addition, mba size variation represents an additional level of phenotypic and genetic variability beyond the serovar category which may provide an important contribution to ureaplasmal pathogenesis. use of kanamicin in the topical therapy of aerobic vaginitis g. tempera, g. bonfiglio, e. cammarata, s. corsello, a. cianci (catania, paternò, i) objectives: demonstrate the efficacy of the topical therapy with kanamicin in a new pathology called aerobic vaginitis (av). methods: eighty-one patients with clinical diagnosis of av in accordance to donders's criteria, have been included in the study. the presence at least of four of the following symptoms were taken as diagnosis of av: objective abnormal yellow vaginal discharge, foul smell (but negative to koh test), elevated vaginal ph (>5), abundant vaginal leukocytes and lactobacillary grade iia, iib and iii in accordance to donder's classification. other characteristics such as vaginal dyspareunia, vulva-vaginal itching, cervical erosion as well as the isolament of vaginal microorganisms were also documented. the patients were randomised treated, 45 with kanamycin (100 mg vaginal ovules per 6 days, consecutively) whereas, 36 with meclocycline (35 mg vaginal ovules per 6 days, consecutively). the patients were visited before starting the study, 1-2 days and 30 days after the end of the study. results: at the first follow-up the patients showed different level of symptoms reduction. particularly, reduction of presence of leukocytes, burning of vaginal mucosa and itching were statistically significant in the group treated with kanamycin with respect to the group treated with meclocycline. moreover, there was also a reduction of isolament of enterobacteriaeae (97%) in the group treated with kanamycin versus 76% treated with meclocycline. at the second follow-up, vaginal homeostasis (normalisation of ph and presence of lactobacilli) was demonstrated only in kanamycin treated group. conclusion: our data suggested that the topic use of kanamycin could be considered a specific antibiotic for the therapy of this new pathology. objectives: establishing the identity of lactobacillus species colonizing the vagina of women is of importance, because clinical studies have demonstrated an association between the presence of h 2 o 2 -producing strains of lactobacillus and a decreased prevalence of bacterial vaginosis (bv). recently, culture based studies using molecular identification methods showed that l. crispatus and l. jensenii are the most common species of vaginal lactobacilli and that colonization by these species was positively associated with a lower frequency of bv. here were report that l. crispatus can be distinguished from other lactobacilli using gram staining of vaginal smears. methods: several approaches were used to characterize 515 vaginal microflora samples obtained from 197 pregnant women at three time points in pregnancy: 1/gram stained smears from vaginal swabs, scored according to modified ison and hay criteria; 2/identification of cultured isolates obtained after anaerobic culture, identified using tdna pcr; and 3/species specific pcr for atopobium vaginae and gardnerella vaginalis. grade i specimens, representing a normal microflora, were further characterized as grade ia when only l. crispatus cell types were present, grade ib when other lactobacillus cell types were present, grade iab when both l. crispatus and other lactobacilli were present and grade ic when either gram-positive rods, small and short, or irregularly shaped gram-positive rods, with clubbing, curved edges and irregular staining (ôdiphteroid cell typesõ) were seen. results: out of the 515 samples, 86.4% showed a normal vaginal microflora (grade i), 8.9% were grade ii, 3.7% grade iii and 1.0% grade iv. based on the presence of different lactobacillus species, grade i specimens were further characterized as grade ia (36.4%), grade ib (40.9%), grade iab (13.5%) and grade ic (8.5%). this classification was supported by the finding that out of respectively 86.0% and 76.6% of grade ia and iab specimens l. crispatus was cultured while this species was present in only 13.1% and 2.6% of respectively ib and ic specimens. in addition, 55.1% of grade ic specimens contained bifidobacterium spp. conclusion: further refinement of gram stain based grading of vaginal smears is possible by distinguishing additional classes of normal microflora. these categories of gram scores may facilitate and improve future studies regarding the interpretation of clinical data and therapeutic outcome. objectives: the importance of the isolation of gardnerella vaginalis in bacterial vaginosis is well known. the involvement in male urogenital infection is uncertain, probably due to low rate of isolation for inadequated specimens process. the objective of this study is to evaluate the possible pathogenic rol of g. vaginalis in male urogenital pathology. semen is a recommended specimen in diagnosing of prostatitis. at the same time it also reflects normal genital tract microflora. although its composition may be affected by the sexual activity, there are no data regarding the changes in genital tract microflora during the sexual debut. objective: to identify possible associations between sexual experience and the reproductive tract inflammatory parameters and microflora in healthy young men. methods: a total of 72 healthy men aged 17-22 years were included and divided into 3 groups based on their sexual experience (virgins n = 13, monogamous n = 11 and polygamous n = 48). a semen sample of each subject was assessed for basic semen parameters (semen volume, sperm concentration, sperm motility), white blood cell (wbc) counts and quantitative cultures for aerobic, microaerobic and anaerobic bacteria, and possible correlations between the type of sexual experience, wbc counts and seminal microflora were calculated. results: basic semen parameters were similar in all groups. 5.5% of men had high (>1 m/ml) and 19.4% of men had medium (0.2-1 m/ml) semen wbc counts. there were no correlations between the type of sexual experience and semen wbc counts. in comparison to sexually active men in virgin subjects the total microbial counts in semen (4.4 vs 5.0 log cfu p < 0.05) and the number of different species (3 vs 5, p ¼ 0.05) were lower, however, no differences between mono-and polygamous men were found. there was a positive correlation between the total microbial concentrations and number of different species (r = 0.54; p < 0.0001). staphylococci, corynebacterium seminale, other coryneforms, gamma-and alpha-haemolytic streptococci and peptostreptococci were the most prevalent organisms. no significant differences in microflora were observed between the groups yet the gram-negative anaerobic rods were more often seen in sexually experienced men than in virgins. the subjects with high wbc counts (>1 m/ml) tended to have higher number of different microorganisms than the rest of the men (median 7 vs 4, p = 0.05). the sexual debut is associated with the enrichment of seminal microflora but not with the influx of wbc or changes in basic seminal parameters. strong positive correlation can be observed between the total microbial concentration and the number of different species in semen. sexually transmitted infections among registered female sex workers in athens under investigation were 122 sexually active non-pregnant women aged from 18 to 40 years with diagnosed cervicitis by cytological examination and presence of muco-purulent discharge. fifty five women were included in studied group -with confirmed c. trachomatis cervicitis and 67 women -in control group (with non-chlamydial/non-gonococcal cervicitis). bv was diagnosed using amsel and nugent (0-3 negative; 4-6 intermediate; 7-10 positive) criteria. by using amsel criteria bv was suggested in 11% and 6% correspondingly among women in studied and control group. by using nugent criteria bv was suggested correspondingly in 5.5% and 3%. these differences were not statistically significant and can be explained by the presence of muco-purulent discharge among studied women. objectives: helicobacter pylori is recognized as an important human pathogen but the differentiation of the species by classical phenotypic methods is really difficult. therefore the objective of this study was the development of an identification method for helicobacter species based on pcr-restriction fragment length polymorphism (pcr-rflp) analysis of the 16s and 23s rrna genes. objectives: since the first identification of helicobacter pylori by warren and marshal in 1983, members of the genus helicobacter increased to more than 26 species, which have been detected in human and animals. the construction of species specific pcr assays is difficult due to the close relatedness between different helicobacter species. the pcr-dgge technique was developed for the identification of helicobacter colonization. the aim of this study was to investigate the effect of multiple regions of the 16 rrna gene on the diagnostic efficiency pcr-dgge. methods: dna was extracted from 40 helicobacter strains, which represent 20 helicobacter species. amplification of 1.2 kb of helicobacter 16s rdna, which contains v3 and v6-7 regions, was done using previously published helicobacter genus specific primers c97 and c05. the pcr product can be used as a template for two helicobacter genus pcr assays, the v6-7 regions 16s rdna was amplified using the primers 1fc254 and 2rc686, the v-3 region was amplified using the primers bsf917 and bsr1114 published at (http://rrna.uia.ac.be/ primers). dgge analysis of the v3 and v6-7 regions was performed on 9% polyacrylamide gels containing urea and formamide gradient from 20-40% or 15-30%, respectively. electrophoresis was performed in a dcode electrophoresis unit (biorad) at constant voltage (200 v) at 60°c for 4 hours. results: dgge analysis of two regions of helicobacter 16srrna gene showed mobility patterns that allowed discrimination of most helicobacter species except those which are closely related such as h. pullorum-h. pametensis, h. ganmani-h. rodentium and h. bizozeroni-h. felis-h. salmonis. conclusions: helicobacter species are widely distributed in the gastrointestinal tract of mammals, birds and other animals. the pcr-dgge technique has proven to be an easy, inexpensive and efficient tool for the identification of helicobacter species and for the detection of colonisation by more than one helicobacter species, without the need for species-specific pcr assays. in addition, the diagnostic efficiency of this technique was increased by analysis of multiple regions of the 16s rrna gene. helicobacter spp. in chronic pancreatitis, pancreatic adenoma and pancreas cancer objectives: helicobacter spp. efficiently colonise various hostile habitats such as the stomach, colon and biliary tract of animals and man. with the exception of h. pylori, many helicobacter spp. are difficult to culture. nucleic acid based detection has thus become a method of choice for analysis of helicobacter spp. in chronic inflammatory gastrointestinal (gi) tract diseases and cancers. pancreatic exocrine cancer, with few established risk factors and very poor prognosis, is a common cause of cancer death. previously, serological studies found an association between h. pylori and pancreas cancer (pc). methods: pancreatic tumour specimens of patients with pc (n = 40), tissues of chronic pancreatitis (n = 6), pancreatic adenoma (n = 8), and benign pancreatic tissue of patients with cancers of the choledochus, colon and duodenum (n = 6), were analysed by semi-nested helicobacter-specific 16s rdna pcr. stomach (n = 23) and gallbladder (n = 12) specimens of the pc-patients were also analysed. were characterized by dna-sequence analysis. results: the helicobacter spp. pcr was positive in 19 of 40 (47.5%) pancreas cancer patients, and in the pancreas in 4 of 6 (66.7%) patients with chronic pancreatitis. adenoma and pancreas tissues of other gi-tract cancers, as well as gallbladder specimens, were all negative. four of 23 (17.4%) stomach samples of the pc-patients were helicobacter positive with sequences homologues to h. bilis by blastn analysis. dna-sequencing of 13 pcr-products amplified in pcs were closely related to h. pylori (n = 9), h. flexispira sp. (n = 3) and h. cinaedi (n = 1). two pancreatitis pcr-products matched h. pylori. conclusions: helicobacter spp. were common in pc compared with benign pancreatic tissue. helicobacter pylori infection and low igg immunoglobulin s. kokkinou, k. pavlou, a. varouta, j. villiotou, k. papaefstathiou, a. moutsopoulou, e. papafrangas (halandri athens, athens, gr) it is established that helicobacter pylori (hp) may cause haematological disturbances such as iron malabsorption and idiopathic thrombocytopenic purpura (itp). the aim of this study is to show that hp is involved in human disease in a more complicated way, producing an abnormal immunologic profile. patients and methods: this work reports the presence of a significant low value of igg immunoglobulin in 52 pts suffering from unidentified iron deficiency (id)or iron deficiency anemia (ida coexisting with megaloblastic anemia(ma), in 12 leukopenic pts and in 3 more pts having thrombocytosis, itp and polycythemia respectively. the ratio male/female was 25/42, and their age ranged from14-80 years. in the blood examination there was a10-fold titre of hp-igg type antibody in all, while in 23 there was also a high titre of hp-iga type using elisa technique. the histopathological examination of an antral biopsy showed diffuse corpus gastritis, atrophic gastritis and achlorydria secondary to hp infection that take place in gastric area. results: 52 pts had unidentified id or ida coexisted with megaloblastic anemia. serum iron, transferrin saturation, serum ferritin and b12levels were found to be significantly low. all of them had also hp infection. their anemia existed for more than 10 years, was unresponsive to treatment and recurrent in nature. twelve pts with undefined eukopenia (l:3.3 · 10 9 /l) lasting for at least 8 years, had also an hp infection. all of our pts had a significantly low value of igg immunoglobulin. mean value ranged from 551 to 868 mg/dl(normal value:850-1517 mg/dl). they subjected to eradication therapy. all but one responded well. conclusions: (1) hp infection was found to coincide with id, ida, ma and leukopenia. (2) the normalization of haematological parameters after eradication started 6 months later. (3) the essential mechanism between the hp infection and low igg is unclear and its value remains low and after the eradication therapy. (4) studies with larger samples should be done for a better evaluation of the hp infection and it's involvement in human disease. (5) hp infection acts in a catabolic way for the humans. it must be considered to search for it in haematological conditions of undefined origin. helicobacter pylori from the dyspeptic patients in thailand: diagnosis, antimicrobial susceptibility and correlation to clinical outcomes c. chomvarin, p. kulsuntiwong, p. mairiang, c. kulabkhow (khon kaen, th) objectives: to evaluate methods for routine clinical diagnostic use of helicobacter pylori infection of gastric biopsies, to study the correlation of h. pylori infection to the clinical outcomes and to study antimicrobial susceptibility by disk agar diffusion in thailand. methods: gastric biopsies, obtained from 210 patients underwent at the endoscopy unit of srinagarind hospital, faculty of medicine, khon kaen university, were diagnosed by culture, rapid urease test (rut, pronto dry) and histological examination. a true positive criteria was indicated when culture or both rut and histology tests were positive. one hundred and fifteen isolates of h. pylori isolates were tested for six antimicrobial agents by disk agar diffusion method. results: the h. pylori infection rate was 44.3% (93/210). the sensitivity vs. specificity of the culture, rut and histology were 88.2% and 100%, 95.7% and 98.3%, and 96.8% and 59.8% respectively. the prevalence of h. pylori in gastritis (gt), duodenal ulcer (du) and gastric ulcer (gu); and gastric cancer (gca) patients were 41.2%, 57.9% and 70.6%, respectively. the chi-squared test showed that gca patients were significantly more often infected with h. pylori than gt patients. the resistance of 115 h. pylori isolates to single antimicrobial agent as metronidazole (mtz), clarithromycin (clr),ciprofloxacin (cip), amoxycillin (aml), and tetracycline (te) were detected in 21.7, 0, 5.2, 1.7 and 0 percent, respectively. the combination resistance to mtz & clr, mtz & cip, mtz & aml, mtz & te; and clr & cip were detected in 1.7, 1.7, 0.8, 1.7 and 0.8 percent, respectively. the 2.5 per cent of h. pylori isolates were resistance to three or more antimicrobial agents. conclusion: rut method was highly sensitive and specific and appropriate for routine laboratory use. the correlation of the gca patients to h. pylori infection was significant difference compared to gt patients. the high resistance rate to metronidazole indicated that the effective for eradication of h. pylori by metronidazole should be considered in the clinical management of h. pylori infection. use of an indirect immunofluorescence test for the detection of caga in h. pylori c. scherer (essen, d) objectives: the cytotoxin associated gene (caga) of h. pylori is a frequently discussed virulence factor which codes for a 128-kda cytotoxin associated antigen (caga). the caga-gene is usually detected by pcr methods. publications about phenotypic methods for the detection of caga are rare. aim of our investigation was to compare a slightly modified, recently published indirect immunofluorescence test (iift) for the detection of caga with commonly used pcr assays. methods: h. pylori atcc 43504 (caga+/caga+) was used as positive control strain, atcc 51932 (caga+/caga+) as negative control strain. nineteen clinical isolates of h. pylori were examined for their caga-and caga-status. caga was detected by iift using monoclonal anti-caga-antibody from mice, which were visualised for immunofluorescence microscopy with fitc-conjugated anti-mouse-antibody from rabbits. dna was extracted by a standard phenol-chlorophorm procedure after proteinase k treatment of the cells from a 4-day old culture. 7 primer pairs and their published protocols were used: f1/b1 and d008/r008 as the most described primer pairs and further five primer pairs. all tests were performed in duplicate. results: 4 from 19 (22%) strains were caga positive in the iift, and also caga positive in each pcr. 13 from 19 (68%) strains were negative in the iift. 10 from these 13 were caga-negative in each pcr and 3 showed varying results in the pcr-assays. 2 from 19 ( 10%) strains could not be interpreted by iift, both showed varying results in the pcr-assays. the iift might be a simple to use tool in the determination of the caga-status, since it showed no false positive result in neither tested pcr. the differing results in iift and pcr suggest that genotypic positive strains may not always express caga in vitro and maybe also not in vivo. therefore the caga-iift may be an interesting parameter for the determination of strain virulence in addition to pcr-assays. evaluation of elisa serology for the primary diagnosis of helicobacter pylori infection in turkish dyspeptic patients: a prospective study from a developing country b. kocazeybek, y. erzin, s. altun, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: hp is recognized as an important human pathogen by virtue of it's association with peptic ulcer disease, gastric cancer and gastric lymphoma and the high prevalence of infection worldwide. both invasive and noninvasive tests are available to diagnose hp infection but there is still no single gold standard. the aim of the present study was to evaluate the diagnostic accuracy of a commercially available anti-hp igg elisa kit for the primary diagnosis of hp infection. materials and methods: a total of 185 patients who were referred to our endoscopy unit were included. according to our gold standard, a patient was classified as being hp(+) if the culture and/or both histology, rapid urease test were positive and as hp()) only if all of these tests remained negative. standard methods were used to calculate sensitivity, specificity, predictive values of positive and negative results and 95% confidence intervals of these values. results: after excluding patients with discordant results131 of 152 patients (86%) were hp (+). the sensitivity, specificity and diagnostic accuracy of the igg-elisa were 97%, 67%, 93% respectively. conclusion: due to it's lack for specificity, we conclude that quantitative anti-hp elisa test may not be a suitable alternative for primary diagnosis of hp in our country, where the prevalence of infection is still very high. comparison of two different stool antigen tests for the primary diagnosis of helicobacter pylori infection in turkish dyspeptic patients b. kocazeybek, y. erzin, s. altun, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: to assess the reliability of two different enzyme immunoassays in detecting the helicobacter pylori(hp) status in stool specimens of turkish dyspeptic patients. materials and methods: 151 patients [74 with non-ulcer dyspepsia(nud), 64 with duodenal ulcer(du),13 with gastric cancer]who were admitted to the endoscopy unit of istanbul university, cerrahpasa medical faculty for upper gastrointestinal endoscopy due to dyspepsia were enrolled in the study. hp infection was confirmed in all patients by histology, rapid urease test(rut) and culture. a patient was classified as being hp-positive if the culture alone or both histology, rut were positive in the absence of a positive culture and as negative only if all of these tests remained negative. stool samples of patients were obtained in order to assess the reliability of a monoclonal (femtolab h. pylori, connex, martinsried, germany) and a polyclonal (premier platinum hpsa, meridian diagnostics inc., cincinnati, usa) stool antigen test and to compare their diagnostic accuracies. the v 2 test was used for statistical comparison of the values. results: using a cutoff 0.19 for femtolab h. pylori and 0.16 for premier platinum hpsa the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy of the former and latter tests were 93%, 90%, 98%, 68%, 93% and 84 %, 67%, 94%, 40%, 81% respectively. the sensitivity, specificity, npv and diagnostic accuracy of the former test were significantly superior to the latter one's (v 2 = 3.98; p < 0.05 for sensitivity and v 2 = 15.67; p = 0.000 for specificity, v 2 = 15.78; p = 0.000 for npv and v 2 = 6.37; p = 0.012 for diagnostic accuracy). the bacterial load did not affect the sensitivity of both tests. conclusions: the monoclonal femtolab h. pylori, using a cutoff 0.19 is a very sensitive, specific and easy to perform diagnostic tool for the primary diagnosis of hp infection in turkish, dyspeptic patients. evaluation of two different enzyme immunoassays for detection of helicobacter pylori in stool specimens of turkish dyspeptic patients after eradication therapy b. kocazeybek, y. erzin, s. altun, s. saribas, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: to assess the reliability of two different enzyme immunoassays(eias) in detecting the hp status in stool specimens of turkish, dyspeptic patients in the post-treatment period. materials and methods: 48 patients with non-ulcer dyspepsia(nud) who were positive for hp underwent a one week regimen of triple therapy. stool samples of patients were obtained 2-6 weeks and 13c-urea breath test(ubt) was performed 6 weeks after eradication therapy in order to assess the reliability of a monoclonal (femtolab h. pylori, connex, martinsried, germany) and a polyclonal (premier platinum hpsa, meridian diagnostics inc., cincinnati, usa) stool antigen test and to compare their diagnostic accuracies. the v 2 and fisher's exact tests were used for statistical comparison of the values. results: using a cutoff 0.19 for femtolab h. pylori and 0.16 for premier platinum hpsa the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy of the former and latter tests were 93%, 67%, 56%, 96%, 75% versus 87%, 33%, 37%, 85%, 50% 2 weeks and 93%, 88%, 78%, 97%, 90% versus 67%, 70%, 50%, 82%, 69% 6 weeks after completion of eradication therapy, respectively. both at the 2nd and 6th weeks in the post-treatment period the diagnostic accuracy of femtolab h. pylori was significantly superior to premier platinum's (75% versus 50%, v 2 = 6.4 ;p = 0.011, and 90% versus 69%, v 2 = 6.316; p = 0.012 respectively). conclusions: the monoclonal femtolab h. pylori, using a cutoff 0.19 is sensitive and specific enough to monitor hp infection in turkish, dyspeptic patients 6 weeks after completion of eradication therapy. carriers develop serious gastroduodenal diseases. so, it is important to define whether strains with specific genotype are associated with the clinical outcome. the aim of this study was to determine the distribution of the caga, cage and vaca subtypes of hp in patients with various gastroduodenal diseases in turkey, and to explore the association between genotype and the clinical outcome of infection. methods: 93 strains of hp were isolated from cultures and pathological archives of 30 patients with non-ulcer dyspepsia (nud), 30 with duodenal ulcer(du) and 33 with gastric carcinoma. the caga, cage and vacas1a/s1b, m1/m2 genotypes were determined by polymerase chain reaction methodology. results: there were no statistically significant difference among three different clinical outcomes according to the positive rates of vaca s1b, s2 and vaca m1/m2. but the positive rates of vaca s1a, caga and cage were 66.7% (20/30), 50% (15/30) and 26.7% (8/30) in nud; 93.3% (28/30), 83.3% (25/30) and 66.7% (20/30) in du; 87.9% (29/33), 84.8% (28/33) and 81.8% (27/33) in gastric cancer group, respectively, showing statistically significant difference (p = 0.015, p = 0.002, p = 0.001, respectively). the prevalence of vacas2 and m2 in nud patients was higher than that in duodenal ulcer group, but this difference was not statistically significant. conclusions: helicobacter pylori caga, cage and vacas1a genotypes have a significant relation with duodenal ulcer and gastric carcinoma in turkish population. there were no statistically significant association of the vaca s1b, s2 and vaca m1/m2 strains among disease groups. clinical relevance of icea1, icea2 and baba2 genotypes of helicobacter pylori in turkish patients with non-ulcer dyspepsia, duodenal ulcer and gastric cancer b. kocazeybek, y. erzin, v. koksal, s. erdamar, s. altun, a. dobrucali, a. oner (istanbul, tr) objectives: the clinical relevance of the helicobacter pylori(hp) adherence factor, baba gene, and the other virulence determinant, icea gene, has not yet been determined in turkish clinical isolates to date. therefore, the aim of this study was to evaluate the prevalence of icea1, icea2 and baba2 hp genotypes in turkish patients with non-ulcer dyspepsia(nud), duodenal ulcer(du) and gastric cancer. methods: 30 strains were isolated from patients with nud, 30 were isolated from individuals with du and 33 were isolated from patients with gastric cancer. bacterial dna was extracted from cultures and hp positive paraffin-embedded biopsy samples and genotyping of icea and baba2 was carried out utilizing polymerase chain reaction molecular technique by using specific primers. results: the icea1, icea2 and baba2 genotypes were detected in 75.3% (70/93), 24.7% (23/93), 53.8% (50/93) respectively, of the hp strains studied. the prevalence of icea1 in gastric cancer patients was higher than that in nud group, 84.8% (28/33) and 60% (18/30), respectively (p = 0.045). the prevalence of icea2 in nud group was higher than that gastric cancer patients, 40% (12/30) and 15.2% (5/33), respectively (p = 0.045). the positive rates of baba2 were 23.3% (7/30) in nud; 46.7% (14/30) in du and 87.9% (29/33) in gastric cancer group, showing statistically significant difference (p = 0.0001). conclusions: baba2 genotype distribution was evaluated in different gastric pathologies in turkish population and was a good marker for the presence of du and gastric adenocarcinoma. thus, our current results confirm previous experimental studies indicating a central role of hp's adhesin and lewis antigens in the pathogenesis of ulcer disease and adenocarcinoma. influences of interleukin 1b and interleukin 1rn polymorphisms on the development of non-ulcer dyspepsia, duodenal ulcer and gastric cancer in turkish population b. kocazeybek, y. erzin, v. koksal, s. erdamar, s. altun, a. dobrucali (istanbul, tr) objectives: il-1b and il-1rn gene polymorphisms (which encode interleukin 1b and interleukin-1 receptor antagonist, respectively) have been associated with development of gastric atrophy and with increased risk of gastric carcinoma. the aim of this study was to determine the influences of host il-1b -31tc, il-1b -511ct and il-1b rn gene polymorphisms on the development of various gastroduodenal pathologies in 93 helicobacter pylori (hp) positive turkish patients [(30 with nonulcer dyspepsia (nud), 30 with duodenal ulcer (du) and 33 with gastric cancer (gc)]. methods: genomic dna was extracted from hp positive paraffin embedded gastric biopsy samples of 30 nud, 30 du and 33 gc patients. il-1b and il-1b rn gene polymorphisms were analysed by polymerase chain reaction-restriction fragment length polymorphism. these polymorphic sites include promoter regions of il-1b at positions-511 (c-t transition) and -31 (t-c transition), and il-1 rn variable tandem repeats (intron2 vntr). results: there were no statistically significant difference among three clinical outcomes in the frequencies of il-1b -511ct + tt and il-1b -31 tc + cc genotypes (as called proinflamatory genotypes) (p = 0.161 and p = 0.077, respectively). the prevalence of the proinflamatory il-1b rn 1/2 + 2/ 2 alleles in gc group was higher than that in nud and du groups. the frequencies of il-1b rn 1/2 + 2/2 alleles were 20.0% (6/30) in nud; 31.0% (9/30) in du; and 57.6% (19/33) in gc group, showing statistically significant differences (p = 0.006). conclusions: there were no statistically significant difference among three clinical outcomes in the frequencies of il-1b -511ct + tt and il-1b -31 tc + cc genotypes, whereas proinflamatory il-1b rn *2 allele (1/2 + 2/2) was associated with gastric cancer in turkey. rapid detection of clarithromycin-resistant helicobacter pylori in patients with dyspeptic by fluorescent in situ hybridisation (fish), in comparison with e-test m. moosavian, s. tajbakhsh, a. samarbafzadeh (ahwaz, ir) objectives: isolation of h. pylori from more than 90% of the patients with peptic ulcer have demonstrated a strong relationship between these bacteria and created ulcers or dyspeptic. recovery of the patients will be accelerated, if clarithromycin be added to therapeutic protocol. the objectives of this study are: 1-rapid detection of the susceptible or resistant strains of helicobacter pylori to clarithromycin, in patients with dyspeptic by fish technique. 2-comparison of the results of fish & epsilometer test ( e-test) techniques. methods: frozen -sections of gastric biopsies of 50 patients with dyspeptic were hybridized in situ by 5 fluorescent oligonucleotide probes (fish). the prepared slides were examined under a fluorescent microscope, after staining with dapi. also, susceptibility and resistance of isolated strains of h. pylori to clarithromycin were determined by e-test. both results of e-test and fish techniques were compared in final. results: 25 out of 50 examined gastric biopsy samples were positive for h. pylori by fish. out of these 25 h. pylori strains, 17 strains (68%) were susceptible, 6 strains (24%) were resistant and 2 strains (8%) were mixture of susceptible and resistance strains to clarithromycin. this study showed no significant different between fish and e-test results, in view of number in susceptible or resistance strains. conclusions: since the patient gastric biopsies are not routinely cultured in the most of clinical laboratories, also, for isolation of h. pylori, it is needed to enriched and selective media and the other way, clarithromycin is an expensive drug, so, it looks like that fish technique is a suitable method for replacing of the culture, antibiotic sensitivity test and or e-test method. diagnosis of atrophic gastritis from a blood sample p. douramani, s. mavrea, a. arkas, a. adamopoulos, e. anastasakou (athens, paros, gr) introduction: the majority of gastritis is related to infectious agents, where h. pylori is the most important and common. h. pylori gastritis could proceed, over time, to atrophic form of gastritis a serious disease that increases the risk of peptic ulcers and gastric cancer. a new test panel was developed for nonendoscopic diagnosis of atrophic antrum and corpus gastritis from a blood sample. aim: to investigate whether atrophic gastritis can be diagnosed and typed non-endoscopically if the serum levels of pepsinogen i (s-pgi) and gastrin-17 are assayed in connection with h. pylori testing. materials and methods: the study population consists of 50 selected dyspeptic outpatients with (cases) or without (controls) advanced (moderate or severe atrophic gastritis) who underwent a diagnostic gastroscopy for dyspeptic symptoms. of the 50 selected patients,28 (cases) had an advanced atrophic gastritis or a resected stomach. of these 28 cases, 4 had an advanced antrum-limited atrophic gastritis, 6 had resected antrum,15 had a corpus limited advanced atrophic gastritis. two patients had an advanced atrophic gastritis in both the antrum and corpus (multofocal atrophic gastritis) and the whole stomach was removed in one patient. the controls comprise 22 patients of whom 10 had a non atrophic h. pylori gastritis; the antrum and corpus were normal and healthy in 12 patients. the sample for ôpostprandialõ gastrin -17 (s-g-17 prand) was taken 20 min after a protein drink. the s-g-17,s-pgi and igg class antibodies to h. pylori were determined using elisa methods. results: a low s-pgi (<25 mg/l) was found in 15 of 18 patients (83%) with and in 2 of 32 patients (6%) without corpus atrophy. a low s-g-17 prand (<5 pmol/l) was found in all 4 patients with h. pylori associated antral-atrophy and in 5 of 7 patients (71%) with resected antrum but in one of 10 patients (10%) with h. pylori-related non-atrophic gastritis. the mean values of both s-g-17 prand and s-pgi decreased with increasing grade of antral and corpus atrophy respectively. among all patients with atrophic gastritis,24 of 28 patients (86%) had a low s-pgi and/or a low s-g-17 prand with positive igg h. pylori antibodies. such low values were found only in one of the 22 (4.6%) control patients. conclusions: low serum levels of g-17 and s-pgi are diagnostic markers of atrophic antral and corpus gastritis, respectively. a low s-g-17prand is a sigh of the multifocal or antrum-limited atrophic gastritis in patients infected with h. pylori. helicobacter pylori in gastric mucosa: an ultrastructural study e. ozbek, a. ozbek, h. dursun, o. vuraler (erzurum, tr) objectives: helicobacter pylori are extraordinary among bacteria in their ability to colonize the human gastric mucosa, an inhospitable acidic environment, and to stay on there for long periods as decades in despite of host immune and inflammatory responses. in this study, we aimed to research where h. pylori are found within the stomach by transmission electron microscopy (tem). methods: gastroscopic biopsy samples from patients suspected with gastritis or peptic ulcer were processed for both of pcr and tem. the presence of h. pylori in biopsy samples was investigated with pcr method by using specific 23 s ribosomal rna primers. the samples for electron microscopic examination were fixed in 3% glutaraldehyde, postfixed in 1% osmic acid, dehydrated in acetone, and embedded in araldite. plastic blocks of positive samples for the presence of h. pylori confirmed by pcr were cut with a nova lkb bromma ultratome. thin sections were stained with uranyl acetate and lead citrate, and examined by using a jeol 100 sx transmission electron microscope. results: we observed that cross-and longitudinal-sectioned h. pylori within the mucus layer overlying gastric epithelium, in the intercellular spaces between gastric epithelial cells (figure 1), and between two microvilli protruding from the apical surface of an epithelial cell into the gastric lumen. apart from the extracellular h. pylori, we found also intravacuolar h. pylori within the epithelial cells of the gastric mucosa. we observed h. pylori engulfed by pseudopod-like structures at the apical part of the epithelial cells. there were also late endosomal vacuoles containing h. pylori within the deeper cytoplasmic parts of the cell. conclusion: although h. pylori is considered generally a noninvasive pathogen, our electron microscopic observations prove that h. pylori is able to invade gastric epithelial cells, and to enter large cytoplasmic vacuoles. that h. pylori is able to be found intracellularly might explain why the eradication of h. pylori infection is difficult with the antibiotics such as gentamicin that cannot easily cross the eukaryotic cell membrane. abbreviations for figure 1: h, helicobacter pylori; n, nucleus of a gastric epithelial cell; e, cross-sectioned epithelial cells; nm, nuclear membrane; is, intercellular space. bar = 0.5 micrometre helicobacter pylori and association with histological findings in endoscopic biopsies in western greece c. petropoulos, e. jelastopulu, m. kardara, s. spiropoulos (erymanthia, patras, gr) objectives: there is evidence that helicobacter pylori (h. pylori) infection is strongly associated with gastric and duodenal lesions in general population. in this study we reviewed gastroscopic and histological records of patients who underwent gastroscopy for multiple reasons in a tertiary hospital in order to evaluate the frequency of h. pylori colonization in biopsy specimens and to examine the association between h. pylori infection and histological findings. methods: the medical records of 251 patients who presented to the general hospital ôagios andreasõ in patras, western greece, during the period 2002-03 for upper gastrointestinal complaints and other specified symptoms were reviewed for the presence of h. pylori. all patients have been submitted to endoscopy with biopsy mainly of the gastric mucosa. the statistical analysis was performed using the spss. results: gastroscopies and biopsies were undertaken in 251 patients (149 men, 102 women; mean age 58.5, range 16-95 years). in 140 (59.1%) of the patients dyspepsia was the main indication for gastroscopy, followed by 44 (18.6%) patients with anaemia, 12 (5.1%) patients with black faeces and 11 (4.6%) patients with reflux. usual macroscopic findings were oedema (70%), hyperaemia (50%), erythema (22.3%), ulcer (16.8%) and erosions (7.5%). the histological examination revealed chronic gastritis in 209 (83.3%) subjects (in 39% of mild form, in 50% of moderate and in 11% of severe form), atrophic chronic gastritis in 8 (3.1%) and adenocarcinoma in 10 (3.9%) patients. the overall colonization of h. pylori was detected in 80 (34.2%) subjects. in case of h. pylori positive patients, chronic gastritis was found in 15.4% of mild form, in 61.5% of moderate and in 23.1% of severe form, whereas in absence of h. pylori infection the histological type of chronic gastritis was 52.5%, 43.2% and 4.3% respectively. in case of chronic gastritis of severe form h. pylori colonization was found in 75% (p < 0.001), whereas regarding the mild form h. pylori presence was observed only in 14% (p < 0.001). conclusions: the study reveals that h. pylori infection is strongly associated with the severe form of chronic gastritis. thus, for the optimal management and treatment of the patients with gastritis a biopsy of the mucosa appears to be necessary. objective: the ultimate aim of the study is to explore potential natural products as alternative for treatment of infections from staphylococcus aureus including methicillin resistant staphylococcus aureus (mrsa). the specific objective of the paper is to assay for molecular changes in the genes of mrsa isolates upon inhibition by natural products. five genes of mrsa isolates study include meca, meci and mecri adab and sav1017 and the non-mrsa isolates were adab and sav1017 genes. the adab gene encodes for methyltransferase activity for dna repair mechanism while the sav1017 is the cell wall protein gene. methods: mrsa and non-mrsa isolates were obtained from patients receiving treatment in malaysian hospitals. these isolates were subjected to growth in methanol extract of marine seaweed. inhibition assay of extract on isolates including several genera of the gram-negative bacteria, were determined by the disc diffusion and minimum inhibitory (mic) methods. treated and untreated isolates inhibited by extract were subjected to pcr amplification, followed by commercial sequencing, rt-pcr assay of the mrna followed by sequencing of the cdna and blastn analysis with the genbank sequences. results: the inhibition assay of methanol extract showed activity only in mrsa and non-mrsa isolates but not in gram negative isolates. the nucleotide sequences changes were seen in four genes of treated mrsa isolates which were the meca, mecri, meci and the adab. the nucleotide changes in non-mrsa and mrsa were only seen in the adab gene but not the sav1017. the blastn analysis showed variation in nucleotide changes in all genes involved in mrsa phenomenon. conclusions: these preliminary results utilizing genomics for study on nucleotide sequences of pathogen can aim at utilizing the biomolecules from natural products to target the affected nucleotides so as to inhibit growth of the organism. the research activity has the potential of speeding up drug discovery programme and the nucleotide changes in several genes treated with the extract indicates the potential target sites of the extracts. the inhibitory effect of extract on the nucleotide of some genes remained unchanged in the pcr and rt_pcr assay, indicating selective effect of extracts on the genes and the extracts has the potential to be applied as antibacterial agents. implication of the findings is directed towards discovery of antibacterial drug target sites, which warrants further study. phenotypic and genotypic characteristics of staphylococcus aureus strains defective in species-specific proteins a. luczak, j. krzyszton-russjan, k. nowak, w. hryniewicz (warsaw, pl) objectives: the objective of this study was to characterise phenotypically clumping factor and/or coagulase defective s. aureus. methods: the study was performed on 170 s. aureus isolates identified between 1996 and 2004 as clumping factor (cf) or free coagulase (fc) defective by clinical microbiology laboratories and submitted to national institute of public health in warsaw for verification. reidentification included detection of clumping factor, coagulase, thermonuclease, and ability to ferment mannitol. antimicrobial susceptibility testing was performed by diskdiffusion method according to the nccls. glycopeptides susceptibility was determined by screening method, mic evaluation and population analysis. pcr reactions were performed to confirm the presence of species specific genes, as cfa, cfb, coa, nuc, spa. to obtain isolates clonality, multiple-locus variable-number tandem repeat analysis (mlva) was applied. results: based on the phenotypic reidentification methods, 114 (67%) isolates were phenotypically defective in respect to clumping-factor or coagulase production. all but one isolate exhibited the presence of the genes encoding cf or fc. seventyfive (67%) isolates were resistant to methicilin (mrsa) and multiresistant. twenty-six (22.8%) isolates showed reduced glycopeptides susceptibility in pap. all hvisa and hgisa isolates were mrsa and all were defective mainly in clumpingfactor production. thirty-one different mlva groups were distinguished. the a and l groups were the most common and variable. the a type was the most prevalent in hvisa/hgisa isolates and found in 18 centres. conclusion: the clonal spread of s. aureus defective in species specific proteins was revealed. the correlation between decreased susceptibility to glycopeptides and defective species specific proteins was observed. detection of panton-valentine leucocidin and other staphylococcal toxins using oligonucleotide arrays s. monecke, r. ehricht (dresden, jena, d) objectives: the recent outbreaks of community acquired mrsa (cmrsa) harbouring panton-valentine leucocidin (pvl) caused serious concern worldwide. in order to screen clinical isolates, we designed and tested an oligonucleotide array which can detect both components of this virulence factor, six staphylococcal superantigens and the two exfoliative toxins as well as several resistance genes and species-specific controls. methods: twenty-two clinical isolates (two suspected cmrsa and twenty randomly selected) from swabs obtained from a dermatological clinic in saxony/germany were cultured. dna was isolated and subjected to a linear multiplex amplification incorporating biotin-labeled dutp into the amplicon. hybridization of the amplicons to the array was detected using an enzymatic precipitation reaction. results: in two cases of chronical furunculosis, pvl-positive cmrsa were identified. one case was a soldier returning from a peace mission to kosovo where he apparently contracted the disease, the other one was his spouse. in three other cases of furunculosis/abscesses, pvl-positive but methicillin-susceptible s. aureus were found. staphylococcal enterotoxins were found in four, toxic shock syndrome toxin in three isolates, and exfoliative toxin a in one isolate. in two cases, multiple toxins were found (pvl + tst + entc and entb + entk + entq + eta). conclusions: while epidemic cmrsa strains often can presumably be identified due to their resistance profile (methicillin, tetracycline, fusidic acid), there is no easy method to detect pvlpositive, methicillin-susceptible s. aureus. therefore, strains carrying pvl are more common than previously suspected. the high prevalence of this virulence factor as well as of other toxins found in the present study warrants further investigations. characterisation of methicillin-resistant staphylococcus aureus harbouring the panton-valentine leucocidine a. anders, m. kaase, s. friedrich, s.g. gatermann (bochum, d) objectives: until now methicillin-resistant staphylococcus aureus (mrsa) have been known only as a major problem in hospitals. lately, a new kind of mrsa has been described. this mrsa is responsible for community-acquired infections (c-mrsa) and harbours the determinant for the panton-valentine leucocidine (pvl).as there are descriptions of c-mrsa possessing the pvl throughout the world with different mlst types we wanted to characterize eleven c-mrsa from our area possessing the gene for panton-valentine leucocidine. methods: the isolates were found from 1999 until the summer of 2004, the gene encoding pvl was verified by pcr.we determined the pfge pattern, the mlst type, the sccmec type and the agr allele type of the eleven isolates. results: so far all isolates belong to the st 80 which has been also described elsewhere in germany and in france but differs from the types found in the us (1 and 8) or in australia (30 and 298). they possess a sccmec element of type ivb and the agr3 allele type. all isolates were resistant to oxacillin, which was determined by meca pcr, and all were resistant to fusidic acid. only two were additionally resistant to erythromycin. no resistance could be found to fluoroquinolones.the average age of patients was 47 years and they all had severe communityacquired abscesses of the skin or soft tissue that caused hospital admission. conclusion: mrsa cannot be longer considered as affecting only hospitalized and elderly patients but we should also be aware of them in community-acquired infections. accessory gene regulator (agr), and its locus of s. aureus is a quorum-sensing gene cluster of five genes (hld, agrb, agrd, agrc, and agra) that upregulates production of secreted virulence factors, including the alpha-, beta-, delta-hemolysins, and downregulates production of cell-associated virulence factors. s. aureus strains can be divided into 4 major agr groups (agr i-iv) on the basis of a polymorphism in agrd and agrc. the purpose of this study is to know the proportion of agr i, ii, and iii polymorphisms and to compare clinical characteristics between group ii and non-group ii polymorphism of methicillin-resistant staphylococcus aureus (mrsa) strains in a tertiary care teaching hospital in korea. methods: isolates were identified as s. aureus by conventional methods. susceptibility to methicillin was determined by the nccls guideline. agr locus was identified using restriction fragment length polymorphisms (rflps) analysis of agr bdc amplicons with drai digestion. the factors assessed included the patients' demographics, comorbidities (diabetes mellitus, congestive heart failure, peripheral vascular disease, dialysisdependent renal failure, cirrhosis, malignancy, and alcoholism), infection site (central catheter-related bacteraemia, bacteraemia of unknown origin, device, endocarditis, intraabdominal, respiratory, skin, urine, and ear), receipt of mechanical ventilation and operation, the presence of nosocomial infection and treatment failure, creatinine level, and mortality. results: a total of 18 strains from 18 mrsa-infected patients (7 males and 11 females) were evaluated. their mean age was 50.5 ± 24.3 years old. there were 4 (22.2%), 12 (66.7%), and 2 (11.1%) strains in agr group i, ii, and iii polymorphism, respectively. only nosocomial infections were statistically significant clinical parameter according to univariate analysis (p = 0.009) and multivariate analysis (or, 22.0 (1.540 ) 314.292); p, 0.023). conclusion: agr group ii was most prevalent in this study and nosocomial infections were correlated with group ii polymorphism. this result suggests virulence and nosocomial spread of mrsa strains are originated from group ii polymorphism. a recruit of more strains will increase and clarify the clinical difference between agr groups. distribution of toxic shock syndrome toxin-1, exfoliative toxins and enterotoxins seg and sei among methicillin-resistant staphylococcus aureus clonal types v. chini, g. dimitracopoulos, i. spiliopoulou (patras, gr) objectives: staphylococcus aureus produces a variety of exotoxins including the toxic shock syndrome toxin-1 (tsst-1), the exfoliative toxins eta and etb and the enterotoxins seg and sei. the seg and sei genes (seg and sei) coexist in the same operon. the aim of this study was to investigate the presence of genes encoding the tsst-1 (tst), eta (eta), etb (etb), seg (seg) and sei (sei) among methicillin-resistant s. aureus (mrsa) clinical isolates, in relation to their clonal types. methods: the mic of oxacillin was determined by the agar dilution method in mueller-hinton agar supplemented with 2% nacl according to the guidelines of nccls. pbp2a production was investigated by a latex agglutination test (biomerieux) in all s. aureus isolates with mic >0.5 mg/l. pcr amplification was performed for the detection of meca gene to the aforementioned isolates, from different patients, during 2001-2003, resulting to 160 mrsa. the presence of tst, eta, etb, seg and sei genes was also investigated by pcr. clonal types were determined by pfge of chromosomal dna smai digests. results: among the 160 mrsa, 35 (22%) carried both seg and sei genes, 24 (15%) isolates carried the tst, 2 (1%) the eta, whereas no strains exhibited the etb gene. precisely, during 2001 the tst gene coexisted in 12 out of 13 seg/sei-positive strains. eleven strains belonged to pfge type a, whereas the other two to type b, mainly associated with wound infections. eight mrsa in 2002 belonging to pfge type a, carried tst, seg and sei. two more strains carrying seg/sei, belonged to pfge type c. two out of 3 seg/sei-positive additional mrsa which were characterized as a new clone f, carried also eta. in 2003, four mrsa carrying the tst, seg and sei belonged to clone a, whereas 5 more carrying the seg/sei were characterized as pfge type c. most isolates were associated with wound infections and abscesses. conclusions: during the study period (2001) (2002) (2003) the incidence of the tst gene carriage among mrsa was reduced from 26% to 6%, while the seg/sei genes were almost constantly present. the distribution of these genes was mainly associated with strains of clonal type a, exhibiting a drift towards clone type c predominant during the last two years. detection of decreased susceptibility to glycopeptides in staphylococcus aureus using tablet (disc) prediffusion s.v. nielsen, j.b. casals (taastrup, dk) objectives: the aim of the study was to develop a screening and confirmatory method to detect staphylococcus aureus with decreased susceptibility to glycopeptides (hvisa/visa and gisa). methods: the glycopeptide resistance of 20 staphylococcus aureus strains from our strain collection including well-known resistant strains (atcc 700788, atcc 700789, atcc 700698, atcc 700699) was examined. we compared: (i) zone sizes of vancomycin 5 lg and teicoplanin 30 lg neo-sensitabs (rosco diagnostica) when prediffused for 2 h (with disc) + 18 h (without disc) on mueller-hinton and bhi, both media supplemented with 5% horse blood, (ii) agar dilution mics (iii) zone sizes around cefoxitin 60 lg neo-sensitabs. the inoculum used was mcfarland 0.5 and the plates were incubated for 24 h at 35°c. results: regression lines were prepared and there was a good correlation between mics for vancomycin and teicoplanin and the corresponding zone sizes. all hvisa/visa/gisa strains tested showed zones less than 15 mm with cefoxitin 60 lg and zones £ 21 mm (teicoplanin) and/or £ 23 mm (vancomycin) on mh blood agar, corresponding to mics > 2 lg/ml for both antimicrobials. on bhi agar the zones were £ 17 mm (teicoplanin) and/or £ 21 mm (vancomycin) for the hvisa/visa/gisa strains corresponding to mics > 4 lg/ml for both antimicrobials. conclusion: zones obtained by agar diffusion methods using prediffusion with vancomycin and teicoplanin neo-sensitabs correlated with mics and predicted hvisa/visa/gisa strains. for screening high resistance to cefoxitin (zones <15 mm) may be used to select strains for further testing in a daily routine laboratory. the system should be evaluated further in the routine diagnostic laboratory. high rate of vancomycin intermediate staphylococcus aureus among methicillinresistant isolates in taiwan from 637 patients in the tri-service general hospital in taiwan were screened for vancomycin resistance and isolates with reduced susceptibility to vancomycin were further studied for their characteristics. methods: brain heart infusion agar plates containing vancomycin (5 lg/ml) (bhia-va5) were using for vancomycin resistance screening. minimum inhibitory concentration (mic) of vancomycin was determined on both bhia and the standard mueller-hinton agar (mha) on screened-positive isolates. for strains with mics equaled to 8 lg/ml, characteristics including population analysis, susceptibility to triton x-100 induced autolysis, van gene and pulsed field gel electrophoresis (pfge) typing were further performed as previously described. background: mrsa isolates with decreased susceptibility to glycopeptides (gisa) have been reported worldwide since 1997. most strains were isolated from catheters and other foreign bodies in patients treated with glycopeptides. we aimed to determine the prevalence of gisa isolates in a belgian hospital where mrsa has been endemic for many years. methods: all mrsa isolates collected between 09/98 and 06/ 04 were screened for reduced susceptibility to glycopeptides on mueller-hinton agar and 5 mg/l teicoplanin (mh-teico) as recommended by the casfm. the macromethod etest (bhi agar, 2 mcfarland (mf) inoculum, 48 h incubation) was performed as secondary screening for all isolates which grew 4 colonies on mh-teico. gisa/h-gisa phenotypes were confirmed by e-test mic determination on mh agar with 0.5 mf and 24 h incubation as well as by vancomycin (v) and teicoplanin (t) population analysis profiles (pap). all confirmed gisa/h-gisa isolates identified were compared by pfge to other strains identified in belgium in order to delineate their clonality. results: among 520 mrsa strains (from 468 patients), 14 isolates showed growth on mh-teico and 8 of these (from 7 patients) yielded a positive macromethod e-test (mic 8 lg/ml for v and t). seven were confirmed by pap as h-gisa and one as a gisa (t mic: 32 lg/ml; v mic: 8 lg/ml). origins of the isolates were: lower respiratory tract (4), urine (2) and wound (2). all h-gisa strains had a similar antibiotic resistance phenotype by disk diffusion (gentamicin, rifampin, erythromycin, clindamycin, ofloxacin) . by molecular analysis, the h-gisa/ gisa isolates clustered in two different pfge groups a (n = 6) and g (n = 1). the a pfge group was previously described in h-gisa isolates in belgium. in this study, it was subdivided in 2 types, both including 3 strains. one pfge type resulted in a small outbreak in 3 patients during a stay in icu. all 5 other isolates were sporadic with no apparent geographic or temporal relationship between cases. conclusion: gisa/h-gisa were found in only 7 out of 468 (1.5%) mrsa-positive patients. none of them presented with severe infection nor had any previous known exposure to glycopeptides. in view of the low prevalence of gisa/h-gisa in our hospital, screening should not be performed systematically but rather on an individual basis taking into account clinical data and risk factors. whenever the occurrence of gisa is considered, teico-mh agar appears as a suitable first screening approach. a case-control study to evaluate the economic outcome of early po linezolid in patients with methicillin-resistant staphylococcus aureus infections oral therapy with linezolid (lzd) has the potential to decrease hospital costs and shorten length of stay of patients with grampositive infections who would otherwise continue to receive iv therapy. objective: to evaluate the impact of early po conversion to lzd in patients receiving traditional iv therapy for methicillinresistant s. aureus (mrsa) infections. methods: patients with documented mrsa infection between 2001 and 2004 were evaluated in a matched case-control study. cases included patients who were converted to po lzd from iv table 1 includes comparisons of cost, duration of therapy and los. predictably, patients receiving lzd in the hospital had higher drug costs; however, a decreased los contributed to a $7000 lower cost of hospitalization. lzd or iv vancomycin (vanc) , and controls were patients who received only iv vanc. patients were paired in a 1:2 ratio of cases to controls and matched based on infection type, age and comorbidities. demographics, antimicrobial agents, concomitant infections, and clinical status were assessed daily from the onset of infection through the end of treatment or hospitalization. antibiotic cost, length of stay (los), and clinical success rates were compared between the groups. results: 78 patients were assessed. demographic characteristics were similar between groups. average age (mean ± sd) was 46 ± 14; apache ii (median, range) was 6 , charlson comorbidity score 2 [0] [1] [2] [3] [4] [5] [6] [7] [8] , and 60% were male. 55% of patients were treated for skin/soft tissue infection, 19% pneumonia, 13% bone/joint infections, 11% bacteraemia, and 2% other infections. clinical and microbiologic outcomes were similar between groups, with 100% clinical success at the end of therapy. objectives: to compare the efficacy and safety of linezolid (lzd) with those of vancomycin (van) for the treatment of infections caused by methicillin-resistant staphylococcus aureus (mrsa) in japan. methods: this was a randomized, open-label study conducted in japan. patients with nosocomial pneumonia, complicated skin and soft tissue infections, or sepsis syndrome, caused by mrsa, were randomized (2:1 lzd: van) to receive lzd, 600 mg q12 h, or van, 1 g q12 h. outcomes were evaluated at end of therapy (eot) and at a follow-up (fu) evaluation 7-14 days after completion of therapy. results: 151 patients received study drug (100 lzd, 51 van); the treatment groups were similar with regards to demographics and risk factors. at eot, clinical success rates in the mrsa-microbiologically evaluable population were 62.9% and 50.0% for the lzd and van groups, respectively; microbiologic eradication rates were 79.0% and 30.0% in the two groups, respectively (p < 0.0001). at fu, the clinical success rates were 36.7% for both groups, and the microbiologic eradication rates were 46.8%, and 36.7%, respectively. clinical success rates in both groups reflect an automatic outcome of failure for patients receiving prohibited antimicrobials prior to fu. reversible anemia (13%) and thrombocyotopenia (19%) were reported as adverse events more frequently in lzd patients. analysis of laboratory data showed that platelet counts decreased more frequently in lzd patients with recovery by fu. the mean platelet count in lzd patients with an adverse event of thrombocytopenia was 101,000/mm 3 . significantly low platelet counts (<50,000/mm 3 ) were more frequently observed in van patients (6 % vs 3%). no difference was observed in mean changes in hemoglobin levels between the treatment groups. conclusions: lzd is as effective as van for the treatment of mrsa infections in seriously ill patients, and may be more effective in achieving microbiologic eradication. although hematologic adverse events were reported more frequently in lzd-treated patients, analysis of laboratory data showed only a mild reversible trend toward lower platelet counts. cost-effectiveness of linezolid versus vancomycin in complicated skin and soft-tissue infection due to suspected methicillin-resistant staphylococcus aureus infection in germany -12, 2003; san diego, calif) . the objective of the present analysis was to evaluate the cost-effectiveness of linezolid compared with vancomycin in the treatment of cssti due to suspected mrsa infection from the german perspective. methods: a decision-analytic model was developed to examine the costs and outcomes of using linezolid versus vancomycin in hospitalized patients with cssti in germany. an expert panel of german physicians experienced in treating cssti provided resource-utilization data through structured interviews. costs from published sources (rote liste, dkg-nt, ebm) were applied to clinical and laboratory tests, adverse events, isolation procedures, intravenous (iv) or oral (linezolid only) drug treatment, hospitalization by ward type (medical, intensive care), and outpatient physician consultation. if appropriate, patients could be discharged from hospital and treated in an ambulant setting. the model assumed 50% of suspected mrsa patients had proven mrsa. outcomes included total costs per patient, cost per death avoided, cost per life-year gained, and cost per cure. results: an additional 4.4% of patients treated with linezolid (98.4%) versus vancomycin (94.1%) were cured. average total cost per episode was 9352 versus 9474 for linezolid-versus vancomycin-treated patients, giving a 121 saving per episode. the model was sensitive to the los in hospital, days in isolation, duration of oral versus iv treatment, percentage of mrsa patients, and price of linezolid. conclusion: in this decision analytic model, more patients with cssti due to suspected mrsa achieved clinical cure in the linezolid group compared with the vancomycin group. linezolid was cost saving versus vancomycin for the treatment of cssti. linezolid resulted in a shorter treatment duration and shorter los that offset the higher acquisition cost of linezolid versus vancomycin in cssti in germany. clinical microbiology and infection, volume 11, supplement 2, 2005 training, scientific output in infectious disease p874 a bibliometric analysis of worldwide trends in research productivity in microbiology p. vergidis, a. karavasiou, k. paraschakis, i. bliziotis, p. papastamataki, m. falagas (athens, gr) background: microbiology contributes significantly to the understanding and control of infectious diseases and has always been a field of extensive research. however, the literature lacks studies estimating the quantity and quality of worldwide research production. we evaluated the contribution of different world regions in research production in the field of microbiology. methods: using the medline database we retrieved articles from 64 journals included in the ômicrobiologyõ category of the ôjournal citation reportsõ database of the institute for scientific information for the period 1995-2002. the world was divided into 9 regions based on geographic, economic and scientific criteria. using an elaborate retrieval system we obtained data on published articles from different world regions. in our evaluation we introduced an estimate of both quantity and quality of research produced from each world region per year using: (1) the total number of publications, (2) the mean impact factor of publications, and (3) the product of the above two parameters. results: data on the country of origin of the research was available for 76,118 out of 77,080 retrieved articles (98.8%). western europe exceeds all other world regions in research production for the period studied, with usa ranking second (table). the difference in production between these two regions increased gradually from 1995 to 2002. however, the mean impact factor for articles published in microbiology journals was highest for the usa (3.34), while it was 2.79 for western europe and 2.31 for the rest of the world (7 regions combined). conclusions: usa and western europe make up a striking 78% of the world's research production in terms of both quantity and quality. all world regions have increased their research production during the period studied. the highest increase was achieved by asia (excluding japan), latin america, and eastern europe. from conference abstract to full paper: differences between data presented in conferences and journals e. rosmarakis, p. vergidis, a. kapaskelis, k. paraschakis, m. falagas (athens, gr) background: on some occasions, we noted differences between data presented in conference abstracts and subsequent published papers. we studied the frequency and the type of these differences in the fields of infectious diseases and microbiology. methods: we reviewed all abstracts from the first session of 7 out of 15 major research categories presented in the 1999 and 2000 icaac. for each selected pair of icaac abstract and related full paper published in journals indexed by index medicus, two independent investigators performed a comparison of all data in the abstract and the corresponding information in the published paper. using cox logistic regression models we analysed variables for association with differences of data. results: from 190 abstracts that were reviewed, 68 (35.8%) were subsequently published as papers by march 2004. from them, 52 referred to the same study period and population for both the abstract and the paper. differences between data presented in conference abstracts and published papers were found in 28 out of 51 pairs which were analysed further (54.9%, 95% c.i.: 41.2%-68.6%). the identified differences were related to the numbers and/or rates of the studied patients (11/28), numbers or rates of isolates (9/28), mics values or ki values (5/ 28), other chemical properties of antibiotics (2/28), odds ratio (1/28), and duration of observation (1/28). the differences were substantial in several pairs. time to publication of full paper was found to be independently associated with presence of differences (p = 0.029, or = 2.043 per year), while the research category, type of presentation (oral or poster), number of publications of the presenting and last author, impact factor of the journal, and country of origin were not. conclusions: while there are several explanations for the noted differences between data presented in conference abstracts and full papers, it is likely that the research community may improve the accuracy of presentation of data. worldwide trends in quantity and quality of published articles in the field of infectious diseases i. bliziotis, k. paraschakis, p. vergidis, a. karavasiou, a. kapaskelis, m. falagas (athens, gr) background: trying to confront with the widespread burden of infectious diseases, the society worldwide invests considerably on research. however, the literature lacks studies estimating the quantity and quality of worldwide research production. we evaluated the contribution of different world regions in research production in infectious diseases. divided into 9 regions based on geographic, economic and scientific criteria. using an elaborate retrieval system we obtained data on published articles from different world regions. in our evaluation we introduced an estimate of both quantity and quality of research produced from each world region per year using: (1) the total number of publications, (2) the mean impact factor of publications, and (3) the product of the above two parameters. results: data on the country of origin of the research was available for 45,232 out of 45,922 retrieved articles (98.5 %). usa and western europe are by far the most productive regions concerning publications of research articles, as displayed in the table. however, the rate of increase in the production of articles was higher in the developing world regions during the study period. the mean impact factor is highest for articles originating in the usa (3.42), while it was 2.82 for western europe and 2.73 for the rest of the world (7 regions combined). conclusions: usa and western europe make up a striking 80% of the world's research production in infectious diseases in terms of both quantity and quality. however, all world regions present a steady increase in the production of infectious disease articles with the developing countries displaying the highest rate of increase. . financial support was obtained from a pharmaceutical company. however, the sponsor had no role in organizing the scientific content of the programme. results: 146 physicians including id specialists were selected from several university hospitals or tertiary care government hospitals where running clinical trials has been a common practice. pre-training queries revealed that among the trainee population, 30.5% never obtained an informed consent from a patient, 47% did not randomize any patients for trials, 44% did not run any phase i-iv trials, 55% did not use placebo for trial purposes and 49% did not report any serious adverse events to local and or international authorities. a 24-question pre-and post-course tests about basic principles of gcp and local and eu regulations for running clinical trials were applied to determine and compare the knowledge of the trainees on these subjects prior and after the course. the percentage of correct answers ranged 35-91% before, and 51-98% after the course was completed (p < 0.01). conclusions: training in gcp and other regulations about running clinical trials may increase the awareness and knowledge of physicians. these educational activities will also help to raise the quality of clinical trials. learning appropriate use of antibiotics (pk/pd and guidelines): a cd-rom course for healthcare professionals and students e. ampe, y. glupczynski, p.m. tulkens, f. van bambeke (brussels, mont-godinne, b) objectives: in a context of growing resistance and limited supply of new molecules, a rational use of antibiotics should be a high priority. our objective is to train healthcare professionals and students in pk/pd and in a correct implementation of guidelines, since this could help to improve antibiotic use in both short and mid-terms. methods: we developed a pk/pd -guidelines course on cd-rom, targeted to both physicians and pharmacists but also usable by students. the course was prepared by a team of 2 pharmacists, 1 clinical microbiologist, and 1 pharmacologist. sources of information were (i) textbooks, review papers and primary papers by internationally recognized experts (ii) materials presented at training workshops of the international society for antiinfective pharmacology (isap; www.isap.org) during the last 3 years, (iii) national and, if not available, international guidelines for the management of respiratory tract or urinary tract infections. results: the course is organized as a series of power point presentations covering in a progressive fashion the followings topics: (1) bases in microbiology (in vitro properties of antibiotics); (2) pharmacokinetics (definition of the main parameters); (3) pharmacodynamics, with (a) the concepts, (b) the methods and pertinent models, and (c) the data, including the parameters to take into account to optimize the dosage of the main antibiotic classes; (4) resistance, including (a) the main mechanisms and (b) the use of pharmacodynamics to avoid the selection of resistance; (5) the appropriate use (including appropriate dosages) of antibiotics in (a) respiratory tract and (b) urinary tract infections. conclusions: this course promotes continuous education in the pharmacology and pharmacotherapy of antibiotics, in a format easily usable for courses and seminars to both students and professionals. background: although macrolides have no intrinsic antipseudomonal activity, these drugs appear effective in controlling infection by p. aeruginosa through mechanisms such as disruption of quorum sensing or anti-inflammatory effects. objectives: the aim of our prospective study was to evaluate the efficacy of macrolides in patients with bronchiectasis infected and colonised by p. aeruginosa. methods: the study included hospitalised patients with ct evidence of bronchiectasis and presenting an acute exacerbation by p. aeruginosa, isolated in sputum. microbiological findings, clinical data and treatment recommendations were recorded at admission and at the end of therapy. patients completed daily diary cards for symptoms and pef values. patients were followed for 1 year. results: twenty-two patients (14 men, mean age 57.3 ± 14.5) with bronchiectasis and p. aeruginosa infection were included. during hospital stay, patients were treated with at least two susceptible antipseudomonal drugs, according to the antibiogram (usually beta-lactam plus aminoglycoside) for a period of 16.9 ± 1.6 days. an oral macrolide (azithromycin 250 mg x 3 days/week in 10 patients, clarithromycin 500 mg daily in 12 patients) was further administrated for 4.9 ± 1.9 months. at the end of long-term therapy, 11 (50%) patients showed no evidence of p. aeruginosa in sputum, 8 (36.3%) patients still presented p. aeruginosa in sputum and 3 (13.6%) discontinued the treatment after less that 1 month because of adverse events. all patients had a significant reduction of sputum volume (83.3 ml/day before therapy vs. 22.5 ml/day at the end of therapy, p = 0.03) and of the mean number of exacerbations during follow-up (2.4 in the previous year vs. 1.2 in the follow-up year, p = 0.01). an increase of the mean pef value was also noted, although statistically not significant (373.2 ± 43.5 l/min before therapy vs. 489.2 ± 23.5 l/ min at the end of therapy, p = 0.3) conclusion: these observational findings suggest that macrolides may have a role in modulation of p. aeruginosa colonisation in patients with bronchiectasis. objectives: there is little information on cap requiring hospitalization in young adults (£ 40 years). we analysed clinical features, causative organisms, and outcomes of cap in this patient population. methods: prospective observational study of non-immunocompromised adult patients with cap (1995 cap ( -2003 . patients with hiv infection, transplantation, and neutropenia were not included. for the purposes of the study, patients were divided into 2 age-groups, £ 40 years and > 40 years. results: we documented 1732 consecutive patients with cap; 150 patients (8.7%) were £ 40 years and 1582 were > 40 years. mean patient ages were 30.3 years in the former group and 69.6 years in the latter group. main reasons for hospitalization in young adults were: hypoxia (po2 < 60) 59 patients, port high severity risk class (iv-v) 13 patients, empyema 9 patients, and/ or shock 7 patients. young patients were more frequently current smokers (63% vs 22%, p < 0.001). comorbid conditions were more common in older patients (20% vs 66%, p < 0.001), mainly diabetes mellitus, copd, and chronic heart disease. multilobar pneumonia was more frequent in young patients (56% vs 32%, p < 0.001). the most common causative organisms were streptococcus pneumoniae (19% vs 24%, ns) and legionella pneumophila (9% vs 7%, ns). atypical agents were more frequent in young adults (11% vs 4%, p = 0.001), while haemophilus influenzae (2% vs 7%, p = 0.026) and gram-negative bacilli (0% vs 1.4%, ns) were rarely identified in this group. the frequency of bacteraemia was similar in both groups (8% vs 12%, ns). icu admission was more frequent in young adults (13% vs 7%, p = 0.006), but no differences were found regarding the need of mechanical ventilation (5% vs 4%). median los was shorter in young patients (7 vs 9 days, p = 0.002). five young patients died; shock (2 patients) and multiorganic failure (3). early mortality (< 48 hours) was similar in both groups (1.4% vs 2.8%). overall mortality (< 30 days) was higher in older patients (3.4% vs 8.7%, p = 0.026). conclusions: cap requiring hospitalization in young adults, mainly smokers, is not uncommon, being respiratory failure the most frequent reason for admission. s. pneumoniae and atypical agents are the most frequent causative organisms in this group. although overall mortality is relatively low, a number of young patients require icu admission and have a complicated course. corticosteroids in patients with severe community-acquired pneumonia: impact on mortality c. garcia vidal, e. calbo, c. ferrer, v. pascual, s. quintana, j. garau (terrassa, e) background: mortality in patients with severe cap has been traditionally related to microorganism virulence and to host characteristics. recently, there has been an increasing interest host-pathogen interaction, and inadequate immunologic response has been shown to be associated with a higher mortality. immune modulation concomitant to antibiotic therapy has been postulated to improve outcome. the aim of our study was to determine the risk factors for increased mortality in patients with severe cap and to evaluate the impact of administration of corticosteroids in outcome. methods: we reviewed the charts of all hospitalized cap patients in our centre between october 2001 and december 2003. severe cap defined by pneumonia severity index (psi) categories 4 and 5 were included. data on demographics, comorbidity measured by charlson score, bacterial aetiology, the presence of immunosuppression, copd, icu admission, antibiotic therapy, use of corticosteroids and mortality were recorded. results: of the 375 severe cap patients included, 258 and 117,were categories 4 and 5, respectively. 256 patients(69%) were treated with standard antimicrobial therapy and116 (31%) received also corticosteroids. mean age (78 vs 77), charlson score (2.5 vs. 2.6), neoplasm (22% vs. 21%), hiv (0.7% vs. 0.8%), chronic liver disease (9% vs.7%), diabetes mellitus ( 19% vs.22%), icu admission (4% vs. 4%), monotherapy (63% vs. 66%), and aetiology (s. pneumoniae 34% vs. 36%; l. pneumophila 3% vs.2%; others 8% vs.11%; unknown 54% vs.51%) were similar. patients receiving corticotherapy had been exposed to another immunosuppressive treatment more frequently (6% vs. 12%; p = 0.04), copd was commoner ( 20% vs. 66%; p < 0.001) and mortality was higher (5% vs. 11%; p = 0.03) than in the group not treated with corticosteroids. in multivariate analysis, severity of disease (or 3.1; ic95% 1.3-7.1; p = 0.07) and the use of corticosteroids (or 3.3; ic 95%, 1.3-8.0;p = 0.009) were found to be related to mortality. conclusions: in our experience corticosteroid treatment in patients with severe cap appears to be associated with higher mortality. use of levofloxacin in community-acquired pneumonia j. olalla, i. escot, j.j. garcía-alegría (marbella, e) introduction and objectives: community acquired pneumonia (cap) is a prevalent and important disease, with an important consume of resources at hospitals. our aim is to study the profile of income patients with cap diagnosis, the year of introduction of levofloxacin in our centre and compare the length of stay in order to different antibiotics treatments, adjusting the results by fine's scale. methods: in our hospital (a second level hospital in marbella, spain) all the diagnosis at discharge are codified and included in the hospital database, so, all the diagnosis of cap between september 2001 and march 2002 were collected, and clinical reports were examined. demographic data were registered, so were all the parameters affecting fine classification, antibiotic treatment, intensive care unit (icu) incomes, deaths and length of stay. results: 129 cases of cap were found (86 males, 43 females), with a mean age of 67 years (ys)-ci 95%: 64-70), no difference between gender. 4 patients (pts) were living in a residence. 11 pts (8.5%) had a previous diagnosis of heart failure -hf-(mean of age 78 vs 66, p < 0.005) and other 11 pts had cerebrovascular disease -cvd-(mean of age 80.7 vs 66, p < 0.01), 12 pts had chronic hepatopathy, 15 -11.5%-any form of cancer, 23-17.8%-pts chronic renal failure (mean of age 74 vs 66, p = 0.049), 41 pts (32%) chronic obstructive pulmonary disease (copd). 3 pts were admitted at icu and 3 deaths were registered. blood cultures were collected in 30% of cases, culture of sputum in 21%, legionella antigen in urine in 13.2%. in according with fine's classification the patients were stratified in group i (10.9%), ii (16.3%), iii (22.5%), iv (27.9%) and v (22.5%). in 40% cases corresponding to fine i and ii levofloxacin was used alone, vs 19% in fine iii, iv and v (p=0.021). when length of stay was analysed in patients fine i and ii, the use of levofloxacin was associated with a non significant reduction (mean + se: 5.86 + 2.7 days vs 7.67 + 4.9 days), in people on groups iii, iv and v use of levofloxacin do not showed any reduction of length of stay (6.67 + 5.06 days in people using levofloxacin vs 6.11 + 4.5 days). no readmissions in relationship with recurrent cap was registered. conclusions: in our experience, use of levofloxacin is associated with less severe cap, in which a non significant reduction in length of stay is observed. telithromycin versus other first-line single-agent antibiotics in the treatment of communityacquired pneumonia: a randomised superiority trial y. mouton, v. thamlikitkul, r.b. nieman, c. janus (tourcoing, f; bangkok, th; bridgewater, usa; paris, f) objectives: macrolides and beta-lactams are commonly recommended for the treatment of outpatients with communityacquired pneumonia (cap). the aim of this study was to demonstrate the superiority of the ketolide telithromycin (tel) to other first-line, oral, single-agent antibiotics (comparators, comp) in achieving clinical cure in outpatients with mild to moderate cap in geographical areas of high pneumococcal resistance (erythromycin resistance rate ‡ 30%). methods: patients with clinical and radiological evidence of cap were randomised centrally to receive either tel 800 mg once daily for 7-10 days or comp (chosen by the investigator in accordance with local treatment guidelines/practices). efficacy was assessed post-therapy (days 17-21). clinical outcomes in this open-label trial were validated by three independent experts who were fully blinded to treatment assigned. results: of the 505 patients enrolled, 482 were included in the modified intent to treat (mitt) and 438 in the per protocol (pp) efficacy analyses. in the comp group, 39% of patients had received macrolides, 44% beta-lactams and 17% fluoroquinolones. resistance to penicillin and erythromycin among streptococcus pneumoniae isolates was 28% and 31%, respectively. clinical cure rates in the tel group were significantly higher than those in the comp group (as shown in the following table). tel and comp were similarly well tolerated. conclusions: in this outpatient cap study, post-therapy clinical cure rates achieved with tel were shown to be statistically superior to those achieved with other usual care first-line antibiotics -in both the mitt and pp populations, and according to evaluations by both the investigators and blinded experts. changing epidemiology, clinical features, and outcome of acute community-acquired pneumonia among adults in india background: acute cap is the third leading cause of mortality in india. two prospective studies from our centre identified common causes of cap in india to be mycoplasma pneumoniae [mp] and legionella pneumophila [lp] by serology in 11% each, and spn in 10% by culture of respiratory secretions/blood/ conclusion: although spn is the most common isolate, the rising numbers of gram negative organisms (38%) and atypical pathogens associated with increasing mortality stress the need for review of initial antibiotic choice for adults with higher port classes. in view of enduring susceptibility of spn to pcn and minimization of resistance with narrow spectrum activity, this should be the drug of choice for spn in india. fqr that has a wide coverage against most respiratory pathogens including atypicals, can be considered as an initial choice in both hospitalized and ambulatory setting. continued surveillance of respiratory pathogens is needed. fluoroquinolones in the treatment of community and hospital-acquired legionella pneumonia objective: community-acquired pneumonia (cap) is a major cause of morbidity and mortality worldwide. inability or failure to comply with standard antibiotic regimens, which may last up to 10 days, may result in patients receiving suboptimal antibiotic treatment. treatment with a single-dose antibiotic regimen maximizes compliance with prescribed therapy. a novel microsphere formulation of azithromycin makes it possible to administer more drug in a single dose while maintaining tolerability. the objective of this study was to test the hypothesis that a single 2.0 g oral dose of azithromycin microspheres was as effective as a 7-day regimen of oral levofloxacin 500 mg/day when used to treat adult patients with mild to moderate cap (fine classes i, ii, iii objectives: many guidelines have been issued for the treatment of cap. we wanted to assess how frequently the national guidelines for treatment of cap were followed in 10 spanish acute care hospitals in patients admitted with cap. methods: retrospective review of the charts of patients admitted to the hospital with the diagnosis of cap over a 1-year period (1nov 01 to 31oct 01) in 10 geographically scattered hospitals in spain. data were available from 3233 patients. results: amoxicillin/clavulanate was the most common antibiotic choice (79.5% was given as monotherapy and 20.4% in association with clarithromycin). levofloxacin (93.1% given only as monotherapy) follows closely. they were followed by third generation cephalosporins (ceftriaxone and cefotaxime) but up to 60% of them were given in association with clarithromycin. clarithromycin was used mainly as a complement to both amoxicillin/clavulanate and cephalosporins since only 8.6% of its 25.8% was given as monotherapy. other antibiotics were very uncommonly prescribed (cefepime 1.9%, and ciprofloxacin 1.4%). conclusions: 1) monotherapy with levofloxacin, followed by amoxicillin/clavulanic acid were the most common first line antibiotic choices in these 10 spanish hospitals. however, amoxicillin/clav alone or in combination with clarithromycin was the most common antibiotic prescribed. 2) monotherapy with clarithromycin was relatively uncommon (2.2%). 3) these choices reflect quite well the recommendations of the different national guidelines issued in our country in an environment of high prevalence of resistance to penicillin and to macrolides in s. pneumoniae in spain and the concomitant coverage of legionella and other atypical pathogens. the microbiology of abecb -potential predictive value of clinical criteria j. kahn (raritan, usa) objectives: to explore the relationship between specific pulmonary disease severity criteria and the microbiology of acute bacterial exacerbations of chronic bronchitis (abecb). methods: this was a double-blind, randomized clinical trial evaluating levofloxacin 750 mg qd for 3-5 days in abecb. all potential subjects had to meet the ats definition of chronic bronchitis but only those patients with anthonisen type 1 or 2 exacerbations (n = 763) were enrolled. stratification by disease severity was determined using parameters suggested by grossman: fev1 % of predicted value, defined co-morbidities, and number of exacerbations during the previous 12 months. because different spectra of etiologic organisms were expected on the basis of the stratification, different comparator agents were used for uncomplicated patients (azithromycin for five days) and those considered complicated (amoxicillin/clavulanate for ten days). results: initial microbiology from all intent-to-treat patients revealed notable differences between the two strata. among isolates from uncomplicated cases, 55% were the ôtraditionalõ triad of s. pneumoniae, h. influenzae, and m. catarrhalis. inclusion of h. parainfluenzae raises this figure to 79%. in the complicated arm the figures were 48% and 68%, respectively. gram-negative bacilli, primarily enterobacteriaceae spp. and several pseudomonads, represented 15% of the uncomplicated and 23% of the complicated isolates. microbiological eradication rates and the percentage of organism persisting after therapy from the uncomplicated stratum were compatible with the unexpectedly large number of macrolideresistant organisms isolated. in the complicated arm, both levofloxacin and amoxicillin/clavulanate had lower, but comparable, eradication and persistence rates. clinical efficacy in microbiologcally-evaluable patients was consistent with these figures. conclusion: while there was clearly overlap among the flora isolated from the two strata defined by application of the grossman criteria, there was separation of the populations. this predictive approach seems to be of value in identifying the optimal antimicrobial regimen, especially for uncomplicated exacerbations. further work to define and validate predictive clinical parameters may help optimize the choice and duration of antimicrobial agents for abecb. objectives: nosocomial pneumonia is the second most common nosocomial infection and, along with primary bacteraemia, the leading cause of death from infection acquired in the hospital. despite the frequency of ventilator-associated pneumonia (vap) and the threat it poses to patient survival, consensus on an appropriate antibiotic treatment of vap has yet to be established. the uncertainty is compounded by a lack of well-controlled comparisons of specific treatment regimens and their impact on relevant outcomes, such as morbidity, mortality, and cost. the comparable analysis of clinical and microbiological efficacy of cefepime and a combination ceftazidime and amikacin in the treatment of vap in severe trauma patients was the aim of present study. methods: this prospective, randomized study was approved by the institutional review board for human research. thirty adult patients who admitted in icu of an 1850-bed emergency municipal hospital with severe trauma complicated with vap were included. the cpis was used for diagnostics of vap. a combination of ceftazidime (2 g tid, iv) and amikacin (1 g once daily, iv) was used as initial empiric therapy of vap in 17 patients and monotherapy with cefepime (2 g bid, iv) -in 13 patients. these regimes of empiric therapy have been chosen according our local microbiological monitoring and p. aeruginosa was the prevalent pathogen of vap. the cost-effectiveness analysis was performed to compare both costs and outcomes of competing regimes. results: there was no difference between groups in patientsõ mean age, average time of mechanical ventilation before onset of vap and cpis. mean apache ii score was 23.1 in the group of combination therapy and 24.0 in the group of monotherapy. the positive clinical outcome was registered in 92.3% patients on cefepime and 88.2% patients on ceftazidime plus amikacin. the rate of eradication was 53.8 and 58.8% respectively. the duration of effective treatment with cefepime was from 7 to 12 days (mean 8.5 days), with combination therapy -from 7 to 18 days (mean 9.1 days). the mean cost of vap treatment with cefepime was 452.8 euro in comparison with 585.4 euro when the empiric therapy was initiated with ceftazidime and amikacin. conclusions: cefepime in a monotherapy regimen has the same clinical and bacteriological efficacy in comparison with a combination of ceftazidime and amikacin in vap patients with severe trauma but the use of cefepime results in lower costs. influence of antibacterial protocol in multiple trauma patients on incidence and mortality of vap d. protsenko, a. yaroshetskiy, s. yakovlev, b. gelfand (moscow, rus) objectives: the aim of present study was the evaluation of antibacterial protocol in multiple trauma patients on incidence and mortality of vap. methods: a comparable analysis of incidence, attributive mortality (chi-square test) and pathogens of vap in multiple trauma patients was performed during two periods: 2001 (before introduction of protocol) and 2003 (after introduction of protocol) years. the developed protocol included: 1. abandoning of antibiotic prophylaxis of vap; 2. intrusion criteria of early diagnostics of vap; 3. exclusion of 1st, 2nd and 3rd generation cephalosporines , aminoglycosides and fluoroquinolones as empiric therapy of vap; 4. cefepime (apache ii <20) or carbapenems (apache ii >20) were used as initial empiric therapy of vap; 5. efficacy of antibiotic treatment was evaluated within 48 hours; 6. carbapenems and/or vancomycin was added if initial therapy was inefficient. in the case of suspected diagnosis of vap microbiological analysis of bronco-alveolar lavage fluid (bal) was performed. a widespread use of broad spectrum antibiotics shifted the structure of nosocomial pathogens. we observed decrease of mrsa rate and significant increase (more than 10 times) in rate of klebsiella pneumoniae (from 0.6 to 18.1%, most of strains were resistant to 3rd generation cephalosporines) and in rate of acinetobacter baumanii (from 1.2 to 12.3%, most of strains were resistant to ceftazidime). conclusions: introduction of strict antibacterial protocol in patients with multiple trauma and vap results in significant decrease of attributive mortality without any change in incidence of vap. antimicrobial prophylaxis in cardiac surgery and postoperative pneumonia rate objective: the purpose of this study was to assess the postoperative pneumonia rate according to antimicrobial prophylaxis regimens in patients after cardiac surgery with cardiopulmonary bypass. patients and methods: cardiac surgery patients were included (n = 86) in the retrospective study. we observed 44 patients with postoperative pneumonia (pneumonia group) and 42 patients without infectious complications in postoperative period. the groups were compared by age, sex, volume of transaction, severity of disease. in a studied period is from 2001 to 2003 two main regimens of antimicrobial prophylaxis were used: ceftriaxone 1-2 g iv once before operation or cefuroxime 1.5 g iv before operation and additional dosage 0.75 mg in 2-3 hours after surgery beginning; then prophylaxis were conducted after operation during 48-72 hours. data were compared by fisher exact; we determined about significantly differences between groups when p < 0.05. results: preoperative prophylaxis by cefrtiaxone was administered more often in pneumonia group than in patients without infection complications (56.3% vs. 33.3%, ns). ceftriaxone was used significantly more often in comparison to cefuroxime in subgroup of patients with cardiopulmonary bypass less than 180 min who developed postoperative pneumonia (63.2% vs 30%, p < 0.05). during cardiopulmonary bypass longer than 180 min we did not reveal difference between antimicrobial prophylaxis regimens. conclusion: cefuroxime is more preferable than ceftriaxone for antimicrobial prophylaxis of postoperative pneumonia in patients after cardiac surgery with cardiopulmonary bypass less than 180 min duration. closed versus open tracheal suction system to prevent ventilator-associated pneumonia objective: the aim of this study was to analyze the incidence of ventilator-associated pneumonia (vap) using a closed tracheal suction system ( objectives: dalbavancin is a novel, second generation lipoglycopeptide that has been shown to have clinical efficacy as a once-weekly treatment for complicated skin and skin structure infections (csssi). the dosage used in clinical studies is 1000 mg day 1/500 mg day 8. in vitro studies have determined dalbavancin mic90s of less than or equal to 0.06 mg/l for target bacteria, principally staphylococci and streptococci, including isolates resistant to other antibiotics. the plasma protein binding of dalbavancin is 93%. as skin infections occur in extravascular space, antibiotic concentrations are assessed in blister fluid as a measure of skin penetration and to obtain pharmacokinetic observations that may correlate with efficacy. methods: a phase i, open label, single-dose study was conducted in 9 healthy subjects. subjects were administered a 1000 mg iv dose of dalbavancin. blisters were induced by applying 0.25% cantharidin ointment to the skin. blister fluid was collected prior to dose, and at 12 hours, day 3, day 5 and day 7. blood samples were collected throughout the study. blister fluid and plasma were assayed for dalbavancin using a validated lc-ms/ms method and pharmacokinetic parameters were determined. area under the concentration-time curve (auc) was determined for blister fluid (aucbf) and plasma (aucp) through the first week. the degree of penetration into skin was determined by aucbf/aucp (%). results: dalbavancin was well tolerated with no serious adverse events; most adverse events were mild and self-limited. maximum observed dalbavancin blister fluid concentrations were achieved by the first collection (12 hours) and concentrations were maintained above 30.3 (±4.4) mg/l through day 7. the mean (sd) aucbf and aucp were 6438 ± 1238 and 10806 ± 1926 mg h/l, respectively. skin penetration was approximately 60%. dalbavancin blister fluid concentrations were maintained well above mics throughout the treatment interval, even when considering the possible effects of protein binding. conclusions: blister fluid concentrations are maintained well above mics of csssi target organisms for a week following a single dose of dalbavancin. these data support a once-weekly regimen and are consistent with the efficacy observed in clinical studies. the safety and pharmacokinetics of dalbavancin in subjects with renal impairment or end-stage renal disease j.a. dowell, e. seltzer, d. krause, t. henkel (king of prussia, usa) objectives: dalbavancin is a novel, second generation lipoglycopeptide antibiotic in late stage clinical development for complicated skin and skin structure infections (csssi). the weekly dosage used in clinical studies is 1000 mg day 1/500 mg day 8. since dalbavancin will likely be used in patients with various degrees of renal impairment, it is important to determine the safety and pharmacokinetics in this population, as well as to evaluate if a dosage adjustment is necessary. methods: single intravenous doses of 1000 mg dalbavancin were examined in subjects with mild (clcr of 50-79 ml/min) and moderate (clcr of 30-49 ml/min) renal impairment. doses of 500 mg dalbavancin were studied in subjects with endstage renal disease (esrd; dialysis-dependent). single doses of 500 mg and 1000 mg dalbavancin were studied in subjects with severe renal impairment (clcr <30 ml/min). subjects with normal renal function were studied and used as controls. pharmacokinetic data were analysed using non-compartmental methods; parameters included maximum concentration (cmax) and area under the plasma concentration time curve (auc). results: a total of 43 subjects received dalbavancin and were included in the pharmacokinetic analysis (6 mild, 6 moderate, 10 severe, 6 esrd, and 15 normal). dalbavancin was well tolerated in each of the renal impairment groups. the majority of adverse events were mild or moderate in severity and unrelated to study drug. there were no related serious adverse events. dalbavancin pharmacokinetics were similar between subjects with mild and moderate renal impairment and subjects with normal renal function. concentrations in subjects with esrd were similar to subjects with normal renal function, indicating compensation in renal insufficiency due to regular dialysis (3 times/week). subjects with severe renal impairment had increased concentrations and exposure. concentrations were increased by no more than 40% through the first week post dose, but differences continued to increase through the rest of the profile. auc was increased almost 2-fold. background: tlv, a novel lipoglycopeptide antibiotic with multiple mechanisms of action, exerts rapid and concentration-dependent bactericidal activity against clinically important gram-positive bacteria, including methicillin-resistant s. aureus. the kidney is the primary route of elimination of tlv in man. phase 3 studies are ongoing in skin and skin structure infections and hospital acquired pneumonia. the lines represents the model-based predictedprobability of the first occurrence of naused. the boxes represent the 25th and 75th percentiles of auc for each dose group. the wiskers extend to the minimum and maximum values. objective: to compare the single dose pk of tlv in subjects maintained on hemodialysis with an aged and sex-matched group of healthy subjects without renal dysfunction. methods: six hemodialysis subjects (5 males, 1 female) received a single 7.5-mg/kg dose of tlv intravenously over 1 hour approximately 2-4 hours prior to a 4-hour hemodialysis session. six subjects (5 males, 1 female) with normal renal function (mean creatinine clearance 94 ml/min) also received the same dose of tlv over 1 hour. blood and dialysate samples were obtained at specified intervals post dose and assayed for tlv with a validated lc/ms/ms assay. pk parameters were determined using non-compartmental methods. results: average values for plasma pk parameters for tlv in both groups of subjects are shown below.the average (range) clearance of tlv via dialysis was 4.6 ml/min (4. 0-5.8 ) and the per cent of dose removed by dialysis was 5.9% (3.0-7.6). subjects tolerated tlv well. conclusions: dose reductions of tlv of 50% are recommended in patients maintained on hemodialysis. supplementation of a tlv dose post dialysis is not needed. pharmacokinetics and tissue penetration of telavancin in healthy subjects background: tlv, a novel lipoglycopeptide antibiotic with multiple mechanisms of action, exerts rapid and concentration-dependent bactericidal activity against clinically important gram-positive bacteria, including methicillin-resistant s. aureus. the mic90 for s. aureus, including mrsa and gisa, from pooled studies is 0.5 lg/ml. phase 3 studies are ongoing in skin and skin structure infections and hospital acquired pneumonia. objectives: to determine the steady-state pk profile of tlv in plasma and skin blister fluid in healthy subjects. methods: 8 subjects (7 males, 1 female) received three daily doses of tlv (7.5 mg/kg) intravenously over 1 hour. cantharidin ointment (0.25%) was applied to the forearms to produce 7 blisters/subject beginning 12-14 hours prior to the third dose of tlv. blood and blister fluid samples were obtained at specified intervals on days 3 and 4 and assayed for tlv with a validated lc/ms/ms assay. pk parameters in both matrices were determined using non-compartmental methods. the average values for pk parameters for tlv in plasma and in blister fluid are shown below. results: after single intravenous infusions of 750 mg equivalent (eq) ceftobiprole for 30 minutes or 500 mg eq ceftobiprole for 60 minutes, mean cmax-values at infusion endpoint are 57.9 lg/ ml and 34.2 lg/ml, respectively. the corresponding mean auc0-inf-values are 154 lg h/ml and 116 lg h/ml. intersubject variability of the parameters auc and cmax of ceftobiprole is below 20%. mean systemic clearance is 4.9 and 4.5 l/h and mean steady-state volume of distribution is 13.7 and 11.0 l for the 750 mg and 500 mg dosing regimen, respectively. after infusion of 500 mg for 60 minutes or 750 mg for 30 minutes, the ôtime above micõ (target mic of 4 lg/ml for methicillin resistant staphylococcus aureus) is 6 and 7 hours for the total plasma concentrations and 3.5 and 5.2 hours for the corrected unbound plasma concentrations, respectively. with twice-daily administration of 500 mg for 60 minutes or 750 mg for 30 minutes, the proportion of the dosing interval above the mic of 4 lg/ml, would be 75% and 87% for the total plasma concentrations and 55% and 65% for the unbound plasma concentrations, respectively. conclusions: ceftobiprole infusions of 500 mg eq for 60 minutes or 750 mg eq for 30 minutes, results in similar time above target concentration of 4 lg/ml well above the minimum efficacy requirement 25-30% required for mrsa. methods: the study was a randomized, three-way crossover study with 30 hours wash-out period in 8 healthy volunteers. each subject received imipenem in three regimens : (i) 0.5 h infusion of 0.5 g every 6 h for 3 doses; (ii) 2 h infusion of 0.5 g every 6 h for 3 doses; (iii) 2 h infusion of 1 g every 6 h for 3 doses. conclusion: the 2 h infusion of 0.5 or 1 g of imipenem both give greater values for t > mic than a 0.5 h infusion and that a 2 h infusion may be a useful mode of administration in tropical countries where drug instability may prevent the use of continuous infusion. pharmacodynamic characterisation of lmb415 against haemophilus influenzae in an in vitro hollow-fibre system a. meagher, p. smith, a. forrest, a. odundele, p. ambrose (buffalo, albany, usa) objectives: lbm415 is a peptide deformylase inhibitor with in vitro activity against those pathogens commonly associated with community-acquired respiratory tract infections. the purpose of these studies was to determine which pharmacokineticpharmacodynamic (pk-pd) measure is most strongly associated with drug response and to examine the relationship between drug exposure and response for lmb415 against haemophilus influenzae (hi). methods: two wild-type hi strains (mic 2 and 8 mg/l) were studied. the hollow-fiber system (hfs) was inoculated with approximately 10e7-10e8 cfu/ml in log-phase growth. simulating human pharmacokinetics (t ½ = 2 hours), bacteria were exposed to escalating free drug lmb415 exposures (auc ranging from 0 to 200 mg · h/l) using a dose fractionation study design and delivering drug q12 hours, q24 hours, and by continuous infusion (ci). serial samples were collected to determine bacterial counts (cfu/ml) and drug concentrations. drug effect was quantified as the log 10 ratio (lr) of the 24 hour area under the bacterial growth/kill curves for drug and growth control (lr = log 10 aucdrug/aucgrowth control). results: overall, the greatest activity (at 6xmic) was seen with the ci regimen (ci > q12 >> q24). due to the short half-life, dosing q24 hours yielded no net kill compared to baseline. neither per cent time above mic, auc:mic ratio, nor peak:mic ratio could co-model the q12 and q24 hour regimens with ci results. separating these into 2 datasets (q12/24 vs ci), only the auc:mic ratio could adequately describe the ci results (emax lr of (2 at an auc:mic ratio ‡50). the q12/24 dataset was reasonably fit by the auc:mic ratio and per cent time above mic (emax lr of (2 to (3 at an auc:mic ratio ‡120 and per cent time above mic of ‡50-60%, respectively). the peak:mic ratio was not informative. conclusion: for intermittent dosing regimens, per cent time above mic and the auc:mic ratio adequately described drug response. percent time above mic and the auc:mic ratio associated with maximal decline were ‡50-60% and ‡120, respectively. ci regimens could not be co-modeled with intermittent regimens, suggesting that neither per cent time above mic nor the auc:mic ratio completely described drug effect. drug effect continued to increase beyond 100% time above mic. q12 hour dosing had more effect than q24 hour dosing, and would probably be an effective lbm415 regimen in humans for hi. pharmacodynamics of antimicrobials for the empiric treatment of nosocomial pneumonia: a report from the optama program objectives: appropriate empiric antibiotic therapy is vital for maximizing patient outcomes in the treatment of nosocomial pneumonia and is dependent on the drug exposure achieved in a patient and the causative pathogen's mic. the purpose of this study was to compare the probability of achieving bactericidal exposures for commonly used intravenous antibiotics against the bacteria most commonly implicated in nosocomial pneumonia. was high, target attainments for fep, caz, and tzp decreased, but carbapenem target attainments remained the same regardless of pathogen prevalence. conclusions: target attainments were greatest for ipm and mem, followed by higher doses of fep, caz and tzp. because these antibiotic regimens provide optimal bactericidal exposure, they would be most suitable for the empiric treatment of nosocomial pneumonia along with an anti-mrsa antibiotic until pathogen identification and susceptibility results are available. minimal inhibitory concentration effect (sme) of 6 antibacterial agents against two strains of b. anthracis. methods: the pa-sub mic and sme were determined by exposing the bacteria to antibiotic concentrations 10 times the mic for 2 h at 37°c. following repeated washings and centrifugations to remove the antibiotic, cultures were divided into four tubes. to three tubes, the tested antibiotic was added to make a final concentration of 0.5 mic, 0.25 mic and 0.125 mic to the fourth tube no antibiotic was added. optical density was determined before exposure, immediately after washing and then hourly up to 9 h. for the determination of sme, the same procedure was performed except that the organisms were not pre-exposed to any antibiotic. the ods were converted to cfus by using a standard curve. the pa-sub mic was defined as pa-sub mic = tpa ) c, where tpa is the time for cultures previously exposed to an antibiotic and then reexposed to different sub-mics to increase 1 log 10 above the counts determined immediately after washing and c is the corresponding time for the antibiotic unexposed control. the sme was defined as sme = ts ) c, where ts is the time for the cultures exposed only to sub-mics to increase 1 log 10 above the counts determined immediately after washing and c is the corresponding time for the unexposed control. methods: serum and csf cefepime pk data was obtained from 7 hospitalized patients with pneumonia and external ventricular drains. the concentration-time profiles in serum and csf were modeled using a three-compartment model with zero-order infusion and first order elimination and transfer. the apparent volume of the central compartment (vc), apparent volume in the csf (vcsf), intercompartmental transfer rate constants (k12, k21, k13, and k31) and plasma clearance (cl) were identified in a population pk analysis (npag) [table 1]. for cefepime 2 g iv q8 h (0.5 h infusion), a monte carlo simulation of 10,000 subjects (adapt ii) was performed to estimate the probability of attaining the targets of free cefepime serum concentration (assumed 20% protein binding) and total cefepime csf concentration 40-100% t > mic for mics 0.25-8 mg/l (table 2) . csf/serum median penetration ratio was calculated. post map-bayesian observed-predicted regression and r 2 for serum and csf were as follows: (serum) observed = 0.984 · predicted + 2.570; r 2 = 0.944. (csf) observed = 0.785 · predicted + 0.868; r 2 = 0.821. the penetration of cefepime as measured by median auccsf/auc serum was 7.8% (25th-75th percentiles 2.9-21.4%). results of serum and csf target attainment analysis for cefepime 2 g iv q8h conclusion: in the setting of non-inflamed meninges, cefepime 2 g iv q8 h does not provide adequate t > mic in the csf for >80% of patients for mics ‡0.5 mg/l. the influence of inflammation on the calculated csf target attainment rates is unknown. the definitive pharmacodynamic target in the csf has not been elucidated and further research is needed. application of microbiological and capillary electrophoresis methods to phenoxymethylpenicillin dissolution assay w. grzybowska, g. pajchel, m. zapasnik, s. tyski (warsaw, pl) objectives: drug absorption from a solid dosage form after oral administration depend on the release of the active substance from the medicinal product. in some cases of drug analysis, especially when the results of tests are out of specification, it is necessary to use another assay, simultaneously with the reference method. in case of phenoxymethylpenicillin tablets, the reference method for dissolution assay is spectrophotometric method. the aim of the study was to apply parallel microbiological and capillary electrophoresis methods and compare the results with those obtained using uv assay. methods: two preparations (tablets), containing 1 00 000 iu/mg of penicillin v: taropen and ospen were examined. the dissolution tests were performed at temperature 37°c ± 0.5°c, in phosphate buffer ph 6.8 (900 ml for ospen and 1000 ml for taropen) using baskets, rotation speed 100 rpm; samples were taken only at: 30 minute or at 10, 20 and 30 minutes in case of profile of dissolution analysis. the amount of penicillin v dissolved was assayed spectrophotometrically at 268 nm. hanson research dissolution system and ce apparatus: quanta 4000 of (waters) were used. pharmacopoeal, microbiological agar diffusion method and staphylococcus aureus atcc 6538 p strain, were applied. results: the dissolution of penicillin v from taropen preparation after 30 minutes was 100%, independently on method applied. in case of ospen tablets these values were different, depending on the analytical assay. the statistical fisher-snedecor test for comparison of dissolution data obtained using three methods was performed. the fisher fcalc. value was lower than theoretical only in taropen case. the dissolution profiles of two examined preparations were also compared and statistically evaluated according to the fda method. the results of these calculations showed that dissolution profiles of phenoxymethylpenicillin from taropen and ospen tablets differ significantly. conclusion: performed analysis proved that capillary electrophoresis and microbiological methods can be used alternatively but only for determination of taropen dissolution. penetration of penicillin g in combination with sulbactam into nasal tissues u. frank, s. wenzler-rö ttele, w. maier, f. knapp, r. trittler, h. egle, k. kuemmerer (freiburg, d) objective: the penetration of antimicrobial agents into target tissues is essential for treatment at the site of infection. we investigated the distribution of penicillin g in combination with sulbactam in human nasal tissues. methods: to determine the pharmacokinetics of penicillin g/ sulbactam, informed written consent was obtained from 22 patients aged between 19 and 73 years scheduled for endonasal operations such as septoplasty and conchotomy. after infusion of 5 m iu of penicillin g and 1 g of sulbactam, the concentrations of these agents were determined in serum and various nasal tissues such as septal mucosa, septal cartilage and bone. serum and nasal tissue concentrations were determined by liquid chromatography-mass spectrometry (lc-ms). results: up to 1, 2, 3, 4 and 5 hours after infusion, the mean serum concentrations of penicillin g were 114.9 lg/ml (sd ± 24.2), 54.0 lg/ml (sd ± 29.8), 33.6 (sd ± 26.9), 12.7 lg/ml (sd ± 9.6), 17.6 lg/ml (sd ± 8.2) and 2.8 lg/ml, while the mean serum concentrations were 17.6 lg/ml (sd ± 8.2) 24.3 lg/ml (sd ± 15.2), 9.3 lg/ml (sd ± 3.5), 3.5 lg/ml (sd ± 0.6), and 1.9 lg/ml (sd ± 0.4). mean penicillin g and sulbactam concentrations in septal mucosa decreased from 141.7 and 128.3 lg/g (1st hour) to 3.0 and 2.7 lg/g (5th hour), respectively. in septal cartilage, the highest tissue concentrations were observed 2 hours after infusion, with mean penicillin g and sulbactam concentrations of 59.4 and 80.1 lg/g, respectively. the mean penicillin g and sulbactam concentrations in bone decreased from 31.2 and 14.8 lg/g (1st hour) to 1.4 and 1.8 lg/g (5th hour), respectively. the regression analysis of the data showed that 3 h after administration of the drug combination, the levels still exceeded the 4-fold mic90 of methicillin-susceptible staphylococcus aureus, streptococcus pyogenes, moraxella catarrhalis and haemophilus influenzae. conclusions: our data support the use of the drug combination in perioperative prophylaxis and the treatment of ent (nasal and paranasal) infections due to common bacterial pathogens. mic vs. kill-curve based pharmacokinetic/ pharmacodynamic modelling of activities of cefpodoxime and cefixime h. derendorf, p. liu, k. rand, b. obermann (gainesville, usa; munich, d) objectives: pharmacokinetic (pk)/pharmacodynamic (pd) modeling of antibiotics usually consists of the comparison between plasma pk and the mic, such as the t > mic, cmax/ mic, and auc/mic. these indices are limited due to the innate inaccuracy of the mic and the fact that it does not reflect the in vivo scenario where concentrations are not static but fluctuate between doses. an alternative is to use time-kill curves that follow bacterial killing and growth as a function of time and concentration. this study provides a systematic comparison of mic and kill-curve approaches to show the potential of both methods for antibiotic evaluation. in an example, we developed a mathematical pharmacokinetic/pharmacodynamic (pk/pd) cefepime population on pk mean (sd) parameter estimates obtained by npag analysis model to integrate the in vitro antimicrobial activity with the pk profile of two oral cephalosporines at the tissue site. methods: kill curves could be described with different combinations of maximum kill rate (kmax), drug concentration at half-maximum effect (ec50) and bacterial growth rate (k0) that resulted in the same mic in each scenario. in the experimental part, bacterial time-kill curves of cefpodoxime and cefixime against four bacterial strains were compared in in vitro kinetic models in which previously measured human pharmacokinetic profiles of unbound antibiotic were integrated. results: different combinations of kmax, ec50 and k0 can yield the same mic and consequently the same mic-based index. however, depending on the combination of pd parameters, kill curves may predict quite opposite outcomes in both scenarios. ec50 values of cefpodoxime and cefixime were consistent with their respective mic values. both antibiotics had similar high potency against h. influenzae (ec50: 0.04 mg/l) and m. catarrhalis (ec50: 0.12 mg/l), while the potency of cefpodoxime against s. pneumoniae strains was about 10-fold higher than that of cefixime (ec50s/ sensitive: 0.02 vs 0.27 mg/l; ec50s/intermediate: 0.09 vs 0.69 mg/ l). simulations showed that cefpodoxime will have higher bacteriological success against s. pneumoniae than cefixime. conclusions: simple comparison of exposure and mic may not be sufficient to evaluate anti-infective efficacy. kill curves provide a more detailed approach in predicting antimicrobial effects. the developed emax model effectively described the pharmacodynamics of cefpodoxime and cefixime. cefpodoxime (200 mg bid) has higher tissue penetration and antimicrobial efficacy than cefixime (400 mg qd) against s. pneumoniae. bacteriologic efficacy of intravenous piperacillin/ tazobactam and ampicillin/sulbactam for infected diabetic foot ulcers objectives: to test the efficacy of single-agent empiric treatment, either intravenously administered piperacillin/tazobactam (tzp) (4 g/0.5 g q8 h) or ampicillin/sulbactam (sam) (2 g/1 g q6 h), of moderate to severe infected foot ulcers in patients with diabetes. in this open-label trial, 314 adults were randomized to receive tzp or sam for up to 21 days. patients with polymicrobial infections involving methicillin-resistant staphylococcus aureus also received vancomycin 1 g q12 h. samples for bacteriologic evaluation were taken at baseline from infected ulcers and blood to document causative pathogen(s) and test for antimicrobial susceptibility; samples were taken as clinically indicated at the end-oftreatment and test-of-cure visits (14-21 days post-treatment). to minimize risk of contamination, samples were to be obtained by aspirate, curettage, or biopsy rather than by swabbing. results: a total of 123 of the 314 patients in the study were bacteriologically evaluable. the most common causative pathogens in monomicrobial infections were s. aureus, streptococcus agalactiae, enterococcus faecalis, and pseudomonas aeruginosa. about 30% of patients in each treatment arm had polymicrobic infections. the combination of s. aureus plus s. agalactiae was most common. the median duration of treatment was 8 days for both groups. the bypathogen bacteriological success rates were similar in both treatment groups for s. aureus, s. agalactiae, and e. faecalis. tzp had an eradication rate of 85.7% for p. aeruginosa; sam is not active against p. aeruginosa, and patients with p. aeruginosa in the sam group (n = 5) were discontinued from the study . conclusions: previous studies have shown that tzp at a dose of 3 g/0.375 g q6 h was effective for treatment of diabetic foot infections. our study confirmed that less frequent administra-tion of tzp at 4 g/0.5 g q8 h was sufficient to attain bacteriologic success rates of 76.9% for s. aureus, 83.3% for s. agalactiae, 71.4% for e. faecalis, and 85.7% for p. aeruginosa in the bacteriologically evaluable population. this study differed from previous studies of infected diabetic foot ulcers, which found gram-negative enterics to be more common than p. aeruginosa as causative pathogens. however, it is consistent with data showing that p. aeruginosa has recently become the gramnegative pathogen most frequently isolated from soft tissue infections in europe, north american, and latin america. penetration of beta-lactamase inhibitors tazobactam and sulbactam in severe acute pancreatitis objectives: despite of high standard intensive care and surgical management, acute necrotising pancreatitis is still related with an extremely high mortality rate. this is determined by local infectious complications, especially in necrotising areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotising pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of beta-lactamases compared to those of beta-lactam antibiotics alone. some bsp has been shown to penetrate rapidly and efficiently into pancreatic tissue. the penetration of bli into inflamed pancreatic tissue has not been investigated yet. methods: addressing the penetration capability of bli, a clinical trial was designed to investigate the penetration of tazobactam (taz, n = 8) and sulbactam (sul, n = 5) in patients with severe necrotising pancreatitis undergoing pancreas surgery. samples were taken from blood, necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of 0.5 g taz or 1.0 g sul. concentrations of bli were determined by hplc/ uv. the aimed concentration for full enzymatic effect of bli should be 4 lg/mg (taz) and 8 lg/mg (sul), respectively. results: mean plasma concentrations at 1.0 h after application were 20.6 ± 9.58 lg/ml (taz) and 48.6 ± 18.8 lg/ml (sul background: uti is one of the most common infectious conditions treated in general practice and represents a major part of abstracts antibiotic use in the community. it is of major importance to know, how we treat these infections correctly, i.e. maximum efficacy with the least amount of drug in order to reduce the risk of development of resistance. materials and methods: uti was induced in anesthetized female nmri mice via intraurethral inoculation of 10-8 cfu of e. coli. one day later, treatment was started with 12-24 hour dosing schedules with 12-16 different doses of each drug securing a wide variation of time > mic and auc/mic. 24 h after the last dose mice were sacrificed and urine collected, and the bladder and both kidneys removed. bladder and kidneys were homogenized before cfu determination. the drugs used were the cephalosporins, cefuroxime (cef, mic = 0 3 mg/l) and ceftriaxone (cro, mic = 0.094 mg/l), and the penicillin, mecillinam (mic = 0.1 mg/l). the pk of the drugs in serum was determined in similar mice as well. the two cephalosporins were studied together (cef has a t ½ of 14 min and cro a t ½ of 56 min, respectively, in mice). pd parameters were calculated from serum total antibiotic concentrations, since protein-binding is a minor issue in the urine, and the relation to cfú s estimated by the hill-equation. results: all drugs reduced cfú s in urine and kidney tissue by 3-4 logs, but little effect was found in the bladder tissue. for the two cephalosporins combined time > mic in % of dosing interval best described the correlation with cfú s in urine (r 2 = 0.69, p < 0.05) and in kidney tissue (r 2 = 0.89, p < 0.05), respectively, while no such correlation was found in the bladder tissue, neither did auc/mic reveal any correlation with cfú s in urine or any organs. the same was found for mecillinam, i.e. no correlation for auc/mic vs. cfú s in urine or organs, while time > mic % significantly (p < 0.05) correlated with cfú s in urine (r 2 = 0.48), and kidney tissue (r 2 = 0.82), respectively. the time > mic% for maximum efficacy was around 30-40% for all three antibiotics. conclusion: the optimal pkpd parameter for efficacy of betalactam antibiotics in uti is the time > mic. maximum effect is seen for time > mic for 30-40% of the dosing interval, when serum total drug concentration is used as surrogate parameter. in humans this would call for tid dosing of mecillinam and cefuroxime, and od dosing for ceftriaxone. comparative performance of different methods to simulate drug exposure variability in a population v.h. tam, g.l. rosner (houston, usa) objectives: stochastic pharmacokinetic (pk) forecasting such as monte carlo simulations (mcs) are increasingly being used to predict the pk variability of antimicrobials in a population, based on data from relatively few subjects. however, various mcs approaches may significantly differ in the accuracy and precision of the predictions. we compared the performance of 4 different mcs approaches using a dataset with known parameter values and dispersion. methods: concentration-time profiles for 10 subjects after an intravenous bolus of 1000 mg were randomly generated using elimination rate constant (k) 0.1 ± 0.025 h)1(mean ± sd) and volume of distribution (v) 20 ± 5 l. normal distribution of parameter values and no correlation between k and v were assumed. system noise was incorporated as a linear function of drug concentration. using these concentration-time profiles, the best-fit parameter estimates were determined by the standard two-stage method. four methods were subsequently used to simulate the auc0-inf of the population by mcs, using the central tendency and dispersion of the following in the subject sample: (1) k and v; (2) clearance and v; (3) dose/clearance; (4) auc0-inf. in each scenario, 10,000 subject simulations were performed with adapt ii, using normally distributed input parameter(s) with means and variances set to the fitted values. results: reasonably good parameter estimates were obtained (k = 0.101 ± 0.034 h)1; v = 18.0 ± 7.2 l). compared to true auc0-inf of the population (581.1 ± 314.9 mg h/l), the simulated auc0-inf by various methods were: (1) 911.1 ± 2416, (2) 1348 ± 23510, (3) 1348 ± 23510, and (4) 713.7 ± 430.1 mg h/l, respectively. conclusions: our results suggest that various mcs approaches may predict pk variability in a population differently. the most realistic approach appeared to be based on the variability of auc0-inf in the subject sample. our observations are consistent with statistical principles concerning estimation. this method did not amplify variability of the model parameters and was the least likely to be associated with model misspecification. in objectives: fluoroquinolone treatment of humans and animals can rapidly select for organisms with increased resistance to these antibiotics. animal models are key to investigating in vivo antibiotic concentrations that prevent selection of resistance (e.g. the mutant prevention concentration (mpc) concept), but use of such models requires validated procedures for extraction and analysis of fluoroquinolones from relevant tissues. here, we report the validation of a method to quantify the concentration of enrofloxacin, and its metabolite ciprofloxacin, extracted from chicken liver, caecal contents and serum following treatment with baytril tm (enrofloxacin). methods: the extraction procedure described by wiuff et al. 2002 was validated for fluoroquinolone analysis, in pig tissues, by fortification of target matrices with enrofloxacin and ciprofloxacin. norfloxacin was added as an internal standard to all samples. samples were homogenised and extracted with acetonitrile (6 ml), centrifuged and the supernatants retained for analysis. the extracts were diluted with distilled water and analysed by hplc equipped with fluorescence detection. the analytes were chromatographed on a c18 reverse phase column and eluted with an isocratic potassium phosphate buffer/ acetonitrile system. results: liver samples were fortified with 0.1, 1.0 and 5.0 lg/g enrofloxacin and provided recoveries of 114.5, 78.6 and 78.2% respectively. quantification of ciprofloxacin at the 0.1 lg/g level was not possible due to interference from sample co-extractives, but the recoveries at 1.0 and 5.0lg/g were 73.7 and 91.1%. for caecal content samples, enrofloxacin recoveries were 70.6 and 69.1% at 1.0 and 5.0lg/g respectively, and ciprofloxacin 65.4 and 63.3%. the recovery from serum was much higher, samples fortified at 0.1, 1.0 and 5.0lg/ml, enrofloxacin and ciprofloxacin yielded recoveries of 94.7, 80.9 and 86.1%, and 91.1, 85.6 and 87.9% respectively. selected samples were also analysed by zonal microbial growth inhibition bioassay and hplc equipped with ms/ms detection; the data were broadly similar between all methods. conclusion: a valid method for the analysis of fluoroquinolones in chicken liver, caecal content and serum has been established, and will be applied to in vivo mpc studies. where sample matrices interfere (e.g. liver, caeca), bioassay or lc-ms/ ms must be used to provide valid results. the ecological effects of norfloxacin and pivmecillinam (piv) on the periurethral and vaginal flora in women with recurrent urinary tract infection objectives: to compare the ecological effects on the periurethral and vaginal microflora and time to normalisation of orally administered norfloxacin (nflx) and pivmecillinam (piv) in women with recurrent lower uti. methods: women with recurrent lower uti participated in a randomized, single blind parallel multi-centre study. twentyfive women, aged 18-55 years, with a positive nitrite test and symptoms (urgency, frequency, dysuria and/or suprapubic pain) of lower uti were included. only patients with an uti caused by e. coli or klebsiella spp were evaluated. key exclusion criteria were; menopause; pregnancy or breast feeding, known hypersensitivity to study drugs, antibiotics within the preceding month, impaired liver or kidney function, known or clinically suspected pyelonephritis, complicated uti and/or gastrointestinal infection. study drugs: seven days treatment of either nflx 400 mg bid or piv 400 mg tid. samples from midstream urine, periurethra and vagina were obtained before start of treatment day 1 and at two follow-ups day 12-4 and 28-35. informed consent was obtained. the ethical review committee and medical product agency approved the study protocol. results: nineteen patients (11 nflx and 8 piv) fulfilled the inclusion and exclusion criteria. no differences of patient characteristics were seen between the two groups. at the initial visit, more nflx patients were colonized with aerobic bacteria, although no differences were seen for anaerobic bacteria. the e. coli strains were suppressed markedly by nflx and piv in both locations. s. epidermidis increased more markedly following piv treatment compared to nflx in the periurethral location. in the piv group e. faecalis was less frequent initially, increased at visit 2, and returned to pre-treatment numbers at visit 3. for the nflx group, more patients were colonized initially with e. faecalis, with a decrease at visit 3. lactobacilli decreased slightly in the periurethra in the nflx group, whilst no changes were seen for piv. bacteroides spp. decreased more markedly for nflx. restoration of the pre-treatment colonization levels occurred gradually, except for e. coli being markedly suppressed by nflx throughout the study period. the bacterio-logical outcome of the urinary tract infection both at the shortterm and long-term follow up was successful. conclusions: limited ecological effects on the microflora were seen following treatment with nflx and piv, a benefit for antibiotics used for treatment of patients with uti. antibiotics influence release of il-6 and il-8 by kb cells and gingival fibroblasts s. eick, m. lausse, s. schwarz, p. wachter, w. pfister, e. straube (jena, d) objectives: in general, antibiotics are used in defense against pathogenic bacteria. nevertheless, side effects such as immunomodulatory activity should be considered. the purpose of this study was to determine the effect of antibiotics normally used in periodontal treatment on the release of il-6 and il-8 by kb cells and gingival fibroblasts. methods: kb cells and primary gingival fibroblasts were infected with actinobacillus actinomycetemcomitans y4 (a.a.) and porphyromonas gingivalis atcc 33277 (p.g.). the antibiotics doxycycline, tetracycline, minocycline, metronidazole, and moxifloxacin were added in concentrations ranging from 0.25 mic to 100 lg/ml. after 1, 6 and 18 h supernatants were obtained and the levels of il-6 and il-8 were measured by elisa technique. additionally, after 18 h the number of surviving bacteria was enumerated. results: minocycline and moxifloxacin in concentrations predicted in serum as well as 100 lg/ml were effective in killing planctonic a.a. and p.g. as well as adherent and intracellular bacteria. low levels of both interleukines released from kb cells and fibroblasts infected by p.g. were found only after addition of tetracycline up to 10 lg/ml. metronidazole, moxifloxacin and tetracycline in subinhibitory concentrations enhanced the release of il-6 from non-infected and a.a.-infected fibroblasts (up to 3200 pg/ml) after 18 h, contradictory 100 lg/ml tetracycline and minocyline reduced the release of il-6. il-6 in the supernatants of kb-cells was detectable only after addition of 100 lg/ml moxifloxacin. each 100 lg/ml of tetracycline and minocycline suppressed totally the release of il-6 and il-8 from non infected and infected fibroblasts, but not from kb cells. metronidazole and moxifloxaxin in high concentrations promoted the release of il-8. conclusions: antibiotics influence the release of il-6 and il-8. besides the good antibacterial effect especially minocycline might suppress inflammation. nevertheless, the killing of bacteria as well as a possible inhibition of virulence factors (bacterial proteases of p.g. by tetracycline) might influence the enhanced or reduced release of these cytokines. exceedingly infrequent infectious complications during orthotopic liver transplantation: tubercular peritonitis introduction: tuberculosis (t) is a very infrequent complication in patients (p) who undergo solid organ transplantation: in liver transplant recipients a 0.9-2.3% rate has been retrieved by a literature update. in these p t occurs within 12 months, and less than 10% of cases are observed after a repeated transplantation. pulmonary localization predominates (50% of p), followed by disseminated t (30%), while extrapulmonary t is an exceedingly rare event, reported only nu anectdotal p descriptions. pathogenetically, a t peritonitis follows disseminated infection or a primary t enteric localization, and is borne by a 80% mortality rate. case report: a 57-year-old female p with a mute t personal and familial history and normal chest x-ray, underwent liver transplantation 9 years ago, because of a hcv-related decompensated cirrhosias. four years later a novel graft was needed, after the development of end-stage hepatic insufficiency due to massive hcv re-infection. one year later (while of cyclosporin treatment), abdominal pain and ascites formation prompted admission, and m. tuberculosis sensible to all tested drugs was repeatedly isolated from an abdominal fluid with an elevated lymphocyte-monocyte count. associated ethambutol, isoniazid, streptomycin and levofloxacin (this one replaced by cycloserine after 2 months), led to a complete clinical and bacteriological cure already achieved after 3 months, although a 3-drug anti-t treatment was continued for 1 year. conclusionas: in our rare case report, the selected anti-t chemotherapy showed a successful efficacy and tolerability profile, with contained untoward events despite a very critical clinical context, due to the severity of t complication, and the broad spectrum of problems related to graft and all associated issues. in order to obtain an early diagnosis, an elevated clinical suspicion should be maintained for this rare complication too, by recommending microscopy and culture search of mycobacteria on ascitic fluid. methods: from january 2000 through december 2001, a prospective cohort study was conducted to determine the rate of bacterial nosocomial infection in renal transplant recipients. the patients were divided in two groups according to the origin of the allograft: cadaver or living donors. enterobacter cloacae strains, the most prevalent multidrug-resistant bacterial organisms isolated from uti were determined using genomic analysis with pulsed-field gel electrophoresis (pfge). results: one hundred sixty-three patients who received renal transplants were reviewed during the hospitalization. one hundred and ten (67.5%) renal transplanted were from cadaver and 53 (32.5%) from living donor. the median of length of stay in hospital was 12 days (range, 9-75 days) from transplant of living donor and 26 days (range, 2-28 days) from transplant of cadaver (p < 0.0001). twenty-one (39.6%) living donors recipients and 68 (61.8%) cadaver donors recipients had bacterial infection episodes (p = 0.019). post-transplant nosocomial infections diagnosed during the hospitalization were uti (44.8%), ssi (11%), pneumonia (6.1%), bloodstream catheter-related infection (4.2%) and others (1.8%). risk factors for uti in the multivariate analysis included cadaver donor recipient; substitution of the initial immunosuppressive regimen; days of urinary bladder catheterization and length of stay in hospital before the infection. substitution of initial protocol of the post-transplant immunosuppressive regimen and the surgeon was also associated with ssi. six different e. cloacae multidrug-resistant to antibiotics dna profiles were detected in uti of our recipients and hospital dissemination was documented. conclusions: uti was the single most important type of hospital infection in renal transplant recipient and a significant difference in the incidence of uti was found comparing living donor and cadaver donor recipient. the high incidence of uti in the early period post-transplant suggested that the operative manipulation of the urinary tract may be an important causative factor for the development of uti. usefulness of cmv antigenaemia assay after liver transplantation recurrence rates were higher (p < 0.001) in symptomatic infections. underlying liver diseases of the recipients, infectious complications other than cmv, rejection rates, and survival rates were not different between symptomatic and asymptomatic cmv infections (p > 0.05).conclusion: about an half of the recipients experienced cmv reactivation, mostly within 180 days post-transplantation. thirty percents of reactivation were symptomatic. peak value and duration of cmv antigenaemia were significantly higher in symptomatic infections than those in asymptomatic infections. toxoplasma gondii infection in bone marrow transplant recipients: the polymerase chain reaction-blood sample combination in diagnosis and early detection a. sensini, r. castronari, c. ferranti, t. aloisi, f. aversa, f. bistoni (perugia, i) objectives: toxoplasmosis is a rare and frequently fatal complication in bone marrow transplant recipients. it presents with local brain lesions or disseminated infection. usually, the diagnosis is based on histologic confirmation and observation of typical lesions on radiologic imaging, which resolve after appropriate therapy. the aim of this study was to stress the pivotal role of the polymerase chain reaction (pcr) in diagnosis and early detection of toxoplasma infection. clinical microbiology and infection, volume 11, supplement 2, 2005 methods: from january 1999 to july 2004 two hundred and thirteen patients underwent allogeneic peripheral blood stem cell transplantation (pbsct). in the presence of symptoms suggestive of cerebral, pulmonary and disseminated toxoplasma infection, 992 biological samples (blood, serum, csf, pleural fluid, bal) were collected and examined by nested pcr. results: fourteen cases of toxoplasmosis were identified, having an incidence of 6.6%. ten patients manifested cerebral toxoplasmosis, two had pulmonary infection and two disseminated. the overall mortality in this group, not necessarily due to toxoplasmosis, was 57% (8/14). the pre-transplant recipient serostatus was known in 9 patients: 5 were seropositive, but it is to be noted that 4 were seronegative, whereas 5 were unknown. mri study was performed in 12 patients. typical lesions were observed in 7 patients (sensitivity: 58%). the pulmonary toxoplasmosis occurred very early (day 9 and 10) and cerebral toxoplasmosis from day 29 to day 240 (mean 80.4) after pbsct. one case of disseminated infection had cns localization (day 21) and then pulmonary (day 42), whereas the second case had first pulmonary involvement (day 8) immediately followed by cns. blood samples demonstrated higher sensitivity (10 pcrpositive/12) than csf (6 pcr-positive/11) in identification of the etiologic agent. all patients with toxoplasmosis were treated with pyrimethamine and sulfadiazine. the study confirms the pivotal role of pcr in early diagnosis of toxoplasmosis after pbsct. in our experience, a positive pcr signal in blood was an early sign of toxoplasma infection, therefore blood appears to be a suitable and reliable biological sample for diagnosis. moreover, the samples were pcr-negative after the start of therapy and therefore pcr can be useful to monitor the effect of treatment. finally, our study indicates that toxoplasmosis is a potential pathology even in pre-transplant seronegative subjects, suggesting primary infection. cryptosporidium associated cholangitis in a livertransplant patient c. denkinger, p. harigopal, p. ruiz, a. tzakis, l. dowdy (wü rzburg, d; miami, usa) cryptosporidium parvum is a parasite of the group coccidia. c. parvum causes self-limiting diarrhoeal illness in immunocompetent hosts and severe long standing diarrhoea in immunocompromised patients. sclerosing cholangitis (sc) caused by c. parvum is frequently found in hiv-infected patients. in the transplant recipients only two case reports exist describing sc due to c. parvum; one in an adult renal transplant patient and the others in three children who underwent liver transplantation. we report herein the first case of c. parvum associated cholangitis in an adult liver transplant patient. this 52-year-old male patient underwent a liver transplantation three years prior to presentation. his initial transplant failed due to acute rejection requiring retransplant within one year. the second transplant was complicated by sepsis, tacrolimus induced hemolytic uremic syndrome, leaving the patient hemodialysis dependent. in 2004, he presented with pruritus, hypotension, severe dehydration and a three-week history of severe diarrhoea. endoscopic biopsy revealed numerous organisms in the stomach and changes in the colon consistent with cryptosporidiosis. stool cultures were negative. in addition, gamma gluytamyl transferase and alkaline phosphatase were elevated while bilirubin and ast were normal. the patient was diagnosed with colitis due to cryptosporidium and treated with azithromycin. immunosuppression with tacrolimus was continued. the patient continued to have diarrhoea, low-grade fevers and fatigue. two weeks later a cholangiogram and liver biopsy were performed. the liver biopsy revealed c. parvum lining the ductal epithelium. sc was diagnosed. the cholangiogram showed no sclerosing changes in the biliary tree, suggesting an early phase of disease. paromomycin was added to the regimen. the patient improved, and was discharged home. we believe this to be the first case of sc associated with c. parvum in an adult liver transplant recipient. objective: human herpesvirus 6 (hhv-6) infection usually occurs during the first 2 years of life, and its prevalence is higher than 95% in adult population. negative serostatus for hhv-6 in solid organ transplant recipients is a risk factor for fungal infection, but the incidence and severity of hhv-6 infections in seronegative patients has not been evaluated. our objective is evaluate prospectively seronegative hhv-6 solid organ transplant patients for hhv-6 infection by means of quantitative pcr for hhv-6. methods: from february to august 2004, we prospectly evaluated all solid organ transplant patients. patients must have a minimum follow-up of 3 months. patients underwent basal determination of igg for hhv-6. seronegative patients were included for follow-up. blood samples were obtained at 7, 14, 21, 28, 45, 60, 75, and 90 days post-transplant. for viral dna analysis, following dna amplification, it was determined by hibridation in microplaque (affigene). results: in the study period, 112 patients were evaluated (58 kidney, 35 liver, 11 heart, 4 kidney and pancreas, liver and kidney 2, pancreas 1, heart and kidney 1). nine patients were seronegative (7 kidney, 1 liver, and 1 heart). we analysed 67 plasma samples. all patients but one had a negative pcr for hhv-6. the patient with primary infection had a maximum of 78,002 hhv-6 dna copies/ml. clinically, the patient suffered from slight abdominal pain and discrete cholestasis in blood analyses; no fever or serious complications were detected. he did not receive pre-emptive treatment with ganciclovir. no fungal infections were detected. conclusion: primary infection by hhv-6 in seronegative solid organ transplant patients is not frequent, and in 1 patient it appeared with slight symptoms. however, a larger study with more seronegative patients is needed. development and validation of a new real-time pcr assay for hhv-6 detection and differentiation of hhv-6a and hhv-6b d. becker, s. ziegelmaier, s. wach, t. laue, t. grewing (hamburg, d) objectives: human herpesvirus 6 (hhv-6) has been identified as the etiologic agent of roseola infantum in infants. in adults hhv-6 causes complications in immunocompromised patients such as aids patients and transplant recipients. two virus variants can be distinguished: hhv-6a and hhv-6b. several studies have shown variant specific clinical outcome. the objective of this work was the development of a reliable, sensitive and specific hhv-6 detection assay, based on real-time pcr technology, which enables the discrimination between hhv-6a and b. this assay ought to run with the same temperature profile as the realart lc pcr assays for hsv1/2, ebv, cmv and vzv (artus gmbh). methods: by sequence alignments, a region of the hhv-6 genome was defined, which allows the specific amplification by real-time pcr, and discrimination between hhv-6a and b by melting curve analysis. assay specificity has been tested by analysing hhv-6 a/b reference material (abi) and cell culture supernatant from clinical isolates. cross reactivity was excluded by testing dna from related viruses and bacteria. analytical sensitivity was determined by probit analysis. a heterologous pcr system was incorporated, which serves as an internal control. a set of quantitation standards was established, for exact quantitation of the viral load. results: with the realart hhv-6 a/b lc pcr assay artus gmbh developed a ready-to-use assay for the detection and distinct differentiation of hhv-6a and b. quantitation of the viral load within a broad linear range is possible for both variants. simultaneous amplification and parallel detection of internal control and specific sample in one reaction tube excludes false negative results. the realart hhv-6 a/b lc pcr assay has the same temperature profile as the realart lc pcr assays for hsv1/2, ebv, cmv and vzv. 0 conclusion: the realart hhv-6 a/b lc pcr assay allows sensitive and specific detection, as well as subtyping of hhv-6. this leads not only to a reliable hhv-6 diagnosis, but also to a better understanding of the role of hhv-6 variants in pathogenesis. due to the same temperature profile, the realart hhv-6 a/b pcr assay completes the realart lc pcr assays for the detection of different herpes viruses. now the parallel detection of hsv1/2, ebv, cmv, vzv, and hhv-6 a/b is possible in a single real-time pcr run. thus, clinicians receive a reliable diagnostic tool for rapid detection of herpes viruses, and especially in transplant recipients. quantitative cmv pcr in allogenic stem-cell transplant patients l. cardeñ oso, c. blazquez, r. rodriguez, j. diaz-regañ on, e. lomas, p. sanchez, r. cámara (madrid, e) objective: to assess the clinical value of a commercial quantitative plasma cmv-pcr assay (cobas amplicor cmv monitor test, roche molecular system) in allogeneic sct patients by comparing the results obtained with the pcr with those obtained with antigenaemia. to evaluate the impact of an automatised dna extraction method and a lower cut-off on pcr sensibility. methods: all patients were monitored until day 100 post-sct with weekly antigenaemia and pcr. a total of 562 blood samples from 30 patients (23 mieloablative and 7 non-mieloablative; 25 hla identical siblings and 5 from unrelated donors) were tested prospectively. twenty-seven cmv seropositive patients (or negative with a seropositive donor) received high dose of acyclovir as prophylaxis. pcr was considered positive when more or equal 600 dna copies/ml were detected. antigenemia was considered positive when one or more positive cells were detected (in 2x105 pmns). positive samples by antigenaemia and/or pcr (n = 54 samples) were tested retrospectively with an automatised extraction method (magnapure). results: all cmv seronegative patients with a seronegative donor (n = 3) were antigenaemia and pcr negative. seropositive patients (n = 27): 14 had a positive antigenaemia and/or pcr, with a total of 23 cmv infection episodes. none developed cmv disease. pcr detected 10 out of 23 cmv episodes. overall there were 44 positive antigenemias (belonging to 13 patients): 23 were pcr+, 20 pcr negative and 1 pcr inhibited. only 1 patient had a positive pcr with a negative antigenaemia. automatised magnapure extraction increased the sensibility of the pcr. with this method pcr detected 19 out of 23 episodes in contrast with only 10/23 with the manual extraction. for samples with less than 600 dna copies/ml a manual calculation of the number of copies was retrospectively done (using a new cut-off of 50 copies/ml). with this lower cut-off, pcr detected 20 out of 23 cmv episodes. none of the negative patients by antigenaemia and/ or pcr gave a positive result with the automatised extraction method or with the lowered cut-off pcr. conclusion: quantitative cmv pcr assay showed a lower sensibility for the detection of cmv infection in allogeneic sct compared with antigenaemia. two technical modifications increased the sensibility without a decrease in the specificity: automatised magnapure extraction, and lowering the cutoff. trichosporon beigelii as a life-threatening pathogen in bone marrow transplantation recipients: the first case report from iran patients undergoing bone marrow transplantation (bmt) are profoundly immunosuppressed as a result of their intensive chemotherapy and are at high risk for opportunistic fungal infections mainly caused by candida spp and aspergillus spp. trichosporon beigelii (t. beigelii) has emerged as a life-threatening opportunistic pathogen in immunocompromised hosts. response to antifungal agents is poor, mortality is high and immunological recovery is the most important factor for a favorable outcome in patients with trichosporonosis. we present the first case of t. beigelii infection in patients undergoing allogeneic bmt in iran. a 12-year-old female presented with aplastic anemia, cough and fever. she had received cytotoxic drug therapy, broad spectrum antibiotics and was neutropenic. t. beigelii was repeatedly demonstrated by appropriate morphological and physiological characteristics in her sputum, nose and mouth ulcers by direct microscopy and culture, and also isolated and histopathologically recognized in post-mortem from lung and liver. trichosporonosis is likely to be recognized more frequently than apparent from the available reports. objectives: trichinellosis is a well-known problem in greenland. in west greenland a number of large outbreaks took place during the 1940's and 1950's, but since then only sporadic cases have been registered. it is unknown whether the decrease in trichinellosis cases reflects the general transition in greenland towards a more western lifestyle with less consumption of meat from wildlife, or whether it reflects insufficient case registration. the objectives of the present study were to determine the prevalence of human trichinellosis from the 1980õies to present times in nuuk, the capital of greenland, and in ammassalik district on the east coast of greenland, where more traditional food is still eaten, and to evaluate if changes in lifestyle had an effect on prevalence. methods: blood samples from 86 persons collected in 1981 and 533 persons collected in 1998 in nuuk and ammassalik district were tested for trichinella-specific igg antibodies using elisa and western blot analyses. background information was obtained from questionnaires from the 1998 study. results: in 1981 the trichinellosis prevalence was 8.7% in nuuk and 23.8% in ammassalik district, while the prevalence in 1998 was 5.2% in nuuk and 19.8% in ammassalik district. persons living in the settlements of ammassalik district had higher trichinella-specific antibody prevalence than persons living in the town of tasiilaq. the following risk factors were significant in a univariate analysis: ethnicity, age, place of living, dietary habits, intake of seal meat, and hunting. a multivariate model was constructed consisting of the variables: ethnicity, age, place of living, diet group, intake of seal meat, and hunting. the variables were removed stepwise in the following order: seal meat (p = 0.73), hunting (p = 0.36), age (p = 0.3), and ethnicity (p = 0.16). the final multivariate model consisted of diet group and place of living, both being significant. thus, living on a greenlandic diet and living in the settlements in ammassalik district implied higher relative risk of being trichinella seropositive than living on a diet dominated by imported food items and living in nuuk. conclusion: as lifestyle in the settlements is more traditional compared both with tasiilaq and nuuk, these results indicate that the decline in human trichinellosis cases in the 19th century most likely reflects the transition from traditional greenlandic lifestyle to more western dietary habits. study on prevalence of toxocara cati in vagrant cats of sari township, iran, 2004 m sharif, p. ziapoor, h. ziaee, s. kholami (sari, ir) objectives: toxocara cati is one of the important and prevalent parasites in cats and common between human and animals. ingestion of larvae of this parasite and lack of it's maturation in human body , the infected persons develop visceral larvae migrans which is known as toxocariasis. considering the significance of transmission of this parasite in human and due to the increase in vagrant cats in sari township, this study was performed in order to determine the severity and prevalence of toxocara cati in these cats in sari township. methods: this cross sectional descriptive study was done on 100 vagrant cats (28 males and 72 females) during april to october 2004. sampling was done randomly at different regions by net. the specification of vagant cats were recorded after hunting and were deeply anaesthetized by chloroform to annihilate them. the intestinal content were examined for the presence of parasites and counting of their number was done finally after fixation and staining by recommended kits of reference book for genus and species identification. results: in all, 42 (42 %) cats were infected with toxocara cati, of which 8(19%)were males and 34 (81%)were females. range of infection was 1-32 and mean severity of infection was 3.14 for each cat. the rate of infection at the west region was more than the other sites, and it was more in the females than males and also it was more in immature than the mature cats. conclusion: considering the merely high prevalence of this zoonotic parasite and it's hygienic significance in causing toxocariasis in human particularly in children, who are in more contact with soil and also lack of control on vagrant cats population, which are the potential risk factors, it is recommended to control personal and dietary hygiene and avoid storing the garbages in unprotected wire boxes on the streets in order to prevent the gathering of vagrant street cats. imaging in 100 patients of pulmonary hydatid disease; review of unusual imaging appearances s. zahirifard, m. bakhshayeshkaram, m. tahbaz, k. kaynama (tehran, ir) objective: to evaluate the chest radiography and ct scan characteristics of pulmonary hydatid disease. material and methods: 100 patients with surgically proven pulmonary hydatid cysts were enrolled for study. we reviewed clinical findings and imaging of patients. the radiological features (localization, diameter, architecture, density and other radiological signs and appearances) were determined. results: on cxr 124 cysts were determined. on 82 available ct scans a total of 112 cysts were detected. no discrete cyst was detected on 11 cxr. on 82 available ct, a total of 112 cysts were detected and in 5 ct scans no cyst was detected. 57 cysts were ruptured and 25 patients with ruptured cysts had hemoptysis. single cysts were in 63 patients while multiple cysts were in 37. median ct density was 24hu. respectively, it was seen on ct and cxr waterlily sign in 18 and 22 patients, air-fluid level in 12 and 17 patients and cresent sign on 11 and 5 of patients. inverse cresent sign and calcification were not observed on cxr s but each one was recorded on 4 ct scans. on ct scan 90% of cysts were smooth and 89% were uniloculated. 19% of cysts were infected. conclusion: ct scan should be done to elucidate cystic nature of the lung masses and for accurate localization in the preoperative period. in endemic regions like iran, atypical imaging presentation of complicated pulmonary hydatid disease such as solid masses should be considered in differential diagnosis of pulmonary lesions (abstract truncated at 250 words) the seroprevalence of coxiellosis in farmers and cattle in north-eastern turkey s. seyitoglu, z. ozkurt, u. dinler, b. okumus (erzurum, tr) objectives: coxiellosis is a worldwide zoonosis caused by coxiella burnetii. the aim of this study was to determine the abstracts prevalence of coxiellosis in cattle and farmers in north eastern turkey. methods: a total of 230 cattle and 92 human sera were collected in 2003, and tested for antibodies against c.burnetii by commercial elisa test. results: the antibodies of c. burnetii were detected in 22 (9.56%) cattle and 18 (19.5%) healthy farmers. seropositivity was found in 12 of 53 (22.6%) cattle with abortion history, and 10 of 177 (5.6%) cattle without abortion history (p < 0.05). there was correlation between animals and human seroprevalance in same district. thirteen of 18 farmers who were antibody positive had seropositive animals, and there were seropositive animals in the villages of remaining 5 cases. it was observed that seroprevalance of coxiellosis was higher in north districts (in animals 15.4%, in humans 32.4%), have drier and warmer climate, than in other districts (in animals 6.5%, in humans 12.1%) both for humans and animals (p < 0.05). conclusions: the study show that coxiellosis was an important health problem both in humans and in animals in our region. an unusual case of gramineae in sublingual salivary gland l. doganci, m. calgurel, y. gungen, g. kiran (ankara, tr) foreign bodies of organic nature and parasitic elements are very rare in oral cavity and in salivary glands in humans because of their structural and functional features. here we report a very interesting case, with sublingual salivary gland involvement of gramineae (brachiaria spp.) which was thought to be a parasitic life cycle in the gland. case: 40 y/o female teacher with a recent history of travel to mountains where she drunk and had an exposure to unsafe spring water. she had a strong stinging pain right after she drunk the water in her mouth, radiating to her neck. she developed fever, pain, lymph node swelling, difficulty of eating, weight lost and remarkable eosinophilia. she also noted a self moving small tail-like object on the orifice of the sublingual salivary gland. removal of the object revealed an interesting unusual living object. then, definitive diagnosis is gramineae (brachiaria spp) after parasitological and pathological consultation. comment: to our best knowledge, this unusual case is the first presented patient related to travel in our region. differential diagnosis has several entities including squid sperm bag sting, marine animal larvae and nematod and trematod life stages. evaluation of echinococcus granulosus prevalence using elisa and abdominal ultrasonography in a group of students staying in a state hostel in turkey objective: cystic echinococcosis, caused by larval form of echinococcus granulosus is one of the most important and widely distributed parasitic zoonotic diseases in the world. echinococcosis is a major problem in all regions of turkey but particularly prevalent in rural areas, where domestic livestock raising is common. the reported epidemiologic findings are usually based on retrospective evaluation pf surgical findings or hospital charts. well planned epidemiologic studies are limited. in this study our aim was to analyse the seroprevalance of cystic echinococosis and prevalance of lesions (with abdominal ultrasonography) in a group of young adult university students who were staying in a state hostel in bornova-izmir-turkey. method: the study group consisted 750 students (360 woman, 390 men, aged 20.92 ± 1.82, min 17, max 29). informed written consent was received from each student and they were requested to fill a questionnaire form. blood sampling was performed by iv puncture and sera were obtained after centrifugation. anti echinococcus granulosus antibodies were detected by using elisa tecnique. all participants were gven an appointment for abdominal ultrasonography. results: of 750 students 99 (13.2%) were seropositive for anti echinococcus granulosus ig g (table 1). a total of 250 students (49 seropositive-201 seronegative) were performed abdominal ultrasonography (3.5 mhz transducer, sonoline, elegra-siemens). two of 250 students (1 in liver, 1 in kidney both seropositive) had cystic lesions and were referred to surgery. well designed epidemiologic studies about cystic echinococcosis are lacking in turkey. a previous study showed seropositivity rate of 13.7% for echinococcus granulosus. our findings suggest that cystic echinococcosis is prevalent in turkey and epidemiologic studies combining elisa and abdominal ultrasonograpy are warranted. objective: anthrax is an epidemic zoonosis in eastern part of turkey. this study was aimed to investigate the epizootiology and epidemiology of anthrax in this region. methods: animal and human anthrax cases during 1992-2004 period were included in this study. data were obtained formal records of health directorate, institute of veterinary control and investigation. the diagnosis of anthrax had based on clinical findings and/or microbiological procedures, including gram strains and culture of materials obtained from lesions. results: in a 13-years period, 464 animal cases and 503 human cases with anthrax were detected. of 464 animal cases, 20 (4.3%) were sheep, others (95.7%) were cattle. most cases occurred between july and september in both animals and humans. anthrax was seen most frequently in erzurum, which is the important centre for animal commencement. when the first 6 years period (1992) (1993) (1994) (1995) (1996) (1997) was compared with last 7 years period (1998) (1999) (2000) (2001) (2002) (2003) (2004) ) the number of animal cases were 161 vs. 230 and human cases were 234 vs. 269. an analysis of the yearly case distribution shows that the incidence of anthrax in this region had not been changed. conclusion: anthrax is an important health problem both animals and humans in this region. the preventive measures should be taken for decreasing the incidence of anthrax. objectives: anthrax is endemic in kazakhstan and occurs among humans at an incidence rate of 0.1 to 1 per 100,000 population per year. during the late 1990s, several epidemics occurred associated with home slaughter of infected animals, and a general increase in incidence was seen among animals and humans. historically, kazakhstan accounted for 15% of the anthrax cases reported from the soviet union; and the plains of central asia may have served as the historical source of b. anthracis for the rest of the world. in kazakhstan, the geographic distribution of anthrax among animals and humans is focal and generally associated with areas considered to be ôhigh-riskõ. the determinants of this focal distribution are poorly understood. in addition, strains of b. anthracis isolated from outbreaks within these foci have not been compared on a molecular or pathogenic basis. methods: work has been initiated to study the geographic distribution and potential environmental preferences of b. anthracis using geographic information system (gis) technologies. results: the gis was used to map a set of b. anthracis strains from several outbreaks between 1997 and 2003 to evaluate geographic patterns. the gis is also being used to derive a historical database of spatially explicit human and livestock outbreaks from 1948 to 2003 for spatial analyses on the nationwide distribution of b. anthracis. this database contains information on the location of the anthrax incident, the year and month of the occurrence, whether the incident involved livestock or human, the outcome of the disease, and biological information about the strains. conclusions: although in a preliminary stage of application, the arcgis software can be used to map loci of anthrax occurrence. in the future, these maps will be used to identify factors that influence the occurrence and spread of the pathogen and to prevent infection of livestock or humans. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency (dtra) under the project «kb0-1950-al-03» and administered by u.s. civilian research and development foundation (crdf). objectives: the anthrax situation in the country was unfavorable from the 1950s through the 1970s. until the 1990s, anthrax in kazakhstan was an occupational disease in animal husbandry. after 1991, the practice of raising livestock for private use increased. this report shows that, at the same time, the incidence of anthrax in persons involved in this practice increased significantly. methods: in order to characterize the epidemic process, we used methods of descriptive epidemiology, using epidemiological variables (who became ill, where and when, trends of disease by time, sources, transmission factors of b. anthracis, and the age, gender, and occupation of persons who become ill with anthrax). results: anthrax occurs in rural areas among private owners of livestock and their families and persons hired to assist with the slaughter. in the last decade, 89.4% of cases of anthrax illness in humans were caused by the slaughter of domestic livestock. of these cases, 62.2% were unemployed rural residents, 2.3% were shepherds working for cooperatives, 11.7% were blue-collar workers, 5.4% were white-collar workers, and 18.1% were students. a single source, path and factors of transmission of b. anthracis are typical of this type of the disease. in recent years, the majority of cases involve infection of several persons during the slaughter of a single diseased animal. the source of infection is cattle in 48.2% of cases, sheep and goats in 15.7% of cases, horses in 31.2% of cases, and soil in 4.7% of cases. the factors of transmission of b. anthracis to humans are contact with the meat of livestock during slaughter and butchering in 91.9% of cases, vector-borne transmission in 2.7% of cases, and soil in 5.4% of cases. a large number of human cases occur in july through september. conclusion: in kazakhstan today, the prevalent sub-type of anthrax disease in humans involves non-professional activity at home. this requires changes in the tactics of preventing the disease. the research described in this abstract was objectives: severe acute respiratory syndrome (sars) is an emerging infection caused by a novel coronavirus known as sars-cov. during the early phases of the disease, the presence of the replicative intermediates of sars-cov has been shown in pbmc from patients. no information is available on the ability of sars-cov to stimulate ifn induction, although ifn-gamma may be activated in sars patients. we investigate the capability of sars-cov to give rise to a productive infection in normal pbmc, in parallel with the ability to activate ifn-alpha andgamma gene expression. methods: normal pbmc were infected with a moi 0.1. virus progeny formation was measured at subsequent time points, by back-titration of cell lysates on vero cells. cell mortality and apoptosis were detected by trypan blue and propidium iodide staining. in order to detect virus-specific plus-and minus-rna strand, total rna extracted from sars-cov-infected pbmc was retrotranscribed in the presence of the sense and antisense primers, respectively, targeting the replicase gene. measurement of sars-cov genomic rna was performed by quantitative real time rt-pcr. ifn induction was performed by exposing pbmc to sars-cov at different moi, ranging from 0.1 to 10. induction of ifn-alpha and -gamma was analysed by measuring their mrna levels by limiting dilution rt-pcr. sensitivity of sars-cov replication to exogenous ifn-alpha and -gamma was tested in vero cells pre-exposed to the individual cytokines of to a combination of both. results: in unstimulated pbmc, no infectious viral progeny formation was detected up to day 13 post-infection. nevertheless, minus-strand and genomic sars-cov rna peaked at 24 hours post-infection. dose dependent induction of both ifnalpha and ifn-gamma mrna was observed, that was most evident at moi 10. a combination of these two cytokines was shown to strongly inhibit sars-cov replication in vero cells. our results show that sars-cov is able to infect normal pbmc. however, this appears to be only a transient phenomenon, followed by a progressive disappearance of both virus-specific rna strands, with no infectious virus progeny production. moreover, sars-cov appears to have the intrinsic ability to activate dose-dependently both ifn-alpha andgamma gene expression in pbmc cultures. our results can be pathogenically relevant to the inflammatory events occurring in the diseased tissues that can be mediated by the endogenous activation of inf system. objectives: trichoderma species are potential candidates for biocontrol of plant pathogenic fungi and cellulase producers of biotechnological importance. on the other hand, they are emerging as opportunistic pathogens of immunocompromised and dialized patients. this study was designed to examine the ability of 10 clinical trichoderma isolates to produce extracellular proteases and to screen them for toxicity to mammalian cells. methods: supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. trypsin-and chymotrypsin-like activities were separated by sephadex g-100 column chromatography and their ph-dependence was studied. sperm toxicity bioassays were performed by exposing boar sperm suspension to trichoderma-methanol extracts, the motility of spermatozoa was estimated by phasecontrast microscopy. mitochondrial membrane damage in spermatozoa was studied by epiflurescence microscopy using the jc-1/propidium iodide staining. results: the production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities was common among the examined strains. separation of trypsin-and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. relatively high activity levels were detected between ph 5 and 9 suggesting that the different isoenzymes have different ph characteristics. more than 90% of the spermatozoa were motile in a non-exposed control sample and in a sample exposed to 5 microliter of methanol. more than 90% of the spermatozoa were immotile in samples exposed to t. longibrachiatum uamh 9515, atcc 201044, cm382 and t. harzianum cbs 102174 in a concentration of 1.25 mg biomass ml-1, indicating that these strains produce heat-stable substances toxic to sperm cells. these four strains were found to inhibit the motility of boar spermatozoa even at a low concentration (50% reduction of motility in the case of 5 microgramm ml)1 of extended boar sperm) and to induce dissipation of mitochondrial membrane potential. other studied strains had no toxic effect. conclusions: production of extracellular proteolytic enzymes and toxic metabolites are among the potential virulence factors of trichoderma strains as emerging fungal pathogens. this work was supported by grants f037663 of the hungarian scientific research fund and 53305 of the academy of finland. occurrence of scabies in patients and in the staff of healthcare facilities objectives: scabies is an itching dermatosis which continues to be a frequent health problem. long-term follow-up of the epidemiological situation confirms a thirty-year cycle for which there is no satisfactory explanation. methods: standard methodology of diagnostics is based on a) subjective sensations in the patient b) objective dermatological finding c) positive epidemiological history d) laboratory demonstrations of causative agent e) remission of symptoms upon specific therapy. cases of scabies diagnosed and reported by dermatologists nationwide are included by the public health service in the central system epidat (niph, prague). results: the last two documented epidemic waves of scabies affecting the czech republic peaked in 1970 and in 1993. specific morbidity is the highest among persons 15-24 years of age (it closely connected with sexual activity-the source of infestation in 60% of adults was a sexual partner and that is confirmed by an almost identical trend in reported scabies morbidity and fresh cases of syphilis in 1965-1999 years), but the morbidity trend has been falling sharply after the year 1993. however, morbidity in persons over 65 years of age has been gradually rising over recent years. group epidemics (86) have been repeatedly reported in such facilities as departments of gerio-psychiatry, institutions for long-term chronic patients, old folks homes, social care institutes and charity facilities. that occurs in bed-ridden subjects hospitalized on a long-term basis, the mentally retarded and the superannuated, in whom the scabies is of a nosocomial character. the affected staff of healthcare institution is very often the source of further transmission to others, namely to immobile patients, as well as to especially vulnerable individuals under higher risk of infection. analysis of the reported cases revealed a 13% share of healthcare workers in this infection. under greater risk are senior nurses (62%), junior nurses (21%), auxiliary paramedical staff (13%)and the last of all physicians (4%). scabies as an occupational disease occurs almost exclusively in the healthcare sector. conclusions: scabies poses on increased risk for the younger population (active scabies) and the elderly population (passive scabies). it poses a great occupational risk in the paramedical healthcare staff carrying out nursing or managing services. it is necessary to consistently observe preventive and repressive epidemiological measures in the population. fire ants, the new public health problem in iran k. akbarzadeh, m. nateghpour, s. tirgari (tehran, ir) ants are probably the most successful of all insects. they are present almost in all countries and all places. a few of them can bite, sting and squirt formic acid. formica rufibarbis and a few the others are secondary hosts of dicrocoelium dentriticum in iran. but the newest public health problem depending on ants in the country is biting and stinging of fire ant pachycondyla sennaarensis (formicidae; hymenoptera) in south and southeastern iran. ecology, biology and morphology of p. sennaarensis were considered in iranshahr county (southeast of iran) where has been much infected because of the ant. according to the survey over 92% of questioned individuals had bitten at least once with the ant. usually the effects of the stings are mild but this ant is capable of multiple stinging and this can induce annoyance people especially in children. objective: tick play as important role in diseases transmission to human being. different tick-borne diseasea including borreliasis, cchf have been reported from iran. method: an attempt was made to determine the fauna and geographical distribution of soft and hard ticks in this area from october 2002 until april 2004. about 10% of the villages were selected randomly and tick collection was carried out using standard method. ticks were collected from indoor resting and hiding places in 70 villages as well as inside stables, rodent borrows, on sheep, lamps, goat and cattel by monthly in each season. result: 3382 ticks were collected and identified using national systematic key. the copmosition and frequencies of them were as follows: ornithodoros lahorensis (11%), argas persicus (23.12%), rhipicephalus bursa (4.3%), rhipicephalus sanguines (1.53%), haemaphysialis concinna (9.6%), haemaphysialis sulcata (26.8%), haemaphysialis punctata (1.54%), hyaloma schulzei (8.16%), hyaloma marginatum (7.21%), hyaloma dentriticum (3.08%), hyaloma asiaticum(1.53%). conclusion: distribution and ferquency of tlck was differ in various season and location. evaluation of the use of saliva to soothe bloodsucking arthropod bites (dedicoat m et al, 2004 , j infect dis 190:1068 . the conditions for hhv-8 transmission are met when i) a child's skin responds to the bite of a blood-sucking arthropods, and ii) an hhv-8-seropositive mother (or caregiver) attempting to relieve the child's itching and to reduce scratching applies her infective saliva to the bite's site. the use of saliva is a traditional behavioural practice in sub-saharan africa, for healing with herbal leaves crushed and mixed with saliva and for medical practices and in the premastication of food (wojcicki jm, 2003 , br j cancer 89:2016 . the human behaviour associated with the transfer of saliva from parents to infants and the use of herbal leaves treated with saliva in relation to arthropod bite could be a risk factor for hhv-8 infection and perhaps other infections too (such as the hepatitis b virus). methods: to evaluate to what extent saliva is used to heal insect bites on children's and adolescentsõ skin, we draw up a questionnaire directed at students of elementary and intermediate school. two groups were tested, one from italy (a school near rome, latium, and schools in veneto) and one from a sub-saharan african country known for high transmission levels (uganda, between kampala and entebbe by lake victoria). results: the frequencies of the use of saliva were not significantly different in latium (12.5%) and in veneto (14.6%) so that an average frequency of 13.5% (96 / 712) has been compared with the frequency of 74.8% (119 / 159) from uganda (p << .0001). the frequencies in the two groups are related also to a different cultural and economic development and improvement in hygiene. conclusion: we suggest that blood-sucking promoter arthropods can play a role in the spread of hhv-8 infection particularly in africa. therefore, a behavioural change limiting the use of saliva on the skin and to prepare herbal leaves, could be an attempt to control the spread of hhv-8 infection. aim of study -to find out in csf some baseline markers of inflammation and redox status for the estimation of development tendences and clinical course of pathological process in tbe. patients and methods: 60 patients with meningeal and meningoencephalitic form of tbe treated at the infectology center of latvia were included in this study. diagnosis of tbe was confirmed by elisa, enzygnost, anti-tbe igm in blood and/or csf. traditional clinical criteria were used for the characterization of tbe severity degree. some nontraditional lab methods were used: c-reactive protein (crp) (immunoturbidimetrically) and reduced glutathione (gsh) concentration (colorimetrically) detection in csf. results: concentration of crp in csf was significantly higher in tbe patients in acute phase (5.55 ± 1.0 mg/l; n = 34) than in reconvalescence (2.41 ± 0.5 mg/l; n = 26), p = 99%. crp and gsh in about 50% of tbe cases in csf was under detectable values. the second part of tbe patients demonstrated tendency of the increasing of gsh during acute stage of disease (1.64 ± 0.20 mg%; n = 32) if compared with reconvalescence (1.41 ± 0.20 mg%; n = 24), p = 61%. no statistically significant differences of crp and gsh concentrations in csf were found in cases of moderate meningitis form of tbe if compared to severe meningoencephalitic form of tbe. conclusion: 1) analysis of data obtained from patients with tbe gives evidence of substantial crp and gsh changes in csf in dependence on disease stage, but not on its severity degree. 2) these objective biochemical data from csf investigation give proof that there are no separately moderate and severe tbe cases in clinic: they have to be interpreted always as equally severe. 3) the origin of crp and gsh in csf is unknown (production in cns and/or from blood due to blood-brain barrier permeability increasing?). background: cholera is a severe disease caused by vibrio cholerae. the natural reservoir of this aquatic pathogenic bacterium is referred to as the ôaquatic environmentõ. our group showed that egg masses of the non-biting midge chironomidae spp. (diptera) harbour v. cholerae and provide nutrient source for their multiplication, thereby offering a new natural reservoir for the cholera bacterium. objective: the presence of viable v. cholerae on flying adult chironomids will suggest that v. cholerae can be carried through the air to other water bodies by the adult flying insects and hence a new route of infestation of water sources by the bacteria is suggested. methods: in field studies chironomid egg masses and adults were simultaneously collected from the same environment. in addition fresh, drinking quality water was left exposed or netted in the vicinity of adult chironomid hatching foci. the exposed water was monitored for chironomid egg masses and v. cholerae presence. in the laboratory those experiments were repeated with v. cholerae o9 tagged with green fluorescent protein. results: to date, more then 30 v. cholerae serogroups have been isolated from chironomids egg masses and adults. in the field experiments, a clear-cut connection between netting and v. cholerae absence was noted implying that a relationship exist, between entrance of large invertebrate to water bodies and presence of v. cholerae in that water. in the laboratory experiments, the gfp tagged bacteria were found on the outer surface of the adult chironomid, in locations prone to microbial attachment. conclusions: the evidence shown here suggests that aerial transport of v. cholerae by chironomids flying adults is feasible and does lead to v. cholerae contamination of unprotected water bodies. objectives: cholera is a severe and potentially life-threatening diarrheal disease caused by certain spices of the gram-negative bacterium vibrio cholerae. many millions of cases of cholera occur annually, in epidemic and pandemic forms due to v. cholerae o1 and o139, and also sporadically due to non-o1 and non-o139 v. cholerae strains. in this context, the survival of v. cholerae in nature is of interest from an epidemiological perspective. although the natural reservoirs for survival and multiplication of v. cholerae are far from compeletly disclosed, our previous study has shown that free-living amoebae can be reservoirs for the seventh-pandemic v. cholerae o1 el tor-inaba strain n16961, which can survive and grow intracellularly in acanthamoeba castellanii.the aim of this study was to examine the ability of different strains of v. cholerae o1 el tor to grow and survive in acanthamoeba castellanii, and to examine whether intra-amoebic survival of bacteria alter their pattern of resistance and sensitivity to different antibiotics. methods: v. cholerae o1 el tor strains were co-cultured with a. castellanii for more than two weeks. the interaction between these microorganisms was followed by viable counts of alone-and co-cultivated microorganisms. intra-amoebic growth and localization of each bacterial strain were estimated by gentamicin assay, viable count, microscopy, and pcr to detect cholera toxin gene and amoebic 18s rna gene disclosing symbiont-host association. results: the results show that examined v. cholerae o1 el tor strains multiplied and survived inside trophozoites and cysts of a. castellanii. the bacterial internalization was in cytoplasmic compartment of the amoebae cells. the relation between these microorganisms in co-cultures could be classified as symbiosis, since presence of the amoebae enhanced growth of bacterial strains, and presence of the bacteria did not affect amoebic growth. the intra-amoebic survival of bacteria did not alter their pattern of resistance and sensitivity to different antibiotics such as ampicillin, gentamicin, and tetracycline. conclusions: this study shows a facultative intracellular behaviour of examined v. cholerae o1 el tor strains, which is in contrast to the general held view, which considers the bacterium to be extracellular microorganisms. the clinical importance of free-living amoebae is their possible role as ôtrojan horseõ. the genetic variation of vp7 and vp4 antigenic proteins was studied in 38 g4 strains recovered in palermo from 1985 to 2003. vp7 sequence analysis showed that 14 strains, recovered during 1999-2003, though cross-reacting with g4-st3:1 mab, belonged in fact to genotype g9 and were 99% identical to g9 strains recovered in palermo from 1999. sequence comparison of 23 g4 strains grouped them into three sublineages (ia to ic) containing respectively viral strains collected in 1990-1994, 1995 and 1999-2003 . amino acid sequences were well conserved within each sublineage and a peculiar single amino acid insertion was observed in the variable region 4 of all sublineage ic strains. both g4 and g9 italian strains exhibited p[8] genotype and their vp4-encoding sequences clustered in previously defined lineages following a temporal distribution: strains collected in 1989-1993 fell within lineage p[8]-1, while those recovered in 1995-2003 clustered in lineage p[8]-3. the variety of their deduced amino acid sequences enabled us to describe, respectively, two (1a and 1b) and six (3a-3f) different patterns of substitutions with regard to lineage p[8]-1 and p[8]-3 reference strains. comparison of vp7 and vp4 gene sequences of italian and european g4 strains indicates that different g4p[8] populations have been circulating in europe over the years and that the latest italian strains are more closely related to recent isolates from other countries than to the reference vaccinal st3 strain. these results will help increase knowledge about g4 rotavirus epidemiology and evolution, and might be useful for future rotavirus vaccine formulation. detection of rotavirus, adenoviruses and astrovirus in gastroenteritis cases among young children, first experience with adenovirus identification by pcr and microplate hybridisation assay , adeno (all human serotypes) and astroviruses antigens (seven human serotypes) was performed by enzyme immunoassay method (dako ideia rotavirus, adenovirus and astrovirus tests, respectively). all specimens positive for adenoviruses were confirmed and identified by a pcr-microplate hybridization assay (pcr adenovirus consensus, argene, biosoft), which used adenovirus genus-specific primers and one generic and six species-specific probes defined in the va rna gene. according to our results: rotavirus was detected in 63 cases (9.2%), adenovirus in 15 cases (2.2%) and astrovirus in 15 cases (2.2%). pcr adenovirus consensus assay identified the adenovirus species from all the 15 cases: adenovirus species f (enteric adenovirus serotype 40 / 41) in 11 cases (1.6%), adenovirus species c in 2 cases (0.3%) and adenovirus species a in 2 cases (0.3%). rotavirus gastroenteritis presented a seasonal distribution in spring, while adenovirus and astrovirus gastroenteritis presented no seasonality. conclusions: rotavirus gastroenteritis was significantly more common than adenovirus and astrovirus infections in young children in our territory. pcr adenovirus consensus technique was a useful method for the identification of adenovirus in stool specimens. eventhought enteric adenoviruses 40 / 41 (species f) caused the majority of acute gastroenteritis due to adenovirus, non-enteric serotypes (species a and c) were occasionally involved in the aetiology of acute diarrhoea. the study continues with the examination of great number of specimens and patients for the investigation of more useful and specific results. immigrant inpatients from 1999 until today: 'pressure' exerted over infectious disease wards r. manfredi (bologna, i) objective: foreign individuals officially living in bologna and province are 39186 with predominant origin from northern africa (31.5%), eastern europe (22%), and far east (12%). the temporal trend of admissions of patients coming from outside of the european union was examined according to a broad series of variables. methods: through the electronic databases of our hospital located in a metropolitan area of northern italy, informations related to all foreign patients (p) hospitalized or followed by day-hospital centres have been collected from january 1999 up to march 31, 2004. after recording all discharge diagnosisrelated group (drg) codes we focused attention on those regarding infectious diseases. results: overall discharges were 339051, with 6670 regarding foreign p:a prevalence of the female sex (64.4%) was noticed. in 49.6% of foreign p,the mean age ranged from 20 to 35 years. the rate of foreigners compared with overall admitted p varied from 0.3 of year 1999 to a. maximum of 0.47% of year 2002. a major involvement was borne by obstetrics-gynecology (39.3%), internal medicine (13.1%), specialistic surgery (7.3%), specialistic medicine and pediatrics (7.3% each), and emergency medicine (7.1%). when considering infectious disease-related drgs, tuberculosis (142 cases), hiv-aids (25), chronic viral hepatitis and related complications (22), septicemia, central nervous system infection, and acute viral hepatitis (12 episodes each), were the most frequent discharge codes followed by other infectious disease drgs (72 cases). the duration of admission of foreign p was very short (1-3 days) in 42.1% of cases,while it reached 4-7 days in 22.6% of p,and 8-14 days in 11.2% of cases, while the mean hospitalization time at infectious diseases ward was 8.6 ± 3.2 days (p < .002). even 549 hospitalizations ended with voluntary discharge which occurred during the first day of admission in 61.8% of p. discussion: our pilot study demostrates that the need for health care expressed by foreign p is increasingly frequent and covers a very broad spectrum of disorders with infectious diseases-associated illnesses gaining a increasing role through time. a significant problem is represented by the tendency to very short duration of hospitalization for foreign p, often ending with voluntary discharge while admissions carried out at our specialized infectious disease ward are significantly more prolonged, although they are expected to give an increased benefit in terms of effective management. change of the characteristics of natural focuses of plague and microbiological properties of plague microbes as a result of consequences of the natural calamity on aral sea enzootics of plague as that of any other transmissible feral herd [feral nidal] infection are determined by the interaction of a disease's causative agent and its warm-blooded carriers under specific ecological environment. introduction and objectives: change of the characteristics of natural focuses of plague and microbiological properties of plague microbes as a result of consequences of the natural calamity on aral sea. methods: conventional microbiological research methods were used, when studying microbiological properties of 116 strains of plague microbe from kyzylkum natural focuses of plague isolated from rodents, ticks and fleas. results: strains from the kyzylkum natural focuses of plague have all determinants of virulence, show characteristics that allow attributing them to the continental variety, i.e. glycerin digestion, do not ferment rhamnose and decompose arabinose; they have nutrient need that is typical for the strains from the central asian desert focus. there are differences in the need for amino acid leucine, whereas strains from ustyurt and gissar focuses are dependant on it. out of 116 strains from the kyzylkum natural focus only 8 turned out to be dependant on luecine. there were identified stains from the kyzylkum natural focus that had essentially different properties as compared to the majority of isolated cultures, in particular, those can not synthesize fraction 1 of the plague agent; have growth factor of different nature, i.e. lower dependence on phenylalanine. plague bacteria strains circulating in rodent populations in the kyzylkum natural focus are characterized by low virulence in white mice and guinea-pig, although they have all the determinants of virulence. when studying resistance to antibiotics, there were identified strains non-susceptible to streptomycin (5%). conclusions: thus, studying the properties of the causative agent of plague is of great importance for solving problems of natural focuses and understanding patterns of fluctuations in the epizootic situation in a certain territory. this understanding is an integral part of determining the epidemic potential of natural focuses, and is used in organizing epidemiological surveillance of the infection. objectives: toll-like receptors (tlr) play a key role in the recognition of products from virtually all classes of pathogenic organisms. amyloid peptides also can stimulate the innate immune system. for this reason, we hypothetized that bacterial compounds and amyloid peptides may jointly stimulate the innate immune system. methods: the interaction of endogenous and exogenous stimulators of innate immunity was examined in primary cultures of mouse microglial cells after application of defined toll-like receptor (tlr) agonists [lipopolysaccharide (lps) (tlr4), the synthetic lipopeptide tripalmytoyl-cysteinyl-seryl-(lysyl)3-lysine (pam3cys) (tlr2) and single-stranded unmethylated cytosineguanosine (cpg) oligodesoxynucleotide (tlr9)] alone and in combination with amyloid beta peptide (abeta) 1-40. supernatants from stimulated glial cultures and unstimulated controls were analysed for nitric oxide (no), tumor necrosis factor-alpha (tnf-alpha) and interleukin-10 (il-10) production. results: co-administration of abeta1-40 with lps or pam3cys led to an additive release of no and tnf-alpha. in contrast, coapplication of abeta1-40 with cpg led to a substantial decrease of no and tnf-alpha release compared to stimulation with cpg alone. cpg was the only compound investigated, which induced the release of the anti-inflammatory cytokine il-10. conclusion: the additive effect of lps and pam3cys and abeta may be one reason for the clinical deterioration frequently observed in patients with alzheimer's disease during infections. the substantial decrease of no and tnf-alpha release after cpg and abeta application compared to stimulation with cpg alone suggests that not all microbial products enhance the stimulatory effect of abeta on innate immunity. the cause for the divergent behaviour after activation of tlr9 versus stimulation of tlr2 and tlr4 probably lies in differences of the signaling cascade. hla-b27 is a marker for susceptibility and severity of reactive arthritis after salmonella, shigella, and yersinia infections but not after campylobacter and e. coli enteritis p. schiellerup, h. locht, k.a. krogfelt (copenhagen, dk) objectives: reactive arthritis (rea) is a well-known complication after infections with salmonella, yersinia, shigella and campylobacter. the presence of tissue-antigen hla-b27 is associated with rea, and hla-b27 may be a marker of a prolonged disease course. we designed a case-case comparison study of rea after gastrointestinal infections caused by salmonella, yersinia, campylobacter, shigella and e coli, to evaluate the impact of hla-b27 on attack rate and severity of rea between these 5 bacteria. methods: between january 2001 and november 2002 consecutive patients were included from the danish registry of gastrointestinal infections, when culture positive for salmonella, yersinia, campylobacter, shigella, or e coli. all patients were addressed by a questionnaire. in this study rea includes reactive arthritis and arthralgia. debut of pain in a previously healthy joint was registered and patients were asked to mark swollen or tender joints on a drawing. a vas-scale was used for assessment of pain caused by rea. all patients were encouraged to donate blood samples for serology and hla-b27 typing (pcr). results: a total of 2106 patients returned the questionnaire (response rate 67%). only subjects with mono-infections were included. the male/female ratio was 40/60. blood samples were obtained from 1558 (74%): campylobacter (n = 693), salmonella (n = 503), e. coli (n = 213), shigella (n = 74) and yersinia (n = 75). the overall prevalence of hla-b27 was 9%, corresponding to the rate within the danish population. odds ratios for contracting rea when the individual was hla-b27 positive were; campylobacter 1.70 (ci: 0.89; 3.43), salmonella 3.4 (ci: 1.83; 6.37), e. coli 1.1 (ci: 0.23; 5.04), shigella 33.5 (ci: 2.48; 452.78) and yersinia 5.1 (ci: 1.03; 25.68). thus, hla-b27 was significantly more frequent among rea-patients with salmonella, yersinia, and shigella compared to campylobacter and e coli. the vasscore for rea was significantly higher in the groups with yersinia and salmonella. there was a significant positive correlation between vas-score and the fractions of patients that were hla-b27 + . conclusions: this large comparative study shows that hla-b27 has a significant impact on the susceptibility to contract rea after salmonella, shigella, and yersinia, but not after campylobacter or e. coli. furthermore the severity of rea as measured by vas differed among bacterial species and was strongly correlated to the presence of the hla-b27 antigen. objectives: the normal gut microbiota triggers experimental colitis and large intestinal manifestations of human inflammatory bowel diseases (ibd). however, the availability of animal models for the study of ileitis is limited. thus, only little is known hitherto in small intestinal ibd. recently, parasiteinduced ileitis in the mouse, mimicking characteristics of ileitis in human ibd, was proposed as a model for crohn's disease (cd). oral infection of susceptible c57bl / 6 mice with toxoplasma gondii induces a severe th1-type immunopathology, which is restricted to the terminal ileum. in order to unravel the mechanisms underlying ileitis, we used this model to monitor microflora changes during ileal inflammation and determined contributions of the gut microbiota to ileal disease. methods: c57bl / 6 mice were infected perorally with 100 cysts of t. gondii (me49 strain). ciprofloxacin, metronidazole, ciprofloxacin plus metronidazole (each 50 mg / kg body weight / d, p.o.), piperacillin plus tazobactam (200 mg/kg body weight/d, i.p.) were applied from day 0 until day 8 after t. gondii-infection. the ileal bacterial flora was analysed on day 8 p.i. by microbiological and molecular methods (16s rdna-targeted denaturing gel electrophoresis (dgge), sequencing of clone libraries). results: microbiological and molecular approaches revealed that ileal inflammation leads to an increased total bacterial load and to drastic changes in the flora composition. in the acute stage of disease, the grampositive cocci and rods -predominant in the ilea of healthy mice -are displaced by gramnegatives, identified as escherichia coli and bacteroides sp., respectively. antibacterial treatment (ciprofloxacin, metronidazole, a combination of both, or piperacillin plus tazobactam) ameliorated disease symptoms and reduced ileal inflammation. this was also seen in spf-mice colonized by only one grampositive bacterial species. conclusions: the fact that gramnegative bacteria accumulate during acute ileitis and contribute profoundly to intestinal inflammation is well in line with similar observations in experimental colitis and in human ibd. this provides evidence that gut flora modulation is a valuable therapeutical strategy for ibd treatment. finally, the contribution of gut microbiota to ileal disease supports the use of the parasite-induced small intestinal inflammation as a model for ibd research. objectives: pathophysiologic mechanisms that contribute to brain injury in neuroinflammatory disorders include breakdown of the blood-brain-barrier, extravasation of leukocytes, cerebral hypoperfusion and vasculitis. matrix-metalloproteinases (mmps) including the collagenases mmp-2 and -9 are crucially involved in all these steps. in this study we aimed to assess the extent of in-vivo collagenolytic activity in cerebrospinal fluid (csf) by determination of the amino acid hydroxyproline, a major and exclusive degradation product of collagen. methods: paired serum and csf samples from patients with bacterial meningitis (n = 11), aseptic meningitis/encephalitis (n = 17), multiple sclerosis (n = 13), and normal csf (n = 12) were assessed. degraded collagen was hydrolysed to dissolve its major component hydroxyproline, which subsequently was determined spectrophotometrically. csf levels of mmp-2 and -9 were studied by gel zymography. in a rat model of pneumococcal meningitis localization of collagenolytic activity was performed by in-situ zymography with intramolecularly quenched gelatin. results: hydroxyproline in csf from patients with bacterial meningitis was significantly increased compared to all studied groups (p < 0.001) while serum hydroxyproline did not differ significantly between the groups. the amount of hydroxyproline in csf correlated significantly with the amount of mmp-9 (r = 0.8; p < 0.001). in the rat model in-situ zymography localized gelatinolytic activity to the subarachnoidal and ventricular space inflammation and in association to cortical lesions. the study documents a significant increase of the collagen degradation product hydroxyproline in csf of patients with bacterial meningitis. the close correlation of hydroxyproline and mmp-9 in the csf validates the assessment of hydroxyproline as an index for csf collagenolytic activity in neuroinflammatory diseases and supports a role for collagenases in the pathogenesis of bacterial meningitis. introduction: toxic shock syndrome (tss), is an acute, noncontagious systemic illness. it is caused by the toxin producing strains of s. aureus and the b-haemolytic streptococci and can occur in any non-immune person exposed to a tss toxin. tss is commonly associated with menstruation and tampon use, however can also be related to skin or soft tissue infections, particularly post surgical, skeletal infections or respiratory tract infection. tss is often non-immunising and recurrent menstrualassociated tss is well-described. literature suggests that tss is extremely rare, but diagnostic difficulties can lead to misdiagnosis and tss can be fatal if left undiagnosed. we report a series of three cases of tss, presenting within a short period of time. case reports: case 1. 17 year old female, presented with sudden onset collapse, diarrhoea, vomiting and abdominal pains. she gave no history of menstruation and an initial diagnosis of severe gastroenteritis was made. she failed to respond to conservative management and required itu support. she was discharged with no firm diagnosis and re-presented one month later with similar symptoms, when a diagnosis of staphylococcal tss was confirmed. case 2. 15 year old female, presented during menses with sudden onset rash, rigors, severe diarhhoea, vomiting and abdominal pains. she was diagnosed with staphylococcal tss on admission. case 3. 28 year old female. presented with sudden onset severe diarrhoea, vomiting, pyrexia and rash. she gave no history of menstruation. she responded poorly to treatment, required itu support, high dose steroids and was eventually diagnosed with streptococcal tss. discussion: diagnostic criteria for tss include high fever, hypotension, erythematous rash and a complicated multisystem disfunction. patients often require aggressive management. the public health laboratory service reports an average of 18 cases of diagnosed tss in the uk per year. however, because of the uncommon and difficult nature of the diagnosis, many cases are misdiagnosed and therefore go unreported. it is essential to maintain a high level of suspicion for patients who are epidemiologically at high risk, but importantly, also the less ill patient with suggestive symptoms who fails to meet all diagnostic criteria but are in an at-risk group. high morbidity and mortality has been reported for undiagnosed and untreated cases. objectives: staphylococcus aureus colonization and infection of burn wounds increases morbidity and delays wound healing of patients suffering from burn wounds. a vulnerable moment in the route of transmission of s. aureus is the exposed burn wound during dressing changes. the aim of this study was to evaluate the effect of dressing changes under laminar flow conditions on burn wound patients with regard to s. aureus colonization. methods: from the first of february 2003 to the first of june 2003, 25 patients were included in this study at our 10-bed burn centre. in 12 of these patients the dressings were changed under laminar flow conditions. all patients were frequently screened for s. aureus in nose, throat and burn wounds. all s. aureus isolates were genotyped with pulsed field gel electrophoresis. results: at admission 17 / 25 (68%) patients had no s. aureus colonization in their nose, throat or burn wounds. dressing changes under laminar flow conditions were carried out on 8 / 17 patients of this group. however, 6 / 8 (75%) patients acquired burn wound colonization with s. aureus during their stay at the centre. six out of 9 patients (67%) whose dressing changes were not carried out under laminar flow conditions acquired burn wound colonization with s. aureus. a total of 12 patients acquired burn wound colonization; these patients also acquired carriage of s. aureus in nose or throat. the same type of s. aureus was carried in the nose or throat as well as the burn wounds in 7 (58%) of these patients. the results of this study suggest firstly that dressing changes under laminar flow conditions does not prevent colonization of burn wounds with s. aureus, and secondly, that the exogenous route plays an important role in the transmission dynamics of this pathogen in burn wound centres. future research should be focused on this mode of transmission of s. aureus in this special circumstance. mupirocin prophylaxis to prevent staphylococcus aureus wound colonisation in patients admitted to a burn centre objectives: staphylococcus aureus (s. aureus) colonisation of burn wounds increases morbidity and delays wound healing. many burn wound colonisations with s. aureus are endogenous in nature. the aim of this study was to evaluate the effect of eradication of nasal s. aureus with mupirocin in patients with regard to s. aureus colonisation of their burn wounds. methods: from september 2000 to march 2001, 42 patients admitted to our 10-bed burn centre were screened for s. aureus in nose and burn wounds. isolates were genotyped with pfge. all patients received nasal mupirocin at the time of admission to the burn centre. fifty-five patients from the same unit who were followed from september 1997 till may 1998 and had not received mupirocin prophylaxis served as a historical control group. results: at admission 29 / 42 (69%) patients in the mupirocin trial had no s. aureus colonisation in their burn wounds. of this group 25 patients were non-carriers and 4 patients were nasal carriers of s. aureus. seven (28%) non-carriers and 2 (50%) nasal carriers acquired burn wound colonisation with s. aureus during their stay at the centre. of the historical control group, 39 (71%) patients had no s. aureus colonisation of their burn wounds at the time of admission. of this group 31 patients were noncarriers and 8 patients were nasal carriers; 18 (58%) non-carriers and 7(88%) nasal carriers acquired s. aureus wound colonisation during their stay. the overall probability of wound colonisation in the patients treated with mupirocin was significantly lower than in the historical non-treated group (p = 0.01, chi-square test with yates correction). conclusion: the results of this study suggest that application of nasal mupirocin to all patients upon admission to a burn centre may reduce but not eliminate the risk of subsequent s. aureus colonisation of burn wounds. other measures including improved infection control practices and eradication of exogenous s. aureus reservoirs, including s. aureus carriage among healthcare workers, may be necessary to further reduce the incidence of s. aureus burn wound colonisation. controlling an outbreak of staphylococcus aureus on a surgical ward results: the characterisation of the s. aureus in february showed a cluster of identical strains in three patients and hcw a. the strains of two patients were similar to the strain of hcw b. three patients had a unique strain. in the surveillance period a total of eighteen isolates in sixteen patients were found. in the surveillance period the epidemic-strains were found in other patients but only in patients who have been on the ward during the epidemic period. two patients had an identical new isolate. that indicates that the exchange between patients of s. aureus may occur without notion. another remarkable observation was that some of the patients lost their original strain and acquired a new isolate of s. aureus during hospitalisation. conclusions: a small outbreak of s. aureus could be traced to 2 hcw who proved to be carriers. relieving them from patient contact stopped the outbreak. molecular surveillance is an appropriate way to evaluate the success of measurements taken and to underscore the importance of hygienic measures. risk factors for s. aureus carriage in health care workers a. widmer, m. lang, r. frei (basel, ch) introduction: s. aureus (mssa) carriage is common among health care workers (hcws)and might be associated with type of care provided and other potential risk factors. transmission of mssa has been observed by ''cloud adults''. objectives: to determine risk factors for mssa carriage in hcws, and the percentage of methicillin-resistant s. aureus (mrsa) in a tertiary care centre. methods: retrospective review of all data of staff health department and the microbiology laboratory. all hcws who were working in a high mrsa prevalence country were routinely screened at the time they were hired by the institution. in addition, data were generated from mrsa screenings of hcws if there was evidence for nosocomial transmission of mrsa in patients. mrsa was defined as s. aureus being oxacillin-resistant and expressing the pbp2' and/or being positive for the meca gene by pcr. data of hcws were collected by chart review in a case-report form that included demographic data, work place, skin diseases and other variables. data were compared by univariate and multiple logistic regression analyses. results: a total of 3474 swabs were taken from 1217 hcws between jan 1997 and dec.2001. data were available from 1182 hcws:50% originated from switzerland, 27% from germany, 4% from france, and 19% from other countries. the overall prevalence of mssa and mrsa was 53% and 1%, respectively. more than one sample was available from 332 individuals: of the repeat samples, 38% were negative, 32% were negative, but had one single positive culture, and 30% were consistently positive. sex, profession, work place, number of years at the institution was not associated with s. aureus carriage (p > 0.2). the only significant risk factor for s. aureus carriage was young age, defined <35years (p = 0.02). the prevalence of mssa increased from 51% (1997) to 63% (2001) . eight hcws were carriers of mrsa: they were young and half of the them reported skin diseases. conclusion: s. aureus carriage among hcws is common: only young age remained a risk factor even after adjusting for other variables. mrsa was rare among hcws. prevalence of nasal carriage of methicillinsusceptible and methicillin-resistant staphylococcus aureus among hospital personnel and outpatients st. fokas, sp. fokas, c. tsamolia, m. skoutari (eghio, gr) objectives: to assess the nasal colonization of staphylococcus aureus among hospital staff in the absence of an epidemic and to compare it with the nasal carrier status among outpatients, focusing on the mrsa rate. methods: the study included 150 hospital personnel and 150 adults admitted to outpatient clinics of our hospital. given that a history of hospitalization is the most important facilitative factor for colonization of staphylococcus aureus we exclude individuals with a history of hospitalization in the preceding year of our study. the specimens were obtained with swabs from the anterior nares-ecological niches of s. aureus-and cultured according nccls guidelines. id 32 staph strips were used for identification and confirmed with slidex staph plus kit (both by biomerieux). mrsa strains detection was achieved with slidex mrsa detection kit of the same company. antibiotic susceptibility of mrsa strains was determined with atb staph method (version 2000) according to the recommendations of the producer (biomerieux). results: among hospital staff the carriage rates of mrsa and methicillin susceptible s. aureus were 2.7% and 20% respectively. none of the outpatients was found to be a nasal carrier of mrsa. in addition mssa carriage in this group was 11%. the prevalence of mrsa nasal carriage rate among personnel working in the different medical wards was also examined. the difference in the rates among personnel working in the surgical ward, operating theaters and the rest of the staff was statistically significant (p < 0.001). all mrsa strains were susceptible to oral antibiotics. conclusions: although the prevalence of mrsa is low among hospital personnel, education is needed as they are one of most important reservoirs for nosocomial mrsa infections. the low incidence of mrsa infections around the time of the study suggests that with usual infection control practices mrsa spread was avoided. all mrsa carriers were treated with intranasal muripocin for eliminating the nasal carrier status. continuous monitoring of mrsa carriage is needed to avoid nosocomial spread. aids patients are a special population related with high risk of infection due to staphylococcus aureus. previous colonization is a recognized predisposing factor for development of infection due to this organism. however, nasal carriage of s. aureus in hivpatients is still not completely characterized. objectives: to describe the epidemiological features of community-acquired s. aureus nasal colonization in out care hivpatients based on the molecular genotypes. methods: hiv-outpatients without previous hospitalization within the last two years were screened for s. aureus nasal colonization. three samples from each patient were collected, and the variables associated with colonization were assessed. nasal carriage was classified as: absent, transient colonization or persistent colonization. the persistent colonization was assessed according to the molecular profiles performed by pulsed-field gel electrophoresis, in: simple (same profile), multiple (different profiles) or combined (two with the same e and one with different profiles). results: 111 patients were enrolled in the study. seventy patients (63%) had at least one culture positive for s. aureus and 99 patients concluded three samples collection. only one isolate was a community acquired mrsa. the rates of colonization was higher when two or more samples were taken, ranging from 45.5% in the first sample up to 65.6% when 3 samples were collected. hiv-patients with aids were more likely to be colonized than non-aids patients (p=0.02). among the patients with s. aureus nasal carriage, 39% were transient and 61% were persistent carriers, whose genomic subtype was: 61.5% simple, 20.5% combined and 18% multiple persistent colonization. previous use of antibiotics was associated with absence of s. aureus colonization (p < 0.05). conclusions: hiv out care patients had high rates of oxacillinsusceptible s. aureus nasal colonization. regardless, the recent literature, only one isolate was community-acquired mrsa. the most common characteristic of colonization is simple persistent colonization showing the same genomic profile. objectives: s. aureus bacteraemia (sab) continues to be a major problem related to both community-acquired and nosocomial infection. we undertook an evaluation of a large cohort of patients with sab to assess features of the infection and predictors of mortality. patients and methods: 238 cases of sab admitted to our hospital (2000-3) were prospectively studied. all patients had at least one positive blood culture for s. aureus and clinical symptoms of bacteraemia. sab was considered to be nosocomial if the first positive blood culture specimen was obtained > 72 h after admission and there was no clinical evidence of infection on admission. microbiological studies were carried under standard protocols. epidemiological and clinical characteristics, antimicrobial treatment and outcome (relapse or mortality) were analysed. results: 434 cases of sab were identified but only 238 were prospectively included in the study protocol. incidence was the highest in year 2001 (4.21 per 1000). out of the 238 cases, 167 (70%) were male with a mean age of 51 year (sd 24.89 have not yet implemented similar guidelines. this study evaluated the detection and reporting of mlsb results in staphylococcus using the bd phoenix tm automated microbiology system and bdxpert system with nccls, sfm or din as interpretative standards. methods: comments regarding mlsb resistance as listed in the nccls m100-s14 were converted into expert rules. special bdxpert rules were also constructed for the detection and reporting of mlsb resistance when applying sfm or din guidelines. a total of 177 strains of staphylococcus (146 s. aureus, 12 s. epidermidis, and 19 coagulase-negative staphylococci) were tested in phoenix panels containing erythromycin (e) and clindamycin (cc). nccls, sfm or din breakpoints were used to interpret the phoenix mic results. the double disk diffusion d-test (e and cc) was used as the reference method for the determination of mlsb resistance phenotypes. results: the phoenix e and cc mic values were interpreted based on the standard selected. the bdxpert rules were executed and applicable expert messages were displayed. the phoenix correctly detected 38 out of 43 constitutive mlsb (cmlsb) phenotypes as compared to the d-test results. the remaining 5 cmlsb strains were interpreted by the bdxpert as potential inducible mlsb (imlsb)/efflux phenotypes. a total of 69 imlsb and 22 efflux phenotype isolates were all reported by the bdxpert as imlsb/efflux phenotype and the users were alerted to perform d-test before reporting the cc results. the cc interpretation was suppressed in these isolates. the nccls, sfm, or din showed identical detection and interpretation of mlsb resistant phenotypes by the phoenix and bdxpert systems. conclusions: the phoenix and bdxpert systems can assist laboratories in rapid detection and accurate interpretation of mlsb results for staphylococcus. special messages can be used to communicate timely and accurate information to clinicians for proper therapy of staphylococcal infections with mlsb resistant phenotypes. in vitro activity of new compounds tested against multi-drug resistant s. aureus isolated in latin american medical centres background: oxacillin-resistant s. aureus (orsa) isolated in lamcs shows high rates of co-resistance (r) with most antimicrobial classes used in the clinical setting. we evaluated the mdr patterns of orsa strains collected in lamc by the sentry antimicrobial surveillance program in 2003. we also evaluated the susceptibilities (s) of new, investigational compounds against these important endemic pathogens. methods: among 1.437 s. aureus collected, 507 (35.3%) were r to oxacillin. these strains were isolated from 10 lamcs (5 countries) and tested against > 30 antimicrobials by nccls broth microdilution methods. orsa strains were grouped by the mdr patterns for the 8 primary drugs (47). conclusions: all newer compounds (lzd, q/d, dap and dal), tei and van were very active against endemic and epidemic orsa in lamcs. these s. aureus having mdr patterns (ave. r to 6 agents) will require greater use of newer compounds particularly in brazil. objectives: coagulase negative staphylococci (cons) represent a part of physiological microflora. however, in recent years, they have also emerged as nosocomial pathogens of infections associated with indwelling medical devices (e. g. pleural drains), mainly in immuno-compromised patients. the aim of this study was to determine dynamics of nasopharyngeal colonization by cons in patients with resectable lung cancer during short-term hospitalization, especially those receiving routinely preoperative antimicrobial prophylaxis. methods: in present study cons species were selected from patients divided into two groups: (i) patients who were undergoing pulmonary resection and receiving preoperative prophylaxis (piperacillin, cefuroxime alone or in combination with amikacin) -ôprophylaxisõ group and (ii) control group. throat and nasal specimens were taken from each patient twice, in four-days interval. all isolates were identified by the biochemical microtests api staph (biomerieux) and tested for drugs susceptibility according to nccls reccomendations. results: 187 strains of cons strains were selected from clinical specimens: 137 strains from ôprophylaxisõ group and 50 strains from control group. the most often isolated species was staphylococcus epidermidis with the predominated api numerical profile 6606113. a detailed analyses of the api numerical profiles and resistance to antimicrobial agents indicated that during four-days interval of hospitalization, changes in phenotypes among cons colonizing nasopharynx were observed in both groups of patients. the changes in drug resistance patterns were found more often in ôprophylaxisõ group compared to those in control group -48 / 54 (88.9%) and 11 / 22 (50%), respectively. this difference was statistically significant (p = 0.0007 -chi2 test with yates correction). conclusion: our data suggest that preoperative antimicrobial prophylaxis routinely used in patients with resectable lung cancer may be regarded as an important factor predisposing to changes in drug resistance patterns among cons isolates colonizing nasopharynx. the susceptibility of coagulase-negative staphylococci in a teaching hospital compared with a secondary care hospital results: in total, six different species were identified in 71 isolates, 72% of which were staphylococcus epidermidis. in the lumc the species diversity was greater than in the mca, with five isolates of staphylococcus haemolyticus found in the first. no difference between the two hospitals was found in activity of the antibiotics tested, except for li. the mic90s were as follows: v 2 mg / l, t 4 mg / l, c 16 mg / l, m 2 mg / l, l 8 mg / l. of li the mic90 was 2 mg / l in the lumc and 1 mg / l in the mca. according to dutch guidelines, in total 7 / 71 isolates were found resistant to t and 21 intermediate resistant. all isolates were susceptible for v. conclusion: the first choice glycopeptide for serious cons infections, t or v, has no influence on the susceptibility of cons for these antibiotics. according to dutch guidelines, the activity of t, c, and l against cons is relatively low, whereas the activity of m and le is in the susceptible range. objective: staphylococcus epidermidis is one of the bacterial species mainly implicated in foreign body associated infections. we have characterized several s.epidermidis clinical isolates for their ability to form biofilms and their resistance to antibiotics. methods: 76 s.epidermidis strains isolated from implantable medical devices have been collected from hospitals of central italy. susceptibility to penicillin, methicillin, erythromycin and tetracycline has been determined in vitro by e-test according to nccls guidelines. the specific antibiotic resistance determinants have been checked by pcr (blaz, meca, erma, ermb, ermc, msra, tetk and tetm). the ability to form biofilms has been determined: (i) by pcr, detecting genes specific for attachment and biofilm development (icaadbc operon, aap, and atle); (ii) by congo red agar (cra) plate test to assay the production of polisaccaridic intercellular adhesin (pia); (iii) by crystal violet (cv) stain to determine the biofilm biomass development on polystyrene microtiter plates; (iv) and by cslm microscopy observations to investigate biofilm structure. results: 94% of the strains under study was resistant to penicillin, 87% to methicillin, 72% to erythromycin and 25% to tetracycline. on the side of biofilm-specific genes detection, 66% of strains was positive to ica operon genes, 82% possessed atle gene, and 42% aap determinant. in 89% of the population, the cra test confirmed the correlation between the presence of ica genes and slime expression. the cv assay classified the quasitotality of our strains (97%) as biofilm producers on plastic surface. in addition, the distribution of optical density values (od540) obtained after cv stain, showed a significant statistical difference in biofilm biomass development between the ica-adbc-positive strains and the icaadbc-negative ones. finally, a correlation, although not always present, has been observed between ability of the strains to develop in a high-structureted biofilm and specific biofilm-formation determinants. conclusions: analysing the origin of colonization or infection in 6 patients with cons isolated from the operative site, the bacteria could only be traced in one patient who developed an infection. transmission most probably occurred by direct contact. the low level of bacterial contamination in the uca together with the lack of correlation of genotypes between isolates found in the uca and in the wounds suggest that airborne transmission does not play a major role in the development of swi. more than 90% of staphylococci found in air samples were not traceable to any investigated sources. most probably, they originated from non swabbed parts of the hcw's bodies. since 50% of the traceable isolates from the uca originated from non scrubbed team members, protection of the operative site by lfv appears to be suboptimal. clonality of endemic methicillin-resistant staphylococcus epidermidis strains isolated in a neonatal intensive care unit n. ben saida, a. ferjani, n. sfar, j. boukadida (sousse, tn) objectives: methicillin-resistant staphylococcus epidermis (mrse) is one of the important pathogen responsible for nosocomial infections particularly in newborns. the aim of our study was to establish if these infections are caused by endemic clones or by incidentally occurring bacterial strains of this ubiquitous species and to evaluate the performance of pcr iner-is256 as a novel method for typing mrse strains using pulsed-field gel electrophoresis (pfge) as the reference method. methods: twenty strains of mrse (meca+) has been collected during the period of survey (december 2003 to september 2004) from different pathological products of newborns hospitalized in a neonatal intensive care unit of a public maternity hospital in sousse, tunisia. identification of strains was done by conventional procedures and the api staph (api-biomerieux. sa). susceptibility testing to antibiotics was determined according to ca-sfm recommendations. all strains have been characterized by genotypage in pulsed-field gel electrophoresis (pfge) variety chef after smai macrorestriction. genomic profiles have been interpreted according to criteria of tenover (j. clin. microbiol 1995; 33:2223 -2239 . strains were also characterized by electrophoretic profiles obtained by pcr-based analysis of inter-is256 spacer polymorphisms. results: these mrse represent 41.6% of the total strain of s.epidermidis collected from the neonatology ward during the period of survey. majority of these strains come from blood sample (75%), other strains from vascular catheter (5%), pus (10%), sound (10%). 17 antibiotypes and 6 genotypic profiles according tenover criteria were individualized: type a (with 5 subtypes), type b, type c (with 1 subtypes), type d (with 1 subtypes), type e (with 2 subtypes)and type f. a total of four pcr patterns were obtained based on the interpretation criteria that pcr patterns exhibiting more than one band difference were considered to represent distinct types, type 1, type2, type3, and type4. conclusion: mrse appear to be endemic and multiclonal with a dominant clone in the neonatal intensive care unit. our study demonstrates that a significant proportion of mrse infections may be attributable to transmission among newborns and that certain strain can become endemic over long periods in this setting. clonality of staphylococcus haemolyticus strains isolated in a neonatal intensive care unit n. ben saida, a. ferjani, j. boukadida (sousse, tn) objective: methicilllin-resistant staphylococcus haemolyticus (mrsh) was increasingly important nosocomial pathogens particularly in critically ill neonates. the aim of our study was to establish if these infections are caused by endemic clone or by incidentally occurring bacterial strains of this species. methods: thirteen strains of mrsh according ca-sfm recommendations and meca+ (murakami k. j.clin.microbiol 1991 , 29:2240 has been collected during the period of survey (april 2004 to october 2004). eleven strains come from different pathological products of newborns hospitalized in a neonatal intensive care unit of a public maternity hospital. tow strains come from paediatric and internal medicine wards were used as control to ensure that endemic strains can be differentiated from outbreak strains. all strains were identified with the api-staph method (api biomerieux). strains have been characterized by genotypage in pulsed-field gel electrophoresis (pfge variety chef) after smai macrorestriction. genomic profiles have been interpreted according to tenover criteria (j. clin.microbiol.1995 ; 33 :2223 -2239 . results: s. haemolyticus was quantitatively the second staphylococci (23.6 %) after s. epidermidis isolated in neonatology ward. mrsh represent 85. 7 % of the total strains of s. haemolyticus collected from the neonatology ward during the period of study. the majority of the strains come from blood sample 7 (63.63%), 4 (36.36%) from pus and 1 (9%) from csf. eight resistant antibiotyps profiles and 3 genotypic pattern according tenover criteria were individualized: type a, type b (with 3 subtypes), and type c. the strains coming from paediatric ward present pattern type b3, but strains coming from internal medicine ward present distinct pattern. conclusion: mrsh is a major bacterium of nosocomial infection in neonatal intensive care unit. this bacterium is responsible as shown in our study, of endemic multiclonal nosocomial infections with a predominant clone. results: s. haemolyticus was isolated from 21% of all positive blood cultures and represented the 37.9% of the coagulase negative staphylococci. twenty-nine cases of shb occurred during the study period (5.6 episodes per 1000 patients-days). all cases were hospital-acquired and occurred after a mean hospitalisation of 24.5 days. the mean age was 68.4 years (range 20-90) and 16 patients (55.2%) were males. twenty-eight patients (96.5%) had a central venous catheter (cvc). all patients received previously antimicrobial therapy. thirteen patients (44.8%) had polymicrobial bacteraemia. in 18 cases (62.1%) the source of bacteraemia was the cvc. all isolates were resistant to oxacillin and gentamicin, 94.3% and 34.3% were resistant to ciprofloxacin and teicoplanin, respectively; all strains were susceptible to vancomycin. sixteen patients (55.2%) had sepsis, 3 (10.3%) severe sepsis, and 7 (24.7%), all polymicrobial, septic shock.; in 3 patients (10.3%) shb had no clinical significance. twenty patients died (69%) and deaths were directly related to shb in 1 patient (3.4%), and partially related in 2 (6.8%). nine patients (31%) survived.the patient who died for shb, developed a persistent cvc-related bacteraemia after a 6-week course therapy for streptococcal endocarditis. s. haemolyticus become resistant to teicoplanin during a teicoplanin-based therapy and the patient died 47 days after the onset of bacteraemia. the 2 patients, in which death was partially related to shb, had a polimicrobial bacteraemia due to c. tropicalis and enterococcus spp, respectively, and died after 22 and 6 days respectively, for septic shock, during adequate therapy. among the 17 patients in which death was not related to shb, 15 (88.2%) received adequate therapy that cleared the blood cultures, 2 patients improved after the removal of cvc without antimicrobial therapy. conclusions: s. haemolyticus is an emerging pathogen, responsible of sepsis, frequently cvc-related; most of strains are resistant to many antistaphylococcal agents currently used as empiric therapy, thus constituting a clinical concern. nevertheless, this pathogen shows a low virulence and is associated with a low related mortality, probably representing a marker of severe underlying diseases. staphylococci from the skin of patients: changes associated with treatment by isotretinoin a. thomas, r. williams, n. hepburn, t. watts, r. dixon (lincoln, uk) objectives: isotretinoin, a retinoid has been used to treat patients with moderate to severe acne despite the adverse effects of mucosal surface drying and the contraindication of use during pregnancy. in clinical practice, patients are frequently prescribed systemic isotretinoin because they have not responded to previous treatment with oral or topical antibiotics. broad-spectrum antibacterial therapy has nevertheless promoted changes in the diversity and antibiotic resistance status of the patientsõ skin microbiota and we are studying the effect of isotretinoin on these changes. methods: the present study investigated the recovery and analysis of skin organisms from 53 patients (mean age 23 y range 15-37y) before, during and after treatment with a 16-week course of isotretinoin (1 mg / kg body weight). the number of aerobic bacterial isolates (presumptive staphylococci) recovered from baird-parker agar from each of three specific sites per patient were compared with age-matched controls of healthy volunteers. results: both patients and controls had generally similar numbers of organisms on the nares, cheek and toe webs before treatment, whereas all patients showed a significant reduction in staphylococci recovered from one site during and after 16 weeks of isotretinoin treatment. the majority of the patients had a greater reduction (1-2 log) for the cheek site than either nares or toe webs both of which showed minimal reduction. conclusions: preliminary results from this study show a pronounced reduction in the number of presumptive staphylococci recovered from the treated group immediately following isotretinoin compared to untreated controls. since isotretinoin has little known antibacterial activity against the staphylococci the observed reductions would suggest that the effect is mediated through changes in the skin nutritional micro-environment. objectives: emergence of mutational resistance to linezolid (lin) in staphylococcus aureus has been associated with loss of erythromycin resistance methylase (erm) genes, both in vivo and in vitro. we tested the general validity of this claim, and examined whether the emergence of lin resistance is delayed in the presence of ery. methods: six lin-susceptible s. aureus strains were used: 2 genetically-related, ery-resistant (ermc) and susceptible pairs of emrsa-15, also the isogenic pair rn4220 and its hypermutable rn422muts mutant, which harbours ermb. in 4 replicated experiments, ery-susceptible and ery-resistant isolates were grown on agar containing increasing concentrations of lin. in addition, ery-resistant isolates were grown with 100 mg / l ery and increasing concentrations of lin. the number of days for isolates to develop the ability to grow in the presence of 6 mg / l lin was recorded. the absence of erm genes was checked by pcr; mics of lin and ery were determined by agar dilution, and pfge profiles of mutants were verified. results: thirty-three mutants were obtained from the 4 experiments; 24 from the 4 clinical emrsa-15 isolates, 3 from rn4220 and 6 from rn422muts. lin-resistance emerged more slowly in ery-resistant clinical isolates grown with lin and ery than with the same isolates grown with lin alone: mean 20 days (sd 26.8) to emerge on 6 mg / l lin versus 30 days (sd 18.4) in the presence of lin and ery. growing the hypermutable strain, rn422muts, in the presence of lin and ery did not delay the emergence of lin-resistance (11 days with sd 8.7, in either case). in the absence of ery selection, loss of erm with the emergence of lin-resistance was only noted for one emrsa-15 isolate in 1 of 4 experiments. all isolates grown in the presence of ery retained erm determinants. ery 100 mg / l did not supress growth of ery-resistant strains and linezolid was stable within the ambit of the long-selection experiments. conclusions: growing the clinical isolates in the presence of ery and lin slowed the emergence of lin-resistance. this merits further study with lower, clinically-relevant concentrations of ery. in vitro selection of linezolid resistance in hypermutable and wild-type staphylococcus aureus objectives: resistance to linezolid (lin) is rare, but can arise via mutations in the 23s rrna genes. we hypothesised (a) that resistance would emerge preferentially in staphylococci with a hypermutable phenotype engineered by mutations in the muts gene, and (b) that hypermutability might be co-selected with linezolid resistance. muts is part of the dna mismatch repair (mmr) system, which identifies and corrects genome alterations post-replication, thereby influencing the net mutation rate. method: linezolid-resistant (linr) mutants of staphylococcus aureus rn4220, its hypermutable muts deletion mutant rn4220muts, harbouring an erythromycin resistance marker gene (ermb), and a genetically-related pair of clinical isolates were selected by repeated passage with increasing concentrations of lin. mutant parentage was confirmed by pfge and susceptibility was tested by agar dilution. an amplified 694-bp fragment of 23s rrna genes was studied by sequencing and by rflp analysis. frequencies at which linr mutants yielded variants resistant to fusidic acid and rifampin were calculated in triplicate experiments. results: seventeen linr mutants (mics 8-64 lg/ml) were raised. ten mutants had a g2447t mutation in their 23s rrna genes; 4, all from rn4220muts, had g2576t, typically seen in linr gram-positive cocci from the clinic; 1 had t2504c; 2 had no identifiable mutations. curiously 5 / 7 rn4220muts mutants lost ermb during the course of the experiment, reverting to macrolide susceptible. linr mutants had mutation frequencies for rifampicin and fusidic acid resistance comparable with those of their parents (10-6 to 10-9), implying no co-selection of stable hypermutability. conclusion: more linr mutants were obtained from the hypermutable (rn4220muts) strain than from the wild type strain, and g2576t mutants were only obtained from the hypermutable organism. these data suggest hypermutability facilitates the emergence of linr, nevertheless, linr mutants derived from wild-type parents did not have elevated mutation frequencies. objectives: the mechanism of resistance in glycopeptide intermediate staphylococcus aureus (gisa) and hgisa isolates is unknown. however the inactivation of certain genes has been shown to confer an increase in glycopeptide resistance. two genes, tcaa and mprf, when inactivated in gisa strains cause a reduction in glycopeptide mic. overexpression of tcaa causes an increase in teicoplanin susceptibility and so both genes may play a role in the development of glycopeptide resistance. this study aims to investigate the expression levels of tcaa and mprf in a set of gisa, hgisa and glycopeptide-susceptible s. aureus (gssa). methods: rna was extracted from the following: 1 set of clinically related (cr) gssa and hgisa (lla and lle), 2 sets of cr hgisa and gisa (pc1 and pc3, lim1 and lim3) harvested during exponential phase growth (ex) and during exponential phase growth in the presence of sub-mic levels of vancomycin (exv). rna was extracted from a further hgisa, mu3 and gisa, mu50 as well as 4 clinical gssa strains. rt-pcr was performed with customised primers and the products visualised by electrophoresis. band intensity was taken as indicative of mrna quantity, and hence expression level, at time of cell harvest. results: the expression of tcaa is similar in all strains tested. each strain within the related strain sets all show similar band intensities, despite differing glycopeptide resistance phenotype. both mu3 and mu50 also exhibited similar expression levels to those found for all other gisa, hgisa and gssa strains. levels of expression were not affected by the presence of sub-mic levels of vancomycin in any strain tested. sequence analysis of tcaa in these strains showed no mutations present as previously thought. expression levels of mprf were found to be strain specific. no difference in gene expression was found in the cr related strain sets. conclusions: no reduction in expression levels of tcaa and mprf were found to correlate with clinical strains exhibiting higher glycopeptide resistance. thus it it uncertain what role tcaa ot mprf play with mediating glycopeptide resistance in staphylococcus aureus. objectives: gisa and hgisa strains all have a characteristic thickened cell wall with reportedly fewer cross-links. fewer peptidoglycan cross-links create increased d-ala-d-ala pentapeptide termini and hence more vancomycin targets. pbp4 has been shown to have both transpeptidase and d,d-carboxypeptidase activity, cleaving terminal d-alanine residues from un-cross-linked muropeptides. reduced levels of pbp4 have been reported in laboratory-generated gisa mutants and a few clinical gisa strains. this study aims to investigate the expression levels of pbp4 in a set of gisa, hgisa and glycopeptide-susceptible s. aureus (gssa). methods: rna was extracted from the following: 1 set of clinically related (cr) vssa and hgisa (lla and lle), 2 sets of cr hgisa and gisa (pc1 and pc3, lim1 and lim3) harvested during exponential phase growth (ex), after overnight growth (on) and during exponential phase growth in the presence of sub-mic levels of vancomycin (exv). rna was extracted from a further 8 clinically unrelated (cu) gisa (including mu50), 8 cu hgisa (including mu3) and 8 gssa (ex). rt-pcr was performed with customised primers and the products visualised by electrophoresis. band intensity was taken as indicative of mrna quantity at time of harvest and hence gene expression. results: the expression of pbp4 varies according to strain. the band intensities of the related strain set lla (gssa) and lle (hgisa) reduce slightly with increasing intermediate vancomycin resistance. however in two other cr hgisa and gisa (pc1/ pc3, lim1/lim3) strain sets no variation in expression was observed. both mu3 and mu50 exhibited lower pbp4 expression than any other strain, with the presence of sub-mic levels of vancomycin reducing expression further. pbp4 expression was found to be reduced in 3 other gisa and 1 other hgisa, which showed lower band intensities than gssa. conclusions: the reduced levels of pbp4 suspected to be associated with reduced susceptibility to vancomycin in s. aureus is not exclusive. the expression of pbp4 is reduced in mu50 and 3 clinical gisa, as reported previously. however 5 other gisa strains exhibit pbp4 expression levels similar to gssa isolates. therefore pbp4 expression levels appear to be strain specific and a lowered expression level is not an essential requirement in the development of the gisa phenotype. objectives: the prolonged use of vancomycin is known to be the most important factor in inducing vancomycin resistance to s. aureus. methicillin-resistant s. aureus (mrsa) colonizing in respiratory tracts might be chronically exposed to subtherapeutic concentrations of vancomycin because the level of vancomycin within pulmonary lining fluids was much lower than the concentration of serum. despite the continuous use of vancomycin mrsas were still found to be presented in respiratory specimens of some patients. methods: all mrsas which had been continuously isolated in respiratory specimens during vancomycin therapy were collected from jul 2003-mar 2004 at seoul paik hospital. mrsas were screened on brain heart infusion plates containing vancomycin 4 lg / ml (bhi-4) and 6 lg / ml (bhi-6) for vancomycin resistance. minimal inhibitory concentrations (mics) were determined by e-tests, agar dilution methods, and broth dilution methods. the patterns of pulsed field gel electrophoresis were analysed. results: nine patients and 27 isolates were assessed. five patients had pneumonia and four patients had other mrsa infections. twelve mrsas were isolated from tracheal aspirates and 15 from expectorated sputum specimens. the duration of vancomycin use ranged from 7-22 days. five isolates and one isolate were cultured on bhi-4 and bhi-6, respectively; the vancomycin mics of these isolates revealed 3 lg / ml by e-tests. the mics from agar dilution methods and broth dilution methods showed from 0.5-2 lg / ml. conclusion: vancomycin resistance during the treatment of patients did not develop. objectives: fusidic acid resistance (fusr) in staphylococcus aureus usually results from acquisition of the fusb resistance determinant, or from mutations in the fusa gene encoding the drug target (elongation factor g). following a recent report of fusr in s. intermedius, and the detection in our own studies of fusr s. lugdunensis (unpublished), we have examined whether resistance in these strains results from fusa / fusb-type mechanisms. methods: standard methodology was employed for strain identification, susceptibility testing, southern hybridization (sh), pcr amplification and dna sequencing. primers for pcr amplification of the previously-unsequenced s. intermedius fusa gene were based on portions of the flanking genes (rpsg, tufa) that are conserved between b. subtilis and s. aureus. results: three fusr s. lugdunensis (fus mic = 4 ug / ml) and two fusr s. intermedius (fus mic = 2 ug / ml) were probed for the presence of fusb by sh alongside their wild-type, fuss counterparts (fus mics of 0.064 and 0.125 ug / ml, respectively). the fusr s. lugdunensis all carried fusb, but this gene was not detected in s. intermedius, or in the fuss strains. furthermore, fusb appeared to be chromosomally-located, since no hybridization was observed with purified plasmid dna. pcr analysis mapped fusb to a chromosomal region upstream of the groel gene, the same locus at which this gene is carried in the epidemic fusr european s. aureus clone. to examine whether mutations in fusa are responsible for fusr in s. intermedius, pcr amplification and sequencing of fusa from the resistant and sensitive strains was performed. relative to fuss s. intermedius nctc 11048, the resistant strains carried six coding polymorphisms in fusa, although none of these correspond to reported fusr mutations in the fusa gene of s. aureus. conclusions: fus resistance in recent strains of s. lugdunensis results from carriage of the fusb determinant on the chromosome, probably acquired horizontally from fusr s. aureus strains. the fusb gene was not detected in the s. intermedius strains, although multiple fusa polymorphisms were identi-fied which may be responsible for the observed fusr phenotype. objectives: methicillin-resistant s. aureus (mrsa) and methicillin-resistant coagulase-negative staphylococci (mrcns) are the most important pathogens that cause nosocomial infections worldwide. one of the few antibiotics which is still effective against mrsa is mupirocin. but mupirocin-resistant s. aureus has been increased since first reported in 1987. and more than 5-10% of s. aureus isolated in tertiary hospitals in korea was reported to be high level mupirocin resistant. in this study, we investigated the restriction fragment length polymorphism (rflp) type for mupa gene loci of plasmid dna and sequenced mupr plasmid containing mupa gene of high level mupirocin resistant (muh) staphylococci from tertiary hospitals. methods: susceptibility to antimicrobial agents including mupirocin was tested by the disk diffusion and mic of mupirocin was determined by agar dilution method. the plasmid-encoded mupa gene was detected by pcr. mupa polymorphisms of plasmid dna digested by hind iii, ecor i, and cla i restriction enzymes was investigated with southern hybridization using mupa probe. s. aureus isolate showing the most prevalent rflp type was conjugated by filter mating method and selected the transconjugant showing only mupirocin resistant phenotype. the mupr plasmid of selected transconjugant was purified and analysed the plasmid dna sequence. results: 9 muh-staphylococci were isolated among the 680 staphylococci from tertiary hospital in korea. muh-mrsa (6 isolates), s. haemolyticus (1 isolates), s. hominis (1 isolates) and s. epidermidis (1 isolates) were isolated. 1.65kb mupa pcr product was detected in plasmid of muh isolate. all isolates contained more than two plasmid molecules that were repeatedly purified with different efficiences. three different polymorphs of the mupa loci were investigated. the most prevalent mupa polymorph was 4.2kb ecori-8kb hindiii-2.1kb clai hybridizing fragment. the mupirocin resistant plasmid dna was about 52kb and is257-mupa-is257 sequence was located. conclusion: the high-level mupirocin resistant staphylococci had the multiple plasmids of various size and the diverse rflp type suggested the variation of mupa gene loci. the mupr plasmid contained transferable element such as is257 with mupa gene. genetic basis of macrolide, lincosamide, streptogramin resistance in coagulase-negative staphylococci s. gatermann, t. koschinski, s. friedrich (bochum, d) objective: in s. aureus erythromycin resistance is almost exclusively caused by erma or ermc genes whereas export of macrolide antibiotics by msra or inactivation of lincosamides by lina is rare. resistance may be inducibly (clindamycin appears susceptible) or constitutively (clindamycin appears resistant) expressed. during therapy of inducibly resistant strains with clindamycin mutations may occur that render them objectives: lysostaphin is an antibacterial enzyme which is specifically capable of cleaving the cross-linking pentaglycine bridges in the cell walls of staphylococci. s. aureus cell walls contain high proportions of pentaglycine, making lysostaphin a highly effective agent against both actively growing and quiescent bacteria. relationship between lysostaphin susceptibility, vancomycin susceptibility and cell wall thickness of passage-selected vancomycin -resistant staphylococcus aureus isolates (vrsa) and their parent strains were studied in order to investigate characterization of isolates with decreased susceptibility to vancomycin. methods: the susceptibility of lysostaphin for six vrsa and their parent strains were determined using the spectrophotometric and the macrodilution methods. spectrophotometric determination of turbidity was measured as a decrease in a620 during incubation of the isolates samples at 37°c. mics were determined by a broth macrodilution method in cationadjusted mueller-hinton broth according to standards of the national committee for clinical laboratory standards. cell wall measurement were determined using transmission electron microscopy. results: determination of lysostaphin activity revealed that there are significant decreases in per cent reduction in turbidity (activity of lysostaphin) of the vrsa. vrsa shown to be more resistant to lysostaphin than parent strains. the mics of lysostaphin for the vrsa strains also increase. there were either a 2 or 4 fold increases in the mic to lysostaphin. electron microscopy of the vrsa showed enhanced cell wall thickness, uneven surfaces and irregular shape in contrast of the thin and regular cell wall morphology of the parent strains. those strains acquiring the highest thickness in cell wall demonstrating the highest resistant to lysostaphin. conclusion: the mechanism of lysostaphin resistance and its relationship to vancomycin resistance remains unclear. it is possible that the increase in cell wall thickness we observed prevented lysostaphin access to all pentaglycine targets or increased the number of cross-linkers requiring cleavage before the strain could lyse. typing and epidemiology of mrsa methods: creation and distribution of a consensus document detailing the needed harmonization of sequencing technology in diagnostic microbiology will be followed by a certification program for sequence based typing, using methicillin-resistant s. aureus (mrsa) as a prototype organism. as typing method spatyping will be used. five strains, 5 dnas, and 5 forward and reverse chromatogram files of well characterized mrsa strains will be distributed to all participating laboratories. an annual proficiency testing for sequence based typing of mrsa is planned. staphylococcus aureus is an important human pathogen related to its ability to develop resistance to many antimicrobial agents. methicillin resistance of s. aureus (mrsa) has become a major public health problem especially in nosocomial infectious. more over community acquired mrsa is a growing concern, the rate of mrsa vary importantly within countries, the highest rates were reported especially in southern european countries. a multicentric study was done to establish antimicrobial resistance of s. aureus isolated from clinical specimens of hospitalized patients over five years period (1999) (2000) (2001) (2002) (2003) . s. aureus was identified by conventional methods and antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. a total of 5186 strains of s. aureus were collected, they were recovered mainly from pus (60%), blood cultures (15%), respiratory samples (8%) and urines (7%). 34% of the strains were from in medicine, 18% from surgery, 16% from orthopaedics, 14% from paediatrics and 13% from intensive care units. the rates of resistance to different antibiotics was 15% to oxacilline,9% to gentamicin, 23% to amikacin,8% to ofloxacin, 23% to erythromycin and 8% to clindamycin. all isolates were susceptible to vancomycin. according to this data the rate of mrsa remained relatively low (< 20%) with no significant change during this time period, however, mrsa presented high rates of associated resistance to gentamicin (48%), to amikacin, (91%) to erythromycin (38%), to clindamycin (20%), to ofloxacin (40%) and to trimethoprim-sulfamethoxazole (33%).mrsa were not of great concern in our country and meticillin remains an effective antibiotic for treatment of s. aureus infections. characterisation of gentamicin-susceptible strains of methicillin-resistant staphylococcus aureus in two spanish hospitals c. potel, m. alvarez, c. lopez-melendez, i. otero (vigo, e) objectives: characterize gentamicin-susceptible methicillinresistant staphylococcus aureus(gs-mrsa)by two molecular typing methods. find out whether some gs-mrsa clones are related to gr-mrsa. describe the spread of these clones. methods: one hundred and twelve samr were isolated in two hospitals between 1997-2001. the epidemiological relatedness was studied by repetitive element sequence-based pcr and coa gene restriction fragment length polymorphism. the genes ant(4) and aac(6)-aph(2õ), that codify two aminoglycoside modifying enzymes, were detected by pcr.we analysed the gentamicin consumption and its relation with the emergence of gs-mrsa. results: thirty nine strains were gs-mrsa. the 80% of these gs-mrsa belonged to two epidemic clones, the clone a was tobramycin sensitive and the clone b was tobramycin resistant. the clone a was only isolated at hospital-i and was replaced by an epidemic gr-samr clone (clone c). the clone b was only isolated at hospital-ii and displaced the clone c. objectives: the long-term care facilities (ltcfs) patients are those with serious underlying disease, poor functional status, wounds such as pressure sores, invasive devices of urinary catheters. there is increasing concern about the emergence of resistance to antimicrobial agents including methicillin resistant s. aureus in ltcfs and mupirocin has been used in ltcfs to prevent the occurrence and spread of mrsa. for inducible mls-resistance. nitrocefin discs were used for beta-laktamase detection after induction with cefoxitin and/or oxacillin. the oxacillin (4 mg / l) agar method was used for mrsa screening. mrsa was confirmed by meca and nuc pcr and further analysed by smai-pulsed-field gel electrophoresis (pfge). results: a total of 102 strains (78%) were beta-lactamase positive. other resistance prevalence rates were: tc (29%), em (19%), cm (19%), ci (11%), gm (11%), ts (0%) and fu (0%). thirteen strains (10%) were mrsa-screen positive, both with the oxacillin-agar test and the cefoxitin disc, and confirmed mrsa by meca and nuc pcr. all mrsa strains were isolated from hospitalized patients and expressed multiple resistances. pfgeanalysis revealed that the mrsa-strains belonged three different pfge-types. type 1 consists of 8 strains from the department of general surgery and icu. the type 2 group consisted of 3 strains from general surgery department. finally, one strain from a patient at the department of pulmonary medicine belonged to the type 3 group. we were not able to type one strain. mlsttyping of mrsa-strains will be performed. results: a total of 793 infants were included in this study. mrsa colonization was detected in 331 infants (42%) during nicu stay and 89% were detected by the first 2 samplings. naris (71%) and umbilicus (60%) were the 2 most common sites of colonization. clinical mrsa isolates were identified from 92 infants (12%). among the 92 infants, mrsa colonization, either preceding or later acquiring, was noted in 84 infants (91%). 121 clinical isolates from 64 infants with 84 episodes of possible mrsa infection were available for genotyping analysis. prior colonization was detected in 68 episodes (81%), among which both the colonized and clinical isolates were indistinguishable in 63 episodes (93%), highly related in 2 episodes and distinct in 3 episodes. among the 16 episodes without prior colonization, colonization was acquired following infection in 9 episodes (53%), among which indistinguishable or highly related colonized strain was acquired in 6 episodes. conclusion: surveillance of mrsa strains as a basis for active antibiotic policy has become of increasing concern to both health care providers in hospitals and community general practitioners. there is a need for better awareness of mrsa infections among both health care professionals and the public. the incidence of mrsa infections in last years in the czech republic shows a slightly increasing trend. very interesting is local distribution of mrsa strains. the development of the incidence of mrsa infections in their spread will be the subject of further study. comparison of phenotypic typing methods and pfge of methicillin resistant s. aureus isolated from two university hospitals in thailand c. chomvarin, k. chaicumpar, s. srigulbutr (khon kaen, th) objectives: comparison of phenotypic typing methods and genotypic typing method to differentiate mrsa isolates obtained from the two university hospital in thailand. methods: seventy-four mrsa isolates were randomly collected from the two university hospital (central and northeastern) in thailand. they were confirmed with the presence of meca gene. antibiograms, phage typing and enterotoxin productions were used for the phenotypic typing analysis. pulsed-field gel electrophoresis (pfge) with smai digestion of chromosomal dna was used for the genotypic typing analysis. results: we found 19 distinct profiles by phenotypic typing methods and 18 pfge types designed as 5 major types (a to e) and 13 subtypes. the most frequently types and their related subtypes found in both hospital, were a and c with represented 54.1% and 27%, respectively. all isolates resistant to penicillin, cephalosporin, erythromycin, gentamicin, kanamycin, tetracycline and oxacillin; and variably resistant to cotrimoxazole, lincomycin, chloramphenicol, ciprofloxacin. all isolates were susceptible to vancomycin and fosfomycin. ten (13.5%) mrsa isolates produced enterotoxin a. forty-five (60.8%) isolates were nontypable by phage typing method. no significant correlation between the phenotypic typing methods and the genotypic typing method were found. conclusions: the phenotypic typing methods were not significantly correlated with the genotypic typing (pfge). our results demonstrated that pfge types a and c were the common endemic mrsa clone of both hospitals in thailand. objective: to investigate the molecular characteristics of methicillin-resistant staphylococcus aureus (mrsa) strains isolated from neonates transferred from primary care obstetric clinics. methods: twelve mrsa strains were isolated from 11 neonates in 2004. ten mrsa strains were also isolated from nurses and facilities in corresponding primary care obstetric clinics. molecular features of mrsa strains were analysed by using multilocus sequence typing (mlst, arcc-aroe-glpf-gmk-ptatpi-yqil), spa typing, and sccmec typing. presence of panton-valentine leukocidin (pvl) gene was investigated by pcr method. results: all 22 mrsa strains analysed in this study contained sccmec type iva. of six isolates from neonates of clinic a, five showed st1 (1-1-1-1-1-1-1) and resistance to only gentamicin and tetracycline. the remaining one showed novel st (25-1-1-1-1-1-1), shared with one isolate from nurse of clinic a. four mrsa strains from neonates of clinic b showed st1 (1-1-1-1-1-1-1) and resistance to erythromycin and gentamicin, which is the same characteristics with three isolates from nurses and environment of clinic b. the other two mrsa strains from neonates of clinic b showed st493 (62-1-1-1-1-1-1), which is shared by six strains from nurses and environment of clinic b. spa type of these 22 mrsa strains was identical as ujebkbp and all strains were negative for pvl gene. objectives: over the last years there has been a dramatic increase in the prevalence of methicillin-and multiresistant staphylococcus aureus (mrsa) leading to a major health problem, especially in the nosocomial setting. to support infection control measures typing of mrsa is essential. the present ôgold standardõ for strain typing is pulsed field gel electrophoresis (pfge), but several sequence based methods including spa typing have recently been introduced. therefore this study was initiated to compare typing results obtained from pfge with those from spa typing. methods: 90 well characterized s. aureus strains of different evolutionary origin including methicillin sensitive and resistant isolates were included. strains were selected from a variety of clonal groups prevalent in germany and middle europe during the last ten years. the collection comprised hospital derived strains as well as isolates from the community, including the recently emerging community acquired mrsa. all isolates were subjected to pfge with subsequent cluster analysis; additionally the polymorphic x-region of the protein a gene was sequenced and a spa type was assigned using the ridom staphtype software. the newly developed algorithm burp (based upon repeat patterns) was applied to the spa sequences to cluster the resulting spa types into different groups. clustering results were compared to those obtained from pfge and mlst. results: overall the results obtained from the different typing approaches were in agreement. in some cases where pfge results were inconsistent despite uniform epidemiological background spa typing was able to group the corresponding strains together. only two strongly related epidemic clones could not be separated by spa typing. conclusion: spa typing in combination with burp analysis is an easy, rapid and reliable method for epidemiological analyses in s. aureus. it is superior to pfge in cases where pfge might be ôoverdiscriminatoryõ. in rare cases a second discriminatory locus (for example a single mlst locus) might be helpful for an ultimate classification. the antibiotic susceptibility of methicillinresistant staphylococcus aureus methicillin-resistant staphylococci (mrsa) are often multidrug resistant and represent a major problem for the antimicrobial therapy. glicopeptides are the golden standard for these infections but resistance and toxicity concerns limit their usage. mrsa antibiotic resistance may be divided into two distinctive profiles: multidrug resistance (probably nosocomial infections) and variable resistance, usually to 1 or 2 non-betalactamic antibiotics (often correlated with community-related mrsa infections results: in total, 4.5 % (98) of s. pn was resistant to erythromycin and 2.9% (8) of invasive and 4.6% (10) of non-invasive s. pyo were resistant to erythromycin. none of the isolates were resistant to clindamycin. among the ery-res s. pn, the most frequent gene was mef(a) 83.7%, while erm(b) was found in 13.3%. in two isolates no resistance genes were identified. 81 out of 82 mef(a) carrying isolates had serotype 14.erm(a) and mef(a) was present in 62.5% and 37.5% of the invasive ery-res. s. pyo isolates. none of the isolates were positive for erm(b). in non-invasive ery-res. s. pyo isolates 40%, 10%, 50% were pcrpositive for erm(a), erm(b) and mef(a) respectively. conclusion: in contrast to s. pyo, ery-res. s. pn is dominated by one serotype, carrying mef(a). the overall resistance profile of s. pn and s. pyo is still favourable, but the resistance to macrolides is of growing concern in denmark. resistance in streptococcus pneumoniae: auc/ mic breakpoints differ between gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin k. laplante, m. rybak, b. tsuji, g. kaatz (detroit, usa) objective: the potential for resistance development in streptococcus pneumoniae (sp) secondary to varying exposure to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin was examined at high inoculum (108.5-9 log 10 cfu/ml) over 96 hours in an in vitro pharmacodynamic model. results: the molecular analysis of one hundred and forty serotype 11a dspn isolates revealed six clonal types. however, the vast majority (95%) belonged to a single clone. the prevalence of this dominant clone during the study ranged from 6.3% to 13.9% and it was disseminated in all day-care centres. conclusions: serotype 11a is not a capsular type with an important representation in drug-resistant collections which are the most commonly studied worldwide. however, it seems to be common among dspn strains. this serotype 11a collection showed little genetic variability. in fact, one genotype was found to be dominant and disseminated successfully in all day-care centres studied. these findings suggest that this serotype 11a clone is frequent in colonization and thus its monitoring is of importance particularly after the introduction of the sevenvalent pneumococcal conjugate vaccine. diseases. an abrupt antimicrobial resistance increase and clonal spread of resistant pneumococcal strains has resulted in serious therapeutic problems in recent years. the aim of this study was to analyze resistance patterns and genetic diversity of s. pneumoniae resistant strains isolated in our region during three years (2001) (2002) (2003) . methods: using e-test method and the nccls criteria for benzylpenicillin (p), erythromycin (e), clindamycin (l), tetracycline (t), cotrimoxazole (s), ceftriaxone (c), chloramphenicol (h), vankomycin (w), imipenem (i), fifty nine resistant or intermediate to at least one drug strains were obtained. strains were submitted to molecular characteristic by pfge with smai restriction enzyme and computer analysis using molecular analyst software application. for each strain resistance pattern and pfge profile was determined. results: resistance to 8 out of 9 determined antibiotics (except vancomycin) was described. strains showed 22 different resistance patterns and tsh (13%strains), psi (11.8%) and elts (10.1%) were the most often. resistance degree reached 7 drugs (5% strains) and 69% strains were mdr. we have found 26 pfge profiles: 15 of them (u 1-15) were unique single isolates, 11 clusters (a-k) were represented by 2-10 strains, which were more than 78% of similar. the most numerous clusters (a-k) consists of strains that were isolated over three years of study and showed the same or very similar resistant patterns: a-tsh (8 of 10 strains), th (2); b-psi (7 of 8), s (1); c-pelshi (3 of 6), ptshi (2), ptschi (1); d-elts (3 of 4), els (1); e-sh (2 of 4), ts (1), t (1). all strains of tsh and psi resistance patterns were found in one cluster a(a1-a9) or d (d1-d6) respectively but strains with elts and pelshi patterns belonged to different (2-3) clusters and unique pfge profiles. another clusters (f-k) were represented by two strains with different resistant patterns each. conclusions: population of s. pneumoniae resistant strains in our region presents high genetic diversity and numerous different resistance patterns. the majority of strains with the same resistance pattern showed different pfge profiles but there were observed some strains of the same resistance pattern, which belonged to only one cluster and were isolated over three years of study. ) hlpr strains showed a profile identical or related to two epidemic clones: spain23f-1 and spain9v-3. the remaining hlpr pneumococci belonged to unique or rare clones. llpr isolates were characterised by more variable backgrounds in terms of pfge patterns and serotypes than hlpr strains. in fact, 27.7% showed an identical or related profile (arbitrarily called d, serotype 15a) previously not described in italy and 10 (15.4%) exhibited the pfge patterns typical or related to the spain23f-1 and spain9v-3 clones. the remaining 37 llpr isolates (56.9%) belonged to 22 different profiles and to 11 distinct serotypes. in this study, the 23f ôitalian cloneõ circulating in 1993-1996 was not found. conclusions: the recent increase of total penicillin-resistance in italy can be ascribed to the emergence of a new clone and to the diffusion of two well known international clones, whose ability to spread is higher than that of the autochthonous italian clone described in 1996. changing antibiotic prescribing habits, including the recent strict limitation to the consumption of parenteral drugs, may also explain the variations described. results: during the study period, 143 strains of s. pneumoniae were isolated: 6 (4.2 %) in csf and 137 (95.8%) in blood. total incidence was 6 cases/100,000 inhabitants/year, with maximum incidence in winter and spring. the largest number of cases, 76 (54 %), were in the over 65s. in children, 11 (7.7 %) were detected, all in the under 5s. the total number of strains whose sensitivity to penicillin fell was 43 (30 %), of which 20 (46.5 %) had an mic 2 mg/ml. 46.5% of the penicillin-resistant strains were also resistant to erythromycin. 16 (11%)/ 143 had reduced sensitivity to cefotaxime, and most of these (63%) had intermediate sensitivity. resistance to erythromycin was detected in 34 (24%). during the study period, resistance to antibiotics decreased gradually in such a way that the resistance percentages each year were: penicillin 41%, 30%, 27%, 23%; cefotaxime 28%, 14%, 4%, 4%; erythromycin 34%, 19%, 29%, 8%. no differences were observed in the resistance percentage in the different age groups. conclusions: the prevalence of csf and blood strains of s. pneumoniae with reduced sensitivity to penicillin in this study was 30%. the treatment of choice in s. pneumoniae invasive disease was third-generation cephalosporins, due to their low level of resistance. resistance to the main antimicrobials from this type of strain has fallen. during the study period, resistance to penicillin fell by 43.9 %, 58.5 % for cefotaxime 63.4 % for erythromycin. the fall in resistance among invasive s. pneumoniae could be due to vaccination in children and the elderly from 2002 onwards, since the vaccination includes the most common and resistant serotypes. the low number of isolates in children could be due to the low number of samples processed. change in distribution of macrolide-resistant streptococcus pneumoniae genotypes over 4 years -data from protekt 1999-2003 objectives: this analysis of data from 39 countries over the first 4 years (1999) (2000) (2001) (2002) (2003) of the protekt global surveillance study was undertaken to investigate longitudinal and regional trends in the distribution of macrolide resistance mechanisms among streptococcus pneumoniae, and the susceptibility of these isolates to antibacterials, including the ketolide telithromycin. objectives: the 7-valent formulation of the pneumococcal conjugate vaccine (pcv7) was introduced in the usa in february 2000. there is a general recommendation for its use in all children <2 years of age, and the vaccine is licensed for children up to 5 years of age. in europe, the vaccine is licensed for children up to 5 years of age, but most countries do not have a general recommendation for use. protekt -a global, longitudinal study of the antimicrobial susceptibility of bacterial respiratory tract pathogens -has now completed its fourth year. a comparison was made between serotype distribution and pcv7 coverage between y1 and y4. methods: analysis was performed only on streptococcus pneumoniae isolates obtained from paediatric patients at the 31 centres common to both years (y1, n = 553; y4, n = 682). serotyping was performed by neufeld's quellung reaction using statens serum institute antisera (ssi, denmark). results: pcv7 coverage decreased from 67.7% and 62.5% in y1 to 51.0% and 41.0% in y4 in the usa and latin america, respectively (chi-squared; p = <0.001 and 0.007, respectively). serotypes that showed the greatest increase (y1, y4) in the usa were 19a (7.0%, 22.2%), 6a (1.8%, 9.9%), 3 (1.8%, 6.2%), 15 (1.8%, 6.2%), and 11 (0%, 6.2%). in brazil, a similar pattern was seen although there was no change in the proportion of isolates with the 19a serotype. in contrast, no change in serotype distribution was observed in canada, western europe, and the far east, where the pcv7 vaccine is not routinely used for paediatric vaccination. conclusions: results from this analysis of protekt data indicate that the proportion of s. pneumoniae isolates from paediatric patients covered by the pcv7 vaccine has decreased significantly over 4 years in regions where the vaccine is routinely used. other serotypes, such as 19a, which is known to be highly resistant to antimicrobials and is not covered by pcv7, have increased in these regions. this study demonstrates the importance of serotyping antimicrobial surveillance study isolates in order to monitor such changes and any potential future implications for therapy and vaccine formulations. background: sequential alterations in pbp sequences constitute the classical mechanism to acquire penicillin resistance in s. pneumoniae, that is reflected in changes in the cell wall peptidoglycan structure. murm gene controls the addition of the first amino acid of the dipeptide bridge of the pneumococcal muropeptide. mutations in this gene are apparently required for high penicillin and cefotaxime resistance. the aim of this study was to compare the murm gene sequence within the earlier and the latest highly penicillin resistant pneumococcal populations recovered in spain. material and methods: a total of 56 s. pneumoniae isolates (10 of them from 1987-88 and 46 from 2002-04) with resistance to penicillin (mic 2-8 lg/ml) were included. susceptibility to different b-lactams, macrolides, tetracycline, levofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole (sxt) were determined by the agar dilution method. all isolates were grouped according to serotype and pulsotype (pfge-smai pattern) and the murm gene was sequenced. results: a high percentage of resistance (intermediate plus resistant) was observed for cefotaxime (100%) and sxt (96.6%). all isolates remained susceptible to telithromycin but not to levofloxacin (93% of susceptibility). all isolates recovered during 1987-88 were genetically unrelated and mainly belonged to serotypes 23f (40%) and 6b (40%). in contrast, the most frequent serotypes during 2002-04 were 14 (30%), 9v (26%), and 6b (17%). in this period, a predominant spain9v-3clone was detected in 10 out of 14 isolates of serotype 14, and in 11 out of 12 isolates of serotype 9v (capsular switch). all 8 isolates of serotype 6b belonged to a single clone (spain6b-2). the analysis of murm sequences of isolates from both periods revealed the existence of different alleles. homologous sequences to the murma (identical to that described for r6 penicillin susceptible strain), were replaced in part by murmb5, and murmb6 (different variants of these alleles were detected in our collection). conclusion: early high penicillin resistant s. pneumoniae isolates in spain were genetically unrelated and corresponding to 23f and 6b serotypes. on the contrary, recent isolates showed dissemination of selected clones, particularly spain9v-3. many s. pneumoniae isolates with high-level resistance to penicillin retain (or reacquired) the murm gene of susceptible strains, but different allelic variants was also detected. objectives: to determine among 712 s. pneumoniae clinical isolates recovered from different spanish hospitals during three different periods (1999-2000, 2001-2002 and 2002-2003) the prevalence of erythromycin resistance phenotypes and resistance genes associated to this resistance. methods: susceptibility testing was performed by the standard microdilution technique (nccls). a pcr assay was carried out to identified erythromycin resistance genes (erm or mef) and a multiplex-pcr was designed to distinguish between mef(a) and mef(e) genes within mef positive isolates. clonality was studied using the sma-i-pfge technique. during the studied period ranged from 48.3% (first studied period) to 45.2% (third studied period) for penicillin, 39% (first studied period) to 32.9% (third studied period) for erythromycin, and 32% (first studied period) to 30.3% (third studied period) for clindamycin. levofloxacin resistance was only found in 2.4% of isolates and were recovered during the third studied period. m phenotype was observed in 3.2% of all tested isolates. considering non-susceptible erythromycin isolates, ermb and mef genes was found in 87.1% and 9.5% of isolates, respectively, as a sole erythromycin resistance gene. the concomitant presence of both determinants was found in 3.3% of isolates. interestingly, among the isolates with mef pcr positive result, only one (3.2%) carried the mef(a) gene whereas the mef(e) gene was detected in the others (96.8%). an increase in mef prevalence was observed between the first and the second studied periods (2% to 5.8%) and a slightly decrease (4.9%) in the third period. pfge analysis revealed a polyclonal structure of mef positive isolates. conclusions: prevalence of mef positive isolates among erythromycin-resistant strains remains low in our country (4.3%) being the mef(e) gene the most prevalent mef determinant among these isolates (96.8%). results: among young children, single ery resistance was most prominent in all countries, (from 6% in sweden to 38% in belgium), except for spain where the proportion of dual resistance was highest (33%). for the other countries dual resistance among young children ranged from 0% in denmark to 13% in belgium. except for ireland (12%) and spain (21%), single pen resistance remained below 5%. conclusion: between countries, large differences in the patterns of s. pneumoniae resistance were found among young children. ery resistance was most common among young children, which may indicate greater use of macrolides in this age group. in order to assess the effectiveness of interventions like vaccination, resistance and serotype data should be monitored carefully. evolution in the pattern of sensitivity to penicillin and cefotaxime of streptococcus pneumoniae background: the increase in resistance to penicillin, and more recently to cefotaxime, means that it is necessary to study the prevalence of resistance to these antibiotics, giving them huge clinical importance. this study compares the sensitivity of s. pneumoniae to penicillin and cefotaxime during 1991,1995,2000,2001,2002,2003 and 2004. methods: a total of 892 strains of s.pneumoniae from significant respiratory tract isolates were studied. these corresponded to the years 1991 (130) conclusions: in 2004, (1) 14.9 % of the isolates were erythromycin-resistant and clindamycin-and miocamycin susceptible (m phenotype), a smaller percentage than in previous studies; (2) there was a significant increase in 2004 of isolates with the mlsb constitutive phenotype; (3) there was a high prevalence of resistance to telithromycin (88.6 %) in the 35 strains with mlsb constitutive phenotype. patterns of macrolide resistance determinants among s. pyogenes and s. pneumoniae isolates in saudi arabia objective: to characterize the macrolide sensitivity of recent isolates of s. pyogenes and s. pneumoniae collected from different hospitals around saudi arabia and to investigate the resistance determinants carried by macrolide-resistant isolates. methods: susceptibility testing was performed using standard nccls methodology on 335 s. pyogenes and 350 s. pneumoniae isolates. macrolide resistance mechanism phenotypes were identified using double disk diffusion. results: all s. pyogenes were penicillin sensitive, while 6.4% were macrolide resistant, the main mechanism of which was of m-phenotype (96%). approximately 51% of s. pneumoniae was penicillin non-susceptible. macrolide resistance in s. pneumoniae accounted for 18.8%, the majority of which was m phenotype (92%). low-level resistance mediated by mef-bearing strains predominated. conclusions: efforts should focus not only on antibiotic resistance surveillance and the development of guidelines but also on appropriate use of antibiotics. strategies have been proposed which include restricting access, compliance promotion and reduction in our prescriptions and inappropriate use of antibiotics. newer macrolides, including azithromycin, are still considered drugs of choice for empirical treatment of respiratory infection in such circumstances. objectives: mlsb phenotype erythromycin resistance (eryr) is often associated with tetracycline resistance (tetr) and in streptococci is mediated by the methylase genes erm(b) or erm(tr). in streptococcus pneumoniae, erm(b) is carried in transposons such as tn1545 which also carry the tetr gene tet(m). m phenotype eryr is associated with the efflux genes mef(a) or mef(e). these genes are carried in genetic elements that do not commonly include tetr genes, but the mef(e) element in s. pneumoniae can be inserted in a transposon that carries tet(m). our objective was to investigate tetr in eryr beta-haemolytic streptococci of groups a, b, c and g. methods: 129 eryr beta-haemolytic streptococci of groups a (26), b (42), c (9) and g (52) were collected over two years. tetr was determined by disc diffusion and mics by e-test. pcr was performed for tet(m), tet(o) and tet(l); eryr genes had been characterised previously. results: the prevalence of tetr amongst eryr mlsb isolates was high in groups a (73%, 11 of 15), b (92%, 34 of 37) and c (100%, 2 of 2) but lower in group g (38%, 18 of 47). the range of mics in tetr isolates was 4 ‡ 256 (mg/l). the tet(m) gene was responsible for most tetr in groups a (100%, 11 of 11), b (85%, 29 of 34), c (100%, 2 of 2) and g (56%, 10 of 18); tet(l) and tet(m) were both found in the same isolates in groups b (2) and g (1). at least 80% of erm(b) isolates and 57% of erm(tr) isolates of groups a, b and c carried tet(m). in contrast, only 21% of erm(tr) isolates in group g carried tet(m). amongst m phenotype isolates, the prevalence of tetr was high in groups b (100%, 5 of 5) and g (80%, 4 of 5) and lower in group c (14%, 1 of 7). tetr was not detected in m phenotypes of group a. in m phenotypes, tet(m) was responsible for 100% tetr in group b (5 of 5) and 25% in group g (1 of 4); 1 isolate of group c carried tet(o). tet(m) and tet(o) were found in mef(e) isolates but not in mef(a) isolates. there were tetr isolates in groups b (5) and g (11) in which no tetr gene was identified. conclusions: 1) the prevalence of tetr in eryr beta-haemolytic streptococci was high in mlsb isolates of groups a, b and c and in m isolates of groups b and g. 2) tet(m) was the predominant tetr gene. 3) tet(m) was strongly associated with erm(b) and to a lesser extent with erm(tr). 4) tet(m) was found in association with mef(e) but not mef(a). results: reduced susceptibility (mic >1 mg/l) was detected in 44 (2.7%) s. pyogenes isolates. a total of 32 (73%) s. pyogenes isolates demonstrated a mlsb phenotype resistance. the dominating gene was erm(a) (n = 27), encoding resistance in 27 strains, whereas erm(b) was detected in only five of the strains isolates. while one of the cmlsb strains carried both erm(a) and the mef(a) gene, another cmlsb strain was reproducibly negative in erm and mef pcr analyses, and will be further investigated. the remaining 11 (25 %) isolates all carried the mef(a) gene and showed a consistent with their m phenotype resistance. thirty (68%) strains were resistant to tetracycline and carried the erm-positive strains (n = 30) genes, while all the mtype strains were susceptible to tetracycline. typing analyses revealed t-type 4 and st39 dominated in nine 9 out of 10 with mtype phenotype, whereas t-type 3 was associated with st46 in 8 of 9 erm(a)-positive and tetracycline resistant strains. results from emm typing supported these clonal observations. conclusions: (i) the prevalence of macrolide resistance among s. pyogenes in norway is low, and the (ii) mlsb resistance, erm(a) or erm(b) was the most prevalent resistance type and co-resistance to tetracycline was frequently observed. rates of resistance to macrolides vary from region to region and these rates are important in defining the role of macrolides in empiric therapy. macrolide and lincosamide resistance are mediated by 3 distinct mechanisms, (1) mefa encodes resistance to erythromycin but not clindamycin, (2) ermb encodes resistance to erythromycin and clindamycin, (3) erma encodes resistance to erythromycin and inducible resistance to clindamycin. methods: consecutive pharyngeal isolates of streptococcus pyogenes isolates were collected between january 2003 and april 2004. isolates were confirmed as s. pyogenes by lancefield grouping using the pastorex strep rapid agglutination kit. erythromycin susceptibility testing was performed by nccls disc diffusion. erythromycin resistant isolates were tested for susceptibility to erythromycin and azithromycin by etest. the mechanisms of resistance was assessed using the erythromycin/ clindamycin disc approximation test and by pcr, with primers specific for the erma, ermb and mefa genes. positive controls were kindly provided by ralf r. reinert. results: four (4) of 360 consecutive isolates (1%) were resistant to erythromycin. erythromycin resistance was confirmed by etest and these isolates were also resistant to azithromycin. all 4 erythromycin-resistant study isolates and 3 of 4 previously collected erythromycin-resistant isolates were confirmed as erma positive by pcr and exhibited inducible resistance by disc approximation. one previously collected isolate was susceptible to clindamycin and was mefa positive by pcr. conclusion: these data show that macrolide resistance in s. pyogenes is very uncommon in this area and is encoded primarily by erma. objective: aim of this study was to evaluate antimicrobial prophylaxis (ap) practice and its cost in intensive care unit of heart surgery depertment in our hospital. method: study was performed prospectively between january-december 2002. ap lasting >1 day was considered inappropriate, unless the patient was in high risk group (heart transplantation or repeated by-pass). in addition ap with two beta lactam agents; beta lactam+aminoglycoside (only beta lactam+gentamicin was allowed); 3rd generation cephalosporins; quinolones or glycopeptides were considered inappropriate. in high risk patients, ap lasting up to 3 days and ap with glycopeptides were considered appropriate. cost of inappropriate ap was calculated by using august-2004 prices in euro. cost of inappropriate ap in patients who received cefazolin or beta lactam/beta lactamase inhibitors (ampicillin/sulbactam or amoxycillin/clavulanate) was calculated by substraction of cost of 1 day lasting ap from total ap cost. in case of inappropriate glycopeptide or quinolone or aminoglycoside usage, one day lasting cefazolin cost was substracted from total ap cost. in high risk patients cost of inappropriate glycopeptide usage was calculated by substracting 3 days lasting ap cost from total cost. infections were diagnosed according to the criteria of center for diseases control and prevention (cdc). data were analysed with chi square and student's t-tests. objectives: the aim of the present study was to investigate the frequency and the type of wound infections, as well as the bacteria involved, after posterior spinal fusion in children, especially with spinal deformities, over a 6-year period (1999) (2000) (2001) (2002) (2003) (2004) . materials and methods: a total of 133 spinal fusions were performed on 110 children, with or without instrumentation, because of spinal scoliosis (120), spondylolisthesis (4), fractures (4), and for other reasons (5 objectives: therapy of chronic wounds with lucilia sericata presents a promising alternative to the classical approach with antibiotics. larvae are placed in wounds free-roaming or in biobags. the therapy often stimulates the process of wound healing. this effect is based on different mechanisms. maggotsõ saliva contains collagenases, trypsin and chymotrypsin-like enzymes which dilute necrotic tissue. furthermore, an antibacterial effect has been described. in our study, we focused on the question whether this effect could be explained by the ingestion of bacteria by larvae. methods: free roaming maggots were placed on agar plates with escherichia coli. these bacteria had been transformed with green fluorescent protein (gfp)-containing plasmids. gfp-free escherichia coli xl1 blue served as a control. two days old maggots were put on agar plates in groups of ten at room temperature. every thirty seconds, a maggot was displaced from the agar plate and washed with sterile saline (0.9%). dead larvae which could not have taken up bacteria served as a control. maggots were tested for flourescence under a fluorescence microscope [zeiss axioskop; hpo50/ac, axiocam mrmzeiss]. larvae were examined by an independent observer as well. results: maggots that had been placed on gfp-labelled escherichia coli showed an intensive green fluorescence, especially at the back of the head and in the intestines. the controls did not show any fluorescence. after three to four minutes gfp-labelled escherichia coli could be detected inside the maggots' body. at this early stage of digestion gfp-fluorescence could mainly be examined in the area of the head. this fluorescence results of the ingestion of gfp-labelled escherichia coli by larvae. we have proven that the uptake of bacteria represents a further mechanism of antibacterial activity of larvae. not only diluting necrotic tissue, but also minimizing the rate of infections, maggot therapy which is also well-known as biosurgery could be a beneficial alternative to the use of antibiotics. as the mechanical uptake is a non-specific process, there might not be a risk of resistance. antibiotic resistance: nosocomial pathogens p1055 antimicrobial resistance patterns of bacterial pathogens from blood culture of cancer patients in a single cancer institution m. faiz, h. aziz, t. bashir, s. asghar (lahore, pak) objective: the widespread emergence of resistance to antimicrobial agents among bacterial pathogens is well known and has an impact on our ability to treat patients effectively. blood stream infections (bacteraemia) among cancer patients that develop during the course of disease are potentially life threatening because of suppression in their immune systems. the changing spectrum in the incidence and epidemiology of microbial pathogens has resulted in an increase in resistance to many antibiotic compounds emphasizing the need to monitor the prevalence of resistance in these strains. methods: susceptibility and resistance pattern of clinically significant bacterial isolates from positive blood cultures collected during 2000-2004 was studied. the isolated strains were tested against a wide range of antibiotics belonging to cephalosporins, aminoglycosides, carbapenems and quinolones derivative groups by disc diffusion method and the results were interpreted according to british standard for antimicrobial chemotherapy. results: a total of 250 bacterial pathogens were isolated with 60% gram positive and 40% gram negative bacteria. the dominating pathogens were staphylococcus aureus, streptococci, pseudomonas , enterobacter and klebsiella. among gram negative strains, highest level of resistance (98%) to third generation cephalosporins was observed followed by carbapenems and penicillin (79%, 80%) respectively. similarly, high resistance to aminoglycosides were found (69% to ampicillin, 50% to tobramycin and 62% resistant to quinolone derivates group of antibiotics. however only 29% were resistant to ciprofloxacin. in gram positive bacteria, high resistance to ciprofloxacin (40%) was observed as compared to gram negative bacteria. a still higher resistance level (100%) was observed for aminoglycosides and third generation cephalosporins (97%) respectively. conclusion: the spectrum of isolates among our patients were shifting towards gram positive bacteria with high resistance to different groups of antimicrobial agents limiting few choices for alternative therapies for infection control. antimicrobial resistance continues to increase and ongoing surveillance of microbial pathogens is essential. this study also warrants the need of infection control measures, rational antibiotic policies and rapid laboratory detection of resistance to prevent the spread of resistance among these strains. (9), and epidemiological swabs from throat and rectum (21). we found two susceptibility groups: group 1 (7 patients) resistant to amox/ clavul.acid, pip/tazobactam, aztreonam, cefuroxime, and group 2 (11 patients) susceptible for these antibiotics. rapd profiles corresponded with the susceptibility testing results. drug resistance profile in group 1 could be attributed to overproduction of chromosomal encoded k1 beta-lactamase. group 1 was from ru and group 2 from nicu, pu and su. analysis of sociodemographic variables and potential risk factors showed differences between analysed groups in mean birth weight, length of hospital stay, antimicrobial treatment and invasive therapeutic procedures usage. the outbreak analysis revealed concurrent isolation of two k. oxytoca clones, normal and k1 overproducer. rapd results were supported antibiotyping results. results: a total of 3077 isolates was analysed. 51.4% of these isolates were non-susceptible to at least one agent, the highest proportion is due to mono-drug resistance to piperacillin (39.6%). 4.8% of the strains were classified as multi-resistant, the most frequent patterns were pip/caz/ctx/gen (2.1%) and pip/caz/ctx/gen/cip (1.7%). two strains were resistant to all six antibiotics. significant differences in multi-drug resistance rates were associated with ward type with highest rates for icu-patients (8.8%). furthermore, multi-resistance rates varied significantly between the centres involved with a range from 0.9% to 7.2%. the centres differed not only in respect to the multi-drug resistance rates, but also in respect to the dominating phenotype. conclusions: the relevance of multi-resistance in k. pneumoniae as a major clinical problem is proved by an overall rate of almost five per cent for german university hospitals and an even higher proportion for icu-patients. among the agents tested piperacillin plays an eminent role in regard to its mono-resistant rate as well as a component of the most frequent resistance patterns. resistance to the combination of pip with tazobactam however is rare. the development of antimicrobial resistance in german hospitals objectives: quinolones (q) are among the most frequently used agents for treating ltcf-acquired infections. consequently, bacteria exhibit increasing resistance to q. a multicentre survey was conducted in order to determine (i) the prevalence and risk factors for colonization with qrgn in greek ltcf (ii) the corresponding antimicrobial resistant rates (á r). methods: a total of 28 ltcf were randomly selected from the public sanitation list of attica province. urine, nasopharyngeal and wound samples were collected from 668 elderly residents. we chose randomly 35% of the existing population from each ltcf (minimum sum 25 residents, age above 65 years). gram negative bacteria were identified by api strips and underwent antimicrobial disk susceptibility testing, following the nccls guidelines. data were also collected on resident factors and institutional variables. univariate and multivariate analyses were performed. odds ratios (or) and p values were calculated. results: 412 gram negative (gn) strains were isolated from 1448 samples (prevalence rate 28.5%). the majority of them were recovered from urine samples (86.8%). 53% of the residents had been taking systemic antibiotics during the preceding month. q were the leading class (38%). á r of gn to ciprofloxacin (cip) were estimated (table) . multi-drug qrgn accounted for 11.5% of the isolated strains. the most prevalent phenotype was resistance to ampicilline, cip and trimethoprim-sulfamethoxazole. in the multivariate analysis, only prior exposure to antimicrobial agents (p < 0.001; or = 2), specifically to q (p < 0.001; or = 14), and the presence of a urinary catheter (p < 0.001; or = 2) were significantly associated with colonization with qrgn. objectives: colonization and resistance dynamics of gramnegative bacteria in the intestinal and oropharyngeal microflora of patients admitted to intensive care units (icu) and general wards (gw) were investigated during and after hospitalization. methods: specimens were obtained on admission, once weekly during hospitalization, at discharge from the icu, at discharge from the hospital and one and three months after discharge from the hospital. five colonies per specimen were selected for identification and susceptibility testing by the vitek 2 system. results: a total of 3316 specimens from 411 patients were collected. in both patient populations, the gram-negative colonization rates in oropharyngeal specimens increased during hospitalization and did not decrease in the three months after discharge. in rectal specimens, colonization rates decreased during hospitalization and increased after discharge. there was a change in species distribution among the dominant microflora during hospitalization. klebsiella spp., enterobacter spp., serratia marcescens and pseudomonas aeruginosa were more often isolated, whereas the frequency of e. coli declined. the percentage of icu patients colonized with ampicillin and/or cephalothin resistant faecal e. coli was significantly increased at discharge from the hospital and did not change in the three months after discharge. emergence of multi-drug resistance was observed in many gram-negative species during stay on the icu. resistance frequencies in e. coli significantly increased with length of stay on the icu. for the gw population, no significant changes in resistance frequencies were found during hospitalization. conclusion: from a population perspective, the risk of dissemination of resistant gram-negative bacteria into the community through hospitalized patients appears to be low in gw patients, but is noticeably higher among icu patients. association of antibiotic resistance phenotypes and the presence of class i integrons in a sample of bacterial isolates from u.s. hospitals objective: we assessed the association between class 1 integrons and patterns of resistance to aminoglycosides, beta-lactams, quinolones, and chloramphenicol in escherichia coli and klebsiella species isolates from u.s. hospitals participating in project icare. methods: a convenience sample of 320 e. coli and klebsiella species isolates was collected between october 2002 and december 2003 from 19 u.s. hospitals as part of phase iv of project icare and tested for susceptibility to a variety of antimicrobial agents. these organisms were submitted in response to a request for organisms resistant to 3rd generation cephalosporins (ceftazidime, ceftriaxone, cefotaxime) and/or fluoroquinolones. the isolates were screened for the presence of class i integrons using sets of primers specific for 5¢ and 3¢ conserved segment of the integrase gene. we obtained measures of association between the presence and absence of integrons and resistance phenotypes. results: of the 320 e. coli and klebsiella species isolates screened, 181 (57%) contained a class i integron. a significant association was seen between the presence of the class i integron and resistance to amikacin, gentamycin, tobramycin, ampillicin, chloramphenicol, aztreonam, ceftazidime, cefotaxime, and cefpodoxime. resistance to ciprofloxacin and cefepime was not significantly associated with the presence of an integron. the presence of class i integrons in 57% of our convenience sample of e. coli and klebsiella species isolates, along with the differential association of those integrons with resistance to some drugs, but not others, suggests that integrons may play an important role in determining how the epidemiology of bacterial resistance results from antimicrobial agent selective pressure. objectives: the main purpose of this study was to investigate the mechanisms of resistance to fluoroquinolones in two isogenic citrobacter freundii clinical isolates, which led us to characterize the acra and acrb genes of this microorganism. methods: two c. freundii strains (1.44 and 1.38) sequentially isolated from the same patient were characterized. the relationship was determined by rep-pcr and pfge. the susceptibility to ciprofloxacin and chloramphenicol was determined by e-test. mutations in the quinolone resistance determining region of the gyra and parc genes, the outer membrane protein profile and the accumulation of ciprofloxacin was also investigated. the expression of genes in both strains was analysed using dna microarrays for e. coli and the expression of the acra and acrb genes was verified by rt-pcr using the gapa gene as the control. the acra gene was cloned and dna sequenced. results: both c. freundii belong to the same clone by both rep-pcr and pfge. the mic of ciprofloxacin was of 8 (strain 1.44) and 32 mg/l (strain 1.38). the mic of chloramphenicol was of 16 and 96 mg/l, respectively. the two strains showed the same substitutions in the gyra and parc (thr-83-ile and asp-87-tyr in gyra and ser-83-ile in parc). no major differences were found between the outer membrane protein profiles. however, differences were observed in the amount of ciprofloxacin accumulated, with strain 1.38 showing less accumulation. eleven genes were overexpressed in strain 1.38 compared to strain 1.44. among these genes the acra was overexpressed. this result was further corroborated by rt-pcr. the nucleotide similarity between the partially sequenced acra (1027 bp) and acrb (420 bp) genes of c. freundii and e. coli was of 81.5% and 86%, respectively. conclusion: the acra and acrb genes of c. freundii are similar to those described in e. coli and in collaboration with mutations in the gyra and parc genes, their overexpression may play an important role in modulating the final mic of fluoroquinolones. high prevalence of aac (3) objectives: multidrug resistance of gram negative bacilli (gnb) has become a major public health problem in tunisia. surveillance of both antimicrobial resistance and antimicrobial consumption has now been recognised as essential for planning future strategies to control resistance. the aim of our study is to examine for the whole hospital and wards with risks the correlation between resistance of gnb and the consumption of third generation cephalosporins (3rd gc) and imipenem for the period from january 2003 to september 2004. methods: a surveillance of antibiotic resistance and antibiotic consumption was established respectively by the laboratory and the department of pharmacy. data on antimicrobial resistance and antibiotic consumption were collected quarterly and antibiotic consumption was calculated in defined daily doses (ddd) using abc calc. results were expressed per 1000 bed-days (bd). statistical analysis was done using spss software for the calculation of the spearman rank correlation (rs) with an alpha risk of 5%. results: for the whole hospital, resistance to 3rd gc ranged from 0.9 to 1.7 gnb/1000 bd. the highest rates were observed in intensive care unit (icu: 4.7-12.7 gnb/1000 bd) and the lowest rates in orthopaedic ward (0-2 gnb/1000 bd all mrsa isolates were resistant to multiple classes of antimicrobials whereas mssa were sensitive (p < 0.001). all esbl+ bacteria were resistant to multiple classes but sensitive to meropenem, colistin, amikacin (82%), cotrimoxazole (50%) and chloramphenicol (50%). resistance in non-esbl+ bacteria was detected to ampicillin (73%), cefalexin (32%), 2nd/3rd generation cephalosporin (0%), chloramphenicol (32%), cotrimoxazole (11%), ciprofloxacin (5%), trimethoprim (21%), gentamicin (5%), netilmicin (53%) and tobramycin (11%). potentially community acquired isolates showed lower rates of resistance (0% to trimethoprim, ciprofloxacin, gentamicin, amikacin, tobramycin, 2nd/3rd generation cephalosporin).duration of hospital stay was not a risk for acquiring either esbl+ bacteria or mrsa whereas having a surgery was associated. exposure to quinolone was a risk factor for mrsa but not esbl+ bacteria. aminoglycoside and cephalosporin were risks for esbl+ bacteria. acquiring both mrsa and esbl+ enterobacteriacae together did correlated with the duration of hospital stay in addition to exposure to aminoglycoside and cephalosporin. conclusion: majority of patients remained free from resistant bacteria implying that cross-transmission alone was not sufficient in absence of risk factors, particularly exposure to antimicrobials. good hand-hygiene and prudent use of antimicrobials are realistic options for resource poor countries to reduce the burden of resistant bacteria. there is a unique opportunity to sequentially introduce specific infection control measure and evaluate its effectiveness. antimicrobial resistance in an intensive care unit before adopting an antibiotic rotation programme for empiric therapy of ventilator-associated pneumonia results: among gram-negative aerobic bacilli, we found p. aeruginosa strains to be meropenem and piperacillin/tazobactam-resistant in 22% and 14.6% of cases, respectively; p. aeruginosa, e. coli and k. pneumoniae were resistant to ceftazidime in 29.3%, 6.25%, and 0% of cases, respectively, and resistant to ciprofloxacin in 54.9%, 12.5%, and 5.6% of cases, respectively. neither e. coli nor k. pneumoniae were resistant to either meropenem or piperacillin/tazobactam. among gram-positive aerobic cocci, oxacillin-resistant s. aureus and cns were 55.8% and 82.7%, respectively; s. aureus isolates were very susceptible to cotrimoxazole (resistance 7.65%) and rifampin (11%), whereas resistance exceeded 40% for clindamycin, gentamicin, and norfloxacin. ampicillin-and penicillin-resistant e. faecalis were 20.8% and 33.3%, respectively. neither s. aureus nor e. faecalis were resistant to vancomycin, whereas one cns strain was resistant to it (1.3%) and 6 out of 76 to teicoplanin (7.9%). conclusion: in our icu, where a careful policy of antibiotic use with no predetermined restrictions has been applied for years, ar among both gram-negative and gram-positive microorganisms is generally lower than in the icus of italy and other mediterranean countries. we expect that the institution of an arp for empiric therapy of vap can further minimize ar in our icu. background: although newer antibiotics have been introduced to the market during the last years, they have not solved the problems arising in the management of infections due to multiresistant gram-negative bacteria. colistin, an antibiotic almost forgotten for decades has proved itself helpful, when used parenterally in patients where a lot of the classic and newer antibiotics fail. methods: we report our experience with the management of the case of a young patient who, after head trauma, had five episodes of meningitis due to multidrug-resistant gram-negative microorganisms. results: a 28-year-old patient was admitted in our hospital because of coma. three months prior to his admission, he sustained acute brain injury due to a fall and was hospitalized elsewhere for this entire period. a computerized tomography scan of his brain at his admission in the first hospital, showed multiple cerebral contusions, and an intracerebral hematoma causing deviation of the midline. he underwent a decompressive craniotomy with removal of the hematoma. during his long-standing hospitalization, he developed 5 episodes of meningitis, i.e. on day 65, 96, 108, 147, and 165 after head trauma. the pathogens isolated from each episode of meningitis were multiresistant strains of pseudomonas aeruginosa (1st and 2nd episodes), acinetobacter baumannii (3rd episode), enterobacter cloacae (4th episode), and acinetobacter baumannii (5th episode). the two episodes of acinetobacter baumannii meningitis were managed only when colistin and amikacin were given by the intraventricular in addition to the intravenous route for long period of time (six weeks for the last episode) and in high doses (40,000 iu of colistin and 10 mg of amikacin results: eight patients received aerosolized colistin. all were admitted to the icu with a mean acute physiological and chronic health evaluation ii (apache ii) score on the day of icu admission and on the 1st day of aerosolized colistin administration of 14.6 and 17.1, respectively. six of the 8 patients had ventilator-associated pneumonia. the responsible pathogens were acinetobacter baumannii (7/8 cases) and pseudomonas aeruginosa (1/8 cases) strains. half of the isolated pathogens were sensitive only to colistin. daily dosage of aerosolized colistin ranged from 1.5 to 6 million iu (divided into 3 or 4 doses) and the mean duration of administration was 10.5 days. seven out of 8 patients received concomitant intravenous treatment with colistin or other antimicrobial agents. clinical response of pneumonia was observed in 7 out of 8 patients [4 cured, 3 improved (they were transferred to another facility)]. one patient deteriorated and died due to septic shock and multiple organ failure. aerosolized colistin was well tolerated by all patients; no bronchoconstriction or chest tightness was reported. conclusions: aerosolized colistin may be a beneficial adjunctive treatment in the management of nosocomial pneumonia (vap or not) due to multidrug-resistant gram-negative bacteria. to date, susceptibility rates to imipenem and colistin of nosocomial strains are reported as high as 88% and >98%, respectively. in particular, resistance to colistin has been rarely described and the mechanisms of this resistance are poorly understood. we report a case of colistin resistant a. baumannii infection in a patient without previous administration of colistin. case report -methods: a colistin resistant (mic >2 mcg/ml) a. baumannii was isolated from 3 sets of blood cultures in a patient admitted in a burn unit without previous colistin exposure. the patient was treated with ampicillin/sulbactam and colistin, despite the documented resistance to all antibiotics. mdr a. baumannii was repeatedly isolated from different sites and the patient died after 61 days of hospitalization. strain identification and susceptibility tests were performed using phoenix automated microbiology system ò . resistance to ampicillin, ampicillin/sulbactam, ceftazidime, imipenem, meropenem, cotrimoxazole, amikacin, ciprofloxacin and colistin was confirmed by disk diffusion test and broth microdilution, in accordance with the nccls guidelines. the susceptibility results were confirmed by a reference laboratory. conclusion: colistin is one of the few effective drug available against mdr acinetobacter infections and acquired resistance is exceptional. our report points out that colistin resistant a. baumannii may be found in hospitalized patients without previous exposure to colistin. nosocomial infections by colistinresistant a. baumannii could represent a new therapeutic challenge. programmes of active surveillance cultures and contact precautions should be implemented to control the spread of this mdr microorganism. intrathecal colistin treatment of central nervous system infections due to multi-resistant acinetobacter baumannii c. suárez fernández, p. fernández-viladrich, f. tubau, l. corral , a. mateu, j. ariza (barcelona, e) objectives: the optimal therapy of cns infections due to multi-resistant acinetobacter baumannii (mrab) is not established. in 1999 we published the first two cases of ventriculitis due to mrab treated with intrathecal (it) colistin. one patient received 10 mg colistin base/12 hours after delayed csf sterilization was documented with initial dosage of 5 mg/12 hours. here we describe our extended experience with this kind of treatment . methods: we retrospectively reviewed all documented cns infections due to mrab diagnosed in our terciary 1000-bed hospital from 1999 to 2004. mrab was defined as those strains with only susceptibility to colistin (mic < 4 mg/l). cns infections were defined as those cases with suggestive clinical features plus ventricular or lumbar csf with both characteristic cytochemical alterations and positive culture. results: there were 7 cases with documented nosocomial cns infection due to mrab. six of them were acquired after neurosurgical procedure through surgical wound infection or local catheter infection. one case was caused by hematogenous spread after urological manipulation. intrathecal therapy was administered through a ventricular catheter in five cases, lumbar catheter in one case and both ventricular and lumbar catheter in one case. the usual doses administered was colistin base 10 mg (equivalent to colistimethate sodium 24 mg) every 12 hours with csf continuous drainage being interrupted for about 3 hours. length of treatment were 8-21 days with any apparent adverse effect. six of these patients received simultaneous intravenous colistin therapy. culture sterilization was documented in all patients. two patients died (one from unclear cause after initial favorable evolution; one after receiving only two doses of it colistin). the rest of them cured with sequela attributable to their previous neurological diseases. conclusions: in our experience, combined treatment with both intrathecal and intravenous colimicin seems safe and effective. when administered by local route to patients with continuous csf drainage we suggest a dosage of colistin base of 10 mg every 12 hours with temporary interruptions of the drainage. bacillus cereus -the forgotten pathogen. an unusual infection in an orthopaedic patient s. thomas, a. pillai, j. arora, a.c. ogden (dumfries, uk) background: infection with bacillus cereus has been well documented in the literature for over a century. infection is generally associated with the gastrointestinal effects of food poisoning and linked to the consumption of infected rice dishes. however, the bacillus genus is extremely heterogenic. it can occupy a wide variety of ecological niches and bacillus spores are found ubiquitously in the environment. even so, bacillus cereus isolated from the hospital and clinic setting in any material other than vomitus or faeces is commonly dismissed since it is a known contaminant of blood cultures and most bacillus bactereamias are transient and usually insignificant. case report: we report a case of bacillus cereus wound infection in a previously healthy 31-year-old male admitted to the orthopaedic ward with a comminuted fracture of the right distal tibia. he developed compartment syndrome one day later and was taken to theatre for fasciotomy. within 48 hours, he developed chest complications and was admitted to the high dependency unit. after three days he was taken back to theatre for wound inspection and change of dressing. seven days later external fixation was applied. subsequently, the wound site became red, inflamed and tender and was associated with an acute rise in inflammatory markers. microbiological reports showed bacillus cereus growth from the suture sites, sensitive to ciprofloxacin. no source was identified. discussion: this report merits concern, because it highlights the risk of wound cross infection with this unlikely pathogenic species. bacillus cereus has been known to be a contaminant of dressings, intravenous catheters, theatre scrub suits and linens pointing to an array of possible infective sources within the hospital. it emphasizes the need for theatre and hospital sterility and stresses the importance of vigilance against this infrequent cause of a potentially serious non-gastrointestinal bacillus infection. this is of particular importance because alcoholbased cleaning solutions are known to be ineffective against bacillus spores and infections dismissed as contaminants may lead to rapid clinical deterioration. antibacterial effect of phenytoin in wound healing objectives: phenytoin is an antiseizure medication, has since been reported to promote wound healing when applied as a topical agent. this effect is due to rapid infiltration of fibroblasts, collagen deposition, new vessel formation as well as antibacterial activity. this study was undertaken to evaluate it's effect on chronic skin ulcers with different causes and compare it with normal saline. methods: fifty inpatients with chronic skin ulcers were included in this case-control study: diabetic foot (20%), fracture and surgery wounds (40%), decubitus ulcers resulting from warrelated (40%). the patients were matched for age, sex, severity and size of wounds and randomly assigned to two group: 25 phenytoin treated (pht) and 25 control (ctl). each group included 15 men and 10 women, age range was between 20 and 60. surface areas of the ulcers was 12-200 cm 2 . the ulcers were debraded of necrotic tissue (if required). cultures of ulcers were taken at the beginning of treatment and on day 7, 14. in pht, thin dusting of phenytoin powder and dry gauze dressing were applied daily after washing with normal saline. ctl group received only daily washing with normal saline and plain dressing. results: bacteriologic culture in both group confirmed some strains (staphylococcus aureus, kelebsiella, proteus and psudomonas). at the beginning of treatment, there was 17 culture positive in pht. 13 case became negative by day 7 and 4 case became negative by day 14. in ctl, we had 15 culture positive that they didn't become negative by day 7 and 14 (seventy-six per cent of pht had negative cultures by day 7 compared to 0% of ctl). the mean time for appearance of granulation tissue was 13.48 days in pht compared with ctl that was 37.5 (p = 0.006). the average time to complete healing in pht was 24.76 days compared to 43.36 in the ctl (p = 0.0001). in pht only 13 patients required to analgesics (mean 1/day) compared with ctl that all patients required to analgesics about 3-4/day (p = 0.0001). age, sex and kind of wounds didn't effect the healing time in both group. conclusions: this study with statistical analysis demonstrated good improvement and efficacy of phenytoin in treatment of chronic ulcers compared with normal saline. 1. prompt pain relief, 2. reduction in ulcer size, 3. changing bacterial culture to negative, 4. more rapid granulation and healing time, characterized the pht group. we recommend wider use of this safe, inexpensive, readily available and easy to use agent because of it's positive effect on wound healing especially in our country that a lot of patients suffer from decubitus ulcers resulting from war-related wounds and limited access to more expensive wound care therapies. recurrent osteomyelitis secondary to different bacteria i. uckay, m. assal, l. legout, p. rohner, r. stern, d. lew, p. hoffmeyer, l. bernard on behalf of the osteomyelitis group study objective: recurrence of osteomyelitis due to the same bacteria from a prior site of infection without evidence of trauma or bacteraemia is an entity well-known in the literature and clinical practice. the objective of this study is to try and determine if all these reactivations are actually due to the same bacteria, as has been assumed in previous case reports. case reports: we report three cases of osteomyelitis recurring in the same bone in otherwise healthy patients. in all three patients, there was no history of illness or trauma to assume another origin. surprisingly, the strains were different from the two infectious episodes. conclusion: reactivation of osteomyelitis can be caused by different strains of bacteria, many years after the initial episode without trauma or evident bacteraemia. former infected and therefore altered bone surface might be a region of diminished resistance for a new infection during silent transient bacteraemia, reminiscent of the clinical entity of (recurrent) infectious endocarditis on altered valve surfaces. objectives: vascular graft infection proved to be the most dangerous complication in vascular surgery patients. the aim of our study was the identification of microorganisms causing vascular graft infections and the evaluation of their antimicrobial susceptibility. 25 patients with infected vascular grafts, treated in vascular surgery department, took part in our research. in 76% of patients, the late type of infection was recognized, in 24% of patients the infection was qualified as early. methods: purulent discharge obtained from the fistula was inoculated on the bacteriological media. antimicrobial susceptibility was assessed by disc-diffusion method. results: staphylococcus aureus and pseudomonas aeruginosa, present in 16,7% of patients, proved to be the most frequently isolated microorganisms. staphylococcus epidermidis and e. coli were isolated in 13.3% and 10% of patients, respectively. mixed infection, caused by two distinct bacteria, occurred in 20% of patients; in all cases one species belonged to gram-positive, and the second one to gram-negative bacteria. in 50% of patients with early type infection different species of gram-negative rods were present, in 37.5% of patients, staph. aureus and staph. epidermidis were isolated, enterococcus faecalis occurred in 12.5% of patients. in late type infection gram-negative rods were isolated from 54.5% of patients and gram-positive bacteria from 31.5% of patients. the most frequently isolated species appeared to be pseudomonas aeruginosa. in one patient candida crusei was isolated. the isolated species of bacteria varied depending on the degree of infection (according to shilagy and samson). in iiia degree of infection staph. epidermidis and e. coli were present in 67.7% and 33.0% of patients, respectively. pseudomonas aeruginosa and staph. aureus were isolated in 21,1% of patients with iiib degree of infection. various species of gramnegative rods were isolated from 80% of patients with iiic degree of graft infection. conclusions: most isolated bacteria appeared to possess the resistance patterns typical for hospital flora; among them methicillin-resistant staph. aureus (mrsa) and methicillin-resistant coagulase-negative saphylococci (mrcns) strains, gramnegative rods producing extended spectrum of beta-lactamases (esbl) or ampc beta-lactamases, and enterococcus faecalis with high level of aminoglicosides resistance (hlar). treatment failure due to emergence of resistance to imipenem during therapy for shewanella algae bacteraemia objective: we describe here a case of bacteraemia caused by s. algae, which was initially susceptible to imipenem, but the bacteria later became resistant during the treatment. in addition, we investigated the propensity of s. algae to develop resistance to imipenem by using a serial passage technique. method: in vitro test for resistance induction 1. bacterial strains resistant variants selection (a) single step resistant variants were obtained from clinical isolate on mueller-hinton agar containing increasing amounts of imipenem (1xmic, 2xmic, 4xmic, 8xmic), (b) serial passage experiment s. algae strain and p. aeruginosa atcc 27853 were grown overnight in mueller-hinton agar and then grown overnight in mueller-hinton agar and then swabbed onto mueller-hinton agar plates containing one-half the mic of imipenem. the surface growth at 24 h was swabbed to mueller-hinton agar containing twice the prior concentration of imipenem. results: single step resistant variants were selected from isolate 1 at up to 4 times the mic, whereas the resistant variant from p. aeruginosa atcc 27853 could be selected at up to 2 times the mic. all the resistant variants of s. algae selected either by a single step or by a sequential stepwise passage exhibited at up to 8-16 lg/ml of mic, whereas those of p. aeruginosa atcc 27853 showed up to 16 lg/ml of mic conclusion: we documented that s. algae, which was initially susceptible to imipenem, subsequently became resistant to imipenem during the treatment. we also demonstrated in vitro that the initial isolate of s. algae could easily develop resistance to imipenem when the organism was exposed to imipenem, suggesting that s. algae organisms have a propensity toward resistance to imipenem. objective: peritonitis remains a common and serious complication of peritoneal dialysis. in this study, we evaluated the frequency of microorganisms isolated from peritoneal fluids of patients on continuous ambulatory peritoneal dialysis (capd). methods: during a 2.5 year period, 321 peritoneal fluid samples were collected from 107 capd patients with peritonitis symptoms. the specimens were inoculated on appropriate media after 10-min centrifugation. gram stained smears and aerobic, anaerobic and broth cultures were performed. the isolates were identified by commercial id panels. mics were determined with a microdilution method according to nccls guidelines. results: fifty two out of 321 samples were positive (16.2%), six of the positives were polymicrobial. the following microorganisms were obtained: pseudomonas aeruginosa (8), staphylococcus epidermidis (7), escherichia coli (7), staphylococcus aureus (6), streptococcus viridans (6), klebsiella pneumoniae (4), enterococcus faecalis (4), stenotrophomonas maltophilia (4), enterobacter aerogenes (2), acinetobacter baumannii (1), enterococcus faecium (1), proteus mirabilis (1), pseudomonas fluorescens/putida (1), acinetobacter lwoffii (1), salmonella group d (1), bacteroides spp. (2). fungi were isolated in three patients: candida tropicalis (1), candida krusei (1) and candida parapsilosis (1). three out of 6 of s. aureus strains and three out of 7 of s. epidermidis isolates were found resistant to methicillin. all saphylococci and enterococci were susceptible to vancomycin and linezolid. gram-negative pathogens were sensitive to used antibiotics. conclusion: thirty five patients (32.7%) developed peritonitis the most prevalent etiologic agents of peritonitis on capd patients were pseudomonas aeruginosa, staphylococcus epidermidis, escherichia coli, staphylococcus aureus and streptococcus viridans. since capd patients are commonly outpatients, the antimicrobial resistance in the gram-negative strains is low compared to the nosocomial isolates. appropriate antibiotic therapy based on microbiologic results needs for the management of peritonitis on capd patients. objectives: cefepime is a fourth generation cephalosporin with a broader spectrum of activity against gram-negative and gram-positive pathogens in comparison with all other cephalosporins. due to the excellent antibacterial activity including pseudomonas aeruginosa and enterobacter spp. together with low rates of resistance and favourable pharmacokinetic and clinical properties, cefepime is a drug of choice for initial empiric treatment of severe nosocomial infections. nevertheless, in addition to recommendations in guidelines, the surveillance of local resistance data is important for empiric treatment decisions in hospitals. methods: in this study, the in vitro activity of cefepime (cep), imipenem (imi), piperacillin tazobactam (p/t), ceftazidime (caz) and cefotaxime (cft) has been investigated against 1130 bacterial strains, isolated in the year 2004. mics have been determined by microdilution method according to din 58940. reference test strains were e.coli atcc 25922, s. aureus atcc 29213, p.aeruginosa atcc 27853, and s.pneumoniae atcc 49619. results: highest in vitro activity against gram negative enterobacteriaceae were determined for cep and imi with susceptibility rates for enterobacter spp. 98%,98%, m.morganii 96%,92%, proteus spp. 98%,96% and citrobacter spp. 96%,100% whereas the susceptibility rates for p/t, caz and cft were much lower (e.spp. 68%,72%,68%; m.m. 88%,72%,66%; p.spp. 96%,96%,66%; c.spp. 68%,76%,72%). for pseudomonas aeruginosa (n = 100), susceptibility rates were 76% (cep), 73% (caz), 72% (imi) and 55% (p/t) with lowest resistance rates for cep (6%) followed by caz (11%), p/t (14%), imi (23%). the study confirmed excellent cefepime activity against gram positive isolates (pneumococci, streptococcus viridans and b-hemolytic streptococci) and high cefepime susceptibility rates for staphylococci (mssa 99% and msse 98%) on contrary to ceftazidime (mssa 44% and msse 42%). conclusion: regarding our in vitro data, cep is a reliable treatment option for empiric therapy in patients with severe nosocomial infections, demonstrating high activity against gram positive isolates, comparable activity in gram negative enterobacteriaceae to carbapenems (imi) and lowest resistance rates in pseudomonas aeruginosa. antimicrobial activity and spectrum of cefepime tested against 65,746 clinical strains from north american medical centres: report from the sentry antimicrobial surveillance program, 1998 program, -2004 h. sader, d. biedenbach, t. fritsche, r. jones (north liberty, usa) objectives: to evaluate antimicrobial spectrum and potency of cefepime (cpm) and selected comparators against clinical bacterial strains collected in north america (na) over a 7-year period (1998) (1999) (2000) (2001) (2002) (2003) (2004) . methods: isolates were consecutively collected from bloodstream (44%), respiratory tract (41%), urinary tract (6%) and skin/soft tissue (5%) infections in 48 medical centres. 75% of isolates were from hospitalized patients. isolates were susceptibility (s) tested by reference nccls broth microdilution methods in a central laboratory. oxacillin-resistant (r) staphylococci (ors) and enterococci were excluded. results: the activity of cpm against the key organisms tested is summarized in the table. overall, 99.8% of gram-positive cocci (gp) tested were s to cpm. imipenem (imp; mic90, 1 mg/l; 99.9% s) was the most active compound tested against ent, followed by cpm (mic90, 0.25 mg/l; 99.5% s) > amikacin (amk; 99.4% s) > ceftriaxone (95.6% s) > aztreonam (95.1% s). the lowest s rate for ent was observed with ciprofloxacin (cipro; 92.8%). imp was also the most active compound against esbl-producing ksp and e. coli (99.3 and 100% s, respectively), followed by amk (81.4 and 97.2% s) and cpm (92.5 and 93.8% s). cpm activity against psa (85.2% s) was similar to that of imp (86.9% s). against ossa, cpm was 4-fold more potent than ceftazidime (caz; mic90, 16 mg/l, 86.4% s) and showed higher activity than cipro (93.2% s). cpm was the most active compound against spn after gatifloxacin and levofloxacin (99.2% s). against vgs, cpm was 8-fold more potent than caz and 4-fold more potent than piperacillin/tazobactam. regionally, mdr ranged from 9.1% in los angeles to 32.2% in south florida. r to pen, ery, and sxt was the most common mdr phenotype in all regions except baltimore/dc (r to ery and sxt was most common). by specimen source, 15.5% of blood, 21.0% of lr, and 29.0% of ur isolates were mdr with r to pen, ery, and sxt being the most common phenotype regardless of specimen source. for the 30 strains tested, the fluoroquinolone mic ranges were: 0.008-0.03 mg/l (gem), 0.12-0.25 mg/l (gat), 0.06-0.25 mg/l (mxf), 0.5-1 mg/l (lfx). conclusions: mdr among sp is a phenotype that is widely dispersed geographically and is likely to be encountered regardless of the site of infection. fluoroquinolones show activity against most mdr isolates. as the use of fluoroquinolone compounds increases, surveillance to monitor the prevalence of mdr and track the in vitro activity of agents such as gem used for these resistant strains must be continued. background: multi-drug resistant (mdr) s. pneumoniae strains are increasing at an alarming rate worldwide. the therapy of respiratory tract infections due to these strains is challenging with an urgent need for antimicrobials with reliable activity against the mdr strains. comparative activity of oral cephalosporins and parenteral cephalosporins against various pneumococcal mdr phenotypes was analysed in a large, multi-year international collection of clinical strains of s. pneumoniae. methods: s. pneumoniae strains (21,605) collected during 1997-2003 from worldwide participants of the sentry program were tested and interpreted using nccls (m7-a6, m100-s14) guidelines. the antimicrobial agents analysed included penicillin (pen), erythromycin (er), clindamycin (cm), tetracycline (tet) and trimethoprim/sulfamethoxazole (ts); cephalosporins monitored included oral (cefpodoxime, cefuroxime) and parenteral (ceftriaxone, cefepime) agents. results: the rank order of occurrence rates of the various resistance phenotypes were: pen only (32.0%) > pen and er (17.6%) > pen, er and cm (8.6%) > pen, er, cm and tet (7.6%) > pen, er, cm and ts (6.5%) > all five drugs (5.7%). the susceptibility rate of all strains to the orally administered cephalosporins, cefpodoxime (77.6%) and cefuroxime (77.3%), dropped to only 16.1% and 14.5% respectively, for the five-drug mdr phenotype. the parenteral cephalosporins retained excellent activity for all mdr phenotypes, with resistance rates being lower for cefepime than ceftriaxone (cefepime, 1.3-1.9%; ceftriaxone, 3.0-4.4%) or the oral cephalosporins (cefpodoxime, 64.4-74.1%; cefuroxime, 69.3-79.1%) using respiratory infection breakpoints (nccls). conclusions: our in vitro findings confirm that the parenteral cephalosporins, cefepime and ceftriaxone, retain excellent activity against the mdr phenotypes analysed, and remain useful drugs in the armamentarium to treat mdr pneumococcal respiratory tract infections. antibiotic resistance surveillance over a 5-year period in spain: results of the mystic programme results: three icus, two neutropenia units and two general wards provided a total of 4.022 gram-negative and grampositive isolates during the period 1999-2003. the most common species tested were escherichia coli (14.3%), methicillin-susceptible staphylococcus aureus (13.8%) and coagulase-negative staphylococci (13.5%), pseudomonas aeruginosa (12.2%), enterobacter cloacae (7.2%), klebsiella pneumoniae (5.7%) and a.baumannii (5.6%). in general, the carbapenems (mem and ipm) were the most active antimicrobial agents tested against the common organisms (range of %s:100% to 71% and 100% to 67%,respectively). among enterobacteriaceae, 100% of enterobacter spp, citrobacter spp and serratia spp were susceptible to carbapenems. e. coli and k. pneumoniae susceptibility to carbapenems were 100% and >98% respectively. the %s of mem was the same as or higher than ipm against every organism tested. cip resistance in e. coli was around 20%. caz and taz were the least active antimicrobial agents against enterobacter sp (74% and 77% s, respectively) and citrobacter spp (67% and 68% s, respectively). mem and taz were the most active agents against p. aeruginosa (89% and 88%s, respectively). a. baumannii were 61-90% susceptible to carbapenems, but only 6-1% susceptible to cip. in this period, around 100% of methicillinsusceptible staphylococci were susceptible to mem. conclusion: there was no significant decrease in susceptibility to the carbapenems throughout the five-year period. mem and imp appear to remain reliable options for the treatment of serious nosocomial infections. the clinical use of mem did not increase bacterial resistance to this agent in the spanish centres evaluated. surveillance of antimicrobial susceptibility of anaerobes in a belgian university hospital y. glupczynski, h. nizet, c. berhin (yvoir, b) background: antimicrobial resistance is becoming a growing concern among anaerobes involved in human infections. since there are only scarce data on the in vitro susceptibility of anaerobes in our country, we aimed to determine the susceptibility and resistance profiles of anaerobic bacteria isolated in our hospital. table. conclusion: b-lactams-b-lactamases inhibitors, carbapenems, and met remain highly active against anaerobes including the more resistant bfg of organism. resistance in cli actually almost reaches 30% and is observed among almost all different species of anaerobes. mox, as a representative of the newer fluoroquinolones exhibits a broad spectrum and potent activity in vitro against all anaerobes tested and looks promising for the treatment of mixed infections involving anaerobes. pharmacokinetics/pharmacodynamics of quinolones objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens, including anaerobes. some of the fluoroquinolones have the potential to prolong qtc. to determine the effect of grn on qtc, a retrospective analysis was performed on manually read ecgs from five phase i studies. methods: serial ecgs were collected in 5 randomized, doubleblind, placebo-controlled studies in healthy adult subjects administered oral (po) or intravenous (iv) grn 50-to 1200mg doses (therapeutic dose £600 mg) with dosing duration 1 to 28 days (therapeutic duration £14 days). the qt interval was corrected for heart rate using bazett's (qtcb) and fridericia's (qtcf) formulas, and the effect of grn was assessed by counts of outliers and linear regressions. single-and multiple-dose pharmacokinetics of po and iv grn were derived from plasma concentration versus time data. results: a total of 149 subjects received grn and 55 received placebo. grn plasma exposure (auc and cmax) increased with increasing dose. no subjects experienced a prolonged qtcb interval (>450 msec for males, >470 msec for females). only one subject experienced a prolonged (>60 msec) qtcb change from baseline which occurred on day 7, but not on subsequent days despite continued dosing. the incidence of borderline qtcb (change between 30-60 msec) was comparable between placebo and grn. means for qtcb max, qtcb avg and qtcb at tmax and their changes from baseline for grn were similar to those for placebo, with the exception of qtcb max, qtcb at tmax, and their changes for 600 mg iv grn on day 14. although 1200 mg po and 800 mg iv grn produced higher plasma exposures than 600 mg iv on day 14, the means for the derived qtcb values were comparable to placebo. all 95% confidence intervals for the linear regression slopes of derived (delta)qtcb on cavg(0-12 h) were equal to or less than 0, except for (delta)qtcb at tmax on day 7, indicating that grn had no effect on qtcb. results obtained for qtcf were similar to those for qtcb. conclusions: grn does not appear to have dose-, route of administration-or concentration-dependent effects on qtc interval in healthy subjects. objective: garenoxacin (grn) is a novel des-f(6)-quinolone being developed for a variety of indications because of its efficacy against a broad spectrum of pathogens, including anaerobes. the objective of this study was to assess the tissue or fluid to plasma ratios of grn following a single oral dose. method: an open-label study was conducted in subjects ‡18 years of age with a body mass index £33 kg/m2 undergoing abdominal surgical procedures that would permit the removal of tissue or fluid without increased risk to the subject. a single 600 mg oral dose of grn was administered based on the scheduled operative time. the tissue or fluid (with corresponding plasma sample) was collected 3 to 5 hours post-dose. concentrations of grn in biological fluids and tissues were determined using validated lc/ms/ms assays. safety was assessed by measurement of vital signs, physical examination, and electrocardiographic and clinical laboratory evaluations. adverse event (ae) monitoring was performed from time of consent until discharge from the study. results: thirty-one subjects were enrolled in and completed the study. mean fluid or tissue grn concentrations greater than that found in plasma (mean fluid or tissue to plasma ratio >1) occurred in large and small bowel tissue, gallbladder, and liver. mean fluid or tissue grn concentrations less than that found in plasma occurred in adipose tissue, bone, sinus mucosa, striated muscle, incisional skin, and subcutaneous tissue. mean grn concentrations in bile and lymph node tissue were roughly similar to that found in plasma. tissue and fluid concentrations of grn exceeded the mic90 of most target organisms involved in skin and soft tissue, bone, and intra-abdominal infections and sinusitis ‡2-fold. no treatment-emergent aes or serious aes were considered to be related to grn. conclusion: a 600 mg oral dose of grn penetrates well into most of the tissues and fluids studied, with concentrations exceeding the mic90 of most pathogens causing sinusitis, skin and soft tissue infections, bone infections, and intra-abdominal infections. these results suggest that adequate concentrations of grn can be achieved to treat infections at these sites. penetration of garenoxacin into lung tissues in patients undergoing lung biopsy or resection objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens, including those commonly found in the respiratory tract (rt). this study was conducted to determine the penetration of grn into lung parenchyma (lp) and bronchial mucosa (bm) following a single 600 mg oral dose. penetration of grn into bone was also assessed. this was an open-label, nonrandomized study in subjects undergoing an invasive procedure to the lung (other than percutaneous lung biopsy) which facilitated the removal of macroscopically normal healthy lung tissue without exposing the subject to undue risk. penetrance of grn into bone was determined when removal of rib bone was part of the normal surgical procedure. a single 600 mg oral grn dose was administered based on the scheduled operative time. lp and, if possible, bm and/or bone samples and corresponding plasma samples were removed at 2 to 4, 4 to 6, 10 to 12, or 20 to 24 h post-dose. concentrations of grn in these samples were determined using validated lc/ms/ms assays. safety was assessed based on the occurrence of adverse events (aes) and changes in physical examinations, vital signs, clinical laboratory results, and electrocardiographic results. results: twenty-seven subjects were enrolled; samples were taken from a minimum of 6 patients at each time interval. mean grn concentration in lp increased between the 2-4 and 4-6 h post-dose, suggesting rapid penetration into lp, then declined similar to the decrease seen in plasma concentration. concentrations of grn in lp exceeded the mic90 of organisms associated with rt infections by 62-to 466-fold over a 24 h period. grn concentrations in bm over a 24 h period exceeded the mic90 of respiratory pathogens 51-to 380-fold. concentrations of grn in bone exceeded the mic90 of the organisms associated with bone infections (except pseudomonas aeruginosa, mrsa, fusobacterium species, and enterobacter species) by 6-to 101-fold over a 12 h period. across the 4 time intervals, mean ratios of tissue to plasma grn concentration in lp, bm, and bone reached 2.80, 0.99, and 0.56, respectively, suggesting adequate penetration. no grn-related aes were reported, indicating that a single 600 mg oral grn dose was well tolerated in subjects undergoing invasive procedures. conclusions: grn penetrates rapidly into lp, bm, and bone tissue, producing sustained concentrations that predict adequate coverage of grn-susceptible pathogens at these sites. bioavailability and safety in healthy volunteers unaltered when crushed garenoxacin tablets are administered via a nasogastric tube with or without enteral feeding r. noveck, r. vargas, g. krishna, d. grasela (new orleans, kenilworth, princeton, usa) objective: the purpose of this trial was to assess, in healthy volunteers, the bioavailability and safety of single doses of the novel des-f(6)-quinolone garenoxacin (grn) administered as crushed tablets with and without concomitant enteral feeding compared with intact tablets. methods: in this randomized, crossover, single-dose study, healthy adult volunteers (aged 18 to 45 y) received grn 600 mg (one 200 mg and one 400 mg tablet) orally in 3 regimens: a) intact tablets; b) crushed tablets suspended in water and delivered via nasogastric (ng) tube; c) regimen b plus concomitant enteral feeding (osmolite, 600 ml at 100 ml/hr). subjects were randomized to receive all 3 regimens in 1 of 6 crossover sequences (abc, acb, bac, bca, cab, cba) separated by 7-day washout intervals. for each treatment, subjects entered the facility 1 day before and were confined for 72 hours after drug administration. subjects were discharged from the study after final assessments on day 4 of the third treatment. post-screening assessments included vital signs; plasma drug levels for pk analysis; and physical, laboratory, and ecg examinations for drug safety. results: 18 male subjects were enrolled (4 white, 14 black; mean age, 30 y). pk analysis for grn administered as crushed tablets with and without concomitant enteral feeding vs intact tablets showed no differences in the adjusted geometric means for cmax (8.5 and 8.3 lg/ml vs 8.3 lg/ml) or auc(inf) (97.2 and 93.4 lg*hr/ml vs 103.3 lg*hr/ml). the 90% ci for logtransformed cmax and auc(inf) for b and c compared to a were contained within the protocol-specified ôno effectsõ limit of 70% to 143%. mean grn half-life was similar among the 3 groups (mean range of 12-13 h). tmax was 1 hour following administration of intact tablets compared with 0.5 hour in the other 2 groups. a single oral 600 mg dose of grn was well tolerated whether administered as crushed tablets suspended in water and delivered via ng tube with or without enteral feeding, or as intact tablets. three adverse events (aes) were reported; 1 (nausea) was deemed probably related to study drug. there were no serious aes, meaningful changes on safety examinations, or discontinuations due to aes. conclusions: the bioavailability of grn was similar for crushed versus intact tablets, regardless of whether an enteral feeding was given with crushed tablets. these results show that grn may be administered as crushed tablets with or without concomitant feeding. garenoxacin pharmacokinetics in subjects with severe renal impairment objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens. the absolute bioavailability of grn after oral intake is 92% and approximately 40% of grn is excreted unchanged in the urine. the objective of this study was to evaluate the pharmacokinetics of grn in subjects with severe renal impairment. methods: this non-randomized, open-label study enrolled patients into 1 of 4 groups: healthy control subjects (hc) with normal renal function (clcr > 80 ml/min); subjects with severe renal impairment (sri) (clcr < 30 ml/min) not requiring dialysis; subjects with sri receiving hemodialysis (hd); and subjects with sri receiving continuous ambulatory peritoneal dialysis (capd). a single 600 mg oral dose of grn was administered on day 1 for all groups except hd patients. hd patients received a single 600 mg oral grn dose both before (hd1) and after (hd2) hd, with dosings separated by a 14-day washout. blood, urine, and dialysate samples were collected up to 144 h post-dose and concentrations of grn were determined using validated lc/ms/ms assays. safety was assessed by physical examination, vital signs, and electrocardiographic and laboratory evaluations. results: twenty-five subjects received grn (hc, n = 6; sri, n = 6; hd1, n = 7; capd, n = 6). six subjects in hd2 received grn. mean grn exposure [auc(i)] was similar in hc and hd1 but was 51%, 15%, and 21% higher in sri, hd2 and capd, respectively, than in hc. however, many of the individual values were within the range observed for hc, and these average increases in auc(i) did not exceed values previously shown to be well tolerated. decreases in cmax were observed in sri, hd1, capd, and hd2, but were not considered clinically relevant. approximately 11%, 1.5%, and 3% of grn dose was removed in the dialysate for hd1, hd2, and capd, respectively. a total of 34 treatment-emergent adverse events (aes) were reported, all mild or moderate in severity; 5 were considered to be probably or possibly related to grn. one treatment-emergent but not treatment-related serious ae was reported. conclusions: a single dose of 600 mg grn was well tolerated in patients with sri, including those requiring hd and capd. there was no clinically significant increase of grn exposure in patients with sri based on the broad therapeutic index of grn. therefore, no grn dosage adjustment is recommended in subjects with sri. the effect of omeprazole on the bioavailability and safety of garenoxacin in healthy volunteers j. kisicki, g. krishna, s. olsen, d. grasela (lincoln, kenilworth, princeton, usa) objective: the novel des-f(6)-quinolone garenoxacin (grn) is more soluble in acidic conditions than at neutral ph. therefore, this study was designed to determine if omeprazole affects the bioavailability of grn. methods: this non-randomized, open-label pharmacokinetic study was conducted in healthy adult subjects. on day 1, the single-dose pharmacokinetics of 600 mg of oral grn (one 200 and one 400 mg tablet) were determined in fasting subjects, with serial blood samples obtained through 72 hours post-dose. omeprazole 40 mg was administered once daily on days 4 to 7 to achieve steady-state inhibition of gastric acid secretion. on day 8, single doses of grn and omeprazole were administered concomitantly. omeprazole treatment was continued on days 9 and 10 throughout the period of pharmacokinetic blood sampling. study assessments included vital signs and physical, laboratory, and electrocardiographic examinations for safety. the gm cmax value for grn and grn/omeprazole co-administration was 9.6 and 9.3 mcg/ml, respectively, with 90% ci of 90% to 104%. half-life (mean range 12.9 to 14 h) and tmax (median range 1.5 to 1.8 h) were similar after administration of grn either alone or concomitantly with omeprazole. concomitant administration of garenoxacin and omeprazole was well tolerated. nine of 14 subjects (64%) experienced a total of 33 adverse events (aes). the majority of aes were mild, and only 12 were deemed possibly or probably related to the study drug. the most frequently cited aes, headache, nausea and abdominal pain, were mild to moderate in severity. two subjects withdrew from the study; neither discontinuation was due to aes. conclusions: the concomitant administration of omeprazole had no effect on garenoxacin bioavailability. these findings indicate that garenoxacin can be administered with omeprazole or other agents that affect gastric ph to a similar or lesser extent. the effect of maalox on bioavailability and safety of garenoxacin in healthy volunteers j. kisicki, g. krishna, s. olsen, d. grasela (lincoln, kenilworth, princeton, usa) objective: garenoxacin (grn) is a novel broad-spectrum des-f(6)-quinolone antibiotic. quinolones are known to chelate with cations such as aluminum, impairing antibiotic absorption. this study was therefore designed to assess the effect of a 20-ml dose of maaloxò (containing 900 mg aluminum hydroxide and 800 mg magnesium hydroxide) on the pharmacokinetics of grn when administered concomitantly with, 2 and 4 hours before, and 2 and 4 hours after, maalox. methods: this was a randomized, open-label, 6-treatment, control-balanced, residual effects design pharmacokinetic study. the 6 oral treatments consisted of 600 mg grn (one 200 and one 400 mg grn tablet) given alone, with concomitant maalox, 2 h before maalox, 4 h before maalox, 2 h after maalox, and 4 h after maalox. each healthy adult subject received 3 of the above treatments, with a 7-day washout period between treatments. in each treatment, serial blood samples for pharmacokinetic analysis were collected before and up to 72 h after grn dosing. study assessments included vital signs and physical, laboratory, and electrocardiographic examinations for drug safety. results: twenty subjects (12 male, 8 females; mean age, 27 years) were enrolled. exposure to grn [auc(inf)] was reduced by 58% when coadministered with maalox. exposure to grn was also reduced when administered 2 or 4 hours after maalox, by 22% and 16%, respectively. in contrast, when administered 4 hours before maalox, grn exposure was not affected. when grn was administered 2 hours before maalox, a small reduction (12%) in grn exposure was observed that is unlikely to be clinically relevant. half-life (mean range 11.5 to 14.2 hours) and tmax (median range 1.5 to 2 hours) were similar across treatment groups. a single oral 600 mg dose of grn was well tolerated. mild adverse events were reported by 2 subjects; 1 subject discontinued due to mild abdominal pain and blood in the stool. conclusions: maalox does not affect grn bioavailability when grn is administered at least 2 h prior to maalox. however, a reduction in grn exposure is observed when grn is administered concomitantly or up to 4 h after maalox. therefore, grn can be administered 2 h before or 4 h after administration of maalox or other products containing a high content of cations, particularly aluminum. objectives: it has been demonstrated that fluoroquinolone treatment of humans and animals can rapidly select for bacteria with reduced susceptibility to fluoroquinolone antibiotics. in recent years, there has been considerably interest in the concept of the mutant prevention concentration (mpc). the mpc has been defined as that concentration of antibiotic at which no mutants are selected from 10,000,000,000 organisms. in previous studies mpcs of ciprofloxacin and enrofloxacin were determined in vitro against salmonella enterica serovar typhimurium dt 104. here the use of a chick model to investigate the mpc of enrofloxacin in vivo is reported. methods: chicks were experimentally infected with s. typhimurium and treated with baytril (enrofloxacin) at 50 ppm/10 mg/kg for 5 days (recommended therapeutic dose) or 2 days or 125 ppm/25 mg/kg for 2 days or 250 ppm/50 mg/kg for 1 day. chicks received the dose in the drinking water (ppm) or by oral gavage (mg/kg). during and 24 hours after dosing, birds were killed and tissue samples (caecal contents, liver and sera) were taken and the levels of enrofloxacin and ciprofloxacin were determined in these tissues by hplc or bio-assay. caecal contents were also monitored for the presence of salmonella during dosing and for up to 4 weeks after dosing. selection of resistance was monitored by replica plating of colonies to media clinical microbiology and infection, volume 11, supplement 2, 2005 containing 4x the ciprofloxacin and nalidixic acid mic of the strain before treatment.results: the only conditions where the antibiotic treatments did not select for reduced susceptibility (e.g. 4x mic of parent strains) were in birds that received the antibiotic at 2.5x the dose for 2 days by oral gavage or 4x the dose for 1 day in the water. this oral gavage treatment also significantly reduced the counts of salmonella compared to all other oral gavage treatments. for the oral gavage study, concentrations of fluoroquinolones in caecal contents were above the mpc for at least 6 hours after dosing for all treatment regimes. conclusion: it was of interest to note that whilst the fluoroquinlone antibiotics are regarded as concentration rather time dependent antibiotics, the 2 day at 2.5x the dose was more effective at reducing the counts of salmonella in both experiments than the 1 day at 5x the dose. these results would suggest that length of treatment as well as dose and route of administration, is critical in minimising the selection of fluoroquinolone resistance in vivo. effect of levofloxacin coadministration on the international normalised ratios during acenocumarol therapy j. de otero, m. payeras, c. carratala, a. artigues, m. sanz, p. nadal (manacor, e) levofloxacin (l) showed no effect on warfarin pharmacokinetics in healthy volunteers but several recent descriptions reveal elevated international normalised ratios (inr) in patients receiving concurrent l and warfarin. to our knowledge no cases of l-acenocumarol (a) interaction have been reported. objectives: to report 6 cases of hypoprothrombotic response resulting from addition of l therapy to chronic a therapy. methods: a 9-month retrospective analysis of adult patients receiving a who were admitted to our ward under l prescription on the basis of clinical diagnosis and judgement. descriptive statistical measurements were performed. results: six patients, 74-89 years old (median 81.5), were included. four were men (66.6%). they suffered from a median of 4 (range 2-6) concurrent chronic medical problems (including coronary heart disease, atrial fibrillation, hypertension, obstructive pulmonary disease, renal insufficiency, cardiomyopathy and heart failure) and took a median of 7 (range 4-10) different kind of drugs (including digoxin, furosemide, antihypertensives and inhaled bronchodilators). types of infection for which the patients were treated with l included acute bronchitis (4 cases) and pneumonia (2 cases objectives: cip is substrate for an mrp efflux pump in j774 mouse macrophages (michot et al, aac 48:2673-82), which reduces its cellular accumulation. we have recently shown that cip-ôresistantõ macrophages can be selected upon chronical exposure of these cells to cip, in which cip accumulation becomes negligible because of an increased activity and/or expression of the efflux transporter (icaac 2004 (icaac , abstract a-1304 . we have now examined whether this reduced accumulation affects cip intracellular activity using a model of intracellular infection by l. monocytogenes (l.m.) and comparing resistant cells to the wild type parent cell line. methods: infection was carried out using a bacteria: macrophage ratio of 7:1, according to the procedure described in seral et al (jac 51:1167-73) . cfu/mg cell protein was determined after 5 h exposure to increasing concentrations of cip ± 10 mm probenecid (inhibitor of mrp transporters). cip cellular concentration was measured by fluorimetry (aac 48:2673-82) . results: in wild cells, an extracellular concentration corresponding to 3 x the mic was sufficient to obtain a static effect, and this value rose to 30 x in cip-resistant cells. this value was decreased to 1 x and 1.5 x the mic, for wild and cip-resistant cells respectively, in the presence of probenecid. in cells incubated with 20 mg/l cip (10 x mic), cip accumulation was 4.8 ± 0.3 and 0.3 ± 0.1 in wild and cip resistant cells, but increased to 21.3 ± 0.8 and 10.6 ± 0.5 in the presence of probenecid. conclusions: increase in expression and/or activity of the cip efflux transporter causes a reduction of the antibiotic efficacy against intracellular infection, which can be reversed upon inhibition of the efflux transporter. objectives: quinolones can cause tendinitis and tendon ruptures. retrospective studies and case reports reveal that an additional therapy with glucocorticoids increases the risk of quinolone-induced tendon disorders. methods: we investigated possible combination effects of quinolones and glucocorticoids in an in vitro model with human tendon cells. tenocytes were cultured for 1, 2, 3 and 4 days as monolayers and incubated with (a) ciprofloxacin or levofloxacin (3 mg/l), (b) 0.1 nm dexamethasone, and (c) ciprofloxacin or levofloxacin (3 mg/l) plus 0.1 nm dexamethasone. immunoblotting was used to quantify changes in the amount of beta1-integrin, activated src homology collagen (shc) and of activated caspase-3 (casp-3). furthermore, ultrastructural alterations were analysed by electron microscopy. results: at 3 mg/l, ciprofloxacin caused a significant decrease of the amount of beta1-integrin from day 3 onwards, while no effect was seen with 3 mg levofloxacin/l medium or 0.1 nm dexamethasone compared to the untreated control. the combination of both quinolones (3 mg/l) with 0.1 nm dexamethasone resulted in an earlier onset of the beta1-integrin reduction: for ciprofloxacin + dexamethasone from the first day of incubation, for levofloxacin + dexamethasone from day 3 onwards. quinolone-induced changes in signalling proteins, such as activated shc, are not fortified by dexamethasone at the concentration tested. interestingly, the time and concentration dependent increase of the apoptosis marker activated casp-3 was intensified with dexamethasone. the results of immunoblotting with respect to the induction of apoptosis were confirmed by electron microscopy. conclusions: our results show that quinolone-induced changes in the amount of the beta1-integrin as well as of the apoptosis marker activated casp-3 are enhanced by dexamethasone in vitro. the addition of the glucocorticoid caused an earlier onset of the quinolone-induced changes. our results support the clinical observations that glucocorticoids are an additional risk factor for quinolone-induced tendopathies. pharmacokinetics and pharmacodynamics of a novel extended release formulation of ciprofloxacin as compared to levofloxacin against extended spectrum beta-lactamase producing enterobacteriaceae s. schubert, h. stass, u. ullmann, a. dalhoff (kiel, wuppertal, d) background: an extended release formulation of ciprofloxacin (cip xr, 1000 mg qd, p.o.) has been developed, delivering peak concentrations which are 40% to 50% higher than following the administration of the conventional formulation (cip ir, 500 mg bid, p.o.) while the areas under the curve (auc) were comparable. objectives: first, cip is used for the treatment of infections due to enterobacteriaceae, frequently being esbl producers. thus, the pk/pd characteristics of cip xr vs. ir against esbl strains was examined. second, cip is used in urinary tract infections (utis). thus, the pk/pd profiles of cip xr vs. ir in serum and urine were studied. third, the pk/pd characteristics in either bhi or artificial urine were compared. methods: genotypically characterised esbl producing e. coli (ec) and k. pneumoniae (kpn) and their wild type counterparts were exposed to the geometric mean plasma and urine concentration profiles following cip 1000 mg xr qd, 500 mg ir bid and lev (500 mg qd). the cip and lev urine concentrations were fitted from phase i study data by compartmental modeling using topfit 2.0. bacteria were cultivated in a modified grasso model at 37°c in brain-heart infusion broth as well as artificial urine acc. to griffith et al.; viable counts were quantitated at 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 24 h. the areas under the bacterial kill curves normalised to the inoculum were calculated. results: first, irrespective of the pk profiles simulated, the wt were eliminated rapidly. the esbl producing ec and kpn were moderately to negligibly affected upon simulation of serum pk profiles of both cip and lev. second, simulation of urine concentrations by using artificial urine ir and xr resulted in an elimination of the esbl producers from the test system. lev eliminated the esbl producing k. pneumoniae but not the e.coli strain, which regrew. cip xr killed the esbl kpn strain more rapidly than cip ir or lev. third, pk/pd surrogates derived from studies in conventional media or artificial urine are significantly different. conclusions: this model and the use of artificial urine predicts a more rapid and pronounced effect of cip xr as compared to cip ir or lev. objectives: the antibacterial spectrum of moxifloxacin includes all the major respiratory pathogens and its pharmacokinetics demonstrates high peak concentrations in plasma as well as at respiratory sites. nevertheless, the tonsillar tissue concentrations have never been investigated. in the present study we determined the moxifloxacin concentrations in plasma and tonsils after the administration of three doses of 400 mg to adult patients with chronic or recurrent tonsillitis undergoing tonsillectomy. methods: this was an uncontrolled, open-label, randomised, parallel group study including 35 patients randomly assigned to 5 groups of 7 patients each, depending on the time between the last dose of moxifloxacin and that of plasma and tissue sampling (2, 3, 6, 12 and 24 h). moxifloxacin was given orally od for 3 days. moxifloxacin concentrations were measured by a validated hplc assay and fluorescence detection. each sample was analysed twice and the mean value obtained used for statistical analysis. pharmacokinetic data were analysed by presenting descriptive statistics of moxifloxacin levels in plasma and tonsillar tissue. results: cmax occurred at 3h in plasma (mean value 3.20 mg/ l) and in tonsillar tissue (mean 8.96 mg/l). tonsillar tissue/ plasma ratios (mean values) were constantly >2 mg/l at any collecting time, ranging between 2.37 mg/l (after 2 h) and 2.93 mg/l (after 24 h), which indicates a prolonged maintenance of moxifloxacin level in tonsillar tissue compared to plasma. variability among pts was present at 6 h, the tonsillar tissue / plasma ratio ranging between 0.8 and 3.4 mg/l. conclusions: moxifloxacin achieves a good penetration in tonsillar tissue, which favourably compares with that reported for other fluoroquinolones. the moxifloxacin concentrations that we observed exceed the mics of the usual respiratory tract pathogens. objectives: acute necrotizing pancreatitis is still related to a high mortality rate, based on local infectious complications, particularly due to bacterial infections of the necrotic pancreatic and parapancreatic areas. limited penetration of antimicrobial drugs in these areas/compartments is considered to be a major cause of failure of therapy of severe local pancreatic infections. however, fluoroquinolones (e.g. ciprofloxacin, levofloxacin) have been shown to penetrate sufficiently into pancreatic tissue. on that score, the value of new quinolones such as moxifloxacin (mxf) has not been investigated yet. using a rat model of acute necrotizing pancreatitis mxf has been demonstrated to penetrate rapidly and efficiently into pancreatic tissue, also into the inflamed and necrotic pancreas. methods: addressing the penetration capability of mxf following intravenous (iv) or oral (po) administration with respect to the human pancreas, a prospective clinical trial was designed using a single iv (20 patients) or po dose (40 patients) of 400 mg mxf for antimicrobial prophylaxis in patients undergoing pancreas resection. samples were taken from blood and from resection area of pancreatic tissue at two time points after application (1st sample at the beginning, 2nd sample at the end of resection). concentrations of mxf were determined by hplc/uv using ofloxacin as internal standard. results: mean plasma concentrations of mxf iv and po at first sampling time (3 -3.7 h) were 1.8 ± 0.5 and 1.2 ± 0.6 lg/ml, respectively. at the end of resection (4.3-5.3 h) 1.5 ± 0.4 and 1.0 ± 0.5 lg/ml were measured. corresponding mean concentrations of mxf in pancreatic tissue were 2 to 3times higher (3.1 ± 0.9 and 2.7 ± 1.4 lg/g 1st sample; 3.6 ± 1.5 and 3.1 ± 1.8 lg/g 2nd sample). the mxf concentrations in pancreatic tissue exceeded the mic90 s of the relevant pathogens encompassed by mxf (e.g. e. coli 0.008-0.06 lg/ml, klebsiella spp. 0.13 lg/ml, and s. aureus 0.06-0.12 lg/ml) for at least five hours at the dosing interval. conclusion: mxf has been demonstrated to penetrate efficiently into human pancreatic tissue following iv as well as po administration. in this study mxf concentrations after po administration were found to be slightly lower than those observed in healthy subjects probably due to diminished or delayed intestinal absorption prior and during surgery. objective: in clinical practice on icu wards moxifloxacin (mfx) may be occasionally administered through a nasogastric (ng) feeding tube. in absence of an oral liquid formulation and since the divalent cations contained in enteral feeds may potentially impair absorption of mfx given via this way, we wanted to study the effect of concomitant enteral feeding on the pharmacokinetics and tolerability of mfx passed as a crushed tablet through a ng tube, firstly in healthy volunteers. methods: in a randomized 3-way crossover study the oral bioavailability of mfx was investigated in 12 healthy volunteers (9 f, 3 m). each subject received separately an intact 400 mg mfx tablet (a), a crushed tablet as a suspension through an ng tube with water (b), and while receiving enteral feeding (c). the concentrations of mfx in serum were measured by a validated hplc. auc and cmax were analysed statistically assuming log normally distributed data using anova. results: all mfx treatments were well tolerated. the auc was slightly, but not significantly decreased in treatments b and c [geo means ( . thus, rate of absorption was not affected by ng tube administration. this route of ingestion seems to be associated only with a slight loss of bioavailabilityindependent of the carrier medium used (water vs. enteral feed). no clinically relevant interaction with the multivalent cations contained in the enteral feed was observed indicating that mfx and enteral nutrition can be administered concomitantly. conclusion: there was no clinically relevant effect of enteral feeding on the pharmacokinetics of oral mfx in healthy volunteers. this results has to be evaluated in patients, especially those from icu, who are characterised by severe infectious and/or concomitant diseases, which might influence absorption of mfx. in the healthy volunteer studies, compliance with dosing regimens was strictly enforced and participants had no significant underlying diseases or concomitant medications that could confound interpretation of safety data. therefore, for the purpose of this analysis, the healthy volunteers served as a control group for the patient population. in both groups, most subjects received pos 800 mg/d in divided doses. results: treatment-related aes (traes) occurred in 44% (196/ 449) of healthy volunteers; the most common were headache (17%), dry mouth (9%), and dizziness (6%). in patients, many aes were consistent with underlying diseases. traes occurred in 38% of patients (164/428); the most common were nausea (8%), vomiting (6%), headache (5%), abdominal pain (4%), and diarrhoea (4%). notably, gastrointestinal (gi) traes occurred in 18% of healthy volunteers and 19% of patients. treatmentrelated abnormal liver function test results were observed in 2% of healthy volunteers and up to 3% of patients. there were no clinically significant differences in mean qtc interval change from baseline in either population. serious aes (sae) considered possibly or probably related to pos occurred in 35 (8%) patients and 1 healthy volunteer. the most common saes in patients were altered drug level, increased hepatic enzymes, nausea, rash, and vomiting (1% each). no significant trends related to age, sex, or race were observed in either group. additionally, no unique traes were identified in patients during long-term exposure (>6 months) compared with those identified during shorter-duration therapy. conclusion: the safety profile of pos in patients was similar to that observed in a controlled population of healthy volunteers and is likely indicative of what will be observed in the clinical setting. headache and gi events (nausea, vomiting, abdominal pain, diarrhoea) were the most common traes observed in patients. given its favorable safety profile, pos should provide an additional treatment option for severely ill patients with ifis. objective: we evaluated the drug interaction (di) potential of posaconazole (pos), an extended-spectrum triazole antifungal agent, in 7 phase 1 studies. pos is an inhibitor of cytochrome p450 (cyp) 3a4 activity, but it does not affect the activity of other cyp enzymes. these studies were conducted to evaluate potential changes in the pharmacokinetics of pos and the coadministered medication, when given in combination. methods: seven open-label di studies were conducted in which subjects received pos (200-800 mg) for 8 to 14 consecutive days, alone or in combination with single or multiple doses of cimetidine, cyclosporine, glipizide, phenytoin, rifabutin, tacrolimus, or antiretroviral medications (zidovudine, lamivudine, ritonavir, and indinavir). in the cyclosporine and antiretroviral studies, patients received established doses (ed) of these drugs, and plasma concentrations were presumed to be at steady state (ss) before pos administration. bioavailability, based on logtransformed auc values, was expressed as the ratio of the combination treatment to pos or concomitant drug given alone. the effects of coadministration on the auc values of pos and each concomitant medication are summarized in the table. conclusion: no dose adjustments are required for glipizide, zidovudine, lamivudine, indinavir, or ritonavir when coadministered with pos, as the small differences in exposure are not considered clinically significant. however, when pos was given with glipizide, glucose concentrations decreased in some healthy volunteers more so than after glipizide administered alone. monitoring of cyclosporine and tacrolimus blood levels is warranted with pos coadministration, and dose adjustments of cyclosporine and tacrolimus should be made accordingly. concomitant use of pos with rifabutin, phenytoin, or cimetidine should be avoided unless the benefits outweigh the risks, due to the decrease in pos concentrations. objectives: the pharmacokinetics (pk) of many medications may be altered in the elderly population. because alterations in pk can potentially affect clinical outcomes, the pk parameters of posaconazole (pos), an extended-spectrum investigational triazole antifungal agent, were examined in healthy elderly persons ( ‡65 years), and the clinical implications of age-related pk differences were evaluated. methods: we conducted a meta-analysis of pos steady-state pk data from 5 clinical studies in healthy volunteers. pk data from elderly healthy volunteers were compared with those of elderly patients with invasive fungal infections ( objective: to develop a bioassay for measurement of csp blood levels using the hypersusceptible c. albicans cell wall sensor mutant delta-mid2. methods: the c. albicans mutant delta-mid2 (mic for csp: 0.008 mg/l) was constructed by targeted deletion of the gene encoding for a cell wall sensor putatively involved in the maintenance of cell wall integrity. the bioassay was validated according to international guidelines (shah et al, 2000; fda, 2001) . standard curve included 8 csp concentrations over the range 0.4 to 25 mg/l. four quality controls were used (0.5, 2, 8, 16 mg/l) to study: i) stability of csp concentrations over time at different pre-analytical storage conditions, ii) accuracy (measured/nominal value x 100, validation range 85-115%), iii) precision (coefficient of variation: sd/mean of measured values x 100, validation range 15%). the validation procedure included 6 intra-run and 6 inter-run measurements. results: the limit of detection and quantification was 0.2 and 0.4 mg/l (corresponding to 5 and 10 ng of csp in a sample volume of 25 ml), respectively. reproducible standard curves were obtained over the clinically relevant concentration range (0.4 to 25 mg/l) (r ‡ 0.99). analytical time was 16 h. csp concentrations with a deviation from nominal values < 15% were measured: i) over 4 days in plasma, and 3 days in whole blood, at 4 c, ii) over 3 and 2 days, respectively, at 21 c, iii) over 6 months in plasma at -80 c, iv) after 5 freeze/thaw cycles. intra-and inter-run validation with the four quality controls (mean % value, ± sd) are shown in objective: to identify the pharmacokinetic and pharmacodynamic (pk-pd) parameters of antifungal therapy predictive for intra-and inter-run bioassay validation i intra (n = 6) inter (n = 6) accuracy 93.1 ± 1.9 96.0 ± 2.5 precision 1.3 ± 1.7 5.8 ± 3.0 invasive fungal infections in patients admitted to an intensive care unit (icu). the medical records of all ptt admitted to a tertiary hospital icu in a 6 month period in 2002 were retrospectively reviewed, and the antifungal therapies were recorded and correlated to the findings of fungi from the ptt. the pk-pdparameters for each pt was estimated from the treatment given and the mic of the isolated candida, i.e. auc/mic-ratio (in h) for the azoles, and peak/mic ratio for the free fraction of amphotericinb. candida isolated from various infectious foci, or from the surveillance samples taken routinely three times a week, were susceptibility tested using e-tests for fluconazole, itraconazole and amphotericinb. results: in total 120 ptt were included.among the 14 ptt with invasive fungal infections, 3 ptt had bloodstream infections at admission. one of the remaining 11 (9%) received systemic prophylactic therapy before getting colonized, and 3 ptt (27%) were prophylactic treated when colonized, 3 ptt with fluconazole (auc/mic: 66-1600), and one pt with liposomal ampho-tericinb (peak/mic = 3 objective: to predict the pharmacokinetic properties of bal4815 a new azole antifungal, in humans.methods: bal8557 (the water-soluble pro-drug of bal4815) was incubated at 10 lg/ml in pooled heparinized rat, cynomolgus monkey and human plasma for 5 minutes at 37°c. bal4815 was incubated at 1,10 and 100 lg/ml equivalent bal4815 with rat, cynomolgus monkey and human liver microsomes for 120 min at 37°c in the presence of 1 mg/ml proteins. the extent of plasma protein binding of bal4815 in rat, cynomolgus and human plasma was measured using the red blood cell partitioning method and 14c-bal4815. rats received a single oral dose of 10 mg/kg equivalent bal4815 and a single intravenous dose of 5 mg/kg equivalent bal4815 as bal8557. cynomolgus monkeys received single oral and intravenous doses of 3 mg/kg as bal8557. serial blood samples were obtained. plasma concentrations of bal4815 and bal8557 were quantified using an hplc/fluorescence method or a lc-ms/ms assay. results: in plasma, bal8557 is converted within minutes to bal4815 in all species investigated. in the presence of rat, cynomolgus monkey and human liver microsomes, less than 10% of bal4815 is metabolized during a 120 min period. bal4815 is bound to plasma proteins with a free fraction of 3.5%, 3.6% and 2.2% in rat, cynomolgus monkey and human, respectively. after intravenous administration, the pro-drug bal8557 is converted to bal4815 within minutes. bal4815 has a large volume of distribution with values greater than the total body water: 15 l/kg and 5 l/kg in rat and cynomolgus monkey, respectively. bal4815 is slowly eliminated with halflives of 5 h in rat and 10 h in cynomolgus monkey. after oral administration of the pro-drug, bal4815 reaches cmax-values between 2.0 to 3.5 hours. we observed high bioavailability of bal4815: 62% in rat and 87% in cynomolgus monkey. conclusions: based on these in vitro results and animal data, we predict that the pro-drug bal4815 is very rapidly converted to bal4815 in man. due to the protein binding and in vitro metabolic stability, bal4815 is expected to behave as a low intrinsic clearance drug in human (clearance < 10 % of liver blood flow). this combined with a large volume of distribution explains the long half-life > 30 hours observed in man. animal data suggest a good oral bioavailability as is confirmed in humans. pharmacokinetics and fungicidal activity of aminocandin (hmr3270), a novel echinocandin in healthy volunteers b. sandage, g. cooper, n. najarian, j. lowther (lexington, usa; romainville, f) objective: aminocandin (ac), an echinocandin, is being developed for treatment of systemic fungal infections. a study was carried out to determine the pharmacokinetic (pk) and fungicidal activity of ac in plasma samples against 2 strains of c. albicans and 1 strain of a. fumigatus following single iv doses. methods: single iv doses of 75-300 mg of ac were administered to 12 healthy, male volunteers in a single-blind, randomized, placebo-controlled trial. individual plasma samples were collected from active and placebo (pbo) treated volunteers, serially diluted in 25% control plasma/75% growth medium, and the fungicidal titre determined on 2 c. albicans strains and inhibitory titre against 1 a. fumigatus strain. titres and pk results are the mean values (n = 3). due to the dilution factor in test medium, the limit of detection was a titre of 4 the germicidal effect of ceiling-and wall mounted ultraviolet c (uvc) light at 254 nm in isolation units with negative pressure ()45 pascal) was examined and compared with disinfection with chloramine during end-disinfection after patient stay. microbial samples were taken from surfaces before and after disinfection with uvc (33-47 min) and chloramine (5%, 1 h exposure) using standard contact plates (20cm 2 ). the uvc-distribution in the isolation units was monitored at 165 positions. the output on the floor varied between 0.08 and 3.2 w/m 2 , with an average ( ± sd) of 2.2 ± 0.5 w/m 2 in the patient room, 2.0 ± 0.7 w/m 2 in the sluice and 1.4 ± 0.5 w/m 2 in the bath/ decontamination room. on other places, the values varied from 0.08 to 6.82 w/ m 2 . the units were uvc-disinfected for 33-47 min, corresponding to doses ranging from 160 j/m 2 in shadowed area to 19 230 j/m 2 at the highest exposed site. according to published uvc-dosimetry, the survival of bacteria and bacterial spores are reduced by 90% with doses ranging from 4-120 j/m 2 and 100-365 j/m 2 , respectively. thus, uvc doses used in this study should be high enough to inactivate most bacterial organisms, including spores, even on surfaces not directly exposed to uvc. uvc-disinfection significantly reduced the bacteria on surfaces directly or indirectly exposed to uvc to a very low number (from ca 30 to 1-2 cfu/plate), as did 5% chloramine disinfection (fom ca 30 to 1-2 cfu per plate) alone; p < 0.001,and p < 0.001, respectively. since cleaning before disinfection may be a risk for the staff in isolation units, disinfection with uvc-or chemicals should always be performed first. the presence of completely shadowed areas in the isolation unit (the bed rail, lockers, matresses etc.) still needs disinfection by chemicals before cleaning. therefore, uvc may not be used alone, but is a good additive to chemical disinfection, to lower the biological burden of infectious agents in isolation units for high risk infectious patients. disinfection of medical equipment, using dry mist of hydrogen peroxide objective: to compare the antimicrobial efficacy before and after disinfection of pine core wood surfaces with surfaces made of polyethylene and synthetic laminate against organisms typically causing hospital-acquired infections. methods: s. aureus (mrsa), e. faecium, e. coli, p. aeruginosa. c.albicans, m. terrae and p camembertii were uses as test organisms. after inoculation of the test organisms on surfaces made of pine core wood, polypropylene and synthetic laminate, samples were collected on rodac plates before and after disinfection with commonly used hospital germicides (alcohol, aldehyde, glucoprotamine and a quaternary ammonium compound). bacterial colonies were counted after 0 min, 30min, 1 h, results: substantially lower colony counts were measured on pine core wood surfaces before application of the disinfectant compared with counts on polypropylene and synthetic laminate surfaces. after bacterial contamination of the surfaces, significant colony counts were not observed on the surfaces after disinfection with alcohol, aldehyde and glucoprotamine. however, after disinfection with a quaternary ammonium compound, significant elevations in colony counts were found on the pine core wood surfaces in comparison to the polypropylene and synthetic laminate surfaces. discussion: the poor efficacy of quaternary ammonium compound on wooden material might be explained by the interaction between wood and the anionic polyphenols and cationic surfactants contained in the disinfectant. the study findings corroborate the antimicrobial effect of pine core wood against organisms typically causing nosocomial infections. with the exception of a quaternary compound, all the germicides displayed good disinfecting properties. from a hygienic point of view, pine core wood is suitable for use in hospitals. survey of the microbiological quality of drinking water supply in hospitals. drinking water cooler vs tap water fed dispenser vs bottled water objectives: hygienic, economic and ecologic comparison of drinking water coolers versus tap (mains) water-fed dispensers versus bottled drinking water. methods: samples were taken from each system without preflow or cleaning of the nozzle. these comprised 2 samples from 20 drinking water coolers each over 2 months, 1 sample per week from the tap water dispenser over 6 months, and samples from 6 different bottles of drinking water. total colony counts were analysed at 22°c and 36°c, respectively. total p. aeruginosa, e. coli, and coliforms, as well as sulphite-reducing clostridia were examined according to european norms (en iso) for compliance with the german drinking water ordinance 2001 (limit value for total colony counts at 22 ± 2°c: 100 cfu/ml, at 36 ± 1°c: 100 cfu/ml and 20 cfu/ml for bottled water). costs per litre were calculated and compared based on the economic data supplied by the manufacturers. the ecological impact of the different systems was evaluated based on expert opinion. results: all the samples from the water cooler system exceeded german drinking water ordinance threshold values. all the samples taken from the tap water dispenser, and, with one exception, all the samples from the bottled water complied with the german drinking water ordinance. if more than 400 l of water are consumed per month, the tap water dispensing system and bottled water are more economical than the water cooler system ( 0.57 per l for water cooler, 0.45 for month tap water dispenser, 0.49 per l for bottled water). in economic terms, the water cooler system can only be recommended for consumption levels below 400 l per month. from an ecological point of view, the tap water dispenser is most favourable (expressed in co2emissions per year for 403.720 l at freiburg university hospital), followed by the water cooler system and finally the bottled water. conclusion: for reasons of hygiene, the use of water cooler systems cannot be recommended in hospitals. furthermore, drinking water cooling systems are only economic for low consumption rates. tap (mains) water-fed dispensers feature the best hygienic, economic and ecologic properties. objectives: the application of antimicrobial finishes to textiles can prevent bacterial growth and might reduce the risk of infection resulting from fabrics that are contaminated with pathogenic microorganisms in hospitals. the main aim of this study was the determination of the antibacterial activities of chemical treatments applied to textiles. comparison of testing methods assessing antibacterial efficiency was conducted. these studies were performed in order to select the right methods of evaluating the antibacterial and bacteriostatic activity of finishes. methods: fibers and fabrics made from cotton (100%) applied with quaternary ammonium salts were tested. finishes treated with bactericidal agent were compared with samples (not treated with the disinfectant). the european standards iso / dis 20645/2002 and aatcc 147/1998 suitable for assessment of antibacterial activity were applied. the bacterial strains recommended by the above standards such as: klebsiella pneumoniae (atcc 4352), staphylococcus aureus (atcc 6538), escherichia coli (atcc 11229) were tested. due to its high resistance to quaternary ammonium salts, additionally p. aeruginosa (atcc 9027) was examined. bacterial suspension 1 -2,9 x 10 8 cfu/ml was prepared according to the standardsõ requirement. the samples impregnated with biocidal products were examined with agar diffusion plate test. the specimens were located transversely across the bacterial inoculum on the nutrient agar. the level of antibacterial activity was assessed by examining the extent of bacterial growth in the contact zone between agar and the specimens. additionally, the extent zone of inhibition (minimal 1 mm) around the specimens was considerate. results: tested fibers did not show antibacterial activity. the fabrics showed antibacterial activity against k. pneumoniae and s. aureus (inhibition zone 4 mm). the examined specimens showed no bacteriocidal activity against escherichia coli and pseudomonas aeruginosa. the results obtained in two applied methods were comparable in assessing antibacterial activity of finishes. the antymicrobial finishes did not fulfill requirements of the standards. conclusion: based on the obtained result the tested antimicrobial finishes may not be considered effective in preventing bacterial transfer at contaminated areas in hospitals. a new interactive approch to improve hand hygiene compliance j. holt, e. tvenstrup jensen, d. buhl (copenhagen, dk) introduction: the compliance of guidelines for hand hygiene is well below 50 % despite several at-tempts to improve. when planning an educational outline to promote a behavioural change in the hand hygiene practice in the aim of lowering the incidence of nosoco-mial infections the mapping of critical factors and implications that affect this practice is of utmost importance. objectives: this study was an empirical investigation of the aspects of the hand hygienic practice as it takes place in the danish health care system today. the study attempted to answer the question:''how can the health staff's lack of compliance with hand hygiene be explained and understood as basis for planning an educational material to support a behavioural change.'' methods: literature studies of hand hygiene, a questionnaire based done survey (1700 per-sons/294 respondents) and qualitative interviews (n = 15) was performed in order to uncover the explanations for this low rate of compliance.the data was analysed and discussed on the basis of theories on action, experience, reflective thinking, control and rituals in order to aim for a broader view and under-standing of this field. results: both the questionnaire and interviews showed that hygiene still is a field that has great implications on the interactions between individuals. furthermore it seemed difficult for the staff to draw a professional and not a private line between ''the clean and the unclean'' and thereby perform hand hygiene without compromising each other. further the interviews showed that it is difficult for the staff to react on something they cannot see and that not gives an immediate result when staff does not act as pre-scribed. introduction: leishmaniasis has increased in importance in recent years as hiv-infected patients have emerged as a risk group for the disease. however, real prevalence in the general population is unknown. objetive: the objective is to know the antibody seroprevalence for infection by leishmania infantum in the general population of castilla-leon (spain). methods: a random sample from the general population (4825 sera) and from hiv-infected patients in the autonomous community of castilla-leon was collected in 1996. seroprevalence of antibodies against l. infantum was determined by an indirect enzymoimmunoanalysis (eia) test designed in the laboratory. results: anti-leishmania infantum antibody seroprevalence in the general population was 4.9%. there is a significant increase in seroprevalence with age (p = 0.001), from 3.96% in the 14-20 years group up to 7.2% for those over 70 years old. there were no significant differences between women and men (5.0% versus 4.9%; p = 0.9534). seroprevalence was significantly higher in people from rural areas than in those from cities (6.0% versus 3.4%; p = 0.001). hiv-infected patients had a seroprevalence against l. infantum of 64.0%. no differences were observed between women and men, nor was there a prevalence increase with age. efficacy of intralesional pentavalent antimony in combination with oral azole for treatment of oldworld cutaneous leishmaniasis results: a 30-year-old man presented with a 6-month history of slowly growing round lesions on his right arm and leg. he had a history of travel in the mediterranean area (including north african and middle east countries) 6 months prior to presentation. examination showed one nodular lesion (1 · 1 cm) with margin induration and depressed central ulceration on the right arm, a second nodular lesion (0.5 · 0.5 cm) on the right leg, and a red papule (0.5 · 0.5 cm) on the left arm. a 52-year-old man with an history of a 4-month stay in iraq until 1 months prior to presentation, showed 6 lesions, which had grown during the last three months: two nodular lesions (3.5 · 1 cm, 4.5 · 2 cm) with erythematous margins and a thick crust on the left arm, 4 round lesion (2.5 · 2.5 cm) with depressed central ulceration, at the left arm, right arm, right leg and second finger of the right hand. a 49-year-old man with a 5-month stay in iraq until 5 months prior to presentation, showed two painless confluent nodular lesions (5.5 · 3.5 cm) with depressed central ulceration at the right elbow, which had grown over 4 months, two round lesions (1 · 0.5 cm and 0.8 · 0.7cm) with a thick crust on the left poplitea fossa and one nodular lesion (1.5 · 1 cm) at right leg. all patients received 12 alternate-days intralesional injections of meglumine antimoniate and 6 weeks of oral fluconazole. all lesions healed completely at the end of therapy and at the 3-month follow-up. none of the patients complained of any adverse events during treatment or follow up. conclusion: treatment with intralesional pentavalent antimony in combination with azole is effective and absolutely safe, in that it reduces the total amount of antimonial exposure. entomological study of sandflies (dipthera: psychodidea) in three foci of endemic cutaneous leishmaniasis in iran human leishmaniasis is a globally widespread parasitic disease caused by members of the genus leishmania. currently, leishmaniasis is considered to be endemic in 82 countries including iran. phlebotomine sandflies the vectors of leishmaniasis have received considerable attention in recent years due to the resurgence of leishmaniasis in some endemic areas of iran ,so extensive studies have been conducted on the ecology of sandflies in different parts of the country in recent years. a total 8026 sandflies were collected by using funnel traps of rodent burrows in rural district of the 3 province of iran (shiraz, yazd, khozestan). these sandflies identified in 3 maine croups: phlebotomus. papatasi(61%) , p. sergenti (10%) , p. sergentomya (29%). these findings indicate that p. papatasi could be a vector of humans and the gerbils (merion libycus). the close contact between vectors and resevoirs have created a very efficient cycle for the transmission of the disease in these areas and the villages around these 3 provinces. erysipelas like cutaneous leishmaniasis: a case report a. erdogan, d. balaban, e. dervis, g. sengoz, g. barut, a. karaoglu (istanbul, tr) cutaneous leishmaniasis is an infectious disorder of the skin caused by leishmania major, tropica, aethiopica and infantum which are transmitted by phlebotomine sandflies. we present here a patient who demonstrated a morphologically rare form of erysipelas like cutaneous leishmaniasis. a 74-year-old woman with a four-month history of erythema and edema on her nasal and left malar area was referred to our clinic for further evaluation. her dermatological examination revealed an erythematous, edematous and fine desquamative plaque on her nose and left malar region resembling erysipelas. other physical and laboratory findings were normal. punch biopsy taken from the lesion and dyed with h.e revealed dense lymphocytic infiltration between the layers of just beneath the epidermis and deep dermis. in addition, a huge number of amastigots were found in macrophages and histiocytes that formed granulomas. in light of these findings the patient was diagnosed with cutaneous leishmaniasis and commenced a regimen of meglumin antimonat 425 mg b.i.d. after two weeks of therapy the lesion was gradually disappeared without any scar formation. in conclusion, cases with cutaneous leishmaniasis are still observed in our country not always with usual clinical appearance, but also like the present case, it may clinically resemble erysipelas and provides difficulties in the differential diagnosis. study of human infection of cutaneous leishmaniasis in a focus of the disease, southern iran ) with scar and 14 cases (1%) with ulcers. the mean of acute lesions and scars per infected cases was 1.64 and 1.87, respectively. totally 129 cases were observed with sores and scars, and 122 out of them were infected in the area. the highest rate of the acute lesion was observed in 0-4 years old age group (4.5%); meanwhile the highest rate of scars was in 10-14 years old age group (12.9%). sores were located on hands (8.7%), foot (65.2%) face (4.4%) and other parts of body (21.7%). in the study of schools, 1201 students (53.5% boys, 46.5% girls) were visited. the infection rate to acute lesion was 0.6% and 22.6% of students had scar. there is no significant difference between males and females based on the acute infection (p > 0.05), but this difference was significant based on scar (x 2 = 9.84, p < 0.05). the highest infection rate was observed in tashkooieh village (2.7%). two injected mice were developed the acute lesion and the agent of the disease was identified as leishmania major by pcr test. conclusion: the infection rate of the sores and scars shows that the disease is located in the studied area in recent years. the disease had one peak during 1995-2000 and has been increased from 2002 up to now. this is the first time that the parasite isolated from human in the area. therefore, this focus must be added to the zoonotic cutaneous leishmaniasis foci of iran. the disease is endemic in the area because more than 95% of cases were infected locally. giardia and cryptosporidium in the netherlands objectives: • the studies were designed to get an insight into the incidence of protozoal-, bacterial-, and viral infections in patients with diarrheal complaints in different groups: patients consulting their general practioner and the dutch population. here we focus on giardia and cryptosporidium • to decrease diagnostic deficit • study the risk factors methods: three studies were designed and conducted : • ôhaarlemõstudy: 1994-1996, general practitioners, haarlem region • nivel: case-control study in sentinel general practitioners practices (1996 general practitioners practices ( -1999 • sensor: prospective population based cohort study with a nested case-control study in the dutch population. (1999)the studies differ in inclusion criteria and the diagnostic laboratory techniques used, esp. virological stool examinations results: incidence of gastroenteritis in the nivel (gp) study (after correction) was 79.5 per 10,000 personyears. this means that 80.000-130.000 persons will consult a gp annually for gastroenteritis. incidence of gastroenteritis in the populationbased study was 283 per 1000 personyears. giardia was detected in 14.8% of the cases in haarlem, in 5.4% of the cases in the nivel study and 3.3% of the controls. for cryptosporidium this was resp 3.3%, 2.1% and 0.2%. the diagnostic deficit decrease substantialy by testing for viral pathogens like nlv. detection of pathogens was influenced by age, season and duration of symptoms. we were able to construct an algoritm for diagnostic workup in gi patients. statistical surveys have been made for the effect of the climate on the epidemic diseases in tropical area, but not so much has been clarified on the relation between the variations of meteorological elements and of the number of patients. in this paper, we apply eof analysis method to time series of diarrhoea patients and meteorological elements in bangladesh, to understand effects of the meteorological variation to the prevalence of the diarrhoea disease. the eof analysis of the time series of patients and meteorological elements averaged every two weeks for 21 years from 1981 to 2001 shows that in the dominating component, the anomaly of the number of the diarrhoea patients has different signs for the periods before and after june, corresponding to the two seasonal peaks of the number of the patients. higher maximum temperature and more humidity in the pre-monsoon period are found to have a tendency to enhance the first peak of the diarrheal occurrence. we will also report the result for the different types of diarrhoea as v. cholera, rota and etec. serologic evidence for babesiosis in the northern and eastern tyrol (austria) and the southern tyrol (italy) methods: leptospirosis was diagnosed by leptospiral igm enzyme linked immunosorbent assay (elisa) and the serovar was confirmed by the microscopic agglutination test (mat) using a battery of 12 live leptospiral strains as antigens. results: most of them were outdoors manual workers (52%), housewives (29%), indoors non-manual workers (16%) and unknown (3%). mean duration of symptoms was 12.4±21.2 days. majority of the patients presented with fever (89%), jaundice (55%), chills and rigors, vomiting (47% each), cough (28%), abdominal pain (25%), diarrhoea, and altered sensorium (19% each), purpura/bleeding (11%). salient laboratory abnormalities included anaemia (50%), leucocytosis (59%), thrombocytopaenia (64%), elevated erythrocyte sedimentation rate (esr) (55%), hyponatremia (50%), hypokalemia (23%). acute renal failure occurred in 47%. hepatic function derangement occurred in 64%. three patients had pulmonary infiltrates and sputum revealed haemosiderin laden macropahges in them. two others manifested exudative pleural effusion which was bilateral and sequential in one of them. the common serovars encountered included l. autumnalis (30%), l. hebdomadis (22%), l. grippotyphosa (18%), l. icterohaemorrhagiae and l. javanica (11% each), l. australis (8%). all patients were treated with parenteral crystalline penicillin, oral doxycycline and were managed conservatively. haemodialysis was required in four patients. two patients died, both of whom developed multiorgan system failure. conclusions: leptospirosis is an important cause of acute febrile illness with renal, hepatic dysfunction and bleeding abnormalities. a high index of clinical suspicion is required confirm the diagnosis early as the condition responds well to conservative management. determination of fr900098, a promising antimalarial agent, in human serum by capillary electrophoresis for pharmacokinetic studies the acetyl derivative of fosmidomycin, fr900098, was demonstrated to be twice as active against plasmodium falciparum in vitro and in the plasmodium vinckei mouse model. fr900098, as fosmidomycin, is an inhibitor of 1-deoxy-d-xylulose-5-phosphate (doxp) reductoisomerase, an essential enzyme of the non-mevalonate pathway of isoprenoids biosynthesis. the biosynthesis of isoprenoids in plasmodium is depending on this doxp pathway, as found in eubacteria, algae and plants, but not in human. in plasmodium, the doxp pathway is localised inside a plastid like organelle, the so-called apicoplast. here, we report on the development of a high-performance capillary electrophoresis (hpce) technique for the determination of fr900098 in serum. various instrumental setting for migration (voltage, capillary temperature) were tested and the buffer properties (ph values, component molarities) were optimized. the assay was submitted to standard quality control procedures: within-, between-days reproducibility, accuracy, limits of detection (lod) and quantification (loq), linearity, short and long term stability. finally, the working buffer used was 14 mm kh2po4 / 56 mm k2hpo4, 15% methanol, and 0.2 mm hexadecyltrimethylammonium bromide (htab). the ph was adjusted to 10.9. the electroosmotic flow was modified by the cationic ion pairing reagent, htab. the assays were linear over the large concentration range tested, from 0.25 to 100 lg/ml. the recovery of the sample pre-treatments was higher than 100%. good precision was obtained, with betweendays reproducibilities resulting in coefficients of variation (cv%) of 2.1, 2.0 and 3.2%, and within-days reproducibilities of 0.6, 1.8, and 4.9% (cv%), for 100, 10, and 1 lg/ml, respectively. the lod was 0.25 lg/ml, and the loq was 0.5 lg/ml. the studies of short and long-term stability of fr900098 in serum showed a good stability of the molecule, at room temperature, +4°c or )80°c. moreover, fr900098 was resistant to numerous cycles of freezing and thawing. indeed, after four freeze-thaw cycles (4 days), 94.9%, 101.0%, and 97.5% of fr900098 were recovered from the 100, 10, and 1 lg/ml samples, respectively. in conclusion, we have developed a convenient ce technique for the determination of fr900098 in serum which offers advantages of speed, sensitivity, and accuracy. at present, the procedure is applied for pharmacokinetic studies in an animal model of gö ttingen mini-pig. present and future of malaria in kahnouj endemic area, southern iran objective: kahnouj district is associated with one of the malaria regions in southeast of iran. the anopheleine fauna does not appear to have changed much over several decades. methods: entomological studies and mosquito collection were performed every 15 days from indoor and out door shelters. malaria surveillance was carried out by health centers, ministry of health. microscopy was performed on out patients with fever or suspected malaria. malaria cases detection were mostly performed passive and rarely actively. the positive cases were treated with chorolquien/primaquen. results: in the present study five vectors species of malaria were found in this study which had been previously recorded 3 decades ago. this district like other malaria endemic areas in iran has been under pressure of anti malaria programmes including case finding and residual spraying insecticide as well as larviciding against the vectors since 1958. annual incidence of malaria declined from 10.27 to 3.86 during ten years. the most transmission occurred in october to december when the temperature was suitable for vector activity in kahnouj area. electricity was recently supplied into the rural area where most malaria cases were found. therefore, most houses equipped with air conditioner and the resident keep windows close, so they are secured from mosquitoes bites during hot season but not in beginning of spring and autumn when temperature is mild enough, in order to save the electricity cost, the residents do not use air conditioners whereas they leave windows open and no other protection against mosquito bites is provided. residual spraying insecticide of indoor shelters have been stopped for five years and the most activity of antimalaria programme set up base on case finding. conclusion; in order to control malaria, indoor residual spraying of insecticide would not assist effectively, so given knowledge to people to use bed net particularly in season people are more expose to mosquitoes bite may be considered as an effective measure in controlling malaria in khanouj district. also development in this area is effectively pushing back malaria in near future. entomological studies and mosquitoes collection were performed every 15 days from indoor and outdoor shelters as well as breeding places with the aid of suction tube and dipper. results: entomological researches were found that five vectors species of malaria in this study had been previously recorded 2 decades ago. anopheles stephensi was recognized as the main vector of malaria in this area with two peaks, one in may and the other in december. the most malaria transmission occurred in june and december. the larval habitats includes draying river bed with pools, rocky river pools, stagnant streams, slow foothill streams, temporary pools, slow moving water with or without vegetation. conclusion: operational of insecticides for adult and larval control, as well as surveillance of malaria cases, would not assist effectively to control of malaria, so given another malaria control methods as impregnation of bed nets as well as repellent particularly in seasons that people are more expose to mosquitoes bite, may be considered as an effective measure in controlling malaria in this area . case report: a 16 year old boy presented with fever and a generalized exanthema at the local dermatology clinic. the mixed appearance of pustules and umbilicated non purulent vesicles led to the suspicion of an orthopoxvirus infection. the patient reported that little red dots had occurred 2 weeks earlier at the extremities after contact with a sick pet rat. 1-2 weeks later a generalized pustular exanthema appeared, which was associated with high fever (>39°c) and a pronounced feeling of sickness. histologically no viral inclusion bodies were found in pustule material but poxvirus particles were detected by elmi in swabs from pustule ground. following the anamnestic suspicion of transmission by an infected rat, which unfortunately had perished soon afterwards, and the experience made at the recent monkeypox outbreak in the usa initiated by imported rats, one of the major aims was the exclusion of monkeypoxvirus. this was accomplished by pcr typing, which like elmi had been established on occasion of the implementation of the austrian plan for poxvirus preparedness (ôpocken-alarmplanõ) and despite the diagnostic means for detection of variola vera also included the ability to discriminate between animal orthopoxviruses. additionally serological diagnosis was performed and presumably due to the protracted course of infection cowpox specific igg antibodies were already present at admission with a titre of 1:800. interestingly the humoral immune response initially seemed to be rather cowpox specific as there were no antibodies crossreacting with vaccinia virus detectable in indirect immunofluorescence. as an exclusive immune reaction with one species alone would be a very rare situation observed with orthopoxviruses we did more extensive testing by western blot analysis. although not exclusively directed against cowpoxvirus antigens a very restricted reactivity of the patient serum was observed in the early course of infection using different recombinant and virus-derived orthopoxvirus antigens. this would suggest that also serological methods might be able to give a hint towards rapid determination of orthopoxvirus species, which certainly is also the case with immunological antigen detection methods. crimean-congo haemorrhagic fever in results: forty veterinerians from endemic region (tokat), and 43 from non-endemic region (aydin) were included. demographic characteristics in two groups were similar, whereas professional activities of veterinarians in non-endemic region were more intense (p = 0.001). the cchf igg positivity (2.5% vs 0%), brucella agglutination titre of >1/160 (27.5 vs 4.6%) were more common in endemic region than non-endemic region. three veterinarians in tokat had malaise, myalgia, and fever. in multivariate analysis to detect the risk factors for serum tube agglutination of > 160, the veterinarians living in endemic area were found to have 7.8 times higher risk of brucella infection than the ones living in non-endemic area (or; 7.8, confidence interval; 1.4-40.9, p = 0.015). the prevalence of coxiella burnetii serology was equal in both regions as 7%, and none of the seropositives had complaints. the history of tick bite was significantly more common in the endemic region than the nonendemic region (35% versus 12%, p = 0.011). conclusions: cchf and brucellosis compose infection risks for veterinarians in endemic region, despite the veterinarians in endemic region perform less riskfull professional activities. veterinarians in cchf endemic regions should be warned to protect themselves against tick bites according to universal precautions. the use of masks should be employed to prevent inhalational transmission of brucellosis in endemic regions. changes in temperature and the crimean congo haemorrhagic fever outbreak in turkey o. ergonul, s. akgunduz, i. kocaman, z. vatansever, v. korten (ankara, istanbul, tr) objective: to investigate the role of the climatic factors that could effect the activation of hyalomma marginatum marginatum population, and consequently the emergence of crimean-congo hemorrhagic fever (cchf) epidemic. methods: the meteorological data were obtained from three meteorology stations (tokat, sivas, and yozgat), where the majority of the cchf cases were reported in the last 2 years. these 3 provinces are located at the northern parts of eastern anatolia and southern parts of black sea region. temperature data have been observed and recorded by the turkish state meteorological service (tsms), and were available for 70 years in sivas, for 40 years in tokat, and for 60 years in yozgat. meteorology stations are located at the city centres, not at the airports. temperature variations and trends for turkey were analysed by using a data set including monthly averages of daily mean, and minimum temperatures. annual rainfall, and the number of days in april with the temperature of > 5°c were also included in the analysis. in order to detect homogeneity in mean annual series, first a homogeneity analysis was performed by using the non-parametric kruskal-wallis (k-w) test. the non-parametric mann-kendall (m-k) rank correlation test (13) the interest in the coordination properties of acrylate acid and its homologues was generated by the facile synthesis of the ômetal-containing monomersõ (mcm) materials. these compounds can be polymerised with a lot of organic monomers leading to various metal-containing polymers. polymeric transformations of mcm led to a new research field of current interest due to the practical importance of the obtained products which exhibit a number of unique features: high catalytic activity, unusual magnetic, electro-physical and biological properties.these polymers are especially appropriate for biological applications (tissue engineering, implantation of medical devices, dentistry, bone repair etc.) because of their molecular weight, compositions and architectures which can be regulated through controlled reactions. we report here the antibacterial and antifungal activity of new complexes of type m(phen)(c3h3o2)2(h2o)y ((1) m: mn, y = 0; (2) m: ni, y = 2;(3) m: cu, y = 1;(4) m: zn, y = 2; phen = phenantroline and c3h3o2 is acrylate anion), representing the first step products in the synthesis of polymeric materials. the in vitro antimicrobial testings were performed by broth microdilution method, in order to establish the minimal inhibitory concentration (mic), against gram-positive (bacillus subtilis, listeria monocytogenes, staphylococcus aureus), gram-negative (psedomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, both concerning the microbial spectrum and the mic value. the mics values widely ranged between 4 mg/ml and 16 mcg/ml. all the tested compunds were highly active against salmonella and listeria (mic = 16 mcg/ml objectives: c. glabrata is innately less susceptible to azole than most other species of candida and it acquires azole resistance after short-term exposure to fluconazole, as recently noted in oropharyngeal isolates. since c. glabrata has emerged as a significant cause of candidemia, we examined the change in azole mic and karyotype in sequential isolates of c. glabrata during the course of fungaemia, and its relationship to antifungal therapy. methods: serial bloodstream isolates of c. glabrata were obtained from 15 patients with fungaemia over periods of up to 38 days. forty-seven c. glabrata isolates from 15 patients (6 patients who received antifungal therapy and 9 patients who did not receive antifungal therapy) were analysed using electrophoretic karyotyping (ek) and tested for antifungal susceptibility to fluconazole, voriconazole, and itraconazole. results: the overall rates of resistance to fluconazole (mic ‡ 64 lg/ml) and itraconazole (mic ‡ 1 lg/ml) for all 47 isolates were 8 and 50%, respectively. for most patients, the sequential strains from each patient exhibited the same or similar azole susceptibilities. however, sequential isolates from two patients showed three-or four-fold increases in the mics of all three azole antifungals, while they retained the same karyotypes. azole-resistant strains were isolated from both patients after fluconazole therapy was discontinued and the intervals after the first blood isolation were 24 and 38 days, respectively. the isolates from one of these patients exhibited increased expression of the cgcdr1 efflux pump. the sequential strains from each patient had identical karyotypes in 10 (67%) patients, but two or four different karyotypes in 5 (33%) patients. the sequential isolates from these five patients exhibited the same or similar antifungal susceptibilities, showed only one or two chromosome band differences, and had no association with previous antifungal therapy. conclusion: this study showed that sequential bloodstream isolates of c. glabrata were able to acquire azole resistance in association with fluconazole therapy, and that they developed two or four different karyotypes in some patients, during the course of fungaemia. results: the next table shows the mics obtained for the tested drugs. from the three isolates with voriconazole mic > 1 mg/l, two of them had a fluconazole mic ‡ 16 mg/l. six isolates with voriconazole mic = 1 mg/l were found: the fluconazole mic for one of them was 4 mg/l, for another four was 8 mg/l and for the last one was 32 mg/l. conclusions: a. there wasn't found any fluconazole or voriconazole ôresistantõ isolate in the haart era. b. the percentage (11.84%) of isolates with voriconazole mic ‡ 1 mg/l is higher than it has been described in other works. c. the 75% of the voriconazole ôresistantõ isolates were fluconazole ôresistantõ too, so a cross resistance mechanism could be implicated. in vitro susceptibiility testing of micafungina (fk-463) and anidulafungin (ly303366) against candida spp. objectives: despite antifungal therapy, mortality in disseminated zygomycosis is still too high. we analysed the in vitro activity of posaconazole (pos), voriconazole (vrc) and caspofungin (cas) against 59 strains of the genera rhizopus, mucor, absidia, and cunninghamella in different media compositions. methods: the following five media compositions were compared: rpmi 1640 ± 2% glucose, am3 ± 2 % glucose and hr-medium. mics were determined by microdilution method following nccls guidelines with minor modifications. each well was read visually, the growth in each well was compared with the growth control. two endpoints were evaluated for each drug: an inhibition of growth >90% war recorded as mic1 and an inhibition of growth >50% was recorded as mic 2. the final concentrations of the antifungal agents were 0.015-8 mg/l for pos and vrc, and 0.03-16 mg/l for cas. results: pos was significantly more active than cas and vor, both in r + g and in hr media (p < 0.05 when comparing the mic1 of pos in hr medium to that of vrc; p < 0.001 for all other comparisons). growth in rpmi and am3 media supplemented with glucose was more robust than in the corresponding media lacking glucose. glucose had little influence on mic values. the agreement when comparing the mic1 evaluated in am3 ± glucose was > 85%, while the use of mic2 endpoint yielded 100% agreement for all genera. when comparing the data obtained in rpmi ± glucose to that in hr, the agreement was good. the percentage agreements using the mic2 and mic1 endpoints were 100% and > 70%, in contrast, the agreement between am3 ± glucose and the other media was generally poor. moreover, the average mics obtained in am3 medium were lower than those obtained in either hr or rpmi which was due to a difference among genera. conclusions: our results suggest the following: a) pos is active in vitro against zygomycetes at clinically relevant concentrations. b) within zygomycetes, there are differences between genera in terms of their antifungal susceptibilies. c) growth medium is an important variable for mic determination in zygomycetes, and the more relevant medium appears to be rpmi supplemented with 2% glucose. objectives: here we describe first clinical case of a caspofungin resistant c. glabrata infection. the patient was a 43 year old male with aml. he received a matched unrelated hsct, 9 months prior to his death. he had a complicated hospital course which included c. glabrata sepsis. c. glabrata was cultured from his stool, from the time of transplant to death. initial blood culture isolates were azole resistant and amphotericin b was started. this was stopped due to renal insufficiency and caspofungin was started (mic 0.12 lg/ml). he had a prolonged duration of therapy which comprised of alternating courses of iv voriconazole and caspofungin. four months after initial fungaemia c. glabrata was cultured which was both caspofungin resistant (mic > 8lg/ml) and azole resistant. despite the addition of amphotericin b the patient died 8 weeks later. c. glabrata was isolated from the bone marrow at autopsy. methods: the series of patient isolates, from time of transplant to death, had susceptibilities performed as per nccls document m27-a. the isolates were typed using cg6 probe and mlst. results: he susceptibilities demonstrated caspofungin resistance in the blood isolate ( > 8 lg/ml) and azole resistance in all but a few of the initial stool isolates. all the isolates were shown to be identical by cg6 and were determined to be mlst group 1, which has previously been shown to be the most prevalent clade of c. glabrata worldwide. discussion: this describes the first caspofungin resistant clinical isolate of c. glabrata. it has been recently reported in c. albicans. this data details an isolate that has developed resistance in the presence of therapy. resistance to caspofungin is not common and is thought to be due to mutations in the beta-1-3-glucan synthase (fks) gene. there are 3 fks genes in c. glabrata. preliminary sequencing data of one of these genes has so far revealed no non-conservative mutations. the 2 remaining genes are yet to be sequenced and the presence of other mechanisms cannot be ruled out. objectives: the aim of this study was to investigate the production of slime factor among candida species which were isolated from hospitalized patients. another aim of this study was to see in vitro activities of antifungal agents and to compare these results with slime production. methods: total 116 candida spp (79 c. albicans and 37 nonalbicans candida spp) isolated from various specimens were included to the study. fluconazole, itraconazole, amphotericin b and caspofungin susceptibilities of these strains were determined by broth microdilution method according to nccls m27-a2 standards. biofilm production of candida spp. was determined by microplate method on polystyrene microtiter plates using brain heart infusion broth supplemented with 0.25% glucose as a growth medium. results: caspofungin and amphotericin b was the most active agents which mic90 values 1mg/ml and 0.5 mg/ml respectively. fluconazole resistance (mics > 64) was obtained from 24 of the isolates (21%). biofilm formation was detected in 33 of the total strains (28%). statistically important difference (p < 0.05) was determined between the biofilm production of c. albicans (21.5%) and nonalbicans species (%48.4). significant correlation between biofilm production and amphotericin b mic values was established (p < 0.05) conclusion: candida species are one of the most important etiologic agents of catheter related infections especially biolfilm producing strains. this study showed us biofilm production rates are too high particularly in non-albicans strains. this will explain the rising incidences of non albicans strains especially c. parapsilosis in serious infections. our study also implicated that the mic values amphotericin b which was one of the most active agent against candida infections had a significant correlation with slime production. this will be a problem in treatment of slime producing candida infections with this drug. conclusions: (i) scedosporium spp. carry on with high mics of antifungal agents, even for new antifungal agent as pos. (ii) s. prolificans is a multi-resistant organism. (iii) s. apiospermum is resistant to itc and tbf, but 15% of strains are susceptible in vitro to amb (mic < 2 mg/l), 75% to vrc (mic < 2 mg/l), and 60% to pos (mic < 2 mg/l). (iv) these findings reinforced the need of continued surveillance programmes that analyze antifungal susceptibility profiles of medically important fungal isolates. assessing susceptibility of fungi isolated from hospital environment to disinfectants objectives: the past decade has witnessed a worldwide increase in serve invasive gas infections. these rapidly progressive infections are associated with high mortality rates despite prompt antimicrobial therapy. the aim of this study was to characterize gas isolates causing severe invasive disease in different regions of poland by emm-typing, multilocus sequence typing (mlst), pfge, virulence genes distribution and their susceptibility to antimicrobial agents. methods: a total of 42 gas isolates from blood (54.7%), pus (33.3%), sputum (7.1%), peritoneum fluid (4.7%) and other sources were examined. susceptibility to penicillin, erythromycin, clindamycin, telithromycin, tetracycline, levofloxacin, chloramphenicol, quinupristin/dalfopristin and linezolid was determined by the microdilution method according to the nccls guidelines. clonality of all isolates was studied by emm typing, pfge of sma i-restricted bacterial dna as well as mlst. the strains were also tested for the presence of spea, speb, spec, spef and ssa genes by pcr. results: resistance to erythromycin was found in four isolates (9.5%), two of them exhibited the imlsb and two the cmlsb phenotype. twenty-one (50%) and five (11.9%) were resistant to tetracycline and chloramphenicol, respectively. all tested isolates were fully susceptible to penicillin, levofloxacin, quinupristin/ dalfopristin and linezolid. twenty different emm types were detected, of which emm1 (23.8%) and emm12 (21.4%) were most common, followed by emm85, emm60 and emm81. all emm1 types and emm12 types corresponded to the st28 and st36, respectively. altogether, 24 different pfge patterns, designated a-y were discerned among the isolates, with two predominant profiles a (n = 10) and b (n = 9). our study showed that all isolates possessed speb and spef genes, while the frequencies of spec and spea were 59.5% and 26%, respectively. conclusion: two clones predominated among gas strains causing severe invasive disease in poland; these clones were of emm type 1 and 12. we present 45 actinomyces spp isolations, their identification to a species level and their clinical sources. in addition, we perform susceptibility testing of 23 of those strains to 13 drugs. the identification of actinomyces spp was done taking into account their cultural features, growthõs atmosphere and biochemical and enzimatical tests, according to schemes proposed by sarkonen n., funke g., moncla b.,hillier s., and bernard k. we studied too, the clinical sources of actinomyces spp, if they were isolated lonely or in association with mucosa's normal flora. the susceptibility testing was performed by the agar dilution method, with mueller hinton agar supplemented with 5% sheep blood. the reading of minimum inhibitory concentration (mics) were done after 48 hours incubation at 37°c in at atmosphere enriched with co 2 . all strains were grown at 37°c on sheep blood agar plates with co 2 added. a. radingae, a. europaeus, and a. odontolyticus were the most frequent isolated species ( 7 each one) followed by a. israelii, a. graevenitzii, a. turicensis and a. viscosus. in 27 isolations, actinomyces spp were recovered as sole microorganism, and in the 18 remainder, in association with mucosa's normal flora. there were not relations between actinomyces species and the clinical sources of the samples. mics for penicilin, ampicilin and cefotaxime were from <0.016 to 0.25 lg/ml. there was a bimodal behaviour with macrolides : erythromycin, azithromycin and clarithromycin (mics from 0.032 to 128 lg/ml) and the same was observed with quinolones: levofloxacin, ciprofloxacin and moxifloxacin (mics from 0.032 to 8 lg/ml). all isolations presented mics for vancomycin <0.125 lg/ml. the identification of the actinomyces genus presents diagnostic difficulties due to its growth requirements. some species are not so infrequent, and the sources from which they are recovered suggest that they may be of clinical relevance, for they are often isolated as sole pathogen. isotretinoin versus tetracycline: a comparative study with regard to efficiency in the treatment of acne vulgaris c. oprica, l. emtestam, c.e. nord (stockholm, s) tetracyclines are most commonly used for treatment of moderate and severe inflammatory acne, and systemically administered isotretinoin has proved to be the most efficient treatment, used in patients with moderate or severe acne that fails to respond to other therapies. objectives: a randomized clinical trial was conducted to compare the clinical efficacy and the antimicrobial susceptibility of propionibacterium acnes strains isolated before, during and after treatment with either tetracycline or isotretinoin in patients with acne vulgaris. methods: 52 male and female patients, 15-35 years of age, with moderate or severe acne, were randomized into two groups of 26 patients each. they received oral tetracycline hydrochloride 1 gram/day together with topical retinoid (differine gel 0.1%) or isotretinoin (roaccutane) 1 mg/kg/day. the therapy was given for a 6-month period. clinical evaluation (leeds acne grading system and lesions counting) and bacterial samples were taken before the treatment started, during the treatment and 2 months after the treatment had stopped. dermatology life quality index questionnaire was completed by patients before and after treatment, in order to determine impairment of life quality. results: acne severity was significantly reduced by both regimens during therapy, and patients in the isotretinoin group continuously improved the acne scores after the treatment had stopped. after 6 months of treatment, isotretinoin produced greater lesion reductions than tetracycline. the mean per cent reduction in the different lesion counts was as follows: 83% versus 45% for non-inflammatory lesions (p < 0.01) and 80% versus 56% for inflammatory lesions (p < 0.01) in isotretinoin or tetracycline group, respectively. in the drug-free period, the group of patients treated with isotretinoin presented significantly less inflammatory lesions compared to the group treated with tetracycline (p < 0.001). both treatments had improved the life quality (p < 0.01), independent of acne severity. resistant p. acnes strains were isolated after treatment in both the tetracycline group (53%) and isotretinoin group (25%). conclusions: both treatments were effective during treatment period. more resistant strains were recovered from the tetracycline group. psoas abscesses. differences between pyogenic and tuberculous abscess objectives: to compare the demographic characteristics, clinical features, laboratory, microbiologic and imaging data, therapeutic options and outcome of pyogenic and tuberculous psoas abscesses. patients and methods: retrospective descriptive study of the medical records of the patients diagnosed of psoas abscess in our institution, in the period between january/1994 and october/2004. results: in this period 14 patients were diagnosed of psoas abscess, all of them secondary to a adjacent source of infection. nine patients had pyogenic abscess, and m. tuberculosis was the causal microorganism in 5 patients. in 3 patients there were bilateral involvement, and all three were tuberculous abscess. an underlying disease was present in 67% patients of pyogenic abscess, and only in 20% of tuberculous abscess. these one were malignancy, intravenous drug use, cirrhosis, use of steroids, and 3 with inflammatory bowel disease. patients with tuberculous abscess were younger (37 vs 49 years), and they presented with a longer duration of symptoms from presentation to diagnosis (145 vs 15 days) than patients with pyogenic abscess. abdominal pain was the most frequent symptom at diagnosis in pyogenic abscess (78%), whereas lumbar pain (80%) was in tuberculous abscess. other clinical manifestations were similar in both groups. the source of infection in pyogenic abscess was gastrointestinal in 7 patients (4 polymicrobial), and in 1 patient infection of an aortobifemoral bypass and sacroilitis (both caused by s. aureus). all tuberculous abscess were secondary to spondilitis. there were no differences between both groups in analysis or imaging alterations. culture of the abscess was positive in all cases practised. drainage was performed in 12 patients (7 percutaneous), without differences in both groups. clinical improvement was more delayed in patients with tuberculous abscess than in patients with pyogenic abscess (18 vs 10 days). there were no deaths in any group. conclusions: tuberculous psoas abscesses presents in younger patients with lesser underlying diseases and a more prolonged clinical presentation than pyogenic abscesses. these tuberculous abscesses are secondary to spondilitis, usually with bilateral involvement. prevalence of micro-organisms isolated from wounds of inpatients versus outpatients in a spanish teaching hospital objectives: hidradenitis suppurativa (hd) is a chronic disorder characterized from dilatation of sweat glands and recurrent bacterial infections. vitamin e was administered in several patients as an antioxidant in an attempt to relieve tissue function probably altered by the locally increased oxidant status. methods: twenty nine patients with hd, 15 male and 19 female, were enrolled over a period of twelve months. all have presented with more than three episodes of bacterial exacerbations for at least two years. a detailed medical history was taken upon first evaluation and patients were examined for areas affected by the disease. they were asked to self-evaluate the severity of their condition on a scale of 1 to 10 (1 representing intact skin and 10 maximum severity). patients were divided into three groups of treatment: a (n = 6), controls; b (n = 8), vitamin e orally 200 mg bid; and c (n = 15), vitamin e 200 mg tid orally. patients were withhold from any antimicrobial regime. patients were followed-up at three-month intervals; they were asked to re-evaluate their condition, and to provide details regarding frequency of relapses before and after the initiation of treatment. results: mean ± se duration of the disease was 11.7 ± 1.9) years and of involved areas 3.84 ± 035, with axillas and groin being involved in the majority of cases. mean interval between exacerbations before initiation of therapy with vitamin e was 33.98 ± 9.43 days and after initiation of therapy 91.35 ± 21.64 days (p: 0.042). for group b, mean ± se time interval between exacerbations before initiation of treatment was 45.00 ± 27.39 days and after initiation of treatment 69.00 ± 38.42 days. for group c, respective values were 36.30 ± 9.42 days and 132 ± 12 days. mean ± se of self-evaluation scores before therapy with vitamin e was 9.00 ± 0.71 and 4.33 ± 1.76 for patients of groups b and c respectively. they were 4.75 ± 1.31 and 2.75 ± 1.55 respectively after twelve months of follow-up. the latter changes constituted a significant improvement (p: 0.027). conclusion: vitamin e seems to improve the overall clinical condition of patients with hd substantially. further placebocontrolled studies are necessary to confirm these results. clonal diversity and toxin genes of staphylococcus aureus causing infections in a clinical ward over a 12-year period (1992-2003) s. pollini, g. zanelli, a. sansoni, s. cresti, c. cellesi, g.m. rossolini (siena, i) objectives: s. aureus remains one of the leading bacterial pathogens worldwide, representing a major challenge for antimicrobial chemotherapy. the aim of this study was to evaluate the molecular epidemiology of s. aureus infections observed in an infectious disease ward during the past 12 years. clinical microbiology and infection, volume 11, supplement 2, 2005 methods: 38 nonreplicate s. aureus isolates were collected from patients with staphylococcal infections (including food-borne infections, osteomyelitis, skin and soft tissue infections [ssti], pneumonia, meningitis and bacteraemia/endocarditis; mostly community-acquired) at the infectious diseases clinic, university of siena, during the period 1st feb 1992-31st march 2003. all isolates were analysed for antimicrobial resistance, clonal diversity and toxin genes (sea-e, eta-d, tst, luks-pv and lukf-pv). susceptibility testing was performed according to the nccls guidelines. clonal diversity was determined by pfge and by analysis of the coagulase (coa) and protein a (spa) genes polymorphism. toxin genes were analysed by pcr and restriction mapping. results: most isolates (35, 92%) were methicillin-susceptible (mssa), while 2 were methicillin-resistant (mrsa, mecapositive) and 1 borderline. genotyping revealed a single mrsa clone and remarkable clonal diversity among the mssa isolates (at least 20 distinct clonal lineages). several clones (including 6 mssa and the mrsa clone) were detected over prolonged times (2-8 years). the most prevalent clone was detected over a 8-year period and was exclusively associated with ssti (mostly bullous impetigo). toxin genes were overall detected in 87% of isolates, the most frequent being sea (45%), tst (32%) and eta and/or etb genes (29%). six isolates (all mssa) harboured the luks-pv and lukf-pv genes. conclusions: mrsa were rare and appeared only since 1997. significant clonal diversity was observed within mssa. a significant relationship was observed between toxin production and some infections (e.g. ssti). severe group a streptococcal infections in romania. a surveillance within the strep-euro project b. luca, m. straut, v. ungureanu, c. schalen, a. jasir on behalf of the strep-euro study group objectives: in the last 20 years, severe invasive streptococcal infections, often afflicting otherwise healthy subjects and yielding high mortality rates, have been increasingly recorded in europe and other areas. our main objective was to investigate the situation in romania lacking earlier data in the field. methods: the strains used in this study were clinical isolates from nine regions in romania. the strains were mainly blood isolates, but some were from other sterile sites. the in vitro susceptibility to antibiotics was tested by disk diffusion on pdm agar following the standard instruction. t-typing was performed by slide agglutination using sera from sevapharma, prague. spe gene detection and emm sequence typing was performed according to provious publications. results: during eighteen months 33 cases were reported. most prevalent t-type was 3,13 followed by types 12 and 1. more than fifteen different emm types were recognised, most of them unusual types; only four were type 1. half of the reported cases were from children below 12 years. out of all strains, 50% harboured spec gene, and 25 harboured spea. erythromycin resistance was uncommon (one isolate), whereas an overall high rate of tetracycline resistance was found (54%). conclusion: this is the first report on severe invasive streptococcal infections since no surveillance has been previously done in romania. the population covered was from eight out of 42 provinces. the emm type distribution might be of concern since many unusual types, not previously associated with severe disease, were found. this may be partly related to the dissemination of new types in populations immune to classical types. since tetracycline is not used in the treatment of streptococcal infections the level of tetracycline resistance among clinical isolates, appeared comparatively high. in contrast to several european countries, macrolide resistance seems not to be common among invasive group a streptococci in romania. acknowledgment: the strep-euro project is funded by the european commission. severe soft tissue infection and secondary bacteraemia due to community-acquired mrsa in a traveller returning from the congo r. padmanabhan, r. shrestha, g. hall, s. gordon, l. saravolatz (cleveland, detroit, usa) objectives: community acquired mrsa is increasingly becoming a global problem. these isolates possess the staphylococcal cassette chromosome (scc) mec type iv in their genome and a different pattern of antibiotic susceptibilities than hospital acquired strains. they are frequently virulent and cause predominantly skin and soft tissue infections by virtue of a panton valentine leukocidin (pvl) toxin gene. a returning traveler, who had spent the preceding four weeks in congo, presented with a staphylococcal bacteraemia and a large cutaneous ulcer of the right lower extremity that progressed to extensive soft tissue necrosis. he first experienced symptoms in kinshasa international airport, while awaiting his return flight to the united states, approximately 48 hours prior to presentation at our facility. methods: mrsa that was isolated from blood was susceptible to vancomycin, clindamycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and gentamicin. pulse field gel electrophoresis (pfge) analysis was performed using sma1 on the patient's isolate along with another strain from our hospital and compared to other strains isolated from the midwestern united states. pcr amplification of the genes encoding panton-valentin leukocidin (pvl) was performed using primers described elsewhere. results: pfge demonstrated that the congo strain was different from any of the other midwestern united states strains. scc mec typing of this isolate revealed that this strain was type iv. pcr analysis did not detect sequences specific for the pvl gene. conclusions: the differential diagnosis of an ulcer in a returning traveler is large and includes bacterial, fungal, helminthic, protozoal, viral and arthropod borne causes. mrsa should be added to this list. the widespread occurrence of ca-mrsa in many parts of the world would support the finding of this organism on the african continent. to our knowledge, this represents the first ca-mrsa with the scc type iv mec gene reported from congo. ten-year of community-acquired methicillinresistant staphylococcus aureus st80-iv clone in denmark objectives: to determine the epidemiology in denmark of the pandemic european ca-mrsa clone st80-iv with respect to subtypes, dissemination, acquisition and types of infection. methods: all methicillin resistant staphylococcus aureus (mrsa) isolated in denmark between 1999-2003 were referred to and stored at the staphylococcus laboratory, statens serum institut (n = 617). the isolates were characterized by macro-restriction (smai) analysis using pfge, sccmec typing, dru sequence typing, antibiotic susceptibility testing and pcr amplification of the panton valentine leukocidin gene (pvl). clinical and epidemiological information from all patients were obtained from discharge summaries and registered. results: between 1999 and 2003, the mrsa st80-iv clone accounted for 28% (172/617) of the total number of mrsa in denmark and 30% (146/490) of infections due to st80-iv had primarily community onset (122/146) and thereby st80-iv accounted for 67% of all community onset mrsa in the period. more than 80% of the st80-iv infections were skin and soft tissue infections. by comparing the antibiogram of st80-iv (streptomycin-, kanamycin-, tetra-cycline-and fusidic acidresistant and gentamicin sensitive) with the isolates stored in our s. aureus bacteraemia collection, we traced the st80-iv clone back to 1993. before 1999 one to five st80-iv isolates were encountered pr. year, which increased to 25, 26, 50, 26 and 45 isolates in the years after. by pfge, the st80-iv isolates exhibited nine different patterns (a1-9), of which 60% were type a1. nine type a1 isolates, isolated 1993-2003 were subjected to dru typing, which has earlier enabled separation of pfge clones into subtypes. this was however not possible for the st80-iv isolates. conclusions: st80-iv isolates cause a large proportion of the mrsa infections in denmark and in particular among the infections with a community onset. the success of st80-iv as an infective clone outside the hospital environment may in part be due to its small sccmec type iv and its ability to cause skin and soft tissue infections probably associated with the expression of pvl. the dru sequence typing could not subdivide the major type of st80-iv found in denmark pointing out the strict clonality of this clone, which may indicate that it is a very well adapted pathogen. epidemiology and management of pressure ulcers in an acute care hospital objectives: the aims of the study were to determine pressure ulcer prevalence in an acute care hospital, to identify risk factors, to evaluate the medical and non medical management of pressure ulcers and to study the microbiological contamination of pressure ulcers. methods: for each patient, a standardized questionnaire was completed. the questionnaire included demographic data (age, sex, previous hospitalizations…) and the risk factors of the braden scale. detection of pressure ulcer was performed by skin examination of patients by two experts in skin care. management of ulcer pressure was evaluated by reviewing the clinical charts of each patient with pressure ulcer. each pressure ulcer was swabbed and inoculated on selective media. all the data were entered and analysed with epiinfo 6.04d (cdc, atlanta, ga). results: a total of 549 patients (59 ± 19 years old) were included : 75 ulcer pressures were observed in 37 patients (prevalence = 6.9%). heel anckle was the most frequent localization (40%), followed by sacrum (20%), elbow (11%), spinous processes (7%) and ischial tuberosities (7%). pressure ulcers were stage i (24%), stage ii (29%), stage iii (31%) and stage iv (16%). eighty percents of pressure ulcers were acquired within the hospital. using univariate analysis, risk factors significantly associated with pressure ulcer were : braden cutoff < 15 (or = 6.56, p < 0.0001), neurological disorders (or = 2.30, p = 0.009), previous ulcer pressures (or = 6.36, p < 0.0001), and hospitalization in an intensive care unit (p = 0.0003). among the criteria used for braden scale, humidity, activity, motility, nutrition friction and shear were significantly associated with ulcer pressures (p < 0.05). among the 75 pressure ulcers, 9.3% were diagnosed only by the experts in skin care and 83.8% were treated. treatment was considered inappropriate in 31.5% (mostly in stage i and iii) according to the french guidelines. microbiological results showed that 24.5 % of ulcer pressures, mostly from stage iii and iv were colonized with multiple resistant bacteria (i.e. methicillin resistant staphylococcus aureus, extended spectrum beta-lactamase enterobacteriaceae). conclusion: this prospective prevalence study led to a better awareness of patients at risk for pressure ulcer. this surveillance also contributed to a better knowledge of the mobile unit of geriatrics recently created within the hospital. background: following surgery for a musculoskeletal infection (mi), a positive suction drainage culture (sdc) is consistent with persistent sepsis and an unfavorable evolution in the majority of cases. the objective of this study was to determine the effect of a negative sdc obtained in a subsequent operation or repeated operations on the outcome of mi. methods: one hundred patients treated surgically for mi utilizing suction drainage for 24-48 hours postoperative and appropriate antimicrobial therapy were enrolled in this prospective study. surgical treatment consisted of the routine practice developed for the treatment of mi, consisting of drainage of purulent material, débridement, and prosthetic exchange or implant removal. the accumulated drainage fluid in the reservoir was cultured. sdc was considered negative if all bottles resulted in negative cultures. patients were placed into one of three possible mi treatment groups according to the sdc results identified after the first surgical procedure, as follows: (i) patients with a negative sdc and no new operation was performed; (ii) patients with a positive sdc and a new operation(s) was performed until the sdc was negative; and (iii) patients with a positive sdc and there was no new operation. the duration of antibiotic therapy for those with osteomyelitis ranged from 6 to 12 weeks, while others with mi (soft tissue infection) were treated for 10-21 days. results: the 3 groups were similar with regards to gender, age, underlying conditions, mi, bacterial organism, surgery, and antibiotic therapy. the majority of patients (84%) had osteomyelitis in the presence of an implant. at final review, a cure was obtained in 90% of patients (45 of 50) in group i, 87% of patients (20 of 23) in group 2, and 44% of patients (4 of 9) in group 3. conclusion: a negative sdc following mi surgery is a strong indication as to the eventual outcome of the infectious process. microbial aetiology of osteomyelitis of the foot in diabetic patients related hospitalization and lower-extremity amputation.acute infections in pts who have not recently received antibiotic therapy are predominantly caused by aerobic gram-positive cocci, often as a monomicrobial infection. although chronic wounds tend to develop polymicrobial flora. objective: to know the microbial etiology of osteomyelitis of the foot in diabetic pts, to determine the best choice empirical antibiotics combination. methods: diabetic pts with osteomyelitis of the foot admitted in our unit between 2002 and 2004 were included. the infection was documented with intraoperative samples and osteomyelitis was histolopathologically prouved. clinical and biological data were collected. results: 87 diabetics pts (64 males, 23 females) with a median age 65 years(23-94) were included.the co-morbidities were chronic renal failure (n = 35), arteriopathy (n = 34), obesity (n = 13), alcoholism (n = 10), immunodepression (n = 10). the clinical data were fever (40%), shock (2%), local signs as erythema (90%), fistula (86%), necrosis (43%) and foul odor (54%). the median rates of neutrophils polynuclear, c-reactive protein and erythrocyte sedimentation were respectively 9.7 g/l, 131 mg/l, 82 mm. 59 pts (68%) had a polymicrobial infection.the intra-operative samples (n = 87) yieled a majority of gram positive cocci [mssa:n = 30;mrsa:n = 15; cns:n = 15; streptococcus sp:n = 34; enterococcus sp: n = 15], following with gram negative bacteria [ pseudomonas sp: n = 12; proteus sp: n = 11; morganella sp: n = 11; e. coli: n = 8; klebsiella sp: n = 6; e. cloacae: n = 6; citrobacter sp: n = 4; serratia sp: n = 3, others: n = 3] and anaerobes (n = 17). 10 pts had positive blood samples. s. aureus is the most frequent pathogen isolated especially in monomicrobial infections and infections with positive blood samples. pseudomonas spp. seems to be correlated with chronic renal failure and immunodepression. no clinical data was significantly associated with a special bacteria , except necrosis and foul odor with gram negative bacteria or anaerobes. conclusion: our results are according with clinical studies in the literature. the major difficulty to treat osteomyelitis of diabetics foot is to well documented the infection. selecting an appropriate antibiotic to treat is particularly important because of the prolonged duration of therapy required and potential resistance of pathogens. the duration of hospitalisation (length of stay) in patients hospitalised with complicated skin and skin structure infections: identifying clinical and microbiologic risk factors in a comparison of tigecycline with vancomycin/aztreonam 42% of patients had polymicrobial infections. about 18% had a gram-negative causative pathogen, mostly escherichia coli. gram-negative causative pathogen prevalence was greatest in asia (39%). conclusions: this analysis showed significant global variations in csssi subdiagnoses, etiologies, comorbidities and causative pathogens. consistent with findings of the sentry antimicrobial surveillance program, s. aureus was identified as the predominant pathogen in csssi, especially in the us. gramnegative pathogens were also identified as causative in a substantial proportion of complicated skin infections. these findings may offer guidance in selecting effective empiric therapy, especially regarding newer agents with expanded broad-spectrum activity. a novel approach for evaluating the microbiological efficacy of tigecycline in patients with complicated skin and skin-structure infections a. meagher, p. ambrose, j. passarell, b. cirincione, t. babinchak, e.j. ellis-grosse (buffalo, albany, collegeville, usa) objectives: tigecycline (t) is a glycylcycline in development for the treatment of patients (pts) with serious infections, including complicated skin and skin-structure infections (csssi). while csssi can be caused by a mixture of grampositive and -negative bacteria, staphylococcus aureus and streptococci are the predominant pathogens. previous analyses combining all pathogens have failed to identify an exposureresponse (er) relationship. a method was developed to create more homogenous pt populations for the microbiological (m) er analysis of t in the treatment of csssi. methods: pts from 3 csssi clinical trials (one phase 2 & two phase 3) with t pharmacokinetic data and classified as both clinically and m evaluable, were pooled for analysis. pts received 100 mg loading dose (ld)/50 mg q12h (100/50) or 50 mg ld/25 mg q12h (50/25). at the test of cure visit, m (eradication or persistence) response was evaluated. indeterminate responses were excluded. non pathogenic baseline isolates were excluded. five homogeneous pt cohorts (c) were created based on baseline pathogens: s. aureus only (c1); s. aureus or streptococci (c2); 2 gram-positive pathogens (c3); polymicrobial (c4); other monomicrobial infections (c5). prospective step-wise procedures for combining c to increase sample size were used. logistic regression was used to evaluate steady-state 24 hr area under the concentration-time curve (auc) to mic ratio (auc/mic) to predict response. results: the dataset included 58 pts with 88 observations. c1 (n = 20) and c2 (n = 9) could not be evaluated due to small sample size. analysis began with pooled c2 + c3. continuous auc/mic ratio was marginally significant (p = 0.1130); a pt was 5.1% more likely to have successful response for every oneunit increase in auc/mic. adding c4, including pathogens with mic values up to 16 mcg/ml, decreased auc/mic, added cures to the lower end of the distribution, and added significant noise to the analysis. adding c5 increased sample size and further decreased the ability to detect a relationship. conclusion: analysis of all pathogens combined could not identify an er relationship. polymicrobial infections with gramnegative and anaerobic pathogens, associated with high mic values, added noise to the analysis and decreased the predictive capability of the model. the approach of creating homogenous populations based on two key pathogens in csssi, s. aureus and streptococci, was critical for identifying significant er relationships. exposure-response analysis of the efficacy of tigecycline in patients with complicated skin and skin-structure infections a. meagher, j. passarell, b. cirincione, s. van wart, k. liolios, t. babinchak, e.j. ellis-grosse, p. ambrose (buffalo, collegeville, albany, usa) objectives: tigecycline (t), the first glycylcycline to reach clinical trials, is in development for the treatment of patients (pts) with serious infections, including complicated skin and skin-structure infections (csssi). pharmacokinetic-pharmacodynamic (pk-pd) relationships, including pt covariates, for microbiological (m) & clinical (c) efficacy of t were evaluated in pts with csssi. methods: pts from 3 csssi clinical trials (one phase 2 & two phase 3), with pk data and classified as both c & m evaluable, were pooled for analysis. only those pts with infections due to staphylococcus aureus and/or streptococci, the predominant pathogens in csssi, were prospectively evaluated. pts received 100 mg loading dose (ld)/50 mg q12h (100/50) or 50 mg ld/ 25 mg q12h (50/25). at the test of cure visit, m (eradication or persistence) & c (cure or failure) outcomes were assessed. indeterminate responses were excluded. steady-state 24 hr area under the concentration-time curve (auc) and auc/mic ratio were evaluated as predictors of response. pt covariates included: age, weight, country, baseline pseudomonas aeruginosa or anaerobes, & comorbidities (diabetes, peripheral vascular disease). classification and regression tree (cart) analyses determined auc/mic breakpoints (bp). logistic regression (one observation/pt) was performed to determine predictors of efficacy. results: the dataset included 35 pts with 40 s. aureus and/or streptococcal baseline pathogens. mic values ranged from 0.06 to 0.5 mcg/ml. clinical cure was achieved in 30 (85.7%) pts and 35 (87.5%) pathogens were successfully eradicated. the median auc/mic ratio was 13.5 and 29 for the 25 and 50 mg dose groups, respectively. covariates were not significant predictors of efficacy. cart identified a significant auc/mic bp of 12.5 (p = 0.0177 for m and 0.0341 for c). the continuous auc/mic ratio was marginally significant based on sample size (p = 0.0563 for m & 0.1960 for c) and was deemed the most informative model. for each unit increase in auc/mic, within the observed range, pts were 3.7% more likely to have a successful c response and 17.1% more likely to have a successful m response. conclusion: pts with auc/mic ratios ‡12.5 were 13.5 times more likely to have successful m response. at the median auc/ mic ratio of 13.5 & 29 for the 50/25 & 100/50 dose groups, the model-predicted probability of c success was 0.66 & 0.96, respectively. t is likely to be an important treatment option for csssi. introduction: vertebral osteomyelitis and/or sacroiliitis due to streptococcus pneumoniae are extremely rare. although it is one of the most common pathogen isolated in blood cultures, there are only about 30 cases reported in the literature. in the main series of vertebral osteomyelitis (vo) spsi represents less than 1% of cases (1/587). material and methods: we report 6 cases of spinal infection due to s. pneumoniae (5 vo and 1 si with psoas abscess) seen between 1999 and 2004 at 4 different acute care hospitals. results: the 6 cases reported were part of a total of 1227 episodes of pneumococcal bacteraemia (0.48%).mean age of the patients was 57.1 years (range 37-82); there were 4 men. alcohol abuse 2/6, immunosupression 2/6 and smoking 4/6 were the most common comorbilities. blood cultures were positive in all cases, epidural pus and vertebral biopsy were also positive in 3/ 6. all strains were susceptible to penicillin. diagnoses were made by mri in 4 and ct scanning 2. all patients recived betalactam agents during a mean of seven weeks (3.8-10.7) in monotherapy (cefotaxime 4/6, ampicillin 1/6) or in combination (with vancomicin+gentamicin in 1 case). one case needed surgery, because of progression of a psoas abscess. other foci were found in three cases (psoas and paravertebral abscesses and acromio-clavicular arthritis). conclusions: spsi is a rare presentation of invasive pneumococcal disease. underlying disease is comnmon and other territories are frequently involved. infective endocarditis must be ruled out. its course appears to be benign. beta-lactam antibiotics are a good option, and surgery is occasionally indicated for progressive disease despite appropriate antimicrobial therapy. lumbar spine spondylitis due to staphylococcus schleiferi following coronary angioplasty: isolation of the pathogen in peripheral blood cultures a. karakolios, c. karagiannidou, t. kallinikidis, f. markou, v. kontos, g. tsinopoulos (serres, gr) a 70-year old man presented with acute, severe lower back pain 3 weeks after having been subjected to coronary angioplasty for ischaemic heart disease. he was treated with nsaids but not antibiotics without any improvement. ct scan of the lumbar spine showed changes in l5-s1 vertebrae that were consistent with spondylitis. mri imaging, as well as confirming spondylitis at l5-s1, it also revealed the presence of a left paravertebral abscess. on admission to hospital, low grade fever was documented. clinical examination of the lower limbs was normal. staphylococcus schleiferi, sensitive to several antibiotics, was isolated from 3 sets of blood cultures taken from both arms. initially, ciprofloxacin 200 mg bd iv was administered for 4 weeks. despite continuous clinical improvement, mri imaging 8 weeks after the commencement of antibiotic treatment showed no improvement of the radiological findings. thus, levofloxacin 500 mg bd p.o. rifampicin 300 mg bd p.o. were given for further 16 weeks. at the end of the treatment, the patient was symptom-free, resumed full activity and mri imaging showed considerable improvement. multiple blood cultures taken from both arms at different time points can help isolate pathogens causing spondylitis averting thus the need for difficult, interventional sampling of the focus of infection. diabetes and use of topical ocular antibiotics: a population-based case-control study objectives: we conducted this population-based case-control study to examine whether patients with diabetes mellitus have an increased relative risk of being treated with topical ocular antibiotics, as compared with population controls. methods: incident cases were defined as 24,559 individuals who redeemed a prescription for a topical ocular antibiotic in 1999 in north jutland county, denmark. ten gender-and agematched population controls per case were selected, using a unique personal identifier. diabetes prior to the topical ocular antibiotic prescription was determined by record-linkage with the county prescription database and hospital discharge registry. we did conditional logistic regression to estimate odds ratios (ors) for ocular antibiotic use among diabetic individuals and population controls, with adjustment for a range of comorbid diseases. results: among individuals treated with topical ocular antibiotics, 2.5% had diabetes as compared with 2.0% among control subjects. the overall adjusted or for use of ocular antibiotics in diabetic individuals was 1.24 (95% confidence interval [ci]: 1.13-1.35). stratified analyses showed that children between 3 and 15 years with diabetes had a 60% increased relative risk for ocular antibiotic use, whereas the effect of diabetes as a risk factor was low in individuals over 65 years and in those with presence of other diseases. conclusions: these results suggest that diabetes is a risk factor for the use of topical ocular antibiotics, especially in younger individuals. objectives: determination of bacterial species and their susceptibility to antibiotics in infectious conjunctivitis in general practice. methods: 179 patients suspected of having infectious conjunctivitis were included from 25 general care centres in the netherlands. from both eyes a swab was taken and pathogens found in the bacterial cultures were tested for their susceptibility using the etest for chloramphenicol, fusidic acid, trimethoprim, ciprofloxacin, and gentamicin (mics according to dutch national criteria). results: 80/220 affected eyes were culture positive. streptococcus pneumoniae (n = 55) was the most predominant pathogen, followed by haemophilus influenzae (n = 19), and staphylococcus aureus (n = 18). only for chloramphenicol and ciprofloxacin the mic90s were in the susceptible or intermediate susceptible range for all three pathogens, whereas this was not the case for the other antibiotics tested, of which at least one mic90 was in the resistant range. conclusion: in only one thirdth of the patients suspected of infectious conjunctivis in general practice a bacterial pathogen was found. although the prescription of antibiotics can be debated on the basis of our results, chloramphenicol and ciprofloxacin seems to be superior above fusidic acid, trimethoprim, and gentamicin. investigation of the aetiology of a trachoma-like condition in a rural population in guangxi province, prc p. cho, m.v. boost, w. ng (hong kong, hk) objectives: to determine the presence of trachoma in primary school children of the zhuang and yiao ethnic minority groups in du¡an county in guangxi, china. method: primary school children were examined using a slit lamp and several were noted to display follicular/papillary conjunctivitis suggestive of trachoma. to confirm the etiology of the condition, children were examined on a follow-up visit. for each subject, after examination with the slit lamp, the upper lid was everted after the application of a topical anesthetic (novesin), and a dacron swab was passed along the upper tarsal lengthwise four times. strict aseptic precautions were employed to prevent cross-contamination during sample collection. swabs were placed in dna-free tubes, transported to hong kong, and tested using the roche amplicor kits for detection of chlamydia trachomatis following the manufacturer¡s instructions. twenty children displayed significant conjunctival signs and photographs of the everted upper lids were taken to determine if these visual signs correlated with presence of trachoma as confirmed by pcr. results: sixty-five of 120 primary school students were willing to be tested, and 29% of these were positive for trachoma by pcr. comparison of photographs and pcr result indicated that not all children who showed signs of conjunctivitis were positive for trachoma. in addition, some children whose eyelids displayed milder form for conjunctivitis were positive by pcr. no obvious corneal involvement was observed for these children. conclusions: it was confirmed that hyper-epidemic levels of trachoma can be found among ethnic minority children in du¡an county. this may be related to the poor hygiene standards and the scarcity of water in this limestone region. pcr was not positive for all children showing signs of follicular/papillary conjunctivitis, which may indicate that some cases were in a dormant stage. it is also possible that failure to detect the organism was due to sampling error or delay in pcr analysis. nevertheless, the high level of infection is a cause for concern as trachoma remains one of the leading causes of blindness, and further investigation and treatment are warranted. clinical and epidemiological considerations about anthrax in constanta county, romania s. rugina, i.m. dumitru, c.n. rugina, e. dumea, e. basca (constanta, ro) introduction: anthrax is by primarily a disease of herbivores but humans can become infected as they come into contact with infected animals or their products. objective: to study the cases of human anthrax diagnosed in the last twelve years and their characteristics: epidemiology, clinical forms and evolution. material and method: it is a retrospective study on a period of twelve years performed in infectious diseases clinical hospital. the diagnosis was based by smear, and culture of vesicular fluid or csf. results: during this period fifteen cases of anthrax in humans were identified. the repartition of cases according to sex groups revealed a high predominance of male cases (13 cases). the disease was most prevalent in 35-44 age group and after 55years old (6 cases), although anthrax can be observed between 15 years and 65 years. in all cases anthrax was occupational disease and the provenience of patients was rural area. the annual incidence of anthrax was characterized by a low value and irregular frequency of the disease. the most cases were in 1994 (6 cases) and 2004 (5 cases). according to the seasonal repartition, anthrax was most prevalent in the summer months (11 cases). the most frequent clinical form of anthrax was the cutaneous anthrax (14 cases), and only one patient achieved anthrax meningitis. under specific treatment with penicillin g, all the patients with cutaneous anthrax recovery complete, only the patient with anthrax meningitis died. conclusions: in constantza county, anthrax remained sporadic in the last years. after a free period (2001) (2002) (2003) 5 cases of coutaneous anthrax were registered in 2004. a good cooperation between veterinary and physicians and individual awareness of the materials is the key to prevention of anthrax. hemolytic activity. all of the 30 strains tested have hemopeptic activity, which is indirect evidence of their virulence. the strains have no phosphatase enzymes or lecithinase. the strains we studied ferment glucose, sucrose, glycerin, and rhamnose, and they form acid without gas. the strains all grew in synthetic "a" medium, although growth was weak. when l-tyrosine, ltryptophan, l-threonine, and l-methionine were added to the synthetic "a" medium, the pathogen grows in the typical manner. two anthrax strains are moderately resistant to rifampicin (mic 1.5-16), and three are not sensitive. the anthrax strains are highly sensitive to tetracycline, benzylpenicillin, gentamycin, and oxacillin. conclusions: regardless of their source of isolation and time of storage, the anthrax strains isolated during outbreaks are virulent, spore-forming, and capsule-forming cultures. this study confirms that virulent forms of b. anthracis are still circulating in kazakhstan. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency under the project «kb0-1950-al-03» and administered by u.s. crdf. the importance of laboratory methods in diagnosing anthrax in humans l. lukhnova, a. aikimbayev, t. meka-mechenko, g. temiraliyeva, s. zakaryan, m. sagatova (almaty, kaz) objectives: isolation of anthrax bacillus using bacteriological methods is slow and frequently misleading. the literature suggests anthrax bacillus is isolated in 13% to 48% of patients infected with bacillus anthracis. the pathogen quickly perishes or transforms into atypical forms. the number of bacilli in a clinical specimen is often low or below the sensitivity of culture. this suggests other methods would be useful in establishing the diagnosis of anthrax, specifically; serological methods would be beneficial in diagnosing infection with bacillus anthracis. methods: we conducted studies to assess various methods for diagnosis of bacillus anthracis infection in patients. we reviewed archival data and our own results on pathological material from patients infected with bacillus anthracis in the southern and eastern kazakhstan oblasts. results: the most informative test for detection of anthrax in patients was the delayed type hypersensitivity (dth) or dermatoallergic test with anthraxin. eighty one per cent of patients with signs and symptoms of anthrax gave a positive reaction with anthraxin. the ascoli reaction complete with scab was positive in 72.1% of patients, and the indirect hemeagglutination (iha) test was positive in 65.6% of patients. the clinicoepidemiological diagnosis of anthrax was confirmed with culture in 44% of cases. serum samples from patients diagnosed with anthrax in the eastern kazakhstan oblast were evaluated using iha in 2001-2004. high titres of specific antibodies (1:640-1:5120) were detected in patients with dermatologic anthrax lesions from which anthrax was isolated. conclusion: our data suggest the anthraxin dth test and iha are good methods for establishing a diagnosis of anthrax. cultures for anthrax bacillus were the least efficient method for establishing the diagnosis. when results of laboratory tests are negative or inconclusive, one still has to rely on the clinicoepidemiological diagnosis. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency (dtra) under the project «kb0-1950-al-03» and administered by u.s. civilian research and development foundation (crdf). objectives: the fluoroquinolones are bactericidal agents frequently used in the treatment of respiratory tract infections. the viridans streptococci (vgs), although associated with disease, generally exist as part of the normal human oral flora. as such, they may exposed to antibiotics with greater frequency than oppurtunistic pathogens. preliminary data suggest that the vgs may act as a reservoir for fluoroquinolone resistance in s.pneumoniae through horizontal transfer. here, we compared the killing kinetics of moxifloxacin (mxf) and levofloxacin (lev), against vgs at simulated saliva concentrations. methods: eight strains of vgs, 4 s. mitis (2 with parc mutation) and 4 s.oralis, (2 with parc mutation) were challenged with mxf and lev in a pharmacodynamic model utilizing mueller-hinton broth supplemented with 5% lysed horse blood. cultures were inoculated at a density of 1 · 108 cfu/ml, incubated at 35ordm;c, and examined for viable growth at 0, 1, 2, 4, 6, 12, and 24 hrs after exposure to the antibiotics at concentrations simulating salivary levels. salivary concentrations of mxf (400 mg), and lev (500 mg) were 3.19 and 3.71 lg/ml respectively. protein binding of mfx and lev were taken to be 50% and 30%. adjusted salivary levels were 1.6 (mxf) and 2.6 (lev) ug/ml. pcr was used to amplify parc and gyra, and mutations detected by dna sequencing. results: both mxf and lev were highly bactericidal against susceptible strains of vgs at salivary concentrations. mfx typically eradicated the vgs by 6 hours and lev by 12-24 hours. for parc mutants, both mxf and lev were consistently bactericidal resulting in eradication after12 hours for mxf and 24 hours for lev. regrowth was observed in both strains of s.mitis (parc) exposed to lev (2 of 10 experiments). the mic of these strains to lev increased 4 fold and dna sequencing revealed the selection of a gyra mutation in each strain. no regrowth was detected in the s. oralis strains. conclusions: both mxf and lev are extremely active against susceptible vgs at concentrations of drug found in saliva, however, superior killing kinetics were consistently observed with mxf. mxf consistently eradicated strains with a parc mutation, however, regrowth was observed on 2 occasions with lev suggesting that mxf may be less likely to drive resistance among vgs. again, this may have implications due to the ability of vgs to exchange its genetic determinants with s. pneumoniae. moxifloxacin and ciprofloxacin uptake by human neutrophils and their influence on viability, phagocytosis, oxidative burst, and chemotaxis a.m. berg, j. altrichter, b. drewelow, r.g. mundkowski (rostock, d) objectives: the influence of antibiotics on the immune defence is of increasing interest. the purpose of this study was to set up a cellular model for correlating the intracellular concentrations of the fluorochinolones moxifloxacin and ciprofloxacin with selected cell functions of early immune response. a human promyelotic cell line which is used for sepsis treatment by the extracorporeal immune support system (eissò, teraklin gmbh) was chosen to establish the model. the second stage involved native human polymorphonuclear neutrophiles (pmns) as target cells. methods: the promyelotic model cell line was differentiated towards neutrophils with all-trans retinoic acid, human pmns were isolated using dextran sulfate sedimentation and density gradient centrifugation. the cells were incubated with antibiotic concentrations within the therapeutic range and above (0.5-2 cmax) for time periods up to 8 hours. the extra-and intracellular concentrations were determined by hplc-uv. the viability of the cells was controlled using the trypanblue-exclusion assay. phagocytosis and oxidative burst were assessed by a combined flow cytometric assay after stimulation of the cells with serum-opsonised fluorescein-isothiocyanate-conjugated e. coli in the presence of dihydroethidium as an indicator of superoxid production. a two-compartement chamber system was used to study the influence of either fluorochinolone on the chemotaxis. results: with either cell type, moxifloxacin accumulated rapidly with peak concentrations within 15 min whereas intracellular maximum concentrations of ciprofloxacin were achieved after 2 h. the accumulation rate of both antibiotics inside the native pmns was more than twice as high as in differentiated eiss ò cells. neither moxifloxacin nor ciprofloxacin showed a significant influence on viability and the immune response parameters phagocytosis and oxidative burst of native pmns; chemotaxis ability was reduced to a minor degree.viability and phagocytosis of the diffentiated eiss ò -cells remain unaffected whereas the oxidative burst appeared decreased. chemotactic acticity of eiss ò -cells could not be stimulated. conclusions: a significant accumulation of moxifloxacin and ciprofloxacin was determined in both cell types. however, no relevant influence on viability, phagocytosis, oxidative burst, and chemotaxis was observed. hence no hints were found suggesting a restricted use of moxifloxacin and ciprofloxacin for immunocompromised patients. prospective, multicentre in vitro study to determine resistance rates and comparative activity of moxifloxacin vs. clinical bacterial isolates from patients with respiratory tract infections (moxiaktiv study) a. dalhoff, g. korfmann, e. jacobs (kiel, leverkusen, dresden, d) objective: to determine the prevalence of resistance in current clinical isolates of s. pneumoniae, h. influenzae, m. catarrhalis, s. aureus and k. pneumoniae as well as the in vitro susceptibility to moxifloxacin and other iv antibiotics in germany. methods: up to 20 isolates of each of the above-mentioned species were cultured on standard media and tested for in vitro susceptibility using etestò in each of the 29 study centres in germany. for validation, an atcc control strain was included for each species. drugs tested were moxifloxacin, levofloxacin, amoxicillin/clavulanate, cefuroxime, clarithromycin and penicillin g. the latter two were not tested for klebsiellae. s. aureus was additionally tested for oxacillin resistance and k. pneumoniae was tested for production of extended spectrum beta-lactamases (esbl). din breakpoints were used where applicable. results: overall, 1859 pathogens of 1,811 hospitalised patients with respiratory tract infections were analysed. results for moxifloxacin are summarized in table 1. of s. pneumoniae isolates 93.3% were susceptible to penicillin g and 85.7% to clarithromycin. with an mic90 of 1 mg/l, the in vitro activity of levofloxacin was markedly lower than that of moxifloxacin. h. influenzae showed almost 100% susceptibility to fluoroquinolones, but only 4.5% to clarithromycin (mic90 32 mg/l). as beta-lactamase production is common among h. influenzae isolates in germany, amoxicillin/clavulanate showed far higher susceptibility rates than penicillin g (87.9% vs. 1.0%). due to a high rate of beta-lactamase production only 9.8% of moraxella isolates were susceptible to penicillin g, compared with 99.1% to amoxicillin/clavulanate. moraxella isolates were fully susceptible to the fluoroquinolones. overall, 13.0% of klebsiella isolates produced an esbl. interestingly, esbl prevalence was 38.8% in eastern germany, but below 10% in western germany. esbl producers of klebsiella were less susceptible to antibiotics other than cefotaxime or ceftazidime than non-esbl producers. conclusion: for all species tested, the fluoroquinolones achieved the highest overall susceptibility rate (92.8%) compared to the other antibiotics (penicillin g: 36.9%, clarithromycin: 60.5%, ampicillin/clavulanate: 85.7%, cefuroxime: 89.6%). moxifloxacin showed a high activity against current respiratory pathogens in germany and was the most active fluoroquinolone, in particular against gram-positive pathogens. objectives: drug combinations have become the best choice in treatment of serious infections with resistant microorganisms including p. aeruginosa (pa). we tested ciprofloxacin (cip) in double and triple combinations with ceftazidime (caz), imipenem (imp), piperacillin (pip), and amikacin (amk) against 20 mdr clinical isolates of pa. methods: the mic for each drug alone was determined by broth microdilution technique described by nccls. double and triple combinations of cip with other drugs were tested by using 96-well plates and by time-kill assay. rows a to g were activity of moxifloxacin on s. pneumoniae (sp), h. influenzae (hi) and m. catarrhalis (mc) clinical isolates collected from the respiratory tract by determination of the mic's using the etest and to compare these results against 9 other antimicrobial agents.these results were also compared for sp versus those of the previous study (winter periods 2000-2002) for interpretive criteria s -i -r (according the nccls -breakpoints). methods: 10 belgian university and non-university medical microbiology laboratories participated during the past winter period in this multicentre in-vitro evaluation of moxifloxacin. each laboratory included 40 strains of sp and 20 strains of hi and mc. the total number of evaluable strains (with the exclusion of duplicate isolates) was 758. testing conditions: method and antibiotics: susceptibility testing was performed in the participating centres by using the e-test. the antibiotics tested were penicillin, ampicillin, amoxi/clav., doxycycline, clarithromycin, cefuroxime, ceftriaxone, ciprofloxacin, levofloxacin and moxifloxacin.medium : sp and mc : mueller-hinton agar with 5% blood; hi : htm agar. inoculum : direct suspension and 0.5 mc farland standard.incubation : 35°c for 24 hours in 5% co 2 . quality control was performed using atcc reference strains (s. pneumoniae atcc 49619 ; h. influenzae atcc 49766). results: see graphs 1 and 2. conclusions: this follow-up belgian multicentre study confirms the excellent in-vitro potency of moxifloxacin against clinical isolates of sp, hi and mc and it demonstrates that it is the most potent fluoroquinolone available in belgium for the treatment of lower respiratory tract infections. according the nccls -breakpoints, there are no major s -i -r differences for sp between these results (2003) (2004) and the results of the previous study (2000) (2001) (2002) . this means that the use of new fluoroquinolones like moxifloxacin and levofloxacin has not led, to date, to an increase of resistance in sp. however, repeated follow-up surveys will continue to be needed in order to assess the activity of fluoroquinolones and to monitor the ecological impact of the recently increased fluoroquinolone usage for respiratory tract infections in belgium in the forthcoming years. objectives: the objective of several experiments was to evaluate the in vitro activity of moxifloxacin against porphyromonas gingivalis (p.g.). methods: mics of moxifloxacin against 16 strains of p.g. were determined by e-test (ab biodisk, solna, sweden). furthermore the spontaneous mutation rate and the induction of resistant strains by the 0.25-fold mic of the antibiotic were determined. to find the target of resistance fragments of gyra and gyrb were sequenced. finally the efficacy of moxifloxacin against p. gingivalis atcc 33277 considering special conditions such as effects on the strain in a biofilm or within epithelial cells (kb cells) was evaluated. results: moxifloxacin had very low mic values (ranging from 0.006-0.032 lg/ml), but subinhibitory concentrations of the fluoroquinolone induced very fast mutations. the spontaneous mutation rate was up to 5 · 10 )8 after the two-fold mic and 1.2 · 10 )8 after the eight-fold mic. often the mutants exhibited a high resistance mics >32 mg/l. all these mutants bore ser-83->phe substitution in gyra. the 5-fold mic eliminated p.g. atcc 33277 within biofilms after 24 h, and the 100-fold mic was able to kill all intracellular p. gingivalis within kb cells. conclusions: moxifloxacin showed a very good activity against planctonic p. gingivalis and p.g. within a biofilm. this antibiotic in a higher concentration was also efficient to intracellular bacteria. but a rapid development of resistance was observed in laboratory. moxifloxacin might be an alternative in the antibiotic treatment of p. gingivalis associated periodontitis, nevertheless clinical studies should focus not only on improvement of clinical parameters but also on occurrence of resistant strains. levofloxacin activity on respiratory isolates of streptococcus pneumoniae with decreased susceptibility to ciprofloxacin in spain j. garcía-de-lomas, j.l. juan-bañ ó n, c. garcía-rey, r. dal-ré on behalf of the spanish surveillance group for respiratory pathogens objectives: to test the activity of levofloxacin against recent respiratory isolates of s. pneumoniae with reduced susceptibility to ciprofloxacin (mic ‡ 2 mg/l; n = 607) belonging to the national surveillance sauce-3 (n = 2721; nov01-oct02) and to identify chromosomal mutations in isolates with a levofloxacin mic ‡ 4 mg/l. methods: the activity of levofloxacin was tested by broth microdilution following nccls recommendations. genotypic sequenciation of the qrdr (gyra, gyrb, parc and pare) was done on all the strains with a levofloxacin mic ‡ 4 mg/l. results: levofloxacin mic distribution, mic50 and mic90 (in bold) are given in the table for the whole sample and broken into ciprofloxacin mic categories. mutations in gyra (e85k) were found in 8 (18.6%) strains. no gyrb mutations were found. mutation in parc occurred in 100% these strains (26 s79f; 12 s79y; 2 s79i; 1 s79r; 1 d831; 1 d83n). finally, mutation of pare was found in 17 (39.5%) strains and all of them had the change i460v. one single parc mutation occurred in 24 (55.8%) strains, (1999) (2000) (2001) (2002) ; >99.9% were at £0.12 mg/l conclusions: gemi continues to be a highly potent fq when tested against the two most commonly isolated gram-negative carti pathogens; h. influenzae and m. catarrhalis. this potency and spectrum was consistent across six years and three continents. norfloxacin as a marker of decreased susceptibility to fluoroquinolones in enterobacteria using the vitek 2 system l. ló pez, j. alcala, f. torres, e.j. perea, a. pascual (seville, e) objective: the detection of nalidixic acid resistance acquires importance in enterobacteria since already resistant strains develop more frequently resistance to fluorquinolones than susceptible strains. many clinical laboratories have incorporated the vitek 2 system because their benefit in routine, but nalidixic testing is not included for enterobacteria cards. the aim of this study was the use of norfloxacin vitek mics value as alternative marker to detect nalidixic acid resistant enterobacteria strains. material and methods: a total of 1013 isolates of enterobacteriaceae were analyzed and routinely inoculated into the vitek 2 id-gnb and ast-n020 cards (biomérieux) and then loaded into the vitek 2 system, as recommended by the manufacturer. ninety-six strains with a higher norfloxacin vitek mic value (1-4 mg/l), but still within the susceptibility range established by the nccls breakpoints, and 100 strains with a norfloxacin vitek mic £0.5 mg/l were selected. they were further tested with a 30 microg-nalidixic acid-disk (nal) (oxoid) according to nccls reference. results: the norfloxacin 1-4 mg/l and susceptible to ciprofloxacin phenotype represented the 9% of total isolates loaded in the vitek system during the study, similar to the prevalence observed in previous analysis in spain. both selected groups, one with the highest and the other with the lowest norfloxacin vitek mic value, included 60% and 58% e. coli isolates, 8% and 20% s. enterica isolates, respectively. among the norfloxacin vitek mic 1-4 mg/l strains 93 isolates (98%) were nal resistant, and 76 (79%) were ciprofloxacin susceptible. the agreement between norfloxacin vitek mic £0.5 mg/l and nal susceptibility was foun in 97 (97%) strains. two isolates (one e. cloacae and one k. pneumoniae) with norfloxacin vitek mic 1-4 mg/l were nal-s and 3 isolates (two e. coli and one p. mirabilis) with norfloxacin vitek mic £0.5 mg/l were nal-r. conclusion: when using vitek 2 system for enterobacteria, the consideration of mic of norfloxacin >0.5 mg/l could be an alternative marker of nalidixic acid resistance (and decreased susceptibility to fluoroquinolones). antibacterial susceptibility studies -i objectives: p. aeruginosa is an important nosocomial pathogen, which causes serious and often lethal infections in immunocompromised hosts. this pathogen is intrinsically resistant to many antibiotics and can easily develop resistance towards many currently available agents. natural resistance can be attributed to the low permeability of the p.aeruginosa outer membrane to a variety of antibiotics including glycopeptides (glys). since glys are powerful antibiotics against grampositive bacteria and resistance is very rarely develop, it seemed interesting to evaluate the effect of combining these antimicrobial agents with antibiotics that might disorganize the structure of the outer membrane allowing the entrance of glicopeptides into the gram-negative cells. in order to verify this hypothesis, ceftazidime (caz) has been tested in association with vancomycin (van) or teicoplanin (tei). the same experiments have been carried out also in the presence of azithromycin (azi), which is normally a non anti-pseudomonal agents but has been shown to interfere with some virulence factors. methods: a bacterial suspension of about 10 9 cfu/ml was seeded on plates containing a fixed concentration of glys (500 mg/l) and increasing doses (2x,4x,8x,16x) of caz. survivors were counted after 48 hr at 37°c. results were interpreted as synergism (99%), additivity (90%), and indifference (10%) of the cfu/ml reduction found in the drugs combination in comparison to the drug alone.the same experiments have been repeated adding azi (16 mg/l) and using glys at concentrations ranging from 500 to 300 mg/l. results: caz in combination with glys reacted synergically in 20 out of 59 cases, additivity was found in 31/59 interactions and indifference was noted in 8/59 tests. preliminary results (12 tests performed) indicated that the addition of azi increased the incidence of synergisms and additivities even when using glys concentration of 300 mg/l. conclusions: caz combined with glys gave additive or synergistic results in the great majority of experiments, while the simultaneous combination of azi, caz and a gly always produce an additive or synergistic effect against p. aeruginosa. these data, given the high concentration of glys employed, could be of particular interest in clinical situations where the drugs could be topically administered. a multicentre correlation study of vitek 2 with reference methods for telithromycin, mupirocin, and daptomycin against staphylococcus spp. k.e. aldridge, a. dizney, k. thomson, g. procop (new orleans, omaha, cleveland, usa) objective: clinical studies have shown that appropriate antimicrobial therapy based on in vitro susceptibility data correlates with better patient outcome. vitek 2 is a fully automated susceptibility testing methodology providing rapid(6-8 h) results for a variety of aerobic and facultatively anaerobic gram-positive and -negative pathogens which have been shown to correlate to established reference methods. the objective of this study was to correlate the susceptibility results of vitek 2 testing of telithromycin, mupirocin, and daptomycin against staphylococcus spp. with those from reference methodology. methods: three geographically distinct clinical microbiology laboratories participated in the testing with each site testing 100 consecutive clinical islates and 84 blinded challenge isolates of staphylococci sent to each site.vitek 2 testing was performed according to the manufacturer's recommendations. the challenge isolates were also tested using a manual preparation of the inoculum. nccls recommended reference agar and broth microdilution methods were used to test telithromycin, mupirocin, and daptomycin. mic results were collated to determine the per cent (%) of isolates susceptible ( in vitro efficacy of tigecycline tested against community-acquired respiratory tract infection and nosocomial pneumonia pathogens conclusions: among r subsets of commonly occurring pathogens, 97% were inhibited by £2 mg/l of tig and >99% were inhibited by £4 mg/l (the current nccls breakpoint for tet). tig is highly stable to most tet-r determinants, including protected ribosomes and efflux mechanisms, and may represent a superior choice among parenteral agents for broad-spectrum coverage, including the most commonly occurring-and problematic-r phenotypes. determination of the minimum inhibitory and mutant prevention concentration of telithromycin against clinical isolates of streptococcus pneumoniae s. borsos, c. hesje, l. blondeau, j. blondeau saskatoon, can objective: telithromycin (tl) is a novel ketolide antimicrobial agent that has recently been approved for use in north america for respiratory tract infections of which sp is a principal pathogen. the global escalation of penicillin (p) and macrolide (m) resistant sp has compromised the use of beta-lactam and macrolide compounds. tl was reported to be active against p and m resistant sp. objectives: garenoxacin (grn) is an investigational des-f(6)quinolone with enhanced in vitro activity against gram-positive cocci. the global escalation in antimicrobial resistance amongst respiratory tract pathogens -particularly sp-has highlighted the importance of quinolone compounds for treating sp infections. the mutant prevention concentration (mpc) is a novel in vitro measurement that defines the drug concentration threshold that would require an organism to simultaneously acquire two or more resistance mutations for growth in the presence of the drug. we measured the mics and mpcs for grn against clinical isolates of sp, including multidrug resistant strains and those with elevated mpcs to levofloxacingrn. methods: microbroth dilution in accordance with nccls guidelines was followed for mic testing using todd-hewitt broth and two-fold concentration drug increments. for mpc testing, ‡1 billion organisms were applied to agar plates containing grn, incubated at 35-37°c and 5% co 2 and screened for colony growth at 24 and 48 hours. the mpc was the lowest drug concentration that completely prevented growth. select organisms with grn mpc values >2 lg/ml were screened for amino acid substitution in the quinolone resistance determining region. great concern nowadays because the therapeutical alternatives are limited. fusidic acid inhibits protein synthesis. it has only a few side effects and is usually well-tolerated by patients as an oral drug. fusidic acid can be considered as an alternative drug for the treatment of infections due to both methicillin susceptible and resistant s. aureus strains. evaluation of synergy between glycopeptides, levofloxacin and beta-lactams against methicillinresistant staphylococcus aureus (imi) + va/tp, lvx + piperacillin/tazobactam (ptz) + va/tp] were considered. synergy was evaluated by means of checkerboard assay against 50 (double combinations) and 25 (triple combination) mrsa strains isolated from respiratory tract infections and by means of time kill curves (5 strains). in checkerboard assay synergy was defined as fractional inhibitory concentration index (fici) of <0.5, additivity as fici >0.5 and >1, indifference as fici >1 and antagonism as fici >2, while in time kill studies synergy was defined as a >2 log decrease in bacterial count of combinations in respect to the most active single drug. moreover, mutational rates of single and combined drugs at antimicrobial concentrations equal to the resistance breakpoints were calculated in 10 strains results: synergy and additivity were the prevalent effects, while no antagonism was observed in checkerboard assay. in particular, lvx + va/tp and ctx + tp/va gave synergy in 16/50, 9/50, 43/50 and 23/50 strains, respectively. combinations of lvx + va + caz/cpm/ptz and of lvx + tp + caz/cpm/ptz yielded synergy or additivity in all the tested strains, with combinations of lvx + tp + caz / ptz giving synergy in 20/25 strains. time kill curves evidenced synergy for lvx/ctx + va/tp after 12 and 24 h of incubation. synergistic triple combinations occurred more frequently with lvx + tp than with lvx + va after 12 and 24 h. for single antibiotics, mutational frequencies ranged between 10-5 and <10-9 for lvx, ctx, amk and imi, and <10-9 for va and tp. when tested in double and triple combinations, mutational frequencies fell below 10-9 for all the combinations. conclusion: the study provide in vitro evidence of synergy between glycopeptides with fluoroquinolones, cephalosporins and beta-lactams and of limitations of occurrence of mutations in mrsa strains responsible of respiratory infections, thus underlining a potential role in therapy of such infections. comparative activity (mic and mbc) of daptomycin, quinupristin/dalfopristin, linezolid, vancomycin and teicoplanin against a large worldwide collection of visa/hvisa a. bolmströ m, a. engelhardt, å . karlsson, e. edling, p. ho (solna, s) objective: the worldwide emergence and possible dissemination of vancomycin (va) resistance in staphylococci involving vana (vrs), visa and hetero-visa phenotypes comprise formidable clinical threats. comparisons of the in vitro activity of newer gram positive agents using larger worldwide collections of visa strains become increasingly important both for individual therapy guidance and for epidemiologic purposes. method: a 10 country collection of 243 staphylococci (217 mrsa, 26 mssa), of which 150 were visa (e-test macromethod and pap-auc, walsh et al, jcm 2001) was assessed. mic of dp, qd, lz, va and tp were determined using the nccls broth microdilution (bmd) and e-test. mbc for all agents for a subset of 46 visa and non-visa strains were determined using an etest mbc procedure and bmd. results: inter-method agreement (n = 2430) for all antibiotics were ±95% ±1 dilution for mic and ±80% for mbc results. the continuous gradient format in e-test allowed detection of subtle upward shifts in mic and mbc distributions of visa, as well as the detection of persister colonies in mbc assays seen with the less bactericidal compounds. conclusion: mic distributions showed that daptomycin and linezolid maintained potency against the large collection of visa while quinupristin/dalfopristin and glycopeptides showed a shift towards reduced activity. mbc results showed daptomycin to be superior to linezolid (bacteriostatic) and the other agents. the potential usefulness of newer agents for visa should be continually assessed with respect to both their static and cidal activities in order to detect subtle shifts in susceptibility as early as possible. performance of a unique e-test daptomycin gradient + calcium gradient on isosensitest compared to nccls broth microdilution in mueller hinton objective: daptomycin (dp) requires physiologic levels of free calcium ions (ca 2+ ) for expression of adequate activity. susceptibility testing methods can be significantly influenced by ca 2+ variations in agar and broth. nccls broth microdilution (bmd) uses a final physiologic level of 50 lg/ml calcium for dp testing. e-test dp is a unique gradient which incorporates a constant level of calcium. e-test dp has been shown to be equivalent to bmd when tested with commercially available mueller hinton (mh) agar. since isosensitest (iso) is used in some parts of europe, and has an inherently low level of calcium (approximately 5-10 lg/ml), it is important to evaluate the performance of e-test dp on iso in comparison to bmd. method: a 3 lab study compared e-test dp on iso to nccls bmd. a total of 760 clinical and stock strains comprising clindamycin (cc), ciprofloxacin (c) and tetracycline (t) and also to determine whether there are any differences in the antimicrobial susceptibility patters among the difference species of the vgs and s. bovis. methods: the 145 vgs and 10 s. bovis were isolated from blood and identified at species level by id 32-strep system (68 s. mitis, 44 s. anginosus, 25 s. sanguis, 7 s. salivarius and 1 s. mutans). mics were determined by the agar dilution method and the percentages of resistance determined according to nccls criteria for streptococci other than s. pneumoniae. results: overall, 64.5% of the streptococci were resistant at least to one of the antibiotic tested and of these, 32% was resistant to 3 or more. the most susceptible species were s. anginosus and s. salivarius (57% susceptibility to all antibiotics tested). the only s. mutans strain was susceptible to all the antibiotics tested. the resistance phenotypes for the different species are shown in the table. conclusions: as expected cc resistance was always associated with e resistance. overall, the phenotype most frequently founded was ecct in 24 strains (15.5%) and it was present in the 70% of s. bovis and 27% of s. anginosus. the pattern eccpt was the second more common phenotype (12.3%) and the most frequent in s. sanguis and s. mitis with 20% and 17.6% respectively. s. mitis was the specie that showed more number of phenotypes (17). heterogeneity in the susceptibility patterns in the species of vgs indicates the need for accurate identification. in vitro antibacterial activity of temocillin against extended spectrum beta-lactamases enterobacteriaceae producing clinical isolates objectives: temocillin is a methoxy-derivative of ticarcillin, active on enterobacteriaceae and stable against b-lactamases, including ampc and extended spectrum b-lactamases (esbl). however data concerning its activity on esbl producing strains are limited. the aim of this study was describe the range of minimal inhibitory concentration (mic) of temocillin in different species of esbl producing enterobacteriaceae from a single tertiary care centre in belgium . methods: esbl producing enterobacteriaceae (172 e. aerogenes, 164 e. coli, 104 e. cloacae, 58 k. pneumoniae and 35 other ) strains from patients hospitalised in our institution during the period of 1 january 2000 to 31 december 2003. esbl production was screened by double-disk test and confirmed by oxoid combined disk method. characterisation of enzymes was performed by multiplex pcr for bla tem, bla shv and bla ctx-m gene families detection and by dna sequencing and/or iso-electric focusing. mics (mg/l) were determined by agar dilution method according to nccls guidelines. susceptibility was determined according to breakpoints provided by fuchs et al: susceptible mic £ 16 mg/l. (eur j clin microbiol 1985) results: isolates originated from screening rectal swabs (28%), urinary tract (25%), respiratory tract (19%), wound (10%), blood (6%), gastrointestinal tract (4%), or other sites (8%) from 135 male and 233 female patients with a mean age of 61 (range, 0-94) years. the antimicrobial activity of temocillin against esbl producing enterobacteriaceae is summarised in the table. conclusions: this study confirmed the good in vitro activity of temocillin against multiresistant esbl-producing clinical isolates of enterobacteriaceae. its activity included enterobacter ampc depressed mutant strains that co-produced multiple esbl. these data suggest that temocillin could be a valuable drug to be considered in the treatment of infections by some esbl-producing enterobacteriaceae. in vitro activity of ertapenem against cefpodoxime-resistant gram-negative bacilli from urine introduction: resistance to commonly used antibiotics for urinary tract infection (uti) is of growing concern. this is due to the emergence of extended-spectrum beta-lactamase (esbl) producing bacteria in the community with reports from different parts of the world. the appearance and dissemination of these resistant bacteria cause serious concerns for the treatment of urinary tract infections (utis). objectives: ertapenem is the latest member of the carbapenem class of antimicrobials. the aim of this study was to assess the activity of ertapenem against a collection of cefpodoximeresistant gram-negative bacilli isolated from urine samples of community and hospital-based patients. materials and methods: between june and october 2004, our laboratory received 10,234 urine samples. of these 2246 gave a positive culture, of which 446 from hospitalised patients and 1800 from the community. of these 258 were gram negative bacilli resistant to cefpodoxime 1 mg/l. the identity of the isolates was confirmed using api 20e or api 20ne (biomerieux, france). esbl production was sought using the mastdd and the identity of the resistant determinant(s) was carried out using a combination of pcr and nucleotide sequence analysis techdifferent antibiotics in order to evaluate alternatives for intrapartum chemoprophylaxis to women allergic to penicillin and colonized by a gbs strain resistant to macrolides and/or lincosamides; (2) characterize the mechanisms of resistance to macrolides and lincosamide; (3) evaluate the clonal relationship between these strains. methods: a total of 610 strains collected in a multicentre study: 131 isolated between 1997 and 2002 from newborns diagnosed of early-onset gbs disease and 479 strains collected in 2002 from vagina or rectum of pregnant women. microdilution method was used to study the antimicrobial susceptibility and disk diffusion to define the macrolide-lincosamide resistance phenotype. pcr was performed to determine the presence of ermb, ermtr and mefa resistance genes. to evaluate the clonal relationship, pfge of total dna was done using smai. results: all the strains were susceptible to penicillin, ampicillin, vancomycin, quinupristin/dalfopristin, levofloxacin and teicoplanin. the 12.45% were resistant to erythromycin and azithromycin, 11.80% to josamycin, 11.31% to clindamycin, 1.80% to telithromycin and 0.32% to fosfomycin. among the macrolide resistant strains, the 90.8% presented a constitutive mlsb phenotype (cmlsb), 5.26% an inducible mlsb phenotype (imlsb) and the remaining 3.94% a m phenotype. three strains were susceptible to macrolides and resistant to clindamycin. the ermb, ermtr and mefa genes were presented in 63.2%, 30.3% and 3.9% of the macrolide resistant strains. two strains did not amplified none of the studied genes. the 100% of the strains harbouring the ermb gene showed a constitutive phenotype. all the strains showing an inducible phenotype harboured the ermtr gene. the 11 telithromycin resistant strains presented a cmlsb phenotype; of them 10 posses the ermb gene and one the ermtr. conclusion: in spain, there is a high rate of resistance to macrolides and lincosamides that makes mandatory to perform susceptibility testing to gbs strains isolated from pregnant women allergic to penicillin. in our region ermb methilase is the main cause of macrolide resistance, followed by the ermtr and the macrolide eflux pump in a low proportion. there is a wide clonal diversity among resistant strains to macrolides, lincosamides and telithromycin. detection of the ermx determinant of macrolide resistance in clinical strains of corynebacterium urealyticum a. ortiz, j.i. garcía-cía, r. fernández-roblas, j. esteban (madrid, e) objectives: to detect the presence of the ermx macrolide resistance gene in clinical isolates of c. urealyticum. methods: 57 c. urealyticum strains were isolated from clinical samples in the fundació n jiménez díaz hospital. the strains were maintained frozen until the studies to evaluate macrolide resistance were performed. antibiotic susceptibility testing was performed by agar dilution assay in 14 cases and by disc diffusion assay in 43 cases. detection of ermx gene was performed by using primers cerm-1 and cerm-2 according to the protocol described by rosato et al. dna was extracted by boiling a suspension of bacteria in distilled water. amplification products were analysed by agarose gel electrophoresis and molecular weight of the amplicons were calculated by using the photocapt software (biogene, usa). results: 46 strains were resistant to erythromycin and clindamycin, and 11 were susceptible to both antimicrobials. we detected the gen ermx in 34 cases (all of them resistant). 12 macrolide-resistant strains gave negative results for ermx detection. the susceptible strains did not show any amplification product. the ermx gene is detected in 73.9 % of macrolideresistant corynebacterium urealyticum strains. however, there were 26.1 % macrolide-resistant strains in which ermx gene was not detected. other resistance determinants must be involved in macrolide resistance among corynebacterium urealyticum. characterisation of phenotype and genotype of macrolide-resistant streptococcus pyogenes objectives: the resistance of streptococcus pyogenes to macrolide is a world wide problem. the aim of present study was to determine the prevalence of antibiotic resistance rate of erythromycin (em) resistant isolates from korea, and evaluate their genetic mechanism, phenotype distribution and clonal relationship. methods: total 51 em resistant s. pyogenes from clinical specimens from 1996 to 2003 were studied; agar dilution method to determine minimal inhibitory concentrations (mics) to 9 antimicrobial agents was performed. resistance phenotypes were determined by triple-disc test and resistance induction experiment with sub-inhibitory concentration of erythromycin (0.05 lg/ml). their genetic mechanisms of resistance were determined by pcr. the genetic relatedness of them was also investigated by means of emm genotyping and random amplified polymorphic dna (rapd) analysis. results: an average em resistance rate was 23% and their cross-resistance rate to clarithromycin, azithromycin, and spiramycin were 100%, 98%, and 67%, respectively. the most common resistance phenotype was inducible mls (imls; 51%), followed by constitutive mls (cmls;31%), and m type (18%). 18 of 26 imls isolates (69% ) showed novel imlsd phenotype, which had small (9-12 mm) inhibition zones around all three discs on triple-disc test and high level resistance to clinamycin and spiramycin after erythromycin induction. all isolates of the cmlsa and imlsd harboured ermb gene, while imlsc and m isolates harboured erma and mefa gene, respectively. all imlsd isolates were emm12 except one while all multidrug resistance cmlsa isolates were emm28 genotype. imlsd isolates made a tight cluster on phylogenetic tree and 78% of them showed a common pattern by rapd analysis. conclusion: imlsd was most common macrolide resistance phenotype in korea and emm gene and rapd analysis was suggestive of its clonal relationship. detection of inducible clindamycin resistance in cutaneous staphylococci clinical isolates by phenotypic and genotypic method , and 17 (14.5%) isolates with constitutive clindamycin resistance (7 s. aureus and 10 coagulase-negative staphylococci). the mrsa gene was present in all the clindamycin susceptible isolates except for 1 cns. among the 40 isolates with clindamycin inducible resistance, 37 isolates possess either erma or ermc while 1 cns isolate possess both ermc and mrsa. one cns and 1 s. aureus isolates were negative for all the genes tested. all 17 isolates with constitutive clindamycin resistance amplified to any of the genes tested. among the s. aureus isolates, 2 have the erma gene, 4 the ermc and 1 both ermc and mrsa genes. all the cns isolates possess the ermc but 3 of them possess also the mrsa gene (table 1) . the disk-diffusion test detected all the clindamycin resistant strains, and none of the clindamycin inducible resistant strains was detected by the microscan. the ermc gen was the most prevalent among the clindamycin resistant, in both cns and s. aureus isolates. ribosomal protein l22 mutations in bacillus anthracis associated with cross-resistance between macrolide, lincosamide, streptogramin and ketolide antibiotics rob chemother 2004; 54: 424) . the aim of this study was to determine ribosomal mutations associated with this resistance. methods: primers were designed and used to amplify and sequence the l4, l22 riboprotein genes and the 11 copies of the 23s rrna gene using b. anthracis str. ames complete genome (genbank accession no. nc_003997). results: (mics in mg/l) two significant mutations associated with resistance were found. a 4 amino acid (aa) insertion (a tandem duplication of 90mgra93) into the l22 gene at position 94 was found in the st-1 strain with a quinupristin/dalfopristin (q/d) mic of 128 selected after 18 passages in q/d. this strain was cross-resistant to telithromycin, erythromycin, clarithromycin and clindamycin with mics of 16, 64, 16 and 8 respectively. an 8 amino acid insertion (mgramgra) into the l22 gene (also at position 94) was found in the sterne strain with a clarithromycin mic of 64 after 18 passages in clarithromycin. this strain was cross-resistant to telithromycin, q/d, erythromycin and clindamycin with mics of 16, 8, 32 and 1 respectively. conclusions: tandem duplications of l22 90mgra93 were found to be important in mlsk resistance in 2 separate strains of b. anthracis demonstrating that this locus is important for resistance development under selective pressure of q/d and clarithromycin. importantly, these mutations were also associated with cross-resistance to other mlsk antibiotics. horizontal transfer of the mlsb resistance gene erm(b) between the human pathogen clostridium difficile and the ruminal anaerobe butyrivibrio fibrisolvens p. mastrantonio, f. barbanti, p. spigaglia (rome, i) objectives: the aim of this study was to investigate the possibility of in vitro transfer of the mlsb resistance gene, erm(b) between the human pathogen clostridium difficile and the ruminal commensal butyrivibrio fibrisolvens. methods: the erm(b)-positive c. difficile strain cd51 was used in filter matings as donor, whereasthe b. fibrisolvens strain 1.230, tetracycline resistant, and the b. fibrisolvens strain 2221r, rifampicin resistant, were used as recipients. the strains were cultured in m2gsc broth, with 30% of sterile rumen, in anaerobic conditions and the filter matings were performed on sheep blood agar plates, supplemented with hemin (0.1%) and vitamin k (0.1%). the same medium, supplemented with erythromycin (20 mcg/ml) and tetracycline (10 mcg/ml), were used to select the transconjugants. mic values were assessed by e-tests. the erm(b) transfer was confirmed by pcr and by hybridisation assays, using an erm(b) probe on transconjugant genomes digested with pvuii and with smai (pfge). results: the transfer average frequency was 5 · 10 )7 . mic values for erythromycin were >256 mg/l both in donor and transconjugants. an internal fragment of the erm(b) gene (730 bp) was amplified in all the transconjugants and a specific band was visualised in hybridisation assays on transconjugant genomes digested with both pvuii and smai. the re-transfer of the erm(b) gene from b. fibrisolvens transconjugants to c. difficile 1977, an erythromycin susceptible strain, was also obtained. conclusion: the in vitro transfer of the mlsb resistance gene erm(b) from c. difficile to b. fibrisolvens and vice-versa, is an important result supporting the possibility that horizontal transfer of resistant genes between human pathogenic bacteria and animal commensal microorganisms could occur, under natural conditions, more easily than expected. candida spp. attach on polymers, create a biofilm protecting the yeast and pose a reservoir for entering the bloodstream. candida spp. in biofilm are less susceptible to the antifungal drugs currently in use. we investigated the in vitro antifungal activity of micafungin (mcf) against biofilms of c. albicans, c. parapsilosis, c. dubliniensis, c. tropicalis, c. glabrata and c. kefyr. methods: biofilm formation was performed in vitro on polystyrene 96-well plates and on hickman catheter discs with minor modifications to kuhn (1). mcf was tested at six concentrations (0.25-32 lg/ml). measurements were done by xtt reduction assay at 490nm, % inhibition was calculated in comparison to the growth control. the biphasic structure of biofilms were imaged by reverse light-through microscopy. results: on polystyrene plates and on hickman catheters all candida spp. produced intermediate biofilm after 24-48h incubation. at all concentrations on polystyrene plates micafungin reached at maximum 50% inhibition in biofilm against all candida spp.: c. albicans (47% at 16 lg/ml), c. parapsilosis (49% at 16 lg/ml), c. dubliniensis (42% at 16 lg/ml), c. tropicalis (28% at 16 lg/ml), except for c. glabrata (73% at 8 lg/ml) and c. kefyr (53% at 1 lg/ml) methods: this multicentre, randomized, double-blind study compared mem with ipm (both 500 mg iv every 8 hours) in patients hospitalized with csssi. the primary efficacy endpoints were clinical and bacteriological responses at follow-up in the fully evaluable (fe) patient population (all patients meeting eligibility criteria and who had an identified pathogen prior to treatment) polymicrobial infections were seen in 38% of all documented infection s. agalactiae (n = 59; 100% s to mem and ipm), and e. faecalis (n = 52; 82% s to mem and 100% s to ipm) pseudomonas aeruginosa (n = 53; 94% s to mem and 92% s to ipm), bacteroides spp. (n = 74; 97% s to mem and 100% s to ipm), and peptostreptococcus spp 3% (mem) and 81.2% (ipm) in patients with polymicrobial infections; bacteriological (documented or presumed eradication, colonization) response rates were 79 we used a cox proportional hazard (cph) model adjusting for potential clinical and microbiologic risk factors, to evaluate los. results: among the 1116 modified intent-to-treat patients, the most common infection diagnoses at baseline were cellulitis (58.9%) and major abscess (27.9%). the most common underlying etiologies were spontaneous infection (52.3%) and trauma (27.6%). about 20.2% of patients had diabetes; 6.9% had peripheral vascular disease. staphylococcus aureus was the most common causative pathogen (about 50% of patients) among 540 patients with confirmed microbiology and complete hospitalization data. however, about 18% of patients had a gramnegative causative pathogen among 540 patients with confirmed microbiology, staphylococcus aureus was the most common causative pathogen but varied significantly in prevalence across regions (about 50% of patients overall, 82% in the us, 67 to 68% in europe and asia, and 39% in south africa). about assigned for the drugs as follows: row a for cip alone, b for second drug, c for third drug, d for cip + second drug, e for cip + third drug, f for second drug + third drug and g for the three drugs together. row h was left for drug-free control. briefly, all wells were filled with 50 ll of cation adjusted muller hinton broth. the drugs at 2x of the highest tested concentrations in 50 ll of the medium (alone, in double or in triple combinations) were delivered to wells a1-g1. two-fold serial drugs. results: the best synergistic effects were observed in combination of amk with caz (80%) or pip (80%) and in combination of caz with pip (60%) or cip (60%) as quinolones are widely used for treating sp infections and quinolone resistance is a concern, we tested gm-a newly approved compound-by mic and mpc against ps and prsp. methods: mic testing was by microbroth dilution in accordance with nccls guidelines. for mpc testing, 10 billion organisms were applied to agar plates containing drug and read at 24 and 48 hours following incubation. the lowest concentration preventing any growth was the mpc. organisms with high mpc values ( ‡2 lg/ml) were screened for amino acid substitution in the quinolone resistance determining region. results: a total of 402 clinical sp isolates were tested: 320 ps, 69 penicillin intermediate (pi) and 13 pr. the following represents the mic50/90 (lg/ml) and range (lg/ml) respectively against pr tr) objective: to determine the in-vitro activities of moxifloxacin and ciprofloxacin against methicillin-susceptible s. aureus (mssa) and methicillin-resistant s. aureus (mrsa) strains. methods: the study was conducted in a university hospital in turkey. a total of 440 non-repeat isolates (196 mssa and 244 mrsa) from various clinical specimens were included in the study. all isolates were identified as s. aureus by phenotypic characteristics, gram staining, catalase and tube coagulase tests. mics of the drugs were determined by an agar dilution method, according to the nccls criteria. the drugs were obtained from their manufacturer (bayer inc). s. aureus atcc 29213 was used as quality control strain. results: twenty-seven (14%) of 196 mssa strains and 239 (98%) of 244 mrsa strains were resistant to ciprofloxacin. mic50 and mic90 values of 27 ciprofloxacin-resistant mssa strains for moxifloxacin were 0.25 mg/l and 4 mg/l, respectively. mic50 and mic90 values of ciprofloxacin-susceptible mssa strains for moxifloxacin were £0.125 mg/l and 0.250 mg/l, respectively. moxifloxacin was 4-fold more active than ciprofloxacin against mssa strains. there was not any difference between these two quinolones against mrsa strains (mic90s: ‡16 mg/l). conclusion: mrsa strains are major causes of nosocomial infections. despite advances in antibacterial therapy, treatment of infections caused by mrsa is still troublesome. new quinolone-derived compounds such as moxifloxacin are reported to have improved activities against s. aureus including mrsa. in this first study on in-vitro activity of moxifloxacin against s. aureus from turkey, the moxifloxacin mics were higher than those reported from different countries objectives: a multicentric study during the winter period of 2003-2004 was performed in belgium to assess the in-vitro two mutations in 15 (34.8%) (3 parc+gyra and 13 parc+pare), and 3 mutations in parc+pare+gyra in 5 (11.6%) strains. conclusions: 1) levofloxacin mic50 and mic90 for pneumococcal strains with decreased susceptibility to ciprofloxacin were 0.5 and 1 mg/l, respectively. resistance to levofloxacin (mic ‡ 8) occurred in 32 isolates (1.2% of the sauce-3 collection). 2) activity of levofloxacin was negatively affected only among pneumococcal isolates with a very high mic of ciprofloxacin ( ‡8 mg/l). in this case, levofloxacin mic50 and mic90 were 4 and 8, respectively, and 44.4% of these strains were resistant to levofloxacin. 3) all sequenced strains with a levofloxacin mic ‡ 4 mg/l had a mutation in the parc gene (single in 56% strains), but none of them in the gyrb. the most common mutations identified were parc/s79f, pare/i460v, parc/s79y and gyra/e85k. 4) one single parc mutation was organism/year (no. tested) gemi cipro levo gati moxi 008/2 (na) methods: the organisms numbered 9,347, a total of 2,004 from np and 7,343 from carti. the most frequent carti pathogens were: s. pneumoniae (spn; 2,865), h. influenzae (hi; 4,011) and m. catarrhalis (mcat; 607) usa) objective: to evaluate the activity and potency of tigecycline (tig) when tested against a large international collection of common bacterial pathogens displaying increasing and worrisome resistance (r) profiles. tig is a novel glycylcycline derivative of minocycline that has demonstrated activity against a variety of gram-positive and -negative pathogens mic 50/90 (mg/l) 275 strains) were collected from 2000 to 2004 in >70 medical centres participating in the global tig surveillance program results: tig results for the r organism subsets are in the table: tig was highly active (mic50 and 90, £0.25 and £0.5 mg/l, respectively) against all resistant sa, cons, esp, spn and vgs with >99% of strains inhibited by £2 mg/l. while potency of tig against r subsets of ent was less (mic50 and 90, £0.5 and £4 mg/l, respectively), the vast majority of tet-r isolates remained s to tig (>98% of isolates were inhibited by £4 mg/l). no geographic differences in tig potency among esbl-producing, cip-r or tet-r ent strains were noted. grn drug concentrations in serum would be expected to remain above the mpc90 value for >24 hours. conclusion: grn exhibits potent activity against sp, as measured by both mic and mpc, and is active against strains with high mic/mpc values for other quinolones. grn is less likely to select for quinolone resistant sp isolates s. agalactiae (119) and s. dysagalactiae (30) were used. interlaboratory reproducibility was evaluated using 25 bias/precision strains tested at all sites and 3 nccls qc strains were used to compare e-test dp on iso values to nccls mh values. the various methods were used according to standard recommendations. nccls susceptible breakpoints ‡1 lg/ml for staphylococci and streptococci and ‡4 lg/ml for enterococci were used as is and as adjusted upwards by 1 dilution. results: percentage essential agreement (ea), category agreement methods: e test strips were used to establish the fici of the antibiotic combination pairs. combination testing was carried out on 125 strains of pseudomonas aeruginosa (pa), 69 strains of burkholderia cepacia (bc) and 29 strains of stenotrophomonas maltophilia (sm) from 96 cf patients. 19, 13 and 6 combination pairs were tested between 10 and 69 times against pa, bc and sm respectively. the fici results were categorised as synergistic (fici £ 0.5), additive (fici = 0.5 to £1.0), indifferent (fici = 1.0 to £2.0) and antagonistic (fici > 2.0). results: 1107 combinations were tested. synergy was found in 3.1% of pa, 9.7% of bc and 8.6% of sm. 31.6%, 32.6% and 25.8% of pa, bc and sm respectively demonstrated an additive effect. 64.2% of pa, 54.5% of bc and 62.4% of sm demonstrated an indifferent effect. antagonism was found in 1.1%, 3.2% and 3.2% of pa, bc and sm respectively results: of the 258 isolates resistant to cefpodoxime, 83 were positive for esbl production and the enzymes produced were: tem-1, shv-5, shv-12, ctx-m-15, ctx-m3 and ctx-m9 singly or in combination. ertapenem proved active against all enterobacteriaceae regardless of production of esbls (mic range 0.003-0.125 mg/l). for acinetobacter sp giamarellou on behalf of the hellenic study group for the susceptibility of streptococcus pneumoniae results: of the 275 strains studied, 76 were ery s and 159 were ery r. all strains but two (mic 4 and 8 mg/l) were susceptible to tel. in particular, results for telithromycin for the ery s strains, showed an mic range for tel between 0.008-0.5, with mic50 of 0.015 and mic90 of 0.06. for ery r in total, mic50 and mic90 were 0.125 and 0.5 (range 0.008-8). with respect to macrolide resistance phenotype, the distribution per type was: m 54.5% (ery mic range 1-32), and cmlsb and imlsb (ery mic range 4 to (256) 39% and 6.5% respectively. the two strains resistant to tel displayed the cmlsb phenotype (ery mic ( 256). for each different macrolide resistance phenotype no isolates demonstrated an inducible mlsb phenotype by this method. forty-nine of these carried the ermam gene (34.3%) and three carried both ermam and mefe genes (2.18%). eighty-seven isolates (63.5%) had the m phenotype (macrolide resistance) and all of these contained the mefe gene. conclusions: our findings are in contrast to european studies and to previous greek studies, where the erm gene prevailed or at least equaled the prevalence of mef gene. this is the first report of isolates carrying both ermam and mef genes in greece. knowledge of the resistant mechanisms to macrolides is crucial for the empiric antibiotic treatment of community acquired infections. p1225 antibiotic susceptibility, mechanisms of macrolide resistance and clonal relationship in group b streptococci microbiological characteristics of anthrax strains l. lukhnova, g. temiraliyeva, t. meka-mechenko, y. pazylov, s. zakaryan, j. denissov (almaty, kaz)objectives: the kazakh science center for quarantine and zoonotic diseases (kscqzd) has a collection of pathogens of especially dangerous infections that have been isolated in kazakhstan. by kazakh government resolution, the kscqzd's collection is the official collection of pathogenic bacteria for kazakhstan. kscqzd has 71 strains of b. anthracis that were isolated in various years . methods: we studied the biological properties of 30 anthrax strains from our collection: 16 from humans, 4 from soil, 5 from sheepskins, 2 from sheep organs, and 3 from the external environment. we tested the anthrax strains using standard identification tests. results: the growth of the bacillus anthracis strains on the hottinger's agar was typical (r -form), and no distinctions based on the origin of the strains were noted. all of the strains form spores and are immobile. when stained with specific antibody directed toward anthrax antigens, all strains stained positive. three of the b. anthracis strains did not have capsules when originally studied, but after 7 cultures on the hottinger's agar, the strains' ability to form capsules in a co 2 atmosphere was restored. the anthrax strains are sensitive to anthrax bacteriophage and penicillin. the strains have lipolytic and background: we studied the application of pk/pd modeling techniques as an alternative method to define pharmacodynamic breakpoints complementary to conventional microbiological breakpoints for fluoroquinolones (fqs). methods: 8 staphylococcus aureus strains with mics ranging from 0.03-8 mg/l were exposed to fluctuating concentrations of levo-(lfx) gati-(gfx) and moxifloxacin (mfx) mimicking their total serum concentration profiles following oral doses of 400 mg mfx, 400 mg gfx, 500 and 750 mg lfx over 24 h using the sedimentation model. strains were cultivated in brain heart infusion broth at 37°c. viable counts were quantitated at 14 sampling points. as pd-measure the aubkcnorm (i.e. area under the bacterial kill curve normalized to the initial inoculum [h] ; aubkcnorm <24 indicates a bactericidal activity, >24 bacterial growth) was determined for the time kill experiment and plotted as a function of mic for each treatment. results: the aubkcnorm vs mic function was similar for all treatments following a bimodal profile. while the slope of all fqs studied was shallow for mics below 1 mg/l with au-bkcnorm <5 h, aubkcnorm started increasing steeply beyond this point. it intercepted the 24 cut off line at concentrations of 1.5 mg/l for lfx 500 mg, 2 mg/l for gfx, 2.4 mg/l for mfx and 3.1 mg/l for lfx 750 mg. this indicates that s. aureus with susceptibilites beyond these mics would no longer be eradicated by the corresponding treatments. thus, according to our investigations a breakpoint for fqs integrating both microbiological and clinical pharmacokinetic relation-ships results in a value of 1 mg/l for lfx 500 mg, and 2 mg/l for mfx, gfx, and lfx 750 mg. conclusion: pharmacodynamic breakpoints determined by pk/pd m+s which are taking into respect the fluctuating concentration time profiles encountered in patients are suitable surrogates complementing the data obtained in microbiological experiments. a multicentre agar and broth dilution susceptibility testing study of fluoroquinolones against the bacteroides fragilis group k.e. aldridge, d. snydman, d. hecht, c. edlund, j. herrington (new orleans, boston, chicago, usa; stockholm, s; west haven, usa)objectives: the development of fluoroquinolones (fq) antimicrobials has resulted in effective therapy for a variety of infections due to aerobic and facultatively anaerobic grampositive and -negative pathogens. fq activity against anaerobes has not been as promising with only trovafloxacin receiving fda approval for anaerobic infections. moreover, more recent reports of increased resistance to fq among the b. fragilis group has prompted interested to determine if these increases are due to susceptibility testing methodology problems. this study was designed to compare the susceptibility results from five laboratories that tested the same 70 isolates against fq using agar dilution(ad) and broth microdilution (bmd) testing. methods: seventy clinical isolates of the b. fragilis group identified by only a sequential number were distributed to each of the five participating laboratories. four of the laboratories performed ad and one laboratory performed bmd susceptibility testing using nccls recommendations including breakpoints where appropriate. serial two-fold dilutions of moxifloxacin (mox), gatifloxacin (gate), trovafloxacin (trov), and metronidzaole (metro) were prepared in the test medium within a range of 0.03 to 64 mg/l. plates were inoculated with 1 · 10 5 cfu per spot or well, incubated anaerobically for 48 hours and the mic read. qc was performed using nccls-recommended atcc strains. results: mic values for each antimicrobial were collated as mic90 and per cent (%) of isolates inhibited at £2 and £4 mg/l: these data indicate that inter-laboratory testing of the same test isolates gave very similar results for each antimicrobial. bmd gave slightly lower mic results than ad but was not significantly different. we conclude that susceptibility methodology is not responsible for unusually high resistance rates. global evaluation of gemifloxacin activity tested against community-acquired respiratory tract pathogens, haemophilus influenzae and moraxella catarrhalis (1999) (2000) (2001) (2002) (2003) (2004) fluoroquinolones are a therapeutic option in treatment of infections caused by non-fermentative gram-negative bacteria.however, resistance to these antibiotics rapidly increase in clinical isolates. the efflux mechanism is the most common cause of multidrug resistance of strains p. aeruginosa, a. baumanii and s. maltophilia. these mdr pumps belonging to the rnd family are inhibited by phe-arg-beta-naphthylamide [mc207, 110] . however, different results of reserpine effect on the activity of rnd pumps was established. in our study, we searched for effect of the efflux pump inhibitors (mc207,110, reserpine and rescinnamine) on the mic of quinolones for mdr clinical isolates of p. aeruginosa, a. baumanii and s. maltophilia. we looked for a interdependence between structure of quinolones and ability of tested inhibitors to inhibit efflux of these antibiotics. results: the mdr strains were resistant to all tested quinolones (nalidixic acid-nal, pipemidic acid-kp, norfloxacin-nor, ciprofloxacin-cip and ofloxacin-of). among studied inhibitors only mc207,110 affected the susceptibility of all tested strains to quinolones, first of all to nal. effect of mc207,110 on the mic depended on its concentration, but did not depend on the temperature. for majority of p. aeruginosa and a. baumanii strains the mic of all quinolones decreased in the presence of mc207,110. the highest decrease of the mic of quinolones was obtained for p. aeruginosa (4-512-fold). for a. baumanii strains the mic of kp, nor, cip and of decreased only from 2-to 4-fold but the mic of nal decreased to 16-fold. moreover, the presence of this inhibitor increased the sensitivity of 80% of s. maltophilia only to nal (4-to 16-fold). reserpine and rescinnamine did not effect on the mic of quinolones for all studied mdr strains. however, reserpine alkaloides showed inhibitors activity against control strains of s. aureus (decrease mic of nor). conclusions: our data confirm that mc207,110 inhibited efflux pumps not only in p. aeruginosa but also in a. baumanii and s. maltophilia strains. additionally, obtained results suggested possibility that for quinolones, depending on their structure, are different binding sites in efflux pumps. furthermore, probably also mc207,110 binds to specific site of pumps depending on the efflux systems. so, the effect of this inhibitor on mic of quinolones depends on genus of bacteria, kind of pumps and structure of agents. in vitro activity of fucidic acid against staphylococcus aureus strains objectives: viridans group streptococci (vgs) resistant to penicillin, macrolides and other antimicrobial are increasingly found. linezolid and quinupristin-dalfopristin exert their mechanism of action by protein synthesis inhibition and are agents with activity against multidrug resistant gram-positive cocci. this study was aimed to determine, by time-kill curves, the activity of linezolid and quinupristin-dalfopristin against five vgs expressing various degrees of resistance to erythromycin and harbouring different erythromycin resistance determinants methods: the species, erythromycin mics (mg/l) and the erythromycin resistance genes present for each of the five isolates tested were: s. anginosus/0.06/none, s. sanguis/4/mef(a), s. sanguis/64/erm(b), s. mitis/256/erm(b) +mef(a), s. anginosus/256/erm(b) +mef(a). quinupristin-dalfopristin and linezolid mics for all strains were 1 and 0.5 mg/l respectively. strains were incubated in cation-adjusted mueller-hinton broth supplemented with 5% lysed horse blood. each of the five isolates were exposed to linezolid and quinupristin-dalfopristin for 24 hours in a shacking water bath in presence of 2 · mic. colony counts were performed at 0, 4, 8 and 24 hours. results: time-kill studies showed that for linezolid, the antibiotic concentration of 2 · mic was bacteriostatic for each strain irrespectively of the resistance to erythromycin and the genes harboured, showing a 90% of killing at 24 h. the in vitro activity of quinupristin-dalfopristin was predominantly bactericidal with a 99.9% of killing at 3 h. the exception were the 2 strains with erm(b) + mef(a) determinants, in one of them, a s. milleri, the bactericidal activity was delayed to 24 h, and in the other strain, a s. mitis, the activity was bactericidal at 3 h but with regrowth at 24 h. conclusions: when vgs are implicated in infections in neutropenic patients and in cases of endocarditis where bactericidal activity is usually considered mandatory, we have to considered that: linezolid was bacteriostatic, at the concentration tested, against all strains and the rapid killing of quinupristin-dalfopristin against vgs was lost when the strain presented the erm(b) and mef(a) genes together. comparative antimicrobial susceptibility patterns in different groups of species of viridans group streptococci and streptococcus bovis isolated fom blood cultures i. rodriguez-avial, c. rodriguez-avial, j.j picazo (madrid, e)objectives: viridans group streptococci (vgs) and s. bovis are part of the normal human microbiota and are implicated as a cause of endocarditis and sepsis in neutropenic patients. because of their resident nature are frequently exposed to the antimicrobial agents. this study was performed to study the resistance phenotypes to penicillin (p), erythromycin (e), key: cord-019347-tj3ye1mx authors: nan title: abstract book date: 2010-02-19 journal: ann allergy asthma immunol doi: 10.1016/s1081-1206(10)61294-x sha: doc_id: 19347 cord_uid: tj3ye1mx nan introduction: the effect of anti ige has been described as a function of complexing free ige to reduce mast cell implantation and consequent reduction of mast cell degranulation upon exposure to antigen. theoretically, as total free ige drops below 10 ng/ml, improvement occurs as free and cell bound ige equilibrate and allergic reactions subside. initial observations ( togias a et al. j allergy clin immunol. 1998 ;101:s171)suggested that prick skin tests consistently reached their low point at 98 days with significant reduction in size. laynadier (leynadier f, doudou o, gaouar h, le gros v, bourdeix i, guyomarch-cocco l, trunet p : effect of omalizumab in health care workers with occupational latex allergy.j allergy clin immunol 2004;113:360-1) noted some reduction in skin testing in sixty percent of patients treated over a six month period with monoclonal antiige. but clinicians since approval of the drug in 2002 have noted that even in the presence of good clinical response to asthma, skin test reactivity is still obvious, and in many cases unchanged. methods: following the suggestions for standard clinical follow up by lanier and marshall ( annals may 2003) , routine skin prick skin testing was completed after informed consent on 24 patients at onset of anti-ige therapy, and repeated at 6-7 months. in all instances were skin tests done and photographed in a standard manner. in a few cases, skin tests were repeated on patients receiving anti ige for as long as five years. results: photographic evidence will be presented. in 22 of 24 patients on current therapy, prick skin testing remained at an almost identical photographic level to the baseline analysis, but there was a correlation between the patients with the lowest serum ige and the likelihood of apparent reduction. there was no correlation to reduction of skin testing and clinical response to anti ige since all 24 patients had experience significant quality of life improvements. conclusion: anti ige has variable effects on reducing prick skin testing, with a greater likely hood associated with low or extremely low initial total ige levels. skin testing is more sensitive for detection of allergen-specific ige than is immunoassay for allergen-specific ige. for some assay methods, the qualitative cutoff of 0.35 ku/l is inappropriately high. this study was conducted in order to determine whether a particular assay system can be used to measure lower levels of allergen-specific ige. the pharmacia unicap system was studied. all reagents were purchased from the manufacturer, and the study was approved by a local human assurance committee. to determine background of the calibration curve, the fluorescent unit (fu) response of 6 replicates of assay diluent measured with an anti-ige solid phase was determined. the background responses of 7 arbitrarily selected allergen solid phases (oak, timothy and short ragweed pollen; cat; peanut; yellow jacket venom; penicillin g) were also measured. lower detection limits (lld) were calculated using the mean background measurement + 3 standard deviations (sd). to examine linearity of a low-range calibration curve, 1:2 dilutions to extinction were made, starting with the 3.5 ku/l calibrator. the mean background of the anti-ige solid phase was 15.4 fu, sd 2.7 and coefficient of variation (cv) of 18%; the lld was 24. this lld corresponded to a level of approximately 0.01 ku/l of ige. the lld of the allergen solid phases ranged from 17.2 fu (yellow jacket venom) to 71.6 fu (short ragweed); this corresponded to about 0.08 ku/l ige. the low level dilution curve exhibited parallelism with a curve constructed using a zero (diluent) calibrator as a 7th point in the assay's reference curve. a low level method is capable of measuring specific ige levels lower than the manufacturer's 0.35 ku/l cutoff. this would seem to be particularly important in research applications, and in the analysis of certain highrisk allergens. the clinical significance of very low levels of specific ige in the serum warrants study. allergists/immunologists are often consulted on patients with rashes or recurrent infections. the differential diagnosis can comprise a variety of disorders, some of which are rare syndromes that require early diagnosis and specific management. a 3 mo old wm developed persistent rash and worsening eczema. later, he had recurrent severe skin infections, skin abscesses, recurrent sinusitis, and chronic otitis media that required multiple placement of tympanostomy tubes, yet had a persistent tympanic membrane perforation. recurrent wheezing was noted from 3-7 yr of age. he had several episodes of pneumonia since 3 yr of age affecting different lobes. he failed to shed the primary teeth and had history of periodontitis and oral thrush. he developed scoliosis and multiple fractures of the upper extremities and ribs. at 12 yr he had staphylococcal bacteremia and osteomyelitis of the acetabulum. in spite of antibiotics prophylaxis, he continued to have recurrent skin infections and lymphadenitis. laboratory evaluation revealed eosinophilia (up to 3138/mm3), normal levels of igg, iga, igm & igg subclasses, except for decreased igg2 (51 mg/dl). he had protective levels of anti s. pneumoniae and h. influenzae, but low anti-tetanus and anti-diphtheria titers. at 4 yr, total ige level was 42, 500 iu/ml which supported the diagnosis of hyper ige (hie) syndrome; it decreased to 197 iu/ml by 12 yr. rast was positive to hd mite, cat, dog, egg, milk, and pollens. flow cytomerty, nbt and phagocytic index were normal; ch50 was low (66 mg/dl). dexa scan showed osteopenia. chest x-ray showed bilateral infiltrate, atelectasis and scoliosis of the thoracic spine. during his hospitalization at 13 yr, his skin was fair, lichenified with widespread maculopapular erythematous rash. the palms and fingers showed open cracks and pustules, but no weeping lesions. in addition to coarse facial features, his face was eczematous. conclusion: hie syndrome is an autosomal dominant disorder that mimics atopic dermatitis but has severe course, multiple infections, and several complications. although a markedly elevated total ige level introduction: mannose-binding lectin (mbl) is a serum protein in the lectin complement pathway. it is important in innate immunity, and is thought to be particularly relevant in young children during the development of adaptive immunity. deficiency in mbl has been reported in population-based studies as a risk factor in children for infection and hospitalization. additionally, age-dependent variability of mbl has been previously noted. this study evaluates the prevalence of mbl deficiency in children with established recurrent infection who were referred for evaluation of immunodeficiency. method: we prospectively evaluated mbl status of children referred for recurrent infection. serum was collected from october 2002 to september 2003. children with known primary or secondary immunodeficiencies were excluded. mbl analysis was performed by standardized elisa using mbl oligomer assays. we performed chart and laboratory review for comorbid diagnoses, quantitative immunoglobulin levels and subclasses, and complement function with ch50 and ah50. results: two-hundred thirty five children were evaluated. mean age was 4.5 years (range 0.08-17 years). mean mbl was 1780 ng/ml (range 17-4806 ng/ml). thirty-one of 235 children (13.1%) were mbl deficient, levels <200, and among this group the mean mbl level was 61.3 ng/ml (range 17-163 ng/ml). mean age in this group was 3.6 years (range 0.3-16 years). of the children with mbl deficiency, 13 (5.1%) children had levels <50. the m:f ratio of children with abnormal mbl levels was 1.08. linear regression analysis showed no correlation of mbl level with age. comorbid diagnoses were variable, including asthma, allergy, and atopic dermatitis. none of the children had symptoms compatible with connective tissue disease. no child with an abnormal mbl level was found to have significant deficiency of igg, iga, or igm. several children had low igg4, which were within normal physiologic range. no other concomitant complement pathway disorders were detected. conclusions: our study did not reveal any correlation between mbl level and age. in children with recurrent infections, mbl deficiency was approximately twice the estimated rate of the general population. contrary to previous studies, no other significant immunologic disorders were identified. therefore, mbl deficiency alone is a risk factor for infection in children. t.b. fausnight * , hershey, pa. introduction: the incidence of perioperative anaphylaxis is estimated to be between 1 in 10, 000 and 1 in 20, 000 procedures. approximately 60% of cases of perioperative anaphylaxis are attributed to neuromuscular agents. very little information is reported in the literature regarding pediatric perioperative anaphylaxis. i describe a pediatric patient with suspected perioperative anaphylaxis to rocuronium. methods: a 10 year-old girl with a history of sacral agenesis and neurogenic bladder was scheduled to have bladder augmentation surgery. the patient was taken to a latex-free operating room. during induction of general anesthesia, she was found to be difficult to ventilate. she also became hypotensive. examination of the patient revealed urticaria on her right arm. she was given epinephrine, diphenhydramine, and dexamethasone. the procedure was aborted. the reaction was believed to be from either rocuronium or propofol. results: because of the high incidence of anaphylaxis to neuromuscular agents, allergy skin testing was performed for rocuronium, vecuronium, and succinylcholine. the patient had negative percutaneous skin tests (1:10) for rocuronium, vecuronium, and succinylcholine. she had a negative intradermal skin test to rocuronium at the 1:1000 dilution. she had a positive intradermal skin test to rocuronium at the 1:100 dilution. she had negative intradermal skin tests to both vecuronium and succinylcholine at the 1:1000, 1:100, and 1:10 dilutions. she underwent surgery 2 weeks after skin testing. she received a test dose of vecuronium and had no reaction. she received 3 doses of vecuronium and multiple doses of morphine without any adverse reactions. propofol was avoided. the surgery was completed without difficulty conclusions: this case illustrates the usefulness of skin testing for neuromuscular agents in a pediatric patient. introduction: extrapulmonary pneumocystis jiroveci infection is rare in non-hiv infected individuals and, to our knowledge, it has not been reported before in a patient with good's syndrome. we report a case of extrapulmonary pneumocystis in a patient with good's syndrome. case report: a 44-year-old african american male patient with a history of good's syndrome presented with left flank pain of 3 months duration. the pain was constant and associated with night sweats, fever, and chills. furthermore, the patient also reported a 20-pound weight loss. on exam, patient was found to have multiple hypopigmented areas over his abdomen and left lower extremity, bitemporal wasting, sunken eyeballs, and oral thrush. the patient was also found to have left costovertebral angle tenderness with a palpable solid fixed mass along the left mid axillary line overlying the splenic area. computedtomography (ct) scan of the chest and abdomen showed a 2.2 x 2.9 cm soft tissue mass at the left lateral aspect of the t11 vertebral body and 7.0 x 5.2 cm soft tissue parasplenic mass. the parasplenic mass involved the inferior anterior aspect of the left 11th and 12th ribs (see figure) . the giemsastained biopsy of the parasplenic mass revealed pneumocystis jiroveci and was confirmed using immunohistochemical stain with monoclonal antipnumocystis antibodies. the patient's symptoms of night sweats and loss of appetite improved within 48 hours following initiation of trimethoprim-sulfamethoxazole therapy. a ct of the chest and abdomen, repeated six months post treatment, confirmed the resolution of both the paravertebral and the parasplenic masses. conclusion: to our knowledge, this is the first case of extrapulmonary pneumocystis presenting in a patient with good's syndrome. although it is rare, extrapulmonary pneumocystis should be considered in the differential diagnosis of patients with good's syndrome and chest or abdominal mass. introduction: t cells are regulated by cellular interactions with the environment during development. they play a critical role in the regulation and development of autoimmune diseases. we report inflammatory myositis in a 10-year-old caucasian female with primary t cell-deficiency since infancy. methods: at nine months of age, our patient developed lymphoid interstitial pneumonitis. immunologic evaluation revealed isolated t-cell deficiency. during the first 3 years of life, she had failure to thrive, recurrent sinusitis, oral candidiasis, fungal skin infections, and pseudomonas pneumonia. at 5 years, autoimmune conditions, characterized by a purpuric rash over the nose; face and exposure area of elbows; knees and fingers; raynaud's phenomenon; and nasal septum perforation, developed. inflammatory markers were persistently elevated and autoantibodies detected. by age 9, she developed proximal muscle weakness with distal joint contractures. dry gangrene of the fingers from a thrombotic incident occurred. results: serial immunologic evaluation revealed low cd4 9-36%, # 153-335 (normal 36-61%, # 900-2860 cell/mm3), low cd8 5-23%, #142-237 (normal 16-34%, # 630-1910 cell/mm3) , decreased lymphocyte transformation to antigens but appropriate to mitogens. igg 1320-2560 (normal 667-1179 mg/dl); iga 470-780 (normal 79-169 mg/dl), and igm 215-340, (normal 40-90 mg/dl) with appropriate antibody responses to vaccine antigens. bone marrow and thymus biopsies were normal. further evaluation was not consistent with known primary or secondary immunodeficiencies. several skin biopsies revealed nonspecific dermatitis without vasculitis. mri of lower extremities showed abnormal signals involving bilateral muscles of thighs and calves. muscle biopsy revealed inflammatory myositis with plasma cell predominance, inconsistent with dermatomyositis or polymyositis. aldolase 8-10 iu/l (< 5), crp 8-16 mg/dl (< 0.5), esr 50-70 (< 20), rf 1:1280 (< 1:40) and type ii collagen ab 26-35 ei/ml (< 20) . ana, anca and ace were within normal range. antibodies for myositis and myositis related-antibodies were negative. diagnosis of nonspecific plasma-cell inflammatory myositis was made. a trial of monthly ivig 2 gm/kg resulted in clinical improvement. conclusion: this novel plasma cell myositis occurring in a child with primary t cell deficiency underscores the role t cells play in the development of autoimmune diseases. introduction the incidence of hypersensitivity reactions (hr) is increased in patients treated with multiple courses of carboplatin. the purposes of this investigation were to evaluate the effectiveness of a 6-hour, 12-step desensitization protocol and to characterize the immune mechanism of carboplatin hr. methods we analyzed ten consecutive patients over a two year period with documented hr to carboplatin who required continued treatment with a platinum agent. the patients were treated with carboplatin using a 6-hour, 12-step desensitization protocol. skin tests were performed on five patients. results ten patients successfully completed 35 planned courses of desensitizations to carboplatin, 31 of which were without reactions. four patients had symptoms during their first (n=3) and third (n=1) desensitizations but tolerated the re-administration of infusions without further reactions. for subsequent courses, the protocol was modified for two patients who had extracutaneous symptoms during desensitization and was unchanged for the patient who had mild urticaria. these three patients tolerated subsequent courses of desensitizations without reactions. the fourth patient with symptoms during desensitization no longer required carboplatin. of the five patients who were skin tested to carboplatin, four had positive wheal and flare reactions. in one patient, the skin test response to carboplatin became negative after desensitization. conclusions the 6-hour, 12-step desensitization protocol is safe and effective for treating patients with carboplatin hr. positive skin tests to carboplatin suggest a mast cell/ige-mediated mechanism. conversion of the positive skin test to a negative response after desensitization supports antigen-specific mast cell desensitization. hypothesis: there is an association between food allergies and acid reflux in atopic adults study design: a retrospective chart review of 60 patients was conducted. 30 people who tested positive and 30 people who tested negative for food allergy were included. the prevalence of gastroesophageal reflux disease (gerd) in the total group and each of the study arms was compared to population prevalence. methods: results of 70 allergen specific ige tests (pharmacia immunocap, nj) run for food allergies were reviewed to help locate charts of 60 atopic subjects, 30 with positive, and 30 with negative food allergy results. we reviewed charts for history of heartburn and acid regurgitation or the diagnosis of gerd, laryngopharyngeal reflux (lpr) or peptic ulcer disease (pud). population prevalence (19.8%; ci 17.7-21.9) of acid reflux was estimated from a historical comparison that studied adults in a similar geographical location. results: in the food allergy positive group, 26.7% of the subjects (95% ci 11.8-41.6) had either a history of heartburn and/or a diagnosis of gerd, lpr or pud. in the food allergy negative group, 40% (95% ci 25.1-54) had acid related disorders. in the total study group of atopic subjects, the prevalence of heartburn, gerd, lpr or pud was 33.34% ). on chi square analysis, the prevalence of acid reflux disorders was significantly higher in the total study group when compared with population prevalence (p=0.013). the prevalence of acid reflux disorders between the food allergy study group and in the population shows no significant difference (p=0.41). the prevalence of acid reflux disorders was significantly higher in the food allergy negative group when compared with the prevalence in the population (p=0.003). there was no significant difference in the prevalence of acid reflux disorders between the two study groups with and without food allergy (p = 0.27). conclusions: the prevalence of acid reflux disorders was higher in people with food allergy when compared to the population, but missed statistical significance. patients without food allergy had a significantly higher prevalence of acid reflux disorders compared to the population. atopic individuals have a statistically significant higher prevalence of acid reflux disorders, but there is no statistically significant difference between atopics with and without food allergy. abstract background: analysis of dispensing patterns of injectable epinephrine offers a method to study the characteristics of school children with allergic or anaphylactic reactions. objective: to analyze the demographics of children prescribed injectable epinephrine in massachusetts school districts with diverse racial and ethnic enrollment patterns. methods: school nurses in 44 schools (grades pk-12) enrolling 21, 870 students recorded the characteristics of students prescribed injectable epinephrine including the number and racial mix of students in each school, student age, grade level, race, sex, and allergic disorder requiring epinephrine. surveyed school districts were two predominately white (98%) suburban districts enrolling 5855 students and one urban area with a minority population of 60% enrolling 16, 020 students. the use of epinephrine for peanut allergy was analyzed in detail. results: a total of 181 of 21, 875 (0.83%) students in three school systems were dispensed injectable epinephrine. males outnumbered females (110 to 71). whites outnumbered non-whites 153 to 28. two thirds (n=116) of children dispensed epinephrine had peanut allergy. the second most common allergy was stinging insect allergy (n=34). the rate of dispensed epinephrine for peanut allergy was lowest in the urban school district (0.30%) versus the two suburban districts, 1.1% and 1.26% respectively. whites with peanut allergy outnumbered nonwhites 99 to 19. males outnumbered females 69 to 49. the lowest rate of prescribed injectable epinephrine for peanut allergy in all three school districts was found in 9612 non-white school children-0.17%. eighty-nine of 118 (75%) school children with peanut allergy were enrolled in grades pk through 5. there were twice as many white versus non-white (32 to 16) school children in the urban system with prescribed injectable epinephrine for peanut allergy. only eight (.11%) of 7209 hispanic and asian students in the were dispensed injectable epinephrine for peanut allergy. conclusions: this is the first study to suggest that there may be a racial disparity in the prevalence of childhood peanut allergy. one possible explanation for this disparity is that varied feeding practices in minority infants and children may induce a state of tolerance and lead to a lower incidence of peanut allergy. f.i. hsu * , h.j. burstein, m.c. castells, boston, ma. introduction: trastuzumab is a humanized igg1 kappa monoclonal antibody specific for human epidermal growth factor receptor 2 protein, her2, used in the treatment of her2/neu positive breast cancer. we present the first report of desensitization to trastuzumab in a patient with an ige-mediated reaction to trastuzumab and documented igg-anti-igg1 human antibodies. meth-ods: a 37 year old woman with metastatic invasive ductal breast cancer (er/pr positive, her-2 strongly positive), unresponsive to conventional therapy, presented with an anaphylactic reaction to trastuzumab and was evaluated for rapid desensitization by skin testing and serum specific igg antibodies. the patient had previously received trastuzumab. results: the anaphylactic reaction to trastuzumab consisted of diffuse erythema, urticaria, respiratory distress and laryngeal edema. premedication with steroids, h1 and h2 blockade, and slow infusion did not prevent a subsequent reaction. skin prick testing with trastuzumab (21mg/ml) was positive with 2 negative controls, confirming an ige-mediated mechanism. igg anti-human igg1 antibodies (haha) to trastuzumab were confirmed by elisa. the patient was desensitized to trastuzumab 2 mg/kg with an intravenous protocol that started at 0.5 mcg/h, and increased in rate every 15 minutes until a final rate of 30 mg/h, for a total infusion time of 6 to 8 hours. premedication included diphenhydramine, prednisone, famotidine, and montelukast. desensitization was confirmed by negative skin prick and intradermal tests (2 mg/ml and 21 mg/ml), with positive histamine control. the patient tolerated a total of 10 weekly doses of trastuzumab 2 mg/kg. intradermal skin testing prior to her 5th and 10th courses showed reactivity, indicating resensitization. serum levels of tratuzumab were undetectable at 1 week after desensitization. conclusion: allergic reactions to trastuzumab are rare but can include anaphylaxis. in patients with documented haha and/or ige-mediated reactions to trastuzumab and clinical symptoms of type i/mast cell mediator-related symptoms, desensitization can allow continued administration of this treatment by providing tolerance. the clinical effectiveness of desensitizations in patients with ige and haha antibodies remains to be defined. in contrast to reports in the medical literature, details of the medical aspects were limited. there were 2 attacks on infants bringing the total to 3 reported infant attacks to date. two of the 3 infants suffered long term morbidity and 1 died. like those reported in the medical literature, the majority of adults were in long term care facilities, although 2 were in hospitals. overall, 6 of the 20 individuals stung died within one week of stings. no significant medical consequences of stings were reported in 6 of 10 of the newspaper reports as opposed to 2 of 10 reports in the medical literature. conclusion: our data suggest that increasing numbers of fire ant attacks are occurring in medical facilities, where chronically ill, frequently immobile patients come in contact with foraging ants. unattended infants in private homes in fire ant endemic areas also appear at risk. the factors that determine why individuals are stung and the severity of injury after attacks remain uncertain. the presence of fire ants inside health care facilities and homes is a harbinger for fire ant attacks of disabled or infant occupants. we hypothesize that morbidity is determined by the number of stings, the condition of the patient and the type of treatment administered. purpose: to determine if gender confers a risk for positive penicillin (pcn) skin test. method: rates of positive pcn skin tests, according to gender, were determined in patients with a history of pcn allergy undergoing an allergy pre-operative evaluation from june 2002 to june 2004. during this period, 19, 429 patients were seen in the pre-operative evaluation clinic in which 1921 had a history of pcn allergy and comprise our study population. a univariate logistic regression analysis was employed to calculate the odds ratio (or) and the 95% confidence interval (ci) for gender differences in the rates of positive pcn skin test and a multivariate logistic regression analysis was used to adjust for age and history of multiple drug allergies. p-value of 0.05 or less was considered statistically significant. results: of the 1921 patients, 1759 underwent skin testing for pcn, 157 patients did not, and 5 charts were not available for review. the mean age of the study group was 60 years. sixtyfour (3.6%) patients had a positive skin test to pcn. of these, 53 (83%) were females and 11 (17%) were males (or 3.6, 95% ci 1.9-7.2, p = 0.0001). of those with a negative/equivocal pcn skin test, 960 (57%) were females versus 720 (43%) males. in a multivariate logistic regression analysis adjusted for age and history of multiple drug allergies, female gender again was more likely to have a positive pcn skin test (or 4.7, 95% ci 2.3-10.5 p < 0.0001). 157 patients did not undergo pcn skin testing, 62 (40%) were male and 95 (60%) were female. conclusion: this is the first report showing that a greater risk for a positive skin test to pcn exists in association with female gender. age and a history of multiple drug allergies are unlikely to be confounding factors to the observed gender risk. further studies are needed to identify other possible confounding factors and/or mechanisms that can explain this risk. a. fiocchi 1* , p.a. restani 1 , s. cucchiara 2 , g. lombardi 3 , g. magazzu' 4 , g.l. marseglia 5 , k. pittschieler 6 , s. tripodi 2 , r. troncone 7 , a. vierucci 8 , 1. milan, italy; 2. roma, italy; 3. pescara, italy; 4. messina, italy; 5. pavia, italy; 6. bolzano, italy; 7. napoli, italy; 8. firenze, italy. background: cow milk substitutes for children allergic to cow milk proteins (cmp) include soy-based formula and cow s milk hydrolysates. neither can rule out a sensitisation risk. objective: prospective assessment of clinical tolerance to a rice-based hydrolysate formula by children allergic to cow milk proteins (cmp) who consume rice openly. patients and methods: ninety-seven children aged 4 to 136 months with immediate reactions to cow milk confirmed during dbpcfc were assessed for clinical tolerance to cow milk by spt with whole milk, -lactalbumin (ala), -lactoglobulin (blg), -and -casein ( -cas, -cas) (sigma chemical, st. louis, mo). whole milk, ala, blg and cas specific ige determinations were performed using cap test (pharmacia, uppsala, sweden) . similarly, sensitisation to rice and hrf were investigated by spt and cap test. patients sera were investigated by immunoblotting for cmp, rice and hrf (heinz-plada, milan, italy). dbpcfc was carried our with 24 g rhf powder masked in neocate tm (equivalent to 180 ml reconstituted formula). results: spt: all patients were positive to cow s milk and/or cmp fractions (>3 mm wheal diameter). ige determinations gave positive results with cow milk and/or cmp fractions (72/84 patients tested), rice (16/81) and with hrf (2/85). immunoblots (n=87) were positive for -cas (n=46), -cas (n=34), ala (n=39), blg (n=34) and bovine serum albumin (n=52). similarly, although patients sera largely recognized rice (78/87), only one weakly reacted with rhf. challenge with rhf was negative in all cases. conclusions: we conclude that, despite their frequent sensitisation to rice, children with cma tolerate both rice and rhf clinically. hydrolyzed rice formulas may thus represent an alternative protein source for children with cma. r.c. cartwright * , w.k. dolen, augusta, ga. introduction: conventional treatment of human seminal fluid allergy includes abstinence, barrier protection, or subcutaneous immunotherapy with fractionated seminal fluid. treatment using local intravaginal desensitization with unfractionated seminal fluid has recently been described, but experience with this desensitization method is still limited and little has been reported as to its long-term results. methods: a 30-year-old woman with a history of allergic rhinitis and asthma experienced anaphylaxis following her first unprotected intercourse since the birth of her first child. ige-mediated sensitization was demonstrated through the use of specific ige testing (pharmacia cap system) and skin prick testing using undiluted seminal fluid obtained from the patient's husband. after approval from the human assurance committee, the patient underwent rush intravaginal desensitization with steadily increasing concentrations of seminal fluid, beginning with a 1:10, 000 v/v concen-tration. results: her specific ige level to human seminal fluid was 0.26 ku/l. skin prick testing was positive with a wheal of 10 mm diameter with pseudopods and a flare of 25 mm diameter. desensitization was successful and the patient tolerated local application of whole semen without significant reaction. following desensitization, the patient reported that she and her husband engaged in unprotected intercourse without local or systemic symptoms. over the last year since the desensitization, she has not experienced further anaphylaxis, but she has developed local symptoms if her exposure to seminal fluid was delayed past 5 days. the longest time period between exposures has been 2 weeks. she became pregnant 5 months ago and has not had any pregnancy complications. conclusions: local intravaginal desensitization was a safe and effective treatment for human seminal fluid allergy in this patient. a delay in seminal fluid exposure greater than 5 days was associated with the return of symptoms emphasizing the need for frequent seminal fluid exposure to maintain desensitization. l. terracciano, t. sarratud, a. fiocchi * , p. restani, s. guerci, milan, italy. background kiwifruit allergy in children has been seldom reported and the reactions observed are usually mild. anaphylaxis has not been described. we document the case of an infant who developed anaphylacic symptoms within minutes of his mother eating two kiwifruits and initiating breastfeeding. case history in his fourth month, the boy was admitted into an emergency department with dysphonia, breathing difficulties, generalised urticaria and angioedema of the lips and face. this episode was treated as an anaphylactic reaction with epinephrine, chlorpheniramine and hydrocortisone sodium succinate administered via a percutaneous catheter and symptomatic control was achieved within 30 minutes. after 15 days a second anaphylactic episode of similar severity occurred under the same circumstances. neither episode required critical care. the boy presented two months later for clinical evaluation in our paediatric allergy unit. skin prick tests (spt) were positive only with egg white and yolk while negative to cow s milk (and protein fractions), beef, chicken, pork, codfish, rice, wheat, soybean, maize, potato, carrot, tomato, bean, pea, celery, peanut, dermatophagoides pteronyssinus and d. farinae, grass and banana. spt with kiwifruit was specifically ordered because exquisite contact was suspected, and induced a positive wheal (>3 mm diameter). positive specific ige determinations (cap-feia from pharmacia, sweden) with kiwifruit (0.71 ku/l), cat dander (1.97 ku/l) and egg white (1.97 ku/l) were returned but determinations with other inhalant and food allergens were all below the cut-off point of 0.35 ku/l and total ige levels were 155 ku/l. strict avoidance of kiwifruit by the breastfeeding mother proved effective and nothing untoward happened in the intervening year. currently aged 2.5 years, the boy is free from food-related symptoms. prick-byprick tests with native allergens and spt carried out with commercial extracts of allergens associated in the literature with kiwifruit allergy were all negative. comment there are no reports of immediate reactions to kiwifruit under two years. in this case, severe reactions via breastmilk in an infant monosensitised to kiwifruit indicate that nursing may represent a hidden source of exposure. in older children monosensitisation without prior sensitisation to pollen has been described as the major risk associated with severity of symptoms. m. sikora * , j.w. sleasman, n. tangsinmankong, st. petersburg, fl. introduction: treacher-collins syndrome (tcs) is an autosomal dominant disorder with an abnormality of craniofacial development during early embryogenesis. there is a known relationship between new bone formation and development of lymphocytes and cytokines. the association of tcs and humoral immunodeficiency has not yet been reported. we present a novel case of a 12 year-old caucasian female patient with tcs and common variable immunodeficiency. methods: our patient presented with midface hypoplasia, micrognathia, microtia, conductive hearing loss and cleft palate with a history of recurrent upper and lower respiratory infections. patient had a 10-year history of chronic sinusitis, recurrent otitis media and pneumoniae which resulted in bronchiectasis. haemophilus influenza and staphylococcus aureus were persistently isolated from bronchial fluids. laboratory work-up for immunodeficiency was initiated. results: immunoglobulin analysis revealed low igg 450 (528-2190 mg/dl); low iga 42 (44-441mg/dl); igm 65 (48-226mg/dl); igg 1 354 (165-1440 mg/dl); low igg 2 59 (71-460 mg/dl); igg 3 85 (28-125 mg/dl); and low igg 4 <8.1 (2-143 mg/dl). patient had no detectable response to protein-derived vaccines (diphtheria and tetanus) and to polysaccharide-derived vaccines (pneumococcal) at baseline and at 4-6 weeks after immunization. t and b lymphocyte enumeration, ch50, ah50, and mannose-binding lectin were all within normal limits. laboratory findings were consistent with the diagnosis of common variable immunodeficiency. patient began receiving monthly doses of 550 mg/kg/dose of intravenous immunoglobulin resulting in clinical improvement. our patient no longer requires antibiotic therapy for her respiratory infections; pulmonary function has improved and bronchiectasis resolved 6 months after initiation of ivig. conclusion: our finding shows that tcs can be associated with common variable immunodeficiency which suggests a link between skeletal dysplasia and immunodeficiency. a.s. hartel * , j.w. sleasman, n. tangsinmankong, st. petersburg, fl. introduction: streptococcus pneumoniae is the most common cause of invasive bacterial infection in humans. however, incidence of s. pneumoniae infections in healthy adolescents is low (5/100, 000 per year). mannose-binding lectin (mbl) is a c-type lectin which plays a central role in the innate immune response by activating the classical complement pathway and acting as an opsonin by binding c1q receptors. several studies have demonstrated an association between invasive bacterial infections, including s. pneumoniae and a homozygous mutation of the mbl gene. however, this association has not previously been described in patients with the heterozygous mutation. methods: a 13-year-old caucasian female presented with a second episode of meningitis over a 4-year span. streptococcus pneumoniae was isolated from peripheral blood cultures on both occasions. lumbar puncture revealed wbc 22, 600; 6% bands; 83% pmns; glucose 3 mg/dl, protein 329 mg/dl; these results consistent with bacterial meningitis. extensive evaluation for predisposing causes revealed benign arnold-chiari type1 malformation. further immunological evaluation was initiated. results: evaluation revealed igg 867 mg/dl (normal 528-2190); iga 67 (44-441); igm 181 (48-226), and normal igg subclasses. antibody responses to diphtheria and tetanus vaccines were adequate. antibodies response to pneumococcal vaccine given 4 years prior were protective to all 10 common serotype tests (>1.4 mcg/ml). lymphocyte subset analysis was within normal range. howell-jolly bodies were not detected from peripheral blood smear. evaluation for secondary immunodeficiency was negative, including hiv antibody by elisa. complement analysis showed ch50 88 u/ml (65-95); ah50 71% (66-129%), and markedly low mbl of 74 ng/ml (>1000). genetic analysis of her mbl revealed heterozygous mutation in codon 54 and mutations in two promoter regions (homozygous mutation on h/l variants and heterozygous mutation on p/q variants). conclusion: heterozygous mutation of mbl codon when associated with mutation of promoter regions can result in severe impairment of mbl protein production, and may lead to increased susceptibility to invasive bacterial infections and meningitis. rationale: patients with similar symptoms may have allergic, non-allergic, or mixed rhinitis triggers and can differentially respond to distinct medications. we assessed st in our clinic patient population to characterize factors that influence appropriate diagnosis and treatment of rhinitis type. methods: we used a validated commercial questionnaire and chart review of 230 patients seen in an academic allergy clinic, to assess the number of allergic vs. irritant st and demographic and historical factors (including pharmacotherapy) associated with differences in symptoms. results: the population (n=189: 138 female, 51 male) consisted of individuals with a mixed rhinitis history. women had higher st scores than men, including: total scores [10.44+/-0.51 vs. 7.20+/-0.65, p=0.0001 (unpaired t-test)]: allergen scores (4.56+/-0.30 vs. 3.51+/-0.37, p=0.0055); and irritant scores (5.58+/-0.29 vs. 3.69+/-0.41, p=0.0003). further, patients treated with both azelastine (az) and intranasal steroids (ins) had lower st scores than patients treated with either alone (total st: az 10.06+/-0.94, ins 10.3+/-0.97, both 8.0+/-0.88, p=0.0348; allergen st: az 4.69+/-0.54, ins 5.05+/-0.37, both 3.11+/-0.50, p=0.0416; irritant st: no significant difference). conclusions: female patients have significantly higher symptom scores; total, allergic and irritant. rhinitis monotherapy (ins or az) is minimally effective in reducing st but is more effective when used in combination. these data demonstrate rhinitis population heterogeneity and address differential responsiveness to similar medications. increased response to combined drug therapy support the heterogeneous pathophysiology of mixed rhinitis features and may relate to a combination of multiple aeroallergen and air pollution exposure in the houston area. introduction: interaction between eye & nose warrants attention with regard to the propagation and treatment of allergic reactions. the role of topical therapy for rhinitis and conjunctivitis is becoming more widely considered and questions of therapeutic route have been raised. purpose: to elucidate the anatomic and pharmacokinetic interactions of conjunctival & nasal mucosa leading to more rational selection of routes of medication administration. methods:our studies have investigated the effect of ocularly instilled allergen inducing signs and symptoms of rhinoconjunctivitis. 1 study compared effects of allergen administered via conjunctival allergen challenge (cac) or nasal allergen challenge (nac) and analyzed tear & nasal secretions for ecp & tryptase. studies have also used the ability of the cac model to induce rhinitis signs & symptoms to evaluate the relative effects of medication routes: 1) ocular v. nasal spray v. systemic; 2) ocular + nasal spray v. systemic + nasal spray; 3) ocular v. placebo. results: the nac/cac study revealed that, following cac (n=34), significant ocular and nasal signs & symptoms were noted; following nac (n=39), only nasal symptoms were clinically significant. nasal symptom scores between cac & nac differed significantly at 1 timepoint, representing lag in allergen & mediator movement from eye to nose. measurable levels of ecp & tryptase were found in tears and nasal secretions for cac, but only in nasal secretions (with exception of 1 subject) for nac. in cac studies: 1) ocular therapy exhibited greater efficacy in ocular itching relief (n=73;p<0.05) and was not significantly different from nasal or systemic therapy in nasal symptom relief. 2) eyedrop+nasal spray combination exhibited significantly greater prevention of overall rhinoconjunctivitis signs and symptoms than nasal+systemic therapy (n=80). 3) ocular therapy offered greater protection from nasal signs & symptoms compared to placebo (n=32;p<0.05). conclusion: nac and cac results support the unidirectional flow from eye to nose, the ability of cac to yield nasal symptoms, and the efficacy of topical therapy. tearing, due to inflammation of the inferior turbinate would be the only ocular symptom resulting from nasal challenge. these results offer insight to the nature of the connection between ocular and nasal mucosa and the efficacy of varied medication administration routes for management of rhinoconjunctivitis symptoms. introduction: asthma disease management programs frequently target high utilizers because they are responsible for a large amount of asthma costs. once identified, such "frequent fliers" usually are offered interventions including case management. programs using this approach usually demonstrate reductions in utilization. though the interventions may cause this decline, it could also be due to regression to the mean (rtm) which is a tendency for outliers to become more like the mean over time. in this study we measured rtm in a medicaid hmo asthma population to determine whether targeted case management is effective independent of this phenomenon. we hypothesized that directed case management provides additional utilization reduction beyond rtm. methods: a weighted asthma utilization score was determined quarterly for members of an hmo with asthma from 2000 to 2004. rtm was measured by determining how many patients were persistent high utilizers quarterly for 1 year after baseline. to determine the effect of utilization-directed case management, the number of frequent fliers at baseline for each quarter also was determined. results: a total of 296 asthma frequent fliers were identified on january 1, 2001. by september 1, 2001 only 15 of these individuals continued to be frequent fliers. similar decreases in utilization were seen for frequent fliers identified at the beginning of each quarter of 2001. the mean decrease in the number of frequent fliers is shown in the table. this represents a substantial regression to the mean. the total number of high utilizers at baseline decreased by 27% after implementation of utilization-directed case management in 2002 independent of regression to the mean. this also represented a decrease from 1.6% of health plan members with asthma to 0.6% by the start of 2004 suggesting that the decline is not a result of diminishing health plan membership. the benefit appears to be the result of early intervention with members before they become frequent fliers. conclusions: utilization-directed case management can reduce overall asthma utilization by preventing members from becoming high utilizers at an early stage. programs that claim to intervene with plan members who already are high utilizers are likely to be taking advantage of regression to the mean. studies indicate a high incidence of asthma(as) among school-aged children. with the prevalence increasing, significant numbers remain unidentified. they experience morbidity, including school absenteeism, which may be preventable, in part, by adherence to national asthma guidelines. we implemented a pilot study to detect as, as control and initiation of appropriate health care among 7th grade students in a suburban population. the study was coordinated with the middle school staff and the local county health department. the screen parameters included: student questionnaires, peak flows, exercise with peak flow assessment (frast)2, and spirometry as indicated. environmental tobacco smoke (ets) was recorded. results: 196 students screened. 131 no as history and negative screen (d). 31 as history and negative screen (e). 5 as history and positive screen (a). 23 no as history with suggestive written screen and negative screen on exercise (b). 6 no as history and positive screen (c). ets in groups a, b, c: 40%, 32%, 60%. ets in groups d, e: 23%, 16%. individual student results were mailed home with medical follow-up recommended for a positive screen. parents of the 34 children who failed the questionnaire or exercise screen (groups a, b, c) were phoned at a 6-12 wk interval. no student in-group a or b had medical follow-up. 2(33%) students in group c had medical follow-up. conclusion: this as screen of 7th grade students identified 6(3%) as having significant, undetected as. 5(14%) with known as had uncontrolled as at the time of screen. 23(11.7%) students with strong suspicion of as based on questionnaire had an acceptable exercise screen. only 2 of the 34 children with a screen suggestive of as or uncontrolled as had medical follow-up. ets was increased in the homes of students with a positive screen and remains a serious health issue. school screening for as has merit, but mail and phone follow-up was insufficient to intervene or initiate acceptable as treatment. references 1. redline s. et. al. development and validation of school-based asthma and allergy screening instruments for parents and students. ann allergy asthma immunol. 2003; 90: 516-528 2. tsanakas.j.n., et al. free running asthma screening test. arch diseases in childhood.1988; 63:261-265 3. american college of allergy, asthma, and clinical immunology screening test ages (8-14) acaai.org. introduction. some laboratory evidence suggests that desloratadine is effective in inhibiting of inflammatory mediators, which play an important role not only in allergic but also in virally induced inflammation. the purpose of this study was to assess its efficacy in acute bronchiolitis developed in young children suffering from concomitant atopic dermatitis (ad). meth-ods. participants were 34 young boys and gilrs aged 18-36 who suffered from acute bronchiolitis as established by wheezing, rhinitis and fever. all patients also had concomitant ad as established by hanifin and rafka criteria. patients were allocated to receive syrup formulation of desloratadine (erius, schering plough) 1, 25 mg/day for 5 days (active group n=17) or no desloratadine treatment (control group n=17) using quota allocation system. we calculated physical global symptom score (combination of cough, wheezing, chest retractions, nasal flaring, blocked nose, runny nose, sore throat -0-5 scale) and respiratory distress assessment instrument (rdai) to examine the patient at baseline and on day 5th after therapy was initiated, day of normalization of body temperature, respiratory rate, heart beat rate, and use of albuterol. results. both group of patients were comparable on age, gender, family history of asthma, time of onset of the acute respiratory illness, extent of medication taken prior to entering the study, global symptom score and rdai index. all children were evenly treated with oxygen, oral theophylline (10 mg/kg/day) and adequately rehydrated. on day 5th global symptom score of patients receiving desloratadine was 2, 51â±0, 51 vs 3, 42â±0, 62 in control group (p=0.035). on day 5th it was also significant difference between active and control groups on rdai index (4, 44â±1, 24 vs 7, 23â±2, 30; p=0.046) especially for significant drop in wheezing score in active group. day when respiratory/heart rate normalized, temperature returned to normal, use of albuterol did not differ significantly in these groups. conclusions. our preliminary study is the first to show that desloratadine is an effective agent in viral lower respiratory tract infections of young children suffering from ad. it encourages further gcp trials to explore action of desloratadine in infantile bronchiolitis and concomitant ad. h.j. su * , w.t. lin, p.j. tsai, c.y. huang, p.c. wu, tainan, taiwan. increasing prevalence of childhood asthma has been observed across the world, and found to be associated with, partly, indoor pollution, including bioaerosol exposure. meanwhile, adequate ventilation is shown to be effective in diluting most indoor air pollutants, while only limited data are available addressing directly how the ventilation rate is implicated with childhood respiratory symptoms. this study aimed to examine the concentration distribution of selected indoor air pollutants, including bioaerosols, in domestic environment, and further to assess the effects of ventilation rate, characterized by a co2 trace gas concentration decay method, on concentration variations of the above-mentioned air pollutants. study subjects were chosen from a prior city-wide questionnaire survey based on positive response to inquiry for physician-diagnosed asthmatic status and wheezing symptoms in the past 12 months. environmental assessments, including ventilation and air quality measurements, were conducted twice, 1 in early winter and the other on early summer. respiratory health diary and pefr (peak expiratory flow rate) were recorded for 1 week concurrent with the sampling activity. increasing ventilation rate is statistically associated with decreasing indoor concentrations of co2 and tvocs. after adjustment for sex, age, and selected housing characteristics, the or between tvocs concentrations and reporting cough of study children is 9.47, and 12.84 between co2 and nasal congestion. the or between indoor bacterial concentration and the morning pefr less 80% is 10.98 in the similar multivariate logistic regression for data collected from winter study. in summer, the only significant relationship is between ventilation rate and the morning pefr less 80% of study children, or= 0.17, in a multivariate logistic regression. this study has identified less reporting of childhood respiratory symptoms, especially for coughing and the morning pefr less 80% are associated with increasing ventilation rate, and higher levels of indoor air pollution appear to be present with greater frequency of the above symptoms. this study suggest quantitatively that proper management of ventilation efficiency may be beneficial for the control of childhood respiratory illnesses. *adjusted for: sex, age, environmental tobacco exposure, use of incense and air conditioner **:p<0.05 ns:no statistical significance with whole model test background: moderate-to-severe allergic asthma can have a substantial impact on a patient's asthma-related quality of life (arql). in addition to asthma symptoms, allergic rhinitis, rhinosinusitis and other related comorbidities are often apparent in patients with more severe disease making it difficult to treat. despite guideline-consistent care, many patients still experience variability in asthma control signaling an unmet need within this population. omalizumab (xolairâ®) has recently demonstrated clinical efficacy and safety in treating asthma. objective: the aim of this paper is to summarize the arql outcomes associated with omalizumab therapy in moderate-to-severe allergic asthma. methods: we performed a systematic review of arql data from the clinical study reports and published clinical trials on omalizumab. arql was measured by the juniper-asthma quality of life questionnaire (aqlq). results: statistically significant results for arql endpoints consistently favored omalizumab over placebo. the magnitude of the changes in arql were consistently aligned with clinical endpoints. moderate to large effect sizes in the omalizumab groups were maintained throughout the 28-week clinical trial program and during the 24-week double-blind extension phase. however, the placebo groups also experienced within-group improvements and moderate effect sizes. a meta-analysis indicated a 1.6 to 2.0 fold increase in large (>1.0 point) improvements in overall aqlq scores in the omalizumab-treated group compared with placebo during the stabilization and steroid-reduction phases of the clinical trials. conclusions: the consistently positive impact of omalizumab on arql outcomes during the clinical trial program provides evidence of its value as an adjunct therapy in patients with moderate-to-severe allergic asthma. significant differences and large effect sizes were observed despite the fact that the control group received active, guideline-consistent treatment producing a substantial placebo effect. improvements were observed in overall, symptom, activity, emotional and environmental dimensions of the aqlq indicating that omalizumab produced benefits in arql in patients with moderate-to-severe allergic asthma. background: omalizumab, a monoclonal anti-ige antibody, significantly improves asthma-related quality of life (arql) for patients with moderatesevere allergic asthma who express symptoms despite moderate-high inhaled corticosteroids (ics) doses. mean scores can mask underlying variability in arql outcomes. this investigation examined variability in outcomes to elucidate the specific impact of omalizumab treatment. methods: aqlq data (n=948) from two randomized, double-blind, placebo-controlled clinical trials were pooled to assess underlying variability in the mean scores and to identify key drivers of arql treatment-effect differences (juniper-asthma quality of life questionnaire (aqlq)) between omalizumab and placebo (active control) patients. results: correlations between aqlq and other clinical outcomes were low to moderate at best (r=0.14 to r=0.60). aqlq assessment captures patient benefit that supplements clinical outcome measures. across all component items of the aqlq patients receiving omalizumab improved more than patients receiving placebo (active control) (p<0.05). omalizumab patients reported the greatest improvement for reducing waking with symptoms in the morning (symptoms domain: 2.06 vs. 1.50; p<0.001), limitations in all activities done (activities domain: 1.18 vs. 0.68; p<0.001), the fear of not having medication available (emotions domain: 0.99 vs. 0.60; p<0.01), and symptoms from being exposed to dust (environment domain: 1.31 vs. 0.88; p<0.001), compared to placebo (active control) patients. conclusion: arql assessment provides complementary and non-overlapping information on clinical benefit that is distinct from other clinical outcome measures. examination of underlying variability in aqlq mean scores and item-level response extend previously published results on omalizumab treatment effect by showing that aggregate arql improvements for omalizumab patients are strongly influenced by symptom and activity improvement. a. brimer 1 , k. malhi, c. adams, c. dinakar, kansas city, mo. introduction: the desire to belong to a group is a very powerful motivator and is exceptionally strong in the adolescent population. asthma is a disease that makes people feel different. this perception may impinge on patient adherence with medication regimens. we hypothesize that belonging to a club where every member has asthma may encourage identification of the adolescent asthmatic with their peers, and mitigate the perception of being different. objectives: (1) to investigate the factors that make the asthmatic adolescent feel different (2) to explore the hypothesis that group activities, such as an asthma club, would help adolescent asthmatics feel less different. methods: as part of an ongoing survey, children with asthma between the ages of 8-18 years were offered an anonymous questionnaire in the primary care and adolescent clinics at our hospital. the questionnaire included both multiple-choice and open-ended questions designed to explore the feelings of the respondents. the responses were numerically tallied and reported as percentages. results: at the present time, 31 surveys out of a proposed 100 have been completed. one third of the youth with asthma answering the survey had negative feelings regarding their asthma. nearly forty percent of the respondents reported that their diagnosis made them feel different from their healthy peers. forty-five percent responded that they have felt restricted or excluded from school activities, athletics, and clubs due to their asthma. over one-third of them feel uncomfortable taking their inhaler in front of their friends. most respondents (93.6%) indicated that they enjoy group activities. the majority of respondents (89.7%) rated playing sports as their favorite group activity. conclusion: almost forty percent of asthmatic youth surveyed indicated that having asthma made them feel different and resulted in restriction/exclusion from school activities, athletics, and clubs. an overwhelming majority expressed a preference for participation in group activities, particularly recreational sports. this social preference towards group activities may be incorporated into an intervention, such as an asthma club, to help asthmatic youth adjust to the disease and its treatment regimen. introduction: airway hyperresponsiveness (ahr) is an exaggerated narrowing of the airways in response to stimuli, such as allergens, histamine, and cold air. ahr is a characteristic feature of asthma linked to chronic airway inflammation. phosphodiesterase 4 (pde4) is an enzyme found in key inflammatory cells involved in the pathophysiology of asthma. inhibitors of pde4 prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde4 inhibitor, which has shown anti-inflammatory activity in vitro and in vivo. this study examined the effect of a single dose of roflumilast on changes in ahr following allergen-induced asthmatic reactions in patients with mild asthma. methods: this double-blind, randomized, crossover study consisted of 2 treatment periods separated by a 2-to 5-week washout period. a total of 13 patients (forced expiratory volume in one second [fev 1 ] 70% predicted) who were hyperresponsive to histamine (provocative concentration causing a 20% drop in fev 1 [pc 20 fev 1 ] 16 mg/ml) were randomized to receive a single dose of oral roflumilast 1000î¼g or placebo on day 1 of each treatment period followed by an allergen challenge 60 min after medication. histamine provocation was performed before and 24 h after intake of study drug. results: there was no change in fev 1 60 min after roflumilast administration, suggesting the absence of direct or acute bronchodilation. allergen challenge elicited early and late asthmatic reactions that were attenuated by a single dose of roflumilast 1000î¼g. the histamine pc 20 fev 1 in patients treated with roflumilast decreased to a lesser extent than in those patients treated with placebo. the change in pc 20 fev 1 from baseline after challenge was 2.51 â± 2.95 mg/ml (mean â± sd) with placebo and 1.23 â± 2.75 mg/ml with roflumilast. the magnitude of the change in pc 20 fev 1 was statistically significantly different between the placebo and roflumilast treatment groups (p=0.002). thus, roflumilast decreased the development of ahr by approximately 1.1 doubling-dilutions. conclusions: a single dose of oral roflumilast 1000î¼g attenuated allergen-induced ahr. these data provide further evidence that roflumilast may provide anti-inflammatory activity in vivo and may be an effective treatment for patients with asthma. introduction for the past 7 years we have been tracking pediatric asthma admissions and evaluations at the huntington memorial hospital. during the fall and winter months (oct-march) from 1996 to 2002, we noted a 1.6 x increase in the number of asthma admissions and evaluations compared with the spring and summer (april -september; 4, 993 patient encoun-annals of allergy, asthma & immunology ters in 7 years). we have analyzed potential causes for this increase. diesel particulate was estimated in 1998-9, and a 2.5-fold increase in the fall versus spring was found in burbank, a city adjacent to pasadena. pollutant particles from the combustion of fossil fuels may act as immuno-adjuvants to allergen exposure as has been demonstrated experimentally in mice and in human nasal exposure studies. methods computerized analysis of asthma admissions and evaluations were calculated according to established codes for acute and chronic reactive airways disease. infectious diseases in patients were analyzed by standard asthma questionnaire. also, pollen and mold counts, as well as diesel particulates and meteorological conditions were studied. results similar numbers of viral infections occurred in the fall of 2001 and spring of 2002. among the most prevalent pollens and molds are chinese elm pollen (sept -oct) weed pollens (july -dec), and a newly recognized aureobasidium mold (nov -march). pollen grains can fragment and release respirablesized debris that are loaded with allergens (taylor et al 2002 (taylor et al , 2004 . the conidia of aureobasidium are also of respirable size and were identified by sequence analysis. fine particulate air pollution (pm2.5), containing diesel particles, is increased in the fall and winter in southern california. con-clusions the incidence of asthma outbreaks may vary with air pollution levels in the immediate environment. pollen fragments and particles from fossil fuel combustion can deposit in similar regions of the lower airways. rainfall increases in the fall and winter and this coincides with increased molds and aureobasidium. aureobasidium was recently discovered in high concentrations in pasadena, is known to be allergenic and could be a factor in increased asthma. a mixture of pollens and molds and fine combustion particles may be relevant to this increased incidence of asthma in the fall and winter months in pasadena. background we have previously noted that tree and ragweed pollen grains can be airborne for most if not all daily periods during their respective seasons (aaaai, 2003) , (acaai, 2003) . because grass levels are lower than those of trees & weeds (aaaai / nab), we chose a longer daily interval to examine (six hours) that we had for trees (one hour) and ragweed (three hours). methods twenty-four hour microscope slide samples were collected using a burkard 7 day air sampler from 05/01/04 to 06/30/04. the slides were examined @ 100 x along a single longitudinal traverse. the presence or absence of grass pollen was noted per six-hour segment (six successive fields). results the presence of airborne grass pollen during the 4 daily segments was sparse the first two weeks of may. it gradually increased during the next 10 days, and then was detected during most of the daily segments through to the end of june. # ammophila arenaria (aa) is a highly resilient grass with habitat in europe, north america, australia, new zealand, south africa and the mediterranean, used predominantly for dune stabilization. patients with atopic diseases including allergic reactions to pollen are transferred to coastal regions in order to minimize pollen exposure. in spite of the presence of aa pollens, patients experience allergy relief at the coast. our study examines the contradiction between the presence of strong grass allergens and the absence of allergic symptoms in atopic patients, and whether allergic cross-reactions among different types of grass pollen include aa pollen. 9 adult patients presenting symptoms of grass pollen allergy underwent prick testing and rast testing to grass pollen mix and extracted aa pollen. 4 subjects showed positive results in prick and rast testing to aa extracts and were subsequently tested via rhinomanometry for reactions to aa using prick test solutions. 2 subjects showed positive reactions to prick tests for aa extract and grass pollen mix, but did not show specific antibodies. all other sera showed elevated specific ige levels, with specific reactions on 8 protein bands of aa. in sera incubated with grass pollen extract, almost all specific bands previously detected with aa were now inhibited. 3 patients with high levels of total and specific ige to aa showed weak bands after inhibition, demonstrating the allergenicity of aa. comparing data from coastal and mainland weather stations in june and july from 1961 to 1990, coastal wind speeds average 6.6 to 6.9 m/s while inland wind speeds average only 3.6 to 3.7 m/s. high wind speeds likely dilute pollen concentrations. based on findings of an inverse relationship between wind speed and pollen concentration in the dispersion of ragweed pollen, we hypothesize that concentrations of aa pollens may be similarly influenced. no sensitized patient reported allergic symptoms in coastal areas inhabited by aa plants, indicating that under natural conditions, either the plant modifies its pollen allergenicity or that environmental conditions including salt aerosols, wind patterns, topography or light exposure cause the modification. as such patterns are likely to become more dramatic as the effects of global climate change transform the natural environment, these findings may also become relevant for other grass allergy types. asthma is the most common chronic inflammatory disorders of the airways with increased prevalence in the past decade. it is characterized by airway obstruction, airway inflammation and airway hyperresponsiveness (ahr) to non-specific stimuli. in this study, we examined the effect of a novel class of molecule, ocid1001, in inhibiting antigen-induced early and late allergic response (ear and lar), ahr, and airway eosinophilia in mouse and guinea pig models of asthma. pulmonary functions were measured in conscious unrestrained animals by whole-body plethysmography. animals were sensitized with ovalbumin (ova; 20mg, intraperitoneally) with 2mg alum followed by treatment with ocid1001 (120mg/kg, i.p. twice daily for 10 days). after the last dose, animals were challenged with ovalbumin. pulmonary functions were recorded for ear and lar and 24 hrs later for ahr. this was followed by bronchoalveolar lavage, blood and lung tissue collection. treatment with ocid1001 (120mg/kg) significantly attenuated lar in ova-sensitized and challenged mice or guinea pigs with no effect on ear. ahr to methacholine in mice and to histamine in guinea pigs were prevented by treatment with ocid1001. ocid1001 attenuated the rise in total number of inflammatory cells in the lung and almost ablated the rise in bal eosinophilia in the lung due to antigen sensitization and challenge. effect of ocid1001 on pulmonary functions and airway inflammation in ova-sensitized and challenged mice were comparable to that of dexamethasone in both models of asthma. these data suggest that ocid1001prevents the underlying pathophysiological changes in allergic airway inflammation and therefore could prove beneficial in the treatment of bronchial asthma. ma; 2. madison, wi; 3. portland, or; 4. milwaukee, wi; 5. normal, il; 6. denver, co; 7. los angeles, ca; 8. fremont, ca. introduction. daclizumab (zenapaxâ®), a humanized monoclonal antibody directed against the il-2 receptor chain (cd25), is approved for prevention of renal allograft rejection and is under evaluation for treatment of asthma. daclizumab inhibits activation of human t lymphocytes by blocking il2induced proliferation, and by reducing production of th2-and th1-associated cytokines. we recently reported that daclizumab improved pulmonary function in a phase ii trial of patients with moderate to severe chronic persistent asthma (jaci, 2004, 113 (2):s286). we now report additional data from this trial on the possible role of daclizumab as an anti-inflammatory agent that affects asthma outcomes. methods. 116 non-smoking asthmatics age 18-70 with baseline fev1 50-80% predicted despite use of 1200 mcg daily inhaled triamcinolone (taa) or equivalent were enrolled in a randomized, multi-center, double-blind, placebo-controlled trial. patients were randomized (3:1) to i.v. daclizumab (2 mg/kg followed by 1 mg/kg every 2 weeks) or placebo added to stable dose taa. starting at week 12, patients underwent 25% reduction of taa every 2 weeks while continuing study drug to week 20. results. patients on daclizumab demonstrated a prolonged time to exacerbation requiring systemic steroid rescue compared to placebo patients (p=0.024). exacerbation rates were reduced in the daclizumab group compared to the placebo group for the 20-week steroid-stable and steroid-taper phases (11.6% vs. 28.6%, p=0.09). patients on daclizumab demonstrated decreased peripheral eosinophil counts from baseline to week 20 (-30â±20 /mm 3 vs. placebo +60â±30 /mm 3 , p=0.004). daclizumab-treated patients with elevated baseline serum eosinophil cationic protein (secp) had a significant reduction in secp from baseline to day 56 compared to placebo patients (p<0.01). peripheral eosinophils decreased significantly in daclizumab patients who had no asthma exacerbations compared to an increase in daclizumab-treated patients with at least one exacerbation (p=0.032). conclusions. daclizumab reduces time to and frequency of exacerbation in treated asthmatics. the mechanisms of this effect remain to be fully elucidated, but initial findings suggest that associated reductions in circulating eosinophil and eosinophil products may play a role in these therapeutic effects. hae is a genetic deficiency causing a decrease in c1 esterase inhibitor levels. it is a rare condition (approximate prevalence 1:10000 to 1:50000 individuals). it is manifest by acute attacks of swelling which can involve the larynx (a potentially life threatening condition), the gi tract (causing a syndrome similar to an acute abdominal catastrophe) or the skin and soft tissue of the patient including the limbs, face and external genitalia. there is no approved therapy for acute attacks in the usa. pathogenesis of this potentially fatal condition is thought due to excessive activity of plasma kallikrein. dx-88 is a specific and highly active (ki 40 pm) recombinant inhibitor of human plasma kallikrein undergoing evaluation in hae and cardiopulmonary bypass. it is an animal free product. a double blind, randomised (5:1 drug to placebo) placebo controlled dose ascending study of dx-88 in acute attacks of hae was run in the usa and the european union. four dose groups of 12 patients were to be included in the study. the doses were 5, 10, 20 and 40 mg/m2. the primary outcome variables were proportion of patients achieving a significant clinical response within 4 hours of administration of therapy, and safety. secondary endpoints included site response, dose response, and median time to significant response by site and dose group. dx-88 met its primary endpoint; 72% of dx-88 patients had a significant improvement within 4 hours, (placebo response rate 25%, difference 47% p= 0.0169). patients receiving dx-88 responded in a median time of 70 minutes. there was no difference in proportions of cases that responded within 4 hours between the three anatomical sites. time to significant response did not significantly differ between dose groups and anatomical sites. the safety profile was comparable for patients treated with dx-88 and placebo, in terms of saes and aes. in conclusion, dx-88 in this double blind study was shown to be statistically superior by 47% to placebo and to be at least as safe as placebo. the drug is effective at treating any hae site, including the larynx. dx-88 represents an important advance in the management of hae. ar is among the most common chronic childhood disorders, and is most effectively treated with intranasally inhaled glucocorticosteroids (ics). recent evidence suggests that intranasal ics therapy may suppress hpa axis function and decrease growth velocity. this study reports 1-year follow-up data on 24 children (12 female, 9 african american), aged 6 to 14 years (mean 10.4 years) at entry, enrolled in a long-term growth trial of intranasal taa for treatment of ar. primary measures included height velocity, bone mineral density (bmd), serum osteocalcin and salivary cortisol levels. all subjects followed their expected age-appropriate growth velocities. average growth velocities were 5.6 and 5.8 cm/year for girls and boys aged < 12 years, respectively, and 5.5 and 7.7 cm/year for girls and boys aged > 12 years, respectively. mean (+ std) bmd was 1.27 + 0.12 and 1.22 + 0.14 gm/cm2, mean serum osteocalcin levels were 89.1 + 25.5 and 114.5 + 32.7 ng/ml, and mean salivary cortisol levels were 16.7 + 8.0 and 11.5 + 6.5 nm/l at entry and 1-year follow-up, respectively. these results demonstrate no significant effect of one year of intranasal treatment with taa on growth velocity or hpa function in children with ar. 13 patients 6 males, 7 females ranging in age from 66 years to 12 years, received (o) in doses ranging from 2 to 17 injections over 4 to 34 weeks. patients were evaluated with symptoms scores, pulmonary function (pft), blood eosinophil count (bec), medication use, and allergy skin test. st were initially performed by prick and if negative, by intradermal (id) (1/1000 w/v) and if further negative by id (1/500 w/v). skin tests were performed immediately before and 20 minutes after the administration of o. there was no change in pft or bec. however there were significant reductions in symptom scores, including nocturnal asthma (<.0001), exercise tolerance (<.0001), sense of well being(<.0001). in addition, parenthetically there was a reduction in nasal symptoms (<.0001). there were also significant declines in medication use especially for albuterol (<.0001). st declined in all patients evaluated and dramatically in many. for several individual allergens st went from prick positive to id (1/500 w/v) negative. moreover st declined in all instances further, 20 minutes after the administration of o except for 2 patients. in addition to asthma one of the patients had allergic bronchopulmonary aspergillosis and another had allergic fungal sinusitis. three of the patients tested positive on prick test to mouse antigen and in each instance this became negative after administration of o with all 3 patients tolerating o without problem. we conclude: (1.) administration of o is associated with an improvement in symptoms and decreased use of medications (2.) in addition there is a prominent decline in st which is more marked 20 minutes after administration of o compared to immediately prior to administration the explanation of this finding is not apparent. (3.) o appears to be safe to administer to patients even if they demonstrate specific ige to mouse antigen by st a.p. baptist * , t.s. tang, j.l. baldwin, ann arbor, mi. introduction: academic medical institutions are under pressure to shorten or eliminate resident elective rotations due to federal work-hour restrictions. it is unknown if this will affect knowledge and referral patterns for resident and faculty physicians. the objective of this study was to examine the factors associated with resident and faculty perceived knowledge and referral patterns towards allergy/immunology (a/i). methods: a questionnaire was sent to 375 primary care physicians at one academic center. it addressed past history of a/i referrals, referral intentions for conditions that may be seen by allergists, and perceived a/i knowledge. independent variables included history of an a/i rotation, gender, department, years in training/practice, and history of referral to an allergist. results: 228 (61%) completed surveys were returned. using logistic regression with forward selection, we found in the resident physician cohort those with advanced years in training or history of an a/i rotation were more likely to have increased past history of referral to an allergist (or = 4.09, or = 3.81), increased referral intention for chronic sinusitis to an allergist (or = 3.51, or = 5.18), and increased perceived a/i knowledge (or = 2. . for the faculty physician cohort, those with a history of an a/i rotation were more likely to have an increased perceived knowledge about a/i (or = 5.64-6.18). in addition, pediatric faculty physicians were more likely to refer asthma (or = 5.62) and chronic eczema (or = 17.09) to an allergist than faculty from other departments. finally, faculty physicians with a past history of referral to an allergist were more likely to have refer asthma (or = 10.64) and allergic rhinitis (or = 9.78) to an allergist. conclusion: the factors associated with resident and faculty physician perceived knowledge, referral history, and referral intentions towards a/i differ. for residents, senior training level and a history of an a/i rotation appear most important. for faculty, history of an a/i rotation, pediatric department, and past history of referral to an allergist appear most important. given that a history of an a/i rotation may increase perceived knowledge and referral patterns towards a/i, eliminating a/i rotations in medical institutions may have important consequences for patients and the field of a/i. because recent anecdotal reports suggest that hbv may be an effective treatment for patients with multiple sclerosis (ms), there has been a movement of patients to zealous lay practitioners to receive multiple and repeated bee stings. since this practice has real risk of possible fatal allergic reactions as well as emotional and economic costs, properly conducted studies of safety and efficacy are needed. the purpose of the present phase i pilot study was to evaluate the safety of hbv extract in patients with pfms. a total of 9 evaluable bee venom non-allergic patients with pfms (21-55 years) were enrolled and were randomly divided into 4 groups, each receiving an increasing allergen dose immunization schedule for one year. hyperreactivity to hbv was evaluated by questionnaire, px and hematologic, metabolic and immunologic tests including skin tests. any possible beneficial responses to therapy were evaluated by questionnaire, functional neurologic tests (fnt) and changes in measurement of somatosensory-evoked potentials (seps) prior to and at the completion of the study. no serious adverse allergic reactions were observed in any of the 9 subjects even at the highest doses of bee venom therapy. although 4 of 9 subjects had worsening of neurologic symptoms during the course of bee venom therapy, requiring termination, this response could not be ascribed to side effects of the hbv or to a spontaneous worsening of the neurologic disease independent of treatment since there was no correlation of these events with the bee venom injections. of the remaining 5, 3 felt that the therapy was beneficial.there were no changes either in fnt or seps measured during the study. the present report represents the first controlled study evaluating the safety of hbv as a possible adjunctive treatment for multiple sclerosis. while this preliminary safety study suggests that administration of repeated injections of hbv in a step-up dosage regimen in appropriately selected non-hbv allergic patients with ms had no serious adverse allergic reactions, because of the small number of subjects studied, it does not permit conclusions regarding efficacy and therefore provides little evidence to support the use of hbv in the treatment of ms. much larger carefully conducted multi-center studies would be required to establish efficacy. i. finegold * , new york, ny. rush immunotherapy (rit) has benefits for inducing immunologic desensitization in shorter periods of time than conventional immunotherapy. however, there is an increased risk of systemic reactions utilizing accelerated schedules. in the past radiocontrast media reactions have been significantly reduced using a standardized protocol. a modification of this protocol was used prior to rit. the oral premedication consisted of 50 mgm prednisone 12 hours, 7 hours and 1 hour prior to rit, fexofenadine 180 mgm at 7 hours and again 1 hour prior to the procedure, and ranitidine 300 mgm, 7 hours prior to rit. patients tolerated the premedications well. a signed consent was obtained prior to rit. patients were desensitized in an office setting with appropriate therapy for anaphylaxis available without an indwelling intravenous line. injections generally began with 1/100 dilution of the anticipated maintenance solu-tions and were doubled every 20-30 minutes for about 2-3 hours and then the patients were observed for 1 hour or more prior to discharge. in this manner patients were desensitized in 2-3 half day sessions. the patient was injected again in 1 week, 2 weeks and then 4 weeks. doses were increased if they were not at a full maintenance dose. if reactions occurred this process was appropriately decreased. utilizing this technique 46 patients were treated. the average age was 35.5 years old (14-56 years of age) 72 % were males.72% of patients had allergic rhinitis, 25% asthma, 3% insect allergy. patients with complicated medical illnesses were excluded. nine patients had systemic reactions requiring an injection of epinephrine. among the symptoms experienced were wheezing, nasal congestion and flushing. no patient required a second injection of epinephrine, intravenous fluids, or hospitalization. the patient reaction rate was 21 % of patients treated or 9 in 1073 injections or 1% of all injections given during the rush protocol. these results are typical of the increased rate of reactions to rit. however, these systemics were mostly very mild and responded to therapy and in only one case was there a delayed 4 hour reaction. thus premedication while not preventing systemic reactions seemed to modify them to make rit in an office setting a practical and effective therapy. we have previously shown that oligonucleotides consisting a novel 3'-3'linked structure and synthetic immunostimulatory motif cpr (r is a synthetic purine moiety), referred to as second-generation immunomodulatory oligonucleotides (imos), induce potent th1 immune responses and prevent ovainduced allergic asthma in mouse models. in the present study, we examined local and systemic immune responses of imos following intranasal (i.n.) administration to naive mice. these studies showed that imos produce higher levels of serum and local il-12 compared with il-6. we next examined the ability of s.c. and i.n. administrated imos to reverse ova-induced th2 immune responses in a murine model of asthma. treatment of ova-sensitized and challenged mice with imos by either route of administration suppressed il-5 and il-13 levels with an increase in ifn-gamma secretion in spleen cell cultures and lung homogenates. imos decreased levels of serum ige and igg1 and induced higher levels of total and ova-specific igg2a titers in serum and balf. while both routes of imo delivery showed similar efficacy on serum and balf immunoglobulin levels, s.c. delivery resulted in stronger systemic effects on the spleen cell cytokine production and the i.n. route of administration produced stronger local effects on the lung cytokines. imos also decreased eosinophils in balf and suppressed inflammatory cell infiltration and goblet cell hyperplasia in lungs. these effects in lung were superior with i.n. administration of imo than did with s.c. administration. further studies to understand the effect of low multiple doses against a single higher dose of i.n. administered imos in naive mice suggest that low multiple doses induce strong th1 responses locally while the single higher dose produces higher systemic responses. these findings suggest that second-generation imos containing cpr dinucleotides potently reverse ova-induced th2 immune responses with strong th1 type cytokine induction in ova-sensitized and challenged mice and i.n. delivery is superior to s.c. delivery in reversing ovainduced airway inflammation and mucosal secretion. showed ait was medically inappropriate, lacked documentation of necessity for initiation or continuation and/or had strong contraindications in 12/18 (67%) of the reviewed records. method: we audited a convenience sample of our own records for documentation of necessity for (continuing) ait, using a checklist based on the oig report which included the following 6 criteria: an appropriate diagnosis; specific justification; exclusion of strong contraindications; signed, informed consent; a written physician order, and at least annual re-evaluation for continuing ait. results: the first 50 charts in our alphabetized file of 80 total ait charts were reviewed. the patients (21 m, 29 f) ranged in age from 9-63 yr (mean, 40 yr). the mean duration of ait was 2.3 yr (range, 0.25-12 yr). indications for ait included allergic rhinitis alone (68%) or with asthma (26%) or chronic sinusitis (2%), asthma alone (2%) and venom anaphylaxis (2%). all had initial documentation of necessity. seven charts (14%) had no written order to initiate ait. nine (18%) lacked a signed consent form. no strong contraindication was found. (peak flow documentation showed the asthma cases were well-controlled. three episodes of ait-induced anaphylaxis requiring epinephrine had occurred, but ait was subsequently resumed and well-tolerated.) seventeen (34%) charts had no documentation of re-evaluation during the preceding 6-12 months, including 3 with no documented re-evaluation during the previous 24 months or more, of ait. discontinuation of ait was "considered" for 3 patients after 1.5, 7, and 10 yr of ait, respectively, but all were still receiving maintenance doses. the estimated time for completing this review was 15-20 min per chart. inter-pretation: an internal chart review for adherence to ait practice parameters using a checklist such as ours can quickly and easily assess documentation for medicare reimbursement and patient safety. objective(s): immunotherapy is an accepted mode of treatment for children suffering from allergic rhinitis and allergic asthma. this study demonstrates that rapid allergen vaccination (rav) can be as safe as conventional allergen vaccination (cav) in children. rav may also improve patient adherence. study design: 148 pediatric patients, 1-18 years of age, diagnosed with allergic rhinitis and/or mild to severe asthma underwent rav over a 2 1/2 hour period in an office-based setting. all patients were premedicated with prednisone and h1 antagonists for 3 days prior to the procedure. patients were monitored for reactions during the procedure and continued on a cav schedule. adherence was reviewed subsequently. results: a total of 148 pediatric patients underwent rav utilizing a 2 1/2 hour protocol. of the 148 patients, 88 of them were male (59.5%) and 60 of them were female (40.5%). 143 (96.6%) of the patients had allergic rhinitis, 118 (79.7%) had asthma, and 23 (15.5%) had chronic sinusitis. during the procedure, 8 patients (5.4%) experienced systemic reaction, and none experienced true anaphylaxis. seven of the eight patients who suffered a systemic reaction were patients with asthma on inhaled corticosteroids, three of the eight patients were male (37.5%) and five of the eight were female (62.5%). every systemic reaction occurred within 15 minutes of the injection. treatment usually included one or a combination of the following: nebulized breathing treatment with albuterol, two sprays in each nostril of azelastine, and diphenydramine taken orally or intramuscularly. every patient that was treated was discharged within two hours, and no one required treatment with subcutaneous epinephrine, treatment for recurrent symptoms or hospitalization. all of the patients continued with a conventional allergen immunotherapy regimen following the rapid protocol to reach their maintenance dose. typically, 6 months of build-up period was saved. patients reached efficacious dosages almost immediately. adherence rates were 95.3%, 90.5%, and 79.7% at 3, 6, and 12 months respectively. conclusions: effective doses of allergen vaccine can be safely reached using a 2 1/2 hour protocol annals of allergy, asthma & immunology for children. advantages of rav over cav include improved adherence with almost immediate clinical efficacy, and decreased costs. introduction: studies involving allergen immunotherapy (ait) have furthered our understanding of cat allergen, proteases, and dust mite allergen. the impact of these data has not been assessed. objective: to evaluate ait prescribing trends over 12 years (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) focusing on new prescriptions before and after published data regarding specific prescribing recommendations. methods: a retrospective review of 38, 400 ait prescriptions from a centralized allergy and extract laboratory database was performed with respect to published literature on findings and recommendations regarding the ubiquitous nature of cat allergen, effective dosing of cat antigen, combining protease containing extracts with those susceptible to degradation, dust mite antigen dosing, and the use of house dust versus dust mite antigen. results: 1) in 1993, cat allergen was included in 10% of new allergy immunotherapy prescriptions reviewed; in 2003, it was included in 45% of new prescriptions, a statistically significant increase (p<0.00001). 2) over the past 11 years, the mean quantity of cat antigen included in new prescriptions has been unchanged at 1ml/prescription. only 5% of new prescriptions were written at the manufacturer recommended maintenance dose of 4ml, significantly less than those written below this level (p <0.00001). 3) there was no significant change in the percentage of extracts combining antigens with proteases with antigens susceptible to degradation. in 1992, 64% of prescriptions containing alternaria and/or cockroach antigen were mixed with one or more antigens susceptible to degradation; in 2003, 56% of these extracts were still being combined. 4) the mean volume of dust mite mix contained in these prescriptions was between 2-2.99 ml/10ml. 5) prescriptions containing dust mite rose from 20% in 1992 to 65% in 2003. the use of house dust was unchanged over the same period at 1.5% of prescriptions. conclusions: although the percentage of cat containing extracts has increased significantly, dosing of cat allergen has remained unchanged despite published literature recommending higher volumes. protease-containing allergen prescribing patterns have not significantly changed from [1996] [1997] [1998] [1999] [2000] [2001] [2002] [2003] young stage of life is a crucial period for aeroallergen sensitization, considering the immaturity of the neonatal immune system which may lead to t-cell anergy or a deviation towards th2-type response in mice. the aim of this work was to evaluate the influence of oligodeoxynucleotides containing cpg motif (cpg-odn) on ovalbumin (ova) and blomia tropicalis (bt) immunization in early life compared to adult stage. three days old a/sn mice were immunized with ova in al(oh)3 and boosted on 10th and 30th day after immunization (dai) and bled on 37th dai. groups of mice received 4mg of cpg-odn or control-odn associated with ova on immunization and boost. some animals from the three groups (ova, ova+cpg, ova+co) were killed at 20 days old and the spleen was collected and prepared to culture. eight to 10 weeks old female a/sn mice were immunized with ova in al(oh)3, boosted on 14th dai and bled on 21th dai. groups of mice received 50mg of cpg-odn or control-odn associated with ova on immunization. animals from the three groups were challenged 3 months after immunization and bled 7 days later. similar protocols were performed using blomia tropicalis (bt) extract in the place of ova. neonate and adult co-administration of cpg-odn with ova or bt were able to significantly decrease specific ige antibody levels and increase igg2a production. moreover, cpg-odn decreased specific igg1 levels in the bt immunization. the co-administration of control-odn in neonates decreased anti-ova igg1 production. after ova-challenge 3 months later of adult immunization, a similar response was detected in the group that received cpg-odn associated with ova, which showed a decreased anti-ova ige and igg1 and enhanced igg2a levels. analysis of cell division of neonate lymphocytes by flow cytometry showed a decreased proliferation in ova+cpg mice group under ova stimulation. this effect was seen either in b cells and t cells. the results showed that immunization with both allergens, ova and bt, associated with cpg-odn decreased the type i hypersensitivity response. the pattern of antibody production suggests th1-type response induced by cpg-odn, and the establishment of memory th1 response. this findings may imply that the use of cpg-odn in early life seems to be beneficial as an strategy to modulate or to prevent the development of allergic diseases. n. horne * , a. capetandes, m. frieri, east meadow, ny. introduction: it has been shown that dust mite allergens can affect the functioning of airway epithelial cells (winton et. al. br j pharm 124:1048 , 1998 . previous experiments showed ca549 aggregate in the presence of dermatophagoides pteronyssinus (dp) (capetandes et. al. am j clin path, 121:25, 2004) . it is hypothesized that epithelial damage is associated with airway remodeling characterized by fibroblast dysregulation. to evaluate the response of fibroblasts to damaged airway epithelial cells, the bioactivity of serum-free conditioned media from dp-treated ca549 cells (dpcm) was assayed with nhlf. methods: all experiments used insulin-transferrin-selenium supplemented dmem (its). dpcm (generated with ca549 treated with 300 au/ml dp; alk-abello) was added to 50% confluent nhlf for 24 hours at 37oc and 5% co2. its from cultured ca549 without dp (its cm) was added to 50% confluent nhlf (control). cell morphology and density were evaluated by microscopy and mtt assay, respectively. data were analyzed by anova followed by student-neuman-keuls (snk) at p<0.05 with power analysis. results: 50% confluent nhlf treated with dpcm showed decreased cell density (figure 1) , and increased aggregation relative to its cm or its alone. nhlf in its alone or with direct addition of 300 au/ml dp to its (dp its) showed no or weak aggregation. aggregated nhlf showed 100% viability and grew to confluence when subcultured to serum-supplemented media. conclusion: nhlf aggregation was greater, and cell density was lower with dpcm than with its, its cm, and dp its. this suggests that dp-treated ca549 cells release an unidentified factor or factors that contribute to nhlf aggregation and possible apoptosis. identification of these factors would increase the understanding of the fibroblast response to epithelial cell exposure to dp which may be involved in airway remodeling, a feature of chronic severe asthma. j.t. zimmermann * , y.c. huang, m. frieri, east meadow, ny. introduction: budesonide has been demonstrated to inhibit il-8, tgf and gm-csf in ragweed and dust-mite (dm) stimulated human alveolar epithelial cells (j allergy clin immunol 209:3a, 2002; ann allergy asthma immunology 90: p79, 2003) . rantes production and expression in dm and il-1 -stimulated a549 cells has recently been shown to be inhibited by budesonide (allergy asthma proc, in press 2004). histamine is known to influence immune response by regulating cytokine synthesis (allergy 47:024-1992) . in this study we examined the effect of budesonide in the presence of histamine on il-8 production and expression by a549 cells. methods: a549 pulmonary epithelial cells were cultured in dmem for 24 hours in 5% co2 in the presence of 1:20 of 10, 000 au/ml dm, 10 -6 m histamine and 10 -7 m budesonide. il-8 production was measured by a sensitive elisa and il-8 mrna was measured by qualitative rt-pcr using standardized primers for il-8 with a gapdh control. results: a549 cells were stimulated by dm (65-101 pg/ml) (p<0.01). budesonide alone decreased il-8 levels to 38 pg/ml (p<0.05), and in combination with histamine further decreased il-8 levels to 23 pg/ml (p<0.001). relative intensity of il-8 mrna expression was reduced 12-fold in dm-stimulated cells and 2-fold in control cells by budesonide. conclusion: budesonide in combination with histamine showed greater inhibition of il-8 production by a549 cells than budesonide alone possibly by increasing cell membrane permeability. c.r. oliveira * , a.e. fusaro, j.r. victor, e.a. futata, c.a. brito, a.j. duarte, m.n. sato, sã£o paulo, brazil. antigen-driven bystander suppression induced by oral tolerance could be an interesting approach in the allergy field, considering the high incidence of new allergens sensitization in atopic individuals. to address the influence of non-related allergen exposure on the type i hypersensitivity response to the mite blomia tropicalis (bt) or ovalbumin (ova) in mice and to verify oral tolerance effect in the bt/ova co-immunization model. groups of mice were immunized with bt extract and two weeks later submitted to ova-immunization, or first immunized with ova or co-injected with bt and ova. ova feeding was performed five days prior co-immunization. ige abs were estimated by means of passive cutaneous anaphylaxis reaction and specific ab and cytokines secretion by elisa. mice sensitized with bt and then exposed to ova developed an enhanced ige response to itself and to ova, but such response has not been observed when ova-immunization was prior to btimmunization. co-injection of bt and ova led to a dominant ige response toward ova over bt and vice-versa for the igg1 response. ova feeding prior co-immunization decreased ige, igg1 and igg2a ab levels against ova in parallel to a bystander suppression, which avoided the outcome of bt-sensitization. these mice showed increased ifn-?secretion levels induced by antigen-specific stimulus. furthermore, effectiveness of oral tolerance was also related with ova amounts employed in the co-immunization since ova feeding prior co-immunization with low amounts of ova led to inhibition of ige ab response to both allergens, whereas inhibition of specific and non-related antigen igg ab response was broken. the results evidenced that, depending on allergenic potential, new allergen exposure may exert an adjuvant effect on the primary sensitized allergen. the bystander effect to non-related allergen by oral tolerance should be an interesting mechanism to control new allerrationale. cd4 + cd25 + regulatory t cells from patients with atopic asthma undergo apoptosis when stimulated by specific allergen. antihistamines are used to control allergic inflammatory diseases, but their influence on treg cells is currently unknown. the present study investigates the influence of desloratadine (d) on apoptosis of cd4 + cd25 + regulatory t cells in atopic patients having dust mite induced allergic disease, including allergic asthma. methods. patients (n=6) with positive skin prick tests to house dust mite allergens and a clinical history of allergic upper and/or lower respiratory symptoms were treated with d 5mg (schering-plough, usa) once daily for 10 days. no patients used systemic corticosteroids, while theophylline and other medications were stopped at least 72 hours before blood collection. blood was sampled before and after treatment with d. the control group was comprised of 6 individuals without allergic respiratory symptoms and with negative skin prick-tests. peripheral blood mononuclear cells (pbmc) were isolated over a ficoll density gradient and stimulated by specific allergen (dermatophagoides farinae) and in a control series by phorbol myristate acetate (pma). pbmc were labeled with anti-cd4, cd25, cd95, and bcl-2 monoclonal antibodies. the annex-inv-propidium iodide (anv-pi) test was done. lymphocyte subpopulations and apoptosis were analyzed by flow cytometry. results. after treatment with d, atopic patients had no significant differences in the number of cd4 + cd25 + cells. during co-culturing of pbmc from patients treated with d with specific allergen a significant increase in cd4 + cd25 + cells was observed. simultaneously, treatment with d led to a significant increase in expression of the antiapoptotic protein bcl-2 in cd4 + cells. this data paralleled the decrease of cd95 expression on cd4 + cells. there was also a decrease in treg cells in the late stages of apoptosis (anv + pi + cells) in d treated patients. conclusions. the antihistamine preparation desloratadine prevented allergen-specific apoptosis of cd4 + cd25 + t regulatory cells in patients with atopic asthma. the type 1 histamine receptor may be involved in the regulation of apoptosis and/or survival of cd4 + cd25 + t cells. a.g. palma-carlos * , m.l. palma-carlos, lisboa, portugal. introduction: solar urticaria porphyrin corresponds to sun sensitivity to a 4000 a wavelengths and is due to disturbances of posphorin metabolism. photosensitivity can cause urticaria, erythema, polymorphic solar eruption and in more severe cases vesicles and bullae. photosensitivity was confirmed by light test. methods: protoporhyrin in rbc, uro and coproporphyrins in urines, copro and protoporhyrins in faeces and porphyrin precursors have been studied un all the cases of solar urticaria seen in the last few years. results: in 10 patients, 8 female, 2 males, a diagnosis of porphyria has been confirmed by laboratory methods. clinically 4 patients presented solar urticaria and 6 solar erythema or polymorphic solar eruption. 7 patients, 8 female, 2 males (21-54) presented also neuro visceral symptoms: abdominal pains (7) vomiting (4) asthenia (7) constipation (3) muscle pains and paresia (3) depression (2) anesthesic reactions (2). in 2 cases the symptoms were associated with anticonceptional drugs. the conjunction of clinical history with laboratory data has allowed to confirm the diagnosis of 2 cases of protoporphyria erytheropoietica, one triggered by anticonceptionals, 6 cases of coporporhyria hereditaria and 2 cases of porphyria variegata. this group comprises one case of erythropoietic protoporphyria induced by estrogens not previously reported. conclusions: research of porphyrins must be done in all the patients with solar urticaria and erythema. the incidence of sun sensitivity in mixed hepatic porphyriaalso presenting neuro-visceral symptoms which can be drug dependent suggests that porphyria study must be mandatory in suspected cases. a.r. narayan 1* , j. kaplan 1 , v. sirvan 1 , a. chandrasekaran 2 , c. goodwin 2 , l. goodwin 2 , l. guida 3 , m. frieri 1 , 1. east meadow, ny; 2. manhasset, ny; 3. bayshore, ny. rationale: our division has reported that nitric oxide (no) from tracheal epithelium can mediate induction of il-8 (american journal of respiratory critical care medicine 151:642a, 1995) . increased levels of il-8, ltb-4, and no in mononuclear cells (mnc) from cystic fibrosis (cf) patients were noted.(pediatric asthma allergy immunology 10: 101-7 1996) . no in mnc from cf was increased by stimulation with aspergillus fumigatus (asp) and rhdnase (j. clin allergy immunol 105:320p 2000). tnf-is produced from bronchial epithelial cells (bec) in the presence of mnc of normal controls (nc). rantes, a chemokine that facilitates leukocyte migration, is associated with airway inflammation in cf, and asp sensitization in cf patients could amplify pro-inflammatory cytokines such as il-8, tnf-, and rantes. methods: we studied the mrna and protein expression of tnf-, il-8 and rantes in a 65 year-old cf patient and a nc. 1x10 6 mncs from the patient and nc were stimulated with 21î¼g/ml of asp with bec at 10x10 5 and 10î¼g/ml rhdnase. mrna expression was studied by real time pcr (taqman chemistry abi prism 7700) and protein levels by elisa. results: tnf-production in supernatants of mnc with bec from nc increased from 8 pg/ml to 64 pg/ml with rhdnase. whereas in cf patients the tnf-mrna expression was greatly enhanced over nc but declined in the presence of rhdnase (fold difference: 8.37-7.70). there was no difference in expression levels of nc (fold difference: 4.0-3.9) with rhdnase treatment. il-8 expression in mnc with bec of patients increased 2.4 fold compared to nc (1.5 fold). rhd-nase decreased asp stimulated levels to 1.72 fold in patients compared to nc (2.9 fold). mnc and bec rantes production in patients increased from 49 to 1025 pg/ml and decreased to 570 pg/ml with rhdnase, but increased from 1134 pg/ml to 1775 pg/ml in nc. in contrast, rantes mrna expression in all groups was higher in nc but decreased with rhdnase in both cf and nc (37-26 fold vs. 14-10 fold). conclusion: rhdnase can increase no and decrease pro-inflammatory tnf-and rantes in cf patients stimulated with asp. rhdnase could lead to augmented bactericidal activity and epithelial defense by enhancing no production and decreasing the expression and production of tnf-, il-8 and rantes. this is a case report of two steroid dependent atopic dermatitis patients who both responded to treatment with omalizumab. patient a is a 39 year-old white male who presented with a history of severe atopic dermatitis for 10 years along with concomitant mild persistent asthma and allergic rhinitis. he had previously received monthly triamcinolone injections, methotrexate and doxycycline with limited response. on presentation he had mild improvement on a regimen of alternate day prednisone 30mg, fexofenadine 180mg bid, and zafirlukast 20mg bid. however, attempts at weaning prednisone were unsuccessful. omalizumab was initiated at a dosage of 150mg every four weeks. the patient remains prednisone dependent yet free from atopic dermatitis. patient a's diagnostic work up determined a total ige of 33.1 ku/l. the patient's specific ige tests were positive for grass mix, weed mix, maple, white pine, peanut, strawberry, gluten, soybean, wheat, oat, and dog dander. specific ige tests were negative for cat dander, egg white, milk, goose feathers, chicken feathers, mold mix, dust mix, and fish mix. patient b is a 23 year-old white male who presented with a history of severe full body atopic dermatitis along with mild persistent asthma and allergic rhinitis. he also was receiving triamcinolone injections with limited success. he responded to alternate day prednisone 30mg, desloratidine 5mg bid, and zileuton 600mg bid. attempts at weaning prednisone failed until initiation of omalizumab 375mg every two weeks. his atopic dermatitis is now under control with once daily desloratidine and omalizumab every two weeks. he was successfully weaned off of corticosteroid medication and has begun an immunotherapy regimen. patient b's diagnostic work up determined a total ige level of 1159 ku/l. patient b's specific ige tests were positive for weed mix, tree mix, maple, peanut, strawberry, dust mix, cat dander, dog dander, egg white, milk, oat, wheat, goose feathers, and chicken feathers. specific ige tests were negative for grass mix, white pine, mold mix, gluten, soybean, and fish mix. these two case reports reveal significant response to omalizumab in severe steroid dependent atopic dermatitis. further research is strongly indicated considering the quality of life issues with severe atopic dermatitis and potential side effects to long-term corticosteroid treatment. eosinophilic esophagitis (ee) has been described in children and is characterized by high levels of eosinophils (>20-24 eosinophils/high powered field [hpf]) in the esophageal mucosa. a recent report indicates that the combined prevalence of the eosinophilic gastrointestinal disorders may be higher than that of inflammatory bowel diseases (ibd). the presenting symptoms of ee mimick those of gastroesophageal reflux disease (gerd), and patients of all ages may experience a delay in time from onset of symptoms to diagnosis of ee. there is a paucity of data in the literature regarding the delay in time from symptom onset to diagnosis of ee. we report the data on four children three years of age and under with ee who experienced a significant diagnostic delay. four young children were referred to the pediatric allergy clinic with a diagnosis of ee (table) . three of the patients were male and one was female. the patients were 17-36 months of age at the time of diagnosis, and the diagnostic delay was 15.5-33 months (average delay 23.6 months). the most common presenting symptom was vomiting, and three of the four patients had a history of respiratory obstructive symptoms. all of the patients had received conventional treatment for reflux disease, and one had undergone a nissen fundoplication prior to diagnosis. in all four patients the esophageal biopsy revealed >20 eosinophils/hpf. three out of four patients had a family history of atopy, and two patients had a known history of a food allergy. skin tests to foods were positive in two patients and rast (radioallergoabsorbent) testing to foods was positive in one patient. recent data suggests that the diagnostic delay in ibd is decreasing as much as 50%. it is hypothesized that this may be due in part to an increased index of suspicion by health care providers. there is little data available regarding the diagnostic lag in children with ee. it is currently believed that chronic ee can lead to progressive esophageal scarring and dysfunction. stricture formation has also been described in children less than two years of age. given the reported increased incidence of this allergic gastrointestinal disease over the past decade, more data regarding the diagnostic delay of these conditions may be instrumental improving health care providers' awareness of this disorder. a. kohli-pamnani * , e. cooney, p. huynh, f. lobo, new haven, ct. introduction: cutaneous hypersensitivity reactions to amprenavir, an hiv-1 protease inhibitor, are reported in up to 28% of treated patients, of whom 4% have severe or life-threatening rashes, with treatment discontinuation required in 3% of cases. we report a case of successful desensitization to amprenavir, for recurrent maculopapular exanthem, in an hiv-infected patient with late stage disease and limited antiretroviral (arv) agent options. methods: the patient is a 40 year-old caucasian female with late stage hiv disease (absolute cd4 count 30 cells/mm 3 , hiv rna 200, 000 copies/ml) and multiple arv intolerances, who developed a severe generalized maculopapular eruption sparing mucous membranes six days following initiation of a regimen comprised of amprenavir, lopinavir/ritonavir, zidovudine, and lamivudine. a similar reaction occurred following re-challenge with amprenavir alone (600 mg oral formulation bid via percutaneous endoscopic gastrostomy (peg) tube), despite concurrent administration of oral prednisone 20 mg/day and loratidine 10 mg/day. results: skin prick testing with amprenavir 0.01 mcg/ml was negative, whereas intradermal testing with 0.1 mcg/ml was positive with a 7 mm wheal and 20 mm flare. percutaneous and intradermal testing with normal saline was nonreactive. the positive histamine control (skin prick only) yielded a 5 mm wheal and 20 mm flare. subsequently, incremental doses of 0.025 mg, 0.1 mg, 0.25 mg, 1 mg, 2.5 mg, 7.5 mg, 25 mg, 50 mg, 100 mg, 300 mg, 600 mg, and 1200 mg of amprenavir oral solution were administered via peg tube at 20 to 30 minute intervals (table) . the patient successfully tol-erated amprenavir desensitization and has remained on therapy without recurrence of rash out to 16 months of follow-up. conclusions: desensitization may permit continued use of amprenavir in patients with a history of amprenavirinduced maculopapular eruptions who have limited alternate treatment options. efforts aimed at characterizing the mechanism of amprenavir cutaneous hypersensitivity reactions seem warranted, given the frequency of reactions and the limited number of arv agents available for patients with late stage hiv disease. doses of oral solution were administered by peg tube at twenty to thirty minute intervals. introduction: beta-lactam (bl) allergy is the most common drug allergy. in most cases, ige antibodies are specific to the bl nucleus. however, sidechain-specific ige (scsige) to bl has been described. despite this, skin testing (st) to bl other than penicillin (pcn) is not commonly performed. we report a case of a 31 year-old female with a selective allergy to pip, and describe the utility of st to this agent to confirm scsige. methods: st to prepen (pp) and pcn g was carried out with percutaneous (pc) testing followed by intradermal (id) testing. st was also carried out with ampicillin (amp; 1 mg/ml), pip (60 mg/ml at pc level; 1 mg/ml at id level-a non-irritating concentration), and pip/tazobactam (tbm; 60 mg/ml at pc level; 1 mg/ml at id level) to exclude the possibility of selective allergy to tbm. case report: a 31 year-old white female with crohn's disease was hospitalized for treatment of intra-abdominal abscesses. she had immediate flushing and urticaria during an infusion of pip/tbm approximately 2 years prior. the allergy/immunology service was consulted for pcn st because her primary service preferred to empirically treat with pip/tbm. she had received imipenem approximately 8 months prior without untoward reaction. st to pp and pcn g was performed with negative responses and adequate controls. she subsequently received pip/tbm, but developed flushing and urticaria during her initial infusion, consistent with an ige-mediated reaction. two months later, st to pp and pcn g was repeated. in addition, st to amp, pip, and pip/tbm was performed. she had positive responses to pip and pip/tbm with negative responses to pp, pcn g, and amp, implying selective ige-mediated potential to pip. she tolerated an oral challenge with pcn vk 250 mg immediately following st without untoward reaction. conclusion: we have described a case in which st to pip was useful for diagnosing scsige in our patient, who had 2 prior reactions consistent with ige-mediated pathogenesis. this information will be helpful in identifying antibiotics she can safely receive in the future. this case supports the utility of st to bl in addition to pcn, in evaluation and management of patients with a history of adverse reaction to bl that may reflect presence of scsige. a.r. vaishnav * , b.s. bochner, baltimore, md. we report the case of a 39-year-old caucasian male with a 15-year history of asthma, two episodes of eosinophilic pneumonia and chronic peripheral eosinophilia with baseline eosinophil counts around 1000/î¼l who developed amnesia and elevated cardiac enzymes in february 2004. another physician had added montelukast in january 2004 and because of a good response advair was decreased from 500/50 to 250/50 bid. in february his eosinophil count increased to 3430/î¼l and soon he developed left arm numbness, diplopia as well as left arm and leg weakness. at his local hospital, he was found to have elevated troponins and cpks. cardiac catheterization showed normal coronary arteries and good left ventricular function. soon after discharge, he developed confusion and global amnesia. brain mri showed diffuse uptake consistent with global inflammation and/or vasculitis. upon hearing this story, we told him to take 60 mg of prednisone and urgently come to our hospital for admission. within hours, his mental status and visual symptoms improved. physical examination was unremarkable except for subungual splinter hemorrhages. montelukast was stopped and 1 gm/day solu-medrol iv was started. endomyocardial biopsy 2 days later showed mild hypertrophy and fibrosis without eosinophils. repeat brain mri and mra were normal. chest ct showed multiple patchy infiltrates with a new central cavitary lesion in the right lobe. other labs included a negative anca, ana of 1:40, normal complements and csf. our differential diagnosis included churg-strauss syndrome (css) versus hypereosinophilic syndrome (ihes) with myocardial, neurological and pulmonary involvement. serum tryptase was normal as was fluorescent in situ hybridization for the fip1l1-pdgfr fusion gene. based on this, along with the new pulmonary cavitary lesion on chest ct, the diagnosis of css was made. after 3 days of iv steroids, he was switched to 60 mg/day of prednisone. upon discharge his eosinophil count was 350/î¼l on 60 mg of prednisone. after all procedures were completed, he was also started on aspirin 325 mg daily to prevent thrombotic and thromboembolic complications. he was seen in follow-up in clinic and cytoxan 100 mg daily was started. his prednisone is being tapered. he is tolerating the treatment well and so far has had an excellent clinical and laboratory response. introduction-c1-esterase inhibitor (c1-inh) deficiency is a rare disorder classified into acquired and hereditary forms. both entities are distinguished by recurrent angioedema without pruritus or urticaria. the upper airway, head, neck, extremities and gi tract are typically involved. the inherited form usually presents in the first or second decade accompanied by a family history. the acquired form more commonly presents after the fifth decade. in acquired c1-inh deficiency, serological evaluation reveals low levels of c4, c1-inh, and c1q and a normal c3 level. levels of c1q are normal in the hereditary form. the acquired form may be associated with lymphoproliferative disorders and autoimmune disease. acquired c1-inh deficiency is typically recognized before the underlying malignant condition is diagnosed. clinical regression has been reported in patients whose underlying disorder responds to treatment. methods-a case report of a 57-year old female who developed repeated episodes of facial angioedema requiring hospitalization preceded by one year of vague abdominal pain and cramping. data-laboratory investigation of this patient revealed c4<10mg/dl (16-47 mg/dl), c1q<3.5mg/dl(5.0-8.6 mg/dl), ch50<20u/ml(31-66 u/ml), and c1-inh at 3 mg/dl(6-25 mg/dl) with a 51% activity (>68% normal). c3 was 138 mg/dl(90-180 mg/dl). other laboratory evaluation was notable for a normal cbc with diff, comprehensive metabolic panel, amylase, lipase and spep pattern. ige was elevated at 173 ku/l. hematology was consulted. ct of chest, abdomen, and pelvis were nondiagnostic. peripheral blood flow cytometry revealed a small distinct pop-ulation consistent with a clonal b-cell lymphoproliferative disorder. a repeat test showed skewing towards lamba light chain expression. bone marrow biopsy and aspirate were essentially normal except for erythroid hyperplasia on danazol treatment. the physical examination was without any masses or lymphadenopathy. the patient remained asymptomatic after beginning danazol although elevations in rbc, hgb, hct and absolute lymphocyte count have developed. conclusion-acquired c1-inh deficiency is rare cause of recurrent angioedema that has been reported in small cohorts and case presentations. we report another case report that reinforces the need for physicians to evaluate for concomitant lymphoproliferative disorders. chronic urticaria is a distressing condition usually associated with poor quality of life and poor response to symptomatic therapy. a wide variety of causes and mechanisms have been described and in some patients the cause remains unknown. rare cases have been associated with thyroid antibodies and some responded to thyroxin, even in the absence of overt thyroid disease. case report: a 54-yr-old obese white female presented with a history of persistent urticaria/angioedema for 4 mo. urticaria involved various parts of the body and individual lesions usually lasted < 24 hr. it was often accompanied by facial edema. oral diphenhydramine 25mg qid caused only little improvement. the patient could not suspect any offending factors. review of systems and past medical history were unremarkable, except for hyperthyroidism at age 12 yr that was treated with "medication" for 3 yr. on physical examination there were several urticarial lesions though she has been taking diphenhydramine. the thyroid appeared normal regarding size, shape and texture. various second generation antihistamines and doxepin were of little help. laboratory evaluation revealed total serum ige of 7 iu/ml (normal <200 iu/ml) and ch50 of 73 u/ml (normal 22-60 u/ml), but high tsh of 15.2 iu/ml (normal 0.4-5.0 iu/ml) and low free t4 of 0.58 ng/dl (normal 1.45-3.48 ng/dl). her antithyroglobulin titer was 32 iu/ml (normal <100 iu/ml) but the antimicrosomal titer was highly elevated at 382 iu/ml(normal <20 iu/ml) consistent with hashimoto's thyroiditis. thyroxin therapy was initiated at a dose of 100 mcg/d which resulted in marked improvement within one week and the patient was able to discontinue doxepin and other antihistamines. because of marked drop in tsh to 0.08 iu/ml, the thyroxin dose was reduced to 50 mcg/d. her thyroid function tests became normal within two months. she did not experience any recurrence of urticaria or angioedema during over 6 mo of follow-up so far. conclusion: patients with chronic urticaria/angioedema, especially women, should be screened for thyroid autoantibodies and if positive, thyroxin therapy might bring impressive remission in urticaria. hypersensitivity pneumonitis (hp) results from an abnormal immunologically mediated response to an environmental antigenic trigger. typically patients with hp experience transient fever, hypoxemia, muscle and joint pain, difficulty breathing, fatigue, weight loss, and cough. there are two clinical presentations of hp differentiated by onset and resolution of symptoms. patients experiencing acute hp experience the onset of symptoms 2 to 9 hours following exposure to a particular antigen and resolve in 1 to 3 days without specific treatment. however, patients with chronic hp experience symptoms that persist for months to years when exposed to a recognized cause of hp. a 55 year-old caucasian male was referred to our practice with acute bronchitis. his referral was secondary to a history of allergic rhinitis, chronic asthma, gastro-esophageal reflux disorder, and sinus surgery. this non-smoking male was a machine operator at a local factory. he was doing well until he began working in the presence of a heat induction machine and metal lubricating spray called multan ea20 (hinkle surface technologies), i.d. no.: 238073. multan ea20 is a product containing naphthenic petroleum distillates, amine salts, amine soap, triethanolamine hexyl, hexylene glycol, ethanol, sodium petroleum sulfonate, and triazine. this lubricant is known to irritate the eyes, skin, and respiratory tract. recent studies indicate that thermal decomposition of triazine, which readily occurs during metalworking, results in the production of formaldehyde. formaldehyde is a known carcinogen, as well as respiratory, skin, eye and digestive tract irritant. the patient developed serious respiratory health problems resulting in work absence and eventually hospitalization. while hospitalized he received a high resolution ct scan which showed diffuse ground glass appearance, and multiple attenuation areas of the lung which are consistent with hypersensitivity pneumonitis. the patient was treated with corticosteroids, and antibiotics with eventual normalization of lung parenchyma by chest ct. this represents the first reported case of hypersensitivity pneumonitis resulting from exposure to the metalworking fluid and respiratory irritant multan ea20. a.j. ham pong 1* , f. chan 1 , s.l. bahna 2 , 1. ottawa, canada; 2. shreveport, la. sexual intercourse has been known as the route for semen hypersensitivity reactions to the seminal fluid protein or to a contaminating drug or food. we report a case of anaphylaxis to cephalexin-containing semen by ingestion. history of present illness: a 37-year-old woman developed a systemic reaction after swallowing semen. since the couple frequently practiced fellatio without any reactions, her husband suspected cephalexin that he was taking over 2 days (500 mg qid). her reaction began in less than 10 min and peaked over 50 min; first as itchy oral mucosa followed by wheezing, flushing of the face and upper chest, and nausea. diphenhydramine 50 mg was administered po at 30 min and the reaction subsided over 1 hr. past medical history: she had generalized urticaria to oral penicillin more than 15 yr earlier and had positive penicillin skin test. she also had allergic rhinoconjunctivitis, mild intermittent asthma, and positive skin test to house dust mite, cat and dog epidermals, and pollens of grasses, trees, and ragweed. she also had oral allergy syndrome to certain fresh fruits and tree nuts; hazelnuts caused also diarrhea and colic. neither the patient nor her husband ate any nut-containing food on the days preceding the reaction. family his-tory: allergic rhinoconjunctivitis in the mother, sister and brother. eval-uation: the patient sought allergy evaluation about 2 yr after the reaction. she and her husband gave a consent. her serum ige was 108 iu/ml and skin test positive to penicilloyl polylysine 6x10-5 mol id, but negative to cefazoline 10 mg/ml for prick and 3 mg/ml for id. she had negative prick test to her husband's seminal plasma both before and after his intake of cephalexin 500mg qid for 2 days. using a cephalexin bioassay sensitive down to 6.25 mcg/ml, the medication level in the husband's urine at 120 min post last dose was 2400 mcg/ml and in his serum at 135 min was 12 mcg/ml, but was undetectable in the semen collected at 90 min. conclusion: this is probably the first case report of systemic reaction to ingested semen. the reaction occurred in an atopic, penicillin-sensitive woman and seems to be caused by cephalexin excreted in the semen or through urine contamination (1 drop = 120 mcg). in addition to her avoiding penicillin and cephalosporins, she was advised to avoid her husband's semen while him taking such drugs and for at least 3 days afterwards. background:asthma causes serious morbidity & mortality all over yet most asthma educational evaluations and programs have an urban rather than rural focus.there is lack of data on rural north dakota(nd). this nd survey aimed to study knowledge & awareness of management strategies for asthma like use of metered dose inhalers, spacer devices and peak flow meters. methods:the qualitative survey addressed attitudes and beliefs on asthma & its triggers;knowledge of medication delivery routes & use of hospitals and provider visits.a simple questionnaire was self-administered at 6 rural clinics in small towns in southeastern north dakota.10 questions explored asthma knowledge, extent of family history of asthma & factors aggravating asthma.it was voluntary & the population surveyed included patients, family, teachers, employees at nursing homes, the local hospital & clinics. results:of 297 surveys completed 61% had no asthma in the family.of those with a family history(39%), 59% had 1 person with asthma, 28.6% had 2 & 12.3% had 3 or more.respondents were aged 18-90 years & 81.1% were female.see table. discussion:our survey was rural & comparison with urban areas may help in planning for asthma education & resource allocation.61% denied asthma in their family. similarly, nonurban alaskan natives have a lower incidence of asthma compared to nonnatives.our results show a lack of awareness in our area.grain dust exposure was perceived as an asthma trigger but without evidence, local research is needed.in our study smoking was considered a trigger by 91.5% & this may be real.there was lack of awareness of peak flow meters & their role in asthma care.a study showed that compared to other areas even rural nurses used peak flow meters less often to assess and monitor asthma.this suggests a need for comprehensive asthma educational programs in rural areas that are based on national guidelines.we had only a moderate followup rate at 60%.in comparison, one study had 21% missed scheduled follow-ups. exercise induced asthma was known to only 50% of our group. conclusion:the need for better patient-provider communication has been highlighted by the lack of awareness of proven strategies to combat asthma.the use of a simple survey has revealed many unknown facts about asthma awareness in rural nd.further study to determine cost effective solutions to achieve national targets for asthma management are essential. aim: in most studies of asthma, the emphasis was on changes in large and middle airways. the aims of this study:(1)to observe the morphologic changes in small airways and lung tissue(salt) in guinea pig asthma models(gpam); (2)investigate the role of vcam-1, eotaxin, nf-b and ap-1 in the inflammation of asthma; (3)explore the functional variation of alveolar type 2 cells in asthma; (4)evaluate the effects of inhaled glucocorticoids on the above annals of allergy, asthma & immunology parameters in salt of asthma models. methods: (1)the gpam were established by ovalbumin challenge. five groups were divided: control, asthma day 4 and day 14, intraperitoneal dexamethasone, and budesonide inhalation group. (2)the expression of vcam-1, eotaxin, nf-b and ap-1 was determined by immunohistochemical technology and rt-pcr; the dna binding activity of nf-b and ap-1 by electropharetic mobility shift assay. (3)the balf phospholipide concentration was measured by phosphorus detection. results: (1)significant inflammatiom with infiltration of eosinophils and lymphocytes in salt was observed in asthma groups. (2)the protein levels of vcam-1, eotaxin, nf-b and ap-1 expressed in the salt were significantly elevated in asthma groups than those in control. (3)the mrna expression of vcam-1 and eotaxin of lung tissue homogenate was significantly increased in asthma group. (4)the dna binding activity of nf-b and ap-1 of lung homogenate was significantly increased in asthma group. (5)the balf surfactant represented by phospholipides was significantly decreased in asthma groups. (6)glucocorticoids in different ways of intake provided significant effects on the inflammatiom of salt. conclusion: (1)widespread and significant inflammation with eosinophilic infiltration existed in the salt in gpam, indicating asthma is a disease involving the whole airway and lung system. not only there were structural changes, but also impaired function of alveolar cells. the role of small airway inflammatiom may be of great importance. (2)eotaxin and vcam-1 actively mediated the process of inflammatiom, nf-b and ap-1 played important roles in the regulation of vcam-1 and eotaxin. the up-regulation of the above mediators in gpam was expressed in salt similar to that in central airways. we have recently reported that immunomodulatory oligonucleotides (imos) consisting of a novel structure and synthetic cpr or r'pg (r and r' are synthetic purine moieties) stimulatory motif effectively prevent ovainduced asthma in mouse models. in the present study we examined whether these novel imos (hyb2055 and hyb2087) can reverse established allergic airway inflammation in mice. balb/c mice sensitized and challenged with ovalbumin (ova) were evaluated for airway hyperresponsiveness (ahr) to methacholine. following ova-sensitization, mice were randomized and treated with placebo or 30 mg or 60 mg/dose of hyb2055 or 2087 s.c. during the following 10 days. two days after the final treatment, mice were rechallenged with ova and pulmonary functions were recorded. mice treated with either imo were significantly protected from both early (ear) and late (lar) allergic response, airway hypersensitivity and hyperreactivity to methacholine, bal and peribronchial eosinophilia, bal and serum il-5, and total serum ige as compared to vehicle-treated ova-sensitized and challenged animals. there was a significant increase in immature lung dendritic cells (cd11c+cd45r+) together with increase in serum il-10 levels and significant decrease in lung dc2 type cells (cd11c+ cd11b+cd8a-) as compared to vehicle-treated ovalbumin-sensitized animals in the lungs following treatment with either imo. these data suggest that both imos are effective and potent in attenuating key features of established allergic airway inflammation in bronchial asthma, and this effect could be mediated via decreasing lung dc2 cells and increasing immature dendritic cells. additionally, imos contain a novel 3'-3'-attached structure that provides higher metabolic stability and may permit lower and/or less frequent dosing. hyb2055 was evaluated for its safety and immunopharmacology in a phase 1 clinical trial in healthy human volunteers. introduction -hemoglobinopathies are common in southern europe and mediterranean area the more frequent being thalassemia and sickle cell disease. aside of the major hematological diseases of homozygotic patients minor forms are frequent, thalassemia minor and sickle cell trait. in these cases mycrocytosis with decrease of red cell volume and mean corpuscular volume or abnormal erythrocytes are found and can lead to hemorheologic disturbances in bronchial circulation and bronchial hypereactivity. the rationale of this study is to evaluate the incidence of asthma in hemoglobinopatic patients allergic to house dust mites.methods-from 4 000 patients seen in the last 4 years in an out-patient allergy clinic 44 cases of hemoglobinopathies have been confirmed by red cell count, hemoglobin electrophoresis, assays of hemoglobin a2, f, and s, and sickle cell test.all these patients had allergic disease characterized by clinical history, skin prick test to aeroallergens total and specific ige (rast-cap-feia) and respiratory function evaluation. results -hemoglobinopathies: betathalassemia 39 cases, betadelta thalassemia 2, sickle cell trait 2, hemoglobin c.1. aside of 4 cases of urticaria, all the patients presented respiratory allergy, rhinitis in 12 cases of thalassemia and 1 hemoglobin c, asthma with or without rhinitis in 25 cases of thalassemia and 2 cases of sickle cell trait. therefore asthma was present in 67, 5%. in a group 491 of respiratory allergic patients without hemoglobinopathies, 57% had asthma and 43% only rhinitis. conclusions -the prevalence of asthma is higher in hemoglobinopathies.(p<005 square chi test). hemorheological changes probably greater rigidity of red blood cells and capillary bed can contribute to bronchial hypereactivity. detection of hemoglobinopathies must be done in asthmatic patients with slight anemia or mycrocytosis. we investigated the role of aspirin-exacerbated respiratory disease (aerd) as a risk factor for the development of airway remodeling. patients with aspirin intolerance develop hyperplastic sinusitis with fibrosis and nasal polyposis. we speculated that similar mechanisms could be acting in the lower airway and that these individuals would demonstrate more severe asthma and evidence for airway remodeling. the epidemiology and natural history of asthma: outcomes and treatment regimens (tenor) study is a multicenter observational study of subjects with severe or difficult-to-treat asthma. baseline data were compared between subjects 18 years who reported asthma exacerbation following aspirin ingestion and those who did not. the primary measure of asthma remodeling was the maximally achieved post-bronchodilator spirometry. adult subjects with aerd (n=469) were compared with aspirin tolerant subjects (n=3009). subjects with aspirin intolerance demonstrated evidence for airway remodeling as shown by lower post-bronchodilator predicted fev 1 . in addition, they were more likely to have physician-assessed severe asthma, to have been intubated, and to have required high-dose inhaled corticosteroids or oral corticosteroids in the previous 3 months. we conclude that aspirin intolerance is associated with remodeling of both the upper and lower airways. asthma is a complex and variable disease with two main components, airway inflammation and smooth muscle dysfunction. according to national and international asthma guidelines, subjects with persistent asthma can be classif ied into one of three categories (mild, moderate, or severe) based upon lung function, symptoms, nighttime awakenings, medications and exacerbations. though it is widely believed that there is a high degree of variability in both pediatric and adult subjects with asthma, few studies have compared variability between them. therefore, an analysis of previously conducted asthma studies was undertaken to evaluate pediatric subjects aged 4-11 years (n=276) and adult subjects (n=85) previously receiving short-acting beta 2 -agoinsts alone in seven double-blind, randomized, 12-week trials. the analysis is limited to subjects who were randomized to placebo in these trials. during the study, subjects exhibited marked fluctuations in asthma severity. despite the fact that all subjects met criteria for moderate or severe asthma at baseline, 27%, 18%, 48% and 8% of weeks for pediatric subjects and 9%, 14%, 71%, and 6% of weeks for adults were spent in the intermittent, mild, moderate and severe categories, respectively. a summary of severity classification based upon symptoms and albuterol use is presented below. in addition, based upon pef % predicted, 62% and 52% of weeks were spent in the intermittent/mild category for pediatric and adult subjects, respectively. however, fluctuations in pef occurred frequently, with 46% of pediatric subjects and 30% of adult subjects experiencing 10 changes in severity based on pef over 12 weeks, indicating more variability in pediatric subjects. this analysis clearly demonstrates that asthma is a variable condition and that both pediatric and adult subjects frequently move between severity categories. furthermore, there are marked differences in severity classifications between pediatric and adult subjects. asthma severity, and consequent optimal therapy, cannot adequately be assessed by discrete, point-intime assessments of lung function, frequency of albuterol use, or asthma symptoms, especially in the pediatric age group. studies suggest that there may be an association between single nucleotide polymorphisms (snps) in the beta 2 -adrenergic receptor gene (adrb2) and the response to beta 2 -adrenergic bronchodilators. polymorphisms at codon 16 have been at the center of this debate. therefore, a retrospective analysis of six large, randomized trials was conducted to evaluate clinical responses to salmeterol administered with fluticasone propionate (fsc) 100/50mcg bid for 12 weeks in patients ( 12 yrs) with moderate or severe asthma and differing adrb2 polymorphisms at codon 16. baseline demographics were similar for all genotype subgroups. all measures of asthma improved over baseline and were similar across arg16/gly subgroups at 12 weeks. pairwise comparisons were conducted between genotypes and there were no differences other than fev 1 as noted in the table. findings from this retrospective analysis show that regardless of arg16/gly genotype, clinical response to salmeterol with an ics was similar during chronic dosing. although prospective studies are needed to fully understand the effect of adrb2 polymorphisms on response to therapy with a long-acting beta 2 -agonist, this analysis suggests that therapy with salmeterol and an ics together is appropriate for caucasian patients with differing arg16/gly genotypes. coronary artery disease(cad) and asthma commonly coexist in adults. iv dipyridamole thallium scintigraphy is considered a safe non-invasive technique in the evaluation of cad when patients can't exercise. dipyrdamole is a purine that blocks reuptake of extracellular adenosine increasing serum adenosine levels after iv administration.this results in a transient coronary vasodilation increasing the sensitivity of the thallium study. methylxanthines reverse the effects of endogenous adenosine by competitively antagonizing adenosine at local purinoreceptors. we report a case of a 69 yr.old female with a 30 year history of stable mild persistant asthma treated with daily salmeterol and low-dose fluticasone. she was referred to cardiology for atypical chest pain. within minutes of a standard iv dose of dipyridamole the patient reported chest tightness, cough, and wheezing. all these symptoms, typical of her past asthma flares, were abolished within 5 minutes of an aminophylline infusion and before albuterol was administered.the stress test was completed and was normal. inhaled adenosine induces bronchconstriction and is used as a probe for bronchial hyper-responsiveness. commercial iv adenosine preparations used in treating supraventricular tachycardia are reported to induce bronchospasm in known asthmatics. since serum levels of adenosine are transiently increased following iv dipyridamole, bronchospastic symptoms during dipyridamole stress tests should not be unexpected. an earlier study found an increased incidence of wheezing in 39% of patients with known copd/asthma undergoing dipyridamole stress tests despite pretreatment with beta agonists. the marked decline of theophylline use for chronic asthma in recent years may actually be increasing the incidence of this risk. cardiologists performing thallium stress tests are familiar with the risk of asthma flares after dipyridamole. they use iv aminophylline frequently to reverse several types of clinical dipyridamole reactions, including bronchospasm. on the other hand, we are impressed that allergists are relatively unfamiliar with this association. allergists and asthma specialists as well as asthmatic patients should be aware of risk of acute dipyridamole induced bronchospasm during elective cardiac stress testing. it has been shown that adhesive molecules are involved in inflammatory diseases of the lungs such as bronchial asthma. the purpose of the study was to measure and establish possible difference in serum levels of soluble icam-1 in 42 atopic patients (patients with allergic rhinitis and patients with bronchial asthma) in comparison with 28 patients without atopy (patients with asthma without rhinitis); whether there is a difference in sicam-1 levels between groups of 26 patients with allergic rhinitis and asthma in comparison with group of 16 patients with allergic rhinitis only and also in comparison with 10 healthy controls. results of the study have substantiated statistically significant difference in sicam-1 levels between all groups of patients in comparison to healthy control, but no statistically significant difference in sicam-1 levels between patients with and without atopy (z=-1.738) or between patients with allergic rhinitis and bronchial asthma in comparison with group of patients with allergic rhinitis only (z=0.00). conclusion: icam-1 is an important marker of inflammation in patients with allergic rhinitis as well as in those with bronchial asthma. atopic status does not influence differences in sicam-1 levels. although mean sicam-1 levels were higher in patients with allergic rhinitis and bronchial asthma (312.71 ng/ml) in comparison with mean sicam-1 levels in patients with allergic rhinitis only (279.69 ng/ml), no statistically significant difference was noted in sicam-1 levels between these groups of subjects, i.e. asthma itself did not contribute to statistically significant increase of sicam-1 levels. k. nadarajah * , g.r. green, m. naglak, abington, pa. objective: to study the various clinical outcomes of penicillin skin testing (pst) in a community-based hospital and to determine the percentage of patients who have an antibiotic modification and the choice of antibiotic used following the result of pst. method: this study is a retrospective chart review of all in-patients who were penicillin skin tested during a period of 6.6 years(jan 1997 to july 2003). information was collected on 101 patients using a detailed data collection form. data was summarized using descriptive statistics including frequencies and percentages. results: of the 101 patients penicillin skin tested, 92 had a negative test, five had a positive test and in four patients the test was indeterminate(histamine controls were negative). eighty six percent of the patients had a history of penicillin allergy, 7.9% had a history of cephalosporin allergy and 5.9% had a history of both penicillin and cephalosporin allergies. the duration of antibiotic prior to pst ranged from zero to 18 days. there was a 72.7% (67/92) reduction in the use of vancomycin, a 24.9% (23/92) reduction in the use of floroquinolones and a 7.6% (7/92) reduction in the use of aminoglycosides following pst. aztreonam was used in 19.6% (18/92) of patients before pst and in zero patients after pst. use of penicillin-based drugs was 49% (45/92) after pst and cephalosporin use increased from 4.4% (4/92 (all third generation cephalosporins)) to 47.8% (44/92( second and third generation cephalosporins)). i.e., 97% of patients with a negative pst received a penicillin or a cephalosporin. vancomycin usage was higher among pst positive patients. endocarditis was the diagnosis in 22% of all patients skin tested and staphylococcus aureus (23.8%) and enterococcus (16%) were the most common organisms on culture. there were no serious adverse reactions to the use of penicillins or cephalosporins when used following a negative pst, and there were no adverse reactions to penicillin skin testing. conclusion: in the population studied, pst lowers the usage of vancomycin, floroquinolones and aminoglycosides and increases the use of penicillins. third generation cephalosporin usage was increased by pst in our study. overall, pst results in antibiotic modification that could lower the emergence of multi-drug resistant organisms and vancomycin resistant enterococcus. the aim of the study was to find a new marker which could be easily done to predict about possible allergy development in infants. preventive procedure is always economically better than treatment itself.food allergens are able to stimulate lymphocytes even in prenatal period. immunological maturity of newborns is still in the center of our interest because its dependence on many different factors. the question was if cd30 activity in cord blood and in 1-year-old children correlates with development or not of allergy? the examined group consists of 120 newborns(58, 2% of boys). breastfed were only 87 children until 3-months-old and 62 until they were 6-monthsold. total ige from cord blood and mothers' blood taken at delivery were measured using unicap. immunological detection was done using fluorocytometry(becton nad dako). additionally parents were questionaired( family atopy). our reaults show that positive family atopy history(group a) or elevated level of ige( group b) or symptoms of allergy in first 3 months of life(group c) does not correlate with cd30 antigenicity. in all subgroups cd30 frequency was 1%. our findings show unfortunately that cd30 cannot be a predictive marker for allergy development. rationale: the etiology of eosinophilic esophagitis (ee) is unknown and the relationship to a type i allergic response is unclear. comparison of patients with positive and negative type i allergy testing may further clarify ee and determine what are the most common food allergens. methods: this is a retrospective chart review of medical records from january 2000 to january 2001 of children 1 year of age with biopsy confirmed ee (n = 42). ee was defined as having 15 eosinophils per high power field on esophageal mucosal biopsy. patients were grouped according to positive (n = 26) and negative (n = 16) allergic responses on skin or radioallergosorbent testing (cap rast, pharmacia). skin testing with commercial extracts (hollister-stier laboratories and greer laboratories) was performed in an allergist's office. a wheal of 3mm greater than the negative control on skin testing or ige > 0.35 ku/l on rast was considered positive. we performed fisher exact analysis to determine if associations existed between a type i allergic response and factors such as age, symptoms, peripheral eosinophilia, and personal and family history of atopy (asthma, allergic rhinits, and atopic dermatitis). results: ee patients with a positive type i allergic response were significantly younger than those with a negative response (mean 4.6 yo, median 4.5 yo, range 1 to 10.3 yo versus mean 8.5 yo, median 7.8 yo, range 1 to 21 yo; p = 0.0065). vomiting as a presenting symptom was significantly increased in the allergic population (85%, 44%; p = 0.014) and abdominal pain as a chief complaint was significantly increased in the non-allergic population (31%, 69%; p = 0.026). personal and family history of atopy and peripheral eosinophilia were similar between groups. the most common allergens were cow s milk (54%), peanut (46%), egg (42%), soybean (38%), and wheat (27%). conclusion: patients diagnosed with ee who present at a young age or who present with symptoms of vomiting may have a type i allergic response contributing to the esophageal eosinophilic inflammation. consistent with food allergies in children, milk, peanut, egg, soybean, and wheat were the most common foods found with allergy testing. because the positive predictive accuracy of food annals of allergy, asthma & immunology testing is only about 50%, the challenge remains to develop a specific plan to confirm causative foods such as through oral challenges or elimination trials. rationale: the relationship between specific ige and igg levels in atopic individuals is unknown. increasing antigen exposure is expected to increase igg production, however, the influence on ige levels is unclear. to further clarify this relationship the following studies were conducted. methods: three hundred and sixty tandem determinations of specific ige and igg levels in 65 individuals were collected using the immunocap 100 instrument (pharmacia diagnostics) and commercially available reagents. only subjects who had ige levels > 0.35 ku/l and igg determinations > 2 mg/l to the same specific allergenic species were included in the study. specific ige and igg determinations were to alternaria alternata, aspergillus fumigatus, penicillium notatum, cladosporium herbarium, felis domesticus, canis familaris, dermatophagoides farina, and periplaneta americana. correlation coefficients were calculated using excel (microsoft). results: the review of this data set yielded 55 tandem determinations of specific ige and igg levels in 25 atopic individuals. the most common ige sensitization was to alternaria. sixtyfour percent of individuals had specific ige directed against alternaria. the lowest ige response rate was for cat and roach. only 20% of individuals had an ige response to those specific species. correlation coefficients (cc) were calculated between specific ige and igg levels and were positive for all eight species tested. the highest cc was for dog (n = 6, cc = 0.92) followed by aspergillus (n = 13, cc = 0.71), penicillium (n = 6, cc = 0.69), alternaria (n = 13, cc = 0.62), cladosporium (n = 8, cc = 0.35), cat (n = 5, cc = 0.31), and roach (n = 5, cc = 0.16). the lowest correlation was for dermatophagoides farina (n = 7, cc = 0.03). conclusion: when individuals with measurable specific ige and igg levels are considered, there appears to be a positive correlation between specific ige and igg levels for many allergenic species. factors that influence the correlation are likely to be both genetic and environmental. identifying this link and a clinical application are our next challenge. recently, protein microarray tests are competing with traditional allergenspecific in vitro assays. while the advantages of microarrays in allergy testing are attractive, microarray analysis alone has not been very effective in reducing analysis time and automated microarray equipment typically is much larger than its benchtop counterparts. we have incorporated microarray technology into an automated microfluidic cartridge that provides rapid allergen testing using a compact, low-cost, desktop instrument. the injection molded microfluidic cartridge (fig. a) contains arrays of miniature pumps and valves that direct reagents independently to a solid phase reaction area. standard ige (nibsc) as well as allergens were immobilized within the cartridge to form a protein microarray. a small desktop analyzer actuated the pumps in the cartridge to automatically carry out a chemiluminescence-based elisa reaction. total ige quantitation was performed in a 10 minute reaction. fig.b shows the resultant image of a dilution series of immobilized ige. concentrations ranged from 0.625 to 1000 iu/ml corresponding to 1.9 fg to 3 ng ige per spot. the average cv value for ige quantitation was 12% showing good linearity (r2 = 0.99) and 6 fg sensitivity. this quantitative ige curve is used to eliminate the effects of cartridge variability. specific ige detection was demonstrated using 3 extracts, d. pteronyssinu(dp), a.fumigatus and b. verrucosa(bv). a 2-step, 20 minute reaction elisa technique was employed. fig. c and d show the resultant images for bv and dp positive serum, respectively. the sensitivity of the dp specific ige test was further investigated using a positive class 1 serum sample confirmed by mast. dilution of the class 1 serum with negative control serum could then be detected on the cartridge down to an 8 fold dilution, resulting in a limit of detection in the range of 0.08-0.31 iu/ml for dp specific ige. we have demonstrated total ige and allergen-specific ige detection with total analysis times less than 30 minutes in a convenient, low cost system using a microfluidic-based microarray cartridge. such rapid diagnosis allows physicians to provide treatment while the patient is still in the office. background: accurate allergen skin tests (ast) are the basis of optimal care of the allergic patient. completing these tests on the first visit can expedite the diagnosis and treatment and offer patients(pts.) efficient use of their time and increase satisfaction. appropriate pre-visit preparation is necessary to reduce the variables that affect ast outcomes. both positive and negative skin test controls are necessary to assure the ast are reliable. methods: we conducted a retrospective chart review of 1, 248 sequential pts. skin tested at an allergy practice, in order to examine the success of our pre-visit instructions and to identify other medications that may affect ast. pts. were given verbal and written instructions to discontinue antihistamines and other histamine-1 receptor antagonist medications (h-1a) 5 days before their visit. they then underwent ast only if adequate positive (histamine) and negative (saline/vehicle) controls were obtained. results: only 53(4.2%) of the 1, 248 pts. had inadequate histamine response (ihr). of these 53 pts., 23(1.8%) had used h-1a within the prior 5 days. 18(1.4%) pts. had taken h-1a in the recent past, but stopped them at least 5 days prior to testing. 12(1.0%) pts. had no exposure to h-1a. the use of psychiatric medications in the pts. with an ihr was also examined. of the 18 pts. who discontinued h-1a for more than 5 days, 13(76%) were also taking various medications drugs used in the treatment of psychiatric disorders (ssris, benzodiazepines, atypical antidepressants & antipsychotics) which are not usually associated with h-1a. the group of 12 of the pts. with no prior use of h-1a included 10 (83%) who were also on various psychiatric medications. the high prevalence of psychiatric medication use in these two groups is in contrast to the to 23 pts. with recent use of h-1a in whom 43% were on psychiatric medications. conclusion: pre-visit patient education about discontinuation of h-1a can lead to successful first visit ast in the overwhelming majority of patients (95.7%).failure to stop h-1a at the appropriate time occurred in only 1.8% of the pts. 1.4% of pts. discontinued h-1 a for the recommended 5 day interval, but still had ihr. another 1% of pts. had an ihr yet no obvious use of h-1a. a few pts. taking psychiatric medications not normally associated with h-1a had ihr. further investigation into this issue is warranted. a. blaziene * , a. chomiciene, l. jurgauskiene, n. ciaponiene, vilnius, lithuania. background: sublingual specific immunotherapy (sit) is accepted as an alternative treatment to subcutaneous sit of seasonal allergic rhinitis. the aim of this study was to evaluate changes in allergen-specific ige and basophil degranulation test (bdt) before and after 1 year of sit with a grass pollen mix. methods: 6 patients (3 male and 3 female, aged 25-44 years) having sensitization to grass pollen with seasonal allergic rhinitis were treated with standardized grass pollen extracts. the blood samples were collected before and after 1 year of sit assessing allergen-specific ige using an elisa method and bdt using a direct immunofluorescent method employing monoclonal antibodies. results: the sit was well tolerated by all patients. an increase in total ige after sit was observed in all patients. specific ige concentration was increased in 2 (33.3%) patients, while the bdt result was greater in 3 (50%) patients after 1 year of sit. conclusions: allergen-specific ige and bdt may serve as markers to indicate the clinical efficacy of sit for seasonal allergic rhinitis patients. a.g. palma-carlos * , s.l. silva, m.l. palma-carlos, lisboa, portugal. introduction -the incidence of primary immunodeficiencies in patients attending an out-patient center for clinical allergy and immunology depends on the recruitment and on patients refered. methods -in the last 3 years 3 000 patients have been observed in lisbon clinical allergy center reporting allergic diseases or repeated infections or refered by specialists (ent, gynaecology, internal medicine, pediatrics, or gp.). screening for primary immunodeficiencies has been done by electrophoresis, assay of immunoglobulins and complement, igg subclasses and flux-cytometry for t, b and nk cells. results -102 cases (3, 4%) of primary immunodeficiencies have been diagnosed. humoral: 13 deficits of iga (12, 7% of immunodeficiencies), 7 of igg (6, 8%) 16 of igg subclasses (15, 7%) 11 of common variable immunodeficiency cvi-(10, 8%), 2 of igm (2, 0%). complement: 4 cases of c1 esterase inhibitor deficiency (3, 9%) cellular: 45 cases of mucocutaneous candidiasis (44, 1%) in fertile females, 2 cases of cd4 deficiency (1, 9%). combined: 2 cases of deficiency of the couple cd40/c40 ligand with igg deficiency (1, 9%).conclusions -in the present series chronic mucocutaneous candidiasis is the prevalent primary immunodeficiency, due to the number of patients refered by gynaecologists. aside of this group where a decrease of nk cells was the more common immunologic pattern, humoral immunodeficiencies are frequent, mainly cvi, iga, igg and igg subclasses deficiencies. the search for immunodeficiencies must be done in all patients with repeated infections attending immuno-allergology departments. a case of atypical c2 complement deficiency. c.c. randolph * , waterbury, ct. introduction:c2 complemennt deficiency occurs in 1 in 10, 000 individuals .it results in decreased opsonization and chemotaxis with increased prevalence ofsle and other rheumatic disordersas well as enhanced vulnerability to pyogenic infection.there are two types of c2complement deficiency :type 1(90%associated with sle)with no protein translation and type ii with absence of protein secretion.we present a 15 year old white female otherwise healthy with recurrent urticaria and angioedema with c2 complement deficiency. method:case report:a 15y/o w/f athlete presented with a two month history of recurrent hives and angioedema which she associated with ingestion of halloween candy .one week before evaluation she had hives with coconut as well.her history was othewise unremarkable except for recurrent uti's, annual sinusitis, pneumonia in 1998 as well as migraines.she denied sexual activity.her physical exam was normal.results:an evaluation for autoimmune disease revealed normal esr, ana, dsdna, mono and hepatitis serology as well as lyme titers however her ch50 was low17u/ml(normal 26-58u/ml)and evaluation of complement revealed c4 14mg/dl(normal 16-47mg//dl)and c2 <1.3mg/dl(normal 1.6-3.5mg/dl)with normal c3, c5-c9.her father had nor-malc4 but c2 was 1.4mg/dl (normal 1.6-3.5mg/dl)her sister had c2 of 1.5mg/dl and normal c4 and her mother had normal c2 and c4.her workup included positive prick skin test to ragweed, ash and grass and she was started on rhinocort and clarinex seasonally.she has been followed for one year with resolution of hives and is asymptomatic.her diagnosis had been confirmed by a pediatric rheumatologist.conclusion;we present an atypical case of c2 complement deficiency in an currently asymptomatic individual. background: we diagnosed a 10 month old male with cgd and aspergillus brain abscesses and pulmonary infiltrates. review of the literature suggests he is one of the youngest cases of cgd complicated by cerebral aspergillosis. case report: a ten month old male previously seen for unexplained persistent pulmonary infiltrates since 4 month of age presented to our hospital with lethargy, fevers, and increased irritability. ct scan of the head revealed severe hydrocephalus. multiple brain abscesses were found on surgical exam and cultured for aspergillus fumigatus. he had a history of failure to thrive and hypotonia since birth. family history was significant for parents that were second cousins. there was no family history of recurrent infections or childhood deaths. physical examination was significant for a child with weight and height in the 25% and 75%, respectively with persistent fevers >38.5 c, hypotonia, hepatosplenomegaly, and left lower lung field rales. initial immune workup was normal except for elevated total ige of 215 ku/l. cd4+ (526/m3) and cd8+ (332/m3) t cells were initially low, but later increased to normal levels without intervention. neutrophil oxidase activity assessed by dihydrorotamine 123 (dhr) oxidation was 0% of control. superoxide production assessed by cytochrome c reduction was zero. the patient was treated with interferon ? for presumed cgd. his aspergillus brain abscesses improved with intravenous voriconazole and caspofungun. the responsible organism for his recurrent pulmonary infections was not identified, but improved with antifungal therapy and broad spectrum antibiotics. the dhr test of both parents and siblings were normal. antibody testing showed a complete deficiency of the p67 protein with normal levels of gp91, p22, and p47 proteins. evaluation of the exons of the ncf-2 gene that encodes the p67 protein were normal. conclusion: mutations due to p67 protein deficiency are responsible for 3% of cases of cgd. there has been only one previously reported case of p67 deficiency without ncf-2 exon mutations in which a single point mutation in an intron of the ncf-ii gene was identified. fungal infections may complicate cgd, however cerebral aspergillosis is seldom encountered. this case annals of allergy, asthma & immunology reports a 10 month old with cgd due to a p67 deficiency without exon mutations complicated by cerebral aspergillosis. introduction: lymphoid interstitial pneumonia is an uncommon condition which is considered both as a disease per se or as an inflammatory pulmonary reaction to various external stimuli or systemic diseases; however, at the present time most of the cases remain idiopathic. we present a case of an infant who developed a lymphoid interstitial pneumonia associated to a deficit of interferon-production. methods: we describe a clinical case and review the medical literature. results: we present the case of a five months old male with a history of cough, respiratory distress and four previous hospital admissions regarded as a 'pneumonic' events from two months of age and four siblings dead (two of them with 'abdominal disease' and one of them with candidiasis) during their first year of life. the parents referred no consanguinity. the physical examination showed a malnourished child with signs of bilateral lung consolidation. oxygen supplementation and intravenous antibiotics were started. our patient didn't have neonatal history related to the present compliant. the chest film revealed a diffuse bilateral interstitial pattern which was confirmed by pulmonary ct scan. sweat chloride test, hiv and epstein barr virus serology test were all negative. an initial immunologic evaluation reported slightly increased leukocytic count, hypergammaglobulinemia, lymphocityc flow citometry and nitro blue tetrazolium reduction test in normal parameters. the open lung biopsy showed a thicked pulmonar interstitium with moderate number of lymphocytes with not atypical pattern. our patient was discharged with clinical improvement, ambulatory oxygen supplementation and inhaled fluticasone-salmeterol. in an ambulatory follow-up we evaluated lymphocytic production of cytokines and we found no levels of interferon-. we started weekly administration of oral transfer factor. the patient showed an excellent clinical improvement evidenced by no more oxygen dependence, weight gaining and absence of further hospital admissions. con-clusions: our patient represent a new linkage between lymphoid interstitial pneumonia and primary immunodeficiency characterized by interferonproduction deficit, maybe with a non previously described molecular defect and a probably autosomic recessive inheritance pattern; also we confirmed the transfer factor usefulness to induce gamma interferon endogeous production. objective: marijuana is a schedule class 1 psychoactive control substance more frequently used by the oriental societies( in the event of jubilation) causing altered state of conscience, euphoria, its prolonged use is followed by addiction and dependence.it contains more than 426 chemical entities with over 60 are of the cannabinoid i.e, cannabidiol (cbd), cannabinol (cbn), and delta-9, tetrahydrocannabinol (thc) the later on use has been associated with craving for further use of narcotics just to enhance the euphorient effects .the active ingredients i.e, delta-9-tetrahydrocannabinolthc) and other cannabinoids(thc) are liquid soluble at high concentrations which alters membrane function, resulting in alterations in immune cell response. cannabinoid has immunosuppressant properties causing impaired cell-mediated and humoral immune system activities, cytokine production, leukocyte migration and natural killer-cell(nk) activity resulting in reduction in the host resistance to bacterial and viral infection, (pms)with hiv infection are at higher risk of developing aids, infection by opportunistic bacteria, fungi, or viruses (pms), when compared to non marijuana smokers, have more respiratory ill-ness.cannabinoids also characteristically as being immunomodulators i.e, generally suppressing but occasionally enhance some immunological responses, some have suggested that the immunosuppressive effects of cannabinoids might be useful clinically; for example, in treating multiple sclerosis.as per clinical response cannabinoids had been found exacerbating existing allergies from antigenic complex (eliciting formation of specific antibodies/metabolites as a hapten, combining with a body proteins). methods .in the follow up studies including 18individuals(all males age25-60 years) with(pms).of more than 6 months having serum level of tch >10um, there have been 20 times reduction in the proliferation of t lymphocytes with a proportionate increase in the proliferation of b lymphocytes, reduction in the cytotoxic activity of t lymphocytes, reduction in macrophage activities i.e, phygocytosis rationale: patients with xla are subject to arthritis and cellulitis. these three relatives have had similar courses, suggesting infectious etiology. methods: two brothers and a cousin, from an african-american family known to carry xla, have been diagnosed with xla and chronically treated with ivig. all three have developed arthritis and cellulitis of a lower extremity. the eldest, now 23 years old, had a 5-month waxing and waning arthritis and cellulitis that was refractory to the oral antibiotics attempted. his younger brother had not yet accomplished pubertal changes at the age of 16, and was below 3rd percentiles for weight and height. he also had several months waxing and waning arthritis and cellulitis refractory to oral antibiotics. he and his cousin have had esophogastroduodenoscopies that show gastritis and duodenitis with heavy growth of an organism visualized by specific staining for h. pylori. the cousin has also experienced more recent weight loss and arthritis/cellulitis. results: the younger brother had a blood culture that grew a curved gram-negative rod. a subsequent culture at the state lab grew a similar organism that was urease-negative. the two brothers are at or near completion of a six-month course of ertapenem and gentamicin; they have had resolution of their arthritis and cellulitis. the younger has gained 10.3 kg and experienced his pubertal changes at the age of 17 years. con-clusions: there are prior reports of several different related organisms that can cause arthritis and cellulitis in patients with xla. these include helicobacter, campylobacter, and flexispira species. the urease-negative organism would not be flexispira, but may be a campylobactor species or helicobacter canis. the results from this family suggest that long-term combination iv antibiotics may be indicated for patients with this syndrome. slow virus infections have been reported to affect the central nervous system. parvovirus b19, herpes and cmv are usually not included in the diagnosis of cns diseases nor has been associated with cns manifestation in an immuno-compromised patient. in this abstract, we report on a neurological manifestation that can be linked to ebstein-barr/herpes virus and parvovirus-b19 in an immune deficient patient. a 50 year old female presented to our clinic with a history of chronic fatigue symptoms, daily arthralgias, frequent sinus infections and idiopathic tremors for the past 6 years. the patients neurological evaluation, including mris was found to be within normal limit, despite worsening tremors of her head and arms. a full clinical and labora-tory evaluation was performed which revealed igg subclass, low t-cell numbers and functioning, low response to specific antibodies and positive igm for ebv, parvo and cmv. the patients common immune deficiency coupled with chronic viral infection and worsening tremors led us to try high dose ivig (2g/kg divided over 3 days on cycles of every 3 weeks). within three months of starting ivig, the patient had a large reduction in her tremors and negative igms for parvovirus, herpes and cmv. given our findings, we suggest that ivig may play a role as a neuro-immune modulator, as has been shown in other neuro-immune diseases. we further suggest that the interaction between parvovirus b-19, herpes and cmv as part of the slow viruses, combined with underlying immune disorder may lead to neurological presentation and high dose of ivig can reverse the syndrome. purpose: to date, only two patients older than fifty years of age have been diagnosed with velocardiofacial syndrome (vcfs); this case represents the third such patient. methods: physical and laboratory findings of the patient are presented as a case report. results: we describe a sixty-six-year-old male, recently diagnosed with vcfs, confirmed by fluorescent in-situ hybridization (fish), was referred by his psychiatrist for comprehensive care. he had a history of cleft palate and learning disabilities as a child, with the onset of psychiatric illness in his late teens. although there was no history of cardiac disease, the patient had agenesis of the left renal artery as well as other vascular anomalies, including hypoplasia of the left a1 segement of the anterior cerebral artery. other findings included hypothyroidism, hypoparathyroidism, sensorineural hearing loss, and typical facies. he also suffered from chronic candidiasis. immunologic laboratory studies revealed over a 2:1 ratio of cd8:cd4 t cells with a decreased cd4 percentage, markedly diminished b cell percentage, decreased t cell mitogen responses, and markedly decreased b cell mitogen responses. nevertheless, total serum igg and specific antibody responses remained normal, with a low serum igm. conclusions: this patient meets the criteria for the diagnosis of vcfs; only the third reported case diagnosed over the age of fifty. though his immunologic findings may be consistent with his diagnosis, they may be due in some part to immunologic senescence given his age. background: the classic presentation for patients with xla and mutations in the btk gene includes marked hypogammaglobulinemia, absent b cells, and significant sinopulmonary infections beginning at 4-5 months of age. typically, mds presents with anemia, leukopenia, monocytosis, and thrombocytopenia with recurrent infections, skin rash, and hepatosplenomegaly. many of these subjects demonstrate chromosomal abnormalities including monosomy 7. we describe a case of mds presenting with some features of both conditions. clinical history: we present a 2 year old male with a suspected diagnosis of xla, chronic sinopulmonary disease beginning at 12 mo, diffuse bronchiectasis, and agammaglobulinemia with absent b cells. he had wbc =3750 cells/ul, with 60% monocytes and platelet count of 122, 000 /ul. he had no anemia, hepatosplenomegaly, or skin rash. bone marrow biopsy showed mds with absent plasma cells and monosomy 7 in 95 % of his cells. btk expression and sequence analysis were normal. conclusion: while xla is the most likely primary immune deficiency that would present in a male with the clinical picture described above, this case illustrates the importance of maintaining an expanded differential diagnosis in patients with presumed primary immunodeficiency. j. wang * , l. mayer, c. cunningham-rundles, new york, ny. background: chronic granulomatous disease (cgd) usually results in acute or chronic infections with a defined spectrum of bacteria or fungi. other characteristics include inflammatory disorders, including genitourinary or mucosal inflammation resembling inflammatory bowel disease. gm-csf has been used in the treatment of mucosal inflammation in glycogen storage disease ib and crohn's disease with some success. objective: to explore the use of a novel therapy in the treatment of mucosal inflammation associated with cgd. methods: the patient was treated with daily gm-csf (0.6 mcg/kg/dose subcutaneous injection) along with parenteral antibiotics and total parenteral nutrition. after 4 days on gm-csf, hydrocortisone enemas were added due to continued pain and swelling. results: rectal and abdominal pain significantly improved, blood streaked stools decreased; he tolerated a regular diet and was discharged home 10 days after starting gm-csf. he now has no rectal or abdominal pain and no blood in the stool. conclusions: gm-csf may be a useful therapy for the management of mucosal inflammation in cgd patients. it appears safe and well tolerated and permits avoidance of immune suppressive alternatives. introduction: management of autoimmune cytopenias in patients with cellular immunodeficiency may be very challenging. treatment of these cytopenias with corticosteroids may potentiate the immune dysfunction leading to further infections. methods: we describe a 15 month-old female with a cellular immunodeficiency and autoimmune thrombocytopenia / hemolytic anemia being treated with steroids and ivig without improvement. rituximab, a monoclonal antibody that binds to b-lymphocyte cd20 surface antigens, was initiated. results: this patient presented at 11 months of age with failure to thrive, eczema and recurrent bacterial pneumonias. she was diagnosed with a t lymphocyte immunodeficiency with decreased absolute numbers of cd3 (493), cd4 (365), and cd8 (80) lymphocytes. t cell function was markedly decreased as measured by pha, cona, and pwm. she developed chronic thrombocytopenia with platelets of less than 20, 000 and coombs positive anemia with hemoglobin of 6 g/dl. she was placed on 4mg/kg/day of prednisone with minimal improvement and continued to develop severe pneumonias. rituximab 375 mg/m 2 /week was initiated for 5 doses. her cytopenias improved with an increase in both platelet count (229, 000) and hemoglobin (15.1g/dl). her absolute cd19 count fell from 1319 to 0. she was weaned off steroids permanently. she has also continued on ivig, prophylactic antibiotics, and as needed rituximab pending bone marrow transplant. conclusions: rituximab, a monoclonal anti-cd 20 antibody, may be helpful in treating autoimmune cytopenias in patients with cellular immunodeficiency in which steroids need to be avoided. ivig antibody replacement substitutes for the subsequent humoral antibody depletion due to induced secondary b cell lymphopenia. introduction: the patient is a 3 month old male born at full term. during his prenatal course he was noted to have a pulmonary abnormality in his left lower lobe at approximately 12 weeks of age. serial ultrasounds and fetal mri were consistent with the diagnosis of congenital cystic adenomatoid malformation (ccam). histology from elective resection of the lesion performed at 2 months of age revealed no evidence of ccam. silver stain revealed florid pneumocystis jiroveci (carinii) infection. the patient was completely asymptomatic. the differential diagnosis for this infection in the newborn period includes hiv infection, severe combined immunodeficiency, and x-linked hyper-igm syndrome. the infection has also occurred in newborns with apparently normal immune systems. methods: immunology evaluation was performed to evaluate the patient for the listed diagnoses. a complete blood count was performed. lymphocyte subsets were performed by flow cytometry. quantitative immunoglobulins g,a, m and e were measured by nephelometry. specific antibodies to tetanus and diptheria were determined by elisa. hiv status was ascertained by western blot and dna pcr. dichlorofluorescein assay was performed. fluorescenceactivated cell sorter technique was used to evaluate cd40 ligand. lymphocyte stimulation to mitogens and tetanus were performed. results: the patient had negative hiv1/2 antibodies and a negative hiv dna pcr. the result of the complete blood count was normal with an absolute lymphocyte count of 6, 750. the patient had normal percentages and numbers of cd3, cd4, cd8, cd19, and natural killer cells. lymphocyte stimulation to mitogens and tetanus was normal. dcf assay was normal. facs revealed normal levels of cd40 ligand. at 5 months of age he had protective titers to diptheria and tetanus titers of 0.52 iu/ml (protective >0.6 iu/ml). at 5 months igg, a, m and e were 235 (normal 218-636), <6, 36, and <5 respectively. conclusion: immunologic testing revealed no evidence of severe combined immunodeficiency, hiv infection or x-linked hyper-igm syndrome. the patient remains well clinically with normal growth and development with no subsequent infections. pneumocystis jiroveci pneumonia can be seen in infants with apparently normal immune systems. commercial preparations of igg produced for iv use (ivig), must fulfill regulatory requirements. the method of preparation, however, may produce alterations in the content or function of igg subclasses such as disturbed composition which can be reflected as a lowered clinical effectiveness of the product. we report 2 patients with specific igg3 immunodeficiency, that, when changed to a new ivig preparation, had lessening of clinical effectiveness of the new product but who also experienced recovery after they were changed to a different ivig product. case 1. a 53 y/o male with hx of cervical lymphadenopathy associated with recurrent uri, fatigue, cognitive alterations and hypercholesterolemia. he was dx in 1993 with a specific igg3 immunodeficiency for which he was started on ivig infusions 400mgxkg q/4 w which brought his igg3 levels to normal values 21 mg/dl, (18-95 mg/dl) with good clinical improvement. when he was changed to a different ivig product with lower iga content his initial symptoms returned with decrease of his igg3 to 17 mg/dl (41-129 mg/dl) he was changed to a different ivig product with higher iga content with disappearance of all his previous symptoms. case 2. a 47 y/o female with history of chronic yeast infection, fatigue, joint pain, asthma, ge reflux, depression, hypothyroidism and shunt for hydrocephaly. she was diagnosed with igg1 and igg3 immunodeficiency and started on ivig 400 mg x kg x q/4 w. when changed to another ivig with lower iga content she experienced worsening of her symptoms during the 2 months that she was receiving it. she was changed to a different ivig product with a higher iga content with complete improvement of her symptoms. although the content of iga in product 1 (50-150 ug/ml) was significantly lower than in prod-uct 2 (720ug/ml), the total igg and igg subclass distribution (including igg3) were comparable therefore, the lack of clinical effectiveness of product 1 would appear to be related to qualitative alterations in the product related to methodological preparation possibly in removal of the iga or other physicochemical alterations affecting biologic activity which contributed to a lessened clinical efficacy of the product. we present these cases to alert the allergist-immunologist to these observations when treating patients with igg or igg subclass deficiencies. background: development of atopy has been known to occur in nonatopic recipients of bone marrow transplants ( bmt) from atopic donors. the reverse condition, with atopic recipients losing evidence of specific ige after bmt from non-atopic donors is quite unique. case report: ph is a 37 year old male referred for recurrent nasal congestion. as a teenager, the patient was treated for chronic allergic rhinitis and asthma, atopic dermatitis diagnosed as severe, and multiple food allergy. skin testing done as a teenager showed 3+ reactions to multiple pollen, dog, cat, most seafood, cashew, walnut and pecan. at 16 years of age, he underwent an allogeneic bmt from his non-atopic brother for acute myelogenous leukemia. asthma and atopic dermatitis were observed to clear almost completely and allergic rhinitis improve after the bmt. his blood type changed from 0+ to 0-. skin testing done on his current visit showed unremarkable responses to all pollen, cat, dog, dust mites and foods with good histamine control. discussion: the negative skin test results suggests that his current nasal symptoms are vasomotor in nature. considering the past severity of his symptoms, it appears unlikely that he lost his specific-ige sensitivity spontaneously. transfer of atopy is known to occur in non-atopic recipients of bmt from atopic donors. in a study of 12 patients undergoing allogeneic bmt for hematologic malignancies, nonatopic recipients developed positive skin tests with profiles similar to atopic donors. 1 a possible mechanism is passive transfer of memory b-cells which initiate specific-ige production with a donor pattern. a reverse of this mechanism could occur with the recipient gaining lymphoid precursors with no atopic tendency leading to clearing of atopy. the literature yielded one report of asthma resolving after high-dose chemotherapy and autologous stem cell transplantation the authors suggested that chemotherapy could have resulted in immune system reconstitution, normalization of the t-cell repertoire and resolution of asthma. introduction: primary central nervous system (cns) lymphomas constitute only a small percentage of central nervous system tumors in adults and are seen more frequently in aids patients and immunodeficiency states. they are extremely rare in children, even those with primary immunodeficiency. we present a 4-year-old female with combined immunodeficiency who developed an eber+ (ebv-associated) large b-cell lymphoma that was confined to the cns. methods: the patient is a 4-year-old female that was being evaluated for combined immunodeficiency who presented to our institution with acute fever and seizure activity. results: the patient had a history of recurrent pneumonia, otitis media, sinusitis, and upper respiratory infections. she also exhibited failure to thrive, chronic diarrhea, and sen-sorineural deafness. she was found to have a combined immunodeficiency with severe neutropenia and lymphopenia, low iga, low igm, and poor specific antibody response to protein and polysaccharide antigens. she had no response to a candida delayed hypersensitivity skin test. hiv tests were negative. she was thought to have acute meningoencephalitis causing the fever and seizure activity, however bacterial, viral, and fungal studies were negative. she received multiple anitbacterial, antifungal, and antiviral agents. she died secondary to brainstem compression due to severe cerebral edema. autopsy revealed an eber+ large b-cell lymphoma that involved about 80% of her brain and spinal cord. no tumor cells were found elsewhere. con-clusions: this case represents a rare manifestation of combined immunodeficiency: the development of a primary cns lymphoma. the tumor cells were found to be eber+ (ebv-associated). ebv is known to be associated with lymphoma, especially in aids and immunocompromised patients. only 6.5% of primary tumor sites in severe combined immune deficiency patients involved the cns in the immunodeficiency cancer registry that was instituted in the 1970s. v. litvinova * , g. muzlaev, krasnodar, russian federation. introduction: glyoma is one of the most prevalent diseases among all brain tumors and more than 35% of them are malignant. in the previous investigations it has been shown the immune disorders in patients with glyoma tumor. at the same time it was suggested the possible anti-tumor activity of some proinflammatory cytokines (tnf, il1, il8), which can penetrate via haematoencephalic barrier and induce the lysis of tumor cells. the aim of this investigation was to study the serum and spinal fluid concentrations of one of the key immunoregulatory cytokines -gamma ifn and il18 in patients with glyoma tumor. methods: the concentrations of the gamma ifn and il18 have been studied in serum and spinal fluid of 16 patients with glyoma by eliza method. in control, the same parameters have been studied in 6 patients with brain trauma. results: it has been shown that concentrations of gamma ifn and il18 in serum and spinal fluid was 1.5-2 times higher than in trauma patients (p<0.001). the serum levels of gamma ifn and il18 in glyoma patients were 82.8 and 28.5pg/ml, accordingly. in trauma patients-44.3 and 16.3pg/ml. the concentration in spinal fluid of the studied cytokines was 1.8 time higher than in control patients. conclusion: proinflammatory cytokines gamma ifn and il18 may be involved in pathogenesis of glyoma and can participate as possible anti-tumor factors of immunity in glyoma patients. a. arrey-mensah * , r.u. sorensen, new orleans, la. rationale: immunodeficiencies need to be ruled out in infants that present with failure to thrive. patients with t-cell lymphopenia, hypogammaglobulinemia and pancytopenia may have a primary immunodeficiency such as scid, or a secondary immunodeficiency that may need to be managed differently. methods: evaluation of cellular and antibody-mediated immunity of a 15 month old aaf admitted with failure to thrive and chronic diarrhea. ohistory of duodenal atresia corrected by creating a blind duodenal loop and anastomosis of the stomach to the jejunum at birth. o mass in hypogastrium. results: immunologic evaluation revealed: " lymphopenia ranging 210 to 350cells/ul " anemia: hemoglobin 7.6g/dl, retic 6.8 " ancs ranging 700 to 2100 " thrombocytopenia 54000l to 26500l " cd4+: 80 " cd8+: 50 " cd4/cd8r: 1.5 " cd19: 290 " cd56: 190 " mitogen responses were normal: " pha 46, 397cpm " con-a, 7, 690cpm " pwm, 36, 168cpm immunoglobulins: igg 102mg/dl iga 80mg/dl igm 55mg/dl ige 20 iu/ml total protein was 3.4g/dl, albumin was 1.2g/dl the immunoglobulin-g half-life study was 7 days in our patient: metabolic imbalance: hypokalemia (1.8mmol/l) hyponatremia (127mmol/l) hypocalcemia (4.6mg/dl) blood, stool, urine cultures normal. stool ova, parasite, virus were negative. hiv-pcr (-) radiological evaluation: superior mesenteric vein thrombosis, chronic malrotation and volvulus, dilated duodenal blind loop with possible lymphatic obstruction. the patient improved clinically and all immunological markers normalized after surgical removal of adhesions and obstruction. conclusion: the patient had a secondary immunodeficiency due to lymphangiectasia. lymphangiectasia should be considered in the presence of a lymphopenia with normal lymphocyte function and hypogammaglobulinemia accompanied by hypoproteinemia and hypoalbuminemia. hypogammaglobulinemia and hypoproteinemia were secondary to gastrointestinal losses. the immunological component of 22q11.2 deletion syndrome (also known as digeorge syndrome;dgs), hypoplasia of the thymus, is quite variable among patients and provides the opportunity to determine the relationship between thymic function and t cell receptor (tcr) repertoire diversity. using a novel measure of repertoire diversity based on cdr3 length polymorphism called hamming distance, tcrbv repertoire diversity in dgs was found to differ from control subjects by having an overall skewing of receptor expression. as expected from clinical outcomes, there was also great intra-individual variability in dgs tcr repertoires. the degree of repertoire diversity was directly correlated with thymic output as measured by t cell receptor excision circles (trecs), i.e. greater thymic output resulted in more diverse repertoires (see figure below). this result demonstrates a quantitative relationship between thymic function and repertoire diversity in humans and may reflect the balance between thymic output and peripheral expansion to maintain an adequate tcr repertoire. in addition, it may suggest a basis wherein limited repertoire diversity mediates both immune deficiency and autoimmunity. tcr repertoire diversity in dgs, as measured by hamming distance, correlates with thymic output, as measured by trecs (plotted on a logarithmic scale) a. sabra 1* , s. sabra 1 , h.j. castro 2 , j. malka-rais 2 , j.i. mendez-inocencio 2 , g. santos 1 , j.a. bellanti 2 , 1. rio de janeiro, brazil; 2. washington, dc. a major investigative effort of our laboratory has been directed to the study of non-ige mechanisms and their role in the immunopathogenesis of food allergy (fa). we have previously reported th1 and th2 cytokine alterations associated with several clinical entities that had overlapping disease manifestations affecting the mucosal-associated lymphoid tissues (malt), of the gi tract (galt), skin (salt), nasal (nalt) and bronchial tissues (balt). the objective of the present study was to evaluate specific immunologic alterations in a group of patients with non-ige fa. peripheral blood cd4 and cd8 lymphocyte subset analyses were performed in 10 patients with fa documented by double-blind placebo-control food-challenge (dbpcfc). all patients were treated with an amino-acid based formula (aabf) and then received an open challenge using a panel of commonly offending allergenic food allergens in a normal diet.the clinical picture of all 10 patients with fa were linked to malt-related manifestations; 8 with galt symptoms of diarrhea, vomiting, abdominal pain and ftt, 4 with balt symptoms of asthma, 4 with salt symptoms of eczema, 3 with nalt symptoms of rhinitis and 2 with edema, ascites and anaphylaxis. all subjects had normal serum ige and eosinophil levels, negative ige food rast tests, normal cd4 (mean 1562 + 368) and very low cd8 (174 + 42) levels in peripheral blood. abnormal cd4/cd8 ratios >3.5 were observed in all patients. age-and gender-matched controls revealed cd4/cd8 ratios ranging from 1.5 to 2.5. both patients with anaphylaxis had cd4/cd8 ratios >6. all patients responded well to aabf. open challenge to common offending foods from a regular diet (milk, soy, wheat, egg, nuts, beef and chicken), revealed multiple food allergies. after two years of follow-up on an aabf regimen, the 10 patients remain well but allergic to multiple foods. very low cd8 levels remain as the unique immunological alteration in all 10 patients.these studies suggest that abnormalities of very low cd8 distribution may play a pathogenetic role in non-ige mediated fa. since cd8 lymphocytes play a role in immunologic tolerance (it), it is tempting to speculate that the very low cd8 levels may signal the failure of development of it in our patients predisposing them to the development of allergies to multiple food components. background: mixed connective tissue disease (mctd) is a disease taht have caused controversy, while some authorities consider it a distinct rheumatic disease, others believe that it's an early stage of a fully defined autoimmune disease. the distinctive feature is the presence of the u1 small nuclear rnp autoantibodies. clinical features involve raynaud's is phenomenon, swollen hands, sclerodactyly, esophageal hypomotility, polyarthritis, and myositis, allof them can be present in others well deined rheumatic diseases. only aew cases have been described in the pediatric population. objetive: to determine the frecuency of mixed connective tissue disease in mexican.children in a tertiary level institution according to kasakawa's criteria, alarcon-segovia's criteria and sharp's criteria and establish if a diagnosis of a well defined autoimmune entity was made during the follow up. methods:medical charts were assessed with the diagnosis of mctd between 1988 and 2003 in our hospital. results: between 1988 and 2004, 9 , 919 cases of autoinmune diseases were treated; 310 with systemic lupus erythematosus(sle), 6544 with rheumatoid arthritis(ra), 1, 128 with scleroderma and 1, 937 with dermatomyositis. we found only four cases, 3 of them did not complete the kasakawa's criteria but the diagnosis was probable according to alarcon-segovia and sharp's criteria. one of them developed sle after one year of follow up. the case that fulfilled the diagnosis criteria of mctd; raynaud's phenomenon, rnp antibody positive, arthritis, lymphadenopathy, restrictive pulmonary disease, sclerodactily and muscle weakness developed a full blown sle at 10 years follow up. discussion: the disease is extremely rare in children. we conclude that at least in pediatric age the disease may not exist by it's own, but it's an early stage of a defined autoimmune disease, and the terminology "un differentiated connective tissue disease" is more appropriate to define this cases. a. baysan * , h.y. song, s. gupta, l. yel, irvine, ca. hereditary angioedema (hae) is an autosomal dominant disease characterized by episodic angioedema of the skin or mucosa of the respiratory and the gastrointestinal systems. hae is caused by a quantitative (type i) and/or functional (type ii) deficiency of plasma protein c1 inhibitor, an early component of the classical complement pathway. attacks of hae may be lifethreatening and even cause death when the airway is involved. here, we report a 67 year-old female patient with hae type ii, who has seven affected family members, two of whom died of laryngeal angioedema. the patient had a history of recurrent swelling of the eyelids, lips, tongue and extremities, and respiratory distress since five years of age. she was hospitalized on several occasions one of which required intubation and assisted ventilation. laboratory studies, between the attacks, revealed normal serum ch50 and complement c3 levels. complement c4 level was decreased to 5 mg/dl (n:16-47 mg/dl). c1 esterase function was impaired, 46% (n: greater than 68%) in contrast to normal quantitative c1 esterase. the patient was on long-term danazol treatment for ten years and experienced side effects, most notably hirsutism. currently, there are limited efficacious and safe treatment options for hae. the treatment of choice for acute attacks and prophylaxis appears to be the c1 inhibitor concentrate, which is not yet licensed in the us. the present patient emphasizes the need to establish the correct diagnosis and an appropriate management plan in recurrent angioedema. j. hajsam * , l. ponomarjev, krasnodar, russian federation. background: the previous investigations indicate on the positive effects of ncir in different chronic inflammatory and infectious diseases. nevertheless, the mechanisms of the clinical efficacy of ncir remain still unknown. the aim of this investigation is the study of the immune disorders in patients with chronic tonsillitis and the immunomodulatory effects of the complex treatment in combination with ncir. methods: 55 children with chronic tonsillitis in the age from 7 to 14 years old were under the observation. the t cell receptors (cd3+, cd4+, cd8+, cd16+, cd25+, hla-dr+) have been studied using flow cytometry method. the cytokines (il6, gamma ifn and il18) have been investigated by eliza method. the treatment include traditional methods in combination with ncir. control group (without tonsillitis) consists of 56 children of the same age. results: it has been shown that in chronic tonsillitis was dcrease of the number of cd3+, cd25+ and hla-dr+ cells. the decrease of cd3+ cells was mainly due to decrease of cd4+ cells. at the same time it has been shown the increase in number of cd8+ cells. in chronic tonsillitis have been determined the increase in serum proinflammatory cytokine il6 level in 3.6 times (p<0.001). at the same time the levels of gamma ifn and il18 were lower than in control up to 3-4 times. the studied treatment method including ncir resulted in increase in the number of cd4+ and cd25+ cells. the level of il6 decreased from 95 pg/ml to 28.4 pg/ml. at the same time the serum concentration of gamma ifn and il18 increased up to 2.4 and 1.9 times, accordingly. conclusion: in chronic tonsillitis it has been shown the immune disorders in t cell's subpopulations and cytokine production. the efficacy of ncir mainly depends on the normalisation in t cellular subpopulations and increase in the concentration of gamma ifn and il18. introduction: viral hepatitis is characterized by suppression of cd4+th1cell function related to disease severity. the aim of our research was the determination of total ige concentration and anti-c1q-autoantibodies levels in patients with viral hepatitis c (vhc) and mixed viral hepatitis (vhb+c) and the investigation of their correlation with t cell parameters. methods: 31 patients with vhc and 11 patients with vhb+c confirmed by pcr were examined. evaluation of serum total ige level was performed by elisa kits produced by "vector-best", russia. immunophenotyping of peripheral blood mononuclear cells (pbmc) was done by cytometry with monoclonal antibodies to cd3, cd4, cd8, cd11b, cd16, cd20, cd25, cd95 and hla-dr-antigens. results: the level of serum total ige in patients with vhc and vhb+c was significantly increased in all groups studied, especially in acute hepatitis c (526.8â±129.4 mu/ml, p<0.05) compared with the control group. analysis of pbmc subpopulations in patients with vhc and vhb+c was done related to the total ige level: group i -0-200 mu/ml; and group ii ->200 mu/ml. in patients with viral hepatitis and high total ige level, the content of cd3+t-lymphocytes, cd4+t-cells and cd4/cd8 ratio were significantly decreased compared with the control group (p<0.05). more notable changes were found in patients with acute vhc and vhb+c. the increase in cd20+-cell content was seen in groups with both low and high levels of total ige. the content of cd16+-cells in patients with high levels of total ige was also increased (p<0.05). conclusion: patients with vhc and vhb+c had significantly greater levels of total ige compared with the control group (p<0.05). the increase in total ige levels in patients with viral hepatitis is associated with imbalances of t-lymphocyte subpopulations, including a decrease in cd4+t-cells, and an increase in b-and nk-cells. title: occupational airway allergy among health care workers (hcw) with latex allergy. introduction: for the last years the frequency of latex allergy has increased. health care workers (hcw) is the risk group of this diseases. latex allergy symptoms occure in different organs -including skin, conjunctive, nose and bronchi. study aim:the aim of this study was to estimate the incidence of airway allergy in group of hcw with latex allergy. materials and methods: the investigations were carried out in a group of 252 hcw, aged 21-67 (average age 36, 3). there were two hundred and eight women and forty four men in the group. investigation consisted of questionaire examinations, skin prick tests (latex), patch tests (rubber additives), sige (latex) and spirometry. results: seventy eight (31%) hcw reported undesirable effects after the contact with latex products. fourty (15, 9%) hcw reported symptoms of airway (nose and bronchi). latex allergy (spt and/or sige and anamnesis positive) was diagnosed in 49 (19, 4%) cases. the symptoms concerned with: skin, nose and conjunctive (12 cases, 25%); skin and conjunctive (2 cases, 4%); nose and conjunctive (2 cases, 4%); nose and skin (5 cases, 10%); bronchi, skin, nose, and conjunctive (2 cases, 4%) and only skin in 26 cases (53%). conclusions: the incidence of occupational airway allergy in group of health care workers with latex allergy is high (43% of all cases). latex allergy symptoms usually affect skin, but sometimes it includes also other organs -conjunctive, nose and seldom with bronchi. the incidence of anaphylaxis is increasing. the food allergy and anaphylaxis network wishes to expand the use of injected epinephrine by first responders to allergic emergencies. in indiana, there are 7, 500 first responders, 14, 900 basic emts, 1, 350 advanced emts, and 2, 500 paramedics. the use of injected epinephrine was only permitted by paramedics and advanced emts (17% of all responders). in 2002 a bill was introduced to expand the use of injected epinephrine by ems personnel in the event of an allergic emergency. it was sponsored by the indiana allergy asthma and immunology society and the emergency medical services commission of indiana. on july 1st 2003 senate bill no. 213 took effect permitting all first responders to administer, without restrictions, injected epinephrine. once the legislation passed it became the responsibility of the medical director (md) of each ambulance service in the state to implement the change. in an attempt to assess the impact of this legislation we surveyed each of the mds in the state 9-12 months after the passage of the bill. mds were notified of the legislation by memo and public meetings. in addition each md received a copy of the legislation. of the 266 mds, 56 (25%) responded to the survey. results: 1. unaware of the legislation: 14 (25%) 2. aware of legislation but no implementation: 9 (16%) 3. aware of legislation with implementation restricted to basic emts: 29 (52%) 4. aware of legislation and implementation at all levels: 4 (7%) conclusions: twenty-five percent of mds were unaware of legislation. among those that implemented changes the majority (52%) permitted use by basic emts only. allergists, lay organizations, and ems commissioners need to assure implementation of legislation with all mds within the state. a. khadavi 1* , b. silverman 2 , a. schneider 2 , 1. great neck, ny; 2. brooklyn, ny. rabbit anaphylaxis is extremely rare, with one documented case upon inhalation only. we describe a patient with severe anaphylaxis upon consumption of a rabbit. a 6-year-old female with a 3-year history of asthma and allergic rhinitis was presented for evaluation. the patient complained of worsening asthma and rhinitis symptoms in the home and upon exposure to outdoor pollen. further history revealed the family had a rabbit as a pet. laboratory testing showed her total ige was 865 ku/l, rast results were normal for tree, ragweed and molds (<0.35 kiu/l). positive results were found for ragweed, dust, cockroach, cat, dog, mouse, peanut and rabbit epithelium (8.43 kiu/l, class iv). among other environmental control measures, the family was advised to eliminate the rabbit from the home environment, as it could be a potential trigger for their daughters allergy symptoms. two days later, the patient presented to the emergency room with wheezing, coughing and angioedema of the hands and face. she was treated with albuterol, diphenhydramine and oral steroids. the parents said they followed all of our instructions and did not know why anaphylaxis occurred. further questioning revealed that the family consumed the pet rabbit on the same night preceding the anaphylactic reaction. this was the first time the daughter ate rabbit. the parents assumed that only exposure to the live rabbit would worsen her allergy and asthma symptoms, never expecting an allergic reaction via ingestion. they were advised not to feed their daughter rabbit again and were given a prescription for self-injectable epinephrine. this demonstrates either complete identity, partial similarity or cross reactivity between inhaled and food allergens, as has been noted previously with certain other foods such as garlic and crustacean proteins. physicians need to advise food allergic patients that an allergic reaction can develop upon inhalation of the food, as seen with peanuts. but also respiratory sensitization to an allergen can lead to allergic symptoms upon its ingestion. t. nsouli 1* , j. scheiner 2 , j. malka-rais 2 , j.a. bellanti 2 , 1. burke, va; 2. washington, dc. the safety of selective cyclooxygenase-2 (cox-ii) inhibitors in patients with known nsaid-induced allergic reactions has not been definitely established and is still open to debate. in the present report, we describe two patients with hypersensitivity reactions to naproxen, the first characterized by a systemic anaphylactic reaction and the second by localized urticaria following ingestion of the drug. the first case was a 56 y/o wf with a history of osteoarthritis who, 15 minutes after the ingestion of 375 mg of naproxen, developed generalized pruritus, urticaria, laryngeal edema and hypotension. she was treated in the er with epinephrine, diphenhydramine and iv corticosteroids. epicutaneous and intradermal skin testing to celecoxib revealed negative results similar to a control. an oral challenge with celecoxib was well tolerated by the patient without any adverse responses. the second case was a 57 y/o wf with a known history of chronic urticaria and osteoarthritis requiring regular use of naproxen. a complete allergic and immunologic workup was negative. upon discontinuation of the drug there was resolution of the urticaria. an oral challenge with celecoxib was well tolerated by the patient without adverse sequelae. these two case reports exemplify two extremes in the spectrum of adverse allergic reactions to naproxen, a non-selective cox-i and cox-ii inhibitor, one systemic, the second localized and suggest that the pathogenesis of these symptoms was most likely pseudo-allergic in nature and not immunologically-mediated. the dissimilar chemical structures of naproxen and celecoxib together with their differing mechanisms of action suggest that the absence of allergic reactions to challenge with celecoxib, a cox-ii inhibitor, may be related to a lack of cross-reactivity between the two drugs. although these findings suggest that oral celecoxib could be a possible safe alternative in patients with naproxen-induced drug reactions, it would be prudent to first conduct careful challenges with the drug in a well-equipped medical setting where clinical acceptability and tolerability could be safely assessed. introduction: most fixed and removable dentures are made from casting alloys. many orthodontic appliances are also fabricated from metallic biomaterials. it has been documented in vitro and in vivo, that metallic restorations release metal ions mainly due to corrosion. those metallic ions may be distributed systemically and locally and could pay a significant role in the induction of oral or/and systemic immunoinflammatory conditions. this study determines the frequency of sensitization to metal salts and clinical characteristics in the group of patients with complaints related to adverse effects of dental alloys. methods: 31 patients (29 women and 2 men) aged 35-71 years with symptoms assumed to adverse effects of dental alloys were studied. all patients were studied with base metal salts, including cu 2+ , co 2+ , cr 6+ , mg 2+ , mn 2+ , ni 2+ , ti 3+ , zn 2+ using patch tests. all patch tests substances were 3% salts in petrolatum. the patch tests were conducted in accordance with recommendations of the icdrg. occlusion time was 2 days, and patch tests were read when removing the patches (finn chambers on scanpor, epitest ltd oy, tulusa, finland) and 2 to 4 days later. the total number, percentage of irritant and allergic patch test reactions were calculated. results: in 25 of 31 patients (80%) sensitivity to base metals used in dental restorations was noted. symptoms included burning mouth (45%), metal taste (19%), electrical sensations (11%), dry mouth (9%), and taste irritation (12%), but 16/31 patients (54%) had no symptoms. local gingivitis (19.3%), anomalies of the tongue (12%), stomatitis (12%) and lichenoid reactions (6%) were often seen. the most frequent patch test reactions were caused by ni (45%), cr (41%) and co (19%) . conclusion: this study demonstrated higher frequency of hypersensitivity reactions to ni, cr and co in this group of patients often having symptoms such as burning mouth, metallic taste, electrical sensation, local gingivitis, and anomalies of the tongue. w.y. mak * , s. kearney, b. silverman, a. schneider, brooklyn, ny. the process of intravenous drug desensitization often involves simple but tedious calculations. miscalculations may arise due to human error. for patients who are truly allergic to the drug in question, such errors can be life threatening, if not fatal. we propose the use of a spreadsheet to minimize such calculation errors. spreadsheets, such as microsoft excel, ibm lotus 1-2-3, and corel quattro pro, are used extensively in finance for tedious and repetitive calculations. this property of a spreadsheet makes it an ideal tool to assist with any drug desensitization protocol. we chose microsoft excel for its availability at our institution. a general template was initially created with equations in specific cells which were based on the protocols listed in patterson's allergic diseases, 6th edition and middleton's allergy: principles and practice, 5th edition. by inputting specific numbers, such as the amount of drug and volume of diluent into key cells of the spreadsheet, the program will automatically calculate the protocol for the given intravenous drug. an example of our pipericillin protocol is listed below. we find that the use of a spreadsheet not only minimizes calculation errors and hence reduces the likelihood of avoidable reactions; but also decreases our preparation time for the desen-sitization protocol. we recommend its use for all allergists performing drug desensitizations. states 25 years ago, the infestation of homes across the northeast, southeast and midwestern states with these insects during the fall and winter months has become increasingly common. in the last 5 years, several investigators have published case reports of patients with allergic rhinitis, conjunctivitis and asthma symptoms associated with suspected inhalational exposure to high levels of proteins from these beetles. we report a series of 5 patients who presented with a spectrum of various allergy complaints ranging from symptoms of allergic rhinitis, asthma, urticaria, angioedema and acute anaphylaxis (with documented elevated serum tryptase) on exposure to high numbers of multi-colored asian ladybeetles(malb.) methods: we performed western blotting with the patients' serum and a whole-body ladybeetle extract made from confirmed h. axyridis obtained from one of the infested homes in that region. results: blots with the patients' serum revealed ige binding to 3 different proteins with molecular weights approximately 8, 28 and 78 kd. the serum from two patients bound a 28 kd protein possibly similar to the 30 kd protein previously identified by yarbrough et al. jaci 1999; 104; 704-5 . ige from four patients bound to an 8 kd protein not previously reported. lastly, serum from two patients revealed ige binding to a 78 kd protein possibly similar to the heavier proteins mentioned in an abstract by magnan et al. jaci 2002; 109; 580 . conclusion: we present five patients with specific ige to malb and allergic symptoms on exposure to high levels of malb. we conclude that malb are likely a significant source of several different allergenic proteins and that malb are increasingly becoming a significant cause of a wide variety of hypersensitivity reactions across the united states. as multiple exposed subjects and several entomologists report that these beetles bite humans, we speculate that a wide range of ige-mediated symptoms including urticaria, angioedema and anaphylaxis can occur by exposure to proteins by inhalation, direct contact and possibly by inoculation of these proteins into the skin by the bite or scratch of this beetle. a case report and literature review. c.s. taylor 1* , s. ramesh 2 , 1. getzville, ny; 2. buffalo, ny. introduction: lentils belong to the legume family and are the staple ingredient of the ethnic indian diet. lentils have been shown to cause allergic reactions and even anaphylaxis. however, little research has been done to distinguish the types of lentils used commonly in the indian diet and their various hypersensitivities. some of these lentils include the black gram (phaseolus mungo), mung bean and split chick peas (bengal gram). case report: we report a 19-month-old indian boy who presented with severe atopic dermatitis since the age of four months. his dermatitis was exacerbated by the ingestion of peas and beans and resolved with the avoidance of such. at nine months of age, the child was introduced to boiled mung beans and subsequently developed lip swelling within a few minutes. a similar reaction occured with split chick peas. physical exam at consultation revealed severe eczema. skin tests using commercial extract revealed 5+ response to peanut (he had never eaten peanuts), soybean, pea and egg. subsequent skin tests at the follow up visit to prepared reagents revealed much greater than 5+ response to mung bean, chick pea, split chick peas, and black gram. the patient developed a systemic response during testing which required epinephrine. discussion: legume allergy (other than peanut, soy and peas) is rare in the unites states but has been reported in asia and the mediterranean area. it has been reported in one case series that patients with lentil allergy react to more than one lentil. there is a five percent cross reactivity between peanut and other legumes such as soy and peas. however, the extent of cross reactivity between peanut and the other legume sub-groups (ie. lentils) is not well documented. there also appears to confusion in the labeling of the various lentils used in ethnic diets. conclusion: this case has been presented to make allergists aware that there are many differnt types of lentils that can cause hypersenstivity reactions and the importance of considering ethnicity and dietary habits in evaulating for food allergy. background: epinephrine is the drug of choice for the treatment of anaphylaxis however it is frequently underutilized. one possible reason is the fear of adverse cardiac effects. the most common side effects of epinephrine are palpitations, tachycardia, sweating, nausea, vomiting, respiratory difficulty, pallor, dizziness, weakness, tremor, headache, nervousness, anxiety and arrhythmias. a previous study showed that administration of epinephrine, in a small number of healthy adults did not cause any significant cardiac adverse events and vitals sign changes were not clinically significant. the purpose of this retrospective analysis is to determine the safety profile of epinephrine in a large number of patients that were administered epinephrine for treatment of anaphylaxis that occurred in a physician's office. methods: all patients in an allergy office, that were administered epinephrine for acute anaphylaxis between 1999-2004 were selected for this retrospective analysis. a chart review for adverse events and vital was conducted. vital signs and side effects had been monitored every 5 to 10 minutes for minimum of 30 to 90 minutes after the administration of epinephrine. results: 105 patients between the ages of 8yrs to 55yrs received 128 injections of epinephrine. after an injections of epinephrine 60% of the patients had a pulse between 50-90, 27% between 72-100, and 13% between 102-110. systolic blood pressure determined 81% of patients to be between 80-140, with 10%, between 142-160, and 7% between 162-170, and 2% between 172-180. concurrently, diastolic blood pressure was found to have 94% between 50-90, 3% between 92-100 and 3% betweem 102-110. the most common reported side effects were tremor, headache and pallor. any rise in blood pressure and heart rate after epinephrine was transient and returned to normal within 90 minutes of observation. there were no serious side effects. all patients responded to the epinephrine and were able to go home. conclusions: this retrospective analysis revealed that the administra-tion of epinephrine is safe and effective for the treatment of acute anaphylaxis in an outpatient setting. the use of epinephrine for treatment of anaphylaxis is life-saving and its use to treat non-cardiac or elderly patients who have anaphylactic reactions should be encouraged. purpose: a clinical pathway was established to decrease the use of vancomycin in surgical patients with self-reported penicillin (pcn) allergy. methods: in 2001, our institution developed a preoperative evaluation (poe) clinic for elective surgical patients. in june 2002, on-site, same-day allergy consultation and penicillin skin testing was made available for preoperative patients with self-reported pcn allergy. we reviewed the antibiotic recommendations, the actual administration of vancomycin, and compliance by the surgeons with our recommendations from july 1, 2002-september 16, 2003. results: during this time, 11, 819 patients were seen for their preoperative medical exam at the poe clinic, and 1204 were evaluated for pcn allergy. of these, 1120 patients met irb standards to participate in the study. the mean age of the patients was 60 years. the top three surgical specialties represented in the poe clinic were orthopedic (28.4%), urology (22%), and neurosurgery (15%). of the 1120 patients, 1031 underwent skin testing for pcn allergy. eighty-five percent (85%) or 951 of these patients with a history of pcn allergy were recommended to use -lactams such as cefazolin, and 164 (15%) were recommended to avoid -lactams. forty-three (43) or 4% had one or more positive skin tests to pcn. of the 1120 patients, 957 patients actually received pre-operative antibiotics. of these 957 patients, 718 (75%) of those received cefazolin, 60 (6%) received clindamycin, 33 (3%) received ciprofloxacin, 25 (2%) received levofloxacin. some patients received more than one antibiotic for prophylaxis. only149 (16%) patients received vancomycin. conclusions: establishment of a clinical pathway in a preoperative clinic that includes allergy testing and consultation reduced vancomycin use to only 16% in surgical patients with a history of penicillin allergy. h. yarmohammadi * , a. nowak-wegrzyn, new york, ny. introduction: anticonvulsant hypersensitivity syndrome is a potentially fatal drug reaction with cutaneous and systemic reactions to the arene oxideproducing anticonvulsants. in most cases, the hallmark features of fever, rash, and lymphadenopathy are accompanied by multi organ-system abnormalities. fatal outcomes are most often associated with liver failure. recognition of the syndrome, which may have variable presentations, is the key to prompt discontinuation of the drug, close monitoring, and management. case 1: a 16y/o male received dilantin for seizure prophylaxis following craniopharyngioma resection. fourteen days later he was readmitted for fever and csf leak from surgical site. he was started on ceftriaxone and vancomycin empirically; an lp and pan culture was done. he continued to have low grade fever and, on the 6th day of admission, developed a maculopapular rash on trunk. on day 8th lfts increased and cbc showed eosinophilia. antibiotics were stopped but lfts continued to rise. on day 10, dilantin was stopped and lfts normalized in 2 days, fever stopped and rash disappeared. case 2: a 17 y/o girl was admitted to the hospital with generalized rash, facial edema and fever (39 degrees c). she developed a pruritic maculopapular erythematous rash over her trunk and face 2 weeks prior to admission. she then developed high fever. three months prior to this visit she had been started on phenytoin (300mg/kg) for control of grand mal seizure. physical exam revealed cervical and axillary lymphadenopathy. she had elevated wbc (13, 400), eosinophilia (20 %), and elevated lfts. phenytoin was stopped upon admission but 24 hours later she continued to have fever, elevated lfts and devel-oped erythema in her mouth. treatment with ivig and prednisone was initiated and resolution of symptoms was seen within 2-3 days after therapy. conclusions: the timely recognition of anticonvulsant hypersensitivity syndrome is important, because accurate diagnosis prevents potentially fatal re-exposure and influences subsequent anticonvulsant treatment options. ivig and prednisone should be considered in situations where prompt improvement of the lfts or clinical symptoms is not observed. the recently published results of a telephone survey about the prevalence of seafood allergy in the us indicate that seafood allergy is a significant health concern. aim of the present study was to retrospectively review our data on the topic. we have interviewed 7391 subiects attending our allergy outpatients about any suspected food allergy. over 95% were 14 years or older. any reaction to food was reported by 33% of the patients, more frequently by women. of these 2419 patients, 125 reported reactions to seafood (5%) with an overall prevalence of seafood reactions of 1.7%: 50 to fish (0.7%), 58 to shellfish (including mollusks) (0.8%) and 17 to both (0.2%). in the case of the reactions to fish it is quite possible that shellfish is also included, as the patients were often unable to tell the role of finfish from shellfish apart. this is most probably due to the fact that seafood dishes or a complete seafood meal in italy generally include both shell and finfish. in only less than half cases there was a convincing relationship between the ingestion of seafood and the reaction. the reactions reported were: gastrointestinal (17), non life-threatening angioedema (15), mild oral allergy syndrome (13), acute urticaria (9), asthma (3), rhinoconjunctivitis (2). the gastrointestinal reactions might not all be true (ige-mediated) allergic reactions, but also toxic. in conclusion, even in a selected population like that attending an allergy clinic and using a broad definition of adverse reaction, seafood allergy appears to be a rare phenomenon and of limited clinical impact, the prevalence of moderate to severe reactions (angioedema and asthma) in the whole selected population being 0.2%. clopidogrel (plavix) is an antithrombotic agent currently used to prevent thrombotic cardiovascular events by inhibiting adenosine diphosphate (adp)dependent platelet activation. clinically, it has been used in patients with aspirin intolerance or who developed neutropenia from ticlopidine. there have been reported cases of clopidogrel causing rash and urticaria. often these patients are told they are allergic to clopidogrel and should not take it again, despite the fact that no workup was ever performed to elucidate whether the reaction was indeed immunologic in nature. a review of the literature revealed no reported attempts at desensitizing a patient to clopidogrel after a presumed immunologic drug reaction. we present the case of a 49 year-old female who initially took clopidogrel (75 mg. p.o. daily) for transient ischemic attacks. after tolerating the medicine for five days, it was replaced with an aspirin/warfarin combination, which she took for the next five days. reintroduction of clopidogrel a week later was uneventful, until she developed a pruritic, maculopapular rash on the 5th, through 7th days of restarting therapy. at that time, she was not taking any other medications, and had no cardiopulmonary or gastrointestinal complaints. she had discontinued the clopidogrel and was asymptomatic during our consultation. skin testing was done using clopidogrel prepared at concentrations of 1 mg/ml and 1/10 mg/ml. at the same time, control skin testing on three non-atopic subjects was performed and produced no reaction to either epicutaneous or intradermal testing. by contrast, the patient had a positive skin reaction at both concentrations on intradermal testing. the patient was hospitalized for an oral desensitization where clopidogrel dilutions were prepared by the hospital pharmacy. the patient tolerated an initial dose of 0.02 mg. serial three-fold increases in concentration were given every 15 minutes, until a total cumulative dose of 77 mg. was reached. the patient tolerated the entire procedure and suffered no adverse reactions upon continuation of the treatment. this demonstrates the utility of oral desensitization to clopidogrel in patients with a positive skin test who require this medication. lamotrigine is a non-aromatic anticonvulsant with desirable pharmacological profile. the most common idiosyncratic reaction in children is rash (10-20%). severe cutaneous adverse reactions and systemic hypersensitivity reactions are uncommonly reported. method: case presentation an 11-yearold caucasian female was prescribed lamotrigine in escalating dose for her first generalized seizure. a week later she developed a pink rash on her cheeks gradually progressing to her trunk, hands and feet. three days later she was seen in the er and treated for possible streptococcal-pharyngitis with fever and rash. oral penicillin-v treatment was initiated. the rash continued to spread all over her body with mild pruritis and sores on lips and mouth. few blisters were noted in left ear, hands and feet. later lamotrigine was discontinued. oral steroids were prescribed. she continued to develope new blisters, rash and hemorrhagic plaques and dysuria. she was later transferred to our institute. the physical examination was significant for afebrile girl without pallor and icterus. bilateral conjunctivitis without keratitis evident. tender cervical lymphadenopathy was palpable. multiple ulcers on lips and gingiva were seen. several targetoid lesions on trunk and extremity were noted along with few hemorrhagic plaques on extremities. superficial sloughing of distal fingers and toes was noted. nikolskys sign was not demonstrable. the systemic examination was unremarkable. laboratory tests: hemoglobin 14.2g/dl, wbc-normal range without eosinophilia. normal pt/aptt and inr. liver function tests showed elevated sgpt-1380iu/ml and sgot-225iu/ml with normal bilirubin. urine analysis was normal. serology for cmv, ebv, hsv and hepatitis a and b was negative. skin biopsy showed full thickness epidermal necrosis and sub epidermal clefts. aggressive supportive therapy was initiated. corticosteroids were continued for five more days. prior to being discharged from hospital the rash had dried and most lesions faded. the blisters and oral ulcers had healed. the liver enzymes had decreased. conclusions: we report an occurrence of stevens-johnson syndrome with lamotrigine in a young child. in the literature severe reactions are associated with higher doses or rapid escalation of the dose, and concomitant use of valproate. early recognition and withdrawal of medication is necessary to improve the outcome. introduction: in patients with a history of penicillin (pcn) allergy and negative pcn skin tests to major and minor determinants, 97-99% of patients will tolerate pcn administration without risk of an immediate reaction. in fact, middletons allergy principle & practice reported that no life-threatening false-negative reactions have been reported when pcn was administered after a negative pcn skin test. we describe a case in which a patient with a history of pcn allergy and negative pcn skin tests to the major and minor determinants experienced life-threatening anaphylactic shock when administered piperacillin/tazobactam. case history: a 38-year-old woman with crohns disease was admitted for treatment of an enterocutaneous fistula. three months before admission, the patient reported a severe reaction to either piperacillin/tazobactam or intravenous (iv) lorazepam needing respiratory support in the intensive care unit. in order to delineate this problem, an allergy consultation was obtained. serum ige antibodies to pcn, measured by a commercial cap system radioallergosorbent test (rast) fluoroenzymeimmunoassay (feia; pharmacia and upjohn, uppsala, sweden), and pcn skin tests to major and minor determinants were negative. a skin test (prick and intradermal) to piperacillin/tazobactam at 4.5mg/ml was also negative. five minutes into receiving 3.375 grams of piperacillin/tazobactam iv, the patient reported feeling lightheaded, flushed, nauseated, and diaphoretic. the blood pressure decreased to 80/50 from a baseline of 110/60, heart rate 150/minute and appeared toxic. no wheezing or rash was noted on physical examination. patient was treated with iv epinephrine, diphenhydramine, and dexamethasone and recovered without sequela. serum tryptase, drawn 1 hour after the beginning of the reaction, was elevated at 56.3 ng/ml but decreased to 17.6 ng/ml 12 hours later. complement levels were normal. conclusion: despite a very high negative predictive value of a negative pcn skin test to the major and minor determinants and reports that no life-threatening false-negative reactions have been reported when pcn is administered after a negative pcn skin test, physicians need to be very cautious in administering piperacillin/tazobactam and other b-lactams in patients with a history of severe reactions such as anaphylaxis to past pcn administrations. a. majmundar * , d.a. khan, dallas, tx. introduction: a variety of adverse drug reactions have been reported after therapy with allopurinol. successful protocols for desensitization to allopurinol have been developed. we report a case of a patient who was successfully desensitized to allopurinol only to develop dress after four months of allopurinol therapy. methods: a 57 year old man was referred to our department with a remote history of rash and fever resulting in hospitalization after initiation of allopurinol. due to severe gouty arthritis unresponsive to colchicine and requirement of chronic steroids, it was recommended to attempt allopurinol desensitization. based on the history of his prior reaction, a slow desensitization protocol was performed beginning with allopurinol 10î¼g and increasing the dose weekly to 25î¼g, 50î¼g, 100î¼g, 200î¼g, 500î¼g, 1mg, 5mg, 10mg, 25mg, 50mg, and 100mg. the patient tolerated the entire desensitization protocol without adverse reaction and was initiated on daily allopurinol at 200 mg per day. results: four months after desensitization and daily allopurinol use, the patient developed fevers and an acute maculopapular eruption on his back and abdomen without mucosal involvement. laboratories demonstrated eosinophilia of 936 cells/mm 3 , elevated ast of 101, alt of 102, and ggt of 163 u/l. he was treated with prednisone 80mg a day which was tapered weekly over 5 weeks with successful resolution of his symptoms, eosinophilia and transaminitis. conclusion: while desensitization to allopurinol can be safely accomplished, allergists need to be cognizant of the potential for delayed serious drug reactions such as dress. wiskott aldrich syndrome is an immunodeficiency characterized by eczema, pyogen infections, and mixed immunodeficiency.we describe a male seven-month old, perinatals antecedents without importance who had an ulcer in site of application of bcg. non-blood relatives, healthy parents. at twomonths-old, he initiated with continuous fever, not quantified, treated with multiples antibiotics without resolution, he was hospitalized, plaquetopenia and anemia were diagnosed, he received a red globules and plateles transfusion, one week after he presented a disseminated dermatosis characterized by erythema and desquamation, at four-month-age presented right axillary adenomegaly, hepatoesplenomegaly and recurrent bilateral media otitis for what was hospitalized in our institute. he was malnutrition, bad general condition, febrile, with disseminated dermatosis affecting the head, trunk and extremities characterized by erythema and desquamation, in addition he had left ankle cellulitis, right axillary adenomegaly, hepatoespenomegaly. during his hospitalization he presented left hand cellulitis, hairy skin abscess, oral candidiasis, required surgical treatment by econdary compartimental syndrome because cellulitis of left ankle. persistent hemogram reported thrombocytopenia with normal platelet volume, blood cultives were positive for grampositive bacterias, isoaglutinines, were normal. immunoglobulines were elevated for the age range, he received antimicrobial and antifungic treatment. but he died. in the autopsy timic alymphoplasia was reported, cortical lymphoid depopulation in lymphatic ganglia and spleen, disease by disseminated cytomegalic inclusion, multifocal pulmonary pneumocystosis, bcgitis, disease graft versus guest. . with the previous features we concluded in a compatible mixed immunodeficiency with wiskott-aldrich syndrome with particular characteristics that make this case interesting. the patient course with cellular immune deficiencie with thrombocytopenia and eczema, even when he didn't have platelet sizing diminished, we consider that the patient had a severe of wiskott s aldrich syndrome and at the moment we are awaiting result of genetic study uasp gene. background : hypersensitivity to mosquito bites (hmb) is a disorder characterized by necrotic skin reaction and systemic generalized symptoms subsequent to mosquito bites. it has been suggested that hmb is associated with chronic epstein-barr virus (ebv) infection and natural killer cell leukemia/lymphoma. we describe here a korean child who had hmb associated with chronic ebv infection and natural killer cell lymphocytosis. case : a 5-year-old male was admitted with well-demarcated necrotic skin lesions and severe swelling on right ear lobe developed after mosquito bites. he had suffered several similar symptoms since last summer, which complicated as deep scars on skin. hepatosplenomegaly or peripheral lymphadenopathy was not detected. laboratory tests showed wbc 7, 280/mm3 (neutrophil 39%, lymphocyte 56%), total eosinophil count 156/mm3, ige by prist above 1, 000 iu/m. immunoglobulin levels were normal. specific ige for aedes communis by cap was negative. lymphocyte subset analysis demonstrated increased nk cells (cd16+cd56, 43%) and decreased cd3 and cd4 cells. igm for anti-nuclear antigen (ebna), igm for viral capsid antigen (vca) and igm for anti-early antigen (ea) dr to ebv were negative. but the levels of anti-vca igg (> 200 u/ml), anti-ea dr igg (> 150 u/ml) and anti-ebna igg (62 u/ml) were increased. type a eb virus was demonstrated in blood mononuclear cells by dna pcr method, and eber in situ hybridization was negative in necrotic tissues. immunostaining with nk-cell marker (cd56) revealed many immunoreactive cells with the perivascular inflammatory infiltrates in tissue. skin patch tests for mosquito allergen (aedes togoi and culex pipiens) showed positive response to c. pipiens. introduction: scimitar syndrome is a congenital anomaly resulting in anomalous pulmonary venous return from the right lung to the inferior vena cava. recurrent respiratory infections have been associated with scimitar syndrome. methods: we report on a 16 year-old adolescent female who presented to immunology clinic with recurrent pneumonias. results: the patient presented with numerous recurrent pneumonias, multiple er visits, and hospitalizations. she complained of intermittent chest pain, cough, fatigue and exercise intolerance. the patient had a large secundum atrial septal defect surgically corrected at 5 years of age. a cardiac echo obtained at 7 years of age revealed rvh, but was normal at 9 years of age. she was a known atopic asthmatic. previous immunologic workup was normal with iga 183 (33-202), igg 1290 (633-1280) and igm 97 . at presentation to our clinic pulmonary functions were fvc of 79% and fev1 of 78%. review of previous chest radiographs showed chronic changes with an opacity partially obscuring the right hemidiaphragm. a high resolution chest ct-scan showed a large irregular venous structure extending through the right chest joining the inferior vena cava above the liver consistent with scimitar syndrome. the patient was referred to pediatric cardiology who recommended surgical correction. conclusion: scimitar syndrome may present as recurrent pneumonias and chronic lung disease. a high resolution chest ct scan may be useful in delineating this disorder. background: laryngomalacia is the most common cause of stridor in infants but only rare reports exist of clinically relevant laryngomalacia in adults. objective: to present a case of laryngomalacia in an adult with significant respiratory symptoms initially attributed to asthma. methods: an 18 year-old female with a history of allergic rhinitis and gastroesophageal reflux disease presented to the allergy clinic for further recommendations regarding a prior diagnosis of asthma poorly controlled on inhaled fluticasone, montelukast and albuterol. the patient was clinically diagnosed with asthma at age 13 due exercise related symptoms. the symptoms progressed and intermittent trials of various inhaled steroids provided minimal relief. on evaluation, the patient described constant wheezing which occurred only on inhalation and originated from the throat. symptoms did not respond to albuterol use three to four times a day, rhinitis control and long-term, high-dose, antireflux therapy. baseline spirometry was normal. histamine bronchoprovacation and fiberoptic laryngoscopy were performed for further evaluation. results: histamine challenge was positive with a 33% decrease in fev1 with 1 mg/ml histamine. however, laryngoscopy revealed redundant airway tissue most notable over the right arytenoid cartilage, consistent with laryngomalacia, which prolapsed into the laryngeal vestibule significantly obstructing the airway on inspiration only. the patient was referred to otolaryngology and surgical excision using a carbon dioxide laser was performed with subsequent improvement in symptoms and decreased asthma medication use. conclusions: we report an unusual case of laryngomalacia in an adult presenting as asthma, which was successfully treated with laser surgical excision. laryngoscopy of the patient revealing redundant airway tissue most notable over the right arytenoid. c. so 1* , s. kuhl 2 , 1. davis, ca; 2. mather, ca. c. so, s. kuhl u.c. davis medical center, sacramento, ca & sacramento va hospital, mather, ca background: wheezing is an uncommon manifestation of phrenic nerve dysfunction. objective: we offer a description of wheezing attributable to phrenic nerve dysfunction to remind clinicians that dyspnea and wheezing can be caused by cor pulmonale which can be caused by phrenic nerve dysfunction. methods: a 71-year old male non-smoker presented with chronic dyspnea and occasional wheezing for the last decade. the patient had a remote history of pericardial stripping for presumed tuberculous pericarditis. he also had a non-productive cough and orthopnea. dyspnea was exacerbated by putting his hands over his head and bending over. he had been treated with inhaled corticosteroids and bronchodilators without relief. results: repeated chest x-rays showed a chronically elevated right hemidiaphragm. pulmonary function tests showed a restrictive pattern with: fev 1 1.2 (40%), fvc 1.59 (36%), tlc 3.23 (49%), dlco/va 4.54 (125%) and no bronchodilator response. left heart catheterization was normal while right heart catheterization showed elevated pulmonary artery pressures (56/20) and he was subsequently diagnosed with cor pulmonale. cardiopulmonary exercise testing showed an increase in minute ventilation which was achieved predominantly by an increase in respiratory rate rather than to an increase in tidal volume suggesting restrictive or interstitial lung disease. chest ct showed left ventricular enlargement and no evidence of interstitial lung disease. a fluoroscopic sniff test was performed and showed paradoxical movement of both diaphragms. he was diagnosed as having diaphragmatic dysfunction as a result of phrenic nerve injury from prior pericardial stripping. conclusions: wheezing and chronic dyspnea can be related to phrenic nerve dysfunction. a review and discussion of various causes of phrenic nerve dysfunction, including autoimmune causes, is offered. patients with a history of prior cardiothoracic surgery who present with recalcitrant dyspnea and wheezing may benefit from evaluation for phrenic nerve dysfunction. background: sudden sensorineural hearing loss (ssnhl) is defined by a loss of at least 30 db in 3 contiguous frequencies over a time course of 72 hours or fewer. etiologies of ssnhl include viral infections, ototoxic drugs, autoimmune diseases, trauma, neoplasms, and vascular occlusion, but viral labyrinthitis is the most common cause. in cases of sudden hearing loss, herpes infections can be reported in approximately 70% of cases caused by viral infections. typically, sensorineural hearing loss is not recurrent. we report a case of recurrent ssnhl is which oral herpes lesion preceded the onset of symptoms on three consecutive episodes. case report: a 55-year-old male with a history of hypertension, hypothyroidism and chronic tinnitus of the left ear (since a gunshot wound 18 years prior) presented to clinic with a complaint of recurrent episodes of sensorineural hearing loss. the first episode of bilateral hearing loss occurred 14 months prior. an otolarnolgologist treated with a steroid taper and valacyclovir and the hearing loss resolved after one day of therapy. since the initial presentation, the patient reports three subsequent episodes of ssnhl, with two of the episodes responding to prednisone and valacyclovir and one episode responding to steroids alone. oral herpetic lesions preceded at least three of the episodes one day prior to the hearing loss. laboratory data was significant for an elevation of varicella-zoster igg, and of hsv igg. hsv igm was not performed. rpr was non-reactive and ana was negative. a mri of the head was normal. audiogram demonstrated 30db increase and speech discrimination improved from 80% to 100%. the rest of the laboratory data was unremarkable. the patient has been maintained on daily valacyclovir therapy and has had no further episodes of hearing loss. conclusion: our patient experienced hearing loss with concomitant evidence of hsv-1 stomatitis. this suggests a cause and effect relationship. we found that the antiviral therapy was effective for treatment of the ssnhl in this patient as demonstrated by the absence of further symptoms while on antiviral prophylaxis and conclude the most likely etiology of the recurrent hearing loss was secondary to the recurrent herpes simplex infections. abstract the illness was described for the first time in 1937 in the chinese literature by kimm, and szeto, the definitive histological description was published by kimura in 1948, this illness is endemic in asia, but rare, about 200 cases had been reported. kimura disease is extremely sporadic in the rest of the world. the etiology of this disease is ignored but it is believed that there is an aberrant immune reaction to an unknown antigenic stimulus, however epstein barr's virus, human herpes virus 8 and candida albicans had been involved in certain cases. the mast cells had been implicated in its pathogenesis and a th2 cytokine pattern with the production of interleukin 4, 5 and rantes which regulate the synthesis of ige and orchestrate the eosinophilic infiltration. on the other hand it is suggested that the eosinophils had undergone an accelerated apoptosis in this illness. case report. it is a 7 year-old boy with a 6 months evolution with the presence of bilateral subcutaneous nodules of 4x5 cm in parotid and submaxillary glands, presenting hypereosinophilia (8172 total eosinophils ) and high ige ( 9187 total ige), with normal renal function and negative mycotic and parasitic tests.the histopathologic findings revealed the presence of eosinophilic infiltrates with capillary proliferation and fibrosis. discussion. for the clinical characteristics of the nodules together with the hypereosinophilia, extremely high ige and the characteristic histological lesions the diagnosis is kimura disease. the usual clinical presentation consists on several indolent subcutaneous nodules that grow very slowly in volume, located in the neck and head, accompanied by satel-annals of allergy, asthma & immunology lites adenophaties, and with increment in the salivary glands, there is renal affectation in half of the patients, and the laboratory detects hypereosinophilia and elevation of the total ige. the histological lesions, shows hyperplastic lymphoid tissue with proliferative germinal centers, with infiltration of eosinophils in their interfollicular and perivascular zones sometimes forming an eosinophil abscess and proliferation of poscapillary venules. at the moment he have been treated with prednisone (1mgkdia), with great improvement in the clinical evolution. this is the first case reported in the literature in mexico. introduction: the incidence of cow's milk protein allergy (cmpa) is approximately 2-3% and presents primarily during the first year of life. manifestations of cmpa in the neonatal period include gastroenteritis, colic, lethargy, metabolic acidosis, and hematochezia or melena and appear to be non-ige mediated. ige mediated reactions in the neonatal period, such as urticaria or angioedema, are unusual. case: a 13-day-old african-american male presented with a 3 day history of rash, swelling, and erythema overlying multiple joints. there was no history of fever, diarrhea, or eczema. he had been on no medications prior to admission. besides mother with a history of childhood asthma, there was no family history of atopy or food allergy. he had been fed cow's milk-base formula (similac â® ) since birth exclusively. physical examination revealed a diffuse erythematous, raised, macular-papular rash, with areas of duskiness and exfoliation. there was angioedema overlying the joints and periorbital areas (figure 1). laboratory evaluation included cbc with diff, hgb electrophoresis, lumbar puncture, urinalysis, c 1 q (qualitative and quantitative), c 2 , c 3 , c 4 , and cultures of the blood, csf, and urine which were within normal. percutaneous allergy skin testing for cow's milk allergy was performed revealing a 3 + reaction to cow's milk extract (greer, lenoir, nc) with positive histamine and negative saline controls. rast testing showed 8.23 ku/l for -lactoglobulin and 5.10 ku/l for cow's milk. he was placed on an elemental formula. skin lesions and swelling resolved completely within 36 hours. at 2 week follow-up the patient was thriving without complaint. conclusion: early sensitization to cow's milk protein in the neonatal period may occur, resulting in ige mediated urticaria and angioedema. the rashes may be misdiagnosed as erythema multiforme or anaphylactoid purpura, since hemorrhagic lesions and cockade pattern are common. physicians should be aware that these reactions may occur so that early recognition and management may be initiated. neonate with periorbital angioedema and exfoliating, urticarial rash. rationale: two patients, presenting with invasive fungal cns infections, were found to have nk cell dysfunction and hypogammaglobulinemia. methods: case reports results: patient #1: a 47 year old caucasian male presented with headache, double vision, periorbital swelling, and bilateral sinus disease on ct scan. biopsies showed invasive rhinocerebral mucormycosis. he continued to deteriorate in spite of iv and intrathecal amphotericin b, hyperbaric oxygen, many debridement procedures, and a left orbital exenteration. immune evaluation revealed low igg, low igm and a barely detectable nk cell killing activity. neutrophil respiratory burst was normal. he continued to worsen despite ivig replacement. he had intolerable side effects to ifn-a. gm-csf (500 mcg qod) was initiated, in order to boost nk cell activity. this resulted in stabilization of his infection. he was discharged on oral anti-fungal therapy (posaconazole), ivig, and gm-csf. sixteen months later, he continues to remain stable clinically and radiographically on ivig and gm-csf. patient #2: a 62-year-old caucasian male presented with headache, mental status changes, and ataxia. a head ct scan showed a left mass effect and edema. he underwent surgical debridement for ventriculitis and zygomycetes (the same family as mucormycosis) was found. he was started on iv amphotericin b and oral posaconazole. his immune evaluation revealed low igg and igm and low nk cell activity. neutrophil respiratory burst was normal. he was started on ivig and gm-csf after which his nk cell function improved significantly. he remains clinically stable on ivig and gm-csf with persistent radiographic evidence of enlarged ventricles. conclusion: hypogammaglobulinemia is usually not associated with invasive cns fungal infections, suggesting that nk cell dysfunction was likely responsible for the clinical courses of these patients. nk cell deficiency has been reported to be associated with recurrent mucosal candidiasis, suggesting an important role for these cells in the control of some fungi. the spectrum of the clinical presentations of nk cell deficiency is not known since it is not normally included in immune system evaluations. these patients illustrate the importance of including functional nk cell assessment in any evaluation of immune function. introduction: to report a case of successful systemic hydrocortisone desensitization, since allergic reactions and systemic desensitization to corticosteroids have rarely been documented. method: we present a patient with multiple medical problems who has a history of both radiocontrast induced anaphylactoid reaction and corticosteroid allergy. this patient had to undergo cardiac catheterization and corticosteroid desensitization was performed prior to the procedure. results: skin testing to radiocontrast is not considered helpful, therefore patients with suspected reactions to radiocontrast are generally premedicated with corticosteroids and antihistamines to decrease the risk and severity of a reaction. 1, 2 since this patient experienced an allergic reaction to a corticosteroid previously, she was skin tested to two different corticosteroids. the least reactive skin test revealed a 3+ positive immediate reaction to hydrocortisone. cardiac catheterization with contrast was considered absolutely necessary in this case and corticosteroid desensitization was performed. the half-life of hydrocortisone is the shortest among the tested corticosteroids (80-118 minutes), so a protocol was developed with short intervals of escalating doses. each dose was diluted yielding a total volume of 75ml to avoid fluid overload because patient has renal failure. during desensitization, patient developed pruritus and erythema between the 3 rd and 4 th dose that resolved immediately with 50mg diphenhydramine. after desensitization, the patient continued to be on hydrocortisone 200mg intravenously every four hours. one hour prior to the procedure, the patient was premedicated with hydrocortisone and diphenhydramine and was administered radiocontrast without any adverse reactions. conclusion: we successfully desensitized our patient to a corticosteroid and premedicated her with hydrocortisone and diphenhydramine before administering radiocontrast. this case illustrates that intravenous desensitization may be a suitable approach to therapy in corticosteroid allergic patients who require systemic corticosteroids administration. autoimmune neutropenia is defined as a decrease in the absolute number of peripheral neutrophils caused by an immunologically-mediated mechanism. autoantibodies directed to neutrophils can lead to the peripheral destruction of neutrophils and/or inhibit myelopoesis in the bone marrow. although recurrent aphthous stomatitis is often the heralding sign of neutropenia, the diagnosis of ain requires the demonstration of specific antineutrophil antibody which acts by promoting the immune destruction and clearance of neutrophils by mononuclear phagocytes. the present case report describes the rare clinical association of ras in an adult. a 66 yr-old black male presented with a 3 year history of recurrent and repeated painful multiple oral ulcers. initially the oral ulcers occurred on a monthly basis then over time the ulcerations on the oral mucosa and tongue began to appear weekly and later continuously. past medical history revealed no drug allergies but a positive history of hypertension. physical exam revealed an otherwise healthy male with no lymphoadenopathy or splenomegaly. laboratory workup revealed : hiv negative, viral culture for h. simplex negative, mild increase in cd8, cyroglobulins negative, anti dsdna negative anti-smith and rnp negative, wbc count 3300/ml, neutrophils 29% (anc 957), lymphocytes 54%, monocytes 11%, and platelets: 243, 000. bone marrow biopsy revealed a normal production of neutrophils. a direct neutrophil antibody assay: revealed an elevated value of 6, 576 (n= <4, 000). although following initiation of prednisone (60 mg) an immediate increase in anc was seen, the levels fell as the dosage was tapered. the figure below shows the time course and dose-response relationship of anc and prednisone dosage. this case report illustrates the importance of recognition of the relationship of ras and neutropenia, an association that can masquerade as other clinical entities. background: dyspnea, wheezing, and decreased fev1 are suggestive of asthma. it is essential for the clinician to consider a broad differential diag-nosis as the outcome could be catastrophic if the correct diagnosis is missed. case presentation: we present a case of a 48 y.o. filipino female who was referred to our clinic for the evaluation of cough, shortness of breath, and wheezy respiration associated with changes in voice quality, nasal and palatal pruritus, and postnasal drainage. her initial evaluation revealed mold spore hypersensitivity by prick puncture testing and spirometry with an obstructive pattern with fvc-3.05 l (99%) and fev1-1.25 l (50%) predicted and a 15% reversibility post nebulized albuterol. an initial diagnosis of allergic rhinitis with adult onset asthma was made and she was started on salmeterol, budesonide, montelukast, and pirbuterol. her symptoms persisted and rabeprazole was added to treat possible laryngopharyngeal reflux. repeat spirometry revealed fev1-0.84 l prompting treatment with systemic corticosteroids again with no improvement. fiberoptic laryngoscopy was within normal limits. a high resolution computed tomography was obtained and showed a mass in the left side of the trachea which was obstructing 60% of the airway. bronchoscopy revealed a tumor 4-5 cm below the vocal cords with the appearance of adenoid cystic carcinoma which was confirmed by pathology. the tumor was resected by removal of 7 cm trachea with re-anastomosis, followed by a 6 week course of radiation therapy. all medications were discontinued. her symptoms of wheezing, dyspnea, and cough completely resolved. repeat spirometry was within normal limits and she remained asymptomatic. surveillance bronchoscopies have been negative for any recurrence. discussion: adenoid cystic carcinoma (acc) is an uncommon form of malignant neoplasm that occurs within the salivary glands. tracheobronchial acc typically presents with symptoms of cough, dyspnea, and hoarseness. due to its slow growth, acc has a relatively indolent course. in a recent study of a cohort of 160 acc patients, survival was 89% at 5 years but only 40% at 15 years. standard therapy is surgical resection often followed by radiotherapy. conclusion: in patients who fail conventional therapies for asthma it is important to entertain other diagnosis and have a systematic approach to establish the correct diagnosis. ipex is an extremely rare, hereditary condition characterized by immune dysfunction, polyendocrinopathy, enteropathy and x-linked recessive inheritance that leads to death without prompt diagnosis. patients usually present by 4 months with severe diarrhea, failure to thrive, and early onset iddm. most children die by one year without a bone marrow transplant. immunologic evaluation is typically normal except for elevated ige, eosinophilia, and autoantibodies. we report a case of ipex in an infant who presented at birth and died at 79 days of multi-organ system failure. this male infant was born at 30 weeks due to chorioamnionitis and prom. at birth, copious green fluid appeared from his rectum and ng tube which evolved into a secretory diarrhea. workup for fistula was negative. at 2 weeks, the patient developed ascites and explorative laparotomy revealed an inflamed appendix. after the laparotomy, the patient had no further stool output and never tolerated enteric feeds. he remained intubated and had problems with apnea and coagulapathy. pathology of the appendix showed an excess of lymphocytes. immune system investigation showed elevated ige (5320) and igg (979). serum anti-enterocyte igg antibody was positive in the patient and negative in his mother. based on this data, ipex was suspected which autopsy seemed to confirm. autopsies are scarce in patients with ipex. the findings revealed many organs affected by fibrosis but lymphocyte infiltration of only the pancreas and gi tract. the pancreas revealed almost complete loss of the exocrine structure, with invasion of fibrosis and chronic inflammatory cells. the mucosa of the gi tract from the stomach to rectum showed columnar epithelium taken over by fibrosis, capillaries and chronic inflammation. these inflammatory cells were cd3+ lymphocytes and plasma cells on immunochemistry. the lymphoreticular system was consistent with lymphoid paucity in the thymus, lymph nodes and spleen. preliminary data suggests this patient has a splice mutation of the foxp3 gene. ipex is an extremely rare disease, often difficult to diagnose while a patient is alive. infants will present in early infancy with diarrhea and endocrinopathies. without prompt diagnosis, death is inevitable. as a result, one must be aware of atypical presentations and considered in patients with total villous atrophy and one other clinical feature such as iddm or autoimmunity. introduction: digeorge syndrome (dgs) is characterized by thymic hypoplasia, parathyroid hypoplasia, and conotruncal cardiac defects, but has a wide variety of clinical manifestations. there is also an increased risk for autoimmune phenomena in later life due to thymic hypoplasia. case report: a 13 year-old aa girl with tetralogy of fallot (repair at age 2), developmental delay, asthma, recurrent sinopulmonary infections/skin abscesses, gerd, arthralgias and seizure disorder (due to hypoxic encephalopathy during cardiac surgery) presented to allergy/immunology clinic for immune workup. there was no history of documented hypocalcemia. at age 10, she developed persistent annular patches on her left leg. a skin biopsy appeared consistent with sarcoid dermatitis. there was no other evidence of sarcoidosis except for slightly elevated ace level. given lack of systemic involvement, the skin lesions were not treated, but resolved spontaneously. at age 12, she developed swelling of the right mandible, and a bone biopsy revealed garre's osteomyelitis (sterile hyperproliferative osteomyelitis), likely triggered by dental caries, with elevated esr (40) and polyclonal hypergammaglobulinemia (igg 2640), but normal crp. immune workup revealed a decrease in cd4 and cd8 cells (cd4/cd8 ratio 2.44), slightly elevated b cells, high-normal range nk cells, normal range t cell cytokine production in response to mitogens and il-12, but excessive production of proinflammatory cytokines in responses to lps. in conjunction with facial dysmorphism, dgs was suspected, and fish analysis revealed 22q11.2 microdeletion. a repeat workup did not reveal evidence of systemic sarcoidosis, and autoantibody screening was negative including lupus anticoagulant. she was treated with a cox-2 inhibitor, secondary to gerd and mild thrombocytopenia, and her joint symptoms resolved with a concurrent decline in esr (to 30) and igg level (to 2390) after 2 months. conclusion: recurrent infections with fragmented care and resultant chronic inflammation (hence overstimulation of the immune system) may have led this dgs patient to develop atypical autoimmune phenomena and other unusual clinical manifestations. this case illustrates the importance of recognizing the phenotypic features of dgs early in life, and of providing close monitoring and coordinated care. rationale: we report a case of a patient with celiac disease who continues to have symptoms of fevers, nausea, vomiting, night sweats and fatigue despite being on a gluten free diet whose symptoms have been responsive to antihistamines. methods: case report. results: in 1998, this 41 year-old white male suffered from mononucleosis and episodes of prostatitis. he began suffering from fatigue and fevers and was followed at a chronic fatigue syndrome center in 1999. in november 2000, patient was placed on famvir alleviating his sore throat. he was later placed on interferon gamma which was discontinued due to increased fevers, nausea and vomiting. after stopping medication, symptoms abated for sometime. in 2002, patient's father was diagnosed with celiac sprue. in april of 2003, the patient developed a pruritic rash on his right arm. biopsy revealed subepidermal vesicles with pmn's at the tips of dermal papillae. in may of 2003, he developed oral ulcers as well as joint pains. patient underwent endoscopy with biopsies revealing intraepithelial lymphocytosis in the duodenum. patient has been on a strict gluten free diet since october 2003. he reports that some symptoms have improved: skin lesions, itchy eyes, and oral ulcers. however, he has begun to suffer from constipation and continues to intermittently have night sweats, fevers, nausea and vomiting. in february of 2004, he had pruritus that was not alleviated by hydroxyzine. a regimen of benadryl and pepcid was started to aid with the pruritus. the patient reported in the improvement of his symptoms of pruritis, sweats, and emesis. conclusions: diagnosis: celiac disease(cd) patient with persistent nausea, vomiting, sporadic fevers, and fatigue despite maintaining a strict gluten free diet has shown improvement of symptoms with antihistamines. patient has history of chronic infections: prostatitis and chronic ebv. patient also reported alleviation of symptoms after interferon therapy suggesting some autoimmune component to his current illness. cd prevalence in the united states is much higher than once thought 0.5-1% of the u.s. celiac disease has been associated with increased risk of lymphoma and malabsorption leading to neurological diseases. this is a rare case of cd associated pruritus responding to antihistamines. rationale: eosinophilic cystitis is a relatively rare condition in children and adults with a varied course. it usually responds to short-term nsaid and steroid therapy. we report a man with a severe case who responded to prolonged oral steroids. case report: a 54 year old caucasian man with a prior esophageal cancer s/p esophagectomy, diabetes, hypertension, allergic rhinitis and chronic obstructive pulmonary disease presented with suprapubic pain, urinary frequency, dysuria, and hematuria. urinalysis showed numerous red blood cells and bacteria but no malignant cells or eosinophils. he was treated with antibiotics with resolution of symptoms. several weeks later he experienced severe suprapubic pain and hematuria resulting in a symptomatic drop in his hemoglobin. he underwent a cystoscopy and biopsy that revealed a chronic cystitis with an inflammatory infiltrate containing numerous eosinophils. he was unresponsive to treatment with nsaid and intravesicular dmso and was then referred to our service. we started prednisone 20 mg/day but the dysuria and hematuria persisted. prednisone was doubled plus a third generation quinolone was added. three weeks later the hematuria and dysuria resolved and he was asymptomatic. the antibiotic was stopped and prednisone was gradually tapered over several weeks. whenever his steroid dose dropped below 20 mg/day he experienced an exacerbation of his symptoms. over the last three years this dose of prednisone has kept his symptoms abated and his renal function stable. conclusion: eosinophilic cystitis is an uncommon diagnosis of unknown etiology. we describe a patient with debilitating suprapubic pain and symptomatic anemia from the hematuria associated with eosinophilic cystitis. for over three years since we first saw him, continued therapy with 20 mg of prednisone/day has prevented exacerbations. this case is unique in the severity of the hematuria experienced and the prolonged relatively low steroid dose needed to suppress exacerbations. we propose that in eosinophilic cystitis patients with severe hematuria who may otherwise be candidates for a cystectomy, a trial of prolonged oral prednisone may be beneficial. rationale: allergic reactions to insulin occurred more frequently in the past, with porcine and bovine preparations. in contrast, allergic reactions to human insulin preparations are now reported in <1% of patients treated with insulin. insulin allergy may be manifested as an immediate-type 1 ige-mediated reaction, delayed type hypersensitivity or as serum sickness, varying in severity from mild discomfort to life-threatening events. we present a case to illustrate that an insulin allergy may complicate hospitalization and often be misdiagnosed. methods: a 66 y.o. diabetic female with a history of "insulin allergy" was evaluated. the patient is a long standing diabetic controlled on oral hypoglycemics but required insulin during acute illnesses and hospitalizations. the patient was unable to recall previous types of insulin she had received. during previous hospitalizations, the patient complained of vague symptoms of fatigue, parasthesia, sweating, anxiety, and palpitations. on one occasion the patient had a syncopal episode with hypotension and on another occasion the patient developed a rash and dyspnea after receiving sq insulin. the physcian intrepreted the findings may be due to hypoglycemic. an allergic reaction was not suspected and the patient never underwent any testing. during a recent hospitalization, the patient's history was reviewed and a formal allergy consult was obtained. she underwent epicutaneous and intradermal testing to human insulin preparations. insulin antibody levels were also obtained. results: the patient had negative epicutaneous testing to all human insulin preparations. however, on intradermal testing, the patient had positive reactions to lispro, nph, and lente and negative intradermal tests to insulin glargine and regular insulin. igg and ige insulin antibody test results were < 1. conclusion: hospitalized patients receiving multiple medications commonly experience pharmacologic, adverse and/or allergic reactions. it is necessary to obtain a thorough history and document all reactions that patients experience to determine what type of event occurred. with a suspicion of drug allergy, skin testing and/or rast assay may provide insight into possible ige mediated reactions. in this case we advised the patient that she may use regular insulin during hospitalizations and that insulin glargine can be used to help achieve optimal glycemic long term control. background: hereditary angioedema type iii (hae iii) is a recently described form of angioedema occurring exclusively in females and characterized by normal c4, c1 inhibitor (c1 inh) protein and function. hae iii is thought to have an x-linked or autosomal dominant mode of inheritance. we evaluated a 13 year old female with recurrent facial swelling, abdominal pain and laryngeal edema with a family history of similar symptoms in several female relatives. case report: a 13 year old african american female presented with a two year history of recurrent lip, tongue and facial swelling. she also had abdominal pain, diarrhea and shortness of breath. episodes were not associated with hives. her symptoms predated menarche and did not correlate with her menstrual cycle. at the time of presentation she described an increase in frequency of her symptoms that did not respond to antihistamines (diphenhydramine, cetirizine) or prednisone. the patient had no other medical problems. family history was significant for recurrent episodes of facial, lip and tongue swelling in a maternal aunt, grandmother and great grandmother. the patient's great grandmother required tracheal intubation with ventilator support for upper airway compromise. the patient's physical exam was unremarkable. laboratory values drawn during an acute episode of swelling revealed: c1 inh function = >68% (normal >68%), c1 inh protein = 323mg/ml (normal 158-413 mg/ml), c4= 21.70 mg/dl (normal 20-59 mg/dl). dna sequencing at exon 8 to investigate the possibility of an unusual c1 inh mutation with normal c1s binding but abnormal kallikrein inhibition was negative. other lab tests included a normal mast cell tryptase of 2.9 mcg/l, a negative rf and ana, a normal angiotensin converting enzyme of 62 u/l (normal 6-64 u/l) and positive skin prick tests to a variety of foods which the patient tolerates. based on two case reports of treatment of hae iii with androgens, the patient was started on danazol 200mg daily with symptomatic improvement. conclusion: hae iii is a rare disease that affects females exclusively. clinically it is indistinguishable from c1 inh deficiency. the mechanism of inheritance remains to be elucidated. it is unclear why danazol appears to ameliorate symptoms even though there is no evidence for c1 annals of allergy, asthma & immunology inhibitor dysfunction in this disorder. we believe this to be the first kindred of hae iii reported in the united states. r. dworski * , m. peters, nashville, tn. a four-month-old female identical twin was evaluated for noisy breathing and recurrent cyanosis. she was delivered at 32 weeks of an estimated gestational age after an uncomplicated pregnancy. at birth she was intubated for 3 hours for respiratory distress but the remainder of her neonatal period was uneventful. at age 3 weeks she developed a noisy breathing often associated with cyanosis, particularly in a supine position or while crying. treatments with inhaled albuterol and prednisolone were ineffective. she had no respiratory infections or symptoms of gastroesophageal reflux disease. her growth was normal. her twin sibling was well. the family history was negative for allergies or respiratory conditions. initially she was diagnosed with tracheomalacia. however, the history of cyanotic episodes prompted a search for a definitive diagnosis. she underwent bronchoscopy which showed tracheomalacia when breathing spontaneously and circumferential narrowing of lower trachea likely due to compression. echocardiogram revealed a double aortic arch. the finding was confirmed by computed tomography angiography which demonstrated the presence of a vascular ring composed of double patent aortic arches, each giving rise to the ipsilateral carotid and subclavian arteries. the airway was normal at the aortic inlet, but narrowed markedly at the level of the two arches. a surgical division of the ductus ligamentous and distal anterior vascular arch was performed. aortic arch abnormalities should be suspected in all infants with hoarse coughing or noisy breathing, especially during inspiration but sometimes also during expiration. the diagnosis should also be considered in older children with recurrent bronchitis or pneumonia. respiratory symptoms are more frequent than gastrointestinal manifestations. diagnosis usually occurs in the first year of life. a surgery is often the treatment of choice. postoperative complications are relatively rare. outcome of surgery should be judged after 12 months, including at least one winter season. malacia can delay extubation and recovery following surgery. surgical cure occurs in approximately 70% of patients. surgery is probably less successful in children with double arches and malacia. a. thatayatikom * , a.j. white, st. louis, mo. background: common variable immunodeficiency (cvid) is the commonest symptomatic primary antibody deficiency syndrome in which b lymphocytes produce low levels of immunoglobulin, leading to recurrent bacterial infection. although hypogammaglobulinemia and susceptibility to the recurrent infection are seen in all patients, other associated conditions such as a non-infectious granulomatous disease have been well described in cvid. corticosteroid therapy has been used with improvement in a subset of cvid with granulomatous disease; however, its treatment remains problematic and a new therapeutic agent is needed. tumor necrosis factor (tnf ) has been demonstrated as a primary mediator in granuloma formation and maintenance. therefore, anti-tnf medications are potentially therapeutic agents of the granulomatous disease. case report: a 22-year-old caucasian male with cvid and severe granulomatous disease was treated successfully with infliximab, a chimeric anti-tnf monoclonal antibody. the patient initially presented with high fever, chills and abdominal pain; subsequently, he developed acute respiratory failure and adult respiratory distress syndrome. the patient was hospitalized and he required intensive care with ventilatory support. his diagnostic tests revealed elevated sedimentation rate and positive epstein-barr virus (ebv) capsid igm antibody. imaging studies demonstrated bilateral diffuse pulmonary infiltrates and hepatosplenomegaly. open lung and liver biop-sies revealed non-caseating granulomatous lesions without evidence of ebv or other infections. high dose corticosteroid therapy was given with partial improvement, then high dose infliximab (10mg/kg) was given weekly with remarkable improvement. the patient was able to wean off ventilator successfully within 3 weeks and his prednisone dose was dramatically decreased. infliximab infusion (5mg/kg) every 8 weeks and low dose prednisone were continued. a follow-up liver biopsy after 6 months of the infliximab showed no granulomatous lesions. infliximab was discontinued after 12 months of the treatment. there was no serious infection or complication during the period of treatment. conclusion: anti-tnf therapy may be a safe and effective treatment and may allow corticosteroid dose reduction. future clinical studies of anti-tnf therapy in cvid with granulomatous disease are warranted. background: chrug-strauss syndrome is a disorder characterized by hypereosinophilia and systemic vasculitis occurring in individuals with asthma. objetive: to present a pediatric case suffering from a systemic vasculitis. this case fulfilled the churg-strauss syndrome clinical criteria and the histophatology findings were compatible with the diagnosis. case: a 14 year female came to our institution with the diagnosis of severe asthma, chronic sinusisits and polyps requiring high doses of steroids. there was no history of administration of antileukotriene receptor antagonists. 2 months before her admission she presented weight loss, fatigue, cephalea and cough. on physical examination, pallor, respiratory difficulty and signs of bronchospasm were evident. tachycardia and hepatomegaly were also documented. the laboratory test showed anemia, eosinophilia 1300/dl, anca+, sgot 246, stgop 253, ige elevated 1345 ui/ml. the chest-x ray showed patchy opacities in both lungs and cardiomegaly. pulmonary scintigraphy reported low perfusion in both lungs, predominantly in the left lung.echocardiography demonstrated signs of myocarditis and eyection fraction of 32%. an open lung biopsy was executed and vasculitis with fibrinoid changes affecting small and medium vessels was reported. treatment was started with oral prednisone 2 mg/kg/d and cyclophosphamide pulses with a satisfactory evolution. discussion: to our knowledge this is the first pediatric case of css reported in mexico. she fulilled the following criteria. asthma, eosinophilia and systemic vasculitis involving the heart, liver and lungs. the disease is extremely rare, specially in mexico. aggressive treatment is necessary as in this case, with a favorable outcome. introduction "all that wheezes is not asthma" is a well-known aphorism among physicians. this same principle exists for patients that present with lip swelling in the allergist's office. we describe a patient who presented with fluctuating lip swelling who was ultimately found to have cheilitis granulomatosa. case history a 26-year-old male with a history of allergic rhinitis and asthma presented to the allergy clinic with lower lip swelling and occasional upper lip swelling. he was receiving allergen immunotherapy for dust mites and trees. the swelling had been waxing and waning for 13 years, but became more persistent for the previous six months. he denied tongue/throat swelling, dysphagia, respiratory distress, or any triggers for the swelling. chapstickâ® and vaselineâ® were used topically on his lips. failed treatments included loratadine, ranitidine, cetirizine, fexofenadine, and montelukast. he was placed on a one-week course of prednisone, which decreased his lip swelling, but it recurred after completion of the treatment. physical exam was significant for diffuse, firm lower lip edema to 3-4 times the normal size. there were no oral lesions or tongue swelling. patch testing to a standard panel, preservatives, oral flavors, and dental acrylate was negative. punch biopsy revealed a noncaseating epithelioid granulomatous inflammation consistent with granulomatous cheilitis. he was placed on minocycline 100 mg by mouth twice daily with little benefit. an 8-week course of oral prednisone resulted in improvement of the lip swelling. conclusion melkersson-rosenthal syndrome (mrs) is a rare syndrome that is characterized by a triad of recurrent facial paralysis, chronic edema of the face and lips, and hypertrophy and fissuring of the tongue. cheilitis granulomatosa is considered a monosymptomatic form of mrs and manifests as a chronic swelling of the lips caused by granulomatous inflammation. the swelling is typically not tender and may be either soft or firm. allergists are often consulted for lip swelling thought due to angioedema. however, as this case illustrates, not all lip swelling is angioedema and one must consider other diagnoses such as melkersson-rosenthal syndrome and cheilitis granulomatosa. progressive multifocal leukoencephalopathy (pml) is a disorder of the nervous system that affects individuals with immune suppression. it has been associated with hiv infection and is present in nearly 10% of patients with acquired immune deficiency syndrome. the jc virus, a common human polyomavirus, causes this demyelinating disorder. progressive symptoms reflect the multifocal distribution of brain lesions, and include mental deterioration, vision loss, speech disturbances, ataxia, paralysis, and, ultimately, coma. in rare cases, seizures may occur. there is no known treatment for pml. we report a 21 year-old (y/o) male with 6 months weight loss and a sudden onset of confusion, lethargy, and progressive loss of cognition requiring hospitalization. upon questioning he was found to have had recurrent upper respiratory tract infections since infancy successfully treated with antibiotics. as a child he had atopic dermatitis, exercise induced asthma, and myringotomy tubes placed twice. at 12 y/o, he underwent a nasal polypectomy. in 2002, at 19 y/o, a squamous cell carcinoma was removed, and he developed benign cervical lymphadenopathy and common warts. a year later he developed hsv esophagitis. the remainder of his history was unremarkable with normal development and growth and no history of drug abuse, multiple sexual partners, or homosexual contacts. on physical examination, he was thin, ill appearing, with oral ulcers, generalized scanty lymphoadenopathy, multiple common warts on both feet, and occasional ronchi. a brain mri showed a demyelinating process consistent with pml. pcr for jc virus was positive, while hiv pcr was negative; total immunoglobulins and cd4 counts were low ( we are reporting a 47 year old female with a 10 year history of moderate persistent asthma, who was started on xolair 150mg sq q 4 wks., and who then presented with a presumed allergic reaction. three hours after her second xolair injection, patient reported developing dizziness, shortness of breath, and felt like she was having an allergic reaction. she was evaluated and observed for two hours in an emergency department. the treating physician reported no wheezing and felt there was no need for treatment. to rule out psychogenic factors, she was given a placebo injection at the next scheduled visit. ten minutes later she reported developing throat tightness and shortness of breath. she had no changes in her peak flows and her lungs were clear. her "symptoms" resolved completely within 5 minutes of receiving placebo epinephrine and nebulized normal saline. patient was informed she had reacted to a placebo injection, as well as placebo epinephrine and albuterol, and counseled. the patient returned to the office every week for the next three weeks to receive blinded injections. she subsequently did not react to either doses of placebo or xolair. she has since tolerated her monthly xolair injections without incident. this case illustrates the importance of ruling out psychologic causes of presumed allergic reactions. introduction :latex allergy, type i ige mediated hipersensitivity, occurs specially in high risk populations, like in patients that have undergone various surgeries. case: a 10 year old boy with asthma and allergic rhinitis since 1996, penicillin allergy and retrospectively, his mother refers lip edema with balloon inflating.at 4 years of age (1998): left orchiorrhaphy because of cryptorchis. between 1999-2004: 4 surgeries because of sacral giant melanocitic naevus.during the fourth surgical intervention (0ct 8 -2004) to collocate a tissue expander, the patient presents perioral and fingertip cyanosis, generalized cutaneous rash and severe bronchospasm.ap:40/22 mmhg, hr 140 x/min. he was treated with iv fluids, steroids, antihistamines and inhaled racemic epinephrine.at the icu his final outcome is satisfactory. laboratory: total ige :27.4iu/l, skin prick test with glove extract, raw and natural latex extract and purified latex proteins(pseudoeveine, molecular hevein, hev b 6.02 and modified hevein)all positive +++. western blot with protein extract of latex positive and elisa with purified latex proteins ( same as above) positive. the last surgery to withdraw the tissue expander was performed with latex free surgical equipment without any problems. discussion: the patient's risk factors for latex allergy are atopy and repetitive exposure to latex articles because of surgery. he presents mild manifestations of latex allergy, till he finally develops full-blown anaphylaxis. diagnosis was made based on clinical history, skin prick test, western blot with protein extract and elisa with purified latex protein. he had a favorable outcome withdrawing latex during the last surgery. introduction: churg-strauss syndrome (css) is a form of primary vasculitis that is a rare diagnosis in an elderly patient. case report: a 75 year old woman presented with a 3 week history of fatigue, vomiting, diarrhea, abdom-inal pain, and right lower leg paresthesia. prior to admission she was being treated for left neck erythema and adenopathy presumed to be cellulitis. she was in good health with no history of atopy until the age of 72, when she developed both chronic sinusitis, requiring bilateral sinus surgery and polypectomy, * and new onset asthma, * that required systemic steroid control. prednisone was tapered 1 month prior to admission. objective findings included coalescent non-blanching petechiae on her abdomen, peripheral eosinophilia of 4800(50%), * normochromic normocytic anemia, rf=124, esr=90, ige=232, igg=1645. ana, p and c-anca were negative. ct showed ascites and pleural effusions. egd revealed duodenitis with ulceration and eosinophilic infiltration on biopsy. echo showed pericardial effusion and septal motion abnormality. troponin of 31 without cad was consistent with subepicardial myocarditis. emg confirmed right peroneal neuropathy.* skin biopsy revealed a dense superficial and mid-dermal perivascular and interstitial eosinophilic infiltrate. additional evaluation excluded malignancy, infection, and abpa. treatment with prednisone, 1mg/kg/day was initiated. a rapid clinical improvement ensued and has persisted. {*acr criteria for css}. discussion: churg-strauss syndrome is a rare form of vasculitis with mean age of onset within the third and fifth decades. it is an uncommon cause (<5%) of systemic vasculitis in patients older than 65. the formes frustes of css is a variant in which early manifestations of the syndrome are hidden by oral and systemic steroids employed in the treatment of worsening asthma often associated with css. this variant makes expedient identification of css more challenging. delayed recognition contributes to a relentless progression of this entity resulting in a systemic vasculitis with multi-organ involvement. early diagnosis, especially in the prodromal and eosinophilic phases, is essential. untreated, css has a high rate of morbidity and mortality. therefore, css and the formes frustes variant must be an integral component in the differential diagnosis of patients presenting with adult onset asthma and/or recurrent sinusitis. common variable immunodeficiency (cvid) is the most prevalent of the primary immunodeficiency diseases. cvid is a heterogeneous group of immunologic disorders of unknown etiology, characterized by impaired antibody responses, hypogammaglobulinemia with normal b cells. the common immunologic defect in patients with cvid is defective antibody formation, and many different immune system defects have been reported in this group of patients. most patients, really, have no identified molecular diagnosis. cvid consists of several different genetics defect. the immunologic defect in cvid is a failure of b-lymphocyte differentiation into plasmacells. b lymphocytes from these patients failed to differentiate into ig-producing cells when stimulated with pokeweed mitogen in vitro, even when cocultured with normal t cells. an overwhelming body of literature suggests that most patients with cvid have intact b lymphocytes of immature phenotype. however the functional classification of cvid patients on the basis of in vitro ig production is time consuming. recently has been proposed a new classification based on the quantitative repartition of memory b cell according to the dual expression of igd and cd27. we present a case of a 14 yr old boy. he presented soon in his life frequent infections, of particular severity: pneumonia, meningoencefalitis, sepsis, bronchitis, otitis, linfoadenitis. he also had a -thalassemia intermedia. this clinical manifestations suggested an immunodeficiency. for this reason at 2 years of age serum immunoglobulin and antibody detection showed a reduction in igg2 subclass and in cd19+ cells, with normal total igg, iga, igm, normal cd4/cd8 ratio, normal cd3, isohemagglutinins and in vitro t cell function. there was also a defective antibody production after tetanus, diphtheria, pertussis immunization. these laboratory findings did not allow, however, a sure diagnosis for cvid. a new immunological evaluation at the age of 11 years old, after the onset of splenomegaly, and enlarged lymph nodes, demonstrated a b memory defect, with a severe deficit of t lymphocytes function in vitro. it was also possible to find a severe deficiency of cd 27 + cells, meaning a defect in memory b cells: we can therfore label this condition as a cvid. in the past two decades there have been conflicting views regarding the clinical importance of igg subclass deficiencies in children. igg2 subclass plays a vital role in the immune response to polysaccharide antigen. isolated igg2 subclass deficiency may be widespread and often asymptomatic in children. however, in association with other subclasses and/or other immunoglobulin classes, there may be a significant, symptomatic outcome. the reported patient was diagnosed with familial dysautonomia (fd) at the age of five weeks, presenting with severe hypotonia and tachypnea. hindered by poor pulmonary function, he was hospitalized over fifteen times for recurrent pneumonias, including four lengthy intensive care admissions. daily inhaled-corticosteroids and brochodilator therapies were initiated, along with chest therapy via a high frequency chest wall oscillator. immunoglobulin levels were measured recently and point to low levels of igg2, igg4 and total iga antibodies: igg1 443 mg/dl (n 330-1065), igg2 57 mg/dl (n 57-345), igg3 50.5 mg/dl (n 7.5-125.6), igg4 <2.0 mg/dl (n 1.8-115.5), and total iga 28 mg/dl (n 33-235). since receiving monthly ivig therapy he had no further recurrence of pulmonary infections and was slowly weaned off daily brochodilator therapy. the currently accepted theory is that low igg2 subclass levels may be associated with increased risk of bacterial infections only in selective groups. fd patients may be in a distinctively susceptible population in which igg2 levels are critical. the older brother, who was also diagnosed with fd, demonstrated igg2, igg4 and iga levels that were slightly higher but nevertheless on the lower end of the normal range. he suffers from less invasive and less frequent bacterial infections. this may support a genetic association between the fd and hypogammaglobulinemia. alternatively, it may signal that fd patients may have a prolonged variant of transient hypogammaglobulinemia of infancy. follow-up immune profile studies, post-ivig trough levels and broader investigations of the fd population are necessary to determine the severity and prevalence of these findings. pulmonary failure is the dominant cause of death in patients with fd. prompt diagnosis and effective treatment of the associated immune deficiency may be proven essential in the effort to enhance and prolong their lives. s. hassan * , j.a. grant, galveston, tx. rationale: common variable immunodeficiency (cvid), a rare primary immunodeficiency presenting in young adults with repeated sinopulmonary infections as a result of profound hypogammaglobulinemia, was first described by suri et al. (ann acad. med. singapore, 1988) . we describe a young man with diagnosed but untreated cvid and its eventual course. case description: a 27-yr-old caucasian male hospitalized secondary to chronic pneumonia and respiratory failure was noted to have non-existent levels of immunoglobulins. history revealed ivig treatment at age 12, stopped after a year due to non-compliance. although untreated, he denied recurrent sinusitis, otitis media, or bronchitis for almost 14 years but notes a recent inability in keeping up with baseball practice. he is the last of ten healthy siblings. patient started monthly ivig infusions but was noted to have hypertension (150/90), tachycardia (120-130), tachypnea (25-30) on the 4th month with o2 saturation of 87-93% and po2 of 49% on room air. with a 2-day history of acute dyspnea, calf muscle and right abdominal pain, he was admitted to the hospital and pulmonary thromboembolism was ruled out. laboratory data: humoral functions (pneumococcal, tetanus toxoid, and hepvac) -undetectable t cell function (mumps, candida, ppd) -normal immunoglobulin (igg, iga, igm) -undetectable flow cytometry -b and nk cell markers normal, mild decrease in the cd4/cd8 ratio high resolution ct thorax -bronchiectasis, bronchial wall thickening, and obstructive changes with airtrapping. 6 minute walk -desaturation to 78% on 4l o2 by nc bnp -462 echocardiogramestimated ef 40-45%; severe pulmonary hypertension. cardiac catheterization -normal coronary arteries, severe pulmonary hypertension (pa pressure 70/40). conclusion: cvid patients have a reasonably good prognosis on treatment. untreated cvid is associated with chronic lung infections, bronchiectasis, pulmonary hypertension and right heart failure. although, lung transplant became available during the early eighties (nejm 1982), this extremely invasive but life saving procedure was undertaken in a patient with cvid and end-stage pulmonary hypertension in 1998 (thorax 1998). lung transplant may be the only way of ensuring survival for this patient. introduction chronic eosinophilic pneumonia (cep) is a rare disorder of unknown etiology characterized as a chronic and relapsing interstitial lung disease with blood or tissue eosinophilia. cep occurs more often in women with preexisting atopic disease. patients with cep respond rapidly to systemic corticosteroids, but often relapse with short, low-dose courses of therapy. while uncommon, extrapulmonary involvement, such as arthralgia, cutaneous purpura, pericarditis, and hepatitis, have been reported. we present a case of a patient who has cep with pericardial effusion. case report the patient is a non-smoking 60-year-old woman with a past medical history significant for allergic rhinitis and asthma who initially presented with a four-month history of worsening shortness of breath, dyspnea on exertion, dry, non-productive cough, and weight loss (approximately five pounds). her symptoms were refractory to increased dosages of inhaled fluticasone. she then developed intermittent fever up to 102â°c. chest x-ray revealed bilateral apical infiltrates. treatment with levofloxacin for seven days resulted in no improvement of symptoms or roentographic findings. thereafter, she presented with chest and abdominal pain, hypotension, and hypoxia. blood work revealed a white cell count of 6800 c/mm 3 with a differential significant for 34% eosinophilia (absolute eosinophil count of 2300 c/mm 3 ). chest ct showed dense consolidation predominantly along the peripheral aspect of the upper and superior segment of the lower lobes, and pericardial effusion. moderate pericardial effusion without evidence of tamponade was confirmed on echocardiogram. left upper lobe wedge biopsy findings included significant tissue eosinophilia, scattered foci of active organizing exudates, and no evidence of granulomatous or necrotizing vasculitis. the patient was treated for cep with a six-month course of prednisone, starting at 60 mg daily, resulting in rapid improvement of her pulmonary and systemic symptoms. background: immunologists are consulted for evaluation of immunodeficiency in patients with recurrent skin infections. disorders of the phagocyte system may be associated with cutaneous infections. methods: case report case: a 15-month-old african american girl (twin a) presents with recurrent skin abscesses. at 13 months of age, she had her first buttocks abscess, which required incision and drainage with oral antibiotics. a month later, she developed another abscess and was found to be neutropenic, with an absolute neutrophil count (anc) of 264/î¼l. cyclic neutropenia was considered and her pediatrician monitored cbcs, which all showed persistent neutropenia (ancs between 68 and 264). at 15 months of age, she was hospitalized for fever with neutropenia. immunology was consulted for evaluation of neutropenia. the rest of the past medical history was unremarkable. she was healthy appearing and growth parameters were appropriate for age. her physical examination was unremarkable. immunoglobulins, b-and t-cell markers, nitroblue tetrazolium, complement assay, hemoglobin, platelets and the peripheral smear were normal. antibodies for hiv, cmv, ebv and parvovirus were undetectable. anti-neutrophil antibodies were positive, establishing the diagnosis of primary autoimmune neutropenia (ain). the abscess healed with oral antibiotics. severe neutropenia (anc 0-280) persisted for three subsequent months without further infections. twin (b) was also found to have persistent severe neutropenia without morbidities, suggestive of primary ain. primary ain is less known among physicians and is typically diagnosed after extensive investigations that exclude other causes of neutropenia. the exact incidence of ain is unknown. it is usually seen in children between 5 and 38 months of age, often with severe neutropenia and self-limiting bacterial infections. the clinical course and presence of antibodies to neutrophil antigens (na1, na2 or cd11b/18) is diagnostic. familial occurrence of primary ain is not reported in the literature. conclusion: primary ain may remain under diagnosed due to lack of characteristic clinical features. although a benign clinical course is likely, severe infections, including pneumonia, sepsis and meningitis, have been reported. genetics may play a role in this disease, as we present primary ain in twins. background: atopic dermatitis (ad) is a chronic inflamatory disease of skin that affects 5% of the wordl population.the natural history of ad in some patients, evolve to the coexistence with other allergic diseases: allergic rhinitis, allergic conjunctivitis and asthma. the sublingual immunotherapy has demonstrated utility in some patients; however, semi-rush immunotherapy to pollens has only showed utility in one animal case published few years ago. case report: a 2-year-old infant was referred to us. he began one year before with skin lesions compatible with ad, six months later began perennial rinhorrea and nasal itching. multiple treatments with topic corticosteroids, moisturizing, antihistamines and topic/systemic antibiotics did not demonstrate utility. our evaluation revealed ad lessions that affected 60% of the total body surface and clinical features of allergic rhinitis (ar).the lab tests revealed eosinophylia (600cell/mm3) in blood cell count, normal levels of total ige but specific ige to dermatophagoides pteronissinus (dpt) was high. the skin prick test reveales the same results. we added environmental control, oral costicosteroids and transfer factor. despite our treatment no improvement was observed. we considered dpt as the principal factor in the maintenance of ad lessions and ar episodes. in the absence of sublingual immunotherapy in our hospital, we decided for semi-rush immunotherapy schedule. we began from 0.1 ml of 1:10, 000 w/v concentrations of dpt until 0, 5 ml of 1:100 w/v concentrations in two months receiving three doses per week. no local or systemic adverse events was reported and the ad lesions and ar symptomatology disappeared in the first month of treatment. at this time, no exacerbations have been documented. conclusion: the semi-rush immunotherapy can be useful and safe for treatmente of ad in some patients in whom specific ige to aeroallergens has been demonstrated. introduction: eosinophilic gastrointestinal disorders are a rare group of disorders that can involve the entire gastrointestinal tract. presentations are varied but may include vomiting, dysphagia, abdominal pain, diarrhea and failure to thrive. the diagnosis is made by endoscopic biopsies which reveals eosinophil rich inflammation in the absence of known causes for eosinophilia. peripheral eosinophils and ige may be elevated but can be normal. we report a patient with eosinophilic gastroenteritis associated with an ampullary tubulovillous adnenoma. methods:a 71-year-old white male with allergic rhinitis and a family history of atopy was admitted for profuse watery diarrhea. he denied any new medications, eating raw foods or recent travels. eosinophils were elevated to 2.4(29% of wbc) and ige was elevated to 3259. multiple stool specimens were negative for ova & parasites and enteric pathogens. serology was negative for strongyloides, trichinella, e. histolytica and toxocara. ast, alt & bilirubin were elevated and a ct scan showed dilated billiary ducts with a possible ampullary mass. biopsy of the ampullary mass revealed a tubulovillous adenoma. biopsies of the duodenum, terminal ileum, colon and rectum were remarkable for focal eosinophilic cryptitis & chronic inflammation in the lamina propria consisting of eosinophils, scattered lymphocytes and histiocytes. all specimens were negative for parasitic infections including duodenal aspirates. he was empirically started on metronidazole and singulair with gradual improvement of symptoms, eosinophils and liver tests. two months after discharge, the patient remained diarrhea free with a normal eosinophil count. conclusion: we report a patient with eosinophilic gastroenteritis and an ampullary tubulovillous adenoma with obstruction of the biliary system. this unique presentation illustrates the diverse nature of gastrointestinal manifestations seen in eosinophilic gastroenteritis. background: zonisamide is an anti-seizure medication chemically classified as a sulfonamide and unrelated to other ant seizure agents. we report a case of hypersensitivity to this agent in a child. method: case report results: this patient is a 22-month-old girl who developed "peeling of her lips" three weeks after starting zonisamide. one week later she developed a rash that began on her face and generalized over several days to her neck, trunk and extremities. there was no respiratory distress, joint complaints and no angioedema associated with the episode, but fever to 103 prompted referral to our hospital on day 6 of the rash. the rash was macular-papular without discrete urticarial lesions. the rash coalesced with underlying erythema on face, chest and neck. the patient has a known history of seizure disorder, asthma, mild eczema, gastro esophageal reflux, development delay, lactose intolerance and failure to thrive. her other medications were, lansoprazole, topiramate, and albuterol. labs revealed a normal white cell count, an elevated sed rate ( 51) and elevated lft's, i.e. sgot 80 and sgpt 180. the only new medication was zonisamide that was discontinued. she was treated with iv steroids and hydroxyzine. the rash started fading by day 2 and the patient's fever resolved by the third hospital day. liver enzymes returned to normal by day 7. conclusion: this relatively new anti-seizure agent can be associated with hypersensitivity reactions in children. introduction: kawasaki disease (kd) is an acute chilhood vasculitis. in addition to the diagnostic criteria a broad range of nonspecific clinical features may be observed including aseptic meningitis, vomiting, diarrhea, abdominal pain, sterile pyuria, arthralgia and arthrtis, pulmonary infiltration, pleural effusion and nonspecific paralytic ileum as manifestation of gastrointestinal vasculitis. we describe a child who developed all features of kawasaki disease included the most rarely reported. patient report: a 2 year-old female presented 15 days of high fever, nonsuppurative cervical lymphadenopathy, petequial rash in legs, swelling of feet, distended abdomen, vomiting, obnubilated and hypoactive, incongruent speech, fisured lips and distended abdomen. lab tests showed: anemia (9.8g/dl), high wbc count (24, 200cells/mm3), thrombocytopenia (47, 000/mm3), hypoalbuminemia (1.1g/dl), lactate dehydrogenase (ldh) 209mcg/l, glutamin transferase (ggt) 190. csf total proteins 129, glucose 60, cells 23, pm 5%, mn 95%, seric complement 88, cultures were negatives. she developed myocarditis, aneurysms in the right and left coronary arteries. on the 25th day presented cardiac failure, pleural effusion, paralytic ileum, hydrops vesicular, mechanic ventilatory assistance was required. the first dose of intravenous immunoglobulin (ivig) (2g/k) was infused, heparin and hydrocortisone. on 45 day a second doses of ivig was infused, because of fever, and abdominal vasculitis. steroids ware discontinued, heparin was suspended and aspirin was added as antiaggregant. discussion: our patient presented an unusual and severe presentation of kd with pleural effusion, nonspecific ileum, cardiac failure secondary to myocarditis, aseptic meningitis and thrombocytopenia; all those manifestations had rarely been reported at the same time. she presented with a devastating evolution. complications were resolved. the lastest studies have shown that treatment with ivig plus corticosteroids significantly reduce serum concentrations of proinflamatory cytokines. in this case antiinflamatory doses of aspirin were contraindicated and steroids were used with satisfactory outcome. limited data is available for nonresponding patients to guide therapy. although multiple doses of ivig are sufficient in some patients, some of them remain refractory to therapy and they need corticosteroids to control the vasculitis process eosinophilia is defined by an absolute eosinophil count above 0.7 x 10 9 and can be seen in association with a broad spectrum of disorders ranging from allergic to malignant. idiopathic hypereosinophilic syndrome (hes) should be considered in any patient with an eosinophil count above 1.5 x 10 9 for more than 6 months without commonly recognized causes of eosinophilia and with evidence for organ damage not otherwise explained in the clinical setting. it is potentially an aggressive disease with mean survival of less than a year without treatment. we present a 39 year old male horse breeder, with eosinophilia lasting more than 10 years. prior to coming to our institution, he underwent extensive medical evaluation including bone marrow and gi biopsies, multiple imaging studies with mri and ct scans of the chest and the head as well as detailed evaluation of his cardiac status. all these tests were normal and the patient was empirically started on treatment for possible hes including trials of prednisone, hydroxyurea, interferon alfa, and imatinib, all without lasting resolution of his eosinophilia and causing significant side effects with profound depression of his immune status. finally, in light of the patient's profession, a strongyloides enzyme immunoassay was done and found to be remarkably positive. duodenal drainage confirmed the infestation with this nematode. within a week of starting treatment with ivermectin, his eosinophil count came down by 50% and eventually normalized. we learn that one should be persistent in excluding all common causes of eosinophilia before considering hes. in case of parasite infestation, premature treatment with immunosuppressors can result in worsening of the infection with potential for poor and even fatal outcome. stool evaluation might not be sensitive enough for detection of parasites and it is therefore necessary to complete a diagnostic work up with appropriate serology. doxil is the pegylated liposomal form of doxorubicin and has been used successfully as a cancer chemotherapy agent in many types of tumors. a hypersensitivity reaction can occur, usually during the first infusion, and appears to be rate related. the symptoms include dyspnea, tachypnea, facial swelling, chills, hives, chest pain, and back pain. we report a case of a hypersensitivity reaction to doxil in a 26 year-old woman with hodgkin's lymphoma. during her first outpatient doxil infusion at 1 mg/min, she developed urticaria, chest tightness, dyspnea, and back pain. these reactions persisted despite being medicated with antihistamines and steroids and immediately resolved during pauses in the infusion. after 23 mg of doxil, the infusion was discontinued. six days later, she was admitted to complete the other half of the dose. she had a negative intradermal skin test to a 1:100 dilution of doxil. she was then premedicated with diphenydramine, dexamethasone, acetaminophen, and famotidine. the doxil infusion was started at 0.4 mg/min and was soon stopped due to flushing of the hands and face. the infusion was decreased to 0.2 mg/min. she was able to tolerate this slow infusion with only mild and tolerable symptoms. when her symptoms worsened, the infusion was stopped for 30 to 60 minutes. she completed the 23 mg infusion of doxil after 13 hours. pre-infusion and post-infusion complement levels were drawn during this second administration of doxil. her pre-infusion c2, c3, c3a, c4, c5, c5a, and bb levels were all normal. her pre-infusion c4a and sc5b-9 levels were high, indicating that she might have had some residual or persistent complement activation caused by her first doxil infusion. her post-infusion c2, c3, c3a, c4, c5, c5a, and bb levels were all normal. however, her post-infusion sc5b-9 levels significantly increased, suggesting complement was activated during the second doxil infusion. given her reaction during her first doxil infusion and a negative skin test to doxil, it is highly unlikely that her doxil hypersensitivity was an ige-mediated process. therefore, in patients with a similar doxil hypersensitivity, we suggest a slow rate of infusion of 0.1-0.2 mg/min, toleration of mild symptoms, 30 to 60 minute pauses during more severe symptoms, and continuation of premedication during the entire lengthy infusion. introduction eosiniphilic esophagitis (ee) is an isolated, severe esophageal eosinophilia. patients are usually young males presenting with vomiting, epigastric or chest pain, dysphagia and obstructive respiratory problems. they are often initially misdiagnosed with and treated for gastroesophageal reflux disease (gerd). distinction between the two diseases can be made with biopsies of the esophageal mucosa. while any eosinophils in the esophageal mucosa indicate pathology, gerd typically presents with up to 7 eosinophils per high powered field (hpf, 400x) while ee most often presents with greater than 20-24 eosinophils per hpf. case report the patient is a 3 and one half year old boy who has experienced severe symptoms of gerd from the age of 6 months. he vomits after meals at least 1-2 times per day. he coughs when he lies down at night and regurgitates phlegm in the early morning. he has complained of discomfort in his lower chest after eating. his weight has remained at 35 pounds for the last six months. the patient was diagnosed with asthma at age 2 and a half; however, there is no family history of asthma or allergies. the patient initially experienced improvement on lansoprazole, but his symptoms recurred when the medication was discontinued and subsequent courses were ineffective. his physical exam was normal barring slightly edematous nasal turbinates. an endoscopic biopsy showed "numerous eosinphils" (later clarified to 38 eosinophils per hpf) in his distal esophagus. the patient showed allergy to milk and wheat on radioallergosorbent (rast) testing. the patient was started on swallowed fluticasone 2 puffs twice daily and advised to see a nutritionist regarding a wheat and milk elimination diet. conclusion ee is an important differential of gerd-like symptoms in childhood. to avoid misdiagnosis it is critical to evaluate the number of eosinophils present in a biopsy specimen to help differentiate between gerd and ee. children with ee are at increased risk of developing esophageal dysmotility and esophageal strictures. patients often have positive skin prick or rast tests to foods and aeroallergens. alternative treatments such as food elimination diets and glucocorticoids (systemic or topical) are effective in treating symptoms which may not respond to reflux medications. introduction in 1992 asherton introduced the term catastrophic antiphospholipid syndrome (caps) to describe patients sharing clinical evidence of multiple (three or more) organ involvement and/or histopathological evidence of multiple vessel occlusions. case report we present a 13 year old female, with lumbar pain, arthralgias, weight loss, fever, malaise, raynaud phenomenom, alopecia, oral ulcers and hepatomegaly. on arrival, she was polypneic with tachycardia, basal hypoventilation, and hepatomegaly was evident. hb: 8.8, leucocytes: 39.300, total lymphocyctes: 8, 300, total neutrophyles: 28, 700, platelets 393, 000. coombs positive. urin exam: proteinuria, leucocyturia and erythrocyturia. creatinine 1.3. diagnosed as sle with pericarditis, cardiac failure and acute pulmonary edema, urinary tract infection and pneumonia, requiring mechanical ventilation and inotropic support, intravenous gammaglobulin and hydrocortisone. acute renal failure and hemodialysis was begun with improvement. suddenly she presented seizures crisis, stuporous, livido reticularis skin and acrocyanosis, external opthamalplexia, bilateral facial diplexia, ocular fundus with arteriolar vasospasm. right facio-corporal hemiparesia, cortical and progressive medular annals of allergy, asthma & immunology segment changes. ct showed multiple left fronto-occipital parietal hypodensities suggestive of lacunar infarcts. diagnosed as neurolupus and caps. initially anti b2 glp1 and anti clp were negative with later positivization. urinary tract infection contraindicated high doses of steroids and, intravenous gamaglobulin and anticoagulation were started. with significant improvement. currently the patient is evolving in a satisfactory way. discussion the literature establishes that catastrophic aps is characterized by elevated mortality. in this patient damage was evident to the cns, pns, kidneys and the skin. with the presence of positive antiphospholipid antibodies with an energic antiinflamatory, immunoregulatory and immunosupressive therapy, the function of each affected organ completely recovered. this case exemplifies that the therapy of caps should be undertaken in a sufficient and early manner given the elevated mortality of this syndrome. patient, a 70-year-old white, male non-smoker physician on high-dose regimen of advair and singulair presents with an 8-day history of progressive chest tightness. pulmonary function tests showed normal (fev 1 of 96%) lung function. in the past, whenever inhaled steroid dosages were lowered, patient experienced a reoccurrence of asthma symptoms, despite consistently normal lung function results. to rule out the possibility of a psychologically induced asthma exacerbation, the patient's fractional concentration of exhaled nitric oxide (feno) levels were measured. the patient's feno level was elevated at 48.9 ppb. values greater than 30 ppb have been described as consistent with airway inflammation. with an increase in the patient's inhaled steroids, the patient had a remission of symptoms within a week. this case is illustrative of an increasingly common clinical picture where a symptomatic asthmatic may have normal spirometry but elevated feno. as devices for measuring feno become more available in the outpatient clinic setting, elevated feno may be an excellent diagnostic marker in assessing whether airway inflammation is being adequately treated in situations where spirometry values are within normal ranges. introduction the autoimmune thrombocytopenic purpura (atp) is characterized by thrombocytopenia and megakaryocytic hyperplasia. the first choice of treatment consists of intravenous gamma globulin (ivig), corticosteroids and anti-d antibodies and the second line measures are immunosuppressant drugs, splenectomy and danazol. case report a 4 years old male presented in the first year of life ecchymosis in several parts of the body intermittently. in april 2003 he presented ephistaxis and lower gastrointestinal bleeding. complete blood count showed platelet count of 45, 000/ul. prednisone was started (2 mg/kg) with no improvement. at that time, danazol, anti-d antibodies, and ivig (2 g/kg) were added. subsequently, the platelet average count diminished to 5, 000. the patient was transferred to our hospital. at his admission he presented cushingoid habitus (figure 1) and acanthosis nigricans. the immunological tests (aan, ch50, c3, c4, immunoglobulins, anticardiolipins and b2 glycoprotein) showed no alterations. bone marrow aspirate demonstrated megakaryocytic hypercellurarity. prednisone dose was tampered when the patient presented secondary glaucoma. patient continued his treatment with danazol and ivig (2 dosages of 2g/kg each), hydroxychloroquine and cyclophosphamide with no improvement. thus, 3 months after the immunosuppressant treatment, splenectomy was performed, obtaining partial improvement. at that time ranitidine and hydroxychloroquine were suspended and cyclophosphamide was changed to azathioprine. last platelet count was 106, 000/ul. conclusion chronic atp in childhood as the present case account for approximately 4-5% of the total atp patients. chronic atp that does not respond to conventional treatment is a therapeutic challenge. in this patient we used first choice and second choice drugs with no improvement, consequently a splenectomy was indicated, not obtaining the desired response at first. the use of immunosuppressant drugs is not common for this disease, but it could be a good alternative for atp resistant to conventional treatment. this are the most important laboratory test of our patient. background: budesonide is the only corticosteroid available for inhalation by jet nebulizer and is indicated for the treatment of asthma in children. objective: to evaluate the distribution and clinical efficacy of inhaled budesonide administered by nebulization with a modified commercial device. methods: a 23 year old male with severe persistent asthma underwent a lefort procedure for multiple craniofacial abnormalities. the external device maintained his mouth open impeding the proper use of controller medications. as a result he developed an increase in his asthma symptoms. he was subsequently treated with nebulized budesonide delivered with a modified jet nebulizer through the end of a plastic tube in a flow-by manner. we then performed dynamic ventilation imaging after administering 33.40 mci of nebulized technetium 99m-dtpa diluted in two ml of normal saline. images were obtained for five minutes at three seconds per frame. spirometry monitoring was not possible given the obstructive nature of the facial device. results: the patient demonstrated improvement of his asthma symptoms. the ventilation scan showed that the patient breathed the technetium dtpa through the specialized device with delivery to his full lung volumes within 12 seconds. conclusions: we report the successful treatment of a patient unable to use the commercially available methods for administration of inhaled steroids. rationale: hp is a non-ige mediated inflammatory response in the lungs due to a variety of organic antigens, including metal working fluids. the wideranging clinical features include acute, subacute, and chronic forms. elevated antineutrophilic cytoplasmic antibodies and platypnea, defined as dypsnea induced by the upright position and relieved with recumbency, have not been previously reported in patients with hp. case: 35-year-old white male tool and dye machinist presented with progressive cough, weakness, dyspnea on exertion, and platypnea. symptoms began 15 weeks earlier with coryza and diffuse myalgias. he had lost 12% of his body weight since symptom onset. examination revealed a pulse of 140, respiratory rate 18, and right basilar crackles. resting pulse ox on room air was 95%, but dropped to 91% upon ambulation. pft's revealed severe obstruction (fev1 42% predicted) with significant reversibility (fev1 +19%), and a dlco of 82% (adjusted for va and hemoglobin). hemoglobin was 17.5 with a normal leukocyte count and differential. c-anca (anti-pr3) was 19.3 (<5), and p-anca (anti-mpo) was 15.9 (<15), both performed by elisa. echocardiogram showed an ef of 50-55% with mild pulmonary hypertension; no shunt was present. chest roentogram was normal, but a chest ct revealed a diffuse ground-glass pattern with scattered centrilobular opacities. biopsy was consistent with hp. he was removed from his work exposure to metal working fluids with full recovery of lung function and resoulution of symptoms. conclusions: hp presents as a constellation of symptoms without a single, unique identifying pattern. platypnea and elevated anca's have been observed in a wide range of disorders, all of which were excluded in this patient. platypnea has been associated with hereditary hemorrhagic telangiectasia, pulmonary avm, hepatopulmonary syndrome, recurrent pulmonary emboli, and patent foramen ovale. elevated anca's has been associated with wegener's granulomatosis, goodpasture's syndrome, churg-strauss vasculitis, drug-induced vasculitis, inflammatory bowel disease, and others. elisa is a more specific modality for anca's than indirect immunofluorescence, but it is less sensitive. the possibility of hp should be considered in patients with either of these two abnormalities. rationale: heart disease is the leading cause of death in america and 70% of persons between 70 and 80 years of age have coronary athersclerosis at autopsy. the presentation in the elderly is commonly atypical. 45% of myocardial infarctions were silent or unrecognized in the framingham cohort. in patients 65 and older, it is estimated that 8-25% will present with dyspnea without any associated chest pain. we present a case of unstable angina presenting as exercise-induced asthma. case: an 81 year-old white male with moderate persistent asthma, hypertension, dyslipidemia, and a long history of allergic rhinitis presented having had an abrupt increase in his usual exercise-induced asthma symptoms four weeks prior. asthma had been diagnosed at age 69 with spiromety showing moderate obstruction and significant but incomplete reversibility. he had a remote history of pipe and cigar smoking, but had quit at age 50. he had been well-controlled since that time with his most recent regimen consisting of fluticasone 100 mcg/salmeterol 50 mcg diskus, one inhalation twice daily. his typical exercise-induced asthma symptoms included dyspnea on exertion and chest tightness without radiation, and were relieved with rest and albuterol. the amount of exertion needed to trigger his symptoms, however, was much less than he had previously experienced, and this remained constant during the four weeks prior to his presentation. one week prior he had a normal ecg evaluation by his primary care provider. six months prior he had a normal nuclear cardiac stress test. physical examination was unremarkale except for moderately decreased aeration and 1+ pitting edema of his lower extremities. cardiology performed a nuclear stress test the next day which was abnormal. catheterization revealed 99% steonsis of his proximal lad. he succefully underwent cabg and is doing well on follow-up. conclusion: the elderly present many challenges to the diagnostician; these include multiple co-morbidities as well as atypical and often late disease presentations. the key to raising suspicion for cardiac involvement in this case was the recognition of the patient's cardiac risk factors in the setting of an abrupt onset of exercise symptoms while lacking other asthma symptoms such as nocturnal cough. cold urticaria is an uncommon form of physical urticaria. this case of a 9-year-old with cold urticaria and angioedema provides additional information regarding an unusual disorder in the pediatric population. an otherwise healthy 9-year-old female presented with a complaint of urticaria precipitated by cold exposure over the preceding 5 weeks. she had no recent illnesses and a past medical history significant only for cat allergy. on multiple occasions the patient noted erythema and pruritus of her arms and face after walking through the freezer aisle of a grocery store. urticaria would then develop on regions where she scratched, spontaneously resolving in 2-3 hours. on one occasion, urticaria appeared diffusely while taking a shower after the patient had been swimming. the urticaria resolved within a few hours after the patient was given diphenhydramine by her mother. three days prior to presentation the patient experienced upper lip angioedema with erythema, globus sensation and difficulty swallowing after drinking a strawberry slushy. the patient denied any respiratory complaints at that time and her symptoms again annals of allergy, asthma & immunology resolved spontaneously. family history was significant for a maternal history of seasonal allergies. upon physical exam the patient was well appearing. she had 2-3 discrete urticaria on each calf. the patient's mother noted that recently these would appear on "cold and rainy" days, attributing them to the fact that the patient's pants left her lower legs exposed. the remainder of her exam was normal and dermatographism was absent. laboratory evaluation consisted of cryoglobulins and strawberry rast, both of which were negative. application of an ice cube to the patient's forearm for 5 minutes resulted in a 9x6 centimeter wheal noted 3 minutes after ice removal. a diagnosis of cold urticaria with associated angioedema was made. the patient's mother opted to use diphenhydramine as needed and an epinephrine autoinjector was dispensed. by 3 months after symptom onset, the patient's only complaint was pruritus of her hands if they became too cold. no urticaria were noted. at 6 month follow-up the patient denied any symptoms for the preceding 2 months and had a negative ice cube test. cold urticaria in the pediatric population is a rare entity and not well understood. this case of a 9-year-old with cold urticaria and angioedema offers additional insight into this unusual disorder. a. khuntia * , m. mcmorris, ann arbor, mi. introduction: chronic granulomatous disease(cgd) is a heterogeneous group of disorders characterized by genetic defects in the ability of phagocytes to generate microbicidal reactive superoxide anions and its metabolites. it manifests early in life, primarily as recurrent infections, caused by catalase-producing bacteria such as staphylococcus aureus, burkholderia cepacia, and serratia marcescens and fungus such as aspergillus fumigatus. the disease may be inherited in an x-linked or autosomal recessive manner, with x-linked disease accounting for 65-70% of cases. the us incidence of cgd is 1/250, 000 live births with an average age at presentation of 3 years for xlinked and 7.8 years for autosomal recessive disease. case report: 18 year old male with history of three separate episodes of pneumonia beginning at age 16. each episode resulted in a hospital admission and intravenous antibiotic therapy after failed oral antibiotic therapy. an extensive pulmonary evaluation was initiated after the third episode of pneumonia, including a chest ct, viral, bacterial and immunodeficiency studies. cbc, complement, immunoglobulins, flow cytometry, viral and bacterial studies were all normal. the ct scan revealed dense nodular opacities in the right upper lobe with surrounding ground-glass opacification and mild bronchiectasis. a subsequent bronchoscopy demonstrated necrotizing granulomatous inflammation. open lung biopsy grew burkholderia cepacia on tissue culture. the clinical history, tissue histopathology and atypical organism found on culture were all suggestive of an underlying immunodeficiency. a neutrophil oxidative burst assay was performed which demonstrated minimal neutrophil activity upon stimulation, suggestive of the diagnosis of cgd. a chemilluscence assay verified the diagnosis of cgd, with minimal fluorescence noted on flow cytometry after neutrophil stimulation. dna analysis demonstrated a p47-phox deficiency, resulting in one of the autosomal recessive and less clinically severe forms of the disease. conclusions: this case of cgd is unusual because of the delayed presentation. it demonstrates the importance of a complete immunological evaluation including an evaluation for neutrophil disorders such as cgd in patients of all ages who present with recurrent and recalcitrant episodes of pneumonia, especially when atypical organisms such as burkholderia cepacia are found on culture. autoimmunity may play a role in the development of premature ovarian failure (pof), but the exact mechanism is not well understood. pof is mainly diagnosed after 19 years of age and most women complain of secondary amenorrhea. pof has been reported in combination with presence of ana and autoimmune diseases, but rarely with jra. here we report two cases of pof and positive ana in pediatric patients, one with clinical features of jra. the first patient, an african american 17-year-old girl, height in 50th percentile, weight in 10th percentile, with tenosynovitis of wrists and arthritis of knees and elbows for the past two years, was referred for further evaluation and management. she did not have her menarche yet and laboratory investigation revealed positive ana. the second patient, an african american 17-year-old girl, 50th percentile for height and weight, was referred to the immunology clinic with chief complaint of primary amenorrhea and delayed development of secondary sexual characteristics and was found to have positive ana without clinical findings of jra. both patients were tanner stage 2 for breasts and tanner stage 2-3 for pubic and axillary hair. both had elevated fsh and lh, karyotype 46xx and small uterus and small ovaries on pelvic ultrasound. antiovarian antibody was not detected in any of the two patients. the bone age was significantly delayed. pof in karyotypically normal women is frequently seen in combination with elevated serum ana. the clinical spectrum of rheumatoid disease associated with pof ranges from asymptomatic ana positivity to typical presentation of jra. women with pof should be monitored for the emergence of autoimmune disorders including jra, and women with jra should be followed for menstrual irregularities and signs of pof. introduction: many u. s. military personnel deployed to the middle east continue to develop infection with leishmaniasis, a parasite transmitted by the sand fly. pentavalent antimonials have been used as an effective treatment for leishmaniasis for many years. in the united states, sodium stibogluconate (pentostam) is the pentavalent antimonial of choice, and is currently being administered under an ind protocol. side effects of therapy include myalgias, arthralgias, rash, malaise, abdominal pain, pancreatitis, and hypersensitivity reactions. the true incidence of hypersensitivity reactions is not currently known. case reports: two u. s. soldiers receiving pentostam for the treatment of cutaneous leishmaniasis were evaluated in our clinic at walter reed army medical center after experiencing urticaria, angioedema, wheezing and dyspnea after medication infusion during the 20-day course of daily therapy. due to the concern of a potential ige-mediated reaction, skin testing was performed. after informed consent, skin testing included a prick test at full strength, followed by intradermal (id)testing. soldier #1 was a 23-year old male reporting lip swelling, throat tingling, dyspnea and chest pain 7-8 hours after treatment #10/20. skin testing to both lots used during the treatment course showed positive values of 8x20mm and 9x14mm respectively at id 1:1000. soldier #2 was a 28-year old male reporting diffuse pruritus, hives, dyspnea, and chest pain 15 minutes after infusion #16/20. skin testing showed positive values of 7x14mm and 8x12mm respectively at id 1:1000. due to clinical symptoms and positive skin testing, therapy was discontinued in each case. one control individual showed negative testing to prick at full strength, id 1:1000, and id 1:100. conclusion: skin testing with pentostam may provide an objective tool for accurate classification of adverse reactions as igemediated. reliance on skin prick testing alone may not be sufficient to detect pentostam skin test reactivity, as both of these patients reacted to id testing only. a prospective study including pre-and post-treatment skin testing should provide more information on the value of skin testing in providing objective evidence for an ige-mediated process and determining the incidence of hypersensitivity to pentostam. introduction: a 3-year-old girl with a history of multiple infections was hospitalized with respiratory distress and hypoxia. her past medical history was significant for hypothyroidism, psoriasis, asthma and multiple pneumonias. a cbc revealed an absence of lymphocytes. laboratory: t and b cell subsets showed no b-cells and very low t cells (<100 cells). inadequate lymphocytes were present for mitogen and antigen studies. serum immunoglobulins were normal and she had antibodies to streptococcus pneumoniae and tetanus. antibodies to rsv, influenza and mycoplasma were absent. chest x-ray revealed interstitial infiltrates bilaterally. a high resolution ct scan revealed septal thickening and confirmed interstitial infiltration. open lung biopsy was consistent with non-specific inflammation with extensive lymphocytic infiltration. bal fluids and biopsy were negative for bacteria, fungi and opportunistic pathogens. clinical course: the patient did not improve with antibiotics and steroids were started. the patient improved rapidly and was discharged to home. biochemical analysis demonstrated a deficiency of adenosine deaminase (ada), a form of severe combined immune deficiency (scid). the patient was started on replacement ada, adagen. a repeat high resolution ct scan showed some improvement. however, the patient continues to be steroid dependant to control her pulmonary symptoms. lymphocyte numbers remain low despite effective ada replacement and the absence of serum datp. she remains clinically stable and receives adagen injection twice weekly. discussion: ada deficiency is a condition, which leads to accumulation of the metabolite datp, which is toxic to lymphocytes. this disease most commonly presents in the first year of life as scid and is fatal unless treated. our case is unusual due to the late onset of severe disease, normal serum immunoglobulin and the presence of some protective antibodies. despite adequate replacement of ada the patient continues to be profoundly lymphopenic, most likely due to steroids. although we do not yet completely understand the underlying lung disease we suspect that the patient has an autoimmune process causing interstitial inflammation. background: atopic dermatitis has a broad range of differential diagnoses. human sarcoptic infestation is characterized by severely pruritic lesions of variable appearance and distribution and may masquerade as eczema. infestation may be difficult to confirm and eradicate. animal transmission has been reported as a source of human infection. objective: to report a case of persistent sarcoptic infestation masquerading as eczema and associated with delusional parasitosis. methods: a 58-year-old female was referred to allergy clinic for evaluation with a two year history of recurrent, pruritic rash thought to be refractory atopic dermatitis. previous ineffective treatments included topical steroid creams, lindane, topical anti-fungals and multiple otc antiitch preparations. at initial evaluation, she had widespread excoriated papules in various stages of healing over 70% of her body. she reputed her dog was diagnosed with recalcitrant mange, which necessitated giving him medicated baths twice daily. results: a clinical diagnosis of subacute sarcoptic infection was made and the patient was prescribed two courses of elimite followed by oral ivermectin. her rash quickly resolved except for post-inflammatory hyperpigmentation. three weeks after resolution of primary lesions, she again complained of pruritic eruptions occurring on easily accessible areas and began bringing in medicine bottles of skin debris and scabs for examination. scrapings, koh preparation and skin biopsy examined microscopically showed no evidence of sarcoptic re-infestation. a diagnosis of delusional parasitosis was made. conclusions: the animal to human transmission of sarcoptic infection seen in this patient is rare and responded quickly to appropriate treatment. despite eradication of the infection, she developed delusional parasitosis, a rare psychiatric disorder characterized by fixed, false belief of an infestation by insects or other creatures. she displayed the classic matchbox sign in which samples of skin, scabs and other detritus are brought in for examination. she is currently receiving psychiatric care. background: thimerosal is a mercury derivative that has been used since the 1930s. it is a commonly used preservative in ophthalmic solutions, otic drops, and vaccines due to its bactericidal property. objective: to report the first case of a generalized reaction to thimerosal found in an influenza vaccine. methods: we present a patient who developed a generalized maculopapular eruption after receiving a thimerosal containing influenza vaccine. patch testing was performed to determine if there was an allergy to thimerosal. results: patch testing confirmed a type iv (t cell mediated) sensitivity to thimerosal, further supported by the prior history of a reaction to a thimerosal containing contact lens solution. the patient was asked to avoid thimerosalcontaining products, including vaccinations, unless the benefit clearly outweighed the potential risk of a reaction. conclusion: physicians need to be aware that thimerosal is found in many products including vaccinations. clinicians should also be aware that allergic reactions do occur with exposure to thimerosal even in vaccines. this is the first case report in the literature of a generalized reactions to thimerosal from an influenza vaccine angioedema is a rare condition that has been described in the literature and exists in both inherited and acquired forms. a defect of the innate immune system, more particularly the complement system, is the inciting culprit. the acquired form of c1 esterase inhibitor deficiency has been divided into two classes and is generally not commonly seen until after the forth decade of life. type 1 acquired angioedema has been described in association with lymphoproliferative disorders, while type 2 is related to excessive complement activation and consumption due to autoantibodies. our case is a 67-year old man referred from an outside physician for further management of idiopathic edema. he first experienced facial edema in april 2002 attributed to sweet myrrh root ingestion. subsequently, in november 2002 he developed diffuse swelling of his upper extremities and tongue without airway compromise. a minimal work-up at that time was inconclusive. he fortunately remained asymptomatic until january 2004 at which time he experienced two episodes of tongue swelling without etiology. he was seen in his local emergency department and treated with diphenhydramine, corticosteroids, and epinephrine on both occasions with gradual improvement of his symptoms. the patient was not on medications commonly associated with angioedema and did not report insect envenomation. subsequently, he was seen by his primary care physician who ordered a number of laboratory tests including a ch50, which was significantly suppressed. in light of this finding, he was referred to our clinic for further evaluation. at the time of presentation to our office, the patient was symptom free. his physical exam was within normal limits. further laboratory evaluation was essentially unremarkable with the exception of comple-ment studies, which are listed in the table. our case demonstrates an elderly patient with evidence of idiopathic swelling. we arrived at our diagnosis of acquired angioedema based on clinical presentation and confirmatory serum complement levels. a low ch50 at time of presentation allowed us to further delineate the etiology of complement deficiency. the fact that our patient did not present with swelling until after age 60 and the paucity of a family history of idiopathic angioedema makes the diagnosis of acquired angioedema probable. measure of serum c1q level confirmed the diagnosis. introduction: certain diseases, widely believed to be of allergic etiology, including atopic dermatitis and episodes of wheezing might be the result of interplay of genetic and environmental factors, at least partially. we describe a child with a rare chromosomal disorder presenting with typical features of atopic dermatitis and recurrent mild wheeze. materials and methods: a new born african-american male was noted to have unusual facial features immediately after birth. the baby was born naturally, without antenatal and neonatal problems. on physical examination, the infant had unusual facial characteristics with tight and taut facial skin, relatively diminished facial pad of fat, pointed chin, and markedly hypertonic extremities. additionally, cardiac exam revealed mumur consistent with uncomplicated asd. frequent reassessments of the infant in the outpatient clinic were done. the child had repeated bouts of wheezing attacks, and facial rashes compatible with the diagnosis of atopic dermatitis. the wheezes were treated in the clinic with nebulaized bronchodilators, and the atopic dermatitis responded reasonably to topical steroid applications and moisturizers as needed. chromosomal analysis of the peripheral blood of the chid confirmed the diagnosis of deletion of long arm (q) of chromosome 1 [46, xy, del (1)(q42q43)]. karyotypic analyses of the mother and father were normal. the child exhibited features of developmental delay, and seizure activities. anti-epileptic drugs (aed) were instituted. initial eeg was normal and the subsequent eeg showed findings of static encephalopathy. ct scan of the brain revealed absent corpus callosum. neurologic evaluations and physiotherapies were requested for improvement of fine motor skills. he did not seem to suffer from any serious infectious or immunodeficient illnesses. his cbc was normal. he continued to have fair gains in his weight, height, and head circumference. his atopic dermatitis and wheezing episodes remained under control. there were a few hospitalizations for break-through seizures and dehydration from gastroenteritis. a subsequent echocardiography confirmed closure of asd. con-clusion: a child with chromosomal anamoly, atopic dermatitis, and mild intermittent wheeze is reported. despite multiple congenital problems, the child continued to have a stable clinical course. etoposide is a chemotherapeutic agent used to treat many solid tumor malignancies. hypersensitivity reactions have been well described and there are a few reported deaths from anaphylaxis. some suggest that the hypersensitivity is an anaphylactoid type reaction as it may occur during the first dose. case reports of cutaneous complications include stevens-johnson syndrome, radiation recall and diffuse erythema. there are four cases in which diffuse erythematous papules developed after etoposide therapy. all rashes spontaneously resolved within three weeks and biopsies demonstrated keratinocytes in a starburst pattern. we report the first case of an immediate etoposide induced skin reaction that evolved into long lasting hyperpigmented plaques. pretreatement was able to prevent this immediate and late reaction on subsequent exposure to etoposide. a 27 year old female with ovarian cancer was treated with bleomycin, etoposide and vinblastine. three hours after initiation of the third dose of etoposide, patient developed pruritic, erythematous macules on her chest, abdomen, face and extremities. the infusion was stopped and decadron administered. over forty-eight hours, the pruritic macules became hyperpigmented plaques on her chest, abdomen, extremities and face. pruritus resolved after a slow taper of prednisone. the darken plaques were treated with multiple topical preparations but persisted for about three months. biopsies demonstrated superficial perivascular infiltration of lymphocytes and a few eosinophils suggestive of a drug eruption. etoposide was considered essential for this patient so allergy was consulted. the literature supports cautious readministration of etoposide with pretreatment and slower infusion rate to prevent immediate hypersensitivity. we could not guarantee prevention of the late hyperpigmented reaction. the patient was pretreated with prednisone and cetirizine. etoposide was administered in an icu with a slower rate of infusion. etoposide was tolerated without immediate pruritus or erythematous reaction. the patient did not develop the delayed darkened plaques. cautious administration of etoposide after premedication and a slow rate of infusion prevented both the immediate and late reaction previously experienced by this patient. eosinophils normally comprise 1-3% of peripheral white blood lymphocytes. peripheral blood eosinophilia is defined as an absolute eosinophil count >500 cells/mm3 and is most often caused by atopy, helminth infections, or collagen vascular diseases. less common causes include adrenal insufficiency and neoplastic processes. eosinophilia can be characterized as mild (<1500 cells/mm3), moderate (1500-5000 cells/mm3) or severe (> 5000 cells/mm3). although severe eosinophilia has been reported in association with adult hiv infection, studies of hiv-infected children have not shown peripheral blood eosinophilia to be a feature of pediatric hiv infection. we report an unusual case of severe peripheral blood eosinophilia in an adolescent male who was subsequently found to be hiv-infected. a previously healthy 15-year-old male presented with 2 week history of fever, diarrhea, cough, vomiting, abdominal pain, anorexia and a 5lb weight loss over the previous 2 months. the patient had recently emigrated from guyana and denied sexual activity, intravenous drug abuse, or other hiv risk factors. there was no known maternal hiv infection. repeated stool specimens were negative for ova and parasites. serologies for e. histolytica, t. canis, and s. stercoralis were negative. an abdominal ultrasound, chest x-ray, serum electrolytes, and liver function tests were all within normal limits. total white blood cell was 16, 500 with 59% eosinophils (absolute eosinophil count of 9, 735 cells/mm3). elisa and western blot were positive for human immunodeficiency virus (hiv-1). cd4+ tlymphocyte count was 1277 cells/mm3 with an hiv-1 rna level of 11, 000 copies/ml. the patients symptoms resolved over the next 10 days without treatment. he was started on combination antiretroviral therapy (zidovudine, lamivudine, abacavir, and efavirenz). absolute eosinophil count continues to slowly decrease with the last count of 3, 605 cells/mm3 three months following the introduction of antiretroviral therapy. severe peripheral blood eosinophilia may be a presenting feature of hiv infection in adolescents. hiv testing should be considered in cases where more common causes of eosinophilia such as atopy and parasitic infections are excluded. triad asthma is well described in adults but not in the pediatric literature. this case highlights the successful treatment of a severe pediatric asthmatic with nasal polyps and aspirin sensitivity. a 13 year-old female presented with severe persistent asthma, eib, allergic rhinitis, chronic sinusitis, nasal polyps, and a history of pneumonia. her asthma symptoms were minimal from age 2 until age 9. she then started flovent and required increasing amounts of inhaled and oral steroids. she required 6 courses of oral steroids per year by age 13. in addition, she had snoring, fatigue, chronic nasal congestion, rhinorhea, and sneezing despite treatment withallegra and rhinocort. she had received immunotherapy from age 8 to 10 with no clinical improvement. since age 8 she had recurrent sinusitis and had required 2 polypectomies. she noted aspirin caused nasal stuffiness and mild wheezing and therefore avoided it. on exam she had allergic shiners, dennie lines, boggy turbinates, nasal polyps, and diffuse wheezing. on evaluation she had severe airway obstruction. her fev1 increased from 36% to 51% with albuterol. her chest ct had a mosaic pattern due to severe air trapping, and her no level was 177. a sleep study showed severe hypopneas, and she had pan-sinusitis on ct. she had multiple positive spts, an eosinophil count of 1100 and an ige of 700. she had a normal sweat chloride and ph probe.the differential diagnosis included churg-strauss, abpa, cystic fibrosis, bronchiolitis obliterans, and triad asthma. after a thorough evaluation, she was diagnosed with triad asthma. with 6 weeks of treatment with oral prednisone, her fev1 improved from 36% to 83%. her inhaled controller therapy was increased and she was placed on xolair. she had a polypectomy and then underwent aspirin desensitization. one year after starting treatment her fev1 remains 83%. she requires albuterol 1x/month and has not required prednisone. her eib, allergic rhinitis and congestion are markedly improved. she has had no further episodes of sinusitis, and a repeat sleep study was normal. in conclusion, we treated a severe pediatric asthmatic with nasal polyps and aspirin sensitivity. this is not frequently reported in the pediatric population, and raises questions about the incidence, optimal long term treatment of triad asthma, and the differences from the adult onset of this disease. a.a. white * , r.a. simon, la jolla, ca. background: human disease from mold has traditionally been isolated to infection, allergic disease, or hypersensitivity pneumonitis. specific diseases or pathologic findings other than those listed above have not been well described. we report a case of acute eosinophilic pneumonia related to mold exposure. case presentation: a 50 year old woman developed dry cough and shortness of breath after stachybotrys mold was discovered in her home. a chest radiograph showed bilateral upper lobe infiltrates which worsened two weeks later. treatment with macrolide and flouroquinolone antibiotics was ineffective. a white blood cell count was 12, 000/cumm with 35% eosinophils. an erythrocyte sedimentation rate was 99 mm/hr. aspergillus ige was negative. treatment with prednisone led to complete resolution of the chest radiographic abnormalities and improvement in pulmonary function testing. this patient was given a diagnosis of acute eosinophilic pneumonia with mold as a likely causal factor. discussion: there is dispute amongst health care professionals regarding the significance of environmental mold contamination in the etiology of human disease. this case represents well characterized disease occurring in the setting of mold exposure. while a causal relationship cannot be established, the temporal relationship of mold contamination, symptom onset, and disease progression is compelling. interestingly, a recent report of mold contamination leading to nonspecific interstitial pneumonia/fibrosis has been described. we have observed a patient in our clinic with similar pathology on biopsy and temporal relationship to mold exposure. to our knowledge, acute eosinophilic pneumonia has not been reported in conjunction with mold exposure. conclusion: this case highlights a new condition in which mold may have a causal role. the mechanism is unknown, but likely is through a non-ige mediated immunologic pathway. public awareness of mold as a health concern is increasing. perhaps similar cases will emerge as a history of mold exposure is offered by patients at the time they are evaluated for lung disease. a stronger correlation may then emerge. scuba diving is a commonly practiced activity which normally carries only minimal risks. any severe allergic reaction such as anaphylaxis, however, occurring during this activity could be a particularly dangerous not only because the swimmer is submerged but also because of the lack of proximity to medical care. we report a case of anaphylaxis occurring during scuba diving resulting from an unsuspected hypersensitivity to a latex component of the scuba diving suit. a 21 yr-old white male developed a severe generalized urticarial rash with angioedema of his lips and eyelids, and difficulty breathing within 5 minutes of his applying the suit and entering the water. after being rescued, he was transported to a nearby emergency department where he received epinephrine, antihistamines, corticosteroids and iv fluids with gradual improvement over a 4 hour period. the patient denied being stung by a marine aquatic organism and there was no prior history of allergy or medications usage prior to his reaction. since subsequent investigation revealed that the scuba diving suit contained latex (brazilian rubber), a specific ige rast was found to be strongly positive (class v) to latex. therefore, the patient was advised to use a suit made of neoprene synthetic rubber (polychloroprene) which is a nonlatex containing product. this case report illustrates the importance of a diligent search for hidden sources of latex products which can produce life-threatening allergic reactions in sensitized patients. there has been considerable debate concerning the safety of immunizing egg-allergic children with the combined mmr vaccine. this concern derives from the possibility of an anaphylactic reaction since the mmr vaccine is prepared from attenuated viruses grown on chick embryo fibroblast cell cultures. we have previously reported the safe administration of the mmr vaccine to 6 severe egg-allergic children without development of an adverse reaction (nsouli, tm, et al. ann allergy asthma immunol. 2004; 90:163) , a finding consistent with the current report of the committee on infectious diseases, american academy of pediatrics, 2003. the present case report describes an anaphylactic reaction in an egg allergic 4 yr-old-white male consisting of generalized urticaria, angioedema, wheezing immediately following the administration of a second mmr vaccine. the history of hives following ingestion of eggs was confirmed by positive specific ige rast testing. the patient's anaphylactic reaction necessitated emergency treatment including epinephrine, diphenhydramine, and corticosteroids in addition to iv fluids. although the administration of mmr vaccine in egg-allergic children is not considered as an absolute contraindication, the present case report suggests that caution should be observed when administering the mmr vaccine in such patients and that careful medical observation be included together with the ready availability of emergency medical equipment. p. buddiga * , r. turbin, a. baisre, l. bielory, newark, nj. introduction: sarcoidosis is a chronic granulomatous disease of unknown etiology that may have a multi-organ system manifestation and is characterized by the histopathological evidence of nonnecrotizing granulomas. case report: a 53 year old african american woman with a 10 year history of type ii diabetes mellitus, hypertension and sinusitis recalcitrant to multiple courses of antibiotics over 3 months was admitted to the hospital with complaints of right eye proptosis, diplopia, headache and right facial numbness.her exam was consistent with an ipsilateral mild optic neuropathy, complete sixth (vi)nerve palsy, trigeminal, ophthalmic and maxillary division numbness.ct and mri of the face, orbit and brain revealed an extensive process infiltrating ethmoid, maxillary and frontal sinuses;orbits and deep facial structures.chest ct revealed bilateral interstitial nodules and symmetric hilar adenopathy. differential diagnoses included sarcoidosis, lymphoma/tumor, wegener's granulomatosis or fungal infection.labs-purified protein derivative test=negative(neg), antineutrophil cytoplasmic autoantibodies=neg. angiotensin converting enzyme=83[8-52 u/l], fungal & anaerobic and acidfast bacilli culture of sinus secretions=neg.ethmoid, adenoid and maxillary sinus biopsies=multiple nonnecrotizing granulomas. on the basis of compatible clinical and radiographic findings, histopathological evidence and exclusion of other diseases with similar findings, the diagnosis of sarcoidosis was established. she was started on parenteral methylprednisolone and subsequently tapered to oral prednisone after 5 days.concomitantly she was started on methotrexate as a steroid sparing agent.most recent chest x-ray after 4 months of treatment reveals near complete resolution of the hilar lymphadenopathy.at 5 months her optic neuropathy and facial dysesthesia had resolved, and she was left with mild persistent vi nerve dysfunction. conclusion:sarcoidosis that manifests itself as sinusitis is an uncommon presentation and the mechanism involved is thought to be a consequence of the destruction of cilia and mucus producing glands by the granulomatous process.review of the literature indicates that this is the eighth case reported and illustrates that a high index of suspicion of other etiologies must be maintained when there is a refractory response to multiple courses of antibiotics in the treatment of sinusitis. j.b. hein * , p. patel, l. bielory, newark, nj. introduction: cid may result in opportunistic infections such as cryptococcal meningitis. we present an unusual case of cryptococcal meningitis in an hiv-negative patient with severe cd4 lymphocytopenia. case: a 40 yearold male with a three year history of sarcoidosis presented with acute onset of right-sided body numbness. the patient had been on prednisone 40 mg/day for 5 months prior to presentation. ct and mri scanning showed no vascular defects, meningeal enhancement, hydrocephalus or mass lesions. gallium scanning revealed normal uptake in the liver and spleen, mild uptake in the lungs and nasopharyngeal region, and asymmetric uptake in bilateral kidneys. the patient's initial wbc was 8, 000 cells/mm 3 , composed of 6970 neutrophils/mm 3 , 50 eosinophils/mm 3 , 50 basophils/mm 3 , 530 lymphocytes/mm 3 , and 400 basophils/mm 3 . repeat studies revealed the total lymphocyte count decreased at 240 cells/mm 3 with cd19 (pan b) cells decreased at 29 cells/mm 3 and cd3 (pan t) cells decreased at 189 cells/mm 3 . cd4 count was 144 cells/mm 3 and cd8 count 50 cells/mm 3 with a cd4/cd8 ratio of 2.9. igg levels were normal at 716 mg/dl. elisa was negative for hiv, and hiv-1 rna by pcr was not detected. serum ace level was 19 u/l and csf ace level was 23 u/l. csf obtained by lumbar puncture stained positive with india ink and culture revealed cryptococcus neoformans. the patient responded to amphotericin b lipid complex 5 mg/kg/day, and his neurological status eventually returned to baseline. conclusions: the severe lymphopenia in this patient caused predisposition to infection with cryptococcus neoformans. the etiology of the profound lymphopenia likely was multifactorial, including mild sarcoidosis activity (lung uptake on gallium scan) and chronic corticosteroid therapy. however, once cryptococcus became entrenched in the patient's csf, the infection itself likely depressed peripheral lymphocyte numbers even further. this case demonstrates the importance of exploring a broad differential diagnosis when faced with lymphocytopenia. background: isosulfan blue 1% is a common dye used in sentinel lymph node dissection. the usage of the procedure and dye has increased in numbers, and although rare, several cases of anaphylactic reaction have been reported. objective: we are reporting a patient who had an anaphylactic reaction to isosulfan blue while undergoing breast cancer excision with sentinel lymph node biopsy. methods: the patient is a 44 year-old woman with breast cancer. she underwent breast mass excision with sentinel lymph node biopsy using the lymphazurin 1% blue dye (isosulfan blue). she has a history of penicillin induced hives but has no other drug allergy. as soon as the dye was injected, she became flushed, hypotensive, and tachycardic. hypotension was refractive to fluid challenge. she was then treated with epinephrine as well as intravenous steroids and cimetidine with relief of symptoms. subsequently, she was evaluated in allergy and skin tests with isosulfan blue 1% at 1:100, 1:10 dilution, and undiluted were performed using histamine as positive control and saline as negative control. this procedure was also performed on two control subjects. results: the patient had a positive skin test (wheal and flare) to isosulfan blue 1% (undiluted) with the control being appropriately positive for histamine and negative for saline. control subjects had negative response to dye and saline and positive response to histamine. conclusions: isosulfan blue 1-% dye may cause anaphylactic reactions in patients undergoing sentinel lymph node dissection. . the positive skin test result to the dye plus the negative skin test responses in the controls suggests that the reaction may be immunoglobulin e (ig e) mediated. eosinophilic duodenitis (ed) and gluten-sensitive enteropathy (gse) or celiac disease (cd) are distinct clinical entities with well-defined clinical and laboratory parameters. ed is a rare condition of unknown etiology, which is manifest by eosinophilic infiltration of the gastrointestinal tract and peripheral eosinophilia. gse or cd is a form of non-ige food allergy caused by immune hypersensitivity to ingested gluten. the simultaneous occurrence of the two entities, however, is a rare event. the following presentation is a case report in which both entities were found in the same patient. an 11 y/o white hispanic male presented with severe, chronic, colicky abdominal pain and headache of 5 months duration. hematologic and immunologic studies were within normal limits. serum ige levels were 356 iu/ml (n= < 200 iu/ml), anti-endosomial ab (+), igg antigliadin: 16 u/ml (n=: 0-12 u/ml), skin tests for food and inhalant allergens were weakly positive (1+). biopsy of the inferior third of esophagus revealed chronic moderate esophagitis; biopsy of gastric antrum revealed lymphatic hyperplasia; duodenal biopsy showed shortening of the villi with the presence of eosinophils. following treatment with esomeprazole 40 mg bid, famotidine 20 mg qd, montelukast 20 md qd, lactobacillus, a diet free of gluten, rofecoxib 25 mg qd and betamethasone for 15 days, betamethasone the patient improved with partial resolution of symptoms. the presence of duodenal eosinophils persisted despite a gluten free diet, and continued to require repeated bursts of prednisone. since to our knowledge this is rare finding in which ed occurred in association with gse, a high index of suspicion for simultaneous occurrence of ed should be raised in any case of gse that fails to respond to a conventional gluten free regimen. we report the evaluation of a 6 month old male presenting with a pustular rash and choking episodes from birth. by age 6 weeks, he had experienced two pneumonias, one with fleeting infiltrates requiring intubation. persistent eosinophilia (5000-9000/ml) and eosinophils on pustule biopsy were noted. persistence of these and subsequent rsv pneumonia and recurrent draining otitis media led to referral. physical examination showed a thriving male infant with multiple erythematous papules and pustules along the scalp, face, axilla, and trunk. eosinophilia was confirmed. quantitative immunoglobulins were abnormal for igg 179 and ige 255 iu/ml. peanut cap rast was 23 kua/l. hib post-vaccination titer, t cell enumeration/stimulation, and nbt were normal. hiv testing, stool eosinophils, o&p, and hemoccult were negative. cxr, hrct, ekg, and echocardiogram were normal. pustule cultures for bacteria, virus, and fungus were negative. skin biopsy revealed numerous eosinophils in the pustule, dermis, epidermis, and perifollicular region. cd1a+ and s-100 staining were negative for histiocytic infiltration. nemo defect/incontinentia pigmenti were considered but testing was negative and karyotype 46, xy. bone marrow biopsy revealed numerous eosinophils at different stages of maturation, and no myeloproliferative or neoplastic changes. bronchoscopy was remarkable for 15% eosinophils on balf. 24-hour ph probe and impedence evaluation was negative. egd with biopsy was normal except for mild eosinophilic infiltration of the proximal and distal esophagus. with the clinical picture of recurrent pruritic crops of sterile pustules and characteristic skin biopsy demonstrating eosinophil infiltration, the diagnosis of eosinophilic pustulosis (ep) of childhood was made. despite no longterm sequelae or other end-organ involvement in ep, the degree and duration of eosinophilia, and presence of eosinophils in the airways and esophagus, raises the concern for other eosinophilic syndromes. following cardiac, cns, pulmonary, and gi systems is warranted. the infants pneumonias were felt to be due to aspiration, and ear infections the result of humoral immunity nadir or draining pustules in the external auditory canals. immunoglobulin levels will be monitored. this case illustrates the heterogeneity of eosinophilic diseases and raises the question of what drives the mechanisms behind malignant and benign disease. introduction: behcet's disease is a chronic, relapsing vasculitic disease characterized by recurrent oral, genital, and gastrointestinal ulcerations, a wide variety of skin lesions, uveitis, and arthritis. pediatric cases of behcet's disease are uncommon with an estimated prevalence of 1 in 600, 000 children under the age of 15. although the disease in children shows similar characteristics as adults, the diagnosis of behcet's disease in the pediatric population remains a challenge. this report describes an 18 year old girl referred to our pediatric immunology clinic for evaluation of recurrent painful oral ulcerations since the age of 7 with no genital ulcerations. the oral ulcers would last for two weeks and heal spontaneously but would reappear 2 to 3 weeks later. the patient had a history of raynaud's phenomenon for the last 6 to 7 years and the recent onset of joint pain. physical examination revealed 4-5 white elliptoid lesions 5-7 mm in diameter on an erythematous base on the tongue and soft palate. skin examination was significant for hyperpigmented patches over the neck, abdomen, and back. there were erythematous, serpigenous lesions on the palms and punctuated necrotic lesions on the finger-tips. pathergy test was positive. laboratory investigation was unremarkable and cultures from the oral ulcers remained negative. the patient was diagnosed with juvenile behcet's disease. she was started on immunosuppressive therapy with low dose prednisone and responded. conclusion: behcet's disease is a difficult diagnosis to make in the pediatric population. this case demonstrates that in children, recurrent oral ulcerations may be the only initial manifestation of behcet, and an important clinical marker for the disease. rationale: relapsing polychondritis is an uncommon severe inflammatory condition with unknown etiology that can present in a variety of manners. we report a 14 year old patient who initially presented with signs and symptoms of anaphylaxis to shellfish and was later diagnosed with relapsing polychondritis. case report: a 14 year old african-american boy with a 6 year history of asthma and fish and shrimp allergy presented to the pediatric intensive care unit on 4/23/03 after an episode of severe shortness of breath. his symptoms started shortly after accidental exposure to shrimp. in the intensive care unit, he was noted to be wheezing and stridorous. an initial chest xray as well as subsequent fiber optic laryngoscopy showed sub-glottic stenosis. he was intubated and received mechanical ventilation for greater than a week. after extubation, he stated that he felt fine, but continued to demonstrate audible stridor. pulmonary function tests demonstrated extra-thoracic obstruction, laryngoscopy continued to show sub-glottic stenosis, and esophagogastroduodenoscopy showed erosive esophagitis consistent with reflux. initial lab tests demonstrated a normal eosinophil count, elevated ige (969 iu/ml), and positive immunocaps testing to multiple foods. the patient was discharged home with minimal stridor and no wheezing. over the next nine months, the severity of his stridor waxed and waned, but in general it worsened in spite of a strict elimination diet and anti-reflux therapy. on 9/25/03 he received an emergency tracheostomy. on 1/26/04, the patient was again admitted to the hospital. at this time he complained of bilateral knee pain as well as significant weight loss. physical exam demonstrated arthritis with effusions of both knees as well as unilateral auricular chondritis and bilateral episcleritis. antibodies against type ii collagen were positive (42.1 eu/ml). the diagnosis of relapsing polychondritis was suggested. conclusion: relapsing polychondritis is a rare inflammatory disorder of the cartilage and connective tissue with an unknown etiology. the wide array of presenting complaints as well as the relapsing nature of this disorder often causes significant delay in diagnosis and treatment. in our case there was a period of nine months between the initial presentation and the time when the diagnosis of polychondritis could be made. d.k. geller * , m. ballow, buffalo, ny. introduction: an 11 yo male previously diagnosed with pandas presented to our immunology clinic for ivig. the patient was healthy until age 8 when he developed facial motor tics associated with group a beta hemolytic strep pharyngitis. he was treated with antibiotics and the tics resolved completely. he has had multiple recurrences associated with strep and other viral infections. the tics improve or resolve completely when he is infection free. he has no history of vocal tics, attention deficit/hyperactivity disorder, or obsessional thinking or compulsive behaviors. laboratory: multiple throat cultures positive for group a beta hemolytic strep, positive antistreptolysin and antideoxyribonuclease b titers. discussion: pandas identifies a subgroup of children with an obsessive compulsive disorder and/or tic disorder whose symptoms seem to be triggered by streptococcal infections. the proposed pathophysiology is an immune-mediated mechanism in which antistreptococcal antibodies, antistreptolysin and antideoxyribonuclease b, cross react with the basal ganglia of genetically susceptible hosts. a few studies have looked at immunomodulatory therapies including ivig for neuropsychiatric symptoms including tic disorders secondary to post-streptococcal autoimmunity. we plan a trial of ivig for this patient given the recurrent nature of his tics and the relation to streptococcal and other viral infections. introduction: the chronic granulomatous disease (cgd) is an inheritable disorder of phagocyte cell respiratory burst that result in life-threatening infections. the pulmonary infection is the primary cause of death in greater that 50% of the cases and the role of surgery in management of this entity remains undefined. case report: a lobectomy was performed in a 2 year-old male who presented a persistent opacity of the medial right lobe with fever, cough, malaise an rapid onset of respiratory failure; skin abcesses were concomitant. his familiar history was unremarkable. a chest tube was placed in a first instance in order to drain empyema, but no progress was observed. a ct scan showed necrotizing pneumonia of the right lung, mediastinal and retroperitoneal limphadenopathy and bronchopleural fistula. a second surgical intervention was undertaken to correct the fistula and pleural debridement was done. at this time, serratia marcenses was isolated from blood culture and lung; a primary immunodefiency was suspected. our evaluation showed nbt reduction on 0% and dyhidrorhodamine assay (dhra) didn't demonstrate and effective oxidative burst. the subsecuent management was based on specific antibiotic to serratia and prophylaxis with tmp-smz and itraconazole. transfer factor to improve the ifn levels was initiated. the patient's mother showed on dhra two granulocyte populations, with and without oxidative burst. a x linked cgd was consistent. medical progress was observed in the next days. conclusion: despite the poor utility showed by the surgical procedures in the complicated pneumonia in cgd patients in many series, attending to the unusual nature of the pulmonary infections and the high mortality and morbidity associated with thoracic surgery; the management of this case showed that an aggressive approach in the diagnosis combined with some procedures used in immunocompetent patients pneumonia may improve the clinical condition in cgd patients. background: takayasu arteritis is a large vessel vasculitis primarily affecting the aorta and its branches. it is most prevalent in women in the second and third decades of life. initial symptoms are systemic and often self-limited, but the disease may progress undetected over years until signs of vascular insufficiency develop. arteritis is commonly seen in the aortic arch and its branches, although the disease is a panarteritis and vascular compromise can occur in many organs. pathologically, involved vessels show intimal proliferation and fibrosis, and scarring of the elastic lamina. case report: we report the case of a 31 year old southeast asian woman who presented with diplopia, ptosis, dizziness, and progressive dyspnea. past medical history was significant for rheumatic fever complicated by aortic valve insufficiency and hypertension. physical findings on admission included hypertension, asymmetric upper extremity blood pressure (left arm 95/50mm hg, right arm 200/50 mm annals of allergy, asthma & immunology hg), wide pulse pressure and a harsh diastolic murmur. admission labs showed anemia (hemoglobin: 11g/dl, hematocrit 33%) and an elevated erythrocyte sedimentation rate of 31mm/hr. transesophageal echocardiography revealed severely dilated aortic root aneurysm with secondary severe chronic aortic valve insufficiency and calcified aortic leaflets not typical of rheumatic heart disease. resection of the aortic valve and aneurysm was performed. pathologic examination of the aortic aneurysm revealed intimal and adventitial fibrosis with focal chronic inflammation and partial loss and fibrosis of media, findings compatible with healed takayasu arteritis. the patient was started on prednisone 100mg by mouth daily for takayasu arteritis and discharged in stable condition. discussion: this is a case of takayasu arteritis masquerading as rheumatic heart disease. the episode of rheumatic fever was likely the initial presentation of takayasu arteritis, as the systemic symptoms of these diseases can overlap. takayasu arteritis is an insidious disease that often leads to a delayed diagnosis with considerable morbidity for the patient. since takayasu arteritis may go undiagnosed until ischemic symptoms develop, physicians should be alert to the possibility of this disease in young women to avert end organ damage and achieve early remission with drug therapy. we present a fifteen year old boy with disseminated papillomatosis and idiopathic cd4+ lymphocytopenia (icl). the warts have been present since he was one year old and have progressively worsened. there are more than fifty warts on each of his hands. his legs have multiple warts and about ten warts recently appeared on his lips and around his mouth. his past medical history is significant for no hospitalizations and no blood transfusions. he has never had problems with other infections, and he had a benign course of chicken pox that spontaneously resolved. he has been on cimetidine for six months without improvement. several topical therapies, including imiquimod, have failed. he has a total lymphocyte count of 1484/ul (normal range, 1288-4512/ul) and a cd4+ count of 301/ul (normal range, 361-1715/ul). the cd8+, natural killer cells, and cd19+ cells were normal. his hiv test is negative. the serum immunoglobulins were normal as well. lymphoctye proliferation studies reveal an absent response to antigens (candida and tetanus) with a normal response to mitogens. further studies showed no other cause of cd4+ lymphocytopenia. this case shows that the diagnosis of idiopathic cd4+ lymphocytopenia should be considered in any patient with widespread viral infection whose hiv test is negative. appropriate evaluation of the cd4+ count should be pursued. j. ko * , a. nowak-wegrzyn, new york, ny. introduction: approximately 40% of children with moderate to severe atopic dermatitis (ad) have food allergies. complementary and alternative medicine (cam) is utilized by an estimated 16-27% of patients with allergies. we present a case of a 4 year-old boy with ad referred after receiving a diagnosis of food allergy by a cam practitioner. methods/case: a 4 year-old boy was referred for evaluation of mild ad and possible food hypersensitivity. his ad started at 1 year of age and was limited to the face. he was initially breast-fed and was switched to milk-based formula at 4 months of age. solids were introduced at 5 months of age without difficulty. he had no immediate reactions or noted worsening of ad due to foods. the patient presented to a naturopath for allergy evaluation. based on electrodermal skin testing results, he was reported to be allergic to milk, egg, peanut, rice, and beef (all of which he had tolerated previously). he was restricted from milk and egg for 4 weeks without noticeable change in his ad, which was limited to the cheeks and controlled on topical pimecrolimus. results: his weight and height were both 75th percentile. physical exam was normal except for 2-3 cm dry, erythematous patches on both cheeks and mildly dry skin on flexor/extensor surfaces of both arms. skin prick testing revealed negative tests to milk, egg, peanut, and dust mite. serum ige testing was also negative. since he had no evidence of ige sensitization to commonly allergenic foods, he was recommended to continue an unrestricted diet and discontinue use of a "sippy" cup. his ad subsequently improved on a skin care regimen of postbathing moisturization and topical pimecrolimus prn. asthma and allergies are the #2 reason for cam use in the us. electrodermal skin testing is a method utilized by naturopathic doctors to detect allergen "sensitivity". however, in two double-blinded trials examining atopic and non-atopic patients, electrodermal testing did not correlate with skin prick testing results, regardless of inter-operator variability. conclusions: allergy patients are seeking cam treatment, and it is important for physicians to be aware of the safety and efficacy of alternative medical practices. the use of electrodermal skin testing has not been proven to detect allergen sensitivity and use of this modality should be discouraged. e.e. mcgintee * , k.e. sullivan, philadelphia, pa. introduction: hematopoietic stem-cell transplantation causes profound tcell immunodeficiency due to the rigorous conditioning involved. following transplant, the t-cell compartment is initially reconstituted through expansion of mature donor-derived t-cells. however, thymopoiesis is necessary to generate naive t-cells with a diverse repertoire. factors affecting thymic function impact the ability of the body to reconstitute the immune system. we report a case of severe t-cell immunodeficiency after two stem-cell transplants for neuroblastoma in a patient with a history of thymic hemorrhage secondary to mediastinal surgery. case: a 5-year-old female with a history of high-risk neuroblastoma was referred for immunologic evaluation due to a significant infectious history since completing her neuroblastoma therapy. she initially presented at 21 months of age with a right atrial mass that was presumed to be an atrial myxoma. she underwent mediastinal surgery to resect the tumor, which was found to be neuroblastoma extending from her right adrenal gland along the vena cava. a ct scan following her surgery revealed an area of hemorrhage in her right thymus. she subsequently received high-dose chemotherapy and two autologous stem-cell transplants. her infection history was significant for multiple episodes of upper respiratory illnesses, sinusitis, and otitis media requiring myringotomy tube placement on two occasions. immunologic evaluation revealed a dramatically low absolute lymphocyte count with deficits primarily in the t-cell compartment, and reduced proliferation in response to mitogens. immunoglobulin levels were normal; she formed protective titers to diphtheria and tetanus but not to any of 14 pneumococcal serotypes. conclusion: stem-cell transplantation often results in severe t-cell immunodeficiency. the thymus plays an important role in reconstituting the t-cell compartment with naive t-cells generated from hematopoietic stem-cells, which restores tcr diversity. as this case illustrates, damage to the thymus can significantly impair the ability to reconstitute the t-cell compartment. a.k. knight * , c. cunningham-rundles, new york, ny. rationale: this is the first case report of oxcarbazepine induced immunoglobulin deficiency. methods: a 49 y/o white female was referred for further investigation of low serum immunoglobulin found as part of an evaluation for chronic bacterial vaginitis. she was taking oxcarbazepine for chronic pain. this was discontinued and immunoglobulin levels and b cell numbers were tested at intervals. specific antibody responses to pneumococcal vaccine were tested. results: at presentation, on oxcarbazepine, immunoglobulin levels were low [igg 576, iga <11, and igm <4 mg/dl] and she had a b cell deficiency [1%, 18 b cells (normal 5-15%, 75-375 cells/cu mm)]. antibody response one month after pneumococcal vaccine was poor (protective antibody to 2/12 serotypes). (7%)] with protective antibody responses to 5/12 pneumococcal serotypes. she continued to have iga deficiency. conclusions: the patient's initial evaluation suggested the diagnosis of common variable immune deficiency with hypogammaglobulinemia and specific antibody deficiency. however, these defects reversed when oxcarbazepine was discontinued. it is unclear if persistent iga deficiency was a pre-existing condition, possibly predisposing her to this adverse reaction to oxacarbazepine, or induced by the oxacarbazepine. immunoglobulin deficiency is a known, though rare, reaction to carbazepine, the parent drug of oxacarbazepine; this adverse reaction can occur with its derivative oxcarbazepine as well. secondary hypogammaglobulinemia should be considered before diagnosing primary immunodefiencies such as cvid and committing the patients to lifelong immunoglobulin therapy. introduction: interferon and glatiramer acetate (copaxone) are indicated for the treatment of relapsing-remitting multiple sclerosis (ms). anaphylactic reaction has been reported as a rare complication of interferon and copaxone use. we report a case of interferon -1a (rebif) and copaxone hypersensitivity associated with positive skin tests and desensitization to interferon -1b (betaseron). case report: the patient is a 28 year-old woman with asthma and allergic rhinitis who was diagnosed with ms in oct '04 after an uncomplicated pregnancy and was subsequently placed on rebif. one month after starting therapy, patient developed wheezing and generalized urticaria after receiving a dose of rebif. the symptoms recurred the next day. her neurologist stopped the rebif and started her on copaxone. two months later she developed episodes of generalized urticaria. the patient was then evaluated in our center and underwent skin testing with interferon -1a intramuscular (avonex), interferon -1a subcutaneous (rebif), betaseron, and copaxone. she had a positive skin prick reaction to copaxone and positive intradermal skin tests to avonex (1:10 strength), rebif (1:1), and betaseron (1:1) after 24 hrs. the intradermal reactions to all 3 interferon formulations continued to progress upto 48 hrs. her neurologist felt that she would benefit from betaseron therapy. the positive intradermal skin test to betaseron was sufficient to warrant desensitization to prevent immediate hypersensitivity reactions. she underwent subcutaneous desensitization as shown in the table. the desensitization was halted at 240 minutes after the patient developed itching of the hands.* she returned in 24 hrs and we restarted by administering 0.1 ml of 1:1 strength betaseron. increasing doses were given until the patient received a full therapeutic dose (0.3 mg in 1 ml). she has since done well with daily doses of betaseron (4.5 mil units). conclusion: we report a ms patient who developed urticaria and asthma exacerbation in response to rebif and urticaria to copaxone. skin tests confirmed an ige-mediated allergic reaction. she underwent successful desensitization to betaseron. to our knowledge, this is the 1st report of desensitization to betaseron as well as extension to previous reported cases of systemic reaction to interferon and copaxone. pentoxifylline (ptx) is a phosphodiesterase inhibitor that has been used for ischemic vascular disease because of its effects on red blood cells and platelets. it has been used for systemic inflammatory diseases such as sarcoidosis because of its ability to inhibit production of cytokines such as tumor necrosis factor (tnf-a). we decided to offer a trial to a patient with chronic urticaria since agents that increase intracellular camp have been shown to inhibit mast cell degranulation. we report a case of a 50 year-old female with a history of allergic rhinitis, hypothyroidism, and diabetes mellitus with recurrent episodes of chronic urticaria since adolescence. she had known allergies to various antibiotics but otherwise had an unremarkable history, family history and social history. she was being treated with fexofenadine 180mg qd, azelastine nasal spray, zafirlukast 20mg bid, hydroxychloroquine 200mg bid, levothyroxine 25mcg qd, doxepin 25mg qhs, metformin 500mg bid, and spironolactone 25mg qd at the time of her initial evaluation. her physical exam was notable only for scattered 2-3 cm urticarial skin lesions as well as areas of hypo and hyperpigmentation from previous excoriations. her nasal turbinates were mildly congested with dull membranes. a previous skin biopsy showed only urticaria with increased numbers of mast cells. her workup included normal cbc, urinalysis, spep and complete chemistry profile, as well as negative ana and anti-thyroid antibodies. pentoxifylline 400mg tid was added to the patient`s regimen. her urticaria resolved within 2-3 weeks and was no longer visible at 2 months follow-up. pentoxifylline is a safe medication with few side effects that may benefit patients with chronic urticaria via several mechanisms, including the inhibition of mast cell degranulation and secretion of various cytokines and chemokines. the beneficial effects of pentoxifylline in the treatment of this patient`s urticaria have been seen in other patients. further study is indicated to determine which patients with urticaria are most likely to benefit from pentoxifylline, as well as elucidate the mechanisms of action by which pentoxifylline ameliorates symptoms of chronic urticaria. c.m. mjaanes * , m. boguniewicz, denver, co. we report the case of an 11-year-old male who since the age of 6 years has suffered from recurrent episodes of left tongue swelling. onset of the swelling is usually abrupt, and generally occurs during the night, awakening him from sleep. he describes a sensation of pain in his tongue but denies any pruritus, throat, lip or eyelid swelling. he has no cough, dyspnea, wheezing, rash, or itchy/watery eyes. the episodes occur infrequently, approximately once every spring . they tend to resolve spontaneously within 4 to 6 hours, however, on 2 occasions, the swelling has lasted for 7-21 days. the patient's current episode has been present for 3 weeks and is progressing. today he reports more pain and increased swelling. the child reports no benefit from oral antihistamines. he was treated with dexamethasone during his initial episode and experienced gradual resolution of the swelling. he has never been treated with topical or systemic epinephrine, or additional courses of systemic steroids. there is no family history of angioedema. the child was referred for evaluation of an immunologic/allergic etiology of his unilateral tongue swelling. laboratory studies obtained early in the course of his current episode revealed the following: c3 level 84 mg/dl; c4 level 14.4 mg/dl; c2 level 22.5 mug/ml; c4d level 0.94 mug/ml; c1 esterase inhibitor function 115% of normal. c1 esterase inhibitor level 17.4 mg/dl; ch50 223 units/ml; ana 1:40, negative; beta-tryptase < 1.0 ng/ml; total tryptase 7.0 ng/ml (all normal). initial evaluation revealed marked swelling along with erythematous and violaceous discoloration of the left lateral and anterior portions of the child's tongue. the remainder of his examination was normal. the patient was referred to the pediatric otolaryngology clinic with a presumptive diagnosis of a glossal hemangioma versus lymphangioma. he underwent a magnetic resonance imaging study which revealed a poorly defined, mass-like enlargement of the anterior and left aspect of the tongue. he was treated with a five day course of prednisolone. after initial regression of the lesion a planned surgical excision was carried out without any complications. in conclusion, this rare case illustrates the unique presentation of glossal hemangioma presenting as recurrent angioedema. highlighted are the differential diagnoses, laboratory studies and clinical features of oropharyngeal swelling. severe tongue swelling. rare skin conditions such as leprosy need to be considered in the differential diagnosis of apparent urticaria and angioedema that is atypical in appearance or response to therapy. a 28 year old male originally from laos presented with chronic recurrent nonpruritic indurated plaques on his face and trunk, and was referred to allergy by his primary physician with a diagnosis of urticaria and angioedema to rule out an allergic reaction to foods. he was otherwise healthy and on no medication, and had a negative personal and family history of atopy. he had moved to the united states from southeast asia in 1990, and visited for several weeks again in 2003. he was treated with prednisone and hydroxyzine without benefit; skin testing to foods was negative, and cbc, differential, sedimentation rate, antinuclear antibody, and liver function testing was normal or negative. a skin punch biopsy from an indurated area on the back showed granulomas with clusters of acid-fast organisms on afb stain consistent with tuberculoid leprosy. as the patient was g6pd deficient, he could not be treated with dapsone and was treated alternatively with minocycline 100mg, rifampin 600mg, and clofazimine 50 mg daily. he was subsequently started on prednisone as he was determined to be having a reversal reaction; his lesions continue to improve. this patient had a rare condition, leprosy, which was confused with urticaria and angioedema. in patients presenting with atypical apparent urticaria or angioedema, especially if response to standard pharmacologic treatment is poor, a skin biopsy is indicated, and may be diagnostic as in this case. introduction: cutaneous flushing about the face of a toddler within minutes of eating specific foods prompts parents and clinicians alike to pursue food allergy testing. auriculotemporal syndrome, also known as frey syndrome, is an isolated and transient facial erythema about the distribution of the auriculotemporal nerve following mastication. we report a child who was referred for food allergy testing and was diagnosed with auriculotemporal syndrome. case history: a 2 yo wf, prior full-term forceps-assisted delivery, has a history of facial flushing within minutes following ingestion of specific foods. the flushing can be induced by a variety of foods, specifically spaghetti, crackers, potato chips and hard candies, and typically resolves within minutes after eating. the child has no respiratory or gastrointestinal distress during the flushing episode. there is no angioedema, urticaria, or other rash elsewhere on her body. within ten minutes of eating a lollipop in clinic, an erythematous, warm macular eruption appeared in a band-like region anterior to her ear extending from her temporal bone to her mandible. this response occurred bilaterally, with a mildly greater intensity on the right side of her face. no sweating or other symptoms developed, and the flushing began to diminish after thirty minutes of observation. discussion: auriculotemporal syndrome is commonly seen in patients who have undergone facial surgery, specifically about the parotid gland. it is believed to be secondary to a disruption in the fibers of the auriculotemporal nerve. it is theorized that the nerve regeneration following an injury may join the parasympathetic fibers of the salivary gland with sympathetic fibers of eccrine sweat glands. mastication then could result in flushing and sweating over the cutaneous distribution of the auriculotemporal nerve that extends from the temporal region to the mandible, anterior to the ear. although rare in children, auriculotemporal syndrome has been associated with forceps delivery. therapy is seldom of benefit or needed, and the syndrome often resolves spontaneously over many years. conclusion: auriculotemporal syndrome leads to a cutaneous eruption temporally related to the ingestion of foods. allergists should maintain an awareness of this rare syndrome to minimize food allergy testing or eliminate unnecessary food provocation challenges or restrictive diets. m. al-ahmad * , s.s. mace, toronto, canada. introduction: ranitidine is a well-known h2-receptor antagonist. anaphylactic reactions are seldom reported despite widespread use of the drug. we report a patient with anaphylactic reaction to ranitidine methods: we report a case of anaphylactic reaction with ranitidine. results: a 45-year-old female with a history of urticaria over one year period, presented with 4 episodes of anaphylactic reactions of increasing severity over 1 year, after ranitidine ingestion. the first two episodes, occurred one hour after ranitidine ingestion, began with a sensation of burning, and itching in the head followed by dizziness, hypotension and near loss of consciousness. the patient had no significant pulmonary or laryngeal symptoms. the fourth episode, occurred 12 minutes after taking ranitidine tablet, was characterized by severe hypotension. two episodes were managed at the emergency department. a skin prick test with a crushed ranitidine tablet demonstrated a positive 20 mm wheal response, which was negative in a control. investigations for carcinoid syndrome and systemic mastocytosis were negative. conclusion: an ige mediated allergy to ranitidine is possible. anaphylactoid reaction to ranitidine is a well recognized entity. however, to our knowledge, few cases of anaphylactic reaction to ranitidine have been described. hypersensitivity reaction to ranitidine should be considered in patients suspected of having drug-related allergy. introduction: severe combined immune deficiency(scid) and cystic fibrosis(cf) may both present in infancy with a history of failure to thrive, diarrhea, and recurrent respiratory infections. although cf is the most common genetic disease occurring in caucasians with an incidence of 1 in 2500 births, scid is a rare condition estimated to occur between 1 in 50, 000 to 1 in 500, 000 births. we report a case of x-linked scid where the diagnosis of cf was originally entertained due to similar presentation and misleading laboratory results. case: 5-month-old caucasian male with 3 month history of recurrent respiratory illnesses, fever, loose stools, thick nasal discharge and failure to thrive. this was his second admission for a respiratory infection. relevant lab results consisted of cultures which grew parainfluenza a virus on np wash and pseudomonas aeruginosa on deep throat culture, chest xray showed patchy infiltrates and a borderline sweat test of 51 mmol/l. despite being treated with broad spectrum antibiotic coverage, albuterol and pulmozyme, his respiratory symptoms were not improving. physical exam revealed a thin infant with palpable though small lymph nodes cervical and groin, diffuse scattered rhonchi and liver edge palpable 3 cm below the costal margin. quantative immunoglobulins were obtained and resulted as an igg of 75, iga of <10, and igm of 13. review of his chest x-ray was significant for absent thymic shadow. lymphocyte count since admission ranged from 400-1000. flow cytometry revealed a cd3/cd4 count of 0%, cd3/cd8 count of 0%, cd56 of 1% and cd19 of 99% of all lymphocytes. t-cell stimulation tests were markedly diminished to both antigen and mitogen. a presumed diagnosis of x-linked scid was made and patient undrewent haploidentical bone marrow transplantation. subsequently, cf gene analysis resulted as negative. discussion: as outlined in the case it is essential to recognize the similarities between the presenting symptoms of cf, a relatively common genetic disease and scid, an uncommon one. although pseudomonas is seen in increased frequency in patients with cf, it is essential to recognize that it is also a leading pathogen affecting patients with antibody deficiency. review of the literature did not reveal any previous cases of individuals with scid reported as having an increased sweat chloride, nor any explanations of why this would occur. while immunodeficiency has previously been associated with some forms of dwarfism, we report the first described case of common variable immune deficiency in a patient with seckel syndrome (bird headed dwarfism). the diagnosis of seckel dwarfism, an autosomal recessive disorder, was made at 6 months of age in this boy based on intrauterine growth retardation, proportional dwarfism, microcephaly, micrognathia, and narrow face with "bird-like" large eyes and beaking of the nose. hematologic abnormalities are reported in an estimated 15% of such patients, and this child developed thrombocytopenia at the age of 3 years. by age 7, he had pancytopenia with hypocellular bone marrow. a low cd4 count (117/ul) was noted and both bactrim and neupogen were begun. his only significant infections, rotavirus and salmonella gastroenteritis, occurred at that time along with frequent otitis leading to pe tube placement at ages 7 and 8 but which subsequently resolved. he has never had opportunistic infections or thrush. by the age of 10 years, he required monthly red cell transfusions for severe anemia. after several months of transfusions, the blood bank reported that he no longer had detectable blood group antibodies during crossmatch; this led to measurement of immunoglobulins. igm was undetectable with low iga (35 mg/dl) and low igg (545 mg/dl). he demonstrated no protective titers to any pneumococcal serotypes after either conjugated or unconjugated pneumococcal vaccines. response to diphtheria, tetanus, rubeola, and influenza vaccines was adequate. total hemolytic complement was normal. he remains lymphocytopenic (alc = 552/ul) with low cd4 (211/ul). after five doses of ivig at 400mg/kg monthly, platelets have remained between 15-20 k/ul and there has not been any improvement in the anemia. although the mechanism for the bone marrow failure in these patients is presumed to be chromosomal breakage, this has not been demonstrated in this child. his karyotype is 46xy with no chromosomal fragility or deletions detected to date. hypogammaglobulinemia should be investigated in any patient undergoing frequent transfusions who loses blood group antibodies, and suspicion of immune deficiency should remain particularly high in patients with severe growth retardation or dwarfism. n.l. rider * , t. craig, hershey, pa. the objective of this study was to assess the effectiveness of physicians at a university primary care clinic in diagnosing and managing adult patients with asthma. a retrospective chart review of 63 adult patients from this clinic was performed. each chart was evaluated using a survey instrument developed from the national asthma education and prevention program (naepp), which consisted of 22 queries. of the charts evaluated 50 met our entry criteria. overall compliance with the naepp recommendations was 34%. adherences to the asthma guidelines in the following areas were: diagnosis (42%), treatment/disease control (60%), monitoring (15%), and education (18%). these data suggest that there is an opportunity to improve the care provided to patients with asthma, especially in the areas of asthma education and monitoring, even in a university outpatient facility. these data also suggest that naepp guidelines have not been effectively incorporated into primary care practices. b.m. wahlers * , l. sherwood, t. craig, hershey, pa. objective: our aim was to evaluate adherence to the national heart lung and blood institute (nhlbi), national asthma education and prevention program (naepp) diagnosis and management guidelines, regarding asthma admissions at a teaching hospital. methods: a retrospective chart review of 197 patients over the age of 18 years admitted to the hershey medical center between 1997 and 2002 with a primary diagnosis of asthma or asthma exacerbation was conducted using a 21-item questionnaire focused on key aspects of asthma care as outlined in the naepp guidelines. results: overall compliance to the guidelines was 61.5%. maximal areas of compliance were noted in regards to pharmacologic treatments utilized (99%, 95%). much lower levels of compliance were noted in areas of patient education (58%), instruction in written action plan prior to discharge (11%), provision of peak flow meter for home use (47%), assessment of asthma symptoms and severity (58%), and inhaled steroid prescription on discharge (69%). conclusion: adequate care in terms of pharmacologic treatments is being given but sub-optimal cares in other areas of the guidelines exist. there continues to be room for improvement in regards to asthma management, most notably in the area of education, documentation of triggers, and preparation for discharge with adequate tools to monitor and control symptoms. background: asthma is one of the most common problems of childhood, responsible for a significant proportion of absence from school because of chronic illness. objective: this study was carried out among the school-aged children (7-18 years) in tehran schools during 2002-2003, in order to determine the frequency of asthma. methods: according to the recommendation of who, a questionnaire was designed, containing 8 standard questions, and the students were given necessary information to complete the questionnaires. the guidance and high school students completed the questionnaires but the parents of primary school students completed them by themselves. results: seven hundred and eight children out of 2000 children had asthma (35.4%); this prevalence was higher in the boys (37.1%), when compared to the girls (33.5%). the prevalence of this disease has been estimated about 39.5% in guidance schools, 35.4% in high schools and 31.6% in primary students. based on this survey, the most common clinical manifestations in asthma were included: prolonged cough lasting more than 10 days (22.4%), and exerciseinduced wheezing or dyspnea (16.9%), followed by repeated dyspnea or wheezing (6.4%). based on the drug responses after receiving solbutamol, the prevalence of asthma was evaluated in the range of 32.2% in primary schools, 16.7% in guidance schools and 22.9% in high schools. conclusions: the prevalence of asthma is high among the students of tehran schools and it needs more careful screening programmes and either more information given to the patients and parents about the disease. in the us, asthma is the most common chronic disease of childhood and affects approximately 5 million children. reasons for inadequate asthma control include: inappropriate therapy, incorrect inhaler technique, poor compliance with treatment, and exposure to environmental triggers. rates for asthma prevalence, hospitalization, and death are highest among children residing in inner cities, and important risk factors for asthma-related mortality include being poor and black. the communities of braddock, north braddock and rankin are among the poorest in allegheny county and are designated areas of greatest health risk. recent statistics have demonstrated an increase in the number of asthma hospitalizations in these communities: 13.6 children per 1000, which is double the average for allegheny county and greater than seven-times the overall us rate of asthma hospitalizations. our goals were to assess asthmatic severity and appropriateness of treatment, provide asthma education, and to ensure access to healthcare for asthmatic children living in these medically underserved and predominantly african-american communities. twenty-seven asthmatic children were identified. of the children assessed 7% were under the age of 2 years, 19% were aged 2-5 years, and 74% were 6-14 years of age. based on nhlbi guidelines the severity of each child's asthma was determined: 41% had mild-intermittent asthma, 15% had mildpersistent asthma, 26% had moderate-persistent asthma, and 18% had severepersistent asthma. the appropriateness of treatment based on severity was also determined. we found that 27% of the children with mild-intermittent asthma, 50% of children with mild-persistent asthma, 60% of children with moderate-persistent asthma and 100% of children with severe-persistent asthma were receiving inappropriate treatment. furthermore, of the inappropriately treated children 92% were undertreated. following the assessment, patients and parents were educated with regard to asthma triggers, pathophysiology, use of peak flow meters, inhaler technique and pharmacologic treatment. patients were also scheduled for outpatient follow-up care. these results demonstrate that inappropriate treatment, especially undertreatment, may be a significant cause of the increased asthma morbidity in this population and underscore the need for aggressive education and innovative methods of ensuring health care. w. li-ling * , t. keng-leong, f.c. stephanie, e. philip, singapore. background: asthma admission is an important indicator of asthma morbidity. little is known about the risk factors associated with asthma admission among high-risk asthmatics. purpose: 1) to characterise the profile of high-risk asthmatics who had previous asthma hospitalization. 2) to identify predictors and risk factors that are associated with asthma hospitalization. methods: data for consecutive high-risk asthmatics who were enrolled into our asthma program from october 2001 to may 2004 were retrieved from our prospective database. high-risk asthmatics were defined as patients who had a clinical diagnosis of asthma and who had at least one hospital admission for exacerbation of asthma in the preceding 12 months or at least one severe exacerbation of asthma in the preceding 6 months requiring unscheduled visit for beta2-agonist nebulisation. a comparison was made among high-risk asthmatics who had at least one or more hospitalization for asthma in the past 12 months prior to the asthma program enrolment with those who were not hospitalized. statistical analyses were performed using spss version 10.1 for windows. results: among 521 high-risk asthmatics, 252 (48.4%) had at least one or more asthma hospitalization in the past 12 months prior to the enrolment into the asthma program. hospitalization was significantly associated with female gender (63.1% with asthma admission v 43.1% without asthma admnission). a significantly higher proportion of asthmatics with no formal education (19.4% v 17.7%), primary education (23.8% v 16.6%) and secondary education (32.1% v 29.3%) were hospitalized for asthma as compared to the those who had tertiary education (24.6% v 36.5%). those admitted were predominantly in lower income group, had poor inhaler technique and have no ownership of an asthma action plan (33.3% v 76.6%). those hospitalized were also younger, median age 37years (19, 62) v 35years (15, 84) and had a lower first peak flow reading, 260l/min (130, 500) v 300l/min (150, 480) . conclusion: among high-risk asthmatics, certain population subgroups are at greater risk for hospitalization for asthma. intervention aimed to reduce asthma morbidity should take the above findings into consideration. introduction an association between asthma symptoms and air quality is well established, but controversy exists concerning the role of various air quality indicators. the association of san antonio air quality with emergency department (ed) visits for asthma at our military medical center has not been defined. the objective of this study is to determine the correlation of selected air quality indicators in san antonio with ed visits for asthma for both the general population and for the pediatric population. methods selected air quality indicators were the 8-hr air quality index (aqi) for ozone, 24-hr aqi for particulate matter < 2.5 î¼m, pollen counts, mold spore counts, and daily high temperature. the number of daily ed visits coded for asthma was obtained retrospectively. pediatric visits (patients < 18 years-old) were evaluated as a subgroup. pearson correlation coefficients (r) were calculated for all data pairs. results during the 331 days of the study period, there were 491 visits for asthma. of these visits, 151 were pediatric. the number of daily visits ranged from 0 to 6 for all visits and from 0 to 3 for the pediatric visits. r values for all visits were -0.067, -0.069, 0.110, -0.011, and -0.218; for pediatric visits, -0.055, -0.036, 0.033, -0.010, and -0.074 for the 8-hr aqi for ozone, 24-hr aqi for particulate matter < 2.5 î¼m, total pollen grains/m 3 , mold spores/m 3 , and daily high temperature, respectively. in addition to calculating r values for same-day air quality indicators and ed visits, r values were also calculated for a 3-day average of air quality indicators with the ed visits. for the 3-day averages, r values for all visits were -0.113, -0.117, 0.127, -0.071, and -0.267; for pediatric visits, -0.081, -0.011, 0.025, 0.017, and -0.063 for the 3-day averages of the 8-hr aqi for ozone, 24-hr aqi for particulate matter < 2.5 î¼m, total pollen grains/m 3 , mold spores/m 3 , and daily high temperature, respectively. conclusion there was no significant correlation between the selected air quality indicators and ed visits for asthma, either for the general population or for pediatric visits. this study provides no evidence that air quality is linked to ed visits for asthma in san antonio but the degree to which air quality in san antonio affects asthma symptoms remains unclear. introduction: the aim of this study was to evaluate the level of no in the exhaled air of patients suffering from chronic cough. methods: the study was performed on 213 young (mean age 28.5 +/-8.2 years), nonsmoking patients with chronic cough referred to the allergy clinic for evaluation. bronchial provocation challenge with histamine was performed according to the method of ryan. exhaled no was measured on-line using a chemiluminescence analyzer (sievers, usa). all patients had resting spirometry within normal values. results: significant bronchoconstrictive response to histamine at a concentration equal to or lower than 8 mg/ml (pc20 8mg/ml) was found in 65 patients (30.5%). these patients had a significantly greater mean concentration of no in the exhaled air (80.6+/-59.4 ppb) than those with a pc20>8mg/ml (28.4 +/-29.2 ppb; p<0.0001). receiver operating characteristic (roc) curve analysis revealed that it is possible to identify from among patients suffering chronic cough those who have significant bronchial hyperreactivity (pc20 8mg/ml). using 40 ppb as a cut-off value for the exhaled no concentration, the specificity for bronchial hyperreactivity was 86.5% and sensitivity 72.3%. conclusion: assessment of no concentration is helpful in the assessment of bronchial hyperreactivity in patients having chronic cough. l. abetz 1* , j. bousquet 2 , e. juniper 3 , e. bateman 4 , h. boushey 5 , w. busse 6 , t. clark 7 , r. pauwels 8 , s. pedersen 9 , 1. manchester, united kingdom; 2. montpelier, france; 3. ancaster, canada; 4. cape town, south africa; 5. san francisco, ca; 6. madison, wi; 7. london, united kingdom; 8. ghent, belgium; 9. kolding, denmark. introduction: the acq has been developed as a measure of asthma control for use in clinical practice or in clinical trials. the reliability, validity and responsiveness have previously been demonstrated for the original 7 item acq, the 6 item (minus fev1 question) and 5 item (minus fev1 and short acting bronchodilator use questions) versions. data from the 1-year gaining optimal asthma control (goal) study was used to assess the predictive value of the acq s 7, 6, and 5 item versions in determining asthma control status. method: receiver operating characteristic curves (roc curves), plotting true positive rates versus false positive rates for different acq cut-points, were used to determine optimum cut points for totally controlled and well-controlled asthma. scores for the 7, 6, and 5 item versions of the acq were examined at 0.25 intervals. results: when predicting totally controlled asthma, the areas under the roc curves (arocc) were 0.88/0.88/0.86 for the 7, 6 and 5 item versions, respectively; and when predicting well-controlled asthma the arocc were 0.86/0.86/0.84 for the 7, 6 and 5 item versions. in both predictive models, the 7 and 6 item versions provided the best arocc. for the 7 and 5 item versions, the best cut-off point to predict totally controlled asthma was <=0.75, with arocc of 0.81/0.79, and corresponding sensitivity of 0.74/0.71 and specificity of 0.89/0.88, respectively. for the 6 item version the best cut-off point was <=0.5, with arocc of 0.82, sensitivity of 0.76 and specificity of 0.87. the best cut-off points for predicting well-controlled asthma were <=1.25 for the 7 item version (arocc=0.77; sensitivity=0.70; specificity=0.83), and <=1 point for the 6 and 5 item versions (arocc=0.77/0.75; sensitivity=0.70/0.69; specificity=0.83/0.81, respectively). conclusion: results indicate the sensitivity and specificity of the acq in predicting asthma control status. a 29 yo black male with asthma and allergic rhinitis developed gradually worsening cough productive of clear to yellow sputum despite treatment with high dose fluticasone, salmeterol, monteleukast, and prn albuterol. asthma history was significant for an intubation at age 10 and pneumonia at age 28. physical exam revealed pale turbinates and expiratory wheezes. pulmonary function test showed a severe obstructive lung defect with fev1 1.52 (38%), improving by 4% after bronchodilator and fev1/fvc ratio 54%. a chest ct revealed significant bronchiectasis in the right middle lobe, lingula and both lower lobes. evaluation for bronchiectasis showed normal quantitative immunoglobulins, subclasses, and humoral responses, rendering innate immunodeficiency less likely. the diagnosis of allergic bronchopulmonary aspergillosis was entertained, but ige was 500ku/l and aspergillus titers were negative. a sinus ct failed to show active disease, yet the patient reported chronic yellow nasal discharge despite courses of antibiotics. although he was sexu-ally active with several male partners, hiv testing was negative 2 times, 6 months apart. evaluation for cystic fibrosis (cf) showed no symptoms of malabsorption and a negative sweat test. other tests revealed normal -1 antitrypsin level, p-anca, esr, ace level, and negative sputum for afb and mycobacteria, helping to exclude other conditions such as 1-antitrypsin deficiency, vasculitis, sarcoidosis, and chronic infection. with no definitive answer, the diagnosis of cf was pursued, but genetic screening as well as mapping were negative. the diagnosis of primary ciliary dyskinesia was then entertained; however, a mucosal biopsy of the nose did not reveal any structural abnormalities. finally, a sperm analysis revealing azoospermia confirmed the diagnosis of young's syndrome, a rare disease with features similar to cf including bronchiectasis, chronic sinusitis and azoospermia. in young's syndrome however, there is an obstructive process rather than a lack of vas deferens resulting in azoospermia and normal sweat chloride, genetic, and pancreatic testing. this case demonstrates the importance of investigating a persistently productive cough in an asthmatic patient and the differential diagnosis as well as the appropriate work up for bronchiectasis. a final diagnosis is important since appropriate management in each situation may affect long term outcome. a.g. palma-carlos * , m.l. palma-carlos, lisboa, portugal. introduction: bronchial challenges are currently done in allergic asthma with allergen, histamine or metacholine.bronchial reactivity to allergens and histamine can probably be variable. purpose to evaluate if the dose of allergen and histamine triggering a significant bronchial obstruction are always correlated in asthmatic patients. methods: a vitalograph model compact has been used throughout the study. allergen challenge has been done with an acqueous solution of house dust mite. bronchial challenges with placebo (sodium chloride) histamine or allergens have been done in all patients. for the provocations with histamine or allergens the threshold dose, inducing a decrease in fev1 greater than 15% has been determined in all the cases, as well for histamine as for allergens. a second challenge has been done in all patients with a supra-threshold dose of histamine or allergen corresponding to the double of the threshold. results: 30 provocations have been done in 10 patients either with histamine or allergen. 6 were allergic and 4 non allergic. the threshold dose for histamine were between 5 and 25 ug. in allergic patients and 250 and 500 ug. in non allergic patients.in allergic patients after histamine threshold challenge mean decrease was for fev1 19, 2% and for fev1/vc 17, 3%. for supra-threshold dose fev1 decreases 37, 8% and fev1/vc 32, 8%. after allergen challenge fev1 decreases 20, 3% and fev1/vc 15, 8%. with supra threshold doses fev1 decreases 28, 6% and fev1/vc 29, 3%. in 4 non allergic patients there was no changes after allergen challenge. a good correlation in constriction induced by histamine or allergen either for threshold or supra threshold doses (p<0.001) was observed but not between the threshold doses of histamine and allergen. conclusions: these results confirm that bronchial obstruction can be triggered by allergens and mediators that the sensitivity of fev1 and fev1/vc for evaluation are roughly comparable after challenge and points to a better sensitivity of a supra threshold dose of allergen or mediators. allergen and histamine challenges don't give the same information. introduction: peak expiratory flow rate (pfr) and forced expiratory volume in the first second (fev1) are currently used in functional evaluation of asthmatic patients but his degree of correlation in obstructive, restrictive and mixed spirometric ventilatory patterns are not well known. the purpose of this study was to evaluate if the correlation between pfr and fev1 2 markers of global airways obstruction is comparable in asthmatic patients with obstruction restriction or both. methods: 152 asthmatic patients, 69 males, 83 females have been studied by spirometry with a vitalograph compact in absence of bronchodilatory or anti-inflammatory therapy. patients have been classified in 4 functional patterns according to vc, and fev1/cv: normal : vc greater than 90% of expected value and fev1/vc greater than 70%, obstructive:vc greater than 90% and fev1/vc greater than 70% restrictive vc, less than 90% and fev1/vc greater than 70% and mixed vc less than 90% and fev1/vc less than 55% presenting a so called functional emphysema. results: according to these criteria 15 patients were normal, 44 restrictive, 15 obstructive and 78 mixed. in all the group the correlation between pfr and fev1 was statiscally significant (p<001) but the correlation coefficient variable. in functionally normal patients r =+0, 74 in restrictive patients + 0, 67 in obstructive + 0, 66 in mixed + 0, 84 and in the sub-group with functional emphysema + 0, 96. for this sub-group the correlation coefficient r allows to define fev1 = 91 + 5, 6 pfr. conclusions: the report between the two most usual assays of global bronchial obstruction depends on the balance between restriction, (either by interstial fibrosis or hyperinflation) and obstruction and is more close when a more marked obstruction coexist with a restrictive pattern (functional emphysema). in practice pfr has the same value that fev1 only in more severe cases of ventilatory failure. the dispersion of values and the standard error or regression do not allow to correlate both data in most patients. introduction: the tenor study longitudinally observes the natural history of subjects with severe or difficult-to-treat asthma. this analysis assessed the association between previous and future asthma exacerbations in tenor. methods: subjects eligible for this analysis were 12 years of age at baseline and had at least one follow-up assessment in year 1 and year 2. recent asthma exacerbations in the past 3 months were defined as: asthma-related emergency room (er) visits, nights of hospitalization, unscheduled physician office contacts; or as a composite measure of at least one of the previous exacerbation events. relative risks (rrs) and 95% confidence limits (95%ci) were generated comparing exacerbations in year 2 relative to year 1. exacerbation rates were defined as the number of events divided by total patient follow-up time. no adjustment was performed for baseline characteristics. results: 2826 subjects were eligible for this analysis. rrs in year 2 vs. year 1 were: 3.9 (95%ci: 3.3-4.6) for unscheduled office contacts, 4.1 (95%ci: 3.48-4.82) for the composite exacerbation measure, 10.2 (95%ci: 7.8-13.4) for er visits and 15.1 (95%ci: 9.8-23.3) for nights of hospitalization. over 37% of subjects with 5 or more exacerbations (composite measure) in year 1 had a similarly high rate in year 2 compared to 3% of patients without an event in year 1 who went on to have 5 or more exacerbations in year 2 (see table) . conclusion: tenor subjects who experienced an asthma exacerbation in year 1 were at statistically significantly increased risk of subsequent exacerbations the following year compared to subjects not experiencing an exacerbation in year 1. this increased risk was consistent for all asthma exacerbation outcomes and most elevated for hospitalizations due to asthma. assessment of recent asthmarelated healthcare use is a critical component of the clinical evaluation of subjects with severe or difficult-to-treat asthma. funded by genentech inc. and novartis pharmaceuticals corp. background: preliminary studies investigating yoga and breathwork for asthma have been promising. several randomized controlled trials have shown a benefit from yoga postures and/or breathing versus control, but the control in these cases involved no intervention other than usual care. this study advances the field by providing an active control. methods: a randomized, controlled, double-blind clinical trial was conducted from october 2001 to march 2003 to determine the effectiveness and feasibility of a yoga and breathwork intervention for improving clinical indices and quality of life for adult patients with mild to moderate asthma. random assignment was made to either a 4-week yoga intervention that included both postures and breathwork, or a stretching control condition. outcome measures were assessed at 4, 8, 12 and 16-weeks and included the mini asthma quality of life questionnaire (mini-aqlq), rescue inhaler use, spirometry, symptom diaries, and health care utilization. results: sixty-two participants were randomized into the intervention and control groups, and 45 completed the final follow-up measures. intention-to-treat analysis was performed. significant within-group differences in post-bronchodilator fev1 and morning symptom score were apparent in both intervention and control groups at 4 and 16 weeks; however, no significant differences between groups were observed on any outcome measures.conclusions: iyengar yoga conferred no appreciable benefit in mild to moderate asthma. circumstances under which yoga is of benefit in asthma management, if any, remain to be determined. in order to investigate asthma mortality trends for the u. s. hispanic population, i have collected and graphed data from the national center for health statistics. mortality data for the hispanic population have been available for the entire united states only since 1997 and readily available for asthma only since 1999. these data indicate decreases in deaths from asthma (j45-j46) from 320 in 1999 to 274 in 2001 with decreases in the non-hispanic population from 4324 in 1999 to 3976 in 2001. rates of death from asthma decreased for the hispanic population from 1.0 per 100, 000 in 1999 to 0.7 in 2001, while that for the non-hispanic population decreased from 1.84 to 1.6. rates for non-hispanic whites decreased from 1.5 to 1.4 in 2001. rates of death from asthma have been twice as high among hispanic women as men. rates of death from asthma for non-hispanic blacks have been more than twice as high as those for non-hispanic whites. (national center for health statistics data dictate racial terminology). data for people of hispanic origin include people of any race. these data are affected by both underreporting of hispanic origin on death certificates and undercoverage in the census and resultant population estimates for a net correction of 1.6%. thus, rates of death from asthma have been lower and have been decreasing faster among hispanics than the rest of the population in the united states. allergic rhinitis and asthma are both highly prevalent diseases and often coexist in patients. rhinitis symptoms have been shown to impact the lower airway. as such, the impact of rhinitis on asthma control may be the greatest in patients with the most severe rhinitis symptoms. therefore, this analysis was performed to determine the impact of baseline rhinitis severity on asthma control in patients with both asthma and allergic rhinitis who received either fluticasone propionate aqueous nasal spray (fpans) or montelukast (mon) added to fluticasone propionate/salmeterol 100/50 mcg (fsc). a total of 863 patients (>15 years) with persistent asthma and seasonal allergic rhinitis received fpans 200mcg qd, mon 10mg qd or placebo (pbo) in addition to fsc 100/50mcg bid for 4 weeks. at baseline, total nasal symptom scores (tnss) ranged from 44 to 400. the analysis was restricted to patients whose pre-enrollment asthma therapy did not include fsc. regardless of baseline rhinitis severity, in patients with both asthma and allergic rhinitis, mon added to fsc resulted in no additional improvements in overall asthma control compared with fsc alone. these data suggest that optimal treatment of the individual conditions should be the goal of treatment for patients with coexistent allergic rhinitis and asthma. (sam40066) introduction recent studies have demonstrated that parainfluenza and coronaviruses are as important triggers of asthma. parainfluenza viruses are one of the main triggers of virus-induced asthma at summer. parainfluenza viruses often cause severe low respiratory tract infections in immuncompromised patients and in patients with bone marrow transplantation. a retrospective study found that 41% of pediatric bmt patients with hpiv infection (47% of viral respiratory infections) developed pneumonia and 6% died. both viruses contributed to fatal asthma. materials and methods for amplification of these viruses, primers which anneal to specific regions of viral genomes: hn-gene for piv1, piv2 and piv3 and spike glycoprotein gene for coronavirus oc43 and nucleocapsid glycoprotein gene for coronavirus 229e were used. pcrdiagnostics was performed on nasal and throat swabs taken from children with atopic asthma exacerbation. results from 02/04 to 05/04 a total of 56 samples (including 28 negative controls) were tested by pcr. 11 positive for respiratory viruses samples noted in children having asthma exacerbations including 23.9% of all tested samples. piv3 was detected in 54.5% of all positive samples, piv2 in 18.2%, and coronavirus oc43 in 27.3%. piv2 and coronavirus 229e were not detected during this study. for negative controls samples were taken from children with atopic asthma in remission. all 10 controls were found to be negative by viral pcr. conclusions respiratory viruses are an important factor in asthma exacerbations in children. piv3 was the most frequently detected among the positive samples. although hpiv-2 was less often positive than hpiv-1 and hpiv-3 infections in this study, the frequency of the detected coronaviruses may have been influenced by the seasonal activity of these viruses. introduction. in budapest all children come under the regular supervision of a specially trained paediatrician. each paediatrician is responsible for an average of 950 children, often seen up to the age of 18 years. methods. a questionnaire was sent to the district paediatricians of budapest in 1995 budapest in , 1999 budapest in and 2003 . the total number of children in their practice and the number of the asthmatics was assessed. the diagnosis of asthma in every case was established in a paediatric hospital, or a special allergy or pulmonology outpatient clinic. results. in 1995, replies were received from 118 paeditricians, who were responsible for the supervision of 104, 087 children, of which 1.88 â± 0.87% had been diagnosed as having asthma. in 1999 replies were sent by 153 physicians, who had a total of 142, 684 children under their care, including 3, 228 asthmatics, a prevalence of 2.26 â± 0.95%. at the end of 2003, 151 paediatricians having 141, 276 children answered, noting 3, 831 asthmatics, a 2.71 â± 1.2% prevalence of asthma. conclusion. the current survey of prevalence of asthma in childhood is by far the largest that has been made. an increasing prevalence of the diagnosis of asthma based on clinical investigations was noted, the differences between the investigated years being highly significant. panel report guidelines for the diagnosis and management of asthma have been available since 1991, to standardize and improve the quality of asthma care. however, asthma remains the leading cause of hospitalization for new york city children. implementation of the guidelines has not been fully embraced in the primary care setting. we attempted to increase primary care practitioners' compliance with the guidelines through extensive training. a hospital based pediatric clinic and 4 school based health clinics in the brooklyn inner city, participated from april 2002 to march 2003 in the program as part of new york city's education and quality improvement project (equip). initially an asthma physician specialist and asthma educator trained the primary care physicians and nurse practitioners in the guidelines. the training was reinforced during monthly luncheon sessions. clinical exam rooms were also supplied with asthma education materials, peak flow meters, and asthma action plans. goals were to identify asthmatics, document asthma severity, give persistent asthmatics written management plans, and utilize appropriate controller medications. compliance was measured by tracking asthma visits with an asthma intake form. prior to this project no consistent attempt had been made in the clinics to regularly classify asthmatic severity, provide written asthma action plans or to place patients on controller medications. after onset of the project a total of 216 asthma visits were evaluated; 104 were from school based health centers and 113 from hospital based pediatric clinic. severity was classified in 83%(179/216) of asthma visits. patients classified as persistent received updated written management plan during 90%(100/110) of asthma visits and 92%(102/110) were placed on controller medications. this asthma management program demonstrated success in improving primary care practitioners' compliance with the naepp asthma guidelines. further studies are needed to determine if compliance with the guidelines persisted after cessation of the extensive training and if such compliance improves asthma outcomes. numerous studies have associated asthma and obesity. although obesity has been identified as a risk factor for asthma, there is limited information on the impact of obesity on childhood asthma. this study examines the effect of body composition on asthma severity and pulmonary function in children. we retrospectively reviewed charts from asthmatic children ages 5 to 18 years, referred to a community-based pediatric pulmonology practice between 1999 and 2003. data were included if asthma was the the primary diagnosis and a baseline spirometry was available. asthma severity was classified and body mass index(bmi) was classified as normal, overweight and obese. spirometry results were reviewed. eighty-five patients were reviewed. thirtyone(36.5%) patients were normal weight; 21(24.7%) were overweight and 33(38.8%) were obese. baseline characteristics such as age, gender and race/ethnicity were similar. asthma severity classification did not differ between normal, overweight and obese children. overall, 34% were classified as intermittent, 53% as persistent mild asthma and 13% as persistent moderate-severe asthma. 21% of children with intermittent asthma were receiving controller therapy compared to 62.5% with persistent mild and 81.8% with moderate-severe asthma(p=0.0001). although healthcare utilization for asthma(emergency room visits/admissions) were significantly different between children with intermittent, persistent mild and moderate-severe asthma(p=0.0004), there was no difference when normal, overweight and obese children were compared. spirometry results showed comparable peak expiratory flow rates(pfr) in all groups; 75% of predicted in normal weight and 79% of predicted in both overweight and obese. mean forced vital capacity(fvc) was 93.9%, 88.5% and 95% and the mean forced expiratory volume in 1 second(fev1) was 85.9%, 81.2% and 86.6% respectively. there was no difference in fev1/fvc; 82.3%, 83.1% and 81.8% and forced expiratory flow in mid-lung volumes (fef25-75); 74.4%, 73% and 76.9% of predicted. in this study we retrospectively reviewed asthmatic children and compared bmi, asthma severity and spirometry. we found no difference in asthma severity classification between normal and overweight/obese patients. regardless of the bmi, all indices of spirometry were similar. further studies are needed to examine the impact of obesity on different asthma related disease outcomes. our objective is to further investigate the effect of budesonide, formoterol, and levalbuterol, alone, and in combination, on tgf-1 production and expression in ragweed (ra) stimulated a549 cells. meth-ods: a549 cells were cultured in duplicate for 24 hours at 37 degrees celcius with 5% co 2 in serum free dmem with or without the following: 10î¼g/ml of ra, 1.0 x 10 -7 m of budesonide, 1.3 x 10 -7 m formoterol, and 1.0 x 10 -5 m levalbuterol. tgf-1 production and expression was measured by a sensitive elisa and mrna assay in cell supernatants and pellets. results: tgf-1 production and expression from a549 cells rose markedly in the presence of ra (952.35 -1268 pg/ml) (59 -71 amol/ml) with significant inhibition following the addition of budesonide, and the combinations of budesonide and formoterol, budesonide and levalbuterol, and budesonide, formoterol, and levalbuterol (p<0.05). combined treatment with budesonide and levalbuterol versus budesonide alone reached significance for production and expression (p<0.05). budesonide and formoterol compared with budesonide alone reached significance for tgf-1 production (p<0.05). conclusion: combination therapy with budesonide and a long or short acting beta-agonist showed greater effect of tgf-1 inhibition compared with budesonide alone. this synergistic effect on the distal airways may prevent a subset of asthmatics from developing airway remodeling. g. tamura 1* , y. sano 2 , k. hirata 3 , s. ishioka 4 , m. nakashima 5 , t. miyamoto 2 , 1. sendai, japan; 2. tokyo, japan; 3. osaka, japan; 4. hiroshima, japan; 5. hamamatsu, japan. tulobuterol tape is the world's first long-acting transdermal preparation of a beta2-agonist designed to release tulobuterol in an optimal fashion. when it is applied once daily at bedtime, the blood concentration of tulobuterol peaks early in the morning and is kept at effective levels for 24 hours. tulobuterol tape has milder and less frequent adverse events than conventional oral tulobuterol preparations, and when used at bedtime can prevent marked drop in peak expiratory flow (pef) early in the morning. consequently, tulobuterol tape has been commonly used in japan as a long-acting beta2-agonist. in the present study, to evaluate its efficacy as a long-acting beta2-agonist and to compare its effects at two different doses, we administered tulobuterol tape at doses of 1 or 2 mg/day to patients with persistent asthma already using inhaled corticosteroids. this study was a randomized, double-blind, double-dummy, parallel-group, multicenter trial of 4-week duration. mean pef in the 1 and 2 mg/day groups were significantly increased from the baseline value by 15.1 and 28.2 at week 1, 19.5 and 32.6 at week 2, 22.6 and 35.3 at week 3 and 23.8 and 35.9 l/min at week 4, respectively. the increase in mean morning pef in the 2 mg/day group was significantly higher than that in the 1 mg/day group at every point of determination. the mean evening pef was significantly increased in both treatments groups compared with baseline values at every point of determination. although the increase in the 2 mg/day group was greater than that in the 1 mg/day group at each point of determination, the difference between groups was statistically significant only at week 1. the safety profiles of the two treatments were similar. in patients with persistent asthma who require inhaled short-acting beta2-agonists while receiving inhaled corticosteroids, tulobuterol tape 2 mg/day significantly improved pef compared with tulobuterol tape 1 mg/day. introduction: on an average, 4% of the 1500 children admitted annually for asthma to children s hospital require admission to the pediatric intensive care unit (picu). these admissions are highest in the months of august, september and october. aeroallergens such as alternaria and ragweed predominate during this season, and allergies to these allergens may account for the increased number of picu admissions for asthma. objective: we hypothesized that the seasonal increase in the admission rate could be related to allergen sensitivity, particularly to alternaria and/or to ragweed that predominate during late summer and fall. method: data was collected from a retrospective chart review of all patients admitted to the picu for asthma exacerbation between 1997 and 2003. skin prick testing (spt) within one year of picu admission was the criteria for inclusion in this study. results: there was a higher prevalence of admissions to the picu for asthma exacerbation during august, september and october (168 out of 416 admissions = 40% of all admissions, p<0.001). sixty-one subjects met the one-year spt criteria for analysis. subjects admitted during august, september and october were categorized as group a, and all others were categorized as group b. sensitivity to a total of 11 aeroallergens (tress, grasses, weeds, ragweed, outdoor molds, indoor molds, alternaria, cat, dog, roach and dust mites) was compared between the two groups. there was no statistical significance in aeroallergen sensitivity to alternaria or ragweed between subjects admitted to the picu in august, september and october (group a) and subjects admitted during the other months of the year (group b). conclusion: asthma exacerbation requiring picu admissions is more prevalent during the months of august, september and october. the increased rated of picu admissions did not correlate with the aeroallergen sensitivity to the prevalent allergens during august, september and october. therefore we suspect other factors such as, viral upper respiratory infections, in addition to aeroallergen sensitivity, which may contribute to severe asthma exacerbation during the late summer and fall months. k. kuzume * , shigenobu, ehime, japan. infantile wheezing may be the result of different phenotypes. the aim of this study was to evaluate the risk factors for allergies and the results of allergyrelated blood tests among infants with different types of allergy symptoms. 2130 infants were examined physically at birth and at 1, 3, 6, 9, and 12 months of age, and questionnaires about number of siblings, the family history of allergy, and feeding methods were given. allergic diseases were diagnosed at 12 months of age, and the subjects were divided into 4 groups: infants with atopic dermatitis (ad) only (a group, n=447), infants with both ad and wheezing (a/w group, n=175), infants with wheezing only (w group, n=155), and infants without allergic symptoms (c group, n=1353). risk factors were then evaluated. 332 patients (198 from the a group, 109 from the a/w group, and 25 from the w group) were given allergy-related blood tests, total serum ige levels, and rast with egg white, milk, and house dust mites at 6 and 12 months of age. the results of the blood tests in 36 control subjects at 6 months of age were compared to the other groups. as shown in table1, there were more male infants in the a/w and a groups than in the c group (p<0.001, a/w vs. c; p<0.01, a vs. c). numbers of siblings were greater in the w and a/w group than in the a or c group (p<0.0001, w vs. a and w vs. c; p<0.01, a/w vs. a; p<0.05, a/w vs. c). the rate of positive family history of allergic diseases was high in the a and a/w groups, compared to the w or c group (p<0.0001, a vs. c and a/w vs. c; p<0.05, a/w vs. w). the rate of breastfeeding at 3 months of age was high in the a group compared to the others (p<0.0001, a vs. c). patients in the w group had lower levels of total serum ige at 12 months of age, compared to patients in a or a/w (p<0.01, w vs. a/w and w vs. a). also patients in the w group had lower levels of rast with egg white at 12 months of age, compared to patients a or a/w (p<0.0001, w vs. a/w and w vs. a). the median of total serum ige levels at 6 months of age was higher in a and a/w but not in w, compared to c (p<0.001, a/w vs. c and a vs. c). in conclusion, there were two different phenotypes of wheezing in infants before 12 months of age. wheezing with ad might be "allergy-related", while wheezing without ad may be related to other factors. as compared to c group: a), p<0.05; b), p<0.01; c), p<0.001; d), p<0.0001 as compared to a group: 1), p<0.05; 2), p<0.01; 3), p<0.001; 4), p<0.0001 as compared to a/w group: f), p<0.05; g), p<0.01; h), p<0.0001 a group, infants with atopic dermatitis only; a/w group, infants with atopic dermatitis and wheezing; w group, infants with wheezing only; c group, infants without allergic symptoms. rast, radioallergosorbent test, n/a, data not available r. amelio 1* , c. capristo 1 , a. capasso 1 , m. miraglia del giudice 2 , n. maiello 1 , f. decimo 1 , 1. naples, italy; 2. napoli, italy. forced oscillation technique (fot) is considered a sensible and specific method to estimate breathing functionality in asthmatic children aged 3 to 6 years, because it doesn't need special collaboration. the aim of our study was to value baseline respiratory resistances with the fot in preschool-aged children with asthma under high dose of budesonide and flunisolide treatment. at this purpose, 40 asthmatic children aged 3 to 6 years were selected: at a first examination (t0) all the patients showed basal value fot at 6hz frequency (rr6) changes major or equal than 35% under 200 mcg of salbutamol treatment. 30 days before the study, children selected didn't take systemic and inhaled steroids, cromons, leukotrienes antagonists, teophylline or anti-histamines and they didn't present upper and lower airways infections; then, two weeks before run-in, all selected patients took beta-2-agonists at least 3 times per week. all the patients were divided in two groups: group i (20 children) under inhalation of flunisolide 40mcg/kg/dose + salbutamol 1500 mcg b.i.d. for 7 days and, for the following two weeks, under inhalation of flunisolide 20 mcg/kg/dose + salbutamol 200 mcg by pmdi+spacer prn.; group ii (20 children) under inhalation of budesonide 0, 5 mg b.i.d. + salbutamol 1500 mcg b.i.d. for 7 days, and for the following two weeks, under inhalation of budesonide 0, 25 mg b.i.d. + salbutamol 200 mcg by pmdi+spacer prn. moreover, we gave diary-card with a simple symptoms score (at t0 and t7 ), to get the real perception of the symptoms in the course of study. however, during the treatment, 4 children have ritired from the study. the results obtained were statistically analysed (t student's test). significant rr6 pre beta-2-agonists and ? value reduction was obtained at t7 and t21 vs. t0. group i had (at t7) such a quick action than group ii reducing airways resistances (p<0, 01). children treated with flunisolide had lower frequency of breathless and nocturnal cough at t7 and lower salbutamol use from t7 to t21 than budesonide group (p=n.s.). in conclusion, fot is actually a safety method to value efficacy of inhaled steroids in preschool-aged children with asthma. introduction: approximately 7.5% of u.s. adults and 6.0% of louisiana (la) adults have current asthma, and over 25% currently smoke cigarettes, according to the 2002 behavioral risk factor surveillance system (brfss). the purpose of this study is to compare asthmatic smokers vs. nonsmokers in la and the u.s. here we address whether asthmatic smokers utilize more medical care services in the er and outpatient clinics, and whether they were able to fully participate in activities of daily living compared to nonsmoker asthmatics. methods: brfss is a state-based, telephone survey of u.s. adults. the survey collects self-reported information about modifiable risk factors for chronic diseases. this study analyzed data from the 2002 brfss survey using sas software. data: out of the current asthmatics, 25% are cigarette smokers in the u.s. in louisiana, 31% of asthmatics are currently smoking, which is significantly higher than the national average (p<0.0001). within the past year, the average number of emergency room visits due to asthma was similar for both locations (la 0.53, us 0.48, p=0.46) . nationally, smokers visited the er significantly more frequently than nonsmokers (s 0.61, ns 0.43, p<0.01). in la, smokers visited the er twice as often as nonsmokers (s 0.80, ns 0.40, p<0 .01) the number of annual routine outpatient checkups for asthma was similar based on location (la 1.39, us 1.66, p=0.06). smokers and nonsmokers had a similar number of checkups nationally (s 1.74, ns 1.64, p=0.32) and in louisiana (s 1.79, ns 1.21, p=0.06). the number of days where asthmatics were unable to perform their usual activities was significantly fewer in la than nationally (la 3.6, us 11.6, p<0.001). smokers had a significantly higher number of missed days nationally (s 17.8, ns 9.6, p<0.0001). in la, there was not a significant difference in the number of missed days based on smoking status (s 4.9, ns 2.9, p=0.47). conclusion: even though louisiana has a lower prevalence of asthmatics compared to the national average, significantly more of our asthmatics are smokers. asthmatic smokers in la and the u.s. utilized the er more than their nonsmoking counterparts. smokers and nonsmokers had similar numbers of routine outpatient visits. asthmatic smokers missed more days, but this was only significantly different at the national level. other research has shown that young age is highly associated with improper use in children using a mdi and holding chamber (arch pediatr adolesc med 2002; 156:378) . the objective of this study was to compare health outcomes achieved by children with asthma using inhaled corticosteroids (icss) delivered by a nebulizer compared with outcomes of children using icss delivered by non-nebulized device. methods: using a managed care organization database (pharmetrics integrated outcomes database), we identified children aged 8 years with an asthma diagnosis and asthmarelated hospitalization/emergency department (ed) visit (july 2000 -june 2002 and with a prescription claim for an ics within 30 days of discharge. we compared relative risk of hospitalization/ed recurrence from day 31-180 (cox proportional hazards regression, covariates=sex, age, current and prior asthma medications, prior oral corticosteroid and short-acting 2 -adrenergic agonist use, initial type of index event) for patients receiving nebulized ics vs other ics by age groups (0-4 years and 5-8 years). results: of 1552 patients with claims for ics, 729 received nebulized ics, of which 480 were aged 4 years; 823 received non-nebulized ics, of which 292 were aged 4 years. postindex hospitalization/ed rates were 12.4%. refill rate was higher for patients using nebulized ics. after model risk adjustment, patients using nebulized ics had a 53% risk reduction for hospitalization/ed recurrence vs those not using nebulized ics (hr: 0.47, 95% ci: 0.28, 0.78). in the 0-to 4-year age group, patients on nebulized ics had a 62% risk reduction compared with patients not using nebulized ics (hr: 0.38, 95% ci: 0.21, 0.69). in the 5-to 8-year age group, patients on nebulized ics had a 52% risk reduction (hr: 0.48, 95% ci: 0.16, 1.46). conclusion: in actual practice, treatment with nebulized ics after an asthma exacerbation is associated with a significant reduction of recurrent hospitalization/ed visits in young children, possibly due to improved technique and compliance. introduction: tenor is a 3-year, multi-center, cohort study of patients with severe or difficult-to-treat asthma. the objective of the tenor study is to better understand the natural history of patients with severe or difficult-totreat asthma. this analysis assessed the frequency of skin testing in this population and characterized the differences between the positive (st+) and negative (st ) subjects. methods: subjects were asked whether they had ever been skin tested and, if so, the test results. those who were st+ were compared to both st and those never tested (stnd) using clinical and other asthma-related characteristics. subjects 12 years of age were included in this analysis. results: 2985 subjects were eligible for this analysis. 85.8% were skin tested in the past, with stnd frequency of 5.1% from allergist sites and 33% from pulmonologist/other sites. of those tested, 93.5% were positive (allergist 95.7%, pulmonologist/other 87.3%). baseline ige for st+ subjects was 104.6 iu/ml vs. 32.4 iu/ml for st ; p<0.0001 (table) . as shown, the age at asthma onset, duration of asthma, rate of atopic disorders, and the rate at which asthma was triggered by aeroallergens differentiated the st+ from the st group. disease severity as evaluated by fev 1 , healthcare utilization, and medication use, however, was similar between the two groups. in general, stnd were more likely to have values closer to the st+ group, suggesting that the majority of those not tested would have been st+, if administered a test. conclusions: the prevalence of st+ subjects from both allergy and pulmonary practices was high, demonstrating that the majority of these severe or difficult-to-treat patients have allergic asthma. st+ patients showed differences in clinical characteristics compared to st , including a greater likelihood of their asthma symptoms being triggered by aeroallergens. these data also show that the clinical profile of stnd patients may be similar to that of st+ patients, suggesting the utility of a more universal allergic evaluation in severe asthmatics. funded by genentech and novartis pharmaceuticals corp. background current national guidelines for the diagnosis and management of asthma such as the naepp epr 2002, classify asthma severity based on frequency of asthma symptoms, medication use, and measurements of lung function by spirometry or peak expiratory flow variability. recently, the utility of spirometry measurements for assigning asthma severity has come into question. here we evaluate the correlation of spirometry measurements with asthma severity in school-aged children who are mostly naive to anti-inflammatory therapy. methods children were participants in a school-based lowincome asthma mobile van program, the breathmobile. recruitment was through referrals by school nurses and community public health clinics, parental response to flyers, and asthma screening questionnaires. spirometry was performed on all children greater than 4 years of age as part of a comprehensive asthma evaluation. asthma severity was assigned based on symptoms only, according to the naepp epr 2002 . results from april 2002 to april 2004 of the 620 children evaluated on the breathmobile, 540 patients were diagnosed with asthma. classification of severity by symptom frequency according to naepp epr guidelines resulted in 28% as mild intermittent (mi), 28% as mild persistent (mip), 32% as moderate persistent (mop), and 12% as severe persistent (sp). the percentage of patients and their mean lung functions at baseline evaluation without bronchodilator challenge are shown in the table. there were statistically significant differences (p<.05) between severity groups which was most pronounced for the fef25-75% when comparing the severe persistent group and the intermittent group. however, mean values appear to be "normal" for all degrees of asthma given current accepted cut-off values, fvc 80%, fev1 80%, fev1/fvc 80%, and fef25-75 60%. conclusion in our study, airflow impairment did correlate with asthma severity, although it was "normal" (fev1 80%) even in children with severe persistent asthma. therefore, a "normal" lung function measurement may be misleading as sole criteria for asthma severity classification. perhaps a higher cutoff point (fev1<90%) might be a better indicator of asthma severity in children. asthma is the most frequently chronic disease in childhood.our main was to know the prevalence of bronchial asthma in children (6-7 and 13-14 years) in the north zone of mexico city and to compare the prevalence obtained by the written questionnaire versus the video questionnaire in the group of 13 to 14 years, according to methodology proposed by isaac. material and methods: by means of a validated and standardized questionnaire (isaac) that was applied to 3500 children from 6 to 7 years and 3899 from 13 to 14. the questionnaires of 6 and 7 years were filled out by their parents. the group from 13 to 14 years answered a questionnaire and a video questionnaire. according to isaac specification and based on a studied population, it was calculated by statcal program the size of the sample. it was choose a haz-ardous sample of 3000 children between 6-7 years and 3000 of the 13-14 group years, both sexes in elementary and high school. measures of central tendency and chi2 were used. results: the final sample size for both groups was 7110 children (3211 of 6-7 and 3899 adolescents), 51.5% men and 48.5% women. the prevalence of the asthma diagnosis for teenagers was of 8 % (p<0.05 ic 7.1-8.7) and it was 4.5% in children (p<0.05 ic 3.8-5.2), wheezing in the last 12 months was 6.8% (p<0.05 ic 5.9-7.6) in children and of 9.9% (p<0.05 ic 9-10.8) for 13-14 years. wheezing induced exercise appeared in 13.1% (p<0.05 ic 12-14.1) in teenager and 3.4 % (p<0.05 ic 2.7-4) in the other group. the severity of asthma showed by nocturnal awakness was 4.3% and 2.6% (p<0.05), number of crisis in the last year 6.3% and 9.3% for children and teenagers respectively (p<0.05). in the video questionnaire of teenagers 7.3% (ic 6.5-8.1) answered affirmative on have displayed a shaken breathing on the last year resting and only 6.8% (ic 6-7.6) in the last month. about exercise question 16.5% confirmed have displayed symptoms, 14.6%(ic 13-15.8) in the last year and 11.1% (ic 10.1-12.1) in the last month. wide-awake at night 7.5% (ic 6.7-8.3) in the last year and 5.2% ) in the last month. conclusions: the prevalence of asthma by diagnosis was higher in teenagers group than in children group. the prevalence of asthma was higher also in this group. rationale: we investigated the relationship of pet exposure and healthcare utilization (hcu) in a cohort of pediatric patients with severe or difficult-totreat asthma in the epidemiology and natural history of asthma: outcomes and treatment regimens (tenor) observational study. we hypothesized that pet exposure would increase hcu due to increased airway inflammation. methods: tenor is a 3-year multicenter cohort study of patients with severe or difficult-to-treat asthma. children ages 6-12 were interviewed at baseline regarding asthma-related hcu in the previous 3 months. patients with pet exposure (ppe, n=370) were compared with patients with no pet exposure (pnpe, n=382). responses were analyzed using fisher's exact test. results: ppe were less likely to have severe asthma by physician assessment (32% vs. 40%; p=0.01). in addition, ppe were less likely to have an emergency room (er) visit (18% vs. 26%; p=0.006) or to have been hospitalized (7% vs. 12%; p=0.02) in the previous 3 months. more pnpe had an ige level >100 iu/ml (74% vs. 63%; p=0.001). ppe and pnpe were similar with regard to presence of allergic rhinitis and skin test positivity. compared with pnpe, fewer ppe with two or more pets had er visits and hospitalizations (table) . among ppe, there were no differences in ige levels or in asthma severity related to the number of pets. conclusions: pet exposure was associated with reduced hcu in the tenor pediatric cohort, with the most pronounced effect seen in reduced er visits and hospitalizations needed by those with multiple pets. ppe had significantly lower ige levels compared to pnpe, regardless of the number of pets. these data support the hypothesis that exposure to pets may paradoxically protect individuals from the development of severe asthma. alternatively, these data may reflect a self-selection in patients with severe or allergic asthma who are likely to avoid having a pet. however, there may be other confounding factors in families with pets that confer a protective effect on asthma severity. introduction: in asthma and other chronic obstructive airway diseases, phosphodiesterase 4 (pde4) is involved in the pathophysiology of the disease and is expressed abundantly in key inflammatory cells. inhibitors of pde4 are investigational, anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde4 inhibitor with demonstrated in vitro and in vivo anti-inflammatory activity, which may translate into clinical efficacy in asthma therapy. this study examined the ability of roflumilast to exert direct or acute bronchodilatory activity in patients with mild to moderate asthma. methods: this was a double-blind, randomized, placebo-controlled, crossover study consisting of three treatment periods of one day each, separated by a 7-to 14-day washout period. during each treatment period, 15 patients with a forced expiratory volume in one second (fev 1 ) of 50% to 90% of predicted received either oral roflumilast 1000 î¼g, roflumilast 500 î¼g, or placebo. the fev 1 was measured twice prior to administration and periodically up to 6 h following treatment. after 6 h, patients inhaled 200 î¼g salbutamol from a metered-dose inhaler; fev 1 was measured 15 min later. adverse events, vital signs, and electrocardiogram results were monitored throughout the study. results: median baseline fev 1 was similar for the three treatment periods. both doses of roflumilast did not lead to statistically significant differences versus placebo in the time-averaged fev 1 over the first or up to 6 h after drug intake (p > 0.05). thus, there was no evidence of a direct or acute bronchodilatory effect with either single doses of roflumilast 1000 î¼g or 500 î¼g. in contrast, inhalation of the short-acting bronchodilator salbutamol led to a distinct improvement in fev 1 versus baseline in all treatments groups with median fev 1 increases of 19%, 21%, and 14% in the roflumilast 1000 î¼g, roflumilast 500 î¼g, and placebo groups, respectively. roflumilast was well tolerated. conclusions: oral, once-daily roflumilast exhibits no direct or acute bronchodilatory activity in patients with mild to moderate asthma as could be achieved with short-acting 2 -agonists. these data support the proposed mechanism of action of roflumilast as an antiinflammatory agent. south africa; 2. guadalajara, spain; 3. munich, germany; 4. weinheim, germany; 5. budapest, hungary; 6. konstanz, germany. introduction: phosphodiesterase 4 (pde4) is found in key inflammatory cells involved in the pathophysiology of chronic obstructive pulmonary disease and asthma. inhibitors of the pde4 enzyme are anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, oncedaily pde4 inhibitor with demonstrated in vitro and in vivo anti-inflammatory activity. this study examined the dose-related efficacy of roflumilast in patients with asthma. methods: patients with stable asthma (forced expiratory volume in 1 second [fev 1 ] 50% to 85% of predicted) were enrolled in this randomized, double-blind, dose-range finding study. after a single-blind placebo run-in period of up to three weeks, patients received oral roflumilast 100 î¼g, 250 î¼g, or 500 î¼g once daily (n = 229, 228, and 233, respectively) for 12 weeks. efficacy was assessed by change from baseline in spirometric lung function fev 1 and forced vital capacity (fvc), as well as morning and evening peak expiratory flow (pef) recorded in patient diaries. safety and tolerability parameters were monitored throughout the study. results: treatment with roflumilast led to dose-dependent and statistically significant increases in fev 1 , fvc (both p < 0.0001), and in morning pef (p < 0.01). at last visit, fev 1 improved from baseline by 260 ml (11%), 320 ml (13%), and 400 ml (16%) in patients treated with roflumilast 100 î¼g, 250 î¼g, and 500 î¼g once daily, respectively. similarly, dose-dependent improvements of 10 l/min, 12 l/min, and 20 l/min in morning pef from baseline were reached. roflumilast was well tolerated at all dose levels tested. the most frequent drug-related adverse events were headache followed by gastrointestinal disorders such as diarrhea and nausea. there were no clinically relevant changes in vital signs, electrocardiogram, or laboratory parameters. conclusions: oral, once-daily roflumilast was associated with dose-dependent, clinically relevant improvements of lung function in patients with stable asthma. roflumilast was well tolerated. j.l. izquierdo 1* , e.d. bateman 2 , p. magyar 3 , u. harnest 4 , p. hofbauer 5 , a. varga 6 , c. schmid-wirlitsch 7 , d. bredenbroeker 7 , t.d. bethke 7 , 1. guadalajara, spain; 2. cape town, south africa; 3. budapest, hungary; 4. munich, germany; 5. weinheim, germany; 6. tatabanya, hungary; 7. konstanz, germany. introduction: phosphodiesterase 4 (pde4) inhibitors are a new class of anti-inflammatory agents for therapeutic use in inflammatory airway diseases. in several studies, the investigational, oral, once-daily pde4 inhibitor roflumilast has provided clinically meaningful improvements in patients with chronic obstructive pulmonary disease and asthma. this study examined the long-term safety and tolerability of roflumilast over 40 weeks in patients with asthma. methods: patients with persistent, stable asthma (fev 1 50% to 85% of predicted at randomization) participated in a double-blind, randomized dose-range finding study and were treated with oral roflumilast 100 î¼g, 250 î¼g, or 500 î¼g once daily for 12 weeks. patients completing the 12-week study per-protocol, then continued treatment in this open-label 40-week extension study. all patients received oral roflumilast 500 î¼g once daily during the extension period. adverse events (aes), clinical laboratory parameters, vital annals of allergy, asthma & immunology signs, and ecg were assessed throughout the extension period. results: a total of 456 patients were enrolled in the 40-week extension study. overall, the most common aes were related to the respiratory system. only a small number of patients (6%) experienced aes that were assessed as at least likely related to study medication. the most frequent drug-related adverse events were headache, diarrhea, and nausea as reported by 3%, 1%, and 1% of patients, respectively. most aes were mild to moderate in intensity and transient in duration; 6% of patients discontinued the study due to aes. most (60%) of these aes leading to discontinuation were assessed as not related or unlikely related to study drug. out of 456 patients, 16 reported serious aes, which were all assessed as not related or unlikely related to roflumilast. no clinically relevant changes in clinical laboratory parameters, vital signs, ecg, or physical examination occurred. conclusions: oral, once-daily roflumilast 500 î¼g administered for 40 weeks was well tolerated in patients with persistent, stable asthma. this study provides evidence that roflumilast is associated with a low incidence of aes, which are mostly mild to moderate in intensity and transient in nature, thus, supporting the potential therapeutic use of roflumilast in asthma. paris, france; 3. madrid, spain; 4. guadalajara, spain; 5. harrow, united kingdom; 6. weinheim, germany; 7. augsburg, germany; 8. munich, germany; 9. kaufbeuren, germany; 10. konstanz, germany. introduction: in asthma, inhaled corticosteroids (ics) have been the mainstay of maintenance treatment. new therapeutic agents are needed that target key inflammatory processes in asthma as effectively as ics but overcome known limitations of ics. phosphodiesterase 4 (pde4) inhibitors are anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde4 inhibitor for potential use in anti-inflammatory asthma therapy. this study compared the standard corticosteroid treatment of twice-daily, inhaled beclomethasone dipropionate (bdp) with oral, once-daily roflumilast in patients with asthma. methods: in a randomized, double-blind, double-dummy study, patients (forced expiratory volume in one second [fev 1 ], 50% to 85% of predicted) received either oral roflumilast 500î¼g once daily (n=207) or inhaled bdp 200î¼g twice daily (400î¼g/day; n=214) for 12 weeks. mean change (lsmean and sem) in lung function parameters fev 1 , forced vital capacity (fvc), and morning and evening peak expiratory flow (pef) from baseline was determined after 12 weeks of treatment. further, symptom score and rescue medication use were assessed. results: both roflumilast 500î¼g and bdp 400î¼g improved fev 1 from baseline to clinically meaningful levels (300 â± 40ml and 370 â± 30ml, respectively; both p<0.0001). similarly, roflumilast and bdp improved fvc (300 â± 40ml and 360 â± 40ml, respectively; both p<0.0001) as well as morning and evening pef (p 0.0003). roflumilast 500î¼g was statistically non-inferior to bdp 400î¼g. roflumilast and bdp led to statistically significant and comparable improvements in asthma symptoms and use of rescue medication. adverse events were generally mild to moderate; the most common drugrelated adverse events reported in the roflumilast group were nausea, headache, and diarrhea. there were no clinically relevant changes in vital signs, ecg, or laboratory parameters. conclusions: roflumilast 500î¼g provided clinically meaningful improvement of lung function parameters, reduced asthma symptoms, and decreased the need for rescue medication. oral, once-daily roflumilast was as effective as inhaled, twice-daily bdp in the treatment of asthma. oral roflumilast 500î¼g was well tolerated. rationale: evaluation of medical claims from commercial health plans permits assessment of therapeutic interventions on resource utilization. this study investigated the impact of controller therapy in children starting asthma maintenance medications. the risk of obtaining prescriptions for oral corticosteroids (ocs), short-acting beta-agonists (saba) or adding another asthma therapy was evaluated. methods: this was a retrospective observational study utilizing medical and pharmacy claims from a large representative managed care plan from 1/1/2000-5/31/2003. children aged 4-17 years old with an asthma diagnosis (icd-9, 493.xx) and an initial pharmacy claim for one of the following regimens: fluticasone/salmeterol in a single inhaler, n=1, 168 (fsc), fluticasone propionate alone, n=2, 473 (fp), montelukast, n=4, 246 (mon), any inhaled corticosteroid plus montelukast, n=1, 009 (ics+mon), and an ics plus salmeterol from separate inhalers, n=296 (ics+sal) were included in the analysis. subjects were excluded if they had received any asthma controller medication in the 6 months prior to the initiation of therapy. regression was used to estimate the incidence rate ratio (irr) for ocs and saba use (negative binomial) and the odds ratio (or) of adding a controller (add) and an ed or hospitalization (ip) event (logistic). all models controlled for demographics, pre-controller asthma-related medications and events, and baseline total health care costs. results: the ratios in the table with a confidence interval excluding unity indicate significantly increased use or likelihood of an event compared to the fsc cohort. controller naã¯ve children started on fsc were less likely to add another controller than mon, fp or ics+sal, and had less use of ocs or saba than the 4 cohorts when studied for 12 months after the initiation of controller therapy. in addition, children treated with ics + mon had a significantly greater or of an ed/ip visit compared to fsc. conclusion: use of fp + sal (fsc) in a single inhaler assures that children are getting more optimal inhaled corticosteroid therapy as well as the benefits from the long acting bronchodilator while avoiding the potential for selective discontinuation of ics in the 2 multiple controller cohorts. a.s. nayak 1* , r. nathan 2 , j. williams 3 , s. kundu 3 , m. lloyd 3 , d. banerji 3 , 1. normal, il; 2. colorado springs, co; 3. bridgewater, nj. introduction: inhaled corticosteroids (ics) are recommended firstline therapy for severe, persistent asthma. systemic exposure to ics may suppress hypothalamic-pituitary-adrenal (hpa)-axis function, particularly at doses required to control severe asthma. ciclesonide (cic) hydrofluoroalkane (hfa) metered dose inhaler (mdi) is a novel and effective ics that is converted in the lungs to its active metabolite, desisobutyryl ciclesonide (des-cic). previously, in short-term studies, cic has been shown to have no effect on serum or urinary cortisol levels, which may be attributed to the low oral bioavailability, high serum protein binding and high clearance rate of cic and its active metabolite. this hpa axis analysis was part of a long-term study evaluating the safety of cic and beclomethasone dipropionate (bdp) hfa-mdi in patients with severe persistent asthma. methods: this was a multicenter, double-blind, parallel-group, 12-month extension of a 12-week double-blind trial of patients 12 years with severe persistent asthma. patients were randomized in a 2:1 ratio (cic:bdp) to receive cic (320 g bid) exactuator or bdp (320 g bid) ex-actuator. after 2 weeks, the doses of both medications could be titrated to 160 g bid as needed for asthma control. hpa-axis function was assessed at selected centers in a subset of patients at baseline and end of study (at month 12 or early termination) by measuring both peak serum cortisol induced by low dose (1-î¼g) cosyntropin and 24-hr urinary free cortisol corrected for creatinine. results: data were available for a small number of patients at 13 centers evaluating hpa-axis (table) . mean baseline levels and change from baseline to end of study values in low-dose cosyntropin peak serum cortisol levels for cic and bdp were comparable. likewise, baseline levels and mean change from baseline to end of study values in 24-hr urinary free cortisol corrected for creatinine were comparable among the two treatment groups. conclusion: these findings demonstrate that cic 320 to 640 î¼g daily for 12 months is safe and has no significant effect on hpa-axis function. a. long 1* , a. rahman 2 , 1. boston, ma; 2. wilmington, de. introduction: little information is available regarding the relationships between asthma severity, disease control, and compliance with medications. methods: physicians' perceptions regarding these variables were determined by internet-based general asthma medication usage survey and patient-specific asthma medication usage surveys between december 18 and 29, 2003, for 406 physicians (100 pcps, 106 pediatricians, 100 allergists and immunologists, and 100 pulmonologists). the patient-specific surveys were based on chart information of 3 randomly chosen patients per physician on controller medication. results: of 1218 patients, 12%, 31%, 43%, and 14% were categorized by their physicians as having mild intermittent or mild, moderate, or severe persistent asthma, respectively. overall, 45% of patients were categorized as having "very controlled" asthma and 58% as being "very compliant" with their current regimen. worse asthma control, decreased compliance, and increased physician visits were all associated with increased asthma severity classification. physicians reported that only 5% of patients with severe persistent disease (n=169) had "very controlled" asthma in contrast to 77% of patients with mild intermittent asthma (n=143). patients with mild intermittent asthma, and mild, moderate, and severe persistent asthma, had a mean of 2.9, 3.4, 4.5, and 5.5 physician visits for asthma, respectively, within the past year. physician perception of patient compliance varied less across the groups, with 55% of patients with severe persistent asthma and 66% of patients with mild intermittent asthma being rated as "very compliant." the incidence of allergic rhinitis was similar among all severity levels (range: 50%-56%), but patients with severe persistent asthma were more likely to have comorbid conditions, including hypertension, chronic obstructive pulmonary disorder, chronic bronchitis, and osteoporosis, compared with patients in other asthma severity levels. conclusions: decreased asthma control and rates of com-pliance, as well as increased physician visits and comorbid medical conditions, may be associated with increased asthma severity classification. rationale: children with asthma frequently have co-morbid allergic conditions requiring medications. the purpose of this study was to assess whether the controller asthma medication selected would reduce the utilization of intranasal steroids (ins) and prescription nonsedating antihistamines (nsa) for children treated for co-morbid allergy. methods: this was an observational retrospective study that utilized the pharmetrics integrated outcomes database that contains administrative medical and pharmacy claims data from over 30 managed care plans across the united states. children age 4-17 years old with a new diagnosis of asthma (icd-9, 493.xx) were identified and observed for 12 months after their initial diagnosis and treatment of asthma (post-index). four cohorts were established based on their controller medication dispensed: montelukast, n=1906 (mon), fluticasone propionate, n=2455 (fp), fp+mon n=287, fp/salmeterol in a single inhaler, n=599 (fsc). results: baseline comorbid diagnosis of allergic rhinitis was observed in 14-20% of the children. the patterns of use of ins and nsa were similar in the 12 months before and the 12 months after the initial asthma diagnosis and treatment. children with a diagnosis of allergic rhinitis were nearly twice as likely to receive a nsa or ins. all 4 treatment cohorts used a similar amount of each allergy medication. the presence or absence of a diagnosis of allergic rhinitis did not alter the findings. the adjusted odds ratio (95%ci) of using a nsa or ins relative to the use of fsc in the post-index period was mon: 0.989 (0.809, 1.208); fp: 0.828 (0.682, 1.007); fp+mon: 1.095 (0.804, 1, 489 ). the table below shows the percent of children dispensed a nsa and/or an ins. the number of units of each allergy medication was also similar across all 4 cohorts. conclusion: the use of mon, fp, fp+mon or fsc for asthma did not alter the rate or quantity of either intranasal corticosteroid or nonsedating antihistamines dispensed to children over the 12-month period. although allergic rhinitis is a common comorbidity of pediatric asthma, the selection of a regimen containing montelukast did not reduce the use of either nsa or ins compared to asthma treatments with fp or fsc. rationale: medication compliance is recognized as a significant challenge in pediatrics.the purpose of this study was to compare inhaled corticosteroid (ics) persistence in pediatric patients using a single inhaler, containing both an inhaled corticosteroid (fluticasone propionate, fp) and an inhaled long-acting beta-agonist (salmeterol, sal) (fsc), to patients receiving fp alone, fp + sal from separate inhalers, or ics + montelukast (mon). methods: retrospective 24-month pre-post database study utilizing medical and pharmacy claims. a total of 4946 subjects 4-17 years of age with a diagnosis of asthma (icd9 493) were grouped into 4 cohorts: fsc [n=1168]; fp only [n=2473] ics+sal [n=296]; and ics+mon [n=1009]. patients were controller naã¯ve for 6 months prior to the index event (the first prescription of the medication of interest). subjects were required to have continuous enrollment of 6 months pre-and 12 months postindex. subjects with cystic fibrosis, copd, bronchopulmonary dysplasia or respiratory distress syndrome were excluded. ics refill rates, as a measure of persistence, were compared between fsc and the ics components of the other cohorts over a 12-month follow-up period. results: mean refill rates over the 12-month follow-up period was significantly greater (p=< 0.05) for fsc (4.48) compared with the mean ics refill rate in the fp alone (3.39) cohort, the ics + sal (3.61) and the ics + mon (3.92) cohort. patients on fsc filled 32% more ics prescriptions than patients on fp alone, 24% more than ics+sal and 14% more than ics + mon. mean refills were generally higher than the median fills as a considerable number of children had only one fill: fsc (35.6%), fp (49.1%), ics (48.3%) + sal (50.0%), ics (36.0%) + mon (18.6%). conclusion: patients using fsc, are likely to fill their inhaled corticosteroids more often over 12-months compared with patients using ics + sal in 2 separate inhalers, ics+mon, and fp as monotherapy. improved persistence with ics has been shown in other studies to decrease morbidity and mortality of asthma. use of fsc, a single inhaler, assures that children are getting more optimal inhaled corticosteroid therapy as well as the benefits from the long acting bronchodilator while avoiding selective discontinuation of ics that may occur when two medications are dispensed. the national health interview survey (nhis) estimates 14.5 million united states residents with asthma. noncompliance with treatment has been estimated as between 20 and 80%. the consequences of noncompliance include absences from work or school, increased emergency room visits, more severe attacks, increased drug side effects, greater cost of care, and death. we questioned compliance in a 47yo man with severe, prednisone dependent eosinophilic asthma because he had persistent symptoms despite treatment with fluticasone propionate/salmeterol xinafoate 500/50 bid, prednisone 20mg to 40mg qd, and budesonide (200 mcg/puff) 2 puffs bid. after discontinuation of budesonide and prednisone and initiation of methylprednisolone 16mg bid for 24 hours and then 16mg qd and beclomethasone dipropionate 2 puffs (80mcg) hfa bid with a spacer, clinical improvement was unexpectedly abrupt. fev1 increased from 58% to 90% of predicted. sputum eosinophil counts decreased from 78% to 2%. because of the sudden improvement with the change to methylprednisolone, compliance to prednisone and fluticasone was questioned. to evaluate compliance, the patient's blood, urine, and sputum were tested for synthetic corticosteroids, without his knowledge, using mass spectrometry. the blood level of methylprednisolone was 2.6mcg/dl and prednisolone was 0.35mcg/dl, documenting use of methylprednisolone and the recently discontinued prednisone. the urine levels were 13mcg/dl of methylprednisolone, 1.3mcg/dl of prednisolone and 0.74mcg/dl of prednisone that further confirmed recent use of prednisone. the sputum testing revealed 0.8mcg/dl beclomethasone, 33mcg/dl fluticasone and 1.8mcg/dl methylprednisolone confirming compliance to inhaled fluticasone and beclomethasone at the time of the test. thus, contrary to our clinical suspicion, the patient was indeed compliant with his inhaled glucocorticoids and prednisone and subsequently with the methylprednisolone. to our knowledge, this is the first case report to document compliance to inhaled and oral corticosteroids by analysis of synthetic corticosteroid concentrations in blood, urine, as well as sputum. if compliance could be documented with certainty, it could potentially minimize the adverse effects of needless escalating steroid doses, reduce the cost of treatment, and minimize morbidity and mortality as well. rationale:the burden of pediatric asthma extends beyond those children with an asthma diagnosis. children with wheezing frequently have a delay in the diagnosis of asthma that may impede instituting appropriate therapy and reducing disease morbidity. this observational study was designed to assess the treatment patterns of children 4-17 years old receiving asthma medication(s) without a diagnosis of asthma compared with children diagnosed with asthma and children without claims for asthma. methods:this was a retrospective cross-sectional study that utilized the pharmetrics integrated outcomes database containing administrative medical and pharmacy claims data from over 30 managed care plans across the united states. three cohorts were selected: children with an asthma diagnosis (icd-9, 493.xx) (dx cohort), children with prescription claims for asthma controllers or rescue medications without an asthma diagnosis (rx cohort) and children with neither an asthma diagnosis nor prescription claims for asthma medications (control cohort). utilization of asthma medications, asthma specific costs and total costs of care were assessed. results: a total of 295, 000 children were identified: 6.7% in the dx cohort, 4.4% in the rx cohort and 88.9% in the control cohort. the potential asthma cohort was 11.1%. total annual non-asthma related costs for the dx cohort was $1, 642 for the rx cohort was $1, 306 and for the control was $802. only 19% and 5% of the total healthcare charges in the dx and rx cohorts retrospectively were asthma related. non-asthma charges were significantly higher in both the dx and rx cohorts compared with the control cohort. in the rx cohort, the ratio of saba prescription claims to asthma controller claims were 1.85-fold higher than in the dx cohort. conclusion: children treated with asthma medications without an asthma diagnosis consume greater health care resources resembling the pattern of health care utilization of children with an asthma diagnosis more closely than controls. further, children with treatment in the absence of an asthma diagnosis appear to be under-treated with controller therapy as recommended by national and international guidelines. background: a recent retrospective study assessed asthma variability in inner-city patients 6 months before and 6 months after enrollment into an naepp guidelines-directed clinical management and educational program. while guidelines-directed care improved asthma symptoms and outcomes, significant variability in disease indices were observed over the 6-month period. the present analysis evaluates resource utilization associated with this variability. method: economic end points, including hospital/emergency department (ed) visits, total unscheduled office visits, sick visits, and days lost from work or school, were collected from 125 inner-city patients aged 18 years (74% female, 69% minority, 80% treated by primary care physicians) enrolled in a guidelines-directed asthma clinical management and education program aimed at minimizing barriers to adherence. patients were stratified into 2 groups: those with high variability in asthma (n=62) and those with low variability in asthma (n=63), with variability defined as number of fluctuations in naepp symptom class in the 6-month postintervention period (high variability = patients with fluctuations higher than the mean; low variability = patients with fluctuations lower than the mean). results: guidelines-directed therapy was associated with improvements in asthma during the 6-month treatment period for both groups. patients in the high-variability group had more ed visits, sick visits, total unscheduled visits, and office visits compared with patients in the low-variability group (see table) . conclusions: despite guidelines-directed therapy and overall improvement in asthma control, patients experienced variability in asthma, indicating that even when their disease is stable, their symptoms continue to fluctuate. as a result, asthma variability may contribute to increased resource utilization. introduction: inhaled corticosteroids (ics) are considered first-line therapy for patients with persistent asthma. ics can lead to oropharyngeal adverse events, which may affect treatment outcomes and adherence. the occurrence of these local adverse events is dependent upon several factors, including dosage and duration of ics treatment. ciclesonide (cic) hydrofluoroalkane (hfa)-metered dose inhaler (mdi) is a novel and effective ics, with relatively low oropharyngeal deposition. cic is converted in the lungs to its active metabolite, desisobutyryl-ciclesonide (des-cic). furthermore, cic undergoes limited conversion in the oropharynx, which may account for its improved safety profile. this oropharyngeal safety profile analysis was part of a long-term study evaluating the safety of cic hfa-mdi vs beclomethasone dipropionate (bdp) hfa-mdi in patients with severe persistent asthma. methods: this was a multicenter, double-blind, parallelgroup, 12-month extension of a 12-week double-blind study of patients 12 years with severe persistent asthma. patients were randomized in a 2:1 ratio (cic:bdp) to receive cic (320 g bid) or bdp (320 g bid) (both ex-actuator). after 2 weeks, the doses of both medications could be titrated to 160 g bid as needed for asthma control.) oropharyngeal adverse events were monitored, and suspected oral fungal infections were verified by positive culture. results: the incidence of oral candidiasis was lower in subjects receiving cic (4.1%), compared with those receiving bdp (10.4%) ( table) . the incidence of pharyngitis and hoarseness was 3.6% and 2.5%, respectively, for the cic group, and 5.2% and 1.0%, respectively, for the bdp group. all local adverse events resolved without sequelae, and there were no withdrawals due to oropharyngeal treatment-emergent adverse events. conclusion: after 12-months of treatment with cic 160 î¼g or 320 î¼g twice-daily, the incidence of oropharyngeal adverse events was low. cic treatment resulted in a much lower incidence of oral candidiasis, compared with similar doses of bdp, reflecting a better local tolerance. objective: to determine the prevalence of asthma and asthma-related morbidity, treatment and asthma-risk factors in a selected population. method: a routine health screening, including tests for asthma, was conducted at the new orleans center for creative arts high school. participants (204) identified their medications and asthma tools in addition to completing the isaac questionnaire. prevalence data included lifetime wheezing, 12 month wheezing, and previous asthma diagnosis. asthma-related morbidity included sleep disturbance, wheezing with exercise, asthma attack rate and night awakening. results: " participants were aged 13-9 years (50.5% african american, 45.1% white) with 31.4% male, 68.6% female. " cumulative asthma prevalence was 18.8%, lifetime wheezing (24%), 12-month wheezing 12.7% with girls reporting 3.36 times more often (95% ci: 1.54, 7.36), and wheezing more than 4 times in 12 months 4.7%. " there was a significant association between race and wheezing in the last 12 months (p < 0.024) with whites reporting 3.78 times more often (95% ci: 2.01, 7.10). asthma morbidity was reported as follows: a. night cough -10.5% b. wheezing with exercise -10.9% " parents with a less education (high school or less) were 8.33 times more likely (95% ci: 7.46, 9.30) to have children who developed wheezing in the past 12 months. " higher body mass index (bmi = 25) was associated with wheezing after exercise (p < 0.008) with a 3.27-fold increase (95% ci: 2.46, 4.31). " among those with current asthma (15/204), 4 were not on any medication, 10 were using bronchodilators, and 5 were on anti-inflammatory medications. three reported using a spacer and 6 reported using a peak flow meter. conclusion: asthma prevalence is higher in this school population than the national average. less parental education, female gender, and bmi = 25 were associated with greater asthma morbidity. the majority of the participants with current asthma reported receiving episodic and inadequate treatment with inhaled corticosteroids. objectives: to determine the prevalence of asthma and asthma related symptoms; to assess its severity among new orleans school children. methods: seven elementary, middle, and high schools in new orleans participated in the screening of 1511 students, 54% female, 45% male, ages 5-18 in the fall, 2000. the 8-item questionnaire was designed by the international study of asthma and allergies in childhood (isaac) to determine asthma prevalence and severity in children. the isaac questionnaire is a validated protocol used on over 500, 000 children in 56 countries. asthma is defined as cur-rent wheezing for the purposes of this study. data was entered using spss 10 and sas 8 software for analysis. results: table: presence of asthma symptoms by ethnicity conclusion: asthma prevalence is significantly higher in inner-city school children in new orleans when compared to the national prevalence (24.6% to 26.4% vs. 7.5%). asthma prevalence and severity do not differ significantly between african-american and caucasian children in new orleans even after removing the effect of gender and age. video is an effective method of teaching pollen identification. projected digital images offer three-dimensional views of unique pollen detail similar to focusing a microscope. the national allergy bureauâ�¢(nab) currently provides pollen counts to the media. these reported levels are obtained by standardized counting methods rather than forecasts based on historical pollination predictions and weather patterns. the american college of allergy, asthma and immunology and the american academy of allergy, asthma and immunology have an interest in ensuring consistent counting and accurate reporting practices. both organizations offer pollen identification training in conjunction with their annual meetings and have used the video for initial training sessions and for experienced counters in advanced courses. there are 112 certified pollen counting stations in the united states and canada with approximately 80 currently active locations. the nab requires the demonstration of ability to count and accurately identify pollen on test slides. an updated recertification process using an interactive web site is currently being developed. ongoing pollen identification training is essential for the continued success of this aeroallergen network. most observers found that the video provided a quality emulation of microscopic viewing and enhanced the depth and detail of individual pollen characteristics. the video received the highest possible ratings by course attendees. in conclusion the video plays a positive role in pollen identification training and the continuing education of experienced counters. the diagnosis and treatment of mold allergies are complicated by the genetic diversity of individual fungal species and the influence of endogenous fungal proteases on extract compositions and potencies. studies examining the biochemical comparability of mold extracts from different sources and their compatibility with other allergens in immunotherapy mixtures are essential to the clinical effectiveness of these products. compositional comparisons of extracts prepared from alternaria, aspergillus and penicillium source materials by 7 u.s. allergen manufacturers revealed variable sds-page protein banding patterns and noticeable differences in total protein and carbohydrate concentrations. alt a 1 levels in alternaria extracts did not correlate closely with total protein levels or with ige-binding potencies measured by elisa inhibition. immunoblot profiles of fungal extracts were more closely related to one another compared to sds-page patterns, suggesting that similar allergenic or antigenic epitopes may be retained in molecules of varying size. parallel elisa inhibition dose-response curves provided statistical evidence that a repertoire of ige epitopes is conserved in many of these products. variations in the potencies of fungal extracts from different manufacturers may result from differences in source materials and/or conditions of extraction and storage. the stability and compatibility of allergen mixtures containing mold extracts were assessed after storage for up to 6 months at 2-8â°c using specific immunoblot and elisa procedures. grass and mite allergens compromised by mixing with mold extracts were stabilized by glycerin. alternaria extracts from different sources produced similar effects on grass allergens when added at comparable strengths. degradative effects caused by aspergillus or penicillium products were more closely related to total fungal protein concentrations than to extraction ratios. structural epitopes on cat, ragweed and fungal allergens were stable after mixing with protease-containing mold extracts, indicating the presence of natural protease inhibitors or allergenic protein sequences distinct from those recognized by these enzymes. allergenic extracts labeled as weight/volume or by pnu have no regulatory requirement for determination of activity. alum-adsorbed allergenic extracts are labeled in pnu and are felt to be depot formulations. the purpose of this study was to develop methods to measure important allergen proteins and specific ige binding capability of these alum-adsorbed allergenic extracts and to begin to assess the potency of these non-standardized extracts. allergen proteins are adsorbed to alum particles making their measurement difficult. in this study allergens were released from center-al â® alum precipitated allergenic extracts using 0.3 m citrate buffer, ph 5. the major allergen contents of several lots of each product were measured using monoclonal antibody elisas for group 5 grasses, cyn d 1 bermuda, bet v 1 birch, ole e 1 olive, and pla l 1 english plantain. these assays were developed and validated by alk-abello and optimized for the citrate releasing buffer. direct binding ige was performed by immobilizing serial dilutions of extract to microtiter plates and then adding specific atopic sera and enzyme labeled anti-ige. the methods used were able to measure in vitro allergen activity in the alum-adsorbed extracts. major allergen content was successfully measured in the extracts and the amount was related to the pnu content. as expected, the variability of the major allergens within a particular extract species was higher than in standardized extracts that are adjusted for potency. the extracts demonstrated specific ige binding ability in the binding assay. the ige binding capability at various dilutions was related to the major allergen content. in conclusion, methods were developed to release adsorbed allergen proteins from alum extracts. the extracts were then analyzed for major allergen pro-teins and the ability to bind ige. major allergen content is currently used to monitor the activity of aqueous and glycerinated extracts and this study demonstrates that implementing these testing procedures will improve the consistency and quality of alum-adsorbed extracts. introduction there are over 5000 species of smut and rust fungi. ustilago maydis, or corn smut, is a basidiomycete fungus that infects ears of corn. it is responsible for a percentage of grain loss in the united states but eaten as a delicacy in mexico. it grows into large tumor-like structures, called galls, dispersing soot-like spores responsible for the common name, "smut." it is readily airborne and believed to be a causal agent for allergic rhinoconjunctivitis. "corn smuts" was added to the standard screening panel for patients presenting for evaluation of rhinitis in the region of middle tennessee. methods using greer laboratoriesâ�¢ corn smut extract 1:20 w/v, patients were tested by prick/puncture method with the hollister-stier quintipâ�¢ device. if negative, intradermal testing was performed. positive and negative controls were assessed. based on current national recommendations, wheals 3 mm greater than negative control were considered positive for prick/puncture testing and wheals 8 mm greater than negative control were considered positive for intradermal testing. results 105 patients were tested between november, 2003 and march, 2004 . 14/105 (13.3%) met criteria for prick/puncture positivity. 52/105 (49.5%) met criteria for intradermal positivity. patients prick positive to corn smut had the following prick positive tests: dust mites 12/14 (85.7%); cat 11/14 (78.6%); local grass mix 10/14 (71.4%); local tree mix 10/14 (71.4%); local weed mix 10/14 (71.4%), mold mix 8/14 (57.1%); cockroach 7/14 (50%); and dog 4/14 (28.6%). discussion allergy testing for aeroallergens is available to a limited number of antigens, of which, only a few are standardized. as time passes, relevant antigens are discovered and their importance further elucidated. this assessment reveals a significant presence of corn smut ige largely accounted for by intradermal positivity which may or may not reflect clinical sensitivity. these numbers are provided so that other clinicians may compare to prevalence in their geographical area. further studies are needed to determine the association of corn smut ige with clinical symptoms. h 1 antihistamines are the mainstay of therapy for allergic disorders, including skin diseases. the most common side-effect of marketed antihistamines is sedation, especially when the clinical symptoms require a higher dose than recommended, a condition commonly encountered in dermatological disorders. the present study describes the pharmacology of a new non-sedating h 1 antihistamine, r129160 (hivenylâ�¢). r129160 binds to the cloned human h 1 receptor with a similar affinity (ki: 19 nm) as the reference antihistamines cetirizine (ki: 50 nm) and loratadine (ki: 37 nm). in vivo, r129160 protects rats and guinea pigs from lethal shock, induced by compound 48/80 and histamine, respectively (ed 50 : 0.06 and 0.055 mg/kg respectively). the compound is at least as effective as cetirizine and loratadine. in rats, r129160 inhibits the histamine-and allergen-induced cutaneous reactions (ed 50 : 1.4 and 2.3 mg/kg) with a similar potency as cetirizine (ed 50 : 0.7 and 2.7 mg/kg) and loratadine (ed 50 : 1.8 and 1.6 mg/kg). even so, in guinea pigs the compound inhibits the histamine-and allergen-induced skin reactions (ed 50 : 0.14 and 1.2 mg/kg) to a similar extent as loratadine and cetirizine. however, in dogs r129160 is more potent in inhibiting the ascaris allergen-induced wheal reaction (ed 50 : 0.024 mg/kg) than cetirizine (ed 50 : 0.14 mg/kg) or loratadine (ed 50 : 0.20 mg/kg). r129160 fails to occupy central h 1 receptors in the guinea pig cerebellum up to 40 mg/kg, in contrast to loratadine (ed 50 : 2.2 mg/kg). in vitro and in vivo cardiovascular safety experiments indicate that r129160 lacks the intrinsic capacity to prolong the qt-interval, even at high doses. as such, r129160 (hivenylâ�¢) is characterized as a potent, non-sedating and cardiosafe h 1 antihistamine. it has been selected for further clinical development, mainly in the field of dermatology. the compound will be a suitable tool to explore the activity of a selective h 1 antihistamine in various indications, without the contamination of the sedative activity often observed with other marketed antihistamines when increasing the dose. ciu represents one of the most clinically perplexing disorders which the allergist-immunologist is faced with. we have previously reported the clinical efficacy of fxt (prozac), an ssri widely used for the treatment of depression, in the management of ciu (nsouli tm, et al. ann allergy asthma immunol 2002; 88:60) . in this presentation we report 2 additional cases of ciu which responded dramatically following the use of fxt. a 41 yr-old female and a 21 yr-old male presented with ciu of 18 and 24 months duration, respectively, requiring oral corticosteroid (cs) therapy for control of their ciu after failure of high dose anti-h1 (hydroxyzine) and anti-h2 (ranitidine) agents (table) . testing for food and latex allergy, viral hepatitis, autoimmune thyroid disease and parasitic infection were all negative. a skin biopsy performed in subject #1 was consistent with an urticarial vasculitis. following one week of oral fxt (40 mg qd [subject #1] 20 mg qd [subject #2]) a striking and complete resolution of urticaria in each patient was observed within 7 to 10 days during which time it was possible to successfully taper the cs. both patients have been maintained on fxt therapy with complete control of their ciu. the very favorable response to fxt therapy in these 2 patients in addition to our first case report suggests that this drug may have a new therapeutic application in the management of recalcitrant ciu. background: ipratropium bromide nasal spray (ib) is indicated for treatment of rhinorrhea caused by common cold (cc), seasonal and perennial allergic rhinitis (ar) in adults and children age 5 and up. symptoms of rhinorrhea from cc or ar in children are similar to those in adults, yet there is little data on the use of ib in children under 5 years of age. objective: evaluate the safety and efficacy of ib nasal spray 0.06% in 2-5 year-old children with symptoms of rhinorrhea from cc or ar. methods: a total of 230 children (43 cc and 187 ar) were treated in an open-label, multi-center study. the cc patients received ib nasal spray (84 mcg per nostril) tid for 4 days, the ar patients received ib nasal spray (42 mcg per nostril) tid for 14 days. effectiveness was measured using a global assessment questionnaire and daily symptom scores reported by the parent. results: from the global assessment questionnaire, 91% and 90% of the parents found ib either "very useful" or "somewhat useful" in the cc and ar groups respectively. regarding effectiveness, 98% (cc) and 87% (ar) of the parents reported that ib had either a "good effect" or "excellent effect" in treating rhinorrhea. moreover, 67% (cc) and 91% (ar) of parents found administration of a nasal spray either "extremely easy" or "very easy." eighty-one percent (cc) and 89% (ar) of the parents reported they would use ib again for their child's allergy symptoms. the daily symptom score (0 = none to 5 = unbearable) for rhinorrhea decreased from 2.9 to 1.3 (-55%, p<0.0001) for cc and from 2.1 to 1.1 (-48%, p<0.0001) from baseline compared to the average on-treatment score, with decreases also seen for stuffy nose and sneezing. the nasal spray was well tolerated with adverse events (ae) reported in 28% of cc and 35% of ar patients. the aes were mostly mild to moderate and no potentially systemic anticholinergic or serious aes were reported. conclusions: ib nasal spray 0.06% administered at a dose of either 42 or 84 mcg per nostril tid is an easy to use, safe, and effective therapy for control of rhinorrhea in children 2 to 5 years of age with common cold or allergic rhinitis. chronic idiopathic urticaria (ciu) represents one of the most clinically perplexing disorders which the allergist-immunologist is faced with. the pathogenesis of the condition derives from the release of potent vasoactive substances including histamine, and products of arachidonic acid metabolism, e.g., prostaglandins and thromboxanes formed by the action of the enzymes cyclooxygenase (cox) of which cox-2 is responsible for pseudoallergic and other inflammatory responses. although a number of therapeutic options exist owing to the plethora of mediators produced, treatment has focused largely on the use of h1 antihistamines and, unsurprisingly, at times without complete resolution of symptoms. in this presentation we report the successful use of a cox-2 inhibitor for the treatment of chronic urticaria. a 10 y/o white hispanic female presented with a 4 month history of chronic urticaria predominantly on the soles of the feet, with unknown precipitating factors, and fail-ure to respond to prior treatment with: cetirizine, steroids, benadryl, ebastine and epinastine. the patient received a blood transfusion 3 years ago and there was no prior history of allergies. skin testing revealed 3+ reactions to dust mite, pollen, cockroach, shellfish, 2+ reactions to corn, milk and ige = 262 iu (nv < 90 iu), aso = 400 ui (nv < 200 ui). after 2 weeks of treatment with rofecoxib (vioxx) 25 mg, and benadryl, the rash resolved. vioxx was continued for 2 more weeks until complete resolution of symptomatology. this case illustrates that the addition of a selective cox-2 inhibitor (i.e., rofecoxib) with an h1 antihistamine may be a more effective regimen for patients with ciu who fail to respond adequately to conventional therapy. introduction: elderly asthmatic patients whose symptoms are controlled by inhaled corticosteroid (ics) therapy may still have breathlessness on exertion. we randomized elderly asthmatic patients stabilized by medium-dose ics therapy into two groups, treated one group with medium-dose ics therapy plus montelukast, a leukotriene receptor antagonist, and the other with increased-dose ics therapy, and compared the effects of the treatment regimens on exercise tolerance. methods:the subjects were 24 patients with bronchial asthma (16 males and 8 females, 70.6â±3.9 years) stabilized by ics therapy (with fluticasone proprionate, fp; 400 mg/day) for three months or more with low peak expiratory flow (pef) rates (<80%predicted, variability<15%). they were randomly divided into two groups (12 patients each) to be treated with ics therapy plus montelukast (400 mg/day fp + 10 mg/day montelukast; m group) or with increased-dose ics therapy (600 mg/day fp; f group). pulmonary function tests, a six-minute walking test, respiratory gas analysis during incremental (15w/min) cycle ergometer exercise were conducted, and exhaled nitric oxide (no) levels were measured before and after two weeks of the study treatment. results: pulmonary function tests showed significant increases in maximal mid-expiratory flow (mmf) and forced expiratory flow at 50% vital capacity (v50) in the m group (p<0.01) but no significant changes in the f group. exhaled no levels decreased significantly in both groups (40.7â±13.9 to 27.5â±9.1 ppb in the m group and 53.1â±17.6 to 38.0â±10.4 ppb in the f group; p<0.01). the six-minute walking distance extended from 531â±64 to 575â±58 m in the m group and from 553â±60 to 570â±58 m in the f group.the peak oxygen uptake (peakvo2) increased significantly in the m group (%peakvo2/w from 76.2â±12.7 to 84.4â±14.6%; p<0.01) but not in the f group (%peak vo2/w from 76.2â±10.7 to 76.4â±10.3%). the peak exercise load also increased significantly in the m group (76.6â±22.0 to 96.1â±23.1 w; p<0.01) but not in the f group (81.2â± 16.2 to 82.2â±14.3w). conclusions: the results indicate that concomitant administration of montelukast is more effective than dose escalation of ics on exercise tolerance in elderly asthmatic patients under medium-dose ics therapy. e. meltzer 1* , y. luo 2 , l. shen 2 , z. guo 2 , c. schemm 2 , y. huang 2 , k. chen 2 , p. king 2 , r. nave 3 , d. banerji 2 , s. rohatagi 2 , 1. san diego, ca; 2. bridgewater, nj; 3. konstanz, germany. introduction: inhaled corticosteroids (ics) are first-line treatment for persistent asthma. ciclesonide (cic) is a novel and effective ics under development. freely circulating, unbound ics is available to cause systemic adverse effects, such as hypothalamic-pituitary-adrenal (hpa) axis suppression. hence, it is important to determine the free fraction of ics in plasma. in separate studies, the protein binding of the active metabolite of cic, desisobutyryl-ciclesonide (des-cic), was evaluated, and the effects of inhaled cic on hpa axis function were determined. methods: human plasma protein bind-ing of des-cic (0.5-500 ng/ml) was determined using equilibrium dialysis. dialyzed samples were analyzed by liquid chromatography with tandem mass spectroscopy to determine free and bound des-cic. in 3 separate clinical studies, the effects of cic (hfa; 320-1280 î¼g daily) and placebo (pbo), via metered-dose inhaler (ex-actuator doses), over 9 days, 4 or 12 weeks, on basal hpa axis function (24-hour area-under-the-curve [auc0-24h] serum or urinary cortisol corrected for creatinine) or stimulated (low-dose [1 î¼g] cosyntropin) serum cortisol were investigated in patients ( 18 years) with persistent asthma. results: the mean % of human plasma protein binding for des-cic was 99%. in 2 studies measuring serum cortisol auc0-24h, there was no difference between pbo and cic (320-1280 î¼g). similar results were observed for 24-hr urinary cortisol corrected for creatinine. in the 3rd study measuring low-dose (1 î¼g) cosyntropin-stimulated peak serum cortisol, after 12 weeks of treatment, there was no significant difference in the mean change from baseline versus placebo for either cic320 (p=0.383) or cic640 (p=0.911). conclusions: the favorable pharmacokinetic profile of cic, in particular the high protein binding of des-cic, may explain the lack of hpa-axis suppression. this appears to result in greater systemic safety. purpose: unscheduled office and ed visits for urgent asthma care are an ongoing point of concern. determining preventive variables regarding these unscheduled visits could have a significant impact on asthma costs and quality of life. objective(s): this research addresses the following question: when stratifying by race, gender, age, metropolitan/rural place of residence and comorbidity, do adults with asthma have fewer ed or unscheduled office visits for urgent asthma care if they: a) have an identified primary care provider, or b) have health insurance? method(s): univariate and stratified bivariate contingency table analyses were performed on weighted 2001 behavioral risk factor surveillance survey (brfss) data. result(s): adults with asthma who had an identified primary care provider were more likely to have no unscheduled office visits (or=1.87) or ed visits (or=2.07) for urgent asthma care. this was also true for adults with asthma who had health insurance (or=1.38 for no unscheduled office visits and or=1.71 for no ed visits). these relationships held when stratifying by race, gender and age. the relationships also held for metropolitan residents. the analysis was inconclusive for rural residency and the existence of co-morbidities. conclusion(s): despite race, gender, age and metropolitan residency, having a primary care provider or having health insurance impact whether or not adults with asthma are more likely to have unscheduled office or ed visits for urgent asthma care. further investigations are needed to examine how these factors impact adults with asthma who are rural residents or who have co-morbidities. d. bukstein 1* , g.a. cherayil 2 , 1. madison, wi; 2. brookfield, wi. introduction: costs for plans to process prior authorizations for non-formulary medications, has been estimated to be $20 to $25 per request. costs for physicians to process these requests has not been extensively studied. methods: dr.bukstein, board certified allergist, developed a data collection tool. the form was utilized by physicians and nurses to document time spent on processing prior authorizations. data collected included, class of medicines requiring the pa, nursing time spent on calling the patient, pharmacist, health plan, nursing time spent completing forms, nursing time clarifying the information for the pa, as well as physician time spent completing pa activities. results: data was collected over 8 weeks in 2003 and 117 requests were processed.the class of medicines most often processed was antihistamines, comprising 66% of requests. nursing calls were tracked and calls to and from patients were the most common call documented. they averaged 5.6 +/-6.5 calls per day per nurse. the nurses spent an average of 17 minutes per patient call. calls to health plans averaged 1.8 +/-1.5 calls per nurse per day and time spent on these call was 8.4 +/-9.5 minutes per call. physician calls documented included 154 calls averaging 1.9 +/-1.2 calls per physician per day. these calls averaged 5.8 +/-5.0 minutes per call.often the results of the prior authorizations were not known on the day of the request. originally 30.8% of requests were granted the same day. retrospective review revealed 98.7% were approved the first time they were processed. salary and benefits were calculated for nurses and physicians. the hourly rate was defined as $21.50 per hour for nurses and $150 per hour for physicians. the costs for the time spent on the 117 prior authorizations were calculated. during the 8 week study period, over 40 hours was spent by nurses on 231 calls and over 8 hours was spent by physicians on 154 calls during the same time period. the total nursing and physician cost in this specialty practice was $17.77 per prior authorization. conclusion: there are substantial costs with processing of prior authorization requests for non-formulary drugs on the physician office side of managed care as well as on the insurance side of the process. specialty physicians should have a different process for obtaining non-formulary medications since almost 100% of their requests are granted. introduction: diabetes mellitus can adversely impact the course and outcome of myocardial infarction (mi). one of the mechanisms underlying this phenomenon is alteration of the course of inflammation and the reparative process following myocardial necrosis. abnormal wound healing, tissue reparation and immune responses in diabetic patients have been intensively studied, but the cellular and the molecular mechanisms are still unclear. transforming growth factor-beta (tgf-beta) is a multifunctional cytokine which plays a critical role in coordination of the course of inflammation and reparation, acting as a potent depressor of inflammation and a stimulator of regeneration. this study investigates the dynamics of serum concentrations of the active form of tgf-beta1 during the period up to the 20th day after the onset of a mi in diabetic and non-diabetic patients. results: in non-diabetics a significant increase was observed in tgf-beta1 on day 2 (5-fold greater than in controls; 1370.0â±306.2 and 269.3â±214.5 pg/ml respectively) with further increases reaching a peak on day 7 (1723.1â±127.6 pg/ml). on day 20 tgf-beta1 decreased to levels less than on day 2, but was still greater than in healthy controls (1265.3â±369.8 pg/ml). in diabetics, concentrations of tgf-beta1 on days 2 (939.7â±155.2 pg/ml) and 7 (965.8â±150.4 pg/ml) after mi were similar to diabetics without mi (724.6â±233.5 pg/ml). only on day 20 was tgf-beta1 increased to levels (1502.8â±285.6 pg/ml) which were 2 fold greater than in diabetics without an mi. thus, in diabetic patients serum concentrations of the active form of tgf-beta1 are much greater than in non-diabetics. mi in patients with diabetes mellitus is associated with a reduced and significantly delayed increase in tgf-beta1. conclusion: tgf-beta deficiency may be a factor associated with low activity of tissue reparation after mi in diabetic patients. introduction helicobacter pylori (hp) is the most common gastrointestinal infection worldwide, but only 10-15% of those infected develop chronic gastritis or peptic ulcer disease. the pathogenesis of ulceration, mechanisms of hp lifelong persistence in gastric mucosa and local immune disturbances induced by this infection are well known, but the mechanisms of resistance and elimination of this infection have not been extensively studied. most study is based only on phenomenological findings, such as absence or low hp colonization in subjects spontaneously producing high levels of il-2. the aim of this investigation was the analysis of the efficacy of the recombinant interleukin (ril-2) roncoleukin (biotech, russia) in treatment of hp-associated gastric ulcer disease. methods 108 patients were randomly divided into two groups. the first group of patients was treated with standard therapy including of two antibiotics (claritromycin and amoxicillin), proton pomp inhibitors and h2 receptor antagonists. patients of the second group were treated with the same therapy, but instead of antibiotics they received 0.1 mg roncoleukin into four to six areas submucously using a gastroscopic method and 0.4 mg roncoleukin dissolved in 400 ml of 0.9% nacl with 4 ml 10% human albumin infused intravenously. this procedure was performed three times at an interval of 72 hours. results immunological findings demonstrated that roncoleukin results in an increase of cd25+, hla-dr+ and cd16+cd56+ cell levels. there was an increase in the serum concentrations of il1 (3 fold), il6 (4 fold) and ifn (more than 20 fold) while the level of tnf and il8 profoundly decreased. one month after the end of treatment, the group treated with ril-2 had hp eradication achieved in 95.4% in comparison to 81.5% of the control patients. in the ril-2 treated group, the ulcer epithelization period was 10.79â±0.46 days while in the normal treatment control group it was 35.23â±1.58 days (p<0.001). conclusion immunotherapy with ril-2 is a more effective method of treatment of helicobacter pylori-associated gastric ulcer disease when compared with traditional methods of treatment employing only antibiotics. introduction the role of different cytokines and the growth factors is now appreciated in progression of essential hypertension. the participation of et-1 and tgf 1 in pathogenesis of essential hypertension especially in the process of fibrosis, hypertrophia of vascular smooth muscular fibers, and cardiomyocytes, and the activation of renin-angiotensin system is now recognized, in addition to effects on myocyte cultures. the aim of the investigation was the study of serum et-1 and tgf 1 levels in patients with essential hypertension. materials and methods 60 patients with essential hypertension (40 males and 20 females) with average age 54.0 â± 5.3 years were studied. all patients suffered from left ventricle hypertrophy documented by echocardiography. the control group consisted of 20 healthy volunteers similar to the investigated patients in sex and age. in all patients the serum concentrations of et-1 and tgf 1 were determined using elisa (biomedica, biosource). results an increase in the serum concentrations both growth factors were noted in the studied group when compared with the control group. the average concentration of et-1 in patients with essential hypertension was 0.64 (0.32-0.91) pmol/l. the level of et-1 in the control group was 0.1 (0.05-0.17) pmol/l (p=0.04). the concentration tgf 1 in essential hypertension patients was 224.2 (125.4-314. 3) pg/ml and in control group was 68.2 (42.5-81.8) pg/ml (p=0.02). there was a positive correlation between the concentrations (spearmen's rank coefficient of correlation was 0.42; p=0.01). con-clusion the increase of the serum concentration of growth factors et-1 and tgf 1 and their co-influence in patients with essential hypertension, suggests a role of these growth factors in the pathogenesis of arterial hypertension. u. kaza 1* , c. lauter 2 , 1. bloomfield hills, mi; 2. royal oak, mi. introduction: few studies have examined the referral patterns for allergy and immunology inpatient consultations in a community hospital. consequently, an invaluable part of physician and housestaff education is missing. our objective was to examine the number of inpatient allergy and immunology consultations, the reasons for consultations and the outcome of the patients in order to improve physician education. methods:we performed a retrospective chart review of all inpatient allergy and immunology consultations in the years 1996-1997 and in 2002-2003 to determine the reasons for consultation, the recommendations made and if they were followed and the outcomes of the patient. results:we reviewed a total of 408 inpatient allergy and immunology consultations. in the 1996-1997 time period 26% of inpatient consults were for asthma, 22% for drug allergy, 12% for rash. in the 2002-2003 time period 25% of inpatient consults were for rash, 21% for drug allergy, 16% for asthma. the top three reasons for consultations remained the same although the order changed. consultations for immune deficiency, angioedema and rashes increased, whereas consultations for asthma and allergies decreased. there were a total of 202 consultations in 1996-1997 and 206 in 2002-2003 . the number of consultations remained the same despite an increase in overall number of hospital admissions from 44, 677 in 1996 to 58, 348 in 2003 . in greater than 95% of consultations, allergists' recommendations were followed. in both of the time periods studied, greater than 70% of patients improved, with less than 9% having no improvement, in the remainder of cases improvement was not applicable. conclusion:in conclusion, we believe that identifying the reasons for inpatient allergy and immunology consultations and examining the most common recommendations, as well as the outcomes of patients will be a valuable guide in the education of our physicians. by incorporating this information into grand rounds and resident conferences, physicians will benefit from learning about when an allergist can be helpful and how to manage some of the more common allergic and immunologic problems in patients that are hospitalized. the high percentage of providers who follow the advice of allergists indicate that allergists have a great deal of educational value to offer other physicians. while complementary and alternative medicine (cam) has generally experienced increased popularity, its utilization by allergy/asthma patients remains uncertain. our private allergy practice surveyed the use of cam in allergy/asthma patients in 1998(1). using a similar questionnaire, we assessed the current interest in cam with our allergy/asthma patients and compared the data to our 1998 survey. we analyzed 103 completed questionnaires from 113 sequential surveys administered. the results were compared to 113 questionnaires reported in 1998. they indicated that in both study periods (1998 & 2004) , the majority of patients wanted to discuss cam (65 and 69% respectively). an equal number (18%) in each study period discussed cam with their primary care provider. sixteen percent (16%) of our respondents sought a cam practitioner for general medical needs in 1998 vs. 19% in 2004. however, there was an increase from 4% to 10% of our patients seeing a cam practitioner for their allergies and/or asthma from 1998 to 2004. sixty-two percent (62%) would like to consider pursuing cam through our allergy spe-cialty practice or other provider. when asked regarding preferred treatment, 62% stated combination traditional with cam, 8% preferred traditional only, 3% cam only, and 27% did not know/doctor s choice. more patients are now seeking chiropractic care (36% to 52%) compared to our 1998 results. acupuncture was the first choice cam modality at 48% in 2004 surpassing vitamin/mineral therapy in 1998. currently, the second and third choices were vitamin/mineral therapy and deep tissue massage. while these numbers were small, we were impressed that currently 10% (two and a half times more since 1998) of patients in our practice were seeking cam allergy/asthma care from outside of our practice. these results demonstrated that more than half of our patients were interested in pursuing traditional with cam options within our office. given this trend, we have begun discussing the concept of integrative allergy, which to us means integrating evidenced-based cam modalities within our traditional allergy/asthma practice. (1) introduction: clinical immunization knowledge is complex and demands ongoing training. nationally, basic immunization and vaccine safety education is limited within traditional medical, nursing, and provider education. project immune readiness (pir), a peer-reviewed, web-based, interactive course, was developed in response to this deficit and the need for standardized resources to provide initial and sustainment training for safe and effective immunization services. it is designed for medical personnel with diverse educational preparation. currently, pir offers 21 course modules addressing 42 hours of instruction on specific vaccines, their respective diseases, and general information on immunization healthcare and vaccination procedures. methods: users completed a pre-test (establishes baseline knowledge), an interactive module, followed by a post-test (to observe change from baseline) for each course in sequence. anonymous user and score data were collected as part of a quality assurance and course validation process. learning gain indices (lgi) were calculated based on average mean pre-test and post-test scores for each module lgi of all modules demonstrated substantial increases in user vaccination knowledge. comparing pre and post-testing is an effective method to assess learning gains. the findings support pir as a successful and valid distance-learning tool that establishes and documents core knowledge of medical personnel administering vaccinations. further research is needed to assess the effectiveness of knowledge acquisition and retention, in addition to variance in vaccine delivery after training. this approach to learning may have value as a resource that supports smallpox and influenza pandemic emergency preparedness plans for just in time training. introduction: variances from practice guidelines for the prescription of auto-injectable epinephrine are well documented among practicing physicians, families, and patients. effective patient education requires provider competency. the current study was designed to survey resident physician perceptions regarding auto-injectable epinephrine education, use, and patient education requirements. methods: residents from primary care disciplines at a tertiary care medical center were invited to complete a voluntary, anonymous questionnaire. a total of 58 surveys were distributed and returned: 22 emergency medicine, 11 family practice, 9 internal medicine, and 16 pediatric residents completed the questionnaire. due to the small sample size, responses from physicians in various disciplines were reviewed as a whole. results: 40 respondents (69%) reported that they had previously prescribed auto-injectable epinephrine for allergic emergencies. the majority (63%) of these prescribers reported that their training was inadequate. 14 respondents (35%) reported no training, while 11 respondents (28%) reported that their training was less than that needed to ensure proficiency. 15 respondents (37%) reported that their training was adequate to ensure proficiency. none reported expertise. only 7 of 40 prescribers (17.5%) reported that either they or their staff always demonstrated proper medication use to the patient. interestingly, 2 of these 7 providers reported they had not been trained on proper use. the table below summarizes resident training and patient education practices. additional physician knowledge deficits included the proper site of medication administration, the proper interval for replacing medication, and the proper place for medication storage. conclusions: of the residents surveyed, who have previously prescribed auto-injectable epinephrine, 63% identified training deficiencies. only 17.5% of prescribers reported that the standard of care requirement to demonstrate proper medication use was always met. there is a clear need to improve auto-injectable epinephrine education in all residency training programs. r. bloebaum 1* , r.k. calabrese 2 , m. 1. houston, tx; 2. new york, ny. introduction: pneumocystis carinii pneumonia (pcp) is a major cause of morbidity and mortality in patients with aids. adverse reactions occur frequently to the most effective medication for both the prevention and treatment of pcp, trimethoprim-sulfamethoxazole (tmp-smx). we looked at the immediate safety and efficacy of three protocols for desensitization in aids patients. methods: by retrospective chart review, we identified 49 patients with aids who had experienced previous mild to moderate hypersensitivity reactions to tmp-smx and required desensitization. patients received one of three desensitization protocols based on illness severity or ward attending preference: a 6-hour intravenous (iv) desensitization, an 8-day oral desensitization, or a 10-day oral desensitization. results: of the 49 patients that received desensitization, 38 (77.5%) completed successfully. of these, 33 subjects had no reaction during the desensitization process; however, seven of these subjects were on steroids for treatment of other diseases. the remaining five successful patients had mild reactions which were treated symptomatically with acetaminophen, antihistamines or both. eleven patients failed to complete the desensitization: six stopped by the attending physician and four dropped out voluntarily. one patient expired during desensitization from extensive disease complications related to the admitting diagnosis. all protocols were equally successful when comparing the immediate success rates, 6/7 (86%) of the 6hour protocol, 4/5 (80%) of the 8-day protocol and 28/37 (75.7%) of the 10day protocol. the 6-hour iv desensitization protocol was most frequently used in the intensive care unit on critically ill patients. two of these patients, counted as successfully desensitized, died 5 and 28 days post protocol completion secondary to extensive comorbid conditions unrelated to the desensitization. conclusion: given the insignificant differences between the success of the 6hour iv desensitization and the oral desensitization protocols, we believe that either may be used effectively. further, in patients with mild to moderate hypersensitivity reactions to tmp-smx, an oral desensitization protocol may be used safely in the outpatient setting if given appropriate lab follow up. a. morales * , e. gonzalez, a. contreras, d. lopez, g. lopez, mexico city, mexico. introduction in 1966 davis describes job syndrome in two women, in 1971 dr buckley reports two children, being known as hyper-ige syndrome (job's syndrome or buckley's syndrome). defined as a primary immunodeficiency, dominant autosomic, characterized by multi-systematic alterations (immunological, skeletal, dermal and dental). it's diagnostic criteria are levels of ige 2000 ui/ml, chronic dermatitis, recurring respiratory infections, cold abscesses, pneumatoceles, infections caused by candida, and finally craniofacial alterations. on another front, extraordinary high levels of ige have been reported in patients with allergic illnesses that increase the risk of anaphylaxis, but do not have the job's syndrome criteria. objetive to determine if allergic patients with ige levels higher than 2000 ui/ml have diagnostic of job's syndrome. material and methods a retrospective revision was realized from may 2001 to april 2004 in files of 27 patients treated in allergy services at the instituto nacional de pediatria with a total ige greater than 2000 ui/ml, by means of a page with recollected dates. results nine women and 18 men were included with an age range of 3 to 18 years, and an average age to 9 years; 22 (81.5%) were diagnosed with allergies; of which 55.6% rhinitis allergy, 51.9% asthma, and 40.7% topical dermatitis of which co-existed in patients. 51.9% presented hereditary antecedents of atopic. the cutaneous tests were positive in 11 (40.7%) with a greater reactivity of dermatophagoids. one syndrome of hyper ige was detected. others diagnosed were found as not allergic were hunter's syndrome and toxocariasis. conclusion there are patients with allergies that have total levels of ige as elevated as 49, 560 ui/ml without correspond to job's syndrome. by which a multi-allergic clinical entity is proposed with levels greater than 2000 ui/ml. introduction home monitoring of lung function in asthmatic patients is used extensively in both clinical and research settings, however, little attention is given to device quality control. objective the purpose of this study was to determine the accuracy, precision and usefulness of the airwatch system (enact health management systems, palo alto, ca, usa). methods the subjects included in this study were submitted to spirometry following american thoracic society guidelines (ats 1995), using collins gs 4g pft system. afterwards, peak expiratory flow rate (pef) and forced expiratory volume in one second (fev1) were determined using airwatch. fev1 and pef measures from both devices were compared, by using two sample t-test and pearson correlation coefficient. results a total of 115 patients (75 females) were enrolled, and their mean age was 43.7 years (9 to 76 years). fev1 measures ranged from 0.54 to 5.81 l (mean 2.43 l), and from 0.62 to 7.44 l (mean 2.51 l), in collins and airwatch, respectively. pef ranged from 107 to 871 l/min (mean 384 l/min) in collins and from 61 to 794 l/min in airwatch (mean 353 l/min). significant difference was noted for pef measures (p< 0.05 and pearson correlation r= 0.61) between the two devices; however, we did not observe this difference when fev1 measures were concerned. regarless the degree of obstruction (high or low flow rates), these results did not change. conclusion airwatch showed great utility for fev1 measures when compared to collins spirometer. although airwatch is able to assess lung function at home, on a daily basis, it is not reliable for pef measures. introduction: epidemiologic data show that poorly controlled asthma is a serious public health problem. the degree of implementation of the naepp guidelines in primary care practice remains to be defined. the objective of this survey was to determine if introduction of an assessment tool into primary care practices along with a specially designed program to implement the guidelines would improve diagnosis and therapy. methods: the asthma care network (acn), a program designed to assist healthcare providers in the assessment and management of their patients with asthma, employs a team of specially trained respiratory care associates (rcas), 100 rns and rts, who visit primary care offices to inform staff about various components of the naepp guidelines and assist in their implementation. .a total of 4901 primary care providers in 2876 sites were recruited as part of the acn program. data from more than 60, 000 patient visits were collected and analyzed between march 2002 and january 2004. the program assessment tool surveyed asthma control and medication prescribing patterns. outcome measures included degree of symptom control, limitation of activity, sleep disruption, use of rescue medication and utilization of urgent care services. these data were collected on an office visit assessment form (ova) completed by both patient and physician. the rcas provided information, education, device training in the use of inhalers and spacers, and a ce course for the staff discussing pathophysiology, assessment and management of asthma. results: a total of 60, 248 ova forms were completed. among all patient including adults (older than 18 years of age) and children (<4-17 years of age), 74% (range 69% to 81%) reported symptoms consistent with lack of asthma control. approximately 70% of the survey group had more than 2 markers of uncontrolled asthma. as a result of this assessment, controller medication use increased by over 30%, 52% of which was an ics-containing medication. conclusion: the information provided to the primary care health care providers resulted in a considerable increase in prescription of controller therapy, and in particular, increased use of ics controller medication consistent with naepp guidelines. background: patients with allergic rhinitis (ar) demonstrate symptoms of allergy to fruits, vegetables and nuts in 48-72% of cases. oral allergy syn-drome (oas), typical for hypersensitivity to plant food, is based on cross-reaction of pollen-allergen specific immunoglobulin e (ige) antibodies with homologous food proteins. the production of th1 and th2 cytokines in allergic rhinitis patients with or without sensitization to food (fruits and vegetables) allergens was assessed. methods: fifty five patients aged 18-38 years with allergic rhinitis were observed. group i -20 patients with ar; group ii -35 patients with ar and oas. sensitivity to pollen allergens was tested by skin prick tests. the allergic reaction to food in patients with oas was proved by a positive history of oral symptoms caused by eating fruits and vegetables and a positive skin prick test with respective food allergens. blood eosinophil counts and total ige levels were determined during the peak of allergic rhinitis symptoms. il-5, il-10 and -interferon levels were measured by elisa. results: 24.5% of patients were sensitized only to grass pollen, 23.3% only to tree pollen and 52.2% reacted to pollens of grasses, trees and weeds. in patients with oas, skin tests were more often positive to birch (57.8%), alder (46.3%), and mugwort (26.7%). the most common food products implicated in oas were hazelnut (69.3%), apple (29.2%), carrot (16.4%), and peanut (12.7%). the allergy to fruits and vegetables was confirmed by positive prick test in 78.4%. during the season blood eosinophil count and total ige levels were elevated in all patients. there was an increase in the production of il-5 to 187.3â±5.4 pcg/ml in group i and to 221.5â±6.2 pcg/ml in group ii (nor-mal=74.3â±3.3 pcg/ml). the levels of il-10 increased to 186â±12 pcg/ml in group i and to 197â±10 pcg/ml in group ii (normal=5.8â±0.25 pcg/ml); the level of -ifn decreased to 287.6â±7.4 pcg/ml in group i and to 272.6â±5.2 in group ii (normal=331â±35 pcg/ml). after sit with pollen allergens the clinical manifestations of allergic rhinitis and oas decreased in 84.6% of patients. conclusion: allergic rhinitis patients' sensitivity to food allergens may cause oas in these patients associated with increased functional activity of th2 responses. patient knowledge and improvement with aller-gen immunotherapy. c.c. randolph * , waterbury, ct. introduction: there is no consensus on objective parameters for improvement in immunotherapy. similarly little is known regarding patient knowledge of immunotherapy. utilizing two published questionnaires (1-2), we assessed clinical improvement based on symptom and medication scoring and knowledge of immunotherapy. methods: the charts of 219 patients of an estimated 530(41%), 109(50%) with allergic rhinitis only and 116 (53%) with concomitant asthma, were retrospectively or prospectively reviewed who had been on immunotherapy to inhalants for 6 months to 6 years, mean 2.7 years, age range 7y-64y, mean 25y, 108 male(49%), 111 female(51%) with 213 caucasian (97%), 2 oriental (1%) and 4(2%) afroamericans .they completed symptom and medication survey (1) every 6 months with range of improvement using a decline in likert scale (+)20 to (-)104, mean 24. 186 (85%) indicated improvement in their symptoms and/or medication .111(50%) completed (2) a 7 question survey of knowledge of immunotherapy. there were 7 questions regarding the outcome of immunotherapy, the years to onset of immunity, the duration until onset of immunity, the danger of immunotherapy and the extract in the vial. the correct responses were recorded to 2/7 (2/111=1.8%), 11(9.9%) 3/7, 19(17%)4/7, 10(9%)5/7, 18(16%)6/7.39(35%) had a perfect response to all questions. 12(11%) had no correct responses. conclusion: the majority of immunotherapy patients improved (85%) by symptom and medication scoring over the mean of 2.7 years but only 35% had complete knowledge of the rationale for immunotherapy. further education and repeated surveying of patients on immunotherapy to assure comprehensive knowledge of immunotherapy and achieve better outcomes is recommended by this investigation. as is unique in this study symptom and medication scoring using the rhinitis /sinusitis questionnaire approved by the acaai and a comprehensive questionnaire assessing knowledge should be conducted periodically ie every 6 months to provide objective parameters for improvement. references: 1.santilli j, nathan r, glassheim j .patient receiving immunotherapy report it is effective as assesses by rhinitis outcomes questionnaire(raq) in private (42)) is a protein involved in the parasite invasion of host erythrocytes and is a leading vaccine candidate for the erythrocyte stage of malaria infection. an increasing number of vaccine clinical trials are being undertaken using various formulations of msp-1(42). comparison of humoral responses among these trials has been limited by the lack of a universal reference standard for specific antibody. the purpose of this study was to develop a human reference standard for msp-1(42) antibody measured in absolute quantity units that could facilitate comparison of interstudy vaccine response. method: we formulated the reference standard by pooling human plasma samples known to contain high titers of msp-1(42) antibody. the specific antibody within the pooled plasma was captured by msp-1(42) adsorbed to nickel resin in a process of immobilized metal affinity chromatography (imac). the intact msp-1(42) antibody-antigen complexes were separated from the nickel resin and total igg in the complexes measured by enzyme-linked immunosorbent assay (elisa). results: our antibody quantitation method yielded a concentration of 48.3 mcg/ml of msp-1(42) antibody in the reference standard. conclusion: the reference standard characterized in this study may be useful as a quantitative working standard for msp-1(42) antibody response in future vaccine clinical trials involving msp-1(42). this standardization may facilitate the clinical development of msp-1(42) as a candidate vaccine for malaria infection. background. specific immunotherapy (sit) is currently one of the most effective and widely used treatment methods of allergic diseases including asthma. efficacy of sit depends on the correct choice of patients, severity of asthma and patient's condition when the sit is begun. according to who recommendations, sit is approved for use in patients with mild to moderate asthma. methods. sit with a saline extract of house dust allergen was administered to 30 patients with mild persistent allergic asthma. injections were performed subcutaneously 2-3 per day. the course consisted of 6766 pnu of allergen and lasted 12-14 days. patients were repeatedly surveyed at 3, 6 and 12 months after sit was completed. dyspnea attacks occurring during daytime and at night were assessed, as was the influence of physical exertion on dyspnea and lung function, and also the number of utilizations of short-acting beta-agonists per day. pulmonary function measurements were performed as was assessment of concomitant allergic rhinitis. 50% of patients received treatment including cromones, and 30% utilized inhaled corticosteroids among whom 13.3% had a dose of 400 mcg per day and 16.7% 200 mcg per day. sit efficacy was assessed by clinical symptoms, pulmonary function measurements and amount of concomitant therapy received. results. the number of daytime dyspnea attacks reduced from 10.70 â± 3.40 per month to 1.20 â± 0.09 during the 3 months following sit, and 76.7% of patients reduced concomitant asthma therapy. at 6 months, dyspnea attacks increased to 2.90 â± 0.75 per month, still significantly less then prior to sit. nocturnal symptoms followed the same pattern, occurring 7.20 â± 1.62 per month prior to sit, 1.01 â± 0.01 per month 3 months after sit and 1.99 â± 0.25 per month at the end of the sixth month after sit. 26.7% of patients had no symptoms of bronchial obstruction during the 6 months after sit and their pulmonary function approached normal values, although 36.7% of patients returned to asthma therapy. nasal congestion decreased from 3.28 â± 1.20 points to 1.98 â± 0.08 (p<0.05), clinical improvement still present for 3 months after sit, but recurring at 6 months after sit. conclusion. sit may be an effective treatment method that improves both asthma and allergic rhinitis for more than 3 months following a brief course, allowing a decrease in the amount of symptomatic treatment. a safe therapeutic vaccine that can alter the allergic response to peanuts would serve a serious unmet medical need. peanut allergy responses are largely associated with downstream events related to antigen specific ige crosslinking of ige receptors and subsequent degranulation of mast cells and basophils. our clinical studies with ragweed have shown that linking immunostimulatory dna (iss) to allergens can decrease ige recognition of the allergen and generate an immunogen that generates protective th1 responses and reduces harmful th2 responses. to test this approach to peanut immunotherapy, we focused on the clinically relevant allergen, ara h 2, as a proof of concept. iss oligonucleotides were linked to ara h 2 at two different ratios: pic (2 iss per protein) and hpic (4 iss per protein). immunogenicity of pic and hpic was evaluated in c3h/hej female mice immunized twice with 5 ug of pic, hpic, or ara h 2. sera were analyzed for anti-ara h 2 igg1 and igg2a responses. spleens were harvested and ara h 2-specific ifng and il-5 responses were measured in vitro. mice were immunized with ara h 2 elicited predominantly igg1 and il-5 responses, indicative of a th2-type response. animals immunized with pic showed significantly enhanced igg2a responses and strong ifng responses, indicative of th1-type responses. hpic immunized mice elicited little antibody response, presumably due to iss blocking b cell epitopes, but did induce ifng responses. to assess allergenicity of pic and hpic, histamine release was measured in blood from peanut allergic donors treated in vitro with pic, hpic, and ara h 2. histamine release was detected at very low concentrations of ara h 2 (0. 01 ng/ml), 10-fold higher concentrations for pic (0.1 ng/ml) and was undetectable at concentrations up to 1000fold higher for hpic (10 ng/ml). an ige binding competition assay also confirmed reduction in allergenicity following the same trend for the iss-linked allergens. linking iss to ara h 2 increases th1 responses to the allergen, blocking ige recognition of epitopes on ara h 2. thus, iss-linked ara h 2 appears to be a promising candidate for a safe immunotherapy product for treating peanut allergic subjects. introduction: immunotherapy has been studied for its adverse effects in some sensitive patients as with worsening of therapeutic response in some amounting to withdrawal of the later. methods: in the allergy center therapeutic trials of immunotherapy(house dust, mixmite) had been undertaken since 1998, in therapeutic dose of 0.02pnu/ml, 0.2pnu/ml, 2pnu/ml, 20pnu/ml, 200pnu/ml2000pnu/ml, 4000pnu/ml(housedust), 0.01au/ml, 0.1au/ml, 1au/ml10au/ml 100au/ml 1000au/ml 2000au/ml(mixmite).patients age 2-65 years, both sex after diagnostic scratch/prick tests with appropriate antigenic preparations reported for local/systemic adverse effects the details of which were as under, results: please see the attached table for details, in some cases worsening of the existing allergic status was noted, which on analysis revealed initially a slow rise in the allergen -specific ige to be later on fol-lowed by rise of igg1 & igg4 & gradual fall of ige.only 1/100 patient could not complete immunotherapy for fear of adverse effects. conclusions: with utmost care/follow-up, no incidence of mortality outcome was reported from 1998to late 2003 the incidence of adverse effects were significantly lowered on pre-medication with anti-histamines significantly more with elderly than younger age. even with all these documented hazards the beneficial effects far exceeded the harmful effects. background: the prevalence of allergic disease in most human population is steadily increasing. seafood allergy is a serious food allergy, although hypersensitive reactions caused by seafood has long been know, biochemical and immunological studies on seafood allergies had only begun lately. shrimp and abalone are the most frequently reported causes early asthmatic response. objective: to investigate cross-reactivity of shrimp, abalone and derp1. methods: shrimp and abalone extracts were prepare from raw seafood. sera from 17 patients from hongkong were studied who had asthma after consumption of seafood. ige elisa analyses comfirmed the combined sensitization to shrimp, abalone and derp1. specific-ige elisa assays were accomplished for shrimp and abalone extracts inhibited by derp1 and derp1 elisa and immunoblot assays inhibited by shrimp and abalone extracts. results: elisa inhibition showed that most ige antibodies against shrimp and abalone were cross-reactive with derp1 and the same time, derp1 elisa was inhibition by shrimp and abalone extract. the elisa inhibition percent (%)of shrimp extract (gm:53.28%) and abalone extract(gm:36.75%) by derp1 were significantly higher than derp1 by shrimp extract(gm:26.64%) and abalone extract (gm:17.59%). (p<0.01). furthermore, and there was a significant correlation of elisa inhibition percent between shrimp extract, abalone extract and derp1 inhibited by each other; sds-page and immunoblot of shrimp and abalone is the 38 and 49kd allergen respectively. conclusion: this indicates that derp1 was the sensitive agent. shrimp, abalone and derp1 demonstrate significant cross-reactivity. these findings confirm that the primary crossreactive allergen of shrimp and abalone is the 38 and 49kd allergen respectively. b. sun 1* , a. wu 2 , n. zhong 1 , 1. guangzhou, china; 2. hong kong. background: the house dust mites (dermatophagoides farinae (derf) are a major source of aeroallergens implicated in the expression of atopic disorders, including asthma, allergic rhinitis, and atopic dermatitis . in particular, strong circumstantial evidence suggests that house dust mites antigens are important precipitating factors of asthma. many house duse mite allergens are proteases that can elicit airway inflammation by stimulating the release of cytokines in bronchial epithelial cells. objectives: we have investigated whether der f allergen proteases induced cytokine production from the epithelial cell line beas-2b. methods: cells were exposed to four different concentrations with serial additions of der f (0.02, 0.2, 2, 20ug/ml) were incubated for 24h to 96h. and compare with those without incubation of allergen. cytokine in the supernatants were assayed by elisa, reverse transcription?pcr was also performed. results: cells treated with der f allergen showed serial changes in the cohesiveness of the monolayer. there was a significant increase in the level of cytokine production compared with the untreaed sample. statistically significantly increased with addition of der f caused the release of il-6 and il-8 in time and concentration-dependent manner (p<0.05, respectively). levels of il-6 and il-8 were elevated 24 h and 48 h after allergen exposure, increasing with time, continued increased levels to be present of il-6 and il-8 in the supernatants at 72 h and 96 h. at the same time show the concentration dependence of induction of il-6 and il-8 expression as well as an increase in the expression of il-6 and il-8mrna. conclusion: hdm-induced airway inflammation may include der f-mediated release of inflammatory mediators, and the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium. suggesting that il-6 and il-8 production by bronchial epithelial cells may play a role in the pathogenesis of allergic asthma. the purpose of this study is to delineate the immune injury mechanisms that involved in the autoimmune inner ear disease by introducing plasmid dna encoding of th1 cytokines (inf-g) into the inner ear. b-tubulin is a microtubular protein which we found as an important autoantigenic in meniere's disease as well as other autoimmune hearing loss. hearing loss was induced in mice and guinea pigs when they are immunized with the tubulin molecules. autoimmune hearing loss could be the results of th1 cytokine responses from autoimmune injury. to test the hypothesis, guinea pigs were immunized with 200mg of tubulin in cfa and boosted once more. two weeks later, we introduced 100ug (5ul) of naked dna encoding inf-g was injected into the left side inner ear through round window. same volume of 0.1m pbs was injected into right side as control. abr was recorded before and after the injection. 15 weeks after the injection, the animals were sacrificed and temporal bones were examined with h-e and inf-g immunocytochemical staining. the ears injected with the plasmid dna-inf-g had an enhanced hearing loss (30 db), and degeneration of the spiral ganglion was found in these ears. however, the injection of the naked dna encoding inf-g did not change the expression of the inf-g in the inner ears. these results suggest that autoimmune hearing loss could be the result of th1 responses to inner ear autoantigens. -tubulin is an important molecule in the hair cells supporting cells within the sensory epithelium of organ of corti and found to be an auto autoantigen in autoimmune hearing loss including mã¨niã©re's disease. the object of the study is to induce hearing loss in mice with varying doses of antigen and evaluate the pathogenesis of autoimmune hearing loss induced by -tubulin in mice. mice were immunized with 100, 200 or 300 î¼g of -tubulin and hearing was evaluated by auditory brainstem responses (abr) and distortion product of otoaccoustic emission (dpoae). all mice had hearing loss by abr and dpoae tests and morphological study of temporal bone showed spiral ganglion degeneration and tunel staining positive cells were noted in these immunized mice. thus this study showed that -tubulin is an autoantigen for hearing loss in animal model as in human autoimmune hearing loss patients including mã¨niã©re's disease. supported by nih r0dc005010-01 introduction: oral polio vaccination (opv) in the united states is currently being replaced with inactivated polio vaccination (ipv) given parentrally. while past studies have looked into the relationship of vaccination and asthma prevalence, none have investigated this relationship with regards to vaccination route namely orally versus parentrally. this study investigates the relationship between vaccination rates for the live attenuated orally administered polio vaccine and parentrally administered vaccines(dtp, mmr)and two potentially dependent factors; asthma prevalence rate and asthma-caused death rate. methods: we looked at data from the national center for health statistics yearly publications of health, united states (1983 -1999 and the morbidity and mortality weekly report surveillance summaries . two databases were compiled to cover the 0-4 age population in the united states since this is the primary period of childhood vaccination. one database contained information for asthma related deaths and vaccination rate (dtp, mmr, opv) covering the years 1970-1999 and the other database compiled information for self reported asthma prevalence and vaccination rates(dtp, measles, rubella, opv) covering the period 1983-1999. a t-test was used for statistical analysis. results: statistically significant correlation was found between vaccination rates and asthma prevalence rates. data for dtp (p = 0.012), measles (p = 0.024), and rubella (p = 0.023) indicated a statistically significant positive correlation with asthma prevalence. the oral polio vaccine was the only one of the vaccines that failed to display a significant relationship with asthma prevalence rates (p = 0.133). statistical analysis proved that a correlation between vaccination rates of united states children ages 0-4 and asthma-caused deaths was insignificant (p > 0.05). conclusions: the opv, which is administered orally rather than parentrally, displayed no relationship with asthma prevalence. this could be due to the fact that live attenuated orally administrated polio vaccine may induce mucosal immunity, simulating a normal pathogen route of entry into the body. childhood vaccination had no relationship with asthma-caused death rates. a.s. alfrayh * , riyadh, saudi arabia. introduction: heredity plays a major role in asthma and other allergic diseases, mechanisms underlying the inheritance of these disorders are poorly understood. this study therefore analyzed the risk conferred by family history of asthma and atopy for having childhood asthma. methods : a total of 1601 children between 1-16 yrs selected randomly in three cities in saudi arabia ( hail, taif and gizan )in 1995-1996 . the questionnaire which is similar to the one used in the international study of allergy and asthma in childhood isaac. were self administerd under medical supervision.apart from the demogrphic details, the questionnaire included questions on symptoms and physician diagnosis of asthma, rhinitis, eczema and family history of these conditions. the family members were grouped as immediate family and relatives. asthma and atopy were defined as ever having had physician diagnosis of such conditions information was also available about exposure to cigarette somke at least one member was a smoker in the household and having pets. relative risk for developing asthma was estimated in terms of odds ratio by bivariate analyses using chi square test and p value was considered significant when less than 0.05. results : history of asthma in the immediate family and relatives conferred a 4 fold and 3 fold risk for development of childhood asthma odd ratio ( or )=4.2, 95% confidence interval(ci)=3.3 to 5.5, p=0.00001 and or=3.3, 95%ci=2.5 to 4.3, p=0.00001 respectively. rhinitis in immediate family and the relatives was associated with 3 folds increased risk for childhood asthma or = 3.0, 95% ci = 2.2 to 4.0, p=0.0001 respectively whereas history of eczema conferred over 3 folds risk or=3.4, 95% ci = 2.4 to 4.7, p=0.00001 for childhood asthma when present only in the immediate family. history of eczema in relatives was not associated with any risk. of the environmental factors, exposure to cigarette smoke conferred 2 folds risk of developing childhood asthma or = 2.6, 95% ci = 2.0 to 3.3, p= 0.0001, whereas exposure to pets was not a significant risk foctor. conclusion : presence of asthma and atopy either in the biological parents or relatives constitute a significant risk for childhood asthma.paricularly in the presence of evironmental risk factors. the murine local lymph node assay (llna) has been developed as an alternative to guinea pig models for the assessment of the contact sensitization potential. however, there is a need to develop a non-radioisotopic endpoint for the llna, because of the radioisotopic method's requiring the use of special facilities. in this study, we investigated to evaluate the lymphocyte subpopulations in the lymph node cells following allergen and irritant treatment. female balb/c mice were treated by the topical application on the dorsum of both ears with sensitizers, 2, 4-dinitrochlorobenzene (dncb), toluene diisocyanate (tdi), and a-hexylcinnamaldehyde (hca), and an irritant, sodium lauryl sulfate (sls), once daily for three consecutive days. the lymph node (ln) cells were harvested 72h after the final treatment. phenotypic analysis of lymphocytes subsets was performed with a flow cytometry. the allergens dncb, tdi, and hca and an irritant, sls increased cell number compared to the vehicle. there was an increase in the percentage of b220+ cells in mice treated with dncb and tdi compared to the vehicle control, but not in those treated with sls. mice were treated with dncb, hca and tdi showed a preferential increase in the percentage of b220+cd40+ cells compared with vehicle and irritant-treated mice. there was an increase in b220+cd86+ cells of mice treated with dncb, tdi and hca, but no significant increases were observed in mice treated with sls. mice were treated with dncb, and tdi showed an increase in the percentage of b220+cd23+ cells compared with vehicle and irritant-treated mice. these results suggest that analysis of b cell activation marker, cd40 on b cells may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice. m. frieri * , y.c. huang, east meadow, ny. introduction: nitric oxide (no) is an important biomarker for inflammation in airway epithelial cells and in exhaled breath of asthmatic subjects. we have previously demonstrated no production in antigen-stimulated human bronchial epithelial cells and an effect of omalizumab (monoclonal anti-ige antibody) on no production in those cells [leyko bt et al. j allergy clin immunol 2004; 113(suppl 193) :a668]. in this study, we investigated the potential role of ige and its receptors on a549 cells by characterizing the effect of omalizumab on antigen, egf, and il-1 stimulation of a549 cells in a medium containing atopic serum. methods: a549 human alveolar epithelial cells were stimulated with 100 iu/ml of il-1 , 10 î¼g/ml of ragweed (ra), 1000 au of dust mite (dm), and 40 ng/ml of egf, and exposed to either 10 -7 m budesonide or 0.1 î¼g/ml omalizumab for 6 or 24 hours. no production was measured in duplicate by a highly sensitive elisa. results: omalizumab but not budesonide inhibited no production at 6 hours in a549 cells stimulated with il-1 (17-13î¼m, p<.05)and il-1 +dm (18-14î¼m, p=.08), but not with ra alone. however, at 24 hours omalizumab and budesonide each significantly inhibited no production stimulated by il-1 (29-9î¼m; 29-17î¼m), il-1 +dm (22-12î¼m; 20-14î¼m), and egf (17-9î¼m; 17-13î¼m) (p<.05). conclusion: no production is a marker for inflammation. omalizumab demonstrated a significant anti-inflammatory effect in distal alveolar cells by inhibiting no production stimulated by antigen, il-1 , and egf in a medium containing atopic serum. as persistent inflammation in asthma may play a role in airway remodeling, treatment with omalizumab may have a beneficial effect on chronic airway inflammation in patients with asthma. oral tolerance trigger regulatory mechanisms able to down-modulate antigen-specific t and b cell response. to address the lasting effect of several regimens of oral tolerance to ovalbumin, in naive or antigen primed mice, b-cell function has been focused. furthermore, we analyze specific antibody response up to eight months of post-immunization, proliferative response, b7 (cd80/cd86) expression on b cells and t cell ctla-4 involvement in oral tolerance. a/sn mice were immunized by intraperitoneal route with 50î¼g of ova/0, 1 mg-alum and boost 10 days after priming (dap). ova feeding was done with 25mg at different days before or after antigen priming. in others protocols, mice were fed twice, before and after priming with a total of 50mg of ova. all groups were boosted at 60 or 120 or 180 or 240 dap. the results showed that only mice fed at naive status and those fed twice before and after immunization demonstrated a long lasting of ige ab response inhibition up to 8 months of immunization. these mice showed a marked inhibition of antigen-specific proliferative response that was restored with anti-cd28 mab in vitro stimulation. evaluation by flow cytometry of spleen cells cultured for 48 hours upon ova stimulation, showed an important decrease of b 7.2 expres-sion on b cells of naive fed mice, which remained inhibited until 8 months of immunization. after addition of anti-ctla-4 mab an enhancement of b7.2 expression was detected on b cells of naive fed mice. ctla-4 molecules expression on cd4+ t cells of naive fed mice remained unchanged following ova stimulation while a peak of expression was detected at 96h of ova stimulation in control group. this finding reinforce the t cell anergic status of naive fed mice, due to the less cell division and consequently a low rate of cd4+/cd44high activated cells. the results showed that antigen feeding before immunization induce a long lasting anergy mediated by an impaired t-b cell cooperation and ab production due to the decrease of costimulatory molecules expression on b cells and negative signaling effects by ctla-4 expression. introduction: vascular endothelial growth factor (vegf), basic fibroblast growth factor (bfgf) and fibronectin (fn) can promote angiogenesis, a putative component of airway remodeling. (s)-albuterol can exacerbate airway hyperresponsiveness, bronchospasm and release pro-inflammatory cytokines from small airway and smooth muscle cells. our study evaluated the effects of (s)-albuterol ((s))-and (r)-albuterol ((r)) on secretion of these factors on normal human lung fibroblasts (nhlf) and myofibroblasts (myonhlf) in the presence or absence of tgf 2, il-1 , or allergens. methods: nhlf were stimulated to differentiate to myonhlf with 1000 pg/ml tgf . dose-dependent effects of (s) and (r) [10 -8 to 10 -4 m] were evaluated for secretion of vegf, bfgf and fn by nhlf and myonhlf with and without 1000 au/ml d. pteronyssinus (dp), 1000 pnu/ml ragweed (ra), 100 u/ml il-1 , or 1000 pg/ml tgf 2 into serum-free media (its) at 37 o c with 5% co 2 collected at 24 hr and assayed by elisa. results: in nhlf the following was observed: vegf secretion was 2-fold higher with 10 -7 m (r) relative to (s), p<0.05; bfgf secretion was increased 50%-100% by 10 -5 m (s) relative to (r), p<0.05. a lower concentration of (s) (10 -6 m) in the presence of either dp or il-1 caused a 2-fold increase in secretion of bfgf relative to (r), p=0.02. in the presence of myonhlf the following was observed: 10 -7 and 10 -5 m (s) caused a 50%-100% increase in secretion of fn relative to (r). at 10 -7 m (s), this effect was further increased with the addition of il-1 , p=0.022). conclusion: in a dose-dependent manner, (s)-albuterol stimulated the release of bfgf and fn by nhlf and myonhlf, respectively. this was enhanced by dust mite and/or il-1 , potentially contributing to the matrix remodeling observed in chronic asthma. vegf over-expression can have a protective effect against chronic hypoxia and can recruit immune cells to the alveoli. increased vegf by (r)-albuterol could contribute to such an effect in vivo in asthmatics. bfgf, in bal and sputum of asthmatics, and fn which contributes to subepithelial fibrosis, can promote angiogenesis. increased bfgf and fn by (s)-albuterol could be detrimental over time by enhancing matrix deposition and remodeling in a subset of asthmatics. o. ozdemir 1* , c. moore 2 , y. ravindranath 1 , s. savasan 1 , 1. detroit, mi; 2. new orleans, la. background: mast cells (mc) have been shown to demonstrate natural cytotoxicity against mouse fibrosarcoma cell line in culture when incubated for 24-48h. this effect has been postulated to be mediated through soluble and/or membranous tnf-. more recently; fasl, mc chymase and serine protease granzyme h with its chymase activity were proposed as mediators of mast cell-mediated cytotoxicity. thus, both 'granule-exocytosis' through chymase, granzyme h and soluble tnf-and 'death receptors' through membrane-bound tnf-and fasl pathways appear to be operative in this process. aims: following our earlier observations on long-term liquid culture-grown human bone marrow mast cell cytotoxicity against human leukemia cells in 24-48h co-incubation experiments, we investigated mast cell-mediated cytotoxicity against natural killer/lymphokine-activated killer cell-sensitive cells in short term (12h) cultures without any stimulation for the first time. methods: human bone marrow mononuclear cells were cultured in methyl cellulose supported with il-3, il-6 and scf. mast cell colonies that developed in six weeks were transferred to liquid medium and maintained for 18 weeks before experiments. cytotoxicity was investigated against k562, raji and daudi cells at 12, 24 and 48 hours of co-incubation by our established flow cytometric cell-mediated cytotoxicity assay. results: after 12h co-incubation, 61% (11% early apoptotic and 50% late apoptotic or necrotic death) and 29% (21% early apoptotic and 8 % late apoptotic or necrotic death) target cell kill was demonstrated in daudi and raji cells, respectively. daudi cell killing has stayed stable at 24h (67%; 27% early apoptotic and 40% late apoptotic). despite a small numbers of experiments, daudi cell kill was statistically significant at 12h (p:0.011) and 24h (p:0.011) compared to control. however, k562 cell elimination (18%) has not occurred until 48h. mast cell-daudi cell conjugates were seen on the wright/giemsa slides (figure) . conclusion: our results demonstrate that human mc can cause cell-mediated cytotoxicity against certain cells in relatively short-term. this further suggests possible contribution of 'granule-exocytosis' pathway to mc natural cytotoxicity, indicating a faster mc response in immune surveillance. o. ozdemir 1* , m. buyukavci 2 , y. ravindranath 1 , s. savasan 1 , 1. detroit, mi; 2. erzurum, turkey. background: the effect of melatonin (mlt) on cellular immunity has been controversial. recently, mlt has been demonstrated to activate t and nk cells through its membrane or nuclear high affinity receptors. it was also shown that pharmacological concentrations of mlt (>nm) could be cytotoxic against different human cancers. aims: our aim was to investigate the effect of mlt alone or in combination with il-2 on peripheral blood lymphocytes (pbl), lymphokine activated killer (lak) cell generation and its cytotoxicity. methods: pbl were cultured for 7 days in the presence of mlt at different concentrations (10 -3 , 10 -5 , 10 -7 m) with or without il-2 (100 u/ml). cell viability was determined by trypan blue exclusion test. cell-mediated cytotoxicity of lymphocytes/lak cells against k-562 and daudi cells was studied using our established flow cytometric cell-mediated cytotoxicity assay. results: although 10 -3 m concentration of mlt did not affect cell proliferation much on day 3, it significantly inhibited proliferation by day 7 (p<0.05) consistent with known anti-proliferative effect of mlt. 10 -5 and 10 -7 m concentration of mlt also mildly inhibited proliferation on day 3; however there was a minimal rebound with 10 -7 m concentration by day 7. consistent with the reported mlt-treated pbl's reduced response to mitogens, il-2 and mlt (10 -3 m) combination suppressed proliferation on days 3 and 7; however, with 10 -5 m mlt concentration pbl counts increased gradually from day 3 to 7. although mlt treatment alone did not enhance cell-mediated cytotoxicity, il-2 and mlt combinational treatment at both concentrations (10 -3 and 10 -5 m) increased it significantly compared to baseline activity (figure) . conclusion: il-2 and mlt combination at 10 -5 m concentration resulted in superior lymphocyte proliferation and lak cell-mediated leukemia cell kill. mlt-induced increase in il-2r-expression of pbl shown earlier might be the mechanism for our observations. mlt can be considered in immunotherapy as an adjunct to il-2 treatment. a.e. fusaro * , j.r. victor, c.r. oliveira, c.a. brito, e.a. futata, m. maciel, a.j. duarte, m.n. sato, sã£o paulo, brazil. the maternal exposure to allergens during pregnancy or even in postnatal period may influence the allergy onset to newborns, through antigen or antibody transmission. we sought to verify the effect of maternal antigen exposure before conception, during gestation or in the breastfeeding period on the offspring type i hypersensitivity response. female balb/c mice were immunized or not with ova extract/alum, boosted twice at 10th and 20 th and mated with normal balb/c male on the 21th day after sensitization (das). others groups of immunized mothers also received oral administration with ova along pregnancy, or non-immunized mothers received ova only during breastfeeding. offspring from immunized or normal mice were immunized intraperitoneally with ova at 25 do and boosted on the 10th das. the results showed a important decreased of tgf-levels in the amniotic fluid and milk from immunized mothers before mating in comparison to obtained from normal mothers. ova exposure during pregnancy of immunized mothers decrease significantly the transference of tgf-by breastfeeding, while both tgf-isoforms were founded at high levels in the amniotic fluid. similar levels of tgf-transference by placenta to the newborn was detected in both immunized mother groups. pups from mothers exposed with ag during pregnancy showed an increased spleen cell number, whereas did not produced il-2, il-4, il-10 e ifn-secretion induced by antigen neither altered responsiveness to anti-cd3 or mitogen. maternal ova-immunization induced a marked inhibition of spe-cific ige antibody response in the immunized pups, contrasting to the enhancement of ige responsiveness detected in the immunized pups from mothers which were exposed to ova only at postnatal period. these results showed that preconceptional immunization exert a protective effect on the offspring ige development and an exacerbation of ige responsiveness due to mother antigen exposure during breastfeeding. the findings suggest that rather than in utero antigen priming occurrence, postnatal period may contribute to offspring early life sensitization. financial support: fapesp and lim56-fmusp introduction: the reasons for the increased incidence of allergic diseases in westernized countries are still unknown. mercury is an important pollution factor to which humans are increasingly exposed. prior studies on the effect of mercury on mitogen stimulated human lymphocytes indicated a th2weighted immune response, but the results were inconsistent and difficult to reproduce. phorbol myristate acetate (pma) is a direct activator of protein kinase c, which has been shown to play a role in mercury induced il-4 production in animals. therefore we investigated the effect of mercury on pmaactivated human peripheral blood mononuclear cells (pbmc). methods: pbmc from 8 individuals were cultured for 4 days in culture medium containing pma and ionomycin in the presence or absence of mercuric chloride (hgcl2). il-4 and gamma-ifn concentrations were measured by elisa of culture supernatants. cell death and apoptosis were determined by 7-aad and annexin staining and fluorescence activated cell sorting (facs). cell-proliferation was assessed by 3h-thymidin-incorporation. results: after 4 days of culture, pma/ionomycin-stimulated a small amount of il-4 compared to untreated pbmc (1.7 +/-0.92 pg/ml versus 0.36 +/-1.0 pg/ml). however, mercury induced a more than 30 fold increase in il-4 production when added to pma-activated cells (57.4 +/-45.2 pg/ml, p<0.01). gamma ifn production was strongly increased in pbmc that were treated with pma/ionomycin (>3500 pg/ml versus 417 +/-1045 pg/ml in unstimulated cells) but dropped markedly in cells treated with mercury plus pma/ionomycin. in addition, mercury induced increased cell death, apoptosis and reduced cell proliferation. conclusions: hgcl2 strongly stimulates il-4 production in pma/ionomycin treated pbmc while cell viability and gamma-ifn production drop significantly. these preliminary results suggest that human exposure to mercury may be playing a role in the observed increased incidence of allergic disease in the industrialized world background: mast cells (mc) have been shown to induce natural cellmediated cytotoxicity in long-term (24-48 hrs.) in vitro assay systems. the cytotoxicity is mediated by at least two pathways: secretory via exocytosis of mc granules containing serine proteases such as granzymes, chymase and soluble tnf-and nonsecretory (cell-to-cell contact) via membranous tnfand fasl. chymase induces apoptosis in neonatal rat cardiomyocytes and human vascular smooth muscle cells. the objective of this study was to investigate mc mediated cytotoxicity against nk/lak-sensitive cells in short term (12 hrs.) unstimulated cultures. methods: human bone marrow mononuclear cells were cultured in methylcellulose supported with il-3, il-6, and stem cell factor. mast cell colonies developed at 6 weeks and were transferred to liquid imdm and maintained for 18 weeks. a flow cytometric cytotoxicity assay was used to determine cytotoxicity against daudi, raji, and k562 cell lines at 12 hrs., 24 hrs., and 48 hours. the controls consisted of cell lines without mast cells at 12 hrs., 24 hrs., and 48 hours. results: rationale: to establish the antibody response rate in children with recurrent infections and fully immunized with the pneumococcal 7-valent conjugate vaccine. methods: we have analyzed 39 patients referred to our clinic with recurrent infections despite complete immunization with the pneumococcal 7-valent conjugate vaccine for age, according to acip guidelines. we assessed the patients by checking their immunization status and the antibody titers to all 7 streptococcus pneumoniae serotypes included in the vaccine (4, 6b, 9v, 14, 18c, 19f, 23f) assessed by standardized elisa. the patients were assembled into 3 groups, a non-immunized group with laboratory data prior to the vaccine, and an immunized group consisting of responders and nonresponders according to their antibody titer (<1.3 or >1.3 iu/ml respectively). the data were analyzed using epi info and spss. results: the mean age was 3.3 years for non-responders and 3.8 years for responders. there was no significant statistical difference between the groups regarding age, race and sex. ten patients were identified who failed to respond to all 7 serotypes included in the pneumococcal 7-valent conjugate vaccine. there was no significant statistical difference between the non-immunized and the immunized nonresponders to all 7 serotypes. conclusions: we have identified a special immunological phenotype of specific antibody deficiency (sad) patients with normal total immunoglobulins and normal responses to protein antigens, but who failed to respond to conjugate pneumococcal polysaccharides. a. yates 1* , r. deshazo 1 , j. butler 1 , g. howell 1 , j. farley 1 , h. liu 1 , n. nanayakkura 2 , g.b.yi 1 , r. rockhold 1 , 1. jackson, ms; 2. oxford, ms. introduction: venom from s. invicta consists of 95% disubstituted piperidine alkaloids and is toxic to insects, birds and farm animals. recent reports of morbidity and mortality in elderly patients after massive fire ant stings suggest the potential for systemic mammalian toxicity. we evaluated the toxic responses to systemic administration of two structurally verified, synthetic s. invicta venom alkaloids, solenopsin a (trans-2-methyl-6-n-undecylpiperidine) and its cis-isomer, isosolenopsin a, in rats. methods: sprague dawley rats were anesthetized with isoflurane, paralyzed with gallamine, artificially ventilated and instrumented to record arterial blood pressure (bp; mm hg), heart rate (hr; bpm) and % change in left ventricular contractility (lvc; p/ t). in addition, a group of rats was chronically instrumented to record bp and hr while the animals were conscious and freely-moving. results: solenopsin a at 3 to 30 mg/kg iv dose-dependently lowered bp, hr and lvc. at 30 mg/kg iv, hypotension (-40â±12 mmhg), bradycardia (-27â±8 bpm) and decreased lvc (-41â±17 p/ t) were marked. isosolenopsin a, 15 mg/kg iv, produced responses similar to solenopsin a 15 mg/kg iv. solenopsin a 30 mg/kg iv elicited tonic-clonic convulsions and respiratory arrest in conscious, freely-moving rats. hematuria was seen with solenopsin a, but not isosolenopsin a. superfusion of a working, isolated, perfused rat heart with 10 um of solenopsin a elicited a marked, reversible decrease in lvc, and cardiac arrest occurred with 100 um of solenopsin a. conclusion: the results demonstrate that these alkaloids possess significant depressant activity on the cardiac and respiratory systems of rats. the neurologic and cardiorespiratory effects can account for lethality to small mammals in the wild, and may contribute to adverse cardiovascular effects noted in humans after massive fire ant stings. introduction: during asthma attacks, the ph of exhaled breath condensate (ebc) decreases two log orders (ph=5.0), returning to normal levels after corticosteroid therapy (ph=7.0). ion channels, once thought to participate only in the transport of ions, are now suspected of mediating airway inflammation in asthma as well. to determine whether allergen directly alters airway mucosal ph and ion function, we measured nasal ph and nasal potential difference (pd) before and after nasal allergen challenge (nac). methods: ten allergic rhinitic subjects (meanâ±sem age 28.3 yearsâ±2.9, 7 females) underwent a crossover, single-blinded, placebo-controlled study, where they were challenged with allergen (dust mite, grass, cat) and control diluent in two different occasions in random order via nasal spray. nasal ph was measured on the surface of nasal mucosa with a ph probe. nasal mucosa pd was measured between nasal mucosa and forearm skin. subjects also filled out a nasal symptom scale. measurements were taken at baseline, 1 hour, 4 hours, and 24 hours after nac. results: the nasal symptom score increased significantly immediately after allergen compared to control challenge (13.0â±2.4 vs. 3.7â±1.1 respectively, p=0.003). there were no statistically significant differences in the nasal ph at 1h and 4h, but a significant decrease at 24h compared with baseline (-0.2â±0.1 vs. +0.14â±0.1, p=0.02). the change in ph from 0h to 24h correlated significantly and inversely with change in symptoms (r=-0.59, p=0.007) and number of sneezes (r=-0.65, p=0.002) after challenges. nasal pd did not change significantly after challenges. conclusion: allergen decreases nasal mucosal ph in the very late phase after challenge in allergic rhinitic subjects. decrease in airway mucosal ph during asthma exacerbations may be caused by aggravation of allergic inflammation. nasal potential difference does not change after allergen challenge. funding: niaid, acaai foundation introduction: uv induces differentiation of t-and b-lymphocytes, suppresses natural killer cells, renders a tolerogenic effect and induces apoptosis resulting in local and system immunosuppression. methods: lymphocytes were studied using indirect immunofluorescence methods employing monoclonal antibodies to cd-markers cd4, cd8, cd16, cd25, cd95, hla-i, and hla-ii, from the blood of 14 volunteers (aged 18-24 years) before uv exposure (control), after an exposure of blood in vitro to a dose 1.12 kj, and after 24 hours of exposure of the medial surface of the elbow joint in vivo (s=200 cm2), to a dose of 1.12 kj. results: the direct exposure of blood results in a significant reduction in the number of lymphocytes, probably due to direct phototoxic effects. significant modifications both of the aggregate number of lymphocytes and amount of t-helpers after uv exposure of the skin were not seen. after uv the number of cells which express the marker cd8 is sig-nificantly increased. exposure to uv in vitro induces reduced number of cells bearing cd16. however on in vivo exposure, a significant increase in number of cells which express cd16 was observed. uv exposure of blood reduced significantly the hla-i (9.67â±0.72 reduced to 0.32â±0.54) and hla-ii (dr) (21.56â±1.19 reduced to 18.45â±1.15) expression. uv exposure of skin increased significantly hla-i to 11.56â±0.81, while hla-ii (dr) was insignificantly decreased to 20.65â±2.01. cd95 expression increased from 22.43â±1.12 to 25.95â±1.08 due to blood uv exposure, while there were insignificant reductions in cd4 (39.33â±2.14 reduced to 36.62â±2.34) and cd8 (36.50â±2.03 reduced to 25.75â±2.74) expression. conclusion: uv starts a cascade-like response, including apoptosis, leading to changes in nk cell, helper and suppression lymphocyte numbers consistent with an immunomodulating effect of uv radiation. background: previous investigations have shown the involvement of histamine and histamine receptors (h 1 , h 2 , h 3 , h 4 ) in ige synthesis in atopic and lymphoproliferative diseases. ige responses may depend on the concentrations of histamine and histamine receptor antagonists and agonists. the goal of this investigation was to evaluate the role of the concentration of histamine and h 3 /h 4 antagonists along with the importance of pre-existing levels of ige in determining ige responses. methods: ige synthesis was studied in mnc cultures of patients (7-14 years old) having mild atopic asthma and rhinitis. all patients had high levels of total ige and specific ige to ragweed pollen, house dust mite, epidermal or mold allergens. patients were divided into two groups according to the serum ige and levels of spontaneous ige synthesis: group a -with low levels (4.8â±1.2 iu/ml) and group b -with high levels (9.7â±1.4 iu/ml). the serum level of total ige in group a was 426.1â±24.3 iu/ml and in group b was 570.2â±26.5 iu/ml. fub 181 hydrogenmaleate was used as an h 3 /h 4 specific antagonist. results: histamine in high concentrations (10 -5 m) suppressed and in low concentrations (10 -8 m) stimulated spontaneous ige synthesis. the h 3 /h 4 antagonist fub 181 activity depends on the pre-existing levels of ige. in high concentrations (10 -5 m), the antagonist increased ige synthesis only in group a, but not in group b having high spontaneous levels of ige synthesis. the synthesis in group a increased 1.36 fold (p<0.001), but h 3 /h 4 blockade cancelled the ige suppressive effect of high concentrations of histamine (10 -5 m). the addition of histamine into the mnc culture stimulated ige synthesis 1.56 fold. fub 181 had no effect on ige-stimulatory effects of low concentrations of histamine (10 -8 m). in allergen (ragweed)-stimulated mnc culture, fub 181 had a co-stimulatory effect on ige synthesis induced by histamine. conclusions: the ige stimulating response depends on the concentrations of histamine and the h 3 /h 4 antagonist as well as pre-existing levels of serum ige and ige spontaneous synthesis. introduction: preliminary data has shown that oral contraceptives can precipitate or worsen attacks of hereditary angioedema (hae). it is thought that estrogens affect the synthesis and degradation of bradykinin with resulting edema. objective: our objective is to determine if there is a difference among the various oral hormonal preparations, namely, combined monophasic, combined multiphasic, progesterone only, and hormone replacement therapy, in regards to their effect on frequency of hae exacerbations. methods: patients in this study consisted of women over the age of 18 diagnosed with hae who receive their care at our institution and women meeting the same criteria who are active in the hae foundation and have access to this association's web page. all patients answered a sixteen item questionnaire about past or present birth control pill or hormone replacement therapy usage and its impact on the frequency of exacerbations. results: of the patients who completed the questionnaire, 94% had taken or were currently taking oral contraceptive pills or hormone replacement therapy. of the women whom had taken monophasic birth control pills, 100% worsened with increased number and severity of exacerbations. however, only 75% of the women that had used combined multiphasic birth control pills worsened. all of the women who had tried progesterone only birth control pills worsened. of those on isolated estrogen for hormone replacement, 67% had an increased number of exacerbations. when androgens were used concurrently with oral contraceptive pills or hormone replacement therapy, exacerbations decreased to 60%. conclusion: we are not able to demonstrate a statistical benefit of one oral contraceptive pill preparation over another because of our sample size. however, our preliminary data supports that the oral contraceptive pill which produces less exacerbations and less symptoms is a combined multiphasic pill with concurrent androgen treatment. introduction atopic dermatitis (ad) is associated with multiple immunological abnormalities including imbalances in the subsets of blood circulating lymphocytes forming cellular infiltrates in inflamed skin. many studies of the functional and phenotypic properties of lymphocytes in ad have been limited to either peripheral blood or skin-infiltrating lymphocytes. the purpose of our study was to evaluate and compare the content and phenotypic properties of cd4+ lymphocytes from blood and inflamed skin of ad patients. materials and methods 15 adult (age 18-43 years) patients with chronic ad were selected by the criteria of hanifin. all patients gave written informed consent. the cd4+ lymphocytes of peripheral blood were phenotyped by flow cytometry. skin biopsies were obtained from eczematous areas, then cryosected and double immuno-histochemistry was performed. for phenotyping of lymphocytes in blood and skin a panel of monoclonal antibodies was used including cd3, cd4, cd8, cd25, hla-dr and cla. results immunophenotype analyses of the peripheral blood lymphocytes showed a predominance of cd3+cd4+ cells (86%â±21.3). most cells were cd4+hla-dr+ (14%â±12.6) and cd4+cd25+ phenotype (26%â±13.5). double immunohistochemistry of the skin biopsies revealed in the epidermis rare but constant presence of cd4+cd25+cells (1.0â±6.4 cells/mm 2 ) and cd4+hla-dr+ cells (1.2â±1.3 cells/mm 2 ). in the dermal inflammatory infiltrates the predominant cells were cd4+cd25+ (21.0â±9.7 cells/mm 2 ) and cd4+hla-dr+ (45.0â±6.7 cells/mm 2 ). the dominant inflammatory infiltrate consisted of cd4+cla+ phenotype (62.9â±23.5 cells/mm 2 ) cells. conclusion in ad skin inflammation is associated with the appearance in the circulation of cd4+ lymphocytes in activated form (cd4+hla-dr+) and lymphocytes with regulatory properties (cd4+cd25+). these lymphocytes are recruited from the circulation, having the skin homing properties (cd4+cla+), and form the main constituent of the dermal inflammatory infiltrate. contact dermatitis (cd) comprises a spectrum of inflammatory skin reactions usually caused by exposure to non-immunogenic low molecular weight substances (haptens). failure to diagnose the condition may result in a chronic and disabling condition with impaired quality of life. a 28 yr-old white male presented with a severe, symmetrically distributed facial maculopapular rash with secondary excoriated lesions. prior to the appearance of the rash, the patient had been applying lubriderm as a skin moisturizer for dry skin. a standardized thin layer rapid use epicutaneous (true) test containing 23 chemical agents suspended in a vehicle and attached to an adhesive backing was applied to the patient's back. at 48 hrs, a positive reaction was observed at the skin site of the paraben mix application. the lubriderm preparation used by the patient contained paraben, in contrast to a newer preparation, advanced therapy lubriderm, which is paraben-free. complete resolution of the facial lesions occurred following discontinuation of the lubriderm and the use of oral and topical corticosteroid therapy. this case report illustrates how a commonly used moisturizer can contain a sensitizing agent that could be detected by standardized patch testing which should be a part of the diagnostic armamentarium of every allergist-immunologist. we report how a commonly used and effective moisturizer (lubriderm) can cause severe allergic cd and that the use of a standardized thin layer rapid use epicutaneous (true) patch test panel can be an effective diagnostic tool for the detection of the offending agent. rationale: this study aimed to establish the possible differences of cutaneous sensitization to common aeroallergens in children under 1 years old. methods:the study was conducted between 2003-2004 and included 120 infants from silesia/southern part of poland/.these infants were refered to our allergy unit due to respiratory symptoms like rhinitis, otitis, pharyngitis, cough, bronchitis recurring;eye's symptoms/ conjunctivitis/ and oral symptoms. the skin prick test/ spt/ to major aerollergens in our environment(hdii, birch, alternaria, cladosporium and trees)were done to 6-months-old and 1-yearold children. a spt of 3 mm or larger was considered as positive. patients were classified into three groups: i:positive family atopy ii:smoking ciggarettemother/ or father iii:cat or dog at home iv: coexisting food allergy results:58, 8 % of examined children were boys. the prevalence of sensitisation found were as follows:latex 38, 1%, birch 32, 8%, dpii-28, 6%, grass pollen 28, 1%, alternaria-8, 4%. positive spts to latex were first seen in the 6-months-old group and this prevelance doubled until they finished 1 year. conclusion: in our study, cutaneuos sensitisation start to appear in 6-months-old chlidren. background: propylene glycol (pg) may induce allergic contact dermatitis (acd) and skin irritant reactions. topical formulations frequently contain pg. it is present in low concentration (5%) in pimecrolimus cream 1%, a non-steroid inflammatory cytokine inhibitor. objectives: this study was designed to assess the incidence of cutaneous responses to pimecrolimus cream in patients allergic to pg and to determine their nature (acd or irritation) and severity. methods: in this double-blind, randomized, vehicle-controlled, withinpatient study, 20 subjects allergic to pg underwent 48-hour patch-testing (pg 30 and 100%, pimecrolimus cream and vehicle) followed by a 7-day repeated open application test (roat). application sites were assessed by the investigator using a scale ranging from 0 (no reaction) to 7 (spreading bullous reaction) at 0, 48, and 120 hours after patch removal and at the completion of the roat. results: pg allergy was confirmed by patch-testing in 16 patients. two patients showed a positive patch-test reaction with pimecrolimus cream and vehicle, indicating an acd. in contrast, no patient demonstrated signs of acd when pimecrolimus cream was applied under normal conditions, i.e. without occlusion (roat). the statistical analysis showed that there were significantly less reactions at pimecrolimus patch-test sites than at the pg patch-test sites (p<0.01 for both pg concentrations, one-sided exact binomial test) and that the median severity was lower with pimecrolimus cream vs. pg (p=0.02 and p<0.01 for pg 30% and 100% respectively, wilcoxon signed-rank test). conclusions: this pilot study suggests that pimecrolimus cream 1%, when applied under normal conditions, can be used safely in patients with pg allergy. introduction: chronic idiopathic urticaria is not always responsive to antihistamine therapy. multiple alternative agents have been tried. we report two cases where tacrolimus has been successful in treating this condition. methods: this is a case report of two patients who have chronic idiopathic urticaria and have been treated with tacrolimus. patient #1 is a 27 y/o wm who pre-sented with urticaria that had been going on several months. the duration of his urticarial lesions was <24 hours his urticaria was treated with several medications including: hydroxyzine, cyproheptadine, ceterizine, montelukast, colchicine, and dapsone. he was intolerant to hydroxychloroquine. montelukast seemed to provide some modest benefit but he continued to have daily urticaria with >50 lesions/day after two months of therapy. tacrolimus was added to montelukast at 1mg bid. patient #2 is a 64 y/o wf with a history of biopsyproven urticaria. she had suffered from episodic urticaria since the age of six that was responsive to systemic steroids. after thirty years of having no urticaria, she developed recurrent urticaria at age 62. at this time, she was treated with hydroxyzine, doxepin, montelukast, loratadine, ceterizine, fexofenadine, colchicine, dapsone, and even prednisone with very little improvement in symptoms. she was then started on tacrolimus 1mg bid as monotherapy while she was having daily urticaria. results: after one month of treatment with tacrolimus 1mg bid, patient #1 had a decrease in the number of urticaria. his dose of tacrolimus was increased to 3mg/day, and after four weeks, his urticaria resolved. he remained on tacrolimus for a total of six months which was tapered and discontinued. he continues to be in remission from urticaria. patient #2 had complete resolution of her hives after just two doses of tacrolimus. after two months, she remains free of urticaria on tacrolimus 1mg bid. conclusion: for patients with severe chronic urticaria unresponsive to multiple therapies, tacrolimus, like cyclosporine, may be efficacious. tacrolimus may also be truly immunomodulatory and capable of inducing remission of urticaria. s.v. gerasimov * , lviv, ukraine. introduction. recent studies suggest that probiotics can be useful in prevention and treatment of atopic dermatitis (ad) in children. as clinical effect of probiotics varies greatly depending on the specific strain, we are currently conduct a search for the most promising probiotic to be used as complementary therapy in infants and young children with ad. methods. we studied 23 infants aged 25-51 weeks (36â±8) with ad established using hanifin and rafka criteria. patients were evenly allocated among probiotic (n=11) and nonprobiotic (n=12) treatment groups using quota allocation system. severity of ad was defined using scorad index and infant`s quality of life was assessed using idqol index. patients were examined before and 4 weeks after the treatment with/without probiotic powder formulation (b. infantis, l. acidophilus dds-1, 10 billion cfu/day, dds-junior, uas laboratories). we compared pre/posttreatment values of indices above and amount of mometasone furoate (0, 1%) used employing a paired or unpaired t-test. results. at baseline, infants in either groups were comparable on age, gender, duration of the disease, scorad and idqol indices, and medication taken for the previous 4 weeks. before entering the study all patients were maintained on allergen elimination diet due to milk and egg white allergy. after the treatment with/without probiotics the mean scorad index decreased from 42, 2 to 32, 7 (p=0, 14) and from 51, 5 to 31, 3 (p=0, 06), respectively. however, the mean amount of mometasone furoate used in non-probiotic treatment group was significantly greater (7, 8 g vs 2, 1 g, p=0, 03), despite the same recommendation on the use of the drug was given. additionally, parents of children taking probiotics reported an improvement in infant`s quality of life as seen on the decrease of idqol index from 21, 1 to 12, 0 (p=0, 04). in non-probiotic group decrease in idqol was not significant (24, 2 to 19, 5; p=0, 14) . there were no adverse events in any of the treatment groups. conclusions. our preliminary results suggest that some probiotics may have a corticosteroid-sparing effect and improve quality of life of infants with ad. there is a clear trend towards reduction of scorad index, which did not change significantly, probably due to a limited number of patients involved. introduction : the aim of this study was to analyze the risk factors of severe atopic dermatitis(ad) in the first 6 months of life. methods : the children aged less than 6 months with ad were divided into two groups according to six area six sign in atopic dermatitis(sassad) score. children with score less than 14 was classified as mild ad(n=90) and those with score above 15 as severe ad(n=44). these patients were fed with breast milk or cow's milk formula, and no allergenic food was given except rice and some vegetables. we analyzed the gender, feeding patterns, family history of allergy, number of siblings, total ige and specific ige to common food allergens (egg white, cow's milk, wheat, soy) by cap-feia assay. total eosinophil count was 1863â±2032 /ul in severe ad and 686â±539 /ul in mild ad(p<0.001). results : 1) total ige was 180.6â±245.1 u/ml in severe ad and 32.2â±50.7 u/ml in mild ad(p=0.002). specific ige to egg white, wheat and soy (19.03â±29.72 u/ml, 4.68â±10.62 u/ml, 7.07â±22.01 u/ml in severe ad; 1.78â±3.54 u/ml, 0.15â±0.99 u/ml, 0.09â±0.30 u/ml in mild ad; p<0.05) were associated with severe ad, but cow milk(4.34â±16.6 u/ml in severe ad, 0.80â±4.17 u/ml in mild ad) showed no difference. 2) gender, feeding patterns, family history of allergy and the number of siblings were not significantly associated with severe ad. conclusion : severe ad is associated with sensitization to food allergens in the first 6 months of life, although they are not fed with those foods. background: atopic dermatitis (ad), an inflammatory skin disease diagnosed primarily in children, has been shown to have a negative impact on quality of life (qol). pimecrolimus cream 1% is a non-steroid, topical calcineurin inhibitor with demonstrated efficacy in the acute treatment and longterm management of pediatric and adult ad. in a 24-week, double-blind, vehicle-controlled study, a secondary aim was to evaluate the impact on parent's quality of life of a pimecrolimus-based or corticosteroid (cs)-based treatment regimen of children with ad. methods: 275 children aged 3 months to 11 years (mean 5 years) with mild to severe ad were randomized 2:1 to receive treatment with pimecrolimus or vehicle cream. emollients for dry skin and pimecrolimus or vehicle bid were applied at the first signs of ad. for severe flares, a mid-potency topical cs (fluticasone or mometasone) indicated for once-daily use in ad replaced the evening study drug application for a maximum of 3 weeks or until all ad resolved. parents completed the parent's index of quality of life-atopic dermatitis (piqol-ad), a 28-item validated questionnaire, which measures parent's needs-based qol, at baseline, week 12, and study completion. change in piqol-ad scores at baseline and week 24 were compared between treatments. a negative change indicated improvement. results: parents of patients in both groups reported improvement in piqol-ad scores at week 24, with greater improvement for the pimecrolimus group. the mean change in piqol-ad score from baseline to week 24 was -3.3 in the pimecrolimus group vs. -2.6 in the cs-based treatment group (37.6% vs. 26.8% improvement, respectively). ancova analysis, using treatment and center as main effects and baseline score as a covariate, compared the change in piqol-ad score in two treatment groups. pimecrolimus treatment demonstrated a more favorable change (-1.2) which approached statistical significance [p=0.056; 95%ci= (-2.5, 0.0)]. conclusion: a treatment regimen utilizing pimecrolimus cream 1% had a beneficial effect on parent's quality of life compared to a corticosteroid-based regimen. this benefit was consistent with other measures of efficacy studied. introduction: atopic dermatitis is a chronic, inflammatory and recurrent skin disorder . its prevalence has increased in recent decades but little is known about it in mexico city. the aim of this study was to evaluate the prevalence and severity of atopic eczema in a pediatric population. methods: a cross-sectional questionnaire survey (isaac phase i questionnaire) was conducted on random samples of schoolchildren aged 6 to 7 years and 13 to 14 years from educational centers of four northern counties from mexico city. those children with a positive response to being questioned about the presence of an itchy relapsing skin rash in the last 12 months were considered to have atopic dermatitis. children whose symptoms resulted in sleep disturbance for 1 or more nights per week were considered to have severe atopic eczema. statistical analyses were done with spss 6.0 for windows, chi square . the size of the sample was determined folowing the isaac specifications and calculated with the statcal program. results: complete data was available for 3211 children aged 6 to 7 years in 45 schools and 3899 children aged 13 to 14 years in 47 high schools. 51.5% males and 48.5% females. response rates were high ( 91.4 % for those aged 6 to 7 years and 100% for those aged 13 to 14 years). the prevalence of symptoms of atopic dermatitis in the last 12 months was 9.6 % at age 6 to 7 and 9 % at age 13 to 14 years. medical diagnose of atopic eczema was 4 % and 2.4 % for children aged 6 to 7 years and 13 to 14 years, respectively. reported eczema accounted for 12.4 % and 11.3 % at age 6 to 7 and 13 to 14 years, respectively. children with symptoms of severe atopic dermatitis accounted for 1 % of all those with symptoms of atopic dermatitis. we also found that the majority of the children begun with skin manifestations of atopic dermatitis before the age of 4 years old. conclusions: the prevalence of atopic eczema was very similar for both groups of ages. our results agree with those found in other two studies acomplished in other mexican cities (cuernavaca and chihuahua) and with those from other countries of latin america like brazil, chile and costa rica. although, we found lowest values than those from sweden, japan and australia. studies that include objective skin examination are required to confirm these findings. a. kaplan 1* , s. meeves 2 , y. liao 2 , s.t. varghese 2 , g. georges 2 , 1. charleston, sc; 2. bridgewater, nj. introduction: the symptoms of chronic idiopathic urticaria (ciu) can have a profound impact on patient health and quality of life. the safety and efficacy of fexofenadine (fex) bid for the treatment of ciu has been previously established in two multicenter, double-blind, randomized, placebo-controlled trials. this study evaluated the efficacy and safety of a qd dose of fex hcl 180 mg, as this dosing schedule could offer advantages in terms of patient compliance and convenience. methods: this multicenter, randomized, double-blind, parallel-group, placebo-controlled study consisted of a single-blind placebo run-in period of 2-5 days, followed by a 28 (â±4)-day treatment period. males and females aged 12 years with a diagnosis of ciu and with active disease were enrolled. patients were randomized 2:1 to receive either fex hcl 180 mg qd or placebo qd. the primary endpoints were change from baseline in mean daily number of wheals (mnw score) and mean daily severity of pruritus (measured on a 5-point scale) over the 28-day treatment period, as assessed reflectively by the patient. secondary efficacy measures included a modified total symptom score (motss), comprising sum of number, frequency, size and duration of lesions, and severity of pruritus. mnw and pruritus severity were also assessed instantaneously at trough drug levels (immediately prior to dosing). results: over the 28-day treatment period, patients treated with fex (n=167) experienced significantly greater improvements in mnw and pruritus scores compared with the placebo group (n=92) (p<0.0001 for both). similarly, over the treatment period, and at individual weekly timepoints, the mean reductions in am reflective, pm reflective and mean daily motss were significantly greater for patients in the fex group compared with those in the placebo group (p 0.0052 for all comparisons). the mean reductions in instantaneous mnw and pruritus scores were greater with those who received fex than those who received placebo (mnw: p=0.0154; pruritus score: p=0.00088). there were no significant differences in the frequency of treatment emergent adverse events between the two treatment groups, and no clinically relevant changes were observed with respect to clinical laboratory data, vital signs or ecgs. conclusion: this study demonstrated that a qd dose of fex hcl 180 mg offers effective and well-tolerated relief from the symptoms of ciu. introduction: the wheals and pruritus associated with chronic idiopathic urticaria (ciu) are often so debilitating that they have a profound impact on patient quality of life. specifically, ciu has been shown to negatively affect patient mobility, sleep, energy levels, social interaction and emotional wellbeing. the purpose of this study was to examine the impact of treatment with fexofenadine hcl (fex) 180 mg on health-related quality of life (hrql) among patients with ciu. methods: as part of a multicenter, randomized, double-blind, parallel-group, placebo-controlled study, designed to evaluate the efficacy and safety of a once-daily dose of fex 180 mg in ciu, the impact of treatment on hrql was also examined. patients were asked to complete the dermatology life quality index (dlqi) and the work productivity and activity impairment questionnaire (wpai) at baseline and at weeks 2 and 4 (final visit or early termination). the primary endpoint was mean change from baseline in dlqi total score, using the mean data of evaluations performed at weeks 2 and 4 as the post-baseline measure. secondary endpoints included change from baseline in individual dlqi domains and wpai scores. additional analyses were conducted to examine the reliability, validity and responsiveness of the hrql measures. results: a total of 254 patients were included in the hrql population: n=163 fex, n=91 placebo. patients in the fex group experienced significantly greater improvements in the mean dlqi total score than those in the placebo group (p=0.0029). this pattern was repeated with respect to the individual domains of symptoms and feelings (p=0.0071), daily activities (p=0.013), leisure (p=0.0425) and personal relationships (p=0.0023). both treatment groups reported improvements in productivity, as measured by change from baseline using the wpai. patients randomized to fex experienced significantly less impairment while working (p=0.0455) and performing activities (p=0.0088) than those who received placebo. the dlqi was demonstrated to be reliable (cronbach's alpha=0.87); both the dlqi and wpai were found to be valid and responsive instruments in this ciu population. conclusion: this study demonstrated that a once-daily dose of fex 180 mg improves the hrql of patients with ciu, as assessed by change in dlqi total score. t. algozzine 1* , a. lee 2 , s. wong 3 , l. anzisi 4 , 1. manchester, nh; 2. centerport, ny; 3. syosset, ny; 4. mamaroneck, ny. symptoms of allergic and non-allergic rhinitis may significantly impact a patient's quality of life, by causing fatigue, headache, cognitive impairment and other systemic symptoms. appropriate management of allergic rhinitis is important in the effective management of coexisting or complicating respira-tory conditions (e.g. asthma, sinusitis, or otitis media). in addition, many commonly used over-the-counter (otc) antihistamines can cause performance impairment that may impact daily activities. we designed a brief ten-question allergy survey in an attempt to determine how patients were being treated for their allergies, evaluate a patient's self-assessed impact of their allergies on their daily activities, and identify any difference in treatment outcomes between primary care and allergist practices. patients were invited to complete the surveys anonymously. completed surveys were collected and entered into a microsoft access database for evaluation. minitab was utilized for statistical calculations. four hundred and thirty seven patients completed the survey. fifty-eight percent of survey respondents were women and 89% were adults (age >18). of those patients surveyed, 53% (n=233) were under the care of an allergist. compared to primary care, patients treated by allergists reported more allergies (3.4 vs. 1.8, p <0.001) and received more medications on average (2.3 vs. 1.4, p<0.001). pollen was the most common allergy type reported among all patients. while the majority of all patients (69%) received prescription medications, more primary care patients received no therapy or only otc treatment when compared to allergist patients (46% vs. 17%, p<0.001). seventy-four percent of primary care patients reported their allergy symptoms were controlled compared to 63% of allergist patients (p<0.05). patients receiving treatment from an allergist reported less impact of their allergies on daily activities (a lower score signified less impact) compared to patients seen in primary care (2.27 vs. 3.84, p<0.001). the results of our survey suggest that allergists treat more complex patients. these patients had multiple allergies, more uncontrolled symptoms, and utilized more prescription medications. despite these findings, patients treated by allergists reported less impact of allergies on their daily activities. introduction the patogenic mechanism of nasal polyps are unknow.they frecuently are associated with aspirin intolerance, intrinsic asthma, chronic sinusitis, young sindrome, cystic fibrosis, kartagener syndrome and churg-strauss syndrome.chemical mediators found in nasal polyps are as follows: histamine, serotonin, leukotrienes, norepinefrine and possibly pgd2.recently, leukotrienes have been implicated in mediation of bronchoconstriction and inflammatory leukotriene levels have also been shown to be elevated in some patients with sinonasal polyposis (figure 1 ) hypothesis antileukotrienes might play a significant role in controlling polyposis and symptoms secundary to sinonasal disease, and they might viable alternative to longterm, oral steroid therapy and repeat surgical debridement purpose this study was undertaken to evaluate the potential role of leukotriene receptor antagonist on recurrent polyposis associated with asthma and improvement of some factor implicated with them design of study:clinical assay, prospective, triple blind. materialsand methods:the study involved 30 patients 12 (40%) males and 18 (60%) women, mean age 20.7 years, with a range 16-45 years selection criteria:inclusion criteria: nasal polyps associated with astha, allergic or non allergic.exclusion criteria: patients with any concurrent illness, use of sistemic or local corticosteroidsor any kind of antileukotriene within 1 month prior to the beggining of the study.we made an alleatory selection 10 patients recived montelukast (10 mgs/day), and nasal steroids, beclometasone (600 mgs/day) for 6 months.10 patients recived loratadine plus pseu-doefedrin5mgs/120 mgs/day and nasal steroids, beclometasone, 10 patints were operated of fees and transnasal endoscipic polypectomy plus montelukast and nasal steroids, beclometasone ige serum levels 2.skin prick test 3.sensitivity in vitro 4.ct scan 5. celullarity on nasal lavage (eosinophils and neutrophils)6.proinflamatory cytokines (il4-il5-il8) on nasal lavege by elisa test. results ther was a tendency for more improvement of the group 3 ( surgery-montelukast-local steroid) of symptoms and objective measurments, in second place of improvement the group 1 montelukast-local steroid) and the worse on improvement subjective and objetive was group 2 local steroid and loratadine-pseudoefedrine use as placebo introduction: sensory perceptions of intranasal corticosteroids (ins) vary among products and can be unpleasant and affect adherence to therapy. methods: we conducted a cross-sectional study of 120 patients across 4 allergy and immunology clinics in the united states. respondents were asked to choose between pairs of hypothetical ins that differed by sensory attribute composition. based on prior research, we measured 6 salient sensory attributes: smell, taste, aftertaste, throat rundown, nose runout, and feel of spray in nose/throat. each attribute was described in 3 intensity levels, such as "no taste" (low), "weak taste" (moderate), and "strong taste" (high) ( table) . other outcomes included an importance score for each sensory attribute and patients' willingness to adhere to an ins having the lowest levels of each sensory attribute compared to one with moderate levels. results: preferences decreased with increasing intensity level of each sensory attribute. the most important attribute was aftertaste in 28% of patients, taste in 19%, throat rundown in 18%, nose runout in 12%, smell in 11%, and feel of spray in 7%. only 5% had more than one attribute tied for most important. if instructed to take ins daily for 3 months, 77% of patients stated that they would definitely be able to follow their doctor's advice (willing to adhere) if given an ins containing the lowest level of each sensory attribute compared with 4% for one having moderate levels (p<0.01). conclusions: patients' preferences decrease with increasing intensity levels of each sensory attribute and affect patients' willingness to adhere. tailoring ins to patient preferences may lead to improved treatment satisfaction and adherence. introduction:a high prevalence of rhinitis and asthma comorbidity has been persistently noticed in epidemiological studies in last years. our objective was to evaluate the prevalence of rhinitis and asthma comorbidity in a portuguese population including both atopic and non-atopic patients. methods: a retrospective study was performed. clinical records from patients attending an immunoallergology outpatients'clinic during 39 months (from january 1998 until june 2001) were reviewed. data were collected concerning clinical history of rhinitis and/or asthma, aeroallergens'skin prick tests and respiratory function evaluation. results: among the 3582 patients attending an appointment during the study period, 2448 (68.3%) had rhinitis and/or asthma (56.2% f; 43.8% m;mean age 26 +/-19 years). patients with respiratory disease included 1983 atopic (81.0%) and 465 non-atopic 19%). diagnosis of asthma, rhinitis or of both diseases was made in 170 (8.6%), 581 (29.3%) and 1232 (62.1%), respectively, in atopic patients and in 56 (12.0%, 265 (57.0%) and 144 (31.0%), respectively, in non-atopic patients. rhinitis was diagnosed in 87.9% of atopic asthmatic and in 72-0% of non-atopic patients with asthma. in patients with rhinitis, asthma was also diagnosed in 67.9% of atopic patients and 35.2% on non-atopic patients. conclusions: in this study population rhinitis was frequently diagnosed in patients with asthma and vice-versa, thereby leading to a high prevalence of this comorbodity. this association was more frequent in atopic patients. our data are comparable to those we found in recent literature. these results suggest that clinical investigation of asthma should be mandatory in management of patients with rhinitis as well as asthmatic patients should be routinely submitted to clinical evaluation of rhinitis. introduction: cetirizine hcl (c) has been shown to be more effective than fexofenadine hcl (f) in controlling seasonal allergic rhinitis (sar) symptoms at 21-24 hr post-dose, however, the efficacy of c and f was comparable between 0-5 hr post-dose. response to treatment in the middle of the dosing interval needed to be addressed. methods: this randomized, double blind, placebo (p)-controlled study was designed to compare the efficacy and tolerability of a single dose of c 10 mg, f 180 mg, and p between 5-12 hr postdose in ragweed-sensitive sar subjects. subjects meeting entry criteria were exposed to pre-determined controlled levels of ragweed pollen in the eeu during priming and double blind treatment. subjects assessed rhinitis symptoms at half hr intervals during pollen exposure; at priming, at the qualifying period, at baseline, and from 5-12 hr post-dose. the primary efficacy endpoint was the change from baseline in total symptom severity complex (tssc) score at 12 hr post-dose. tssc score was the sum of severity ratings (0=absent to 3=severe) for 4 symptoms: runny nose, sneezing, itchy nose/palate/throat and itchy/watery eyes. results: a total of 599 subjects (mean age 32.8 y; 58.9% females) were randomized: c, 249; f, 250; p, 100. baseline characteristics were comparable among groups; tssc: c=9.2, f=9.2, p=8.9. c produced a 26% greater reduction in tssc score at 12 hr (-4.3, p=0 .001) and a 14% greater reduction overall (i.e., average over the 5-12 hr post-dose period) (-5.0, p=0.006) compared with f (-3.4, -4.4, respectively). both c and f reduced tssc score more than p (12 hr, -1.9; overall, -2.3, p<0.001) including all individual symptoms (p<0.05). c however, was more effective than f for runny nose and sneezing at 12 hr and overall, itchy/watery eyes at 12 hr, and itchy nose/throat/palate overall (p<0.05). rates of discontinuation due to adverse events (aes) were low: c, 0.4%; f, 0%; p, 1.0%. the incidence of treatmentemergent aes was similar: c, 25.3%; f, 29.6%; p, 35.0%. somnolence occurred in 0.8% of subjects on c, and 0% on f or p. conclusion: c produced a greater improvement in rhinitis symptoms compared with f and p, at 12 hr post dose and over the 5-12 hr post-dose period. all treatments were safe and well tolerated. background: medication utilization patterns of patients suffering from seasonal allergic rhinitis (sar) are not well documented, and although many anti-allergic medications are prescribed for daily use, actual usage is unknown, but recognized to be quite variable. methods: 1821 subjects with positive ragweed skin tests were mailed a survey during the third week of ragweed season, soliciting the nature and severity of sar symptoms, usage patterns and reasons for choice of anti-allergic medication. results: 550 subjects completed the survey (30.2%). the prevalence of symptoms were, in decreasing order: sneezing (91.5%), runny nose (82.4%), itchy/gritty eyes (80.4%), stuffiness (78.5%), itchy nose (71.3%), watery eyes (64.7%), itchy palate/throat (56.9%), post-nasal drip (55.6%), red/burning eyes (49.1%), headache (36.5%), itchy ears (34.0%), cough (30.0%), shortness of breath (15.7%), and wheeze (15.5%). sar patients used antihistamines most frequently (94.7%), followed by decongestants (63.1%), combination (antihistamine/decongestant) products (52.0%), and intranasal corticosteroids (42.5%). medications were mostly taken intermittently rather than daily (antihistamines 68.0%; nasal corticosteroids 71.8%), conclusion: a constellation of nasal symptoms were the most common seasonal allergic manifestations, followed by ocular, palatal and ear irritation. antihistamines were the most frequently used medication to treat symptoms, succeeded by decongestants (alone or in combination). a significant proportion of subjects took their allergy medication, including nasal corticosteroids, intermittently rather than regularly, underscoring the relevance of single-dose evaluations of drug efficacy. a.k. ellis * , e. rafeiro, j.d. ratz, j.h. day, kingston, canada. background: traditional assessment of seasonal allergic rhinitis (sar) medication efficacy utilizes randomized controlled trials over 2-4 weeks in season. an additional study method employs single-dose responses using controlled allergen challenge such as the environmental exposure unit (eeu). a comparison of allergic symptoms generated by controlled allergen challenge to those occurring in ragweed season symptoms has not been done. methods: 1821 subjects with known sar to ragweed were mailed a survey during the third week of ragweed season, soliciting the nature and severity of sar symptoms. subjects participating in a subsequent controlled allergen challenge study using the eeu, were again asked to complete a similar survey that documented symptoms generated in this model. those who completed both surveys comprised the primary analysis group. results: 550 subjects completed the ragweed season survey, 516 subjects completed the eeu survey, and 270 completed both. symptoms generated by eeu exposure were similar to those elicited during ragweed season, with the exception of cough (68% vs. 27%, respectively, p <0.01). subjects reported that symptoms were more severe in the eeu than those experienced on a typical ragweed season day, but less severe than those during peak ragweed season days. conclusion: allergic upper respiratory tract symptoms produced during controlled ragweed pollen exposure in the eeu were similar in nature and degree to those expe-rienced during ragweed season, supporting evidence that the eeu is a valid model for studying sar. r. nave 1 , m.a. wingertzahn 2* , s. brookman 3 , s. kaida 4 , t. shah 2 , 1. konstanz, germany; 2. florham park, nj; 3. princeton, nj; 4. tokyo, japan. rationale: ciclesonide (cic) is a new corticosteroid under development for treatment of allergic rhinitis (ar). cic is a pro-drug that is hydrolyzed to the active metabolite desisobutyryl-ciclesonide (des-cic) in the target tissue. cic has low oral bioavailability and is highly bound to plasma proteins; therefore cic administered as a nasal spray is expected to have minimal local and systemic effects. objective: the primary objective was to evaluate the safety and tolerability of repeated escalating doses of cic (50-800 mcg/day) given as a nasal spray for 14 days to healthy and asymptomatic subjects with sar. secondary objectives were to determine pk of cic and des-cic and to evaluate the effect of cic on endogenous cortisol. methods: this was a single-center, randomized, placebo-controlled, double blind, modified sequential dose study. six cohorts were randomized. cohorts i-v consisted of healthy subjects given doses of cic up to 400 mcg bid. cohort vi (asymptomatic sar subjects) received 400 mcg bid. each cohort was comprised of 6 subjects who received cic and 2 subjects who received placebo. safety assessments were conducted by recording adverse events (aes), clinical laboratory, eye, and nasal examination findings. serum and urine samples were taken for evaluation of cortisol levels. cic and des-cic serum concentrations were determined by lc-ms/ms. results: no trend was observed for aes when comparing cic and placebo treatment. additionally, no subject experienced a serious ae or withdrew from the study due to an ae during the trial. cortisol levels showed no differences between the dose groups or between activetreated subjects and placebo-treated subjects. additionally, serum concentrations of cic and des-cic in the majority of serum samples were shown to be below the lower limit of quantification (lloq; 25 pg/ml for cic and 10 pg/ml for des-cic), therefore no descriptive statistics could be calculated. con-clusions: ciclesonide nasal spray, at the doses evaluated, was safe and well tolerated in healthy and asymptomatic sar subjects with no detectable effects of cic on serum or urinary free cortisol concentrations. additionally, no pk parameters could be calculated for cic nasal spray, as it was virtually undetectable in serum despite the use of a sensitive assay. the preferred treatment for allergies is avoidance. air filtration is logical but room air purifiers have been of limited efficacy. zephyrâ�¢ is a new device which creates an envelope of air 99% free of allergenic particles around the head of the sleeping person. allergic rhinitis frequently causes daytime somnolence. this is a pilot study of the effectiveness of zephyr on symptoms of seasonal allergic rhinitis (ragweed hay fever) and on daytime sleepiness. methods: subjects age 12 to 45 with ragweed hay fever were studied in the ragweed season using each subject as his/her own control. usual allergy medicines were not allowed, except loratadine for rescue. outcome measures were a symptom score, juniper rhinitis quality of life questionnaire, a tolerability rating, and epworth sleepiness scale. during the first week subjects qualified by symptom scores, then entered the one-week treatment period with zephyr. the third week was a post-treatment observation period. results: of 13 participants, 10 (77%) showed symptom improvement. the whole group averaged 26% reduction in morning symptoms and 24% reduction in evening symptoms. sleepiness scores improved 29%. rhinitis qol improved 33%. the zephyr system was well tolerated as evidenced by the response to several statements regarding zephyr use and tolerability: (scoring: 1 = strongly disagree, 5 = strongly agree) "the system did not bother me while i was sleeping." (mean score 4.8) "the noise level did not affect my ability to sleep." (mean score 4.7) "the temperature was just fine for me." (mean score 4.8) "the system did not get in my way during sleep." (mean score 5.0) conclusions: zephyr significantly reduced seasonal hay fever symptoms and daytime sleepiness, improved quality of life, and was well-tolerated by subjects. zephyr may provide maximal environmental control of bedroom allergen exposure irrespective of ambient airborne allergen levels in the room. nasal congestion is an important symptom that is associated with significant morbidity in the rhinitis sufferer. disrupted sleep leading to daytime fatigue, loss of concentration, and decreased productivity are potential results of this symptom. a study employing online interviews with 1200 rhinitis sufferers from harris interactive's online database was conducted in order to understand the symptoms they identify with most and to determine the impact nasal congestion had on their daily activities. respondents were at least 18 years of age and experienced nasal congestion from seasonal allergic, perennial allergic, and/or perennial non-allergic rhinitis. the results showed that 60% of the respondents agreed nasal congestion is the most bothersome symptom of rhinitis; 37% experienced nasal congestion daily, while 35% experienced it several times per week. not surprisingly, sleep was the most important factor affected by nasal congestion. two-thirds (66%) of the respondents felt their sleep had been moderately or significantly impacted by nasal congestion, and more than half (56%) felt it was difficult to get a good night's rest because of congestion. sleep was interrupted or disturbed by nasal congestion approximately 3 nights per week, on average. furthermore, sleep disruption from nasal congestion contributed to daytime fatigue; 62% of the respondents indicated that they were typically tired or fatigued during the day when they experience nasal congestion. in addition, 42% felt that activities requiring concentration, such as reading, were moderately or significantly impacted by nasal congestion. this study confirms that nasal congestion causes the rhinitis sufferer significant problems beyond that of just a stuffy nose. consequently, healthcare providers need to ensure that their rhinitis patients who have nasal congestion as their primary complaint are managed effectively. desloratadine (dl, clarinex â® ) is a non-sedating oral antihistamine that is metabolized to 3-oh dl. however, a phenotypic polymorphism has been observed in some patients that results in reduced formation of 3-oh dl. the prevalence and safety profiles of such poor metabolizers of dl were examined in pharmacokinetic and clinical trials. a poor metabolizer was defined as a subject having a 3-oh dl to dl auc ratio of <0.10, or a dl half-life of 50 hours. in pediatric studies, where a sparse sampling approach was uti-lized to screen for poor metabolizers, a plasma concentration ratio of 3-oh dl to dl of <0.10 at 12 hours classified a subject as a poor metabolizer. a total of 3, 748 adult and pediatric subjects (2-70 years old) were phenotyped with a single dose of dl or loratadine. the overall prevalence of the poor metabolizer phenotype was 6% (228/3748). this prevalence was comparable for adult (70/1194, 6%) and pediatric subjects (158/2554, 6%), and greater in both populations among blacks (16% pediatric, 18% adult) than caucasians (3% pediatric, 2% adult). pharmacokinetic analysis found that exposure to dl was approximately 6-times higher in poor metabolizers than in normal metabolizers. there was no apparent difference in dl exposure among poor metabolizers in different age groups (6-months to <2-years, 2-to <6-years, 6-to <12-years, and 12-years) when treated with age-appropriate doses. the multiple-dose (7-35 days) safety profile of dl was examined in pediatric poor metabolizers (2-11 years) in 5 placebo-controlled trials, and in adult poor metabolizers (20-70 years) in pharmacokinetic trials. pooled ae rates were low and comparable between the poor metabolizer and placebo subjects, as shown in the table below with all aes that appeared in >2% of subjects in any treatment group (or >3% in adult poor metabolizers). there was no difference in cardiovascular safety profile or ecg results (including qtc interval) among these groups. in conclusion, (1) expression of the dl poor metabolizer phenotype is independent of age, but higher in blacks than in caucasians, (2) exposure to dl in poor metabolizers is independent of age when administered at age-appropriate doses, (3) the safety profile of dl poor metabolizers is not different from that of placebo at all ages down to at least 2-years old. these results are consistent with the high therapeutic index of dl. desloratadine (dl, clarinexâ®) is extensively metabolized to 3-oh dl and subsequently glucuronidated. the enzyme responsible for the formation of this active metabolite is unknown. poor metabolizers of dl represent a subset of the population that has a reduced ability to form 3-oh dl. a poor metabolizer was defined as a subject having a 3-oh dl to dl exposure ratio of <10%, or dl half-life of 50 hr. pk parameters from adult and pediatric poor metabolizers following repetitive administration of dl were characterized in clinical pharmacology trials and are summarized in the table below. exposure to dl [auc(0-24hr)] in poor metabolizers was approximately 6-fold greater than the corresponding values in normal metabolizers; cmax was 3to 4-fold greater. the magnitude of the reduction in the formation of 3-oh dl and the concurrent increase in exposure to dl associated with the poor metabolizer phenotype was similar in pediatric and adult subjects at age-appropriate doses. despite the increased exposure to dl in poor metabolizers, there was no increase in adverse event frequency or changes in electrocardiographic parameters. a: dose normalized to 5 mg. b: least squares mean ratio: anova of log-transformed data extracting sources of variation due to age group and metabolizer status. the ratio is a contrast of metabolizer status. c: lower and upper 90% confidence interval based on log-transformed data. introduction: many patients with seasonal allergic conjunctivitis (sac) complain of symptoms of dry eyes. we have previously reported that patients with sac experience discomfort from dry eyes anywhere between 2 to 6 days per week and that the overall severity of the dry eye symptoms tend to range from mild to severe. this preliminary cross over study evaluated the benefits of nedocromil sodium 2% ophthalmic solution used twice daily, compared with nedocromil sodium 2% ophthalmic solution used with refresh (ocular lubricant), for the treatment of dry eye symptoms in association with sac during 8 weeks. methods: patients who had a minimum of two-year history of sac and dry eyes, a positive skin prick test towards grass pollen and between the ages of 18 to 65 were enrolled. patients were evaluated on four visits and completed a daily diary, which included scales for grading allergic conjunctivitis and dry eye symptoms. at each clinic visit they completed the osdi and the rqlq (rhinitis quality of life questionnaire). the osdi (ocular surface disease index) was developed to assess dry eye symptoms and the impact on vision related functioning. the rqlq evaluates quality of life in patients with allergic conjunctivitis. at the last visit, both the patient and the physician assessed the treatment and the effectiveness, if any, of the addition of refresh to nedocromil sodium 2% ophthalmic solution. daily grass pollen counts were preformed using a burkhard sampler. results: 19 patients (7 male and 12 female) were enrolled and 3 dropped out. patients experienced minimal symptoms at the start of the season due to low concentration of grass pollen. preliminary analysis carried out using a paired t-test was preformed on the ocular average scores for the two treatment periods. there was no significant difference between the 2 treatment groups for redness, light sensitivity and tearing of the eyes. a reduction in dry eye symptoms was reported in all patients using refresh eye drops and the number of drops required varied between patients. during the treatment periods there was improvement in patients rqlq. conclusion: patients preferred the addition of refresh eyedrops in alleviating sac and dry eye symptoms. further studies need to be carried using refresh eye drops in larger number of patients with sac and dry eye symptoms. c. laforce * , raleigh, nc. introduction: the objective of this study was to determine the ability of azelastine nasal spray to improve rhinitis symptoms and quality of life parameters in seasonal allergic rhinitis patients remaining symptomatic after treatment with fexofenadine. methods: this placebo-controlled, double-blind study began with a 1-week, open-label lead-in period, during which patients received fexofenadine 60 mg bid. after 7 days, patients who improved less than 25% to 33% on fexofenadine were randomized to treatment for 2 weeks with: (1) azelastine nasal spray, (2) azelastine nasal spray plus fexofenadine, or (3) placebo. the primary efficacy variable was the change from baseline to day 14 in thetotal nasal symptom score (tnss), which consisted of runny nose, sneezing, itchy nose, and nasal congestion scores recorded twice daily in patient diary cards. in addition, quality of life was assessed using the rhinitis quality of life questionnaire (rqlq). results: after 2 weeks of treatment, azelastine nasal spray (p<.01) and azelastine nasal spray plus fexofenadine (p<.01) significantly improved thetnss compared to placebo. based on 90 patients with complete tnss and rqlq data, the overall rqlq score also was significantly (p<.01) improved compared to placebo. conclusions: azelastine nasal spray was an effective treatment for patients with seasonal allergic rhinitis who did not respond well to fexofenadine and significantly improved quality of life parameters compared to placebo. the results of this study indicate that azelastine nasal spray is an important alternative to oral antihistamines and should be considered in the initial management of seasonal allergic rhinitis. w. berger * , mission viejo, ca. objective: to evaluate improvement over time with azelastine nasal spray in the treatment of patients with moderate-to-severe seasonal allergic rhinitis (sar) who remained symptomatic after treatment with loratadine or fexofenadine. methods: the studies were 2-week, multicenter, double-blind, placebo-controlled trials that began with a 1-week, open-label lead-in period in which patients received either loratadine 10 mg qd (study no. 1) or fexofenadine 60 mg bid (study no.2). patients who improved <25%-33% with loratadine were randomized to treatment with: (1) azelastine nasal spray 2 sprays/nostril bid, (2) azelastine nasal spray 2 sprays/nostril bid plus loratadine 10 mg qd, (3) desloratadine 5 mg qd, or (4) placebo. patients who improved <25%-33% with fexofenadine were randomized to treatment with: (1) azelastine nasal spray 2 sprays/nostril bid, (2) azelastine nasal spray 2 sprays/nostril bid plus fexofenadine 60 mg bid, or (3) placebo. the primary efficacy variable was the change from baseline to day 14 in the total nasal symptom score (tnss), consisting of runny nose, sneezing, itchy nose, and nasal congestion. symptom severity was recordedam and pm in diary cards on a 4-point scale (0=none; 1=mild; 2=moderate; 3=severe). results: in both studies, patients treated with azelastine nasal spray experienced increasing improvement intnss over 14 days of treatment.the improvements were approximately 2-fold greater than placebo at each day of the study, and the differences from placebo were statistically significant (p<.05) at days 2, 7, 14, and overall. in study no. 1, 33% of patients treated with azelastine had >30% improvement in tnss compared to 17% in the placebo group. in study no. 2, 29% of patients treated with azelastine had >30% improvement in tnss compared to 12% in the placebo group. conclusions: azelastine nasal spray was effective in treating patients with moderate-to-severe sar who remained symptomatic after treatment with either loratadine or fexofenadine. azelastine demonstrated first-day effectiveness, and patients treated with azelastine experienced increasing improvements in rhinitis symptoms over the 14-day study periods.azelastine nasal spray is an effective treatment alternative to oral loratadine or fexofenadine and an appropriate first-line therapy in the management of sar. introduction: a large, open-label, azelastine (astelin) nasal spray patient experience trial was conducted in patients with sar, vmr, or mixed rhinitis (allergic rhinitis with nonallergic triggers). this analysis evaluated the effect of azelastine nasal spray in treating rhinitis symptoms in a subset of patients with a history of asthma or sinusitis. methods: patients were entered into an open-label protocol and treated for 2 weeks with azelastine nasal spray at a dosage of 2 sprays per nostril bid. after 2 weeks, the patients completed a questionnaire that assessed onset of action, symptom improvement, satisfaction with therapy, and quality of life. results: from a total of 4364 rhinitis patients who received azelastine monotherapy during the 2-week study period, data were analyzed for patients with asthma (n=260) or sinusitis (n=346). a greater percentage of these patients had severe rhinitis compared to the overall population. nasal congestion and postnasal drip were reported as the most bothersome rhinitis symptoms. after 2 weeks of treatment with azelastine nasal spray, >80% of patients with asthma and >85% of patients with sinusitis reported some or complete control of congestion and postnasal drip. in addition, >62% of patients with asthma reported chest tightness, shortness of breath, and wheezing were somewhat or completely controlled, and >77% of patients with sinusitis reported that headache and facial pain were somewhat or completely controlled during treatment with azelastine nasal spray. conclusion: azelastine nasal spray provided effective control of rhinitis symptoms, including nasal congestion and postnasal drip, in patients with a history of asthma or sinusitis. introduction: epinastine is an antihistamine with mast cell stabilization and anti-inflammatory properties. epinastine 0.05% ophthalmic solution was evaluated for treatment of allergic signs and symptoms elicited by feline dander in a cat exposure room. methods: participants (n=30) were aged 18 years, with a history of ocular allergy to cats, a positive skin prick reaction to cat dander, and an ocular itching score of 2 (on a 0-4 scale) within 30 minutes of entering the cat room. subjects wore a tb mask while in the cat room to reduce the effects of inhaled allergen. after 30 minutes of exposure, 1 drop of epinastine hcl 0.05% was instilled in one eye, and olopatadine 0.1% was instilled in the fellow eye. environmental exposure to cat dander continued for another 60 minutes. prior to instillation, and at 5, 15, 30, 45 and 60 minutes after instillation, conjunctival hyperemia and chemosis (scales of 0-3), and ocular itching, tearing, ocular burning, nasal itching and rhinorrhea (scales 0-4) were assessed. results: instillation of a single drop of ophthalmic epinastine significantly reduced ocular itching from a mean pre-instillation score of 2.27 to a mean score of 0.37 at 60 minutes after instillation (p<.001), despite continuous exposure to cat dander during the entire period. similarly, ocular burning, tearing, and hyperemia were significantly reduced by epinastine treatment (p<.002). chemosis was only weakly induced by cat room exposure, with a mean pre-instillation score of 0.167; however, epinastine treat-ment decreased that to 0 (p=.023). instillation of epinastine also significantly reduced nasal itching and rhinorrhea scores (p<.043). results for olopatadine were not statistically different; however, the change from baseline in itching scores in the epinastine-treated eyes was greater than that seen in the olopatadine-treated eyes at the majority of timepoints. conclusion: ophthalmic epinastine is indicated for the prevention of itch associated with allergic conjunctivitis.this study shows that treatment of cat-sensitive subjects with ophthalmic epinastine following environmental exposure to cat dander significantly reduced ocular itching and other signs and symptoms of allergic conjunctivitis. objective: the objective of this study was to evaluate azelastine (astelinâ®) nasal spray, cetirizine (zyrtecâ®), fluticasone (flonaseâ®), and placebo in the treatment of patients with symptomatic seasonal allergic rhinitis. methods: this was a double-blind placebo-controlled pilot trial in 60 patients with seasonal allergic rhinitis. the study began with a 1-week, placebo lead-in period, followed by a 1-week blinded treatment period (day 1 to day 7). efficacy variables were: (1) change from baseline to day 7 in the total nasal symptom score (tnss; consisting of rhinorrhea, sneezing, itchy nose, and nasal congestion); (2) onset of action based on tnss over the 4 hours following initial administration of study drugs; and (3) change from baseline to day 2 (24-hour change) in tnss. tnss was scored twice daily (am and pm) on a 4-point rating scale (0=none, 1=mild, 2=moderate, 3=severe). patients recorded a minimum 12-hour tnss of 8 on at least 3 days during the lead-in period, and a congestion score of 3 on at least 3 days to qualify for entry. qualified patients were randomized to treatment with: (1) azelastine nasal spray 2 sprays per nostril bid plus placebo capsules qd; (2) cetirizine 10-mg tablets qd plus placebo nasal spray; (3) fluticasone 2 sprays per nostril qd plus placebo capsules qd; or (4) placebo nasal spray plus placebo capsules. results: azelastine significantly (p<.05) improved the tnss compared to cetirizine, fluticasone, and placebo beginning 30 minutes after initial administration; cetirizine significantly improved tnss versus placebo at 150 minutes; fluticasone showed no significant differences from placebo over the 4-hour evaluation period. azelastine significantly (p<.05) improved the tnss at day 2 (24-hour change from baseline) compared to cetirizine, fluticasone, and placebo. conclusions: azelastine nasal spray improved the tnss in patients with moderate-to-severe seasonal allergic rhinitis. in addition, azelastine nasal spray demonstrated a 30-minute onset of action and significantly improved tnss versus cetirizine, fluticasone, and placebo 24 hours after initial administration. introduction: epinastine, an antihistamine with mast cell stabilization and anti-inflammatory properties, has been developed for the treatment of allergic conjunctivitis. the efficacy and tolerability of epinastine was assessed and compared with levocabastine. methods: eligible patients for this randomized, double-masked, parallel-group, active-controlled environmental clinical trial were 18-65 years old with a recent diagnosis of seasonal allergic conjunctivitis. patients instilled 1 drop epinastine hcl 0.05% or levocabastine 0.05% ophthalmic solution in each eye bid for 6 weeks. patients and investigators assessed efficacy and tolerability at study visits on days 0 (baseline), 7, 14, 28, and 42, and assessed overall efficacy and tolerability at study exit. adverse events were monitored. results: epinastine provided superior itch relief compared with levocabastine (p=.048; see figure) ; mean ocular itch scores over 4 treatment visits were 0.79 for epinastine and 0.97 for levocabastine (0-3 scale; 3=worst; baseline scores were 2.08 for epinastine and 2.06 for levocabastine). the mean summed score (ocular itching, tearing, and foreign body sensation) over 4 treatment visits was significantly better for epinastine than levocabastine (p=.010).at study exit, 53% of epinastine-treated patients rated overall efficacy "very good" (the highest possible rating), versus 34% of levocabastine-treated patients. at 5 minutes postinstillation, 77% of epinastine-treated and 75% of levocabastine-treated patients rated tolerability "very good". at study exit, 80% of epinastine-treated and 75% of levocabastine-treated patients rated overall tolerability "very good", with investigator ratings being similar. treatment-related adverse events occurred in 7.1% of epinastine-treated patients (most frequent: eye pain and skin itching, 1.2% each) and 10.3% of levocabastine-treated patients (most frequent: double vision, 3.4%; influenza-like symptoms, 2.3%); most aes were mild or moderate. conclusion: our results confirm the therapeutic potential for epinastine as an efficacious treatment suitable for long-term use throughout the allergy season. ophthalmic epinastine was superior to levocabastine for relief of ocular symptoms in patients with allergic conjunctivitis and was well-tolerated. since chronic inflammation is the histopathologic landmark of otitis media with effusion, clinical observations have led us to believe that the combination of a cysteinyl leukotriene receptor antagonist montelukast with an oral antibiotic may be more efficacious than monotherapy with an oral antibiotic in the treatment of serous otitis media. we studied twenty pediatric patients (age 3 years to 6 years) in a randomized open labeled 2-week trial to compare the efficacy of the combination montelukast (4 mg or 5 mg chewables tablets qd dosed according to patient's age) with an oral antibiotic amoxicillin/clavulanate potassium (90 mg/kg/day in 2 divided doses every 12 hours) to a monotherapy with an oral antibiotic amoxicillin/clavulanate potassium for the treatment of otitis media with effusion. the efficacy of treatment options was assessed using pneumatic otoscopy, impedance tympanometry, and audiometry to monitor the clinical course of the middle ear effusion in both treatment groups. in the combination group montelukast and antibiotic a resolution of otitis media with effusion occured at the 7th day. in contrast in the group treated with monotherapy with the oral antibiotic the resolution of otitis media with effusion occured on the 14th day. in conclusion, the combination of montelukast plus an oral antibiotic is more effective than monotherapy with an oral antibiotic.the combination of montelukast plus an antibiotic may be a safer and shorter therapy given the safety issues with long term use of systemic antibiotics. introduction: a randomized, double-blind, placebo-controlled study was conducted in an environmental exposure chamber (eec) to compare the efficacy of three doses of olopatadine nasal spray, a topical anti-allergy treatment for seasonal allergic rhinitis (sar), versus placebo spray. methods: patients aged 17-65 years old with a history of sar were screened, consented, evaluated for a positive skin test to ragweed allergen, and enrolled in this irbapproved study. a total of 320 "primed" patients were exposed to ragweed allergen in the eec and randomized to olopatadine 0.2% (n=80), olopatadine 0.4% (n=80), olopatadine 0.6% (n=80), or placebo (vehicle) (n=80) spray, 2 sprays/nostril once in the morning. symptoms were self-assessed using a 4point scale (total nasal symptom score, tnss, comprised of sneezing, runny, itchy and stuffy nose) via diaries at periodic intervals during the 12-hour study period. safety was also assessed. results: obvious trends indicated a dosedependent response to olopatadine 0.2%, 0.4% and 0.6%, though concentrations were not statistically different from each other. all three concentrations of olopatadine were clearly more efficacious than placebo spray at the first time point, 30 minutes, continuing to the end of the 12-hour session. olopatadine exhibited a safety profile comparable to placebo. conclusions: olopatadine nasal spray 0.2%, 0.4% and 0.6% exhibited dose-dependent responses. onset of action for all three concentrations of olopatadine was apparent at the first post-dose timepoint, 30 minutes, and efficacy was maintained throughout the next 12 hours. olopatadine nasal spray was safe, well-tolerated, and effective for the treatment of sar in the eec chamber. c. slonim * , tampa, fl. objective: to assess patient subjective responses to the treatment of allergic conjunctivitis using azelastine hydrochloride ophthalmic solution. methods: participating physicians selected 20 patients from their practice to receive azelastine hydrochloride 0.05% ophthalmic solution 1 drop per affected eye twice daily for 5 days. patients on prior ocular allergy medications were allowed to participate. after 5 days of treatment with azelastine hydrochloride, patients (n=2, 887) rated their experiences with azelastine hydrochloride via a self-administered survey. not all questions were answered by each patient. results: at baseline, 88% of patients reported that their ocular itching affected their work, school, and/or leisure activities at least "somewhat". after using azelastine hydrochloride, 70% of patients achieved either "moderate" or "complete" relief from ocular itching, including 30% who reported "complete" relief from itching. seventy-one percent (71%) of patients had been treated previously with a topical prescription anti-allergy medication for their ocular itching. seventy percent (70%) of patients (n=836) who had not been previously treated with a topical prescription anti-allergy medication treatment and 71% of previous medication users (n=2, 192) experienced at least "moderate" relief of itching when treated with azelastine hydrochloride. sixty-five percent (65%) of the previously-treated patients rated azelastine hydrochloride as "somewhat better" or "much better" than their previous medication. conclusions: results suggest that azelastine hydrochloride ophthalmic solution is an effective treatment for the ocular itching associated with allergic conjunctivitis as rated by the patient, regardless of whether they had been treated previously with a topical prescription anti-allergy medication. objective: to determine an association between food allergy and acid reflux in adults. method: we conducted a retrospective chart review of 60 atopic adult patients in an academic otolaryngic allergy practice. 30 adults who tested positive and 30 adults who tested negative for food allergy were included in the study. charts were reviewed for a diagnosis of gastroesophageal reflux disorders (gerd). this included a history of laryngopharyngeal reflux (lpr) and peptic ulcer disease (pud). population prevalence (19.8%; ci 17.7-21.9) of acid reflux was estimated from a historical study (locke gr et al. prevalence and clinical spectrum of gastroesophageal reflux: a population-based study in olmstead county, minnesota. gastroenterology 1997; 112:1448-56) that had a similar subject population. the prevalence of gerd in each study arm, as well as in the total study group of atopic adults, was compared to the historical control. results: subjects testing positive for food allergy had a diagnosis of gerd in 26.7% (95% ci 11.8-41.6) of cases. those subjects who did not demonstrate a food allergy were positive for gerd in 40% (95% ci 25.1-54) of cases. in the total study group, the prevalence of gerd was 33.34% (95% ci 22.8-43.8). on chi square analysis, the prevalence of gerd was significantly higher in the total study group when compared with the historical control (p=0.013). those subjects who tested negative for food allergy had a statistically significant higher prevalence of gerd than the control population (p=0.003). the prevalence of gerd between the group testing positive and the historical control did not show a significant difference (p=0.41). when comparing the study arms to each other, there was no significant difference in the prevalence of gerd (p=0.27). conclusions: the prevalence of acid reflux disorders was not higher in subjects with food allergy when compared to a historical control. although a statistical significance was noted between adults who tested negative for food allergy and the control population with regards to the prevalence of gerd, this may reflect the fact that individuals examined in an otolaryngologist's office are more likely to have acid reflux disorders than the general population. overall, the atopic subjects did have a higher incidence of acid reflux disorders, but there was no statistical significance between subjects with and without food allergy. a. suryadevara * , d.l. hamilos, boston, ma. introduction: recent studies suggest that crs without nasal polyposis (crssnp) and crs with nasal polyposis (crscnp) represent distinct pathologic entities. we wished to determine whether these conditions differed in their clinical presentation. methods: over a two-year period, new patients coming to a university based specialty clinic meeting criteria for crs were enrolled in an outcomes study. patients indicated which of four major (facial pain/pressure/headache, nasal obstruction, nasal purulence/discharge, and hyposmia/anosmia) and four minor (fever, halitosis, dental pain, cough) criteria for crs they were experiencing. rhinoscopy was performed to look for nasal polyps or polypoid tissue in any sinus area. the prevalence of each symptom was compared in the groups by chi square analysis. results: the population (n=126) had a mean age of 46 +/-13.6 and was 59% female, 86% caucasian, 8.7% african-american, 1.6% asian and 1.6% hispanic. most patients (87%) were non-smokers. all had at least 2 major criteria or 1 major + 1 minor criteria for crs at enrollment. forty-one patients (32.5%) had crscnp. the mean number of major criteria was greater in the crscnp than crssnp (3.27 vs 2.91, p=0.017). nasal obstruction and hyposmia/anosmia were more prevalent in crscnp (p= 0.05, 0.025 respectively). facial pain/pressure/headache was more prevalent in crssnp (p=).025). the most prevalent symptoms in crscnp were: nasal purulence/discharge (97.6%)>hyposmia/anosmia (85.4%)>facial pain/pressure/headache (82.9%)>nasal obstruction (61%). in contrast, the most prevalent symptoms in crssnp were: facial pain/pressure/headache (95.3%)>nasal purulence/discharge (88.2%)>hyposmia/anosmia (64.7%)>nasal obstruction (42.4%). none of the symptoms were absolutely distinguishing of these conditions. no differences were found between the two groups for fever, halitosis, dental pain or cough. conclusion: we conclude that patients with crscnp have a greater burden of symptoms of crs and a much higher prevalence of hyposmia/anomsia. these findings are consistent with other studies showing that, in comparison to crssnp, crscnp tends to be more difficult to treat and have a higher rate of relapse after intensive medical therapy (subramanian et al, am j rhinology 2002;16:303). l.e. mansfield 1* , e.e. philpot 2 , c. posey 1 , 1. el paso, tx; 2. research triangle park, nc. daytime sleepiness is a common complaint in sar. patients often complain of mental slowness, difficulty in concentrating, and thinking. the present study evaluated whether effective therapy of sar would decrease dss and improve an objective measure of cp. dss was measured using the epworth sleep scale (ess). objective cp was measured using the test of variables of attention (tova), a validated test. thirty two adults (15 males, 17 females with a 2 year history of sar, a compatible physical exam, and corresponding positive allergy testing) volunteered for this 3 week randomized double blind placebo controlled study. after a one week inf placebo (pl) baseline, the subjects received either active inf or continued pl. they maintained daily nasal symptom dairies and ess. the subjects took the tova test at the end of week1 and week3. weekly nasal symptom scores significantly improved with the inf, but not the pl. w1 vs. w3 nasal congestion inf 29, 24 p=.03, pl 29, 26 p=ns; runny nose inf 22.5, 19.9 p=ns; pl 21.75, 21.38 p=ns; sneezing inf 26.5, 19 p=.01; pl 24.9, 21.1 p=ns. total weekly ess was abnormal and decreased significantly in the inf group, but not in pl group. w1 vs. w3 inf 60.5, 46.5 p=.001, pl 52.6, 46.8 p=ns. response time of the tova testing, initially somewhat slow, significantly decreased in inf but not pl treatment. w1 vs. w3 inf 435 msec, 385 msec p=.02; pl 360 msec, 359 msec p=ns. these results demonstrate that sar is associated with dss and cp problems. the mechanism is likely to be sleeping disordered breathing associated with nasal congestion and obstruction. effective treatment of nasal congestion with inf led to decreased dss and improved cognitive performance. l.e. mansfield 1* , c. graham 2 , 1. el paso, tx; 2. new york, ny. there is increasing recognition that sleep disturbance and daytime tiredness occur during active ar. the mechanism appears to be nasal congestion and obstruction leading to sleep disordered breathing and resultant poor quality of sleep. in our practice, as part of the initial history, questions regarding fatigue, snoring, sleep problems, and tiredness are addressed. sleep problems and tiredness are graded according to the following scale: effect on daily activity ; 0=not troubled;1= a little trouble; 2=somewhat troubled ;3= trou-bled a lot ; 4= total disruption. we reviewed 272 consecutive charts of patients with allergic rhinitis documented by history, physical examination and allergy testing. there were 169 females and 103 males; age range 5y to 84y. 127 (47%) of patients or parents recognized they commonly snored. 40 (15%) stated they were chronically fatigued. the graded answers concerning sleep problems and tiredness were even more revealing of ar patient's perception of their problems see table in general, the higher sleep problem responses were associated with higher tiredness scores. national surveys of unselected populations suggest that about 10 percent of adults consider themselves to have sleep problems. the high frequency of sleep related problems and tiredness in our sample has prompted us to add more detailed questions regarding sleep related events to our intake history. it is our opinion that questions specifically related to sleep and daytime tiredness should be included in all evaluations for allergic rhinitis. we conclude that sleep related problems and tiredness may be more common in patients with allergic rhinitis than previous reported. r.w. weber 1* , j. garcia 2 , r. faruqi 2 , d. banerji 2 , g. georges 2 , the study 405 investigator group 3 , 1. denver, co; 2. bridgewater, nj; nj. introduction: the intranasal glucocorticosteroid triamcinolone acetonide (taa) is a safe and effective treatment for persistent allergic rhinitis (par). a new hydrofluroalkane-134a (hfa) propellant delivery system (taa-hfa) has recently been developed. this study primarily assessed the long-term safety of taa delivered via this new device, as well as its long-term efficacy. methods: 396 patients aged 12-69 yrs (mean=32) with par enrolled in this 1-yr, open-label study at 10 centers in the us. patients received taa-hfa 220 î¼g once daily for a 2-week run-in period before adjusting the dose to 440 î¼g or 110 î¼g once daily based on symptom severity. doses were standardized to 440 î¼g once daily across all patients at ~4 months to ensure sufficient long-term safety data at the maximum dose. physical exams, including measurement of vital signs and laboratory measurements were taken at baseline, 6 months and study end. independent patient and physician global symptom evaluations were performed at baseline, week 2 and months 1-12 thereafter. patients recorded any adverse events (aes) on daily diary cards. results: of the 396 patients included in the study, 349 (88.1%) reported aes. the incidence of aes was similar to that of other comparable allergic rhinitis long-term studies. the most frequently reported aes were pharyngitis, rhinitis, local reactions, headache, epistaxis and sinusitis (table 1) . most aes were mild-to-moderate in intensity; 34 patients withdrew from the study due to aes. there were no clinically relevant changes in physical exams, vital signs or laboratory measurements. a total of 4 serious aes (saes) were reported; breast carcinoma (n=1), depression (n=1), post-operative spinal fluid leak (n=1) and staphylococcal infection (n=1). saes were thought to be not related to the study drug. at final visit, 83% of patients had either moderate or marked/complete relief of symptoms using global symptom scores. similarly, 86% of physicians rated their patients as having either moderate or marked/complete relief. conclusions: long-term administration of taa-hfa 440 î¼g exhibited a good safety and tolerability profile, while providing moderate-to-complete symptom relief in more than 80% of patients treated for par. introduction: second-generation, 'non-sedating' antihistamines (ahs) have lower tendency to cross the blood-brain barrier and cause central nervous system side effects than first-generation agents. however, studies have suggested differences between second-generation ahs regarding cognitive function impairment. methods: a medline literature search was performed using the search terms 'antihistamine and impairment', 'antihistamine and psychomotor' and 'antihistamine and central effects', as well as with individual ah names (acrivastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, mequitazine and mizolastine). the findings for second-generation ahs were reviewed and well-designed, placebo-and positive-controlled studies in humans using objective measures of cognitive impairment were included. results: 43 publications were identified as the inclusion criteria. all included objective assessments such as driving performance, critical flicker fusion and divided attention tasks. there was a large variation in the numbers of available well-designed studies; the most rigorously assessed agents were fexofenadine and cetirizine. a number of the ahs (ebastine, acrivastine, loratadine, mequitazine and mizolastine) were not impairing at recommended doses, but were at higher doses. in a small number of available studies, the newer ahs desloratadine and levocetirizine produced no impairment at recommended doses (5 mg); however, higher doses were not investigated. cetirizine studies were variable; impairment at the recommended 10 mg dose or higher was seen in a number of studies. in contrast, fexofenadine hcl, up to a dose of 360 mg, was non-impairing in a large number of studies. only one study indicated impairment in one task (critical tracking) and this was only observed with the first doses of fexofenadine hcl (120 and 180 mg) and not subsequent doses; however, the authors concluded that doses up to 240 mg/day should be safe for patients who drive. conclusion: while second-generation ahs are less impairing than the first-generation, some newer ahs produce impairment at or above the recommended dose. these differences become important to the patient when agents cause sedation or if patients over use beyond the recommended dose. in conclusion, fexofenadine was the only ah found to be non-impairing in all studies, even at double the recommended us dose. s. shaver 1 , r.b. berkowitz 1* , c. lutz 1 , p. jones 1 , c. qiu 2 , s. meeves 2 , s.t. varghese 2 , g. georges 2 , 1. woodstock, ga; 2. bridgewater, nj. introduction: fexofenadine (fex) is a h1-receptor antagonist, with proven efficacy in the treatment of allergic rhinitis (ar). to date, no live cat-room challenge studies have assessed the efficacy of fex in cat-allergen induced ar. this study assessed the efficacy of a single dose of fex hci 180 mg in preventing and controlling cat-allergen induced ar using the cat-room challenge model. methods: this single-center, prospective, randomized, doubleblind, placebo-controlled, two-way crossover study consisted of a screening visit, one or two priming visits and two treatment periods, separated by a 14 (â±3)-day wash-out. qualifying subjects were randomized to treatment sequence 1 (placebo followed by fex) or 2 (fex followed by placebo). baseline endpoints were obtained prior to study drug administration, and 5 minutes before entering the cat challenge room for allergen challenge. allergen challenges were initiated 1.5 hours post-dose, for both treatment periods. the primary endpoint was the change from baseline in total symptom score (tss; sum of rhinorrhea, itchy nose/palate/throat, sneezing and itchy/watery/red eyes) after 30 minutes of allergen exposure at 2 hours post-dose, compared with placebo. other endpoints included changes in individual symptom scores, including nasal congestion. levels of airborne felis domesticus allergen 1 (fel d 1) were determined. results: of 211 subjects screened, 66 were randomized and 63 completed the study; 32 and 31 in sequence 1 and 2, respectively. mean change in tss from baseline was significantly less with fex compared with placebo at 30 minutes after initiation of the cat allergen challenge (p=0.0327). significantly greater percentage reductions in the individual symptom scores for sneezing (p=0.0037) and nasal congestion (p=0.0455) were observed with fex compared with placebo, 30 minutes after challenge. although levels of fel d 1 varied widely, they were balanced between treatment groups. the overall incidence of treatment-emergent adverse events (aes) was low and comparable between groups; no serious aes occurred. conclusion: this study demonstrated that a single dose of fex hci 180 mg is effective and well tolerated as a prophylactic agent for alleviating the ar symptoms induced by exposure to cat allergen. further large-scale clinical trials are warranted to confirm these findings. rationale: allergic rhinitis in children is thought to be associated with several co-morbid disorders. we conducted the following study to assess whether children with diagnosed hypertrophy of tonsils and/or adenoids evaluated by an allergist were more likely to demonstrate objective evidence of allergen sensitization than children similarly evaluated without evidence of these upper airway obstructions. methods: records from the past ten years were identified in the hospital database by presence of an allergy clinic visit and an icd-9 code for hypertrophy of adenoids and/or tonsils. we reviewed these records to confirm that the diagnosis was accurate. we included subjects if reliable skin prick or in vitro testing for specific ige to aeroallergens was performed. for comparison, we randomly obtained an age and testing-type similar sample. first group subjects were excluded from the second group. skin testing was performed with commercial allergen extracts applied with a dermapikâ©. in vitro testing was by unicapâ© feia. positive skin testing had at least a wheal 3 mm or flare 10 mm greater than the negative control. positive in vitro values were 0.7 ku/l or greater or a positive pollen mix. additionally, we performed a search in an insurance database to determine how many local children had a code for allergic rhinitis. results: of 202 pediatric allergy patients with adenoid/tonsillar hypertrophy tested, 41.6% (95% ci +/-6.8%) had at least one positive test. in 802 without adenoid/tonsillar hypertrophy, 50.7% (95% ci +/-3.5%) had at least one positive test. the observed difference was 9.1 % (95% ci +/-7.6%). the standard error of the difference in percentage was 3.94; the z score was 2.3. the corresponding p value is 0.02. the percentage of 70, 356 children with an allergic rhinitis icd-9 was 15.6% (95% ci +/-0.3%). conclusion: the number of allergist-referred children with adenoid and/or tonsillar hypertrophy testing positive for an aeroallergen is slightly less than the number of similar children without these diagnoses testing positive. it is not clear that adenoid/tonsillar hypertrophy is associated with a higher risk of allergic rhinitis. a prospective study with allergy testing of all children with adenoid and/or tonsillar hypertrophy might provide information that could alter referral patterns. w.e. berger 1* , w. storms 2 , s. kimura 3 , m. beck 4 , s. galant 5 , t. westbrook 3 , 1. mission viejo, ca; 2. colorado springs, co; 3. pensacola, fl; 4. miami, fl; 5. orange, ca. background: patients having allergic rhinoconjunctivitis are often treated with nasal spray or systemic allergy therapy, forgoing therapy specifically targeting ocular symptoms. the rhinitis quality of life questionnaire (rqlq) and allergic conjunctivitis quality of life questionnaire (acqlq) instruments can be used to quantify the relative benefit of varying medication regimens. objective: to determine the extent of benefit gained in quality of life when an eye drop treatment for ocular allergy (olopatadine) was added to rhinitis patients' preexisting regimen of nasal or systemic allergic treatment. methods: this was a four week prospective, multi-center, open-label crossover, quality of life study occurring during allergy season. at visit 1, patients completed the rqlq and acqlq questionnaires to assess baseline quality of life. patients were randomized to receive ocular allergy therapy (olopatadine, patanol bid) concomitant with their systemic or nasal rhinitis treatment(s) for 2 weeks between either visit 1 and visit 2 (group a) or between visit 2 and visit 3 (group b). at visit 2 and visit 3, patients completed the rqlq and acqlq. results: a total of 200 patients completed this study: 97 in group a, 103 in group b. of these, 181 (90.5%) experienced eye allergy symptoms at least 1 day during the previous week as reported in the baseline acqlq. baseline scores of the rqlq and acqlq for both groups were comparable. clinically significant improvement from baseline in global rqlq and acqlq was seen following addition of ocular therapy for both groups (rqlq: -1.07, -0.94; acqlq: -1.2, -1.2). similar improvement was seen across all domains of both questionnaires. the acqlq correlated with the rqlq in the applicable domains. conclusion: many allergic rhinitis patients using nasal or systemic medication also suffer from ocular allergic symptoms. for these patients, quality of life improvement is not maximized; the addition of a topical antiallergy eye drop can result in beneficial effects on quality of life. in this study, the addition of olopatadine eye drops to these patients' medication regimens resulted in significant improvement in not only eye symptom related quality of life domains but in overall quality of life. h. milgrom 1* , r. lanier 2 , f.c. hampel 3 , b. kittner 4 , 1. denver, co; 2. fort worth, tx; 3. new braunfels, tx; 4. bridgewater, nj. introduction: fexofenadine, a non-sedating, selective h1-receptor antagonist, is currently indicated for use in children aged 6-11 years with seasonal allergic rhinitis in a number of countries, including the us, and has an excellent safety profile in this age group. this study was designed to assess the safety and tolerability of fexofenadine in children aged 2-5 years with allergic rhinitis (ar). methods: the study had a multicenter, double-blind, randomized, placebo-controlled, parallel-group design. children aged 2-5 years (n=453) with ar were randomized 1:1 to either placebo twice daily (bid; n=231) or fexofenadine hcl 30 mg bid (n=222), for 2 weeks. both treatments were given orally as granulated powders (capsule content) mixed with two teaspoons of apple sauce. treatment-emergent adverse events (teaes) were recorded for all subjects by parents/caregivers and assessed by investigators. clinical laboratory variables, physical examinations, vital signs and ecg evaluations were also assessed in a subgroup of children (placebo, n=79; fexofenadine, n=71). results: baseline demographics were similar in both treatment groups. while approximately 50% of children in both groups experienced at least one teae, no unusual or unexpected teaes were observed in the fexofenadine group. when the total group was analyzed for teaes by body system, or assessed separately by age groups of 2-, 3-, 4-and 5-year-olds, no clinically meaningful differences were observed between the two treatment groups. in both treatment groups, the majority of subjects overall and in each age group experienced teaes rated as mild or moderate in intensity. few subjects experienced teaes considered possibly related to study medication (placebo: 8.2% [19/231]; fexofenadine: 9.5% [21/222]). the percentage of discontinuations due to teaes was also comparable between treatment groups (placebo: 6.9% [16/231]; fexofenadine: 5.0% [11/222] ). in the subgroup assessment, no clinically relevant changes were seen from baseline for laboratory variables, vital signs, ecgs or physical examinations in either treatment group. introduction: the efficacy and safety of fexofenadine hcl 30 mg bid has been proven in two large phase iii studies in pediatric subjects aged 6 to 11 years with seasonal allergic rhinitis. in addition, the safety of fexofenadine has been demonstrated in pediatric subjects 2 to 5 years of age with allergic rhinitis (ar). subsequently, two studies (t/3001; t/3002) have assessed the pharmacokinetics, safety and tolerability of fexofenadine 15 and 30 mg bid in pediatric subjects 6 months to 2 years of age. methods: both studies were of multicenter, randomized, double-blind, placebo-controlled, parallelgroup design and enrolled pediatric subjects aged 6 months to <1 year weighing 10.5 kg and aged â±1 year to <2 years weighing >10.5 kg. all subjects had a diagnosis of ar as assessed by previous medical history, pattern, or suggestive physical findings. subjects were randomized to receive fexofenadine hcl 15 mg (t/3001), or 30 mg (t/3002) granulation powder twice-daily, or placebo for a minimum of 7 days. safety was evaluated based on adverse events (aes), vital signs, 12-lead electrocardiograms (ecgs), and physical examinations. results: a total of 174 and 218 subjects were randomized in studies t/3001 (fexofenadine hcl 15 mg bid: n=85, placebo: n=89) and t/3002 (fexofenadine hcl 30 mg bid: n=108, placebo: n=110), respectively. in t/3001, 40.0% (34/85) of children receiving fexofenadine and 42.7% (38/89) receiving placebo experienced at least one treatment-emergent ae (teae). in t/3002, the incidences of teaes were 35.2% (38/108) and 52.7% (58/110), respectively. in both studies, most of the teaes experienced were mild or moderate in intensity. the incidence of possibly-related teaes was also similar for both treatments in each study. no clinically relevant changes from baseline to study end were observed for vital signs, ecgs and physical examinations. conclusions: the findings of this study show that fexofenadine 15 mg and 30 mg bid are well tolerated and have a safety profile comparable to placebo in pediatric subjects aged 6 months to 2 years. for seasonal allergic rhinitis (sar) patients that remain symptomatic on an h1-receptor antagonist, cetirizine, and a nasal glucocorticosteroid, mometasone furoate, the addition of omalizumab, a recombinant, humanized, chimeric, anti-ige monoclonal antibody, may provide additional efficacy in sub-optimally controlled seasonal allergic rhinitis patients. in this open labeled 12week trial, 20 patients with symptomatic sar currently using cetirizine, 10 mg qd, + mometasone furoate, 100 mcg/nostril qd, were randomized to continue the existing therapy cetirizine + mometasone or to add-on omalizumab subcutaneously (every 2 weeks to 4 weeks) dosed according to patient's weight, and baseline ige levels to the existing therapy cetirizine + mometasone furoate. the endpoints of the trial include: rhinomanometry, nasal symptom score (composite score of nasal congestion, rhinorrhea, sneezing, post nasal drip and itching) and flexible rhinopharyngolaryngoscopy examination. mean efficacy measurements at the end of the 12-week trial revealed a significant improvements in all parameters examined in the treatment group receiving omalizumab (as add-on to the existing therapy), compared to the other group. in conclusion, the addition of omalizumab to the combination of cetirizine plus mometasone furoate, is more effective than the combination of cetirizine plus mometasone furoate, for the treament of seasonal allergic rhinitis patients. it appears that when omalizumab is added to the combination h1receptor antagonist, cetirizine, and nasal corticosteroid, mometasone furoate, the primary end points (rhinomanometry and symptom scores) are significantly improved. background: based on the official statistic data, the prevalence of allergic diseases in russia has increased more than 3 times during the past 10-15 years, but still the lowest of all european countries. in some studies done in some regions of russia based on isaac program, it has been shown that the prevalence allergies is significantly higher than indicated in official statistics of health departments. the goal of this investigation was to study the prevalence of allergic diseases, in children of south russian region. meth-ods: this study was done in two steps according to isaac program guidelines. in the first step a questionnaire was given to a total of 6558 school children, ages 7 to 8 (group a) and 13 to14 years old (group b), from the two cities of krasnodar and novorossiysk in southern russia. in the second step a physical exam, skin prick tests with different allergens (pollens, molds, cat, dust mites, foods etc.), and pulmonary function tests were performed. results: wheezing was reported in 12.9% of group a and 15.3% of group b children within the past 12 months. the severity of asthma reported was mild in 72% (group a)-84% (group b); moderate in 16.7% (group a)-10.7% (group b) and severe in 3.4% (group a)-5.8% (group b) in studied children). majority of the cases of asthma was noted to be allergic asthma. in both groups of children there was a high incidence of allergies to perennial allergens such as dust mites, cats, molds and other indoor allergens, 18.7% (group a) and 77.3% (group b). the percentage of allergies to pollens was 19.1% and 33.0%, accordingly. allergic rhinitis symptoms were noted in 26.6% (group a) and in 38, 6% (group b). atopic dermatitis symptoms with skin itching observed in 8.3% (7-8 years old)and 5.3% (13-14 years old). family history of atopy was noticed in 34.6% of group a and 41.6% of group b children. conclusions: the prevalence of allergic diseases in south russia is similar to the ones in most of the european countries. the prevalence of asthma has been associated with increased incidence of allergies, due to indoor and pollen allergens. the prevalence in rhinitis and atopic dermatitis in south russia is parallel to asthma. acknowledgement: we would like to thank hollister-stier lab., greer and antigen lab. for allergen samples. objective: to examine the cost and effectiveness of telithromcyin vs. azithromycin for treatment of mild acute sinusitis under current levels of antimicrobial resistance. methods: we considered an adult with mild acute sinusitis, and symptom duration of 7 days. a decision analytic model was created to compare 2 strategies of 1st-, 2nd-and 3rd-line therapies: azithromycin/amoxicillin-clavulanate/levofloxacin (azi/amc/lev), and telithromycin/amoxicillin-clavulanate/levofloxacin (tel/amc/lev). we considered 3 outcomes: response to initial therapy, cost, and time to completion of all antibiotic therapy. clinical resolution was due to response to antibiotic, or to spontaneous resolution. we assumed bacteria that were resistant in vitro would only be resistant in vivo 50% of the time. resistance levels were based on the 2000-2001 us protekt surveillance study. model parameters, such as prevalence of bacterial infection, frequency of causative pathogens, and rates of spontaneous resolution, were based on the published literature. those failing to improve after 5 days were switched to next line therapy. we analyzed claims data from 8 managed care organizations to estimate non-drug charges for initial and follow-up care, and applied a 60% cost-to-charge ratio. drug costs were estimated using the wholesale acquisition cost plus $8 for overhead and dispensing. all costs were adjusted to year 2004 $us. results: the model predicted the tel/amc/lev strategy would result in 71.8% improving by 5 days compared to 65.4% for azi/amc/lev. the telithromycin strategy had lower mean cost, $172 vs. $176. although telithromycin cost more than azithromycin, the tel/amc/lev strategy had slightly lower total drug costs ($95 vs $96) due to less need for additional antibiotic courses. mean time to completion of therapy was 8.4 days for tel/amc/lev and 9.1 days for azi/amc/lev. sensitivity analyses showed that tel/amc/lev had superior health outcomes even when only 5% of in vitro resistant organisms were also resistant in vivo. tel/amc/lev also had lower cost if at least 30% of cases were bacterial, or if in vitro resistant organisms were at least 30% resistant in vivo. conclusion: based on results obtained using this decision analytic model, initial treatment of mild acute rhinosinusitis with telithromycin may result in improved outcomes and lower cost than initial treatment with azithromycin. rationale: "delta crud" is the term used to describe rhinosinusitis syndromes in the mississippi delta region. local perception is that it is associated with chemical crop dusting, regional produce, especially cotton, or high rates of mold allergy. the climate is extreme with mild winters, hot humid summers, and large volume rain. thirty-five percent of inhabitants live below the poverty level, 62% are african american. patients from the agriculturally intensive delta suffer from asthma at almost twice the rate of those in the neighboring hills. rates of pesticide use are among the highest in the nation. there are no prevalence estimates for rhinosinusitis in this region. "delta crud" is chronic in many patients with exacerbations occurring in the summer and fall, especially during chemical spraying and defoliation. we conducted this cross-sectional questionnaire based pilot study to begin characterization of rhinosinusitis in this population. method: the sinusitis treatment outcome questionnaire, with two modifications, was given to consecutive patients presenting to the north sunflower regional hospital er and the sunflower outpatient clinic one month prior to the beginning of summer crop dusting. the question, "do you have delta crud?" was added. the survey was anonymous, allowing irb exemption. results: 91 consecutive patients returned completed questionnaires. 46(50%) admitted to having "delta crud". patients who claimed to have delta crud had significantly more sinus headache, nasal pruritus, conjunctivitis, and chest symptoms (see table) . there was no significant difference in antibiotic prescriptions, er visits, and missed work. six patients with delta crud had been hospitalized for "allergy reasons" compared with three patients without delta crud. despite severe symptoms just 4 patients received sinus ct scans. only 10/46 patients were prescribed intranasal corticosteroids. conclusion: in our pilot study, the prevalence of chronic rhinosinusitis symptoms (delta crud) is 50%, with severe sinus, pulmonary and ocular symptoms. evaluation and treatment may be suboptimal in this economically disadvantaged rural region. further characterization of "delta crud", pollen counts, ige mediated disease, environmental contributions and comparative studies of related regions are needed. introduction: patients with rhinitis commonly experience sinus pain and pressure (sp+p). many patients with recurrent acute bacterial sinusitis (rabs) base the presence of a recurrence on the severity of sp+p and other nasal symptoms. the ability of patients to differentiate between rhinitis and rabs was evaluated by reviewing the subject screening logs of 4 previously reported clinical trials of flonase (fluticasone propionate) nasal spray, 50 mcg (fp). methods: the efficacy of fp was studied in two trials in subjects with sp+p due to allergic rhinitis and in two trials of subjects receiving cefuroxime axetil to treat an acute episode of rabs. sp+p screening logs were examined to determine how many subjects who thought they had sp+p were excluded from the study for an upper respiratory or sinus infection. rabs screening logs were examined to determine how many subjects with symptoms of an acute episode of rabs were excluded from the study for a negative sinus x-ray or ct scan. rabs symptoms experienced at the screening visit by subjects who qualified for the study were also evaluated. results: of 505 subjects screened in the sp+p studies, only 4 (0.8%) were excluded for evidence of sinus or upper respiratory tract infections, as clinically diagnosed by the investigator. out of 1502 subjects with screening log data in the rabs studies, 649 (43.2%) were excluded for a negative sinus x-ray or ct scan. of the 704 subjects who qualified for the rabs study, the most prevalent symptoms included nasal congestion (100%), mucopurulent discharge (97%), facial pain or tenderness in the sinus area (93%), sinus headache (90%) and malaise (90%). only 15% of subjects had a fever. only one symptom, nasal congestion, was rated by clinicians as severe for greater than 40% of subjects in either study (46% and 43%). conclusions: while most subjects can determine when sp+p is due to allergic rhinitis, many cannot determine when symptoms progress to a sinus infection. symptoms experienced by subjects with rabs were consistent with inflammation, but did not include fever as may be expected with an untreated bacterial sinus infection. under appreciation by patients for the role of inflammation in sinus disease may result in over self-diagnosis of sinus infection and the demand for antibiotics. liposomes are small particles consisting of lipid bilayer membranes, which are used to deliver drugs including amphotericin b, more efficiently and with less toxicity. very few cases of allergy to liposomal amphotericin b (lab) have been reported to date. although there is a report on conventional amphotericin b (cab) desensitization, to our knowledge no case of lab desensitization has been described yet. a 75 y/o patient with lymphoma was treated with lab for pulmonary aspergillosis. within 30 minutes of infusion he developed urticaria, hypotension and tachycardia for which he was treated with the appropriate drugs for anaphylaxis. a second attempt to re administer lab resulted in a similar anaphylactic reaction. desensitization to lab was then successfully completed using a modified protocol based on desensitization to cab (jaci 1995; 96:425-7) : 0.0005 mg infusion over 10 minutes 0.005 mg infusion over 10 minutes 0.05 mg infusion over 10 minutes 0.5 mg infusion over 30 minutes 5 mg infusion over 30 minutes 50 mg infusion over 30 minutes 100 mg infusion over 30 minutes 200 mg infusion over 120 minutes conclusion: desensitization to lab, a lifesaving drug for invasive fungal diseases, can be performed successfully. adverse events (aes) were recorded throughout the study. therapeutic comparability was defined a priori as a clinical response within 30% for hfa vs cfc for the primary efficacy measures. results: baseline demographics were similar between treatments for the intent-to-treat population. mean 24-h baseline symptom scores for nasal stuffiness, nasal discharge and sneezing were 2.4, 2.3 and 2.1, respectively, out of a possible score of 3. all taa-cfc and taa-hfa doses significantly (p<0.05) reduced 24-h, and am and pm 12-h reflective scores for nasal stuffiness, nasal discharge, sneezing and ni vs placebo (except cfc 14 î¼g for sneezing). taa-hfa and taa-cfc were statistically comparable (within 0.70 and 1.3 for the 2 one-sided 90% confidence interval) at doses of 110 î¼g and 440 î¼g for the primary variables of nasal discharge, sneezing and ni for the 24-h, as well as the am and pm 12-h reflective assessment, over the 2-wk period 36 * , 66 * agrawal p27, p35, 41, p58, p105, p106, p110, p180 41 * , p21, p27, p58 * p105, p106, p110, p180 p7 * finegold, i. 42 * fink p21 * 26 * sienra monge 45 * , 55, 62 * so, c. p52 * soane 70 * , p143 * staveren, a.m rates of cardiovascular mortality are higher during peak pollen times. (brunkreef, lancet 2000) . for this reason, mast cell stabilizing agents and leukotriene antagonists are under development for the treatment of myocardial infarction. conversely, ige mediated disease may be protective against sudden cardiac death.(szczeklik, coron art dis 1993) ar has not been studied in this context. we conducted a retrospective case control pilot study of va patients who were diagnosed with allergic rhinitis and had an acute myocardial infarction. methods:the electronic medical record of a large va hospital was queried for patients diagnosed with an acute myocardial infarction (ami) in the past six years. patients were divided into groups with and without ar. patients with chronic urticaria, asthma, and eczema were excluded from this study. primary outcome was all-cause mortality. secondary outcomes included lv systolic function and peak troponin levels. results: 424 patients were diagnosed with ami, 63 (15%) of which were also diagnosed with ar. patients with and without ar did not differ in terms of age, ethnicity, tobacco status, hypertension, diabetes, or lipid levels. 14/63 (22%) patients with ar died, compared to 152/361 (42%) control patients, (p-value: 0.0028). ar patients had lower mean peak troponin values (7.7 vs. 11.8), but these were not significant. both groups had similar lv function. conclusion: this pilot study suggests an association between allergic rhinitis and a decrease in ami allcause mortality independent of other factors. atopic patients have been shown to have prolonged bleeding times, reduced platelet aggregation, and delayed thrombin generation which can result in delayed clot production. (szczeklik, thromb haem 1986) possibly, patients in our study diagnosed with allergic rhinitis were more likely to monitor their health symptoms or had physicians who carefully addressed multiple issues. rigorous prospective studies are needed to determine the role of ar in myocardial infarction. purpose and methods. a formulation of olopatadine hydrochloride ophthalmic solution (olopatadine 0.2%) was evaluated in a randomized, placebocontrolled, double-masked, hybrid environmental study intended to determine efficacy and safety in 240 subjects with histories of seasonal allergic conjunctivitis or rhinoconjunctivitis. in this 12-week trial, subjects regularly assessed their ocular signs and symptoms. additionally, subjects evaluated both the frequency and severity of their nasal symptoms. daily throughout the study, ragweed pollen counts were obtained from each investigative center. repeated measures analysis of variance was used to compare treatment differences in the slopes for nasal symptoms as a function of pollen counts. results. the nasal results are presented herein. specifically, relative to placebo, olopatadine 0.2% significantly reduced the frequency of pollen effects on sneezing (p=0.0355) and itchy nose (p=0.0032), and reduced the severity of pollen effects on sneezing (p=0.0451), itchy nose (p=0.0178), and runny nose (p=0.0327). in this study, the most frequent ocular adverse event that was related to therapy with olopatadine 0.2% was ocular dryness, occurring at an incidence of 1.7%. no treatment-related, clinically relevant changes were observed for visual acuity, intraocular pressure, ocular signs, or fundus parameters. conclusion. olopatadine ophthalmic solution, 0.2% is safe, well-tolerated, and effective in reducing some effects of pollen on the nasal symptoms associated with allergic rhinoconjunctivitis. author index key: cord-022650-phsr10jp authors: nan title: abstracts tps date: 2018-08-14 journal: allergy doi: 10.1111/all.13539 sha: doc_id: 22650 cord_uid: phsr10jp nan (either in men or women) between metabolic syndrome and incident asthma. conclusion: this study confirmed the significance of obesity as a risk factor for incident asthma. moreover, obesity appeared to be a stronger risk factor than metabolic syndrome. 0638 | relationship between helminth infection, blood eosinophils and asthma symptoms in a rural community from the tropics peñaranda d; alvarez l; sierra n; lopez j; zakzuk j; caraballo l institute for immunological research. university of cartagena, cartagena, colombia background: immune response to helminths shares many features with the allergic response. in tropical regions where helminths are highly prevalent, asthma is still a major public health burden. large clinical cohorts suggest that high blood eosinophils (hbe=>400 cells/ mm 3 ) are associated with asthma exacerbations. however, the association between hbe and asthma severity in rural communities with prevalent helminthic infections is unclear. method: patients with wheezing symptoms in the last year living in a rural tropical community (santa catalina, colombia) where helminths are highly prevalent, were recruited for this study. blood eosinophils were assessed by complete blood count. parasitic infection was evaluated with two serial coprological exams (kato-katz method) and skin prick tests were conducted to determine reactivity to ascaris. results: seventy-three patients (mean age: 21; range: 2-64 years old) were recruited in this study. a. lumbricoides and t. trichuria active infection (47.9% and 16.4%, respectively) were not related to age or gender. a positive spt to ascaris extract, aba-1 and d. pteronyssinus was observed in 23%, 18.4% and 34.2%, respectively. mean eosinophil count was 621 cells/mm 3 ; 43.9% had hbe. rate of patients with at least one emergency department visit was 61.9% and hospitalization, 21.9%. blood eosinophil counts (as a continuous variable) were inversely associated with age (p = 0.03) and higher in helminth infection (p = 0.002). in crude univariate analysis, exacerbations (er and/or hospitalization) were associated with age (or: 0.96; 95% ci: 0.93-0.99, p < 0.01) and hbe (or: 2.9; 95%ci: 1.1-7.8, p = 0.04), but not with helminth infection. for a better definition of asthma, multivariate analysis done in those >7 years old indicated that hbe, helminth infection and positive ascaris spt were not associated with asthma exacerbations. conclusion: uncontrolled asthma is common in rural places of the tropics. since helminth infection influences eosinophilia, the clinical value of hbe to predict exacerbations is limited in helminth-endemic populations. castro mc 1 ; ferreira j 2 ; sarmento d 2 ; carvalho c 2 ; matos a 2 ; bicho m 2 1 chln-immunoallergy; lisbon medical school-genetic department, lisboa, portugal; 2 lisbon medical school-genetic department, lisboa, portugal background: the bioavailability of no and endothelial homeostasis depends on the functional polymorphism of 19-bp del/ins within intron-1 of dhfr (dihydrofolate reductase enzyme) (rs70991108) that could interfere in the regeneration of bh4 (tetrahydrobiopterin) from bh2 (7,8-dihydrobiopterin) and contributes to endothelial dysfunction in asthma. method: asthmatics (n = 123) compared with control group (n = 50).the polymorphism was analyzed by pcr. control of asthma assessed by (acq7 and paqlq). statistical analysis with spss 23.0 establishing a significance level of p < 0.05. results: there are 80 women and 43 males in asthmatics and 39 women and 11 males in controls (p = 0.137). in asthmatics: age ( x ± sd): 38.26 ± 19.24 ; and in control group: age ( x ± sd): 51.50 ± 13.34. the genotype frequencies in asthmatics are: dd (6.5%); id (66.7%); ii (26.8%); in control group: dd (12.0%); id (64.0%); ii (24.0%); there is no statistical difference between groups (p = 0.478). the allelic frequencies in asthmatics are: allele d (39.8%); allele i (60.2%); in control group: allele d (44%); allele i (56%); there is no statistical difference between groups (p = 0.476). the genotype frequencies in the uncontrolled asthmatics are: dd (0.0%); id (61.1%); ii (38.9%) ; in the controlled asthmatics are: dd (9.4%); id (68.2%); ii (22.4%); there is statistical difference between groups (p = 0.047). genotypes id and ii are more frequent in the uncontrolled asthmatics. the allelic frequencies in the uncontrolled asthmatics are: allele d (30.6%); allele i (69.4%); in the controlled asthmatics are: allele d (43.5%); allele i (56.5%); there is a trend to have differences between groups (p = 0.059). allele i is more frequent among uncontrolled asthmatics. the uncontrolled asthmatics are older than the controlled asthmatics (p < 0.001). there is no differences in gender distribution (p = 0.903). the genotype ii confers a risk of being uncontrolled asthmatic of 2.950 times when compared with controlled asthmatics and adjusted for age: or b : 2.950 [1.117-7.789 ]; p = 0.029. physiopathology and the emergence of evidence-based clinical guidelines. however, variation still exists among some diagnostic aspects of asthma in real life. it is unknown to what degree diagnosis is affected by the treating physician's medical specialty. results: a total of 62 gps, 239 pediatricians, 364 allergists, 161 pulmonologists and 34 otolaryngologists (orls) replied. although for general application of diagnostic clinical criteria all physicians rated similarly, in general accordance with the mag suggestions, a third of non-pulmonologist practitioners don't recognize chest discomfort as one of the clue symptoms of asthma, but they erroneously believe crackles are (p = 0.01). we found agreement in almost half of all physicians to erroneously believe that viral illness' induced wheezing in non atopic children predisposes asthma. conversely, 75-85% are aware that allergic sensitization predisposes to asthma. most specialists -except pulmonologists (p = 0.02)-incorrectly listed fev1 as the best parameter to identify airflow obstruction (ao) and fev1/fvc to assess ao severity. 20% of gps do not know peak expiratory flow (pef) measurements could be valuable, and 75% of all specialists are not aware that changes in pef can also be used to confirm ao reversibility. to classify asthma, only pulmonologists adequately considered the level of control in similar proportion than severity (81% and 83%, respectively), which is uniformly the preferred method by most other specialists. conclusion: although in general many clinical aspects of asthma diagnosis seem to be accurately assessed, there is a wide specialityspecific variation regarding some aspects of phenotyping and classification, diverging from mag's recommendations. as such, our results can help to detect knowledge-gaps and to guide the development of more focused specialty-specific learning tools to improve clinical impressions, process medical evidence, and apply it to patient care. 0652 | issues, continuous medical education on treatment of acute asthma, exercise induced asthma and asthma in pregnancy should include, per medical specialty background: to unify and improve the management of asthma, including asthma exacerbations, the mexican guideline on asthma (25.5%) of 51 employees who stated increased symptoms with flour exposure. among all workers 9 (5.5%) employees were diagnosed as asthma and 7 (4.3%) workers were diagnosed as ba. conclusion: wheat flour sensitivity is high among workers who are exposed to wheat flour, however the prevalence of ba is similar to the previous data in the literature. johnsen cr 1 ; callesen kt 2 ; jensen bm 2 ; poulsen lk 2 1 clinic of allergy, dept. of dermato-allergology, gentofte hospital, copenhagen, denmark; 2 laboratory of allergy, gentofte hospital, copenhagen, denmark background: enzymes are well known as sensitizers and causes of occupational allergy primarily in the industries producing and using the products. we present a case of occupational contact urticaria, rhino-conjunctivitis and asthma in a 32 year male chef who was using a transglutaminase enzyme powder obtained from fermentation of streptomyces mobaraense as meat glue in processing of fine culinary dishes. this transglutaminase has been used for protein food preparation in industrial settings since 1992 to improve the texture of protein rich foods such as surimi or ham. in this case it was used in small scale in a gastronomy restaurant kitchen spraying enzyme powder with a sieve over raw meat without any protective equipment in contrast to the producer's recommendation. the chef was also found allergic to dried, edible mushrooms also forming part of the meat dish prepared with the transglutaminase enzyme powder. in one occasion he experienced an oral reaction with itching and swelling of the mucosa in the mouth, stridor, angioedema of the face, and urticaria after ingestion of beef meat treated with transglutaminase and rolled in horn of plenty dried mushroom powder. no other symptoms of food allergy were reported but a known cat allergy was. background: formaldehyde and xylene are occupational skin and respiratory irritant and/or sensitizer, exposure to those may be associated with dermatitis, rhinitis and asthma. health care workers, as nurses, laboratory technicians, doctors could be exposed in different tasks in operating rooms, endoscopy and in pathology laboratory. we describe three cases of work-related rhinitis in technicians employed in the same unit of hospital pathology . first case: a woman of 38 years old underwent medical examination in our occupational allergy unit because allergy respiratory symptoms. she has been working for 8 years in pathology laboratory and was exposed to xylene and formaldehyde. she developed rhinitis, rhinosinusitis, hyposmia and cough with sputum after 5 years started work. she had negative skin prick test for common aeroallergens. lung function was normal with a fev1/fvc ratio of 80% of predict. blood cells count reveled 15% of eosinophils (980/mmc) with 6550 total leucocytes. second case: a woman of 40 years old was affected by moderate persistent allergic rhinitis with positive skin prick tests to house dust mite, dog and cat. in the last year rhinitis symptoms worsened in relation to work and improved during vacation. when she was exposed mostly to formaldehyde during shift at the end of it she usually experienced face skin and conjunctival erythema. she developed work-related symptoms after 14 years of exposure in the pathology unit. third case: a woman of 38 years old, who has been working for 14 years in the pathology unit and was exposed to formaldehyde and xylene, in the last year developed moderate-severe persistent rhinitis with hyposmia and chronic cough. she referred to otorhinolaryngologist and an irritant induced rhinitis was diagnosed. she had negative skin prick test for common allergens and normal lung function. results and conclusion: the workers experienced respiratory symptoms in relation to work exposure to formaldehyde and xylene. the suspected causal agents were monitored in the work environment and an exceeding of the recommended limit values was found. preventive measure were adopted with a reduction of exposure and symptoms improve only in the second and third case. challenge test with mannitol is considered to be more specific than test with methacholine. also, duration of procedure is shorter and safer. therefore the study aim was to compare the usefulness of these two tests in monitoring of sict. method: four bakery workers with suspicion of oa underwent single-blind, placebo-controlled sict with workplace allergens accompanied by evaluation of nsbhr with mannitol and methacholine before and after sict. clinical examination, spirometry, skin prick tests (spts) to common aeroallergens and occupational allergens, serum specific ige antibodies to occupational aeroallergens were also performed. results: positive spts results to occupational aeroallergens were found in all bakery workers, specific ige to flours were detected only in two subjects. three out of the four patients displayed positive sict reaction (in two cases early spirometric response). in all of these 3 patients, airway response to methacholine increased significantly. in the first two patients also airway reaction to mannitol was significant, whereas in one subject with early reaction there was no increase in nsbr after mannitol inhalation. the patient with negative sict results did not reveal any changes in nsbr before and after the test, neither to methacholine nor mannitol. 0668 | rice-induced occupational anaphylaxis and socio-economic impact-case report method: in this prospective study, a total of 240 students completed a self-administrated questionnaire that comprised different questions and gave information about the participants and their glove use, working habits, signs and symptoms related to these gloves, precautions taken to minimize it, etc. skin prick test is performed through commercial extract latex gloves (stallergenes), while patch test is prepared through latex gloves and adhesives. two types of gloves are used: gloves that contain latex and gloves without latex (vinyl gloves), which are used also as e negative control. results: questionnaire items and diagnostic tests revealed that one-fourth of subjects were suspicious for latex gloves hypersensitivity. their mean value for skin reactions like irritant or allergic dermatitis or contact urticaria was between 10% and 14%, while for other symptoms the mean value was under 5%. logistic regression analysis revealed an association between different questionnaire items and positive allergy tests among suspected cases and diagnosed cases of latex allergy. approximately 20% of people who work with laboratory animals experience some allergic symptoms and about 10% of animal technicians go on to develop serious symptoms of asthma. uk government guidelines state that employers must prevent or adequately control exposure of employees to animal allergens and should undertake monitoring to ensure that suitable controls remain effective. the most widely used monitoring method is personal iom filters. however, these need to be attached to a pump and carried by the technician which can be cumbersome and awkward. previous data has demonstrated that allergens from dust mite, cat, dog and pollen could be captured and quantified by a novel type of nasal filter. in this current study, we sought to assess the feasibility of using the nasal filters for the assessment of exposure to mouse allergen in a laboratory facility. method: technicians working in a laboratory animal facility were asked to wear the filters during normal routine work. for comparison, they were also asked to wear an iom filter for the same duration. allergen was extracted from nasal and iom filters by gentle rocking in pbs-tween for two hours. levels of the major mouse urinary protein (mus m 1) were quantified using our multiplex array technology, which is highly sensitive and allows for quantification of mus m 1 down to 0.01 ng/ml. results: significant levels of mus m 1 were detected in the nasal filter extracts and these levels correlated with the type of activity that was being performed by the technician, as well as the housing environment of the mice. levels were compared to the suggested 'safe' limit of allergen exposure of 5 ng/m 3 . we also found that the technicians grew accustomed to the nasal filters quickly and found them far more practical for every day monitoring that wearing the iom filter and pump. conclusion: these data indicate that nasal filters may be considered a simple and easily wearable method for monitoring laboratory animal allergen exposure. future studies are planned to assess the feasibility of wearing the filters for analysing exposure to other laboratory animal allergens from rat and guinea pig. havana university lower co 2 emission, water and feed consumption and limited waste production. insects are currently allowed both for human and animal feeding in some eu countries, including italy and the risk profile related to production and consumption of insects as food and feed, including risk of allergenicity, is currently under evaluation by efsa. both food and feed products derived from insects require multiple manipulations by the breeder and/or by the workers who transform the insect into the commercial products, thus the occupational exposure have to be considered too. the aim of this work is to evaluate the allergenicity of tenebrio molitor, one most used species for animal feeding. method: t. molitor proteins were extracted from intact dried larvae and from flour of dried larvae. the protein extracts were separated in one-dimensional electrophoresis conclusion: according to these results, the larva flour seems to be less immunoreactive than the intact counterpart, probably due to the processing that causes the degradation of protein bands over 50 kda. working in gastronomy is associated with exposure to many factors with an irritating and allergic potential influencing respiratory system. food products and organic dust are the source of inhaled allergens which may cause sensitization during apprenticeship. the study aim is a prospective observation of incidence of sensitization to selected environmental and occupational allergens among culinary school apprentices and identification of work-related allergic diseases in this group. method: the cohort comprised 374 apprentices. they were examined in the first and the second year of education. questionnaire and allergological tests [(skin prick test) spt to common and occupational allergens, ige level evaluation (total and specific for occupational allergens) and pulmonary tests] were performed]. results: the most frequent symptoms reported by examined apprentices were rhinitis (12.8%), conjunctivitis (7%), skin symptoms (6.7%), dyspnea (5.6%) and cough (2.7%). 10 subjects developed nasal symptoms during the second year of education, while in 6 cases the skin symptoms and in 4 subjects conjunctivitis appeared. in 7 cases the work-related symptoms were reported. the most frequent positive results of spts were obtained with dermatophagoides pteronyssinus 24.4%, dermatophagoides farinae 21.6%, grass pollens 18.6%. positive spt to rye and barley flour were found in respectively 2.2% and 1.4% apprentices. 4.1% of apprentices had specific ige to flours. the preliminary results indicate that work-related allergy symptoms and hypersensitivity to occupational allergens are rarely found among culinary school apprentices in the first years of education. the further observation will allow to evaluate the trends in incidence of allergy to occupational allergens, as well as the clinical presentation of allergy in that group. 0677 | how multifaceted the clinical presentation and etiology of allergic diseases could be? method: the study was done in children from west georgia randomly and on based of questionnaire of representative cohort. (2014) (2015) (2016) . the cohort was 1500 children, 1-16 years old, risk factors were studied by way of interviewing, clinical-laboratory dates. for assessing the risk factors, was used 'case control' method. the statistical processing of material was done with computer program sps/sv12. inclusion criteria for enrolment were: collectors of dust, gender, existence of moisture and mold consuming of tabasco, atopic dermatitis and seasonality. results: the groups, which we have studied, prevalence of acute respiratory viral infection was 61%, bronchitis −29.3%, allergic rhinitis 33.9%, atopic dermatitis 9.1%, food allergy 5.4%. the reliability was high (p < 0.05) in families with bronchial asthma compared with healthy population. bronchial asthma was detected in 5.7% of population. the hereditary load of allergic diseases in patients with bronchial asthma was 9.7% and in healthy cohort it was 5.7% (p < 0.001). conclusion: based on the results, we can conclude that, ecological factors and genetic predisposition significantly influences on prevalence of sensibilisation of house dust mite, molds and formation of bronchial asthma. as the genetic and environmental factors that act on an immune system are better elucidated and their roles established, the implementation of more enduring preventive efforts will be developed. however, at present, the best approach to the child at high risk for the development of allergies is to institute dietary and environmental control measures early to decrease sensitization, and to recognize and appropriately treat the evolving signs and symptoms of allergic disease. background: plantation of road-side avenue trees has become a major part of the urbanization programme in kolkata metropolis of india for megacity beautification and environmental management. due to evergreen habits, gulmohor (delonix regia) and chhatim (alstonia scholaris) are frequently selected for plantation programme to generate green belts. however, an increasing incidence of seasonal pollinosis was observed among the inhabitants living in close vicinity to these trees suggesting a possible link between the airborne pollen load and the concomitant respiratory hazards. this prompted us to investigate the allergens in the pollen of these two dominant avenue trees. method: aerobiological surveys were conducted at multiple sites of kolkata for a period of two years using seven-day volumetric burkard sampler to record the pollen concentration in the outdoor ambient air. clinical data and residual blood of pollinosis patients were collected from a public hospital. allergens were detected in the pollen proteome fractionated in 2d gel by ige-serology. the major igereactive proteins were partially purified by ammonium sulphate fractionation followed by ion-exchange chromatography. the allergenic activity of the fractions was tested by histamine release assay. results: a clear correlation was observed between the pollinosis related morbidity and the aeropollen load especially during the peak flowering period of these two trees. about 41% and 52% of the patients displayed positive spt response and ige-reactivity using pollen extracts of gulmohor and chhatim respectively. immunoproteomic analyses revealed the presence of 7-8 ige-reactive components in the 2d pollen proteome of these species. hierarchical cluster analysis with patient immunoblot data identified a 31 kda and a 25 kda protein as major allergens of gulmohor and chhatim respectively. the purified fractions containing each of these two major allergens induced histamine release from granulocytes within a range between 42 and 77%. method: immortalized human keratinocyte cell line (hacat) and primary normal human epidermal keratinocytes (nheks) were differentiated with calcium chloride for 1 and 3 days, respectively. following the differentiation, the cells were treated with il-4 (100 ng/ml), il-13 (100 ng/ml), and/or hcho (4 × 10^-4%) for 2 hours. the mrna expression of flg, ivl, lor, dsg1, dsg3, dsc1, dsc3, as well as tslp was analyzed using quantitative real-time pcr. results: hcho exposure decreased the mrna expressions of structural components (flg, ivl, and lor) and cell adhesion molecules (dsg1, dsg3, dsc1, and dsc3) in a short-period time of exposure (2 hours). we also found that hcho exposure significantly enhanced il-4-and/or il-13-induced tslp production in nheks as well as hacat. interestingly, exposure to hcho alone is enough to increase the tslp mrna expression in both cells. conclusion: our results suggest that hcho exposure might synergistically damage the skin barrier function with il-4 and il-13 by increasing tslp expression and decreasing structural components as well as cell adhesion molecules. 0685 | skin prick test reactivity to aeroallergens in adult allergy clinic in a tertiary hospital: a 12-year retrospective study results: five different human sera were screened for specific ige level against 29 different allergen sources using test methods of three different suppliers. the sensitivity of the three different methods can be arranged in the ascending order manufacturer a < manufacturer c < manufacturer b. with the test of manufacturer a, 45% of the measurements were below the detection limit (0.35 ku/l), with the test of manufacturer c, 16% of the measurements were below the detection limit, whereas the test of manufacturer b leads to values below the detection limit in 10% of the cases. in terms of variation coefficient, the test system of manufacturer c had the best performance. test systems of manufacturers a and b exhibited comparable variation coefficients, which were considerably higher than that of manufacturer c. conclusion: based on these test results, only the test of supplier c is recommendable for determination of levels of specific ige for diagnostics of allergic patients. with the test of manufacturer a, elevated levels of specific ige antibodies for many allergens cannot be detected due to the poor sensitivity of the test system. the test system of supplier b exhibits a good sensitivity but the coefficient of variation is rather high for a diagnostic test. this drawback could be circumvented by multiple determination of one test parameter. although this is an advisable strategy in general, the routine in diagnostic laboratories is incompatible with this approach, since throughput would decrease while costs would increase. this study is another good example for the need of the implementation of a characterized standard material with known values of sige, as demanded by wojtalewicz et al. method: cd203c and cd63 expression on basophils were monitored upon exposure of whole blood samples (<24 hours) to anti-ige and/or allergenic extracts. staining was conducted on exposed samples using dry room temperature stable antibody panels (dura innovations format) coated in 96 well plates, eliminating all antibody pipetting steps from the workflow. red blood cells were lysed and data was acquired (without further wash steps) on a cytoflex flow cytometer (beckman coulter). staining and lysing were automated using a biomek (beckman coulter). results: the described no-wash preparation protocol, already established for manual preparation mode in tubes, could be transconclusion: the hr-test was significantly less likely to be positive, if a patient suffered from monosymptomatic ae than in ae patients with concomitant urticaria. this could signify a higher likelihood of treatment response to antihistamine and other anti-allergic medication in the latter group. background: pathogenetic mechanisms of allergy are polymorphic. they include ige-dependent and ige-independent, allergen-specific granulocyte-mediated and lymphocytic reactions, as well as nonspecific hypersensitivity, which are realized through a variety of mediators: histamine, tryptase, etc. allergen bucal challenge mimics the natural situation and is useful for understanding the mechanisms of allergic airway inflammation and airway hyperresponsiveness (ahr).saliva used as a non-invasive readily available bio-sample for diagnosis instead of blood. biomarkers in saliva are associated with the pathogenesis and clinical outcome of allergic diseases method: aim: to examine mediators for ahr with buccal(mucosal) challenge tests. we examined 14 patients with allergic asthma(the history, positive skin prick test, serum specific ige) and 12 healthy volunteers. saliva were collected. then, both groups were subjected to buccal(mucosal) allergen challenge by a water-salt solution of the mite allergen dermatophagoides pteronyssinus. saliva was recollected in 30 minutes and 24 hours after the provocation. the level of myeloperoxidase, elastase, tryptase in saliva were determined by the elisa. that provocative test did not cause clinical symptoms development or reduction in nasal bronchial patency in any patient. results: in patients with allergopathology, an initially increased level of myeloperoxidase and tryptase in 30 minutes after the provocation, elastase increased in 24 hours (table 1) . tryptase in saliva after 30 minutes increased till 0.07 (0.04; 0.19) (me, pg/ml (lq;uq)), p = 0.0006. increased tryptase is presence of increased cellular inflammation, e.g. mast cells. its ige-dependent hypersensitivity, because there was the correlation between the elevated level of tryptase and positive prick tests. elevated levels of myeloperoxidase and elastase in saliva may be the criteria for the neutrophil hypersensitivity and ige-independent reactions. in healthy volunteers this increase was not observed. the identification of tryptase, myeloperoxidase, elastase can be used for diagnosis of types of ahr. tryptase is a mediator of early (immediate) response to allergen. increased myeloperoxidase and elastase indicates the involvement of the eosinophils and neutrophils in the oral mucous membrane in the allergic process. these mediators have additional roles in the late phase response. elevated levels of myeloperoxidase and elastase in saliva may be the criteria for the neutrophil hypersensitivity. conclusion: safe and acute in vitro methods allow to conduct early etiological diagnosis of allergy, which contributes to the effectiveness of therapy; the detection of polyvalent sensitization dictates the need for molecular diagnostics to single allergens, which has a higher prognostic level and the clinical significance of predicting the appropriateness and effectiveness of allergen-specific therapy; laboratory diagnostics of the allergy allows to reveal sensitization at the 0 (0, 0) 0 (0, 0) 0 (0, 7.28) *p = 0.002; **p = 0.008; ***p = 0.038. subclinical level, which increases early diagnosis and identify persons with a predisposition to allergy; the establishment of causal allerbackground: antibacterial chemicals like parabens and triclosan have been associated with allergic disease in children. parabens are also suspected to affect metabolic functions, possibly due to their weak endocrine disrupting properties. furthermore, a possible link has been suggested for eczema and adiposity, and thus, how body burden of chemical exposures affect both of these outcomes are of interest. we aimed to describe the association between exposure to parabens and eczema and body mass index (bmi) in an adult population in norway. method: urine biomarkers of butyl-, ethyl-, methyl-and propylparabens were quantified by mass-spectrometry in 496 adult participants (median age=29 years) from the rhinessa study in bergen, norway. linear regression models adjusted for gender, age and bmi (for eczema outcomes) and with clustering for siblings, were applied to model possible association between specific gravity standardized urine biomarker concentrations of parabens with bmi and eczema. results: propyl-(ppb) and methyl-parabens (mpb) were detected in 95% of the urine samples; ethyl (epb) in 62% and butyl (bpb) in 38% of the samples. in women, epb and bpb were detectable in 73% and 61%, respectively. participants with current eczema (15%) had lower level of several parabens compared to those without eczema (bpb for both genders; epb in women only and sum of all parabens in men only). body burden of epb (geometric mean (gm)) was 6.7 μg/l in women with current eczema compared to 22.3 μg/l in women without eczema (p = 0.03). body burden of parabens (mpb and epb) were inversely associated with obesity (bmi>30, (11.4%) ), as compared to normal range bmi (bmi=18. 5-25 (56.4%)) in both men and women. the concentration of mpb for obese women was gm=2213 μg/l compared to 11503 μg/l in women with normal range bmi. for men, the gm for mpb was 35.3 μg/l in obese compared to 134.4 μg/l in normal weight men (p = 0.03). conclusion: person with eczema or obesity had lower paraben levels in urine. we speculate that these chemicals might be stored in adipose tissue, and therefore excreted in urine in lower levels among the obese. eczema and obesity was not strongly associated in the current study. method: we retrospectively analysed medical records of 75 patients who were patch-tested with our dental screening series of 23 substances. adverse reactions to dental materials were suspected based on subjective complaints in the oral cavity and/or objective conditions of the oral mucosa. square plastic chambers on hypoallergenic tape were used. patch tests were applied to the upper back and removed by the patient after 48 hours. readings were performed 3 and 7 days after application (d3 and d7). results were evaluated according to the international contact dermatitis research group guidelines. positive patch test reactions fulfilled the criteria of at least a one plus (+) reaction on d3 and/or d7. the term »contact allergy« is usually used for such reactions. we prefer the term »contact sensitization«. clinical relevance of positive reactions to dental materials was not systematically assessed in this analysis. conclusion: we report a high frequency of positive reactions on d7 that were not seen on d3. this finding demonstrates the importance of an additional late patch test reading in patients with suspected contact allergy to dental materials. background: psoriasis is a chronic inflammatory skin disease. its etiopathogenesis is not exactly known. it is believed that the disease occurs in people with genetic tendency with the effect of a triggering factor. in some studies it is observed that contact dermatitis in psoriasis is increased with respect to normal population. for this reason it is proposed that allergen materials could trigger psoriasis. in this study it is aimed to determine contact allergy frequency in psoriasis cases using patch test. results: 62 of the cases were plaque, 5 of them were guttate, 10 of them were palmoplantar and 3 of them were inverse type. more positivity rate is observed in psoriasis cases (26.25%) than control (12%). the positively responsed materials with respect to decreasing number of patients are found as follows: nickel sulphate (16.25%), thimerosal (7.5%), peru balsam(5%), p-phenylenediamine(2.5%), colophony(2.5%), n-isopropyl-n-fenil-4-fenilendiamin(2.5%), mercaptobenzothiazole(2.5%), benzocaine(2.5%), most frequently plaque type and following guttate type positive responses are observed in evaluations with respect to clinical types. no statistical significance is found between patch test results and pasi values in psoriasis cases. conclusion: patients with psoriasis should be carefully evaluated. sometimes some materials may trigger psoriasis. the composition of the pigments that professional tattooists use are varied inorganic salts of metals or organic vegetable pigments. red tattoos, especially those that contain mercury, are the most common cause of late reactions. method: 35 year-old male patient with no previous allergy history known, who gets a tattoo on his right leg and develops within months, cutaneous erythema and pruritus on the same location as the tattoo. true test ® for skin allergy patch epicutaneous testing is performed. results: 48 and 96 hours reading: showed positiveness for mercury ++, with no late positive reactions after that. conclusion: as allergists we should be familiar with the different types of tattoos available, and know the possible cutaneous complications that each of these decorative techniques can present. it is our responsibility to be able to diagnose any complications at an early stage, establish the most appropriate treatment and, if possible, prevent them by informing the possible users. background: within otorhinolaryngological pathology chronic eczematous otitis externa is one of the most common, usually treated with topical medication successfully; however, there are cases in which the poor response to treatment, or the recurrence thereof, may be due to causes secondary to the medication itself, as observed in cases of allergic contact dermatitis caused by these drugs. case report: we present a 68-year-old male patient without relevant pathological antecedents or known allergies, who consulted the otorhinolaryngology service of our center for otorrhea of 15 days of evolution, bilateral external otitis is diagnosed and a topical otological combination is recommended (beclomethasone dipropionate 0.025% and clioquinol 1%, excipient: macrogol) with improvement. however, during the following 3 years the patient presents exacerbations and remissions of the condition, with negative or inconclusive microbiological studies. during all that time he was using the prior topical treatment and other combination treatments of topical antibiotics, corticosteroids and local antiseptics. more aggressive causes of external otitis such as malignant external otitis were ruled out. during the third year of follow-up, a clear relationship of exacerbations was observed with the use of the first combination of topical drugs, so it was decided to investigate allergic sensitization. material and methods: we perform patch tests using true test ® , standard spanish series (geidac -spanish group for investigation of contact allergy dermatitis), topical corticosteroid battery, antiseptic, as well as topical medications used by the patient. results: from the first reading on day two, positivity was observed for: mixture of quinolines ++ patient's otological combination ++ and chlorquinaldol ++; being confirmed in the reading at day four. eczematous external chronic otitis is diagnosed with allergic sensitization to quinolines (clioquinol, chlorquinaldol). we conclude that in the case of chronic external otitis, allergic contact dermatitis should also be investigated as a possible cause, and it is important to perform epicutaneous tests with the patient's own products to evaluate non-common or hidden allergens that may be relevant to their current pathology. most common causes of acd. it is important to distinguish local findings of infection from acd caused by topical antibiotic treatments. here we present a patient with acd with topical use of bacitracin and neomycin combination therapy due to recurrent blepharitis. an 11-year-old male patient presented with the complaints of itching, redness, swelling of the eyelids and facial edema. he had used various topical ophthalmic antibiotherapy and eye shampoo for 7 years due to recurrent blepharitis. five days ago, due to the redness of the eyelids, burning sensation in the eye, itching of the eyes; he was examined by an ophthalmologist. the eyelids and eyelashes were scaly and dry. the patient was treated with warm water soaked cloth dressing, mechanical eyelash cleaning and topical antibiotherapy (neomycin-simple combination therapy). after the second day of treatment, the patient's topical ophthalmic antibiotherapy was discontinued due to an augmentation of the redness in the eye0723 | contact allergy after exposure to ivy (hedera helix l) potent steroid treatment associated with systemic antihistamines, but with no improvement. on dermatological examination a small, well delineated, eczema-like plaque was noticed on a digital finger, as a new finding striking with her old burn scars. she denied any symptoms and was in good health condition. a 4 mm punch biopsy was performed and histological report established the diagnosis of squamous cell carcinoma. the patient was transferred to oncology department for further investigation and treatment. conclusion: early diagnosis and prompt surgical therapy are recommended to all patients with chronic wounds and scars who develop malignant transformation. *written informed consent for the publication of potentially identifiable personal details of patient (gender, age, illness, location) was obtained. **in relation to this presentation, i declare that there are no conflicts of interest. ertugrul a; hizli demirkale z; bostanci i dr sami ulus maternity and children training and research hospital, ankara, turkey introduction: the incidence of contact sensitization among adolescent has been increasing. nickel is one of the important causes of allergic contact dermatitis (acd) in this age group. increased exposure to nickel and deterioration of the skin barrier are among the important risk factors in children. the gold standard for diagnosis is skin patch test. we report here an adolescent patient who has allergic sensitization to nickel and cobalt. case: a 17-year-old female patient admitted in our clinic with a complaint of edema on her face. the patient had applied chickpea water to her face at least once a day for one week because of her acnes. her medical history revealed that she had experienced similar edema on her face after applications of clay mask one year ago. she was diagnosed with cellulitis and she had been treated with antibiotics for five days. on her physical examination, angioedema was observed on her face, especially on the glabellar region. eosinophilia was not found on her laboratory data. c-reactive protein (crp), c4 and c1 esterase inhibitor protein levels were also normal. the skin prick test was performed with aeroallergens, chickpea, lentil, bean and nuts, and no reaction had been observed. the patch test was performed with 'thin-layer-rapid-use-epicutaneous' (t.r.u.e) test and chickpea. the patient had positive reactions to nickel and cobalt. detailed questioning disclosed that the patient was preparing the chickpea water in a metal pot. result: chickpea water and clay mask contain varying amounts of nickel. it was thought that the edema of the patient is due to nickel allergic contact sensitization. an increased exposure to nickel and cobalt raises the frequency of sensitization. nickel allergy can cause different clinics ranging from localized lesions to systemic reactions. we want to emphasize that a detailed medical history and the patch test would enable clinicians to demonstrate hidden allergens and then make a correct diagnosis. case report: autoimmune progesterone dermatitis is a condition of hypersensitivity to progestogens. it is not an easy diagnosis given the variety of clinical presentations it may have, ranging from eczema, urticaria, erythema multiforme, folliculitis, to angioedema or even anaphylaxis. manifestations are cyclical, occurring when the levels of progesterone are higher, this is, at the luteal phase of the menstrual cycle, and disappear during menses, with the physiological decrease of the hormone. it can also be triggered by exposure to exogenous progestins. we report the case of a 49-year-old woman with a cyclical erythematous and violaceous rash related to the menstrual period. the symptoms typically began 4-6 days before the onset of menses and ended 1-2 days before. the diagnosis was based in the clinical history and intradermal skin tests: skin prick testing with levonorgestrel and medroxyprogesterone were negative, but the intradermal skin test with medroxyprogesterone was positive at a concentration of 50 mg/ml. we performed intradermal testing with the same concentration in three other women with no symptoms to exclude an irritative reaction, which were negative. autoimmune progesterone dermatitis is, perhaps, not so rare, but rather poorly recognized and reported, and thus, underdiagnosed. clinicians should be aware and include always this condition in the differential diagnosis, especially in cases of atypical or intractable skin eruptions. case report: a 46 year old male was referred to a community allergy clinic for assessment of chronic urticaria (cu). allergy assessments for foods, inhalant and inducible physical triggers revealed no association. an autoimmune workup followed, with treatment consisting of standard dose antihistamines (h1 and h2). blood work revealed a persistently low hemoglobin with low-normal ferritin. hematology consulted and followed attempted iron replacement to no avail. skin biopsy revealed neutrophilic rich urticaria with the presence of eosinophils. serum protein electrophoresis (spep) revealed a monoclonal gammopathy with elevated igm, felt to be of undetermined significance (mgus). c-reactive protein (crp) was consistently elevated (183, 256) in conjunction with anemia. rheumatology consulted and cleared of any evidence of vasculitis. hematology considered the anemia to be of chronic disease linked to cu. the cu was resistant to treatment including high dose antihistamibackground: isolated head and neck angioedema (ae) can be mediated by bradykinin (bk) or histamin (hi) . the objective of our study was to determine which etiology was most frequent in cases of death by asphyxiating ae in france. we sought all cases of death by isolated asphyxiating ae reported in france between 2000 and 2014 via death certificates and/or the national pharmacovigilance database. results: the overall mortality by asphyxiating ae for all causes was 0.36 / million inhabitants. the death rate of bkae per million inhabitants was 0.14 and lethality of 0.27 per thousand patients per year. the death rate of hiae per million inhabitants was 0.09 and lethality of 0.006 per thousand patients per year. we found a 45 times higher risk of death in case of bkae than hiae. conclusion: consequently, particularly severe episodes must be initially considered as bradykinin mediated and quickly reassess any first-line treatment that is inappropriate. case report: we present the case of a 72-year-old man who suffered recurrent abdominal pain since age of eight, leading to 3 unnecessary emergency surgical interventions and 5 endoscopies before hereditary angioedema due to c1 inhibitor deficiency (c1-inh-hae) was diagnosed at the age of 70. rare subcutaneous swellings were considered allergic reactions preventing proper diagnosis. family history, positive for recurrent abdominal pain and swellings was totally neglected until diagnosis of c1-inh-hae type i was established through appearance of severe oro-facial symptoms in the propositus' grandson. the diagnosis was suggested by the boy's mother, directed by educational materials available in the international hae patients' association website (www.haei.org). this report highlights and emphasizes the importance of accurately evaluated personal and family history to suspect condition that are scarcely known to the majority of physicians. highlights: diagnostic delay in hae and iatrogenic procedures are an underestimated problem, hiding undefined consequences, possibly destructing an entire lifetime. correct, publically available information provided by patients' associations raise awareness about the disease and could put the milestone of establishing correct diagnosis. de luque v 1 ; lara p 1 ; guardia p 1 ; jimenez ar 2 1 virgen macarena hospital, seville, spain; 2 hospital virgen macarena sevilla, seville, spain background: in the protocol for the study of patients who consult for recurrent acute angioedema with facial involvement, the contactant battery is included (epicutaneous test). we review the results in our patients with facial angioedema to evaluate the contactants to which these patients present sensitization, some of them coexisting with contact dermatitis clinic. we reviewed the patients referred to the clinic for recurrent acute angioedema with facial involvement and to whom a standard battery epicutaneous test was requested. in all these patients, other habitual triggers included in the diagnostic protocol (food, medications, autoimmune diseases, bradyinergic aea/complement deficit …) were ruled out. conclusion: it seems to be profitable to continue including in the diagnostic battery of patients who consult for aea with facial affectation, study of epicutaneous with standard battery. it is a small sample, but the data correlate with what has been published, being more frequent the sensitization to contactants in women and the contactant more frequently involved nickel sulphate. 0734 | ace inhibitor-related angioedema-the value of history taking background: angioedema is a well-recognized side effect of angiotensin-converting enzyme (ace) inhibitor therapy. although it occurs in <1% of the patients who take these drugs, it seems to be responsible for 40% of the episodes of angioedema. this entity is underdiagnosed and failure to recognize it leads to recurrence of episodes, with an impact on morbidity and increased risk of serious reactions. our objective is to analyze the clinical, therapeutic and orientation approach of patients diagnosed with ace inhibitor-related angioedema, evaluated at the outpatient consultation (oa) of immunoallergology (ia). a 3-year retrospective study was performed by analyzing the clinical files of all patients diagnosed with angioedema observed in oa of ia. the following variables were analyzed: gender; age; clinical data; evaluation in emergency department (ed); therapy in the episode; evolution and orientation. the chi-square test was used to study the association between categorical variables: "established therapy"/"disease evolution" and "place of reference"/"withdrawal of ace inhibitor". results: review of 62 cases of patients referred for angioedema. only in 29% the final diagnosis was "ace inhibitor-related angioedema". the mean age of the patients was 63.7 years and 67% were male. the location of angioedema occurred in the tongue in 33% and in the remaining sites (lip, hemiface, tongue and hemiface, tongue and lip) appeared in the same frequency, 17%. none of the patients had airway obstruction. during the episode of angioedema, 22% of patients were not referred to ed and the therapeutic approach was done with antihistamines in 75%. in patients who were referred to ed (78%), antihistamines and corticosteroids medications were administered in 85%. regarding the evolution, it was verified that the duration of the episode was independent of the established therapy (p > 0.05). regarding the place of reference, 60% of the patients were referred form hospital (ec or ed) and, in these, the ace inhibitor was suspended in 73%. in patients referred from general practitioners (40%), in none of them the ace inhibitor had been withdrawal. a causal association between the use of ace inhibitors and the episode of angioedema becomes crucial, since drug withdrawal is indicated. a reference for ai oa should be weighed. therapy with antihistamines and corticosteroids has no proven efficacy. hereditary angioedema (hae) seen by physicians belonging to the hae scientific committee of the aaaeic background: patients with c1-inh-hae frequently suffer from anxiety and stress. the impact of prophylactic treatment on anxiety and stress in c1-inh-hae patients is largely unknown. here, we analyzed data from the apex-1 study, a phase ii study that investigated the effects of the oral kallikrein inhibitor bcx7353. method: c1-inh-hae patients with a history of at least 2 hae attacks per month were randomized to receive four different doses (350, 250, 125, 62.5 mg) of bcx7353 or placebo for 28 days. the depression anxiety stress scale (dass) was administered at baseline and at day 29. the dass consists of three self-reported scales designed to measure the negative emotional states of anxiety and stress. subjects used a 4-point severity/frequency scale to rate the extent to which they have experienced each state. results: baseline dass total scores as well as anxiety and stress domain mean (sd) scores for the 125 mg treatment arm (n = 13) were 20.9 (23.7), 5.0 (5.9), and 8.9 (9.1) points respectively. placebo scores were generally similar or slightly lower at baseline than for the 125 mg treatment arm. the dass questionnaire data showed statistically significant improvements in total score vs. placebo at day 29 (−11.4 method: a two-phase mixed methods approach was used to develop the hae-rt tool including: phase 1: delphi study [hae specialists (n = 9) and national patient advocacy group members (n = 3)] was conducted to reach consensus (80% agreement) on predictor variables to include in the tool. phase 2: retrospective chart review was conducted to assess the predictive findings of the decided variables. a convenient patient sample presenting with angioedema (with and without hae) between january 2012-january 2017 were included in the study. results: nine of 12 invited experts (75%) participated in the delphi study. of 8 hae-specific predictive variables, 4 reached consensuses including: (i) recurrent angioedema; (ii) absence of urticaria; (iii) recurrent abdominal pain/swelling; (iv) lack of response to allergic therapy. the retrospective study included 85 patients (n = 46 with hae; n = 39 non-hae; overall 72% female). hae patients were significantly more likely to have a family history of hae (72% vs 0%; p < 0.0001); previous recurrent angioedema (96%; p < 0.009); present with no hives (91%; p < 0.036); previous recurrent abdominal pain (80%; p < 0.0001); and 2% responded to allergy treatments (p < 0.0001). a regression analysis categorized observed frequencies (actual patient outcomes from chart review) versus predicted (by model); plotted on a 2 by 2 table and calculated the sensitivity and specificity of the hae-rt which resulted in one hundred percent for both. conclusion: our study demonstrated that expert involvement led to the identification and prioritization of variables that when included an hae-rt tool, were associated with a high level of sensitivity and specificity when applied to known patients. the next step is to observe the effect of the hae-rt tool on patient care in the ed. method: evaluation of cardiovascular manifestation included morphology, serum level of troponin t, electrocardiography (ecg) and echocardiography. evaluation of pulmonary manifestation included spirometry, diffusing capacity of the lung for carbon monoxide (dlco) and evaluation for mastocytosis included bone marrow biopsy and serum total tryptase measurements. results: in the study there were 55 patients -35 women and 20 men between 21 and 71 years old (the average age was 45). there were 7 (12.73%) patients with mpcm, 4 (7.28%) with bmm, 42 (76.36%) with ism and 2 (3.64%) with ssm. the average level of serum tryptase was 45.1 μg/l (6.9-270) . troponin levels was within the normal range in all patients. one patient had lowered the ejection fraction (eflv=23%). no one patient had restriction. the average value of a forced lung capacity was 3.33 l (104%) and a total here, we describe a case series of twelve mis patients seen at our department over a 3-year period and report how many of these patients have sm. common phenotypical manifestations of acute hae1 episodes in this region, to review therapeutic challenges in a rural setting in comparison with world standards, and lastly to evaluate the socio-economic burden inflicted by the disease. method: a sample of 13 individuals from a total of 28. the exclusion criteria was the inability to attend booked appointments more than 3 times in 1 year (2017). the following methods were used: an interview to formulate a family tree identifying affected individuals in 4 contiguous generations, and review of the acute presentations in the past year (2017) . a questionnaire to obtain the relevant hae1 associated socio-economic burdens. a chart review to identify the therapeutic strategies in this region. c1inh levels, and complement c4 to confirm the diagnosis. results: polygamy as a local culture was found to be an important factor that perpetuated the genetic burden of the disease. c1inh and c4 levels confirmed hae1 in all participants individually. clinical features during acute attacks included swelling of extremities (100%), facial swelling (69%), neck swelling (23%), and laryngeal swelling (8%). therapeutic strategies for acute attacks included fresh frozen plasma or fresh dried plasma. danazol was used for prophylaxis. hae1 has had a significant negative impact upon the socioeconomic status of the affected individuals. conclusion: hae1 is a newly identified disorder in the broad spectrum of allergy medicine in kwazulu-natal. the diagnosis is simple to confirm but requires an initial high index of suspicion, and therapeutic management still poses a challenge in this region due to lack of resources. genetic counselling is of paramount importance during intervention since polygamy forms part of most cultures in this region. a support strategy is highly recommended in order to help alleviate the socio-economic burden posed by the disease in this region. the socio-economic burden secondary to hae1 (10 participants/5 identical questions each) question 1 (0-5 points) 3 question 2 (0-3 points) 2 question 3 (0-3 points) 1 question 4 (0-3 points) 1 question 5 (0-1 point) 1 method: a total of 172 medical faculty senior year students were included to study on a voluntary basis. students are divided into two groups. one group was given visual user guide that has not been modified, and a visual user guide on which we have modified to the other group ( figure 1 ). then they were asked to show how to use the inhaler spacer. results: the mean age of the volunteers was 25.3 ± 126 years and 104 (60.5%) were male. there were 82 students in the group without modification of the visual user guide and 90 students in the other group with modified the visual user guide. sixty-four per cent of the modified user guide group showed correct use of the inhaler spacer, while 15% of the unmodified group showed correct use (p = 0.001). the group that given modified visual user guide was more successful in all of the display steps of the inhaler spacer. conclusion: modification of the currently available visual user guide of inhaler spacer in our country will increase the correct usage rate. results: mean fev1, fvc, fev1/fvc z-score were 1.8 (1.1-3.9), 1.7 (0.9-3.7), 0.9 (0.8-0.9), respectively. restriction had 31 (57.4%) and obturation 3(5.3%) patients. fev1 (p < 0.001, r 2 = 0.37) and fvc (p = 0.001, r 2 = 0.38) decreased with age (%pv and z-score). background: children who were treated for leukemia are known to have developed long term impairment of lung function. the reasons that complication are only partially known. the aim of this study was to asses pulmonary function in children treated the lower dlco is the most frequent abnormality in childhood leukemic survivors. hsct and pulmonary infection (in particular cmv pneumonia) is a strong risk factor for impairment of dlco in children. clinical manifestation of dlco impairment is poor exercise tolerance. a screening for respiratory abnormalities in survivors following treatment for childhood haematologic malignancies, seems to be of significant importance. 0746 | phenotyping allergic respiratory diseases: an unsupervised classification using latent class analysis allergen groups was significantly associated to uawi (aor[95% ci]:2.1 [1.3-3.6] ), compared to uasi. results: 80.8% of br and 49% of py were women, median age was 32 years, 35% br and 52% py reported having more than four years of training. although they recognized the main symptoms of ar, 26% br and 100% py never asked whether the patient had a medical diagnosis of ar; 20.5% br and 100.0% py did not ask whether the symptoms occurred when close to animals or allergens; 55% br and 76% of py did not ask if the patient had a medical diagnosis of asthma; 59% br and 70% py did not ask if rhinitis worsens asthma symptoms and 51.3% br and 84.3% py did not ask whether symptoms of rhinitis interfere with their daily activities. results: there was a predominance of female (br: 73%, py 50.4%, uy: 70%) median age 31 years old, 124/235 worked in the community and 75/127 in the emergency departments, 34% of the br had more than 10 years of education, 67% from py had between 1 and 5 years, and 82% from uy had been graduated for less than 1 year. br/uy recognize the main symptoms of ar, however 67% of those from uy do not ask: if the patient has physician diagnosis of ar, 72% present shortness of breath, and 93% a medical diagnosis of asthma, 94% if rhinitis worsens asthma symptoms and 90% if symptoms of rhinitis interfere with the patient's daily activities. the prescribed treatment varied a lot, the intranasal corticosteroid use rate was: bd: 78%, pd: 92% and ud: 54%. 100% of doctors in py, 73.4% in br and 78% of uy never refer the patient to the specialist. 22.9% of pcd of br, 62% of py and 6.0% of uy are aware of aria guideline. conclusion: although ar is largely attended by pcp, recognition of symptoms and their impact on asthma, as well as the knowledge about aria guide is low and treatment is not always prescribed according to best practice. allergy education programs, with an emphasis on ar and aria guide, need to be directed to pcp in la for the better assistance of ar patients. 0749 | assessing knowledge of allergic rhinitis among final year medical and pharmacy students in croatia-curriculum change necessity? the two factor structured questionnaire was formed by the authors regarding the topics mentioned. t-test was used for statistical analysis. the global results were formed as composites of (1) ar general characteristics, (2) ar treatment approach, and (3) the participants' overall knowledge. of the 201 respondents, 143 (71.1%) were female and 58 (28.9%) were male (p < .0001). medical students had a median score of 6 of 10 correct answers on (1), 4 of 10 on (2), and 10 of 20 on (3), whereas pharmacy students had median score of 7 of 10 correct answers on (1), 4 of 10 on (2), and 10 of 20 on (3). there were no significant differences in knowledge between two student groups. the results indicate an inadequate level of knowledge of ar in both groups, especially regarding the therapy approach. since general practitioners and community pharmacists have a major role in providing treatment to patients suffering from ar, it is important to develop advanced knowledge on this topic during medical and pharmacy degree courses. despite a relatively small study population, it would be advisable to introduce change by improving the core curriculum regarding ar with more emphasis on treatment, but additional research on this topic is necessary. tan r 1 ; cvetkovski b 1 ; kritikos v 1 ; price d 2 ; yan k 3 ; smith p 4 ; bosnic-anticevich s 5 1 woolcock institute of medical research; university of sydney, sydney, australia; 2 observational pragmatic research institute pte ltd, singapore, singapore; 3 royal prince alfred hospital, sydney, australia; 4 clinical medicine, southport, australia; 5 griffith university, sydney, australia background: people with allergic rhinitis symptoms frequently selfselect over-the-counter medications from community pharmacies without seeking advice from a health care professional. this increases the incidence of complications due to delayed diagnosis and suboptimal treatment. this study aims to (i) compare the demographics, clinical characteristics and medication selected, between pharmacy customers who choose to self-select and those who interacted with a pharmacist when purchasing medication for allergic rhinitis symptoms, and (ii) identify the key factors associated with allergic rhinitis patients' medication self-selection behaviour. a cross-sectional observational study was conducted in a convenience sample of community pharmacies from the sydney metropolitan area. data were collected using a researcher administered questionnaire that included: demographics, pattern of allergic rhinitis symptoms, their impact on quality of life, factors triggering allergic rhinitis symptoms and medication(s) selected. logistic regression was used to identify key factors associated with participants' medication self-selection behaviour. results: of the 296 recruited participants, 202 were identified with allergic rhinitis, of which 67.8% were female, 54.5% were aged more than 40 years old, 64.9% had a diagnosis of allergic rhinitis, and 69.3% self-selected medication(s). significant differences were noted in allergic rhinitis symptoms, impact of allergic rhinitis on quality of life and medication(s) selected between participants who chose to self-select and those who interacted with a pharmacist. participants who experienced moderate-severe wheeze were 4 times more likely to self-select allergic rhinitis medication(s), and those who had allergic rhinitis symptoms impacting on their quality of life were 0.4 times less likely to self-select allergic rhinitis medication(s). conclusion: there is a high incidence of self-selection of over-thecounter treatments for allergic rhinitis symptoms in community pharmacy, with the majority of allergic rhinitis sufferers failing to seek pharmacist advice. this research identified predictors of medication self-selection behaviour in community pharmacy among people with allergic rhinitis, which can inform the design of tools/strategies and targeted interventions, aimed at improving pharmacist engagement and future practice in optimising allergic rhinitis management. the weir family health clinic, cork, ireland; 2 university college, cork, ireland background: allergic rhinitis is a common condition that is predominantly managed in primary care. the incidence of allergic rhinitis is increasing. it is frequently under diagnosed, misdiagnosed and mistreated. it has a significant impact on patients' health related quality of life and represents a huge cost both to healthcare systems and society. the aim of this study was to implement appropriate guidelines regarding the management of allergic rhinitis in primary care and evaluate the effect on patients' health related quality of life. method: patients with a history of allergic rhinitis were selected from three general practice bases in west cork, ireland and quality of life of patients was assessed initially in year one and followed up one year later in a general practice setting using the standardised rhinoconjunctivitis quality of life questionnaire (rqlq). allergic rhinitis and its impact on asthma (aria) guidelines and appropriate prescribing were implemented during this year and patient education and structured follow up was arranged in the intervention group. this was compared with the control group who received usual care. results: 93 valid responses were received, 34 from the control group and 59 from the intervention group. the study demonstrated a statistically significant difference in quality of life in the intervention group. in the adult intervention group the quality of life score decreased between 2015 and 2016 representing an improvement in their quality of life, (t = 4.13; df=56; p < 0.01). the difference in the score between the control and intervention groups in 2016 was also statistically significant.(t = 6.11; df=54; p < 0.01). the numbers in the adolescent groups and paediatric group also demonstrated an improvement in quality of life but the sample size was too small to demonstrate a statistically significant difference. conclusion: as the majority of patients rely on their general practitioners for treatment and diagnosis of allergic rhinitis, primary care represents an important area to target in the management of allergic rhinitis to improve patients' quality of life. the implementation of guidelines has been shown to improve patients' quality of life. this study demonstrates this care can be delivered in a primary care setting with an improvement in patients quality of life but substantial investment in education and resources available to primary care physicians is needed. 0752 | the predictive value of allergy tests in the diagnosis of peanut allergy in adults rey-garcia h; gunawardana n; wheeler k; scadding g; durham s; skypala i royal brompton hospital, london, united kingdom background: adults presenting with either new-onset symptoms attributed to peanuts or with early-onset peanut allergy, often wish to know whether they should continue to avoid peanuts. clinical history and standard tests may be sufficient to provide an answer, but for many the tests are inconclusive and an oral food challenge is required. this review was undertaken to determine the most accurate tests. conclusion: these data suggests that peanut spt and ara h 2 provide the most accurate prediction of the outcome of oral food challenge in adults. should components not be available, then spt would be the test of choice being more accurate in all aspects than sige. combining spt and sige improves the sensitivity and negative predictive value of spt alone. however, the best combination is spt and ara h 2, which increases the overall accuracy to 87%. further studies are needed before it can be determined whether peanut diagnostic tests can replace the oral food challenge in adult patients. method: retrospective chart review was carried out in a community allergy clinic. patients with rap, bloating and altered stools who underwent bt were characterized by age, gender and atopic status. a separate study to assess patients' outcome post-dietary counselling was carried out to determine impact on symptom management. results: thirty-four patients were assessed for fi from january 2014 to december 2017. female gender predominated (31/34, 91%) with an average age of 31 years at presentation. results of fi were positive in 15/34 (44%), borderline in 2/34 (6%) and negative in 17/ 34 (50%). the average age of patients with a positive, borderline and negative tests were 30, 20 and 34, respectively. of the patients who tested positive for fi, 5 (33.3%) had comorbid inhalant allergies alone, 1 (6.7%) had comorbid (unrelated) food allergies alone, 2 (13.3%) had inhalant and food (unrelated) allergies, and 7 (46.7%) were non-atopic. of the patients who tested negative for fi, 7 (41.1%) had comorbid inhalant allergies alone, 0 (0%) had comorbid (unrelated) food allergies alone, 1 (5.9%) had inhalant and food (unrelated) allergies, and 9 (53.0%) were non-atopic. conclusion: patients investigated for carbohydrate intolerance with rap, bloating and altered stools were predominantly female (91%). fi was confirmed in half. atopic status did not help differentiate between the fi positive or negative groups. results of a fod-map elimination diet are separately reported. conclusion: post-bt, 88% of patients reported symptom improvement. patients who implemented fructose or fodmap avoidance reported symptom improvement. one patient who tested negative for fi reported symptom improvement with a low fodmap diet. patients suspected as being fructose intolerant may benefit from a fructose restriction or fodmap diet, while awaiting bt confirmation. this form of dietary intervention may assist and shorten the natural history of non-specific chronic gi symptoms. inappropriate referrals to a uk paediatric tertiary allergy clinic demonstrate lack of allergy education and knowledge in primary care marriage de bristol royal hospital for children, bristol, united kingdom background: up to 8% of children have a food allergy. allergy has become an explanation for all manner of nebulous symptoms and self-diagnosis is common. sham allergy tests are easily available giving incorrect results and resulting in unnecessary, potentially harmful abstracts | 423 parentally-imposed dietary exclusions. there are 100 million allergyrelated google searches per year. the rising prevalence of perceived allergic disease has led to an increase in health service utilisation, including increased referrals to secondary care. clinic waiting lists are long and children with severe food allergies have to wait longer than necessary to be seen method: uk paediatric tertiary allergy clinic referrals were prospectively reviewed over three months. five inappropriate referrals deemed most reflective of poor knowledge in primary care were selected as case summaries to highlight this gap in knowledge. results: 1: schoolchild referred for investigation of allergic cause for a red, watery eye after splashing juice in her eye whilst cutting a kiwi, despite having a co-existent dendritic ulcer. 2: schoolchild referred for peanut allergy testing after inhaling a peanut and developing wheeze, with all respiratory symptoms resolving following peanut removal. 3: young child referred for peanut allergy testing after developing a rash on leg following skin contact with faeces 24 hours after ingestion of peanut butter. 4: teenage boy referred for investigation of likely peanut allergy despite eating peanut butter and tree nuts almost every day. the family were concerned he was allergic to peanut butter. 5: toddler referred for milk allergy investigation after developing urticaria lasting 24 hours 15 minutes after drinking a bottle of milk. the child had consumed cow's milk formula since birth, and continued to consume milk daily for a further five months following the episode of urticaria. conclusion: provision of allergy services in the uk is poor and lack of investment in allergy services has led to suboptimal recognition and management of food allergy in primary care. allergy education provision for primary care practitioners is inadequate and fails to empower healthcare professionals to discern between allergy requiring full investigation and management, parentally-diagnosed allergy or symptoms which clearly have no association with allergy. progress to improve primary care training for allergy needs to be optimised to prevent further unnecessary referrals and lengthening clinic waiting lists. background: in case of allergic reactions to food or insect venom, quick and adequate treatment, based on clear instruction for use of emergency medication and calling for help, is necessary. however, daily practice shows that patients do not use the prescribed emergency medication because they are afraid to use the epinephrine auto-injector or they do not know how to use it. information and instruction offered by a reliable app could be a useful aid. we aim to develop an app for adult patients and children older than 12 years with allergy to food or insect venom, which offers a step-wise approach to support patients, their relatives or acquaintances in case of an allergic reaction. method: first, the content of the app, including a step-wise approach to treat the allergic reaction has been determined, based on literature, a survey about needs of patients and on consultations of healthcare professionals. subsequently, a web-based prototype has been developed with an adult profile and children profile. the content and flow of this prototype was tested by the project group, as well as by selected healthcare professionals and patients and improved according to the test results. next, the revised prototype was submitted to representatives of patient and professional organizations for final approval. currently the procedure for ce approval is ongoing. finally, the app will be built and offered to the market for ios and android. results: a web-based prototype of the allergy app is available with two profiles: adult and older children. the app is useful for patients with a doctor's diagnosed allergy to food or insect venom, who received emergency medication and instructions to use prescribed medication in case of an allergic reaction. based on severity of complaints, the user is informed about the steps to treat the allergic reaction. in case of a moderate to severe reaction, the patient is advised to use an epinephrine auto-injector, to call the emergency number and, if prescribed, to use medication such as antihistamines, prednisone or inhaler. besides that, the app provides links to websites of expertise centres and patient organisations and includes instructions how to use the epinephrine auto-injector. conclusion: the allergy app will help patients, their relatives or acquaintances to adequately treat an allergic reaction to food or insect venom. involving patients and professionals in the development of the app will contribute to its acceptability and usability. 0757 | electronic documentation of drug allergies in a tertiary hospital in singapore: are we relying too much on it? choo kjl; garuna murthee k; naing cs singapore general hospital, singapore, singapore background: singapore has 26 hospitals shared between public and private healthcare system. its healthcare system, ranked #6 in the world by who in 2010 serves a multi-racial population of 5.2 million of which 9% are above 65 years old. drug allergy alert cards (medik awas) were started in 1970s by singapore medical association to improve patient safety. work on computerisation of drug allergy and medical alerts started in the 80s, a precursor to today's critical medical information system (cmis). cmis serves as a platform across all public hospitals in singapore. it promotes uniform reporting of drug allergy and notification of adverse drug events to the health science authority. yet, we found that there is a lack of awareness of one's own drug allergies. method: all patients admitted to ward 73 (general medicine ward) at singapore general hospital from 17 july to 31 oct 2017 were screen for any previously documented drug allergies. consenting patients who had previously documented drug allergies on cmis were interviewed to document their demographics, education level, current medications, knowledge of their own drug allergies and possession of a drug medication alert card. the answers were then compared with their electronic documentation of drug allergies for accuracy. we interviewed 192 patients aged 20-94 with documented drug allergies during the recruitment period. 74% had secondary school education or higher. the majority (76%) spoke english and (58.5%) mandarin. almost half had medical problems and are on long term medications (mean 5.17 medications); hypertension and diabetes being the top two common diseases. 48% of the patients could accurately relay their drug allergies; antibiotics and analgesia being the most labelled. only 24% had a drug allergy alert card while the rest both rely on the hospital's electronic documentation and/or their caregivers to record and relay their allergies to future prescribers. about 21% received prescription from multiple healthcare sites in both the public and private healthcare system. we found patients' knowledge of their own drug allergies dismal. the cmis electronic documentation provided a false sense of security. unfortunately, the cmis platform is not available to all private hospitals, increasing the risk of mis-prescription due to the lack of information. unless this is made available nationally, patients with drug allergies should be given some written documentation, either a letter or medik awas. how frequent are they and how are they treated? method: for this cross-sectional study, participants were recruited in the waiting rooms of local doctors in the rural bavarian forest region of southern germany (q1/2017). a paper questionnaire was handed out to the participants, asking for allergies (pollen, animal hair, bee and wasp venom, drugs, food, house dust mites, contact allergies and other allergies) and how or rather by whom (e.g. general practitioner, specialist, self-treatment) these allergies are treated. results: 718 participants with a mean age of 50.61 years (sd=15.15) and 59% women were included in this study. 38.2% indicated to have at least one allergy, including pollen allergy most frequently (17.4%). women had significantly more often at least one allergy than men (rr= 1.552; ci [1.243;1.937] ) and for almost all examined allergies a significant higher risk of disease. younger age groups indicated more often to have at least one allergy (18[.506;1.151 ]) seemed to be affected less. participants indicated most frequently that their allergy was treated by a general practitioner (37.5%), except of the 18-29-yearold young adults who indicated "no treatment" most frequently (28.2%). conclusion: there is a high self-reported prevalence of allergies in the examined rural bavarian region, that increases with decreasing age and is significantly higher among women. moreover, the data on the absence of an appropriated treatment for allergies is alarming. therefore, medical care needs to be improved in rural regions to lower the burden of allergies. 0759 | the prevalence of the burnout syndrome among medical professionals involved in allergology education programmes astafieva n 1 ; kobzev d 2 ; gamova i 1 ; perfilova i 1 ; udovichenko e 1 ; skuchaeva l 1 ; michailova i 1 1 ≥10) and rpa (low ≥0-11, subscales were calculated and analyzed. results: on average students demonstrated: moderate/high ee scores (24-26); moderate/high dp scores (9-12) and moderate rpa scores (17); higher rpa scores were common (41.4%) among junior students, which is also linked with their levels of engagement, and lower (28%) among senior students. junior specialist (starting specialization) had very low scores in all 3 subscales and expressed a very high motivation in their course and new profession. clinical allergologists with significant experience demonstrated moderate / high ee scores (22-26); low dp (1) (2) (3) (4) (5) and rpa (below 10) scores. high ee scores associated with pressures of service were compensated by a substantial loyalty to their profession and positive assessment of the outcomes of their work. clinical academics demonstrated the highest level of ee scores (29 + among 70 + % of the group) with low to moderate dp and rpa scores, the latter being associated with a loyalty to their profession. it was also possible to identify a correlation between engagement in research activities and lower rpa scores. conclusion: burnout is a complex and multifaceted phenomena, which requires further investigation. however, this research identified that students and junior specialists involved in allergology and clinical immunology programmes with higher levels of engagement and motivation to acquire new specialist knowledge had lower levels of ee, while loyalty to the profession and positive assessment of the outcomes of clinical and research work allows to compensate high levels of ee among experienced practitioners and clinical academics and to reduce burnout effects overall. 0760 | appeal (allergy to peanuts impacting emotions and life): the first pan-european study to evaluate the psychosocial burden of living with peanut allergy deutscher allergie-und asthmabund (daab)/german allergy and asthma association, mönchengladbach, germany; 8 food allergy italia, padua, italy; 9 aepnaa asociación española de personas con alergia a alimentos y látex, madrid, spain; 10 afpral association pour la prevention des allergies, paris, france; 11 asthma-allergy denmark, roskilde, denmark; 12 anaphylaxis campaign ireland, cork, ireland; 13 brainsell ltd, london, united kingdom; 14 aimmune therapeutics, london, united kingdom background: peanut allergy, one of the most common and rapidly growing food allergies, is most frequently a lifelong condition. current management is limited to avoidance and symptomatic treatment of allergic reactions when accidental exposures occur. peanut allergy can affect the quality of life (qol) of individuals and also that of parents/caregivers and family members. appeal was designed to assess the impact of peanut allergy on qol in peanut-allergic individuals and their parents/caregivers and families. method: the first, quantitative part of appeal is described here and consisted of a pan-european, cross-sectional online survey of approximately 30 minutes in length. the study was conducted in the uk, republic of ireland, france, spain, germany, italy, the netherlands and denmark. over 1800 participants were recruited via patient advocacy groups or directly through a specialist survey recruitment panel. ethics committee approval was obtained and all participants provided their informed consent. eligible participants were: (1) parents or caregivers of a child/adult with peanut allergy; (2) parents or caregivers responding as a proxy for a child aged under 18; (3) adults. all allergic subjects had self-reported diagnosed peanut allergy. after several screening questions, eligible participants answered a set of clinical questions about their (or their child's) allergies and other conditions, details on the peanut allergy diagnosis, contact with healthcare professionals, worst allergic reaction to date and use of emergency medicine. depending on whether they were an allergic adult, a parent responding on behalf of the child, or a parent/caregiver recounting their own experience, they then answered specific questions on restrictions on life choices, coping strategies and the impact of peanut allergy on feelings and emotions of families, friends and other people. sociodemographic questions completed the questionnaire. the results of the survey are being summarized using descriptive statistics and the data are being analysed on a pan-european level, by country, and according to the participant's perspective (parent, caregiver or individual). conclusion: this comprehensive, pan-european online survey has been specifically designed to uncover the psychosocial burden and effect on qol of peanut allergy in terms of individuals' lives and those of their families. results: 246 pediatric patients were included in the study. 65.9% (n = 162) were male. the median age of onset of symptoms (interquartile range) was 4 months (2-6).the median age (interquartile range) of diagnosis was 6 months (4-7). initial reactions associated with cmpa were observed in 95.8% of patients before 12 months old. patients' diagnoses were atopic dermatitis (39%), urticariaangioedema (24%), anaphylaxis (12.2%), proctocolitis (10.2%), atopic dermatitis and urticaria (8.9%), food protein induced enterocolitis (4.9%) and eosinophilic esophagitis (0.4% method: the study involved 147 patients, who are two years old (and above), diagnosed with cow milk allergy and are observed for at least six months in our hospital. socio-demographic features of the patients, their symptoms, symptom-start ages, age of diagnosis, clinical findings at diagnosis and during observation were recorded in data collection questionnaires. results: the samples were 91 (61.9%) boys. the average symptomstart age was observed to be 5 months , average diagnosis age 6 months (1-54) and average age of final check 36 months . when symptoms at entry were observed, 85.7% of the patients had dermal system, 25.2% gastrointestinal system, respiratory system disorders, and 15% were detected to have developed anaphylaxis. among the patients diagnosed with cow milk allergy, 31.3% showed food reaction to nutrients with lge agents, 51.7% to mixed types, and 17% to nutrients with non-lge agents. it was also observed that, in the end of 30.4 ± 19.8-month observations, sensitivity to cow milk was observed to continue in 34 (23.1%) of our patients. when tolerance improvement rates among the patients were compared, anaphylaxis (p < 0.001) during entry were observed to be influential in continued allergic state. 5 (3.4%) patients were able to consume yoghurt, 10 (6.8%) patients could consume dairy products and 19 (12.9%) patients could not consume dairy products. conclusion: in the end of our investigation, it was observed that 57 (50.5%) of the 113 patients developed cow milk tolerance before the age of 2. when the factors enabling the continuation of sensitivity in cow milk allergy were investigated, anaphylaxis during entry, entry specific lge and pasteurized milk antigen as well as high skinprick test results were detected to be significant. 0765 | initial lower threshold was a risk factor of severe adverse reaction during oral immunotherapy for cow's milk anaphylaxis results: before oit, median age was 5.7 years old, median threshold to induce initial reaction was 2.1 ml, to induce anaphylaxis was 14.6 ml of cm and median milk specific ige was 56.4 ku/l. twentyseven subjects (24%) dropped out from the protocol, 69 subjects 0766 | investigation of heat and matrix effect on milk proteins' allergenicity and the development of hypoallergenic food products reduce some milk proteins' allergenicity (ß-lactoglobulin) . in this project we aimed to investigate the effect of heat and matrix on different milk protein fractions through maillard reaction and eventually develop hypoallergenic food products that have milk protein with low reaction risk. method: milk cake matrix is prepared in different flour/sugar (f/s) ratio (2f/1s, 1f/1s, 0.5f/1s) and baked 30 minutes at 180°c. proteins that cake contains are separated using sds page and stained with coomassie blue to check total protein. in parallel specific proteins are detected by western blotting using pooled sera from patients with milk specific ige>60ku/l for incubation results: in normal milk cake recipe (2f/1s) ß-lactoglobulin bands are disappeared but casein bands did not differ in size. in order to investigate the matrix effect f/s ratio is changed and it is found that when this ratio decreases, with the affect of heat and maillard reaction, milk casein bands' intensities also decrease in sds gel coomassie staining. in western blot experiments it is also shown that milk specific ige bound weakly to casein bands in low f/s ratio cake (0.5f/1s) whereas in cakes that have high f/s (2f/1s) ratio it bound significantly higher. and ß-lactoglobulin proteins' structure and lower the milk specific ige bindings to milk proteins in low f/s ratio cake through maillard reaction. 0767 | extensively hydrolyzed formulas for the management of cow's milk protein allergy in infants: is extensive hydrolysis sufficient to guarantee success? method: to better understand the range of ehfs, we aimed to analyse samples of commercially available ehfs from 11 countries and various manufacturers, with a focus on suitability for cmpa management. samples were de-identified and coded for the analyses. molecular weight (mw) distribution of hydrolysates and residual proteins and peptide profiling were assessed with sds-page gel and size exclusion-high-performance liquid chromatography (se-hplc), as they reflect both the design of the formula and the quality management applied during production. osmolarity, nitrogen fractions, lactose content, total and free amino acids, β-lactoglobulin, and casein content were quantified and β-lactoglobulin residual allergenicity was assessed. results: peptide mw distribution displayed significant variation, with the percentage of peptides with mw >1.2 kda varying from 1% to 36%. mw distribution was shown to be positively correlated with β-lactoglobulin specific in vitro degranulation. twenty % of samples had non-measurable β-lactoglobulin content (smaller than or at the limit of quantification (loq): 0.010 mg/kg); however, 80% of samples had β-lactoglobulin content greater than the loq, with high variability from 0.020 to 36 mg/kg. surprisingly, even in samples featuring a high degree of hydrolysis, significant levels of residual β-lactoglobulin were quantified. conclusion: lack of consensus over the definition of 'extensively hydrolysed' is reflected in the wide range of degree of hydrolysis in commercially available ehfs, and can result in products that are mislabelled as 'extensively hydrolysed' and may be high-risk or even unsuitable for the management of cmpa. results of these analyses also highlight that degree of hydrolysis alone is not sensitive enough to characterise ehfs, and that whilst a high degree of hydrolysis is desirable, further quality control measures are essential to ensure clinically safe and suitable products. actionable guidelines to better define hypoallergenic formulas based on extensively hydrolysed milk proteins are warranted. background: assessing the effect of baked milk products on accelerating unheated milk tolerance in patients with cow's milk allergy. method: a randomized clinical trial was done on 84 patients (6 months-3 years old) divided randomly to case and control groups matched for age and sex. baked milk in form of muffin for 6 months followed by baked cheese in form of pizza for next 6 months was given to the patients in case group. skin prick test and serum ige (sige) levels (immunocap) of milk, casein and betalactoglobulin were measured before and after the study. the ones having milk sige less than 3 ku/l and being asymptomatic during the study underwent oral food challenge test for evaluating unheated milk tolerance. chualalongkorn university, bangkok, thailand; 8 kk women's and children's hospital, singapore, singapore; 9 university of the philippines, philippine general hospital, manila, philippines background: problems in recognising cow's milk allergy (cma) and lactose intolerance (li) in infancy may lead to a delayed or incorrect diagnosis, as well as inappropriate dietary interventions. method: between january and november 2017, a survey was conducted online in china, india, singapore, thailand, mexico, kuwait, united kingdom, australia, and paper-based in the philippines. the survey consisted of 12 multiple-choice questions on cma and li in infants aged under 12 months, two case scenarios (non-ige cma and anaphylaxis) and 10 questions on educational needs (likert scale [1] [2] [3] [4] [5] . data on the type of medical practitioner and clinical setting were collected. responses were summarised as percentages and categorised by country. results: 1663 responses were received from general practitioners (22.2%), paediatricians (39.7%), paediatric allergists (6.9%), paediatric gastroenterologists (11.2%) and other specialities (20.0%). there were significant misconceptions about the clinical importance of primary li in infancy. while primary li rarely manifests before 3 years of age, 73.7% of participants felt it was a significant clinical problem in the first year of life. regarding secondary li, 44% of respondents recommended lactose restriction for viral gastroenteritis, and 36% for cow's milk protein-induced enteropathy. while the management of ige cma was relatively well understood, there were greater knowledge gaps for non-ige cma. 59% of practitioners appropriately identified extensively hydrolysed formula (ehf) as first-line treatment of cma in formula-fed infants. however, the distinction between lactose-free and lactose-containing ehf appeared to be an area of uncertainty. in india, 34.4% used soy-based formulas as firstresults: patch tests were positive in 62 (79.5%) and negative in others. positivity to milk was seen in 29 patients (37.2%), to soy in 41 (52.6%), to egg white in 7/72 (9.7%), to wheat in 12/66 (18%), to potatoes 6/48 (12.5%), to corn (maize) in 3/38 (7.9%), to rice in 1/2, and to peanut in 3/37 (8.1%). patients were requested to withdraw the suspected food(s) from their diets during a 4 months period. preliminary follow-up data show the improvement of one or more symptom in 19/20 patients (gastroesophageal reflux in 10, appetite in 5, stool consistency in 1, respiratory symptoms in 10, pain in 3, eczema in 1). conclusion: patch tests are informative, easy to use tools in order to identify potential causes of common lasting symptoms in children with negative or weak rast results and introduce beneficial changes in the daily diet. longer follow-up is necessary in order to refine and assess the benefit of such strategy. background: currently in the us, 1 in 13 children suffer from food allergies. at present, there is no cure and strict avoidance of the relevant foods is the only way to prevent allergic reactions. elimination diets put infants and children at risk for nutritional deficiencies and impaired growth. we examined the role of the registered dietitian (rd) in advising patients and families of food allergic children. method: a retrospective review of clinical notes was performed for the first 13 consecutive children who required a dietetic consultation in a dedicated food allergy clinic. we examined common questions from parents that were addressed by the dietician during the consultation. results: patients were aged 10 months -9 years (median: 16 months) and were diagnosed with the following food allergies: cow's milk: 69.2%, egg: 62%, tree nut: 54%, peanut: 39%, wheat: 31%, soy: 31%, fruit/vegetable: 15%, legume: 8%, fish: 8%, sesame: 8%. the most common questions for the dietitian included: ways to meet nutritional needs following a prescribed allergen-restricted diet (31%), meeting vitamin d and calcium requirements on a milk protein-free diet (23%), suitable oral supplements and recommended serving sizes (14%), appropriate order of solid foods introduction in food protein-induced enterocolitis syndrome (fpies) (14%), cautionary food ingredient statements (7%), baked milk protein introduction (7%), cross-reactivity risk of milk protein with soy (7%) and crossreactivity of nuts in retail bakeries (7%). conclusion: parents of children with food allergies have multiple questions with regards to nutrition. dietetic input in the food allergy clinic addresses important issues for children and families including successful avoidance of allergen-containing foods while ensuring optimal nutrition, decreased exposure to high-risk situations and avoidance of allergen cross-contamination. 0773 | multicenter prospective study of a stepwise single dose oral food challenge of egg background: oral food challenges (ofcs) are necessary for allergy management. we previously reported that a low-dose ofc can avoid complete elimination, even if patients react to higher doses of causative foods. nevertheless, this approach has only been validated in a retrospective single-center trial. we have previously reported that the median time for initial symptom onset is 50 minutes for egg ofc using a single exposure. therefore, this study aimed to confirm the safety and effectiveness of a stepwise single-dose ofc in a multicenter, prospective study. who showed a positive reaction to low-dose ofc, only 1 patient (2%) showed a severe reaction: barking cough immediately improved with adrenaline inhalation. among 8 patients with a positive reaction to medium-dose ofc, none had a severe reaction. the median times to symptom onset were 60 and 50 minutes following low-dose and medium-dose ofc, respectively. patients in the three groups, divided according to threshold doses, differed significantly in sige levels against egg white and ovomucoid. conclusion: this multicenter prospective study confirmed that stepwise single-dose ofc to egg will help to clarify the severity of egg allergy, and will contribute to improved food allergy managemethod: the study design was a retrospective cohort study extracting data from the electronic chart of children older than 4 years who visited our out-patient clinic for egg or milk allergy and who underwent an oral food challenge test (ofc) twice within 24 months between november 2013 and december 2017. the patients were divided into five groups according to their treatment schedule, which consisted of those who: a) started from 1/10 of the first ofc reaction threshold and maintained 1/10 till the end of oit; b) started from 1/100 of the threshold and maintained 1/10; c) started from 1/10 000 of the threshold and maintained 1/10, d); conventional slow oit (started from just below the first ofc reaction and increased 1.2-1.5 times every few weeks); or e) continued elimination. we determined the presence or absence of an increase in threshold reacted to the allergen, any adverse events during oit, and food-specific ige reduction. results: the number of participants was 217 and their median age was 6 years. the number of patients in groups a, b, c, d, and e was 17, 51, 33, 95, and 21, respectively. the percentage of patients in groups a, b and c showing an increase in reaction threshold to the allergen was higher than that in group e (p < 0.05), and that in group b was higher than that in group d (p < 0.001). the number (percentage) for group a, b, c, d, and e was 12 (70.6%), 43 (84.3%), 23 (69.7%), 54 (56.9%), and 5 (23.8%), respectively. there was a significant difference in the frequency of adverse events during oit between group a-c and d, which was as follows: 5 (29.4%), 9 (17.6%), 8 (24.2%), and 67 (70.5%), for the respective groups (p < 0.0001). there was no significant difference in the percentage of patients showing a decrease in food-specific ige in each group. conclusion: the regimen starting from 1/100 of the ofc reaction threshold and maintaining the dose at 1/10 was safer and more effective for increasing the threshold reacted to the allergen than the 'conventional slow oit' regimen. elimination continuation was not effective for increasing the threshold reacted to the allergen. legumes allergy was presented in different clinical features; urticaria and angioedema in 32 (50%) patients, anaphylaxis in 23 (35.9%) patients, atopic dermatitis in 5 (7.8%) patients, eosinophilic esophagitis in 3 (7.8%) patients and as food-related enterocolitis in 1 (1.5%) patient. thirteen (35.1%) of the patients had asthma, 9 (24.3%) had allergic rhinitis. fourteen (58.3%) of the 24 patients with single legume allergy showed improvement. the patients who developed tolerance, of these 7 (29.1%) had peanut allergy, 4 (16.6%) had lentil allergy and 3 (12.5%) had chickpea allergy. two of 13 patients with multiple legumes allergies, it developed tolerance to all the legumes they are allergic. conclusion: peanut and lentils were the most frequent legumes that displayed allergic reactions in our study. in these patients the rate of allergy to non-legumes food is high. in patients who were allergic to single legumes, the symptoms were ameliorated in 58.3%. conclusion: cashew nut is a potent allergen and can cause quite severe reactions. avoidance of pistachio nut and other related allergens should be advised to patients after allergologic investigation. in the majority of the patients, presence of atopic dermatitis with food allergy is noteworthy. therefore, it would be useful to investigate these patients for cashew and other tree nut allergy before they present with a serious clinical reaction. and jellyfish sting. serum allergen-specific ige test was negative; skin prick test was positive for natto and pork. we performed an oral food challenge with natto, pork, crustaceans, and wheat, and she developed a general itchy rash after 7 hours of eating natto. h1blocker was administered and she recovered soon. however, the general itchy rash relapsed after 4 hours. hence, we intramuscularly injected epinephrine, h1-blocker, and steroids; then, her symptoms did not relapse. based on these findings, we inferred that anaphylaxis caused by natto could be associated with a jellyfish sting. discussion: although association between japanese fermented soybeans (natto) allergy and jellyfish sting has been previously reported, its anaphylaxis is a rare event. in this case, we suggest that anaphylaxis was caused by natto allergy, which was perhaps related to jellyfish sting. hence, further investigation is essential to elucidate the association between fermented soybeans allergy and jellyfish sting. introduction: non-celiac gluten sensitivity (ncgs) is a syndrome characterized by intestinal and extra intestinal symptoms related to the ingestion of gluten-containing food, in subjects that are not affected by either celiac disease (cd) or wheat allergy (wa).once the gluten-containing foodstuff is removed from the diet, the patients will have relief of their symptoms. case: a 6-year-old girl was referred by his general practitioner with history of occasional constipation and abdominal pain (especially after main meals and defecation), short stature and low weight. the growth indices were proper for her age till she was 5. then after there was a stunting. she had short stature and low weight. despite different types of supplementation, there was no improvement in growth indices, so she was referred to a pediatric endocrinologist for gh therapy. primary investigations and anti-ttg, iga, anti-ema all were normal. after a consultation with a pediatric gastroenterologist, a genetic study of hladq2 and 8 were done because of the highly suspicion of celiac disease. the results were also negative. at last she was referred to immunology-allergy clinic for evaluation of probable food allergy. ige level was checked and a prick test was performed which they were not indicative of any suggestive food allergy. because of the history of the abdominal pain and constipation which was more prominent after meals, negative results of genetic study, spt to wheat, and serologic markers, a gluten free diet was suggested for her with the suspicious of non celiac gluten sensitivity. a significant improvement in her symptoms was noticed within 2 weeks of starting gluten free diet. she has 2kg of weight gain and height improved from 116 cm to 120 cm in 4 months. she continued to improve on a gfd and when seen in the follow-up clinic 6 months later reported complete resolution of symptoms and another 2 cm and 1 kg gain in her height and weight. conclusion: non-celiac gluten sensitivity syndrome is a diagnosis made by excluding celiac disease and wheat allergy. it should be taken into consideration especially in patients who have the suspicious symptoms of celiac without supporting lab data, and also negative spt to wheat. the young man in question along with his parents were keen to proceed, so with some hesitation we proceeded to a hazelnut oral provocation challenge, having very carefully explained the risks of undertaking such a challenge. he successfully completed the challenge and experienced no allergic symptoms and is now able to have hazelnuts in his everyday diet. discussion: this young man wanted to confirm if indeed he was allergic to hazelnuts. not being hazelnut allergic would mean that he would be no longer allergic to any nut and would not have to take precautions prior eating products. positive results to both cor a 9 and cor a 14, hazelnut storage proteins are associated with the patient possibly experiencing systemic reactions, at a higher risk of experiencing anaphylaxis it they were to ingest hazelnut. these facts in conjunction with his specific ige to hazelnut would have prevented us from proceeding to challenge was it not for this young man's persistence that he wanted to proceed to challenge despite the risks. conclusion: appearances are deceptive, as this case demonstrates; allergen-specific ige and component testing can only predict the probability of an allergic reaction, the final test in the diagnostic process is the oral provocation challenge. the patient and his family were happy for me to share the above with other health care professionals. method: we present the case of a female of 29 years old diagnosed of acu with poor control of the symptoms at maximum doses of antihistamines. we decided to associate omalizumab treatment. the patient had a good control of the symptoms with omalizumab at dose of 300 mg/4 weeks, but in the 6th month she presented an erythematous, raised and pruritic lesion in the area of injection together with localized abdominal edema at 12 hours of the administration, with two weeks of evolution without symptomatic treatment. we decided to discontinue omalizumab alter a second episode with half doses. results: we performed a skin biopsy of the lesion and epicutaneous tests with the drug. immediate hypersensitivity tests were not taking due to the impossibility of stopping antihistamines. skin biopsy showed a perivascular lymphocytic inflammation of the superficial and deep dermis with frequent presence of perivascular and interstitial eosinophils, suggestive of a hypersensitivity reaction. results: nine months before presentation at our clinic, the patient had been hospitalized and treated with imipenem and tmp/smx for pulmonary nocardiosis. once discharged, she had been prescribed oral tmp/smx alone, according to antimicrobial susceptibility. at our first evaluation, the patient presented with fever, macular erythematous non-pruritic (vasculitic-like) skin lesions on the upper limbs, polyarthralgia and bilateral ankle arthritis. tmp/smx was transiently stopped. after four days, there was a dramatic improvement, with resolution of all signs and symptoms. she was tentatively diagnosed with a viral infection and thus tmp/smx was started again. however, after three days, the symptoms (fever, arthritis and skin lesions) recurred. laboratory investigations showed increased levels of inflammatory markers. complete blood count with differential, serum creatinine, urinary sediment, liver enzymes, rheumatoid factor, antinuclear antibody, c3, c4, immune complexes, serology for rickettsia, borrelia and coxiella were all negative. hence, tmp/smx was stopped again and cutaneous lesions, fever and arthritis resolved spontaneously in five days. conclusion: given the clinical course and the resolution after the withdrawal of tmp/smx, we diagnosed a sslr due to sulfonamides. to the best of our knowledge, this is the first case of sslr occurring after a nine-month therapy with tmp/smx and allergists/immunologists should be aware of the possibility of such a reaction even after months. case report: * we received written informed consent for publication of these clinical details and/or clinical images included in my abstract was obtained from the patient. drug rash with eosinophilia and systemic symptoms (dress) syndrome is a severe adverse cutaneous reaction that usually appears 2-6 weeks after treatment with the causative drug. this syndrome is characterized by severe dermal rash, fever, eosinophilia, and internal organ involvement, and clinically, diffuse maculopapular eruption, exfoliative dermatitis, and facial edema are often observed. we performed blood tests and laryngeal fiberscopy for the diagnosis of the patient. intradermal test with delayed reading and patch test were performed 2 months after the end of treatment. a 53-year-old man had begun treatment with carbamazepine for epilepsy. after 4 weeks of treatment, he observed skin rash with pruritus on both lower extremities, and after 6 weeks, his skin lesions had begun to spread over his whole body, and he complained of several new symptoms, including hoarseness, dyspnea at rest, and dysphagia. an examination revealed maculopapular rash, facial edema, and bilateral cervical lymphadenopathy. laryngeal fiberscopy revealed both arytenoid and epiglottic swelling. laboratory studies revealed eosinophil counts of 2100 /μl and increase in alanine aminotransferase level to 65 u/l. a diagnosis of dress syndrome was definite according to the regiscar group criteria. carbamazepine, the suspected culprit drug, was withdrawn, and systemic corticosteroid was initiated. the patient experienced rapid improvements in hoarseness, dyspnea, and dysphagia. after 5 days of treatment, laryngeal fiberscopy revealed complete resolution of both arytenoid and epiglottic swelling. to the best of our knowledge, our case is the first reported case of dress syndrome to manifest with laryngeal edema. case report: bortezomib (velcade ⓡ ), a targeted therapy works by blocking the action of proteasomes in side cells, is commonly used to treat newly diagnosed as well as relapsed/refractory myeloma. bortezomib has been reported to have gastrointestinal symptoms, peripheral neuropathy, neuropathic pain and thrombocytopenia as its most common side-effects. although several cases of skin lesion caused by bortezomib have been reported, severe cutaneous adverse reaction (scar) such as stevens-johnson syndrome (sjs) is very rare. we here report a case of bortezomib induced sjs. a 71-year-old female patient, who was diagnosed with multiple myeloma, received bortezomib and melphalan /dexamethasone therapy. after the 4th dose of bortezomib, she presented with fever and maculopapular skin rashes spreading from face to the trunk. erosive lesions in the oral mucosa and corneal ulceration with conjuntival injection were observed. she was diagnosed as sjs. the symptoms of sjs improved after bortezomib was discontinued and systemic steroids and intravenous immunoglobulin were administered. drug patch test was performed, the result was positive in bortezomib. this is the first case report of bortezomib induced sjs in this country, which was diagnosed by a patch test. although the scar by bortezomib is generally considered very rare, we suggest that clinicians be aware of potential adverse reactions, including sjs. case report: we report the case of a healthy 46-year-old woman with history of red erythematous macules in both hands, one hour after taking a fluconazole (150 mg) tab for a vaginal candidiasis. it faded spontaneously. she didn't recall if she had ever taken that medicine, but denied known drug allergies. although fde is primarily a clinical diagnosis, we conducted an oral challenge test with fluconazole (150 mg). two hours after intake of the drug the patient started complaints of pain and erythema in both hands and the challenge was stopped. two days after the challenge, she developed red painful erythematous macules on the same sites of the first episode. due to the specificity of the challenge, local patch testing was not performed. introduction: the classic form of a fixed drug eruption is one or more anular or oval erythematous patches as a result of systemic exposure to a drug. these skin lesions normally resolve with hyperpigmentation and may recur in the same location with re-exposure to the drug. other types of fixed drug eruptions have been described, being fixed drug urticaria a rare form of presentation. (1, 2) case report: in the last 2 years, a 48 year old woman has developed more than 15 episodes of a wheal in the right supraciliary region 5 minutes after taking 600 mg of oral ibuprofen. the symptoms resolved in less than 6 hours without treatment and without leaving residual lesion. after the last episode, she refers good tolerance to 1 g of oral paracetamol. she denies local traumas. she also refers mild spring rhinoconjunctivitis well controlled with antihistamine, and sneezing with house dust. a 37-year-old-m patient had psoriasis vulgaris for 15 years, and had been using methotrexate at intervals of 4 years. despite the addition of phototherapy, he underwent a new treatment with biological agent (antitumor-necrosis-factor; anti-tnf), since the disease control was insufficient. before anti-tnf, preventive treatment against latent tuberculosis (tb) activation was indicated with positivity in tuberculin skin test (20 mm). he was given inh 300 mg/day, and at the 20th day of treatment, desquamation, erythema, and subsequent exfoliation developed in his hands and foots dorsum. inh was withdrawn. in order to distinguish the lesions from psoriasis attack, skin biopsy was performed and reported as erythema multiforme-like dermatitis with no relation to psoriasis. the lesions were completely improved at 3 weeks of topical steroids, and inh was re-initiated at the same dose. a week after the initiation of the drug, skin lesions similar to previous reoccurred with more severity and progression from distal to proximal extremities. cell counts, renal and hepatic function tests, and hepatitis markers in blood were in normal limits. skin lesions were retracted after 4 weeks of topical steroids, and withdrawal of inh. there was positivity in skin patch test with inh at 72 hours. finally, for tb prevention an alternative drug rifampicin (10 mg/kg/day) was given, and the patient successfully completed with no adverse event. his psoriasis lesions were improved with anti-tnf which was started after 1 month of tb prevention with rifampicin. in these days which the use of biologic agents is increasingly widespread, inh use will be more prevalent than the past. even tough, it is effective and safe in most of the patients, its adverse event dermatitis may be a reason to withdraw in patients with dermatological diseases. in this case, diagnostic drug allergy evaluation should be performed to optimize the second-line treatment of tb infection, in addition to early withdrawal of the culprit drugs. background: around 50% of cancer patients will receive radiotherapy (ionizing radiations) as a treatment, either as a single therapy or as an adjuvant to chemotherapy and surgery. several side effects have been described due to radiotherapy, of which we can mention erythema multiforme and stevens johnson syndrome, but in lower prevalence. erythema multiforme can be described as an acute skin condition and may be present within a wide spectrum of severity. erythema multiforme minor represents a localized eruption of the skin with minimal or no mucosal involvement. the papules evolve into pathognomonic target or iris lesions that appear within a 72-hour period and begin on the extremities (see the following image). lesions remain in a fixed location for at least 7 days and then begin to heal. it is considered to be a type iv hypersensitivity reaction associated with certain infections, medications, and other various triggers precipitating factors and complex interactions may trigger the appearance of signs and symptoms. these include especially recurrent herpes simplex virus (hsv), epstein-barr virus (ebv), histoplasmosis, alcohol, systemic diseases and immunological factors. method: 69-year-old male diagnosed with prostate adenocarcinoma who underwent transurethral resection and was taking trinomia (ramipril, atorvastatin, acetyl-salicylic acid) after his 8th rte external radiotherapy session, he presented erythematous maculo-papular lesions in the suprapubic area with some vesicles. therefore, withdrawal of treatment was decided and the performance of a skin biopsy. 15 days later, regarding the improvement of the lesions, rte was continued, presenting incipient exacerbations of the lesions but it allowed us to end the cycle of treatment. results: skin biopsy results (anatomical pathology): basal keratinocytes, which blur the dermoepidermal interface, with lymphocyte exocytosis at this level, associated with isolated images of spongiosis. the dermis shows a superficial perivascular lymphocytic inflammatory infiltrate of moderate intensity. compatible with erythema multiforme. conclusion: radiotherapy is a technique of increasing use, so it is important to recognize the associated cutaneous lesions that appear less frequently and are sometimes underdiagnosed. diagnosis is both clinic and pathological and is usually late in most cases so it is vital to take into account this skin disease complication in order to be properly managed. including chinese herbal medicine is usually considered to be without any allergic and adverse reaction. method: visits were made to pharmacies in hong kong and luoyang, china and a martial art monastery/temple in dengfeng, china. some cam were found to have ingredients with potential allergic and adverse reaction. results: three cam, one from hong kong (1), one from shaolin martial art monastery/temple in dengfeng (2) and one from luoyang (3), china were found to contain chinese herbal medicine with potential allergic and adverse reaction. (1) cordyceps ling-zhi complex ingredients: cordyceps sinensis "caterpillar fungus", tremella fuciformis "snow fungus", ganoderma lucidum (ling-zhi) "reishi mushroom" and others 30 years old female patient who has ulcers in oral mucosa and purple, itchy lesions on her right hand palmar area, little finger, index finger, on her left hand palmar area, pollex finger. in her history, she has relapsing vaginal yeast and she hasn't any hypersensitivity reaction with fluconazole before 1 month ago she started to take fluconazole because of vaginal candidiasis. after using fluconazole she started to itch from described areas and dark redpurple eruptions appeared. she was prescribed oral methylprednisolone and topical pomade which included corticosteroid for four days but she didn't aware of fluconazole related drug reaction. lastly four days ago she took fluconazole and metronidazole for severe vaginal yeast. 12 hours later pruritus, same eruption appear on the same area, lip and tongue angioedema than she had dyspnea, dizziness, hypotension, arrhythmia and consciousness. she had admitted to the emergency department and performed adrenalin. after a day bullae and ulcers came into existence in her oral mucosa. in her blood analysis there was mild increase in white blood cell count (13.300 /mm 3 ), eosinophil count was normal (300 /mm 3 ), biochemistry parameters were in normal limits, crp and sedimentation rate were in normal limits, total ige was 439 iu/l. introduction: tuberculosis is a disease that most commonly affects the lungs, which is transmitted by the respiratory tract and drugs are the most important factor in the treatment. non-resistant tuberculosis infection is usually treated with hrze. in rare cases, a hypersensitivity reaction may develop against one or more of the drugs during treatment. case: a 37-year-old female patient was diagnosed with culturepositive pulmonary tuberculosis and hrez treatment was started by the related department. seven days later she referred to the policlinic with edema and itchy erythematous lesions which are common in her extremities, which developed after 6 hours of taking her medication. liver and kidney function tests and eosinophil count were normal. drug eruption was considered with current physical examination findings. the treatment was interrupted, short time systemic corticosteroids and antihistamine treatment started. desensitization planned. there was no feature in the prick and patch tests with drugs. desensitization was performed with isoniazid, no reaction was observed during the procedure. six hours after the procedure, the patient applied to the emergency department with painful edema and pruritic erythematous lesions in the extremities. desensitization procedures with rifampicin, ethambutol, pyrazinamide were performed without any problems after the lesions were regressed. isoniazid was withdrawn from the treatment protocol. outcome: we would like to present on this case that drug eruption may develop in the form of maculopapular rash after desensitization. this study aims to compare ethmoid mucosa and nasal polyp regarding density of tissue eosinophil and its sensitivity, specificity, and correlation with clinical characteristics for diagnosing ecrs. method: patients with crs with polyps scheduled for endoscopic sinus surgery were enrolled. specimens were collected from polyp apex, polyp pedicle and ethmoid mucosa. tissue eosinophil from these three sites in the same patient were compared. using eosinophilic mucin as a reference, sensitivity, and specificity of each site for diagnosing ecrs was assessed. correlations between tissue eosinophilia (defined as greater than 10/ hpf) and clinical characteristics of ecrs including asthma, serum eosinophilia, and eosinophilic mucin were analyzed using each site of specimens. results: thirty patients with crs with polyps were enrolled. polyp apex, polyp pedicle and ethmoid mucosa gave similar results regarding tissue eosinophilia in 16 patients (53.3%). eleven (36.7%) patients were ecrs (having tissue eosinophilia at all sites) and five (16.6%) were non ecrs (no tissue eosinophilia at any sites). median tissue eosinophil was significantly greater in polyp apex (84, and polyp pedicle (96, iqr: 80-320) than ethmoid mucosa (21, iqr: 10-220), p = 0.04. sensitivity of polyp apex, polyp pedicle and ethmoid mucosa for diagnosing ecrs were 100%, 60% and 80% respectively. specificity were 36%, 40% and 48% respectively. correlations between tissue eosinophilia and asthma were significant when assessing ethmoid mucosa (p = 0.04), and polyp pedicle (p = 0.05) but not polyp apex (p = 0.21). correlations with serum eosinophilia, and eosinophilic mucin were not significant (p > 0.05) when assessing any specimens. gov/pubmed/) was performed using the following key words: "obstructive sleep apnea syndrome"; "allergy rhinitis"; "hypoxia"; "intermittent hypoxia"; "fluctuating hypoxia"; "cyclic hypoxia"; and "hif-1α" results: osas may affect the prognosis of ar patients based on the following evidence: 1) ar is thought to be a cause of osas. 2) exposure to hypoxia could mediate immune activation in ar and affect the response to treatment. 3) hif-1α expression may be a risk factor for ar. 4) intermittent hypoxia can induce robust expression of hif-1α. conclusion: first, improvement of ventilation during sleep represents an efficient strategy for treating ar. therefore, continuous positive airway pressure or nasal surgery to resolve a nasal obstruction could be added to ar treatment. finally, medications that target hif-1α, such as digoxin, can be tested as adjuvant therapy. method: forty patients diagnosed with allergic rhinitis and olfactory dysfunction were recruited in current study in the group 1 and 2. patients of group 1 were administered with no treatment and patients administered with the traditional chinese acupuncture therapy were incorporated into the group 2. before the treatment, all of them underwent t&t olfactory testing, nasal sinus computer tomography scanning and visual analog scale (vas; 0-100), and repeated the assessment after four-week treatment. results: improved total t&t olfactory testing scoring averages and vas scoring averages was observed in eleven patients treated with traditional chinese acupuncture compared with four patients in the observation group. no side effect was found. no significant differences in olfaction recovery were found according to age, gender, or duration of disease between the two groups. the observation group underwent nasal endoscopic sinus surgery and the control group underwent external approach surgery, and the therapeutic effect of the two groups were investigated. results: the total effective rate was 94% in observation group and 74% in control group, the total effective rate of observation group is significantly higher than control group (p < 0.05). the recurrence rate was 3% in observation group and 24% in control group, the recurrence rate of observation group is significantly lower than control group (p < 0.05). complication occurrence rate of observation group was 6% which is significantly lower than control group 24% (p < 0.05). the therapeutic effects of endoscopic sinus surgery on chronic sinusitis in geriatric patients are better than conventional external approach surgery which is worth clinical application. results: (descriptive). the equick app is user-friendly even for vkc patients with sub-optimal reading ability. home use between clinic appointments allows responsive temporal data gathering of qol, symptoms, medication scores and impact of medical interventions. equick may be used in future as a research tool in gathering outcome data following interventions for vkc. ectoine, a substance deriving from halophilic micro-organisms, is a strong water structure forming solute exerting cell protective antiinflammatory and antiallergic properties. method: purpose of our study was to assess the efficacy of the preventive administration of 2% ectoine eye-drops (3 times a day for 6 months) to shorten the duration of vkc relapses (which begin, in our country, very early in spring and usually end in october), or to mitigate the attacks, which are only controlled by topical corticosteroids or cyclosporine resulting in an important burden of side-effects. in this retrospective study, we included 192 children of both sexes (148 males and 44 females), under the age of 10 years (mean age 7.8 years), affected by vkc from more than 3 years/seasons and treated for more than 4 months during a year, with cyclosporine eye-drops. these patients underwent, from february to september 2017, the additional-to-the-usual protocol treatment with 2% ectoine eyedrops. results: 8% of the included subjects astonishingly had no relapse of vkc, 38% needed topical cs or cyc treatment but it was started 2 months later compared to previous years, 29% needed the topical drugs 3 months later and 25% had a similar to previous years course (no ectoine efficacy). the treatment was well tolerated and only 1 child had to stop it because of local allergy to the eye-drops. the preventive administration of 2% ectoine eyedrops was able to stabilize and to delay vkc attacks in more than 50% of the selected patients showing the importance of anti-inflammatory and anti-allergic properties of this product. following international criteria we considered normal levels of vitamin d the levels between 50 nmol/l (20 ng/ml) and 125 nmol/l (50 ng/ml), a potential deficiency between 30 nmol/l and 50 nmol/l and a severe deficiency less than 30 nmol/l. results: 58.8% (30 children) of akc group patients presented vitamin d low levels, among them 18 children showed a potential deficiency and 12 a severe deficiency. 24.3% (18 subjects) of vkc patients suffered a deficiency in vitamin d which was mild in 13 and severe in 5 patients. 40.3% (25 children) of sac group showed a deficiency in vitamin d which was potential in 17 and severe in 8 subjects. conclusion: our study shows that in different forms of allergic conjunctivitis many children are suffering a vit. d deficiency and it can be supposed that a correlation between the severity of the allergic form and the level of vit. d deficiency exists. we recommend allergists and ophthalmologists to check vit. d levels in children suffering from allergic conjunctivitis because its deficiency is very common and many are unaware of it; in case of a vit. d insufficiency it is fundamental to give a vit. d suitable-to-the-case supplementation. method: 83 children (57 males and 26 females, mean age 7.3 ± 5 months) affected by vkc and allergic rhinitis from more than 2 years were treated with mometasone furoate nasal spray 1 spray bid × 2 weeks in a month, for 3 consecutive months as a co-seasonal treatment at the beginning of eye allergic symptoms. other systemic or topical treatments did not vary compared to the previous 2 years. results: a quick questionnaire administered to children and their care-givers showed that nasal symptoms regressed after a mean period of 9.2 days from their beginning but, impressively, in more than 30% of them, these patients did not show a vkc typical relapse along the 3 months of mometasone treatment, moreover the following summer period was milder in subjective ocular symptoms in more than 25% of the patients. our experience pointed out that incs adjunctive treatment was positively associated with a regression of eye and nose symptoms in children suffering from vkc, confirming previous literature data which concern milder forms (seasonal allergic conjunctivitis or allergic rhino-conjunctivitis) compared to the severe forms (like vkc) we analyzed in our work. one of the involved mechanisms of action can be the alleged effect on the reduction of substance p in tears; it is supposed to reflect the neuropeptides levels in ocular tissues. 0816 | patient response to mp-azeflu in an allergen exposure chamber onset of action (ooa) timing may impact treatment adherence. mp-azeflu, intranasal azelastine hydrochloride (aze) and fluticasone propionate (fp) in a single device, has proven to have greater efficacy and faster ooa than a combination of oral loratadine and intranasal fp (lora/infp), but the clinical relevance for patients is unclear. this single-center (ontario, canada), randomized, double-blind, double-dummy, three-period crossover trial examined by which extent mp-azeflu provides clinically relevant symptom improvements according to different efficacy parameters. method: ar symptoms were induced in asymptomatic, ragweedsensitive patients via ragweed pollen challenge in an environmental exposure chamber. patients received a single dose of mp-azeflu, lora/infp, or placebo and were monitored for 4 hours. symptoms were assessed using total nasal symptom score (tnss) and total ocular symptom score. responder analyses included the number of patients to achieve relevant response (rr) to therapy (30% or 50% reduction in tnss), time to rr (ie, first time point at which rr was reached), and minimal clinically important difference (mcid) in ooa. background: nasal allergen provocation test (napt) is a standardized diagnostic tool indicated in the diagnosis of allergic rhinitis, to design and monitoring allergen immunotherapy, and to study the pathophysiology of airway allergy. unfortunately, until now very few studies have evaluated its reproducibility and safety. in this study we wanted to analyse the safety and reproducibility of napt in a large group of rhinitis patients and healthy controls. unit until december 2017. a bilateral saline challenge followed by a bilateral napt were performed in symptoms-free individuals. the response was assessed by nasal-ocular symptoms and acoustic rhinometry. all subjects signed a written informed consent. the safety of napt was checked by the occurrence of extra-nasal/ ocular reactions (enor), severe adverse events (sae), and use of rescue medication (rm). enor was assessed by clinical symptoms, physical examination, cardiopulmonary auscultation, spirometry, and oxygen saturation. the reproducibility of napt was tested by comparison of the results in 2 or more sessions with≥1-month interval. background: nasal hyperreactivity (nhr) is self-reported by a majority of patients with allergic rhinitis (ar) and is likely mediated by neural-immune interactions. the combination of fluticasone propionate (fp) and azelastine (aze) hydrochloride administered in a single spray (mp-azeflu) has been shown to be superior to fp or aze alone in patients with seasonal ar (sar). we hypothesize mp-aze-flu may reduce neuro-immune mediators in ar with nhr. in a post hoc analysis of three pivotal studies of mp-azeflu, we analyzed the efficacy of mp-azeflu, fp, and aze in patients with ar with and without nonallergic triggers. method: in three randomized, double-blind, controlled trials, patients with sar were randomized 1:1:1:1 to mp-azeflu, fp, aze, or placebo (pbo). patients self-reported sensitivity to nonallergic triggers. change from baseline in total nasal symptom score (tnss) and treatment differences between active agents and pbo were calculated. results: across 3412 patients in three studies, mean age was 36.3 years and mean age at ar symptom onset was 15.6 years. overall, 89% reported ≥1 nonallergic trigger, which included sudden temperature/humidity change (72%), tobacco smoke (61%), perfumes/fragrances (57%), incense/candles (38%), and cleaning products (38%). change from baseline in tnss for patients with ar and nonallergic triggers was greater with mp-azeflu than with fp or aze (table) , and patients with nonallergic triggers improved slightly less than patients without nonallergic triggers in both the mp-azeflu and fp groups. background: in low-income countries (lics), assessment of phenotypes, prevalence and risk factors for allergy-related diseases (ards) using allergen-specific ige may be complicated by environmental exposures such as helminths. these exposures may also induce cross-reactive carbohydrate-specific ige profiles that could inhibit allergic effector responses. we sought to elucidate the molecular basis of ige sensitisation among individuals in uganda, using a component-resolved approach to ige measurement. we employed the isac ® allergen microarray to assess plasma ige reactivity to 112 purified natural and recombinant allergen components in participants of three studies: a trial of intensive versus standard anthelminthic treatment in the rural helminth-endemic lake victoria islands (n = 126), a parallel urban survey of allergy outcomes in a lower helminth exposure community (n = 60) and a study on asthma risk factors in children from the urban setting and from nearby rural schools (n = 100). data on sensitisation to crude allergen extracts were obtained by skin prick testing (spt) with cockroach and house dust mites (hdm), and by immunocap ige testing (cockroach, hdm, and peanut). results: the rural setting was characterised by high prevalence (≥34%) of sensitisation to crude extracts (immunocap ige>0.35 ku/ l) but low sensitisation to the major, established, allergenic components on the microarray (≤2%, ige>0.30 isu). however, sensitisation to cross-reactive carbohydrate determinant (ccd)-bearing components and venoms was more common in rural (up to 14%) versus urban (up to 8%) individuals, and was associated with helminth infection. urban individuals mounted higher responses to allergenic components of dust mites but responses to other components were similar between the two settings. sensitisation to allergenic components was higher among asthmatics and spt+ children but ccd sensitisation profiles were similar between asthmatics and nonasthmatics, and between spt+ and spt-school children. conclusion: we show that, in lics, ige to crude allergen extracts (detected in standard immunocap assays) reflects sensitisation to a myriad of environmental exposures (absent in more developed countries), such as ccds expressed by helminths, and may not accurately define ard phenotypes in this setting. however, our data does not seem to indicate that ccd-specific ige detected by isac ® microarray protects against ards. considered minor allergens. due to their sequence homology and conserved structure, they show a high cross-reactivity. the objectives were to study the ige/igg binding properties of polcalcin in relation to the calcium ions, and the ige cross-reactivity between purified polcalcin from olea europea (ole e 3) and two recombinant polcalcins (rphl p 7 and rbet v 4). method: ole e 3 was purified by immune-affinity chromatography using polyclonal antibodies anti-rche a 3. serum samples were obtained from 6 patients allergic to grasses recruited at hospital de guadalajara (spain), all of them positive to phl p 7 with sige values ranging from 12.1 to 92.6 ku/l. equal volumes of all sera were used to prepare a pool. calcium binding assay was performed either by addition or not, or depletion of ca 2+ . ole e 3 was incubated with 0.1 mm cacl 2 or with 1 mm egta ph 7.5 (ca 2+ chelator agent) at the same time as the antibody in immunoblot or elisa assays with the pool of sera or with anti-che a 3 polyclonal antibody. crossreactivity assay was performed by immunocap inhibition. aliquots of the pool of sera were previously incubated with amounts of ole e 3 ranging from 0.02 to 12.5 ng. the same dilution of the pool of sera without ole e 3 was used as a control. after 2 hours of incubation, sige (ku/l) binding to rbet v 4 or rphl p 7 was determined. results: a 9 kda protein was purified from the o. europea extract and identified by lc/ms-ms as ole e 3. in the calcium binding assay there were no differences between the samples with or without ca 2+ . however, the addition of egta to the reaction completely inhibited the binding of the polyclonal antibody by immunoblot and also produced a 32.3% reduction of ige binding by elisa. in the cross-reactivity assay, a 50% inhibition of ige binding was obtained with 2.8 ng of ole e 3 for rbet v 4 and 3.9 ng for rphl p 7. the maximum rate of achieved inhibition was 68.6% for rbet v 4 and 61.6% for rphl p 7. conclusion: native purified ole e 3 contains the ca 2+ necessary to bind to the specific antibodies and the depletion of ca 2+ inhibited this binding. high cross-reactivity of ole e 3 with rphl p 7 and rbet v 4 was demonstrated. 0826 | effect of glutathione-s-transferase pi on the cysteine protease activity of the house dust mite allergen der p 1 background: environmental proteases have been proposed to be involved in the pathogenesis of allergic disorders via different mechanisms, such as the disruption of epithelial tight junctions, the cleavage of surface proteins, the activation of damage and pathogen-associated molecular patterns receptors, and the alteration of redox status. der p 1 from house dust mite is one of the most clinically relevant indoor allergens worldwide, which exhibits cysteine protease activity and has been linked to allergenic rhinitis and asthma. however, it is unknown whether the host microenvironment could regulate der p 1 activity once it reaches the mucosal surface. glutathione-s-transferase pi (gstpi) is an anti-oxidant and detoxification enzyme. gstpi is the predominant gst in human lung epithelial cells, where it is expressed in high levels. polymorphic variants of gstpi have been associated to various inflammatory lung disorders such as allergic asthma. more recently, gstpi has been identified as a redox regulator through protein s-glutathionylation, a post-translational modification where glutathione (gsh) is conjugated to cysteine residues. method: this work aimed at determining if gstpi affects the cysteine-protease activity of der p 1, compared to gstmu -a different gst isoform-by using different in vitro approaches. results: we found that gstpi increased der p 1-activity, but not gstmu. our results suggested a potential role of gstpi in upregulating the protease activity of der p 1 allergen. however, the clinical implications of these findings in allergic airway diseases needs for further investigations. 0827 | cari p 1, a novel polygalacturonase allergen from papaya acting as respiratory and food sensitizer biswas sarkar m; sircar g; ghosh n; das ak; jana k; dasgupta a; gupta bhattacharya s bose institute, kolkata, india background: papaya was globally reported to elicit ige-mediated hypersensitivity. certain papaya sensitive patients with food allergic symptoms were found to experience recurrent respiratory distresses at peak flowering period of papaya even after quitting the consumption of papaya fruits. the immunoreactive protein present both in pollen and fruit proteome was detected by ige-serology and identified by mass spectrometry. one such allergen, designated as cari p 1 was cloned, and purified as recombinant protein. the ige-reactivity of rcari p 1 was examined by immunoblot using patient sera. the allergenic activity of rcari p 1 was evaluated by histamine release assay from ige-sensitized granulocytes. the aggregation and folding pattern of rcari p 1 was assessed by size exclusion chromatography and circular dichroism spectroscopy respectively. the presence of cari p 1 in papaya fruit was searched by igg-immunoblot using allergen-specific rabbit antisera. a mouse model of papaya allergy was established to study the role of rcari p 1in eliciting respiratory and food hypersensitivity. results: a 55 kda ige reactive protein commonly present in pollen and fruit proteome of papaya was identified as endopolygalacturonase. recombinant cari p 1 remained monomer and the cd-spectra revealed predominantly β-sheet characters. the melting curve of the allergen showed partial refolding from a fully denatured state indicating the possible presence of conformational ige-epitopes in addition to the linear ige-epitopes of food allergens. 7 out of 7 papaya allergic patients displayed ige reactivity to rcari p 1. rcari p 1 at 1 μg/ml, induced histamine release from challenged granulocytes within a range of 30% to 72% (i.e. 50 ± 9.2%; n = 4 patients). expression of cari p 1 was detected in the peel and pulp tissues of papaya fruits at two edible stages of fruit maturation. in mouse model, rcari p 1 exhibited a comparable level of eosinophil infiltration and goblet cell hyperplasia in lung and duodenum histology. conclusion: cari p 1 the first major allergen reported from papaya with a dual role in respiratory sensitization via pollen inhalation and sensitization of gut mucosa via fruit consumption. the recombinant allergen can be used as marker allergen for molecular diagnosis and immunotherapeutic management of papaya allergy. background: lipids can be potent stimulators of the immune system, and their role in allergy is highly investigated and debated. since many allergens bind lipids, one question that arises is the relative importance of the lipids versus the lipid-allergen complex in eliciting the immune response. also of interest is an evaluation of the importance of the allergen-lipid complex. in our characterization of the structure of the cockroach allergen bla g 1, we discovered that it could promiscuously bind a variety of lipids in a large central cavity. this suggested that bla g 1 could be used as a prototypical allergen and lipid delivery vehicle to test in various models of sensitization. method: cd spectroscopy. nmr spectroscopy. molecular modeling. we have developed an hplc procedure to strip the phospholipids derived from the e. coli-based expression system, and reconstitute the allergen with a variety of lipids. using cd spectroscopy and nmr, we have verified that the protein conformation is highly similar in the presence and absence of lipids. temperature dependent cd spectroscopy revealed that unloaded bla g 1 is the least stable, and the melting temperature increased with increasing fatty acid chain length up to c20. similar cd melting experiments revealed that bla g 1 could bind lipoteichoic acid (lta) from gram positive bacteria, but did not interact with lipopolysaccharide (lps) from gram negative bacteria. molecular modeling studies have suggested that the stoichiometry of phospholipid binding is likely 4 phospholipids per bla g 1 and give insight as to the different binding characteristics that would allow bla g 1 to bind lta but exclude conclusion: these biophysical studies will allow the design of bla g 1-lipid systems to test a variety of sensitization models. 0829 | sal k 7, a new allergen from salsola kali sola jp; pedreño y; fernández j; cerezo a; peñalver m probelte pharma, murcia, spain background: the polcalcin from salsola kali was identified and sequenced (genbank kt254655) and the recombinant protein was characterized as a minor allergen with a prevalence of 40% of patients with a spt positive to s. kali. the objective of this study was to purify the polcalcin from s. kali pollen and to include the allergen in the website for the systematic allergen nomenclature (www.allergen.org). method: the native polcalcin from s. kali (npsk) has been purified from pollen after a first step of protein extraction and then diverse chromatographic steps: a size exclusion chromatography to remove particles minor than 5 kda, an ionic exchange chromatography, a hydrophobic interaction chromatography and a final step of size exclusion chromatography to obtain the purified sample of polcalcin. the purity of the npsk has been determined by sds-page and the binding capacity to a specific polcalcin antibody from rabbit serum was tested by immunoblot. the specific antibody had previously been obtained by immunization with the recombinant polcalcin from s. kali. the allergenicity of the npsk has been assayed by immunoblot with a pool of sera of patients sensitized to s. kali. the identity of the purified npsk has been analyzed by peptide footprint in hplc-ms/ms after digestion with trypsin. all the information about the polcalcin from s. kali was sent to who/iuis allergen nomenclature sub-committee. the npsk showed a high purity in sds-page with a molecular weight of approximately 9 kda and this purified protein reacted with the specific polcalcin antibody from rabbit serum. the ige binding capacity of the npsk was confirmed by immunoblot using a pool of sera from patients sensitized to s. kali. the analysis of peptide footprint confirmed that the purified protein is a polcalcin. the who/iuis allergen nomenclature sub-committee included the polcalcin from s. kali in the website for the systematic allergen nomenclature as a new minor allergen named sal k 7. conclusion: the polcalcin from s. kali has been purified from pollen and tested for its ige binding. it is included in the website for the systematic allergen nomenclature as the new allergen sal k 7. background: alt a 1 protein is the major allergen from the fungus alternaria alternata and responsible for chronic asthma, yet little is known about its physiological role and immunological activity. our main purpose was to investigate the mechanism through which alt a 1 induces an allergic response in bronchial epithelium. method: although alt a 1 has a unique topology, we studied the structural relationship by in silico procedures consisting of three distinct structural alignment methods in order to understand its nature. the immunological properties of the allergen were investigated by using monocyte cell line thp1 and human peripheral blood mononuclear cells. results: its crystal structure has been recently reported and claimed to be exclusively in fungi without equivalent in the protein data bank. data obtained in silico show that this allergen shows some structural relationships with a number of other β-barrel proteins such as human lipocalin 2 (lcn2). besides, our experimental data demonstrate that alt a 1 is also able to interact with lcn2, human lipocalin. in this way, the results obtained from several immunological assays showed that alt a 1 is able to produce a response of the immune system through different immune innate receptor pathway inducing the th2 cytokines. background: increasing evidence of cross reactivity syndromes between pollen grains and fruits, with immediate or delayed reactions, has been reported. while some syndromes such as the birch pollen/apple syndrome are well documented, some other such as the cypress pollen/peach syndrome remain to be understood. for the latter, significant progress has recently been made with the discovery of a new allergen family, the gibberellin regulated proteins (grps), which has been shown to be responsible for the observed cross reactivity i.e. pru p 7 and bp14 (1, 2) for the peach and the cypress pollen respectively. grps are small cationic proteins with anti-microbial properties and have been shown to be over produced in response to a stress. herein, the case of a patient, born and raised in the south of france but currently living in paris, has been studied. this patient has been suffering since childhood from allergic rhinoconjunctivitis to cypress pollen and from some oral symptoms to peach and other fruits (including pomegranate). method: in addition to the clinical exploration and cutaneous tests, a very thorough biological characterization of the patient samples has been performed through various specific ige quantitation techniques, western blotting after one and two-dimensional gel electrophoresis and flow cytometry based basophil activation testing (bat). results: specific iges to cypress pollen, birch pollen, peach, orange and apple have been found. pr10 allergenic proteins are recognized by iges but no ltps. the presence of specific iges to cypress pollen bp14, peach peamaclein (pru p 7) and a cationic 14 kda protein from pomegranate has been shown through western blotting after gel electrophoresis separation of the protein extracts. the use of bat finally enabled to demonstrate that the basophils of this patient were, ex vivo, strongly activated with protein extracted from orange and cypress pollen and also with purified proteins such as bp14 and pru p 7. conclusion: these results unambiguously show that the cypress pollen grp, bp14, is clinically relevant, similarly to its homologous protein in peach, pru p 7. it can be proposed that these two allergens are at the basis of the observed cross-reactivity syndrome. the search for new cross-reactive allergenic grps in pollen, fruits or vegetables may enable to better understand other pollen/food associated syndromes that still remain unexplained. background: nine allergens of phleum pratense have been described until now (iuis database) and classified into groups based on their function and cross-reactivity. group 1 and 5 allergens are considered the most immunodominant, due both to their greater ige-binding capacity and the number of patients ige-reactive to them. previously published studies have estimated that group 1 is recognized by almost 95% of grass pollen-allergic patients, and group 5 by 80%. however, until now a comparative of the ability of these allergens to provoke an immune response has not been performed. the objective was to study the immunogenicity of the major allergens phl p 1 and phl p 5, by analyzing the ability of the recombinant forms (rphl p 1 and rphl p 5a) to induce a humoral immune response. method: five mice were immunized with the same amount of each recombinant protein: rphl p 1 and rphl p 5a (indoor biotechnologies) (60 μg plus two boosters of 30 μg). the specific igg antibodies produced by each mouse were tested against the recombinant proteins by direct elisa and the title of each of them was determined by optical density (o.d.). additionally, the recognition of both allergens in native and depigmented-polymerized (dpg-pol) extracts of p. pratense was studied by direct elisa using these generated antibodies. results: preimmune sera were negative. all mice produced antibodies against the corresponding recombinant protein. the immune response (sigg) was statistically significant higher in mice immunized with rphl p 5 than in those immunized with rphl p 1; it was needed 8 times more rphl p 1 serum than rphl p 5a serum to obtain the same o.d. values. the difference in responses was higher in the group of mice immunized with rphl p 1 than with rphl p 5a. differences in the recognition of phl p1 and phl p 5 in native and depigmented-polymerized extracts of phleum pratense was also observed. it was necessary 8 times more rphl p 1 serum to produce the same signal than rphl p 5a serum in native extract and it was necessary 4 times more rphl p 1 serum to produce the same signal than rphl p 5a serum in dpg-pol extract. conclusion: rphl p 5a is more immunogenic than rphl p 1, which was also probed with native and dpg-pol extracts. background: glioblastoma (gbm) is an incurable primary malignant brain tumour with a median life span of less than 15 months despite multimodal treatments. therefore, there is a serious need for the development of innovative medications. several epidemiological studies underlined an inverse correlation between pre-existing igemediated allergy and gbm risk, where having such an allergy decreased the odds of developing gbm by 20 to 40%. we aim to delineate the intrinsic immuno-biological and molecular mechanisms that can be responsible for these correlations, based on the hypothesis that allergies may promote a state of increased immuno-surveillance in the brain through the presence of immunological factors such as immunoglobulins, cytokines and cells involved in th2-driven allergic reactions. we consider that as the major immune cell type of the brain, microglia should be implicated in this beneficial association and may favour the elimination of the nascent tumour in brain parenchyma in an allergic context. we implemented a long term allergic airway inflammation by repeated nasal instillation of house dust mite (hdm) extract in a syngeneic orthotropic mouse model of gbm. we followed animal survival and the tumour growth by mri. in addition, we purified microglia from allergic vs non-allergic mice in order to assess their cytotoxic function against the gbm cell line ex vivo and their secretory capacities. finally, we investigated immunoglobulin reactivity against gbm antigens in the context of allergic reactions by reverse phase protein array (rppa). we demonstrated an increase of the animal survival that was correlated with a delayed tumour engraftment and a reduced tumour growth. these phenotypes were associated with functional modification of microglia from sensitized mice. indeed, these microglia showed a rise in the production of il-6 and tnf-a as well as an increase in cytotoxic functions against a gbm cell line ex vivo. in parallel, we observed an increase in serum igg1 reactivity against gbm antigens in mice sensitized with hdm compared to control mice. results: in 23 patients (48%) with cvid we recorded at least one temporary platelet count decrease below 150 × 10 9 /l compared to only 1 patient (10%) with xla (p = 0.024). more importantly in 18 patients (38%) with cvid this decrease was observed in a period longer than 6 months compared to 1 patient (10%) with xla (p = 0.077). in 10 patients (21%) with cvid we recorded at least one temporary platelet count decrease below 100 × 10 9 /l and only in 3 patients (6.5%) with cvid this decrease was observed in a period longer than 6 months. we did not record any platelet count decrease bellow 100 × 10 9 /l in patients with xla however the difference with cvid did not reach statistical significance. no thrombocyte count decrease bellow 50 × 10 9 /l was observed in either group. none of patients required immunosuppressive treatment for immune thrombocytopenia (itp). conclusion: although the statistical significance was documented only in temporary platelet count decrease below 150 × 10 9 /l it is obvious that numbers of thrombocytes commonly fluctuate in some patients with cvid. the mechanism leading to these temporary decreases is unclear. monitoring of complete blood count is a basic follow-up investigation in patients with cvid. introduction: wegener's granulomatosis (wg) is a systemic disease that may affect all organs, most frequently the ears, noses, throats, sinuses, lungs and kidneys. it is a rare autoimmune disease, also called granulomatosis with polyangiitis, and characterized by necrotizing granulomatous inflammation in small and medium sized blood vessels. anti-neutrophil cytoplasmic antibody against to proteinase 3(c-anca) is thought to be responsible for autoimmune inflammation. the coexistence of wg and common variable immunodeficiency (cvid) is extremely rare. in this report, we describe a patient with wg and cvid who was treated with immunosuppressive drugs and intravenous immunoglobin concomitantly. case report: a twenty-four-year-old male patient was referred to our clinic for immunological evaluation due to recurrent infections, fever of unknown origin and neutropenia. the patient had been diagnosed with wg and taking immunosuppressive therapy for three years. he had chronic renal failure due to wg and had also been on peritoneal dialysis for three years. serum igg, iga levels, peripheral blood cd19 + b cell percentage and absolute count of the patients were found to be low according to reference limits. he was diagnosed with cvid after excluding secondary reasons for hypogammaglobulinemia and he started to receive 600 mg/kg intravenous immunoglobulin (ivig) therapy once in a month. also, the treatment that consists of mycophenolate mofetil (mmf) and glucocorticoids was continued to decrease c-anca levels in serum. he has been accepted as a candidate for kidney transplantation, and prepare for this purpose. discussion: the management of the patient with cvid and wg may be complicated. it is considerably difficult and needs competency and courage. moreover, the cases similar to ours, are extremely rare. therefore, the authors should share their own experiences on cvid and discuss them by comparing the data obtained from other cases. background: leukocyte adhesion deficiencies (lads) are a group of three genetic disorders leading to defective leukocyte adhesion to the endothelium and as a consequence decreased leukocyte recruitment and immune defense. lad-i is caused by mutations in the gene encoding the ß2-integrin cd18 on chromosome 21.lad-iii is a rare primary immunodeficiency syndrome, characterized by homozygous mutations in the kindlin-3 gene (official symbol fermt3). we have aimed to evaluate our patients who were followed up with lad for the last 15 years, retrospectively. method: all data of the cases were obtained from the file records of age at diagnosis. results: seven patients from separate 6 families were included in the study. four patients were lad-iii and 3 patients were lad-i. the female to male rate was 2/5. the age of diagnosis is ranged from 16 days to 4 years. the median umbilical cord detachment was 21 days (7 -53 days groups: up to 6 times (6 people) and from 6 to 12 times (6 people). 10 healthy donors were examined as a control. flow cytofluorometry method was used to study peripheral blood and assess the parameters of innate and adaptive immunity results: it was found that at a frequency of edema up to 6 times a year there are changes in the t-system of adaptive immunity, which are shown by a decrease in the expression of late activation markers (cd3 + hladr+ 3.46 ± 0.60%, in control 8.04 ± 0.14%), an increase in the number of cd3 + cd8 + cytotoxic lymphocytes (0.60 ± 0.17x10 9 /l, in control 0.39 ± 0.01x10 9 /l) and as an increase in their functional activity (cd8 + gr+ 0.53 ± 0.02x10 9 /l, in control 0.16 ± 0.01x10 9 /l). the nature of disorders of cellular factors of the innate immunity is manifested by decrease in the adaptive resources of neutrophils (kstnbt 1.62 ± 0.13u.e., in control 2.15 ± 0.02u.e.). patients with hae with a frequency of edema up to 12 times a year, we observed the disorders of the humoral link of adaptive immunity, which consist in an increase in the number of circulating b lymphocytes (0.27 ± 0.11x10 9 /l, in control 0.11 ± 0.01x10 9 /l). in addition, with the strengthening of the hae clinic, changes in the system of innate immunity progressed very fast and consisted in increasing the amount (cd16 + 0.37 ± 0.05x10 9 /l, in control 0.21 ± 0.01x10 9 /l), and functional activity (cd16 + gr+ 0.32 ± 0.16x10 9 /l, in control 0.14 ± 0.0210 9 /l) of natural killer cells results: we included 261 children, mostly males (54%), aged between 1 month and 16 years. 37.5% of patients (n = 98/261) showed abnormal absolute results of lymphocyte count for age. we found more patients evaluated in the age group of 2 to 5 years (31.8%), followed by 5-10 years (24.5%), lymphopenia was found in 15.4% of patients. b lymphocyte deficiency was the most common pattern (37%) followed, in decreasing order, by low cd4, t cd3, tcd8 and nk. many patients have more than one affected population (12.6%) . some patients were affected in all three series (4.2%). the cd4 / cd8 ratio decreased in 28.7% of the patients. the majority of the children were males between the ages of 1 month and 16 years. 37.5% of patients showed abnormal absolute lymphocyte count for age. b-cell deficiency was the most common pattern followed, in decreasing order, by low cd4, t cd3, tcd8 and nk. many patients have more than one affected population. 0847 | indicators of the humoral immunity in the mechanical jaundice of benign genesis the aim of the investigation was to study the indices of humoral immunity in patients with benign mj, depending on the level of bilirubin. method: 62 patients with mj and 125 practically healthy volunteers were examined. patients with a level of bilirubin less than 60 μmol / l -9, with a bilirubin level of 60-200 μmol / l -37 and with a bilirubin level of more than 200 μmol / l -16 patients. the concentration of immunoglobulin classes a, m, e and g in serum was determined by enzyme immunoassay. the statistical significance of the differences was determined using the ranked mann-whitney test. the critical level of significance in checking statistical hypotheses was assumed to be p < 0.05. results: of the contacted dermatologists, 136 participated (53 women, 83 men; mean age 53.2 ± 8.5) which results in a response rate of 27.3%. the guideline compliant prescription rate of biologicals in patients with csu was 6.9%. the most prevalent barriers in the prescription were the high cost of the treatment (64.7%), low reimbursement for doctors (62.5%) and the fear of a recourse claim (52.9%). however, a lack of evidence or an insufficient efficiency were not concase report: eosinophil associated gastrointestinal disorders (egids) including eosinophilic colitis are commonly associated with atopy. aeroallergen sensitization may accompany food allergy in these patients. a case with eosinophilic colitis responsive to anti-ige monoclonal antibody (omalizumab) treatment is presented. an eleven-year-old boy had bloody diarrhea lasting nearly one month in autumn for last 3 years. this year diarrhea lasted more than 3 months. colonoscopic biopsy revealed lymphoplasmacytic inflammatory cells including eosinophils leading to a diagnosis of ulcerative colitis. corticosteroid and mesalazine treatment was started with a good clinical response. recurrence of diarrhea during corticosteroid dose reduction suggested corticosteroid dependent ulcerative colitis. eosinophilic/allergic colitis was an alternative diagnosis when seasonal recurrence, lack of weight loss, eosinophils in biopsy and high serum ige level were considered. colonoscopy done after cessation of therapy for one month, revealed exudative ulcerous lesions, lacerations, loss of haustration compatible with colitis (inflammatory/allergic?). presence of significant mucosa associated lymphoid tissue in biopsy supported any inflammatory, reactive process. he had recurrent bronchiolitis until age six and allergic rhinitis in spring for three years. total ige and mix aeroallergen specific ige were high (518 iu/ml, 56.1 kua/l), absolute eosinophil count was normal (210/mm3). food skin prick and patch tests were negative. he had positive skin reactions with dermatophagoides, grass and olea pollens (induration diameter: 9, 10, 6 mm, respectively). pulmonary function test was normal. he was considered as eosinophilic/allergic colitis and omalizumab was started according to manufacturer's dosing table (300 mg/ 2 weeks). rectal bleeding decreased after first dose and ceased after the second dose. early colonoscopy examination after 3rd month of therapy showed that exudations disappeared and haustrations became evident. microscopy revealed mild nonspecific colitis. few patients with eosinophilic colitis improved with omalizumab were reported before. ige-mediated processes are responsible from eosinophilic inflammation in egids, making anti-ige therapy as a promising treatment option. 0859 | design of liposomal carriers modified by glycoconjugates for liver cell delivery of nucleic acids used. the surface of liposomal nanoparticles can be modified to increase the selectivity of intracellular delivery. it is well known that asialoglycoprotein receptors of hepatocytes have a strong affinity to galactose carbohydrate. therefore, the aim of this study was to assess the effect of the modification of the liposome surface by glycoconjugates on the selectivity of intracellular transport of nucleic acids into the liver cells. method: liposomes based on ornornglu(c 16 ) 2 were chosen previously as the effective nucleic acid delivery system. we modified liposomes with novel lactose-based derivatives. every of four glycoconjugates was added to ornornglu(c 16 ) 2 in an amount of 5, 10 and 15%. as a result, 12 variants of modified liposomes were obtained. to determine the cytotoxicity, an mtt test was used. using luciferase test, the selectivity of penetration was evaluated on nonspecific 293t (human embryonic kidney) and specific hepg2 (human liver cells) cell lines. results: modified liposomal compositions ornornglu(c 16 ) 2 -4 + lacc 16 (5%) and ornornglu(c 16 ) 2 -4 + lacggg16 (15%) had the lowest cytotoxicity similar to that for unmodified ornornglu(c 16 ) 2 . the ic 50 , calculated based on the data of mtt test, was 0.14 and 0.26, vs. 0.16 mg/ml, respectively. ornornglu(c 16 ) 2 -4 + lacggg16 (15%) showed a 1.5-fold increase in transfection activity on the nonspecific 293t cells, compared to unmodified ornornglu(c 16 ) 2 , whereas the modification of ornornglu(c 16 ) 2 -4 + lacc16 (5%) resulted in a 6-fold decrease in transfection activity. however, the ability of these variants to penetrate the specific liver hepg2 cell was significantly higher by 15 and 25 times, respectively, than for unmodified ornornglu(c 16 ) 2 . results: the greatest inhibitory effect of sbfhd was observed in mdm infected with hiv-1 bal: 90% and 50% suppression of hiv replication was achieved at concentrations of 1.5 μg/ml and 0.4 μg/ ml, respectively. the activity in pbmc and dc was less pronounced (the respective ic90 values were 5.8 μg/ml and 19.7 μg/ml). studies in endometrial hec-1a cells demonstrated that sbfhd suppressed cd4-independent entry of hiv-1 (10 3 tcid 50 /ml) by 33%, 54%, and 98%, respectively, at 1, 10, and 100 μg/ml. the effect was also observed after increasing the dose of the virus. at 10 4 tcid 50 /ml, sbfhd suppressed hiv infection by 45% (10 μg/ml) and 97% (100 μg/ml). the cytotoxicity of sbfhd in this system was low. similar results were obtained with colorectal caco-2 cells. sbfhd exhibited no spermicidal activity at concentrations of up to 3 mg/ml. combining within a single microbicide two agents that target distinct steps of hiv life cycle will maximize its efficacy (via synergistic effects and/or interference with multiple stages of the transmission). we therefore explored the synergistic potential of combinations of sbfhd and azt, the classical nucleoside rt inhibitor. in these experiments, 50% suppression of hiv infection was reached at concentrations of sbfhd and azt, which were significantly lower than the respective ic50 values of each component (determined in parallel experiments). the synergistic effect was most pronounced for the combination of 0.1 μg/ml sbfhd (which is 60 times less than the ic50) and 0.16 nm azt (which is 45 times less than its ic50). cd80 expression was increased after the co-culture with reishi, shiitake and boletus mushrooms (c -5.6(1.4-9.2)%; pma -60.5 (27.2-70.9)%; )%; shiitake -9.6(4.3-15.5)%; boletus -16.6 (14.0-24.0)%). method: the study included 38 men (mean age 34 ± 5.9 years) before and immediately after staying in countries with a hot climate. results: the development of lymphopenia observed in the first week of observation. this was accompanied by a decrease in the number cd3 + lymphocytes expressing the markers of late activation (cd3 + hladr+ 4.8 ± 1.09x10 9 /л и 3.2 ± 2.04x10 9 /л). revealed significant decrease of cd4 + cd25 + foxp3 + regulatory cells in the first week after returning from the area of adverse climatic conditions, as well as a significant sustained decrease in the number cd3 + cd8 + hladr+(p < 0.05). change of the effector link of innate immunity was determined in significant reliable decrease in relative (cd16 + 13.2 ± 2% and 4.6 ± 2.1%, respectively, p < 0.05) and absolute (cd16 + 0.3 ± 0.02 x109/l and 0.1 ± 0.03%, respectively, p < 0.05) in the number of a population of natural killer cells in the first week of observation. in the context of acute stress marked a significant increase in relative and absolute numbers of b lymphocytes (8 ± 1.16% (0.15 ± 0.04 × 109/l) before a trip to countries with a hot climate and 19 ± 3.1% (0.4 ± 0.07 × 109/l) in the first week after returning, p < 0.05). the activity is the production of antibodies was not changed. (ast) which is the intramuscular injection of patients own serum, is a promising therapy with a substantial efficiency on ciu patients. in this study we aim to assess the efficacy of ast on chronic urticaria patients by dlqi questionnaire. method: this was a single-blind randomized clinical trial which evaluated the efficacy of autologous serum therapy compared to oral antihistamines in patients with ciu. 49 ciu patients received the ast. every session 5 cc of each patient's blood was centrifuged at the speed of 2000 rpm for 10 minutes and 2.5 cc of the serum was injected intramuscular into the patient's deltoid muscle weekly for 9 weeks. the control group consisted of 51 ciu patients took 10 mg of cetirizine daily for 9 weeks. patients answered the dlqi questionnaire at the first session of treatment as baseline and 7 weeks after the last session(week 16) as response to treatment. the mean baseline score of dlqi for ast group was conclusion: pharmacotherapeutic and inpatient costs for patients with prevalent ar and asthma were lower in those prescribed ait than in those not prescribed ait in all years, both with and without including the cost of ait itself. this indicates that treatment with ait is associated with lower cost burden for health services. background: immunotherapy with peptides rather than conventional whole allergens is being developed to improve the benefit/risk balance of subcutaneous immunotherapy (scit). lolium perenne peptides (lpp) demonstrated reduced allergenicity following ex-vivo analyses, allowing higher doses to be given over a shorter period to improve treatment adherence and compliance. such treatment resulted in significant reduction in symptoms and rescue medication intake during the grass pollen season. here we report the safety of lpp immunotherapy in adults. background: a new allergoid from alternaria alternata was characterized to determine its reduced allergenicity in vitro. the objective of this study was to determine the skin response to the allergoid and to evaluate the clinical tolerance of the immunotherapy with the allergoid product using a rush schedule. method: to assess the skin response (sr) two groups of patients were included: group 1 with patients sensitized to a. alternata and with respiratory disease caused by this mold; group 2 (control) with patients sensitized to others allergens and non-atopic patients. the sr was determined by spt using three concentrations of the allergoid: p1 (lowest concentration), p2 (four times higher than p1) and p3 (estimated to obtain a wheal area similar to histamine 10 mg/ml). in spt was also used a native extract of a. alternata (n) and histamine 10 mg/ml (h). all products were tested in duplicate in all patients and the sr was evaluated by comparing the median of the wheal area produced by different products. to evaluate the clinical tolerance to immunotherapy the patients of group 1 were treated with the allergoid product using a rush schedule consisting in a dose of 0.2 + 0.3 ml the first day and 0.5 ml after one month (maintenance dose). the clinical tolerance was determined as the percentage of adverse reactions (ar) to the treatment and the classification of ar was established according to eaaci. the number of patients included to evaluate the sr was 46 (group 1: 25; group 2: 21, 16 atopic and 5 non-atopic) with an average age of 34.8 (range . the spt data from group 1 were expressed as median and interquartile range of wheal area (mm 2 ): h: 19.58 (15.3-25.2); n: 22.71 (11.1-33.1); p1: 0.99 (0-5.7); p2: 6.44 (0-12.5); p3: 19.78 (12.0-30.4 ). it was determined that sr of allergoid was reduced in 87% respect to the native. the products n, p1, p2 and p3 did not produce any response in patients of group 2. to evaluate clinical tolerance, 21 patients of group 1 were treated with the allergoid product with a rush schedule and only two ar were registered (3.2% of doses). these were retarded local reactions with a wheal diameter higher than 5 cm. no systemic reactions were registered and all patients continued the treatment. the allergoid from a. alternata produces a significant reduced response to spt due to its reduced allergenicity. the treatment with an allergoid product in a rush schedule is safety and clinically well tolerated. background: in our study we aim to determine the more effective, the total cost of 3 years of patients using scit was 15665 tl per person whereas the total cost of 3 years of patients using slit was 15271 tl per person. when we compare the total cost data of both groups, we found that they are close to each other. while the greatest portion of the cost data of patients with scit treatment was direct costs associated with the treatment itself (72%); the remaining part of the total cost was indirect (16%) with non-medical expenses such as transportation (12%). in the slit group, direct costs including drug expenditures have a larger percentage (92%) and it was significantly more costly compared to the direct costs of the scit group (72%). transportation costs were found to be more costly in the scit group (12%) when compared to the slit group (4%). similarly loss of parent work days in the scit group(%16) was found to be significantly more expensive compared with slit group (4%). our study results show that slit is a similar treatment for clinically and laboratorially and has a similar efficacy to scit to reduce the patients' complaints and to the need for medication. for cost-effectiveness however medicines for treatment of scit are less costly; when long term total treatment costs are calculated slit and scit treatment are economically close treatments. the protein content of the new acd was 182.28 μg/mg and the protein profile in sds-page and sec-hplc confirmed the presence of proteins with high molecular weight and the absence of smaller proteins. the content of free lysine in acd, involved in glutaraldehyde modification, was reduced in 91.96% respect to ncd and it can be considered as the polymerization degree. regarding to the allergenic profile, through elisa inhibition was determined a reduction of 18 times in the capacity to bind ige of the proteins in acd respect to ncd, whilst the igg binding capacity was maintained. in immunoblot there was no reaction of acd proteins to specific ige from sera. the analysis by peptide footprint determined the presence of fel d 1 and others allergens in acd. the content of major allergen fel d 1 in acd was determined as 11.9 μg/mg. the new developed and characterized allergoid from cat dander has an excellent safety profile and will allow a safer immunotherapy to treat the allergy to felis domesticus. results: the protein content of the new aaa was 65.6 μg/mg and the protein profile in sds-page and sec-hplc confirmed the presence of proteins with high molecular weight and the absence of smaller proteins. the content of free lysine in aaa, involved in glutaraldehyde modification, was reduced more than 85% respect to naa and it can be considered as the polymerization degree. regarding to the allergenic profile, in immunoblot there was no reaction of aaa proteins to specific ige from sera and by elisa inhibition was determined a reduction of 91% in the capacity to bind ige of the proteins in aaa respect to naa. the igg binding capacity in aaa was maintained. the analysis by peptide footprint determined the presence of alt a 1 and others allergens in aaa. the content of major allergen alt a 1 in aaa was determined as 2.4 μg/mg. a. alternata shows an excellent safety profile and allows a safer immunotherapy to treat the allergy to this mold. she was an otherwise healthy woman: she took no drugs and she did not have any remarkable concomitant diseases. the distribution and appearance of the remaining body hair was normal and the hormonal level profiles (lh, fsh, estrogens, progesterone and testosterone) did not show any significant alteration according to her age. a 30 year old woman with allergic rhinitis underwent sq glutaraldehyde-modified ait to house dust mites (d pteronyssinus and g domesticus) without any incidences and complete tolerance to maintenance dose without local reactions during a 5 year period. two years after ait discontinuation, patient first experienced a local urticarial reaction with multiple hives at previous sq ait injection sites 40 minutes after 600 mg of ibuprofen intake. these symptoms recurred at least in seven occasions when patient was exposed to ibuprofen (in five) and metamizol (in two). results: case 1: dermatologist diagnoses localized hypertrichosis. case 2: a single blind, placebo controlled oral challenge (sbpcoc) with ibuprofen 600 mg was performed and elicited multiples hives in the circumscribed area in the arm where ait was conducted. subsequently, sbpcoc with aspirin was carried out showing the same reaction although a controlled challenge with celecoxib was negative. conclusion: local hypertrichosis is a very rare injection-disease associated with injected allergen vaccine treatment. we also firstly described a recall urticaria phenomenon after allergen immunotherapy which has been only elicited after different nsaids intake. results: there were included 47 patients, in five spanish hospitals. following aria 2010 guidelines, 95.7% of patients were diagnosed of persistent moderate/severe rhinitis. the mean age was 37.7 ± 11.8 years, being 59.6% female. moreover, 57.4% of the patients had concomitant mild/moderated asthma. the period between the diagnosis of rhino-conjunctivitis and the informed consent signing was 9.8 ± 7.5 years. according to international 2006 guidelines, eight systemic reactions were registered, representing 1.9% of the administered doses: five reactions grade 0, (described as nonspecific ocular pruritus, nasal herpes, general discomfort, localized non-specific pruritus plus nausea and non-specific pruritus in throat), a grade i reaction described as rhinoconjunctivitis and two reactions grade ii, registered as generalized urticaria and asthma. all reactions were classified of mild or moderate intensity and only two required symptomatic treatment. there were five clinically significant delayed local reactions, which were higher than 10 cm or involved modifications in next dose. regarding efficacy parameters, immunoglobulin titers between baseline and final visit according to specific igg and igg4 significantly increased. cutaneous reactivity also decreased significantly in the dose response skin prick test. results: 47 patients were included, 24 to accelerated and 23 to polymerized cluster group schedules. according to aria criteria, 89.4% of patients presented persistent moderate/severe rhinitis. the mean age was 31.1 ± 9.9 years, being 40.4% male. moreover, 61.7% had concomitant mild/moderated asthma. immunoglobulin titers method: the quantification of total proteins in the products was carried out by means of a colorimetric technique using the bradford reagent (sigma-aldrich™, us) in accordance with the manufacturer's instructions. the absorbances of each standard and samples were obtained in a scinco™ s-3100 spectrophotometer (seoul, korea) at 595 nm. all samples were analyzed in duplicate. the electrophoretic profile of the proteins in the tested allergens was obtained according to the procedure described by laemmli, under denaturing conditions in a polyacrylamide gel at 12.5% concentration and stained in silver. in each lane approximately 30 μg of total proteins were applied. commercial extracts of the main allergens marketed in mexico were obtained, rossel ® , alk ® , alerquin ® , alergomex ® , allerstan ® , ipi asac ® ; and they were assigned randomly with the numbers 1, 2, 3, 4, 5 and 6. results: the following protein concentrations were found in the various extracts analyzed: see table 1 conclusion: differences were found in the protein profiles anabackground: a new allergoid from cat dander was developed and characterized to determine its reduced allergenicity in a 95% and the maintenance of igg binding capacity. the objective of this study was to develop an immunogenicity assay in mice with the new allergoid and a native extract from cat dander. the study included 24 female balb/c mice separated in three groups of 8 mice each: group 1, immunized with a mold allergen extract (control); group 2, immunized with a native extract from cat dander with a fel d 1 content of 0.525 μg per dose; group 3, immunized with the new allergoid from cat dander with a fel d 1 content of 2.36 μg per dose. all mice were immunized four times by subcutaneous injections with a volume corresponding to 1/10 of the recommended human maintenance dose with an interval between injections of 2 weeks. one week after the last injection the mice were sacrificed and the serum was obtained. to determine the specific antibody title indirect elisa were performed using a cat dander extract as antigen, sera from mice as primary antibody and antimouse igg or igg1 as secondary antibody. elisa assays were performed using serial dilutions of sera or a simple dilution by duplicate to determine the specific antibody title as arbitrary units/ml (au/ml). the data were analyzed by one-way anova and tukey hsd test to compare the averages of specific antibodies in each group. results: the immunization with both the native extract and the allergoid from cat dander produces specific igg and igg1. regarding to igg, a higher title was observed in group 3 respect to group 2 in a curve obtained after elisa with serial dilutions of sera. the specific igg title obtained in terms of au/ml was 18.6 ± 2.6 in group 1, 105.0 ± 15.0 in group 2 and 139.9 ± 16.6 in group 3. concerning to igg1 the au/ml obtained was 5.7 ± 0.4 in group 1, 73.5 ± 16.8 in group 2 and 97.5 ± 18.8 in group 3. the increase of specific igg or igg1 in mice from group 3 respect to mice from group 2 and control group was statistically significant (p ˂ 0.01). the safety profile of the allergoid from cat dander allows a treatment with higher dose of allergens to produce a greater response to immunotherapy to induce formation of specific this was an open, multicenter clinical trial, in patients aged between 18 to 60 years with rhinoconjunctivitis with or without concomitant mild asthma sensitized to house dust mites (hdm). the aim was to evaluate the safety and tolerability of the vaccine. secondary endpoints included were: changes in immunoglobulin levels (specific ige, igg and igg4) versus d. pteronyssinus and d. farinae and changes in cutaneous reactivity. patients were under study treatment for 17 weeks: five for the induction phase (weekly injections) and 12 for the maintenance phase (monthly injections). results: 42 patients were included. there were 6 withdrawals from the trial; no one was related to treatment. the patients mean age was 33.6 years, being 50% female. 71.4% were diagnosed of persistent moderate/severe rhinitis according to aria guidelines and 31.0% presented concomitant mild asthma. regarding to safety results, 22 systemic adverse reactions were registered which corresponded to 7.9% from a total of 277 administered doses. the most of systemic reactions were grade i, (5.4%) described as rhinitis or urticaria, grade 0 or nonspecific (2.2%) and 1 reaction (0.3%), was grade ii. all of them were mild or moderate and only 4 needed treatment. among local reactions, 21 (7.6%) were clinically relevant late local reactions, meaning a wheal at injection site >10 cm and /or requiring a dose readjustment in the next administration; 6 (2.2%) were clinically relevant immediate local reactions meaning a wheal >5 cm. concerning the efficacy parameters, cutaneous reactivity at the final visit versus baseline was, in average, significantly decreased, and specific titers of igg and igg4 against tested hdm increased significantly at final visit. 162 patients completed the study. mean values in rqlq questionnaire (total score) decreased from 2.56 to 1.24 points (51.6% score reduction) in final visit, reflecting a statistically significant improvement (p < 0.01). annual episodes of rhinoconjunctivitis decreased from 13.7 to 9.7 (p < 0.01). 43.8% of patients improved from persistent to intermittent rhinoconjunctivitis (p < 0.01) and 46.9% from moderate/severe to mild intensity (aria) (p < 0.01). moreover, 16 .1% of asthmatic patients at baseline, did not have any bronchial symptoms after 1-year treatment (p < 0.01). mean value of treatment satisfaction was 7.2 (sd=1.8) and 7.2 (sd=1.7) for patients and physicians respectively. 0884 | evaluation of safety and tolerability of "allergovac poliplus" in polysensitized patients with allergic rhinitis-rhinoconjunctivitis with or without asthma: an observational prospective study (apolo) background: the objetive of this study was the safety and tolerance assessment of "allergovac poliplus" scit treatment, with 2 allergen combination-mixtures in polysensitized patients, as well as the evaluation of the clinical improvement and patients' satisfaction after treatment. method: this is a prospective observational clinical study. allergovac poliplus treatment is being administered in a "1-day" or in an abbreviated schedule. polysensitized patients (to pollens or mites), with rhinitis or rhinoconjunctivitis, with or without asthma, and between 5-60 years have been included. all adverse events are being recorded. visual analog scales (vass) are being used to evaluate clinical improvement, tolerance and satisfaction after treatment (10 months). results: a total of 147 patients have been included, with an averresults: in all groups prevailed severe forms of the disease and the phenotype of frequent exacerbations. groups were comparable in age composition and structure of severity. observations in the group of vaccinated pcv13 continue the dynamics of decreased dyspnea up to 1.66 (1.11;2.21) results: of a total of 631 pts under scait, 110 were excluded due to data unavailability, and 521 included (♀283 (54%), mean age 32 ± 13 years (minutes: 7 max 73 md30), age range [18-30] being most prevalent (40%). the most frequent diagnosis was rhinitis/rhinosinusitis (97%), followed by asthma (43%), diagnosis coexisting in abstracts | 479 214 pts (41%). other diagnosis such as conjunctivitis (24%), atopic eczema (15%) and food allergy (9%) were also found. mite sensitization occurred in 422 patients (81%) of which 199 (47%) were monosensitized. the pollen sensitization was verified in 288 (55%) with 88 monosensitized pts (31%). the double sensitization mitespollens was displayed in 189 (36%). sensitization to epithelia and fungi occurred respectively in 100 (19%) and 42 pts (8%). it was found that 20 pts (4%) presented sensitization to the 4 groups of allergens (mites, pollens, fungi, dander). an average of 58 ± 23 pts started this treatment per year. prescription included 10 laboratories with the following %: a-39.6; b-29.3; c-13.2; d-5.4; e-4.7; f-4.2; g-2.5; h-0.9; i-0.1; j-0.1. option for extract of physical modification (5%), physical-chemical (16%) and chemical (79%). table 1 shows the frequency of distribution of scait composition. conclusion: in this population sensitization to mites was predominant being the most prescribed scait followed thru sensitization to grasses with the respective scait. the majority of the population was polysensitized. however, in composition preference the choice of 1 group of allergens prevailed and only 5% had more than one sort of pollen and 4% pollen+mites. polysensitization is a reality, nonetheless the choice of ait composition should be guided thru scientific criteria and not through the availability of mixtures encouraged by laboratories. background: allergen immunotherapy (ait) has been proven to be an effective treatment of allergic diseases in numerous studies. however, its use in seniors remains limited and questionable, due to common comorbidities and limited evidence of efficacy and safety of ait in aging population. the aim of presented study was to assess the safety of ait in patients over 55 years of age undergoing subcutaneous immunotherapy (scit) and analyze the potential risk factors of adverse reactions in this population, compared to younger adults. we followed subcutaneous immunotherapy in a group of 1302 patients treated in the outpatient clinic of medical university of lodz, of whom 163 were aged 55 and older (118 between the age of 55-60, 31 aged 61-65 and 14 patients above the age of 65). we recorded detailed information of each administration and corresponding adverse reactions over the period of 2 years. we compiled results of our observations with patients' medical records to compile a database, which we then analyzed using statistical software. method: a total of 46 cases with seasonal allergic rhinitis undergoing pre-seasonal immunotherapy and 28 cases followed with conventional drug treatment were included in the study. immunotherapy and control groups were divided into monosensitized (only pollen) and polysensitized (at least 1 additional allergen except pollens) patient groups according to skin prick test reactivity. all patients were followed between march-september 2013 with symptom and medication scores, and visual analogue scale (vas). the quality of life was assessed using the mini-rqlq questionnaire. phleum pratense (phl p) specific ige and specific igg4 (uni-cap 100, phadia) measurements were performed before and after 7 weeks of immunotherapy in all patients. gramineae pollens were counted during the grass pollen seasons. results: mean age was 34.9 ± 10.6 and 34.2 ± 12 years, female/ male ratio was 29/17 and 17/11, the number of monosensitized/polysensitized patients were 37/9 and 20/8 in immunotherapy and control groups, respectively. in the immunotherapy group, june-july symptom scores, may-june-july-august vas scores and june combined symptom-medication scores were lower than the control group (p = 0.005). furthermore, improvements in activities-practical problems and other quality of life scores were significantly different between two groups (p < 0.05). in immunotherapy group, phl p specific ige and phl p specific igg4 levels measured after immunotherapy were significantly higher compared to those before immunotherapy (p < 0.001, p < 0.001, respectively). phl p specific igg4 levels measured after immunotherapy were also significantly higher in the immunotherapy group than in the control group (p < 0.001). there was no difference in terms of clinical and immunologic parameters in monosensitized and polysensitized patients (p > 0.05). conclusion: clinical improvement with pre-seasonal allergoid immunotherapy is accompanied by an important increase in specific igg4 blocking antibodies despite short-term injections. our findings show that pre-seasonal allergoid immunotherapy has similar clinical efficacy and b cell response in polysensitized subjects compared to monosensitized patients. 0891 | the safety trial of sequential sublingual immunotherapy with japanese cedar droplet and house dust mite tablet matsuoka t 1 ; kuroda y 1 ; igarashi s 1 ; fukano c 2 ; natsui k 2 ; ohashi-doi k 2 ; masuyama k 1 1 university of yamanashi, yamanashi, yamanashi, japan; 2 torii pharmaceutical co. ltd., tokyo, japan background: sublingual immunotherapy (slit) is recognized as the only treatment option with the potential to provide long-term posttreatment benefits. in japan, the prevalence of japanese cedar (jc) pollinosis is very high, about 30% of the population, of which the majority are co-sensitized to hdm. slit is now well established, safe and convenient treatment form for allergic disease, and recently, jc slit-droplet and hdm slit-tablet products were approved in japan for treatment of jc and hdm induced allergic rhinitis, respectively. however, the safety of sequential jc slit-droplet and hdm slittablet has not yet been investigated. therefore, we investigated the safety trial on slit combined with jc droplet and hdm tablet in allergic patients. method: eleven subjects with jc pollinosis and hdm rhinitis were enrolled. patients were treated once-daily with jc slit-drops for 4 weeks, followed by 24 weeks of sequential slit treatment where the jc slit-drops and the hdm slit-tablets were administered daily with a 5 minute interval (1st: jc-slit drops, 2nd: hdm slit-tablet). the primary endpoint was the frequency and severity of adverse events (aes) during sequential slit by common terminology criteria for adverse events (ctcae) v4.0 and slit grading system. serum antibodies were measured as the secondary endpoint. results: eleven patients were recruited. aes after jc slit-drops administration were found in 9 patients out of 11 cases (82%). aes after sequential slit were found in 8 patients out of 10 cases (80%). all aes were graded 1 or 2. no severe aes were observed during the study period. the levels of jc-and hdm-specific ige and igg4 in serum were increased during treatment. conclusion: sequential-administration of jc slit-drops and hdm slit-tablets was well tolerated by patients suffering from both jc pollinosis and hdm rhinitis. background: according to the ema guideline on the clinical development of products for specific immunotherapy products should be tested in phase ii at different doses in several study-arms to establish a dose-response relationship for clinical efficacy before confirmatory trials can be initiated. allergen exposure in an aec may be used as primary endpoint. the study was a single-center, randomized, double blind, placebo-controlled, phase ii trial, treatment duration 10 months. 168 grass pollen allergic patients (18-65 years of age) with seasonal rhinitis/rhinoconjunctivitis (arc) with (mild, gina i) or without concomitant asthma were randomized to three different dosages of a liquid phase iii study is in preparation. as part of an effort to prepare the analysis plan using the csms as primary endpoint, the grass pollen data of the european aeroallergen network (ean) was used to identify the window within the grass pollen season (gps) with optimal correlation between the grass pollen counts and the csms. method: ean currently includes information from more than 400 active and 300 historical pollen-monitoring stations in europe including 39 countries. the ean database used for analysis included grass pollen data collected during 2009-2016. the daily allergy symptoms and medication were recorded spontaneously using an app questionnaire on the subject's smart phone. the csms was re-calculated using the ean database, using the recorded symptom scores with estimation of the medication score using similar methods as recently published. the correlation between the daily grass pollen count and the daily csms was analyzed with a mixed effects model accounting for patient-specific correlations and symptom levels. conclusion: these results confirm a statistically significant correlation between grass pollen counts and the csms. importantly, these findings suggest that the optimal window to observe treatment effects after immunotherapy may be a short interval after start of the gps and during the peak gps, due to generally higher csms values. this provides sufficient basis to consider additional sensitivity analyses to evaluate the treatment effect of grass mata mpl scit on the primary csms endpoint during a shortened window after the start of the gps and to consider excluding the overlapping period between the bps and gps from the primary analysis. 0894 | combo-vas as a tool to assess efficacy of allergen immunotherapy ciprandi g 1 ; silvestri m 2 ; olcese r 2 ; tosca ma 2 1 ospedale policlinico san martino, genoa, italy; 2 istituto g. gaslini, genoa, italy background: allergen immunotherapy (ait) is at present the unique cure for respiratory and venom allergy. usually, ait lasts for some years, but its efficacy is longstanding. criteria for assessing ait efficacy are mainly based on symptom severity improvement and saving of symptomatic medications. in this regard, there are different score grading for both measuring symptom severity and drug use. visual analogue scale (vas) is a well-defined and validated method widely used in many diseases, including allergic disorders. vas is a psychometric tool measuring the patient's perception of symptoms, emotions, pain, drug use, etc. recently, it has been published an eaaci position paper concerning the recommendations for the standardization of clinical outcomes used in ait trials for allergic rhinoconjunctivitis, but it is complex. so we would propose a simpler way to measure ait efficacy by vas, in particular a combo-vas based on one vas for symptom and one for medications. results: globally 150 patients were retrospectively evaluated. all of them were treated with a 3-year ait course: 120 were defined as responders and 30 as non-responders. in responders group the combo-vas mean value was 14 (iqr 12-15) at baseline and 4 (iqr 3-6) after ait treatment. in non-responders group combo-vas mean value was 13 at baseline and 11 (iqr 9-12.5) at the end of ait. the difference was significant (p = 0.012). the d combo-vas was −66.67% in responder group and −10% in non-responders group (p < 0.0001). conclusion: combo-vas, i.e. the sum of vas for symptoms and medications, could be an easy and quick tool for assessing ait efficacy and reflects the patient's perception. therefore, it could be very fruitful in clinical practice. 0895 | rapid up-dosing in sublingual specific immunotherapy is safe, well-tolerated and effective in patients suffering from tree pollen allergic rhinitis background: an optimised up-dosing period of specific immunotherapy (sit) is desirable for better patient compliance because a long or complicated up-dosing scheme is sensitive to disruption. the aim of this study was to compare the safety, tolerability and effectiveness of an optimised up-dosing scheme with two preexisting schemes of sublingual sit (slit) in patients under standard medical care. method: this was a prospective, open, active controlled, multi-center non-interventional study in germany and austria to document the treatment of children and adults with allergic rhinoconjunctivitis and/or allergic asthma treated with a slit containing purified, aqueous extracts of birch, alder and hazel pollen. the investigators were free to select an up-dosing scheme for included patients: scheme a consisted of an up-dosing period of up to 12 days at the patient's home using three different solution strengths to reach the maximum dose; ultra-rush scheme b performed only with the highest solution strength at the physician's office within 2 hours, and the optimised scheme c which was initiated at the physician′s office and continued at home using exclusively the highest solution strength within 2 (long-term) or 4 (pre-seasonal) days. data on up-dosing and maintenance treatments were documented by physicians during 5 patient visits and by patient diaries. the study was approved by ethic committees, and all patients or parents gave their informed consent. results: in total, 164 patients aged 3-76 years were included into this study. scheme a was applied by 90 patients, 29 patients decided on regimen b, and 45 patients on the optimised scheme c. conclusion: one-day ur-scit conducted in an outpatient clinic was safe and well-tolerated in patients with ad sensitized to hdm. ur-scit can be a safe and useful option to start a subcutaneous allergen immunotherapy for ad. 0899 | factors affecting on adherence to allergen specific immunotherapy results: among 1162 enrolled patients, 228 (19.6%) patients failed to complete at least 3 years of ait, which were regarded to be nonadherent in this study. univariate analysis revealed that male, younger age group less than 40 years, cluster and ultra-rush schedules, atopic dermatitis, the absence of associated diseases, and follow up of other department were found to be associated with nonadherence to ait. in multivariate analysis, younger age group less than 40 years (or 2.31, 95% ci 1.55-3.45), cluster (2.37, 1.56-3.60) and ultra-rush schedules (6.03, 3.18-11.42) , and absence of follow up of other department (2.11, 1.31-3.40) were independently associated with non-adherence to ait. no association was found in gender, diagnosis of allergic diseases, kind of allergen extracts, and patients' distance from hospital. conclusion: various factors are related with ait non-adherence to interfere the effectiveness of immunotherapy. clinicians need to be aware of the factors associated with non-adherence to ait and consider them when choose to maximize ait adherence. 0900 | cost-effectiveness of allergen immunotherapy to grass in patients with allergic rhino-conjunctivitis and asthma background: allergen immunotherapy (ait) has been shown to reduce symptoms and medication use in subjects with rhino-conjunctivitis and asthma. however, long-term cost effectiveness of this therapy needs to be evaluated. our aim was to assess cost effective of ait, both subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit), vs. pharmacotherapy alone in subjects with rhino-conjunctivitis, with or without allergic asthma, to grass pollens. method: a markov cohort state-transition model with a time horizon of 9 years was used to assess the costs and effects of 3-year ait in adults. relative efficacy of the treatments expressed as standardized mean difference was estimated using an indirect comparison on symptom and medication score extracted from available meta-analyses. the rhinitis symptom utility index was used as a proxy to estimate utility values for symptom score. the societal perspective, through the human capital technique, was used to estimate indirect costs, to represent the scenario of a country with nationalized medicine. data on drug and other medical costs were derived from published sources as well as ait duration and asthma occurrence. additional sensitivity analyses were performed to test the robustness of our results. results: in the base case analysis, using italy clinical practice patients with moderate-to severe allergic rhino-conjunctivitis (ss ranging from 6 to 15 points) and a mean age at entry of 21 years, both scit and slit were associated with increased cost but superior efficacy compared to pharmacotherapy alone. the results were most sensitive to variation in efficacy estimates and ait persistence rates. conclusion: this analysis suggests that ait is cost effective relative to pharmacotherapy alone. scit, despite significantly higher indirect cost burden, seems to be the most cost effective option. the results should be interpreted in the context of the data input and modelling assumption used. 0901 | ielisa as a tool to measure ige binding towards single modified peanut allergens background: immunotherapy has shown to be a potential treatment for food allergies but needs further research to improve safety. modification of peanut allergens to reduce their allergenicity is a promising approach to develop a safe and effective immunotherapy as shown by the successful completion of a first-in-human safety and tolerability study using hal-mpe1 in adult patients with peanut allergy (eudract 2013-004238-13) . in order to assess the impact of modification on individual peanut allergens and to assess its impact on ige binding by individual patient sera, we have developed peanut allergen-specific inhibition elisas. with this methodology we are able to identify patients with residual ige binding to modified peanut allergens. method: ige inhibition elisas (ielisas) were developed and performed to test ige binding towards purified ara h2 and ara h6 and their reduced and alkylated (modified) versions, using the individual responses of single patient sera. results: ara h6-specific ielisas showed that modification of ara h6 results in >95% reduction in ige-binding for all individual sera tested. ara h2-specific ielisas showed that modification of ara h2 also results in >95% reduced ige-binding for most of the sera, but some sera were identified which showed residual, 10%-20% ige binding to mara h 2. in some of the latter sera, the presence of ige binding to a linear hydroxyproline-containing peptide could be confirmed as a possible source for the residual ige binding to mara h2. we have developed a methodology to assess residual ige binding to modified peanut allergens. the sensitivity of the allergen-specific ielisas allowed us to discriminate between patient sera in which ige binding to mara h2 and to mara h6 was virtually completely absent and sera in which 10-20% residual ige binding to ara h2 was observed. the clinical importance of these observations is yet unknown. future clinical studies will need to reveal whether the patient-specific ige binding profiles to individual modified peanut allergens do correlate with the adverse events profile of immunotherapy with modified peanut extract. 0902 | design of a phase ii allergen immunotherapy study to determine the optimally effective and safe dose of subcutaneously administered tyrosine adsorbed modified grass allergen+mpl (mpl) adjuvants for the treatment of allergic rhinoconjunctivitis (arc) due to grass pollen. there is increasing evidence that the effectiveness of allergy immunotherapy to control arc symptoms is related to the cumulative allergen (or allergoid) dose administered. previously, two clinical studies have been conducted using a conjunctival provocation test (cpt) as primary efficacy measure for a similar scit mata mpl product for birch allergy [eudract 2012-004336-28 and 2015-000984-15] . these studies showed a 5.5 fold increase in cumulative dose to achieve~50% increase in efficacy, with a relative reduction in total symptom score (tss) of 32.3% compared to placebo and no safety signals of concern. the shape of the dose response curve was curvilinear, where this high dose almost reached plateau. method: this is a multi-center (~47 clinical study centers across europe), randomized, double-blind, placebo-controlled, parallel-group study in~440 adult patients with moderate to severe seasonal arc with or without mild asthma. a positive cpt is to be achieved at screening and verified prior to randomization. the primary outcome is the post-treatment tss following cpt. a wide range of cumulative dose regimens is used (5100, 14400, 27600 and 35600 su) applied over 6 weekly injections to establish the shape of the dose response to support dose selection for phase iii. the design of the current phase ii grass allergoid scit study will be discussed, including the rational of using 4 cumulative dose regimens and placebo and the pre-selected shapes of the dose response curves. in addition, the number of patients screened and randomized will be presented by country, gender and/or age category and screen failures will be categorized. conclusion: this phase ii study was initiated to establish the dose response of a grass mata mpl scit product, using cpt to measure the effect of a wide range of cumulative dose regimens. the achievement of its aim will be an important milestone in the development of an efficacious and safe state-of-the-art grass scit. conclusion: we observed that the specific nasal challenge with house dust mite generates an inflammatory response within the first hours, but we did not demonstrate any correlation with the response to immunotherapy after six months. 0905 | tolerability of a two week rush updosing with modified allergens in pollen allergic subjects in the day-to-day practice background: in two phase iv studies the tolerability of a subcutaneous rush up-dosing, using three injections in two weeks, has been tested and proven to be save in adults. in the course of a non-interventional study (nis) now the tolerability of this treatment scheme was tested in the day-to-day practice. conclusion: over 97% of the patients could reach the highest dose of 0.5 ml. the overall tolerability is very good. the data from daily practice confirm the data that were previously obtained in two phase iv studies. siges from 40 patients, evaluated during the 1st semester of 2017 at an outpatient clinic. all patients presented persistent moderatesevere allergic rhinitis, in pollen season and had not been submitted to it. all patients had positive spt for grasses (grass) and olive (olea). sige-tot for phleum pratense and olea europaea and some sige-crd (rphl p 1, rphl p 5, rphl p 7, rphl p 12, role1 and nole7) were determined. physicians were divided into 2 groups (group 1 if <10 years of practice and group 2 if≥10 years of practice) and were asked to choose which it to prescribe for each patient (none, only grass, only olive or both grass and olive), according to spt and sige results. results: fifteen physicians (60% with ≥10 years of practice) participated in the survey. considering only the sige-tot results, the it choice (group 1/2) was: no vaccine in 10%/3%; grass vaccine 50%/ 58%; olive vaccine 5%/10% and both grass and olive vaccines in 35%/30% of the patients (p = 0.42), the intergroup agreement was 70% (kappa 0.612). according to the sige-crd results the physicians chose (group1/2): no vaccine at 10%/13%; grass vaccine in 63%/60%; olive vaccine in 10%/5% and both vaccines in 18%/23% of patients however, data on control of allergic rhinitis (ar) after discontinuation of therapy are insufficient. the aim of our study was to assess sustained control of ar in three consecutive years after grass-pollen slit discontinuation. method: a total number of 35 patients [24 (68.57%) males; mean age 32 years, age range 15-56] well-controlled after a three-year course of slit with grass pollen extract were prospectively evaluated in three consecutive years after discontinuation of therapy. conclusion: a three-year course of grass-pollen slit seemed to have a long-term effect on control of symptoms in patients with ar. the authors declare no conflict of interest. results: all patients showed a positive sensitization profile by skin prick test to either betula and/or alnus. in 40% of patients this profile was furtherly confirmed with serum specific ige levels to betv1, (mean 13.56 ku/l). allergic symptoms in patients with birch/alnus pollen allergy after ingestion of certain food can result from crossreactivity of bet-v1-specific ige to homologous pathogenesis-related proteins, particularly the pr-10 protein. conclusion: within the allergy history we emphasize on focusing on sao symptoms as many patients under-recognize them. among the sensitization profile of these patients it is quite important to highlight cross reactivity between bet v1 and alnus. the other patient suffered from anaphylaxis(grade ii) induced by minimum amount of lettuce consumption without co-factors. both patients suffered from oral allergy syndrome to peach and allergic rhinitis. spts to foods and pollens were performed with commercial extracts, prick-through-prick with fresh plant foods, while specific ige was determined accordingly. ltp syndrome was defined as a sensitization to pru p 3 and symptoms elicited by at least 2 unrelated plant foods. co-factors were also investigated. results: the first patient was sensitized to lettuce, peanut, hazelnut, sunflower's seed, peach and banana, and plane tree, olive tree, grasses, parietaria and mugwort. sige to lettuce was 2.04kua/l, to pru p 3 was 20.8kua/l and total ige was 703.4u/ml. co-factors, such as exercise, were involved. the second patient was sensitized to peanut, walnut, hazelnut, almond, sunflower's seed, cashew, lettuce and peach, and, plane tree, olive tree, grasses, parietaria, mugwort and willow. sige to lettuce was 27.6kua/l, to pru p 3 was 37 and total ige was 1705.9ku/l. no co-factors were identified. background: garlic (allium sativum) is a vegetable that belongs to amaryllidaceae's family. hypersensitivity to garlic is not very common. it has been mainly reported in occupational allergy but it also may cause contact dermatitis, rhinoconjunctivitis, asthma, urticaria, gastrointestinal symptoms and anaphylaxis after its ingestion. some studies have identified alliin lyase, a 56 kda protein, as a major garlic allergen and it seems to be a heat-sensitive allergen. we report on a 9-month-old infant who presented, 15 minutes after an accidental ingestion of garlic sauce, generalized erythema and cough. she was still breastfeeding and she had never abstracts | 491 eaten garlic before (although the mother usually consumed garlic). the patient had never tasted other vegetables belonging to amarylidaceae's family either but zucchini, with good tolerance. we performed skin tests and specific ige (sige) to different vegetables. a raw garlic extract was also carried out and analysed in the patient by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). results: prick by prick with garlic was positive (10 mm) and negative to onion, leek, asparagus, zucchini and saffron. skin prick tests to commercial extracts of mugwort, grass pollen, peach ltp and profilin were negative as well. specific ige to garlic was 3.15 ku/l (out from a total ige 32 ku/l) and 0 ku/l to onion and asparagus. sds-page immunoblotting assay with patient′s serum revealed ige reactivity with proteins of 6 kda and 8 kda. conclusion: we report a garlic ige-mediated anaphylaxis case in an infant with proteins of 6 and 8 kda as the relevant allergens. the mechanism of sensitization in the present case remains unclear. the authors hypothesized that breastfeeding, cutaneous contact or inhalation might be possible mechanisms involved. chong kw 1 ; saffari se 2 ; chan n 3 ; seah r 3 ; tan ch 3 ; goh sh 1 ; goh a 1 ; loh w 1 1 allergy service, department of paediatric medicine, kk women's and children's hospital, singapore, singapore; 2 centre for quantitative medicine, office of clinical sciences, duke-nus medical school, singapore, singapore; 3 yong loo lin school of medicine, national university of singapore, singapore, singapore background: the predictive decision points for both peanut skin prick test (spt) wheal size and serum ige concentrations, in peanutsensitized children, have not been evaluated in singapore. we aim to assess these for purposes of risk stratification and prediction of oral food challenge (ofc)s' outcomes by means of a retrospective chart review. results: the number of patients evaluated was 328, of which 269 had clinical diagnosis of peanut allergy based on recent immediate reaction to peanut (pa group) and 59 were tolerating peanuts regularly (pt group). the mean age of both groups were similar, 3.9 ± 3.2 and 3.7 ± 3.3 years in pa and pt groups respectively. there was a high prevalence of atopic diseases in both groups, with atopic dermatitis (75.8% in pa, 79.7% in pt), and other food allergies (55.4% in pa, 44.1% in pt). presence of rhinitis was statistically higher in the pa group compared to the pt group, with odds ratio of 2.52 (95% ci: 1.42-4.47) . a wheal size of ≥8 mm and a peanutspecific ige of ≥6 ku/l provided for a 95% positive predictive value. the larger the wheal size on spt, the higher the probability of a clinical reaction to peanuts. the results will help us in deriving preliminary cut-off values when conducting future prospective studies with ofcs in our peanut-sensitized cohort (whom had no prior peanut exposure), and to eventually reduce the need for expensive and potentially risky food challenges. 0913 | ginger: flavory, spicy …allergenic? a report of four patients with allergy to ginger background: ginger (zingiber officinale) belongs to the family zingiberoidae, along with cardamom and turmeric. the edible portion is the horizontal rhizome, and it is very appreciated for its aroma and spicy flavor. it also presents great interest for its therapeutic and culinary use. hypersensitivity to ginger is rare and has been scarcely reported. we report 4 cases (p1, p2, p3, p4) of adverse reactions to ginger after its ingestion and with good tolerance to cardamom and turmeric. method: skin prick tests (spt) to environmental allergens and prick-by-prick with ginger were carried out. total ige, and specific ige to ginger were also determined. a raw ginger extract was prepared. this extract was analyzed in all the patients by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). background: food allergy is divided into 3 groups according to pathophysiology: ige-mediated, ige-and non-ige (mixed type), and non-ige (cellular type). however, in clinical practice, patients who fall under more than one group may be observed. method: patients who were diagnosed with food allergy at our clinic from january 2012 to december 2016 were included in the study. the medical files of patients were retrospectively evaluated, their symptoms and findings after consumption of foods were recorded, and they were categorized into 3 groups (ige-mediated type, non-ige type, and mixed type) according to their symptoms and findings. results: a total of 587 patients (63.5% male) with food allergies were included in the study. according to categorization via symptoms and findings, the distribution of patients was as follows: 140 (23.8%) ige-mediated type, 44 (7.5%) non-ige type, 195 (33.2%) mixed type. the remaining 208 (35%) patients were found to show various combinations of symptoms and findings that fit more than one group. in this study, we observed that food allergy symptoms and findings were distributed in a broad range which caused difficulties in the categorization of more than one-third of our patients. background: fish allergic patients suffer a lifetime of strict dietary restrictions. crocodile meat is a nutritious alternative choice in many countries around the world; however, it has recently been reported to also trigger severe allergic reactions. in these two case reports from 2017 pediatric patients were sensitised to the major fish allergen parvalbumin (pv), a potential cross-allergen. bony fish contain predominantly pvs of the β-lineage, which are the most common trigger of allergic reactions in fish allergic patients. in most other vertebrates, pvs of the α-lineage are most abundant, which have been reported as a causal allergen in frog, cartilaginous fish, and chicken allergies. we aimed to evaluate the allergenicity of crocodile meat in fish allergic children, with focus on pv. method: over 70 children with clinical history of ige-mediated fish allergy were identified, skin tested to commonly consumed fish species using commercial and in-house preparations, and serum samples were collected. a sub-cohort was skin tested to crocodile using heat-treated tail muscle tissue from saltwater crocodile (crocodylus porosus). extracted proteins and purified pvs were analysed by sds-page, immunoblotting, and mass spectrometry. serum from all fish allergic children was analysed for ige reactivity to the crocodile pv. this reactivity was compared to those of raw and heated crocodile protein extracts as well as protein extracts and purified pvs from frequently consumed fish species. results: more than 5 fish allergic children were positive on skin testing to crocodile (wheal size>3 mm), demonstrating its clinical reactivity. in vitro analyses revealed ige reactivity to crocodile pv in serum from more than half of all patients. two βand one α-crocodile-pv isoforms were identified. pvs constituted approximately 90% of total proteins in heated crocodile extracts, with β-pv (11 kda) being 10 times less abundant, but up to 500 times more ige-reactive than α-pv (13 kda). αand β-pvs from crocodilians, including alligators and crocodiles, share more than 96% and 74% of their amino acid sequence, respectively. conclusion: crocodile pv is a new allergen as per the iuis guidelines. fish allergic patients may be at risk of severe allergic reactions upon ingestion of crocodile meat due to strong ige cross-reactivity of β-pvs. this study suggests that fish allergic individuals and health professionals need to be aware of potential allergic reactions to meat from crocodilians, termed 'fish-crocodile syndrome'. background: we present a 39-year-old nonatopic woman that in december 2016 after eating a seafood paella with green pepper presented asthenia, nasal obstruction, incoercible vomiting and diarrhea. later she ate a grilled loin sandwich in a bar and she had the same symptoms (she asked the waiter at the bar and the chef had cooked her sandwich in the same pan where he had cooked green pepper just before). after that, she suffered from abdominal pain, nausea, abdominal distension without diarrhea two hours after she ate an omelet sandwich with certain flavor of green pepper. at present even the casual smell of pepper causes her nausea. the woman eats everything including spices and just avoids pepper. method: skin prick tests were performed using extracts from food (nuts, fish, mollusk, fruits, vegetables, legumes), aeroallergens (mites, abstracts | 493 pollens and epithelia) and purified proteins (pru p 3, profilin, polcalcin, alfalactoalbumin, betalactoglobuline, casein). we also performed prick by prick to raw and cooked green pepper. sds-page immunoblotting according to laemli under reducing conditions (with 2-mercaptoethanol) was performed to study the molecular mass of the ige-reactive proteins. extracts from green pepper and green pepper seed were used. the prick tests were all negative and the prick by prick test to raw and cooked green pepper was positive in both cases. blond was taken from all patients to specify the levels of allergenspecific ige against 112 allergen components of immunocap isac test, the result≥0.30 isu-e was assumed as positive. results: in the study group, in 12 patients (24%) specific antibodies against ltp were detected, the isu-e level range 0.3-34 average 5.26 isu-e on average, in patients with detected ltp ige was detected for 3.5 components belonging to the ltp, however the highest number 41% patients were detected ige only for 1 ltp. in 9 subjects (75% of respondents with detected ltp), ige was detected against art v 3 and this is the only ltp component whose occurrence was statistically significant (p = 0.044). in 6 patients ige was detected against pru p 3, jug r 3, pla a 3,; 5 patients ige to ara h 9; 4 patients cor a 8; 3 patients ole e 7, 2 patients tri a 14, and in 1 person para j 2. background: the birch pollen-associated oral allergy syndrome (oas), an ige-mediated local allergic reaction, is the most common manifestation of pollen-associated food allergies. its origin is explained by cross-reactions between birch pollen-and food-allergens belonging to the pathogenesis-related protein subfamily 10 (pr-10). [1] so far, there is no marker available for its detection and no standardized test established to evaluate objectively the subjective feelings experienced by oas. the diagnosis is based on a characteristic history and on detecting the sensitization to triggering allergens in skin prick test (spt) and laboratory examination. method: the aim of this study was to evaluate whether the food skin prick test could be a helpful marker in the diagnosis of a birch pollen-associated oas. for this exploratory study, data from 2016-2017 was collected retrospectively at the dermatological outpatient department of the ordensklinikum linz elisabethinen. patients with positive spt results for birch pollen were included. the variables age, gender, tree pollen-(birch, alder, hazel) and food-spt, laboratory tests (ige, bet v 1, bet v 2, gly m 4) and symptoms (oas, rhinoconjunctivitis allergica, atopic dermatitis, anaphylaxis) for statistical analysis. results: there was an association between food-spt and oas but also between the negative oas patients and food-spt (p = 0.9-0.1). all of the bet v 1 sensitized patients with positive gly m 4 results had also a positive food-spt result. conclusion: there was no evidence for a possible role of food-spt as helpful markers in the diagnosis of birch pollen associated oas. maybe gly m 4 could be a helpful marker, but more data are needed. bet v 1 seems to be the cause of birch-pollen associated oas, due to the dominant sensitization pattern to major allergens in austria. background: eggs are among the foods most frequently causing allergy. the most common one is hen egg, although we may consume other bird's eggs such as duck's, those of goose, quails and seagulls. clinical and serological crossreactivity between hen egg proteins and those of other birds eggs have been described. allergy to other species eggs are less frequent and are usually described in patients allergic to hen eggs. we report a case of food allergy after ingestion of duck egg in an adult patient without hen egg allergy. the patient was a 50 year-old man who had symptoms of generalized itching, swelling uvula, erythema neck and deglutition difficulty immediately after he ate eggs from duck and hen. he claimed to have eaten hen eggs almost daily without clinical symptoms and he had not previously ingested duck egg. he denied allergic reactions to any other food but did complain of seasonal allergic rhinoconjunctivitis in the spring. we performed skin prick test with extracts of egg and feathers, prick by prick test with cooked and fresh yolk and white from duck and hen egg and oral challenge with hen eggs. specific serum ige was measured to hen proteins and we carried out a western blot with the proteins of allergenic extracts from different eggs (white and yolk of quail, chicken, goose and duck) and an inhibition of western blot with ovalbumin as inhibitor allergen. results: skin test with extracts of eggs and feathers, cooked and fresh hen and duck eggs were positive. total serum ige was 29.2 ku/l. specific ige to hen's egg was class two for hen egg white, ovoalbumin and class one for yolk and ovomucoid. oral challenge with heated egg yolk negative and with egg white was positive. the patient's serum recognized mainly and intensely several proteins of white and egg yolk of quail and duck with a molecular mass around 45 to 50 kda respectively. on the other hand a protein around 45 and 50-52 kda was unique recognized in white eggs of hen and goose. the western blot inhibition revealed ovalbumin inhibited the protein recognized in the egg white but not egg yolk involved in. background: management of tree nut allergy is usually based on the avoidance of the suspected tree nut (tn), as well as peanuts and seeds, either because of the risk of cross-reactivity and/or contamination, or due to the clinical severity. objective: to assess the sensitization pattern and clinical reactivity patterns to different tn, peanut and sesame seeds (ss) in patients with a history of reaction to at least one of these foods. results: a total of 47 patients with confirmed nut allergy were included; 70% female, median age [interquartile range] of 28 years; 72% were atopic. the most frequently involved foods were walnuts (53%), hazelnuts (40%), almonds (32%) and peanuts (38%). anaphylaxis was the clinical presentation in 51% of the patients. in those with a history of reaction to only one nut (24), the most prevalent was peanut (42%). in the 23 patients that reacted to more than one nut, the most frequent combinations were walnut/hazelnut (15), walnut/almond (12) and almond/ hazelnut (10). of these patients, 5 tolerated other nuts. two had sesame seed allergy, one reacted only to ss. nine patients (19%) were sensitized to foods that they tolerated. fourteen (30%) patients were sensitized to ltp and 8 of them reacted to more than one nut. conclusion: these data concur with the existence of different sensitization profiles (primary, concomitant or cross-reactive), which may predict different clinical reactivity patterns and therefore, influence dietary recommendations. background: buckwheat (fagopyrum esculentum) is a polygonaceae weed, not a cereal, which is increasingly been consumed and used as an alternative food in the diet of celiac patients. despite its wide use, allergy to buckwheat has unfrequently been reported in our setting. method: two female patients, aged 37 and 58, suffered an immediate severe allergic reaction after eating a bread and a pancake containing buckwheat among its ingredients. the first patient presented generalized urticaria, palpebral angioedema and pharyngeal occupation. the second one showed those same symptoms, as well as abdominal pain, nausea, dyspnea, dizziness, hypotension and loss of consciousness. skin tests and specific ige determination to aeroallergens and food allergens were carried out, including prick-prick test with buckwheat and all other components contained in the food involved. buckwheat allergens were studied by sds-page and ige-immunoblotting of one of the patients. results: prick-prick tests yielded strongly positive results to the food itself and buckwheat, and negative to the remaining food components in both cases. cap was positive to buckwheat (11.30 ku/l and >100 ku/l, respectively). basal serum tryptase levels were normal. both patients were not sensitized to cereals, ltps, profilins or pr-10 proteins. for the first patient, the skin tests were negative for other foods, seeds and nuts. the cap was negative for ltps, profilins and storage proteins of peanut and other nuts. the second patient who had the most severe reaction, was also sensitized to hazelnut(cap 13.7 ku/l), pistachio (1.44ku/l), almond(0.66ku/l), walnut(29.7ku/l) and sesame(4.47ku/l), which were not included in the pancake. the buckwheat immunoblot of the first case, under non-denaturing conditions, revealed a ige-binding protein of 60-kda. ige-immunoblotting under reducing condition showed protein bands of 30, 20, 17 and 14 kda in the buckwheat extract. conclusion: two cases of anaphylaxis by buckwheat flour contained in two frequently consumed foods are presented. the absence of sensitization to ltps, together with the pattern of specific ige binding in the immunoblot in one of the cases suggests that the responsible allergen could correspond to a storage protein of buckwheat, without cross-reactivity with other seed and nut allergens. buckwheat must be taken into account as an unsuspected food allergen capable of causing severe allergic reactions. background: allergy to linseed (linum usitatissimum) has infrequently been reported despite of its wide use in bread and in a range of "health food" products. linseed contains potent allergens which have not yet been characterized. we studied three patients who presented allergic reaction after eating different foods which contain linseed. two of them (patient p1 and patient p2) had anaphylaxis and the third one (patient p3) had oral syndrome, abdominal pain and diarrhoea. p2 was also allergic to mustard and p3 to sesame seed. all of them tolerated the remaining seeds, nuts and food. baseline tryptase levels were in normal range in all patients. skin tests and specific ige determination to inhalants and food allergens were carried out. linseed allergens were studied by sds-page and ige-immunoblotting. skin tests: patient 1: prick tests were positive to pollens and linseed, and negative to ltp, profilin, nuts, seed and the remaining food; patient 2: prick tests were positive to linseed and mustard and negative to other food and inhalants; patient 3: prick tests were positive to linseed, pollens, sesame and nuts (peanut, hazelnut, almond, pistachio). we present three cases of severe allergic reactions to linseed. the pattern of specific ige binding in the immunoblot seems to lead to storage proteins as the responsible allergens. 0925 | pr-10 sensitization-looking it up in food allergy background: pr-10 protein group sensitization is found in patients with respiratory allergy, mainly in areas inhabited by trees of the betulacea or fagacea families. its role in food allergy is, however, more frequently described in the context of cross reactivity. our aim was to characterize the pattern of molecular sensitization of food allergic patients sensitized to pr-10 protein group with immu-nocap isac ® (isac). the remaining patients, with more severe reactions, were all co-sensitized to either ltp and/or storage proteins (sp). only 2 of the 7 patients without pfa were co-sensitized to ltp or sp versus 7 of 9 with pfa (p < 0.05). conclusion: pr-10 sensitization is rare in our population. approximately half of the patients had allergy to plant foods, but the majority were co-sensitized to ltp and/or sp. the few patients only sensitized to pr-10 had minor reactions. among the patients without plant food allergy, co-sensitization to ltp and sp was significantly less common. according to these results, in our population, pr-10 seems to be less relevant to food allergy, when compared to reported results from other european countries. gür çetinkaya p; uysal soyer ö; esenboga s; sahiner üm; sekerel be hacettepe medical school, ankara, turkey background: pistachio is a tree nut belonging to "anacardiacea" family, and constitutes 7% of tree nut allergies. this nut most often abstracts | 497 cross-reacts up to 95% with cashew which is located in the same family. pistachio allergy is mostly seen in iran, turkey, the united states, and china where this tree nut is frequently consumed. in this study, we analyzed age and cut-off values for development of tolerance to pistachio. method: children who had reported allergic reactions with pistachio, and who have not consumed pistachio, but had positive spt and/or specific ige levels were enrolled into the study. spt and specific ige levels were measured in all patients. oral provocation(op) tests with pistachio were performed by 82 patients, and 21 of them had positive test result. the median age of tolerance development was 60 months (iqr: 48.0-89.0 months). the most commonly involved systems during op tests were skin (90%, n = 19), gastrointestinal system (33%, n = 7), and lower respiratory tract (24%, n = 5). concomitant allergic diseases were atopic dermatitis (70%), asthma (40%) and allergic rhinitis (17%). there was a positive correlation between skin prick test diameter (spt) and specific ige levels (spige) (r = 0.331, p = 0.001). spt of≥8.25 mm to pistachio nut was found as highly predictive of clinical allergy (auc: 0.83, 95%ci: 0.73-0.94, p < 0.001). any relation was not determined between eosinophil, basophil counts, triptase levels, and op test positivity. conclusion: pistachio allergy is one of the frequently seen nut allergies in turkey which may cause serious allergic reactions including anaphylaxis. op test showed that tolerance was achieved by the median age of 60 months, and a cut-off level of 8.25 mm was best predictor for positive reaction. in an oral challenge test, the patient responded with generalized urticaria, swelling of the lips and difficulty breathing at a cumulative dose of 0.1 g peanut protein, however, without blood pressure drop. (grade 2, moderate symptoms). the patient was treated with anti-ige for 3 months (with 450 mg every 4 weeks sc). oral desensitization began with ingestion of 500 μg peanut, escalating to 500 mg, on the first day and escalating weekly doses of peanut from 500 mg to 4000 mg (8 peanuts). then anti-ige was discontinued while the patient ingested 8 peanuts every day. we have followed the patient's sensitivity to peanuts from before anti-ige treatment to after anti-ige washout with basophil testing. results: the patient completed desensitization without side effects, and continues to ingest 8 peanuts a day. basophil sensitivity was reduced 13-fold by anti-ige treatment, but returned to baseline levels after anti-ige washout. conclusion: anti-ige allows rapid desensitisation of peanut allergic subjects with peanut oral immunotherapy. in the majority of subjects, this desensitization is sustained after anti-ige is discontinued. additional studies will help clarify which patients would benefit most from this approach. the return of basophil response to pre-treatment levels suggests that the patient is desensitised, and depends on the daily ingestion of allergen. what do we (not) know? background: the use of biologic prosthesis is a well-established surgical procedure. acute and delayed complications may occur, but accurate epidemiologic data about allergic reaction to graft tissue is lacking. methods: a 9 years old boy referred to our unit for urticaria and gastrointestinal symptoms which developed a few years before. he received a biologic porcine vascular duct during a cardiovascular surgery at 20 days of life. at the age of 5 urticarial episodes occurred, and a diagnosis of beef allergy was made. after the exclusion of beef meat from the diet, most symptoms resolved, but the child began to complain about occasional episodes of vomit and diarrhoea. results: skin prick tests confirmed the beef meat sensitization and prick-prick test resulted positive against raw pork meat but not against cooked pork meat. in vitro tests demonstrated the presence of pork meat specific iges (14.1 kua/l). component-resolved diagnostic tests revealed allergy sensitization toward bos d 6 (bovine serum albumin, bsa; porcine serum albumin was not tested due to lack of a specific test). after accurate exclusion of pork meat from diet, a complete remission was achieved, and the diagnosis of pork meat allergy was confirmed. conclusion: bsa is a major beef allergen, responsible for the raw beef-cow milk cross-reactivity but with a scarce importance in milk allergy. it is highly homologous with human serum albumin and other mammalian serum albumins, including porcine albumin. porcine albumin is also highly homologous with cat serum albumin and therefore responsible for cross-reactivity in patients affected by cat-pork syndrome. we hypothesize that the implanted porcine tissue was the trigger for pork meat allergy in our patient, as this condition is exceptional in childhood and that our patient never owned a cat. few cases of pork allergy due to porcine tissue implantation have been reported so far. of interest, pig sensitization was recognized as a rare but possible cause of blood-culture negative endocarditis in patients with porcine bioprosthesis according with anamnesis, increased ige level against pork, tissue eosinophilia during autopsy. background: desensitization to foods is assuming a new paradigm in food allergy. this technique is becoming widespread especially for patients with egg and milk allergy, but the effect of desensitization on the consumption of similar but not identical foods is still uncertain. material and methods: we describe the case of a 16-year-old patient, with a history of chicken egg allergy, who had been successfully desensitized tolerating cooked and raw chicken eggs for a year. the patient came to the office after presenting an episode of anaphylaxis immediately after eating fried quail eggs. an immunological study was carried out. we performed skin prick test with chicken egg′s proteins (white, yolk, ovoalbumin and ovomucoid). an immunoblotting to detect specific ige to egg proteins was also performed. for this purpose, the following extracts were used: chicken egg white and yolk (commercial extract form alk) and quail egg white and yolk prepared following a similar procedure (extracted in 10% phosphate buffer (w/v)). conclusion: we present the case of a patient with specific allergy to quail egg white and yolk, probably through ovotransferin, but not to chicken egg. the desensitization against chicken eggs does not allow the consumption of eggs of other species of birds, such as quail eggs, and this indication must be made specifically to patients after a protocol of desensitization against chicken′s eggs. case report: seven years ago we presented the case of a 32year-old male, who suffered two acute episodes of oral pruritus, lip angioedema, epigastric pain, generalized urticaria and dizziness after ingesting lettuce. he previously tolerated salads (including lettuce). he was later diagnosed with non-ltp-dependent lettuce anaphylaxis and an aspartyl protease was identified as a new lettuce allergen with cross-reactivity with other members of the compositae family. since the diagnosis he has avoided lettuce and all the other compositae. we present the patient's curious outcome after 7 years of follow up. methods: total ige, basal tryptase, specific ige to lettuce by immu-nocap, prick-prick and oral challenge tests with lettuce and other compositae. sds-page, immunoblotting and molecular characterization of ige binding bands by mass spectrometry. skin prick tests and specific ige to lettuce were repeated on several occasions in the following years. after 7 years an oral challenge test with lettuce was carried out. results: prick-prick test was positive to fresh lettuce (11 × 15 mm) and to other compositae (raw endive, chicory, thistle, artichoke and chamomile). prick-prick test was negative with these boiled compositae. basal tryptase was 7.07 μg/l. total ige in serum was 189 ul/ml. specific ige by immunocap was positive to lettuce (7.35 ku/l) and negative to profilin, ltps and thaumatin. oral challenge test with endive was positive and negative with cooked compositae (artichokes and thistle). sds-page and immunoblotting detected an intensely binding ige band about 46 kda, common for endive and chicory, which was identified by mass spectrometry as an aspartyl protease. no bands were detected at ltp (9 kda) and profilin (16 kda) . in the first two years after the diagnosis, prick-prick test and specific ige to lettuce remained positive (cap 1.91 ku/l and 0.44 ku/l, respectively). after 7 years, prick-prick test and specific ige to lettuce became negative (cap 0.08 ku/l). with this findings a new oral challenge test with lettuce was carried out which result turned out negative. we report the first case of spontaneous tolerance to lettuce in a patient who previously presented lettuce anaphylaxis and identify an aspartyl protease as the causative allergen. introduction: eosinophilic esophagitis (eoe) is an emergent allergic inflammatory disease that is triggered by food allergens and characterized by progressive esophageal dysfunction. recently, it has been seen that eoe develops in up to 2.7% of patients with ige-mediated food allergy undergoing oral immunotherapy (oit). ingestion of baked milk and egg was associated with increased development of tolerance to regular milk and immunologic changes have been reported in subjects ingesting baked milk and egg, similar to those seen in food oral immunotherapy studies. case description: we present the case of a 13-year-old girl, with history of ige mediated cow′s milk allergy and rhino conjunctival and bronchial asthma symptoms. prick test against milk proteins showed: milk 6 mm, alpha-lactalbumin 6 mm, casein 3 mm and to beta-lactoglobulin: negative. total ige: 469 ku/l. specific ige to milk: 3, 15 alpha-lactalbumin: 3, 16 ku/l; casein: 2 ku/l and beta-lactoglobulin: 0.10 ku/l. we performed an oral food challenge with baked milk which was well tolerated. then, we performed an oral food challenge with fresh milk and she presented facial urticaria and pharyngeal pruritus. after 3 months eating baked milk every day, she had symptoms of dysphagia and esophageal food impaction. for this reason, we performed an esophagogastroduodenoscopy (egd) and biopsies which showed white exudates and vertical furrows. the histological study showed eosinophil count>20 per high-power field. eosinophilic esophagitis was diagnosed and she started treatment with esomeprazole 20 mg. the egd was repeated 8 weeks later with similar results in the biopsy. she was treated with a comprehensive diet free of cow′s milk proteins. after 8 weeks she was asymptomatic and endoscopy and biopsy findings were normal. we report the case of a cow′s milk allergic patient who developed eoe after introduction baked cow′s milk which apparently was tolerated in her diet. the avoidance proved efficacy in inducing the remission of eoe. method: we examined 87 bottle-feeding infants with food allergy aged from 1 to 12 months. serum vitamin levels were measured by immunoassay methods (retinol binding protein (rbp), vitamin b6, 25hydroxy vitamin d), biochemistry methods (vitamin c, vitamin e) and microbiology methods (vitamin b1, vitamin b2). as criteria for complete sufficiency standards adopted in the russian federation were used (the lower limit of normal levels: rbp -0.52 μmol/l; b6-8 ng/ml; 25-hydroxy vitamin d-15 ng/ml; vitamin c -0.4 mg/dl, vitamin e -0.8 mg/dl; vitamin b1 -28 μg/ml; vitamin b2 -6 ng/ ml). results: complete sufficiency were observed in 21 infants (25.9% cases), one vitamin deficiency in 33 infants (40.7%), two vitamins deficiency in 23 infants (28.4%), three and more vitamins deficiency in 6 infants (7.4%). should be used after vitamin status asses, mainly using monovitamin medication. results: patients were followed with the diagnoses of cma and asthma. age at the beginning of symptoms and start of oit were in the range of 3-6 months and 4-19 years, respectively. they had high total ige (139-843 iu/ml), milk spige (2.9-418 kua/l), casein spige background: food allergies may affect up to 6% of school-aged children. it has been shown that approximately 20% of all anaphylactic reactions caused by food allergy are firstly presented at (pre) school. therefore, it is of high importance that (pre)schools have a policy on food allergy management and the use of an epinephrine auto-injector (eai). to our knowledge, limited is known on the policies of food allergy management at (pre)schools. the aim of this study was to investigate the policy on food allergy management in preschools and primary schools in the northern part of the netherlands. results: we included 87 preschools and 110 primary schools in this study. we showed that 80.5% of the preschools and 73.6% of the primary schools had a child(ren) with food allergy. only 13.8% of the participating preschools and 27.2% of the primary schools had a policy on allergen avoidance and only 35.6% of the preschools and 35.8% of the primary schools had a policy for the use of an eai. the majority of the pre-and primary schools in the northern part of the netherlands have children with food allergy. however, only a limited number of (pre)schools do have written guidelines for food allergy management in (pre)schools. additionally, there is limited experience how to use an eai at (pre)schools. therefore, an evidence-based policy on food allergy management in (pre) schools is needed. background: food allergies are the most common cause of anaphylaxis in childhood. here we present 3 cases had anaphylaxis due to cow 's milk allergy, and treated with specific oral tolerance induction (soti̇) to cow' s milk. method: soti protocol was administered according to previously published by longo et al. skin prick test were performed according to standard methods with allergens. cow's milk specific ige was investigated with immunocap system. in all cases, the wheal size of the cow's milk in skin prick tests or the specific ige levels was higher than level of positive predictive value of 95%. results: case 1: a 7 years old male patient who had 3 anaphylaxis after milk consumption. soti treatment was started one year ago. there was no complications during the dose increasing phase. however, he had two episodes of anaphylaxis during the maintenance phase. in the final visit, we observed that he could drink 200 ml milk and could consume dairy products. case 2: eight-year-old male patient has an intensive care-of-hospitalization story due to anaphylaxis three times after milk consumption. his accordance to strict diet was bad, and had frequent asthma attack. anaphylaxis developed 2 times during the dose increasing phase in soti protocol. after soti treatment, he could consume 200 ml cow's milk and dairy products without problems. case 3: a 7-year-old male patient followed-up for asthma and cow's milk allergy. it was learned that anaphylaxis developed 2 times after milk consumption. he was fed in accordance with the milk-free diet. he has used fluticasone nebules, montelukast, mometasone nasal spray, and cetirizine. anaphylaxis developed 4 times during the dose increase phase of soti administration. there were many mild to moderate anaphylactic episodes during the maintaining phase. after the soti treatment, he could consume 150 ml milk and dairy products. background: cow's milk protein allergy (cmpa) is one of the most common food allergies in early childhood. small dietetic group sessions for parents of infant with non-ige mediated cmpa were held to meet increasing demands and reduce waiting times. parents were given information on cmpa, advice on weaning and milk reintroduction using a locally designed milk ladder. parents were also advised to contact the dietitians via telephone if they had further questions. we aim to evaluate the sustained effectiveness and patient satisfaction of the group sessions. method: parents and carers who attended the group dietetic sessions held between november 2015 and july 2016 were included in the survey. feedback were obtained via a self-designed questionnaire using a likert-type scale, rating several questions from 1 (least satisfied) to 5 (most satisfied). initial feedback was obtained directly after the session. we followed these patients up a year after the initial session via telephone and postal questionnaire. results: overall attendance rate of the 9 group sessions held was 58% (n = 40). during the initial survey, participants found the group session useful (mean score 4.7 out of 5) and felt more confident in managing cmpa (mean score 4.8). we successfully obtained follow up feedback from 13 participants. majority agreed that the group sessions have been informative (mean score 4.8). they also said they felt confident weaning their child on milk-free diet (mean score 4.8), and in reintroduction of cow's milk in diet (mean score 4.3). 30% (n = 4) said that they would have preferred an individual session. 38% (n = 5) have contacted the dieticians via telephone after the initial session, and 15% (n = 2) had requested individual consultations. 69% (n = 9) have attempted reintroduction of cow's milk in their child's diet using our local milk reintroduction guide. the mean age at first challenge was 14 months (age range 6 to 26 months), with average of two attempts. 31% (n = 4) have been successfully challenged and are managing well on a normal diet. we recognised the limitation in obtaining feedback via telephone and postal questionnaire, which resulted in the poor follow-up response rate. overall, parents felt more confident in managing cmpa and the positive responses were sustained a year on, highlighting the success of these group sessions. follow up opt-in sessions could be offered to provide additional support and allay parental anxiety in challenging their child with cow's milk at an appropriate age. results: goats and rabbits were immunized with specific allergoids, the allergoid-specific igg titer determined and sera pools produced. the allergoid reference material was comprehensively characterized. while ige reactivity of the allergoids was not detectable anymore, igg reactivity was maintained. allergoid-specific assay parameters as serum dilution, reference dilution and sample dilution factor to obtain at least six data points within the pseudo-linear range of the inhibition curve were determined. with these set parameters the evaluation of the analytical method was performed. the assay showed very good results in terms of linearity, accuracy, precision, reproducibility and robustness for all investigated allergoids as well as aluminum-adsorbed allergoid-preparations. conclusion: with the developed immunological inhibition assay, it is possible to determine the specific igg reactivity of allergoids in different preparations. the performance of the analytical method met all pre-defined acceptance criteria, which will be confirmed in the next step by a validation procedure according to ich-guidelines. case report: we present the case of a 32-year-old patient, diagnosed with rhinitis and asthma due to sensitization to pollens, as well as dyshidrosis and allergic contact dermatitis to cobalt. the patient presented a cutaneous pattern consisting of erythematous papules, some scaly, very pruritic, of initial appearance in the upper limbs 2-3 days after initiation of administration of specific immunotherapy extract (depigoid forte grasses, leti ® ). subsequently generalize lower limbs and neck. she presented them repeatedly and late after 2-3 days of the first 3 doses administered. she referred partial control of pruritus with oral antihistamines, without total resolution of lesions for weeks. immunotherapy was suspended persisting the skin lesions for 3 more weeks. according to the personal history of sensitization to metals, and the clinic presented in a temporal relationship with the use of an extract of immunotherapy with aluminum hydroxide, a study was requested with epicutaneous tests with aluminum hydroxide as well as with epicutaneous tests with immunotherapy extract. aluminium hydroxide and depìgoid forte grasses extract epicutaneous test were negative. in subsequent visits the patient reported that coinciding with the start of immunotherapy, presented at home and mainly in her bedroom a plague of cimex lectularius, popularly known as bedbugs, proving that they had been the cause of bites on their skin, and later skin reaction. patient reported that with the elimination of said pest the skin lesions disappeared. the administration of its immunotherapy extract was tolerated. cimex lectularius, commonly known as bed bugs, is a hemiptera insect of the family cimicidae. the clinical picture usually corresponds to multiple pruriginous lesions from the prurigo type, to multiple erythematous plaques, some infiltrated and others with a urticarial appearance, or even bullous. the lesions last for 3 to 6 weeks without treatment, and while the older ones heal, new ones may appear. in our patient it was not considered as an initial diagnosis, having considered immunotherapy as an etiological factor, but we must not forget that although in our country it is not a reason for frequent consultation, either due to underdiagnosis, because of the transitory nature of the pathology or because of scarce number of causative agents, it is important to consider insect bites in the differential diagnosis of dermatosis. di cara g; salvatori c; testa i; pacitto a; bizzarri i; isidori c; tarsia m; esposito s università degli studi di perugia-dipartimento di scienze chirurgiche e biomediche, perugia, italy background: house dust mites (hdm) are one of the most important allergens involved in childhood respiratory diseases, and the most frequently prescribed extract for sublingual immunotherapy in children (slit). despite the improvement of standardization methods for the production of slit, the differences in cultivation and purification processes used to produce raw materials for specific immunotherapy extracts may still impact on the final composition of mite allergen extracts. our study investigated the total protein and main allergen content of five commercial hdm sublingual immunotherapy extracts using sds-page and immunoblotting. recombinant allergens of group 1 and group 2 major allergens were used to test the immunogenicity of such extracts. method: hdm slit extracts were purchased from five italian suppliers (alk-abellò, allergy therapeutics, anallergo, lofarma, stallergenes). the protein composition of extracts was evaluated analysing equal volumes (15 ml/lane) by sds-page (12% separating gel) and subsequent immunoblotting. for identification of allergens in the extracts, western blot analyses were performed with rabbit monoclonal antibodies (raybiotech) against der p 1 and der p2. the total protein content in the five tested commercial extracts showed a relevant variability. the protein contents ranged from 32.9 to 44.2 μg/mg for what concerns der p1, while der p2 showed a greater variability, ranging from 4.2 to 14.6 μg/mg. sds-page showed a similar pattern of distribution in 3 of the tested extracts, which showed protein bands of comparable intensity, while 2 extracts showed a lower total protein count. extract 2 showed a higher intensity band corresponding to the molecular weight of tropomyosin. western-blotting showed a similar concentration of der p 1 in most extracts, while der p2 was more variable. conclusion: our analysis of five commercial extracts commonly used for sublingual specific immunotherapy against hdm showed important variations in term of total protein content. a less evident but still relevant difference was also evidenced when testing the major allergen content, with up to 25% variation in der p1 and up to a 3-fold variation in der p2 concentration. this differences, likely related to the different production and extraction methods, could still be responsible of a different immunological response in children who underwent slit. method: we evaluated 29 children who had completed their immunotherapy treatment. along with demographic data we were able to record skin prick test (spt) results and mrqlq at start and end of treatment. we only included patients who had completed pre and post treatment questionnaires in the study to allow a comparison. the scores were evaluated using a student t test. results: patients' starting age ranged from 6 to 17 years (mean 11 years). 29 of the 48 children had completed pre and post treatment questionnaires. all 29 had grass pollen allergy confirmed on spt at the start of treatment. 10 patients (34%) had isolated grass pollen allergy on spt and 19 (66%) had multiple allergies. mean start treatment score for all patients was 37 on mrqlq. mean score at end of treatment was 24, indicating a 35% reduction in total mrqlq score (p value <0.05). for those with multiple allergies the mean total mrqlq scores were 33 at start of treatment and 25 at end of treatment, indicating a 27% reduction (p value 0.16). for those with isolated grass pollen allergy scores were 44 at start of treatment and 23 at end of treatment indicating a 50% reduction (p value <0.05). conclusion: for children with uncontrolled symptoms of allergic rhinoconjunctivitis, grass pollen immunotherapy is associated with statistically significant improvement in quality of life. this improvement is most beneficial for patients with isolated grass-pollen sensitivity on spt. those with multiple aeroallergen sensitivities on spt did show an improvement (not statistically significant) post-treatment. grass-pollen immunotherapy is an effective treatment for rhinoconjunctivitis to offer patients in a rural dgh setting. background: allergic asthma is a common clinical refractory disease, most patients with asthma are accompanied by varying degrees of allergy. in clinical practice, treatment of this disease using specific immunotherapy has proven effective. in the current study, we examined the effectiveness of specific immunotherapy in a total of 99 patients admitted to our hospital from 2015 to 2016. method: to investigate the clinical efficacy of allergic asthma-specific immunotherapy. 99 patients were selected, of which 49 were males, aged 22 to 61 years, 50 females, aged between 23 to 64 years old, all patients were clinically diagnosed only as allergic asthma. the 99 patients were randomly divided into two groups, including the observation group containing 50 cases, the control group of 49 cases. all patients were first treated with conventional basic treatment. the observation group was subsequently treated with specific immunotherapy. both groups were followed-up and the treatment efficiency were analyzed. results: after treatment, both two groups of patients showed improvement, in the observation group, the effective rate was 93%, while for the control group, the effective rate was 75%. observation group showed significantly better outcomes than the control group. conclusion: in allergic asthma treatment, adding specific immunotherapy on the basis of routine treatment is beneficial and could be widely used in clinic. case report: atopic dermatitis(ad)is the most common itchy dermatosis that affects millions of children and adults. during recent years, diagnosis and treatment based on component resolved diagnostics (crd)is recommended. we report a 9-year-old boy with severe atopic dermatitis. he had positive family history of atopy. the atopic dermatitis was developed since infancy. he was referred to our clinic when he was 5 years old. he had generalized xerosis with ulcerative eczematous lesions on his neck, popliteal and antecubital areas. he had mild eosinophilia and his serum total ige level was 590 iu/ml. daily bleach bath, moisturizing agents, topical steroids and systemic antibiotics in addition to antihistamine were prescribed. he had multiple food and aeroallergen sensitization in skin prick test (spt). he started to eliminate some foods according to the spt results. he was suffering from recurrent relapse even after strict food avoidance; so treatment with cyclosporine was initiated for him, with partial response. crd showed sensitization to alternaria alternata (alt a1 specific ige:10.97 ku/l). allergen immunotherapy by alternaria alternata was started. after accomplishment of buildup phase, he had significant improvement and we were successful to taper and finally discontinue cyclosporine. now he is on maintenance phase of immunotherapy, his skin is in optimal condition only by hydration and moisturization. result: a 15-year-old thai girl is a known case of severe asthma since one year old. her asthma was uncontrolled asthma even treatment with high dose combination of inhaled corticosteroid and long acting beta agonist (ics/laba), montelukast and omalizumab. spirometry revealed the force expiratory volume in one second (fev1): 34% predicted, fev1/forced vital capacity (fvc): 48% predicted and 14% improvement of fev1 after salbutamol 400 ug inhalation. allergic sensitization showed specific ige to cat: 2.04 kua/l. slit with cat allergen started at the dose of 450 au per month and increased to 1500 au per month (scit dose is 1000 au per month) for three-year-and-six-month course. after slit, her asthma symptom improved significantly. she can exercise without exacerbation and plays sport at school. her last episode of asthma exacerbation was 1.5 year ago. her fev1 (% predicted) was improved from 34% predicted to 48% and the fev1 bronchodilator response decreased from 14% to 6%. conclusion: an improvement of pulmonary function and asthmatic symptoms of the presenting case would support the efficacy of slit of cat allergen in a patient with severe asthma. ra developed during pollen scit in this case might be related with immunomodulation effect of immunotherapy. background: we report a case of 20-year-old woman with allergic rhinoconjunctivitis and mild persistent asthma due to sensitisation to seasonal pollens and molds with bad clinical evolution and not response to conventional drug therapy. we decided to start subcutaneous allergen specific immunotherapy with alternaria extract in our immunotherapy unit in accordance with the guidelines of the european academy of allergology and clinical immunology (eaaci) and we used a cluster regimen. the immunotherapy was not well tolerated: the patient had two grade 2 systemic reactions with the first dose in two attempts. method: sensitisation was diagnosed through skin prick test with aeroallergens standard panel and serum specific ige by elisa. due to the bad tolerance to immunotherapy, molds molecular diagnosis by immunocap, study of the molecular weight to specific ige binding proteins by sds-page ige immunoblotting, and cross-reactivity study by means of immunoblotting-inhibition assay were performed. a. fumigatus extract was able to produce a total ige binding inhibition on the 38 kda band of a. alternata extract when ige immunoblotting assay was performed. conclusion: respiratory allergic disease due to alternaria is difficult to control, the use of subcutaneous specific immunotherapy could be of significant benefit. most of the allergic patients to a. alternata are sensitized to alt a1, major allergen from a. alternata. however, our patient is sensitized to a 38 kda alternaria protein due to cross-reactivity with a. fumigatus allergens, this sensitization could explain the bad tolerance to the alternaria immunotherapy. background: the association between natural pollen exposure, clinical symptoms as well as allergen-specific immune responses has not been investigated at a molecular level. our aim was to monitor the effect of seasonal birch and grass pollen exposure on clinical symptoms as well as specific b cell and t cell responses to defined allergen molecules in sensitized subjects during two consecutive years. method: grass pollen sensitized (n = 10) and birch pollen sensitized (n = 13) subjects were included in this study and were followed for two consecutive years (2013) (2014) . subjects were taking part in a clinical trial for the recombinant grass pollen vaccine (bm32) but did not receive immunotherapy for the allergen they were sensitized to. before, during and after the respective seasons ige and igg levels as well as t cell responses to the major birch pollen allergen bet v 1 and the major grass pollen allergens phl p 1, 2, 5 and 6 were measured. pollen counts were recorded throughout the year and patients kept a daily diary including symptom medication score (sms) and visual analogue scale (vas). results: we noted that ige levels specific for bet v 1 and the grass pollen allergens increased most in the seasons in which patients experienced the highest peak symptoms according to vas and sms but not depending on cumulative pollen counts. increases in allergen-specific t cell responses were observed in the pollen seasons as compared to shortly before the pollen seasons in the grass pollenallergic patients also in association with vas and sms but not in the birch pollen allergic subjects. no relevant changes of allergen-specific igg levels were observed during the two years observation in grass and birch pollen allergic patients. we found an association of increases of allergen-specific ige increases shortly after the pollen season with clinical symptoms in the pollen season as reflected by vas and sms which was not necessarily reflected by cumulative pollen counts in the season. these results may be important for the analysis of allergen-specific immunotherapy trials. background: the morbidity and mortality of severe asthma is much higher than that of mild to moderate asthma. this study was performed to understand the clinical characteristics of severe asthma in korea. results: data from the questionnaire showed that bronchial asthma was diagnosed before pregnancy only in 193 women (4.83%). 43 patients (1.1%) were diagnosed with chronic bronchitis at the pregestation stage. asthma attacks were experienced repeatedly during a lifetime in 4.2% of patients, 12.7% of patients noted long periods of dry cough at night, among them 8.1% had wheezing. the cold did not precede the wheezing breathing in 5.1% of patients. difficulty in breathing on waking was noted in 2.6% of patients, at night-4.3%. after examination, the diagnosis of asthma was confirmed in 14.65% of the respondents (586 people). symptoms of rhinitis are noted in 58% of women surveyed, 18% of rhinitis was allergic. before examination, the diagnosis of ar was only in 3.5% of patients. the incidence of symptoms of asthma and ar in pregnant women is significantly higher than the reported cases of these diseases, which leads to untimely initiation of treatment. method: a total of 117 nonsmoker asthmatic patients without concomitant pulmonary pathology are recruited to our study. all patients underwent spirometry tests, measurement of fraction of exhaled nitric oxide and sputum induction to asses sputum cell counts, demographic features and current medications were recorded. using the variables of age at onset, bmi, allergy status, fev1%, fev1/fvc, asthma severity and induced sputum cytology cluster analysis is performed. results: 4 clusters are identified. cluster1: (n = 43) early onset atopic asthma, consists of mild asthmatics with a good asthma control and lower bmi. cluster 2: (n = 14) severe atopic asthma, consists of lowest spirometry measurements with a least act scores. induced sputum cytology shows a neutrophilic character, while having also the highest percentage of eosinophils. cluster 3 (n = 27) late onset obese asthma, nonatopic asthmatics having high spirometry measurements, with a lower act scores. cluster4 (n = 33) nonatopic mild asthma, consists of patients with the best respiratory functions and least inflammation in means of lowest total ige, feno, sputum cell counts. conclusion: identification of asthma phenotypes in different countries will improve our understanding on the heterogeneity of the disease among the different geographies. results: results and discussion. in the course of analysis, obesity was more common in children with bronchial asthma −25% than in the comparison group-in 9.3%. obesity of the 1st degree was diagnosed in 10 patients of the main group, ii degree-in 8, and iii degree-5 and iv degree-in 2 patients e diagnosis of obesity, the sds indices of body mass index (bmi) were determined. obesitymore than +2.0 (i degree: sds bmi 2.0-2.5, ii degree: sds bmi 2.6-3.0, iii degree: sds bmi 3.1-3.9, iv degree: sds bmi ≥4.0). conclusion: thus, the results obtained indicate a high prevalence of constitutional-exogenous obesity in children with bronchial asthma and precedes the formation of the underlying disease e diagnosis of obesity, the sds indices of body mass index (bmi) were determined. obesity-more than +2.0 (i degree: sds bmi 2.0-2.5, ii degree: sds bmi 2.6-3.0, iii degree: sds bmi 3.1-3.9, iv degree: method: postal questionnaires were distributed to an unselected group of asthma patients (n = 190). healthy non-asthmatic volunteers were recruited amongst university and hospital co-workers (n = 53). the presence of self-reported nhr, the type of triggers evoking nasal symptoms, asthma phenotype, medication use and environmental factors were evaluated. results: 114 patients and 53 controls completed the questionnaire (responder rate of 60% and 100% respectively). nhr was reported in 71% of asthma patients and 22% in non-asthmatic controls (p < 0.0001), with changes in temperature being the most important inducer of nasal symptoms (74% of asthmatics), followed by strong odours (62%) and cigarette smoke (61%). interestingly, nhr was more prevalent in patients with severe (79%) compared to mild (50%) asthma symptoms (p = 0.015), and more prevalent in atopic (77%) compared to non-atopic (55%) asthmatics (p = 0.028). most asthma patients reported more than one trigger evoking nasal symptoms, with 44% of patients reporting 3 or more triggers evoking nasal symptoms. results: the mean score of cbcl questionnaire in case group with 28.12 ± 2.06 was significantly higher than in comparison with a control group with 17.33 ± 9.7 (p = 0.01). the mean scores of the subscales of social isolation (the case group: 5.83 vs control: 1.18, p = 0.006), anxiety-depression (4.99 vs 2, p = 0.22), intellectual problems (4.8 vs 2.3, p = 0.000), and aggressive behaviors (5.1 vs 2.3, p = 0.003) were significantly higher in children with asthma than in healthy children. the study showed a significant correlation between the mean duration of asthma and a general score of cbcl (p = 0.012, cc=0.8). moreover, there was also a significant correlation between asthma severity and cbcl scoring (p = 0/001, cc=0.03). conclusion: behavioral disorders in children with asthma are significantly more than healthy children. the duration of asthma and the severity of asthma, are related to and can predict behavioral disorders in children with asthma. background: assessment of asthma control is an integral part of the management of asthma. whilst asthma control test (act) is a commonly used questionnaire to assess symptom control, its utility in predicting long term risk of exacerbation has not been well studied. aim: to analyze the factors associated with uncontrolled asthma symptoms using act and its impact on predicting future exacerbation. method: severe asthma patients on at least step 4 background: most of the asthma-scoring tools detect the asthma severity from patients' symptoms but there is no scoring tool using parameters to define risk of asthma exacerbation. thus, this study use factor analysis to evaluate the relationship of parameters in childhood asthma. method: the descriptive study using factor analysis in asthmatic children aged less than 15 years old, who attended thammasat university, the center of excellence for allergy, asthma and pulmonary diseases, thailand. the participants or caregivers were inter the factors which have the major impact on asthma control are changing bed sheets less than once per month and using dust mite-proof bed sheets. this study is supporting non-pharmacological strategies but further studies are needed to create a more efficient asthmatic symptom checker. were not different between the controlled and uncontrolled group. the act score in the controlled group was significantly higher than the uncontrolled group (p < 0.001). the study showed that cigarette smoke is one of the significant factors that can trigger asthma exacerbation (p < 0.001) and mosquito repellent coil smoke is also significantly associated with asthma exacerbation (p < 0.03 background: experimental studies have demonstrated that tumor necrosis factor family member 14 (tnfsf14/light) plays an important role in airway remodeling. there is little data available concerning in vivo regulation of tnfsf14/light expression in humans. the aim of this study was to evaluate serum concentration of tnfsf14/ light in different subsets of asthmatic patients. the study was performed on 36 nonsmoking asthmatic patients (a), including 24 mild-moderate-severe asthmatics controlled on inhaled corticosteroids (aics) and 12 asthmatics evaluated twice during asthma exacerbation (aex) and during subsequent remission (arem). in addition age and sex matched 24 nonsmoking healthy controls were included (hc). serum tnfsf14/light concentration was evaluated using elisa method. in asthmatic patients lung function tests, exhaled nitric oxide concentration (feno), serum total ige concentration (t-ige), allergen-specific ige concentration (s-ige) and peripheral blood eosinophilia were evaluated. (250 + /-134 pg/ml) was significantly greater than that in hc (72 + / -32 pg/ml; p < 0.001). among all asthmatic patients studied the greatest tnfsf14/light serum concentration was demonstrated in aex (357 + /-79 pg/ml), which was significantly greater than that in aics (194 + /-123 pg/ml p < 0.001). during resolution of asthma exacerbation a significant decrease in serum tnfsf14/light concentration (118 + /-70 pg/ml; p < 0.001) was demonstrated. in arem the mean serum tnfsf14/light concentration was comparable to that seen in aics (p = 0.06) but was still significantly greater than in hc (p < 0.01). no significant correlation could be demonstrated between serum tnfsf14/light concentration and baseline lung function parameters, exhaled nitric oxide concentration, serum t-ige or s-ige concentration or peripheral blood eosinophilia. conclusion: enhanced production of tnfsf14/light seen in asthmatic patients, which is further upregulated during asthma exacerbations may play an important role in asthma pathogenesis. method: two models of aspergillus fumigatus-induced allergic airway inflammation were used in the study: long terms (4 weeks) and short terms (2 weeks background: chalcone 4 is identified as an inhibitor of the interaction between cxcr4 or cxcr7 and their ligand cxcl12. therefore it is called a neutraligand. the chemokine cxcl12, interacting with the cxc-receptor4 (cxcr4) can play a role in the progression and development of bronchial asthma. asthma is defined as a chronic disease characterized by episodes of obstructive events which affects about 300 million people over the world. the aim of this study is to approach the mechanism of the anti-inflammatory effect of the cxcl12 neutraligand chalcone 4 and also to assess its impact on the migration of dendritic cells in a murine model of allergic airway inflammation. method: chalcone 4 is administered intranasally to balb/c ovalbumin (ova) asthma mice and control groups as well. our results indicate that the cxcl12 neutraligand chalcone 4 can modify the inflammatory reaction in an airway allergic hypereosinophilia model. furthermore, found out that cxcl12 neutraligand chalcone 4 prevents dc migration to the airways and airway jnc ganglia during allergic airway inflammation. the detection of the cxcr4-cxcl12 pathway and its role in the pathophysiological actions of asthma offers a promising target for allergic diseases treatments. method: four groups of balb/c mice were defined: control and asthmatic, with and without treatment. asthmatic groups were sensioverexpression of ptgdr in pulmonary cells associated to a generalized increase of cytokine expression. conclusion: in a mouse model we confirmed the involvement of ptgdr in allergic asthma by the increase of its expression levels after ovalbumin sensitization. we also identified a reduction of ptgdr levels in response to dexamethasone treatment. the in vitro model suggests that ptgdr induces an inflammatory response, increasing the cytokines levels. 0979 | immune imbalance between transcription factor t-bet/gata3 and allergic asthma results: t-bet mrna expression of peripheral blood lymphocytes in patients with allergic asthma was lower than that of the normal control group, and the expression level of gata-3 mrna was higher than that of the normal control group (p < 0.05). the th1 percentage of peripheral blood lymphocyte subsets was lower than that of the normal control group (p < 0.05), the percentage of th2 cells was significantly higher than that of the normal control group (p < 0.05), and the changes in t-bet/gata3 expression and th1/th2 ratio was highly correlated. our objectives were to assess the changes of bmp4 and bmp7 serum levels in the response to allergen and methacholine challenge tests and the correlation between bmp4 and bmp7 serum levels and fev1 before and after allergen and methacholine challenge tests. method: study group consisted of 59 patients with asthma and 48 healthy volunteers. spirometry, skin prick tests, allergen and methacholine challenge tests were performed in compliance with eaaci, ers and ats guidelines. personalized clinic surveys including act ™ were performed. venous blood was collected before and after 1 hour, and 24 hours afterwards the provocation to edta-ke-filled test tubes. evaluation of bmp4 and bmp7 serum protein levels was performed using specific elisa immunoassay kits according to the manufacturer's protocol. results: the increase in bmp4 and bmp7 serum level 24 hours after provocation test correlates significantly with the concentration methacholine during provocation time (p < 0.05). bmp7 serum level before the provocation, 1 hour and 24 hours after provocation, correlates negatively with fev1 change (p < 0.05). the median bmp7 level 24 hours after provocation was significantly lower in patients with negative methacholine challenge test compared to the control group (p = 0.03). the median bmp4 level 24 hours after provocation was higher in patients with positive allergen provocation test than in patients with negative test results (p = 0.03). the bmp7 serum level 24 hours after positive methacholine test is lower and correlates inversely with fev1 change in every time point, which could indicate that serum level of bmp7 is a predictive factor of fev1 change. the higher bmp7 serum level, the lower fev1 change was observed. this could suggest the protective influence of bmp7 in patients with obstructive pulmonary disease, i.e. asthma. the higher bmp4 serum level 24 hours after positive allergen provocation test result shows that the bmp4 could be an indicator of the response to a specific trigger. background: inflammation and coagulation are closely linked events. thrombin is the key enzyme in coagulation system. besides its well-known functions in hemostasis, thrombin plays a role in inflammation. the aim of our study was to evaluate thrombin generation in children with mild asthma and demonstrate associations between thrombin levels and control of asthma. method: forty-two children with mild asthma and forty-nine healthy children included in the study. asthmatic children had no asthma exacerbation during the last 6 months. patients (n = 27) who had mild persistent asthma, were using either inhaled steroid or montelukast. all patients performed spirometry. thrombin levels were measured by thrombin generation test. thrombin peak levels, endogenous thrombin potential, thrombin lag time, time to thrombin peak and thrombin tail time were recorded. results: thrombin lag time was significantly longer in children with asthma (3.98 ± 1.2) compared to those in control group (3.29 ± 0.6) (p < 0.01). children with asthma also had longer thrombin tail time compared to control group (19.5 ± 8.9 vs 16.7 ± 2.9, p = 0.02). thrombin peak was inversely correlated with fef 25-75 (-0.41, p < 0.01). thrombin lag time was inversely correlated with fef 25-75 (-0.39, p < 0.01). thrombin generation parameters did not show difference according to asthma control treatment, asthma control scores and having atopy. conclusion: coagulation/anticoagulation balance is disturbed in mild asthma but this disturbance may not be as strong as to increase thrombin levels. factors increasing inflammation may cause an increase in lag time, and increase in inflammation and excessive fibrin deposition may contribute to airway narrowing. background: cytokines represent key mediators in the onset and persistence of inflammatory process, in both asthma and copd. il-31, which belongs to il-6 family, it might act in a similar way with il-6 at the beginning of the inflammatory process. its role in atopic skin diseases has already been demonstrated, but there are conflicting results related to its role in respiratory allergic diseases. the aim of the study was the evaluation of il-31 plasmatic level in patients with asthma and copd and the its correlation with clinical and lung function parameters. method: fifty consecutive patients with bronchoobstructive diseases were included in the study. thirty-two patients presented asthma and 18 patients had copd. the evaluation included: number of exacerbation in the last year, disease's severity, spirometry. plasmatic levels of il-31 and il-10 were determined in all patients. results: the mean age was higher in patients with copd dermatophagoides pteronyssinus [house duste mite (hdm), 100ug/ mouse] were administered oro-tracheally on days 0, 7, 14, 21, 28, 35, 42 and 49 . at was performed in a treadmill during 4 weeks in moderate intensity, from day 24 until day 52. results: at inhibited hdm-induced total cells (p < 0.001), eosinophils (p < 0.01), neutrophils (p < 0.01) and lymphocytes (p < 0.01) in bronchoalveolar lavage (bal), and eosinophils (p < 0.01), neutrophils (p < 0.01) and lymphocytes (p < 0.01) in peribronchial space. at also reduced bal levels of il-4 (p < 0.001), il-5 (p < 0.001), il-13 (p < 0.001), cxcl1 (p < 0.01), il-17 (p < 0.01), il-23 (p < 0.05), il-33 (p < 0.05), while increased il-10 (p < 0.05). airway collagen fibers (p < 0.01), elastic fibers p < 0.01) and mucin (p < 0.01) were also reduced by at. at also inhibited hdm-induced airway hyperresponsiveness (ahr) to methacholine 6.25 mg/ml (p < 0.01), 12.5 mg/ml (p < 0.01), 25 mg/ml (p < 0.01) and 50 mg/ml (p < 0.01). mechanistically, at reduced the expression of stat6 (p < 0.05), stat3 (p < 0.001), stat5 (p < 0.01) and jak2 (p < 0.001), similarly by peribronchial leukocytes and by airway epithelial cells. socs1 expression (p < 0.001) was upregulated in leukocytes and in airway epithelial cells, socs2 (p < 0.01) was upregulated in leukocytes and socs3 down-regulated in leukocytes (p < 0.05) and in airway epithelial cells (p < 0.001). conclusion: at reduces asthma phenotype which is followed by positive modulation of socs-jak-stat signaling in peribronchial leukocytes and in airway epithelial cells. rodolfo a 1 ; paciência i 2 ; rama t 1 ; leão l 1 ; silva d 3 ; rufo j 2 ; mendes f 4 ; padrão p 5 ; oliveira fernandes e 6 ; moreira p 7 ; delgado l 8 ; moreira a 9 porto, portugal; potentially irritating chemicals that may have a cutaneous drying side effect. this study aimed to evaluate if skin barrier function, as measured by transepidermal water loss (tewl), is affected by a training session in swimmers compared with football players. environment impact on the human respiratory health (clinicaltrials.gov identifier: nct03017976) and football players were invited to participate. due to the lack of prior information no sample size calculation was possible and all athletes that provided informed consent were included in the analysis (n = 58, 29 females, aged 12 to 21 years). tewl was measured using the tewameter ® tm 300 before, immediately after, and 30 minutes after a 2 hours training session. the probe was held on the dorsum of hand, the volar forearm and the antecubital flexure for 60 s. the average of two consecutive measurements was recorded. non-parametric statistic was used were appropriate. ethical approval was obtained from the university clinical research ethics committee and informed consent provided. results: mann-whitney u test showed significantly higher baseline median tewl level on football players hand's dorsum compared with swimmers, median (p25-p75) respectively 17.1 (14.7 to 21.7) and 13.7 (11.9 to 16.2); p = .001. friedman test revealed a significant effect of swimming on tewl on the hand's dorsum, volar forearm and antecubital flexure (p < .001) while football training affected only the hand's dorsum (p = .008). differences in changes after swimming and football training were significant only for tewl in volar forearm (p = .028). in conclusion, our exploratory findings do not provide support for a specific deleterious effect of swimming, compared with football training, on the training induced changes in tewl. background: exercise-induced bronchoconstriction (eib) is defined as transient, reversible airway narrowing occurring during or after exercise, is common among elite athletes and associated with epithelial damage. however, little is known about the existence of eib in young athletes. the goal of this study is to investigate the presence and to evaluate potential (bio)markers of eib in young high-school elite athletes in different sport disciplines versus age-matched control subjects. method: high-school selected elite athletes (12-13 years) from different sport disciplines: basketball (n = 26), football (n = 44) and swimming (n = 47) performing at least 12 hours of sport per week (median=13 h) and 17 control subjects (performing less than 6 hours of sport per week) were recruited. the eucapnic voluntary hyperventilation (evh) test was performed according to ats guidelines and adapted for this age group. lung function was measured before, immediately after and 5, 10, 15 minutes after the evh test. the test was considered positive if a maximal fall in fev1 of 10% was measured on at least one time point and exhaustion was excluded. a blood sample was obtained at baseline. sputum induction and skin prick test for the most common allergens were performed after the evh test. results: fifteen swimmers had a positive evh test (33.3%), which is higher than in basketball players (18.2%), football players (17.95%) and controls (17.65%). 40.4% of the swimmers were atopic which is also higher than in basketball players (23.1%), football players (31.8%) and controls (23.5%). serum clara cell secretory protein (cc16) levels are significantly higher in swimmers (7.6 ± 2.5 ng/ml) compared to indoor athletes (5.1 ± 2.2 ng/ml) and controls (5.2 ± 1.2 ng/ml). a significant positive correlation was found between the magnitude of maximal fall in conclusion: young elite swimmers have a higher prevalence of eib compared to basketball and football players. atopy and/or chlorine is a risk factor for the development of eib in young elite athletes. cc16 levels and sputum uric acid levels are increased in athletes compared to control subjects suggesting the presence of epithelial damage already at young age. this is especially observed in young elite swimmers, pointing to a probable role of exposure to chlorineby-products in combination with intensive exercise. results: data from 237 subjects (134 females, 56.5%) were analyzed. frast was positive in 97 (40.9%) patients (55 females, median age of 14 years (iqr 11-18)). in this group, 55 (56.7%) had a previous diagnosis of asthma, 27 (27.8%) practiced federated sports, 18 (18.6%) had smoke cigarette exposition and 3 (3.1%) had a bmi >30 kg/m 2 . 82.5% of patients showed a δfev1% >10% in the first 5 minutes after finishing the challenge. median fev1 reduction was 13.4% ) and 390 ml . frast was more frequently positive in patients with previous diagnosis of asthma (p < 0.01). there were no differences related to conclusion: frast is an important tool to diagnose exerciseinduced bronchospasm without asthma (eib wa ), as well as to diagnose asthma. in our study, frast was fundamental to access eib wa in 28% of patients with rsee, and confirmed asthma diagnosis in 53% of cases with previous asthma diagnosis and negative sbt. there was no difference in the prevalence of atopy between patients with positive and negative frast. patients older than 9 years-old presented higher δfev1% compared to younger patients (p = 0.04). higher levels of feno were observed in patients with positive frast (p = 0.032, p = 0.045), both in patients with and without previous diagnosis of asthma. a positive correlation was observed between feno levels and δfev1% in the whole sample (r = 027, p = 0.009); when these data were analyzed considering a previous diagnosis of asthma, only patients with this condition showed a positive correlation of feno and δfev1% (r = 0.29, p = 0.031). conclusion: our results evidenced that higher feno was associated with atopy and a positive frast, both in patients with and without previous diagnosis of asthma. higher feno seems to correlate with δfev1% in patients with previous diagnosis of asthma. background: specific immunotherapy is the casual treatment for allergic rhinitis. a 28 year old professional footballer suffer from severe allergic rhinitis since two years. during may, june and july his level of playing, concentration and durability decreased about 20%. patient was complaining of runny nose, nasal blockage, each eyes, sneezing, tearing. method: we did skin prick tests -which showed greatest allergy to grass pollen. we confirmed the allergy by specific ige and nasal provocation tests. spirometry was done-fev1 116%. the patient was qualified to undergo specific immunotherapy. however, because of his profession, it was hard to find a day without trainings to get the vaccine. after long discussion, patient decided to start specific immunotherapy-scit. results: the patient start the immunotherapy. he was attuning very irregularly, because of matches, injuries, trips, trainings, and lack of time. several times we had to call the patient to remind him about the immunotherapy. after one year of scit the patient felt big improvement. during grass pollen season he suffered from mild allergic symptoms, and just for few days. after next year of immunotherapy, the patient had no symptoms of allergic rhinitis during the grass pollen season. however, it was the reason for him, to stop sit, before 3rd year of immunotherapy. conclusion: such a treatment-specific immunotherapy-is a burdensome method for both, for professional athletes and doctors. such a patients need to be on special observation, and cooperation with trainers must be obtained, if we want to see results. to improve compliance we have to keep in touch with patients, to remind them about next visit. gherasim a 1 ; choual i 1 ; radu c 2 ; khayath n 2 ; beck n 1 ; jacob a 1 ; schoettel f 1 ; domis n 1 ; de blay f 2 1 alyatec, strasbourg, france; 2 hôpitaux universitaires de strasbourg, strasbourg, france background: late allergic response (lar) is a good asthma model. it has been shown, in individual challenge tests that mite allergen induces more frequently late allergic responses (lar) than cat allergen. the aim of this study is to compare the frequency of lar in asthmatic subjects allergic to mite with asthmatic subjects allergic to cat. method: 24 asthmatic subjects allergic to mite were compared to 21 subjects allergic to cat (gina 1 or 2). the subjects had prick tests≥5 mm compared to the negative controls and specific ige ≥ 0.70 ku/l. the dose selected for the mite and cat allergen was the airborne allergen concentration inducing the most frequently early asthmatic response (ear) (a 20% drop in fev1) and lar (a 15% drop in fev1). results: the frequency of lar with mite allergens was 66.6% and 20% with cat allergens (p = 0.007). the frequency of ear for mites was 78.2%; of 91.3% for ear or lar, and 50% for ear and lar. in contrast, with cat allergens, 55% of patients had an ear, 60% had ear or lar and 25% had an ear and lar. no significant differences was observed between cat and mite allergen regarding the severity and the time necessary to obtain an ear and lar. no significant differences was observed between cat and mite allergen regarding the severity and the time necessary to obtain an ear and lar. the frequency of lar in asthmatic subjects allergic to dust mite exposed in alyatec ® eec was higher than in asthmatics sensitized to cats. our results confirmed previous results with individual bronchial challenge. therefore, it appears that the mite model is more interesting in the study of asthma. exposure chamber in strasbourg (alyatec ® ) in asthmatic patients allergic to cat allergens gherasim a 1 ; choual i 1 ; radu c 2 ; khayath n 2 ; beck n 1 ; jacob a 1 ; schoettel f 1 ; domis n 1 ; de blay f 1 1 alyatec, strasbourg, france; 2 hôpitaux universitaires de strasbourg, strasbourg, france background: as recommended by the task force on environmental exposure chamber (eec), allergenic and non-allergenic exposure must be better controlled in eec. it is the aim of alyatec's eec. the aim of the study is to validate alyatec's eec by determining the concentration of fel d1 inducing 50% of early asthmatic response (ear) and/or late phase asthmatic response (lar) in subjects sensitized to cat. method: it was a randomized, double blind, cross-over study including group a:20 asthmatic subjects allergic to cat and group b:10 asthmatic subjects allergic to another allergen. all subjects were first exposed to placebo. group a was exposed to 2 fel d1 concentrations. the number and size of particles were recorded online during the exposure. group b was exposed to the concentration of fel d1 which fulfills the objective of the study. the mean age of subjects was 29 years (±8). for the 2 concentrations of fel d1, we obtained more than 50% ear and/or lar. the mean time necessary to obtain an ear was: 59.7 ± 8 minutes and 138.6 ± 90 minutes for the lar. the mean fall in fev1 during ear and lar was −29.22% and −17.64% respectively. we didn't observe any severe reaction. no subjects in group b experienced any symptoms during exposure. we have validated alyatec's eec in asthmatic subjects allergic to cat allergens. we also demonstrated its specificity. background: the best test and strategy for diagnosing asthma especially in those patients with negative bronchodilator reversibility tests still remains unclear. in this study we aimed to investigate the diagnostic yield of peak expiratory flow (pef) variability for the patients with symptoms suggesting asthma but negative bronchodilator reversibility tests. method: subjects referred to our outpatient clinic with suspicion of asthma were enrolled in this study. demographics and referral symptoms were recorded, asthma control test (act) scores and health related quality-of-life scores (aqlq, sf36) were calculated. monitoring of pef variability during 2-weeks and bronchial challenge test with methacholine (bpt) were analyzed. asthma was diagnosed by having pef variability ≥20% and/or positive bpt. results: thirty out of 50 enrolled patients were diagnosed as having asthma. when we compare asthmatic patients with nonspecific respiratory symptomatic subjects there were statistically-significant differences regarding to wheezing (p = 0.020), activity limitation (p = 0.058), total symptom score (p = 0.028) and basal fef (p = 0.050) in the favor of asthma cases. multiple logistic regression analysis revealed that lower basal fef 25-75 was an independent predictive factor of asthma diagnosis (p = 0.05). when the bpt positivity was assessed as gold standard for the diagnosis of asthma, the sensitivity and specificity of pef variability for different cut-offvalues (≥20%, >15% and >%10) were 61.5-83.3%, 88.5-62.5% ve 100-16.7%, respectively. conclusion: fef 25-75 is an important diagnostic parameter for asthma. although current guidelines recommend pef variability of 10% for the diagnosis of asthma in general, this cut off level may not be appropriate for this defined group of subjects. our results suggest to use a cutoff level of >15% while excluding asthma and ≥20% while confirming the diagnosis of asthma for patients with asthma suspicion but without shown reversibility. de barayazarra s background: in recent years obesity has been considered as a factor that contributes to the development of asthma, increases exacerbations and leads to poor control of it due to resistance to drugs to control this pathology. it is known that obesity produces chronic systemic inflammation; one of the markers that are affected is the levels of c-reactive protein (crp), which are increased. objective: evaluate, lung function, the use of medications to control asthma and systemic inflammation, after bariatric surgery. results: 15 obese asthmatic patients with surgery, 15 non-asthmatic obese patients with surgery, 16 obese asthmatic patients without surgery. a significant difference was found between the severity of obesity and forced expiratory volume in patients with asthma and without asthma of 1 second (fev1) before surgery with an average of 87.9% at the beginning of the study and 105.5% at 12 months (p:0.0004). in the non-operated group, fev 1 at the beginning was 69% and 83.04% at 12 months (p: 0.0018). the crp, before surgery in all operated patients had crp: 9, at 12 months after surgery they became negative, crp: 0 (p: 0.004). in obese asthmatics with surgery at the beginning, 100% used medication, and at 12 months only 20% in obese asthmatic patients without surgery, 93.75% used the medication at the beginning, at 12 months 62.5% (p:0.0006). method: 20 patients with asthma aged 18 to 80 and a predetermined positive methacholine pc20 were recruited and underwent a single challenge to cause bronchoconstriction of~20% comparing the outcome of the device with spirometry. the subjects were monitored at baseline, after a~20% fall in fev 1 and after bronchodilation back to baseline. the study protocol allowed for an interim analysis of the initial 8 subjects at which point the sensor was calibrated to optimise sensitivity. a further 12 subjects were studied using the optimised sensor. results: all 20 subjects successfully completed the study. the device was found to be straightforward to use by both operator and subject with no concerns regarding safety. the initial sensitivity of the device was found to be suboptimal in the first eight patients to reliably detect changes in lung function. after adjustment to the device the tests results of the remaining 12 subjects were analysed. 66.6% of subjects were female. the mean age of all subjects was 45.8 years. an average baseline fev 1 value of 2.575 (s.d. 0.881) was observed. changes in lung function were detected in 100% of subjects. a baseline value, drop in lung function and reversal were measured in 66% of subjects. the mean percentage drop observed in 66% of subjects using the investigational device was 13.04 %. the mean percentage increase observed using the investigational from drop to reversal was 25.36 %. the device (using ebc 1 ) was able to detect changes in lung function tracked using fev 1 . this provided proof of concept that the device could potentially be used to monitor lung function more effectively in the home than peak flow and supports further development to optimise the device and demonstrate functionality in clinical asthma. method: ninety four patients under 18 years of age seen in the allergy department due to common asthma symptoms (wheezing, dyspnea, cough, chest tightness) with normal spirometry and negative bronchodilator response, underwent mct during 2016 and 2017. the variables studied were: sex, age, body mass index (bmi), asthma symptoms, exercise symptoms, rhinoconjunctivitis, family history of atopy, sensitization to respiratory allergens, spirometric data and fractional exhaled nitric oxide (feno). results: of the total sample, half were women (51.06%) and the other half were males (48.94%). mean age was 11.8 years. bmi was normal in most of them (with an average of 19.55 kg/m 2 ). the most common symptom among the patients with positive mct was cough (79.8%), followed by dyspnea (62.76%), wheezing (34%) and chest tightness (11.7%). 43.6% had symptoms of asthma with exercise and 70.2% had rhinoconjunctivitis. 37.23% had a family history of atopy. 74.4% were sensitized to aeroallergens, mainly to pollens (grass and olive tree). 75.5% of the mct′s were positives, with a mean pc 20 of 1.89 mg/ml. 62.7% had a moderate-severe result (pc 20 ≤4 mg/ml), 7.4% mild (pc 20 4-6 mg/ml) and 5.3% bordering (pc 20 6-8 mg/ml). the mean feno was 19.62 ppb. conclusion: in our series, the completion of a test of hrb was decisive to confirm the diagnosis of asthma in most patients of a pediatric population with symptoms of suspicion (cough, mainly), normal spirometry and negative bronchodilator response (with normal feno in most of them). therefore, we consider it important to include in the routine clinical practice hrb tests in the pediatric population with suggestive symptoms of asthma, despite normal functional and/or inflammation tests. 1000 | cut-points of the ′control of allergic rhinitis and asthma test′ (carat) asthma subscale based on an international survey patients with asthma) in kashan, iran. the data collection tool was a questionnaire with 38 questions, designed to gather information on demographic asthma patients, the current use of mobile functionalities, and the willingness to use these functionalities to receive selfmanagement services, which was distributed among patients with informed consent. the collected data were analyzed by descriptive statistics method using spss software. results: the most use of patients from mobile phone functionalities was to receive information about asthma symptoms and allergens and irritants via mobile internet (42.1%). patients were most likely to use social networking (31.7%) in comparison with other mobile phone functionalities, to receive reminders about appointments and medication. the respondents were most likely to use social networks through mobile phone functionalities, to receive asthma self-management information (53.1%), to communicate with other patients (55.9%), to receive reminders about medication use, and to perform a peak flow meter test (52.4%) and to get an alert when the asthma is not controlled (53.1%). the findings show that asthma patients are currently using the internet search for educational information and they have a tendency to use social networks to receive asthma-related services. patients believe that mobile health is an appropriate intervention for providing educational information, reminders, and alerts and communication with other patients. 1002 | concordance between the determination of asthma control through the gina 2015 guidelines and the act questionnaire-results of the efimera study background: the exacerbation of asthma, progressive worsening of acute episodes, is one of the most frequent attending reasons at hospital emergency unit 1 . several factors causing poor control of asthma, such as inadequate therapy, have been described. in the present study, estimations of asthma severity by researchers were assessed by comparing the concordance between the assessment of asthma control through the gina 2015 guidelines and the act questionnaire method: cross-sectional observational study on the evaluation of factors related to treatment that influence the poor control of asthma was assessed through the gina guidelines and the act questionnaire. patients referred to a pneumologist or allergist by a primary care for the first time were evaluated. two variables were collected for the assessment of asthma control: one derived from the gina 2015 guidelines and another derived from the act questionnaire. regarding the gina assessment, researchers' evaluations guidelines were compared with the scoring calculated from the variables registered in the crd. both measures were compared in terms of sensitivity-specificity to determine their ability to classify patients. the patients included in this study (n = 1682) had a mean age of 45 ± 17 years, with a 64% of women and an average disease evolution of 14.9 ± 14.1. the control of asthma according to "gina results: pts were reasonably representative of those in sls asthma (at sls baseline: 43.8% male; mean age 48.4 yrs; mean asthma control test [act] score 16.5) . the most frequently reported symptoms during sls asthma for these pts were cough/ breathlessness, followed by wheeze, phlegm and chest tightness; breathlessness and wheeze were perceived as the biggest impactors on pts' lives. the aspects of daily life most impacted by asthma were reported as walking at a hurried pace, strenuous physical activity, and asthma-related frustration. since sls began, 50% of pts in this subset reported improvements in overall asthma (44% no change; 6% worsening). perceived changes in symptoms are shown (table) . most pts (66.0%) reported avoiding places with dust, smoke or fumes. most pts (57.5%) perceived no change in overall qol; 36.3% reported improvement. being an act responder during sls (total act score ≥20 or ≥3 change at end of sls) was associated with reported improvements in overall asthma symptoms, lower impact of asthma on qol, and higher perceived confidence/control in managing asthma. more pts (65.8%) in the ff/vi arm reported an overall improvement in asthma vs uc (34.3%); the most evident differences between treatment groups were for breathlessness, wheezing and chest tightness. improvements in confidence/control in managing asthma were reported by 53.8%/49.3% of pts (ff/vi) vs 31.3%/25.3% (uc). conclusion: breathlessness and wheezing were key symptoms in sls asthma and had the biggest impact on pts' daily lives. this patient-centred study enriches the findings of sls asthma. funding: gsk (study 204500) 1004 | asthma and copd treatment adherence and breach using tai questionnaire suarez-vergara m; fuentes-soltero f; garcia-nunez i; ignacio-garcia j background: adherence is defined as medication (inhalator) intake following the dosage and schedule prescribed. adherence mistakes are a public healthy problem according to the big morbi-mortality presented in patients with an incorrect intake. our aim is to evaluate the adherence level and fulfillment in patients with asthma or copd using tai (inhalators adherence test) questionnaire. method: patients with a diagnosis of persistent asthma or copd were selected. we used to size adherence and breach type the tai questionnaire. adherence is defined as good when patient′s test reaches 50 points, medium (46-49 points) and bad (less than 45 points). breach type is defined as erratic when points between questions 1 to 5 are less than 25, deliberate when questions 6 to 10 are less than 25, and unconscious when questions 11 and 12 are less than 4 points. a correct fulfillment is defined when questionnaire reaches 50 points plus 4 points of conscious fulfillment. results: fifty-five patients more than 14 years old (mean age 50.61 years and 50.9% males) were selected. a 67.27% of them were asthmatics, 30.9% copd and 1.81% a mix phenotype. a 21.81% presented a correct fulfillment with conscious fulfillment, and the other 78.18% presented good, medium or bad adherence with a breach type. according to adherence level, a medium adherence was defined in 16 patients (29.09%), with an erratic mistake in 8 patients (50%). bad adherence was seen in 21 patients (38.14%) , with the three breach types in 9 patients (40%). good adherence with unconscious breach type was defined in 6 patients (10.9%). conclusion: tai questionnaire confirms a good adherence and fulfillment in less than 30% patients. an erratic mistake is the most frequent breach type defined in our patients. educational protocols should be applied to improve adherence and fulfillment. 1005 | what is adhesion to treatment of asthmatic patients like in argentina according to the tai questionnaire? background: asthma is a chronic inflammatory disease of the airways, which requires an adequate treatment and control. the adhesion of a patient to an asthma treatment is a critical factor in order to achieve and maintain control. this adherence arises from a consensual agreement of the doctor-patient relationship; it is a complex multifactorial variable in which the variability in human behaviour in relation to its environment influences. background: the association between ambient pollen and asthma has been studied intensively with inconsistent results, attributed to differences in study population, geographic factors (geoclimatic features), data sources, measurement of pollen (different types of traps), and different outcome occurrence (hospitalizations or emergency department visits). we investigated the associations between daily sales of short-acting β2-agonists (saba) and outdoor pollen concentrations in the central france area. the relationship between daily changes in pollen concentrations and daily saba sales obtained from the social security database was analysed with generalized additive models, taking into account confounding factors such as air pollution, weather conditions, and day of the week. results: the daily saba sales (mean, sd) rose from 46. 5 (23.6) conclusion: this study indicates that outdoor pollens contribute to asthma morbidity in the general population. it confirms the highly allergenic role of fraxinus, betula and quercus pollens, but also shows a relatively unknown association between treated asthma and carpinus and platanus pollens, despite their counts being less than 1% of overall pollen concentration. results: of 15 437 asthma patients (mean age 47.8 years, female 62.6%), regular ocs use was identified for 223 patients (1.4%), periodic ocs use for 3054 patients (19.7%), and no ocs use for 12 160 patients (78.7%) 1-year post-index. regular ocs users had a greater mean age, were more often male, and had greater eosinophil counts, lower lung function, and greater prevalence of comorbidities than did the periodic and no ocs users (p < 0.001). total yearly cost was greatest for the regular ocs users (€ 5509), followed by periodic ocs users (€ 2892) and no ocs users (€ 2042) (p < 0.001). among regular ocs users, hospital admissions were the main cost driver (41.5% of total cost), while gp consultations were driving the total cost in periodic and no ocs users (48.0% and 52.9% of total cost, respectively). conclusion: in this sample of patients with asthma in sweden, the total yearly cost of health care resource utilization for a regular ocs user is twice as high as for a patient with no ocs use, demonstrating substantial economic and clinical burden in asthma patients on regular oral steroid treatment. method: children with physician-diagnosed asthma who attended to an outpatient pediatric allergy and asthma center were enrolled in the study along with control subjects. asthma severity and control status of the patients were evaluated according to recent gina guidelines. laboratory investigations including skin prick tests, complete blood counts with differential, total ige levels, serum periostin levels and pulmonary function tests were performed. results: a total of 158 children (125 with asthma and 33 age and sex-matched control subjects) with a median age of 10.2 years (range 5.9-17.0) were enrolled. asthma severity was mild in 41 (32.8%), moderate in 63 (50.4%) and severe in 21 (16.8%) children. children with asthma had significantly higher periostin levels than controls (53.1 ± 13.1 vs 43.0 ± 11.2 ng/ml; p < 0.001). the mean serum periostin levels of children with severe asthma (63.8 ± 10.8) were significantly higher than in children with moderate asthma (53.3 ± 12.7) and mild asthma (47.4 ± 11.1) (p < 0.001). serum periostin levels were found to be significantly correlated with asthma severity (spearman's rho [r]=.41, p < 0.001). analysis using roc curves identified the role of periostin levels in determining children with severe asthma (auc: 0.77, 95% ci: 0.67-0.87, p < 0.001]. conclusion: serum levels of periostin, a novel asthma biomarker, were higher in asthmatic children, and were associated with asthma severity. adam i 1 ; selevestru r 1 ; rogut v 2 ; sciuca s 1 background: nowadays, data from several epidemiological studies confirm the important role of fungi in respiratory disease in the indoor as well as in the outdoor environment. in general, exposure to fungi occurs via inhalation, skin contact, or ingestion. alternaria alternata is one of the most common fungi associated with presence asthma and persistence and severity of asthma. although exposure to a. alternata is also may represent a risk factor for development of asthma. in ukraine has been an increase in the number of the mold sensitized children for the last few years. at the same time we can see increasing frequency ba at the children of pre-school age. method: thirty five children aged 3-7 years with allergic rhinitis and high level of asthma predictive index (api) sensitized to a. alternata were included in a 2-year cohort study of the efficacy and safety of slit (diater laboratories, spain) using standardized sublingual extracts containing molds (alternaria alternata). treatment efficacy was analyzed using the score of symptoms such as difficulty in nasal breathing, rhinorrhea, sneezing, itching of the nasal mucosa (upper palate) and discharge from the nose and recurring wheezing. we also have analyzed the level api during the period investigation. symptoms were measured before starting treatment, and at 4, 12 and 24 months after starting immunotherapy. results: slit significantly reduced both symptoms and medication score: nasal symptoms (38% vs. control group) and the use of rescue medications (38% vs. control group), and improved fev1 (in children aged≥5 years). in the slit group, api decreased by 15% for the first year, by 31% for the second year. no patient had a systemic reaction during therapy. our results have shown that slit is an effective treatment in pediatric patients suffering from allergic rhinitis and high api with significantly improved clinical outcomes (less symptoms and less medication intake) in comparison with children treated with symptomatic drugs only. in this study, large and statistically significant differences in symptom and medication scores were demonstrated in patients receiving slit compared to control group. sublingual immunotherapy is effective for allergic rhinitis in children especially early age and is generally advantageous because of the convenient administration and safety profile and ensure prevention of developed ba. bednarek a background: the classification of asthma based on the severity of its clinical course has been recommended by gina since 2008. this division is useful for the patient's initial assessment when asthma is being diagnosed and essential decisions concerning an appropriate therapy are made. the objective of the work is to evaluate the influence of a clinical form of asthma on vaccine immunity in preschoolers following three years after the programme of mandatory vaccination has been realised. the study encompassed 172 preschool children (mean age of 5.22 ± 0.34 years old) with asthma being newly diagnosed, including 140 patients with mild asthma and 32 ones with moderate asthma, whose vaccine immunity (igg specific antibody titer) was assessed after the mandatory early childhood vaccines had been administered. monovalent vaccines (hbv+ipv+hib) along with a three-component combined vaccine (dtwp) were given to 86 children while a six-component vaccine (dtap+ipv+hib+hbv) was given to the remaining 86 children. the vaccine doses were consistent with the polish immunisation programme and manufacturers' recommendations. the elisa immunoenzymatic method was applied to assess titer of specific antibodies to diphtheria, tetanus, pertussis, poliomyelitis and h. influenza type b. the level of hbv antibodies was measured chemiluminescently. the immunity class for particular vaccinations was assessed according to the test manufacturers' instructions. results: children suffering from mild asthma had considerably more frequently vaccinations on time (p < 0.001) and the type of vaccines (monovalent, highly-combined) administered to them did not have a significant influence on a clinical form of asthma in the children examined (p > 0.6951). apart from the vaccines against hepatitis b and rubella where considerably more frequently a high antibody titer occurred in children with mild asthma, the titers of antibodies to other vaccines, namely diphtheria, tetanus, pertussis, hib and mumps, were not associated with a clinical form of asthma. the protective antibody titers in the children with asthma were found in 100% after vaccinating them against poliomyelitis (≥12u/ ml) and measles (≥300 ml u/ml). significantly higher current weight was solely found in the children with mild asthma (m = 20.80, sd=3.77; p < 0.05). conclusion: there are some clinical and cultural differences among the four southern chinese cities within the canton province. this study identifies potentially modifiable environmental and treatment factors associated with poor asthma control and qol for healthcare interventions. having a smoker in the family is independently associated with poor asthma control and qol. were classified into two groups (levocetirizine group (l) and montelukast group (m)) and we treated each group for another 10 week. to evaluate the therapeutic effectiveness, we used symptom score (ss) and ebc leukotriene e4 (lte4). ebc samples were collected with rtube. each parameter was checked at 0, 2, 12 week therapeutic period. results: most ar patient showed clinically improvement with 2 and 12 week fluticasone therapy (0 wk ss=6.6, 2 wk ss=3.2, 12 wk ss=1.9 p < 0.01 in l group; 0 wk ss=6.8, 2 wk ss=3.6, 12 wk ss=2.0 p < 0.01 in m group). lte4 levels of ar were higher than control (0 wk 77 vs. 12 pg/ml), and were reduced after 2 week flutimic were: md allergic rhinitis, wheezing apart from colds, eosinophilia ≥4%. outcome was defined as md asthma and at least 1 episode of asthma during the previous year or more than 3 episodes of wheezing during the 12 months regardless of asthma diagnosis. results: from a total of 144 of parents approached, 86 (58%) agreed to participate in a phone interview. 18 (21%) children were diagnosed with asthma. the age at the time of admission (mean, abstracts | 533 (sd)) was 27.0 (9.9), at the time of phone survey 77.6 (10.9) months, respectively. positive loose api at 2-3 years of age had sensitivity of 77.8%, specificity 75%, positive predictive value (ppv) 45.2%, negative predictive value (npv) 92.7%. positive stringent api at 2-3 years of age had sensitivity of 50%, specificity 91%, ppv 64.3%, npv 85%. background: asthma is the most common chronic airway disease in childhood, with a high unmet need for new treatments due to insufficient symptom control in a relevant percentage of patients. ethics and resource factors limit the feasibility of large, long pediatric trials required to assess outcomes such as exacerbations and symptoms. for diseases like asthma, where the disease process is largely similar in children and adults, with the same expected therapy outcome, the international council for harmonisation advise extrapolating adult data to those of a younger age, reducing unnecessary pediatric trials. here we assess the partial extrapolation used in the clinical development of tiotropium. phase 3 trials in adults (aged 18-75), adolescents (aged 12-17) and children (aged 6-11) with symptomatic severe (primotina-/pensie-tina-/vivatina-asthma) or moderate asthma (mezzotina-/rubatina-/ canotina-asthma), respectively. trials lasted 12-48 weeks, all with tiotropium respimat 5 μg add-on vs placebo as two puffs once daily. results: in adult trials, lung function, symptoms and exacerbation endpoints were evaluated in a confirmatory manner: tiotropium significantly improves lung function and asthma control, and reduces risk of exacerbation, vs placebo ( conclusion: based on similarities in disease profile and magnitude of treatment responses between age groups, it is reasonable to expect tiotropium add-on to produce clinically meaningful improvements in exacerbation and symptom endpoints in children and adolescents, as in adults. the robust tiotropium clinical program supports using a partial extrapolation to avoid overly long and large trials in pediatrics. 1019 | clinical state of treatment and examination during last 3 years before remission about asthmatic children in long-term remission cases method: remission cases (no symptom and no therapy) for 3 years of asthmatic children were studied. clinical background and treatment (drugs) was studied during last 3 years before remission annually. acetylcholine inhalation test by standard method was performed, and respiratory threshold of acetylcholine (rt-ach) was obtained. fev1%, and serum ige also examined. these data were compared before remission with 3 years after remission. results: mean age of 25 cases at 3 year before remission was 9.2 years old. male to female ratio was 1.3. severity of asthma was all mild type, and number of attack was 2 to 8 times in a year. there was no admitted case during this study. the long-term therapeutic drugs were leukotriene receptor antagonist (anti lt) in 20 cases, and/or inhaled corticosteroids (ics) in 12 cases, but 5 cases had no treatment for the control. geometric mean of rt-ach (after then: 3 years before and after remission) was 2900 μg/ml and 5800 μg/ml. the mean fev1% was 83% and 90%. geometric mean of serum ige level was 360 iu/l and 410 iu/l. complicated cases of atopic dermatitis decreased after remission, but the incidence of allergic rhinitis increased slightly. conclusion: characteristics of asthmatic children during last 3 years before remission were mild type, had several times of attack in a year, and the treatment was mainly anti lt and/or ics. fev1% was within normal range, and serum ige level was not changed after remission. rt-ach had the tendency to improve during 3 years before and after remission. these data is supposed that airway hyperresponsiveness is one of the indicators for quitting treatment. 1020 | clinical aspects of polyvalent mechanic bacterial lysate (pmbl) treatment in children with uncontrolled asthma our results indicate that long-term treatment with omalizumab in children can help to achieve better asthma control and reduce the amount doses of basic therapy. method: allergic rhinitis (ar) and allergic rhinoconjunctivitis (arc) diagnosed-patients' demographic information, accompanying-asthma, the allergic history of the family, the onset of symptoms, types of aeroallergens sensitivity were noted from patients' files in our hospital's pediatric allergy clinic. results: in this study, 675 patients were evaluated. the mean age of the patients were 11.65 ± 3.85 years and 61% (n = 412) were male. 452 (67%) patients had ar and 223 (33%) patients had arc. background: allergic rhinitis (ar) is a disease characterized by symptoms of nasal discharge/congestion, sneezing, and pruritus, and is caused by an ige-mediated immunological response to inhaled allergens. we aimed to evaluate pollen season and out of pollen season pulmonary function tests (sft) of patients with ar in our study. method: in our study, the demographic characteristics and aeroallergens were recorded from patients' files with ar diagnosed. in addition, pollen season and out of pollen season sfts were evaluated and compared. conclusion: in patients with ar, fev1 and fvc values are seen to be lower during the season even though there is no lower respiratory symptom. therefore, sfts of patients with ar should be evaluated during pollen season. results: among the clinical manifestations, the most common combination of allergic rhinitis (ar) and conjunctivitis (ac) is noted in 83.80% of adults and 55.11% of children, but in children aged 3-7, the combination of ar and ac is observed only in 48.40%, among 8-12 years old-51.25%, while in the remaining age groups it is encountered in more than 80%. higher percentage of isolated ar is also observed among young children-17.55%, and those of the results: allergic rhinitis was a main symptom in 69.8% of children with pollen-food sensitization. in all of them concomitant allergic disorders were noticed: bronchial asthma (77.7%), atopic dermatitis (77.8%). only in 26.7% temporal association between ingestion of pollen-related foods and nasal symptoms was observed (mainly apple and peanuts); occurring also outside the pollen period. the simultaneously sensitization to animal origin food allergens was stated in 63.3% of children with sar, but only in two of them milk and white egg proteins were an additional exacerbation factor of nasal symptoms. in 18.5% anaphylactic reactions to food allergens were registered. 36.7% of children were asymptomatic despite pollen-food sensitization. the statistically significant differences were noticed in comparison to the control group. conclusion: 1. allergic rhinitis in children, similar to adults, is a common manifestation of pollen-food syndrome and this type of sensitization should be taken into account regardless to age. 2. children with pollen-related food allergy have the predisposition to multiorgan clinical manifestation. 3. the lack of association of symptoms with plant-origin foods in the majority of cases and the asymptomatic course of food sensitization in more than one third of patients indicate the need for follow-up. 1026 | clinical benefit of the screening of suspected food allergen using multiple allergen simultaneous test in the patient with pollen-food allergy syndrome (pfas) background: the quantitative fluoresce enzyme immunoassay immunocap (ic) system has been widely used for detection of allergen-specific ige for the diagnosis of allergy. however, the system can only detect ige against a single allergen, the multiple antigen simultaneous tests has been developed such as the fluorescence enzyme immunoassay view allergy 39 (va) or chemiluminescent enzyme assay mast iv (ma) and both assay detect more than 39 allergen-specific ige. in this study we examined the diagnostic capability of these two systems for screening test in the patient with pfas. method: total number of participants are 37 (male/female: 20/17), aged 12.0 ± 3.9 (range 3~20) years old. all the patients showed oral allergy syndrome (oas) to rosaceae family plants (apple, peach) and/ or kiwi and/or banana, also showed tree pollen allergy. specific ige assay were performed using ic, ma or va. results of greater than class 1 were to be regarded as positive, and the concordance rates between the assays were assessed. results: the correlation of sensitivity between pr-10 (rbet v1, rmal d1, rpru p1, measured by ic) and specific ige to apple (measured by va), specific ige to peach (measured by ma) in 27 oas patients to rosaceae family plants were assessed. rbet v1, rmal d1, rpru p1 were found to be 96.0%, 96.3%, 92.6% positive measured by ic while the specific ige to apple (supposed to be including pr-10) were found to be 100% positive measured by va. on the other hand, the specific ige to peach (supposed to be including pr-10) were found to be only 33.3% positive measured by ma, this detection rate was lower than that of va (p < 0.0001). also, the correlation of sensitivity between pr-10 (ract d8, measured by ic) and specific ige to kiwi in 17 patients with oas to kiwi were assessed. ract d8 were found to be 76.5% positive measured by ic while the specific ige to kiwi (supposed to be including pr-10) were found to be 64.7% and 17.6% measured by va and ma, respectively (p < 0.005). additionally, all the 3 oas patients to banana found to be positive for the specific ige to banana measured by va, but only 1 patient was detected as positive measured by ma. conclusion: in this study, we found that va showed better agreement of sensitivity and specificity with ic compared to ma in the oas patients to rosaceae family plants, kiwi, or banana. therefore, it may be clinically useful for screening of allergen specific background: the hygiene hypothesis for autoimmune and allergic diseases, which exists nowadays, shows that human immune system is dependent on various environment factors. we consider the effects of humic substances (hs) to be important in understanding the hygiene hypothesis. due to urbanization, the amount of human interaction with hs found in soil has significantly dropped. the goal of our work was to study allergenic potential and antimicrobial activconclusion: hs appear to be exogenous immunocorrectors, and also to have an ability of suppressing propagation of allergic reactions and sensibilization, which leads to conclusion that they seem to play a major role in hygiene hypothesis. moreover, hs selectively interact with bacterial cell wall, and this effect could be used in order to create antimicrobial drugs based on hs. background: peach tree pollen has been identified as having relevant allergens (the third most prevalent after olive tree and grass pollen) in areas of high cultivars (murcia, east-spain). when analyzing molecular components in sensitized patients, along with pru p 3, we have identified other relevant inhalant allergens one of which was named pru p x. because pollen of different species share allergens and with plantderived food, we have also studied peach tree pollen sensitization in a non-exposed population (madrid, central-spain). the aim was to study the association between peach tree pollen and several panallergens, as well as the relevance of pru p x in our area (madrid). method: a total of 328 patients who came to our allergy unit in those patients with positive spt to at least one pollen we also performed peach tree pollen spt. if positive, we tested pru p 3, pho d 2, pho d 3 and pru p x. to study the clinical relevance of these findings, we also performed nasal provocation test (npt) with peach tree pollen and pru p x. results: a total of 57 patients were sensitized to peach tree pollen. from these, 33% had also positive spt to pru p 3 and none of them to pru p x. positive spt to polcalcin were found in the 33% of the cases and to profilin in the 13%. in 7 patients sensitized to peach tree pollen npt was performed being 4 cases positive to peach tree pollen and none to pru p x. conclusion: peach tree pollen sensitization in non-exposed patients with allergy to other pollens is high although primary sensitization is unlikely. these patients present clinical response when exposed to that pollen that needs further evaluation. in our study, one third of the patients were also sensitized to polcalcin and pru p 3 and none to pru p x. we have not found clinical response to this new inhalant allergen identified in highly exposed peach tree pollen population. results: the bet v 1 elisa 2.0-ep complete kit format (including pre-coated plates and all buffers and reagents) allowed for the consistent measurement of bet v 1 in birch pollen extracts within the same lab (intralab cv=5.5%) and between different labs (interlab cv=13.5%). the average recovery from matrix spiked samples (crs in birch pollen extracts) ranged from 75-112%, with an average recovery of 91% (n = 10). assay time was reduced from several days to two hours compared to the original method. the performance of the bet v 1 elisa 2.0-ep kit was comparable to that of the stallergenes greer candidate standard method and has been successfully cross-validated. this will enable allergen manufacturers and regulatory authorities to adopt a standard method for bet v 1 determination, which, ultimately, may be included in the european pharmacopoeia. the development of a certified elisa represents a major step forward in the standardization and quality control of allergen products. 1031 | an isoform of the ole e 7 allergen assembled by proteomics could explain the cross-reactivity with pollen and food nsltps results: a total of 457 peptides were obtained by de novo sequencing. ten of them allowed the completion of the full-length amino acid sequence of the allergen. after purification, role e 7 was obtained with a yield of 1.5 mg/l of cell culture. immunological assays confirmed that the recombinant isoform of ole e 7 shared most of the allergenic and antigenic properties of the natural allergen. moreover, we observed its implication in cross-reactivity with pollen extracts, and plant-derived food extracts. conclusion: these results suggest that the presence of this isoform in the olive pollen could explain the co-sensitization observed in some allergic patients between ole e 7 and nsltps from foodderived extracts and might be used for a more effective clinical diagnosis of olive pollen sensitized patients. background: penicillium oxalicum, one of the prevalent airborne fungi in india, was selected to detect its spores as potential source of allergens and also to identify and characterise its major ige-reactive component. the airborne spores of penicillium oxalicum was detected by andersen 2-stage air sampler at different parts of west bengal. the allergenic potency of p.oxalicum was tested by spt, elisa and immunoblotting. total protein was resolved in 1-d and 2-d gel electrophoresis and allergens were identified by 1-d and 2-d immunoblots. identification of major ige-reactive protein spots was made by mass spectrometry based maldi-tof-tof. major allergen was partially purified by ion exchange chromatography. results: aerobiological investigation clearly indicated the predominance of p. oxalicum spores (56 cfu m −3 ) in the air of west bengal, india. sensitivity of patients to spore antigens was highly correlated with rhinitis. in sds-page, 80 bands were detected with molecular weight range of 16-180 kda. the allergenic potency of spores was confirmed by skin-prick test, elisa and dot-blotting. eleven ige-reactive proteins were detected as allergens by 1-d and 2-d immunoblots, of which 43% patients were sensitized to 22 kda allergen. this 22 kda protein was found to be the major allergen which was further characterized by mass spectrometry based maldi-tof-tof. this major allergen (pi 6.08) was partially purified by ion exchange chromatography. the eleven allergens were identified from spore of penicillium oxalicum fungi for the first time from india. immuno-proteomic identification of major ige-reactive protein (22 kda background: airway epithelium (ae) is one of the largest cellular surfaces exposed to the environment. ae constitutes a physical barrier due to the presence of intercellular apical junctional complexes between neighboring cells. in the past years evidence indicates an association between epithelial airway dysfunctionality and allergic asthma. it is still unclear if an impaired epithelial barrier could be the cause of allergy development as opposed to the consequence. one of the most common comorbidities of asthma is house dust mite (hdm) allergy. it has been shown that hdm allergen der p 1 can disrupt the epithelial airway due to its protease action against cellular apical junction complexes damaging the epithelial monolayer. in the last decade, metabolomics has been successfully employed as a new approach to describe metabolic changes in biological systems. metabolomics focuses on describing and identifying small molecules to explain complex biological processes. we theorized that metabolomics could be used as a new tool to detect damage of epithelial barrier in vitro after der p 1 exposure. method: human cell line calu-3 cultured at air-liquid interphase (ali) was used as an in vitro model of bronchial epithelium. ali culture system allows establishing 2 different compartments, mimicking the conditions found in the human airways: a basolateral compartment in which basolateral surface of the cells is in contact with the culture medium, and an apical compartment where the apical cellsurface is exposed to air. after 7 days in ali, the cells were exposed to either der p 1 or pbs as a control in the apical side for 24 hours. then, apical and basolateral media were collected and processed for metabolomics analyses. results: metabolic profiles from samples were obtained, these were composed by 248 and 397 features for apical and basolateral media, respectively. of these, using mann-whitney unpaired test as statistical analysis, 108 and 15 features were found changed within the apical and basolateral compartments, respectively. specifically, in the apical compartment there were 104 signals significantly increased and 4 decreased after der p 1 exposure; whereas for the basolateral compartment, 11 signals were found to be significantly decreased and 4 increased after exposure. background: mites are one of the major causes of allergies. it is known that allergen concentration varies depending on the species of mites and the degree of allergy induction is different, but the difference in microbiota according to mite species is not known. in addition to allergen, endotoxin or bacterial dna, adjuvants of allergen derived from the microbiota in the mites, are also present in the feces. bacterial endotoxin is found in gram-negative bacteria, acting on tlr4 and acting as an adjuvant to allergies. method: three species of mites (d. farinae, d. pteronyssinus, and t. putrescentiae), known to cause allergies, are cultured in same condition(autoclaved media, 80%rh, 25°c)and analyzed for microbiota of each species. using the next generation sequencing that complements the existing sanger sequencing, we analyze the difference of microbiome according to the dust mite species and measure the level of endotoxin. method: six hundred and thirty five patients (51.8% males and 48.2% females, mean age 30.2 years old, range 3 to 82 years old) were included. all of them referred respiratory symptoms (rhinitis, conjunctivitis or bronchial asthma) and had skin prick tests positive with any pollen. patients were skin prick tested with a battery of common pollens in our area, including three species of chenopodiaceae: chenopodium album, salsola kali and salsola oppositifolia. results: three hundred and forty tree (54%) patients were sensitised to pollen of any chenopidaceae species: 255 (40.2%) to chenopodium album, 245 (38.6%) to salsola kali and 220 (34.6%) to salsola oppositifolia. the prevalence of skin sensitisation to pollen of salsola oppositifolia was 34.6% in the population studied and 64.1% in patients sensiresults: in patients aged 1-60 years of age in 80.5% of the cases ige reactivity was at least to one allergen tested. the majority of patients (more than 2/3) had a complex sensitization profile and reacted on average to more than 5 allergens. the highest frequency of sensitization in ukraine among patients who turned to the clinic among adults was found phl p1 (34.1%), amb a1(33.3%), fel d1 (33.3%), bet v1 (30.2%) and children (29.2%, 34.6%, 38.5%, 36.9%), respectively. when analyzing the results of tests for the source of the allergen, most often among house dust mites (hdm) allergens in adults and children is sensitization to fel d1 (35.9%), as well as to hdm: in adults (der f2-15.1% der p2 −15.1%, der f1-12.7%, der p1-11.9%) and in children (12.3%, 13.1%, 9.2%, 10 .8%), respectively. among fungal allergens the most common is sensitization to alt a1 and varies from 2.4% in adults to 26.9% in children. among pollen allergens in adults is sensitization to phl p1 (34.1%), amb a1 (33.3%), bet v1 (30.2%), cynd1 (23.8%), art v1 (22.2%), bet v2 (11.1%) and in children (29.2%, 34.6%, 36.9%, 19.2%, 16.9%, 21.5%), respectively. tests for food allergens in adults and children are more common on pr-10 proteins. in children, sensitization to milk and egg proteins is more common than in adults. conclusion: most patients who came to the clinic have a complex ige reactivity profile in which pollen sensitization predominates. among hdm allergens, more than 1/3 of the examined have sensitization to the cat's proteins. sensitization to mold alternaria alternata in children occurs 10 times more often than in adults. results: total 130 children were examined, aged 1-16 years (median 5 years). 72% children were sensible to two and more components 57.7%to 5 and more components. the frequency of sensitization to inhalation components was 65.4%, to food abstracts | 545 components-33.1%. among the most frequent inhalation components were feld 1-39%, betv1-37%, amba1-35%, phlp1-29%, alta1-27%, the sensitization to house dust mites (hdm) was most often observed to der p2-13%. however, the analysis of these protein by the level of isu showed that the highest levels were for der f1median 19 (iqr 9.4-47.0), whereas for fel d1-6.9 (iqr 2.6-15.0). among food allergens, sensitization was most commonly observed to pr-10 proteins -42%. children sensitized to pr-10 proteins were in most cases sensitized to -mal d1(28%), cor a1.0401(27%), pru p1 (22%).this co-sensitization was accompanied by a high correlation of isu levels among these components. sensitization to celery and kiwi was less common, the level of these proteins was also low. the frequency of sensitization to storage proteins was 38%, among which the highest level of isu was in ara h6 median 13 . sensitization to ltp proteins was detected in 22% of children, among which the most commonly detected pru p3 protein was 9.2%. the sensitization to profilins, which was evaluated at the level of bet v2, was found in 21% of children, but the levels of these proteins were not high. among the food products of animal origin, the most frequent was sensitization to egg component gal d2 −13.1%, however, isu levels were the highest to milk component bos d4 −9.8 (iqr 1.2-21.0). the most frequent causative inhalation allergens were epidermal allergens and weed pollen, however, the highest level of isu was to hdm and mould. among food allergens, the most commonly observed sensitization was to pr-10 proteins. hypereosinophilia of peripheral blood was observed in 87 children under study, which was 50% (4.7%). as a result of testing patients with a wide panel of allergens, 98% of the patients had diagnostic levels of antibodies to allergens siged1, 91.2%to allergens siged2. in 50% of cases, a significant level of antibodies to plantain allergens sige w8 was detected, 30.8% to dandelion allergens sige w9, 35.7% to evergreen trees sige t23, to maple sige t11 to 33.3%, to allergens of olive tree sige t9 -40%, to the banana allergens sige f92 -63.6%, to the egg protein sige f1 in 24.5%, in 34% to the milk allergens sige f2, to the food mixture sige f x5 -37.7%, to allergens of mold fungi mx2 −17.8%. among the leading household allergens were registered in the 1st group and in the 2nd group of the investigated children -d pteronyssinus (80.3%, 97.8%), and d. farinae results: the prevalence results are expressed in the table 1 . we have not observed any significant association in allergic rhinitis patients group with any ltp or pr-10 molecules. for atopic dermatitis only rara h 9 (or with 95% ci -10.2 (1.9-54.6) and njug r 3 (or with 95% ci -6.7 (1.6-29.5)) were associated significantly. for asthma, the most important molecules were rbet v 1, raln g 1, rcor a 1.0101, rcor a 1.0401, rmal d 1, rpru p 1 and rapi g 1 (p-values for or less than 0.05). conclusion: future studies focusing on the evaluation the association of cross-reactive molecules with allergy phenotype should be done. background: the fuzzy/green kiwifruit (actinidia deliciosa), widely grown commercially, contains various pulp allergenic molecules, including the major allergen cysteine protease actinidin. methods. this case report is about a 40-year-old male patient with house dust mite allergic persistent rhinitis and intermittent asthma, presenting a convincing history of anaphylaxis immediately after eating a kiwifruit on empty stomach, followed, a few months later, by a severe oral allergy syndrome after licking a slice of raw kiwi. previously, the patient ate kiwi without any problems and had no manifestations of pollen or latex allergy. skin prick testing was done with commercial allergen extracts, while prick-prick testing was performed with raw kiwifruit, avocado and banana. molecular approach consisted in assessment of serum specific ige to native extracts and molecular allergen components using patient-friendly allergen nanobead array multiplex test and singleplex capsule-enclosed activated cellulose solid phase fluorescence enzyme immunoassay. results. regarding kiwifruit allergy, the patient presented positive prick-prick tests with raw edible kiwifruit components: outer pericarp and inner pericarp (each 15 mm wheal) and columella/core (19 mm wheal) and negative with kiwifruit whole seeds, avocado and banana, and pollen extracts. serum specific ige to kiwifruit were detected (0.5 ku/l), but specific ige values were negative (≤0.01 fiu/ml) for actinidin act d 1, thaumatin act d 2, kiwellin act d 5, nsltp type 1 act d 10, bet v 1-like major latex/ripening-related protein act c 11, act c chitinase_iv, act d 9 cross-reactive profilins bet v 2 (birch pollen profilin) and hev b 8 (latex profilin), and also negative (<0.1 ku/ l) for pr-10 ract d 8. moreover, specific ige to avocado were nor found (≤0,01 fiu/ml). although ige against seed proteins cupin/11s globulin act d 12 and 2s albumin act d 13 were not determined, this was not considered of great importance since allergic symptoms were also induced by licking kiwi pulp, in which abundantly expressed actinidin enzymatically degrades seed storage proteins, and prick-prick test was negative to kiwifruit seeds. conclusion: in a patient with anaphylaxis to kiwifruit, positive skin tests to its pulp and detectable serum specific ige to actinidia deliciosa, a detailed molecular allergy diagnosis is necessary, including assessment for act d 3 glycoallergen or other molecules, not performed in this patient. 1049 | is pr-10 sensitization a portuguese phenomenon as well? background: bet v 1, a major allergen found in birch pollen, belongs to the pr-10 protein group. in our practice, some bet v 1 sensitized patients have been identified, residing in areas without this tree genus in its flora. our aim was to characterize a portuguese patient population with pr10 sensitization. method: a group of patients in whom immunocap isac ® (isac) study was performed, between january 2009 and june 2017, were analyzed. all subjects with one or more pr-10 sensitizations were selected, and their clinical records reviewed. a sequential sample of the last subjects (n = 80) who underwent isac study, was then used for comparison. results: out of 234 isac studies performed, only 16 were positive for pr-10 protein group. median age was 18.3 years, 68% (n = 11) were male. pr-10 sensitized individuals were more likely to live in portalegre district compared to the control group (7/16 vs 1/80; p < 0.001). 15 patients were positive for pr-10 family pollens (93.7%), frequently bet v 1 (n = 13), followed by aln g 1 (n = 10) and cor a 1 (n = 8). 14 out of the 16 patients were sensitized to pr-10 foods, mostly cor a 1.0401 (n = 13) and mal d 1 (n = 12). skin prick tests revealed birch as the main sensitizing pollen as well (14/ 16). moreover, only four patients were skin prick tested for fagaceae trees which were positive for oak (4), chestnut tree (3) and cork tree (1) . all patients were co-sensitized to other pollens, namely grass and all had respiratory allergy. nine patients were food allergic, although seven of them were co-sensitized to other cross reactive (ltp/profilin) or species specific proteins. conclusion: although pr-10 sensitization is known to be rare in our population, mostly alto alentejo inhabitants showed sensitization to this protein family in our sample, either by in vitro and/or in vivo methods. this phenomenon is consistent with the native plant species of this region, which should be taken into account when studying the allergic profile of these patients. in our sample, all pr-10 sensitized patients had respiratory allergy while this protein didn't seem to be relevant when it comes to food allergy. further studies are needed to characterize which plant species belonging to this protein family are more significant for our country's aerobiology context and to determine its clinical relevance. included. allergic asthma, rhinitis, conjunctivitis and eczema allergic symptoms were diagnosed. all patients were tested by immunocap with mugwort pollen extract and the natural components nart v 1, nart ar 2, nart v 3, and nart an 7. results: the positive frequency and sige levels of the four components in the artemisia allergic patients from southwestern china were significantly lower than that from the north. art v 1 and art an 7 were the highest recognized allergens, followed by art v 3 and art ar 2. patients from northern china were more likely to have abstracts | 549 asthma (50%) than patients from southwestern china (3%), and being sensitized to more than two allergens increased the risk of asthma. sensitization to art v 1, art v 3 and art an 7 played a significant role in the development of asthma. artemisia pollen allergic patients is helpful to assess the potential risk of asthma. conclusion: a small but significant part of the population react to ragweed pollen extract and are not identified as disease-positive by standard sige tests. there is a need for targeted tests towards a larger spectrum of allergen molecules. in ragweed allergic individuals, this allergy can be the main cause of overall sige levels and also of in vivo reactions (tested by spt). 1052 | molecular profile of pollen sensitization of tashkent residents with respiratory allergy background: in paediatric cohorts, a correlation between specific ige (sige) levels to house dust mite extract or allergen components and the occurrence of asthma has been shown. higher levels of sige to mite extract were associated with a higher risk of wheezing. moreover, asthmatic children recognized more allergens and had higher sige levels to nder p 1 as well as rder p 2, 5 and 23. we sought to investigate potential differences in sige levels or sensitization patterns between asthmatic and non-asthmatic patients in a mixed paediatric and adult house dust mite allergic cohort. method: total ige and specific ige against house dust mite extracts (dermatophagoides pteronyssinus and farinae) and allergen components (rder p 1, 2, 10, and 23) were determined in 190 house dust mite allergic patients. 35 patients had diagnosed asthma ("asthmatic", 54% females, mean age 27 ± 15 years, 31% younger than 18 years), whereas 155 had rhinitis (and conjunctivitis) without respiratory symptoms ("non-asthmatic", 54% females, mean age 28 ± 15 years, 29% younger than 18 years). results: total ige levels were markedly higher in asthmatic compared to non-asthmatic patients (315 vs. 144 ku/l, p = 0.022). positivity to rder p 1 (71 vs. 52%, p = 0.039) as well as rder p 10 (9 vs. 1%, p = 0.044) differed between both groups. specific ige levels to house dust mite extracts and allergen components (rder p 1, 2, 10, and 23) and positivity to rder p 2 and 23 did not differ between both groups. conclusion: in contrast to previously published data, sige levels to house dust mite extracts or allergen components were not statistically different between asthmatic and non-asthmatic patients in our mixed paediatric and adult house dust mite allergic cohort. only higher total ige levels and a higher reactivity to rder p 1 and 10 were found in asthmatic patients. however, larger studies are needed to confirm clinical relevance of these findings. results: prior treatments reported at baseline (bsl) included: 17.3% of pts were receiving 1 or more second-generation h 1 -ah at approved dose (recommended first-line), 23.9% were receiving them at increased dose (second-line); 8.5% were receiving omalizumab (third-line); 29.9% had no treatment. the majority of pts (78.2%) had uncontrolled csu (uct<12) at bsl (table) . treatment changes were most evident at the bsl visit, with an increase in pts receiving omalizumab (21.4%) and a decrease in those receiving no treatment (12.8%) vs. prior therapy. these changes were associated with improvements in rates of hives and/or angioedema, uct and qol scores at month 3, but only modest improvements thereafter (table) . a sub-analysis of 528 pts with uct<12 and who were receiving the approved (36.9%) or increased dose h 1 -ah (63.1%), revealed that few pts had recommended escalation from the approved to increased dose h 1 -ah (0.0-6.7%) or from increased dose h 1 -ah to omalizumab (0.0-11.1%) (table) . conclusion: poor physician adherence to guidelines was evident throughout aware. initial improvements in disease activity and qol plateaued after month 3, possibly owing to fewer changes to recommended therapies. greater physician adherence to guidelines is needed for better symptom control in pts with uncontrolled csu. results: we revealed that in russians urticaria is associated with rs20541*arg/gln genotype of the il13 gene (p = 0.02) and rs5743794*cc genotype of tlr6 (p = 0.0011) gene polymorphism. in tatars the association with disease development was shown for rs5743571*tt genotype of tlr1 gene snp (p = 0.0054). the rs5743794*c allele of tlr6 gene polymorphism is associated with acute and chronic forms of urticaria (p = 0.0209 and p = 0.0063, respectively) and rs2243250*c allele of il4 gene polymorphismwith acute urticaria (p = 0.0247). method: 100 csu patients from the urtica cohort (clinicalttrials.gov number: nct01940393) participated in the study. a questionnaire was carried out evaluating the triggers identified by the patients, the comorbidities and the treatments received. patients with a self-report of skin exacerbation by foods, nonsteroidal antiinflammatory drug (nsaid) or physical triggers were subjected to a controlled provocation test with the suspect food, medication or physical stimuli report by the patient. the levels of anti-tpo ige were measured during a period of clinical control and during two exacerbations in all patients. results: 30% of the patients had at less one inducible urticaria demonstrated by provocation tests (24% dermographism, 10% cold, 8% pressure). self-reported exacerbation for a food (60%) or medication (30%) were high, but positive provocation tests were low (1% and 14% respectively). 30 patients had (+) anti-tpo ige during the baseline period. among them, 60% presented a significant elevation of anti-tpo ige during at less one of the two exacerbations. 18.5% of patients (n = 13) with (−) anti-tpo ige, presented elevation of anti-tpo ige one of the two exacerbations. conclusion: foods, drugs and physical triggers must be verified by challenge tests to avoid unnecessary lifestyle restrictions in patients with csu, nevertheless self-report is usually greater than positive provocation tests. increase concentrations of anti-tpo ige seems to be implicated in urticaria exacerbations in some patients with csu. brzoza z 1 ; adamczyk k 2 ; wcislo-dziadecka d 3 ; zbiciak-nylec m 2 ; brzezinska-wcislo l 2 adipokines. the aim of the study was to evaluate the possible contribution of leptin to chronic spontaneous urticaria pathophysiology. the study included 48 chronic spontaneous urticaria patients and 41 healthy subjects. the leptin level in both examined groups was measured. results: no statistically significant difference in leptin level was determined between the studied subgroups. we are among the first to present the effects of exploration aimed at assessment of the possible role of adipokines in chronic spontaneous urticaria pathogenesis. in this study we did not prove any difference in leptin level. in our opinion it is valuable to perform further studies in this area. the microorganisms were inactivated with phenol, and the concentration was adjusted to 1 000 000 microbial cells/ml (labeled as a 1/ 1). dilutions 1/2 and 1/4 were made from the product labeled 1/1. the dot blot technique was used to detect the presence of specific ige to the different microbial antigens and controls (anti ige 1/1 and 2 fold dilution ½ and ¼). the dot blot images were processed with a documentation system (gel doc ez, bio-rad), and the different microbial antigens in different dilutions were compared with the positive anti-ige controls. results: all patients have specific anti ige to microbial antigens (see table below). the presence of microorganism-specific ige could explain, the relationship between the infections and / or microorganisms in ciu, as well, the urticaria control by omalizumab, even when it has not been detected ige sensitizations to common allergens. finally, these findings, showed that the bacterial allergy could be one line of research to understand the unresolved etiology of urticaria. background: dermographism is the most common form of inducible urticaria. it shows itself as hives made by scratching or rubbing on the surface where it has been produced and with the same morphology. the pathogenesis has not been clarified nor has it been associated until now with the sensitization to allergens. we have studied the relationship between the presence of dermographism and domestic mites sensitization. we have selected 95 patients older than 14 years old. all of them had symptoms compatible with dermographism at the moment of medical evaluation. at least one third of patients additionally showed rhinitis and/or asthma symptoms. we performed:: -skin prick tests with our basic neumoalergens (mites d. pteronyssinus y lepidoglyphus destructor, pollen, molds, dog, cat and horse dander, latex and anisakis simplex). -determination of specific ige levels for dermatophagoides pteronyssinus, lepidoglyphus destructor, and anisakis were measured in serum by using the immunocap (thermo fisher scientific). -blood count, serum immunoglobulins, antithyroid antibodies, serine tryptase and proteinogram. results: blood count, serum immunoglobulins, antithyroid antibodies, serine tryptase and proteinogram were normal. we divided patient in different groups. background: urticaria results from the appearance of pruritic papules and/or erythematous plaques caused by substances from mastocytes present in the skin, notably histamine. chronic urticaria is defined as flare-ups that occur at least two or three days per week over a six-week period. in addition, affected subjects are often prone to an atopic or auto-immune profile that promotes urticaria [1] . the association of polyphenols (ambora, green tea) and the soothing active ingredients slow down the itching biological process from the outset by reducing the release of pruritic mediators (e.g. histamine, cytokines, etc.) of immune cells such as mastocytes and lymphocytes, involved in urticaria. in this context, the purpose of the study was to evaluate the efficacy and the tolerance of an anti-pruritic spray containing the polyphenols and the soothing ingredient. the tested product aims to quickly calm the itching in subjects with chronic urticaria. results: on average, the product was applied 2.3 times per day with a significant decrease of 5d-pruritus scale (-35%) and sensations of itching (-58%) between d0 and d21. in terms of quality of life, a significant decrease of the skindex score was observed (-48%). the product soothed the pruritus within 60 seconds for all subjects and the anti-pruritic effect lasts at least 2 hours for 80% of subjects. the product also showed very good cosmetic properties and was well tolerated; no intolerance case was reported. showed near complete remission. in the week before omalizumab and for a few days after, her urticaria flared but on 3 of 4 weeks she was largely asymptomatic (uas7 0-5). after 3 years of successful treatment she reported an increase in csu activity. no trigger factors could be identified. add-on treatment with cyclosporine was refused, montelukast showed no, and prednisolone only transient benefit. over a period of 2 months wheals occurred almost daily and a maximal score of 42 was achieved on uas7. we replaced omalizumab with cyclosporine but this was subsequently discontinued due to side effects. 3 months later the patients' csu remained poorly controlled with up to 100 wheals occurring almost daily despite rupatadine 40 mg/d. due to the good initial response to omalizumab and lack of good treatment alternatives, a trial of re-treatment was considered. results: within 1 week of re-commencing omalizumab she once again achieved near complete remission of csu with uas7 ≤ 3 on 3 of 4 weeks. the mechanism of action of omalizumab and the development of resistance to it in csu, are incompletely understood. our case shows that some csu patients developing resistance to omalizumab may benefit from a subsequent trial of re-treatment, particularly if treatment alternatives are poorly tolerated. manipulate and store data by electronic means. this includes e-mail, sms text messaging, video chat and online social media as well as all the different computing devices that perform a wide range of communication and information functions. a rapid increase in the use icts in recent decades is an enormous contributing factor in the development of a number of novel clinical and public health intervention strategies. the aim of the present study is to assess the level of ict use and to examine patterns of preferences among patients with chronic urticaria (cu). method: we will conduct an anonymous multicentre cross-sectional study, starting from january 2018, to investigate the use of icts in patients with cu, using a questionnaire as a survey method. this questionnaire will assess the frequency of use of social media and icts in patients, and their preferences for receiving and asking disease-related information. the survey will consist of 20 items, evaluating demographical information, time with disease, medication currently used, and additional aspects of social network use. results: we will use a chi-squared test to assess the association between internet access or owning a cell or smartphone, and age, gender, type of urticaria, educational level and number of years since diagnosis. we will employ the same test to assess the association between the independent variables previously introduced and the frequency of use of each ict type (short messaging service [sms], facebook, twitter, youtube, email, internet, linkedin and skype) as well as agreement in receiving and seeking information (i.e. asking questions to the practitioner) through such icts. we will perform adjusted regression analyses between categories of age, gender, educational level, type of urticaria, years since diagnosis and the use and level of interest shown in communicating through icts. our aim is to report on remarkable findings from a registry of a large sample of patients, potentially providing clues for its approach and results: 285 patients with a median length of 14 months suffering from urticaria were registered, being 71% women; mean age 35.9 years. in 72% of patients no causal agent was identified. parasites were found in 9.3% and thyroid peroxidase antibodies in 8.9%, while autologous serum skin test was positive in 47% and igg to mycoplasma in 42% of evaluations. two thirds of patients reported wheals on uas7, with just 1/4 having concomitant angioedema. almost 2/3 reported significant affection on quality of life because of itch by cu-q2ol. just 14% of patients achieved total control on first anti-histamines provided, and less than half had good control of urticaria. cetirizine was the first choice in 44%, followed by fexofenadine (15%) and first generation anti-histamines (16%). method: cases at 18-65 years of age which were being followedup in our clinic with diagnosis of chronic urticaria and were not receiving any antihistaminic medication for last one month were included in the study. cu-q2ol, uas-7, psqi and psg results of the patients were evaluated. correlation of data with each other in regard to sleep disturbances was evaluated. results: 21 patients were included in the study. patients' mean total score in cu-q2ol was 36.25 ± 13.23. patients' mean uas-7 value was 16.71 ± 9.41. mean total psqi was 9.75 ± 3.92, the ratio of total scores ≥5 and those with poor quality of sleep was 87.5%. mean epworth sleepiness scale (ess) score was 9.71 ± 2.05, with total score ≥10 in 52.4%. in psg, mean apnea-hypopnea index (ahi) was 11.93 ± 12.6, with 44.4% of the patients having ahi ≥5. when patients having ahi<5 were compared with patients having ahi ≥5, no significant difference was determined in regard to total cu-q2ol score, mean score for questions concerning status of sleep, uas-7 and psqi. when correlation analysis was performed between cu-q2ol and total score for questions concerning status of sleep, a positive correlation was determined with psqi (p = 0.037). conclusion: it was demonstrated in our study that patients with chronic urticaria had poor quality of sleep and this disturbance was independent from ahi. omalizumab was discontinued due to absence of improvement in csu symptoms after three consecutive doses. the plasmapheresis without intravenous immunoglobulin replacement was initiated. results: the symptoms were relieved during the first procedure and the disease improved shortly thereafter. the following weeks the symptoms still occurred but with lower intensity and severity. (angioedema was gone). the second attempt with omalizumab was successful after this course (5 procedures of plasmapheresis). case report: rosacea is a chronic skin disorder associated with flushing, erythema, dryness, burning and stinging, and inflammatory papules and pustules. new treatments available or in development target the inflammatory and erythematous components of the disease. these agents include the selective alpha-2 receptor agonist brimonidine. allergic contact dermatitis to brimonidine is an unusual condition. in addition to this, urticarias due to brimonidine are rarely reported. we report on a 35-year-old woman who, immediately after apply a thin layer of brimonidine gel as preparation for a rosacea treatment on her face developed facial urticaria, which reverted in approximately four hours with systemic steroids. she had previously tolerated this product without any problems, but has not use it again ever since. skin prick-tests with brimonidine (0.03 mg/ml) and latex were realized in the patient. skin prick-tests with brimonidine were realized in eleven healthy control subjects. results: skin prick-tests with latex was negative in the patient. skin prick-tests with brimonidine were positive in the patient (9x5 mm). the prick-test with brimonidine was negative in teen healthy control subjects. we report on a case of immediate urticaria due to brimonidine and triggered by an immediate, probably ige-mediated, hypersensitivity mechanism. we highlight this case because it is the only case described in the literature with a positive prick-test. method: the study was in accordance with the helsinki declaration and was previously approved by the national comity of ethics. this was a one dose study conducted on 16 fasting young healthy volunteers, of which 11 were females and five males. the mean age was 21 ± 1 years old and the body weight 61. 13 + 9.44 background: cetirizine is a potent h1-receptor antagonist indicated in the treatment of allergic rhinitis and urticaria. cetirizine is widely used due to its potent antihistaminic effects in yielding strong and fast relief of itchy sensation, sneezy and rhinorrhea and its unlikely probability to manifest anticholinergic side effects in therapeutic doses. histamine flare and wheal inhibition by anti-h1 are widely used as a standard to test and compare the effect intensity and duration. our study aimed to test these effects of cetirizine in young healthy adults. method: this was a double-blind, single dose study in healthy young adults, previously approved by the national comity of ethics. eleven females and five males with a mean age 21 ± 1 years participated in this study. histamine skin pricks were tested before and after they received a tablet of 10 mg cetirizine as previously scheduled. twenty minutes after each test flare and wheal were drawn in a transparent paper which was then scanned and measured with a software. wilcoxon signed ranks test two-sided with significance at 5% level was used to analyze the differences. claims that the preparation relieves itch within 60 seconds of its application. we performed a simple study to verify this claim. we used irp in 20 consecutive subjects, 15 males, median age 12, range 6-49 years, whose workup implied ast. their preliminary diagnoses were "asthma" (6 subjects), "allergic rhinitis" (10 subjects), "atopic dermatitis" (2 subjects) and "food allergy" (2 subjects). all of them had refrained from systemic antihistamines for at least one week. standard skin prick tests (spt) were applied as appropriate, including histamine controls to assess the level of their skin sensitivity. subjects were asked to mark their sense of itch in the area of the skin to be tested on 100 mm visual-analogue scales (vas) starting from "0"-"no itch" to "100"-"unbearable itch". vas assessments were repeated 20 minutes after ast was done; then irp was applied according to the manufacturer's instructions, and the vas assessments were repeated after 60 seconds and 20 minutes. results: there vas assessments are shown in table format: table 1 irp did not affect the wheal and flare of the histamine control, nor did it abolish positive spt. no differences were outlined between subjects with different diagnoses. the commercially available itch relieving preparation not containing defined pharmacological antihistamine is effecting in relieving itch associated with allergen skin testing. before ast (1) 4.9 ± 2.6 vs (2) p < 0. represent the first-line treatment for osteoporosis-related mastocytosis. we report a case of sm with bone pain and with an area of osteolysis in the femur as first sign and symptom. we had to consider the risk of adverse reaction when we decided to treat the patient with bp, but the patient was under antihistaminic treatment and also we made a premedication to reduce the risk. the pk/pd model available was informed by data from 12 clinical trials. the pd endpoint data was available from two studies and used to characterize the effect of bilastine on wheal and flare. moreover, food effect had been characterized in 2 pk studies and the data was used to model the effect of food in the pk of bilastine. the pk parameters relative to the fed state were then used to simulate the temporal evolution of the wheal effect using the pk/ pd model. all analyses were conducted by nonlinear mixed effect modeling (nonmem v 7.2). using the pk model developed (food effect model) and the pk/pd model already available, monte-carlo simulations for plasma concentrations and pd over time were performed for both the fed and the fasting states. results: . a reduced bioavailability (f) and a slow absorption constant characterized the pk of bilastine when administered concomitantly with food (f = 77% relative to the fasting state and ka = 0.51 hour −1 , a 3-fold reduction compared to fasting conditions). the rest of the pk parameters remained unchanged. onset of action was 1 hour for bilastine both in fed and fasted conditions. maximum wheal inhibition occurred at 3.5 hours (fasted 78% and fed 77%). from 2 to 12 hours, the percentage reduction with bilastine for both fasted and fed was between 75% and 86% after the third day of treatment. a 50% inhibition in wheal effect was maintained during 23 hours for both conditions after the third day. the results of the simulations show that even if the pk is altered with food, the pd is maintained unchanged. conclusion: even if a significant food effect was described for bilastine at a pk level, the difference is not translated directly into the pd. therefore, the antihistaminic effect of bilastine remains unaffected by the concomitant administration with food. the results of these simulations will be further confirmed in a dedicated clinical trial. results: we also found no correlation between the different tgt parameters and other clinical and analytical parameters associated with uc (table 1) results: both cetirizine products have no differences in respect to the pharmacodynamic and pharmacokinetic parameters analyzed. the 95% confidence interval of the mean ratios of the auc 0-24 , auc 0-inf , cmax, auce 0-24 , and e max , between the test and the reference, were within the bioequivalence ranges (80%-125%) in both cases. no statistical difference was revealed when comparing the respective t max and te max too. the two cetirizine products tested were bioequivalent. the bioequivalence was evident even when tested with the pharmacodynamic parameters. there is strong evidence that supports the use of histamine skin prick test for the bioequivalence evaluation of different cetirizine products. 1089 | bradykinin-mediated angioedema associated with combination of angiotensinconverting enzyme and dipeptidyl peptidase iv inhibitors: a disproportionality analysis from the who database method: we performed a disproportionality analysis using data from the who pharmacovigilance database by a case-noncase study, until the 14/12/2017. we extracted all individual cases safety reports (icsrs) included in the high level term "angioedemas", according to the medical dictionary for regulatory activities classification. given the absence of term "bma", we selected only the icsrs of angioedema without associated symptoms evoking another underlying mechanism, such as histamine angioedema (e.g. pruritus, urticaria, rash, etc.). drug class exposure was "acei" and "dpp4i", considered suspect or concomitant, using the atc classification. we results: there was no correlation between mother's disorders such as periodontitis, rhinitis, diabetes etc. and the onset of ar (p > 0.05). a multivariate analysis showed, neonatal jaundice (p < 0.001), respiratory system infection (p < 0.001), diarrhea (p < 0.001), eczema (p < 0.001) in the first 6 months of life and home environmental factors (house decoration (p < 0.001), mold environment (p < 0.001), keeping flowers (p < 0.001), passive smoking (p < 0.001)) increased the risk of ar. besides, there was no significant difference in current height and birth weight of the participants between ar and control group. however, ar group had significantly lower current weight (p = 0.003) and age (p < 0.001) compared with the control group. paternal age and maternal age in the ar group were significantly higher than the control group (p < 0.01). conclusion: diseases in the first 6 months of life and home environmental factors increased the risk of sequential ar. the older parents increased the possibility of ar in the offspring. the data of general characteristics of participants were statistic analysis by z text analysis. *significance at p < 0.05. results: anosmia was more frequent in crs than in rhinitis (28.1% vs 3.9%, p < 0.001) and in crswnp than in crssnp (40.6% vs 13.4%, p < 0.001). lms was higher in crs than in rhinitis (8 [4-15] vs 0 [0-0], p < 0.001) and in crswnp than in crssnp (10 [6-18] vs 5 [1] [2] [3] [4] [5] [6] [7] , p < 0.001). in addition, lms was associated with loss of smell in patients with hyposmia (or = 2.66 [1.27, 5.53 clinics. patients were submitted to confirmatory exams including oral provocation test with aspirin. nasal polyps were removed by functional endoscopic sinus surgery and eosinophils in this tissue were quantitated. eosinophil counts in peripheral blood was obtained. serum periostin was measured by elisa and total ige was determined using immunocap. as control groups, 12 (9f/3m) patients with par and 23 healthy subjects (14f/9m) were selected. samples of nasal tissue and blood were collected from these subjects during elective surgery for correction of anatomical variations, and compared with the patients with aerd. results: ar symptoms were significantly improved in the treatment group compared with the control group (76.6% (36/47) vs 21.7% (10/46); p < 0.05). furthermore, the mean total vas score for patients in the treatment group was reduced from 6.21 ± 1.83 before treatment to 1.36 ± 1.51 after treatment (p < 0.05). moreover, the reduction in free ige levels was greater in the treatment group than in the control group. the results of this study suggest that the chinese herbal medicine ber may be effective for improving the symptoms of ar. a multicenter clinical trial is needed to confirm this finding. results: in patients with "eosinophilic" polypoid rhinosinusitis, mucociliary transport was 35.2 ± 0.80 minutes, ph 7.4 ± 0.01, suction-88.6 ± 6.5 minutes, excretory-57.9 ± 0.9 mlg and in patients with "neutrophilic" polypous rhinosinusitis, mucociliary transport was 34.5 ± 0.65 minutes, ph 7.3 ± 0.01, suction-76.2 ± 5.0 minutes, excretory-54.9 ± 0.8 ml. the study showed that disruption of the transport function, changing the concentration of hydrogen ions method: this prospective controlled study was carried out on crs patients underwent ess. patients participating in the study were divided into two groups-group 1: partial middle turbinectomy (n = 22) and group 2: partial middle turbinectomy and middle turbinate fenestration (n = 23). objective assessment of olfactory function using the university of pennsylvania smell identification test (upsit) and subjective assessment of symptom using visual analogue score (vas) were performed before and 3 months after surgery. results: there were significant improvement comparing postoperative and preoperative upsit in both group 1 (35.23 ± 2.96 vs 32.23 ± 2.54, p = 0.000) and group 2 (36.09 ± 2.35 vs 32.04 ± 2.64, p = 0.000). the vas were also significantly improved postoperatively compared to preoperatively in both group 1 (5.77 ± 0.75 vs 6.73 ± 1.08, p = 0.000) and group 2 (5.13 ± 0.81 vs 6.78 ± 1.28, p = 0.000). patients undergoing partial middle turbinectomy and middle turbinate fenestration were more likely to show improvements in upsit (4.00 ± 1.20 vs 3.00 ± 1.11, p = 0.014) and vas (1.59 ± 0.21 vs 0.95 ± 0.15, p = 0.000) compared to those with only partial middle turbinectomy. conclusion: partial middle turbinectomy and middle turbinate fenestration during ess is an effective method for improving postoperative olfactory function. 1109 | nasal irrigation for the alleviation of nasal symptoms in pregnant women with allergic rhinitis we sought to determine specific ige responses to bacterial pathogens in sera from cystic fibrosis patients and analyze their kinetic during disease course. genes, respectively. in contrast, most of healthy donors had normal homozygous genotype with tt-60.0 ± 8.9%(n = 18) and cc-56.6 ± 9.0%(n = 17) with low frequency of mutations; gg-16.6 ± 6.8%(n = 5) and tt-23.3 ± 7.7%(n = 7) and heterozygous genotype tg-23.3 ± 7.7%(n = 7) and ct-20.0 ± 7.3%(n = 6) for il-2 and il-4 genes, respectively. following a 2 month treatment, there was a significant reduction of cytokine levels in the il2-29.5 ± 0.5 and increased in the il4-16.6 ± 0.4, when compared to the beginning of therapy and after 2 months (p < 0.001) results: at baseline the 1st group had serum levels of il-2 (43.6 ± 0.8) pg/l; il-10 (34.8 ± 0.8) pg/l and ifn-γ (108.2 ± 1.1) pg/ l; 2nd group had il-2 (39.6 ± 1.5) pg/l; il-10 (38.6 ± 1.2) pg/l and ifn-γ (103.3 ± 1.4) pg/l vs il-2 (21.6 ± 0.8) pg/l; il-10 (50.2 ± 1.2) pg/l; ifn-γ (63.8 ± 2.2) pg/l in the control group. after 2 months, there was a significant decrease in pro-inflammatory cytokine levels in the 1st (il-2: 35 ± 0.9; ifn-γ: 75.6 ± 1.9) pg/l and 2nd group (il-2: 28.6 ± 1.3; ifn-γ: 66.7 ± 3) pg/l, respectively. conversely, il-10 increased in 1st and 2nd groups to 41.9 ± 0.9 pg/l and 46.0 ± 1.7 pg/l (p < 0.05). conclusion: prior to the study initiation patients with tuberculosis had higher il-2, ifn-γ and lower il-10 content than healthy controls. two-month chemotherapy produced significant reduction in proinflammatory cytokines and increase in anti-inflammatory il-10, with levels approaching those of healthy controls. thus, tuberculosis drugs appear to have the anti-inflammatory effect in tuberculosis patients, which was predictive of positive clinical outcome. 1114 | antibiotic resistance: ligands of innate immunity take the challenge in this work, we aimed to perform an ex vivo hrsv infection in precision-cut lung slices (pcls) from human, rhesus, and cynomolgus macaques, comparing whenever possible the response with the viral surrogate poly i:c. method: pcls containing airways were prepared from lung sections of human, rhesus, and cynomolgus macaques. the slices were inoculated with hrsv-a2 10 6 iu/ml, uv-inactivated hrsv, or vehicle control for 48 hours. macaque slices were also incubated with poly i:c 100 μg/ml with and without the immunosuppressive dexamethasone 50 μg/ml. viral replication, tissue viability, and immune response assays were assessed in supernatants, lysates, or slices. the inoculum infectivity of 10 6 iu/ml as well the uv-inactivation were confirmed by plaque-assay on hep-2 cells. immunofluorescence staining using a fitc-labeled anti-rsv showed the presence of infected macrophages in pcls, but not in mock infected samples. hrsv stimulation slightly decreased tissue viability, as seen by live/dead staining and ldh assay. the viral infection increased ip-10 production in pcls of human, rhesus, and cynomolgus macaques, reaching respectively 13.3, 3.4, and 1.7 fold-increase in comparison to the vehicle controls. poly i:c stimulation caused ip-10 response comparable to hrsv in rhesus and cynomolgus pcls. the ip-10 production ratio comparing hrsv/poly i:c was 1.1 in rhesus and 0.9 in cynomolgus pcls. conclusion: hrsv infects ex vivo pcls of human and non-human primates, inducing the release of the pro-inflammatory chemokine ip-10. this response is comparable to the viral surrogate poly i:c. in the future, these systems can be used to further investigate host response to hrsv, especially in the context of asthma development. however, a relatively small number of reports are related to the association of ebv with allergic diseases, in particular atopic ones. we found that among patients with activated ebv infection, polysensitization was found to be 2.2 times more frequent, chest syndrome was 1.32 times more common and hyper-ige syndrome occurred 1.5 times more frequently. in most of these patients, atopy was not detected in medical history. method: we evaluated the laboratory test results of five boys (45.4%) and six girls (54.6%), 11 children (6 with hbov and 5 with cov). their average age at the study time was 59.2 ± 45 months. nasal swab specimens were taken from these patients who admitted to our hospital with respiratory symptoms between 2015-2016. patients are recalled after an average of 21 ± 2.5 months. isaac questionnaire and skin prick test to common inhalated allergens were performed. results: only one patient had family history of atopy. forty percent of the patients with cov and 50% of the patients with hbov developed rhinitis. one patient with cov and one patient with hbov developed recurrent wheezing. one patient with cov developed atopic dermatitis. all skin prick tests were negative. it was noteworthy that 72.7% of the patients were passive smokers. conclusion: hbov and cov may be associated with rhinitis but there is a need for more patient groups for a clear result. rna_lig (ccg-agg-aug-cga-ggc-uug-uu) . to study chemotaxis in vitro, a boyden96 chamber was used -wellfiltrationplatemultiscreentm -mic with a pore size of 8 μm (millipore, usa). chemotaxis was studied in dynamics after 10, 60 minutes and a day using the above ligands. as control, rpmi-1640 medium without glutamine was used (paneco, russia). the statistical analysis was carried out using the computer statistical program biostat2009 conclusion: with all the data provided, a drug induced hypersensitivity was diagnosed. we present a case of immediate allergic reaction with eosinophilia due to carbapenems, with tolerance to other beta-lactams antibiotics. written informed consent of patient has been obtained in the two cases. discussion: the first case shows cutaneous immediate hypersensitivity response to infbeta1a. literature reports a few cases of urticaria and anaphylaxis but this is the first for the pegylated formulation. polyethylene glycol (peg) confers to a drug modified pharmacokinetics, solubility and immunogenicity. immediate reaction to peg (macrogol) have been described when combinated in vaccines or drug pils. dmf is a known cause of contact dermatitis related to footwear, wallets and furniture. flushig is a reported side effect of dms in ms managed with dose reduction. this case shows the possibility to immediate sensitization to dmf. as the armamentarium to treat ms now combines immunomodulatory and biologic drugs, the avaliability of diagnostic and desensitization protocols for hypersensitivity reactions must be keeped in mind. case report: drug rash with eosinophilia and systemic symptoms (dress) syndrome is an uncommon but serious hypersensitivity drug reaction, manifested with rash, fever, lymphadenopathy and visceral organ involvement. table) . drug withdrawal and prednisolone treatment leaded to attenuating of mentioned skin symptoms within 2 days, associated by occurrence of a exfoliative dermatitis. one week after admission, the patient developed fever that lasted for 4 days with enlarged lymph nodes on submandibular, paracervical, axillar and inguinal regions. a preventive antibiotic therapy is started and 2 weeks later, the lymph nodes were not palpable and the skin got the normal appearance. corticoid therapy is reduced gradually according to symptoms resolvement. case 2: a 56-year old woman presented to our department with a 6-day history of pruritic, macular rash, periorbital swelling, cheilitis and fever. she had started some weeks ago the allopurinol for asymptomatic hyperuricemia, had longer history for treatment of arterial hypertension and type-2 diabetes mellitus (olmersartan, nitrendipine, methyldopa, furosemide, regular and glargine insulin), and experienced nephrectomy and cholecystectomy. the patient was febrile, while blood tests revealed eosinophilia, increased seric creatinine/urea levels (due to nefrectomy), and severely-altered liver parameters (see table) . the allopurinol withdrawal, topical and systemic corticoid therapy, and the liver protectors attenuated serologic transaminases levels and patient's skin lesions within few days, followed by substantial improvement of laboratory findings one week after therapy start. the treatment dosage was gradually tapered and finally stopped within a period of 2 months in accordance with attenuating and complete resolvement of the clinical and laboratory abnormalities. our case demonstrated that dress syndrome is a severe drug reaction, but the immediate introduction of treatment and supportive measures can improve disease's outcome even after a temporary exacerbation or severe affection of internal organs. case report: a 40-years-old woman, diagnosed of ischemic cardiopathy, developed an anaphylactic shock 5 minutes after the administration of 2 ml sulphur hexafluoride intravenous during an echocardiogram. she was treated in emergency room with a total recovery. 2 months earlier, she had developed an extensive erythematous-maculopapular rash converging in plaques in relation with adhesive dressings which had been placed during a hospitalization due to thoracic pain. an allergic contact dermatitis was suspected and recommendations thereon were given. interestingly, an arteriogram with iodixanol (icm) was carried out one week before skin reaction with good immediate tolerance. methods: blood test: blood count and serum chemistry were done during both reactions to contrast media. serum tryptase level was not measured during the anaphylaxis, but its baseline level was quantified later. conclusions: we present a patient with a double sensitization to parenteral contrast media: an anaphylactic shock due to sulphur hexafluoride and an atypical delayed exanthema related to iodixanol, and diagnose was obtained with st in both cases. this is the first documented case with a positive immediate st to sulphur hexafluoride. with the culprit drugs mixed with 10% and 30% petrolatum resulted negative. patient was suspected to have behcet's disease, and consulted to rheumatology department. oral colchicum dispert twice a day was prescribed. afterwards, patient achieved to take oral amoxicillin-clavulanate for a week without any hypersensitivity; and has been following by oral colchicum dispert maintenance therapy since then. the reported patient had one anaphylactic perioperative reaction to morphine and another anaphylactic reaction to tramadol during her diagnostic investigation. remain the question if this patient had two allergic anaphylactic reactions with cross-reaction between morphine and tramadol, or two non-allergic anaphylaxis due to "hypersensitive" mast cells. case presentation: a 61-year-old female was diagnosed with rectal adenocarcinoma. one year after radical surgery, progression with pulmonary metastasis was shown. in first line of systemic therapy she received premedication with pantoprazole, metoclopramide, clemastine and dexamethasone, followed by cetuximab infusion. during first minutes of infusion, grade 4 anaphylactic reaction occurred. a reaction started with generalized pruritus, urticaria, rhinitis, followed by hypotension, bradycardia and loss of consciousness. she was treated with fluids, clemastine and methylprednisolone. next day she received same premedication followed by panitumumab. during first 30 minutes she had grade 1 reaction with generalized urticaria. the third day she had generalized urticaria 10 minutes after metoclopramide application. skin prick tests with cetuximab (5 mg/ml) were negative, but intradermal test were posibat response was highly positive for both cetuximab and alpha-gal, with comparable values and dose response curves. thus, we showed 70%, 63%, 58%, 35% and 4% of cd63 positive basophils for stimulation with cetuximab (500-0.1 μg/ml), and 67%, 40%, 17%, and 1% for stimulation with alpha-gal (33.3-0.033 ng/ml). bat response to panitumumab was negative (<5%; 500-0.1 μg/ml). drug provocation with panitumumab was negative and patient received treatment with panitumumab. in the operating theatre, the skin is disinfected using povidoneiodine and pupil dilation is carried out with tropicamide (showing no immediate reaction in the surgery). method: as we are dealing with a late cutaneous reaction, the study of the medicine involved is carried out by means of epicutaneous medicine testing. in order to do the study of aflibercept, we wore gowns, two sets of gloves, a mask, eye protection and in a containment hood in the outpatients hospital. the patient diagnosed himself with dermatitis when in contact with povidone-iodine and despite the fact that the cutaneous provocation was negative, it is known that when there is surgery involved, there needs to be moistness and occlusion for it to show up clinically. the application of this antiseptic seems to lose its irritation and allergic properties when it dries on the skin and therefore tends to give a negative result in these patients, but this does not mean that they are not allergic to this antiseptic. we report the case of a 60 year old man who experienced erythema and pruritus immediately after an intravenous injection of ranitidine and hyoscine butylbromide given for gastric pain treatment. results: spt and idt were performed for ranitidine (10 mg/ml and 0.01 mg/ml respectively) and hyoscine butylbromide (0.5 mg/ml and 0.005 mg/ml respectively) being exclusively positive for ranitidine at idt dose with a 15 × 25 mm papule (histamine control 23 × 35 mm). oral provocation test for hyoscine butylbromide was negative. bat for ranitidine and famotidine were carried out, being negative for both drugs. conclusion: skin tests for h2ra are the best option when studying a suspected reaction to h2ra and are also useful for assessing cross-reactivity between other h2ra. the sensitivity for bat in diagnosis of drug allergy is about 50%, and the specificity up to 93%, although these percentages make reference to the common drugs studied (beta-lactams, quinolones, pyrazolones, etc). specific studies for h2ra are still to be done. in our case we had a negative result for the bat test, although we proved ranitidine was responsible for the reaction. conclusion: gentamicin is an aminoglycoside antibiotic used systemically for septicemia and as prophylaxis during surgery. immediate type allergy (type i) to gentamicin is rarely reported. since 2016, approximately only five cases have been reported in literature. in our case, initial theories were pointed towards cefazolin as beta-lactams report a higher rate of allergic reactions. after an exhaustive allergological study, results disproved our initial theory indicating gentamicin as the responsible drug. giangrande n 1 ; bobadilla-gonzález p 1 ; garcía-menaya jm 1 ; cámara-hijón c 2 1 allergy department, infanta cristina university hospital, badajoz, spain; 2 clinical immunology department, san pedro de alcántara hospital, cáceres, spain background: polyethylene glycol (otherwise known as macrogol or peg) is a polymer with a wide application as an excipient, solvent and dispersing agent in food, cosmetic and pharmaceutical industry. it presents distinct length polymer chains with a molecular weight from 200 to 10 000 000 g/mol conferring them specific properties. macroglol with a molecular mass between 3500 and 4000 g/mol is commonly used as osmotic laxative previously to colon endoscopy and radiologic examinations. after the introduction, anaphylactic reactions to macroglol are rarely reported, considering it safe and well tolerated. we report on a 56-year-old man who, immediately after of the topical application of benzindamine in left inferior limb developed acute urticaria in this limb, which reverted in approximately 2 hours with systemic steroids. she had previously tolerated this product without any problems. skin prick-tests with benzindamine (0.06 mg/ml) and latex were realized in the patient. skin prick-tests with benzindamine were realized in eleven healthy control subjects. results: skin prick-test with latex was negative in the patient. skin prick-test with benzindamine was positive in the patient (6 × 5 mm). the prick-tests with benzindamine were negative in eleven healthy control subjects. we report on a case of contact urticaria due to benzindamine and triggered by an immediate, probably ige-mediated, hypersensitivity mechanism. some of the drug used in daily clinical practice can cause allergic contact urticaria and should therefore be borne in mind. background: the use of new oral anticoagulants which act as direct inhibitors of activated factor x is constantly increasing, due to lower rates of serious and fatal bleeding events than warfarin/acenocoumarol. rivaroxaban, the first commercialized drug in this group, is the most used for prevention of thromboembolic events. however, <10 cases of hypersensitivity reactions have been described so far, most of them delayed and severe. to present a case of delayed hypersensitivity to rivaroxaban, diagnosed by a positive ltt (lymphoblastic transformation test). a 79 year old woman with hypertension and chronic atrial fibrillation (af) was referred to our clinic for suspected drug allergy. she reported that 2 months before, for af she was started on oral amiodarone and rivaroxaban, presenting on the seventh day with both of them generalized erythema, pruritus, micropapular rash and facial angioedema. no oral or other mucosal were observed, neither pustules, vesicles or blisters. blood eosinophilia, enlarged lymph nodes, renal and hepatic injury were discarded in emergency, where the new drugs were discontinued and replaced by acenocoumarol. the rash subsided one week later, with oral antihistamines. before and after the episode the patient also has been taking losartan and hydrosalurethyl, with good tolerance. she denied other adverse reactions. in allergy department we performed skin prick tests and intradermal tests with amiodarone (0.05 mg/ml and 0.5 mg/ml) and rivaroxaban (0.1 mg/ml and 1 mg/ml), and a ltt with both drugs, 3 months after the reaction. background: patients with history of beta lactam allergy, often self-reported, are commonly encountered in the hospital setting. this frequently leads to increase use of broad spectrum and more expensive antibiotics that may be unnecessary or even less efficacious at times due to fear and concerns about potential disastrous outcomes. nonetheless, with increasing awareness, many patients are now being referred to allergy service for formal evaluation. we aim to look at patients who underwent evaluation for beta-lactam hypersensitivity and determine the number of patients that were successfully de-labelled. method: a retrospective analysis was conducted with patients referred for evaluation of questionable beta-lactam allergy to the allergy service in our institution from the years 2016-2017. initial evaluation process included a thorough history to determine the type of hypersensitivity reaction and suitability for further testing. patients underwent skin prick test (spt) and intradermal (idt) with either (a) both major and minor determinants of penicillin, benzyl penicillin, amoxicillin and ampicillin, and/or (b) the culprit drug itself. if skin testing was negative, oral or intravenous (iv) drug challenge was then performed after informed consent. clinical details and reactions were documented. patients were also contacted post challenge to ensure no delayed reaction had occurred. results: a total of 130 patients were evaluated for beta-lactam allergy in the 2 year period, of these 80 were females and 50 were males. 107 of the referred patients had presumed penicillin group allergy and 29 had cephalosporin group allergy (3 patients had both penicillin group+cephalosporin allergy). 95 cases (73%) were successfully de-labelled. beta-lactam allergy was confirmed in 26 patients (20%); identified by positive spt in two patients, positive idt in six patients and positive drug challenge in 18 patients (15 patients developed rash/urticaria, 1 had respiratory symptom and 2 patients developed anaphylaxis). 14 patients were referred before any drug allergy labelling was done, out of which 10 were confirmed not to have beta-lactam allergy. conclusion: in our study, 80% of patients were confirmed not to have true beta-lactam allergy. we were able to successfully remove beta-lactam allergy label from the electronic record for 73% of the patients. results: a total of 66% referred amoxicillin-clavulanic acid (ax-clv) as trigger for the hypersensitivity reactions (hrs), followed by ax (21%), penicillin (6%) and cephalosporins (5%). almost 80% of hrs were immediate (<60 minutes). positivity of skin tests was observed in 65% subjects, of bat in 61% and of rast in 68%. in conclusion: the label of penicillin allergy is quite often erroneous. this involves using of more expensive and less effective therapeutic alternatives, which also facilitate the emergence of multi-resistant micro-organisms. hence the importance of confirming the diagnosis of allergy. finally, we did not find differences in the study of penicillin allergy in patients older than 60 years compared with the general population. background: severe cutaneous delayed drug reactions (toxic epidermal necrolysis -ten-, stevens-johnson syndrome -sjs-, acute generalized exanthematous pustulosis -agepand drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome -dress/dihs-) among others, are a rare but potentially fatal complications of drug treatment. although its epidemiology has been described in different latitudes, it is unknown in latin america. our aim was to describe the epidemiological characteristics of severe cutaneous reactions to drugs in countries of latin america. method: an online questionnaire was designed to report new and old cases (since 2013). it was a modified and adapted version of enda questionnaire for drug allergy interesting group. sociodemographic data, type of reaction (ten, sjs, dress-dihs, agep), culprit drug (s), treatment, complications, mortality and sequelae, were described. three centers from colombia, one from argentina, one from brazil and one from paraguay were included. an excel database was created, in which cases were recorded and analyzed. results: thirty seven cases were reported. 24 (65%) were women. the median age was 47 years. 19 (51%) had dress/dihs, 6 (16%) ten, 3 (8%) sjs, 3 (8%) agep, 3 (8%) other not classified scars, and 1 (2.7%) overlapping ten/sjs. the main culprit drugs were aromatic anticonvulsants in 17 cases (46%), beta lactam antibiotics in 6 (16%), non-beta lactam antibiotics in 3 (8%) and allopurinol in 2 (5.4%). in 100% of the patients the suspect drug was withdrawn. thirty one patients (83.7%) received systemic corticosteroids. complications occurred in 17 cases (49%) and death in one patient (2.7%). seven patients (19%) had some type of sequelae. countries, dress/dihs was the most frequently reported clinical entity, and the anticonvulsants were the main triggers. complications were frequent, but mortality was low. 1155 | drug-induced cough: analysis of nationwide spontaneous reports in korea over ten years using who-adverse reaction terminology (who-art) indicative of cough. results: from 856 524 cases of spontaneously reported adverse drug event cases, a total of 9003 cases (4.5%) were identified as drug-induced cough. most cases occurred in adults (93.4% of the subjects) and females were more common than males (54.9% vs 45.1%). regarding severity, only 629 cases (7.0%) were classified as serious based on who criteria. the most common causative drug category was antineoplastic and immunomodulating agents (24.8%), followed by cardiovascular drugs (24.2%). the most common causative drugs were ace inhibitors including perindopril and ramipril. conclusion: in the nationwide spontaneous reports of adverse drug events, many cases of drug-induced cough have been reported so far. much attention is needed to find new causative drugs of cough in the future. background: allergological assessment to determine the mechanism of the perioperative reaction and to identify the agent responsible and recommendation of a range of drugs or agents likely for future surgery is essential, but it often poses a significant challenge. in this study, we analyze our experience in the investigation of adverse reactions during anesthesia in the last 4 years. method: a total of 15 patients who attended our allergy unit with suspected perioperative reactions between january 2014 and december 2017 were reviewed retrospectively. the severity of the perioperative allergic reactions was graded according to ring and messmer system. results: grade iii, ii and i reactions were observed in 9, 1 and 3 patients, respectively. in 2 patient we didn't know the reaction suffered. tryptase measurements were available for 11 patients. of those, 3 and 4 patients had elevated and normal levels respectively and suffered grade 3 reaction. ige mediated reactions was diagnosed in 9 patients (60%): 3 for ßlactam antibiotics (33.3%), 2 for patent blue (22.2%), 1 for neuromuscular blocking agents-nmbas (11.1%), 1 for latex (11.1%), 1 for colloids (11.1%) and 1 for ranitidine (11.1%) . cefazolin was the ß-lactam antibiotics causing the largest number of reactions. non-ige-mediated reactions was diagnosed in 6 patients (40%). the allergy tests were negative and tryptase levels were normal. conclusion: in our series, among the 9 patients who suffered allergy reactions during anaesthesia and the cause was subsequently identified, ß-lactam antibiotics were the most common causative agent (33.3%), followed by patent blue (22.2%), nmbas, latex, colloids and ranitidine (11.1% each agent). in contrast, data from other authors indicated that nmbas were the most common cause of anaphylaxis, followed by latex, hypnotics, antibiotics, plasma substitutes and opioids. these differences might be due to the small size of our study, which was limited to our centre over the last 4 years and thus may not be representative. diagnostic evaluation. all patients signed an informed consent. we made a retrospective analysis of their clinical records and excluded 11 patients whose records were missing or incomplete. it was analyzed each patient's medical history (focusing allergic disease) and clinical reaction to the suspect drugs. signals/symptoms at pcc were characterized. we also studied the variation of the dpt's results when it was performed after a pcc. aim: to define and quantify the ongoing pharmacy needs in sustaining a large drug allergy assessment program. method: a retrospective review of pharmacy files was used to identify and quantify the drugs and dosages most frequently used and to determine prescription trends within the allergy testing program over the last 3 years. results: initially, this reaction was thought to be a result of a drug allergy, but upon further review and the onset of fever, we determined that it met the diagnostic criteria of jhr. his twin brother was diagnosed with penicillin and betalactamic allergy. neutrophilia 83% was to be underlined in the blood test. after this, drug oral challenge with penicillin was performed, ruling out penicillin allergy. conclusion: it is not uncommon to confuse drug allergy with jhr. jhr should be an anticipated reaction to early doses of antibiotic treatment for treponemal diseases, such as syphilis. antibiotic treatment should be continued; it is not a warrant to stop treatment. clinicians should be aware and anticipate jhr as a potential complication to early doses of antibiotic for spirochetal diseases such as syphilis or lyme, leptospirosis. the patient was unresponsive in oral drug provocation tests with amoxicillin-clavulanic acid, clarithromycin and trimethoprim sulfamethoxazole for 6 months. the patient could use these drugs. results: chronic abacterial inflammation of the prostate gland was accompanied by a significant increase in concentration of slpi, il-8, tnf-α, il-17 in the seminal plasma and serum concentration, and a decrease in the concentration of il-6 and tgf-β1 compared to healthy men (p < 0.05). there was no statistically significant difference between slpi, il-8, tnf-α, il-23, il-17, and tgf-β1 in the ejaculate of patients with inflammatory and non-inflammatory forms of cap (p < 0.05). the concentration of il-6 in ejaculate of patients with inflammatory forms of cap was significantly greater than in patients with non-inflammatory form of cap (p = 0.01). the inflammatory and non-inflammatory forms of cap are pathologically similar with changes in the concentration of the studied cytokines except for il-6 in both forms with signs of inflammation. the terms "leukocytic" vs "non-leukocytic" chronic abacterial prostatitis are more correct than "inflammatory" and "non-inflammatory" when describing chronic abacterial prostatitis. results: the status of all patients after dc immunotherapy was evaluated as satisfactory. heart rate, blood pressure in patients remained within the age norm. skin had normal color without rash or peripheral edema. there were no local or systemic allergic reactions. the body temperature after the injection did not exceed 37°c. conclusion: these results show, for the first time, that among mastocytosis patients, besides the already known periodontal disease risk factors that include diabetes, age, osteoporosis and alcohol consumption, the bone marrow mast cell burden is also associated with increased periodontal disease severity. results: metformin at relatively low doses (1-10 μm) was shown to mildly suppress ige-mediated responses, including degranulation (34% reduction, p = 0.0135), tnf-α (23% reduction, p = 0.0139) and il-13 (38% reduction, p = 0.0015) secretions in bmmcs. importantly, metformin at the same doses potently inhibited mast cell responses in all parameters (100% reduction, p < 0.0001 for degranulation; 87% reduction, p < 0.0001 for tnf-α; 90% reduction, p < 0.0001 for il-13) in mast cells treated with an ahr ligand, 5,11-dihydroindolo[3,2-b]carbazole-6-carbaldehyde (ficz). mechanistically, its inhibitory effect was mediated through the suppression of ficz-induced mapk activation, intracellular calcium release and ros generation. metformin also blocked ahr-mediated pca in vivo (90% reduction, p < 0.0001). conclusion: metformin, a common anti-diabetic agent, was shown to exert inhibitory effect on ahr-mediated mast cell activation in vitro and in vivo, suggesting its potential utility as a newer form of therapy for asthma and allergic diseases; this is particularly relevant when considering the adverse effect of the exposure to environmental polycyclic aromatic hydrocarbons. gasser p 1 ; brigger d 1 ; zbären n 1 ; jardetzky t 2 ; pennington l 2 ; eggel a 1 results: in one of affected family members, we were able to identify the c.9886a>g mutation in the plasminogen (plg) gene that was recently described to be associated with hereditary angioedema. this mutation leads to a missense mutation with an amino acid exchange p.lys330glu in the 3rd kringle domain of plasminogen. there is no direct relationship between the earlier described cases with this mutation and the family we report here. in all affected members of the family, the symptoms manifested in early adulthood, with swelling of the face, the tongue and the larynx. the frequency of attacks was variable, between once in a year to once in a month. in one of the three family members, we found a slightly decreased level of coagulation factor xii and of plasminogen. icatibant proved to be very effective for the treatment of acute attacks in the affected family. the occurrence of the same c.9886a>g (p.lys330glu) mutation in the plg gene in many families with no or only unknown distant relationship suggests that the disease might have been inherited through the generations without being purged from the population. the mutated amino acid exchange appears to be significant for the function of plasmin or plasminogen. we found a decrease in plasma levels of coagulation factor xii and plasminogen, which may be beneficial markers for diagnosis and monitoring of this disease. several biomarkers are useful in the diagnosis (fibrin degradation products (fdps), d dimer (dd), and fragments of prothrombin 1 + 2). also, a correlation between the levels of biomarkers and activity phases of the disease has been detected. alterations in coagulation parameters have an etiopathogenic role in the ae attack, but have not been considered as biomarkers of activity phases. tgt is a global coagulation test which quantifies in vitro the ability of plasma to generate thrombin and estimates alterations in coagulation parameters. the objective is to assess the usefulness of the thrombin generation test (tgt) to characterize patients with hereditary angioedema (hae). method: seventeen hae patients from hospital la fe were recruited to obtain blood samples in remission and during ae attacks. none of them experienced thromboembolic events. plasma was collected in citrate tubes to obtain platelet rich plasma. hemostatic parameters were analyzed:. tgt was conducted using a calibrated automated thrombogram (cat) method and a fluoroskan ascent as a reader. results were analyzed via thrombinoscope v5. citrated plasma was incubated with calcium, tissue factor, phospholipids and a fluorogenic substrate. a thrombin generation curve is generated, obtaining parameters: latency time (lagtime), thrombin generation maximum speed (vo), maximum peak of thrombin generated (peak), time to generate the maximum peak of thrombin (ttpeak), total quantity of generated thrombin (etp), and the end time of thrombin generation (starttail). tgt parameters from 322 healthy donors were used as controls. results: thirty-eight samples were collected from seventeen hae patients (58.8% female). fifteen (39.5%) samples were collected during ae attacks. tgt parameters and fdps were significantly higher in hae patients compared with controls (p < 0.0001), although no significant differences were found in tgt between acute attacks and remission. a decrease trend in tgt is observed in ae attacks. fdps were increased during ae attacks, but normalized at remission periods. these results support the involvement of coagulation in the pathophysiology of hae, although no increase in prevalence of thrombosis is observed during acute attacks. method: the repeated measures design study included 108 patients in two groups: the slit group, 63 patients-67 follow-ups per allergen (p), and the vit group, 45 patients-54 p. the slit group had patients treated for hdm (41 p), and patients on pre-coseasonal pollen ait (grass 11 p, ragweed 10 p, birch 5 p). the vit group had 28 patients on rush protocol (18 for bee and 9 for wasp) and 18 patients on conventional protocol (9 bee, 2 wasp, and 7 for both). the ige and igg4 levels were measured by the immunocap method. the friedman test was used to compare data. results: when compared to placebo group, slit+vitamin d group therapy was more effective in the reduction of nasal symptoms (p = 0.04), asthma symptoms (p = 0.001) and combined symptommedication score (p = 0.001); there was no significant difference between groups in medication and ocular scores. we observed a significant improvement of fev1 (vitamin d group p = 0.014, placebo group p = 0.015) and fev1%vc levels (vitamin d group p = 0.004, placebo group p < 0.001), within both groups, between visits. feno results did not differentiate statistically significantly the study participants in terms of receiving slit along with vitamin d or placebo. significant increase in the percentage of cd4 + cd25 + foxp3 + and in tlr positive cells in children receiving slit+ vitamin d was observed compared to placebo group. increase in cd4 + cd25 + fox-p3 + induction, and in tlr positive cells recruitment were independently associated with better clinical effect of slit in children. conclusion: overall, ait with a high-polymerized ash pollen extract was well tolerated. as ash pollen are supposed to be an important allergen during spring time, it is recommended to include spt and npt with ash pollen in the test panel for allergological diagnostic. additionally, determination of ash pollen specific ige could be applied. furthermore, appropriate ait should be considered for ash pollen allergic patients. 1194a | impact of sublingual immunotherapy with a five-grass pollen tablet on grass pollen allergic rhinitis and asthma: a real-life, long-term analysis in france background: data on the fulfilment of prescriptions of symptomatic medications in patients with grass pollen allergy were analysed to evaluate the long-term effectiveness of sublingual immunotherapy (slit) on allergic rhinitis (ar) and asthma. method: by using data in the lifelink ™ treatment dynamics database (iqvia, paris, france), we compared two cohorts of patients with ar: a group treated with oralair® (stallergenes greer, antony, france) slit tablets (n = 617), and a matched control group having received symptomatic medications only (n = 10 990). oralair®'s effectiveness was assessed as the change in symptomatic medication fulfilments between the pre-index period (before the initiation of slit) and the follow-up period (after slit), and as the onset of asthma or the progression of pre-existing asthma (based on fulfilments of prescriptions for asthma medication). the number of fulfilments per year was calculated for each patient and each period. results: in line with prescribing guidelines, the mean duration of treatment with oralair® was 6.3 months per season for either 2 seasons or 3 seasons. the mean number of symptomatic medications for ar fulfilled per patient and per year in the pre-index period was 6.7 ± 5.9 in the slit tablet group and 9.6 ± 7.7 in the control group. in the follow-up period, this value fell for the slit tablet group (to 2.6 ± 4.2) but did not change significantly in the control group (8.6 ± 8.1). when considering individuals not taking any asthma medications in the pre-index period, asthma onset during the treatment period was observed in 12.1% of those in the slit tablet group and in 15.8% of those in the control group. the corresponding values for the follow-up period were 1.2% in the slit tablet group and 5.8% in the control group. when considering individuals already taking asthma medications in the pre-index period, the mean ± sd number of asthma medication fulfilments in the pre-index period was lower in the slit tablet group (4.7 ± 4.6) than in the control group (8.1 ± 7.9). the corresponding values for the treatment period were 4.1 ± 5.4 and 10.2 ± 9.5, respectively. in the follow-up period, the number of asthma medication fulfilments fell more in the slit tablet group (to 2.3 ± 4.1) than in the control group (7.2 ± 9.0). oralair® tablets have long-term effectiveness by relieving allergic rhinitis and slowing a progression to asthma. 1194b | a real-life, retrospective analysis evidencing slower long-term progression of asthma in grass pollen allergy patients treated with sublingual immunotherapy tablets 1195 | an examination of the reasons for treatment discontinuation and non-compliance to allergen immunotherapy background: allergic rhinitis (ar) patients treated with subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit) may be non-compliant or discontinue treatment too early, which can negatively impact efficacy. therefore, understanding the reasons for non-compliance and treatment discontinuation is vital to help improve compliance, persistence and thus outcomes. this study reported reasons for treatment discontinuation to scit and slit and non-compliance to slit in patients with ar in published real-world studies. method: a literature review was conducted in embase, medline, ebm reviews, psycinfo and econlit (1998-2017) using key search terms for allergic rhinitis, scit, slit, non-compliance and non-persistence. across all studies,~20% of patients were non-compliant, and 1-year drop-out rates ranged from 23% to 74%. reasons for noncompliance and treatment discontinuation in this subset of patients were stratified according to the who dimensions for adherence (patient-related, treatment-related, or socio-economic). results: from the 428 publications identified, six studies reported reasons for non-compliance to slit (n = 3) or treatment discontinuation (n = 3) to scit or slit, and the results were grouped for analysis. the majority of patients cited treatment-related factors as the primary reason for discontinuation (66% for slit, 50% for scit). common reasons were a length of treatment for slit and frequency of injections for scit. 45% of patients discontinued scit due to patient-related factors such as travel to doctors and waiting time for administration. only 24% of slit patients discontinued due to patient-related factors. socio-economic reasons for discontinuation were low for both therapies (10% slit and 5% scit). conversely, for non-compliance to slit, socio-economic factors were the most frequently cited reasons (50%), and included taking time off work and financial concerns. conclusion: of patients who discontinued therapy, treatmentrelated factors were the most cited reasons for scit and slit, reflecting concerns with administration and treatment length. noncompliant slit patients cited socio-economic factors as common reasons for non-compliance, suggesting financial concerns over a long treatment course. differences in reasons for non-compliance and treatment discontinuation may be due to patients assigning differing importance for compliance (a day-to-day decision) compared to the long-term decision to discontinue treatment. results: 53% of patients had monosensitization to rbet v1 component. the rest 47% had combinations ige to rbet v1 and ige to one, two or even three minor allergens (35%, 10%, 2% accordingly). after 2 courses of slit by standardized pollen extracts symptoms of arc and pfas decreased in 90% and 63% patients accordingly. in group 54 patients with monosensitization to rbet v1: 49 patients had a reduction of arc (85% had 3-4 degree by ado); 41 patients had reduction of pfas. 3 patients hadn't finished treatment due to allergic reactions. among 26 patients with sensitization to rbet v1/v6: 24 patients had a reduction of arc (75% -3 to 4 degree by ado); 17 had reduction of pfas. 1 patient hadn't finished treatment due to allergic reactions. in 9 patients with sensitization to rbet v1/v2: 7 patients had a reduction of arc (57% -3 to 4 degree by ado); 3 had reduction of pfas. 10 patients with sensitization to rbet v1/v2/v6 showed the similar results: 10 patients had a reduction of arc (57% -3 to 4 degree by ado); 3 had reduction of pfas. 2 patients had sensitization to all cra, and only 1 patient who also received slit with grass allergens had reduction of arc only (2 degree by ado). as the result of the study it was identified that beneficial effect of slit is highest in patients with monosensitization to rbet v1. the increase of sige sensitization profiles to minor birch allergens caused less efficacy of slit treatment. dermatophagoides pteronyssinus immunotherapy is independent of sensitization to blomia tropicalis among children with allergic rhinitis and asthma method: 95 children (5-17 years old) with allergic rhinitis and asthma sensitised to both dp and bt received 3 years dp-scit. clinical symptom and medication scores, serum specific ige and specific igg 4 were evaluated during dp-scit. in order to investigate whether the treatment outcome was dependent on the sensitisation pattern between dp and bt, patients were further grouped into dp and bt co-sensitisation and cross-reaction, according to positive or negative ige against bt major allergen (btma) blo t 5 and blo t 21. btma+ group, with specific ige to either blo t 5 or blo t 21, was defined as the co-sensitized group; btma-group, with no detectable ige to both blo t 5 and blo t 21, was defined as the cross-reactive group in this study. results: all the recruited 95 patients completed 1 year of dp-scit, 74 (78%) patients completed 3 years of treatment. after 3 years of dp-scit, compared to baseline, all patients had significant reduction in symptom and medication scores. lung function (fev 1 ) was significantly improved as well. 65% of the patients were free of medication use and asthma symptoms, 3% of them were free of rhinitis symptom, and the fev 1 % in all patients were higher than 95% of predicted. dp-scit induced significant increases in dp and bt specific igg 4 . in 50% of patients, dp specific igg 4 increased more than 67 fold and bt specific igg 4 increased more than 2.5 fold. further investigation in btma groups showed moderate correlation (spearman r = 0.48, p = 0.004) between specific ige against dp and bt in the btma-group (n = 34), indicating specific ige cross-reactivity. no specific ige correlation (spearman r = 0.03, p = 0.82) was found in the btma+ group (n = 61) indicating co-sensitisation to both dp and bt. the two groups showed almost identical change in clinical responses. dp and bt specific igg4 significantly increased during dp-scit, no difference was found between the two btma groups. conclusion: dp-scit can induce specific igg4 cross-reacting with bt allergens. patients with specific ige sensitisations to both dp and bt may have clinical benefit from dp-scit treatment. moreover, the clinical benefit of scit was independent of ige cross-reactivity or co-sensitisation to dp and bt. method: we investigated 16 allergic rhinitis children who were basically sensitized to house dust mite and received house dust mite slit for 1 year and 6 months. among 16 patients, 9 patients were mono-sensitized to house dust mite (group 1) and 7 patients were poly-sensitized aside from house dust mite (group 2). we also assigned another 7 allergic rhinitis children who were only treated by medication as control group. nasal symptoms (rhinorrhea, sneezing, nasal obstruction, nasal itching, sleep disturbance) and anti-allergic medications use were assessed at every 6-month visit. results: the symptoms of allergic rhinitis started to improve after 6 months of slit and significantly improved after a year and a half in group 1 and group 2 compared with control group. there was no significant difference between group 1 and group 2. anti-allergic medication use in group 1 and group 2 significantly decreased after a year and a half compared with control group and there was no significant difference between group 1 and group 2. conclusion: house dust mite slit was more effective than treatment only by medication. the effect of house dust mite slit was similar between mono-sensitized and poly-sensitized allergic rhinitis children. house dust mite slit could also be recommended to polysensitized allergic rhinitis children. method: a prospective, randomized, double-blind, controlled, multicenter phase ii study was conducted with four different concentrations of cluster allergoid clustoid wiesenlieschgras (group 1: 2000 tu/ml; group 2: 10 000 tu/ml; group 2: 30 000 tu/ml; group 4: 50 000 tu/ml). out of 103 patients screened, 83 grass pollen allergic patients (18-59 years) were randomized. the cluster build-up phase was followed by four monthly maintenance injections of 0.5 ml. the efficacy was evaluated by the change of the threshold concentration step needed to induce a positive reaction in a titrated nasal provocation test (tnpt) before start and after end of the study (pre-post analysis). the safety profile was assessed for each treatment group by analyzing treatment-related adverse events. background: allergen immunotherapy relies on the consistent administration of allergen extract, therefore compliance to these treatments (subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit) tablets and drops) is vital for efficacy. as scit is administered as an injection by a healthcare professional, and slit is self-administered, compliance to scit may be perceived as superior. therefore, a review of real-world studies investigating compliance to scit, slit-tablets or slit-drops was conducted. real-world studies, instead of clinical trials, were included in this review as they are more likely to reflect actual clinical practice and patient compliance. method: a literature review was conducted in embase, medline, ebm reviews, psycinfo and econlit (1998-2017) using key search terms for ar, scit, slit-tablets and slit-drops, and real-world compliance. compliance was reported according to ispor medication compliance and persistence work group definitions. results: from the 428 publications identified, eight studies (seven slit [one slit-tablets, two slit-drops, four unspecified], one both scit+slit-tablets) reported compliance rates and were included in the analysis. real-world compliance rates ranged from 80% to 81% for slit administration and 83% for scit administration. only one study compared compliance of slit to scit, with similar rates reported over three years (81% and 83% respectively). three studies reported "good" compliance (physician-reported or patients consuming >60% of allergen extract) to slit-drops or slit-tablets. the good compliance rates were higher for slit-drops (90%-99%) compared to slit-tablets (77%-80%). observed compliance to slit-drops or slittablets did not vary by country or geographical region. the percentage of patients defined as having "good" compliance to slit-tablets or slit-drops did not vary by study length or patient population. conclusion: whilst compliance to scit may be perceived as superior to slit-tablets and slit-drops, comparable compliance rates between scit, slit-tables and slit-drops were identified across real-world studies. differences between perception and real-world results may be explained by a lack of direct comparisons between scit and slit administration. limitations included discrepancies in definitions of compliance, as well as methodology between studies. however, these are common to reviews analysing compliance, regardless of therapy area. conclusion: in the allergic rhinitis patients, successful compliance for 3-year slit compared with control was approximately 52%. method: igg inhibition elisa: rabbit igg antibodies specific for grass allergen allergoids are pre-incubated with different concentrations of alum-adsorbed grass pollen allergoid. the mix is added to an allergoid coated microtiter plate. unbound igg will bind to the allergoid coat and is subsequently incubated with anti-igg hrp labeled conjugate and stained with tmb. results are expressed as percentage inhibition relative to the uninhibited value. the concentration of alum-adsorbed allergoid that is required to inhibit 50% igg is used as read-out. circular dichroism: far-uv cd spectra (190-260 nm) were recorded on a j-815 spectropolarimeter. a cuvette with a stirring compartment was used to keep the suspension homogeneous during measurement. results: the igg inhibition elisa assay is specific for grass pollen allergoids (not for other allergen allergoids), has a good inter-and intra-assay precision and is robust for assay variation. thermally stressed alum-adsorbed grass pollen allergoids were used to show that the igg inhibition assay can be used as a stability indicating method. severe thermal stressing resulted in a higher 50% inhibition value, indicating a loss of igg epitopes. furthermore, far-uv cd analyses showed that there is a close relation between the decreasing igg binding capacity (50% inhibition values) and the loss secondary protein structures by unfolding (cd-ratio 207/222 nm values). the igg inhibition assay was demonstrated to be a valuable method to determine the stability of alum-adsorbed grass pollen allergoid preparations. in addition, a relation was shown between the igg binding capacity and the change in secondary protein structures. 1205 | design of a pivotal phase iii trial of allergen specific immunotherapy (ait) using a high-dose house dust mite (hdm) allergoid in patients with allergic bronchial asthma method: 1038 male and female outpatients (age 12-65 years) asthmatics allergic to hdm are enrolled. during the baseline phase, the patient's minimal dose of ics required to achieve asthma control will be assessed. after the baseline period, approx. 446 patients will receive double-blind placebo-controlled treatment for approx. 8 months, followed by a 2nd period of 16 weeks to assess the minimal ics dose and further 2 months of observation for the assessment of asthma exacerbation. based on the results of the dose finding study regarding the efficacy endpoints and the safety profile, the optimal allergoid dose is considered to be 5400 pnu. results: competent authorities and ethic committees in all participating 12 eu countries, serbia, russia and ukraine approved the study design. the primary endpoint of the trial is the change in predefined dose steps of the minimal daily ics dose required to achieve asthma control after approximately 8 months of subcutaneous ait. all efficacy data will be determined using daily questionnaires and the acq6 by e-diary for 4 months from october to january. the aim of this clinical trial is to demonstrate efficacy and to evaluate safety of ait with an allergoid preparation of major allergens of dermatophagoides pteronyssinus in patients suffering from allergic bronchial asthma caused by house dust mites. asthma increases the burden of allergic disease and health care costs, especially when uncontrolled. with the development of a high-dose preparation we intend to treat asthmatic patients highly efficiently. romantowski j; jassem e; lata j; wasilewska e; chelminska m; specjalski k; niedoszytko m background: specific immunotherapy (sit) is the only causal treatment in patients allergic to airborne allergens. it has been proven to be widely effective in allergic populations, but individual patients vary in terms of response to the therapy. the aim of the study was to assess the factors that might affect the efficacy of sit. method: patients treated with sit for grass pollen or house dust mites were included. the efficacy of sit was assessed with the use of allergy control score (acs), performed before and at least after one year of sit. the following variables were assessed as potential risk factors for a poorer response to sit: age, gender, type of allergy, type of allergen, type of vaccine, type of sit and smoking history. background: specific immunotherapy (sit) is a suitable treatment option for asthma and allergic rhinitis (ar), but it is not commonly used in korea. in the achievement of the treatment, it is important that immunotherapy is applied with ideal dose and regular intervals and it is essential for the patient compliance. the aim of this study is to investigate evaluate compliance with immunotherapy protocols of patients who were treated with sit in clinic and their satisfaction of the treatment. we performed a multicenter, cross-sectional survey using a specially designed questionnaire that was given to allergy specialists and patient in korea. a member of the trained research group conducted face-to-face questionnaire interviews with each respondent. conclusion: this study shows that most patients are ar with asthma. in our study sit compliance and satisfy are found to be high in both groups. aim: to identify the frequency of regress claims in a dermatological setting and to assess its impact on general prescription behaviour and immunotherapy. method: all physicians of the psoriasis-praxisnetz süd-west e.v. (n = 222) were invited to participate in a web based questionnaire study on the topics of dermatology and medical law. the survey was separated into two sub-polls which were carried out after a first poll deciding whether the topic of medical law is of any interest for the dermatological practice. the topic of interest was located in the second poll. results: overall, 66 dermatologists participated in this study. most participants were form bavaria, baden-wuerttemberg or rhineland-palatinate and had more than 10 years of experience as a dermatologist. out of the 66 participating physicians 28.8% (n = 19) already experienced a previous regress claim. of these, 73.7% (n = 14) stated, that the experienced regress claim changed their prescription behaviour. half of these participants (n = 8) further stated, that the fear of a possible recourse affects their prescription behaviour, whereas only 4 out of the 47 other participants declared a possible influence. missing values excluded, this leads to a substantial hesitation in physicians who experienced a prior recourse (50.0% vs 16.7%). nevertheless, this seems not to affect the usage of allergen immunotherapy, as all 19 physicians who already experienced a regress claim, stated to use allergen immunotherapy. the fear of a possible regress can change physicians' prescription behaviour but does not seem to have an effect on the prescription of allergen immunotherapy. therefore, the topic should be addressed from another perspective such as providing trainings on relevant regulations for physicians who experienced a prior recourse claim. this approach could also improve patient centred care related to modern treatments. results: the sds were significantly reduced in patients subjected to slit (p < 0.01) 1 year after the onset it. vas also was significantly reduced (p < 0.05) with satisfied control of sar and the same time with translation from moderate-severe to mild-moderate sar, after slit. nbh was also significantly reduced (p < 0.05) 1 year after the onset slit. in patients receiving pht only, sds, vas and severity of sar did not change and significantly higher (p < 0.01) from the value obtained in the experimental group. nbh also remained unchanged and significantly higher (p < 0.05) then in experimental group. precoseasonal slit added to pht shows short-term beneficial clinical effects in polysensitized patients with sar and scuad phenotype. results: ten studies (248 children, 99 adults; median sample size, 28) met the inclusion criteria. the risk of bias was moderate to high in all but one studies. low strength evidence supports the assumption that ait is effective in reducing symptoms and medication use, with only 5 out of 10 studies reporting higher benefit in the ait group vs comparator group. subgroup analyses of studies sharing similar characteristics did not explain inconsistency. safety does not appear was not major concern for alternaria ait. conclusion: this is not enough strength of evidence to suggest that mold ait is efficacious for the treatment of respiratory allergies. high-quality studies with an adequate sample size are needed. abstracts | 627 1213 | tolerability of a two week rush updosing with modified trees, modified grasses or modified grasses/trees mixture in pollen allergic subjects in the day-to-day practice table) severe systemic reactions (grade iii and iv) did not occur. conclusion: rush immunotherapy is an effective therapeutic method for patients with allergic rhinitis. it seems that in cases requiring faster response to treatment, this immunotherapy can be considered as a substitute for conventional immunotherapy. 1217 | design of a pivotal phase iii allergen immunotherapy study to assess the efficacy and safety of subcutaneously administered tyrosine adsorbed modified birch allergen+mpl results: the design of this study, including sample size and primary and secondary endpoints, will be discussed based on prior experience gained in two dose finding studies. in addition, the number of patients screened and randomized will be presented by country, gender and/or age and screen failures will be categorized. results: the primary analysis showed an absolute difference in tcs between placebo and 12 du of 3.02 (40%, p < 0.0001). the odds of experiencing a severe day during the bps were approximately doubled in the placebo group compared to the 12 du group (or = 2.12, p < 0.0001) and the odds of experiencing a mild day were halved (or = 0.52, p < 0.0001). similar results were seen for the tree pollen season (tps), covering both alder, hazel and birch pollen seasons. the total rqlq score was improved for 12 du compared to placebo during the bps and tps (p < 0.05, except for the last week of the tps), with the most pronounced effects during week 2-5 of the bps (absolute difference: 0.47-0.58, p < 0.0001). treatment was well tolerated. the most frequent adverse reactions were mild or moderate local reactions related to the sublingual administration. no deaths were reported and no serious adverse events were assessed as related to the sq tree slit-tablet. conclusion: treatment with the sq tree slit-tablet improved arc symptoms and need for symptomatic treatment. the 12 du group had less "severe days" and more "mild days" during the pollen seasons. the quality of life was similarly improved. these findings substantiate the clinical relevance of the sq tree slit-tablet for patients with arc induced by pollen from the birch homologous group. nagaraju k 1 ; nagaraju k 2 ; katare s 3 ; kapatkar v 3 ; shah a 3 ; rathod r 3 results: total n = 57 subjects (mean age 4.6 ± 2.09 years, 61.4% males) completed entire study. the mean incidence of artis reduced from 7.7 ± 2.34 episodes at baseline to 1.4 ± 1.23 (p < 0.001), with 31.6% subjects not suffering from any episode. the mean duration of episodes reduced from 8.2 ± 3.58 to 2.5 ± 1.23 days (p < 0.001). 64% of episodes (vs 95% at baseline, p < 0.05) required antibiotics for mean duration of 2.3 ± 0.98 days (vs. 6.6 ± 1.76 days, p < 0.001). none of arti episodes in follow-up period required hospitalization as against 22.8% episodes, (mean duration 5 ± 2.06 days; p < 0.05) before pidotimod therapy. the number of school days lost & work days lost showed reduction of 3.6 ± 5.34 days(p < 0.001) & 0.1 ± 4.07 days(p = 0.871) respectively. the average expenses incurred in treatment of artis shows significant reduction of rs. 13 193 ± 1541 (p < 0.001). 11 adverse events were reported in 8 (14%) subjects, which were mild in nature. a statistically significant increase in absolute counts of t-& nk cells was seen in explorative assessment of immune markers. the study shows pidotimod to be well-tolerated effective therapy in reducing the incidence and severity of recurrent artis, thereby providing additional benefit of reduction in discomfort & healthcare cost due to recurrent artis. thus, pidotimod can be considered as potential therapeutic option for treatment of recurrent artis in children. martignago i 1 ; ridolo e 1 ; incorvaia c 2 1 department of medicine and surgery, university of parma, parma, italy; 2 cardiac/pulmonary rehabilitation, asst pini/cto, milan, italy background: two registered sublingual immunotherapy (slit) products are available to treat grass-pollen induced rhinoconjunctivitis, consisting of the 1-grass (phleum pratense) and the 5-grass pollen tablets. no study of direct comparison of the efficacy of the two products was performed. we report the case of a patient who was treated in different years with the 5-grass or the 1-grass tablets with contrasting efficacy. the patient was a 18-year old woman suffering from 3 years of grass pollen induced rhinoconjunctivitis. in 2015 slit was started with the 5-grass pollen tablets, but in 2016, due to unavailability of the product, slit was performed by the 1-grass pollen tablets. in the third year of treatment slit with the 5-grass pollen tablets was resumed. for the 5-grass tablets slit was initiated before the pollen season and stopped after 7 months of treatment, while for the 1-grass tablets the treated was prescribed to be continuous. the efficacy of slit was evaluated by symptom-medication scores as reported in diary cards by the patient during the month of may, when the grass pollen usually reach the higher concentration in the atmosphere in lombardy, where the patient lives. results: the mean symptom-medication score in the first year of treatment (5-grass tablets) was 4.35, compared with a mean score of 7.2 in the second year (1-grass tablets). the patient was unsatisfied of the symptoms control and asked to resume for the last year of slit the 5-grass tablets. the mean symptom-medication score in such year was 0.77. no clinically relevant adverse event was reported with any slit product. conclusion: based on the momentary unavailability of the 5-grass pollen tablet, it was possible to assess in a same patient the clinical outcome associated to either of the two registered slit products. a significantly different efficacy of slit with the 5-grass tablets compared with the 1-grass tablets was observed. 1222 | fusion proteins consisting of bet v 1 and phl p 5 form ige-reactive aggregates with reduced allergenic activity najafi n 1 ; hofer g 2 ; gattinger p 1 ; smiljkovic d 3 ; blatt k 3 ; selb r 4 ; stoecklinger a 5 ; keller w 2 ; valent p 3 ; niederberger v 4 ; thalhamer j 5 ; valenta r 6 ; flicker s 6 background: bet v 1 and phl p 5 representing major allergens in birch and grass pollen, occur as monomeric proteins with high allergenic activity as assessed by clinical provocation testing in patients. she did not suffer more hymenoptera stings after the last reaction. one bee sting several years before bst resulted in no reaction. she has no symptoms with honey, vegetable or other food ingestion. methods: total ige and specific ige were determined using inmu-nocap system (thermofisher, scientific inc). apis, vespula and polistes (hørsholm, denmark) were also performed. prick tests showed negative results for all extracts tested. intradermal skin tests were positive for apis at 1 μg/ml, but negative for vespula and polistes. our patient was diagnosed of anaphylaxis due to apis venom, thus bst was contraindicated and an epinephrine autoinjector was prescribed. she rejected hymenoptera venom immunotherapy. conclusion: to our knowledge, this is the first case of anaphylactic reaction after bee sting therapy. bee sting therapy should be considered a risk factor for anaphylaxis. patient reported good control of their disease, improved their quality of life, tolerating contact and exposure to numerous horses as well as contact with clothes of people who had been exposed. although ita is absolute contraindication on uncontrolled asthma with a degree of evidence ia, our case had only "transitory" con and immunocap among wasp allergen components-i3, i209, i211 were 88%; 0.5, 90%; 0.6 and 68%; 0.4 respectively and honey bee allergen components-i1, i208, i217 were 95%; 0.9, 93%; 0.7 and 97%; 0.9 respectively. agreement between polycheck and immuno-cap i1 and i3 allergen components were 85%; 0.7 and 66%; 0.3 respectively. agreement between polycheck and euroline i1 and i3 allergen component were 86%; 0.7 and 61%; 0.2 respectively. based on wasp and bee components in all three systems, sensitization pattern was analyzed. similar test results were found between euroline and immunocap systems. the comparative studies carried out showed a markedly higher compliance of results with the euroline tests compared to polycheck with the immunocap system. percent agreement was extremely high and kappa value was substantial or almost perfect in the case of bee venom allergy between euroline results: a total of 113 patients were included; 80 (71%) males, with a mean age of 38 (±15) years; 23 (21%) beekeepers, 25 (23%) were atopic, 4 (4%) had asthma, 14 (13%) rhinitis and 18 (16%) cardiovascular disease, and 14 of these patients were on ace/beta blockers. vit with honeybee was proposed in 73 (64%), wasp 38 (34%) and polistes 2 (2%). the mean duration of vit was 45 (±16) months. however, 23 completed less than 36 months. of the total, 28 patients (25%) were not treated with vit. eighty-eight patients (78%) participated in the telephone interview: 49 completed vit (56%), 15 were still on vit (17%) and 24 did not undergo vit (27%). of those who completed vit, 14 (29%) were restung and 3 went to the emergency department (er). twenty-four patients (36%) were stung while still on vit. of those never on vit, 12 (50%) were re-stung and 9 went to er. the severity of the reactions according to mueller of the patients who completed vit (mean follow-up time was 45 months (1-110 months)) and were stung again was: local reaction in 11 (79%), grade i in 1 (7%); grade iii in 1 (7%). one had a toxic reaction after multiple stings. in those who were stung during vit, 20 (87%) had local reactions, 1 (4%) grade i and 2 (9%) grade iii. of those who were not treated and were re-stung: 3 (25%) had grade i, 4 (33%) grade iii and 5 (42%) grade iv. in this series, the patients who did not undergo vit presented a greater number of systemic reactions when re-stung as well as more severe reactions (p < 0.01). conclusion: in this group with indication for vit, the reactions of the re-stings were less severe in the patients who had completed or who were on venom immunotherapy, as expected. three quarters of those who did not undergo treatment had severe anaphylactic reactions when they were stung again. this study reinforces the importance and the efficacy of immunotherapy in the treatment of hymenoptera venom allergy. method: this is a retrospective, descriptive study of cases diagmethod: data were issued from the reference centre in mastocytosis of toulouse university hospital. ms diagnosis was determined using world health organization diagnostic criteria. hymenoptera venom immunotherapy was performed with an ultra-rush protocol (table) . results: seven patients were included (4 women, 3 men), median age 47 years old. during the anaphylactic reaction, cutaneous signs missed in all cases. the reaction was most often severe: grade 2 (n = 1), grade 3 (n = 5), grade 4 (n = 1). three patients suffered from digestive symptoms and one from respiratory manifestations. basal tryptase in serum reached 10.1-41.7 μg/l. hymenoptera venom specific ige were low (0.11-1.51 kui/l) except for one patient (35.6 kui/l). ait was initiated with vespula venom in 3 patients, polistis in 1 patient, apis mellifera and vespula in 1 patient, vespula and polistis in 2 patients. no reaction was observed during ait. four restringing accidents led to increase the cumulative dose to 150 μg and 200 μg in 2 patients. in these patients, the diagnosis of mastocytosis was made due to the resting. conclusion: hymenoptera venom ait using ultra-rush protocol seems well tolerated in patients with systemic mastocytosis. specific studies are necessary to determine the real tolerance profile of this protocol. collaboration with reference centres for mastocytosis should be considered for all patients with mastocytosis associated to hymenoptera venom allergy. dose (μg/ml) results: see table. conclusion: there is a shift or immunomodulation in terms of sige to vespids. even in patients double sensitised who were receiving venom of only one of the vespids. albanesi m background: slit has been suggested as an alternative route for allergen-specific immunotherapy. aim of this study was to investigate allergen-specific antibody responses in birch pollen allergic children who had received slit for two years using recombinant allergens. method: children (n = 28; 5-12 y o) with respiratory symptoms of birch pollen and oral allergic syndromes (oas) were studied. ten children received slit with staloral, 10 were treated by slit with microgen, and 8 children received only symptomatic therapy (control group). sige and sigg levels to rbet v 1, rbet v 2, rbet v 4 were measured twice (before therapy started and after two years) using quantitative immunocap and a panel of more than 170 microarrayed allergens using immunocap isac technology. clinical efficacy of slit was evaluated by recording symptoms upon allergen contact and need of rescue medication. results: all 28 children were sensitized to the major birch pollen allergen, bet v 1 and one patient from each of the groups showed to bet v 2, no patient had sige to bet v 4. after two years of slit clinical improvement was observed in the slit patients. in the staloral group there were no respiratory symptoms in 3 patients and a decrease of symptom severity in the other 7 cases as well as a partial or complete tolerance to pr10 allergen-containing food in the 10 patients. microgen treatment had no influence on oas symptoms but decreased of pollinosis severity in 8 children. however, there were no statistically significant differences of bet v 1-specific levels measured before and after treatment in the slit and control groups (mann-whitney, p > 0.05). in this real-life study we found that birch allergic children who had been treated with slit showed a reduction of clinical symptoms but we did not find a significant induction of allergen-specific igg levels in the slit-treated group when compared with children who had only symptomatic treatment. conclusion: our study confirms the scarcity of food additives allergy. it also suggests that even when the diagnostic of allergy was excluded with a negative oral food challenge, families remain suspicious about industrials feeding products containing food additives. these results should reassure health professionals and parents who incriminate too frequently food dyes and conservators when a manifestation which mimics allergic reactions occurs. background: autumn/winter birth has been reported to be a risk factor of food allergy (fa) development. a putative mechanism is that dry/cold weather causes and exacerbates infant atopic dermatitis (ad), which is a major risk factor for food sensitization through inflamed/damaged skin. we investigated prevalence of fa among infants under well skin care in relation with seasons of birth (sob). we recruited full-term newborn infants without perinatal diseases at an obstetric/pediatric clinic. participants were followed up for skin status and food allergy symptoms until 12 months of age. sob were defined as spring (march-may), summer (june-august), autumn (september-november) and winter (december-february). ad was diagnosed based on the united kingdom working party's criteria. use of moisturizer (mo) and topical corticosteroids (tcs) was recorded. primary outcome was fa based on apparent immediate allergic reaction after ingestion of causative food. we classified infants who avoided any food because of sensitization or mother's anxiety as suspected fa. results: six hundred and thirty-one infants were screened for 12 month-period and 531 infants were enrolled in this study. of them, 277 infants were born in spring-summer (s-born) and 254 infants were born in autumn-winter (w-born). fa developed in 24 (4.5%) infants and 31 (5.8%) infants had suspected fa. there was no difference (p = 0.68) in prevalence of fa and suspected fa between s-born and w-born. multivariate analysis revealed ad at 2 and 7 months of age was a significant risk factor for fa with or=2.6 (95%: 1.1-6.0) and or=3.5 (95% ci: 1.4-8.9), respectively. prevalence of ad at 2 months of age was higher in w-born than s-born but prevalence promptly decreased thereafter and stayed low with early use of mo and tcs. prevalence of ad was rather higher at 7 months in s-born than w-born. results: 160 cases and 126 controls were included. the median age was 44 years, (q1-q3 33-53). men and women were almost equally represented (50.35% males). alcohol consumption associated with the intake of mammalian meat or innards as the trigger factor. the overall prevalence of a positive result of sige to α-gal was abstracts | 645 15.7% ic95% (11.5, 20 .0); cases _26.3% ic95% (19.4, 33.1) controls _2.4% ic95% (0.0, 5.1)_. among cases sige anti α-gal positivity rate ranged from 55.8% (rural), to 35.7% (half-urban) and 12.6% (urban). the rates of positivity were 69.5%, (northern) 1.4% (center) and 0% (mediterranean). a positive result of sige to α-gal was more frequently observed among men (24.31%) than women (7.04%) and associated with history of tick bites, practice of outdoor activities, pet's ownership and the antecedent of having eaten mammalian meats or innards previously to the development of symptoms background: a special challenge in the 21st century for allergists is allergy to food, which is considered "the second wave" of epidemics of allergic diseases. panallergens occur in unrelated organisms and perform a similar function in them. in their structure, they have highly conserved amino acid sequence regions and a similar three-dimensional structure, and thus meet the requirements for cross-recognition by ige. results: in 46 patients (92%) isac test has been shown to have specific ige for panallergen components. mostly, the presence of ige for pr-10 proteins has been shown in 41 patients. in 12 patients ige to ltp; 11 patients ige to ccd; 6 patients to profilin; 5 patients to tropomyosin; 4 patients to serum albumin, 1 person to tlp. an important aspect is undoubtedly the occurrence of simultaneous sensitization to several panallergens. analysis of data from the study group showed that isolated sensitization to one panallergen concerned only pr10 proteins (24 patients), tropomyosin (2 patients) and profilin (1 patient). in the remaining 19 patients, the analysis of the isac test results showed that two or more panallergens were allergic. in the study group, asige for the component responsible for the occurrence of real food allergy was detected in 13 (26%) patients. mostly, the presence of ige for jug r 2 has been shown in 9 patients. in the study group, panallergens were more likely to be responsible for food intolerance than specific food allergens. results: of 43 children, 14 children had peanut allergy only, 14 children had tree nut allergy only, and 15 children had both. the mean age was 3.3 ± 1.9 years in peanut allergy, 5.0 ± 3.6 years in tree nut allergy, and 4.2 ± 3.0 years in both. male to female ratio was significantly higher in tree nut allergy (78.6%) than peanut allergy (42.9%). among tree nut allergens identified, walnut (75.9%) was most frequent, followed by almond (55.2%), hazelnut (34.5%), pine nut (20.7%), chestnut (13.8%), cashew (6.9%), pistachio (3.4%), and macadamia (3.4%). mean serum total ige level was 888 kua/l in tree nut allergy and 1440 kua/l in peanut allergy. mean serum specific ige level to peanut, walnut, almond, hazelnut, and pine nut was 30.0, 21.9, 14.2, 22.4, 5.6, and 6 .3 kua/l, respectively. children with peanut allergy had higher rate of co-sensitization with soybean and higher soybean-specific ige levels than children with tree nut allergy. however, there was no difference in co-sensitization rate with tree pollen between peanut and tree nut allergy. children with peanut allergy showed significantly increased co-sensitization rate with egg white and wheat compared to children with tree nut allergy. a 42.8% of the children with peanut allergy and 28.6% of tree nut allergy showed co-sensitization with aeroallergens. a total of 75% of the children with peanut allergy showed decreased specific ige levels within 1-5 years. conclusion: prevalence of peanut and tree nut allergy is similar. tree nut allergy develops later than peanut allergy and more common in male. children with peanut allergy showed higher co-sensitization rate with soybean, egg white and wheat compared to children with tree nut allergy. 1257 | natural history of egg allergy in a large cohort of infants with food allergy shows its high prevalence but also its transient nature in a 36 months of follow-up background: the cohort of 80 infants (55 boys,25 girls,3-36 months) with the food allergy has been followed for 36 months. as more than 70% of infants manifested atopic dermatitis (ad), a condition closely linked to egg sensitisation, we focused our attention on egg allergy, following its natural history as well as a development of atopic march. method: the diagnosis of food allergy was based on a personal history, clinical examination, skin prick tests and/or atopy patch tests with native foods. laboratory tests were performed within 1 year of age the latest. the specific ige levels against food allergens were measured using immunocap or immulite. patients with ad were scored according to scorad system. the oral food challenges (ofcs) with cooked/baked egg were done in children at the age of 12 months except for children at risk of anaphylaxis. results: within the whole cohort the allergy to cow milk proteins was confirmed in 70 pts (87.5%), to egg in 35 pts (43.7%), to wheat in 8 pts(10%), to lentil in 3 pts (3.75%) to banana in 3 pts (3.75%), to soya in 2 pts (2.5%) and to potatoes in 1 pt (1.25%). in a cohort of 35 egg allergy patients we found out that: 83% of pts presented the early onset of allergy-up to 3 months of age, 80% of pts presented severe ad (scorad >50), 46% of pts showed cosensitisation to peanuts, 34% of pts had early sensitisation to inhaled allergens, and majority 54% of pts presented with early onset allergic rhinitis and/or asthma. we proved that egg allergy is closely linked with the early onset of allergy symptoms, with severe forms of ad, co-sensitization to peanuts, early sensitisation to inhaled allergens and an early onset of allergic rhinitis and/or asthma. we also proved that the egg allergy in infancy is transient. the tolerance to baked/cooked egg was achieved in about 75% of pts at the age of 3 years, unlike previously published results claiming the reach of tolerance in 75% of pts at the age of 7 years. in these patients we studied: sex, personal history, type of reaction they presented, time of onset of symptoms and food involved. results: out of a total of 1479 patients over 60 years of age (from 60 to 99 years old) who have been attended the consultation for the first time during these period, 70 patients (4.7%) ask about possible allergic food allergies. of these patients who came for possible allergic pathology, 28 patients (40%) presented positive results. these are the other item we have studied: 1. sex of patients: 55% of the patients are women. these patients because of that, it is important to remember that food allergy can also appear in old people. the food that is mainly involved in our population is fish and seafood. a much higher percentage than in other populations, probably due to the mediterranean diet of spain. the symptoms mainly involved are itching and skin lesion, which is the characteristic symptom of a mild allergic reaction. in our population, there was 3 patients with a anaphylactic shock, a much higher percentage than in other studies. the experience with this group of patients is still limited. more studies are needed to know better this patient profile. background: cow's milk allergy (cma) is the first atopic disease in children. diagnosis suspicion in the emergency room (er) is increasingly frequent, however, further assessment by an allergist is often difficult to schedule. therefore, screening for cma through a blood test (specific ige) while the infant is still in the er has gained momentum in recent years. we set out to analyse (a) symptoms which had led the emergency physician to prescribe specific ige, (b) the prevalence of confirmed cma among infants screened in the er, and (c) the long-term outcome of the screened infants. method: a retrospective study of medical records and laboratory results was performed. patients were infants under 6 months, without a previous diagnosis of cma, attending one of the two pediatric er of the university hospitals of marseille, france. allergy blood tests were specific ige to cow's milk extract (immuno-cap, thermofisher, sweden). in infants with specific ige to cow's milk extract of 0.12 kua/l or higher, ige directed to the main three individual proteins (casein, alpha lactalbumin et beta lactoglobulin) were also measured. results: 482 infants were included from december 2011 to june 2016. the sex ratio was 1.45. 40% of infants were atopic et 48% were currently or had been breastfed. the most prevalent symptoms were vomiting and reflux. one third of infants were hospitalized after the er visit. following the er visit, 25% of infants attended a specialized consultation with an allergist. 15% of infants with a follow-up visit were diagnosed with an ige mediated cma. 18 infants with cma developed further food allergies (egg, nuts, cashew…). it is difficult to diagnose it. the emergency pediatrician are increasingly confronted to infant with symptoms evoking cma. thus they prescribe sige and extensively hydrolysed proteins because they know that ige-positive infants can be ige-negative during the interval between the er visit and the follow-up one. after bad results interpretation of blood assay after er visit, cma was probably over diagnosed without prick test for ige positive allergy and no eviction/ reintroduction test for non ige. the lack of allergist is probably leading to over prescription of blood assay in er to diagnose cma and prolonged eviction of milk. results: a total of 96 patients were included (59% female) aged from 18 to 82yo with an average 33.9 ± 15 years. 77% of patients had history of atopic disease: 60% rhinitis, 30% asthma, 9% prior food allergy, 8% eczema, 6% drug allergy, 5% eosinophilic esophagitis (ee) and 3% chronic urticaria. mean serum total ige was 441.9 ui/ml. sensitization to aeroallergens was present in 63% of patients, the most common were dust mites (22.9%), pollen (15.6%) or both (25%). in 69(72%) patients, first symptoms of fa appeared ≥18 yo, with an average age of 35 ± 15.4 yo. in this group, 4 were diagnosed with ee, 1 with eosinophilic colitis and 2 with eosinophilic gastritis. from the remaining 62 patients, 20 had history of reaction with more than 1 food group (fg). cutaneous reactions were referred in 60% of patients followed by anaphylaxis (20%) and gastrointestinal symptoms (10%). the fg most commonly implied were: fresh fruits (n = 26), seafood (n = 19) and tree nuts (n = 6). fa diagnosis was confirmed in 66% of patients, the remaining had negative ofc. in 27(28%) patients, their symptoms started under 18 yo, with an average age of 11.5 ± 5yo. from this group, 10(44%) were diagnosed ee. from the remaining 17 patients, cutaneous complaints were the most frequent (47%) followed by gastrointestinal (35%) and respiratory symptoms (17%). the most common fg implied were: fresh fruits(n = 9), seafood(n = 4) and tree nuts(n = 3). only one anaphylaxis was referred. fa was confirmed in 82%, the remaining had negative ofc. in patients with history of anaphylaxis 13 of 14 had positive st and/ or sige; one had negative sp and sige, with ofc positive. the 401 blood donors were classified based on their clinical symptoms related to possible as contact via fish intake: allergic to as (1%), chronic urticaria (0.5%), unspecific dyspepsia (2.8%) and asymptomatic (95.5%). the prevalence of sensitization (anti-as ige >0.35 kua/l) were 12.7% (ic: 9.6-16.4%; mean 0.69 kua/l; median 0.03 kua/l) with a maximum value of 40.70 kua/l. raw fish consumption was the only variable associated with statistical significance (p < 0.05) to as sensitization (20.4% vs 9.5%, respectively). albacore and codfish were the most consumed species associated to seropositive results (15%), followed by hake (10%). coastal population (13.6% vs 9.3%), non-previously frozen fish consumption (13.7% vs 9.2%) and >3 times per week fish consumption (51.2%) were other seropositive associated factors. background: oral allergy syndrome (oas) is an ige-mediated allergy caused by raw fruits and vegetables in patients with pollen allergy, which is known as the most common food allergy in adults. however, there has been no nation-wide study on oral allergy syndrome in korea. the aim of this study is to investigate the prevalence and clinical manifestations of oas in korea. method: twenty two investigators from 19 hospitals and 2 private clinics participated in this study. the patients with allergic rhinoconjunctivitis and/or bronchial asthma with pollen allergy were enrolled to the survey. the questionnaires include demographics, a list of fruits and vegetables, and clinical manifestations of food allergy. pollen allergies were diagnosed by positive results of one or more pollen allergens including birch, alder, hazel, beech, oak, willow, poplar, bermuda, meadow, orchard, rye, timothy, mugwort, ragweed, hop japanese on allergy skin prick tests (allergen/histamine ratio ≥3+) and/or serum specific ige levels using multiple allergen simultaneous tests (mast ≥2+) or immunocap (≥0.35 ku/l). conclusion: this is the first nation-wide study for oas in korea. the prevalence of oas in korea was 41.7%, in which substantial proportion had anaphylaxis. these results will provide useful information for clinicians to apply in clinical practice. we conducted a self-administered, questionnaire-based survey in 2013-15 during the 18-month checkup. children were considered to have food allergies if they were diagnosed by a physician or if they had been instructed to avoid a causative food after medical examination by interview. we divided the year into three periods. the months of march-june were considered spring, july-october as summer/fall, and november-february as winter. while the season of onset for the 4095 boys occurred in 8.2%, 12.7%, and 15.3% in spring, summer/fall, and winter, respectively, it was 6.9%, 10.5%, and 12.2%, respectively, for the 3922 girls. thus, the onset rate was the highest in winter for both genders. in boys whose mothers did not consume folic acid (fol − ), the food allergy onset rate was significantly higher for boys whose mothers ate no eggs and for boys whose mothers ate 2-5 eggs per week than for those whose mothers ate eggs daily according to the dunnet multiple comparison test. however, no relationship was observed with egg intake if the mother had consumed folic acid (fol + ). on the basis of seasons, fol − and egg intake by mothers affected only children born in winter, with a significant difference in the dunnet multiple comparison. among mothers who did not eat eggs, fol + was 15.2% and fol − was 28.2%; for mothers who ate 2-5 eggs per week, fol + was 16.5% and fol − was 12.9%; and for mothers who ate eggs every day, fol + was 17.9% and folwas 4.9%. thus, consumption of folic acid seemingly annulated the effects of eating eggs. however, for girls, neither folic acid nor eating eggs had any effect on the onset rate. conclusion: since this effect varied according to the birth season, consumption of folic acid, a methyl group donor, appeared to affect the allergy onset in children. results: skin prick test with commercial extracts of tuna (7 mm), cod (5 mm), rooster (8 mm), hake (6 mm), salmon (4 mm), trout (7 mm) and anisakis (7 mm ige to shrimp, lobster, crab and mixed seafood were all undetectable. dermatophagoides pteronyssinus 10, to assess for tropomyosins was negative. outcome: the patient continues to react to both hdm and shrimp, despite undetectable ige levels to tropomyosin associated components. this is the only testing available in south africa currently and hence we are unable to look at other proteins. the relationship between tropomyosins in shellfish allergy and mite allergy has been well documented and investigated, but other allergens are now also being implicated in cross-reactions. we also established the level of ige specific to allergen components using the immunocap isac method. allergen-specific ige was not elevated to any shrimp allergens available in immunocap isac: n pen m1 (tropomyosin), n pen m2 (arginine kinase) and n pen m4 (calcium binding sarcoplasmic protein). the patient was diagnosed with a shrimp allergy. the molecular diagnostics used did not explain which allergen component is the patient allergic to. it is possible that the patient is allergic to hemocyanin, which can also cross-react with house dust mite allergens, but confirmation of this diagnosis requires further investiresults: the groups were identical in terms of the age and sex (table 1) . ara h 2 ige correlated (spearman test) with the cumulative protein dose (threshold dose) r = −0.439 (p = 0.023) but not with reaction severity r = 0.091 (p = 0.348), or the use of adrenaline r = 0.300 (p = 0.093). patients with ara h ige < 10 ku/l had higher threshold doses (644 vs 71 mg) than children whose ara h 2 ige was ≥10 ku/l (p = 0.016). there were no significant differences in severity of the reaction or in use of adrenaline (table 1) . the level of ara h 2 ige is relevant in predicting the threshold dose at peanut exposure. a low reaction threshold dose increases the risk of reaction at an accidental exposure leading potentially to a severe reaction. flaxseed allergy is uncommon and most of the cases reported involved anaphylaxis. cross reactivity has been described with other seeds. case report: a 9-year-old atopic girl diagnosed with egg allergy and rhinoconjunctivitis and asthma due to pollens. when she was eight, she presented two reactions consisting of conjunctival, periorbicular, malar erythema and abdominal pain after eating egg free french toasts cooked with flaxseed. she was treated with oral antihistamines. the allergic workup included prick-by-prick test with flaxseed which was positive and skin prick tests with mites, molds, cockroach, cat, dog, profilin, ltp and pollens with positive results for olive and grass pollens. the serum total immunoglobulin (ig) e was 476 u/l, and specific ige to flaxseed was 7.23 kua/l. the flaxseed extract was resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and an ige immunoblotting was performed under nonreducing conditions. the patient's serum showed specific recognition of a 18-kda band in the immunoblot. proteins were identified using mass spectrometry (maldi-tof) that showed results highly consistent with conlinin, a 2s storage protein of flaxseed. we described for the first time a patient with allergy to flaxseed due to conlinin, a 2s storage protein of flaxseed. background: cyperus esculentus is an herbaceous plant that has edible tubers called tiger nuts. in spain, they are used mainly in the elaboration of the well-known "horchata" or tiger nut milk, which is obtained by macerating tiger nuts with water and sugar. tiger nut allergy has rarely been reported, despite of its widespread consumption. case report: we present the case of a 19-year-old male with a history of oral syndrome allergy with several fruits (peach, melon, banana, kiwi, apple, pear and plum) and seasonal allergic rhinoconjunctivitis due to grass pollen. he reported oral pruritus, vomiting and cutaneous itching in both arms and hands immediately after drinking tiger nut milk. he became asymptomatic without treatment after 3-4 hours. the allergic workup included skin prick tests with profilin, ltp, latex and fruits that were positive to melon and watermelon and negative to profilin, ltp, latex and the rest of the fruits. prick-by-prick tests with melon, banana, kiwi, apple, pear, tiger nut and tiger nut milk were positive. the serum total immunoglobulin (ig) e was 68.7 μg/l, and specific ige was negative to profilin, ltp, bet v1 and all the tested fruits. background: specific blood ige tests for food allergens are mainly used to confirm a suspect food allergy than to diagnose such an allergy. this is due to the low positive predictive value and the high negative predictive value they have. nevertheless, they are very helpful when they are interpreted in the context of medical history by an experienced allergist. we analyzed the prevalence of sige in children with suspected food allergies. we retrospectively analyzed the all the consecutive laboratory tests of sige for food allergens during the two-year period (2015) (2016) (2017) . tests were of children diagnosed or suspected to have food allergies. a quantitative immunoblot assay was used to measure the circulated 30 different sige. t-test, wilcoxon signed rank test, and chi-square were used to make comparisons. results: fifty-six (55.4%) men and 45 (44.6%) females, 3.4 + 3.2 years old with a maximum of 16 years and a minimum of 2 months old were part of 101 children whose tests were analyzed. one-third of the tests (30.7%) reveals more than one sige present and 55.4% of the tests resulted negative for the sige for the 30 allergens tested (table) . only 3 (3.0%) children have very high concentrations (>100 iu/ml) of egg whites sige, and 2 (2.0%) have egg yolk sige. concentrations of 79.27% of sige positive cases were 0.35-3.5 iu/ml. in our study more than half of the children suspected of food allergies resulted negative for sige for 30 most common food allergens. seventy-nine percent of the positive cases had relatively low sige. only a few positive cases have higher sige than 100 iu/ml. our data support the recommendation that sige couldn't be decisive in food allergic diagnosis, but they may help if they are interpreted cautiously. method: seven patients with a mean age of 61.4 years and female to male ratio of: 3:4, with a background history of hypertension treated with an acei presented with oro-pharyngeal irritation (itch, tingling), face and tongue angioedema and laryngeal constriction, on ingesting fresh fruits (cherries, apples, plums, peaches, apricots, strawberries, grapes), vegetables (parsnips) and/or nuts (peanuts, hazelnuts). two patients required admission to emergency department and three received adrenaline auto-injector. six out of seven patients underwent skin prick testing to common aeroallergens and the index foods. in five cases, immunocap/isac testing was undertaken. in one case the diagnosis was based on the history and in one other case it was based on history and skin prick tests. results: a diagnosis of pfas was confirmed in all patients through the clinical history, spt and/or specific ige serology to the offending food confirming predominant sensitisation pr-10 allergens. primary food allergy and spontaneous angioedema was excluded in all patients. in the cohort studied, the pfas symptoms were unusually severe. we therefore postulate that this was secondary to concurrent use of acei. the management of these patients to sensitization to a β-casein with high homology between only the first 3 milks. more precisely, the allergen candidate could be γcasein, which is derived from β-casein by proteolysis, whose abundance increases during cheese production from fresh milk, and which is absent in cow's milk. a lactoglobulin specific to buffalo's milk may also be responsible. in case of ewe's and goat's milk allergy without cow's milk allergy, sensitization to buffalo's milk should systemically be seeked out. we recommend inclusion of all mammalian milks in the list of the 14 mandatory allergens for declaration on food products. method: prick tests with fruit battery, ltp and profilin was made. analytical with blood count, immunoglobulins, triptasa, total ige and specific ige to banana, apple and orange. finally, open oral provocation with fruits was performed. the diagnostic key was given by the mother of the patient who attended consultations because the infant had erythema in the temporary zone after drinking sea water on the beach. associated with salivation, the patient presented erythema in the malar area lasting a few seconds many times. the prick tests with fruits, ltp and profilin were negative. hemogram: 100 eosinophils/μl, total ig e: 4.9 iu/ml, specific ige to fruits were negative. triptase: 5.1 μg/l. method: here we describe a variety of cases of nsltp allergy presenting to a tertiary allergy centre in the north west of england. results: nsltps have been found to be major allergens in various foods and they are likely to produce severe and systemic allergic reactions. this is reflected in the cases we present here. these proteins are highly cross-reactive due to extensive sequence homology and are panallergens. nsltps are remarkably heat stable and retains its allergenicity in processed foods. it is assumed that nsltps may sensitise both by inhalation and ingestion. an intriguing aspect in nsltp hypersensitivity is the extreme variability of its clinical expression. co-factors are often needed for the clinical expression of nsltp hypersensitivity. conclusion: patients regardless of where they are from, presenting with multiple severe/systemic food allergies need to be investigated for nsltp allergy. these patients require specific dietary advice on foods to avoid and a tailored management plan on how to deal with their allergic reactions. cow's milk as well as cow's milk products are tolerated. material and methods: skin prick tests with different sorts of milk, cheese and milk proteins were performed, specific ige antibodies were measured, a basophil activation test with cow's and goat's milk was performed and an oral provocation test (opt) with cow's milk, cow's milk cheese, raw milk and raw milk cheese was conducted. results: skin prick tests were positive for sheep's milk, goat's milk, goat's milk casein, feta, pecorino and parmesan cheese. elevated specific ige against goat's milk (12.7 ku/l) and sheep's milk (11.7 ku/ l) were detected. activation of basophil granulocytes after incubation of the patient's blood with cow's milk and goat's milk was measurable but also in the non-incubated control blood. all cow's milk products were tolerated in the opt. conclusion: despite consistent homologies between whey and casein proteins of mammals and high cross-reactivity between cow's, goat's and sheep's milk an isolated goat's and sheep's milk allergy with tolerance of cow's milk is possible. skin testing and specific ige help to distinguish from allergy against cow's milk proteins. diet counseling is possible after opt. introduction: salmon roe's allergy, without concomitant fish allergy, is rarely described in western countries. there are few studies on its allergenicity. objective: to report of a case of salmon roe allergy without concomitant fish allergy in a western country. case report: a 26-year-old male with house dust mite allergic rhinitis and asthma, describes for the 1st time, in 2015, an acute episode of dyspnoea, rhinorrhoea, ocular pruritus, epigastric pain and nausea, a few minutes after the ingestion of a sushi meal with rice, salmon, salmon roe, wasabi, soy and ginger. these complaints motivated observation in the emergency room, where it was still documented uvular oedema. he was prescribed intramuscular adrenaline, intravenous steroids and anti-histamines with complete symptoms resolution. the patient declares not had eaten other foods, taken any drugs including nsaid, been infected or practised exercise. skin prick tests with food extracts (salmon and other fish, shellfish, soy, rice, egg total, egg white, egg yolk, ovalbumin and ovomucoid) were negative. skin prick-prick tests were positive for salmon roe (17 × 10 mm) and negative for egg (white and yolk), ginger, salmon, flying fish roe (tobiko), sturgeon roe (caviar) and black scabbard fish roe. specific-ige (sige) to salmon roe extract was 0.28 kua/l (immu-nocap-phadia) and negative against extracts from salmon fish and other fish (<0.1 kua/l). sds-page immunoblotting with salmon roes extract showed a 20 kda-ige binding band, that may correspond to a lipovitelin. after the allergic reaction the patient have tolerated abstracts | 659 salmon fish and other fish roes (tobiko, caviar and black scabbard fish). no oral provocation test with salmon roes was performed given the severity of the reaction. conclusion: this report is an example of a severe allergic reaction to salmon roe without concomitant fish allergy, where the clinical history and the in vivo and in vitro tests were important to an accurate diagnosis. the authors believe this is the first report of a salmon roe anaphylaxis in our country and highlight the importance of this allergen in the western countries, given the increase of sushi consumption in these countries. case report: the prevalence of food allergy is increasing worldwide and consumer habits are changing. pomegranate (punica granatum) was commonly consumed in the mediterranean area but in the last few years became also popular in the different parts of europe. a 5-year-old boy was admitted to our emergency department suffering from allergic reaction within 10 minutes after consumption of pomegranate. he presented with skin pruritus, generalized urticaria and eyelid edema. symptoms resolved within an hour after oral intake of cetirizine. prick-to-prick test with pomegranate was positive (wheal diameter 13 × 7 mm). he had a history of anaphylaxis with egg (urticaria and wheezing) at the age of 9 month, meanwhile consumption of egg is well tolerated. an episode of anaphylaxis with unknown origin appeared at the age of 4 years (urticaria, wheezing and abdominal pain). skin prick tests with aeroallergens revealed birch pollen allergy and he was diagnosed with allergic rhinoconjunctivitis. dietary elimination of pomegranate was suggested and adrenaline auto-injector was provided. we have analyzed omalizumab effectiveness and safety in patients with csu from our database. clinical response was categorized as: no response, partial or complete response by using the urticaria activity score 7 (uas 7). furthermore, the dosage, administration frequency and any side effects were recorded. results: effectiveness: 23 out of 25 patients (92%) achieved complete response. 15 (65.2%) after a single dose of 300 mg (8 patients) or 150 mg (7); 5 patients (21.7%) after two doses of 300 mg (4 patients) or 150 mg (1) results: multi-ethnic adolescents accounted for approximately 1.2% of the total sample of adolescents. prevalence of asthma was significantly higher in multi-ethnic group than non multi-ethnic group. we examined if maternal or paternal foreign born status had a differential effect: in multi-ethnic family with foreign-born father, prevalence of asthma was significantly higher. parental region of country at birth had a significant influence on the prevalence of asthma. adjusted logistic regression analysis was used to determine risk factors for occurrence of allergic disease. residential area, perceived household economic status, parental region of country at birth, and body mass index (bmi) had a significant effect on prevalence of asthma. conclusion: population admixing appears to have significant effect on the prevalence of asthma. further study will be needed to clarify the effect of population admixing on prevalence of allergic disease. several studies have aimed to explore the possibility of mirs as biomarkers for various diseases. in our study we examined six different mirs, previously shown to be involved in eosinophil development and other immune responses, in serum from non-allergic and allergic asthmatics and healthy control subjects in order to determine their potential ability to be used as biomarkers for varying forms of asthma. method: serum from healthy individuals as well as age matched non-allergic asthmatics (naa) and allergic asthmatics (aa) were utilized. additionally, the naas and aas subjects had high eosinophila (≥0.4 × 10 9 cells/l) compared to healthy controls (≤0.1 × 10 9 cells/l) and eosinophil cationic protein (ecp) in serum was measured. asthmatic subjects were included irrespective of inhaled corticosteroid usage. rna was extracted from serum, reverse transcribed and subjected to qpcr analysis. expression changes in six candidate mirs, mir-126, -145, -146a, -155, -223, and -374, were investigated. results: two mirnas, mir-155 and mir-146a, were significantly upregulated in aas as compared to naa or healthy subjects. additionally, mir-223 was upregulated in naa, but not aa or healthy subjects. furthermore, the expression change observed in the aa mirs appeared to correlate with the use of inhaled corticosteroids, but not in the naa mirs. finally, mir-223 and mir-374 expression levels were altered based on the number of eosinophils, which correlated to ecp levels, in naa subjects. conclusion: using six mirs found in the literature to be involved in eosinophila or immune responses, we were able to detect expression changes in the serum of healthy and asthmatic individuals. moreover, were able to distinguish between healthy individuals, aas, and naas on inhaled corticosteroids or with differing eosinophil levels, leading to the possibility that these mirs may be valuable future biomarkers for asthma. background: some studies report that certain sensitization profiles may increase the risk of a more serious allergic respiratory disease. the aim of this study was to describe the sensitization patterns to major allergens of dust mites in our area and investigate the association of these patterns with a specific clinical picture. method: multicenter study performed in 3 hospitals for 4 months. we recruited 133 patients older than 5 years with rhinitis and/or bronchial asthma, with a history of allergy to dust mites and both skin test and specific ige to d. pteronyssinus, d. farinae or l. destructor positive. der p1 and der p2 were determined to all of them. we analyzed 133 patients with an average age of 28.25 years, 60.9% women, 9.7% smokers, 69.9% rhinitis and asthma, 17.3% only rhinitis and 12.8% only asthma. 94% of patients presented sensitization to d. pteronyssinus, 83.5% to d. farinae and 63.2% to l. destructor. the detected sensitization patterns were: both der p1/der p2 positive 73.9%; der p1 positive 8.4%, der p2 positive 11.8% and both der p1/der p2 negative 5.9%. it was observed that patients with higher specific ige levels had more severe forms of respiratory disease, with isolated asthma or associated with rhinitis. for d. pteronyssinus, d. farinae, der p1 and der p2 > 50ku/l there is a greater number of cases of asthma associated with rhinitis, while for l. destructor >3.5ku/l greater number of cases of asthma. no relationship was observed between a specific sensitization pattern and an increased risk of asthma. conclusion: 1. specific ige values greater than 50 ku/l for d. pteronyssinus, d. farinae, der p 1 and der p 2, were significantly associated with a higher probability of asthma and this association was significant for l. destructor>3.5 ku/l (class 3). 2. there are four well-defined sensitization patterns in our population that are influenced by geographic location, being the double sensitization to der p 1 and der p 2 the most prevalent and allowing the correct characterization of 94% of the cases. none of them increased the risk of asthma. kobori t 1 ; nagao m 1 ; ekenkrantz t 2 ; borres m 2 ; sjölander a 2 ; fujisawa t 1 1 national mie hospital, tsu-city, japan; 2 thermo fisher scientific, uppsala, sweden background: reliable biomarkers for diagnosis and management of asthma in young children are needed since pulmonary function test and exhaled no measurement, good biomarkers for asthma in older children and adults, are difficult to perform in young age. we have developed a sensitive and stable assay system to measure eosinophil-derived neurotoxin (edn), an eosinophil granule protein, that is released upon activation, and that may serve as a marker for eosinophilic inflammation also in young children. method: volunteer children from 0-6 years old were recruited and an isaac-based questionnaire was filled out by their caregivers. venous blood was obtained to measure serum and plasma edn and eosinophil count. edn was measured with a research assay developed on the immunocap ® platform. conclusion: blood edn may be a reliable biomarker for diagnosis of asthma in preschool children. conclusion: feno measurement as an add-on option in asthma management to identify asthma patients with th2 driven airway inflammation is less costly than the use of standard diagnostic methods. new biologics may have an additional impact on overall asthma treatment costs. our model demonstrates that incorporating feno measurement may help to optimize asthma medication and reduction in physician visits as well hospitalizations due to severe exacerbations. 1294 | peripheral airway inflammation assessed by fractional measurement of the exhaled breath temperature is a leading feature of asthma background: airway inflammation is considered to be a hallmark of asthma. the potential clinical benefits of assessing it non-invasively has led us to develop a method and device for measuring the temperature of the exhaled breath (ebt=exhaled breath temperature) reflecting the thermal state of the airway mucosa. studies have demonstrated that ebt is increased in asthma, proportionately to the level of control of the disease. in an attempt to further increase the usefulness of this approach, we have further developed a device to allow the assessment of the relative contribution of the central and peripheral airways (caw and paw). now we present the frebt data gathered from patients with suboptimal control of their asthma and from healthy subjects. method: in this cross-sectional study we included 120 volunteers: 71 patients with suboptimally controlled asthma of mild to moderate severity (median age 44, range 18-68 years, 31 men) and 49 nonsmoking subjects without respiratory disease (median age 49, range 18-70 years, 17 men). we measured the fractions corresponding to caw and paw sampled with a fast reacting inflatable balloon valve system operated by a computer during a single breathing cycle. it allows steering of the expired airflow through channels with sensitive temperature sensors. during an initial deep inhalation, the inspired volume is measured and the sequence of valve openings is adjusted so as to yield volumes of air characteristic of caw or paw during expiration. the ratios between [pawebt-cawebt] over the total ebt [%] measured during the same manoeuvre (fractional ebt, frebt) were calculated and compared between asthmatics and controls. results: there was high statistically significant difference between the frebt ratios of asthmatics and controls: 16.25 ± 0.51 (mean ± sem) vs 12.66 ± 0.28, p < 0.001. as the magnitude of the ratio depends on the difference between pawebt and cawebt, higher values of the frebt ratio point to bigger contribution of the peripheral lung tissues, presumably indicative of peripheral inflammation. multiple regression analysis with frebt ratio as dependent variable identified only asthma diagnosis as significant predictor (p < 0.001) and excluded all other anthropometric indices. conclusion: peripheral airway inflammation assessed by frebt measurement appears to be a leading characteristic in asthmatics compared to healthy subjects. method: we examined 142 outpatients (30% male, aged 18-82 year, mean age 54.7 years) with severe asthma according to ers/ ats (2014) definition treated with high dose of ics/laba± tiotropium, antileukotrienes and omalizumab. some patients (n = 21) had orally steroid-dependent asthma. they referred to our secondary care center by gps. pulmonary function tests were measured by dry spirometer (2120, vitalograph ltd., uk). skin prick tests or serum specific ige to common inhalant allergens (house dust mite, animal dander, pollen) were used to assess atopic status. results: seventy five percent (n = 107) of patients with severe asthma had fao in 50% of those was diagnosed concomitant copd. duration of asthma was 14.6 years in patient with reversible airway obstruction (rao) and 14.5 years in those with iao (p > 0.05). early (before age 12 years) onset of asthma was established in 13% of patients with rao and in 9% of patients with fao (p > 0.05). prevalence of atopy did not differ between both groups (81% vs 81%, p > 0.05) but total ige level in serum was higher in severe asthmatics with rao than fao (924 me/ml vs 330 me/ml respectively, p < 0.01). most of atopic patients with severe asthma both with fao (91%) and roa (83%, p > 0.05) were sensitized to house dust mites (d. pteronyssinus and d. farinae). hypersensitivity to pollen was diagnosed in 36% patients with foa and in 17% with rao (p > 0.05), to cat and dog dander in 49% and 25% respectively (p < 0.05). the majority (75%) of patients with severe asthma had fao. hypersensitivity to house dust mites was most common in severe atopic asthmatics with foa and roa where as sensitization to animal danger was associated with presence of foa. 1296 | loss of smell as a clinical marker of severe asthma and its association with upper airway inflammatory diseases background: asthma is frequently associated with rhinitis and chronic rhinosinusitis (crs) while severe asthma is more associated with crs with (crswnp) than without (crssnp) nasal polyps. loss of smell (los) is associated with crs, mainly with crswnp. we aimed to assess loss of smell as a clinical marker to discriminate crs from rhinitis and severe from non-severe asthma. method: in a cross-sectional multicentric study, asthmatic patients (n = 383) were evaluated by pulmonologists and ent specialists using gina, aria, and epos definitions. los was evaluated by severity [vas scale, 0-100 mm, median (iqr,inter-quartil range)] and by prevalence of anosmia (hyposmia vas >0-70 mm, anosmia vas>70 mm). results: los was present in 55.4% of asthmatics (hyposmia 41.5%, anosmia 14.9%). los was more severe [22 mm (0-75), p < 0.001] and anosmia more frequent (26.4%, p < 0.001) in severe persistent asthma than in moderate [10 mm (0-50); 11.4%] mild [0 mm (0-28); 10.7%], or intermittent [0 mm (0-45); 8.6%] asthma. in addition, los was more severe [38 mm (2-76) vs 0 mm (0-20), p < 0.001] and anosmia more frequent [28.1% vs 3.9%, p < 0.001] in crs than in rhinitis patients. in those asthmatic patients with crs, los was even more severe [50 mm vs 20 mm (0-56) p < 0.001] and anosmia more frequent (40.6% vs 13.4%, p < 0.001) in crswnp than in crssnp. conclusion: loss of smell and specially anosmia may clearly discriminate severe from non-severe asthma and crs (specially with np) from rhinitis alone in asthma patients. thus, los may be considered a significant clinical marker of severe asthma and its association with upper airway inflammatory diseases. 1297 | last station in the eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis march: eosinophilic asthma with radiological findings associated with blood eosinophilia yilmaz i 1 ; nazik bahçecioglu s 2 ; türk m 3 ; tutar n 1 ; oymak fs 2 ; gülmez i 3 1 erciyes university school of medicine, kayseri, turkey; 2 department of chest diseases, kayseri, turkey; 3 division of immunology and allergy, kayseri, turkey background: eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis (eacrs/np) is a subphenotype of adult-onset eosinophilic asthma. blood eosinophil levels are shown to be highly elevated in patients with ea-crs/np and have potential for tissue infiltration. we aimed to demonstrate the clinical features of the patients who have a blood eosinophil level above 10% and have thorax computed tomography findings due to blood eosinophilia. results: we identified 36 patients who met the above criteria. we defined this group as "eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis with radiological findings related to blood eosinophilia" (earr). the mean age was 44.9 ± 11 years and 64% was female. nasal polyps, aspirin exacerbated respiratory disease and atopy was present in 81%, 47% and 25% of the patients, abstracts | 665 respectively. the mean blood eosinophil count was 1828.6 cells/mm 3 (19%). the majority of earr patients had upper lobe dominant ground-glass opacities. the mean follow-up period was 3.2 ± 2.5 years. earr patients did not evolve into eosinophilic granulomatous polyangiitis in the follow-up. method: we aimed to identify features more probably associated with asthma in a unselected group of patients with diagnostic criteria for aco. we consecutively selected the first 10 consecutive patients with diagnostic criteria for aco. all patients were evaluated by accurate clinical history interview, assessment of asthma control test (act) and copd activity test (cat), lung function and exhaled nitric oxide (fe no ) measurements, sputum cytology, blood eosinophil count, serum total ige and periostin levels, methacholine and adenosine-mono-phosphate (amp) bronchial challenges. all these measures were repeated after an oral corticosteroid (ocs) trial of methylprednisolone 32 mg/day for 14 days. we defined 9 parameters that we expected improved after the ocs trial, and therefore considerable as markers of asthma: fev 1 , fef 25-75 , fev 1 /fvc, fe no , act, sputum and blood eosinophilia, methacholine and amp challenges. patients with improvement of at least 4 of these parameters after ocs trials were defined as "responders" to the treatment, and therefore more likely to be asthmatic than copd or aco. results: five (50%) patients were classified as responders and they were characterized by having basal higher fe no values (102.8 ± 34.2 vs 13.6 ± 3.5 ppb, p = 0.032), greater bronchial reversibility basal values of serum periostin and total ige, and blood eosinophils were higher in responders but without reaching the statistical significance. conclusion: fe no and the degree of bronchial reversibility (and possibly also the degree of response to an amp challenge) are reliable biomarkers to distinguish asthmatics among those with suspect aco. method: the preliminary case-control study included 52 obese persons with asthma who were matched for age and sex and 48 nonobese asthma subjects. non fasting serum levels of adiponectin, and leptin were measured by commercially available immune assay kits, and routine biochemical parameters were analyzed in both the study groups. the results show statistically significant lower levels of serum adiponectin and higher serum leptin levels in obese asthma subjects with respect to non-obese asthma patients (p < 0.001). moreover, an inverse correlation was also observed between serum adiponectin and serum leptin in obese asthma subjects (p < 0.05). our results indicate the association of these hormones might act as a significant predictor in the progression of asthma. moreover, the role of serum adipokines is promising and might potentially act as a meaningful drug target in the pathogenesis of asthma. background: overweight/obesity is known to be a possible factor for poor asthma control. the aim of the study is to determine the serum concentrations of leptin in atopic asthmatic patients and its relationship with body mass index (bmi), asthma severity defined by medical treatment and asthma control defined by the asthma control test (act). method: we randomly selected 33 adult patients previously diagnosed with allergic asthma based on gina (global initiative for asthma) guidelines, returning for follow-up to an outpatient allergy/ immunology clinic during november 2017. following an informed consent, the patients were asked to fill act, their bmi was recorded, and elisa blood assays for leptin were drawn. exploratory data analysis, spearman's correlation (95% ci by bootstrapping), and partial correlations were performed. results: female/male ratio was 24/9, mean bmi was 27.2 ± 4. conclusion: leptin was significantly associated with overweight/ obesity in asthmatic subjects, and showed higher values for women. leptin inverse correlation with act did not reach statistical significance, likely owing to underpowered estimates, in a small sample characterized by an elevated mean bmi and severe allergic asthma. background: immunoglobulin lowering may be associated with recurrent wheezing symptoms and clinic by increasing the tendency to viral respiratory tract infections. in this study, it was aimed to investigate the frequency of immunoglobulinemia in preschoolers with wheezing. the study was conducted between 01.01.2013 and 01.01.2016 between. university of health sciences, ankara child hospital, the children allergy and immunology clinic included patients who had been followed up and treated for at least one year with recurrent wheezing attacks within younger than 48 months. the immunoglobulin (g, a, m) values of the patients were retrospectively analyzed. immunoglobulin levels were determined to be normal and low according to age limits. the study included 585 patients (65.6% male, 34.4% female) under the age of 6 years with a mean age of 26.9 months. the mean follow-up period of the patients is 2.2 years. in 33.7% of these patients, at least one immunoglobulin was found to be low. none of these patients had any signs or symptoms of immunodeficiency. immunoglobulin a was low in 21% of the patients, immunoglobulin g in 18%, and immunoglobulin m in 7.5% of all patients. conclusion: immunoglobulin was found to be low in these patients when there was no immunodeficiency and preschool wheeze was diagnosed. this should be etiologically investigated as to whether if this is a special group in preschoolers with recurrent wheezing and hypogammaglobulinemia combination. method: asthma severe unit is formed by allergists, pneumologists, pediatricians and otorhinolaryngologists. hematologists and immunologists make specific collaborations. we present the partial results of our data collection, which include 75 patients with severe asthma according to ers/ats task force, selected by peripheral eosinophils >220 according to wagener et al. we followed them up to assess control for 1 year. we obtained cellularity in sputum using induced sputum technique. values of il of th2, th1, th17 pathway, periostin and ilc were not yet available. results: median age was 36 ± 24, feno 45 ± 34, exacerbations previous year 1.8 ± 2.4, act 17.7 ± 5, fev1 75% ± 21 and dose of inhaled corticoids (budesonide equivalent) 1235ug ± 618. most of the patients were sensitized (78%) and 15.9% were polysensitized. the most frequent sensitization was dust mites (81%). 27% had received immunotherapy of whom 35.3% with lack of response. not sensitized patients were older. sputum cell analysis of 54 patients was performed, 37% had sputum eosinophils>3%, mean sputum eosinophil value was 4.1 ± 4.7 and peripherally 518 ± 384. correlation among sputum and peripheral eosinophilia was 0.097 (p = 0.485). the peripherally eosinophil value >220 had a sensitivity of 80% and a specificity of 54% for the detection of sputum eosinophils >3%. no differences were observed in sputum cell count depending on allergic sensitization. 64% had an uncontrolled asthma. presence of polysensitization, rhinitis or polyposis were not statistically related with the control. different patterns were observed in function of cause of poor control: patients with obstructive pattern (fev1 < 80%) were older and received more inhaled treatment. patients with high rate of exacerbations had more sputum eosinophilia and neutrophilia. both groups had worse act and received more oral steroids. patients who received oral steroids were more often sensitized to fungi in some follow-up visits. not significant differences were observed in control according to the act. asthma had more sputum neutrophilia, were older, received higher inhaled steroids dose and had adult onset asthma. the only control variable related with sputum eosinophilia was exacerbation. fungi sensitization was more frequent among patients with oral steroids. method: a randomized, double blind, placebo controlled study with 80 patients from the de la salle university medical center with a mean age of 44 with partly or uncontrolled asthma. they were assigned to either cm glucan or placebo group for two months. act score and %fev1 postbronchodilator were assessed at the 1st visit, 4th week follow up, and 8th week follow up visits. an independent and paired t-test were used to determine mean changes in act scores and %fev1 between the groups. results: in the two treatment groups, those in the cm glucan group had a greater % fev1 mean change of −6.95 compared to placebo which had only −14.1, a mean difference of −7.15, and a trend toward significance with a t-test p value of 0.061. in terms of changes in act score, those in the cm glucan group had a mean change of 10.12 and 9.75 for placebo, a mean difference of 0.37 and was not significant at t test p value of 0.718. the result of the emanuel trial showed a trend of improvement among patients on both groups in terms of act score and %fev1 postbronchodilator. however, it was not statistically significant. 1306 | lung function improvements with tiotropium in patients across all ages: impact of episodes of asthma worsening during phase 3 trials vogelberg c 1 ; casale tb 2 ; bleecker er 3 ; goldstein s 4 ; szefler s 5 ; engel m 6 ; el azzi g 6 ; dewberry h 7 ; hamelmann e 8 method: post hoc analyses involved 6 phase iii, randomized, double-blind, placebo-controlled trials: 4 in patients aged 6-17 years (rubatina-/canotina-/vivatina-/pensietina-asthma) who received tiotropium (5 or 2.5 μg) or placebo, as two puffs once-daily via the respimat, as add-on to ics ± other controllers; and 2 in adults (pri-motina-asthma replicate trials) who received once-daily tiotropium 5 μg or placebo, as add-on to ics/laba ± other controllers. we analyzed change from baseline for peak fev 1(0-3h) and trough fev 1 at week 12 in vivatina-and pensietina-asthma, and week 24 in pri-motina-/rubatina-/canotina-asthma, comparing patients with and without episodes of asthma worsening during the trials. asthma worsening was defined as an episode of progressive increase in dayto-day asthma symptoms (recorded by patients and confirmed by the investigator) or a decrease of patient's best morning pef ≥30% from mean for ≥2 consecutive days. as a post hoc analysis, p values are nominal. results: there were no differences in baseline disease characteristics between those who experienced episodes of asthma worsening and those who did not, within specified age and asthma severity groups. placebo-adjusted lung function improvements were observed with tiotropium 5 μg in patients who experienced episodes of asthma worsening and those who did not during the trials (table 1) . there was some variability in subgroups with low numbers of patients. conclusion: once-daily tiotropium add-on had a similar efficacy in adult and pediatric patients with symptomatic asthma, irrespective of whether they experienced episodes of asthma worsening or not during the trials. these data support the broad efficacy of tiotropium and show largely consistent improvements in lung function even in patients who experience episodes of disease worsening. 1307 | cochrane review of the use of antibiotics for acute exacerbations of asthma method: we searched the cochrane airways trials register, trial registries and reference lists of primary studies. we extracted outcome data and assessed risk of bias in duplicate and used current cochrane methodology throughout. our primary outcomes were intensive care unit (itu) admission, duration of symptoms/exacerbation and adverse events. we included six studies, including a total of 681 adults and children. trials were of varied methodological quality and we were able to perform only limited meta-analysis. one study reported a single itu admission but no other studies reported admissions to itu. two studies investigating macrolides reported diary card symptom score and showed antibiotics improved symptoms (md −0.34, 95% ci −0.60 to −0.08). one study including 40 participants reported more symptom-free days in the macrolide group than usual care. one study of a penicillin including 69 participants reported asthma symptoms at hospital discharge; the between group difference was reported as non-significant. serious adverse events were rare; 10 events were reported across the three trials (n = 502). the pooled effect estimate for all adverse events from three studies was imprecise (or 0.99, 95% ci 0.69-1.43). no deaths were reported. conclusion: our results confirmed that omalizumab significantly improves disease control and is a safe add-on therapy. also in appropriate patients with controlled disease over time, efforts to stepdown other asthma medications will be appropriate. (45) aerd: aspirin exacerbated respiratory disease; sd: standard deviation. data are n (%), mean ± sd or n/n (%). c-act: asthma control test for children, fev1: forced expiratory volume in one second; fev1/fvc: the ratio of forced expiratory volume in one second to forced vital capacity, pef: peak expiratory flow; feno: fractional exhaled nitric oxide, vas: visual analogue scale *these included allergic rhinitis, asthma, eczema, atopic dermatitis, food allergy, etc. **there were 11, 13 and 18 missing data in treatment a, b, and c, respectively. this study examines the potential treatment effects of sq ® hdm slit-tablet on qol measured by sf-36v2 in people with aa and ar. the analyses are based on data from the mt-04 trial (eudract no. 2010-018621-19) and utilize data from the 12 sq-hdm treatment group (282 subjects) and the placebo group (277 subjects). throughout the trial, qol was measured at each of visit 3-13 via sf-36v2. this yielded psychometrically-based physical and mental health summary measures, as well as a sf-36v2 total score. according to trial design, the use of inhaled corticosteroid (ics) was reduced by 50% for a three months period (visit 10 and 11) and completely withdrawn for the last three months of the trial (visit 12 and 13). results: by estimating a simple regression on differences in sf-36v2 total score from baseline measurements (visit 3), a positive and statistically significant treatment effect on the overall qol of the 12 sq-hdm treatment compared to the placebo group in visit 9 and 10 was found. further analyses show that the qol improvements are mainly driven by increases in the general mental health score, which are carried through to visit 12. in particular, the mental health and role emotional domains show statistically significant improvements. the results show that the sq ® hdm slit-tablet improves qol measured by sf-36v2 in patients with hdm induced aa and that this effect is driven by improvements in the mental health domains. 1315 | impact of treatment prescription, adherence to treatment and use of inhalers in asthma control-results of the efimera study method: cross-sectional multicenter observational study conducted with patients who use any type of medication with inhaler devices. patients referred from primary care and seen by a pneumologist or allergist for the first time were evaluated. the following data was collected in a single visit: adequate prescription according to gina2015 guidelines (gina); specific and general treatment adherence using morisky-green questionnaire (mg) and inhaler adherence test (tai); disease control with asthma control test (act) and assessment of inhaler use technique were measured with the extended tai. results: patients included in this study (n = 1682) had a mean age of 45 ± 17 years, an average disease evolution of 14.9 ± 14.1 years, 64% of which were women. according to gina recommendations, 35.9% of patients have insufficient or inadequate prescription. when measured by the mg test the 68.5% of patients showed bad adherence, meanwhile measured by the tai test adherence was 76.8% measurements of inhaler use technique resulted in 17% of patients having one or more mistakes regardless of whether the device was a mdi or dpi. several factors showed to be related with bad asthma control: inadequate prescription (or: 8.05 [5.74-11.27 background: it is well known that the constant and prolonged tobacco smoking affects the natural history of asthma. vaping is the act of inhaling and exhaling the vapor produced by an electronic device called e-cigarette (e-cig), whose basic structure includes a power source and an atomizer. two types of vaping are the most popular ("mtl" and "cloud chasing"). we have created a web-survey with questions concerning epidemiological data, quality of life and symptoms worsening in asthmatic vapers. the survey has been advertised through various social networks and local press. 2403 people responded, including 437 asthmatics (18%). the asthmatics were: males 88%, under 18 3%, 18-25 years 36%, 26-30 years 19%, 31-65 years 42% and over 65 0%. 70% used ecig-only, 30% smoked and vaped together, 0%. those who preferred mtl-type of vape were 43% and "cloud chasing" were 57%. results: to the question: "has vaping ever worsened asthma symptoms?" 90% answered no, 10% yes. to the question: "as asthmatic, would you suggest to an asthmatic smoker to start vaping instead of smoking?" 1.3% answered no, 98.7% yes. to the question: "how much nicotine do your vaping liquids have?" 15% answered 0 mg/ml, 4% 1.5 mg/ml, 62% 3 mg/ml, 11% 6 mg/ ml, 6% 9 mg/ml, 1% 12 mg/ml and 0% 18 mg/ml. to the question: "do you take medications for your asthma?" 57% declared to use a drug as needed, 0% used a single drug daily, 16% used more than one drug daily and 27% declared "i don't take any asthma medication". we related (χ2 test) the worsening of asthma symptoms with the nicotine content (p = 0.313), the type of vaping (p = 0.305), the current therapy (p = 0.123) and we did not find a statistically significant correlation. vaping has undoubtedly shown an advantage in terms of improvement of symptoms compared to cigarette smoking (p = 0.016), in particular 82.5% subjects who smoke and vape did not have a worsening of symptoms, while 17.6% of them had a worsening. the vaper-only users who never worsened were 271 (90.6%) and 28 (9.4%) had a worsening. conclusion: despite the limits related to the online survey as a data source, e-cigs seem to be a useful tool in the pathway to quit smoking. in fact, 98% of the asthmatics who smoked traditional cigarettes would recommend switching to e-cig and 90% did not worse their asthma symptoms. background: despite the success of pharmacotherapy, more than half of patients with persistent bronchial asthma (ba) do not achieve disease control. in recent years, the issue of approaches to treatment based on the identification of phenotypes of the disease has been increasingly discussed. this approach becomes the key to optimizing therapy for asthma, allowing the personification of treatment. anti-ige-therapy using omalizumab is one of the most researched variants of phenotype-specific treatment. method: aim of our study was to investigate of the causes of uncontrolled predominantly atopic asthma, the frequency and effectiveness of the personalized therapy in real clinical practice. 54 patients with uncontrolled severe atopic asthma were examined in outpatient department of the city hospital during 2017. all patients underwent physical examination, pulmonary function testing, and total serum ige evaluation. results: 35% of patients had uncontrolled asthma due to inadequate basic therapy of the disease. the change in therapy allowed them to achieve control of the disease. obstructive sleep apnea syndrome (osas) was revealed in 11.1% of patients. these patients underwent cpap (continuous positive airway pressure) therapy. 13% of patients had gastroesophageal reflux disease (gerd). 25% of patients had an elevated level of serum ige level and needed anti-ige therapy. in 7.4% of cases, the initial serum ige level was more than 1500 iu/ml which was a contraindication to therapy of omalizumab. 8 patients received omalizumab therapy. this therapy led to relief of symptoms and decreased frequency of asthma exacerbations. results: it was found that the prevalence of obesity among the 367 patients with asthma and being treated in inpatient conditions in 2013-2015 was 44.9% of patients, which is comparable to the prevalence of obesity among the population in general. the data of the patients suffering from asthma and obesity treated both in inpatient and outpatient conditions, was analyzed and it is set that obesity does not affect the severity of the clinical course of asthma. it is shown that obesity does not affect the control of symptoms of asthma. thus, the control of asthma symptoms depends on timeliness of diagnosis, the adequacy and terms of appointment of basic asthma therapy, the presence, severity and adequate treatment of concomitant diseases, psychoemotional background of patients, their compliance and adherence to therapy. results: the causes of smoking in asthmatics were not significantly different from the control (p > 0.05). patients most often used smoking as "support for emotional stability". the motivation to smoking cessation was higher in the asthmatics group (58%) than in the control group. the main reason for smoking cessation was a deterioration in health -60%. the majority of smokers -85%, performed attempts for smoking cessation. low level of br was revealed in 90% asthmatics (27% non-smoking asthma patients and 18.5% of cases in the control group, p < 0.001), cf had low values and was lower in asthmatics group in compare to the control group (p < 0.05). the pac values correlated with the level of br: a low level was determined in 100% in smoking asthmatics, in 50% in nonsmokers with asthma and 7.4% in smokers of the control group obstructive sleep apnea (osa). all of the patients were on regular treatment with low dose inhaled corticosteroids for at 12 months and start treatment with continuous positive airway pressure (cpap) .to assess quality of life, we used asthma symptom control tools (asthma control test) .patients performed daily peak flow meter and spirometry (once a week) during period of 4 weeks after start using cpap. results: during the study, 47 of the followed patients had no exacerbation of asthma. four of patients during this period had exacerbation, due to upper airway infection so they were excluded from study. results of following showed that there was improvement in quality of life in all 47 patients included in study but there no statistically significant improvement in pulmonary function tests fpt. huang y 1 ; yao t 1 ; huang y 1 ; chiu c 1 ; tsai z 1 ; kao p 1 ; lu k 1 ; fang h 1 ; lin c 1 ; gau c 1 ; lee w 1 ; tsai h 2 results: the rate of preterm birth among the study subjects was 18.2%. the prevalence of physician-diagnosed rhinitis was 50.6%. there was no significant association between preterm birth and physician-diagnosed rhinitis (p = 0.43). when stratifying by atopy status, we found that preterm birth was associated with physiciandiagnosed rhinitis among children without atopy (adjusted or [aor] = 0.33, 95% ci = 0.12-0.93, p = 0.04), but not among children with atopy (p = 0.77). when further classifying by gender, greater protective effect of preterm birth on rhinitis was only found in boys without atopy (aor = 0.12, 95% ci = 0.03-0.56, p = 0.007). the results suggest that preterm birth may have a protective effect against the development of childhood rhinitis in our study population. the protective effect is only observed in boys without atopy. further investigations will be merited to confirm these findings and to investigate underlying mechanisms. background: folic acid supplementation (fas) during pregnancy has been suggested due to its protective effect against neural tube defects. at present the effect of fas during pregnancy on childhood rhinitis has remained unclear. we aimed to investigate the relationship between fas during pregnancy and childhood rhinitis. logistic regression analysis with covariate adjustment was performed. adjusted covariates included sex, age, number of older siblings, breast feeding duration, maternal smoking during pregnancy, maternal allergy, maternal education level, maternal age and socioeconomic status results: the prevalence of physician-diagnosed rhinitis was 55.3%. there is a significant association between fas and physician-diagnosed rhinitis (adjusted odds ratio [aor] = 2.07; 95% confidence interval [ci] = 1.25-3.43 for fas ≥3 months) compared to the group of never use. in the stratified analysis by atopy status, maternal fas during pregnancy was significantly associated with physician-diagnosed rhinitis in the atopic group (aor = 1.91, 95% ci = 1.07-3.40 for fas <3 months; and aor = 2.34, 95% ci = 1.19-4.58 for fas ≥3 months), but not in the non-atopic group. when further stratified by gender, significant association between maternal fas during pregnancy and physician-diagnosed rhinitis was only found in boys with atopy (aor = 3.23, 95% ci = 1.39-7.50 for fas <3 months; and aor = 4.02, 95% ci = 1.51-10.72 for fas ≥3 months). the results demonstrate that maternal folic acid supplementation during pregnancy might increase the risk of childhood rhinitis, especially among boys with atopy. further investigation will be needed to validate our findings and to understand potential underlying mechanisms. according to sequence data 13 from 16 detected adv (in all groups of patients) belongs to species f 41 type and 3 samples to species c 1 type (rei group). bv type 1 was identified in 3 strongly positive (ct ≤ 25) swab samples in ari group. conclusion: simultaneous testing of respiratory and stool samples together shown that at least 6.6%/7.9% of 151 study subjects had dual/mixed infections, respectively, including 11%/9.5% of 63 respiratory disease patients, 3.6%/7% of 56 gastroenteritis patients and 3.1%/6.2% of 32 patients with combined respiratory/enteric infections. we found no virus combination specific for different groups of patients. 1327 | neonatal respiratory supports and future asthma-like presentation in prematurity with bronchopulmonary dysplasia results: of all the tests analyzed 54.4% were males and 45.6% females with a mean age 5.7 ± 4.8 years old. half of the tests (49.2%) reveals positive specific-ige to more than one allergen and 38.6% (66) have no serum specific-ige for the tested allergens (table 1) . sixteen patients (9.4%) have very high concentrations (>100 iu/ml) of derm. pteronyssinus specific ige, 13 (7.6%) of derm. farina, 7 (4.1%) of rey pollen and 6 (3.5%) of oak and timothy grass pollen. further studies are needed in order to elucidate the effect of these cytokines on allergy development and protection. shinohara m 1 ; matsumoto k 2 1 department of pediatrics, ehime university hospital, toon, japan; 2 department of allergy and clinical immunology, national research institute for child health and development, tokyo, japan background: probiotics consumption during perinatal and postnatal periods reportedly reduces the risk of atopic dermatitis in the offspring, whereas such probiotics consumption did not affect ige levels or the risks of other allergic diseases; the precise mechanism how probiotics consumption reduces the risk of atopic dermatitis remains unknown. we hypothesized that probiotics consumption may reduce skin hypersensitivity to histamine. to test this hypothesis, we investigated whether perinatal/postnatal consumption of yogurt associates with skin hypersensitivity to histamine or not. method: this was a cross-sectional study enrolled 256 motherinfant (≥6-months-old) pairs. physician-diagnosed allergic diseases and food consumption, such as milk, fermented drinks, and yogurt, by mothers during the third trimester of pregnancy and by infants during the first 6 months of life were assessed using self-questionnaires. skin prick tests (spts) to saline and 1 mg/ml histamine were performed using bifurcated needles, and wheal sizes were measured 15 minutes after the puncture. the spt wheal sizes in infants with eczema/atopic dermatitis (n = 44) were significantly larger than those in infants without eczema/atopic dermatitis (n = 156; 4.6 ± 1.9 mm vs 3.8 ± 1.8 mm, respectively, p = 0.015), and thus these infants were excluded from the further analyses. the spt wheal sizes to histamine in infants with daily yogurt consumption during the first the aim of this study was to evaluate the prevalence and clinical relevance of sensitization to profilins in atopic patients with food allergy. the study was performed on a group of 43 children age 2-14 years with sensitization to at least one plant-derived food allergen (ige > 0.7 ku/l). the included patients had never been treated with allergen immunotherapy before the study. the presence of ige to recombinant (r) rbet v 2, rart v 4 and ramb a 8 in serum was evaluated using elisa method as previously described (jbc 2016; 291:15447) . in addition serum level of igg4 to rbet v 2, rart v 4 and ramb a 8 was also evaluated. results: sensitization to profilins was found in 8 out of 43 (18.6%) patients (p+). sensitization to all 3 studied profilins was demonstrated in each p+ patient. the remaining 35 children, with pollenfood sensitization, were not sensitized to any of the studied profilins and they served as a comparator group (p−). analysis of the clinical status revealed that asymptomatic patients in regard to plant-derived food hypersensitivity were found more frequently among p+ (75%) than p− (37.1; p < 0.01) patients. sensitization to profilin was associated with positive ige to the same food allergens as in the control group. clinical manifestation of pollen-food sensitization expressed as allergic rhinitis, bronchial asthma and atopic dermatitis was comparable between groups, except of oral allergy syndrome, which was not seen among p+ children. similarly, history of anaphylaxis to plant-derived foods was registered only among p− (11.4%) patients. interestingly, all patients with sensitization to profilins had also elevated level of serum igg4 against rbet v 2, rart v 4 and ramb a 8. results: no significant difference of physician-diagnosed eczema (p = 0.88) or current eczema (p = 0.82) was observed between children born full-term and preterm. after stratifying by atopy status, we found that children born preterm had a more than three-fold higher risk of having physician-diagnosed eczema (adjusted or (aor) = 3.92; 95% ci = 1.25-12.29; p = 0.02) and current eczema (aor = 3.16; 95% ci = 1.06-9.41, p = 0.04) than their counterpart in the non-atopic group. no statistical significance was observed for the association between preterm birth and eczema in the atopic group. no association between preterm birth and eczema was found when stratifying by gender. our results reveal that non-atopic children born preterm have a higher risk of developing eczema. the results suggest potential modifiable effect of atopy on the association between preterm birth and eczema. further studies with a larger sample size are needed to validate the findings in this study. background: there is a need for more knowledge about factors of importance for a successful transition from childhood to adulthood among adolescents with allergic disease and especially those with severe allergy. therefore the aim of this study was to describe experiences of living with severe allergy from the adolescents and their parent's perspective and thereby identify factors of importance for transition from pediatric to adult care. method: a qualitative study was performed based on six focus groups interviews, two with adolescents and four with their parents. in total 11 adolescents (age 10-16 years old) and 21 parents participated. the interview guide contained questions about experiences of living with severe allergy. the transcribed data was analysed using systematic text condensation. results: in total four themes were presented, two themes occurred in both the adolescent and the parent's focus groups, to be special and to be prepared. for two themes there was a difference between the adolescents and their parents. the theme, the importance of the parents, only occurred in data from the adolescents and the theme the meetings with health care only occurred in the parent's data. the adolescents felt that they had low priority in the class and several stated they were teased at school and their parents felt that focus on their child often was in a negative way. the adolescents described that they took responsibility for their diseases while their parents expressed a need to protect. the adolescents stated that one of the parents were always present or had been during the years, the reason being safety and security. only the parents mentioned experiences from healthcare. parents who described that they had continuity in healthcare meetings and where met by high competence and with a professional approach were more satisfied with the support from the health care. one factor that was felt to be important was whether the doctor involved the youth in the conversation or not. the teenagers in this study relied on their parents while also taking responsibility for their illness at the same time. parents, on the other hand, showed a tendency to overprotect their adolescents. for healthcare professionals it is important to involve the adolescents in the care to facilitate the transition. results: 50.9% of the children used antibiotics currently and 21.0% out of them used antibiotics ≥3 times yearly. current wheeze (w) was established in 6.5%, sleep-disturbing w in 3.6%, exerciseinduced w in 1.7%, dry night cough apart from a cold in 12.2%, and asthma in 2.3%. current antibiotics use ≥3 times yearly was positively associated with current w (aor: 13.37; 6.14-29.11; p < 0.001), sleep-disturbing w (aor: 7.87; 3.34-18.57; p < 0.001), exercise-induced w (aor: 5.44; 1.89-15.61; p = 0.002), dry night cough (aor: 3.80; 2.29-6.29; p < 0.001), and diagnosed asthma (aor: 5.68; 1.96-16.50; p = 0.001) while antibiotics use <3 times yearly was positively associated only with current w (p = 0.003) and dry night cough (p = 0.011). the results suggest an aggravating role of antibiotics use on asthma in school age thus further supporting the recommended restriction of antibiotics exposure. results: after questioning 31.6% 95% ci, 23.2-40.9 were suffering from respiratory diseases, having symptoms of chronic disease: cough-86.1%, wheezing-41.66%, tightness in the chest-27.7%. the risk factors (passive smoking, open fire house warming and no air conditioning) were commonly met in major cases at ill children rather than healthy ones (68.4% 95% ci, 59.1-76.8). as a result of studies made of the equal to 17.5 ± 0.3, comparative the end of lessons equal 19.2 ± 0.2 (p ≤ 0.05); air relative humidity varies during lessons equal with 62.9 ± 1.4 (norma toilet 30%-60%); co2 concentration exceeds allowable limits −0.3 ± 0.02 (mac −0.1%). conclusion: respiratory morbidity in high school examined has a tendency to increase. we noticed deviations from the hygienic norms: the indoor temperature and relative humidity was lower and the co 2 level was twice higher than the normal one. the "asthma ever" outcome was reported in 32 cohorts. 28 cohorts defined this as parental reported asthma (with or without specifying that it was doctor-diagnosed), 2 cohorts used gp records as the only source of diagnosis, and 2 used parental report or gp records. the "current asthma" outcome was reported in 40 cohorts. there was little consistency with how current asthma was defined or worded, with 23 different definitions used. the most common definition of current asthma, reported 16 times, was "asthma ever and either asthma symptoms in the last 12 months or asthma medication in the last 12 months". other criteria included in asthma definitions were bronchial hyper-responsiveness, reversible airway obstruction, positive exercise test, and asthma symptoms reported at a previous questionnaire. only one "current asthma" definition was based exclusively on prescription data: "dispensed two asthma medication during the past year". nine cohorts reported asthma outcomes without specifying how it was defined, and were categorized as "asthma unspecified". conclusion: "asthma ever" and "current asthma" are two main asthma outcomes used to define asthma in child cohort studies. definitions of asthma vary substantially across cohorts. case report: thereby we present two case reports of two children with impairment verbal communication as part of asd and allergic diseases. the first patient was a 3 year old boy with sneezing, rhinorrhea night cough and eye redness. he had been suffering for almost 2 years from the above mentioned symptoms. he had family history for atopic diseases and was for 7 month breastfed. specific ige revealed sensitization to birch, alder, hazel, oak, mugwort pollen and dog epithelia and dermatophagoides farinae. specific ige resulted positive for nuts and rye flour. the second patient was almost 3 year of age in the tame that he presented in our hospital. he cried and screamed all the time because of severe atopic dermatitis and typical symptoms such as itching all over the body and his impairment of verbal communication. specific sensitization showed sensibilization to egg white and egg yolk, to nuts, rye and wheat flour. the food specific ige leaded to positive results to alder, birch, hazel and oak pollen, but also to grasses, ragweed and mugwort. prick by prick test showed positivity to egg white and egg yolk. atopy patch test to pollens resulted negative. results: the first patient symptoms were well controlled after treatment with antileukotrienes. his verbal communication was also improved after a year or more. the second three year old patient after required a combination of specific treatment with antihistamines, corticosteroids, immunosuppressive drugs and diet recommendation. afterwards he had a reduced level of itching and anxiety but compared to other children he had a severe eczema. erythema multiforme (em) is an acute, immune-mediated, mucocutaneous condition that is most commonly caused by infection and drugs. it is characterized by targetoid lesions, sometimes accompanied by oral, genital or ocular mucosal erosions. there was no pediatric patient that had previously been reported in the literature with development of type 4 reaction after omalizumab treatment. we presented a case who developed em to omalizumab therapy. an 16 year-old female patient was admitted to pediatric allergy clinic with complaints of fever and rash. she had been diagnosed with chronic spontaneous urticaria (csu) 8 years ago and she was planned to treat with omalizumab (75 mg, subcutaneously every 2 week) because of the inadequate response of antihistamines at a medical center. her complete blood counts, liver, renal, thyroid function tests and serum c3,c4,c1 esterase inhibitor protein levels introduction: celiac disease is an autoimmune disease triggered by exposure to gluten in genetically predisposed individuals and characterized by chronic inflammation of the small intestine. chronic urticaria is a skin disease, characterized by the appearance of pruritic wheals with or without angioedema, whose underlying mechanism cannot be identified. objective: to report a sporadic case of an 11-year-old boy with chronic urticaria associated with celiac disease. methods: an 11-year-old boy(weight 31 kg, 3rd-10th percentiles) was admitted to our clinic with a 6-year history of chronic urticaria. during the first three years, he was under antihistamine treatment(of incremental doses)and occasionally received preparations of cortisone according to the eaaci guidelines. he was asymptomatic for 2 years until treatment was discontinued. eight months earlier, after a viral infection, a recurrence of urticaria, involving the trunk and extremities without angioedema was noted. subsequently, he was under antihistamine treatment with cetirizine but had an uas-7 score of 11. total laboratory investigations were performed. results: laboratory control was negative except for positive antibodies to celiac disease(anti-transglutaminase >200 u/ml, anti-endomysial, gliadin antibodies).further control with colonoscopy and biopsies (from duodenum and stomach) were obtained. the histopathological findings along with the clinical findings indicate celiac disease, type 3b marsch-oberhuber and grade b1 corazza-villanacci. in the past, similar cases have been reported. efforts have been made to associate chronic urticaria with celiac disease, although the mechanism remains unclarified. evidence suggests that the duration of gluten exposure, among otherwise asymptomatic patients with celiac disease, is related to the development of other autoimmune mechanisms. this can be explained by resolution of urticaria manifestations after the onset of gluten-free diet. in our case, three months after gluten-free diet, an improvement of urticaria with decreased uas-7 score of 5 was observed. conclusion: he specific case of subclinical diagnosis of celiac disease in a child with chronic resistant urticaria further reinforces the suggestion that screening for celiac disease should be included in the diagnostic approach of chronic urticaria. 1350 | allergy to gingival balm in an infant with cow's milk protein allergy we report a case of an infant with a diagnosis of cmpa with an allergic reaction to a gingival balm caused by the presence of cmp in its constitution. furthermore, it is important to reinforce that milk proteins were labeled in an unusual form which might increase the risk of misunderstanding. these findings illustrate the difficulty in implementing total avoidance of common food allergens as well as the need to improve their labeling, particularly in non-food products. bakiri ah results: after specific treatment with corticosteroids, antihistamines, emollient creams, disinfectants and antileukotriens he was feeling better, he was smiling again and wished to have the chance to play with his classmates again. conclusion: this case report shows an association between level of stress and risk for atopic dermatitis. as previously showed children with low educational level parents and boys with higher stress have increased risk of having severe atopic dermatitis as compared to "no stress" boys. so early treatment and diagnoses are key important factors improving the children`s social life. results: the data cover 69 immigrants (mean age 39.0, range 8-62) and 232 locals (mean age 40.8, range 9-80). a slight difference in male prevalence (30.4% vs 47%, p = 0.01), and pet possession (20.2% vs 41.8%, p = 0.01) were found between immigrants and locals, respectively. no differences were find in term of age and symptoms at presentation. the pattern of sensitization to the different allergens showed no statistically significant differences between migrants and controls. the rate of monosensitization resulted slightly higher in migrants (27.5%) than controls (21.1%). pollen-only sensitization was statistically higher among migrants than control (39.1% vs 22.4%, p < 0.01). monosensitization was more frequent among patients who have been living in italy for less than 4 years (52.6% vs 20%, p = 0.05). the opposite phenomena can be seen among polysensitized patients. conclusion: migrants are more frequently monosensitized than locals and tends to cluster towards either a pollen or dust mite sensitization. sensitization to house dust mite tends to appear early (< 4 years of stay). pollen or mixed sensitization is more frequent the longer the residence time. 1353 | allergenonline.org: update of comprehensive allergen and celiac protein searchable databases for risk assessment of novel food proteins goodman re 1 ; baumert jl 1 ; taylor sl 1 ; ebisawa m 2 ; ferreira f 3 ; bohle b 4 ; van ree r 5 ; kleine-tebbe j 6 ; abdelmoteleb m 1 ; koning f 7 ; amnuaycheewa p 8 conclusion: allergen and cd databases have been updated following a described review process. they can be used to identify proteins that might represent risks of food allergy or cd for affected consumers. han dh 1 ; lee jw 1 ; yim hj 1 ; ko yk 1 ; kim d 1 ; rhee c 2 1 seoul national university hospital, seoul, south korea; 2 seoul national university bundang hospital, seoul, south korea background: stress can change the immune response and aggravate various allergic diseases. we already demonstrated in previous allergic rhinitis cohort (arco) kids study that stress might be a risk factor for pediatric allergic rhinitis (ar). the aim of this arco study is to investigate relationship between stress intensity, symptoms severity and quality of life as well as allergic markers in adult ar patients. results: as stress intensity increased, the proportion of moderatesevere ar patients was significantly increased. ar patients in high stress group was likely to belong to moderate-severe group (or, 1.60; 95% ci, 1.01-2.50). global vas of ar symptom was 7.0 ± 1.9 in high stress group and 6.7 ± 2.0 in low stress group, respectively. the each 7 rqlq domain score was significantly higher in high stress group than in low stress group. total rqlq scores were 75.3 ± 35.5 in high stress group and 57.4 ± 36.0 in low stress group, respectively. however, as the level of stress increased, there were no significant changes in serum levels of allergic markers. our results suggest that stress may affect ar symptom severity and quality of life in ar patients. 1355 | skincare and synbiotics for the prevention of atopic dermatitis or food allergy in newborn infants: a 2 × 2 factorial randomized non-treatment controlled trial dissanayake e 1 ; tani y 2 ; sahara m 2 ; mitsuishi c 2 ; nagai k 3 ; sato y 4 ; suzuki y 5 ; nakano t 1 ; yamaide f 1 ; shimojo n 1 (n = 118). the skin care group was advised to apply an emollient 2-3 times/day especially on cheeks and peri-oral area. the synbiotics group consumed a mixture of fos (1 g) and bifidobacterium bifidu-mol6378 (7 × 10 9 )/day. the last group received both. emollient application was not prohibited in the no-intervention group. interventions were carried out from birth to 6 months of age. the development of ad was assessed at 1 month, 6 months and 9 months by a pediatrician and at 1 year by a questionnaire. ad was diagnosed using guidelines of the japanese society of dermatology. sensitization to food allergens was assessed by allergen-specific ige levels at 9 months of age. results: skin care and synbiotics, alone or in combination, did not prevent the development of ad at 1 year of age or the sensitization to food allergens at 9 months of age. conclusion: our data suggest that skin barrier protection using emollients may be insufficient to prevent the development of ad as other factors affecting skin barrier integrity and trans-epidermal water loss such as the method of skin washing may have an additional effect. the probiotic bacterial species used may also affect the outcome as lactobacilli have been shown to be more beneficial. more studies are required to confirm the effects of skin care and synbiotics on ad. results: in the population number of girls exceeded the one of boys (p < 0.001), especially within the age group from 4 to 15 years. questioning, for 12 months, symptoms of allergic rhinitis (rhinorrhea, sneezing, nose itch, nasal obstruction and eyes' itch) were identified in 19.5 (p < 0.05); symptoms of bronchial asthma (wheezing (14%), episodes of cough at night (8.3%), intolerance to physical load (2.5%), indoor and outdoor (13.6%), coughing and rales in response to stimulus (7.2%)) in 9.8% of the population; atopic dermatitis (dermatitis, itch, revelation in early age, involvement of large areas in early age, damage of extremities bending and stretching surfaces in adults)-5.7% (p < 0.01); food allergy-3.7% (p < 0.001) etc. at the second stage of clinical studies, on the basis of prick-testing, average ige, in our case, was 3-5 times greater than normal level. results of study of allergens showed sensibilization to domestic dust (d.f. and d.p.) (64, 43%) (p < 0.05). in 25.46% of cases there was stated sensibilization conditioned by cat and dog epidermal allergens results: among the 2624 women with available serum, 39.3% were sensitized of whom 12.5% were monosensitised, and 25.3% polysensitised (to two or more allergens). sensitisation to inhalant allergens dominated (38.9%), with grass being most common (25.5%). only 4.0% were sensitized to food allergens, most often to peanuts (2.6%), while among the 11.4% who reported ddfa, ige reactivity to foods were identified in 17.5%. compared to women with no asthma, women with dda (14.9%) were in a significantly higher background: regular exercise has been known as beneficial that it reduces the risk of chronic diseases including allergic diseases. however, little has known regarding the relationship between exercise and allergic diseases in korean adolescents. we analyzed the national data whether exercise is related to the prevalence of allergic diseases in the population of korean adolescents method: data from sixth korean national health and nutrition examination survey (2013-2014) that included 1272 adolescents from 12 to 18 years old was analyzed. we defined regular exercise according to physical activity guidelines for americans. multivariate regression analysis was performed to find whether lack of exercise could be a risk factor for allergic diseases. results: the prevalence of asthma, allergic rhinitis (ar) and atopic dermatitis (ad) were 1.3%, 9.3% and 5.2% in korean adolescents, respectively. after adjusting for factors, lack of exercise was not associated with asthma and ar, but was significantly related to ad in korean adolescents (adjusted odd ratio 3.254, 1.202-8.810 , results: it was found that over 43% of ch up to 14 y.o. having the ad within allergic disease (ads). the most significant symptom was a long-lasting itchy rash lasting for 6 month in 12.42 ± 0.56% of 1g and 9.23 ± 0.49% of 2g. the first morbidity of ad was noticed at the age of up to 2 y.o. among 56.29 ± 1.27%. at the age of ch 2-4 y.o. and older than 5 y.o. the skin ads onset was noticed for 31.12 ± 1.11% and 12.58 ± 0.57% accordingly. the ad sl was determined as follows: 52%moderate (mo), 12%severity (s), 36% were 13 ± 10 kua/l, 344 ± 132 iu/l respectively. skin prick tests were positive in 43.4% of the patients (61.7% multiple allergens). grass pollens (53%) and dermatophagoides (45.2%) were the most common allergens. average vitamin a and d levels were 469.8 ± 108 μg/l (257-832), 54.8 ± 21.3 (13-187) respectively. thirty percent of the patients vitamin d levels were mildly low, 8.6 percent was low. in control group 20% was mildly low, vitamin a levels was low in 6.7% of the patients. none of the children in control group had low vitamin a levels. we didn't find any statistical significant difference for both vitamin levels between patient and control groups. vitamin a deficiency was mostly found in asthma patients whereas vitamin d deficiency was mostly in allergic rhinitis and asthma groups. passive smoking and vitamin d deficiency was significantly related (p = 0.09). there wasn't any relation between asthma attacks and vitamin levels. conclusion: in conclusion vitamin a and d levels weren't found significantly related with allergic diseases but was found lower than control group. patients having chronic diseases are one of the population groups that are chronically exposed to drugs. this study aims at evaluate the impact of this factors in developing drug allergies in the medical staff. method: this was a cross-sectional study that included 639 nurses from the uhc "mother theresa" of tirana. they were asked to fill up a questionnaire where questions about chronic diseases and drug allergies were included. 92.18% were females and the mean age was 43.28 (+10.71) years old. relative risks with 95% ci were calculated for different groups. results: 40.69% (260) nurses reported to have at least a chronic disease. the most common non-atopic disease was hta followed by the groups of autoimmune and thyroid diseases. nurses who had one chronic disease have a rr of 1.82 (95% ci = 1.03-3.21, p < 0.05) to develop a drug disease higher than those who didn't had any chronic disease, and those who have more than one chronic disease have a rr of , p < 0.05) to develop a drug disease. the presence of chronic diseases can be a risk factor to develop a drug allergy probably through the increased risk to drug exposure. these patients may be exposed to drugs not only through therapy but also through hospitalizations and other forms of health care. lapeere h 1 ; oosterlinck p 2 ; vermeir p 3 ; vermeire i 2 ; coppens m 3 ; gevaert p 3 1 ghent university hospital/ghent university, ghent, belgium; 2 ghent university hospital, ghent, belgium; 3 ghent university hospital/ghent university, ghent, belgium background: the key to managing latex allergies in healthcare professionals and patients lies in correct recognition and appropriate action. 7.7 million people are employed in the health care sector. while there are no overall statistics on the prevalence of latex allergy in that work force, studies do indicate that 8%-12% of health care workers regularly exposed are sensitized, compared with 1%-6% of the general population. latex allergy is defined as an immune mediated reaction to latex products (e.g. balloons, contact dermatitis for gloves, condoms, surgical catheters); these encompass immediate and delayed hypersensitivity reactions. method: based on the experience of the belgian dutch pathway network, a 7-phase method to develop, implement, evaluate and continuously follow up a care pathway for latex allergy was designed and implemented. the purpose of the study was to develop and implementation of latex allergy clinical care pathways to provide all staff at ghent university hospital with appropriate knowledge and skills to identify and manage patients who have a known latex allergy or those at risk of developing latex allergy. results: care pathways, also known as clinical pathways, are used all over the world to implement and monitor patient-centered care processes in a transparent way. care pathways are defined as a complex intervention. 7 -phase method consists of: 1) screening phase; 2) project management phase; 3) diagnostic-and objectification phase; 4) development phase; 5) implementation phase; 6) evaluation phase and 7) continuous follow-up phase. this phased approach is based on the deming cycle, better known as the "plando-study-act" (pdsa)-cycle. conclusion: this method can offer support to multidisciplinary teams (re)designing and implementing safe, efficient, effective, person-centered, timely, equitable, continuous and integrated care processes. however, the method is no guarantee to success. the key to success is the collaboration and critical attitude of the entire multidisciplinary team when implementing pathways. background: cord blood ige (cb-ige) were considered to be a useful predictive tool for allergic symptoms especially in early childhood. there is only sparse knowledge about their importance for health in later life. the aim of our work was to determine the importance of cb-ige for allergic symptoms in young adults. we also studied the possible modifying factors for cb-ige concentration. results: resutls shown as daily mean, pollen grains/m³: table 1 . the daily means of pollen concentrations of cupressus arizonica, platanus acerifolia and plantago lanceolata in our area differs from other sites in madrid city. although cupressus arizonica and platanus acerifolia counting were lower, plantago lanceolata counts were higher, representing a relevant pollen in our area. the clinical relevance of these findings is under evaluation by our group. method: grass pollen counts were performed since 1979-2016 using a burkard 7 days spore trap located in our allergy center in madrid. the beginning of the algid period of pollination was considered the first of three consecutive days with more than 10 grains/m 3 and the end, the last day of three consecutive days with more than 10 grains/m 3 . madrid, barajas meteorological station data, was used. skin prick tests (pt) to grass pollen was also studied in comparison conclusion: total grass pollen concentration did not suffer any increase or decrease in its counts despite the dramatic increase of the temperature. an advance at the beginning and the end of the season was seen. these changes significantly correlate with the temperature increase during may and july. discrete decrease in the sensitization prevalence. since several years, the reference method to monitor the biological particles concentrations has been the hirst method: a volumetric pollen trap, located on the roof of building for background measurements, sucks continuously 10 l of air per minute, particles depositing by impaction on a coated tape. the tape is then analyzed by optical microscopy. the hirst method produces accurate but past data. nowadays, many researches are focused on the development on new devices to get real time information. method: rapid-e from plair sa is a device using red laser beam to determine the size and the shape of sucked particles and an ultraviolet ray to measure the fluorescence of these particles. the results: the correlation coefficients got between rapid-e and hirst trap are higher than 70% for most of calibrated pollens, this correlation reaching 86% for all pollen taxa: • plane 99% • pine 99% • birch 96% • oak 82% • plantain 75% • dactylus 73% • urticaceae 55% conclusion: new calibrations are planned for 2018 and a real time information will be set up. results: the quinquennial media concentrations since 1979-2016 were 3.866; 4.903; 6.610; 13.454; 9.501; 7.248;11.027 and 19.406 grains/m 3 . the quinquennial media temperatures were 14.2; 13.8; 14.3; 14.9; 14.2; 14.7; 14.9 and 15.8°c. increase of 1.4°c (r s = 0.9 p < 0.05). the beginning and the end of the actual season advanced 5 days respectively in regard to the period from 1979 to 1983. the annual prevalence of positive pt to platanus in 1979 was 2% an 52% in 1994. the quinquennial media from 1999 to 2016 was 50, 38, 26 and 29%. conclusion: platanus pollen counts had a dramatic increase that meaningfully correlates with the dramatic increase of the temperature. a discreet advance at the beginning and the end of the season was seen. these changes did not influence in a longer duration of the season. we observed a significant increase in platanus pollen sensitization prevalence whiting madrid pollinosis patients. results: the quinquennial media concentrations since 1979-2016 were 1963, 4813, 5455, 6948, 6985, 5976, 6727 and 9298 grains/m 3 . the quinquennial media temperatures were 14.2; 13.8; 14.3; 14.9; 14.2; 14.7; 14.9 and 15.8°c. increase of 1.4°c (r s = 0.9 p < 0.05). the actual season beginning advanced in 51 days and the end has results: over 60% of house dust samples collected between april and may from central european countries were found to contain bet v 1 allergen at levels well above the limit of detection of 0.0039 μg/g for elisa 2.0 ep kit and 0.001 μg/g on maria. samples were found to have much higher levels of bet v 1 allergen from midto-late april, particularly those that were collected in germany, belgium and hungary. samples taken from outside of the pollination season were tested and found to be negative for bet v 1. in conclusion, we found that bet v 1 allergen can be detected and quantified in house dust samples. these data suggest that household dust is a source of pollen allergen and could therefore be contributing to asthma and allergic rhinitis symptoms in individuals affected by pollen allergy. household dust may also be considered as a source of bet v 1 allergen which could contribute to allergic sensitization. 1383 | cupressaceae pollen in the atmosphere of alentejo: disruption of pollen grain during air transport spring, depending on the temperature. despite being considered moderately allergenic, it might be responsible for winter allergic outbreaks. as ornamental trees, they are found scattered throughout the territory but are more abundant in pockets of wild forest, outside alentejo. despite being more common in mountain, this pollen type is captured in considerable amounts in alentejo, portugal, where its aerobiological features and allergenic impacts are poorly characterized. the aim of this work is to characterize the aerobiology of cupressaceae pollen, to evaluate the effect the meteorological conditions and the source of this allergenic pollen type in the atmosphere of evora, alentejo. method: pollen were collected using a hirst type 7-day pollen trap and pollen was identified following standard methodology. background: allergic rhinitis caused by pollen is one of the most common allergic diseases. the presence of pollen in the air is currently centrally monitored at roof top levels, and not in the direct living environment of sensitized subjects. in the current project we aimed to develop a handheld pollen sampler, called pollensniffer, that can collect pollen in the living environment of the allergic subjects. as a first step this device was validated against the standard burkard pollen sampler and used to monitor local pollen concentrations at street level in the city of leiden. method: rooftop level pollen were monitored routinely by a hirst type pollen sampler (burkard, uk). the pollensniffer ( 1385 | does the allergy risk due to pollen exposure information is useful for the allergy sufferers? sindt c; oliver g; thibaudon m background: in france the information for the allergy sufferers is not made with pollen counts, which have not a real signification, but with the allergy risk due to pollen exposure. method: since more than 20 years, rnsa (réseau national de surveillance aérobiologique), the french aerobiology network, has measured the pollen exposure in the main cities of france, using background: the effect of environmental factors on allergic sensitizations is still unclear. rural areas vs cities have different exposure levels to pollutants and aeroallergens. these differences could give clues on the causes of higher allergic sensitization rates in children exposed to city air. method: two studies with children aged 5 years old were initialized to analyse the airborne drives of allergic sensitization: seal (günzburg, 350 children) and ae2r kids (munich, 200 children). capillary blood was collected and the parents filled in a questionary. sensitization rates were quantified using the immunocap ® isac sige array. pollen data were measured at both locations. results: in günzburg more children were sensitized to aeroallergens, however munich children showed significant higher sensitization to phl p 1 (p < 0.05), despite the lower concentration of pollen. in günzburg 66% children had no sensitization at all compared to 58% in munich. 80% of the children in munich spend at least 1 hour per day outside and 91% of the total have no animals at home. 23% felt symptoms of hay fever in the last 12 months, the majority between march and june, which correlated with the pollen flight. results: the total rate of atopy in crd patients was 36.1%, and asthma patients was the highest (45.9%). the positive rate of phadiatop in urban asthma patients (56.0%) was significantly higher than that in rural areas (25.0%, p < 0.05) and the phadiatop positive rate of office staff (62.5%) was significantly higher than that of outdoor workers (37.5%, p < 0.05). the total rate of atopy in copd patients was 32.7%, and in patients with acute exacerbation was 42.9%. beside, atopy is a risk factor for dyspnea (or = 1.22, p < 0.05), and the fvc levels in copd patients with atopy were significantly lower than those without (2.06 l vs 2.63 l, p < 0.05). optimal scaling analysis show that, there were a correlation between the tige and smoking coefficient (cronbach's alpha = 91.1%). in addition, the correlation between the level of tige and phadiatop sige was so strong in the patients with mild to moderate asthma (r s = 0.709, p < 0.001), but it was weak in severe asthma patients (r s = 0.486, p < 0.001), and up to 35.0% of the gold iii iv patients with low phadiatop level (≤10 ku/l) had a high level of tige (≥1000 ku/l) compared gold i ii (5.5%). conclusion: the rate of atopy in patients with crd is high, and atopy is an important factor affecting the process of crd. the patients with severe copd or asthma is likely to has high serum tige level but the level of common allergen sige is low, so the allergy screening strategy should be adjusted and we should pay attention to those patients, therefore, it is necessary to screen the sensitization situation of crd patients at first, and the results can guide the treatment, management and prevention of crd. background: due to a limited amount of epidemiological data [1] it has been thought that many severe allergic asthmatics in germany remain unidentified and are therefore not adequately treated. a pilot project demonstrated that more than 50% of patients, having been mean total ige (sd) was 366.4 (692.2) ku/l. 26.5% of the patients had no sensitization towards any of the specific iges tested, whereas 24% were positively tested on 5-10 allergens and further 23.5% showed sensitizations towards >10 allergens. conclusion: approximately 75% of 392 online recruited (severe) asthmatics had a total ige level of >30 ku/l and ≥1 sensitization (allergen-specific ige) towards atopic allergens. this further supports the high prevalence of atopy in asthma. results: 875 patients (mean age: 26 ± 11.9 years, range 2-64 years, m/f ratio: 0.89) who suffered from allergic rhinitis or allergic rhinoconjunctivitis enrolled in this study. highest rate of skin sensitivity was for weeds/grasses pollen including salsola kali, amaranthus retroflexus, chenopodium album and compositae family (74.3%, 62.5%, 50.9% and 39.3% respectively). among tree's pollen; ash (49%), walnut (46.9%) and mesquite (29.3%) were the most common. less than 20% of patients showed skin reactivity to indoor allergens and storage mites, mix of cockroaches and house dust were the most common (17.6%, 17.3% and 9.8% respectively). the results of current study confirmed the importance of weed/grass and trees pollen as the major source of allergic sensitization in our area. interestingly the rate of sensitization to indoor allergens was low which can be explained by geo-climatic situation. background: there are few studies of cutaneous sensitivity to gramineae in our region. mostly of them use allergens of foreign species. the study aims to estimate the prevalence of skin sensitivity to widespread grasses in our region. method: this is a retrospective observational study of 894 patients with seasonal allergic rhinitis. patients were studied using skin tests with pollens extracts from pooideae, chloridoideae and panicoideae grass species. results: the prevalence of positive reaction to pollen from pooideae subfamily was 86.8% (ic: 84.4%-88.9%). in turn, prevalence of allergy to panicoideae subfamily pollens was 69.6% (ic: 66.5%-72.5%) and positive reaction to chloridoideae subfamily reach 48.1% (ic: 44.8%-54.1%). cochran test suggests that prevalence in those three groups is different (χ2 = 319.11, p < 0.01). when comparing just the groups of allergens from pooideae and panicoideae differences are also significant (χ2 = 71.43, p < 0.01). in particular, 52.5% (ic: 49.2%-55.7%) of patients were allergic to paspalum notatum. regarding cross-reactivity between subfamilies, we find a no crosscorrelation between pooideae and panicoideae (χ2 = 2.197, p = 0.138). conclusion: in bahia blanca, patients with seasonal rhinitis are sensitive to pooideae, chloridoideae and panicoideae. paspalum notatum, belonging to panicoideae, has a significant prevalence, high reactivity and low cross-reactivity within the group of species studied. this last species is relevant because it is a native grass from the northwest region of our country, paraguay and the south of brazil. prevalence of grass positive skin tests in patients with seasonal rhinitis by species. allergen frequency percentage 95% ci results: correlation analyzes were performed between sige and spt and area results. the concentration with the highest correlation by diameter and area for blo t was 30 μg/ml and for der f of 400 μg/ml. in the case of der p the concentration with the highest correlation for the diameter was 30 μg/ml and for the area of 3 μg/ml. when evaluating the reproducibility of the results according to the area and the greater diameter of the spt, a strong agreement was observed for blo t in the concentrations of 3 μg/ml and 0.3 μg/ml. results: among the 94 patients, the majority of allergens-positive was t20, accounting for 60.6%, followed by f14 (59.6%), f13 (58.5%), ds1(57.4%) and ccd (56.4%). the prevalence of plant-related allergens (t20, w1, f14, f13, w6 and u80) in ccd-positive patients were significantly higher than those in ccd-negative patients (all results: with our new point-of-care methods using a selected recombinant protein e other markers, we were able to detect the disease early as 10 days post-infection and more than 95% of positive cases from chronic and low endemicity areas (which are characterized by hard to detect patients with extremely low parasite load, <10 eggs per gram of feces) were obtained. plus, chromatography poc-cca ® test was improved by our group with a urine concentration step that turned its sensibility from 6% to 56%. conclusion: monoclonal antibody and recombinant protein technologies allowed superior detection methods when comparing it to the conventional ones. in conclusion, data showed 100% of sensitivity of chronic patients and 98% of acute patients. marton c county hospital, oradea, romania background: allergic rhinitis is a disease that affects about a quarter of the population, a disease with an important negative impact on daily activities, both on learning and working ability, as well as spending leisure time or sleeping. in the western part of romania, the most popular and blamed allergen is ambrosia, in the late summer months. it is a plant of the compositae/asteraceae family, along with goldenrod, sunflower, dandelion, cocklebur, chamomile, wormwood, daisy, etc. allergen identification is important for applying prophylactic measures, but especially for determining the allergen to be desensitized. considering the possible cross-reactivity within the compositae plant family, as well as the possibility of co-sensitization, as well as the number of patients sensitized to these pollens, which is steadily increasing, i considered is necessary a broad screening for a more precise identification of the allergen and increase chances for a successful desensitization. method: the observational study includes 37 patients who presented on october for testing with standardized allergen extracts, as recommended. criteria for inclusion: patients with specific symptoms of rhinoconjunctivitis in august and september, with or without asthma symptoms, who returned for allergic prick test after the end of treatment. criteria for exclusion: patients who disagreed with cutaneous testing, who did not discontinue antihistamine treatment or who had been treated for other diseases with drugs that influence skin testing. background: in recent years, cationic liposomes are thought to be the most effective and non-toxical nucleic acids`transport system, so most of gene therapy drugs are developed on their base. however, lipoplexes are quickly captured by reticuloendothelial system cells after the injection and taken out of a blood stream. there are many modification methods of liposomal surface for liposomes with prolonged pharmacokinetic properties production. addition of hydrophilic polymers (peg) is seemed to be the most promising approach, that is able not only to create steric barrier on the particle`s surface and prevent the interaction with blood plasma lipoproteins, but also inhibits the protein adsorption, opsonisation and subsequent degradation in human body. the aim of this study is the evaluation of liposomal surface modification by hydrophilic polymers influence on nucleic acids`lipoplexes conjugation and on their physico-chemical and biological properties. method: liposomes preparation (including peg-modified liposomes), size determination by photon-correlation spectroscopy, examination of transfection efficacy by luciferase assay. (c16h33)2) were obtained. also the modified liposomes were produced by addition of 5% of peg (by mass) during thin lipid layer preparation step. the size distribution was analysed by photon-correlation spectroscopy. it was shown that peg addition does not increase the parconclusion: it can be noted that addition of peg can change the lipoplex formation but the cationic liposomes still remain an effective rna delivery system. and peg modification will be able to impart prolonged properties for the vehicle in bloodstream. foundation (grant № 17-74-10111). ory c4 may cause a cross reaction with fel d4 (cat), can f6 (dog), equ c1 (horse), mus m1 (mouse) and rat n1 (rat). february 2017 eight patients that were treated at our institution were diagnosed with rabbit allergy. results: all eight patients with the diagnosis of rabbit allergy presented with signs of upper respiratory involvement. two patients had itching teary eyes, watery nasal discharge and sneezing while feeding farm rabbits. one of those also presented with dyspnea. four patients developed problems whenever in contact with domestic rabbits. one patient developed allergic rhinoconjunctivitis whenever she was home-her parents own a rabbit, but while away in her college room she had no problems. another patient had dyspnea whenever visiting his girlfriend's house. she owned a rabbit. two patients developed asthma-like symptoms, one also presented with angioedema. the other two had developed allergic rhinoconjunctivitis. two patients have problems in contact with cats, one of them also with cows, however skin prick tests were also positive to rabbit. three out of eight patients developed allergic asthma with a positive methacholine test. six patients had a positive house dust mite prick test. all patients were diagnosed with a positive prick tests to rabbit allergens. all were treated with a nasal steroid and antihistaminesic. they were also advised to avoid contact with the animal. conclusion: domestic rabbit-induced asthma and/or allergic rhinoconjunctivitis is possible, however it is still rare in our environment. it is very important to always ask the patient about their pets in general, not just focusing on cats or dogs. only with a thorough examination and history we can find the true cause of the patient's allergy where pets play an important role. korea. changes of protein and major allergen concentration were measured over one year by bradford assay, two-site elsia, and sds-page after reconstitution of the lyophilized allergen extracts in various buffer (normal saline, 0.3% phenol saline, and 10 or 50% glycerol with saline) and stored at room temperature (rt, (18) (19) (20) (21) (22) (23) (24) (25) (26) or refrigerated (4°c). results: more than 90% of the initial protein concentration in all four extracts examined was detected over one year when 50% glycerol was added and refrigerated, whereas 57.9%-94.5% remained in the extracts at rt. the addition of 50% glycerol to the storage buffer was found to prevent protein degradation at rt. all four extracts were found to be stable when reconstituted in 50% glycerol. amb a 1, a major allergen of ragweed, was almost completely degraded in 9 weeks at rt when reconstituted in a buffer without 50% glycerol. however, 55.6%-92.8% of amb a 1 content was detected after one year of incubation at 4°c in all buffer conditions except 0.3% phenol. conclusion: addition of 50% glycerol as well as refrigeration was found to be the important to increase the shelf-life of allergen extracts from pollens of allergenic importance. results: this is the first genetic study of the bulgarian hae patients. genetic defects were identified in 10 hae families are: 3 nonsense, 2 splice-site defects, 2 frameshift mutations, 1 indel non frameshift, 1 missense, and 1 large deletion of exon 4. novel mutations, not previously reported in human gene mutation databases were discovered, and were predicted to be deleterious due to the expected effect on dna transcript and protein. descriptive statistics were used to summarize the eq-5d-y descriptive system responses and vas scores by treatment and visit. results: twelve patients with hae type i and a median (range) age of 10.0 (7) (8) (9) (10) (11) years were enrolled, 7 (58.3%) of whom were female. during bop, treatment with 500u c1 inh, and 1000u c1-inh, ≤33.3%, ≤22.2%, and none of the patients, respectively, reported having problems with mobility, self-care, doing usual activities, pain or discomfort, and feeling worried, sad or unhappy. the mean [sd] eq-5d vas scores increased from 78.3 (13. results: overall, the model-derived median exposure and peak concentration across all weight ranges in paediatric patients is predicted to be higher with 5 wb vs weight-based dosing (table) . the effect was most pronounced in patients aged 2-5 years, where the 5wb dosing achieved approximately 30% higher values than weight-based dosing for median auc 0-6 (1251 ng hour/ml vs 853 ng hour/ml, respectively) and c max values (777 ng/ml vs 529 ng/ml, respectively). the 5wb levels are closer to those in adults receiving 30 mg icatibant (median auc 0-6 2975 ng hour/ml; median c max 1254 ng/ ml) but never exceed them. results: samples from 124 patients were analysed. lanadelumab concentrations in plasma increased with higher doses and dosing frequencies. steady state was reached around week 10 (range week 8 to week 14 as evaluated by predose concentrations). at baseline, mean (sd) chmwk levels were 49. 9% (28.8), 47.7% (27.1), 53.7% (24.5) and 48.4% (30.0) for patients in the placebo and lanadelumab 150 mg q4 wks, 300 mg q4 wks and 300 mg q2 wks treatment arms, respectively. by week 14, mean (sd) chmwk levels decreased to 24.0% (13. 3), 28.7% (17.2), and 22.4% (11.5) following treatment with lanadelumab 150 mg q4 wks, 300 mg q4 wks, and 300 mg q2 wks, respectively, and remained reduced throughout the treatment period. conversely, chmwk levels remained elevated at 52.3% (28.1) at week 14 in patients who received placebo. patients in the placebo group had the highest attack rates over the 26-week treatment period (mean 2.46 attacks/month), whereas the rates were markedly lower in patients treated with lanadelumab 150 mg q4 wks (0.48 attacks/month), 300 mg q4 wks (0.60 attacks/month) and 300 mg q2 wks (0.31 attacks/month). dose-and frequency-dependent manner. exposure to lanadelumab was associated with decreased chmwk levels (indicating inhibition of plasma kallikrein activity) and lower hae attack rates, corroborating the efficacy findings and utility of chmwk as a bioactivity marker in the help study. with cyklokapron (tranexamic acid) since she was 9 years old and took the medication very irregularly due to lack of efficacy. one year before presentation at our clinic, she married and moved to vienna. we started a treatment with the bradykinin-2 receptorantagonist icatibant sc at the beginning of the menses and if needed a second time at the time of ovulation. she responded well until she got pregnant. during pregnancy, she developed weekly attacks with increasing severity. therefore, a weekly treatment with humanplasma-derived, pasteurized, nanofiltered c1-inhibitor (inh)-concentrate, 1000 units iv once a week was started and had to be increased to twice per week after one month of therapy due to increasing number and severity of attacks. results: with this treatment attack frequency and severity attenuated. in january 2016 she had a normal delivery at term and gave birth to an otherwise healthy son. treatment had to be continued during 1 year of lactation period and also thereafter due to persistent attack severity. conclusion: there are only limited data for the use of humanplasma-derived, pasteurized, nanofiltered c1-inh concentrate during pregnancy and lactation period. this case confirms the safety and efficacy of the named drug during these periods. wheals are yet classified. the best characterized stem from hereditary or acquired c1 inhibitor deficiency (c1-inh-hae and c1-inh-aae) . last year, the french angioedema network (creak) joined the registry of angioedema without wheals (cloud-r hae). here we present the contribution of the grenoble alpes university hospital (chuga) to this disease registry. the study population is composed of patients with a proved diagnosis of c1-inh-hae/aae. the following items are collected: patients' personal-demographic data, clinical/laboratory/genetic characteristics, major comorbidities, treatments (prophylaxis/acute attacks). data from existing registries at chuga are merged into cloud-r hae and missing data obtained at follow-up visits. as from cloud-r hae structure, patients can directly provide information on angioedema attacks and their treatment through a dedicated electronic app, web connection or paper support, which is then transferred into the registry at chuga. method: in a retrospective study, we included a total number of 48 patients, suffering from a chronic skin disease, whose lesions did not improve or even worsened under immunosuppressive treatment (18 chronic ulcers/pyoderma gangrenosum, 21 bullous autoimmune diseases, 9 skin lymphomas). ffpe tissue was examined for the presence of cmv dna by pcr. next, within the framework of a small prospective study (n = 29) we analyzed the seroprevalence of cmv as well as the presence of cmv dna in lesional skin in patients that had been diagnosed with a chronic skin disease and in whom longterm immunosuppressive therapy had been initiated. results: in the retrospective study cmv dna could only be detected in 1/18 chronic ulcers/pyoderma gangrenosum (5.6%), but not in bullous autoimmune diseases and skin lymphomas. 21/29 patients (72.4%) of the prospective study group were seropositive for anti-cmv-igg, as compared to 55/87 patients (63.2%) in an ageand sex-matched control group. anti-cmv-igm could be detected in method: in this study, we aimed at testing the diagnostic potential of skin function measurements in ss. sixteen patients with conformed diagnosis ss were enrolled in the study. skin fibrosis was assessed by conventional rss and involvement of inner organs and serum inflammation parameters were determined. four objective criteria, namely transepidermal water loss (tewl), corneometry, ph and elasticity, were assessed at nine predefined sites of the body. results were compared to patients with atopic dermatitis (n = 19) and acne vulgaris (n = 22). method: a multicenter prospective observational study was conducted to investigate the clinical significance of serum scca2 in children as a biomarker for ad. patients with ad younger than 16 years old and age-matched healthy children without any allergic disease were enrolled in this study. the severity of ad was evaluated using the objective scorad (o-scorad). the serum levels of scca2, tarc and total ige were also measured. results: in total, 176 patients with ad and 159 non-allergic healthy children were recruited. the serum levels of scca2 had the strongest significant correlation with o-scorad, compared with tarc and ige (r = 0.622, 0491 and 0.407, respectively). after standard treatment with topical steroids and emollients resulting in an improvement of symptoms, the serum levels of scca2 and tarc decreased significantly. the area under the curve (auc) for the roc curve was higher for scca2 (0.929) than for tarc (0.871) or ige (0.820). the difference in aucs between a single cut-off value and age-dependent cut-off values was not significant for scca2, compared with that for tarc (0.042 and 0.064, respectively). conclusion: scca2 is a more reliable biomarker than tarc for the diagnosis of ad and for determining the clinical severity of ad in children. challenges in predicting severity of atopic eczema patients results: before modeling, we checked for significant differences between patients and controls. these were detected for the levels of ccl17, ccl22, cxcl10, ige and ldh. next, we assessed whether single serum proteins already explain disease severity by calculating correlations. twelve of the proteins, namely gcsf, il-5, il-13, il-22, ccl22, il-1ra, cxcl8, ifng, ccl3, il-1ß, ccl17, and il-6, significantly correlate with severity (r 2 range: 0.3-0.45). finally, we built a model for the severity of ae based on all measured serum proteins. ten of the proteins are included in the best-fit model (adjusted r 2 =0.47). the overall correlation between original and predicted severity scores is high (r 2 =0.759) nevertheless the cross validation prediction error is substantial with 19%. conclusion: applied in daily practice, a prediction error of 19% translates to a possible miscalculation of 19 scorad points in both directions and therefore the model is of no practical use. aside from using model-based quality measures like cross validation prediction errors to infer the usefulness of predictive models, testing them in independent cohorts could validate these models. collaborations among scientists working on similar approaches would lead to an increase in statistical power and ideally to more robust models. only robust and validated models are going to have the chance to take the step forward from being a result of computational modeling to being applied in the clinical practice of assessing disease severity in patients. jargosch m 1 ; lauffer f 1 ; pätzold k 1 ; krause l 2 ; garzorz-stark n 1 ; schmidt-weber c 3 ; eyerich s 3 ; eyerich k 1 preclinical studies in cell cultures, mice, guinea pigs and rabbits, comprising sterility, cytotoxicity, systemic toxicity, skin irritation, delay contact sensitization and phototoxicity tests, demonstrated safety of this therapeutic agent. we next conducted a single-blinded, intra-individually controlled, 2phased clinical trial on patients with keloids. the aim was to determine the effects of 1-month therapy on keloid volume and symptoms of pain and itch. two similar keloids on each subject were selectedone was treated with once-daily, self-administered application of triamcinolone-loaded (0.015 mg/patch) microneedles for 4 weeks, while the other served as control with no intervention. outcome measures were (a) keloid volume using a 3-dimensional high-resolution (0.1 mm) scanner and (b) pain and itch scores on 0-10 numerical rating scales. evaluations were performed at baseline, 4 and 8 weeks. in phase 2 of the trial, the whole process was repeated using microneedles loaded with a higher dose of triamcinolone (0.1 mg/patch). case report: a 15-year-old girl was admitted to our department with a single round-shaped lesion in the popliteal fossa which spread to extremities, trunk and face and persisted for several weeks and then faded slowly to residual hyperpigmented patches. courses of antihistamines, antibiotics, cyclosporine a, fluconazole, hydroxychloroquine, prednisolone and topical steroids were ineffective. also patient has had history of itchy urticarial rash and angioedema since 5-year-old, suffered from the flares triggered by physical exertion, stress, cold air and water, spicy food, which resolved within 3-4 hours. the patient's father and 7-year-old brother also had chronic urticaria induced by the same stimuli. the physical examination revealed multiple pink-to-red non-scaly, non-pruritic papules coalescing into annular, arcuate, polycyclic plaques (4-5 cm) with central clearing, centrifugal spread, indu1448 | the role of humoral immunity in the pathogenesis of psoriasis results: we found significantly increased levels of iga in the serum of treatment-naïve psoriasis patients correlating with disease score. however, iga was only observed in dermal vessels of skin sections. we next performed in-depth analysis of peripheral b cell subsets using flow cytometry. among all investigated subsets, we only found a moderate positive correlation of cd138 + plasma cells with iga levels and disease score in untreated psoriasis patients. however, in the group of treated psoriasis patients, neither did iga levels drop nor did plasma cells correlate with iga levels and disease score, rather hinting at an epiphenomenal finding. confirming our hypothesis that psoriasis can develop in the absence of proper humoral immunity, we present a patient who suffered concomitantly from both psoriasis and a hereditary common variable immune defect (cvid). conclusion: here, we provide new insights in the immunology of psoriasis, demonstrating the clear dominance of t cells over shifts in b cell subsets. conclusion: allergic diseases show an increasing incidence in geriatric age. this is partly due to the growing emphasis on a more accurate and careful diagnosis of the aging population. we must also take into consideration the influence of other factors, besides comorbidities and therapeutic regimens in elderly that might affects the immune response, such as environmental pollution as well as food contamination and changing dietary habits of elderly such as easy access to exotic food. one of the challenges in the decades to come is recognizing and fulfilling the need for accurate and timely diagnostics of allergic manifestations in elderly patients, as important part of achieving the best possible quality of life for this growing age group. method: sixty-eight patients with various forms of psoriasis and 30 healthy subjects (healthy control group) were assessed after informed consent was obtained. all subjects were asked to complete a questionnaire including age, gender, duration of psoriasis, concomitant diseases and medications. in the group of patients psoriasis was with only skin involvement with skin plus joints involvement ranging from moderate to severe. psoriaticplaques were evaluated by a specialized medical team using the psoriasis area and severity index (pasi). all patients were seen by a dermatologist and clinical immunologist, who collected data considering the demographic, health status and any other relevant details. blood samples included serum levels of 25-hydroxycholecalciferol and tnf-α using an elisa kit (germany). method: 30% ethanolic extract of sp (sp etoh ) and its five major chemical constituents are prepared. to elucidate whether human orai1 modulated by sp etoh and its chemical constituents, conventional whole-cell patch clamp performed in horai1-overexpressing hek293t cell. we also assessed whether sp etoh and its constituents could inhibit mast cell degranulation and t cell activation. results: in jurkat t lymphocytes, we found that 3 mg/ml sp etoh inhibited orai1 current (i orai1 ) by 81.0 ± 11.1%, while one of its constituents (compound v (com v ); 100 μm) inhibited i oria1 by 48.9 ± 8.71%. investigation of human primary t cell proliferation induced by co-stimulation with antibodies to cluster of differentiation 3 and 28, and of rbl-2h3 mast cell degranulation following ige-antigen complex stimulation, revealed that 100 μm com v inhibited both t cell proliferation (by 34.8 ± 6.08%) and mast cell degranulation (by 36.7 ± 0.07%); these effects were concentrationdependent, and no cytotoxicity was observed. conclusion: considering that most regional plants have not been investigated chemically or pharmaceutically, they remain as untapped potential sources of topical agents for drugs and other application. our findings suggest that com v , which derived from sp etoh , represents a promising candidate compound for the development of therapeutic agents for the prevention and treatment of allergic diseases. results: the cohort consists of 14 caucasian patients. eight of them (57%) are women. the mean age (and range) at the clinical presentation of disease was 33 years (4-51 years). the mean age at diagnosis for men was 23 years and 48 years for women. all patients have a positive history of recurrent and/or persistent lip swelling, 3 of them (21%) report oral ulceration, 5 cases (36%) have history of previous or current facial palsy and 4 patients (28%) present tongue fissuring. concurrent cd has been diagnosed in one patient. biopsy reports were available for 10 patients (71%); in 6 cases (60%) non-caseating granulomas were seen. various therapeutic approaches have been described: intralesional corticosteroids had a good response in 6 patients, infliximab was partially effective in 3 cases; oral corticosteroids and/or methotrexate seem to cause a partial symptoms improvement. conclusion: this is the first attempt, in our knowledge, to (a) centralize all data of patients with ofg in a national registry with the aim of carrying out epidemiological data and (b) develop italian guidelines including a diagnostic-therapeutic flow chart, shared by the participating centers. the registry will guide the clinicians in the identification and management of the ofg patients, reducing the diagnostic delay and hopefully improving quality of life. case report: a 10-year-old girl without personal history of atopy, got a temporary tattoo with henna. after three days, she developed a local exudative, erythematous eruption with painful blisters lesions that followed the contours of the tattoo. she had neither fever nor other lesions. she was treated with topic methylprednisolone-gentamicin showing an important improvement 10 days after. as a liquenoid scar remained in tattoo area, trofolastin ® (centella asiatica, αtocopherol, hydrolysed collagen, elastin) patch was prescribed to be placed on the scar. forty-eight hours later, the patch was removed and was newly observed an exudative, erythematous and painful wound that required oral treatment with amoxicillin-clavulanic. after three days, the girl developed on a maculopapular, generalized and itching rash. she was treated with dexchlorpheniramine and methylprednisolone with a complete resolution in 4 days and she was referred to our allergy unit to be studied because of a suspicion of drug allergy to amoxicillin-clavulanic acid. an allergy workup was performed after obtaining an informed consent. case report: it may be sometimes difficult to find the causing allergen in allergic contact dermatitis. face is a region on which various materials contact. in this manuscript a woman case is presented who shows patch test positivity to her husband's shaving product. a 35 years old woman applied because of allergic contact dermatitis on her face. it is learnt that lesions have been continuing for a long time, occasionally getting well with corticosteroid creams; but continuing again. patch test was performed with european standard series and cosmetic products she was using. negative result was observed. following, patch test was performed for the products her husband was using. 2 positive results were obtained for the shaving cream of her husband was using. in detailed anamnesis, it is learnt that the lesions developed approximately 1 month after her husband started to use this cream. it is advised not to use this product to her husband. the disease did not repeat again. it should not be forgotten that cases with allergic contact dermatitis could get in touch with allergenic materials via individuals in close contact. gül ü akdeniz university faculty of medicine, department of dermatology, antalya, turkey case report: tnf-alpha plays role in etiopathogenesis of allergic contact dermatitis (acd). in mice which lack tnf-alpha, the response of late type hypersensitivity is spoiled. in addition, tnfalpha blocker are also used in some cases with acd. in this poster the results of european standard patch test is given in which acd is observed and tnf-alpha blocker are used without dermatological indication. 3 cases who use tnf-alpha blocker applied because of acd: there was lesion in one case on face, in other case on face and hand, and in the last case only on hand. european standard patch test was performed to patients who were continuing to use tnf-alpha blocker. in one case no positive response was observed, while in two cases positive response to more than one allergen were obtained. in conclusion, tnf-alpha blockages cannot suppress the response of delayed type hypersensitivity. 1459 | case of allergy to nickel on the background of its intake in food peredelskaya m case report: nickel is one of the most commonly used metals; it is used for the manufacture of jewelry, plates and dishes, and medical products. a patient n, 32 years old, female, complains of pruritic rash on the body skin with the itch intensity up to 8-9 points and the number of lesions more than 50. allergic background: for quite some time now the patient noted occasional eruptions on her skin after a contact with jewelry made of non-precious metals. previously patch skin tests with nickel showed a positive reaction. the patient sought emergency medical care with complaints of a number of itchy lesions erupted on her whole body during the last 24 hours. on admittance: state of moderate severity, the patient was emotionally labile, focused on her body sensations, tearful. on the skin of face, upper and lower extremities and torso a punctuate purpura with lesions up to 0.5 cm diameter, prone to confluent. a physical status was within normal limits. in order to control the itching, as well as to sedate the patient, antihistamines of the first generation were administrated parenterally; but the eruptions kept to progress and to intensify; lesions were spread throughout the whole body, merged in gigantic areas. system glucocorticosteroids therapy was administrated, with 120 mg of prednisolone, but then new lesions kept appearing in a large number, including after-meal rash. water, tea, bakery products, thin yoghurts did not impact the skin condition, whereas the intake of pasta, cereals, and similar products provoked intensifying of eruptions. the patient observation revealed a sharp increase in the rash after such manipulations as intravenous injections or blood sampling from the vein, the process spreading from the injection site to the entire arm. a detailed anamnesis of the disease: on the eve of the start of hives, the patient purchased a coffee machine (with metal nickel-plated parts) and started to use it. diagnosis: a systemic contact dermatitis. an allergy to nickel. the injection treatment was discontinued and a therapy with per oral gcs and antihistamines of the second generation was administrated. a recommendation was given to cook and to eat food using ceramic or wooden utensils. three days later marked positive dynamics of the skin process has been noted. the episode of systemic contact dermatitis has developed due to exposure to nickel from ingestion in food, as well as during the parenteral treatment. background: anaphylaxis reactions during anesthesia can have a mortality of 3%-6%. 2/3 of the anaphylaxis in the operating room are due to the use of neuromuscular blockers. rocuronium is frequently involved because is oftenly used. we present a case of a 41 years old man with an anaphylaxis shock due to the administration of rocuronium. method: 41 years old man with no personal history of interest that is going to undergo vertebral surgery. minutes after anesthetic induction with fentanyl, propofol and rocuronium he started with lowering of oxygen saturation. orotracheal intubation is performed and, with the suspicion of anaphylaxis shock, adrenaline, antihistamines and corticoids were administered. after 20 minutes without improvement, 500 mg of sugammadex was administered, given the possibility that the condition was secondary to the use of rocuronium. tryptasa level was 63. results: skin test to fentanyl, propofol, látex and rocuronium were done 6 weeks after and only rocuronium test was positive. conclusion: in summary, the occurrence of anaphylactic shock after neuromuscular blockers is widely described in medical literature. there are conflicting data about the use of sugammadex as coadjutant treatment in case of anaphylaxis due to the use of rocuronium. we believe is a good option when conventional treatment is not useful. case report: a 31-year-old woman with past history of allergic rhinitis and hypertension was admitted to the obstetrics service in labor of first child in april 2016. epidural anesthesia with ropivacain and sufentanil was administered. as there was no labor progression, eighteen hours later she was admitted to undergo cesarean section and epidural anesthesia was re-administered. metoclopramide, ampicilin and ranitidine were given intravenously. during ranitidine perfusion, the patient presented general cutaneous erythema and pruritus, tongue, lips and eyelids angioedema and dyspnea. perfusion was suspended and hydrocortisone and supplementary oxygen administered. she denied any type of previous adverse reaction to drugs and any symptoms with use of latex-containing material. allergic evaluation revealed negative latex skin prick test (spt) and negative penicillin, amoxicillin and ampicillin specific ige assay. skin prick and intradermal tests with sufentanil, ppl, mdm, amoxicillin and ampicilin were negative. oral amoxicilin and metoclopramide provocation challenge were negative. spt and subcutaneous provocation challenge with ropivacain were negative. spt with ranitidine was negative but skin intradermal test proved to be positive. the patient was taught to avoid histamine h2 receptor antagonists and use as a safe alternative proton pump inhibitors. conclusion: anaphylaxis during anesthesia is an unpredictable, severe, and rare reaction. the identification of responsible drugs is a complex task. we report a case in which a commonly used and generally safe drug caused a severe reaction, which demonstrated that even the least obvious culprit should not be disregarded. epidemiologic data suggest that the number of cases of chx allergy appears to be increasing. background: chlorhexidine is a synthetic chemical with excellent antiseptic and disinfectant quality frequently used in everyday products and medical devices. the prevalence of allergic reactions towards chlorhexidine is rare, though there is increasing evidence for its allergenic potential. in this case we report about a patient with serious perioperative anaphylaxis. next to multiple potential allergens that he was exposed to, a chlorhexidine containing lubrication gel has been used for urinary catheterisation. within minutes post-exposure, the patient developed generalized urticaria, bronchospasm, tachycardia and hypotension. material and methods: we performed skin prick tests and intradermal tests with all substances documented in the anaesthesia chart, further we analysed specific immunoglobulin e (sige) antibodies and performed oral provocation challenges for exclusion. results: in the skin tests all substances except for chlorhexidine (spt: 7 mm wheal diameter/31 mm erythema) were negative. a sensitization for chlorhexidine was further corroborated by chlorhexidine-specific ige antibody (4.08 ku/l) in the patient's serum. in addition, the challenges for the drugs without sensitization (cefuroxime, lidocaine) were tolerated. considering all potentially relevant allergens that the patient was exposed to and the proof of specific sensitization, we diagnosed an immediate-type allergy towards chlorhexidine. conclusion: with the ubiquitous use of chlorhexidine an increase in hypersensitivity reactions including immediate-type allergic reactions is observed. anaphylactic reactions are rare, but potentially life-threatening, the diagnosis is crucial. as a warning declaration in medical devices is missing, the diagnosis of chlorhexidine allergy might be easily under-recognized or misdiagnosed. unfortunately, until now validated provocation tests are not existent, but the evaluation of combined skin tests and sige is sensitive and specific. 1464 | an approach to incidence of death due to anaphylaxis in spain (1998 spain ( -2011 background: reports about death due to anaphylaxis are still scarce because of its rarity and limited information to few countries. also, data source analysis is usually not included. we report incidence of death due to anaphylaxis in spain using two databases. method: we used a hospital series of anaphylaxis deaths from the spanish hospital system and a series from the national institute of toxicology and forensic sciences (intcf) predominantly formed by extra-hospital deaths. deaths from the spanish hospital system were extracted using codes from icd-9-cm, related to anaphylaxis among all deaths occured in the 1998-2011 period. for extracting deaths due to anaphylaxis at the intcf in the same period, two allergist researchers identified these deaths among cases with suspicion of anaphylaxis cause. a regression logistic was run to discriminate the probability of anaphylaxis death belonging to each database. incidence rates were calculated for the different groups (age, sex) using the spanish population as the denominator. temporal trends were calculated from the hospital database using poisson regression models with the number of cases of anaphylaxis detected each year as the dependent variable, and age and sex as covariates. results: there were four positive predictors of fatal anaphylaxis after the logistic model (usual allergen, positive specific ige, suggestive symptoms and previous reaction to the same allergen case report: we were informed that a girl was admitted to the pediatric endocrinology department due to early breast development. she had been diagnosed as central precocious puberty (pp). later, triptorelin acetate (ta) therapy had been started monthly. within 20 minutes after first sc injection of ta at home, she had developed shortness of breath, decreased air entry, and coughing for ten minutes and lastly she had developed vomiting for 15 minutes. her symptoms were accompanied by a pruritic blanchable maculopapular rash on her ears, cheeks, lips, and eyelids approximately for two hours. although they had applied emergency department of the local hospital. based on the diagnosis of anaphylaxis she was immediately treated with adrenalin. she was subsequently hospitalized for possible recurrence and discharged next day without any further events. treatment with another preparation, leuprolide aseptate(la-lucrin), as an alternative treatment was started with premedication against anaphylaxis risk only at first time and the patient did not develop any reactions. the patient is still on this treatment with no complications. anaphylaxis is diagnosed in the presence of a detectable allergen accompanied by symptoms of two systems. our patient had symptoms of the three systems as described above, that is, dyspnea with coughing, hives, nausea, and vomiting. main treatment of anaphylaxis is the epinephrine use. early usage maximizes the likelihood of survival. diagnostic tests with culprit drug were not performed in our hospital if the patient had the anaphylactic drug reaction and grouped as "physician diagnosed anaphylaxis". there has been only one report regarding anaphylaxis to ta treatment in cpp in turkey. in the literature, anaphylactic reactions against ta have been reported only in few pediatric cases. gnrh analogues are important to ensure the physiological growth in precocious puberty. because anaphylaxis can be lethal, and gnrh analogues are similar structure; the present case suggests that one should bear in mind the possibility of anaphylaxis in all patients who receive gonadotropin-releasing hormone and anologs and monitor such patients carefully as needed. furthermore, we must provide sufficient information of adverse reactions, including anaphylaxis, to patients. hence, managements against anaphylactic shocks should be recognized and treatment should be given immediately. 1467 | an anaphylactic shock induced by the rocuronium anesthesia: a case report cabrera v; barrios j; callero a; gonzález ce; pérez e; martínez ja hospital universitario nuestra sra de la candelaria, santa cruz, spain background: the anesthetic act is a unique pharmacological situation, where the patient is exposed to a multitude of substances.among them, neuromuscular blocking agents are the leading cause of preanesthetic anaphylaxis, with a frequency of between 50%-70%.followed by latex in second place and antibiotics in third place. among the neuromuscular relaxants, most reactions are due to suxamethonium or succinyl-choline in 60.6%, followed by atracurium, rocuronium and verocuronium.the one that produces the least reactions is cisatracurium. method: a 61-year-old woman presented a type iii anaphylaxis of the brown classification during the anesthetic induction in a surgery scheduled for laparoscopic cholecystectomy. for which adrenaline, dexchlorpheniramine, hydrocortisone, ranitidine and sugammadex was administered and was transferred with orotracheal intubation to the anesthetic resuscitation room. due to good evolution of the patient, she was extubated within three hours. the drugs involved in the reaction were: rocuronium, amoxicillin-clavulanic, fentanyl, propofol, midazolam, lidocaine and atropine. there was a high suspicion by the anesthesiology and resuscitation service that the abstracts reaction could have been due to the neuromuscular relaxant used, in this case rocuronium, since the reaction was reversed with sugammadex. the patient had undergone surgeries under general anesthesia previously without incidents. a specific allergy study was performed with laboratory tests with tryptase, skin tests with drugs and basophil activation test for rocuronium, sugammadex-rocuronium mixture and cisatracurium. • serial measurement of serum tryptase: 12.9 u/l, 10.9 u/l y 3.42 u/l there is no activation of basophils for sugammadex-rocuronium mixture and cisatracurium. the patient is diagnosed with rocuronium allergy. sugammadex not only acts as an antidote to reverse the neuromuscular block against rocuronium, but also has antiallergic properties by inhibiting mast cells. as an alternative for future interventions, the patient can use cisatracurium, as the skin tests and the basophil activation test are negative. unal d yedikule chest disease, istanbul, turkey case report: tetracycline hydrochloride may rarely cause hypersensitivity reactions. (hrs). immediate type reactions are at the level of case presentations and anaphylaxis is reported. we report a patient with late onset anaphylaxis caused by tetracycline. a 47-year-old woman referred to our allergy outpatient clinic because of urticaria due to an antibiotic that she does not remember the name of. the patient reported that many years before she had presented urticaria on her arms and legs one hour after taking the drug. to confirm drug allergy invivo and invitro testing have to performed. for many drugs there was no validated skin test. for all that invitro tests are often less sensitive and more expensive. therefore single blind placebo controlled drug provocation tests (sbpcdpt) is the gold standard in the diagnosis of drug hypersensitivity reactions. we did not know which group of antibiotics were allergy to the patient. because the patient had history of asthma and atypical pneumonia we were performed the allergy tests with clarithromycin and she had tolerated. it was necessary to use tetracycline because of patient had vaginal infection. skin tests have not yet been validated for tetracyclines. for skin prick tests of tetracycline that is only available as tablet, not in a soluble form. therefore, the tablet was smashed and diluted with 0.9% nacl. it was also tested. 10 healthy controls to exclude irritation. because of skin prick tests with tetracycline negative. sbpcdpt was planned. sbpcdpt was performed by progressively increasing four divided doses at 30 minute intervals. two hours after last dose the patient experienced dyspnea, palpitations, and hypotension. as the reaction was considered to be anaphylaxis, she was given 0.5 mg of intramuscular epinephrine, intravenous 45 mg of pheniramine, and 40 mg of methylprednisolone. the reaction resolved within 3 hours. blood tryptase level was 14.9 ug/l taken at the 2nd hour of the reaction approximately 2 months after the anaphylaxis, serum tryptase level was 1.59 ug/l the serum tryptase level and the patient's clinic confirmed anaphylaxis due to tetracycline. we had proved late onset anaphylaxis due to tetracycline with the patient's clinic and serum tryptase level. anaphylaxis due to tetracycline is limited to case reports and small series but to our knowledge, there is no previous report of late onset tetracycline anaphylaxis. 1469 | case series of ige mediated anaphylactic shock due to polysorbate case 2: an 86-year-old male patient with hypertension, hypothyroidism and episodes of sustained monomorphic ventricular tachycardia (smvt), developed an anaphylactic shock after the administration of injectable amiodarone due to smvt. serum tryptase levels reached 34.8 μg/l during the reaction (baseline 6.59 μg/ l). skin tests were positive to injectable amiodarone (prick 50 mg/ ml, intradermal 0.5 mg/ml) and polysorbate 80 and 20 (prick-prick). skin prick-prick to amiodarone and dronedarone tablets were negative. the patient tolerated oral amiodarone. we report an anaphylactic reaction during the first intravenous administration of amiodarone in a female patient being treated for supraventricular tachycardia. bat was positive, suggesting a direct effect on basophil activation, as the patient was not previously exposed to the drug. 1472 | anaphylaxis during labor: don't forget to think of an amniotic fluid embolism case report: a 32-year old primigravida (40 weeks of gestational age) was admitted with signs of pre-eclampsia and labor was induced. benzylpenicillin and ropivacaine (epidural anesthesia) was administered >3 hours before the event. eighteen minutes after starting an infusion with oxytocin (15 ml/h) and a vaginal toucher, the patient developed a decreased level of consciousness, generalized edema/erythema and thoracic pain, followed within 3 minutes by fetal bradycardia and maternal collapse. after resuscitation, an urgent sectio was performed, and a baby girl was born. patient was extubated the same day. serum tryptase, 1 hours after the event, was 11.2 μg/l (basal tryptase level 3.4 μg/l). allergy workup demonstrated negative specific ige and skin tests for latex and chlorhexidine, negative skin and provocation testing for ropivacain. however, skin testing was hampered by dermographism: intradermal (idr) testing of benzylpenicillin (10 000 iu/ml, 1/1000-1/1) and oxytocin (10 ie/ml, 1/10 000-1/100) showed extensive erythema. idr testing of oxytocin in healthy volunteers showed pallor around the injection site (n = 3). intravenous provocation with benzylpenicillin was uneventful. a basophil activation test with oxytocin (patient and control) was negative. an additional bone marrow evaluation showed no evidence for mastocytosis. although clinical criteria for anaphylaxis were fulfilled, a diagnosis of an amniotic fluid embolism (afe) was concluded. no drugs were prohibited. patient gave consent for publication. conclusions: afe is one of the most devastating conditions in obstetrics, occurring typically during labor and delivery or immediately postpartum. the pathogenesis remains incompletely understood, however, it has been suggested that afe involves an anaphylactic reaction to fetal tissue exposure associated with breaches of the maternal-fetal physiological barrier, supported by transiently increased serum tryptase levels. the diagnosis is primarily clinical, and generally one of exclusion. no specific antemortem diagnostic tests are available to confirm afe. postmortem identification of fetal squames in the maternal pulmonary circulation gives final diagnosis. differential diagnosis includes drug-induced anaphylaxis or mastocytosis, which were ruled out in our case. method: the patient presented after hymenoptera stings dyspnoea, generalized erythema with pruritus, edema of the face that required emergency therapy in 3 episodes. results: an angio-ct was performed at the inferior limbs with optiray and 5 minutes after the end of the investigation, the patient presented an anaphylactic shock requiring admission to the icu for 4 days. conclusion: the patient's progression was slowly favorable. results: thirty seven cases were reported. 24 (65%) were women. the median age was 47 years. 19 (51%) had dress/dihs, 6 (16%) ten, 3 (8%) sjs, 3 (8%) agep, 3 (8%) other not classified scars, and 1 (2.7%) overlapping ten/sjs. in 100% of the patients the suspect drug was withdrawn. thirty one patients (83%) received systemic anti-inflammatory treatment. twenty six patients (70%) received intravenous (iv) corticosteroids alone, 3 (8%) iv corticosteroids plus ivig, 1 (2.7%) iv corticosteroids plus ivig, infliximab and colchicine, and 1 (2.7%) iv corticosteroids plus infliximab and cyclosporin. there were complications in 17 cases (49%), and death occurred in the patient with overlapping ten/sjs who had received corticosteroids plus immunoglobulin. in this study, our aim was to evaluate severe ihr to icm. method: we retrospectively analysed patient who consulted to our allergy unit between july 2011 and july 2016 reporting symptoms within 1 hour after icm administration. from a total of 109 patients, we selected eight that had suffered an anaphylactic reaction. a written informed consent had been obtained for diagnostic procedures. introduction: immediate type hypersensitivity reactions to pemetrexed have been reported as very rare case reports. as limited availability of alternative therapies in chemotherapeutic allergy, desensitization plays an important role in ensuring reuse of the culprit drug. we report a case of pemetrexed anaphylaxis and successful desensitization. case: 43 years old female patient with lung adenocarcinoma had been treated with cisplatin-pemetrexed as second-line therapy. during the 5th cycle within 5 minutes after the end of pemetrexed infusion she had chest pain, shortness of breath, cough, swallowing difficulty, erythema on face and body, nausea and vomiting. she was diagnosed as anaphylaxis and adrenaline was administered besides antihistamine and methylprednisolone. symptoms and findings of the patient were improved within 15 minutes. oncologists decelerated no suitable alternative therapy for the patient. although skin tests (prick test with 1/1 concentration, intradermal test with 1/10 000-1/10 concentration) were negative with pemetrexed, taking into account the severity of the reaction, pemetrexed desensitization was applied with the consent of the patient. no reaction was observed during the procedure result: desensitization is a successful and safe method of reusing the culprit drug. successful desensitization of pemetrexed with immediate type hypersensitivity reaction is described. the 28 years old man was admitted emergency department with fever, rash (maculo-papular) and pain in joints. it was the 9th day of taking of amoxicillin. the hematological abnormalities were revealed -eosinophilia, increased erythrocytes sedimentation rate. the level of serum ecp was 183 μg/l. the liver functional tests were increased too. hepatomegaly and cervical lymphadenopathy were observed. the patient was treated as a dress syndrome (infusion therapy, systemic steroids) and discharged after 2 weeks with improvement. all hematologic parameters were in normal limits. lymphadenopathies were resolved. the level of ecp was retaken -84 μg/l. patient was prescribed oral steroids till normalization of limits of ecp. it lasted 2 weeks after discharging. the serum level of ecp can play key role in the management of dress syndrome and in the making of diagnostic processes. until now, allergic or anaphylactic reactions to peg have been rarely reported. although patient with hypersensitivity to peg should avoid peg-containing drugs or products, patient who needs colonoscopy has few alternative bowel cleansing methods. no successful desensitization to peg has been reported to date. we report a case of successful desensitization and subsequent safe colonoscopic examination in patient with allergic reaction to peg. method: a 50-year-old woman developed generalized urticaria, pruritus, throat swelling, and shortness of breath immediately after taking a bowel preparation solution for colonoscopy. she had the first symptoms 3 years ago, and has had 2 more experiences so far. the symptoms appeared within 20-30 minutes of taking cleansing solution, and the endoscopy was no longer possible. seven years ago, she underwent endoscopy with no specific symptom. when the last symptom occurred a year ago, she was treated at emergency room because of severe dyspnea and dizziness. the patient came to our clinic for the proper diagnosis of allergy reaction and possible colonoscopic evaluation. objectives: to describe a successful desensitization to vedolizumab in one patient diagnosed with ulcerative colitis, refractory to infliximab and intolerant to azathioprine and sulfasalazine. methods: our patient was a 38 year old woman receiving treatment with intravenous vedolizumab (300 mg/cycle). cycles 1 and 2 were well tolerated, but in cycles 3, 4 and 6 she experienced hypotension and dyspnea, in spite of premedication with oral dexamethasone and metoclopramide. during cycle 6, she also showed facial angioedema, systemic urticarial reaction and oropharyngeal pruritus treated with methylprednisolone and ebastine. the results of prick (vedolizumab concentration 60 mg/ml) and intradermal skin tests (1:10 and 1:100) with vedolizumab were negative in our patient and in ten healthy controls. total ige level was 26.4 ui/ml and specific ige against dermatophagoides were positive, being negative for hamster epithelium and latex. since vedolizumab was the only therapeutic alternative, the patient was planned to undergo vedolizumab desensitization according to an 8-step protocol. patient informed consent was obtained previously. premedication consisting of ebastine, acetylsalicylic acid, montelukast and methylprednisolone one hour before desensitization was administered. desensitization protocol was performed with a total duration of 4 hours and 35 minutes and a total dose of 293 745 mg. dose steps were 0.015, 0.03, 0.3, 0.6, 1.2, 9, 18 and 264.6 mg. conclusions: our 8-step protocol desensitization to vedolizumab resulted safe and effective in our patient and it has allowed the continuation of treatment with vedolizumab for her ulcerative colitis. montelukast, anti h1 and h2 blockers were used for the pretreatment of desensitization. all procedures (skin and blood tests, desensitization) were carried out with the informed consent of the patient. we present an exceptional, non-immediate case of fever after cisplatin and etoposide infusion with positive skin test. case report: a 63-year-old man, recently diagnostic of lung cancer stadium iv, in first line of treatment with cisplatin and etoposide, started 2 hours after finishing the 2nd infusion: facial erythema that becomes generalized after 4-5 hours from infusion. twelve hours later, developed warmth sensation, shivering and fever (39°c) that persisted despite the use of several oral antipyretics treatment. infectious disease was discarded, so he was referred to our department in order to assess further administration of cisplatin and etoposide. methodology: skin testing was performed 30 days after the last reaction to minimize false-negative results, as follows (a) cisplatin prick test (1 mg/ml) and intradermal tests (0.1 mg/ml); (b) etoposide prick test (20 mg/ml) and intradermal tests (2 mg/ml); with histamine as the positive control and nacl-diluent as the negative control. the results of skin test were negative for immediate reading. but two hours later, intradermal test for cisplatin turned into red and itchy and 12 hours later, still associated a wheal. the patient was classified as high-risk (lung diseases, forced expiratory volume in 1 second <1 l) and underwent programmed inpatient desensitization according to the standardized birmingham women's hospital protocol. desensitization was performed in the medical intensive care unit. the patient received only standard oncology premedication. he tolerated the final dose of cisplatin with no breakthrough reactions followed by etoposide standard infusion. two additional desensitization procedures were performed, with no breakthrough reactions. therapy ended when the disease worsened. the importance of this case, lies in the fact that fever has not been described as a clinical hypersensitivity reaction for cisplatin but for oxaliplatin. although a non-immediate reaction at the 2nd infusion of cisplatin could scarcely suggest a hypersensitivity reaction, the positive skin test and successful desensitization with this drug, could suggest it. introduction: propylthiouracil is commonly used as the first treatment option in patients with hyperthyroidism. although it is generally a well-tolerated drug, it may lead to some side effects including liver damage, leucopenia and skin rash. among skin rash findings, urticaria is considerably common. nevertheless, in cases that developed urticaria, a rapid desensitization protocol specific to propylthiouracil has not been encountered. we represented a case in which we applied successful oral desensitization via a scheme in accordance with general desensitization principles in a case that developed propylthiouracil-induced urticaria. case report: propylthiouracil at a dose of 50 mg/day was initiated for a 36 year-old female patient with diagnosis of hyperthyroidism in internal diseases clinic. the patient developed widespread itching and swelling in the body 5-6 hours after she took the first dose of the drug. she had experienced a similar reaction with use of propylthiouracil in 2012. the patient who was breastfeeding a baby and did not have any treatment option other than propylthiouracil was referred to us with pre-diagnosis of drug allergy. the patient was thought to have propylthiouracil-induced hypersensitivity reaction and desensitization was planned. we prepared a desensitization scheme in accordance with general desensitization principles (table 1 ). in accordance with this prepared scheme, we successfully applied the desensitization protocol with propylthiouracil for the patient. the patient gave informed consent before testing and desensitization. results: spt was negative, but idt reaction was positive at 1:1000 method: we present a desensitization protocol to intravenous etoposide used in a 35-year-old male for non-hodgkin's lymphoma who was referred to the department of allergy at sotiria general hospital of athens. within 5 minutes after receiving the first dose of the drug, the patient complained for flushing, retrosternal pain, difficulty in breathing and weakness. the infusion was ceased immediately and the patient received proper treatment with gradual recovery of the symptoms. the next day, skin prick test (spt) and intradermal test (id) were performed with etoposide at dilution 1:10 (2 mg/ml). both of the tests, spt and id, were negative. histamine and nacl 0.9% were also used as positive and negative controls, respectively. a desensitization protocol of three-day cycle with intravenous etoposide was conducted. premedication for 3 days was administered including methylprednisolone, cetirizine, ranitidine, paracetamol and montelukast. results: the desensitization protocol of the first day consisted of 11 steps of rapid pulses administered at increasing infusion rates every 15 minutes, and 1 step of drip infusion at a final rate of 60 ml/hour (372 mg/232.5 ml) until completion of the infusion. the following 2 days, the patient received a modified rapid protocol consisting of the administration of the calculated dose of 400 mg in only one step of infusion rate of 60 ml/hour completing in only 4 hours and 15 minutes. the same protocol was applied in another three-day cycle with no adverse reactions. conclusions: hsrs to etoposide are rarely described in the literature. we propose a three-day modified rapid desensitization protocol to intravenous etoposide that could be particularly useful compared to other time-consuming desensitization protocols. case report: imatinib, a tyrosine kinase inhibitor, sometimes causes cutaneous reactions that can be of various severity. we present a case of a patient who was started on imatinib 400 mg daily and after 3 months developed diffuse mildly pruritic rash with some desquamation of palms of the hands. the dose of imatinib was reduced to 300 mg daily and therapy with prednisone 30 mg was started. after resolution of rash, the dose of prednisone was tapered to 10 mg daily, but the rash reappeared, although milder in intensity. the dose of prednisone was increased and levocetirizine added and rash resolved. prednisone was slowly discontinued and rash did not appear. in the case of reactions to imatinib the dose of drug can be reduced and short course of oral corticosteroid given. milder reactions can be treated with antihistamine or topical corticosteroid. therefore, when adverse skin reaction to imatinib occurs, induction of tolerance to this important drug should be attempted. method: the exosomes were collected from in vitro primary human sinonasal epithelia cell, which derived from three different groups (normal control, chronic rhinosinusitis and chronic rhinosinusitis with asthma). generation of exosomes in epithelia was confirmed by nanosight, tem and western blot. the proteins of exosomes were identified by proteomics analysis. the cellular proliferation and ciliogenesis were analyzed by cck8 and qpcr.the ciliary beat frequency was detected by sava system. we found that epithelial cellular exosomes from chronic rhinosinusitis and chronic rhinosinusitis with asthma could reduce the multiplication rate of normal epithelial cell at a certain concentration (≥10 μg/ml).we found that exosomes from chronic rhinosinusitis with or without asthma could interrupt the cellular ciliogenesis and ciliary beat frequency. using mass spectrometric analysis we demonstrated that the epithelial exosomes contained different proteins in different disease states. conclusion: our findings first identified that exosomes could be secreted by nasal epithelial cells. we also demonstrated exosomes from chronic rhinosinusitis with or without asthma could be a pathogenic factor in the remodeling of sinonasal mucosa. it could be considered as a significant biomarker for detecting the progress of chronic rhinosinusitis and a alternative therapy target. background: mucociliary transport (mct) is a major respiratory tract host defense mechanism and chronic exposure to allergen can deteriorate the these defense mechanism. the aim of this study was to investigate the effects common allergen (dp/df) on human nasal mucociliary transport in allergic rhinitis patients, and to determine the pathophysiology of ciliary beat frequency (cbf) during allergeninduced change method: allergic nasal mucosa cells of allergic rhinitis patients were exposed to common allergen (dp/df), and cbf was analyzed using an optical flow technique with the peak detection method results: the allergen(dp/df) exposed group showed a decreased cbf when compared to the control group. in the cytotoxicity assay, difference in survival rates was not found between the two groups. in the allergen(df/df)-exposed group, protein kinase c (pkc) activity was increased during a pkc activity assay. the broad pkc inhibitor, calphostin c abolished the allergen(dp/df)-induced decrease of cbf. the allergen-induced decrease of cbf was abolished by gf 109203x, a novel pkc (npkc) isoform inhibitor, whereas the decrease was not attenuated by g€o-6976, a specific inhibitor of conventional pkc (cpkc) isoform. conclusion: allergen may inhibit cbf via an npkc-dependent mechanism. therefore, we have confirmed that chronic exposure to allergen could decrease cbf by increasing pkc activity. method: ova-alum allergic rhinitis mouse model (ar model) and poly(i:c) induced il-17 dominant mouse model (neutrophil dominant model) were used. both mouse models were exposed to tio2 particles for 2 hours twice daily for 7 days, while the controls (n = 5) were not. sirius red staining for eosinophil infiltration, immunohistochemistry for neutrophil and il-17a, serum immunoglobulin (ig) g and e were assayed by using enzyme-linked immunosorbent assay. in addition, the expression of interleukin (il)-4, il-17, and interferon (ifn)-γ in the nasal mucosa and cervical lymph nodes was measured by immunohistochemistry, and real-time reverse transcription-polymerase chain reaction (rt-pcr), il-17 monoclonal antibody (secukinumab) was administered in vivo to evaluate il-17a dependency. results: tio2 exposure did not influence eosinophil infiltration in both ar and neutrophil dominant model. however, tio2 exposure increased neutrophil infiltration in both models and neutrophil infiltration was correlated with il-17 expression in the nasal mucosa. serum igg and ige levels were changed significantly in the tio2exposed group. th2 cytokines (il-4, il-5) and th1 cytokine, ifn-γ were not changed significantly in both models after tio2 exposure, however, il-17 were increased in tio2 exposure group. and these increased type 17 pathway and neutrophil infiltration were reversed after il-17 monoclonal antibody administration. conclusion: exposure to airborne tio2 induced neutrophil infiltration in the nasal mucosa. the type 17 response seems to play a dominant role in the nasal immune response following airborne tio2 exposure. 1501 | toll-like receptor 9 ligands increase type i interferon induced b-cell activating factor expression in chronic rhinosinusitis with nasal polyposis results: first: paf-r mrna expression was very low in fibroblasts from nm and np (data not shown). paf-r mrna expression was detected in whole sinonasal tissue, submerged and ali epithelial cell cultures from both controls nm and np. paf-r mrna was also detected in peripheral blood eosinophils. although no differences were found between nm and np tissues and cultures, paf-r mrna expression was significantly higher (p < 0.001) in eosinophils than in upper airway tissues and cells. second: protein paf-r was found expressed in whole tissue (predominantly in the epithelium and submucosal glands), submerged and ali epithelial cell cultures from both nm and np. peripheral blood eosinophils also showed paf-r protein expression. conclusion: both paf-r mrna and protein expression was found in sinonasal nm and np tissues (epithelium and submucosal glands) and in peripheral blood eosinophils. these findings suggest the paf/ paf-r system could play a pathophysiological role in crswnp through the modulation of structural and inflammatory cell functions. "this study was funded with a research grant from uriach group". background: allergic rhinitis (ar) is an increasingly more common nasal inflammatory disease in which an antigen such as pollen or dust mites triggers symptoms such as itching, sneezing, and rhinorrhea, which can lead to nasal obstruction. ar is mediated by thelper type 2 cells together with mast cells, eosinophils, and several inflammatory cytokines and chemokines. for example, recent abstracts | 757 research indicates that hypoxia-inducible factor 1α (hif-1α) is involved in the mechanism of ar development. the anti-heart failure drug digoxin has a specific inhibitory effect on hif-1α, and thus, the aim of the present research was to explore the anti-hypertensive effect and mechanism of digoxin in ar. method: an animal model of ovalbumin-induced ar was established in guinea pigs. the experimental group was treated with digoxin through the tail vein. for the comparison of symptoms between the experimental and control groups, the incidence of sneezing was recorded, and the eosinophilic interleukin il-4 and il-5 levels in nasal secretions were measured by enzyme-linked immunosorbent assays. western blotting and reverse transcription polymerase chain reaction analyses were conducted to evaluated hif-1α expression in guinea pig nasal mucosa. results: the ar symptoms of guinea pigs in the experimental group were significantly improved after administration of digoxin. specifically, the experimental group exhibited a significantly lower numbers of sneezing times(average 36.0 ± 1.10 vs 7.4 ± 0.78, p < 0.05) and lower il-4 and il-5 secretion levels (p < 0.01) compared with the control group. moreover, guinea pigs of the experimental group showed less severe nasal mucosa edema, lower hif-1α production, and reduced eosinophil infiltration in nasal mucosa compared with the control group. conclusion: the anti-heart failure drug digoxin may ameliorate the symptoms of ar by inhibiting hif-1α production. campo p 1 ; eguiluz i 1 ; bogas g 1 ; gomez f 1 ; ariza a 2 ; espino t 1 ; torres mj 1 ; rondon c 1 1 allergy unit-regional hospital of malaga-ibima, malaga, spain; 2 allergy laboratory_regional hospital of malaga-ibima, malaga, spain background: similarly to what has been described in allergic rhinitis, there is an important association of local allergic rhinitis (lar) with lower airway symptoms suggestive of asthma, being selfreported in 24.4% of lar patients after five years of follow-up, and increasing to 30.7% after 10 years. however, clinical suspicion alone it is not enough for asthma diagnosis and could overstate its real prevalence. the aim was to evaluate the real prevalence of asthma in lar patients based on validated objective methods. method: seventy-five patients (29 with lar, 20 with non-allergic rhinitis (nar), 18 with allergic rhinitis (ar)), and 8 healthy controls (hc) were included. all patients had perennial history of rhinitis and bronchial symptoms suggestive of mild-moderate asthma for at least two years. non-specific airways hyperresponsiveness (methacholine challenge test, using tidal breath method following ats guidelines) was performed in all subjects. results: subjects were mostly young females, non-smokers. median μg/day of inhaled corticosteroids (budesonide/equivalent dose) was similar in all groups. median fev1% in ar group (75.5%) was significantly lower compared to lar (90%, p = 0.002), nar (85%, p = 0.007) and hc (88%, p = 0.005). in the lar group, 15/29 (51.7%) had a positive methacholine, 12/20 (60%) in the nar, 15/18 (83.3%) in ar group and 0/8 (0%) in hc. patients with lar had a significant lower percentage of confirmed asthma than ar (p = 0.031) and similar to nar (p = 0.771). no differences were detected between ar vs nar (p = 0.155). conclusion: presence of objectively demonstrated asthma was lower in lar compared to ar, and with better lung function. conclusion: ambient air pollution influenced the hospital visit of patients with rhinitis, even, the level of pollutants, below the national standard. so2, o3, no2, and pm10 could increase an incidence of rhinitis and/or induce an aggravation of rhinitis symptoms. health care provider might expect upraising patients with rhinitis in the clinic with increase of air pollutants, even under the standard levels. results: during relapse of erosive oral lichen planus mononuclear cells obtained from peripheral blood of patients showed increased number of nk-cells cd3 + cd56 + in acute (15.5 ± 4.5%) and chronic (14.9 ± 6.3%) disease periods, and cd16 + grb cells in acute (18.1 ± 1.5%) and chronic (14.5 ± 2.5%) disease periods, p < 0.05. in patients with the non-erosive forms of olp there were cd3 + cd56 + and cd16 + grb cells in acute (8.9 ± 2.5%) and (7.3 ± 1.3%) and chronic disease (9.5 ± 2.2%) and (8.2 ± 3.1%), p < 0.05. the number of cd3 + cd56 + and cd16 + grb cells in the controls were (9.8 ± 2.1%) and (6.2 ± 5.4%), p < 0.05. conclusion: acute relapse of erosive oral lichen planus, unlike nonerosive forms, is characterized by increases in the number of cd3 + cd56 + and cd16 + grb cells. chronic disease in patients with erosive oral lichen planus showed a steady increase in the number of cd3 + cd56 + killer cells and cd16grb lymphocytes. bite", were observed in 6 patients (85%). typical hyper-and hypopigmentation were observed in six patients mainly on the fingers and the cheekbones. fibrosis of the skin of the fingers often leads to flexion contractions, which we observed in 6 patients. we were watching two-sided swelling of the fingers, but it was very pronounced in 5 patients (71%). 86% of our patients have impaired motility of the esophagus. accelerated esr and c-reactive protein were found in 5 patients as follows-2 intensively accelerated and 3 moderate. in our patients with positive ana, we observed 4 patients -at low titer 1:40 at 2 and titration 1:80 in 2 patients. the spectrum of ana found by us in raynaud's syndrome patients is closer to scleroderma than to lupus. we underline the importance of ana (57%) and anti-cc antibodies (27%) for the early diagnosis of raynaud's syndrome and scleroderma, which is also seen in our patients. anti-scl-70 antibodies were observed in 3 patients coinciding with other publications describing about 40% of the patients. low levels of complement were observed in 2 patients. low hemoglobin levels were observed in 1 patient, with no iron deficiency. conclusion: 1. we observed a typical fibrinoid necrosis and polymorphonuclear infiltration, and collagen accumulation in the walls of small and medium-sized blood vessels. results: statistically significant increase of il4 level (3.23 pg/ml [2.18; 5.02]; 3.42 pg/ml [2.8; 4.18] respectively) was determined in patients with uc both in acute stage and remission compared to controls (1.87 pg/ml [1.4; 3.2] , (p = 0.002; 0.009 respectively). statistically significant increase of il17a level (15 pg/ml [12.11; 23.38] ); 14.68 pg/ml [11.29; 17.19 ] respectively) was also observed in patients both in acute stage and remission compared to controls (7.36 pg/ml [5.18; 8 .06], p = 0.00007, p = 0.00029 respectively). besides statistically significant increase of ifnγ both in acute stage (176.15 pg/ml [65.15; 359.84] ) and remission (42. 6 pg/ml [29.4; 64.45 ]) compared to controls (16.5 pg/ml [12.3; 23 .2], p = 0.00107; 0.0118 respectively) was revealed. background: the presence of antinuclear antibodies (ana) is commonly associated with a broad spectrum of connective tissue diseases. low titres might be detected rarely also in healthy individuals, especially in higher age. an indirect immunofluorescence (iif) detection of ana antibodies on hep-2 cells is the most frequently used laboratory method in this respect. the method is quite reliable regarding sensitivity, however the specificity of this test is lower. we would appreciate a biomarker for clinical discrimination of anaother autoantibodies were tested in relation to basic diagnosis. results: a cohort of patients was divided into 6 groups according to main diagnosis: immunodeficiency, connective tissue diseases, bronchial asthma and allergic rhinitis, recurrent infectious diseases, gastrointestinal diseases, endocrinopathy and others and the last group was generated from healthy subjects. the presence of anti dfs70 antibodies was highest in the group of recurrent infections, mostly in females. in these subjects homogenous pattern of ana antibodies by iif was also detected quite often, probably induced by non-specific activation of immune system. on the other hand, in a group of connective tissue diseases, we have not found any anti dfs70 positive patient. the clinical impact of anti-dfs70 antibodies is not yet finally confirmed, but their low frequency in connective tissue diseases and presence in 5%-10% of healthy subject suggests their potential role as a new biomarker to be used as a negative predictive factor in non aard. confirmation of presence or absence of anti-dfs70 antibodies seems to be helpful to exclude potential diagnostic errors in iif ana positive patients. background: multiple sclerosis is a debilitating autoimmune and degenerative condition of the central nervous system, that predominantly affects young adults. both genetic and environmental factors are associated with increased risk for this disease. we propose that the effect of environmental factors, particularly latitude of childhood, is mediated through epigenetic mechanisms. specifically, we propose that unfavourable gene methylation predisposes individuals to multiple sclerosis, that this is set in childhood and adolescence, and transmitted from haematopoietic stem cells to progeny. method: cd34 + , cd14 + and cd56 + cell subsets were isolated from peripheral blood of healthy controls. libraries enriched for cpg islands and promoter regions were generated using modified reduced representation bisulfite sequencing and subjected to next generation sequencing. site specific methylation profiling of genome wide cpg islands, including ms susceptibility genes was conducted using methpipe software. results: genomic coverage was consistent with other published methylomes using modified reduced representation bisulfite sequencing. the methylation signature of peripheral blood derived subsets showed greater differences in methylation compared to buccal cells than with each other. individuals displayed differences in cd34 + methylomes, and these were recapitulated in the progeny cd56 + and cd14 + cells for those individuals. methylation of specific genes regions (e.g. prf1), were consistent with the known biological function of these genes and their potential contribution to ms risk. the vast majority of cpg islands interrogated show recapitulation of their methylation signature from cd34 + to progeny. however, individual differences and cell subset differences identified, likely reflect the known biological function of these genes in progeny cells. our preliminary results are consistent with the hypothesis that the epigenetic signature (that predisposes to ms risk) is set in childhood and adolescence. the physiological basis underlying the setting of this epigenetic signature is still to be elucidated, but may involve uv light and/or vitamin d, and may provide novel therapeutic targets, especially at a personalised level, for treatment of ms. background: auto-inflammatory diseases are rare disorders characterized by recurrent episodes of fever/inflammation affecting serosal surfaces, joints, eyes and skin without autoantibody production or an underlying infection. innate immunity is implicated in their pathogenesis and the underlying genetic defect has been identified in a fraction of the syndromes. during last years, the increased knowledge about auto-inflammatory diseases and the difficulty in their characterization aroused great interest to better understand these pathologies. the acidic soluble fraction of salivary proteome of patients and controls (hc) were analyzed by rp-hplc-esi-ms. 60 known salivary proteins (salivary acidic proline-rich phosphoproteins (aprps), histatins (hst), salivary cystatins s, sn and sa, statherin, p-b peptide, α-defensins 1-4, cystatins b, c, thymosin β-4, s100a7, s100a8, s100a9, and s100a12 proteins) and several derivatives (acetylated, glutathionylated, phosphorylated, and oxidized forms) were searched in the chromatographic profiles by xic (extracted ion current) procedure. 21 adult patients (mean age ± sd: 34.4 ± 10.1; 15 f, 6m) were enrolled and compared with 27 sex/age matched healthy controls (mean age ± sd: 33.4 ± 9.6; 18 f, 9m). patients are classified on the base of clinical manifestations as follows: 6 patients with fmf (mean age ± sd: 33 ± 7.9; 5 f, 1m), and 15 with unclassified fever syndrome (uc) (mean age ± sd: 34.9 ± 11.1; 10 f, 5m). results: fmf patients showed low levels of α-defensins 2, 3 and 4, this last was absent, with respect hc, and high levels of the glutathionylated proteoforms of cystatin b, and s100a9, and of antileukoproteinase (slpi). similar results were obtained on saliva of unclassified patients, which showed also levels of cystatin c higher than controls. interestingly, proteins and peptides typically secreted by salivary glands (cystatin c, histatins, statherin, aprps) were found more abundant in uc patients than in controls, and in some cases also than fmf patients (see table) . an evaluation of relative abundance of phosphorylation of phosphorylated proteins/peptides highlighted a significant hypophosphorylation of hst-1, prp-1 and prp-3 in uc patients with respect to controls, probably due to a less active fam20c kinase responsible for their phosphorylation conclusion: we show by a top-down proteomics approach a wide salivary modification, highlighting dysregulation in neutrophil-derived proteins and significant differences between fmm and uc patients. the control group consisted of 10 healthy donors aged 40-50 years. immunological methods of investigation included determination of membrane antigens b cells: cd3 − cd19 + cd45 + , cd19 + cd5 + cd45 + , cd19 + cd23 + cd45, cd19 + cd25 + cd45 + , cd19 + cd40 + cd45 + , cd19 + cd86 + cd45 + , cd3 + cd4 + cd40l + cd45 + , cd19 + cd45ra + cd27 + cd45 + , by flow cytometry. results: in the study subpopulation composition of lymphocytes in seropositive variant form of ra visceral a statistically significant increase in relative amount as b1-cells with immunophenotype cd19 + cd5 + cd45 + (0.6 ± 0.01% 0.12 ± 0.03%) and b2 lymphocytes with the phenotype cd19 + cd5 − cd3 − cd45 + (16.4 ± 1.2% and 7.9 ± 0.5%). in the analysis of the processes of maturation and differentiation of b2 cells detected statistically reliable increase of the relative number of mature cd19 + cd3 − cd45 + naïve b2 cells cd19 + cd45ra + cd27 number of mature cd19 + cd3 − cd45 + naïve b2 cells cd19 + cd45ra + cd27 − cd45 + (11.8 ± 0.9% and 5.7 ± 0.5%) compared to the control group. in the study of surface markers b2 lymphocytes revealed an increase of expression of costimulatory cd19 + cd40 + (19 ± 1.3% and 7.0 ± 0.42%) molecules and increasing the relative amount of cd40l 0.9 ± 0.2% (0.3 ± 0.05%) ligand on cd3 + cd4 + cd45 + subpopulation of t-lymphocytes. analysis of surface antigenic receptor b2 cells in the visceral form of ra showed an increased expression of early markers of cd19 + cd23 + cd45 + (2.9 ± 0.08% and 0.91 ± 0.1%), cd19 + cd25 + cd45 + (1.6 ± 0.4% 0.05 ± 0.01%) activation in comparison with the control group. case: a 44 year-old male patient who works as a dental technician with a history of lung silicosis and recurrent sinusitis applied to an orthopedics clinic for left hip pain and difficulty in walking. he has a history of keeping a dog during childhood. hip mri revealed a 5 × 3 cm sized mass on left iliac wing extended to gluteus muscle and subcutaneous tissue. incisional biopsy was reported as chronic granulomatous osteomyelitis. the lesion was considered as tuberculous abscess. despite anti-tuberculous (fourdrug regimen) treatment for one year, the lesion showed no regression. excisional biopsy was carried out by the same orthopedics clinic. chronic inflammatory reaction and fibrosis was considered to be due to cyst hydatid in the detailed evaluation. antiechinococcus igg and igm was performed with elisa and found positive. no other lesion was detected in lungs and liver. albendazole 400 mg twice a day was initiated and substantial regression observed after three months. atypical and sustained infections made us think of primary immunodeficiency disorders. immunoglobulin subgroups were as follows: iga: <1 mg/dl (70case description: 31 year-old male was firstly admitted to gastroenterologist due to intermittent diarrhea, abdominal pain and reactive lymphadenopathy. celiac disease was suspected as genetic test showed hla dq8 (hla-dqa1*03 and hla-dqb1*0302), histological evaluation of duodenum biopsy provided picture of lymphoid hyperplasia and marsh iiia variant. however, laboratory testing for celiac disease showed very low amount of antibodies against transglutaminase. gluten free diet for almost one year was ineffective as patient had a continuous problem of gaining weight due to chronic diarrhea. additional questioning revealed recurrent respiratory tract infections with a need of antibiotics more than two times/year during last decade. lymphocyte phenotyping by flow cytometry showed that cd3, cd4, cd8, cd19 are in normal ranges, but amounts of all immunoglobulins are low: igm <0.22 g/l, igg 2.53 g/l and iga 0.09 g/l. based on clinical symptoms and immunological evaluation diagnosis of cvid was confirmed, and replacement therapy with subcutaneous immunoglobulin (400 mg/kg/month) was initiated. after six months of treatment patient affirmed reduction of gastrointestinal symptoms; he gained 12 kg of weight, has no more infections and stable sufficient level of igg (7.9 g/l). conclusions: this clinical case shows the importance of immune testing for primary immunodeficiency in all subjects (despite age) with unusual symptoms of autoimmune and/or infectious disorders. cvid may have manifestation of various symptoms, which can lead to misdiagnosis, as well as inadequate treatment. results: in our sample, all patients who progressed to hypogammaglobulinemia were receiving lymphomas. there is no immunoglobulin dosage record prior to treatment. of the 9 cases, mean age was 52 years (4 men and 5 women), 2 lost follow-up, and 1 of them also presented neutropenia. seventeen patients who continued in followup required ivig replacement, due to infectious exacerbations, mainly pneumonia and sinusitis. the mean serum igg dosage at the time of onset of ivig replacement was 491 g/dl. the mean time between the first dose of rtm and the need for ivig replacement ranged from 2 to 8 years, with an average of 5 years. the iga dosage was used as a parameter for the recovery of hypogammaglobulinemia, and it was observed that only 1 of the 7 patients presented recovery of the condition up to the moment. conclusion: given the data, we considered the immunoglobulin dosage to be important before initiating rtm treatment and periodically, in order to indicate the replacement of ivig or igsc in a timely manner avoiding complications such as potentially serious infections. background: steinert's disease, also known as type 1 myotonic dystrophy (md1), is the most common dystrophy of the adult. it is inherited with an autosomal dominant mechanism. it causes myotonia, progressive muscles atrophy, muscular weakness, and problems at the heart's conduction tissue and at the respiratory muscles. in patients with myotonic dystrophy, hypogammaglobulinemia is frequently described. the associations and the pathogenesis between those affections are not totally clear, but it is recognized an increased catabolism of the immunoglobulin in these patients. in most of the cases, hypogammaglobulinemia affects only the igg class and does not become clinically manifest. however, replacement treatment is not always successful in these patients. we report the case of a patient with myotonic dystrophy and hypogammaglobulinemia. case report: a 44-years-old man with md 1 came to our attention for a history of recurrent infections of the upper respiratory tract and persistent infection by helicobacter pylori. at the laboratory tests, we documented low serum igg levels (449 mg/dl), normal igm and iga levels and protective antibodies against tetanus consisting with the diagnosis of hypogammaglobulinemia. due to the recurrent infections, he started replacement therapy with ivig (0.4 g/kg/ months), switched one year ago to facilitated subcutaneous ig (fscig) with achievement of protective serum igg levels (>600 mg/dl) and significantly reduction of infectious episodes. conclusion: hypogammaglobulinemia is frequently reported in patients with md1. in literature most of the cases described does not become clinically manifest, but in our case, the patient was symptomatic with recurrent infections. the replacement therapy with fscig showed both clinical effectiveness and safety. 1532 | real-world experience of a novel, highly purified 10% liquid iv human immunoglobulin for the treatment of antibody deficiencies guidelines for immunoglobulin use (july 2011). a highly purified 10% liquid iv human immunoglobulin (ig), with low levels of iga, anti-a and anti-b haemagglutinins, factors xia, xiia, kallikrein and aggregates (i10e) was recently approved for use in the uk. here i report our centre's experience in using this novel 10% i10e in three patients with antibody deficiencies. case presentations: a patient who presented in clinic with a first diagnosis of pid, was initiated on 10% i10e at 30 g infused every four weeks. after starting i10e, they experienced a decrease in the rate and frequency of infections, in line with expectations for igrt. a young patient on home therapy with a 20% subcutaneous ig for pid presented in clinic with low trough igg levels. non-compliance was identified as the cause of these low trough levels and therapy was switched to 10% i10e at 40 g infused every four weeks in a clinical setting. both the rate and severity of infections reduced and trough igg levels normalised. an older patient on igrt for sad was reviewed in clinic due to discontinuation of their current igrt product. they were switched to 10% i10e at 20 g infused every four weeks. the efficacy and tolerability of i10e was comparable to their previous therapy. a detailed analysis of patient, clinical and safety parameters associated with the initiation of 10% i10e will be presented, including infection rates, white cell counts, c-reactive protein levels, tolerability and infusion-related adverse events. conclusion: these cases highlight the real-world use of 10% i10e in two patients with pid and one patient with sad. they show that i10e was well-tolerated and efficacious in one treatment-naïve, and two previously-treated patients. method: prospective study of families with one or more members with c1-inh-hae followed in 7 hospitals in the northern area of spain. a cohort of 105 patients from 28 families with c1-inh-hae was evaluated for familiar diagnosis of c1-inh-hae one or several patients from the same family were chosen and were given a questionnaire to identify the total family members from the family branch affected by c1-inh-hae that had been already studied (members with diagnosis of c1-inh-hae and healthy members) and those that had not been previously studied. we also register the difficulties for obtaining these data. family members not previously studied and that consent to be contacted were asked for study of c1-inh-hae. for c1-inh-hae screening we use c4 blood levels results: we have studied 28 families with c1-inh-hae, 24 (87%) type 1 and 2 (7%) type ii; 13 families had all their known members already studied for c1-inh-hae (50%): 12 families have all their members studied (42.8%), 18 families have 90% or their known members studied (64.2%) and 10 families had less than 50% of their total known members studied. we have identified 85 members from 11 unrelated families that had not been previously studied for hae, 10 healthy, 4 had low c4 levels and had presented symptoms of hae; 1 had not presented symptoms of angioedema, had normal c4 levels, and low antigenic and functional c1-inh levels. difficulties for a complete family testing study have been: family dispersion, scarce or no family relationship, do not wish to know their possible pathology conclusion: it is crucial to insist on the study of the relatives of patients with hae. we propose to include a questionnaire to identify all patient's relatives at medical reviews of hae patients. case: a-4 year old boy was admitted to our clinic with the history of recurrent respiratory tract infections. his all immunoglobulins were low (igg <154 mg/dl, iga <25.4 mg/dl, igm <17.8 mg/dl) associated with the absence of b cells. his aunt cousin also had xlaa missense point mutation, c562c>t in exon 17 of the btk gene was identified in both affected cousins. the patient was commenced on regular ivig treatment every 3 weeks. at the age of 8, he suffered from intermittent fever attacks, abdominal pain and weight loss. tests for giardia lamblia, clostridium difficile and cryptosporidium, noro virus or parasites were negative. mr-enterography revealed intra-abdominal fluid and thickened walls of his jejunum and cecum. histopathological examination of the biopsy material obtained from terminal ileum, colon and cecum showed crohn disease. initially, he was treated with prednisolone and infliximab. because of the lack of response, infliximab treatment was switched to adalimumab. terminal ileum was resected to relieve obstruction complication. although he had been treated with adalimumab for 1 year, a significant improvement was not observed. vedolizumab (entyvio ™ ), is a humanized monoclonal antibody α4β7 integrin-receptor antagonist, was commenced. induction dosing was 300 mg infusions at 0, 2, and 6 weeks followed by a maintenance phase at 8week intervals. at the 6 month of the treatment, fever and abdominal pain attacks reduced, while his weight and oral intake increased. no side effects were observed. discussion: vedolizumab is effective for inducing and maintaining remission in adults with inflammatory bowel disease (ibd); however, there is limited pediatric data. this is the first immunocompromised child treated with vedolizumab. the symptoms of the patient receded and no side effect observed during 6 months of the treatment. results: louis-bar syndrome is a multisystem progressive disease with polymorphic manifestations which varies by age. the locomotor disability of these children is determined by neurological disorders, the exitus being caused by respiratory infectious and malignancies. the children involved in the study, had frequent episodes of respiratory infectious (bronchitis, pneumonia, atelectasis, empyema, lung abscess), ent infections (otitis, mastoiditis, sinusitis), chronic pulmonary disease (pulmonary fibrosis, bronchiectasis). index of death in this group is high (66.7%). in one of the boy, the pulmonary ct showed lymphadenopathy, later was confirmed non-hodgkin lymphoma, with subsequent death. another child died from pulmonary and systemic infectious complications. results: in this paper we present the clinical and morphological analysis of children with nezelof syndrome diagnosed post-mortem. clinically were predominantly the generalized intrauterine infections or their development in the postnatal period. at macroscopic examination all patients had thymic hypoplasia. later on the microscopic study of the thymus specimens determined dysplastic changes, defined by the presence of concentrically arranged epithelial cells. in all patients, was determined the total lack of hassall corpuscles and its predecessors. besides the above-mentioned modifications in all specimens, there was no cortico-medullary segregation. thymic parenchyma outside pseudorrhagia was made up of a reticular stroma with total lymphocyte depletion. conclusion: nezelof syndrome is a severe primary immunodeficiency associated with thymic dysplasia and alymphocytosis, which is manifested early with generalized infections and major risk of death in neonatal and infant. background: the study was aimed to evaluate the cytokine profile in nasal secretion and blood serum in patients with seasonal (sar) and perennial allergic rhinitis (par) with a potential for additional sensitization with microbial allergens. method: the inclusion criteria for ar were as follows: a diagnosis of ar for more than 2 years, the absence of nonallergic disorders of the nasopharynx, age of patients from 4 years to 60 years.control group: healthy volunteers at the age of 3-43 years without any allergic disorders at examination.in order to evaluate the innate and adaptive immunity, the cytokine profile of blood serum (il-4, il-10, and tgf-β) and nasal secretion (tslp, il-1β, tnf-α, and gm-csf) was determined. to determine tslp, tgf-β, il-10, and gm-csf concentrations, enzyme-linked immunosorbent assay kits were used (ebioscience, bender medsystems, r&d systems, mn, usa). we have noticed a significant correlation (r = 0 46, p = 0 014) between the tslp concentration in nasal secretion and as-ige level to staphilococcus aureus enterotoxin (allergen component m80) in patients with par. there was a significant correlation conclusion: staphylococcal superantigens might be one of the stimuli of local tslp hyperproduction by the epithelium. there was a significant correlation between gm-csf concentrations in nasal secretion and the intensity of sensitization to a staphylococcal enterotoxin (seb) in the patients with ar. seb is one of the polyclonal t cells activators, which may account for increased concentrations of cytokines such as gm-csf locally within the system of mucosal immunity. the patients with ar and additional high sensitization to ses demonstrated a higher tnf-α production profile due to macrophage and tcell activation by these toxins. 1547 | evaluation of circulating osteopontin level as potential biomarker of allergic asthma in patients with caucasian and south-east asian ethnicity background: osteopontin (opn) is a pleomorphic cytokine known to influence a wide range of immune cells; allergic asthma was previously associated with high circulating opn levels. in the present study, we aimed to verify if opn may qualify as biomarker of activated immune response in allergic patients belonging to two different ethnic groups: caucasians and south-east asians. method: serum opn levels were measured by elisa test (human osteopontin duoset, r&d systems) in a series of 121 italian adult patients affected by extrinsic asthma, allergic rhinitis, hymenoptera venom allergy, food allergy, allergic contact dermatitis and ige mediated hypersensitivity to beta lactams. 116 healthy subjects served as controls. 576 ethnic chinese subjects were recruited at the national university of singapore (nus) as cross-sectional cohort of an ongoing epidemiological study on the national prevalence of allergic diseases, and opn levels were detected by luminex (milliplex map, merck) and elisa assays (r&d systems). results: in the italian cohort, opn levels were significantly higher in cases compared to controls (p = 0.0010 by the mann-whitney test). statistically higher opn levels were found in asthma (p = 0.0269) and food allergy (p = 0.046) groups in comparison to controls. no significant differences were found (p = 0.597) between singaporeans with lifetime asthma and healthy controls, only the highest opn levels were heterogeneously found to correlate with asthma. however, a strong gender effect was shown, in both cases (p < 0.0001) and controls (p < 0.0001), with males presenting higher opn levels in comparison to females. consequently, we checked the mrna expression levels of opn gene (spp1) with illumina chips in whole blood of males and females, and no difference was found (p < 0.05). several experiments with western blots and different gel types were performed to verify if possible post-transcriptional/posttranslational modifications of opn could explain these findings. conclusion: opn seems to be a promising biomarker for current, active allergic asthma in caucasians even though technical difficulties, due to opn intrinsically disordered structure, the complex enzymatic metabolism, and the low circulating levels, significantly affect the experiments. further studies are needed to confirm these data. 1548 | mortality, intubation, and healthcare cost in patients with allergic bronchopulmonary aspergillosis in a hospital setting: a nationwide study fan x; luo y; yue b background: abpa is a complex hypersensitivity reaction to aspergillus fumigatus that colonize in airways, it is almost exclusively seen in patients with asthma or cystic fibrosis(cf). this study is to estimate hospitalization outcomes and healthcare cost of hospitalized patients with abpa. method: we conducted the study using data from national inpatient sample(nis) from 2010 to 2014. diagnosis were identified using icd-9-cm codes. hospitalization with a primary diagnosis of abpa and hospitalization with a primary diagnosis of acute respiratory failure/acute and chronic respiratory failure/respiratory distress/ asthma/cf and a secondary diagnosis of abpa were included. the study population was divided into groups including abpa with asthma, and abpa with cf. mortality and intubation rate were the primary outcomes; length of stay and total hospitalization cost(adjusted to cost in 2014 based on medical care cpi) were secondary outcomes. student t-test and chi-square were used for univariable analysis, linear and logistic regression were used for multivariable analysis. results: a total of 6308 hospitalizations with abpa were included, with 2896 hospitalizations with abpa and asthma, and 2793 hospitalizations with abpa and cf. the overall mortality rate was 0.82% (95% ci: 0.44%-1.50%), the mortality for abpa with asthma was 0.69% (95% ci: 0.26%-1.83%) and for abpa with cf was 0.56% (95% ci: 0.19%-1.63%). the overall intubation rate was 4.83% (95% ci: 3.77%-6.18%); the intubation rate for abpa with asthma was 5.51% (95% ci: 3.91%-7.72%) and 84.0% were early intubation (<3 days); the intubation rate for abpa with cf was 1.95% (95% ci: 1.13%-3.35%) and 36.6% were early intubation. the overall mean length of stay(los) was 8.7 (95% ci: 8.0-9.3) days, while the los for abpa with asthma was 5.6(95% ci: 5.2-6.1) days and the los for abpa with cf was 12.1 (95% ci: 11.1-13.0) days. the overall total cost was 147 million usd, the total cost for abpa with asthma was 33.4 million usd with a mean of 12 069, while the total cost for abpa with cf was 101 million usd with a mean of 38 005. conclusion: mortality among hospitalized patients with abpa is low<1%. intubation rate is relatively low, intubation, especially early intubation (<3 days), is more common in patients with asthma. although abpa is not a common disease in inpatient population, it does have a high health care cost and despite lower intubation rate, patients with abpa and cf generally have a longer hospital stay with a higher hospitalization cost. 1549 | frequent exacerbations of bronchitis with wheezing in adults: is it possible to predict and prevent asthma? case report: frequent episodes of bronchitis, accompanied by wheezing and dry with a prolonged duration in adults, the clinical course may be similar to bronchial asthma. the aim is to assess the risk of asthma in adult patients with 2 or more episodes of acute bronchitis per year, had a prolonged duration and accompanied by a dry wheezing. for 5 years in two regional clinical pulmonology centers were observed in 46 patients (19 men and 27 women) with average age 34 ± 6.8 years. each had at least 2 episodes of acute bronchitis per year, which was accompanied by prolonged cough and presence of wheezes. average number of acute episodes per year was 3.2 ± 0.8. in the course of the observation the patients were divided into 2 equal groups. the first group consisted of 23 persons treated in acute episodes of the disease symptomatic therapy, including inhaled β2-agonists short-acting short course. in the second group to the corresponding treatment added montelukast 10 mg per day lasting for 1 month. in all cases of exacerbation had a viral nature. held in the period of remission of allergic sensitization, the survey revealed. starting from the first year of follow-up all patients were vaccinated against influenza annually. by the end of the fifth year of observation in the first group in 5 cases was diagnosed of bronchial asthma-4 cases easy persistent asthma and 1 case moderate. the diagnosis was exhibited in accordance with the gina criteria. in the second group, the diagnosis of bronchial asthma were exposed to 1 patient (hazard ratio of 0.18). prospective observation suggests that the use of anti-inflammatory potential antileukotriene medicines in complex therapy of recurrent acute episodes of bronchitis accompanied by a dry wheezing in adults may be a factor preventing the development of asthma. for more conclusive results require more extensive research. background: chemokine receptors play an important role in regulating the migration of t lymphocytes, monocytes and neutrophils from the peripheral blood into inflamed tissue, such as lung. however, little is known about their expression on natural killer (nk) and natural killer t (nkt) cells in patients with chronic obstructive pulmonary disease (copd). therefore the aim of the study was to determine the chemokine receptor profile of peripheral blood nk and nkt cells of copd patients. method: for analysis of lymphocytes subtypes the flow cytometry method was used. the study population consisted of 57 smokers with copd, 12 healthy smokers and 12 healthy non-smokers. results: we observed an increase in blood nk cells expressing cxcr3 receptors in smokers with copd compared to healthy smokers (p = 0.003) and healthy non-smokers (p < 0.001). the percentage of nkt cells containing cxcr3 receptors was also significantly higher in blood of smokers with copd compared to healthy smokers (p = 0.021) and healthy non-smokers (p < 0.001). copd smokers had significantly higher proportion of ccr5 + nk cells than smokers without copd (p = 0.002) and healthy non-smokers (p < 0.001). increased proportion of blood nkt cells expressing ccr5 on their surface was observed in smoking copd patients compared to healthy smokers (p = 0.002) and healthy non-smokers (p < 0.001). there were no significant changes in the percentage of cxcr3 + and ccr5 + nk and nkt cells between healthy smokers and non-smokers. in addition, no differences were seen in the proportion of nk and nkt cells expressing cxcr4, cxcr6, ccr6 and ccr7 among all studied groups. method: 58 patients with copd in stable condition (gold stage 2) aged 40-70 years old, smoking history of ≥10 pack-years, were studied. bmi of patients were divided into 2 groups: obese (n = 31) (bmi-30.0-39.9 kg/m 2 ) and non-obese (n = 17) (bmi-18.5-24.9 kg/ m 2 ). ten subjects with normal lung function and bmi were the control group. the level of il-26 assessed in induced sputum (pg/ml) was measured using an elisa (raybiotech ® ). serum levels of crp were measured using the "vector-best" (russia federation). spirometry was performed according to american thoracic society and the european respiratory society (ats/ers) guidelines. results: obese copd patients had significantly increased concentrations of il-26 compared with healthy subjects and non-obese copd patients by 2.6 fold (139.0 ± 85.4 pg/ml vs 53.15 ± 18.5 pg/ ml) (p < 0.008) and 1.15 fold, (139.0 ± 85.4 pg/ml vs 120.6 ± 60.15 pg/ml)(p < 0.04), respectively. non-obese copd patients had higher levels of il-26 by 2.3 fold compared with healthy subjects (120.6 ± 60.15 pg/ml vs 53.15 ± 18.5 pg/ml) (p < 0.008). results: among these three groups, the level of cer in lung cancer patients (0.35 ± 0.10 g/l) was significantly higher than that in ild patients (0.31 ± 0.25 g/l) and healthy individuals (0.25 ± 0.04 g/l) (p < 0.05). meanwhile, the levels of c3 and c4 in healthy individuals, which are 1.70 ± 0.29 g/l and 0.27 ± 0.34 g/l respectively, were both significantly higher than that in lung cancer patients (c3: 1.04 ± 0.26 g/l, c4: 0.24 ± 0.09 g/l) and ild patients (c3: 0.97 ± 0.25 g/l, c4: 0.21 ± 0.09 g/l), (c3: p < 0.05, c4: p < 0.05). results from optimal scaling demonstrated that lung cancer was closely associated with immune factors including crp, cer, c3 and c4 (cronbach's alpha = 84.7%). conclusion: for ild patients, when the level of crp and cer is increased and the level of c3 and c4 is decreased simultaneously, the risk of the development of lung cancer should be considered for these patients. results: among pbmc subpopulations, endurance exercises impacted the number of nkt and activated t cells with nkt cell numbers greater in male bobsledders vs bullet shooting and biathlon (42.4% and 44.3%, respectively). the number of activated t cells (cd25 + ) was greater in bullet shooting and bobsleigh athletes vs the biathlon group (34.9% and 22.7%, respectively). in female athletes the number of cd25 + cells in the shooting and bobsled groups was greater by 46.1% and 25.5%, respectively vs the biathlon group (p < 0.05). increased il-10 occurred in bobsleds in comparison to bullet shooting and biathlon: 32% and 80% in male and 70.5% and 83% in female, respectively (p < 0.05). the concentration of il-18 in male bobsledders and biathlon was 30% and 34% greater, respectively, compared with bullet shooting (p < 0.05). serum concentrations of ifnγ in male as well as female athletes showed an increase of 41.6% and 39.5%, respectively vs biathletes (p < 0.05). increased il-4 occurred in the male biathlon group by 39.7% and 13%, respectively, vs the bullet shooting and bobsleigh athletes (p < 0.05). il-6 was increased in the male biathlon group, compared to bullet shooting and bobsledders by 76.7% and 70.3%, respectively (p < 0.05). conclusion: prolonged endurance exercises impacts secretion of pro-and anti-inflammatory cytokines in athletes of different sport specializations. concentrations of studied cytokines did not exceed reference values perhaps due to specialized sport nutrition, which may restore immune function during endurance exercises. background: oral immunotherapy (oit) is a promising therapeutic approach to treat food allergic patients. recently, we have shown that the use of a mixture of short-chain-and long-chain fructo-oligosaccharides (scfos/lcfos) improves the efficacy of oit in cow's milk and peanut allergic mice. however, concerns with regard to safety and long-term efficacy of oit remain and there is a need to identify novel biomarkers (panels) that predict, monitor and/or evaluate the effects of oit. here we present a method for the selection of candidate biomarkers by using the computational approaches bayesian networks (bn) and topological data analysis (tda). method: data were used from scfos/lcfos diet-supported oit studies performed in 2 independent cow's milk allergy (cma) and 2 independent peanut allergy (pna) experiments in mice. first, a subset of the data was used for learning the data structure and their interactions in terms of a bn. this bn was used to compare the key parameters in both experimental food allergy models. finally, the relations within the dataset in combination with the bn were explored to identify and rank candidate biomarkers for the effect of oit by applying tda. the bn was able to predict the efficacy of oit in the cma and in the pna model with 82% and 80% accuracy respectively, thereby identifying a set of 5 parameters (allergen-specific ige and igg1, body temperature, mmcp-1, earswelling) being key in the mechanisms involved in both scfos/lcfos-aided oit food allergy models. the tda zoomed in on the full set of 67 previously analyzed parameters and identified clusters of biomarkers closely linked to biologically relevant clinical symptoms but also unrelated and redundant parameters within the network. taken together, this enables the prioritization of candidate biomarkers. moreover, the tda indicated differences between pna and cma models in how the data are related to each other. here we provide promising bioinformatics methods to compare mechanistic features between two different food allergies and to determine the biological relevance of biomarker (panels) of oit for food allergy. we have shown that the key drivers that influence pna and cma are similar, but that these phenotypically similar diseases show mechanistic differences in their subnetworks. these new insights provide excellent starting points to generate new hypotheses to explain why cma has a different disease pattern than pna and to select biomarkers that are useful in future clinical studies. 1562 | functional and immunoreactive levels of igg4 correlate with clinical responses during the maintenance phase of house dust mite immunotherapy basophils were identified as ssc low cd193 high , and cd63 was used as an activation marker. reactivity was confirmed by anti-ige as a positive control. results: in 4 patients, basophil reactivity and sensitivity was comparable for grass pollen extract and recombinant phl p5, while phl p1 only caused a lower basophil activation. in one patient, recombinant phl p5 did not cause any basophil activation, while phl p1 elicited an even higher sensitivity and reactivity than grass pollen extract. conclusion: in 4 patients, basophil reactivity was comparable for grass pollen extract and recombinant phl p5, while phl p1 only caused a minor basophil activation. in one patient, recombinant phl p5 did not cause any basophil activation, while phl p1 elicited an even higher sensitivity and reactivity than grass pollen extract. reactivity of extract and the main sensitizing components correlated, while sensitivity did not. 1564 | in vitro assessment of hypersensitivity to allergen before and after allergen immunotherapy with whole blood basophil histamine release assay wbbhr assay in these patients was performed 7-10 days before and 7-10 days after ait. heparinized whole blood samples (8 ml) of each patient after substitution of plasma with pipes buffer were incubated one hour at 37°c with different concentrations of birch pollen extract (t3) in u-shape 96-well micro-titer plates. after incubation plates were centrifuged and supernatants from each well of the plate were directly analyzed for histamine content by reversedphase high performance liquid chromatography with electro-spray ionization mass-spectrometry (rp-hplc-esi-ms). results were expressed as ng/ml released histamine. sensitivity (limit of quantification) was 5-7 ng/ml. to compare results of histamine release in patients before and after ait data were calculated as area under the curve (auc) values. results: in contrast to pre-immunotherapy activity of blood basophils there were significant decreases in hr induced by t3 extract after ait. according to auc values all patients demonstrated decrease in hr after ait in compare to hr before ait in a range of 25%-65% demonstrating a decrease of hypersensitivity to birch allergens. analysis of basophil hr in patients received s.c. or s.l. the asthma and rhinoconjunctivitis symptom scores during and after pollination season decreased significantly and showed correlation with histamine release by t3. 1565 | immunotherapy with the recombinant b cell epitope-based grass pollen allergy vaccine bm32 induces a biphasic allergen-specific igg1 and igg4 response background: immunotherapy with the recombinant b cell epitopebased grass pollen allergy vaccine has been shown to reduce symptoms of grass pollen allergy in a multicenter, double-blind, placebocontrolled study. aim of this study was to investigate the levels and kinetics allergen-specific igg responses in a double-blind, placebocontrolled phase iib combined field and exposure chamber trial studying the effects of three, four and five pre-seasonal injections of bm32 as compared to placebo. method: a quantitative elisa assay based on purified human monoclonal allergen-specific igg 1 as well as igg 4 antibodies as standards was developed to measure allergen-specific igg 1 and igg 4 concentrations induced by ait with bm32. results: we found rises in levels of both tested allergen-specific igg subclasses in the actively but not placebo-treated patients. phl p 1-and phl p 5-specific igg 1 levels up to 83 μg/ml and 784 μg/ml, respectively and phl p 1-and phl p 5-specific igg 4 levels of up to 468 μg/ml and 1423 μg/ml, respectively were measured in bm32treated patients. five pre-seasonal injections induced the highest allergen-specific igg levels. interestingly, allergen-specific igg 1 and igg 4 antibodies showed a biphasic response with early rises of allergen-specific igg1 which declined quickly after the pollen season and a delayed but very sustained allergen-specific igg4 response. conclusion: treatment with bm32 induces a biphasic allergen-specific igg response consisting of an early igg1 and a sustained allergen-specific igg4 response which may be responsible for early and sustained protection against allergic symptoms. 1566 | t reg cd4 + cd25 high in peripheral blood in patient with grass pollen allergy during sublingual specific immunotherapy slit the aim of this study was to evaluate t reg cd4 + cd25 high in peripheral blood in patients with grass pollen allergy during sublingual immunotherapy slit. method: we examined 27 adult patients, aged 20-41, 13 female and 14 male. patients were qualified to slit after confirmation of allergy-by skin prick tests, specific ige and nasal provocation tests. we determined t reg cd4 + cd25 high from blood sampling of those patients (by flow cytometry method), before the slit, after reaching the maintenance dose, before the grass pollen season, during the grass pollen season, and after one year of slit. results: during slit the percentage of t reg cd4 + cd25 high increase after reaching the maintenance dose, then it decreased before and during the grass pollen season, and again increase after one year os slit. we observed, that in the group with significant improvement of symptoms, t regcd4 + cd25 high decreased during grass pollen season, comparing to group without clinical improvement. conclusion: slit as a method of immunotherapy influence on levels of t reg cd4 + cd25 high cells. the observed decreased levels of these cells during the grass pollen season might be consider as a prognosing marker of clinical improvement. 1567 | t reg cd4 + cd25 high from peripheral blood during subcutaneous specific immunotherapy (scit) for grass pollen hofman a; hofman j; hofman t centrum alergologii, poznan, poland background: specific immunotherapy is the only causal method for grass pollen allergic rhinitis. however, we don't observe in every patients satisfying clinical effects. because the treatment of allergic rhinitis takes 3-5 years, and is quite expensive, everyone is constantly looking for a perfect parameter, which may prognose the effectiveness of specific immunotherapy (sit). the aim of this study was to evaluate t reg cd4 + cd25 high lymphocytes from peripheral blood in patients during subcutaneous specific immunotherapy (scit) for grass pollen . method: we examined 99 adult patients (36 female, 63 male), age 18-60, who undergo scit for grass pollen allergy. we have done skin prick tests and specific ige in those patients. additionally, we confirmed the allergy by nasal provocation tests. we have determined lymphocytes t reg cd4 + cd25 high from blood sampling from those patients, with flow cytometry method: before immunotherapy, after reaching the maintenance dose of scit, before and during the grass pollen season, and after one year of scit. our control group was represented by 12 adult healthy volunteers. after one year of scit we divided patients into two groups-with and without clinical improvement. results: in allergic patients we have observed decreased levels of t reg cd4 + cd25 high compared to control group before the start for scit. during immunotherapy, the percentage of t reg cd4 + cd25 high increased after reaching the maintenance dose, however it did not reached the level of healthy volunteers. again, levels of t reg cd4 + cd25 high decreased before and during the grass pollen season, and increased after one year of scit. we compared also patient with significant improvement of clinical symptoms, and without. and we observed that the level of t regs cd4 + cd25 high decreased during pollen season in improved group, and increased in the group without clinical improvement. conclusion: in patients with grass pollen allergy, during the grass pollen season, decrease of t reg cd4 + cd25 high cells might be conresults: in the group of patients treated with sit gene expression analysis revealed significant change in ifng expression (p = 0.03) (comparison between sample a and b). comparison between samples a and c showed significantly different expression in genes: afap1l1 (p = 0.006), commd8 (p = 0.001), pik3cd (p = 0.027), and twist2 (p = 0.0003). duncan's multiple range test confirmed difference between sample a and c for commd8 (p = 0.004) and also revealed new significant difference in tbx21 in samples a and b (p = 0.035; in wilcoxon's test p = 0.08). k nearest neighbors algorithm was built based on ifng, pik3cd, commd8 expression. the results of the study indicate, that there is a significant change in the expression of a few genes during the build-up phase of sit. it may be suspected, that this change contribute to the mechanisms involved in the building tolerance to allergen. k nearest neighbors algorithm may be useful for sit efficacy prediction. 1569 | regulation of cytokine thymic stromal lymphopoietin (tslp) in modulating tgf-ß1induced interstitial inflammation and cellular fibrosis background: thymic stromal lymphopoietin (tslp) has previously been linked to allergic inflammatory diseases, tissue fibrosis and organ dysfunction. it remains unclear, however, whether tslp plays any role in the occurrence of renal fibrosis, so this study investigated that underlying mechanism. method: an in vitro fibrosis model was established by treating normal rat kidney fibroblast (nrk-49f) cells with transforming growth factor-β1 (tgf-β1), after which the levels of various fibrogenic markers (e.g., fibronectin) and downstream fibrogenic signal proteins (e.g., smad 7) were investigated. also, tslp shrna was used to silence the effects of tslp, while an elisa was conducted to evaluate the fibronectin secretions. results: the level of fibronectin in the nrk-49f cells was doseand time-dependently increased by the administration of exogenous tslp (p < 0.05). tslp also significantly increased the level of fibrosis signaling, in addition to inducing a marked decrease in the down-regulation of smad7. interestingly, the application of tslp shrna caused a dramatic reversal of the tgf-β1-induced cellular fibrosis while simultaneously leading to the suppression of fibronectin and fibrogenic signal proteins. conclusion: taken together, these observations provide insights into how extracellular matrices develop and could lead to potential therapeutic interventions for the suppression of renal inflammation and fibrosis. abstracts | 779 1571 | effects of two years treatment with the recombinant b cell epitope-based grass pollen allergy vaccine bm32 on allergen-specific b and t cell responses background: bm32 contains recombinant fusion proteins of nonallergenic peptides from ige-binding sites of the four major timothy grass pollen allergens phl p 1, 2, 5 and 6 and pres protein from the hepatitis b virus as a carrier. in a multicentre, double-blind, placebocontrolled trial, 181 grass pollen allergic subjects were treated for two years either with bm32 or placebo. here we investigated in detail the effect of immunization with bm32 on allergen-specific t and b cell responses. during the study from 35 subjects treated in the vienna centre (bm32: n = 25, placebo: n = 10) were investigated regarding proliferation using 3 h thymidine incorporation and cytokine production in response to various recombinant allergens at 9 different time points. grass pollen allergen-specific ige, igg 1 and igg 4 levels were determined by immunocap and elisa. results: a significant increase of allergen-specific igg 1 and igg 4 levels was found in the bm32-but not in the placebo group in both years (year1 > year2) after treatment. there was no difference regarding t cell proliferation in response to phl p 1 and phl p 5 after first grass pollen season between actively and placebo-treated patients whereas proliferation in particular of phl p 1-specific responses seemed to be blunted in the active group in the second year. no significant differences regarding allergen-specific th1, th2 and tolerogenic (i.e., il-10) cytokines were observed between bm32and placebo-treated patients. the findings indicate that the bm32 induces high levels of allergen-specific blocking antibodies which may reduce allergen-specific t cell proliferation but does not induce significant increases of regulatory cytokines in t cells. this study was supported by grants f4605, f4613 and dk 1248-b13 of the austrian science fund (fwf). 1 hospital universitario ramón y cajal, madrid, spain; 2 department of immunology iis-fundación jiménez díaz, uam, madrid, spain background: shiitake mushroom (sm) (lentula edodes) is an edible fungi native to east asia. it is traditionally cultivated and used in many asian countries and its consumption is increasing worldwide. direct skin exposure to sm can cause cutaneous reactions, including allergic contact dermatitis and urticaria, while its oral intake may prompt "shiitake flagellate dermatitis" (sfd), which is a distinctive itching linear erythematous eruption. sfd is usually considered a toxic reaction to lentinan, a thermolabile polysaccharide that increases interleukin-1. we report 3 cases (p1, p2, p3) of shiitake flagellate dermatitis studied in our centre. method: skin prick tests (spt) to environmental allergens-including moulds -, prick-by-prick and patch test with raw and cooked sm were carried out. total ige and specific ige to mushroom, white mushroom and environmental moulds were also determined. a raw and cooked shittake mushroom extracts were prepared. both extracts were analyzed in all the patients by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). results: skin prick tests, prick-by-prick, patch tests and specific ige were all negative except for p1, who had positive prick-by-prick to raw shiitake mushroom. sds-page ige immunoblotting assays with the patient's sera revealed ige-reactivity with proteins ranging from 16 kda to 32 kda for p1, p2 and p3. we report 3 cases of shiitake flagellate dermatitis with demonstrated ige-sensitization. physicians should take into account that some cutaneous reactions considered as toxic might be allergic reactions. vorozhko i 1 ; sokolnikov a 1 ; sentsova t 2 ; donnikov a 3 ; ilyenko l 2 ; denisova s 2 background: allergic diseases such as asthma, rhinitis and food allergy have increased in recent decades in tropical countries. the tropics has climatic, environmental and ecological peculiarities that allow us to emit several hypotheses that could explain this phenomenon. in one of them, it is postulated that the increase of the sensitization to food, is due to the presence of lower serum levels of vitamin d, product of the adoption of a western lifestyle, with lower sun exposure, which in its turn diminishes the immunomodulatory action of this vitamin at intestinal level, favoring the sensitization against food antigens. we evaluate differences between the titers of serum antibodies against food antigens between two populations with african ancestry but different environment (rural vs urban) and investigate the influence of vitamin d levels. method: an observational, cross-sectional and descriptive study was carried out on 200 afro-descendant children living in san basilio de palenque (rural) or in the city of cartagena, bolivar (urban). the sensitization was determined by a positive skin prick test to allergen extracts, including foods, and, specific ige, iga and igg4 to egg, milk and peanut extract, as well vitamin d, were measured by elisa. antibody and vitamin d titers were correlated by spearman's test and comparisons between groups were done using the wilcoxon rank sum test. a p < 0.05 was considered significant. results: atopy was more prevalent in the urban population (24% vs 7%, p < 0.001). however, none participant tested was positive for food allergens. regarding vitamin d levels, these were found to be higher in the rural population compared to the urban group (p < 0.001). among the antibodies analyzed, only ige against peanut showed differences, which were higher in rural population (p < 0.001) as well as those of iga to peanut, which were higher in the urban population (p < 0.001). we observed only a significant correlation between peanut specific ige (rho 0.2127259, p < 0.005) and iga (rho -0.342434, p < 0.001) response and vitamin d. conclusion: in our study, we found differences between the peanut specific ige and iga response in urban and rural populations. the correlation between the levels of specific ige and iga to peanut and vitamin d, suggest that this vitamin may influence peanut sensitization in this population, besides other components like diet and genetic and environmental factors. 1580 | evaluation of inhaled allergen sensitivity in patients with food allergies younger than two years of age kulhas celik i 1 ; aldemir es 2 ; buyuktiryaki b 1 ; ginis t 1 ; toyran m 1 ; dibek misirlioglu e 1 ; kocabas cn 3 ; civelek e 1 carbohydrates and pyruvate was observed in the severe group compared to the rest of allergic groups. in addition, an increment in lactate was noticed. these metabolites were closely associated with the energy metabolism. other metabolic changes included increased levels of fatty acids such as myristate, palmitate and laureate. these fatty acids might be precursors of arachidonic acid, a key molecule in inflammation. finally, alterations in some amino acids and adenosine were found method: to evaluate these critical processes, 22 five-week old germ-free c3h/hen mice were split into two groups; 12 were intraperitoneally sensitized to the peanut allergen ara h 2 and 10 remained ns. upon reaching 8 weeks of age, mice were intragastrically challenged with purified ara h 2. mice were harvested in two groups: 30-minutes and 60-minutes post-gavage. upon harvest, the left lobe of the liver was collected and sera were removed. sera and livers were evaluated for drp-ara h 2 using an in-house quantitative sandwich enzyme-linked immunosorbent assay (elisa). a sample of the proximal small intestine was monitored for drp-ara h 2 using immunohistochemistry (ihc) and for mast cell degranulation using toluidine blue stain. results: sensitization does not have a large effect on the concentration of allergen present in the sera or liver. however, s mice allowed to digest ara h 2 for 60-minutes were more likely to display tissues positive for detection of drp-ara h 2 than ns mice at the same time point. conclusion: there is drastic biological variation among mice in their capacity to absorb and transport allergens. the elisa used in these analyses proved effective in the quantitative detection of drp-ara h 2 in both liver and sera samples, while ihc provided inconsistent results for the detection of drp-ara h 2 in tissues. however, in positive ihc samples, staining was indicative of paracellular transport across the epithelial barrier. 1583 | eczema induces a high ovalbuminspecific ige/igg1 ratio and affinity maturation during the lactation period irahara m; kido h; shinahara w inst. for enz. res., tokushima university, tokyo, japan background: recent articles have revealed that ingestion of foods induces oral tolerance and cutaneous sensitization induces food allergy. relationships with levels of immunoglobulin subclasses, affinity of allergen-specific ige, and development of food allergy have also been indicated. however, relationships with levels and affinity of specific immunoglobulins and eczema during early infancy remain poorly understood. therefore, the present study aimed to elucidate these relationships. method: this study enrolled women who visited naruto hospital (tokushima prefecture, japan) in late pregnancy and their children. blood samples and information on skin condition were taken every 2 months from neonate to 6 months old. egg white and milk allergen-specific immunoglobulin subclasses and affinity of ovalbumin (ova)-specific ige levels were measured using the densely carboxylated protein (dcp) microarray with 20 μl of serum. results: this study included 84 infants whose parents agreed to join this study. of these, 42 infants (50%) were diagnosed with eczema by 6 months old. egg white (ew) and milk-specific igg4 were detected in a few subjects at 6 months old. however, these specific ige and igg1 were detected in some subjects at that time ew-and ova-specific ige levels and ige/igg1 ratios were significantly higher in participants with eczema than in those without eczema at 6 months old. moreover, subjects with high ova-specific ige/igg1 ratios showed higher affinity ova-specific ige antibodies than subjects with low ova-specific ige/igg1 ratios. these results were not reflected in milk-specific ige levels. the milk-specific ige level differed between breast feeding and formula-fed infants, with no difference in the ige/igg1 ratio. conclusion: eczema contributed to high ew-and ova-specific ige levels and ige/igg1 ratios. high ova-specific ige/igg1 ratios involved high affinity ova-specific ige antibodies. however, the milk source during early infancy had no effect on the specific ige/igg1 ratio with eczema. these results suggest different sensitization routes provoke different results in levels and affinity of immunoglobulins. 1584 | purification and characterization of naturally occurring post-translationally cleaved ara h 6, an allergen that contributes substantially to the peanut allergome background: the 2s albumin ara h 6 is one of the most important peanut allergens. a post-translationally cleaved ara h 6 isoform has been described in the past but had not been characterized in detail, nor had its relevance for peanut allergy been investigated. method: post-translationally cleaved ara h 6 (para h 6) and intact ara h 6 (intact ara h 6) were purified from virginia type peanuts and the cleavage site was mapped using high-resolution mass spectrometry. biochemical characteristics were determined by sds-page, uv absorbance spectroscopy, far uv cd spectroscopy, and immunochemical reactivity of both forms of ara h 6 was compared by igg immunoblotting and ige-elisa using sera from individuals sensitized to peanut. reversed-phase liquid chromatography was applied to study the occurrence and abundance of para h 6 in various peanut types. results: compared to intact ara h 6, para h 6 lacks a 5-amino acid stretch, resembling amino acids 43-47 (uniprot accession number q647g9) in the non-structured loop. consequently, para h 6 consists of 2 chains; a n-terminal chain of approximately 5 kda, and a c-terminal chain of approximately 9 kda, held together by disulfide bonds. intermediate post-translationally cleaved products, in which this stretch is cleaved but not removed, are also present. the secondary structure and ige-binding of para h 6 resembles that of intact ara h 6, indicating that the loss of the non-structured loop is not critical for maintaining conformational ige-epitopes. both forms of ara h 6 were reactive with several commercially available igg antibodies. the peanut cultivars runner, virginia, valencia, and spanish contained para h 6 at equivalent levels, suggesting para h 6 is a consistent and important constituent of the peanut proteome. conclusion: a post-translationally cleaved form of ara h 6 is abundant in the main peanut market types, and has ige-binding comparable to intact ara h 6. this should be taken into account when ara h 6 is investigated in peanut-containing products. 1585 | release of major peanut allergens from their matrix at various ph and at saliva conditions; ara h 2 and ara h 6 are quickly bioaccessible background: the oral mucosa is the first immune organ that encounters allergens upon ingestion of food. peanut is often consumed in solid form, and it is not known if peanut allergens are released from the food already in the mouth. we set out to investigate the solubility of individual peanut allergens at conditions that mimic the first exposure site, i.e. the mouth. method: light roast peanut flour was suspended in buffers of various ph mimicking saliva. protein concentration was measured in supernatant, and release of major allergens ara h 1, ara h 2, ara h 3, and ara h 6 was assessed by sds-page. also, the allergen profile of un-dissolved material was assessed. results: peanut protein solubility is poor in the ph range 3-6, while at low ph (1.5) and at moderately high ph (>8), the solubility is higher. at all conditions tested, there was a substantial amount of un-dissolved protein. this indicates that the ph range of saliva, between 6.5 and 8.5 in healthy individuals, may be critical for the release of peanut protein from its matrix. in this ph range from 6.5 to 8.5, ara h 2 and ara h 6 are readily released, while ara h 1 and ara h 3 are poorly released. increasing the ph from 6.5 to 8.5 slightly increased the release of ara h 1 and ara h 3, but still the recovery was low (approximately 20% for both ara h 1 and ara h 3) compared to that of ara h 2 and ara h 6 (approximately 100% and 65%, respectively). this remarkable difference in extraction kinetics suggests that ara h 2 and ara h 6 are the first allergens an individual is exposed to upon ingestion of peanut-containing food. conclusion: based on our observations, we conclude that the peanut allergens ara h 2 and ara h 6 are quickly bio-accessible in the mouth upon ingestion of peanut. this new insight may contribute to the understanding of the extraordinary allergenicity of ara h 2 and ara h 6 compared to other peanut allergens. background: in proven cases of non-ige mediated cow's milk allergy clinical response can be partial even when treated with amino acid formulae e.g pain in infants. residual intestinal symptoms can be related to ongoing nerve hypersensitivity, changes in microbiome or motility disturbance. mast cells are thought to play crucial role in non-ige mediated food allergy. ketotifen is a first generation h1 antihistamine which has mast cell stabilising properties with pain blocking and anti tnf-a effect. hence ketotifen could have important role in symptom resolution where diet elimination has not been successful. we aim to find out effectiveness of ketotifen to unresponsive/partially responsive symptoms such as pain in non-ige mediated cow's milk allergy infants. method: children who presented to single specialist centre over 11 years had their case notes reviewed retrospectively. inclusion criteria were those children with confirmed non-ige mediated cow's milk allergy by elimination of cow's milk with improvement of symptoms and worsening of symptoms on reintroduction of dairy. where symptoms partially responded e.g pain, ketotifen was used at 0.5-1 mg once at night for 4 weeks and symptoms were reassessed. statistical analysis was performed using r v3.3.3 with significance was set at p = 0.05. results: 1031 patients were identified with 675 patients excluded due to unconfirmed non-ige mediated allergy. of the 358 case (206 males, age 3-50 months), atopic co-morbidities were found in 62% children. common symptoms were abdominal pain (51%), vomiting (47%), back arching (37%), constipation (35%), bloating (33%), food aversion (29%) and diarrhoea (26%). we compared the children who had symptom improvement on ketotifen and cow's milk elimination against children who improved on cow's milk elimination alone. significant difference of symptom improvement was found with abdominal pain; 69% using ketotifen compared to 53% who did not use results: a total of 3548 analysis for ttgiga have been performed for a total of 2965 patients during the study period (2010, 2012, 2014, 2016) . 128 patients showed at least once a positive ttgiga. among these, 11 had a negative result at first testing, and were positive at the second (n = 10) or third testing (n = 1). despite an increasing number of ttgiga requests, the number of positive results decreased. wige were rarely requested but were positive in about 45% of tested sera (table 1a and 1b). the amount of laboratory requests for ttgiga has increased, while those for wige remains stable and is rare. wheat allergy seems to be rarely investigated in our center and may deserve more attention. case report: eosinophil-associated gastrointestinal disorders (egids), including eosinophilic gastroenteritis (eog), are a inflammatory diseases, characterized by gastrointestinal symptoms and eosinophilic infiltration. patients with eoe have an increased incidence of allergy, with increased ige mediated food and inhalant sensitivities. use of either a targeted food allergen avoidance approach (based on allergy testing) or untargeted approach (based on food allergen avoidance) results in the resolution of eosinophilia in the gastrointestinal tract of 50%-70% of adult. we describe a case of a 27-year-old patient diagnosed with eosinophilic enteritis, associated to protein-losing enteropathy. the patient experienced severe diarrhea, nausea, vomiting and weight loss, that caused a severe dysproteinaemia and electrolytes abnormalities. an upper and lower endoscopy was performed, showing an ulcerative ileitis. the histological pattern was characterized by eosinophilic infiltration of ileum and duodenum>40 hpf. she presented also high levels of total ige (800 k/ui), high serum tryptase (20 μg/l, n.v. ≤9.0) and sensitization to the lipid transfer protein (ltp) of peach. the patient was prescribed to a six-food elimination diet (sfed) and underwent high doses of oral and intravenously corticosteroids, but a satisfactory therapeutic response was not achieved. we hypothesized that ige has a role in the mechanism of aeg and that blocking ige would have improved disease symptoms and reduced allergic inflammation, as measured by a decrease in intestinal tissue eosinophilia. we started off-label administration of omalizumab 300 mg/month subcutaneously, the same dosage schedule used in allergic asthma and, by other authors, in eosinophilic gastrointestinal disease after achieving informed consent by patient. except for an exacerbation of symptoms occurred 9 months after starting the therapy, when a further endoscopy, showing a gastrointestinal eosinophilic infiltration>50 hpf, was performed, a significant improvement of both gastrointestinal and cutaneous symptoms was observed during therapy, together with a normalization of laboratory parameters. after 30 months a clinical remission of disease was obtained and administration was stopped. although a histological remission during the first few months of treatment was not obtained, in a subset of aeg patients, ige plays a role in the pathophysiology of the disease and that anti-ige therapy with omalizumab may result in disease remission. 1590 | fullerene c60 reduces the allergic inflammation in food allergy mouse model background: a food allergy (fa) is an abnormal immune response to food. the signs and symptoms may range from mild to severe. they may include itchiness, swelling of the tongue, vomiting, diarrhea, hives, trouble breathing, or low blood pressure. food allergy is becoming increasingly common. fullerene c60 has the unique electronic properties making it an attractive candidate for allergic diseases therapy. the main purpose of our research was to assess therapeutic effect of fullerene c60 in a mouse model of fa. method: new efficient method for producing a water-soluble fullerene c60 has been developed. fa experimental model was induced in balb/c mice by the intragastrical (ig) ova administration after subcutaneous (sc) sensitization. fullerene c60 was administrated ig once a week, or twice a week, or daily. ova-specific antibodies were assessed by elisa. splenocytes cytokine production upon ova in vitro stimulation was detected by elisa. samples of jejunum of the small intestine were removed for histological examination immediately after the last ig allergen administration. results: it was shown that ova-specific ige and il-5 level were significant decreased in groups treated with water-soluble fullerene c60. the greatest effect was observed in mice receiving fullerene c60 daily. the ifn-gamma level was significantly higher in ig c60treated groups. the histologic analysis of jejunum of the small intestine samples showed that c60-therapy improved the histologic picture. the greatest effect was observed in mice receiving fullerene c60 daily too. conclusion: taken together, these results demonstrate that the water-soluble fullerene c60 exhibits a significant anti-inflammatory effect in a mouse model of fa, and possesses a high therapeutic potential. background: a 47-year-old male reported eight episodes of anaphylaxis after exercise. all the ingested food eight hours before each episode was analyzed. before each episode, he had always eaten chicken or turkey meat, and he tolerated these foods without exercising. in one of the episodes the patient had taken a tablet of dexketoprofen a few hours before. since several years, he referred chest tightness after eating some fish (emperor, salmon and whiff), however he tolerated others. the patient denied having eaten fish before any of the episodes of anaphylaxis. method: commercial skin prick tests (spts) and prick by prick tests (pp) with all food ingested and fishes were performed. tryptase, total serum ige (ige) and specific ige (sige) (immunocap. thermo-fisher scientific, uppsala, sweden) to the foods involved were determined. a controlled oral provocation test (opt) with dexketoprofen was performed. results: spt was positive to tuna extract (3 mm) and negative (0 mm) for the rest of fish extracts. it was also negative for chicken meat extract and other foods tested. pps were positive for raw and cooked turkey meat (3 mm), raw and cooked tuna (6 and 8 mm), raw emperor (11 mm), raw and cooked whiff (9 and 8 mm) and raw hake (18 mm). pps were negative to raw and cooked chicken meat. ige was 700ui/ml and tryptase 2.95 ng/l. sige was slightly positive to hake, cod and chicken meat. dexketoprofen opt was negative. at that moment, we recommended the patient to avoid chicken and turkey, as well as the fishes which he had symptoms with. since then, he has not suffered any new episode of anaphylaxis despite exercising daily. protein extracts from turkey meat, tuna, emperor, salmon, hake and whiff were prepared and analyzed by sds-page. conclusion: recently, triosephosphate-isomerase (28 kda) has been identified as a new chicken meat allergen. this allergen could be responsible for the cross-reactivity between bird and fish meat and the episodes of anaphylaxis after exercise in our patient. the triosephosphate-isomerase has not been implicated previously as a cross-reactive allergen involved in the fish-chicken syndrome. results: twenty-three patients were included, 61% male, median age 30 years (iqr 12.5), 48% atopic, 30% asthmatic. non-specific lipid transfer proteins (nsltp) were implicated in 70% (n = 16) and ω-5-gliadin in 30% (n = 7). eighteen (78%) patients referred anaphylaxis in the reaction with co-factor, 7(22%) urticaria/angioedema, 2 had both depending on the co-factor. all patients in which ω-5-gliadin was the allergen involved had anaphylaxis in the presence of co-factor, with tolerance to wheat without it. in patients in which nsltp was the allergen involved, 12 (75%) had anaphylaxis in the presence of co-factor. reaction with co-factor was more severe than without in 13 (81%) patients; 4 patients had no previous history of reaction and subsequently tolerated the culprit food. only 1 patient had anaphylaxis in the absence of co-factor; the remaining presented oral allergy syndrome and/or urticaria. exercise was the main co-factor, present in 22 patients. nonsteroidal anti-inflammatory drugs (nsaids) were the only co-factor in 6 patients; all of them had anaphylaxis, 4 with allergy to ω-5-gliadin, 2 to nsltps. all subsequently tolerated the nsaids involved. conclusion: nsltps and ω-5-gliadin were the most frequently involved allergens in cefa, with exercise being the most frequent co-factor. nsaids were relevant co-factors, even when ω-5-gliadin was the allergen involved. several patients subsequently tolerated culprit foods and nsaids, difficulting the diagnosis and further emphasizing the importance of a correct cofactor evaluation. molecular allergens had an important role in the diagnosis, avoiding unnecessary ofc. information about co-factors must be included in all patients with allergy to nsltps and ω5-gliadin. 1594 | allergy to wheat-dependent exerciseinduced anaphylaxis (wdeia) proteins, without? 5-gliadins as responsible ferreira a 1 ; castillo m 2 ; martins s 1 ; pineda f 2 1 unidade de imunoalergologia hospital das forças armadas., lisbon, portugal; 2 departamento de aplicaciones. diater laboratorios, madrid, spain background: the second well-characterized form of allergy to wheat proteins is wheat-dependent exercise-induced anaphylaxis (wdeia), with the ω5-gliadins (part of the gluten protein fraction) being the major group of proteins which are responsible, but other forms of food allergy have also been reported, with the proteins responsible including gluten proteins, cm proteins and non-specific lipid transfer proteins. the patient was a 38-year-old man who visited the hospital with acute urticaria just eat bread before run (30 minutes). according the components of bread. it was formed by a mixture of wheat, rye and barley. with a history (for several years) of episodes of severe urticaria after intake a mixture of cereals and/or different kinds of beer. results: prick test and specific ige with wheat, rye and barley were negative and the proteins from allergenic extract from these cereals, and also the gliadins and glutenins fractions were transferred onto a pvdf membrane to carried out a western blot technique with the patient's serum. the patient's serum recognized several proteins from wheat and millet gliadins not compatible in molecular mass with a ω5-gliadins. the association of w5 gliadin as responsible for the symptoms produced after the intake of products containing wheat and exercise is well referenced but in the case of this patient could have other proteins involved as triggers of their symptoms. suksawat y phramongkutklao hospital, bangkok, thailand background: food-dependent, exercise induced anaphylaxis (fdeia) is an anaphylactic condition that develops in patients who ingest specific food followed by exercise. a variety of foods have been described to be the cause including shellfish, wheat and vegetables. the mechanisms of fdeia is believed that exercise increases allergen absorption or decreases threshold of mast cell. the investigations such as skin prick test or specific ige for food are useful because food sensitization is demonstrated. however, a challenge test including ingestion of suspected food followed by exercise is the only method to diagnose this disease. we report a case of fdeia in a 17-year-old adolescent male. result: he presented with generalized urticaria and hypotension after eating a barbecue buffet which was one hour followed by playing taekwondo. after treatment with intramuscular adrenaline, antihistamine and systemic steroid, his condition was improved. the barbecue buffet consists of many kinds of food including shrimp, squid, salmon and pork meat which were previously tolerated. he had no past history of anaphylaxis or drug allergy. he was referred to our allergy unit for investigation. we performed skin prick test with food allergens and many kinds of fresh foods that he ate on that day and the result was positive to shrimp (6 mm. in diameter). three-day challenge protocol was set up a month after recovery and we used aspirin as a cofactor. on the first day, open challenge for 500 gram of shrimp was administered and the result was negative. on the second day, exercise challenge test based on the american thoracic society guideline was also negative. however, on the last day, he developed generalized urticaria five minutes after the same exercise challenge test which was 1 hour preceded by aspirin intake and 500 gram of shrimp ingestion. but his vital signs appeared to be stable. the patient was administered intramuscular adrenaline and antihistamine with full recovery. he was strongly advised to avoid shrimp for 4-6 hours before exercise and carry an adrenaline autoinjector. the-three day challenge protocol is a definite tool to confirm the diagnosis of fdeia. a correct diagnosis is important to avoid unnecessary restricted diet. 1596 | food dependent exercise induced anaphylaxis in peach allergic patient-case report consumption of different types of food (pancakes with cream cheese and fruit, peach, chinese dish, sandwiches-all eaten on other occasions without symptoms) and co-occurring physical exercise (dancing, shopping, walking). during diagnosis we performed spt with inhaled and food allergens (allergopharma), prick by prick tests with peach, banana, apple, pear and bread. we established the concentration of allergen specific ige (peach, wheat flour, peanuts, hazelnuts) and the level of ige specific to allergen components (immunocap isac). we performed exercise provocation test and open food challenge with peach. results: spt were negative with all tested food and inhaled allergens (inc.egg; milk; cocoa; tomato; carp; apple; banana; strawberry; rye flour; wheat flour; peanuts; hazelnut; citrus, d. farinae; d. pteronyssinus; grass; weeds; clad. herbarium; alt. tenuis; dog; cat; poplar; hazel; alder; birch; mugwort). prick by prick tests were positive with fresh peach. concentration of peach specific ige was 0.55 ku/l. in immunocap isac we found elevated levels of ige specific to ltps from different allergen sources (jug r 3 -1.3; pru p 3 -0.8; pla a 3 -0.6; tri a 14 -0.5 [isu-e]). open food challenge with a medium size peach was negative. exercise provocation test without allergen exposition was negative. exercise provocation test after eating a medium size peach concluded with severe lip and eyelids edema, followed by whizzing, dyspnea and urticarial. patient received adrenaline 0.5 mg im, steroids and antihistamines with good clinical effect. conclusion: patient was diagnosed with food dependent exercise induced anaphylaxis (fdeia) due to ltp allergy. she was advised to eat peeled fruit and vegetables, avoid cofactors of allergic diseases and carry rescue set (adrenaline, steroids and antihistamines). cases reports: we report 1case of fdeia and 3 cases of wdeia. case 1:24-year-old woman with intermittent severe allergic rhinitis and food allergies since childhood. in the last 7 years she registered weekly episodes of fdeia (urticaria, angioedema, wheeze, drop of bp) especially when during effort and after alcohol intake. prick tests were positive for grass pollen, mugworth, ragweed, dust mites, celery, soy, sesame, pistachio, mango, honey and shellfish. she followed 3 years of subcutaneous immunotherapy for grasses pollen. the fdeia episodes frequency and severity diminished during it. provocation test for celery and mango were positive, for alcohol, soy, sesame, pistachio, honey and shellfish were negative. case 2:23-year old man with a 7-year history of acute gluten induced urticaria, recently developed 2 episodes of wdeia (flushing, severe urticaria and angioedema, wheeze) when he went for gym. skin test was highly positive for wheat flour and dust mites, in vitro tests for 5 omega gliadin and wheat-specific ige were positive. food challenge for wheat was positive. he followed oral immunotherapy for wheat. the wdeia episodes were rare and mild. case 3:30-years old female with mild allergic rhinitis and controlled asthma, with wdeia (urticaria, angioedema, wheeze, drop of bp) in the last 3 years. skin tests showed positive results for wheat flour, dust mites and dog hair. omega-5 gliadin and wheat specific ige were high, but the provocation test for wheat was negative meanwhile combined wheat and effort provocation test was positive. 25-year-old female athlete, otherwise healthy, experienced three episodes of fdeia following running sessions. two reactions were preceded by intake of salad containing lettuce, tomato and sunflower seeds and the third one occurred after eating celery salad. the patient denied occurrence of any symptoms with physical exertion or food ingestion alone. physical examination and blood testing did not reveal any abnormalities. the differential diagnosis was performed. in skin prick test and specific serum ige antibodies sensitization to house dust mite, grass and mugwort were found. the spt and specific ige assay to culprit and most common food allergens were negative. the molecular diagnostic has been applied (faber, caam, rome, italy). the test scored positive for art v, blo t, der f, der p, eur m, lol p, phl p. no positive results for available food molecules including celery, tomato, sunflower seeds and lettuce were found. the detection of culprit food in fdeia is of crucial meaning as the syndrome can be life-threatening. the molecular diagnostic has been applied already in diagnosis of wheat-dependent exercise-induced anaphylaxis (wdeia) proving ω-5-gliadin sensitization in the majority of the cases. as the presented case did not reveal sensitization to culprit food in traditional allergy tests the molecular diagnostic was performed. this test did not show sensitization to culprit food either. however, not all of the molecules are available in molecular assays yet. cross-reactivity reaction to mugwort and grass has to be considered. the pathophysiological components of physical exertion has to be taken into consideration as well. this could contribute to the assessment of reasonability of molecular approach in diagnostic work-up for fdeia and to the establishment of standardized protocols for diagnosis and management of that syndrome. case report: food allergy to wheat is rare in adults, often reported in exercise-induced anaphylaxis. food-dependent exercise-induced anaphylaxis (fdeia) is a form of food allergy induced by exercise. fdeia symptoms can include urticaria/angioedema, respiratory and gastrointestinal manifestations and hypotension/shock. a 61-year-old male patient presented to the emergency department, was admitted after an episode of hives, hypotension and loss of consciousness. his consciousness was restored after treatment with epinephrine, glucocorticoids as well as fluids, and thereafter, the patient reported that the anaphylactic episode occurred when he started rapidly walking 1 hour after eating a slice of pizza. he mentioned that the offended food was tolerated always when it was not followed by a physical exercise. review of his past medical history and family one were non-contributory with respect to this episode. the allergy skin prick testing for common foods revealed a positive response only to wheat, while and other laboratory test values were within normal ranges. the patient is discharged after instructions on the use of epinephrine auto-injector. he was also advised to avoid wheat containing products up to 6 hours prior to physical exercise. our case demonstrated that fdeia can be characterized by the onset of anaphylaxis soon after physical exercise, when preceded by the ingestion of the responsible food. avoidance of the combination of the exposure to respective allergen and exercise is the most efficient precautive measure toward subsequent fdeia episodes. 1600 | residual exercise-induced allergic reactions after successful rush oral immunotherapies for milk and wheat method: we conducted roit for 55 children (median 8.7 years old) with milk allergy and 46 children (median 6.8 years old) with wheat allergy during 2010-2015. after 5-12 days of the rush phase in the hospital and a slow-increasing phase at home, patients consumed the maintenance dose (6.5 g milk protein or 5 g wheat protein). after at least three months of the maintenance phase without allergic symptoms, we conducted an exercise provocation test (ept) after eating the target food. if the ept was positive, we repeated it after a couple of years to check for remission. the presence or absence of eiars was based primarily on the results of epts but also on the clinical history in some cases. results: as of december 2017, 44 milk-and 41 wheat-allergic patients were able to continue ingesting the maintenance dose (desensitization). in these patients, 38 milk-and 38 wheat-allergic patients underwent the first ept at a median of 635 (228-2538) days after roit, and the result was positive in 19 (50.0%) and 19 (50.0%) patients, respectively. among these ept-positive patients, 10 milk-and 9 wheat-allergic patients conducted a second ept at a median of 504 (119-1120) days after the first ept. the result of the second ept was positive in 7 milk-and 7 wheat-allergic patients. in addition, clinical histories of eiars were subsequently observed in 4 milk-and 4 wheat-allergic patients after negative results on an ept. altogether, 19 (43.2%) milk-and 21 (51.2%) wheat-allergic patients still had eiars even after getting desensitization as of december conclusion: patients with persistent milk and wheat allergy often have residual eairs even after three to five years of desensitization due to the administration of successful roit. abstracts | 791 1601 | anaphylaxis caused by omega-5-gliadin initially diagnosed as idiopathic anaphylaxis: a case report tziotou m 1 ; syrigos k 2 ; syrigou e 1 ; sinaniotis a 1 1 department of allergy,, athens, greece; 2 gpp,, athens, greece case report: we report the case of a 59-year-old man who experienced two episodes of wheat dependent exercise induced anaphylaxis, initially diagnosed as idiopathic anaphylaxis. first episode: the patient woke up in the morning and drove to his resort. while driving, he ate a piece of cheese and ham pie. when he arrived, he walked some meters to the garden and started feeling pruritus and dizziness. he lost consciousness and recovered by himself. he was carried to hospital where his vital signs were normal. the patient has a history of atrial fibrillation and has been on flecainide bid and aspirin at noon for the last four years. a month after the first episode he visited an allergist. skin prick tests to aeroallergens and prick to prick tests to the ingredients of the pie were negative. tryptase levels were within normal limits and skin biopsy was negative for mastocytosis. an endocrinology workup was also negative. the patient was prescribed an epinephrine autoinjector and was asymptomatic for eight months. second episode: that morning he had a cup of milk and two slices of toast for breakfast and started working in the garden. two hours later he experienced pruritus and urticaria and fell unconscious. his wife had to administer two epinephrine autoinjectors before he regained consciousness. after the second episode it was decided to start treatment with omalizumab. two months later he experienced an episode of urticaria while working in the garden. he could not recall what he had eaten before. based on history, we thought that a cofactor might contribute to the occurrence of anaphylaxis. we performed skin prick tests with peach (ltp) and gliadin, allergens associated with food dependent exercise induced anaphylaxis in our region. the test to gliadin was positive. specific ige in serum to omega-5-gliadin was also positive, while specific iges to all ltps tested were negative. the patient was advised to avoid wheat and has been asymptomatic ever since. cases diagnosed with idiopathic anaphylaxis may actually be cases in which the culprit allergen has not been identified. detailed history and extensive workup may contribute to the successful management of these patients. written informed consent has been obtained from the patient. 1602 | wheat-dependent exercise-induced anaphylaxis (wdeia) and nsaids: clinical history is crucial conclusions :we present a case clinically compatible with wdeia with nsaids intake as augmenting factor. this case emphasizes that a carefully and thoroughly taken medical history is of crucial importance, otherwise wdeia can easily be unrecognized. as a result, non-allergic hyperreactivity to nsaids could be excluded and the diagnose of selective allergy to arylpropionic acids was made. exercise challenge test could not be performed in our case. case report: kounis syndrome (ks) has been defined as an acute coronary syndrome that manifests as unstable vasospastic or non-vasospastic angina, and even as acute myocardial infarction. it is triggered by the release of inflammatory mediators following an allergic insult. a 77-year-old woman with type ii diabetes and hypertension, and unstable angina pectoris as cardiovascular risk factors, has consumed chamomile tea. thirty minutes later, she developed generalized itching, skin rash, swelling of the face and the throat, chest tightness, dyspnea and syncope. the patient was transferred immediately to the emergency department, and sodium chloride, 50 mg prednisolone, 8 mg dexamethasone, 80 mg methylprednisolone, 5000ui heparin, 75 mg voltaren were intravenously administered along the subsequent 60 minutes under simultaneous treatment with oxygen therapy. an ekg examination is performed based on the patient disease's history, showing a 2.5-3 mm st-depression on d1-d3 leads, 1 mm on v3, and 3.5 mm on the v4-v6 ones. in addition, 2.5 mm st-elevation on the avr and 1 mm on the v1 derivation was observed, associated by a negative t-wave on the v1, v2, and avr leads. blood tests revealed a normal troponin i level (of 0.07 ng/ml). five hours later in the ekg was noticed: isolined st-segments, and negative t waves on the d3, avr, and v1 leads. ultrasound examination revealed normal heart kinetics and function. following the heart changes, the patient was administered sol. heparin 5000ui twice i.v., nebivolol 5 mg, plavix 75 mg, atorvastatin 80 mg, abstracts | 793 monocinque 20 mg, ordinary insulin 10ui s.c., glargine insulin 20ui s.c., and sol. furosemide 20 mg i.v. the patient progressed favorably, and four days after the anaphylactic episode the ekg revealed a 0.5 mm st-depression on leads v4, and v5, negative t-wave on avl lead, and normalized one on the v3 one. this case emphasizes the role of serious allergic reactions as cause of acute coronary syndrome in patients with altered coronary arteries and food intake as cause of kounis syndrome. 1605 | recall urticaria in two young patients with alpha-gal-syndrome after tick bites case report: patient a (m, age 25) had suffered from 7-8 anaphylactic reactions (hives, nausea, dyspnea and dizziness) within the past 15 months. all episodes occurred 3-5 hours after ingestion of red meat, once with alcohol as a co-factor. all episodes started with a wheal measuring about 0.5 cm in exact the same spot where he had been bitten by a tick one year before. specific ige to galactose-alpha-1,3-galactose (alpha-gal) was positive (2.55 ku/l). skin prick testing using raw pork kidney suspension and intradermal testing with gelafundin ® 4% diluted 1:10 also showed positive reactions. we performed an oral challenge with cooked pork kidney under careful monitoring being able to reproduce the recall urticaria as described above with a cumulative dose of 8 g pork kidney. we stopped the challenge and treated the patient with antihistamines and corticosteroids. patient b (m, age 22) reported on several anaphylactic reactions within the past 4 years with symptoms including abdominal pain, diarrhea, as well as dyspnea and loss of consciousness in one of the episodes. all episodes occurred several hours after ingesting food. furthermore the patient remembered a tick bite about 2 years before the first anaphylactic reaction, which repeatedly became inflamed and only healed completely over months. every episode started with pruritus and a wheal in the area of the former tick bite (in loco). specific ige to galactose-alpha-1,3-galactose was positive (4.37 ku/l) as well as the skin prick testing with cooked pork kidney and intradermal testing with gelafundin ® 4% diluted 1:10. this patient refused performance of oral challenge tests. an elimination diet of red meat for 12 months resulted in the absence of the symptoms as described. the diagnosis of alpha-gal-syndrome with recall urticaria in loco was made in both cases. this symptom may also be useful in evaluating results of oral challenge tests as well as an important clinical sign in medical history. pali-schöll i 1 ; meinlschmidt p 2 ; purschke b 2 ; hofstetter g 1 ; einhorn l 3 ; mothes-luksch n 3 ; jensen-jarolim e 3 ; jäger h 2 background: insects have gained interest as alternative nutrient source for humans and animals. however, being a "novel food" in the industrialized part of the world, several safety aspects, like allergenicity, need to be thoroughly addressed. in the present work we evaluated the cross-recognition of ige from patients allergic to crustaceans, house dust mite or stable flies, using house cricket acheta domesticus (ad), desert locust schistocerca gregaria (sg) and mealworm tenebrio molitor (tm). we further investigated changes of immune-recognition in terms of ige-binding in differently processed insect extracts. method: migratory locust locusta migratoria (lm) was subjected to different extraction methods, enzymatic hydrolysis or thermal processing, whereas tm larvae (tml) were evaluated after different centrifugation modes and ph levels. results: we revealed that ige from patients with crustacean allergy shows cross-recognition of acheta domesticus, schistocerca gregaria and stable flies. ige from house dust mite allergic individuals binds to acheta domesticus and schistocerca gregaria. importantly, the cross-reactivity to lm can be deleted by enzymatic hydrolysis with different enzymes or heat treatment (cooking, autoclaving), but not by different extraction methods. changes of ph and varying centrifugation steps are not sufficient to reduce ige-binding to tml. our results show that patients allergic to crustaceans, house dust mite or stable flies-allergic patients cross-recognize desert locust and house cricket proteins, and crustacean-allergic patients also flies proteins. furthermore, we confirm that the appropriate food processing method of insect proteins can reduce the risk of cross-reactivity for crustaceans-and house dust mite-allergic patients. the study was supported by the austrian science fund fwf (grant sfb f4606-b28 to ejj). results: the mean age at diagnosis was 10 years in children and 34 years in adults, more frequent in males (3:1). in the pediatric group, three had first-degree relatives with eoe and three had celiac disease. two children had performed milk oral immunotherapy and five adults aeroallergens subcutaneous immunotherapy. most of them were atopics with sensitization to aeroallergens (77.5% of children and 82.5% of adults) and food allergens (75% of children and 82.5% of adults), without statistically significant differences. the most frequent foods were fruits and nuts in both groups. we found significant statistical differences in fruits (35% of children abstracts | 795 and 57.5% of adults; p = 0.007) and cereals sensitization (5% of children and 47.5% of adults; p < 0.001). in the clinical presentation we observed significant statistical differences in impaction (22.5% of children and 82.5% of adults; p < 0.001), dysphagia (42.5% of children and 77.5% of adults; p < 0.001) and abdominal pain (25% of children and 7.5% of adults; p = 0.034). in the endoscopic findings children had more frequently exudates (92.5%; p < 0.001) and adults had esophageal trachealization (50%; p < 0.001). significant statistical differences were found in the treatment with topical corticosteroids (30% of children and 77.5% of adults; p < 0.001) obtaining a variable positive response. 77.5% of patients in both groups received food elimination diet, 45% with four or more foods. conclusion: eoe presents differences in the sensitization profile, clinical manifestations and endoscopic findings according to the age of presentation. the response to pharmacological treatment is variable and a high percentage of patients receive food elimination diets. it is a pathology difficult to control, therefore new non-invasive techniques would be useful in order to facilitate its management. 1610 | modulation of gut microbiota in patients with nickel allergy and ibs after diet and probiotics supplementation mb 1 ; garcía-figueroa be 2 department of allergy1 department of allergy fang l 12 united states 1431 | bcx7353 improves health-related quality of life in hereditary angioedema with c1-inhibitor deficiency bygum a 4 fang l 16 former yugoslav republic of; 6 division of clinical immunology huissoon a 2 bygum a 3 ; panovska vg 4 united states callejas fdb 1 alobid i 1 ; muñoz-cano r 1 izquierdo i 2 icahn school of medicine at mount sinai method: the case report form was developed by experienced allergists, and the web-based registry was established in cooperation with a professional medical software team. twenty-two departments from 16 hospitals took part during the first year 8%), whereas in adults, drugs (54.5%) were more common than foods (31.4%). the most common food triggers were eggs (27.1%), milk (15.8%), and walnut (7.9%) in children, and shrimps (21.1%), wheat (15.8%), and crab (7.9%) in adults. among drug triggers in adults, antibiotics (50.0%) were the most common cause followed by nsaids (16.7%), and h2-blockers (13.6%). the onset time was≤10 minutes in 48.6%. in children, home was the place of occurrence in more than half of the cases, whereas adults experienced anaphylaxis in out-of-home settings more often than children. cofactors were present in 19%. among the 289 cases registered via the emergency department of participating hospitals, epinephrine was administered in 66.8% (62.5% in adults, 69.8% in children) and the route of administration was im in 90.8%, iv in 6.3%, both im and iv in 2.1%, and subcutaneous in 0.7%. the number of epinephrine administration was single in 83% conclusion: this multicenter prospective registry would provide a better understanding of anaphylaxis, and provide visionary modalities to improve the management and prevention of anaphylaxis in future a case of kounis syndrome after chamomile tea consumption characterized by symptoms related to esophageal dysfunction and esophageal mucosal infiltration by eosinophils objective: characterize patients (pts) with eoe diagnosis and analyze the differences between pts with diagnosis at pediatric (ch, <18 years old) and adult age epicutaneous tests(epict)], serum total ige and eos, findings in upper digestive endoscopy (ude) and biopsies. the correlation between food sensitization, clinical severity (visits to er services or hospitalization due to complications of eoe, sclin) or severe histology results: 74 pts (81% male, average age 27 ± 17 years) ad 31 and 34, respectively. 96% ch and 67% ad were atopics. the most frequent symptoms of eoe were dysphagia (73%) and gastroesophageal reflux(46%) in ch; impaction(85%) and dysphagia(46%) in ad. 92% ch and 71% ad had aeroallergens sensitization. 77% ch and 69% ad had food sensitization. the most frequent positive tests were for ch: spt to milk(25%) and shellfish(19%), epict to shellfish(50%) and meat(19%); for ad: spt to milk(21%), fresh fruits and nuts both 18%), epict to shellfish(35%) and meat(13%). ude showed: 65% striation and white plaques in 50% ch shist (46%) was associated with sclin (35%), p = 0.001 in ch; but this was not observed in ad group there was no correlation between food sensitization and sclin or shist in both groups(p > 0.01). the average values of serum total ige (kua/l) were 653 in ch and 458 in ad; eos were 679 and 413, respectively in ch and ad allergy unit-fondazione policlinico universitario a. gemelli, università cattolica del sacro cuore conclusion: ltp with a fixed dose (2000 iu in 4 ml) of ready-touse shp616 led to fewer severe attacks, a higher proportion of attack-free patients, and a clinically meaningful and statistically significant reduction in cumulative attack severity and daily severity in hae patients relative to placebo. background: c1-inh-hae is a rare, potentially life-threatening disease characterized by episodes of subcutaneous and/or submucosal swelling. apex-1 was a phase 2, double-blind, placebo-controlled study to evaluate the prevention of attacks with bcx7353, a once daily oral kallikrein inhibitor, in patients with c1-inh-hae.method: patients with c1-inh-hae with a history of at least 2 hae attacks per month were randomized to receive four different doses of bcx7353 (350 mg, 250 mg, 125 mg, 62.5 mg) or placebo for 28 days. blood samples for bcx7353 concentrations and kallikrein inhibition were obtained from patients before dosing and for 24 hours post-dose on day 14. pk analyses and pk-pd modeling were done in phoenix winnonlin v 8.0 and sas v 9.3. the pk population included 16, 14, 14, and 7 subjects in the 350 mg, 250 mg, 125 mg, and 62.5 mg groups respectively.after daily dosing achieved steady state, c max was reached at a median of 3-4 hours after dosing. there was a greater than dose proportional increase in exposure (auc tau and c max ) over the 62.5-mg to 350-mg dose range, with an approximate 14-fold increase in exposure with a 5.6-fold increase in dose. at doses ≥125 mg, which showed statistically significant and clinically meaningful reductions in hae attack rates, geometric mean plasma trough concentrations (c tau ) were maintained at or above the minimum target concentration (4-fold ec 50 ) estimated to be required for adequate plasma kallikrein inhibition. percentages of study subjects at steady-state with bcx7353 plasma concentrations>4-fold ec 50 were 0%, 57%, 100% and 100% in the 62.5, 125, 250, and 350 mg dose groups, respectively. a 125-mg dose provided a mean c tau of slightly above 4.0fold ec 50 , with a corresponding reduction in hae attack rate of 69% (p < 0.001) compared with placebo. consistent with the exposure data, a dose dependent inhibition of kallikrein was observed with bcx7353 treatment over the dose range. the drug effect on kallikrein inhibition was highly correlated with exposure (r = 0.867). in patients with c1-inh-hae, bcx7353 treatment at doses ≥125 mg resulted in clinically meaningful reductions in the mean weekly hae attack rate. concentrations of bcx7353 at doses ≥125 mg were maintained at or above a c tau of 4-fold the kallikrein inhibition ec 50 in most patients, and kallikrein inhibition was highly correlated with bcx7353 plasma concentrations.background: c1-inh-hae is a rare, life-threatening disease characterized by recurrent episodes of subcutaneous and/or submucosal swelling that lead to considerable morbidity and a poor quality of life (qol). apex-1 was a phase 2, double-blind, placebo-controlled study to evaluate the prevention of attacks with bcx7353, a once daily oral kallikrein inhibitor, in patients with c1-inh-hae.method: patients with c1-inh-hae with at least 2 hae attacks per month were randomized to four different bcx7353 doses (350 mg, 250 mg, 125 mg, 62.5 mg) or placebo. subject-reported qol assessments were conducted at the start and end of treatment using the disease specific angioedema quality of life (ae-qol) questionnaire that measures 4 domains (function, fatigue, nutrition, fear/ shame) and has minimal clinically important difference (mcid) of 6 points. the changes from baseline in total and domain scores was compared between the treatment and placebo groups. modified angioedema activity score (aas) values across 4 domains (daily activities, appearance, physical discomfort, overall severity) were calculated for each attack and a total score was derived for each subject by summing scores from each attack. total scores were compared to placebo using an ancova model with adjustment for qualifying attack rate. reduction of attacks was statistically significant for all 3 top doses and there was a dose related increase in adverse events.results: in the 125 mg dose group, qol assessed by ae-qol was significantly improved after 4 weeks of treatment compared to placebo for ae-qol total score (-26.0, p < 0.001) as well as across all 4 domains (function: -28.2, p = 0.002; fatigue: -12.7, p = 0.05; fears/shame: -36.5, p < 0.001; nutrition: -23.4, p = 0.012). all other treatment groups showed a trend towards improvement. qol improved the most in the125 mg group, and 92% of subjects in the 125 mg group showed ae-qol reduction of more than 6 points. disease activity as assessed by the aas was significantly reduced in the 350 mg, 250 mg and 125 mg dose groups as compared to placebo, whereas there was no significant reduction in the 62.5 mg dose group. results: there were no marked differences in the age, sex, total ige titer, comorbidity of bronchial asthma and atopic dermatitis or the starting dose of rush oit among the groups. all patients in the low-bmfi group achieved ≥100% of the target dose, whereas 28.6% of the middle-bmfi group and 50% of the high-bmfi group failed to achieve the target dose (p < 0.05). results: a total of 473 infants were diagnosed with food allergy (ige-mediated and mixed type) at our center during the study period, 305 of these patients underwent prick test for inhalant sensitivity, and 220 (64.9% male) of these were younger than 2 years of age. conclusion: sensitivity to inhaled allergen were found in 14 of 292 (4.9%) patients with food allergy. therefore, we believe that inhalant sensitivity should be evaluated in these patients. there is also a requirement for further studies to identify the influence of inhaled antigens on the disease activity of patients with allergic conditions. method: in this study, we aimed to perform the metabolic profiling of severe profilin mediated food allergic patients looking for biomarkers that might both, predict the prognosis of the disease and understand the molecular mechanisms of inflammation underneath. other allergic patients (mild and moderate) and non-allergic were recruited in the study as comparative groups. the allergic patients class was predicted using a mathematical algorithm from non-allergic vs severe model results: plasma samples from non-allergic subjects, mild, moderate and severe allergic patients were measured using gas chromatography coupled to mass spectrometry (gc-ms). the samples were from 4 different hospitals in spain covering the areas with the highest pollen exposure. the metabolic profile was composed of 95 metabolites for each sample. results after the statistical analysis showed differences between the groups. firstly, a clear reduction of several abstracts conclusion: we found, as expected, a predominance of males with eoe diagnosis. ch were more frequently atopic and had aeroallergen and food sensitization. impaction and esophageal stenosis were more frequent in ad than ch. shist was associated with sclin only in ch. method: three children with ee, boys aged 1.5-11 years, were assessed over 6 months to 3 years. results: 2 cases developed in infancy. one had dyspepsia and low weight gain in infancy soon after feeding began. another had symptoms in infancy but not diagnosed until age 6 with dysphagia and esophageal stricture.. the third case was diagnosed at 11 years old after 2 episodes of food impaction in the esophagus beginning at age 9. all cases had allergic comorbid diseases including atopic dermatitis in all 3 and allergic rhinitis in 2. skin prick tests were positive to several food allergens (cow's milk, egg protein) in 2, to dust mite in 2 and to pollens in 1. serum total ige levels ranged from 490 to 1039 iu/ml. eosinophils in peripheral blood were elevated in all 3, reaching 10%-13%. treatment included restricted diet and topical budesonide 1-2 mg daily depending with periodic endoscopic biopsy.in all cases clinical improvement occurred by one month of treatment, with endoscopic confirmation. morphological improvement folthe study aimed to evaluate the effects of probiotic supplementation, in addition to diet, in ibs and snas patients, in terms of modulation of faecal microbiota population, reduction of gi and cutaneous symptoms, increase of patient's quality of life and modification of gut dysbiosis.method: forty patients aged between 18 and 65 years, affected by ibs, ni sensitization and ltp sensitization were enrolled to evaluate gut dysbiosis. dna extraction method (next generation sequencing) with commercial kit (microbiopassport ® ) was performed on stool samples. ibs patients were divided in two groups, according a gluten free diet prescription or a low fodmaps diet prescription for three months. similarly, (suspected) snas patients (confirmed by 5% ni sulfate in petrolatum patch test) were prescribed a low ni diet (100 μg/kg nickel content during the first four weeks and then up to 200 μg/kg up to three months). two ltp (lipid transfer protein) sensitized patients underwent a ltp free diet.gut dysbiosis was re-assessed after a fixed probiotic supplementation. a sex-age matched group of individuals without history of ibs, snas or any gastrointestinal disease was considered as control.gastrointestinal symptoms were evaluated using the visual analogue scale before and after treatment. conclusion: our preliminary findings suggest that probiotic implementation could be useful in patients with snas on a low-ni diet to increase population diversity, which could contribute to restore the intestinal homoeostatic conditions. key: cord-023364-ut56gczm authors: nan title: education day monday: plenary session 1 monday: parallel sessions date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00651.x sha: doc_id: 23364 cord_uid: ut56gczm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023346-8sqbqjm1 authors: nan title: monday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00652.x sha: doc_id: 23346 cord_uid: 8sqbqjm1 nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023354-f2ciho6o authors: nan title: tuesday plenary session 3 tuesday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00654.x sha: doc_id: 23354 cord_uid: f2ciho6o nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-006229-7yoilsho authors: nan title: abstracts of the 82(nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the 18(th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: 2016-02-06 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-016-1213-y sha: doc_id: 6229 cord_uid: 7yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise 80% of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only 6.5% of the volume, but 40% of the total cell number (kmiec 2001) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. 2010) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr3 breast cancer cells were exposed to the anti-cancer drug herceptin (20μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm-13 cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around 35% of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr3 cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm-13 cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu 7th health program grant agreement no 279288. deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases 1 and 2 (erk1/2) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk1/2 activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine 188 as a prerequisite for nuclear erk1/2 signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk1/2 signaling: since activation of erk1/2 triggers erk dimerization, a prerequisite for erk t188autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t188 -phosphorylation. first, we investigated the impact of monomeric erk2 on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk2 ∆174-177 and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk2 ∆174-177 overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆174-177 in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk2 and prevents its dimerization. moreover, the peptide effectively inhibited erk t188 -phosphorylation and nuclear translocation of yfp-tagged wild-type erk2 after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype 9 (aav9)-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i2knockout on cardiac function and survival in a murine heart-failure model of cardiac β 1adrenoceptor overexpression. methods and results: β 1 -adrenoceptor overexpressing mice lacking gα i2 (β 1 -tg/gα i2 -/-) were compared to wild-type (c57bl/6) mice and littermates either overexpressing cardiac β 1 -adrenoceptors (β 1 -tg) or lacking gα i2 (gα i2 -/-). at 300 days of age mortality of mice only lacking gα i2 was higher compared to wild-type or β 1 -tg, but similar to β 1tg/gα i2 -/mice. beyond 300 days mortality of β 1 -tg/gα i2 -/mice was enhanced compared to all other genotypes (mean survival time: 363±21 days). echocardiography revealed similar cardiac function of wild-type, β 1 -tg and gα i2 -/mice, but significant impairment for β 1 -tg/gα i2 -/mice (e.g. ejection fraction 14±2% versus 40±4% in wild-type mice). significantly increased ventricle-to body-weight ratio (0.71±0.06% versus 0.48±0.02% in wild types), left-ventricular size (length 0.82±0.04 cm versus 0.66±0.03 cm in wild types) and anp and bnp expression (mrna: 2819% and 495% of wild type, respectively) clearly indicated hypertrophy. gα i3 was significantly upregulated in gα i2 -knockouts (protein compared to wild-type mice: 340±90% in gα i2 -/and 394±80% in β 1 -tg/gα i2 -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β 1adrenoceptors in β 1 -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than 2-fold higher (b max : 1425 ± 68 fmol/mg) than on the otherwise used c57bl/6 background. accordingly, fvb/n-based β 1 -tg mice showed a significantly impaired cardiac function at an age of 300d while c57bl/6-based β 1 -tg mice did not. conclusions: gα i2 -deficiency combined with cardiac β 1 -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c57bl/6 background β 1adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day 300 while there was overt cardiomyopathy in mice additionally lacking gα i2 . we propose an enhanced effect of increased β 1 -adrenergic drive by lack of protection via gα i2 . the observed gα i3 upregulation was not sufficient to compensate for gα i2 deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i3 is currently addressed in a subsequent study using β 1 -tg and gα i3deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; 10 mg/kg per d) for 6 and 24 hours and 7 days. overall 7 days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; 7d iso 156±1) vs. wt cardiomyocytes (7d iso 143±2). at this time point a 29 % decrease of cardiac output and a 16 % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after 6 h (75-fold), and declined after 24 h (29-fold) to 2.5 fold increase after 7 days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: 55 regulated genes (up-regulated: 45 %) in untreated; 103 altered genes (up 35 %) after 6h; 1437 changed genes (up 97%) after 24 h and 131 altered genes (up 19%) after 7 days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after 7 days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at 24 h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m3 receptors, while m2 receptors appear to exert a heart-specific function only at later stages. continuous m3 blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m3 inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m3 receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m3 receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m3 inhibition. taken together, we postulate that muscarinic m3 receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m3 receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir-21 has proved effective against fibrosis of the myocardium and in other organs. mir-21 has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir-21 in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir-21 fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir-21 in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav9-icre vector allowed for specific and almost complete deletion of mir-21 in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir-21 in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir-21 gene in mice. aav9-mediated deletion of mir-21, albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir-21 demonstrated that mir-21 exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir-21 therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c81 acts as a multikinase inhibitor. results of a thermal shift assay revealed that c81 shows by far the highest binding affinity to the bmp-2-inducible kinase (bmp2k/bike). bmp2k represents an as yet largely uncharacterized protein, which is not regulated by bmp-2 in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c81 and (ii) the role of bmp2k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c81 affected the viability of human umbilical vein endothelial cells (huvecs). both c81 and the knock-down of bm2k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec-1). also the proliferation of hmec-1 was reduced by c81 treatment (ic 50 : 7 µm). a tube formation assay on matrigel demonstrated that c81 significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c81 treatment nor by bmp2k gene silencing. in regard to inflammatory processes, c81 treatment or bmp2k silencing of huvecs decreased the adhesion of thp-1 cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c81 or bmp2k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c81 treatment, the knock-down of bmp2k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p65 translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c81 in vitro. the precise role of bmp2k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp2k silencing and c81 treatment will be further elucidated. moreover, since the inhibition of bmp2k seems not to be responsible for all actions of c81, we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). cxcr4 seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr4 agonists or antagonists could result in reduced tlr4 signaling. however, the role of cxcr4 and the influence of different cxcr4 ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr4 activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose (5 mg/body weight) in mice. the plasma stable cxcl12 analog ctce-0214d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. 24 hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr4, cxcl12 and tlr4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce-0214d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately 35%, 50% or 70%, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce-0214d diminished the lps induced expression of cxcr4, cxcl12, tlr4, nf-κb, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce-0214d in all organs. the cxcr4 is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce-0214d attenuated tlr4 signaling, induced heme oxygenase 1 activity and mitigated apoptosis. thus, the administration of cxcl12 analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c57bl/6j mouse strains with a loxp flanked exon 4 of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t2 ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef 2-1 (fxn -/-) and fx-mef 2-8 (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over 12 days, with medium and substance changes at day 4 and 8, which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day 12 of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day 3, 5, 7 and 10 and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into 96well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor 2 which leads to disruption of protein synthesis and finally causes cell death [1] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c2 toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [2, 3] . in detail, we confirmed the involvement of the host cell chaperone hsp90 and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [4, 5, 6] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp40 and fk506-binding protein (fkbp)51 as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp90, cypa, cyp40, fkbp51 and fkbp52. the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [1] murphy (2011) toxins 3, 294-308. [2] barth (2011) naunyn-schmied arch pharmacol 383, 237-245. [3] kaiser et al. (2012) cell. microbiol. 14, 1193-1205. [4] dmochewitz et al. (2011) cell. microbiol. 13 , 359-373. [5] ratts et al. (2003) j. cell biol. 160, 1139-1150. [6] schnell et al. (2015) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins (1). prompted by these result, we found that only human α-defensin-1 (hnp-1) but not human β-defensin-1 (hbd-1), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin-1 prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp-1 causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy1-asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a 42.6% knockdown (p<0.05 student's t-test, n=6) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n=9). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p450 (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s9 mix), as was the synthesized ba-epoxide (-s9 mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type 2 diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o 6 -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr5-positive colon stem cells, which coincided with atr-chk1-p53 signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h2ax, suggesting the usefulness of γ-h2ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [2] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type 2 diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines (1). in particular, the chemokines monocyte chemoattractant protein (mcp-1, ccl2), interleukin-8 (il-8/cxcl8) and stromal cell-derived factor 1 (sdf-1a/cxcl12) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl14 was shown to attenuate obesity in mice (2) . the cc chemokine ccl2 is known to stimulate ccr2 receptors, and the cxc chemokines cxcl8 and cxcl12 to activate cxcr1 and cxcr2 or cxcr4 and cxcr7, respectively. the receptor(s) activated by cxcl14 is currently unknown. a role of cxcl14 in modulating cxcr4 signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr1 and the related receptor fprl1, and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr2 receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr2 receptors were absent (3) . yet, the analysis revealed appreciable levels of mrna encoding ccl2, cxcl12, and cxcl14, and ccr1, cxcr2, cxcr7, fpr1, and fprl1, and cxcr4. of interest, cxcr4-and cxcr7-mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl2, cxcl12, cxcl14. while sgbspreadipocytes and adipocytes did not accumulate ccl2-mcherry upon stimulation, they showed a small accumulation of cxcl12-mcherry, and a strong accumulation of cxcl14-mcherry in the endosomal compartment. similar results were obtained in murine 3t3l-1 preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl14-binding putative receptor protein(s) in murine 3t3l-1 preadipocytes. (1) makki, k. et al., (2013) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic 3', and to some extent of the corresponding cytidylyl analogue ccmp 1,2 . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown 3 . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp 1 . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity 2 . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac1-camps 3 in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. 1 kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell 157, 163-186 (2014) . 2 koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. 14 (2): p. 190-9 (2010) . 3 nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem.279, 37215-37218 (2004) cardiovascular pharmacology 039 hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel (70%:30%:0.4 mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology (1% strain, 1 hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen 0.03; collagen + cms 0.03; collagen + cms + cfbs 0.09, collagen + cms + ffbs 0.2. we next generated ehm with cms and ffbs or cfbs. after 4 weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p<0.01) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm 2 : 1.8±0.1, n=29 vs. 0.3±0.1, n=20). cross sectional area (csa) of tissues was greatly increased (p<0.01) in cfb-ehms (csa in mm 2 : 1.6±0.1, n=20 vs. 0.6±0.03, n=30) and nonmyocyte content was higher in cfb-ehms (5.6±0.7, n=9 vs. 3.1±0.4, n=15; x10 5 cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase 2 (has2) enzyme was significantly (p<0.01) upregulated. based on these findings, we pharmacologically intervened with has2 mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all 4 weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p<0.01) reduced csa (csa in mm 2 : control 1.8±0.4, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.9±0.2, n=4) with a concurrent, statistically significant (p<0.01), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm 2 : control 0.2±0.05, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.4±0.08, n=4). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type 2 receptor (vegfr2), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac1 and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with 5 µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with 30 µm of the camp analog 8-pcpt-2'-o-camp (007) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac1 in huvec decreased the basal and vegf-induced sprouting. surprisingly, 10 µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than 007, indicating an additional role of pka. in accordance, 1 µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, 50 ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac1 and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh5cre ) were created using the cre/loxp system. mr cdh5cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received 1 % nacl with drinking water for 6 weeks. the mineralocorticoid deoxycorticosterone acetate (doca, 2.5 mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype (141.7 ± 8.0 vs. ctrl 97.8 ± 3.2 mmhg, p<0.001) and mr cdh5cre (150.0 ± 3.0 vs. ctrl 104.1 ± 4.7 mmhg, p<0.001) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh5cre mice (ventricle weight 162.4 ± 4.2 mg vs. mr wildtype 189.0 ± 10.9 mg, p<0.05). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh5cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam1) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: 2.4 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) and mr cdh5cre (2.3 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) mice. mrna expression of the fibrosis marker gene col3a1 (mr wildtype 4.3 ± 0.9-fold; mr cdh5cre 3.9 ± 1.1-fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype 20.4 ± 5.7fold; mr cdh5cre 34.3 ± 12.5-fold vs. ctrl). in the kidney vcam1 mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca 2+ concentration due to ca 2+ entry from the extracellular space is critical for platelet activation and aggregation. ca 2+ entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a 2 (txa 2 ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca 2+ entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca 2+ entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc3 and trpc6 in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca 2+ signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc3/c6 proteins for procoagulant activity. in addition, we detected the expression of s12 trpc1 transcripts in mouse platelets. therefore, we tested trpc1/c3/c6-deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc1/c3/c6 inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc1/c3/c6-deficient mice and we observed that the aggregation was reduced after adp (3µm) or txa 2 -analogue (1µm) stimulation, but not after collagen stimulation (10µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca 2+ response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/0 mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/0 mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/0 mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/0 mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/0 mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp2b10 (1200-fold in females, 700-fold in males) in 6-day old offspring of adult female mice exposed one week prior mating to tcpobop (3 mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop (3 mg/kg) prior to mating. the analysis of hepatic cyp2b10 expression in 6-day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a 700-fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e18.5 followed by crossfostering proved transmission to be mediated predominantly via lactation (f1 hepatic cyp2b10 induction 3000-fold) and only to a minor part via intra-uterine exposure (300fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f0 and f1 generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp2b10 catalytic activity. in both tcpobop-pretreated f0 and in their f1 descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by >50%. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by 45% in 12-weeks-old offspring. in summary, the car-mediated cyp2b10 induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb 761 ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with 96 alzheimer´s disease patients indicated that the combined treatment with donepezil and egb 761 ® had less side effects than donepezil alone (yancheva et al., 2009) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb 761 ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment (15 days of pretreatment) with donepezil (1,5 mg/kg p. o.), egb 761 ® (100 mg/kg p. o.), or the combination of the two drugs, or vehicle in 18 -20 month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally 30 minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb 761 ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb 761 ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. (2009) . ginkgo biloba extract egb 761(r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, 13 (2) , [183] [184] [185] [186] [187] [188] [189] [190] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations (3-28 µm; just et al. expert opin emerging drugs 20: 161-164, 2015) observed in patients treated with dantrolene. although not yet proven directly, oat3 may be involved in renal secretion of dantrolene and 5-oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp4. since oat3 was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. 483: 133-138, 2004) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr1, by influx through oat3, where it inhibits calcium efflux by ryr1 thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat3. in addition, we identified, besides dantrolene and 5-oh dantrolene, several further fdaapproved drugs such as tyrphostin ag 1478, ceefourin 1, glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat3. controls 1 with other defects and controls 2 with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥50mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with 95% confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls 1 and additionally adjusted for maternal age in controls 2. results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were 65/464 (14.0%) for cases, 512/6033 (8.5%) for controls 1 and 130/1564 (8.3%) for controls 2 and adjusted ors (95%cis) were 1.71 (1.29-2.26) and 1.77 (1.27-2.46), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-(2.41 vs. 1.61 for controls 1, and 2.55 vs. 1.67 for controls 2). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being 2 and 1.6 times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac1 protects non-transformed cells from topoisomerase ii-(top2)-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac1-mediated regulation of topoisomerase ii activity. however, the link between rac1 and top2-poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top2-poison-induced cytotoxicity. here, we investigated the relevance of rac1-regulated actin cytoskeleton integrity as well as mitochondrial integrity in top2-poison-induced dna damage responses as well as cytotoxicity under situation of rac1 inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h5v) as well as cardiomyocytes (h9c2) were chosen as in vitro model systems for top2-poisoning. the cells were pre-treated with rac1 inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh2ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top2-poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc1-assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac1 inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h2ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac1-inhibitor. top2-poison-induced raise in mitochondrial mass was slightly enhanced by the rac1-inhibitor and statins. interestingly, inhibition of rac1 counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top2-poisoninduced cytotoxicity in h9c2 and h5v cells. the data point to a role of rac1-regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top2-poisons. poly(adp-ribose) polymerase 1 (parp1) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [1] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp1 as well as by non-covalent poly(adp-ribose)-wrn interaction [2] [3] [4] . furthermore, pharmacological parp inhibition as well as a genetic parp1 ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [5] . here we report a novel role for parp1 and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h 2 o 2 -induced oxidative stress, parp1 is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp1 activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp1 interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp1 and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp1-dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp1 in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [1, 2] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [3] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [4] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [5 -7] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases 5methylcytosine and 5-hydroxymethylcytosine as well as their derivatives (5formylcytosine and 5-carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene 9, , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb2, xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the 17 members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp1 is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle 2011, mangerich and bürkle 2015) . here we generated a genetic knock out of parp1 in one of the most widely used human cell systems, i.e. hela parp1 ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp1 ko cells with a series of artificial and natural parp1 variants, we analyzed structure-function relationships of parp1 in a cellular environment without interfering with endogenously expressed wt-parp1. we confirmed that the parp1e988k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp1l713f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp1-par interaction via at least one specific par binding motif located in zinc finger 2 of parp1. expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp1l713f itself triggered apoptosis, parp1e988k expression led to a strong g2-arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt888 mitigated effects of the e988k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp1 ko cells with a natural cancer-associated parp1 snp variant (v762a), as well as a newly identified parp1 mutant present in a patient of pediatric colorectal carcinoma (f304l-v762a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp1-dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp1 variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle (2011) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer128 (2): 251-265. mangerich, a. and a. bürkle (2015) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: 125-179. leukemic cells frequently overexpress the transcription factor wilms tumor 1 (wt1) and the persistence of wt1 expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt1 determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt1 and cell death. in addition, we show that an rna interference-mediated elimination of wt1 sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt1 suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt1 also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt1 is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt1 is a novel biological target of hydroxyurea and they suggest that wt1 has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß 1 ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res 97:472, 2013) and stably transfected to hek 293 cells (hekß 1 -fret). to monitor the camp response the epac1-based fret sensor of camp, was stably transfected alone (hekwt-e1) and together with a moderate level of native ß 1 ar to hek293 cells (hekß 1 -e1; nikolaev et al. jacc 50: 423, 2007) . fret-activity was measured with recently developed fluorescence detectors (12 channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using 438 nm for excitation and 483/540 nm for the emmission ratio. cells were cultivated in 96-format 12-well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß 1 -fret cells exhibited ec 50 -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: 6.9±0.1, adrenaline 5.7±0.1, noradrenaline 6.2±0.1). ßar-expression levels were controlled by radioligand binding with [ 3 h]-(-)-cgp12,177 resulting in different densities of ~4x10 6 and ~1.4x10 4 receptors/cell in hekß 1 -fret and hekß 1 -e1, respectively, whereas in hekwt-e1 cells only ~1000 ß 2 ar were found. surprisingly, the low level of ßar in hekwt-e1 cells allowed the measurement of the action of ß 2 -sympathomimetics (ß 2 sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~6 .7) to an effective regulation of fret activity in the presence of 0.06 mm ibmx (pec 50~8 .3±0.1), nearly matching the ~100-fold amplification of iso (pk d~6 .9; pec 50~8 .9). in order to determine ß 1 ar-mediated side effects of ß 2 sym, hekß 1 -e1 cells characterized by a 14-fold higher level of ß 1 ar over ß 2 ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec 50~8 .4 whereas the high affinity ß 2 sym salmeterol acted a partial agonist (~75% of iso maximum, pec 50~8 .6), both compounds being rather insensitive against the highly effective ß 1 ar-blockade with 1 µm cgp 20,712 a. for that reason hekß 1 -fret cells were used characterizing fenoterol as partial agonist (~25%) whereas salmeterol activated less than 10% of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß 2 ar present in hekß 1 -e1 cells precludes the exact determination of ß 1 ar-mediated side effects of ß 2 sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β 1 -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β 1 -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek293 cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek293 cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β 1 -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β 2 -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β 2 -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β 2 ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. the affinity of arrestin-3 to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin-3 and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin-3 dissociation from the β 2 ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin-3 into the bleached region was prolonged between two-and fourfold for the β 2 ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β 2 ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek293t cells transfected with the appropriate cdnas. here we focused on muscarinic m1-, m2-, and m3-receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/11-proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m3-and m1-achrs was found to be 10-fold faster in comparison to gq. similarly, we observed a 15-fold right shift of the concentration-response curves of go proteins binding to m3-achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m3-achr by using a fret-based assay. our results showed that gq activation required 10-fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m2-achr for gi family members by comparison of dissociation kinetics of gi1-, gi2, gi3-, and go-proteins from m2-achr under nucleotide depleted conditions. k off of gi1 and gi3 were found to be two-fold higher in comparison to gi2 and go proteins, indicating the higher affinity of the latter ones to m2-achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising 15 receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone 1 receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone 1 receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine 375 that is predominantly catalyzed by g protein-coupled receptor kinase 5. as a consequence, the selective morphine-induced s375 phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s375 but also drive higher order phosphorylation on the flanking residues threonine 370, threonine 376, and threonine 379 in a hierarchical phosphorylation cascade that specifically requires grk2 and grk3 isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels 061 hcn4 in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn1 and hcn2 isoforms for brain function and epilepsy has been demonstrated, however the role of hcn4, the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn4 in controlling oscillations in the thalamocortical network. hcn4 is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn4-deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn4 null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn4 is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc4 and 5 channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc3/6/7 channels are directly activated by diacylglycerol (dag), trpc4 and 5 channels are commonly regarded as daginsensitive. in contrast to trpc3/6/7 channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) 1 and 2. interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc5 confers dag-sensitivity on trpc5 channels. trpc5 channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip 2 depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf1 and 2 proteins and overexpression of a nherf1 mutant incapable of trpc5 binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip 2 depletion itself caused slight trpc5 current increases while during pip 2 depletion, the membrane permeable dag analogue oag evoked even higher trpc5 currents suggesting that pip 2 depletion induces an active and dag-sensitive channel conformation. receptor mediated pip 2 depletion also resulted in dissociation of nherf1 and 2 from the c-terminus of trpc5 thereby eliciting a dag-sensitive trpc5 channel conformation. thus, our findings suggest that dag-sensitivity of trpc5 is the result of an activation cascade starting with pip 2 depletion and subsequent dynamic dissociation of nherf1 and 2 from the c-terminus of trpc5. altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm3 is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm3-deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm3 may exert antinociceptive properties. fluorometric ca 2+ assays and a compound library containing approved drugs were used to identify trpm3 inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura-2-loaded single cells was applied to monitor [ca 2+ ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm3 cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm3 inhibitors. the compounds exhibited half-maximal inhibitory concentrations of 0.6-6 µm. the selectivity profiles of maprotiline and primidone for trpm3 were promising with no inhibitory effects on trpm2, trpm8, trpa1, trpv1, trpc5, trpc6 and p2x7 receptor channels. primidone inhibited pregs-induced [ca 2+ ] i signals in rat drg neurones, indicating a block of native trpm3 channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm3 at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm3-related effects by acutely challenging the channel´s function. pharmacological interference with trpm3 applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm3 in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il-6) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e103/k308 can be crosslinked efficiently in both states, the closed-and open state of the p2x2r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e84/r290, e103/k308, e167/r290 and e167/k308 and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p2x2r. the comparison of the closed-state and open-state model of the rat p2x2r showed that atp promotes a marked rearrangement of the side chains of the residues r290 and k308 to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r290 and k308 with atp competitively releases e84, e103 and e167 from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. 1 in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem1, tmem2 and tmem4. expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem2 is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem2 proteins exhibit 6 to 10 plasma-membrane spanning domains, but fluorescently labeled tmem2 fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem2-eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem2 -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem2-deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura-2 or indo-1 indicate higher resting ca ++ concentrations in tmem2-/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem2-yfp-knock-add-on mice we identified tmem2 in organelles of the apical acinar cell pole and a partial colocalisation with lamp2 proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh 4 cl application. the role of tmem2 for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin 2pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck-8 stimulation indicating that tmem2 inactivation leads to an enhancement of the globalization of cck-8 evoked ca2+ release from intracellular organelles. taken together, our study identifies tmem2 as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [1] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the 3rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [1] gundert-remy, u. et al. (2015) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol 89: 1881-93. during the past decades, considerable progress has been made in implementing 3r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster 3r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. 3r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes (4 x 4 gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed 24 h after the last irradiation by γh2ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht1864, a small molecule inhibitor of the small rho-gtpase rac1, was applied in vivo, pointing to an involvement of rac1 in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas9 is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas9 allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh3-interacting domain death agonist) knockout cell line in neuronal ht-22 cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas9-knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht-22 cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin-1 and the bid inhibitor bi-6c9 on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas9-bid-knockout cells. bi-6c9 inhibited glutamate-induced morphological changes of ht-22 cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin-1 in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi-6c9 abolishes lipid peroxide formation in the erastin model and ferrostatin-1 in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi-6c9 and ferrostatin-1 in the respective model systems. investigating the crispr/cas9-bid-knockout ht-22 cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type 2 (aav2) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange 13 and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of 1 mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange 13 at the azo-and other weak bonds in our experimental set-up (fig.1a) . amongst other substances, the carcinogens aniline (max. conc. 1.01 ± 0.12 µg/ml) and 3,3-dichlorobenzidine (max. conc. 0.88 ± 0.18 µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. 1b) . here, 1,2-benzenedicarbonitrile (max. conc. 1.11 ± 0.12 µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. 35.8 ± 4.32 µg/ml) and the human carcinogen benzene (max. conc. 0.19 ± 0.06 µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥50 µg/ml evolving upon ruby laser irradiation of >1.5 mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference 1 schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep 5, 12915 (2015) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween 20 and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco-2 model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). 2015; 10 (21) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [1, 2] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > 120 °c [3] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [4] . we here present results of a nine-day human intervention study with 14 healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [5] . the study design included washout periods with an aa-minimized diet (21 -41 ng /kg bw), a low aa intake day (0.6 -0.8 µg /kg bw) as well as a high aa intake day (1.3 -1.8 µg /kg bw). after a three-day washout period an aama baseline level of 93 ± 31 nmol/d was determined. low aa intake led to an aama excretion within 24 h of 225 ± 37 nmol/d, high intake to 404 ± 78 nmol/d corresponding to an aama excretion rate of about 30% of the ingested aa dose within 24 h, whereas aama output within 72 h corresponded to 58% of the respective aa intake, the aama baseline after 3 days washout corresponds to a net exposure level of 0.2 -0.3 μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [1] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste 2013 , 2013 doi: 10.1002 iarc, iarc monographs on the evaluations of carcinogenic risks to humans 1994, 60. [3] tareke et al., j. agric. food chem. 2002, 50, 4998-5006. [4] efsa panel on contaminants in the food chain (contam), efsa journal 2015; 13(6):4104 [321 pp.] . [5] ruenz et al., arch. toxicol. 2015 doi: 10.1007 /s00204-015-1494 heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct116 colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf2/skn-1, daf16), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + 57%). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn-1 (homolog to mammalian nrf2), but not daf-16 (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct116 cells, baicalein activates nrf2; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf2. the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf-16 transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn-1 but not daf-16. experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf2/skn-1 activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [1] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in 12 healthy postmenopausal women (50-70 yrs).in a randomized doubleblind protocol each subject received four treatments, 2 doses of intranasal progesterone (4.5mg mpp22; 9.0 mg mpp22), 10mg of zolpidem and placebo. the 4 conditions consisted of 2 experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from 23:00 to 07:00. simultaneously blood was collected every 20 min between 22:00 and 07:00 by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after 4.5 and 9.0mg mpp22. univariate f-tests in the manovas pointed to the following results (significant p-values at α=0.05). sei was higher after zolpidem than after the other three treatments. after 9.0mg mpp22 sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after 9.0mg mpp22 and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after 9.0mg mpp22 and after zolpidem. after all active treatments with mpp22 and zolpidem the time spent in sleep stage 2 was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp22 resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after 9.0 mg mpp22. no other changes of hormone secretion were found. our study show sleep promoting effects of 9.0 mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after 9.0 mg mpp22. no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january 2013 and june 2015 from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of 2 or 3 and possible anticholinergics with a score of 1). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of 3. a patient's individual score of 3 or higher is considered to be clinically relevant. in total, 130,186 geriatric patients (median age 82 years, 66.3% female, median no. of drugs 9) were evaluated. of these, 41,456 (31.8%) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in 10,941 (26.4% of the patients taking anticholinergic drugs) patients. 11,241 (27.1% of the patients taking anticholinergic drugs) patients had a score of 3 or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine (58), amitriptyline/quetiapine (41) and amitriptyline/carbamazepine (35). 2,885 (7.0%) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct2 and mate1 expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. 1,2 although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to 3 the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. 4 however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α 2 -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct1 (encoded by slc22a1), oct2 (slc22a2), oct3 (slc22a3) and mate1 (slc47a1). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek293 stably expressing the organic cation transporters oct1, oct2, oct3 or mate1. 5 intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct2 and mate1 in a time-and concentration-dependent manner, but not by oct1 or oct3. timolol is only transported by mate1, but not by the octs. as shown by immunolocalization studies, the oct2 and mate1 transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct2 and mate1 may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: 1. zhang et al., nat rev drug discov. 2012 jun 15; 11(7) :541-59. 2. lavik et al., eye (lond). 2011 may; 25(5):578-86. 3. gaudana et al., pharm res. 2009 may; 26(5):1197 -216. 4. kraft et al., invest ophthalmol vis sci. 2010 51(5):2504 -11. 5. nies et al., plos one. 2011 6(7) :e22163. supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz 1 , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel 600mg, prasugrel 30mg or prasugrel 60mg (n=300). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day 1 after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s =0.18; p=0.002), hipf (r s =0.18; p=0.002), ipc r s =0.26; p<0.001). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p<0.001). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p450 2c19 polymorphisms (*2 and *17), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s21 was the strongest predictor of on-treatment platelet reactivity (partial η 2 =0.045; p<0.001) followed by intrinsic platelet reactivity (partial η 2 =0.034; p < 0.002). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp 2c19*2 polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [1] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [2] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [3] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki-67 index 23-fold in the cscc model (p£0.001). while the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [4] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for 2.6-fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [2] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb3r with integrated graduate education was launched in 2014. the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the 3rs and to find alternatives to animal experimentation within the next years. a panel of 3r experts has been set up to provide advice and assistance and to raise awareness in society for 3r-related issues. research in bb3r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb3r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on 3r including modules on ethics and legislation. currently, 14 phd students are qualified for management positions in professional areas related to the 3rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb3r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd 442c and 442d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz-3-derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after 14 days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer 2,4-dinitrochlorobenzene (dncb) induced il-6 and il-8 secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd83 and pd-l1 in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine (3rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the 21st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than 10,000 compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing 647 compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. 20 %) fail in the late drug development due to toxicity and adverse effects [1] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about 51,000 compounds and 115 cancer cell lines from the national cancer institute's (nci) nci-60 growth inhibition data [2] with focus on five immune cell lines (rpmi-8226, ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [1] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [2] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [3] , and encourage the reduction of in vivo testing according to the principles of the 3rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype 1e1 activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure 1 ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult1e1 ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for 3d pharmacophore development. eight specific 3d pharmacophores were created that allow identification of sult1e1 substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over 6,500 experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult1e1 inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult1e1 for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. (2011) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. (2011) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p=0.013). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p=0.010). 50% of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in 2013. by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like 3d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the 2-organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd86 and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex 3d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the 2-organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused 2-organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd86-values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the 2-organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past 10 years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [1, 2] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) 14 clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck14, which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il-1 alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [1] vávrová, k., henkes, d., strüver, k., sochorová, m., školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3d skin construct. the journal of investigative dermatology. 134, 746-753 (2014 bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive 2010/63 has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the 3-rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the 3-r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c57/bl6 j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for 45 minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored 30 and 150 minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for 24 hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the 82 nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb3r is funded by bmbf. in 1959 the 3r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the 3r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the 3r-concept is the overall goal of scientific animal welfare. article 4 of the 'directive 2010/63/eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [1] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately 70 % [2] [3] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [4] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from 0.05 to 2.5 mg/kg per bodyweight [5] [6] [7] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [8] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [1] rl 2010/63/eu. [2] bmel (2014) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [3] eu commission (2013) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [4] carbone l (2011) pain in laboratory animals: the ethical and regulatory imperatives. plos one 6, e21578. [5] gv-solas, a.f. a.d. (2013) . empfehlung zur schmerztherapie bei versuchstieren. [6] carpenter, j.w., t.y. mashima, and d.j. rupiper (2001) . exotic animal formulary, 2nd edition, w.b. saunders co., phila. [7] flecknell, p. (1996) . laboratory animal anaesthesia, 2nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a 64-slice scanner were collected from 18 female minipigs, consisting of 6 animals aged 12 months (group 1, n=6) and 12 animals (group 2; n=12) examined at the age of 17 and 21 months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than 50 parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to 65% where the largest volume was 13,4 ml and the lowest 4,7 ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between 12 and 17 months of age. comparing 17 and 21 months old minipigs, no significant difference in distance could be observed. from the age of 17 months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between 12 and 17 months of age indicates ongoing anatomical changes of this parameter until the age of 17 months. therefore animals should be older than 17 months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [1]. worldwide it is estimated that over 85 million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [2] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [3] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals (3r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin (5-ht) system, in particular the 5-ht 1a receptor, playing a major role. activating 5-ht 1a receptors by the 5-ht 1a receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic 5-ht 1a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the 5-ht 1a receptor in serotonergic projection areas, bert et al. (2008, pmid: 18396339) revealed exaggerated 8-oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic 5-ht 1a receptors to thermoregulation, more precisely to the hypothermic effect of 8-oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of 8-oh-dpat (0.1 mg/kg -4 mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the 5-ht synthesis inhibitor parachlorophenylalanine (pcpa; 100 mg/kg, i. p. on four consecutive days) would alter the effects of 8-oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the 5-ht 1a receptor. results: 5-ht 1a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: 36.0 °c; nmri wild-type mice: 37.4 °c). in both genotypes, systemic administration of 8-oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (-2.8 °c compared to -1.5 °c in nmri wild types). dose response curves of 8-oh-dpat revealed an ed 50 = 0.4 mg/kg in transgenic and an ed 50 = 0.57 mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to 8-oh-dpat in mice. the dose-response curves indicate a higher potency of 8-oh-dpat in transgenic mice. the exaggerated hypothermic response to 8-oh-dpat in mutant mice implies that postsynaptic 5-ht 1a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that 8-oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing 5-ht 1a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg389gly in the human beta1-adrenoceptor (adrb1). the adrb1 carrying arginine at position 389 in helix 8 in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta1-adrenoceptor variants arg389-adrb1 and gly389-adrb1 is poorly understood. despite its hyperfunctionality, we found the arg389-variant of the adrb1 to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly389-variant. using adrb1 sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg389-adrb1 exerted faster activation speed and arrestin recruitment than the gly389-variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb1 mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb1 finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb1 variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved 10-residue sequence, 370 trehpstant 379 , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine 375 (s375) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase 5 (grk5). by contrast, high-efficacy opioids not only induce phosphorylation of s375 but also drive higher-order phosphorylation on the flanking residues threonine 370 (t370), threonine 376 (t376), and threonine 379 (t379) in a hierarchical phosphorylation cascade that specifically requires grk2/3 isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk2 and grk3 to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk2/3. in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk2/3 isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk2 or grk3. conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk2 or grk3. mutation of s375 to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all 11 carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/11, and g12/13, which interact with distinct cellular effectors to shape the final cellular response 1 . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs 2 . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation 3 . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/11/14 inhibitor fr900359 4 on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m1 acetylcholine receptor (cho-m1). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor 1,5 . we show that fr900359 can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m1 membranes. in contrast to this, bim-46187, a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation 3 . interestingly, inhibition of high affinity agonist binding by fr900359 was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr900359, combined uncoupling of both gi and gs proteins from m1 was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m1 receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [1] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [2] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [2] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m1 machr which preferentially activates g proteins of the g q/11 type but can also promiscuously stimulate g s proteins. g q/11 -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m1 machr in ip1 and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper-6-naph completely lost intrinsic activity for both g q/11 and g s activation at the m1 machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper-7-phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper-8-phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m1 machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/11 -mediated signaling, activation of the g s protein in m1 machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m1 machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades 3-(1h-imidazol-4-yl)propylguanidine (sk&f-91,486 (1) [1] ) is known as the prototypic pharmacophore of highly potent histamine h 2 -receptor (h 2 r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [2] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f-91,486, we characterized 78 newly synthesized derivatives including several bivalent compounds (e.g., 2) by using different pharmacological in-vitro methods. [3] the potential h 2 r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf9 cells [4] (hh 1,2,3,4 r). compounds were also functionally characterized in the [ 35 s]gtpgs assay (hh 2 r, sf9 cell membranes). [5] selectivity vs. hh 1,3,4 r was determined for selected derivatives also using this technique. organ bath studies (gph 1 r (ileum), gph 2 r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f-91,486 analogues displayed partial or full agonism via hh 2 r and gph 2 r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine 2, was a partial agonist (e max = 88%) and 250-times as potent as histamine vis-à-vis the gph 2 r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa 2 values for the antagonist (5.87 -6.38) which are in accordance with literature data. however, in the functional in-vitro assay on gph 2 r, the positive chronotropic response evoked by sk&f-91,486 was surprisingly resistant to antagonism by cimetidine and other typical h 2 r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h 2 r agonist. this behaviour is unique within the large series of sk&f-91,486 analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [1] parsons, m.e. et al.; agents actions 1975, 5, 464. [2] buschauer, a.; j. med. chem. 1989 , 32, 1963 -1970 [3] pockes, s.; dissertation, univ. regensburg 2015. [4] seifert, r. ; j. pharmacol. exp. ther. 2003, 305 (3) , 1104-1115. the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine 351 (s351) and threonine 362 /serine 363 (t362/s363), which enabled us to selectively detect either the s351-or the t362/s363-phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch221510 and ro64-6198 induce a stably phosphorylation at s351 and t362/s363 followed by a profound internalization of the receptor. the nociceptin-induced s351 and t362/s363 phosphorylation can be blocked by selective nop receptor antagonists such as j113397 or sb612,111. nnc63-0532, buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s351 phosphorylation occurred at a faster rate than phosphorylation at t362/s363 indicating that s351 is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol 12-myristate 13-acetate facilitated receptor phosphorylation only at s351 but not at t362/s363, indicating that s351 can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t362/s363 in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch221510 failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling (1). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation (2) . in man, two cc chemokine receptors ccr2a and ccr2b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin (2) . interested in differential signaling of ccr2a and ccr2b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk1/2) in the presence of ccl2, ccl8, ccl7, and ccl13. all these chemokines have been shown to interact with human ccr2 receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr2 receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr2b receptors by ccl2 induced both rho-dependent srf activation and receptor internalization, while ccl8-stimulation resulted in srf but little if any receptor internalization. in contrast, ccr2a-expressing cells showed ccl2dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr2 receptors for coupling to g proteins revealed that arginine 313 within the putative 'eighth helix' of the carboxyl-terminal portions of ccr2a and ccr2b is not involved in ga i -mediated induction of erk1/2 and plays a minor role in ccr2b receptor internalization, but is specifically required for the ccr2a/ and ccr2b/ga q -mediated activation of srf. serotonin 5-ht 2c receptors (5-ht 2c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). 5-ht 2c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express 5-ht 2c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa2-10 cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where 5-ht 2c r in vivo expression has been detected. thus, we tested mhypoa2-10 cells for putative 5-ht 2c r expression by performing single cell calcium imaging. we observed that serotonin and the specific 5-ht 2c r agonist, way161,503 induced robust calcium transients, which were strongly inhibited by two unrelated 5-ht 2c r-selective antagonist (sb206,553, rs102,221). serotonin and way161,503 also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases-1/2 in a sb206,553 and rs102,221 sensitive manner, providing further evidence for functional 5-ht 2c r expression in mhypoa-2/10 cells. intrinsic activity of way161,503 ranged between 0.3 and 0.5 compared to serotonin in all assays, defining way161,503 as a partial 5-ht 2c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa-2/10 cells endogenously express 5-ht 2c r and thus represent the first cell line to analyse 5-ht 2c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub 1 1 walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with 3 h-adenine and calculation of the conversion rate to 3 h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α 2a or µ 1 opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek293 cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc5 or heteromeric kir3.1/kir3.2 or kir3.1/kir3.4 channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp2a mice leading to double transgenic mice (h 2 xpp2a = dt). pp2a mice (j biol chem 2004; 279:40827) overexpress the catalytic subunit of protein phosphatase 2a (pp2a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about 240 days we noted reduced ef in pp2a (33.1 ± 3.5 %, n=16) compared to wt (54.4 ± 4.5 %, n=10) and tg (59.8 ± 2.9 %, n=10). interestingly, in dt the ef (43.9 ± 4.9 %, n=11) was higher than in pp2a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp2a is involved in h 2 -receptor signaling and we tentatively conclude that the h 2 -receptor is able to ameliorate systolic but not diastolic cardiac function of pp2a mice. serotonin (5-ht) can exert positive inotropic and chronotropic effects in humans via 5-ht 4 -receptors. we have generated transgenic mice (tg) which overexpress the human 5-ht 4 -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with 30 µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and 3, 7 and 24 hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to 58.8 ± 20.7% seven hours after lps in wt and to 61. [4] [5] [6] [7] [8] [9] [10] [11] [12] p<0 .05 vs pre-lps value). however, 24 hours after lps, diastolic function, measured as e/a, amounted to 1.86 ± 0.43 in p<0 .05 tg vs. wt). moreover, after 24 hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after 3 and 7 hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from 1158.3 ± 212.2 to 782.5 ± 154.3 in wt and from 1208.4 ± 235.1 to 798.8 ± 170.1 in tg (tg vs. wt, p>0.05, n=10-12; after 7 hours). we tentatively conclude: 5-ht 4 -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec 50 = 34 nm) or amthamine (ec 50 = 10 nm), a more selective and potent h 2 -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to 44 mm potassium chloride. thereafter, histamine (100 µm) induced a pie (31.1 ± 10.4 % of control, n=7) in tg but not in wt preparations whereas isoprenaline (10 µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound 48/80, a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine (10 µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h 2 -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk2, a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek293t cells transfected with yfp-tagged µor and mturquoise-tagged grk2 as well as non-fluorescent gα i1 , gβ and gγ subunits showed a robust increase in fret upon superfusion with 10 µm [d-ala 2 , n-mephe 4 , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine (30 µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately 15-20% of that of the corresponding damgo signal. grk2 binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk2 affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i1 and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i1 and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk2 on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk2 interaction with that of the µor-gβγ interaction in the presence of grk2 revealed similar time constants both for the on-and off-kinetics (grk2: k on 0.16 s ). this suggests that, in the absence of grk2, the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk2, however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk2-gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor in the absence and presence of grk2 and compared the kinetics with the kinetics of grk2 binding and unbinding to these receptors. in both cases, we found that grk2 and gβγ in the presence of grk2 associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c57bl/6 mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin-1 (et-1) for 90 sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in 3d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et-1 treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et-1 had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin-3 was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn1 and ccn2 to be moderately up-regulated after 24h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et-1 treatment. finally, we could show that the perinuclear localization of cav3 and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production (1). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs (2) (3) (4) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac1-camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine 1-phosphate (s1p) is generated by sphingosine kinase (sk)-1 and -2 and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s1p 1-5 . after activation, s1p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s1p 1 mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru-486. in addition, in vivo studies showed that dexamethasone downregulated s1p 1 expression in glomeruli isolated from c57bl/6 mice treated with dexamethasone (10 mg/kg body weight). functionally, we identified s1p 1 as a key player mediating s1p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s1p-induced migration of mesangial cells. this effect was again reversed in the presence of ru-486. conclusion: we suggest that dexamethasone inhibits s1p-induced mesangial cell migration via downregulation of s1p 1 . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. 2015; 396: 803-12) . the g protein-coupled receptor mrgd is a receptor for angiotensin-(1-7) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-(1-7) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-(1-7). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek293 cells, we provided final pharmacological proof for mas to be a receptor for ang-(1-7). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-(1-7). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type 2 receptor at2 as a receptor for the heptapeptide, but discovered that the at2 blocker pd123119 can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-(1-7) but excluding the at2 receptor, and by enforcing the revisit of such publications which concluded at2 function by using pd123119 as a specific at2 blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within 1 ms 1 . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells 2 . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between 30-80 ms 3 . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor 1 (mglur1) sensors by fret after uncaging of an inert glutamate derivative. 4-methoxy-7-nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur1s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur1 protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur1 activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α 2aadrenergic receptor, among others receptors, has been extensively studied in single cell assays (1) (2) . the activation kinetics of the labeled α 2a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α 2a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology (3) . additionally, we used halotag in combination with the nanoluc (4) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α 2a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec50 value in accordance with the previously published data. this suggests the functionality of the fret-based α 2a -halo/cfp sensor. similar results were obtained with the α 2a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α 2aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin2 biosensors. upon stimulation, β2-adrenergic receptors bound β-arrestin2 with a time constant τ = 1.3 ± 0.17 s, indicating that β-arrestin2 binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin2 with a τ = 2.2 ± 0.22 s. stimulation of β2-adrenergic vs. m2 muscarinic or ffa4 receptors resulted in different patterns of conformational changes in the various β-arrestin2 sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin2 activation. upon agonist removal, first the interaction (delay = 1.9 ± 0.51 s) and only then the active state of β-arrestin2 (delay = 4.2 ± 0.85 s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β2-adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about 1,000 fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi10463 (historical strain), strain 1479 (serotype f, variant tcdbf), strain r20291 (hypervirulent strain, ribotype o27), and strain r9385 (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain 1470) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi10463. in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p63rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in 2d and 3d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p63rhogef-knockout mice were adenovirally transduced for 48 to 72 h with recombinant adenoviruses or directly used. for 2d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p63rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p63rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p63rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p63rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p63rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p63rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p63rhogef implies that p63rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p63rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p63rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il-6 and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/11 , g 12/13 and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr1 (r206h), a bmp (bone morphogenic protein) type 1 receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c2c12 cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp4-induced osteoblast differentiation in c2c12 cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr1 r206h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p63rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p63rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p63rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for 48 h in culture. for activation of the g q/11 -signaling the cells were treated with endothelin-1 (et-1), angiotensin ii (ang ii) or phenylephrine (pe) for 90 s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p63rhogef is involved in the regulation of the et-1-induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p63rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor 3 (kdelr3) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p63rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin-3 the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p63rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p63rhogef in the transport of membrane proteins, we overexpressed p63rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr3 and its co-localization with p63rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr3 was found in control-transduced cells. further, p63rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p63dn and detected similar changes with respect to kdelr3 localization and golgi structure suggesting that this regulatory function of p63rhogef is not dependent on its activity. conclusion: p63rhogef mediates the activation of rhoa from gpcrs coupled to g q/11 proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/11, g12/13) exist in mammals and these must suffice to convey signals from about 800 g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. 1 understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr900359, a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/11/14 over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek293 cells that lack functional gq/11 alleles by crispr-cas9 genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/11 using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") 2 . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. 2009 ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept2, 6, and 7 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc42 and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb1 thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc42, borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [1] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [1] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [2] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at 24 hours post fertilization, larvae at 5 days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [3] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac10). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [4] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [1] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences 40, 8-15 (2015) . ccmp, 3',5'-cump and 3',5'-cimp were ineffective. to further characterize the action of 3',5'-cgmp on hut-78 cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). 3',5'-cgmp significantly increased apoptosis (ec 50 = 75 µm) and inhibited proliferation (ec 50 = 63 µm) of okt3-activated hut-78 cells. interestingly, also 2',3'-cgmp exhibited comparable effects on apoptosis and proliferation with ec 50 values of 92 µm and 75 µm, respectively, while 2',3'-camp, 2',3'-ccmp and 2',3'-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested 3',5'-cgmp degradation products under the same experimental conditions and found that both 5'-gmp and guanosine increased apoptosis and inhibited proliferation with ec 50 -values between 50 and 100 µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of 3',5'-cgmp, 2',3'-cgmp, 5'-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [1]. 3',5'-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut-78 cells (e.g. enpp1), yielding 5'-gmp, which could be further degraded to guanosine by the 5'-ekto-nukleotidase cd73. a similar pathway may lead from 2',3'-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms 3a and 3b as well as the cgmp-selective pde9a also degrade the emerging second messenger cump [1, 2] . we analyzed the enzyme kinetics of pde3b-mediated cump-hydrolysis using recombinant gst-tagged pde3b and a highly sensitive and specific hplc-ms/ms method. our data show that pde3b is a low-affinity enzyme for cump with a cump k m -value of >100 µm. the pde3-selective competitive inhibitor milrinone inhibited pde3b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde3b is highly expressed in adipose tissue [3, 4] . thus, we differentiated murine 3t3-l1-mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde3b expression and basal cnmp-concentrations. in both differentiated and undifferentiated 3t3-l1 cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by 66 % and 60 %, respectively, while ccmp was reduced by 78 % and cump even by 85 %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde3b was upregulated ~1000-fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde9a-mediated cump hydrolysis. pde9a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi500. they comprise a "green" membrane-targeted version (mcgi500) and a "red" variant (red cgi500) that contains the fluorophores tsapphire and dimer2. methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi500 sensor showed a good signal-to-noise ratio, an ec 50 value of ≈1.1 µm for cgmp, and a high selectivity for cgmp over camp (>100-fold). flincg3, a non-fret-based cgmp sensor, showed similar properties as cgi500. the new membrane-targeted mcgi500 and the new red cgi500 displayed ec 50 values of ≈0.4 µm cgmp and a high selectivity for cgmp over camp (>300-fold). in vsmcs, the red cgi500 showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de5. the "green" fret-based camp sensor, epac1-camps, showed a signal-to-noise ratio comparable to that of cgi500, an ec 50 value of ≈3 µm for camp and a selectivity for camp over cgmp of ≈6-fold. finally, imaging of cells expressing both the epac1-camps and the red cgi500 demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi500 would be useful to investigate membrane-associated cgmp compartments and red cgi500 to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at1-receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [1] . whether this causes an activation of cgks is not known. these enzymes exist in 3 different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [2] , [3] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet (0.02% na + ) and enalapril (10mg/kg/d) for 3 weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay41-8543, a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw1/lrmp (lymphoid-restricted membrane protein) is a type 2 membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a 539 amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [1] , [2] . jaw1 and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw1 and irag share the highest homology [3] , [4] . the coiled coil domains of irag and jaw1 are important for the interaction with ip 3 rs. as already known, irag forms a trimeric complex with cgkiβ and ip 3 r1 and gets phosphorylated by cgkiβ [5] . hence we examined if jaw1 is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw1 possible phosphorylation sites can be found. our in vitro studies with jaw1 and cgki showed that jaw1 gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw1 was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw1 and cgkiβ was detected. to examine the importance of jaw1 in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw1. mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw1 was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw1 influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw1. hence, the function of jaw1 in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (camp) and guanosine 3',5' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp 1 and 2, hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine 3',5'-cyclic monophosphate (ccmp) and uridine 3',5'-cyclic monophosphate (cump) also fulfill the criteria of second messengers [1, 2] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [3] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap9 (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [4] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap9) were examined as potential ccmp and cump target proteins. hek293 cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor 2 (npr2, also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase 2a (pde2a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde2a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor 3 (npr3) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e12.5 embryos. because npr3 binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr3 could lower the activity of the cnp-npr2-cgmp signaling cascade in drg neurons. in the absence of npr3, the majority of sensory axons showed normal bifurcation, but »13 % of the axons turned only in rostral or caudal direction. this study shows (1) that pde2a is important for the degradation of cgmp in embryonic drg neurons, (2) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde2a, and (3) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi500). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d-2-amino-5-phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac1-camps fused to a phosphodiesterase (pde4a1). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than 100 different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families 3, 4 and 5) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde4. however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde2a3 (v max = 120 µmol/min/mg) to a camp fret sensor (epac1-camps) we monitor camp concentrations in direct vicinity of pde2a3. in comparison to pde4, we show that pde2a3, albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j774 macrophages, arglabin strongly induced accumulation of lc3 type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc3-ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp3 and pro-interleukin (il)-1β, we further analyzed whether arglabin inhibits the nlrp3 inflammasome. arglabin reduced expression of nlrp3 and pro-il-1β, inhibited activation of caspase-1, and release of mature il-1β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp3 inflammasome. intraperitoneal injection of arglabin into female apoe2.ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il-1β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) 2 stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par2 activating peptide sligrl (100µg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), losartan (3 mg/kg) and furosemide (40 mg/kg) in conscious wild-type (wt) and par2-deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet (0.6% nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par2-deficient mice than in wild-type mice (190 ± 35 versus 380 ± 91). renin mrna expression was 50 ± 9% of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par2-deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par2 (-/-) and wt mice. administration of sligrl increased renin secretion about 6-fold (p<0.01). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par2 (-/-) and wt mice. the absolute changes (delta prc) were similar (3780 ± 770, 2230 ± 400, 2780 ± 70, 1390 ± 460 in wt, and 2320 ± 270, 2530 ± 250, 3010 ± 210, 1230 ± 470 in par2 (-/-)). in conclusion, chronic absence of par2 reduces basal renin expression and renin release. however, par2-deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par2 does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca 2+ sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin-3-gallate (egcg), which has been shown to decrease myofilament ca 2+ sensitivity, on cardiac myocyte contractility and force-ca 2+ relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc3-targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca 2+ transients at 37 °c under 1 hz pacing using the ionoptix system in the absence or presence of egcg (1.8 µm). furthermore, force-ca 2+ relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg (30 µm). results: in baseline settings and absence of fura-2, ki cardiomyocytes displayed higher sarcomere shortening ( type-1 serine/threonine protein phosphatase (pp1) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca 2+ handling and contractility. all pp1 heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor 2 (i-2). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i-2 resulted in an improved basal ca 2+ handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i-2, regulated by gsk3-dependent phosphorylation at thr 72 , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, 30 mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with 0.9% nacl (n=5). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca 2+ transients were measured in isolated, indo-1-loaded myocytes. the peak amplitude of [ca] i was reduced by 39% in tg nacl compared to wt nacl (p<0.05), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by 25% in tg iso compared to wt iso (p<0.05). consistently, sr ca 2+ load was diminished by 28% in tg nacl compared to wt nacl (p<0.05). chronic iso stimulation led to an unchanged sr ca 2+ content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than 4-fold higher phospholamban phosphorylation at ser 16 in tg (p<0.05). thus, these findings suggest that overexpression of i-2 is able to reduce the progression of heart failure by an improvement of myocyte ca 2+ handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., 168 derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of 56 virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp-1 macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a1 (abca1) expression in human thp-1 macrophages by this leoligin derivative. the transporters abcg1 and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock1 and rock2 are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock1 and rock2 are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock1 and rock2 by 75% was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with 10 μm fasudil or 3 µm h1152p for general rock inhibition and 3 µm slx-2119 for inhibition of rock2. protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock1 and rock2 influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock1 and rock2 in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock2, but not of rock1 led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock2 in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h1152p and qpcr analysis of cf with a downregulation of rock1 and rock2 showed that both rocks are involved in the regulation of ecm proteins, such as collagens 4a2, 6a, and 8a1, biglycan, decorin, elastin and its respective degrading enzyme mmp12. conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, 2011] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, 2014] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p1 mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm-1 as a nucleus-specific marker was used to enrich cm nuclei to >95%. proteins were extracted from nuclear lysate with 2% sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from 3 murine cell lines served as silac (lys8/arg10)-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified 1041 proteins, 688 of which are annotated to the nucleus. 21%/23% (adult / p1) of nuclear proteins are annotated as dna-binding. 1.5%/1.4% belong to transcription factor complexes; 7.3%/7.5% are able to bind transcription factors. 7.9%/8.3% have chromatin modification functions; 4.3%/4.4% modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. 71% of proteins are shared between p1 and adult nuclei. 142 proteins are exclusive to p1, 34 are only found in adult. 93 proteins are enriched (1.3-fold increase in abundance) in p1 hearts, 89 proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p21 cip1/waf1 , senescence-associated ß-galactosidase (sa-ß-gal) and p16 ink4a were increased 2-, 8-and 20-fold (n=5-7; p < 0.01), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β1-adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin (92 ± 0.9%), platelet derived growth factor receptor α (94 ± 0.9%) and α-smooth muscle actin (71 ± 2.3%), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor 1 (cb1) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb1 receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, 1,5 mg/kg/day) with osmotic minipumps for 14 days. treatment with cb1 antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb1 antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb1 antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb1 ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna-181a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb1 antagonist treatment and foxb1 (a direct target of mir-181a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb1 receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb1 antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir-181a/b family seems to be sensitive to cb1 antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb1 inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f2α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type 2 diabetes mellitus (t2dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t2dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: 10-week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or respective placebo for 20 weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in 3t3-l1 cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd11c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il-6 from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using 3t3 l1 cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca 2+ -atpase (serca2a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca2a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. 1 using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca 2+ cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca 2+ kinetics were measured in isolated, fura2-loaded and electrically paced (0.5hz) cardiomyocytes as the time to 50% decay of the ca 2+ signal (t 50% ). the time to 50% baseline of sarcomere length (t 50% baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed 3.3-fold stronger phosphorylation signals if pentamers were absent (p<0.0001). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t 50% [ms]: 92±8 and 122±8, respectively, p<0.05). notably, t 50% of pln-ko myocytes was 79±5ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca 2+ clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t 50% baseline [ms]: 32±8 in tgpln versus 7±4 in tgafa-pln and 5±7 in pln-ko, p<0.05). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca 2+ kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the 3' end of mrnas (3' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous 3' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih-3t3 fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor-2 (par2) regulates inflammatory responses including monocyte migration and cytokine release. par2 is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine-1-phosphate (s1p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s1p on the expression of par2, tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp1 and u937 cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par2, tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with 50 ng/ml pma (phorbol 12-myristate 13-acetate) over 72 h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp1 and u937 cells s1p induced a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of par2 mrna and total protein expression. par2 total protein was upregulated maximally (about 1.5-fold, n=7) with 1 µm s1p after 16 h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp1 monocytes and tm was not regulated by s1p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s1p resulted in a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of tf expression within 3 to 6 h of incubation. conversely, par2 total protein expression was reduced by about 50% after 24 h s1p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp1 cells as tf source. timedependent incubation with s1p (1 µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from 8.0±1.6 to 6.5±1.4 min and elevated total the thrombin generation capacity from 814±130 to 1286±129 nm. peak thrombin formation was elevated from 66±31 to 130±30 nm/min (control versus s1p for 6h, mean±sd, n=5, respectively). these data suggest that s1p induces an enhanced expression of par2 in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s1p does upregulate tf expression but attenuates par2 levels. since par2 is involved in regulation cell migration, s1p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin-6 (il-6) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il-6 expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il-6 mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp-1 cells was determined by western blot. immunostainings for il-6 and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa (30 nm) induced a time-dependent (3 -16 h) increase in il-6 mrna expression. maximum expression was observed within 6 h to a 7.2±3.3 fold increase (mean±sd, n=5, p<0.05). incubation with an inhibitor of p38 map kinase (sb203580, 10 µm) or pi3k (ly294002, 10 µm) significantly attenuated fxa-induced il-6 mrna expression (n=5). inhibition of p42/44 mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for 24h resulted in a markedly increased il-6 secretion into the smc culture media from 0.6±0.2 to 1.1±0.3 ng/ml (p<0.5, n=4). stimulation of thp-1 cells with il-6 (1 ng/ml) induced a time dependent (6 -24 h) increase of up to 2.1±0.6 fold (p<0.05, n=5) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il-6 expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il-6 regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il-6 expression and secretion in venous smc which may be regulated via p38 and pi3k signaling. il-6 enhanced tf expression in thp-1 monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il-6 release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p300). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week 5-16 with vpa (0.71% in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week 7 and 16). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week 7: tg veh : 33±2%, wt veh : 47±1%; week 16: tg veh : 19±2%, wt veh : 44±1%, p<0.05). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week 7: 40±2%, tg vpa at week 16: 34±1%, p<0.05 vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p<0.05) 998 proteins were significantly changed while 854 proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p<0.05). 109 proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p<0.05), whereas vpa affected 525 proteins in tg atria (tg vpa vs. tg veh , p<0.05), out of which 43 proteins were common. 295 prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h 2 -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine (1 mm), shifted the concentration response curves for the pie of histamine from ec 50 = 110 nm to 37 nm (p<0.05). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine (10 µm), shifted the pie of histamine from ec 50 = 70 nm to 38 nm and increased the efficacy of histamine for the pie (p<0.05). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp1, pp2a, pp2b (calcineurin) and pp5 in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase 2cβ (pp2cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp2cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased (4.24 ± 0.14 mg/g) compared to wild type (wt) littermates (3.78 ± 0.20 mg/g; p<0.05) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg (4.78 ± 0.17 mg/g) vs. wt (4.13 ± 0.22 mg/g; n=8-12; 10-11 months of age; p<0.05). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: 58.33 ± 3.16 % (tg) versus 76.94 ± 0.92 % (wt); n=12-16; 8-11 months; p<0.05 and fractional shortening: 30.84 ± 2.02 % (tg) versus 44.94 ± 0.87 % (wt); n=12-16; 8-11 months; p<0.05). the left ventricle was dilated (systolic diameter: 2.78 ± 0.21 mm (tg) versus 1.84 ± 0.06 mm (wt); n=12-16; 8-11 months; p<0.05; diastolic diameter: 3.97± 0.19 mm (tg) versus 3.33 ± 0.07 mm (wt); n=12-16; 8-11 months; p<0.05). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp2cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein 5 (igfbp5) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein 5 (igfbp5) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp5 functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp5 in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp5 in murine cardiac tissue at different developmental stages by qpcr normalized to tpt1 (tumor protein, translationally-controlled 1). this analysis showed temporal changes of cardiac igfbp5 expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp5 expression in different heart cells showed a very low igfbp5 in adult cardiomyocytes in contrast to a high expression in undifferentiated sca-1 positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp5 was found up-regulated (p<0.05). further we found an increased igfbp5 expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p<0.01). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp5 upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p<0.05). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp5 expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp5 in angiotensin 2 or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin 2, showed the opposite results, namely a reduction in its expression (p<0.01; p<0.05, respectively). summary and conclusion: our results show active igfbp5 transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp5 in a conserved manner. we hypothesize that igfbp5 may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp5 is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c57bl/6j mice fed a high-fat diet (hfd) for 22 weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last 4 weeks with crataegus extract ws® 1442 (150 mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® 1442, neither in pvat nor in aorta. phosphorylation at serine 1177 is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine 1177 in pvat, but not in aorta. ws® 1442 treatment significantly improved enos serine 1177 phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine 1177 phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® 1442 treatment. in addition, ws® 1442 treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin-1 (sirt1), known also as a longevity gene. the activity of sirt1 depends, among others, on the intracellular content of its cofactor nad. ws® 1442 treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt1 is enos. deacetylation of enos at lysine residues 497 and 507 by sirt1 enhances the activity of the enos enzyme. currently, we are studying the effect of ws® 1442 on nad synthesis, sirt1 activity, and enos (de)acetylation. in conclusion, crataegus extract ws® 1442 reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine 1177 and upregulation of sirt1. the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk2) and the raf-erk1/2 pathway. these two functions of rkip could counteract each other. while grk2 inhibition is cardio-protective, inhibition of the pro-survival erk1/2 axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. 1). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp1) under control of the alpha-mhc promoter. two different rkip-transgenic lines with 2.7-fold and 3.4-fold increased cardiac rkip protein level were generated (ref. 2, and jax id number 911819). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as 10 weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of 32.9±2.3 % (n=6; ±s.d.) were documented at an age of 8 months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk2 and raf-erk1/2 axis by the human rkip gene (pebp1) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp1), which does not seem to inhibit the raf-erk1/2 axis in vivo but instead confers grk2 inhibitionheterodimerization between the at1 receptor (at1r) for the vasopressor peptide, angiotensin ii, and the b2 receptor (b2r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at1r--b2r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at1r--b2r heterodimer in vivo, we generated a transgenic model of at1r--b2r heterodimerization (tg-b2r+) by transgenic expression of the b2r gene (bdkrb2) in the b2r-deficient tg-b2r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at1r and b2r at a distance of less than 9 nm in tg-b2r+ mice whereas the at1r--b2r heterodimer was absent in tg-b2r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b2r in the aorta of tg-b2r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b2r. the efficient at1r--b2r heterodimerization in tg-b2r+ mice was accompanied by an enhanced angiotensin ii at1r-stimulated vasopressor response relative to that of tg-b2-/-mice, which lack the at1r--b2r heterodimer. as a control, the endothelin-1-stimulated vasopressor response mediated by the etar, which did not dimerize with b2r, was not significantly different between tg-b2r+ and tg-b2r-/-mice. together these findings provide strong evidence that at1r--b2r heterodimerization occurs in vivo and enhances the angiotensin ii at1r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase 2 (grk2) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf24 extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk2, grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk1/2 axis and involved the erk1/2-dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine-273. as a consequence of erk-dependent phosphorylation of pparg on serine-273, the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine-273 phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine-273a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk2 inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as 5 weeks of age in homozygous mice and 10-12 weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac2 inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from 12 weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for 7 weeks with vpa (0.4 mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by 7 mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of 90.2±8 v/s (tg: n=33 cells) vs. 109.8±5.1 v/s (wt: n=30, p=0.05). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at 50% (tg: 11.6±1.4 ms, n=35 vs. wt: 6.9±0.5 ms, n=30, p<0.01), at 70% (in ms: 21.2±2.5 vs. 13.6±1, p<0.05) and at 90% (in ms: 46.8±4.5 vs. 34±2.5, p<0.05) repolarization. vpa treatment reduced ap amplitude in wt mice by 6 mv (n=22, p<0.05) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced (50%: 7.3±0.8 ms, 70%: 13.8±1.5 ms, 90%: 30.9±3.2 ms, n=21, p<0.05 vs. untreated tg), the amplitude was increased by 5 mv (n.s.) and the slope of depolarization was increased by 21% (p=0.16, n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: 104±7.2 pf, n=27 vs. wt: 55±5.4 pf, n=30, p<0.001), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: 64.8±7 pf, n=17 n.s. vs. wt; tg-vpa: 88±7 pf, n=14, p<0.05 vs. wt-vpa; p<0.01 vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac2 inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca 2+ exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca 2+ imaging revealed that the transduction rate of spontaneous sub-threshold ca 2+ -waves into supra-threshold transient-like ca 2+ -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca 2+ transport rate (r serca ) was enhanced in tg vcms potentially limiting ca 2+ extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during 10 -6 m isoproterenol challenge, tg: 3.65*, ctl: 0.4; n=20/condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx1, serca2a and decrease of kchip2 protein levels. however, the respective mrna levels (slc8a1, atp2a2 and kcnip2) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir-369 (fold change in tg: 0.04*) and mir-1 (fold change in tg: 0.13*) (n=10/condition). atp2a2 is a predicted target of mir-369 and mir-1 has recently been shown to regulate ncx1 and i to related potassium channel subunits. (*p<0.05 vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. 8 days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from 6.2(mg/g) in untreated control animals to 10.9(mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of 7.7 (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr (8.4 mg/g) compared to saline treated animals (6.8mg/g) and pka mutants (7.1 mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: 6.5 vs 7.9 mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh6/7 ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged (6.9 mg/g) or a strongly elevated hbr (13.5 mg/g). however, additional hypertrophic factors including anp, myh6/7 ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of 100 µm after 24 and 48 h (ctb assay) nor increase in the apoptosis rate after 24, 48, or 72 h up to 100 µm. measuring adenosine triphosphate (atp) levels revealed that 30 µm soraphen a does not alter the atp levels in huvecs after 24 h treatment. in contrast, a 48 h treatment significantly lowered the atp levels by 12 %. also gene silencing of acc1 in huvecs attenuated the atp levels by 11 %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels (10 %) in soraphen a-treated cells after 24 h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic 50 value of 34 µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g 0 /g 1 phase by 26 % and increases the number of cells in the g 2 /m phase by 50 %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, 30 µm soraphen a lowered the migration of endothelial cells by 65 %. gene silencing of acc1 in huvecs strongly decreased endothelial migration, whereas a knockdown of acc2 had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by 34 %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by 60 % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type 9 (pcsk9) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october 2015. we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor 15 (klf15) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf15 and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w2 domain containing protein 2 (bzw2), a phylogenetically conserved protein, as a β-catenin and klf15 interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw2 mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw2 is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p<0.05, cm n=4, cfb n=4, cpc n=2), and skeletal muscle. bzw2 was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw2, containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw2 protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf15 knockout (ko) mice **p<0.01 n=3, and in a cardiac specific β-catenin stabilized mouse model, **p<0.01 n=6). a mouse model with constitutively bzw2 loss of function (bzw2 ko) showed cardiac specific upregulation of β-catenin on rna level (***p<0.001, p<0.05, ctrl n=4, bzw2 ko n=5) and on protein level (**p<0.05, ctrl n=7, bzw2 ko n=9). echocardiography analysis in eight-weeks-old bzw2 ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw2 expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p<0.05 ctrl n=4, angii n=3) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p<0.05 ctrl n=3, dcm n=11, icm n=10). conclusion: these data demonstrated that bzw2 is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine-1-phosphate (s1p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s1p lyase [1] . mice that lack s1p lyase are characterized by the accumulation of s1p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [2] [3] [4] . on the other hand, embryonic fibroblasts from s1p lyase-deficient mice (sgpl1 -/--mefs) are resistant to chemotherapy-induced apoptosis [5] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [6] . interestingly, s1p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [7] . the aim of the present study was to analyze the link between s1p lyase deficiency and altered cholesterol homeostasis using sgpl1 background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine-2 (p-ser2) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser2-pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after 4 weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm1 antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> 0.062 fpkm, gene expression rank < 12,653) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser2-pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type 1 (tnni1) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni1 gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin 1 (acta1) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta1 after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase 1 is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt1 in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt1 gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt1 fl/fl mice with heterozygous dnmt1 fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl7-cre) resulted in embryonic deletion of dnmt1 (ko). embryos without myl7-cre served as controls (ctl). embryos were dissected and genotyped at e11.5, e12.5, e13.5 and e14.5. rna-seq and pyrosequencing of genomic dna was performed on e11.5 hearts, histology on e12.5 hearts and electron microscopic imaging on e13.5 hearts. for deletion of dnmt1 in adult mice, homozygous dnmt1 fl/fl mice expressing an inducible cre recombinase (myh6-mcm) were given tamoxifen i.p. over 4 days. homozygous dnmt1 fl/fl mice not carrying myh6-mcm as well as myh6-mcm carrying mice without dnmt1 fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl7-cre mediated loss of dnmt1 resulted in progressive embryonic lethality with absence of living ko embryos after e14.5. ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt1 in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt1 in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt1 is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet3 involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet3. methods: female mice homozygous for tet3 flox and male mice heterozygous for tet3 flox and heterozygous for myl7-cre were mated and offspring were genotyped after weaning. mice homozygous for tet3 flox and heterozygous for myl7-cre (ko) or homozygous for tet3 flox without myl7-cre (controls) were sacrificed at 12 weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly 50 %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet1 and tet2. cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp2a2 gene and at the binding site of the transcription factor gata4 but not near the promotor and the binding site of the transcription factor tbx5. higher methylation levels were not accompanied by changes in atp2a2 expression. however, both myh7 and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet3 is involved in dna demethylation in cardiomyocytes. loss of tet3 expression resulted in embryonic lethality. compensatory upregulation of tet1 and tet2 isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet3 in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor 16 (pi16). a recent gwas analysis showed an association of a genetic variant in the pi16 genomic locus (rs1405069) with chemerin plasma levels. here we tested the hypothesis that pi16 determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi16, which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi16-deficient animals compared to littermate controls. to investigate whether pi16 and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi16 to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi16 would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi16. when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi16 to potently inhibit cathepsin k activity. taken together, we propose pi16 to act as a regulator of chemerin processing. the transient receptor potential canonical 6 (trpc6) is a second messenger-gated cation channel, which mediates depolarization and ca 2+ entry. it is known to be activated by diacylglycerol derivatives (dag, 1-oleoyl-1-acetyl-sn-glycerol oag) [1] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc6 gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [2] thus, the discovery of potent inhibitors of trpc6 may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc6. [3] larixyl acetate, another component of the resin showed an even higher potency in trpc6 inhibition (ic 50 = 0.26 µm) and a 12-fold selectivity compared to trpc3. these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc6. hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c1 position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc6 with similarly low ic 50 values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c1 position, however, seems to play no important role for the inhibition of trpc6 currents. larixyl methylcarbamate lead to trpc6 inhibition with an ic 50 value of 0.15 ± 0.06 µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc6, this compound bears the benefit of high subtype selectivity towards trpc3. even with concentrations up to 50 µm of larixyl methylcarbamate, no complete inhibition of the ca 2+ influx via trpc3 channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc6 in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α7 nachr using whole-cell patch clamping under voltage-clamping conditions (-70 mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα7 nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α7 nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, 4-tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes (1). among the 28 mammalian trp channels, the canonical channel 5 (trpc5) is a ca 2+ -permeable ion channel, which is predominantly expressed in the brain (2) . many aspects of trpc5 function are still elusive although behavioral experiments with trpc5-knock-out mice suggest a role in innate fear-response (3) and some studies indicate a trpc5-mediated down regulation of neurite outgrowth in nerve cells (4, 5) . to elucidate trpc5 function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc5 modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo (6, 7) . thus, there is still a need for identifying novel and efficient trpc5 modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc5 activator. hek293 cells heterologously expressing trpc5 upon tetracycline induction (hek trpc5 ) show a btd-induced concentration-dependent activation in both ca 2+ assays (ec 50 = 0.71 µm) and in electrophysiological whole cell patch clamp recordings (ec 50 = 1.1 µm). btd elicits currents with an n-shaped i/v curve, typical for trpc5. the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc5 inhibitor (8) . mtt assays revealed that incubating hek trpc5 cells for 24h with btd concentrations above 1 µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca 2+ -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to 10 µm. ca 2+ assays showed no influence of btd on closely related trpc4 channels, as well as trpc3/6/7 at concentrations up to 10 µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc5 subunits can also assemble to heteromeric channel complexes with their closest relatives trpc1 and trpc4 (9) . trpc1/5 and trpc4/5 heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc5 subunit. transient receptor potential canonical channels 3/6 and 7 are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc3, based on the recently available structural information on trpv1, we performed structure-guided mutagenesis and identified a single residue in transmembrane domain 6 (g652), which is conserved within the canonical family of trp channels. single point mutations at position 652 in trpc3 largely eliminated lipid sensitivity. trpc3 g652a expressed in hek293 cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc3/6/7 channels (gsk1702934a) activated wild-type trpc3 and trpc6 channels as well as the respective lipid insensitive mutants (trpc3 g652a, trpc6 g709a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g652 to gsk recognition by trpc3. our results demonstrate the existence of two different mechanisms of trpc3/6 activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc3/6 involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s6 provided by g652. lipids and synthetic activators of trpc3/6 may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb327 (1,1'-(propane-1,3-diyl) bis (4-tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c 1 -c 10 ) of mb327 were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by 100 µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c 7 ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of 100 µm carbamoylcholine in the functional assay. mb327 and several bispyridinium structure analogues (mainly c 2 -c 4 linker) exhibited no regular displacement curves at [ 3 h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the 4-tert-butyl-substituted bispyridinium compound mb327 showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb327 or mb420, could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations (1-300 µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field (20, 50, 100 hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb327 (1,1′-(propan-1,3-diyl) bis (4-tertbutylpyridinium) di(iodide)) and mb420 (1,1′-(propan-1,3-diyl) bis (2-ethylpyridinium) di(iodide)) in the tested concentration range (1-300 µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low (20 hz) and high (50 and 100 hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p2x3 receptor-induced current by the opening of asic3 channels g. stephan 1 , p. illes 1 1 universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p2x3 receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel 3 (asic3) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p2x3 and asic3 subunits has to be taken into consideration. we transfected rat (r)p2x3r and rasic3 constructs individually or together in a ratio of 1:1 into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p2x3r-and asic3-constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from 7.4 to 6.7 caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to 6.5. the opening of asic3 channels failed to facilitate the p2x3r current when 2-guanidine-4-methylquinazoline was used to stimulate a non-proton ligand-sensor of asic3. then, we substituted ca 2+ in the extracellular medium by ba 2+ or decreased the intrapipette concentration of egta, to modify the free intracellular ca 2+ concentration. in cells individually transfected with the receptor-channels, external ba 2+ increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p2x3r-and asic3-specific currents in a similar manner as external ba 2+ . in cells co-transfected with p2x3r/asic3, the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic3 activation. taken together, our results suggest that p2x3r and asic3 interact with each other, since the activation of asic3 had a marked impact on the p2x3r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca 2+ and the proton sensor of asic3 appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned-19. in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc1 specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc1 null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned-19 support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv4 channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv4 has been identified in endothelial cells (1), epithelial cells (2) and arterial smooth muscle cells (3) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in 4). it is a matter of debate however if trpv4 activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc6 (5) which is also expressed in both cell types. to analyze the potential role of trpv4 on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after 90 minutes of ischemia in trpv4-deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv4/trpc6 double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc6 deficiency protected lungs from iri-induced edema (5) . moreover, we identified reduced expression levels of aquaporin 5 in trpv4-deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv4 might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily 3 (trpm3) has been shown to be expressed in human kidney 1, 2 . using newly developed anti-trpm3 antibodies, we are able to visualize trpm3 protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm3 (trpm3 the atp-gated p2x7 receptor (p2x7r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p2x7 -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p2x7r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p2x7r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p2x7r are overexpressed under the control of the endogenous p2x7 promoter. the egfp-tagged p2x7rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p2x7-egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map2-and neun-positive cells indicating that under non-pathological conditions, the p2x7 receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd68 protein was observed in the p2x7r-overexpressing mice. these results suggests that overexpression of p2x7rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd68 primarily localizes to lysosomes and endosomes, this further supports a role of the p2x7r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc1 and tpc2 -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca 2+ channels or such as trp channels. due to their biophysical properties, tpc1 and tpc2 are assumed to be involved in the efflux of ca 2+ from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc1 for vesicle fusion & fission processes we investigated a potential impact of tpc1 on the uptake of bacterial toxins. first we determined the precise localization of tpc1 in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc1 deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc1-deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm3 ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland 1,2 . the trpm3 gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists 3, 4 . variations include the presence or absence of five stretches of 10 to 25 amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini 5 . furthermore, three different aminotermini of human and mouse proteins have been described 5 , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified 12 variants that differ in exons 8, 13, 15, 17 and 20 . however, only variants bearing the short, ca 2+ permeable pore loop were detected. 3´ rapid amplification of cdna ends (3´race) revealed that trpm3 proteins of the pituitary gland carry truncated c-termini exclusively. 5´race identified five independent regions of transcription initiation within the trpm3 gene implying the presence of five independent promotors. the different transcripts encode four trpm3 amino termini α, β, γ and δ of 1-155 amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~76 %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to 16 different bilayers at a time. here we utilize the "orbit" multichannel system with a meca16 chip by ionera to measure tpc1 channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav1) and potassium channels (like kv1, kv4). the voltage-gated na + channel nav1.5 initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv4.3 is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav1.5 and kv4.3 have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) 10 is known to interact with the kv4.3 channel complex, modulating kinetics and voltage dependence. the overexpression of dpp10 in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv4 channels. to study the potential influence of dpp10 on nav1.5 channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k1 cells, expressing scn5a alone or with dpp10. surprisingly, we observed significant effects of dpp10 on nav1.5 channel voltage dependence and kinetics. thus, the co-expression of dpp10 significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav1.5 channels alone. in addition we analysed the effects of dpp10 on the kinetics nav1.5 currents. while time to current peak was not affected in cells co-expressing nav1.5 and dpp10 compared to nav1.5 alone, dpp10 slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav1.5 and dpp10 compared to nav1.5 alone. in summary, we provide first evidence that dpp10 not only interacts with kv4 channels, but also influences nav1.5 channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv4.3 and nav1.5 behave very differently depending on the expression of its associated proteins like dpp10. cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, 2009 ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip 3 ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc1/c4-deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca 2+ -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc1 and trpc4 (camacho londoño et al., 2015) . nevertheless, the role of trpc1/c4 for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal 6 days. our results show that there is no difference in 100 nm at-ii induced ca 2+ -release or ca 2+ -entry in trpc1/c4-deficient cf compared to wt. to evaluate whether the lack of trpc1/c4 can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca 2+ -release and ca 2+ -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca 2+ ] i like isoproterenol, 5-ht, thrombin and endothelin-1. cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv7.1 channels, which are known to be expressed in vascular smooth muscle cells. k v 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l3 (k v 7.1 opener), hmr1556, chromanol-293b (k v 7.1 inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq1-/-mice to determine whether k v 7.1 plays a role in the regulation of arterial tone. we found that r-l3 produces similar concentration-dependent relaxations (ec 50 ~1,4 µm) of wild-type (kcnq1+/+) and kcnq1-/-arteries pre-contracted with either phenylephrine or 60 mm kcl. this relaxation was not affected by 10 µm chromanol-293b, 10 µm hmr1556 or 30 µm xe991 (pan-k v 7 blocker). the anti-contractile effects of pvat were normal in kcnq1-/-arteries. chromanol-293b and hmr1556 did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq1-/-mice exhibited normal peak k v currents. the k v 7.2-5 opener retigabine caused similar relaxations in kcnq1-/-and wild-type vessels. we conclude that k v 7.1 channels are apparently not involved in the control of arterial tone by alpha 1 adrenergic vasoconstrictors and pvat. in addition, r-l3 is an inappropriate pharmacological tool for studying the function of native vascular k v 7.1 channels in mice. introduction: according to international guidelines [1] [2] [3] [4] [5] [6] [7] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [8] [9] . material and methods: tissue the skin samples are descended from female pigs of german landrace (50 kg weight, approx. 4 month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from 10 different skin area (fig. 1) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of 1.000 µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of 30 mm were punched out from the frozen skin stripes and stored at -20°c. hplc and skin absorption waters corporation hplc containing 2767 sample manager, 2545 binary gradient pump, 2998 pda detector (optional: 3100 electron spray mass spectrometer); column: nucleodur® 100-5 c18 ec 50mm x 4.0 mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, 6-cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of 48 h at 32°c. the dosage compartments of each cell were filled with approximately 300 µl of the caffeine solution (10 mg/ml). samples of 1 ml are taken after 0, 12, 24, 36 and 48 hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. 2) . thickness was in the order of magnitude between 897 to 1.230 µm and intra variations were less than 10 %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [10], demonstrating intact barrier properties of the membranes after 3 month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps1000 provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in 2012, the mak commission derived a new threshold value of 0.3 mg/m 3 for gbs with a density of 1, recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio 2 dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day 3, 28 and 90) and the lung toxicity after intratracheal instillation in rats (day 3 and 28): tio 2 (rutile, micro), tio 2 (anatase, nano), eu 2 o 3 (micro-nano mixed), baso 4 (micro), zro 2 (micro) and amorphous sio 2 (nano). two doses of 0.5 and 1.5 µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio 2 (rutile) and baso 4 (pmn < 5% after 3 days in the low dose group; < 15% in the high dose group; full recovery after 28 days). in contrast, the tio 2 (anatase) showed a stronger response (pmn > 30% after 3 and 28 days). the rare earth eu 2 o 3 (micro-nano) dust showed the strongest effect (approx. 40% pmn after 3 and 28 days) including a red-coloured lung lavage fluid. µ-zro 2 and amorphous sio 2 showed a strong acute response after 3 days, however, mostly complete recovery after 28 days. the low solubility criterion was met by the following dusts: tio 2 (both) and zro 2 . -similar volumetric lung burdens were deposited in a parallel validation experiment (14-day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group 1 carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, 25 µm of pyr and 20 µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp1a1 gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp2b6 in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p450 enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p450 enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use 8 compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the 3 test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from 1) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and 2) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of 10 µm (bpa), 3 µm (fen) and 0.01 µm (keto), for concentrations in target organs resulted in estimated oral loels of 16, 4 and 0.04 mg/kg. when calculations were made for plasma levels oral loels were estimated to be 608, 77 and 16 mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of 375 mg/kg bw day for bpa (1), 50 mg/kg day for fen (2) and 6 mg/kg day for keto (3) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal4/uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp3a4 promotor activity was investigated using a pxr-dependent cyp3a4 reporter gene assay. cyp3a4 induction was analyzed at the mrna and protein levels in hepg2 cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp3a4 promotor activity could only be detected for echimidine. cyp3a4 induction by echimidine was verified at the mrna and protein level in hepg2 wildtype and hepg2 pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases (7 isoforms are known, nox 1-5, duox 1 & 2) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox 1-, nox 2-and nox 4-deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600 ng/kg during 28 days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox 2 and nox 4 could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox 1-knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox 1-knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox 4-knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox4. conclusion: separate eliminations of 3 nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last 50 years about 60 drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across 5 species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin 2, podocin, synaptopodin, retinol-binding protein 4, clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of 1,2-intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c3 exoenyzme (c3) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c3 promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c3 lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c3. knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c3. moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c3. proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c3-induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c3 compared to wild type cells. as vimentin knock out did not completely inhibit c3 uptake into cells, an additional uptake mechanism or additional receptor for c3 is likely. nevertheless, our data reveals that c3 employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl 2 on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl 2 was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl 2 . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an 80 compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for 24 h before toxicants were added to the cells. migration and viability of the cells were then measured after another 24 h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, 26 compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed 23 of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein 1 (rip1) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) 1 , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht-22 cells erastin promotes activation of rip1 and subsequent rip1-rip3 necrosome formation which has been investigated as a hallmark of regulated necrosis 2 . in fact, silencing of rip1 by sirna or by the rip1 inhibitor necrostatin-1 prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin-1 fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein-1 (rip1) and rip3 kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) 2, 3 . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip1/rip3-dependent necroptosis 4 and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip1-rip3 complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns5a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns5a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns5a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi-6202, was from medchem express. murine embryonic stem cells (es-d3) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of 0.1% in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after 10 days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between 0.1 and 30 mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of 10 mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in 23, 22 and 2 wells out of 24 wells in total at concentrations of 1, 3 and 10 mg/l. at 30 mg/l no differentiation was observed. effects on cell viability were observed at 30 mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of 1 mg/l this nsa5-inibitor showed a clear impact on differentiation with 6 out of 24 wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi-6206 at concentrations up to 30 mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg 416) and developmental toxicity studies (oecd tg 414) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the 2-generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some 21-days old pups. for all effects clear noaels were determined. in the 2-generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies (1-generation toxicity study and a 3-week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in 2006, dimoxystrobin was classified with r63 (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r63 (which has been translated into the ghs classification repr. 2, h361d) was based on offspring body weight and heart effects seen in the 2-generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in 2009 and a restriction program for pfoa was initiated in 2013. therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek-293t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist 17β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of 17β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by 17β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t47d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a 6-fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a 6-fold repeated estrogen response element (ere). both constructs were stably transfected into t47d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e2 and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone-2 and 4-mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of 17betaestradiol (e2) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) 81. furthermore, rats fed idd until pnd74 were fed ird for 7 days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e2 and estrone (e1) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e2 and diet (idd vs ird and idd vs idd+ird). e2 levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e1 and ratios e2/e1 in mg and ratio of e2 levels in pl/mg were not affected by diet. in contrast, diet tended to affect e2 concentrations in pl (p=0.1211) due to an increase in the ird group (p=0.0056) whereas e2 levels in the idd+ird group only tended to be elevated (p=0.0788). in mg, ird and idd+ird increased e2 levels only weakly (p=0.0788 each). likewise, besides significant changes in transcript levels of cyp1a1 and 1a2, putatively decreasing oxidation of e2 to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e2 levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp53, mt1a) was observed in the idd+ird group. e2 mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e2 levels but not diet were significantly associated with gata3 transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt4) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e2 (increase in gdf15, hgf, igf1r, wnt5a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e2 and even decreased not significantly by idd+ird. taken together, despite an increase in e2 levels in pl, less er activation was observed after dietary exposure to if. whereas e2 and transcript levels of enzymes involved in e2 (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le 1329/10-1. august copenhagen irish (aci) rats with 17β-estradiol (e2)-releasing implants are an accepted model to study the etiology of breast cancer, but neither e2 (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) 75 and 175, placebo (-e2) or silastic implants containing 4 mg e2 were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd 285. palpable mg tumors (pt) and 1-2 mg per animal without pt were characterized histologically and categorized into normal (-e2 group, n=12), hyperplasia and non-pt and pt with and without solid tumors (+e2 group, n=32). e2, estrone (e1), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e1 and e2 in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of 49 transcripts involved in (biotrans)formation of e2 and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e2, plasma e2 as well as e1 and (borderline) e2 levels in mg were higher in ird. plasma e2 as well as e1 and e2 levels in mg were lower in the -e2 group than that in the +e2 group. e2 levels as well as e2/e1 and e2 mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki67). ird increased e2/e1 ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata3) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd1) tissues. levels of e1 and 2-meo-e1 were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd17b2 (conversion e2 to e1) and cyp1a1. transcript levels of gstm1 and gstm2 were decreased in the whole +e2 group and of gstt1 and gstt3 in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp53 and mt1a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of 2-meo-e1 nor cellular stress (gclc, mt1a, tp53). of note, neither 4-meo-e1, nor e1 catechols, nor e2 catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e1 or e2 were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e1 or e2. taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e1 and cellular (oxidative) stress, pt exhibited highest e2 levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e2/e1 ratio in pt. supported by dfg le-1329/10-1. level of 17beta-estradiol (e2) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from 37 mammoplasty specimen obtained from women (age 18-66 years old) not taking estrogen active drugs. 14 of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for 7 days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n=12) and bacterial metabolites (n=7) as well as the estrogens estrone (e1)-sulfate, e1, e2 and 2-methoxy-e1 were determined by uhplcand gc-ms/ms, respectively and transcript levels of 19 enzymes involved in e2 (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e2/e1 ratio with agl(+con) was observed in glandular tissues (r=0.49, p=0.002), accompanied by a significant negative correlation of e1 levels with agl (r=-0.35/p=0.032), possibly due to reduction of 17beta-hydroxysteroid dehydrogenase 2 (conversion of e2 to e1) expression as indicated by a weak negative correlation of transcript levels of 17beta-hydroxysteroid dehydrogenase 2 with agl+con (r=-0.25, p=0.080). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e1/e2 ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le-1329/10-1. allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp-1 cells were used as a model system and treated with the sensitizer 2,4dinitrochlorobenzene (dncb; 5, 10 and 20 µm) and the irritant sodium dodecyl sulfate (sds; 100 µm). samples were taken after 4, 8 and 24 hours. thp-1 activation was analyzed by measuring the established activation markers cd86 and cd54 after 24 hours. a targeted lc-ms/ms approach was used to analyze 188 metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., 2015) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp-1 were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp-1-macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp-1 cells. evaluation of hepatic cell stress showed the strongest effect on thp-1-macrophages when phh were incubated with apap or dic for 4 h.treatment of kc and thp-1-macrophages with supernatants of phh challenged for 4 h with hepatotoxic compounds indicates that thp-1-derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp-1 derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of 4 h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a 90day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to 0, 0.1, 0.3, 1 and 3 mg/cm³ ceo 2 as well as 50 mg/m³ baso 4 nanoparticles (6 h/day, 5 days/week, 13 weeks). animal dissections were conducted at five time points (exposure day 1 and 28; recovery day 1, 28 and 90) aiming for endpoints mandatory according to oecd guideline 413. additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo 2 and baso 4 retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to 90 days was detected by balf and histopathology examination. based on our current results effects of ceo 2 nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt-90 project is funded by the german federal ministry of education and research (bmbf) -03x0149a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco-2 model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco-2 cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana 2.0 expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the 25 market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana 2.0 project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana2.0 provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana 2.0 is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around 13% of total pm10 emissions are emitted by wood burners (uba 2006) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a549) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted 1:40 and directly delivered to the exposure chamber. after 4h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp9 cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. 2014 part fibre toxicol 11:59), or amosite asbestos at 3 µg/cm2 for 24 h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed 3788 significantly differentially regulated genes for mwcnt a, 1680 for mwcnt d, 145 for amosite, and 4 for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht29). for this purpose ht29 cells were exposed for 24 hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of 2-10 µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of 1.39 pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to 40 % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about 50 % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about 12%; control about 6%), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about 20%; control about 6%) for concentrations above 8 µg/ml. induction of apoptosis was determined for silver particles (up to 24% early apoptotic and 20% late apoptotic cells) as well as for titanium dioxide and zinc oxide (10% each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to 90%). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to 100nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [1] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of 45nm and 16µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in 48-well plates and exposed to doses of 1, 5, 10 and 25µg/cm² growth area of the respective cnt type for 24h. the wst-8 assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h 2 so 4 /hno 3 mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [2] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph 2 the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to 48h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union 7 th framework program (grant agreement no. 310187), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of 50µg/cm 2 (24h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il-8 liberation (elisa) as primary endpoints, and quartz dq12 and al 2 o 3 as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) 2 nanoplatelets (mg1; mean lateral size: 1,5-2 µm; mean thickness: 15-20 nm) represented the least, and pristine few layers graphene nanoplatelets (gr1; mean lateral size: 2 µm; mean thickness: 3 nm; graphene layers: 8 ± 0.5) the most biologically active nm. clear concentration dependencies were detected for gr1 in follow-up experiments. mg1 and gr1 were further tested in other lung-relevant cell types, i.e. mrc-5 primary human lung fibroblasts and a549 lung adenocarcinoma epithelial cells. interestingly, mrc-5 cells were less sensitive towards biological effects of gr1, compared to am, whereas a549 cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for 24 h to 12.5 or 25 µg/cm 2 of gr1 on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr1, i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc-5 cells and limited uptake in a549 cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc-5 cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) for two years; a control group was exposed to clean air. after one year exposure, 42 µg/lung was found in animals exposed to 0.1 mg/m³ and 2.6 mg/lung in animals exposed to 3 mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of 0.1 and 0.3 mg/m³ ceo 2 . neither pre-neoplastic nor neoplastic changes were observed after 12-months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) and barium sulfate (nm-220; 50 mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and 50 animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally 6 additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo 2 lung burdens were maximally 3.5 mg/g lung tissue at the highest exposure concentration of 3 mg/m³. in comparison, highest ceo 2 burdens in organs remote to exposure were liver and spleen with maximally roughly 1 x 10 -3 g/g tissue. in brain, maximum ceo 2 levels were 7x10 -6 mg/g lung tissue. baso 4 lung burdens were comparatively low (1 mg/g) within the first 13 weeks of exposure and steeply increased to 6 mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df4nano) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df4nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df4nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. the df4nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df4nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df4nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df4nano for 24 nanomaterials were validated against grouping by results of existing in vivo data and demonstrated 23 concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a549 cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo 2 ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo 2 nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo 2 nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo 2 np with an average diameter of 15-30nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations (0.01, 0.1, 1 and 10%) and were used to expose human lung adenocarcinoma cells (a549) for up to 24 hours. at 100µg/ml ceo 2 np showed little to no toxic effecton growth arrested a549 cells at fcs concentrations of 1% or below, but cell viability was decreased to about 80% in proliferating cells in cultures with 10% fcs. the proinflammatory effect of ceo 2 np was investigated through measurement of il-8 mrna expression after 3 hours and cellular il-8 secretion after 24 hours. the qrt-pcr showed that the expression of il-8 mrna in cells treated with 100 µg/ml ceo 2 np in 10% fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il-8 secretion, which showed a stronger increase by cells treated at 10% fcs. differences in cellular uptake of ceo 2 np was determined by fluorescence-activated cell sorting (facs) after 2 and 24 hours of exposition. after 2 hours, the cells treated with ceo 2 np in 10% fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after 24 hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo 2 np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about 1030nm (10% fcs) to 4090nm (0.01% fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr8383 rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio 2 np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr8383 rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase-1 (ho-1) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)-1β, il-6, il-18 and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il-1β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio 2 np by wst-1 assay overall revealed only minimal toxicity in comparison to sio 2 np which were used as reference material. ho-1 and g-gcs mrna analyses indicated that specific tio 2 np triggered a moderate induction of oxidative stress. il-6 was only induced after sio 2 treatment, whereas il-18 was not affected by any of the tested np. in contrast, various tio 2 np caused a significant induction of il-1β mrna expression. however, no significant induction of il-1β and tnf-α protein secretion was observed for any of the tio 2 np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio 2 np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp7 programme. specific types of tio 2 particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase 1 (nat1) activity j. lichter 1 , b. blömeke 1 1 trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase 1 (nat1), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat1 enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat1 has not been analyzed yet. to address whether human nat1 is a target of silver nanoparticles and released irons on protein, purified nat1 was exposed to silver ions (agno 3 ) and silver nanoparticles (ag10-cooh, average size 10 nm, carboxyl functionalized), and nat1 enzyme activity was analyzing the n-acetylation of the nat1 substrate para-aminobenzoic acid (paba). therefore, purified nat1 (1 ng/µl) was co-exposed for 20 min to paba (1 mm) and agno 3 or ag10-cooh (0.01, 0.1, 1, 10 and 100 µg/ml each), resulting in a nat1:silver relation of 1:0.01-100 (w/w). both, agno 3 and ag10-cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat1 (w/w, 1 µg/ml) enzyme activity was reduced about 98±0.2% (agno 3 ) and 82±0.6% (ag10-cooh). the lowest concentration analyzed (0.01 µg/ml) reduced nat1 activity about 24±5% (agno 3 ) and 17±5% (ag10-cooh). fifty percent activity reduction was caused by 0.11 ± 0.01 µg/ml of agno 3 and 0.21 ± 0.04 µg/ml of ag10-cooh, which is 10-fold lower compared to the published ic50 values for other metal oxide nanoparticles (3-15 µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat 28-days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of 60 and 6 mg/kg body weight for nanosilver and corresponding amounts of silver acetate (9.3 and 0.93 mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by 2-d gel electrophoresis and differentially expressed spots were identified by maldi-ms. 374 unique proteins, showing a log 2 ratio of ≤ -0.3 for downregulation and ≥ 0.3 for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf2-mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated 14-3-3 protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso 4 -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso 4 -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below 40 % for 1 mg/ml after 72 h). in general the effects of both nps were almost similar. additionally the effects of the baso 4-nps were compared more in detail. uptake of the baso 4 was quantified by icp-ms after 24 h and 72 h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso 4 of plc and nhbec to 0.1 mg/ml over 24 h and 72 h leads to ~200 µg baso 4 /1 mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il-8 and il-33 could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after 72 h. summing up baso 4 -nps showed toxic effects in primary human lung cell cultures after 72 h for concentrations under 1 mg/ml. c3 exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht22 revealed a c3-mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., 2012) . former results of studies on map kinase signalling indicated c3-induced modulations of downstream signalling modules. therefore, ht22 cells treated with 500 nm c3 for 48 h were applied for screening of the activity of 48 various transcription factors followed by luciferase reporter assays. five transcription factors namely sp1, atf2, e2f-1, cbf, stat6 were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp1 target genes p21 and cox-2 as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd153, a target gene of atf2, was decreased. our results suggest that c3 is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of 1.077g/cm 3 , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure 1 ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from 400 to 500 xg resulted in higher yields. in general, large variations in yield (46.8% ± 17.1%) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl-2-naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or 2-tert-butyl-4-methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule 1 chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine 34 -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system (4000 qtrap) and a hybrid quadrupole time-of-flight instrument (tt5600 + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the 20 th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group 2a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. (2011 ) chem res toxicol 24, 1957 resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d 7 -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of 5 fmol per injection (5 pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and 3-monochloropropane-1,2-diol (3-mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and 3-mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and 3-mcpd. the metabolite 2,3-dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is 3-mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary 3-mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound 13 c 2 -dhpma. the limit of quantification is 10 µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = 45) of one male volunteer collected over ten days was 154 ± 21 µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing 1.1 mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c 3 -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b6c3f1 mice and nasal neoplasms in male f344/n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic 2-sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n 2 -[(furan-2-yl)methyl]-2′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase 2 (nat2) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat2 genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in 772 bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n=207), dortmund (n=167) and neuss (n=398). nat2 was genotyped using seven characteristic polymorphisms (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208, rs1799931). haplotypes were reconstructed using phase v.2.1.1. we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat2*5b/*5b and *5b/other slow haplotypes as well as between the ultra-slow *6a/*6a and *6a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to 5 yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of 371 (49%) patients showed a relapse within the first 5 yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators (51% vs. 46%, p=0.154). this frequency is even higher in ultra-slow acetylators (61%, or=1.81, p=0.019) but not in slow *5b/*5b genotypes (49%, p=0.609). ultra-slow acetylators had a significantly shorter relapse free time within 5 yr after bc diagnosis than rapid acetylators (median 0.66 vs. 0.94 yr, hr=1.57, p=0.009). this trend was not that pronounced in all slow acetylators combined (0.78 yr, hr=1.19, p=0.127) nor in the subgroup of nat2*5b/*5b genotypes (0.79 yr, hr=1.20, p=0.255). the effect of ultraslow nat2 is even more pronounced in smokers (hr=1.78, p=0.003) but absent nonsmokers (hr=0.89, p=0.781). ultra-slow nat2 seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat2 in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand 17β-estradiol (e2). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf-7 cells treated with e2 or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e2 and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [1]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; 2011/12). urine specimens from children aged from 20 to 80 months from 23 different day-care centers were analyzed. in total, 253 preschool children were recruited with mean age of 54 months. our study results show that nearly all children (>95 %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, 5oh-mehp, 5oxo-mehp, 7oxo-minp), for bisphenol a and methylparaben. triclosan was detected in 16 % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about 5-50 µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about 1000 µg/l. median urinary concentration for methylparaben and bisphenol a were about 48 µg/l and 2 µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were 1770 µg/l, 72.4 µg/l and 55,6 µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study (2014/2015) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of 2 µg/kg benzo[a]pyrene and 12 µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [1]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl 3 ). first, the time-dependent internalization of hemin and fecl 3 into hct116 cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after 1 h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to 24 h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco-2 and hct116-cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion 8-oxoguanine (8-oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue 1 (mth1), which protects the nucleotide pool by hydrolysis of 8-oxogtp, 8-oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct116 and caco-2 cells using western blot analysis, which was followed by a reduction in cell viability at high doses after 72 h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o 6 -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o 6 -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between 0.3% and 95%. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [1, 2] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [3] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta 100 for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for 24 h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [4] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [1] göggmann, schimmer (1983) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. 121, [191] [192] [193] [194] wiseman et al. fatty acid esters of 3-chloro-1, and of 2-chloro-1, are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free 3-mcpd and 2-mcpd. 3-mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class 2b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which 10 mg/kg b.w./day of either 3-mcpd or 2-mcpd were orally administered to rats over a period of 28 days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis (2de). among others, the redox sensor protein dj-1 was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with 3-mcpd or 2-mcpd. up to six different isoforms of dj-1 were identified by 2de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either 3-mcpd or 2-mcpd predominantly led to a shift of the abundance between two dj-1 isoforms in the rat tissues. this effect was more pronounced with 3-mcpd compared to 2-mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys106) of dj-1 to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either 3-mcpd or 2-mcpd. dj-1 is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys106 was shown to be crucial for the activity of dj-1, and the irreversible oxidation of cys106 to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of 3-mcpd and 2-mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. 1,2-unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure (24h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure (14d) comparatively to short-term exposure (24h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after 14d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp7a1 (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in 1998 in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab 5 protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp78, which results in cell stress and caspase 3/7dependent cell death. recently, we discovered that higher concentrations of suba (10 mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [1] . in contrast to hela and caco-2 cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of 3-mcpd and its esters. high amounts of 3-mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related 2-mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of 3-mcpd, pointing to kidney and testes as main target organs. 3-mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for 2-mcpd, showing similar organ toxicity compared to 3-mcpd and identifying heart as additional target organ. no such data exist for 2-mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for 3-mcpd, 2-mcpd and their esters. hence, an oral 28-days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by 3-mcpd and 2-mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of 3-mcpd + 2-mcpd (10 mg/kg body weight), and 2-mcpd-dipalmitate (53 + 13.3 mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by 2-d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of 270 unique proteins deregulated at a log 2 ratio of ≤ -0.5 for downregulation and ≥ 0.5 for upregulation at p ≤ 0.05 were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by 2-mcpd versus 3-mcpd. dipalmitate treatment even caused more deregulation than 2-mcpd. upregulated cytochrome b-c1 complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after 2-mcpd and 2-mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after 3-mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after 3-mcpd treatment. protein dj-1 (park7), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with 3-mcpd and the higher dose of the 2-mcpd diester. tropomyosin beta chain (tpm2) was commonly upregulated after 3-mcpd and 2-mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of 2-mcpd-and 2-mcpd dipalmitate-induced proteomic changes exist compared to 3-mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as 7α-hydroxy-chol (7α-ho-chol), 7β-hydroxy-chol (7β-ho-chol), 7-keto-chol (7-o-chol), 5,6α-epoxy-chol (α-epoxy-chol) and 5,6β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where 7β-ho-chol (milk) and 7-o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: 5α-cholestan-3β-ol), respectively in breast adipose tissues of 48 healthy women undergoing mammoplasty. furthermore, tissue levels of 25 fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: 0.36 nmol/g tissue, range: 0.06-1.55 nmol/g), followed by α-epoxy-chol > 7-o-chol > 7α-ho-chol> 7β-ho-chol (0.009 nmol/g, range: 0.002-0.11 nmol/g). tissue levels of chol (2.8 micro mol/g, range: 1.88-3.87 micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of 7α-ho-, 7β-ho-, and 7-o-chol (r = -0.29, p=0.024-0.044) median oxychol/chol ratios ranged from 0.0119 (5, to 0.0003 (7β-ho-chol). 7-o-chol and 7-ho-chols correlated strongly with each other (r=0.81-0.91, p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, 7β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r=0.30, 0.30, and 0.31, respectively, p=0.037, 0.034, 0.028, respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products (1) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin (2) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them (3) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation (4) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument (3). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently (1,2). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points (3). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas 134-62-3), icaridine (cas 119515-38-7) and ebaap (cas 52304-36-6) (4). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than 10% as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than 1,000 international publications have been transferred to a database, with more than 120,000 entries. according to the database, pharmaceuticals have been found in the environment of 71 countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than 600 active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in 50 countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than 33000 discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, 3585 chemicals (ca. 11%) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a 4 and 32 days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet (1 -100 mg/l) and els study (0.0003 -0.03 mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat-1 (slc7a1) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat-1 for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat-1 expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat-1 in human ea.hy926 endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat-1 mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb (5,6-dichloro-1-β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat-1 gene responsible for this regulation. to our surprise and in contrast to data published by others 1 the chromosomal region up to 5 kb upstream of the first hcat-1 exon exhibited no promoter activity in either endothelial or dld-1 colon carcinoma cells that exhibit a very strong endogenous hcat-1 expression. we could however identify a promoter element within the first intron of the hcat-1 gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat-1 expression. our results indicate starvation-induced transcriptional regulation of hcat-1 through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc47-family consisting of the two multidrug and toxin extrusion transporters mate-1 (slc47a1) and mate2-k (slc47a2). while mate-1 is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate2-k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with 26 different anti-neoplastic agents comparative transport experiments with the model substrate 1-methyl-4-phenylpyridinium (mpp) have been carried out. therefore stably transfected hek293-cells expressing mate-1 or mate2-k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek293-cells were used. the interaction analyses were carried out by determining the uptake of [ 14 c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) 2b1 that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp2b1 substrate estrone 3sulfate (e 1 s), and observed a significantly lower k m for mdck-hoatp2b1 compared to .00 ± 10.23 µm, *p<0.05 student´s t-test) whereas there was no difference in v max (mdck-hoatp2b1 119.4 ± 11.3 fmol/min/µg protein; mdck-roatp2b1 102.3 ± 12.8 fmol/min/µg protein). the human oatp2b1 exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [1] . to identify whether rat oatp2b1 owns the same characteristics, the cellular accumulation of 50 µm e 1 s (low affinity site) or 0.005 µm e 1 s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp2b1. as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e 1 s in mdck-hoatp2b1 cells (110.0 ± 14.6 fmol/min/µg protein vs. 60.7 ± 7.5 fmol/min/µg protein, *p<0.05 student´s t-test), there was no inhibition of e 1 s accumulation in mdck-roatp2b1 cells (53.2 ± 13.2 fmol/min/µg protein vs. 49.0 ± 17.4 fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp2b1 while human oatp2b1 is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per 1000 ng of total rna) in different tissues of rat revealed a high expression of oatp2b1 in liver (159.6 ± 45.5), a moderate expression in small intestine (14.9 ± 4.0) and colon (57.0 ± 12.3), and a low expression level in kidney (2.3 ± 0.8) . the latter is in accordance with previous findings showing that oatp2b1 is abundant in rat kidney as quantified by absolute proteomics technics [2] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of 10 drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of 754 mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v.5, targetscan7.0) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of 754 mirnas, 248 were detected in all tissue types. out of ten transporters five showed significant inverse correlations with 10 putatively targeting mirnas (e.g. pept1 and hsa-mir-27a, r= -0.498, p=0.002). reporter gene assays indicated interactions of mir-193a/b with pept1 3'-utr (p = 0.0025 and p = 0.0012) as well as of mir-27a with abcb1 3'-utr (p = 0.0006). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept1 and abcb1. background: the human uptake transporter nact [gene symbol slc13a5; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc13a5 gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc13a5 gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g219r, -p.t227m and -p.l488p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of 24 tested) inhibited nact-mediated citrate transport at the tested drug concentration of 100 µm. from these, benzbromarone shows the strongest inhibition with an ic 50 value of 83.2 µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g219r, -p.g219e,p.t227m, -p.l420p and -p.l488p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc13a5 gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member 1 (abcb1) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb1 in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb1 deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol-4,5-bisphosphate (pip 2 ) is a known regulator of abcb1. the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip 2 . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb1 activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht-29 served as a model. we employed various in-vitro methods for the measurement of abcb1 activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb1 activity and re-sensitize our cell model against chemotherapeutic agents like 5-fluorouracil which are known to be substrates of abcb1. taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb1 might be a new approach to target resistant tumors without interfering with the natural function of abcb1 in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ 3 h]-labeled homoarginine as substrate and human embryonic kidney (hek293) cells stably overexpressing human cat1 [gene: slc7a1 (solute carrier family 7)], cat2a (slc7a2a) or cat2b (slc7a2b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat1 (7-fold), cat2a (1.6-fold) or cat2b (2.2-fold) after 2.5 min using 100 µm substrate (each p<0.001). apparent k m values for cellular net uptake of homoarginine were 174.6 µm for cat1, 3640 µm for cat2a and 522.8 µm for cat2b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat1, cat2a and cat2b. compared to other cat substrates, the cat1-and cat2b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter 1 (oct1, alternative name slcc22a1) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b1), known oct1 substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct1 has been shown to have strong inter-species differences. in rodents oct1 is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct1 is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct1 orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct1 orthologs. methods: overexpression of oct1 orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ 293. the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct1 orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct1. human oct1 showed a 5fold higher v max for the uptake of asp + and 2-fold increase of v max for sumatriptan in comparison to the rodent oct1 orthologs. conversely, human oct1 showed a 4-fold lower v max for the uptake of fenoterol compared to rodent oct1s. there was no difference between rodent and human oct1 in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct1 constructs and by comparing the effects of key point mutations in mouse and human oct1 orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct1 orthologs. therefore oct1 pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct1. ranitidine is a histamine h 2 -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately 30% of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct1 [1]. oct1 is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct1 alleles are known [2] . thereof 12 alleles confer partial or complete loss of oct1 activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct1 on the uptake of ranitidine. we used hek293 cells stably transfected to overexpress wild type or variant oct1 isoforms. the variant oct1 alleles oct1*1a (met408val), oct1*1c (phe160leu), oct1*1d (pro341leu/met408val), oct1*2 (met420del), oct1*3 (arg61cys), oct1*4 (gly401ser), oct1*5 (gly465arg/met420del), oct1*6 (cys88arg/met420del), oct1*7 (ser14phe), oct1*8 (arg488met), oct1*9 (pro117leu), oct1*10 (ser189leu), oct1*11 (ile449thr), oct1*12 (ser29leu) and oct1*13 (thr245met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct1 isoforms. wild type oct1 showed a time and concentration dependent uptake of ranitidine with a k m of 62.91 ± 4.32 µm and a v max of 1125.41 ± 86.12 pmol/min/mg protein. variants oct1*5, oct1*6, oct1*12 and oct1*13 completely lacked ranitidine transport activity. variants oct1*2, oct1*3, oct1*4 and oct1*10 showed v max values decreased by 64, 77, 91 and 50 %, respectively. oct1*8 variant showed an increase of v max by 25 %. there was no significant changes in ranitidine uptake by variants oct1*1a, oct1*1c, oct1*1d, oct1*7, oct1*9 and oct1*11 compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct1 substrate and demonstrated that genetic polymorphisms in oct1 can strongly affect ranitidine uptake. the effects of oct1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of 5-ht 2a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate 5-ht 2a receptors, we here compare lsd and dmt with regard to processes of 5-ht 2a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the 5-ht 2 selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective 5-ht 2a antagonists ketanserin and mdl100907. after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ 35 s]gtpуs binding) is paralleled by a desensitisation of frontocortical 5-ht 2 receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha-5-ht 2a transfected) hek293 and (endogenously 5-ht 2a expressing) c6 glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise 5-ht 2a receptors, fails to activate the phospholipase d (which is needed for 5-ht 2a internalisation), and fails to inhibit the synthesis of 5-ht 2a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of 5-ht 2a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of 5-ht 2a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., 2010) and sunset (schmidt et al., 2009) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div 60. in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor-2 kinase inhibitor a 484954 and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a 484954 and spermidine had no effect on de novo protein synthesis in juvenile neurons (div 20) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div60). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin 1a receptors (5-ht 1a r) apparently play a pivotal role in the development of depression. the involvement of an altered, 5-ht 1a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located 5-ht 1a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic 5-ht 1a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic 5-ht 1a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment (15 days) with the selective 5-ht 1a r agonist 8-oh-dpat (0,5 mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; 50 mg/kg/day). mice are sacrificed either one day (proliferation) or 21 days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic 5-ht 1a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia (33 days), but not after spontaneous remission (age 90 days). as indicated by unaltered homeoboxprotein nkx 2.1 (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc2 and cavii mrna, used as markers for the gaba-switch, were unchanged in 18 and 33 day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha1 subunit). by stereological determination, we found a 46% decrease in alpha1 subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in (32%) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha1 gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem (2.0 and 10.0 mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam (0.5 and 1.0 mg/kg i.p.) in the dt sz hamster. examinations of αlpha2 gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha1 gaba a r subunits. changes in αlpha1 gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. 2005) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: 250 µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh-018 (5 x 10 -6 m) and jwh-210 (5 x 10 -6 m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh-018 was not affected by pregnenolone (10 -7 m), the inhibition by jwh-210 was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone (10 -7 m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ 9 -tetrahydrocannabinol (2 x 10 -5 m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ 9tetrahydrocannabinol were not changed by pregnenolone (10 -7 m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, 10 -6 m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol 521: 437-449, 1999) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ 9 -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β3-antagonist sr59230a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor 1 (trh-r1) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp1 mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr59230a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr59230a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par-1, par-3 and par-4. in human adipose tissue, par-1 is found in vascular stromal cells and par-4 in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in 3t3l1 cells. results: differentiation of 3t3l1 preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par-4 is time-dependently upregulated in maturing cells while par-1 expression is detectable but not altered. in preadipocytes, thrombin (3 u/ml) activates the mitogenic kinase erk1/2, promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki67 and the inflammatory cytokine interleukin (il)-6 are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par4-activating peptide (gypgkf, 100 µm) and cathepsin g, a proteolytic par-4 activator released from neutrophils and mast cells. repeated exposure of maturing 3t3l1 cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il-6 expression. pretreatment of mccm with a par-4 inhibitor further drives lipid accumulation, the induction of il-6 by contrast is suppressed. conclusion: par-4 activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par-4 expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr4 antagonist amd3100 exerts deleterious effects in endotoxemia in vivo s. seemann 1 , a. lupp the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). although a blockade of the cxcr4/cxcl12 axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr4 seems to be part of the lipopolysaccharide sensing complex, cxcr4 agonists or antagonists may have a positive impact on tlr4 signaling. additionally, cxcr4 is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr4 blockade on endotoxemia by applying a sublethal lps dose (5 mg/body weight) in mice. the selective cxcr4 inhibitor amd3100 was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. 24 hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp91 phox as well as of heme oxygenase 1 in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd3100 plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than 200% or 35% when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about 11% in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd3100 and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp91 phox to occur above average, whereas heme oxygenase 1 expression was distinctly decreased. our results indicate that a blockade of the cxcr4 in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd3100 and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr4 antagonist cannot be recommended, hence indicating the activation of cxcr4 to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr2 by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr2 expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr2, tlr1 and tlr6 mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr2 expression in a time-dependent and concentration-dependent manner and reduced tlr1/6 expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il-1β further increased tlr2 mrna levels. gene expression of mapk phosphatase-1 (mkp-1) was also upregulated by dexamethasone. glucocorticoid-induced tlr2 expression was negatively regulated by p38 mapk signalling under inflammatory conditions through mkp-1 induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr2 signalling as demonstrated by reduced il-8, il-1β, mmp-1 and mcp-1 levels. however, the expression of traf6, a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr2 agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il-1 cytokines and t helper 17 (th17) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th17 activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il-1β, propranolol induced the th17 priming cytokines il-23 and il-6 in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il-36γ and lps failed to increase il-23 release in molc and modc in the presence of propranolol but further induced secretion of il-1β. autophagy has been linked with the secretion of il-1 family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p62 and lc3-i to lc3-ii conversion, induced accumulation of lc3 positive vesicles, as well as expression of il-1 signalling downstream adapter molecule traf6, indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz-3 (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr2 expression and responded robustly to tlr2 engagement, confirmed by increased cd83 and cd86 expression, upregulated il-6, il-12p40 and il-23p19 mrna levels and il-8 release. tlr2 activation reduced ccr6 and elevated ccr7 mrna expression and induced migration of mutz-lcs towards ccl21. similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il 1β whereas ligands of tlr3 and tlr4 failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr2-activated mutz-lcs, co culture with naive cd4+ t cells led to significantly increased ifn-γ and il-22 levels indicating th1 differentiation independent of il-12. tlr2-mediated effects were blocked by the putative tlr2/1 antagonist cu-cpt22, however, no selectivity for either tlr2/1 or tlr2/6 was observed. computer-aided docking studies confirmed non-selective binding of the tlr2 antagonist. taken together, our results indicate a critical role for tlr2 signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep19-2.5, a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep19-2.5 and the structurally related compound pep19-4lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr2 (fsl-1) but not tlr4 (lps) activation by increased il-8 production, as determined by elisa. western blot analysis showed that both salps inhibited fsl-1-induced phosphorylation of nf-κb p65 and p38 mapk. furthermore, the peptides significantly reduced il-8 release and gene expression of il-1β, ccl2 (mcp-1) and hbd-2 as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below 10 µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il-6 secretion, downregulated expression of the maturation markers cd83 and cd86, as analysed by flow cytometry, and inhibited ccr7-dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il-6 levels and cd83/cd86 expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep 19-2.5 affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as 1 ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf2 exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf2 in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf2-deficient (nrf2 -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for 30min (v t =16ml/kg, f=90min -1 , peep=2cmh 2 o, fio 2 =0.3), before 50 µl hydrochloric acid (hcl) with ph=1.5 or ph=1.3 were instilled intratracheally, controls received nacl. mice were then ventilated for further 6h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph=1.5 induced mild lung injury, indicated by hypoxemia (po 2 /fio 2 ~300mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf2 -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf2 -/mice. in contrast, hcl ph=1.3 caused more severe lung injury (po 2 /fio 2 ~200mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf2 -/mice. conclusion: we conclude that the presence of nrf2 augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany 2 fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [1] [2] [3] [4] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [5] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [6] . adma is degraded by dimethylarginine dimethylaminohydrolase 1 and 2 (ddah) enzymes [7] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide-3 kinase (pi3k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh1] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah1 and ddah2 enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi3k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah 1 and 2 enzymes. the pi3k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-(1-7) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-(1-7) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-(1-7) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-(1-7) infusion provided best protection from experimental ali when administered by continuous infusion starting 30min after oa infusion till the end of the experiment (30-240min). both pretreatment (-60-0min before oa) and short-term therapy (30-90 min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-(1-7) 90min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-(1-7) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y-27632 and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of 10 µm y-27632 under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens-1 (zo-1). under conventional culture, y-27632 induced higher growth rate of primary ebec and increased the passage number up to 5 passages with retained epithelial cell behavior. in the presence of y-27632, ebecs under ali showed higher teer values. expression of zo-1 correlated with the increase in teer, but in y-27632-treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y-27632 facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic 50 values for the viability in the low micromolar range [1] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe 2+ . the radicals were trapped by 2-methyl-2-nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = 16.8 g, and a h = 1.8 g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic 5,5-dimethyl-1pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe 2+ a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = 16.1 and a h = 24.6 g. according to previously published data [2] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [1] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine 21:1048-1052, 2014. nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca 2+ -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the 5' untranslated region (5'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld1. using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 5'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld1 cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c101a-enos). normal enos (enos-tg) or a novel dimer-destabilized c101a-enos described previously (antioxid redox signal. 2015 sep 20; 23(9) :711-23) were introduced in c57bl/6 in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for 4 weeks or treatment with sod mimetic tempol. results: c101a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos 1176/79 phosphorylation and amp kinase (ampkα) phosphorylation at thr172 in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c101a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd31. exercise training increased phosphorylation of enos at ser 1176/79 and of ampkα at thr 172 in wt but not in c101a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c101a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c101a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp-1 cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka (0.1 mg/ml) and mya (1 mg/ml) treatment down to 24% and 16% resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka (3 mg/ml) treatment by 26% and 25% resp.; after mya (0.1-1mg/ml) treatment by 17% and 44% resp. lps-induced tnfα release from thp-1 cells was inhibited concentrationdependently by my (ic50 = 60.65 μg/ml; 97% inhibition), ka (ic50 = 439 μg/ml; 71% inhibition) and cc (ic50 = 1886 μg/ml; 44% inhibition). furthermore, ka (200 µg/ml) and cc (500 µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to 100-fold (for cxcl13). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a2a receptors (a2ar) and adenosine a2b receptors (a2br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a2ar agonist (cgs 21680) and an a2br antagonist (psb-1115) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw 5 with a2ar as a possible mechanism of the protective effect of stw 5 in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a1, a2b, and a3 receptor with specific agonists reduced the acetylcholine (ach, 10µm)-induced contractions, while activation of a2br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs 21680 (10µm) or with psb 1115 (100µm). the same effects with smaller intensity were found for stw 5 (512 µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw 5 interact with the a2ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw 5 are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw 5 as a therapeutic approach in ibs. (allescher 2006) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw 5(ottillinger et al. 2013) . stw 5 (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw 6) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw 5 and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than 150 pharmacological tests, is needed. all data from studies including stw 5 alone, or stw 5 and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of 2d histograms (lorkowski et al. 2015) . results: more than 150 pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw 5 contribute differently to the overall effect of stw 5. altogether, the evaluation of the data shows that stw 5 is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw 5 manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw 5 ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw 5 (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw 5 consists of a mixture of 9 standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw 5 ii lacks the last 3 components. stw 5 was shown to be effective clinically to treat functional dyspepsia (1) and irritable bowel syndrome (2) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis (3) and in the management of ulcerative colitis (4) . the present study was initiated to show whether stw 5 ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with 5% dextran sodium sulfate in drinking water for 1 week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw 5 in doses of 2 and 5 ml/kg or stw 5 ii in a dose of 2 ml/kg for 1 week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of 300 mg/kg. conclusions: the modified preparation, stw 5 ii thus proved to be as effective as stw 5, thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. (1) schmulson mj (2008) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor 1 (maropitant) and serotonin receptor 3 (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk1) and channel opening (activation of 5ht3) on the intracellular calcium homeostasis (using fluorometric ca 2+ analysis) and cell proliferation rates in endogenously nk1 and 5ht3 receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk1 or 5ht3 cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk1 receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk1 and 5ht3 receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every 4-5 days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b2 and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b2 in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b2 signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal1 controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal1 in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal1 gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal1 in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh1 promoter, further corroborated that rasal1 expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal1 as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa-5ht], urb597) on a549 lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, 2-ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (timp-1). using sirna approaches, a causal link between the timp-1-upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp-1 expression. inhibitor experiments point toward a decisive role of cb 2 and transient receptor potential vanilloid 1 in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa-5ht and urb597. as underlying mechanism of its anti-invasive properties an upregulation of timp-1 was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb597 and arachidonoyl serotonin (aa-5ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p42/44 mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p42/44 mapk activation, a blockade of pparα left p42/44 mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p42/44 mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes 9 and 22 leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k562. an inverse correlation of mir-212 expression and protein levels of the efflux transporter atp-binding cassette transporter g2 (abcg2) was observed in cells resistant to different im-concentrations, pointing to a relation of mir-212 to im-resistance. hence, we investigate in current studies, how the influence of mir-212 on im-sensitivity could be explained. methods: we transfected k562 cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir-212 or inhibitory anti-mir-212, challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst-1-, caspase glo 9-assay and cell counting. in addition, we analyzed changes in abcg2 expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k562 showed an effect of mir-212-inhibition using anti-mir-212. this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p<0.01) and cell membrane integrity and reduced capase-9 activity (p<0.05). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg2 expression, we found that 2 µm im-resistant k562 do not express higher amounts of abcg2, but showed higher transport rates of im or the abcg2-substrate hoechst 33342. conclusions: overall, these experiments indicate that mir-212 does not only affect abcg2-expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg2. in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than 50 % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe(1%)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of 1-3 gy using a 120 kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around 1 nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [1] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [2] 1 to 5 % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to 30% of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h 2 s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h 2 s production in the nociceptive system are only poorly understood. here we investigated the expression of the h 2 s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h 2 s. this work was supported by the deutsche forschungsgemeinschaft (sfb815-a14) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany 2 heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt-112) and mesenchymal-like (j-82) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt-112 r , j-82 r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh2ax and 53bp1 foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j-82 and j-82 r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt-112 cells showed an about 2-3-fold higher resistance to cispt than j-82 cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt-112 r and j-82 r revealed a 2-3-fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt-112 r cells disclosed a reduced apoptosis and enhanced g2/m arrest following cispt exposure as compared to rt-112 wild-type cells. by contrast, induction of cell death was similar in j-82 and j-82 r cells. notably, j-82 r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j-82 r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt-112 r and rt-112 cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j-82 r and rt-112 r . unexpectedly, j-82 r and rt-112 r shared the upregulation of xaf-1. treatment of j-82 r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk-1 and, moreover, re-sensitization to cispt by chk-1 inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt-112 cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j-82 cells. furthermore, our findings indicate that chk-1 might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her3 expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her3 impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein 1 (satb1) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb1 in the upregulation of her3 after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb1 expression in gastric cancer cells, and the downregulation of satb1 by sirnas completely prevented the induction of her3 upon c-met inhibition. in contrast, her1 or her2 expression levels were not affected by satb1-specific sirnas. the function of satb1 as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her3 expression after c-met inhibition. interestingly, the upregulation of her3 in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb1 and pkc are critically involved in the regulation of her3 expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her3 plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb1 may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb-231 breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki-67 proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb-231 breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to 47 h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for 1406, pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb-231) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam-1, vcam-1, e-selectin, n-cadherin, and galectin-3. moreover, the chemokine system cxcl12/cxcr4 was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl12 in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl12 could be excluded, since blocking the cxcl12 receptor cxcr4 on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β1-integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β1-integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse (1). based on an evolutionary cancer model (2) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc9 and hcc827 cell lines; both erlotinib sensitive) (3). plasma concentrations from two single-dose groups, 30mg/kg and 200mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens (15mg/kg daily, 30mg/kg daily, 200mg/kg every 2 days, 200mg/kg every 4 days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem 7.3. results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the 200mg/kg dose. a 1-compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: 13 and 52 days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec 50 =0.53μm for both cell lines), and on the partially sensitive transit phase (ec 50 =1.24μm and 3.00μm, for hcc827 and pc9 cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and 5-lipoxygenase (5-lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of 30 min whereas the increase in vocalisation threshold persisted over a period of 90 min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox-2 and 5-lo but not of cox-1. we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox-2 and 5-lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. 2,5 million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., 2001 and buchner et al., 1998 documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > 0,6. in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over 7000 purified compounds and over 500 extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of 0,05 %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d 2 receptor partial agonist 2bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than 21 million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d 2 receptor partial agonist 2-bromoterguride (2-bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [1]. to determine its atypical character in vivo, the ability of 2-bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of 2-bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: 10 week old male sprague-dawley rats were injected with 2-bt (0.1 or 0.3mg/kg; i.p.) followed by pcp (1.5mg/kg; s.c.) or apomorphine (0.5mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of 2-bt (0.1 or 0.3mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp (5.0mg/kg; i.p. twice daily for 7 days) was assessed in the nor task consisting of a 3min acquisition trial and a 3min retention trial separated by a 1h inter-trial interval. clozapine (5.0mg/kg; i.p) or haloperidol (0.1mg/kg; i.p) were used as positive controls. results: the dopamine d 2 receptor partial agonist 2-bt (0.3mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly 2-bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that 2-btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that 2-bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of 2-bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of 2-bromoterguride [1], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of 2-bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid-1 receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb2 possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb2 for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in 1. 2-1.8 year old cb2 -/and control mice, fed with a standard diet and assessed effects of the cb2 agonist, hu308 during high fat diet in 12-16 week old mice. results: the cb2 -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb2 agonist hu308 prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m1-like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb2 agonists may fortify cb2-mediated anti-obesity signaling without the risk of anti-cb1 mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic 50 : xan 23 µm, res 161 µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic 50 : xan 7 µm, res 14 µm) while had less influence on the viability of pmm (ic 50 : xan 68 µm, res > 438 µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin (5-ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph1), or the raphe nuclei of the brainstem (tph2) and the respective 5-ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral 5-ht is mainly produced by tph1-expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, 5-ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about 37000 small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic 50 in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral 5-ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco2 permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg2 as a hcc tumor cell line and the fibroblast cell line fi301 as stroma simulation. additionally caco2 cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of 0.01 -100 µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: 0.5 µg/ml -cr: 1 µg/ml -es: 5 µg/ml. performance of the caco2-transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with 5-fluorouracil (5fu), which is used as a chemotherapeutic agent, caused a reduction of its ic50 values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of 5fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin-1, -3, -5, -12. claudin-5 is known to be the key tj protein tightening the bbb. therefore, claudin-5 has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin-5 and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin-5 containing barriers. the claudin-5 targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin-5 and immortalized brain endothelial cells (bend.3). the peptides decreased the amount of claudin-5 and zo-1 at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein (376 da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, 547 da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin-5 (cldn5) which tightens the bbb for molecules <800 da. consequently, cldn5 is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn5 assay, the barrier opener 1 (bo1) was identified as a cldn5 modulator. initially, a significant removal of cldn5 from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw 521 da) demonstrated the effect of bo1. concentration dependent treatment (50-150 µm) of cell monolayers with bo1 reduced tightness of the tjs between some hours and 24 h. applying 2-hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo1 became detectable in mice. due to short stability (< 2 h) of bo1 in the bloodplasma repeated administration (1.5 mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw 376 da). the small molecule bo1 is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo1 is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about 16 h of incubation with concentrations varying from 100 to 150 µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow (457 da) and fitc-dextran (10 kda); the localization of tric elongated from ttjs towards btjs and cldn1 was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c57bl/6 mice. c57bl/6 mice without transplantation were used as normal controls. donor c57bl/6 mice to recipient c57bl/6 mice were served as the isograft group. 42 days after transplantation, mice were scanned using an in vivo small animal µct (skyscan 1176). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf-1 pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf-2 (igf1r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf-1 pathway is the transcription factor daf-16 (hfoxo3a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf-1 pathway. recently, a cohort study showed that a decreased mortality rate in humans between age 78 -90 correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac1 or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about 20%) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf-16 overactivity. consequently we found an increased nuclear localization of daf-16 in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf-16-ko mutant background. rt-qpcr brought jnk-1, a known activator of daf-16, into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf-16 dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf-1 pathway and particularly the transcription factor daf-16. the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. (2005) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance (1). in hfd/stz model, the rats are fed with high-fat diet for 2-4 weeks or for a relatively long time (≥ 3 months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz (30-35 mg/kg), some of the diabetic rats receive treatment (2) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for 4 weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats (234.40 ± 42.71 mg/dl vs. 464.00 ± 23.88 mg/dl, p < 0.05). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats (249 ±6.00 gvs. 288.00 ±4.41 g, p < 0.05). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for 4 weeks. further details can be found in table 1 . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency (3) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type 2 diabetes (4). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type 2 diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: 1. srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. (2005) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. pharmacol res 52 (4): 313-320. 2. oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. (2015) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem 90 (7): 506-515. 3. franz, m. j. (2007) . the dilemma of weight loss in diabetes. diabetes spectr 20 (3) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt1 dystonia, which is caused by a gag deletion in tor1a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d2 receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d2 receptor mediated acetylcholine release has not been examined yet. the aim of present study was to (1) generate more powerful tests to detect behavioural alterations in the dyt1 knock-in mouse and to (2) examine the behavioral effects of the d2 receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt1 knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt1 knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole (0.25 -1 mg/kg i.p.) were tested in dyt1 knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt1 knock-in mice showed increased thigmotaxis at a dose of 0.5 mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt1 mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt1 model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt1 knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt1 dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d2 dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay 41-8543 after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay 41-8543 was (4mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay 41-8543 administration influenced the activity of the ecm degrading matrix metalloproteases (mmp2 and mmp9) and their inhibitor timp-1, the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor-1 (pai-1)) and the secretion of cytokines, e.g. il-6. thereby, bay 41-8543 increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay 41-8543 might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly1 (sh3-domain protein expressed inlymphocytes 1) is essential for the generation of a fully functional t-cell response. the lack of sly1 leads to reduced survival rates of infected mice. the increased susceptibility of sly1 ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly1 ko t cells displayed a dysregulation of forkhead box protein o1 (foxo1) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. interestingly, we observed a similar regulation for the adapter protein sly1, where tcr stimulation results in sly1 phosphorylation and sly1 export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly1 to 14-3-3 proteins. altogether, this study describes sly1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly1 regulates t-cell proliferation (schäll et al., eur j immunol. 2015) . the catalytical isoforms p110γ and p110δ of phosphatidylinositol-4,5-bisphosphate 3kinase γ (pi3kγ) and pi3kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p110γ and p110δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi3kγ and pi3kδ in p110γ ko δ d910a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. here we analysed p110γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il-5 and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p110γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p110γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p110γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p110γ and p110δ causes eosinophilia and ige hyperproduction, p110γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type 2 receptors (b2) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med 2015:372; 418-425) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually 20-30 min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a 200,000 kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately 200 µm below the surface. after stabilization of the green fluorescent signal, a 10,000 kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b2 changes physiologic endothelial permeability. this newly developed transgenic mouse line (b2 tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment. we observed that b2tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the 10,000 kda dextran at each time point (fig. 1) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b2 increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type 2 (b2). using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b2 was generated and backcrossed to c57bl/6 for more than 10 times (b2 tg ). lung mrna using primers specific for the human or the mouse b2 cdna revealed a 12.5-fold stronger expression of human b2 in b2 tg (n=6), while the expression of murine b2 mrna was unchanged and similar to transgene negative littermates (b2 n ). we have evaluated the specificity of several antibodies directed against b2 and found that a rabbit monoclonal anti b2 antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b2 -/mice. however, this antibody primarily stains rodent b2 and has only little cross-reactivity to human b2. hence, we were not able to detect a significant increase of b2 protein in tissues of b2 tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at 1 µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b2 activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b2 n , we found a significant icatibant sensitive aortic dilation in b2 tg following preincubation with diclofenac which indicates functional overexpression of b2 in conductance vessels of b2 tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of 30 µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching 4.41±0.11 fold at 18.9 nmol bradykinin in c57bl/6 (n=6 each, p<0.0001 vs. vehicle). a similar increase was found in b2 n (4.45±0.25 fold, n=7, p<0.0001 vs. vehicle), while there was a stronger response in b2 tg (5.50±0.16 fold, n=7, p<0.0001 vs. vehicle) which was significantly different to b2 n (p<0.01) and c57bl/6 (p<0.01). in another set of experiments the specific and ace-resistant b2 agonist labradimil (1.89 nmol) was used instead of bradykinin. labradimil increased extravasation by 3.736±0.121 fold in c57bl/6 (n=6 each, p<0.0001 vs. vehicle), by 4.51±0.11 fold in br2 n (n=7 each, p<0.0001 vs. vehicle) and 4.88±0,21 fold in b2 tg (n=6, p<0.0001 vs. vehicle) which was significantly different to c57bl/6 (p<0.01) but not to b2 n (p>0.05). the effects of bradykinin and labradimil were largely blocked by 10 nmol icatibant (i.v.) in c57bl/6, b2 n and b2 tg mice (p<0.0001) and hence mediated by activation of b2. these data suggest that overexpression of b2 in b2 tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b2 tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller 1 1 universitätsklinikum, innere 1, nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = 0 a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = 0.5772 …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd19+ and cd20+ b-cells completely persists for 6 month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = 100 mcg/ml and the infusion time t = 2 hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t1/2 = 2 hours (not shorter than the time t of infusion). with the terminal half-life of 460 hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t1/2 < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: 1) are the observed metabolites ruminant specific and formed directly in the rumen? 2) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro >7 days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph 6.6) and redox potential (~-300 mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh3 and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least 7 days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating 14c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within 72 h to 1.2.4.-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides (12c-polydatin, octyl-14c-β-d-glucopyranosid) were cleaved within 1 hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd503 and contribute significantly to animal welfare (3r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. (1982) and including an empiric correction factor, mrd for the training set was 2.49 whereas bias was 2.80 µmol/l. by applying the developed pbpk model to the test set the respective values were mrd=3.92 and bias=1.43 µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco-2 cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco-2 cell system under different experimental conditions. the permeation characteristics of hypericin (5 µm) in absence or presence of hypericum extract 145, 62.5 µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from 0 to 7.5 %). the majority of hypericin was found after cell extraction (44% in absence and 76% in presence of the extract). the recovery was in the range of 90 %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in 10 healthy foals (5 ♂ and 5 ♀, age: 42-63 days, body weight: 100-177 kg) which were treated once with rif alone (10 mg/kg s.i.d., p.o., a) followed by the administration of gam (6 mg/kg once weekly, i.v., b) for 3 weeks. study period 3 ("rif-gam acute", c) includes the administration of gam and rif for 7 days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for 2 weeks. all periods were completed with blood sampling for pharmacokinetic analysis for 48 ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c 24 h : 1.2 ± 0.5; balc/c 24 h : 2.01 ± 1.24). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c 24 h -ratio was unaffected (peters et al. 2011) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c 24 h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. 1 1 institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp2d6. direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp2d6. however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct1 and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about 9% of caucasians are compound heterozygous carriers of loss-of-function oct1 polymorphisms. therefore, oct1 polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct1 function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct1 alleles. methods: 50 mg desvenlafaxine (pristiq®) was orally administered to 41 healthy subjects preselected according to their oct1 genotypes. oct1*1 allele was regarded full active, oct1*2 to *6 alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to 60 hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the 41 subjects 14 carried two active, 13 one active, and 14 zero active oct1 alleles. age, height and weight were 26.9 ± 6.4 years (mean ± standard deviation), 1.75 ± 0.11 m and 70.9 ± 12.1 kg with no significant differences among the oct1 genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc 0-infinity of desvenlafaxine varied between 52.8 and 282.2 min*mg/l and auc 0-infinity of didesmethylvenlafaxine between 3.5 and 30.7 mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct1 genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average 14% greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct1 genotype groups. conclusions: our results suggest that oct1 genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct1 genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly (1). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression (2) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september 2013 and december 2013 using the medical records of elderly patients (≥65 years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss 16.0 statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of 541 elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n=135). median age was 72 (min:65, max:98), 84 patients were female (62.2%). according to medical records of 135 patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug (69.6% vs. %30.4) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics (65.4% vs. 33.3%) and angiotensin receptor blockers (36.9% vs. 23.5%) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women (58.8% vs. 38%, 68.6% vs. 20.2%, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over 20% of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e14 guideline [1], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [2] the ability to detect qtc-prolongations of about 10 ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [3, 4] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like-1 (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin-12, -13 and -17. apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in 23 patients with nstemi, 4 stemi patients and 14 controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd62 and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap1) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for 30 sec to 5 min reduced platelet aggregation in lta studies by up to 20 % for apelin-17. this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name (300 µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group (56,84% ± 9,28 % in mi versus 100 % ± 19,35 %in controls; p = 0.029). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin-17 in patients with mi (14.95 ± 0.6 pg/ml versus 16.98 ± 0.6 pg/ml; p = 0.035). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = -0.46; p = 0.03). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin-17. upon platelet activation, apj is internalized and surface expression is reduced by about 50 %. in mi patients, plasma levels of apelin-17 and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin-17 levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m 1 receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m 1 muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m 1 receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from 2004 to 2011. inclusion criteria comprised the initial absence of dementia and age of 75 years or older in 2004. anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m 1 receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn 1,2 , s. schmiedl 3, 4 , k. janhsen 3, 5 , p. thuermann 3, 4 , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of (1) drugs with a known association to a certain adverse event (=positive controls) and (2) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for 100% of the positive control drugs, and for none of the negative controls. ryan et al. 2013 developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. 2013 ;36 suppl 1:s33-47) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in 2010 to 2013 were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of 50% (relative risk 1.5) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with 80% statistical power. results: from a total of 182 index drugs in the reference sets of ryan et al., 142 (78.0 %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of 5,485,722 index drug initiators were included in the analysis. for a total of 16 index drugs, a relative risk of 1.5 could be detected with 80% power. the numbers of index drugs for each of the outcomes of interest were: ami (3 positive controls; 6 negative controls); aki (3 positive controls; 1 negative control); ugib (2 positive controls; 1 negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with 80% power to detect a relative risk of 1.5 was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of 11-nor-9-carboxyadministration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ 9tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study 11-nor-9-carboxy-∆ 9 -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of 16 patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > 0.05). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of 440 lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term 2014/15) at technische universität münchen (tum). the course consisted of a 28-day teaching period, followed by a 12-day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of 224 out of 393 (57%) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. 1). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n=194 3 rd -year medical students, n=213 4 th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of 60 items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the 60 items were presented as statements for restudy, 20 items were tested using single-choice questions, and 20 items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of 30 single-choice questions, 15 questions of which had not been used before (as a standard of comparison). the remaining 15 questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of 5 of these reworded 15 questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [1] . in order to address the persisting prescribing deficiencies [2] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [3] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of 67 indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least 0.3% of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [4] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising 8 indications, 28 drugs representing 25 mechanisms, and up to 120 adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july 2014 by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november 2015 and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are 15 chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in 2014 1 it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. 1907/2006 chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above 1000 tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, 1932 dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, 43% to 82 % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., 2015) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category 4. this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of 10000-100 000 t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to 800 ml/m³ and in mice in response to 200 ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category 4 with a mak-value of 5 ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl-60 cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm (900 mhz) and umts (1950 mhz). additionally, lte (2.535 mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from 0 to 4 w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in 2014 who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ 10% (m) in 5% (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in (1) a simple non-probabilistic way, (2) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or (3) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector (2) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between 0.1 and 3.0 µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox21, which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s81 assays in addition to the h295r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some 60 substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than 750 different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized 4 week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than 80 control groups with each 10 males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of 10 control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., 2012) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = 0.1, the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately 1000 to 3000 mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca 2+ -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of 389 and 778 mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca 2+/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: 610,8 mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to 3000 mg per day over the period of up to 12 weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with 24 male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of 3000 mg of hca for 12 weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer 1 1 universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include 0 and 1, respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to 100 µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only 10 µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg 431 and 439 especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt4 to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt4 cells were either continuously incubated for 72 h (approximately 1.5 cell cycles duration) with methyl methanesulfonate (mms; 50-200 µm), benzo[a]pyrene (b[a]p; 0.1-1 µm), vincristine (3-20 nm) , and colcemid (10-20 nm) or cells were irradiated with xrays (0.5-2 sv) and then cultured for 72 h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation (75 w, 20 min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for 3 out of the 5 treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤2-fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt4 cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt4 cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk-52e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s139 phosphorylated h2ax (γh2ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk-52e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p53, p-chk1, p-chk2 and p-kap1. furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk-52e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann 1 , g. fritz 1 1 institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a 3´-5´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic 10 ) and moderate toxic (ic 40 ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy926 cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic 10 and ic 40 were determined after 24h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy926 cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, 24h after permanent tbhq treatment. pictures were taken at the time points 0h, 4h and 8h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht-29 cell line on the ea.hy926 monolayer was investigated. wrn-deficient or non-deficient cells were treated with 100 µm and 160 µm tbhq or with tnfα. results: for ea.hy926 cells, 100 µm and 160 µm tbhq were determined as ic 10 and ic 40 , respectively. performing the migration assay, ea.hy926 cells showed 75% gap closure, whereas wrn-deficient cells showed a closure of only 35% after 8h. the gap was closed of 65% and 55% after 100 µm and 160 µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after 100 µm tbhq treatment, whereas the treatment with 160 µm tbhq showed a slight decrease in migration of about 10% compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after 160 µm tbhq treatment a slight increase of adhesion was detected in ea.hy926 cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy926 cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht-29 adhesion to ea.hy926, which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl60, jurkat and tk6) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk6 cells showed similar increasing trends, whereas hl60 cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration (100 nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl60 cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk6 cells. the results for the highest investigated concentrations (31.6 nm and 100 nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk6 cells. in contrast the hl60 cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p53 status of hl60 (null), jurkat (multiple mutations), tk6 (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc-1 were applied. accumulation of p53 and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p53 and pakt analysis, and quantification of the oxidized dna base 8-oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than 1000 bacterial species and population densities of up to 10 7 cfu per cm 2 . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., 2014) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of 20-30 hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and 6-4 photoproducts (6-4pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [1] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc3b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct116. similar results were obtained in sw48 and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p62 as observed by flow cytometry and western blot analysis in hct116, sw48 and hcec. cdt-and irinduced autophagy rates in hct116 increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein 1 (lamp-1) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser473. pharmacological inhibition of akt in hct116 cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc3b and lamp1 after 48 h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after 72 h and significantly lower apoptosis/necrosis rates after 48 h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n7-guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative 2chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n7-ethylthioethyl-2´desoxyguanosine (n7-ete-dg) and n3-ethylthioethyl-2´desoxyadenosine (n3-ete-da) and their thermal hydrolysis products n7ethylthioethyl-guanine (n7-ete-gua) and n3-ethylthioethyl-adenine (n3-ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh2ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone 2ax (gh2ax), which can be visualised as gh2ax foci by immunocytochemistry. in the present study, we tried to assess if gh2ax is a reliable biomarker for detecting the cellular response to dna damage. we selected 14 well-characterised genotoxic compounds and compared them with 10 non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh2ax by manual and automatic scoring of γh2ax foci, and by flow cytometry counting of γh2ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh2ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh2ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh2ax positive cells showed a significant correlation. further we compared dna damage induced by 4 selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh2ax assay detects a specific response to dna damage which can persist longer. the γh2ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of 3 stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh2ax), nuclear division (phospho-h3, dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk6 cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for 4 and 24 hrs in triplicates on a 96 microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide 3-chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c6 (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h3 events at 4 and 24 hrs and polyploidy at 24 hrs time point. eto induced a clear increase of yh2ax with a simultaneous reduction of phospho-h3 at 4 and 24 hrs. finally, cccp caused a reduction of phospho-h3 events, increased cleaved parp events and did not influence γh2ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh2ax and phsopho-h3 pilot study results as well as cleaved parp with caspase 3/7. in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage 2, additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih3t3, mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g0/g1 = ~70 %) and semi-confluent cultures, similarly arrested in the g0/g1 phase by serum-starvation or the mek inhibitor u0126, ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc6 staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk2. however, inhibition of atm, the kinase responsible for chk2 activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip1, since the chemical rip1 kinase inhibitor necrostatin-1 (nec-1) blocked cell death up to averagely 50 %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp-1 and p53 are probably not involved. deeper analyses could give evidence that nec-1 did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec-1, possibly rip1, acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca 2+ signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc1 and mtorc2) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c 3t3 cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for 3 days with the tumour initiator mca (3-methylcholanthrene) followed by 13 days with the promotor tpa (12-o-tetradecanoylphorbol-13-acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with 10 nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi-027. surprisingly, an incubation with 3 µm osi-027 led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s6. our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi-027, which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi-027 by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past 50 years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type 2 diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c 3t3 cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator 3-methylcholanthrene (mca) and the tumour promotor 12-otetradecanoylphorbol-13-acetate (tpa). in the first experiment several metformin concentrations (0.1-1 mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using 1 mm metformin. this effect was not caused by growth inhibition of metformin itself since 1 mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the 2 phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using 1 mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, 55131 mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to 5, 10, and 20 ppm for 2 years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [1]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: 0.5 ppm) after whole body exposure to vapor [2] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of 0, 5, 10, 20 ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate 200 mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp4a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, 2-pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [3] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day 0 (e0) at 37.5°c and 82% humidity. on e3, the eggs were cracked open and transferred into petri dishes. on e10, cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to 48 hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with 3-methylcholanthrene as a tumour initiator and 12-o-tetradecanoylphorbol-13-acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in 96-well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march 2013). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between 5-10%. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's (3r), described by russel and burch in 1959. in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches 1 , resulting in a refinement of in vitro testing. in this study, human tk6 cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of 19.3 (± 1.7) h was observed while fbs supplemented cells showed a doubling time of 13.5 (± 0.8) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg 442c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by 1) determining reactivity class cut offs based on published data on 199 substances for the dpra performed according to oecd 442c to predict un ghs sensitizer classes, 2) a variant of the dpra assessing reaction kinetics (time and concentration) for 12 substances or 3) an extended protocol testing several test substance concentrations for 50 reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of 6.38% (ec6.38%). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes 1a and 1b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between 5 min and 24 hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the 3 rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. 1, 10, and 100 mm) indicated that potency classes could be assigned using the ec6.38% value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec6.38% value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase 1 (nat1) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u937 and thp-1) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p450-dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of 27 putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). 18 of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or 5-amino-2-methylphenol, respectively. in contrast, covalent peptide adducts were identified for 12 putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a '2 out of 3' prediction model provided a high sensitivity of 81% for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the '2 out of 3' prediction model in a set of 95 direct haptens was comparable (sensitivity = 87% when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than 1 tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf2 luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and 86 two-test and 360 three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. 1) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [1] [2] [3] [4] . in recent supplement, 2-methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [5, 6] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [7] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of 1 mg/ml (1%), finite dosing and 30 min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [8] and electrostatic potentials using gamess [9] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. 1). toluene-2,5-diamine shows a higher accumulated mass due to increased lipophilicity (fig. 1) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [1] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [1] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in 2004 the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba1] contains data for about 600 substances on dermal absorption which has been found through the echemportal [3] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. 165 chemicals have been investigated in a comparable test system; from these 79 were shown to have a low dermal absorption of less than 1% and 51 compounds showed a high absorption rate of more than 50%. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for 15 substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than 1% to more than 50%. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in 2015 [5] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [1]who (2006) ehc 235 dermal absorption [2] scholz et al (2014) naunyn-schmiedeberg´s arch pharmacol 3 (suppl 1):s86 [3] www.echemportal.org [4] http://edetox.ncl.ac.uk [5] jung et al (2015) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p450 variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb3r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett 233:172-86, 2015) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology 79: 63-73, 2004) . we modelled the concentration time profile exploring the dose which would lead to a concentration at 1 hour and at 24 hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between 3 and 15 µm 3 /sec (truisi et al., 2015) and an absorption of 100% and an absorption rate of 1/h (cristofoletti and dressman, j pharm sci 103: 3263-75, 2014). results: the data of the in vitro study with 100 µm ibuprofen could well be modelled. when assuming a clearance of 15 µm 3 /sec the dose of 1480 mg resulted in an 1 hour concentration of 66.5 µm in the hepatic vein of pbhkm equal to 133.0 nmol/well (volume of the well = 2 ml) in the in vitro study in which the measured concentration was 138.75 nmol/well. the concentration at 24 hours of 8.7 µm (equal to 17.4 nmol/well) corresponded with the in vitro concentration (16.5 nmol/well). the modelling approach was less successful with in vitro dosing of 1000 µm. the 10 fold higher dose of 14800 mg lead to nearly double the concentration at 1 hour than measured in vitro. with a dose of 8500 mg/kg an approximation was feasible resulting in 396.7 µm in the hepatic vein at 1 hour which is equal to 793.4 nmol/well whereas the measured concentration in vitro was 782.85 nmol/well. even with a clearance value as low as the 2.5 percentile (3 µm 3 /sec), the concentration at 24 hours was modelled to be lower than the in vitro measured value (in vivo model: 98.7 µm which corresponds to 197.4 nmol/well; measured in vitro concentration: 979.2 nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro (100 µm) corresponds to a dose of 1480 mg (note: the highest approved daily dose is 2,400 mg). for the high in vitro dose modelling was successful only for the concentration 1 hour after dosing and a dose of 8,500 mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and 3.5 fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up 12 acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score (4 positive out of 8 possible genotoxicity categories) was assigned to 9 of the 12 tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [1], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of 12 acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even 200 years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin 1 (trpa1) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk1/2, are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa1 activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk1/2 and increased protein biosynthesis of sre-regulated genes in hek293 cells overexpressing htrpa1. cees increased perk1/2 levels already after 2.5 min which could be prevented by the trpa1 blocker ap18. activation of target genes through perk1/2 signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa1 channel activation. thus, trpa1 might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst-1) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort ("3rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a549), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a549 cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a549 cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg2 is often used as a model for liver toxicity studies. however, under two-dimensional (2d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for 21 days in a three-dimensional (3d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg2 cells. in our present study we extended previous studies and compared hepg2 cell cultivation in three different 3d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p450 monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. our results show that the previously reported increase of metabolic competency of hepg2 cells is not primarily the result of 3d culture but a consequence of the duration of cultivation. hepg2 cells grown for 21 days in 2d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all 3d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg2 is not due to 3d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about 55 different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [1]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [2] , whereas asfas were less toxic [3] . a substantial transfer across an intestinal barrier model (caco-2) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [4] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [5] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after 48 h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic 50 ~ 7-12.5 µm) in a concentration range comparable to that of arsenite (ic 50 ~ 7.5 µm), whereas asfas were less cytotoxic (ic 50 > 100 µm). after incubation with ashcs the cellular arsenic concentrations increased 10-20fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [1, 2] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic 50 values of 0.67 ± 0.02 µm for hache and 81.28 ± 0.04 µm for hbche. ic 50 values with chloroquine were 28.37 ± 0.02 µm for hache and 55.62 ± 0.02 µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after 60 minutes, only paraoxon-inhibited hache (50%) and cyclosarin-inhibited hbche (10%) were reactivated by 500 µm chloroquine. on the contrary, 10 µm amodiaquine reactivated all tested ops after 60 minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with 100 µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [1] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., 2012, 49, 229-238. [2] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., 2015 , 16, 2205 -2215 bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional (3d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a 3d model was used consisting of the permanent breast epithelial cell lines mcf10a (er -, estrogen receptor negative) and mcf12a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the 3d culture of mcf10a and mcf12a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf12a) and er-independent (mcf10a and mcf12a) effects. the 3d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg2) cells and (b) co-culture systems of human monocytic or macrophage-like (thp-1) and hepg2 cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg2 and thp-1 monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about 20% cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg2. we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator (3-methylcholanthrene) and promotor (12-o-tetradecanoylphorbol-13-acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day 42, morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that (1) several substances have a different impact on the transformation process, (2) it is possible to identify for each substance the phase with the greatest effectiveness and (3) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c 3t3 assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et50 protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg 405) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg 437) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg 438) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et50 (exposure time reducing viability of treated tissue to 50%) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg 438, one of the five severe ocular irritant formulations (un ghs cat 1) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat 1 formulations was just above the un ghs cat 1 classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et50 predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et50 protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins-1e) and exocrine (ar42j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, 2014 . 9(12): p. 2809 . briefly, pancreas was infused and embedded with agarose. tissue sections of app. 200 µm were prepared using a vibratome and maintained in cell culture medium for up to 6 days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in 10 % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins-1e cells) or amylase/lipase (ar42j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins-1e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar42j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of 6 days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg 1 , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd86 assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers (2,4-dinitrochlorobenzene, 2-mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd86 on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd86 after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology 2008; 249:146-1529) . dendritic cells generated in fcs supplemented medium were cd1a+/cd1c+, whereas dendritic cells from serum free culture conditions were cd1a−/cd1c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd1a+/cd1c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp-1 cells, as model for dc, enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p450 (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp-1 cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp-1 cells by microarray technology. we identified 6 differentially expressed mirnas in cocultured thp-1 cells compared to monocultured thp-1 cells irrespective of the treatment (medium, 0.2% dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after 48h) 8 additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp-1 cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp-1 cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp-1 and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa1 expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm5 and 6. trpa1 has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in 1). although recently two reports identified trpa1 in pulmonary epithelial and endothelial cells (2, 3) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa1 protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa1-deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa1 on a cellular level we cultured a hek293 cell line stably expressing trpa1 (4) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca 2+ -influx in this cell line, which was blocked by the specific inhibitor a96079. in the future, we will utilize the isolated perfused lung model (5) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa1-deficient mice after exposure to potential toxic inhalation hazards (tih see 6) to challenge the hypothesis of trpa1 as an important toxin sensor in the lung. by this strategy we hope to understand trpa1 function in lung cells and to evaluate trpa1 proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for 28 days to male rats including blood sampling for metabolic profiling after 7, 14 and 28 days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole-3-acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole-3-propionic acid for aminoglycosides, taurine for fluoroquinolones, 3indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., 2011; luo, schomaker, houle, aubrecht, & colangelo, 2014; yamazaki et al., 2013) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low (30 mg/kg) and a high (80 mg/kg) dose. rats were sacrificed following 3, 7, or 14 consecutive daily doses, or after recovery 10 days following 14 consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) 4 and 15 days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide 1 (oatp1) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/11 proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c58 protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann 1 1 background: in a 28-day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp2r1, gc, cyp3a, ugt1a) in liver was also affected, while initial indications for modulation of renal cyp24a1 and renal and hepatic cyp27b1 could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n=5 for single substances, n=10 for combinations) were treated for 28 days at dose levels tested based on noaels from 90-day subchronic feeding studies and ranged from noael/100 to noaelx10. quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total (25-oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n=30) were 64.7±8.3 ng/ml (min/max: 48/78 ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c (1000 ppm in food, n=5), there was a statistically nonsignificant reduction of vitd levels to 71.3±17.6% of the concurrent control (n=5). however, for 4 of 5 animals of this group, measured vitd level were below the range observed in pooled controls (n=30). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p21 for c, e and pz; cdk1 and gadd45a for e; cdkn1c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp3a1/2 and ugt1a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with 1000ppm cyproconazole, there were indications for a moderate reduction of 25-oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a 26-year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the 9th week, and she has been taking a multidrug therapy (dapsone 100 mg/day, rifampicin 600 mg/month, clofazimine 50 mg /day and clofazimine 300 mg/month) for the past 8 months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about 20 weeks. in the 8th, 22nd, 28th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at 28 weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the 39th week of gestation without perinatal complications. the baby was also healthy (apgar 8-9,3300 g,51 cm), and its growth and development were normal during a 6-month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was 15 months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after 3 months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in 2009 in berlin, germany, to evaluate kidney function in people ≥ 70 years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure 1 illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table 1 shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january 2014 until april 2015). the probability of dabigatran use rose with increasing age (+12%), and the probability of phenprocoumon use rose in case of egfr < 60 ml/min/1.73m 2 (+54%) or male sex (+82%). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ 65 years in 12 nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > 20 mmhg and/or dbp decrease of > 10 mmhg within 3 min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > 139 mmhg and/or mean dbp >89 mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with 96 nhr (mean age = 84.5 ± 7.3 years). in total, 15 subjects (15.6%) had oh. the mean change in sbp from sitting to standing was 19.2 ± 15 mmhg (range +8.5 to -52.5 mmhg) in patients with oh and 1.5 ±10.9 mmhg (range +44.5 to -17 mmhg) in patients without oh. mean sbp was significantly higher (143.6 ± 17.1 mmhg) in people with oh than in those without (131.5 ± 20.1mmhg). all of the nhr with oh were hypertensive compared to 89% of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p>0.05). medication data was available for 89 patients. all individuals with oh and 60 nhr without oh (80%) had antihypertensive medication. more than 2 different antihypertensive drugs were present in 11 patients with oh (78.5%) and in 43 patients without oh (57.3%). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than 75% of patients in both groups reached 80 points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around 50% of patients with oh as well as patients without oh needed more than 19 sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz 1 , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than 1000 metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ 30 %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer (60:40) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from 50 mg/d clom citrate to up to 150 mg/d in case of non-ovulation. however, therapy outcome is variable and approximately 10 -30% of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via 4-hydroxylation of trans-clom by the highly polymorphic cytochrome p450 (cyp) 2d6 leading to the major active metabolite trans-4hydroxyclomiphene (trans-4-oh-clom) [1] . recently, we identified a less active trans-3-oh-clom which is also formed by cyp2d6. besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of 100 mg clom citrate. results: incubations with human liver microsomes revealed an almost 60-fold higher glucuronidation rate for trans-3-oh-clom, which is exclusively catalyzed by ugt2b7, compared to the more potent trans-4-oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans-4-oh-clom to its sulfate is 16-fold higher compared to 3-oh-clom. for both metabolites a participation of sult1a1 and sult1e1 was identified. these results were in line with previous studies, which identified the same sults [2] and ugts [3] responsible for the conjugation of the structurally related trans-4-hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans-3-oh-clom and trans-4-oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom-3-o-glucuronide. conclusion: our results suggest a new metabolic route via trans-3-oh-clom which appears to be a potential inactivation pathway of clom. 1 1 institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of 0.07 µm sm (ic 10 ). today the cells are able to tolerate a concentration of 7.2 µm sm (ic 90 ), which reflects to a concentration of which 90% of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h 2 o 2 , glutathione, ca 2+ -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening 49 significant up-regulated and 20 significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of 11 different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than 35 weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg2 cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp1a1 expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm1-mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg2 cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka 1 1 institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp7b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp7b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in 2006 as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in 2008. the program was accredited in 2011 by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates 30 places per year to an average of 60 applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about 75% of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler 1 , m. hermanns-clausen 1 1 uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from 2010 to 2015 in total 85 cases of human exposures to chlorine developed from mixtures of hypochlorite and acids (0.8 of 1000 cases) were registered. in 55 cases the exposure was due to mixtures of products intended for domestic use. 94 % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough (45 %), dyspnea ( 33 %), irritated upper airway (26 %), abdominal discomfort (pain, nausea, vomiting) (21 %), thoracic pain (20 %), irritated eyes (11 %), dizziness (8%), and bronchospasm (6 %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in 12 patients (14 %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( 83% ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were 42 patients in the same period exposed to hypochlorite evaporation alone. 36 of them (86 %) had symptoms of which in 30 cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway (33 %), nausea or vomiting (30 %), cough (23 %), irritated eyes (20 %). dyspneoa was less fequent than in the mixture group (10 %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh2ax assay for genotoxic and nongenotoxic agents: comparison of h2ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant 1322-530 is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [1] mürdter t, et al. hum mol genet, 2011, 21:1145-54 [2] nishiyama t. et al., biochemical pharmacology, 2002, 63:1817 -1830 [3] sun d. et al., drug metabolism and disposition, 2007, 35:2006 background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: 10 bd patients (5/5 m/f, 43.4±5.3y, tbsa 40 .9±5.9%) and 5 patients (74.4±3.9y) from the icu were included in this observational study. blood samples were taken within the 3 rd interval of the 8h-lasting dosing period of piperacillin/tazobactam (4/0.5g within 0.5h) at 1, 4 and 7.5h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least 1xmic but preferably 4xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic 16 mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at 1, 4, and 7.5h, were as follows: c max 69.6±7.9 vs.116.3±10.4 mg/l, p<0.05, half-life 1.8±0.3 vs. 2.3±0.3h, p>0.05, clearance 12.6±1.9 vs. 6.5±0.4 l/h, p<0.05, volume of distribution 27.8±2.0 vs. 20.9±1.3l, p<0.05. free concentrations (which were included in tdmx calculations) were 87±2 vs. 81±2% (p<0.05) of total concentrations. duration per day while concentrations exceeded 1xmic (15.6±1.9 vs. 22.0±1.1h, p<0.05) or 4xmic (5.4±1.3 vs. 9 .4±0.7h, p<0.05) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for 4xmic could be enhanced to 16.2±1.5h if the piperacillin amount will be increased to 4x8 g/d and the infusion duration to 3h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as >1000mg mtx/m 2 bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the 1950s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years 2004-2009 were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. 29 patients aged 2-18 years (male = 19, female = 10) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, 103 mtx treatment cycles corresponding to 300 mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph <6.5, trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [1] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [2] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [3] , we speculated that repression of gene expression as the predominant transcriptional response to ota [4, 5] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using 13 c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [6] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk-2) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h2a, h2b, h3 and h4. we further selected acetylation at histone h3 lysine 9 (h3k9), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [7] and which was reduced from ~ 3% in controls to < 0.1% in response to ota, to establish a link between loss of h3k9 acetylation and expression of genes consistently shown to be down-regulated in response to ota [4, 5] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h3k9 acetylation at promoter regions of the selected genes (% of controls: amigo2: 45%, clasp2: 60%, ctnnd1: 54%). overall, these data provide first evidence for a mechanistic link between h3k9 hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays (7 most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of 3 ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced 3d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from 500 in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to 80-90% (average prediction accuracy was 70%). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> 30 µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [1] [2] started in 2010 and is a public-private partnership project within the european innovative medicines initiative (imi) [3] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to 4 week) repeated dose studies, mostly in rodent. overall more than 7000 study reports for approximately 1400 investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [4] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than 13 million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately 150 prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [5] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than 400 active substances approved for being used in pesticides and over 100000 chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg2, heparg, rat: h4iie) and adrenal gland (human: h295r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a 28-day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg2, heparg, hek293). cytoxicity has been analysed by wst-1 and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p450 (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp-1 as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens (hennen et al., 2011) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp-1 with b[a]p and eugenol in coculture with hacat cells augmented cyp1a1 and/or cyp1b1 mrna levels, while this was not found for thp-1 monoculture. augmentation of cyp1a1 mrna needed continuous presence of hacat cells. in coculture, levels of 3-oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase 1 enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of 3-oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp-1 cells by 2,4-dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp-1 cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci 2011 123:501-510.toxicology -toxic pathway analysis/aop 395 background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel 600mg or prasugrel 60mg (n=200). adp-induced platelet reactivity was assessed by impedance aggregometry 30 to 120 minutes and day 1after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at 120min (r=0.21; p<0.001). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations 120min/day 1: p=0.57 for clopidogrel, p=0.76 for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so1861) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc-3 and miapaca-2 cells in comparison to the non-target cell line nih3t3 with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc-3 cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so1861 by subcutaneous injection at the neck. after the tumor reached a diameter of 2 to 3 mm in size 6 treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so1861 in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k1492). complete blood count analysis was done by labor 28 gmbh, berlin. results: the adenine release mediated by dianthin-egf was 47.8 pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic50 value of approximately 1 nm for egf receptor expressing miapaca-2 and bxpc-3 cells as compared to 100 nm for non-target nih3t3 cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from 10 pm to 1 µm. toxicity studies in balb/c mice revealed 0.4 µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas 40 µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of 28 days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be 80% less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet 50% less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc-3 cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so1861 proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp3a4 and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp3a4 at different concentrations with and without nadph to determine the ic 50 shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr1 -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version 2014.11 and admetsar. results: fractions (f1-f3) indicated ic 50 shift reduction ≥5 post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = 2.46) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic 50 = 35.45 µg/ml) across mdr1 -mdckii cells. niaziminin indicated 85.57% bioavailablity via the human intestinal membrane with 61% chance of inhibiting cyp3a4. βsitostenone showed strong p-gp inhibition (83.27%) with 100% absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp3a4 or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in 2000 to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than 180 departments of all 51 berlin hospitals from october 2002 until december 2011. through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than 2 mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all 198 cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases (5.1%). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table 1 . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient 1, pelargonium sidoides in patient 6), and eight times as possible (valeriana in patients 3, 4, 8, 9, 10 , mentha piperita in patient 5, hypericum perforatum in patient 2, eucalyptus globulus in patient 7). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient 6). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil 2 as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. 1). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. 1, fig. 1 ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil 2 for one of our patients ( fig. 2 & 3 ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for 61 drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for 20% of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, 3rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, 2015) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg 428 under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from 4 different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n=4 per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at 32°c throughout the experiment and electrical resistance barrier integrity testing was performed at the start (0 h) and end of the experiment (24 h). four test preparations at final bpa concentrations of 2.4, 12, 60, and 300mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ 14 c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca 1%, v/v), uridine 5'-diphosphoglucuronic acid (udpga, 2 mm) and 3'-phosphoadenosine-5'-phosphosulfate (paps, 40 µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with 20 successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration (300 mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between 1.7 and 3.6% of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between 16 and 20% of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z 225 and 237 instead of the molecule ions (m/z 294 and 306) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were 1.4 ng/ml and 0.5 ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over 24 hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter 4 (mct4), encoded by the slc16a3 gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct4 and its epigenetic regulation by slc16a3 promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct4/slc16a3 might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct4 protein expression was analysed in 130 paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v.3.6 (definiens ag). dna methylation in the slc16a3 promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in 82 paraffin embedded metastasis samples by maldi tof-ms. mct4 protein expression data and dna methylation at the specific cpg site in the slc16a3 promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct4 protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately 28% and 14% in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc16a3 promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the 5'-regulatory region of mct4 may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) 4-hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in 390 postmenopausal breast cancer patients of european origin who were treated with 20 mg/day of tamoxifen for at least 6 months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method 1 . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of 313 premenopausal breast cancer patients mainly of european descent 2, 3 . twelve snps close to or above genome-wide significance (p <5e-08) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes 1, 2 and 16 for (e) metabolite e and to chromosomes 15 and 18 for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < 5% were confirmed as putative candidates: rs662106 was associated with a 21-39% variant allele-dependent increase of (e) and (z) metabolite e isomers (p< 0.05), and rs3731872, mapping to a gene encoding zinc finger protein znf124, was associated with increased risk of reccurrence or death (hr carriers 2.6, 95% ci: 1.3 -3.4; p < 0.005). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of 15 known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water (1:1) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from 7.8 nm to 10000 nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the 15% tolerance range for all analytes in all matrices. matrix effects were between 39-104% (plasma) and 31-95% (urine), apparent recoveries in plasma were above 97%. basal plasma ba level (mean ± sd) in fasting healthy subjects were 667 ± 574 nm (unconjugated bas), 935 ± 629 nm (glycine-conjugated bas) and 104 ± 62 nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were 193 ± 225 nm (unconjugated bas), 89 ± 29 nm (glycine-conjugated bas) and 6 ± 2 nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord-023095-4dannjjm authors: nan title: research abstract program of the 2011 acvim forum denver, colorado, june 15–18, 2011 date: 2011-05-03 journal: j vet intern med doi: 10.1111/j.1939-1676.2011.0726.x sha: doc_id: 23095 cord_uid: 4dannjjm nan clinics ãã also see infectious disease abstracts id-1 -id-14 (thursday, june 16, 2:15 pm -6:15 pm) ãã also see pharmacology abstracts p-1 -p-5 (thursday, june 16, 5:00 pm -6:15 pm) ãã also see gasteroenterology abstracts gi-1 june 17, 8 hypertrophic cardiomyopathy (hcm) is the most commonly observed myocardial disease in cats. beta-blockers and calcium channel inhibitors are frequently administered drugs to cats with preclinical hcm despite the fact that neither drug category has been proven to slow disease progression or improve survival. ivabradine (procorolan s , servier, france) is a novel negative chronotropic agent used in the treatment of ischemic heart disease in people. little is known about its efficacy and safety in cats. the purpose of this study was to determine the short-term effects of ivabradine on heart rate (hr), blood pressure, left ventricular (lv) systolic and diastolic function, left atrial (la) performance, and clinical tolerance in healthy cats after repeated oral doses. ten healthy laboratory cats were involved in the present study. physical examination, systolic blood pressure measurement, and transthoracic echocardiography were performed in all cats at baseline and after oral administration (4 weeks each) of ivabradine (0.3 mg/kg, q12 h) and atenolol (6.25 mg/cat, q12h; 1.0-1.7 mg/kg) in a prospective, double-blind, randomized, active-control, fully crossed study. a-priori non-inferiority margins for the effects of ivabradine compared to atenolol were set at 50% (f 5 0.5) based on predicted clinical relevance, observer measurement variability, and in agreement with fda guidelines. variables were compared by use of 2-way repeated measures anova. ivabradine was clinically well tolerated with no adverse events observed. hr (ivabradine, po0.001; atenolol, po0.001; ivabradine vs. atenolol, p 5 0.721) and rate-pressure product (ivabradine, p o 0.001; atenolol, p 5 0.001; ivabradine vs. atenolol, p 5 0.847) were not different between treatments. at the dosages used, ivabradine demonstrated more favorable effects than atenolol on echocardiographic indices of left ventricular (lv) systolic and diastolic function and left atrial performance. ivabradine is non-inferior to atenolol with regard to effects on hr, rate-pressure product, lv function, la performance, and clinical tolerance. clinical studies in cats with hcm are needed to validate these findings and further assess safety. the aim of this study was to compare outcome from cpa in dogs following initial administration of either epinephrine or vasopressin during cardiopulmonary resuscitation (cpr). dogs having cpa in the er or icu of a university hospital were randomized to receive either iv epinephrine (0.01-0.02 mg/kg) or vasopressin (0.5-1.0 u/kg) in a blinded fashion immediately following establishment of iv access and again three minutes later. a standardized cpr protocol was followed. other vasopressors were not permitted during the six minute study period, at the end of the study period additional cpr interventions were at the discretion of the managing clinician. the primary end point was return of spontaneous circulation (rosc) within the study period; secondary end points included rosc at any point, survival to 20 minutes, and survival to one hour. sixty dogs completed the study, 31 received epinephrine and 29 received vasopressin. rosc within six minutes was 53% (13 vasopressin, 19 epinephrine p 5 0.2), rosc at any time was 60% (15 vasopressin, 21 epinephrine p 5 0.2). survival to 20 minutes was 32% (6 vasopressin, 13 epinephrine p 5 0.077), survival to one hour was 18% (2 vasopressin, 9 epinephrine p 5 0.027). five dogs survived to 24 hours, one survived to hospital discharge. of animals dying after rosc, 13/35 were euthanized and 22/35 rearrested. no advantage of routine substitution of vasopressin for epinephrine was seen for rosc, a small survival advantage at one hour was seen in the group receiving epinephrine. the study also demon-strated that prospective clinical cpr research in animals is both possible and practical. three dogs were evaluated in 3 phases. phase-1: single-dose diltxr at approximately 6 mg/kg po. phase-2: same dose q12h for 4.5 days. phase-3: after a 14 day wash-out the single-dose protocol was repeated using cut tablets to assess affect on extended release properties. blood pressure (bp), 6-lead ecg, echocardiogram, and 24h-ambulatory ecg were performed at baseline, and conclusion of phase-2. blood samples and bp was obtained 0, 1, 2, 4, 8, 12, and 24h after final dosing. peak median plasma diltiazem concentrations (mcg/ml) measured by hplc for each phase were 0.0979, 0.016, and 0.07 respectively. diltiazem concentrations were below the limit of detection in the majority of samples in phase-2. median diltiazem concentration reached purported therapeutic concentrations (0.05-0.2 mcg/ml) by 2 h post-pill in phase-1 and 1h post-pill in phase-3. therapeutic concentrations were maintained for 24 h in phase-1, but only 2h in phase-3. median bp (mmhg) was 142.7 at baseline and 126.0 at peak concentration in phase-1. median heart rate (bpm) was 127.1 at baseline. 24h-ambulatory ecg analysis revealed the median hourly heart rate was 101.5 at baseline and 102 during phase-1. median heart rate at peak concentration in phase-1 was 127.5. lack of detectable plasma diltiazem during phase-2 may be due to up-regulation of drug metabolism via p-glycoprotein (abcb1-1) mutations. ongoing data collection and analysis will include mutation testing. adiponectin (adpn) is a cytokine produced by fat cells which has been shown to be correlated with adverse cardiac conditions in humans. in the heart, adiponectin activates several pro-survival reactions, including the ampk pathway and cox2 receptors, which protect the heart following ischemic injury. recent studies have shown that higher levels of adpn influence cardiac remodeling signaling, inhibiting protein synthesis and suppressing pathological cardiac growth. in humans, adpn plasma levels rise with decreased activity of the sympathetic nervous system and b-adrenergic agonists inhibit adpn at the level of gene expression. in contrast c-reactive protein (crp), a marker of systemic inflammation is elevated in humans with congestive heart failure (chf) and correlates to the severity of disease. first, we hypothesized that dogs with chf would have reduced adpn and elevated crp compared to normal dogs and that cytokine concentrations would predict severity of chf. second, we hypothesized that adpn receptor-1 (r1) and adpn protein would be elevated in the myocardium of chf dogs reflecting a compensatory process. we collected serum from 32 dogs (18 healthy and 18 chf). circulating adiponectin and crp levels were quantified using a mouse/rat adiponectin elisa and a canine crp elisa. we found lower mean crp concentrations in normal dogs (3.5 ae 2.3 mg/ ml) than dogs with chf (17.9 ae 17.1 mg/ml), however, the results were not statistically significant due to the large variability seen among the chf dogs (p 5 0.07). we found greater mean adpn concentrations in normal dogs (11.46 ae 3.3 mg/ml) than chf dogs (9.2 ae 3.2 mg/ml) (p 5 0.05). in general, the greater the severity of the heart failure, the lower the level of serum adpn. when the 2 tests the purpose of this study was to determine if there are any clinically important differences between the approaches (including devices) used in non invasive transvascular (interventional) closure of patent ductus arteriosus (pda) in dogs in our institution. initial and follow up records from all dogs (n 5 112) that underwent attempted transvascular pda occlusion from january 2006-december 2009 were examined. dogs were placed into 4 groups depending on the device and route of vascular access (transvenous or transarterial). group 1: amplatz s canine ductal occluder (acdo) (transarterial) -36 dogs; group 2: gianturco s or mreye flipper s detachable embolization (flipper) coil (transarterial) -38 dogs; group 3: amplatzer s vascular plug (avp) (transarterial) -23 dogs; group 4: flipper coil (transvenous) -15 dogs. statistical comparisons were made using the kruskal-wallis test with mann-whitney tests to compare pairs of groups when significance was detected. p o 0.05 was considered significant. there was no significant difference in ages between the 4 groups. there was a significant difference in body weight between groups with dogs receiving a coil either transarterially or transvenously (groups 2 and 4) being significantly smaller than dogs receiving an acdo or avp. this was by design since the acdo and avp cannot be used in small dogs. overall, the success rate of the total procedure (including vascular access and satisfactory pda occlusion) was high (92%) with success rates being comparable between groups (87-97%). there was a significant difference in complication rate between groups (p o 0.0001) with the acdo group having a markedly lower complication rate than the remaining groups (3% for acdo versus 24-33% for the other groups). total fluoroscopy time ranged from 3-78 minutes (median 8 minutes). fluoroscopy time for the transvenous method was significantly longer (median 13 minutes; range 10-78 minutes) than in the remaining groups (median 6 minutes; range 3-33 minutes) (p o 0.0001). number of dogs with residual flow immediately following the procedure and 24 hrs later was significantly less in the acdo group than in the remaining groups (2 dogs from group 2 and 3 from group 3 had moderate persistent flow while 1 dog from group 2 and 1 from group 4 had severe persistent flow 24 hours after the procedure). the acdo appears superior in ease of use, complication rate, completeness of occlusion and fluoroscopy time than other devices. the remaining limiting factor with this device is patient size. until a smaller acdo device is marketed, coils remain the only choice for interventional closure in very small dogs ( o 2.5kg). previously presented at the university of california davis, house officers seminar day. subvalvular aortic stenosis (sas) is one of the most commonly reported canine congenital heart defects and is inherited in newfoundland dogs and human beings. the golden retriever and rottweiler are breeds over-represented in dogs with subvalvular aortic stenosis; however, a genetic cause of this disease in these breeds has not been described. we performed genome wide association analysis in both normal and sas affected rottweilers and golden retrievers to identify chromosomal regions of interest that could implicate a causative mutation by high density single nucleotide polymorphism (snp) array. 48 (24 unaffected/24 affected) adult golden retrievers and 48 (20 unaffected/28 affected) adult rottweilers were included in this study. criteria for affected included a subcostal continuous-wave doppler aortic velocity ! 2.5 m/s and presence of a left basilar systolic ejection murmur; criteria for unaffected included a doppler aortic velocity 1.8 m/s. dna samples were obtained from anticoagulated blood. genotypes were obtained using high density (173,662) snp arrays, and genome wide association with sas was evaluated for each breed. significance cut-off was set at p 5 5 â 10 à5 , and all snps meeting this criterion were plotted within each breed and compared across breeds using plink. affected golden retriever data implicate the most significant region of genetic variation on chromosome 21 at location 27384260 (p 5 1.15 â 10 à5 ; odds ratio 7.58) with 11 other significant surrounding snps . affected rottweiler data also implicate the most significant region of genetic variation on chromosome 21 at location 27895300 (p 5 8.98 â 10 à6 ; odds ratio 23.39) with 3 other significant surrounding snps . other regions of statistical significance were on chromosomes 4 and 7 in the golden retriever and 11 and 38 in the rottweiler. genome wide association with subvalvular aortic stenosis in the golden retriever and rottweiler implicate overlapping chromosomal regions of interest for causative mutations on chromosome 21. the different secondary chromosomal regions of interest (chr 4, 7 in golden retrievers and 11, 38 in rottweilers) supports the known familial nature of this disease within different breeds and may suggest the presence of multiple mutations or breed specific disease modifiers. these data highlight the need for candidate gene evaluation on chromosome 21 in golden retrievers and rottweilers with sas. heart valves share developmental signaling pathways with bone and cartilage. degenerative aortic valve disease in humans is characterized by valve stenosis and calcification. recent evidence suggests that degenerative aortic valves are undergoing pathologic processes that mimic osteogenesis. degenerative mitral valves in dogs and humans are characterized by valve regurgitation, and rarely undergo calcification. we tested the hypothesis that canine and human degenerative mitral valves might be undergoing pathologic processes that mimic chondrogenesis. to test this hypothesis, expression of bone morphogenic protein 2 (bmp2), a chondrogenic growth factor; sox 9, a chondrogenic transcription factor; aggrecan, a proteoglycan abundant in cartilage; and type ii collagen were evaluated utilizing immunohistochemistry. normal canine mitral valves, different stages of canine degenerative mitral valves (early, intermediate, and late), and late-stage human degenerative mitral valves were studied. canine and human degenerative mitral valves showed focal areas that co-expressed all four markers of chondrogenic signaling and phenotype. valve interstitial cells and surrounding extracellular matrix in these focal areas adopted a morphologic appearance reminiscent of cartilage. focal chondrogenesis was present in all stages of canine degenerative mitral valves, but not normal canine mitral valves. focal areas of chondrogenesis did not coincide with nodular areas of glycosaminoglycan accumulation on the leaflet edge, but rather seemed to occur at points of chordae attachment to leaflets. in conclusion, canine and human degenerative mitral valves undergo pathologic processes that mimic chondrogenesis. this finding suggests that mitral valve degeneration may be recapitulating developmental signaling pathways shared by heart valves and cartilage. the triggering events for chondrogenesis in mitral valves remain unknown; as does the reason why aortic and mitral valves appear to be undergoing different pathologic processes. the fact that humans exhibit degeneration of both the aortic and mitral valve, and that dogs commonly exhibit only the latter could eventually provide insight into both processes. arrhythmogenic right ventricular cardiomyopathy (arvc) is a familial cardiomyopathy characterized by right ventricular fibrofatty infiltration and ventricular ectopy of left bundle branch block morphology (vpc) . a deletion in the striatin gene has been associated with arvc in at least some boxer families. syncope and sudden death (sd) occur in some affected dogs, although many affected dogs survive for years. the objective of this study was to define clinical characteristics of arvc in boxers that experienced sd, and compare them to those of a contemporaneous group of arvc boxers that had not died suddenly (nsd). data for both groups were collected from adult boxers enrolled in a long term prospective study of arvc in which echocardiograms and 24 hour ambulatory ecg (aecg) are evaluated annually. aecg are quantitated for vpc numbers and arrhythmia grade (1-4). arvc diagnosis requires at least 100 vpcs/ 24 hours in the absence of other disease. forty three adult boxers that entered the study had died suddenly at the time of analysis (sd defined as the absence of observed clinical signs within 24 hours prior to an unexpected and unexplained death). striatin genotype was available for 28 of the 43 sd dogs (17 heterozygotes, 11 homozygotes); 19 were female (9 intact) and 24 were male (14 intact). sd occurred at a mean age of 8 years (range, 1-12); 27 sd dogs (63%) had no prior history of syncope. twelve sd dogs (28%) were on antiarrhythmics at the time of death (metop-p0oooooooooooprolol (1), sotalol (4), amiodarone (1), procainamide (2), mexiletine & atenolol (3), atenolol (1)). eleven sd dogs (25%) had decreased myocardial systolic function defined by a shortening fraction (%fs) o 25% (range 11-45, mean 5 27) on the most recent echocardiogram prior to sd. median vpcs/24 hours on annual aecg was 7,700 (range 106-91,000) with a median arrhythmia grade of 4 (range 2-4). twenty one contemporaneously entered arvc boxers that had survived to at least the median age of the sd group with nsd were available for comparison; 15/21 were genotyped (10 heterozygous, 1 homozygous, 4 negative), 13 were female (3 intact) and 8 male (2 intact). twelve nsd dogs (57%) had no prior history of syncope. median nsd group age was 10 years (range, 8-13); 11/21 (52%) were on an antiarrhythmics (sotalol (9), mexiletine & sotalol (1), mexiletine & atenolol (1)). one nsd dog had decreased %fs (nsd group %fs range 20-42, mean 5 32). the nsd median number of vpcs was 5,342 (range 622-62,622), median arrhythmia grade was 4 (range 2-4). striatin genotype was significantly associated with sd. no significant differences were found between groups with respect to vpc numbers or arrhythmia grade. shortening fraction was significantly lower in the sd group (p o 0.01). sd in arvc appears to be associated with the presence of the striatin mutation and reduced % fs, it does not appear to be associated with number of vpcs or arrhythmia grade. coughing in the small breed dog may be related to cardiac causes associated with myxomatous mitral valve degeneration (mmvd) including pulmonary edema and compression of the mainstem bronchus by a severely enlarged left atrium, or due to respiratory causes such as tracheal and/or bronchial collapse or chronic bronchitis. the purpose of this study was to evaluate the association between left atrial enlargement and large airway collapse in dogs with mmvd and chronic cough. we hypothesized that airway collapse was independent of degree of left atrial enlargement. twelve dogs with mmvd and a chronic cough in the absence of congestive heart failure were prospectively evaluated with thoracic and cervical radiography, echocardiography, fluoroscopy, bronchoscopy and bronchoalveolar lavage (bal). group 1 dogs (n 5 8) had moderate to severe left atrial enlargement based on an echocardiographically calculated left atrial:aortic surface area [la:ao(a)] 4 6. group 2 dogs (n 5 4) had no to mild left atrial enlargement [la:ao(a) 6]. the site and severity of airway collapse was graded on bronchoscopy and bal cytology was assessed for evidence of inflammation or infection. the occurrence of bronchoscopic abnormalities was compared between groups using fisher's exact test. p o 0.05 was considered significant. age and body weight did not differ between groups. left atrial size was interpreted radiographically as moderately to severely enlarged in 7 of 8 dogs in group 1 and as moderately enlarged in 2 of 4 dogs in group 2. fluoroscopy revealed variable degrees of airway collapse during normal respiration and induced cough in both groups. radiography and fluoroscopy were not accurate in identifying site and degree of collapse in either group when compared to bronchoscopy. cervical tracheal collapse was identified during bronchoscopy in both group 1 (2 of 8) and group 2 (3 of 4) dogs but was subjectively less severe in group 1 dogs. bronchial collapse 4 50% was evident at multiple sites in both groups of dogs with no difference between groups. all dogs had suppurative and/or lymphocytic inflammation on airway cytology. infection was not present in either group of dogs, although non-specific light bacterial growth was detected in 6 of 8 group 1 dogs and 1 of 4 group 2 dogs (p 5 0.22). preliminary results failed to identify an association between left atrial enlargement and airway collapse in dogs with mmvd but did suggest that airway inflammation is common in affected dogs. further studies are needed to identify factors contributing to airway collapse in dogs with and without mmvd. atenolol is often used empirically in cats with asymptomatic hcm, even though clinical and experimental evidence of efficacy is lacking. cardiac biomarkers play a critical role in the early detection of subclinical cardiac disease, in the prediction of long-term prognosis, and in monitoring the response to therapy in humans. we hypothesized that serum concentrations of the biomarkers, nterminal pro-brain natriuretic peptide (nt-probnp) and cardiac troponin i (ctni), would improve following the chronic administration of atenolol po to asymptomatic cats with hcm. six maine coon or maine coon cross cats with severe hcm from the research colony at ucdavis were administered atenolol (12.5 mg po twice a day) for 30 days. no cat had severe left ventricular dynamic outflow tract obstruction due to systolic anterior motion of the mitral valve. the concentrations of nt-probnp and ctni were assayed prior to drug administration and on the last day of drug administration. there was no statistically significant difference identified in nt-probnp [median before: 394 pmol/l (range: 71-1500 pmol/l), median after: 439 pmol/l (range: 24-1500 pmol/l); p 5 0.63] or ctni [median before: 0.24 ng/ml (range:0.10-0.97 ng/ml), median after: 0.28 ng/ml (range: 0.09-1.0 ng/ml); p 5 0.69] concentrations before and after drug administration using the wilcoxon matched pairs test. the ctni finding suggests that atenolol does not reduce chronic myocyte death in cats with hcm. the lack of improvement in nt-probnp suggests that atenolol does not improve myocardial wall stress in cats with hcm. a clinical trial is warranted to confirm or refute the findings from this study. therefore, leptin-gene expression was investigated in blood samples of dogs with congestive heart failure (chf; n 5 8) in comparison to dogs presented for cardiac screening (n 5 8) without abnormalities. additionally myocardial samples (interventricular septum, right and left atrium and ventricle) of 10 dogs with no cardiac abnormalities (controls), seven dogs with acquired and three with congenital cardiac diseases were investigated using quantitative rt-pcr. leptin blood levels were significantly higher in dogs with chf in comparison to dogs without diseases (p 5 0.013). there was an association with gender with higher myocardial leptin levels in female dogs with cardiac diseases (p 5 0.001). differences between cardiac regions were present (p o 0.05) and cardiac diseases resulted in an increase in atrial leptin levels in both sexes (p 5 0.006). interestingly, a significant reduction of myocardial leptin was present in dogs with congenital cardiac diseases (p 5 0.016), whereas acquired cardiac diseases resulted in an increase in leptin (p 5 0.035) in comparison to controls. these results suggest that the heart might be a target of leptin action in the dog and myocardial leptin production might play a role in regulating cardiac function in an auto-and paracrine manner. predicting risk of chf in asymptomatic dogs with mitral valve disease (mvd) is challenging. we examined ability of nt-probnp to identify asymptomatic dogs at high risk for chf. dogs with isachc-1b (la:ao 4 1.6) were prospectively recruited; dogs with current or previous chf or diuretic therapy were excluded. physical examination, radiography, echocardiography, and nt-probnp were performed at 2-9 mo intervals for 66 dogs (median follow-up 5 276 d, range, 17-1334 d). thirty-one patients reached a study endpoint of radiographic pulmonary edema; 35 remained asymptomatic. parameters from the visit immediately previous to onset of chf (future-chf) or prior to the most recent visit without chf (remain-asympt) were analyzed. median nt-probnp of future-chf (3001pmol/lpmol/l, iqr 2255-3001) was significantly different from remain-asympt (1600 pmol/l, 984-2863; p 5 0.0004). median time to chf of future-chf was 86d (iqr, . groups also differed in median la:ao (future-chf 2.20 [2.00-2.50]; remain-asympt 1.96 [1.85-2.13], p 5 0.0014); vhs (future]; remain-asympt 11.3 [10.75-12.0], p 5 0.003); and lvidd:ao ]; remain-asympt 2.33 [2.15-2.64], p 5 0.0001). roc analysis to predict if chf would occur prior to the next visit yielded auc 5 0.753 (95%ci, 0.632-0.851). sensitivity was 90.3% or 77.4% and specificity 48.6% or 68.6% for nt-probnp 41490 pmol/l or 42150 pmol/l, respectively. mean increase in nt-probnp between penultimate visit and two visits prior to endpoint: future-chf 5 1440.1 pmol/l vs. remain-asympt 5 110.5 pmol/l. within 6 mo, 4.5%, 10.7%, 17.4%, and 36.7% of dogs with nt-probnp o 1000, , and 43000 pmol/l developed chf. nt-probnp and heart size helped assess risk of chf in asymptomatic mvd. increasing the assay's upper limit of detection would likely improve utility of nt-probnp. piiinp is a serum biomarker of collagen biosynthesis and is described as a marker of myocardial fibrosis in human patients. we hypothesised that piiinp concentrations would vary according to the degree of remodelling demonstrable in dogs with naturallyoccurring myxomatous mitral valve disease (mmvd). serum piiinp concentrations (mg/ml) were measured in dogs with mmvd and healthy controls using a validated commerciallyavailable radioimmunoassay. results are reported as (mean ae sd). non-normally distributed variables were logarithmically transformed. comparisons of continuous variables were made between groups using t-tests and one-way anovas with tukey's post-hoc comparisons. univariable analyses were used to evaluate associations between piiinp and clinical characteristics (age, breed [cavalier king charles spaniel (ckcs) yes/ no], sex, weight, heart rate [measured from ecg], treatment with acei [yes/ no], treatment with diuretics [yes/ no] and echocardiographic measurements [la/ao, lvedd/ lvfwd, lveddn, lvesdn]). multivariable analysis was initially performed with all dogs included and then repeated excluding all dogs receiving therapy. dogs with mmvd were divided into those with no cardiomegaly (nc) (la/ao o 1.5 and lveddn o 1.8), those with cardiomegaly (la/ao ! 1.5 and/ or lveddn ! 1.8) but no clinical signs (c) and those dogs with cardiomegaly requiring treatment for congestive heart failure (chf). one hundred and fifty-four dogs with mmvd and 23 control dogs were studied. there was no difference in age (p 5 0.870) or weight (p 5 0.606) between the mmvd and control groups. there was a significant difference in serum piiinp (p 5 0.034) between normal (11.2 ae 3.66), nc (11.6 ae 4.57), c (10.1 ae 3.44) and chf (9.4 ae 3.06) groups. post-hoc comparisons demonstrated a difference between nc and chf groups (p 5 0.038). there was no difference in serum piiinp between genders (p 5 0.228). in the univariable analysis ckcs (yes/ no) (p 5 0.016) was positively associated with serum piiinp. age (p o 0.0001), log (la/ao) (p 5 0.002) and lveddn (p 5 0.002) were negatively associated with serum piiinp. in the multivariable model including all dogs, lveddn (p o 0.0001, b 5 à4.04 (95%ci 5 à6.11 to à1.97)), age (p 5 0.006, b 5 à0.28 (95%ci 5 à0.47 to à0.08)) and ckcs (yes/ no) (p 5 0.003, b 5 2.01 (95%ci 5 0.67 to 3.34)) were independently associated with serum piiinp. in the multivariable model including only dogs not receiving therapy (n 5 141), lveddn (p 5 0.006, b 5 à4.30 (95%ci 5 à7.32 to à1.29)), age (p 5 0.011, b 5 à0.31 (95%ci 5 à0.55 to à0.07)) and ckcs (yes/ no) (p 5 0.034, b 5 1.75 (95%ci 5 0.13 to 3.37)) were independently associated with serum piiinp. in conclusion, serum piiinp decreases with age and with increasing lveddn. ckcs have higher serum piiinp measurements independent of age and lveddn, which may reflect a difference in collagen turnover in this breed. left atrial (la) chamber dilation and congestive heart failure (chf) are common consequences of cardiac conditions in cats. in some cases chf is manifest as right-sided chf (r-chf) or pleural effusion, in other cases chf manifests as left-sided chf (l-chf) or pulmonary edema. it is not always readily apparent as to which cats will develop what form of chf. a general impression has been that la enlargement is associated with the average burden of elevated filling pressures, but little attention has been paid to the function of the la chamber itself. since chf is classically preceded by abnormal atrial chamber dilation and alterations in atrial chamber function, we want to understand how these changes may help us manage or predict chf in the cat. we hypothesized that cats manifesting r-chf have la failure with the la acting primarily as a conduit, resulting in greater pulmonary hypertension, whereas l-chf cats maintain some booster pump and reservoir function. we measured la maximum and minimum areas from right parasternal long axis four-chamber views on 2d echo, and la m-mode dimensions at maximum, minimum, and beginning of atrial contraction. la area change, fractional shortening, active emptying fraction, and expansion index were calculated from these measurements. right ventricular internal diastolic diameter was also measured on m-mode views. preliminary data revealed that maximum left atrial size is not significantly different between r-chf and l-chf cats on 2d or m-mode measurements due to high variability. however, total left atrial fractional shortening is significantly reduced in r-chf cats (11.32% ae 4.9) compared to l-chf cats (19.7% ae 5.1)(p 5 0.001), and r-chf cats have reduced left atrial active emptying fraction (6.12% ae 3.5) as compared to l-chf cats (13.7% ae 3)(p o 0.001). left atrial expansion ability is poorer in r-chf cats (13.11% ae 6.7) than in l-chf cats (25.0% ae 8)(p 5 0.002). these findings may suggest that atrial stiffness and poorer atrial function is associated with a greater degree of pulmonary venous and thus secondary pulmonary arterial hypertension resulting in pleural effusion (r-chf). right ventricular diameter on m-mode was increased in r-chf cats (4.8 mm ae 2.1) when compared to l-chf cats (2.89 mm ae 0.8)(p 5 0.002) and normal cats (2.74 mm ae 0.8)(p 5 0.002), which may also be evidence for a greater degree of pulmonary arterial hypertension in these cats. episodic weakness and syncope are common in boxer dogs. reported causes include rapid ventricular tachycardia (vt) and exertion-excitement triggered neurally-mediated bradycardia (nmb) .the purpose of this retrospective study is to describe the features of presumed nmb in boxers. to be included in the study, each dog must have been overtly healthy with a history of exertion-excitement triggered syncope or presyncope; had a normal echocardiogram (ec); had absence of vt and fewer than 500 ventricular premature complexes (vpc) on an initial and subsequent 24 hour holter recordings; and been alive and overtly healthy for at least six months following the initial evaluation. a total of 27 boxers were identified. sixteen were male and 11 were female. most (90%) dogs were either less than 4 or more than 7 years of age. most dogs had multiple, but infrequent, episodes and heart rhythm was documented at the time of an episode in only 8 (30%) and only once (bradycardia) on the first holter recording. owners were instructed to attempt to precipitate episodes. bradycardia related episodes were subsequently recorded in 5: during the 2nd (1), 3rd (2) or 4th (1) day of 120 hour holter recordings and during the 5th day of an event recording (1). collapse and bradycardia were documented during auscultation in 2 additional dogs. the heart rate during syncope was never documented in 19 (70%) dogs. a presumptive diagnosis of nmb was based on the absence of initial and follow-up of ec abnormalities and the presence of no or few vpc during extended ecg monitoring. multiple holter recordings (48-168 hours) were performed in 8 of 19 (42%) dogs and event monitoring of 5 days (1) and 7 days (1) was performed in 2 additional dogs. in conclusion, documentation of the heart rhythm during episodes of collapse was difficult, accomplished in only 30% and was unlikely during the first holter recording. in boxers with suspected nmb, extended ecg monitoring and implantable loop recorders may be best for hr documentation. arrhythmogenic right ventricular cardiomyopathy (arvc) is an inherited myocardial disease with high prevalence in the boxer dog population, and is associated with sustained monomorphic ventricular tachycardia, sudden cardiac death, and replacement of myocardium with fatty or fibro-fatty tissue. though several genes have been linked to the disease both in humans and in boxers, the etiology of arvc is still unclear. several mechanisms for the development of arvc have been suggested, including dysfunction of the canonical wnt pathway, which results in an arvc phenotype in the mouse. the canonical wnt pathway has been linked to many cellular functions, including growth and differentiation of adipocytes. with the recent discovery that the gene encoding striatin, a protein involved in wnt signaling, may be involved in the development of boxer arvc, we hypothesized that changes in the wnt pathway may also play a role in the etiology. here, we show changes in the localization and decreased amount of proteins affiliated with the canonical wnt pathway. afflicted boxers were identified by 24-hour holter monitoring and histopathological examination of the heart. samples from the right ventricle rv) of 15 arvc afflicted boxers, and 5 unafflicted dogs (1 beagle, 2 mongrels, and 2 german shepherds) were collected, fixed in 10% formalin, processed, treated with antibodies recognizingcatenin, striatin, and calnexin, and examined using confocal microscopy. western blots were performed on 3 unafflicted rv samples, and 2 arvc afflicted rv samples. frozen tissue samples were homogenized in laemmli buffer, and 10 mg of protein was loaded into each well of a 8-16% gradient gel. -catenin, an integral modulator of the wnt pathway, and striatin were colocalized with the endoplasmic reticulum (er) marker, calnexin. in the unafflicted animals, -catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern, with no detectable localization in the er. in contrast, in the 15 arvc boxers, bothcatenin and striatin were colocalized with calnexin in an er pattern. in the afflicted samples, -catenin and striatin were not visualized to the intercalated disc and intracellular space, respectively. western blots of striatin and -catenin revealed no changes in the amount of protein. interestingly, a western blot for the wnt protein revealed a decrease in the amount of protein in arvc samples, compared to unafflicted samples. our preliminary data suggest that disturbances of the canonical wnt pathway may play an etiological role in the development of arvc in the boxer dog. there are numerous benefits of omega-3 fatty acid supplementation in human heart disease, including reduction in arrhythmias, decreased incidence of sudden death, and improved survival in heart failure. antithrombotic effects of omega-3 fatty acids have been demonstrated in people, which may have particular benefit in cats given their risk of thromboembolic complications with cardiac disease. benefits also have been found in canine heart disease, and reduced serum concentrations of the omega-3 fatty acids, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) have been found in dogs with congestive heart failure (chf) secondary to dcm. to the authors' knowledge, no studies to date have investigated fatty acid concentrations in cats with cardiomyopathy. the purpose of this study was to measure serum fatty acid concentrations in normal cats and cats with cardiomyopathy. serum fatty acid concentrations were measured in normal cats and cats with cardiomyopathy using gas chromatography. cats with cardiomyopathy and at least mild left atrial (la) enlargement (la to aortic ratio 4 1.5 on two-dimensional echocardiography from a right parasternal short axis view) were candidates for study. normal cats had a normal history, physical examination, echocardiogram, packed cell volume, total solids and platelet count. cats with evidence of other systemic disease or those receiving anticoagulants were excluded from the study. normally distributed and skewed data were compared between the cardiomyopathy and control groups with independent t tests or mann whitney u tests, respectively. statistical significance was set at p o 0.01. thirty cats with cardiomyopathy (23 neutered males and 7 neutered females) and 27 healthy controls (13 neutered males and 14 neutered females) were enrolled. median age was 8 yr (range, 2-16 yr) in the cardiomyopathy group and 5 yr (range, 1-10 yr) in the control group (p 5 .009). cats in the cardiomyopathy group were classified in the international small animal cardiac health council stage 1b (n 5 10), 2 (n 5 8), 3a (n 5 3) and 3b (n 5 9). compared to control cats, cardiomyopathic cats had higher concentrations of palmitic acid (p 5 .002) and dha (p o .001), and lower concentrations of linoleic acid (p 5 .005). among cats with cardiomyopathy, there was no significant correlation between any serum fatty acid concentration and left atrial size or age. these findings warrant further investigation into the role of fatty acids in cats with cardiac disease. platelet mapping is an application of thromboelastography that relies on the generation of at least three tracings: ma thrombin (maximum platelet activity),ma fibrin (fibrin activity only), an-dma aa or adp (platelet activity not inhibited by arachidonic acid or adp receptor antagonists, respectively). using these three tracings, the % inhibition of platelets can be calculated. the purpose of this study was to evaluate the platelet mapping assay in normal cats and assess platelet inhibition in cats receiving clopidogrel. employee-owned cats with normal history, physical exam, echocardiogram, thromboelastography, packed cell volume, total solids and platelet count were eligible. clopidogrel (18.75 mg po q24h) was administered for 10 days with platelet mapping performed on days 0 and 10. platelet mapping values were compared using a paired t test, with significance set at p o 0.05. seven cats (4 fs, 3 cm, aged 2-10 years) were enrolled. compared to day 0, ma adp (p o .001) and ma fibrin (p o .001) were lower on day 10. the latter unexpected result prompted measurement of fibrinogen concentrations at day 0 and 10 in the last 5 of these 7 cats. fibrinogen was not different from day 0 to 10 in these 5 cats. these results suggest that platelet mapping may be a simple, outpatient clinical tool to measure antiplatelet activity in cats receiving clopidogrel. this clopidogrel dose resulted in significant platelet inhibition as measured by ma adp in all cats. further studies correlating antiplatelet effects measured by platelet mapping with clinical outcomes in cats with cardiomyopathy are warranted. this study investigated the hemodynamic effects of application of an itd in a canine model of cardiopulmonary arrest. 8 laboratory beagles which were part of a separate terminal study were anesthetized and instrumented for continuous measurement and recording of right atrial pressure, arterial pressure and carotid blood flow. following euthanasia, cpr was performed for one minute, then a pause of one minute followed by a second one minute period at a compression rate of 100-120/minute, ventilation with 100% oxygen was delivered at eight breaths/min and 15 ml/kg tidal volume. cpr was performed with the itd in place (itd-cpr) and without the itd (s-cpr) for one period each in a randomized fashion with the rescuer blinded to its application. baseline, s-cpr and itd-cpr data were assessed for normality, a kruskal-wallis one-way anova on ranks was used (baseline v cpr). when appropriate a pairwise multiple comparison procedure (dunn's method) was used. percentage of baseline s-cpr v itd-cpr was assessed using the student t-test. the right atrial diastolic pressure was significantly more negative with the itd attached than without (p 5 0.02), the coronary perfusion pressure and carotid blood flow were significantly higher during cpr with the itd than standard cpr (p 5 0.015, p 5 0.017). no significant differences in diastolic, mean or systolic arterial pressure or end tidal co 2 were seen. application of the itd resulted in significantly improved hemodynamics during cpr in dogs. clinical evaluation of the device is warranted to examine whether this translates into improved success in resuscitation and survival. left ventricular (lv) systolic dysfunction is a common problem in dogs and can be due to a variety of etiologies. one potential etiology for systolic dysfunction is persistent or paroxysmal tachyarrhythmias, leading to tachycardia-induced cardiomyopathy (ticm). in humans, ticm carries a relatively good prognosis in that remodeling may be reversible with normalization of heart rate. differentiating between primary and secondary tachyarrhythmias in dogs with systolic dysfunction is critical for prognostic purposes as primary tachyarrhythmias may be associated with a better outcome. the goal of our study was to describe a population of dogs with ticm and to determine if treatment of arrhythmias was associated with reversible cardiac remodeling as indicated by standard echocardiographic parameters. medical records of dogs referred to the cardiology service of ksu vmth from 2008 to 2010 were reviewed. ticm was defined as the presence of severe tachyarrhythmias that were reversible with treatment, systolic dysfunction or ventricular enlargement that improved with treatment of the arrhythmia, or dogs with severe tachyarrhythmias and systolic dysfunction of breeds with that are atypical for idiopathic dilated cardiomyopathy. exclusion criteria were dogs with congenital heart disease, severe mitral regurgitation, and endocarditis. transthoracic echocardiography, thoracic radiographs and electrocardiography (ecg) were performed in all dogs. ventricular enlargement and systolic dysfunction were defined according to standard echocardiographic parameters. arrhythmias were confirmed with a holter monitor in 7 dogs. a total of 12 dogs were included in the study. mean age was 7 years (range 2-12 years) with 8 males (4 intact, 4 castrated) and 4 females (3 spayed, 1 intact). four dogs had pulmonary venous congestion or pulmonary edema and two dogs had ascites. at initial presentation, the meanaesd values were as follows: heart rate 219ae75 bpm, m-mode fractional shortening (fs) 17.29ae9.05%, ejection fraction (ef) using the area-length method 30.69ae13.45%, and left atrial to aortic root ratio (la/ao) 1.92ae0.37. initial meanaesd m-mode derived lv internal dimensions corrected for body weight were as follows: diastolic 1.92ae0.37 and systolic 1.49ae0.38. at least one of the following tachyarrhythmias were identified in each dog: atrial fibrillation (4), supraventricular tachycardia (5), junctional tachycardia (2), and ventricular arrhythmias (3). ten dogs were available for follow up. seven dogs improved in at least one of the following parameters: resolution of tachyarrhythmia (3), improved systolic function (3) antidiuretic hormone (adh) has been shown to be elevated in humans with congestive heart failure (chf). recently, antidiuretic hormone antagonists were successful during investigational treatment of refractory congestive heart failure in humans. adh levels have been only modestly investigated in dogs with cardiac disease, primarily due to the technical difficulty in measuring adh levels via radioimmunoassay. based on the homologous structure of canine and human adh, we aimed first to determine the feasibility of measuring adh in dog plasma using a human elisa kit, and secondly to investigate the level of adh in dogs with congestive heart failure due to acquired cardiac disease. elisa assay kit validation was performed using six healthy dogs with normal clinical and echocardiographic examinations. pooled canine plasma was spiked with synthetic adh and intra-assay precision, dilutional parallelism, and linearity were assessed. to address the second aim of the study, samples were collected from normal dogs and dogs with heart failure due to one of two types of acquired cardiac disease: chronic degenerative valve disease (cdvd) or dilated cardiomyopathy (dcm). patients underwent clinical, radiographic, and echocardiographic examination to confirm diagnosis, assess severity of disease, and determine presence of pulmonary edema. whole blood was collected into edta tubes containing protease inhibitors, cold centrifuged, and plasma was stored at à801 until analysis. following ether extraction, plasma adh in each sample was measured in duplicate using a human elisa kit. statistical analysis included a d-agostino and pearson test for normality; group results were compared using a nonparametric mann-whitney test. adh was measured in canine plasma using the human elisa kit with acceptable intra-assay precision, linearity, and dilutional parallelism. intra-assay coefficient of variation was 11%. twenty-four dogs were recruited for the second phase of the study. six normal dogs and twelve dogs with radiographic evidence of pulmonary edema due to either cdvd or dcm were selected for participation. the remaining six dogs were excluded due to lack of definitive radiographic evidence of congestive heart failure. median adh values were 3.67 ae .38 pg/ml for the normal group (n 5 6) and 6.155 ae .94 pg/ml for the heart failure group (n 5 12). median adh values for the two groups were statistically different (p 5 0.0057). our preliminary results indicate that measuring canine adh using a human elisa kit is feasible and provides results with an acceptable coefficient of variation. we also showed that dogs with congestive heart failure due to cdvd and dcm have elevated adh levels in comparison to normal dogs. our findings motivate further investigation to assess the degree of plasma adh level elevation and the possible use of adh antagonists as an adjunct treatment for refractory congestive heart failure in dogs. aortic thromboembolism (ate) occurs in both cats and dogs. whereas ate in cats is strongly associated with structural heart disease and typically has an acute catastrophic presentation; the pathogenesis and presentation of ate in dogs is less well known or understood. further, an effective antithrombotic strategy for ate in dogs has not been reported. medical records of dogs diagnosed with ate between 2003 and 2010 were examined retrospectively. diagnosis of ate was based on ultrasonography, doppler flow studies, and diminished or absent femoral pulses. dogs were treated with various acute and chronic antithrombotic therapies. the severity of ambulatory dysfunction was graded as none, mild, moderate, severe, or non-ambulatory at presentation and after therapy. a cohort of dogs in this study received a standardized protocol of chronic warfarin therapy with or without antiplatelet drugs. target international normalized ratio for warfarin therapy was 2 to 3. twenty-six dogs were diagnosed with ate. all had an apparent mural aortic thrombus caudal to the renal arteries with most having evidence of embolization to the iliac and femoral arteries. none had structural heart disease at diagnosis. twenty dogs (77%) were still ambulatory at diagnosis. the median duration of ambulatory dysfunction prior to presentation was 7.8 weeks (range 1 day -52 weeks). a majority of dogs (58%) had no concurrent conditions at diagnosis. nine dogs (33%) had protein-losing nephropathy. four dogs (15%) were hypothyroid. fourteen dogs were treated with a standard warfarin protocol for a median period of 22.9 months (range 0.5-53 months). eight dogs were treated concurrently with aspirin, 2 dogs were treated concurrently with clopidogrel, and 4 dogs were treated with warfarin only. ambulatory function improved between 1 and 3 grades in dogs treated with chronic warfarin. the median period until clinical improvement was 13.9 days (range 2-49 days). two dogs treated with chronic warfarin therapy had documented resolution of ate, and 5 dogs had complete resolution of ambulatory dysfunction. none of the 14 dogs treated with chronic warfarin became nonambulatory, died, or underwent euthanasia because of ate. the median period of freedom from an adverse event was 24.2 months. no serious hemorrhagic events were reported. four dogs were treated with tpa. three of these had an unfavorable outcome. two dogs were ambulatory before tpa and become non-ambulatory after treatment. two dogs underwent surgical thrombectomy. one had a favorable outcome until ate recurred 14 months after surgery. in conclusion, the pathogenesis of ate in dogs is not associated with structural heart disease or left atrial thrombus formation. the presentation tends to be for chronic ambulatory dysfunction. most dogs are still ambulatory at presentation. warfarin, with or without concurrent antiplatelet therapy, is an effective antithrombotic treatment strategy for dogs with ate. information is known. through previous work, investigators have encountered norfolk terriers (nt) with echocardiographically apparent dmvd in the absence of a heart murmur. in order to more fully understand dmvd in this breed of dog, we sought to characterize findings from the physical and echocardiographic exam, biochemical, biomarker, and nutritional profile, and select environmental variables from a cohort of apparently healthy nt. overtly healthy nt ! 6yrs old were recruited by 3 different veterinary hospitals and underwent historical, physical, ecg, and 2d/color-flow doppler echocardiographic exam. anterior mitral valve length, maximal thickness, area, and prolapse were measured from 2d images. presence of dmvd was defined as thickened, prolapsing, or flail mitral valve leaflets in the presence of color flow doppler evidence of mitral regurgitation. blood samples were obtained for serum biochemistry and serotonin, plasma nt-probnp, amino acid profile, c-reactive protein, and cardiac troponin-i. forty-eight dogs were entered into the study (median age, 8yrs iqr [7-10]; gender, 29f, 19m; median bcs, ). of the 48 dogs, 23 (48%) had murmurs, 2 (4%) had mid-systolic clicks, 11 (23%) had ecg p-pulmonale, and 41 (85%) were deemed to have echocardiographic evidence of dmvd, including 18 nt without a murmur. seven (15%), 28 (58%), and 13 (27%) dogs were classified as isachc 0, 1a, and 1b, respectively. mean indexed echocardiographic mitral leaflet length (p o 0.0001), thickness (p 5 0.019), prolapse (p 5 0.0005), and la:aod (p 5 0.01) were significantly different between isachc 1a/b vs 0. between isachc 1a/1b and 0, there were no differences in serum amino acids, c-reactive protein, troponin, diet, or environmental factors; however 6 different amino acids (ala, gly, phe, pro, trp, tyr) were significantly higher in isachc 1b vs. 1a. median serum serotonin was increased in dogs with 1a/b vs. 0 (p 5 0.025). dogs whose diets contained some canned food (p 5 0.12) and dogs residing in suburban environments (p 5 0.03) had higher serotonin concentrations. nt-probnp tended (p 5 0.07) to be higher in isachc 1a/1b vs. 0. dmvd appears to be relatively common in nt and echocardiographic changes consistent with mild dmvd can be seen in dogs without a heart murmur. the results of this study establish a foundation of useful information upon which additional prospective studies can be developed. left ventricle (lv) evaluation is one of the most important contributions of echocardiography in the assessment of cardiac function. however, lv analysis can be made from images obtained by different modes and views of the heart. the aim of this study was to compare lv measurements, shortening fraction (sf) and ejection fraction (ef) obtained from four methods: m mode in short-axis and in long-axis, bidimensional mode in short-axis and in long-axis views of the heart. forty normal adult german shepherds were selected. echocardiographic study of lv of each animal was performed by the four methods described above. ancova test was used to examine the effects of axis, mode, weight and gender over lv measurements. isolated effect of the axis was observed for lv end-diastolic diameter (lvedd), with greater values obtained from short-axis views. there was isolated effect of mode over ef and sf, with greater measurements derived from bidimensional mode methods. weight correlated with all linear lv measures at least in one method, but not with ef and sf. weight had positive effect over lv endsystolic diameter and lv end-diastole posterior wall thickness in all methods, except from m mode in short axis in the last one. gender had isolated effect over lvedd, males showing greater values than females in bidimensional mode in short and long axis. the combined effect of axis, gender and weight was identified in interventricular septal end-diastolic thickness. we concluded that normal reference values obtained by different echocardiographic modes and planes should not be used interchangeably. abstract c-29 assessment of left ventricular diastolic func-tion by color tissue doppler imaging echo-cardiography in maine coon cats tested for mypbc-ap31 mutation hypertrophic cardiomyopathy (hcm), characterized by increased cardiac mass and diastolic dysfunction, is the most common feline heart disease. myocardial analysis by color tissue doppler imaging (tdi) is more sensitive than conventional echocardiography. this study evaluated diastolic dysfunction in various stages of feline hcm. maine coon cats (n 5 57) were screened for the mybpc-a31p mutation and examined with both echocardiography and tdi. then, were phenotypically classified in: normal (n 5 45), suspects for hcm (n 5 7) and hcm group (n 5 5); and genotypically classified in: negative (n 5 28), heterozygous (n 5 26) and homozygous group (n 5 3). myocardial velocities, measured in the basal and mild ventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw) and in radial segment of left ventricular wall, was compared among different groups. a significant decreased (p 5 0,01) longitudinal em/am at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. a significant increased (p 5 0,01) longitudinal e/em at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. and a significant decreased (p 5 0,02) longitudinal sm at the basal segment of the lvw was observed in heterozygous cats compared with negative cats, both without hypertrophy. there was a significant positive correlation between summated early and late diastolic velocities (emam) and heart rate (p o 0,001); and a positive correlation between sm and em velocities and heart rate (p o 0,01). the mybpc-a31p mutation is not consistently associated with ventricular hypertrophy and negatives cats can also develop hcm. the tdi alone is not able to identify cats with mutation before myocardial hypertrophy. diastolic dysfunction occurs in many cats with hypertrophic cardiomyopathy (hcm) but less is known about systolic function in various stages of hcm. myocardial strain analysis by tissue doppler imaging is a noninvasive echocardiographic method to assess systolic function. this study evaluated systolic function in various stages of feline hcm. maine coon cats (n 5 57) were screened for the mybpc-a31p mutation an examined with both echocardiography and strain. then, were phenotypically classified in: normal (n 5 45), suspects for hcm (n 5 7) and hcm group (n 5 5); and genotypically classified in: negative (n 5 28), heterozygous (n 5 26) and homozygous group (n 5 3). peak myocardial strain, measured in the basal and mildventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw), left ventricular anterior wall (lvaw), left ventricular posterior wall (lvpw) and radial segment of left ventricular wall, was compared among different groups. whereas conventional echocardiography demonstrated an apparently normal contractile state based on fractional shortening, myocardial strain (at mildventricular segment of ivs) in hcm cats was significantly decreased compared with normal group (p 5 0,01). myocardial strain (at basal segment of lvaw) also was significantly decreased in heterozygous cats compared with negative group (p 5 0,00); and was significantly decreased in heterozygous cats compared with negative group, both without ventricular hypertrophy (p 5 0,019). there was a significant negative correlation between strain values and wall thickness (p o 0,05). this method allows detection of abnormal systolic deformation in maine coons cats with hcm mutation despite apparently normal systolic function. the abnormal systolic deformation already can be present in heterozygous cats without hypertrophy and increased with progressive ventricular hypertrophy. recently, multiple advanced resting electrocardiographic (ecg) techniques have been applied in humans for detection of cardiac autonomic and repolarisation function. this has improved the diagnostic and/or prognostic value of short-time ecg in detection of common human cardiac diseases even before onset of symptoms or changes in the standard ecg. therefore, this study investigates, if advanced ecg can predict the severity of mitral regurgitation (mr) in dogs with myxomatous mitral valve disease (mmvd) and thereby improve the diagnostic value of ecg. the study included 77 privately owned cavalier king charles spaniels (ckcss) (age 6.0 ae 2.7 years; 30 males and 47 females). all dogs were examined by echocardiography and a short-time (3-5 min) high-fidelity 12-lead ecg, with the dog in a resting position and in sinus rhythm. dogs were divided into 5 groups according to the degree of mr estimated as the percentage of the left atrium area using color doppler mapping (0%; 0% o jet 15%; 15% o jet 50%; jet 4 50%; jet 5 100% and with clinical signs of congestive heart failure). ecg recordings were evaluated via custom software programs to calculate 76 different parameters, including heart rate variability (hrv), qt variability (qtv), t-wave complexity, wave morphology and 3-d ecg. one-way anova determined 21 ecg parameters, which were significantly different (p o 0.05) between the 5 dog groups. principal component factor analysis identified a 5factor model with 83.2% explained variability. qrs dipolar voltage and two repolarization indices of qtv increased significantly with mr severity, whereas total power of the frequency spectrum of rr interval and the standard deviation of qtv decreased significantly with mr severity. for the 5 selected parameters the prediction of mr jet value was tested by multiple linear regression. a correlation coefficient (r) of 0.65 indicated that the prediction value was significant (p o 0.01). if age was included in the multiple linear regression the prediction value was further increased (r 5 0.80). our results indicate that for a cut-off criteria of mr ! 50% jet the five selected ecg parameters could predict the severity of mr caused by mmvd in ckcss with sinus rhythm with sensitivity 65% (78% with age inclusion) and specificity 98% (92% with age inclusion) (p o 0.05). nt-probnp concentration is increased in canine patients with heart disease. relatively little is known about the stimuli for release of nt-probnp in dogs. physical activity independent of cardiac disease and the stress of being in the hospital could influence nt-probnp release and affect diagnosis and management of patients. we hypothesized that nt-probnp concentration in healthy dogs would not exceed the normal reference value (900 pmol/l) following a period of exercise. the goal of this study was to examine whether physical activity could elevate plasma nt-probnp and cause false positive results in healthy dogs. the study population included healthy dogs 41 yr of age without heart murmur or known systemic disease, and normal 2d/color flow echocardiographic exam. plasma samples for nt-probnp were obtained before, immediately after, and 1 hour after a standardized 5-minute submaximal exercise regimen. the study included 14 dogs with a median age of 5.3yrs and included 6 females and 8 males. there was no statistical difference in median plasma nt-probnp concentration across the three time points (baseline median, 617 [iqr, immediately post, ; p 5 0.11). the average coefficient of variation of nt-probnp concentration across the exercise regimen was 23.0 ae 18.9%. in 1 of 14 dogs (7.1%), nt-probnp increased from 790 to 1054 pmol/l immediately after exercise. the results of this study demonstrate that submaximal exercise does not significantly change median nt-probnp concentration and the incidence of false positive results is low. further studies should investigate effects of exercise on nt-probnp concentrations in dogs with heart disease. obesity is an increasing problem in veterinary medicine. obese human patients are shown to present lower levels of natriuretic peptides, regardless of an increased volume and pressure load, what raises the possibility that the natriuretic response is impaired in these individuals. considering the controversial findings in obese humans, and the lack of studies reported in dogs, this study proposed the evaluation of nt-proanp concentration in obese dogs. nt-proanp concentration was determined prospectively in 39 obese dogs (27 females; 12 males; 6-108 months) and in 23 non-obese dogs (controls; 13 females; 10 males; 12-108 months) from a veterinary hospital population. obesity was determined by body condition score [2 (4/9); 21 (5/9); 1 (6/9); 3 (7/9); 19 (8/9); 18 (9/9)]. dogs were excluded if they had any primary cardiac disease, renal insufficiency, endocrine disease, or if they were receiving diuretics, vasodilators, antiepileptic drugs or corticosteroids. commercial kits were used (vetsign s canine cardio screen nt-pro-anp vc3167 -guildhay/biomedica). mann whitney test was used for group comparison. results are presented as median; interval; p25 and p75). nt-proanp was significantly lower in obese dogs [413.56fmol/ml (0.0-1287.14); p25 5 228.673; p75 5 595.205] than in controls [647.98fmol/ml (34.99-1596.82 ); p25 5 490.785; p75 5 957.055]; (p 5 0.004). results were similar to what has been found in obese humans. lower levels of natriuretic peptides are also seen in obese heart failure patients. this study provides important information regarding nt-proanp concentration in obese dogs, which should be better explored characterizing the behavior of natriuretic peptides after weight loss, and also in obese dogs with primary heart disease. left-to-right shunting patent ductus arteriosus (pda) is one of the most common canine congenital cardiovascular defects. human studies have shown that bnp and nt-probnp concentrations are elevated in patients with pda, and can be used to detect hemodynamically significant pda. the purpose of this study was to measure nt-probnp concentrations in dogs with pda, and to assess whether additional indicators of hemodynamics correlate with ntprobnp. we hypothesized that nt-probnp will serve as a simple non-invasive marker of hemodynamic significance in dogs with pda prior to and following transcatheter ductal occlusion. nt-probnp was measured in 30 client-owned dogs with echocardiographically normal hearts. ten dogs with pda were initially evaluated with thoracic radiographs, transthoracic and transesophageal echocardiography, pulmonary capillary wedge pressure (pcwp) and nt-probnp. nt-probnp and echocardiography were repeated at 1 day and 3 months following ductal occlusion. pcwp was repeated at 3 months. baseline nt-probnp was significantly higher in pda dogs compared to control (1877 ae 2081 pmol/l (mean ae sd), 651 ae 301; p o 0.0025). at 1 day and 3 months following ductal occlusion, nt-probnp was 1347 ae 1256 and 466 ae 380, respectively. the following decreased significantly from baseline: pcwp (11.8 ae 4.7 to 6.4 ae 3.2 mmhg; p 5 0.018), and indexed left ventricular internal dimensions in diastole (2.26 ae 0.47 to 1.67 ae 0.19; p 5 0.006) but not significantly in systole (1.40 ae 0.31 to 1.20 ae 0.19; p 5 0.13). nt-probnp is elevated in dogs with pda and transductal closure is associated with a reduction in nt-probnp, pcwp and left ventricular size. cardiac biomarkers, particularly nt-probnp, are becoming more commonly used in dogs and cats as part of a diagnostic work up. multiple studies already have documented the correlation of this peptide with cardiac disease status and potential clinical implications. in a portion of these reports the manner in which samples were handled was placement of whole blood into an edta tube, followed by centrifugation and decanting of the supernatant that was ultimately stored at à201c or à801c prior to shipment, either with or without protease inhibition. our objective was to compare the nt-probnp concentrations in feline plasma collected using the previously reported methods to the california animal hospital (cah) collection method using tubes containing a protease inhibitor. this study compared nt-probnp concentrations using the protease inhibitor tubes vs. edta tubes from 18 privately owned feline patients, with confirmed cardiac disease, and 6 control feline patients. for all study participants, we performed a full history and physical examination, a hematology and chemistry panel, thoracic radiographs, ecg, and echocardiogram. in each study participant, at least 4 ml's of whole blood was drawn from a peripheral vein, and transferred to a plastic edta tube. the sample was centrifuged within 1 hour after collection. 1 ml of plasma was then transferred to a tube containing a protease inhibitor, which was stored at 41c until being shipped within 24 hours of collection. the remaining plasma was placed into 2 separate microtubes, which did not contain a protease inhibitor. one microtube was then stored and shipped as previous studies have reported (à201c, styrofoam container, shipment within 24 hours), and the second microtube was frozen at à801c. all samples were shipped, received and analyzed within 24 hours of collection. results of this study showed that no difference was found between the 2 frozen sample methods (663 pmol/l and 650 pmol/l p 5 0.40). it was determined that both frozen methods had lower nt-probnp levels (655 and 646 pmol/l) when compared to plasma samples shipped in protease inhibitor tubes (756 and 794 pmol/l). the findings of this trial demonstrate that the nt-probnp levels are significantly different between samples placed in edta tubes vs. contain protease inhibitor (p 5 0.008 and p 5 0.0006). utilizing protease inhibitor tubes allows more accurate measurement of plasma nt-probnp. as for its relevance for future research and publications, authors should take care to investigate the manner in which blood samples were handled and the conclusion/results of these studies should be taken in light of the methodologies used in collecting, storing, shipping and analyzing the samples. degenerative mitral valve disease (dmvd) is one of the most common heart disease and is present approximately 75% of the canine heart disease. although the high prevalence exists in small dogs, the underlying molecular mechanism of its pathophysiology is rarely known. dmvd is functionally and pathologically similar in humans and dogs, thus, there will be a common pathogenesis in human and dogs with naturally occurring dmvd. serotonin and serotonin-related mechanisms have been implicated as a cause of valvular disease in human and animals, including spontaneous dmvd in dogs. increased circulating 5ht concentration as a potential source of heightened 5ht signaling is demonstrated in small dogs with dmvd. the aim of this study was to investigate whether serum 5ht concentrations were associated with severity of naturally occurring dmvd in small dogs and to investigate potential associations of dog characteristics on serum 5ht concentrations in our study population. forty-eight dogs were included in this study and were classified into control and dmvd groups according to the results of physical and echocardiographic examinations. based on the la:ao ratio, dogs with dmvd were classified as follow: control (la:ao ratio 1.5 and no mr), mild (la:ao ratio 1.5 and mr), moderate (1.5 o la:ao ratio 1.8 and mr), severe (la:ao ratio 41.8 and mr). serum serotonin concentrations were measured by elisa. an overall significant difference (p o .05) was found among 4 groups and 5ht concentrations (control, 72.38 ng/ml [51.34-95.11 dmvd, ). significantly higher 5ht concentrations were observed in dogs with moderate (p o .05) and severe (p o .05) dmvd, compared with concentration in control group. additionally, 5ht concentration in dogs with moderate dmvd were significantly higher (p o .05) than concentration in dogs with mild dmvd. also, dogs with severe dmvd had significantly higher 5ht concentration than dogs with mild (p o .05) and moderate (p o .05) dmvd. there was no significant association of age, platelet, and lvidd, on serum 5ht concentration, however, weak correlation between serum 5ht increased significantly and la:ao ratio (r 2 5 .211, p o .05) was observed. the results of this study indicate that serum 5ht concentrations were higher with increasing severity of spontaneous dmvd, which may be the potential cause to advance the progression of dmvd. further studies should be performed to reveal the role of 5ht in inducing and accelerating spontaneous dmvd and to investigate if lowering serum 5ht concentration could alter the progression of dmvd. the objective of this prospective study was to evaluate the utility of cardiac troponin i (ctni) in differentiating between underlying etiologies of pericardial effusion in the canine patient. patients were prospectively recruited at time of diagnosis of novel pericardial effusion. serum samples were collected prior to pericardiocentesis. patients were evaluated by echocardiography and classified with the diagnosis of hemangiosarcoma (hsa), heart base tumor (hbt), or unknown etiology at the initial evaluation based on established characteristic echocardiographic findings. idiopathic pericardial effusion (ipe) was defined by histopathology, echonegative for a mass lesion with no recurrence of pericardial effusion 46 months, or symptom free 412 months from time of enrollment. patients were excluded from analysis if a diagnosis could not be established based on above criteria or concurrent moderate azotemia (creatinine 43.0 mg/dl) was present at time of sample collection. serum samples were frozen and analyzed in batches within 60 days of collection by a ctni assay with a 0.2ng/ml lower limit sensitivity. sixty-three patients were recruited over a one year period with 15 patients excluded due to lack of diagnosis (13) or azotemia (2). median ctni levels of dogs with hsa (n 5 24), hbt (n 5 19), and ipe (n 5 5) were 14.8 ng/dl (interquartile range (iqr) o 0.2-11.3), 0.23 ng/dl (iqr o 0.2-0.29), and o 0.2 ng/dl (iqr o 0.2-o 0.2) respectively. concentrations of ctni differed significantly between dogs with hsa and hbt (p 5 0.001) and ipe (p 5 0.0029). there was no difference between ctni concentrations between hbt and ipe dogs (p 5 0.911). receiver operating curve analysis to determine the optimal cutoff for differentiation of dogs affected with hsa and both hbt and ipe revealed a significant (p 5 o 0.001) area under the curve (0.79). a cut-off point of ctni of 40.78 yielded a sensitivity of 67% (95% ci, 45-84%) and specificity of 95% (95% ci, 79-99%). utilizing a higher cut-off point of 43.0 yielded a lower sensitivity of 50% (95% ci, 29-71%), but a higher specificity of 100% (95% ci, 86-100%) which may have more clinical utility given the disparity in prognoses of the etiologies compared. in conclusion, this study supports the diagnostic utility of ctni concentrations to delineate between patients with hsa and other etiologies of pericardial effusion, but does not reliably differentiate between dogs with ipe and other neoplastic etiologies. the pathogenesis of degenerative mitral valve disease (dmvd) in dogs remains to be fully elucidated. the high sheer stress caused by mitral regurgitation damages the endothelial surface of the valve, and a previous study demonstrated increased transcription of intercellular adhesion molecule-1 (icam-1) and e-selectin in affected mitral leaflet tissue. we hypothesized that this may be responsible for platelet recruitment and adhesion, and initiation of a proliferative cascade, resulting in further myxomatous changes. the goal of this study was to compare plasma levels of icam-1 and e-selectin in healthy dogs and those with dmvd. the study population included dogs with echocardiographic evidence of dmvd and healthy control dogs 41 year old with no heart murmur or known systemic diseases. dmvd dogs underwent 2d/color-flow doppler echocardiographic exam. blood samples were obtained for plasma icam-1 and e-selectin analysis using commercially available elisa kits. the study included 34 dogs, of which 20 had dmvd and 14 were normal. the dmvd group had a median age of 9.5yrs ) and included 6 females and 14 males. two (10%), 13 (65%), 2 (10%) and 3 (15%) dogs were classified as isachc 1a, 1b, 2 and 3a, respectively. of the control dogs, median age was 4.5yrs [2-6.5], with 5 females and 9 males. there was no statistical difference in plasma e-selectin between control dogs (median 2.71 [2.05-7.66]) and those with dmvd (2.46 [1.54-3.55]); p 5 0.35. plasma icam-1 concentrations were higher in dmvd dogs (1.58 [1.20-10.58 ]) than controls (median 1.31 [1.11-1.65], but this difference did not reach statistical significance (p 5 0.22). linear regression analysis showed no significant correlation between icam-1 or e-selectin and serum serotonin level, nt-probnp or echocardiographic measures of dmvd severity (la:ao, lvidd:ao, lvids:ao). the results of this study demonstrate no significant difference in circulating adhesion molecules icam-1 and e-selectin in dogs with dmvd as compared with healthy controls. further studies investigating adhesion molecules within the mitral valve tissue itself are likely needed if icam-1 and e-selectin play a role in the pathophysiology of dmvd. the rate of glucose utilization in the heart is greater than in other tissues, and impaired glucose uptake may play a major role in the pathogenesis of heart failure (hf). glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (gluts), which includes glut-4, the major cardiac isoform, and glut-12, a recently discovered isoform, the role of which is unknown in the heart. in addition, despite the wellknown regional differences in myocardial structure and function, potential regional patterns in glucose transport have not been investigated. thus, we hypothesized that glut-4 and -12 protein and gene expression would be chamber specific in healthy dogs and during chronic hf. using a canine model of tachypacing induced chronic hf, glut protein and messenger rna in both ventricles and atria were investigated by immunoblotting and real time pcr. in control dogs, glut-4, but not glut-12, protein expression were significantly higher in the atria compared to the ventricles, with the highest content in the right atrium (ra, p o 0.001). glut-4 and -12 mrna were homogeneously expressed in all the cardiac chambers. during chronic hf, glut -4 and -12 expression was highest in the left ventricle (lv, by 2.5 and 4.2 fold, respectively, p o 0.01), with a concomitant increase in glut-4 and -12 mrna (p o 0.001). glut -4, but not glut-12, was decreased in ra during chronic hf (p 5 0.001). our data suggest that glut-4 protein was differentially expressed across the cardiac chambers in the healthy heart. during chronic hf, lv was the primary site dependent on both glut4and glut12-mediated glucose transport, which was transcriptionally regulated. in addition, the paradoxical decrease in glut4 content in ra may induce perturbations in atrial energy production during chronic hf. some obese dogs are suspected to have cardiac disease because they have enlargement of the heart on thoracic radiograph. it has been reported in cats that the fat increases the cardiac silhouette, while echocardiograms revealed normal cardiac dimensions. the purpose of this study was to determine whether obesity overestimates cardiac dimension in radiographs compared to echocardiographic findings in dogs. twenty three obese dogs and 20 controls were included based on a 1-9 body condition scoring (bcs). computerized radiography was obtained and vhs measurement was performed as previously described. echocardiographic measurements were interpreted based on reference values according to lean body weight regression equations. results for echo and vhs measurements were classified in scores as normal, mild, moderate or severe increase. student's t test was used for comparison of vhs between groups. mann-whitney rank sum test was used to assess echocardiographic scores between groups. spearman rank order correlation was used to assess relationships between any pairs of variables between echo and vhs scores, echo vs bcs and vhs vs bcs. groups were similar regarding age [obese (69ae24); control (62ae27); p 5 0.368], breeds and gender distribution. obese dogs had significantly higher vhs and echo scores compared with controls [vhs: (10.58ae0.69) vs (9.77ae0.54); p o 0.001; echo score: range (1-4) vs (1-2); p 4 0.05]. there were no relationships between any pair of variables analyzed. these results show that there are changes both in echo and radiographic appearance of the heart in obese dogs, but vhs overestimates cardiac silhouette compared to echo, probably related to pericardial fat accumulation. heart rate variability (hrv) is an indirect measurement of the autonomic modulation of heart rate (hr). reduced hrv measured from short-time electrocardiography is seen in dogs with heart failure (hf) secondary to myxomatous mitral valve disease (mmvd). however, hrv is suggested to increase with disease severity at early stages of mmvd. the aims of this study were 1) to associate hr and hrv with severity of mmvd in cavalier king charles spaniels (ckcs) and 2) to compare hr and hrv between ckcs and other dog breeds in a group of dogs in hf secondary to mmvd. one-hundred dogs were examined by echocardiography and 24hour electrocardiography. the dogs were divided into five groups: 1) ckcs with no/minimal mitral regurgitation (mr) (mr jet 15% of the left atrial area using color doppler mapping) and no murmur, 2) ckcs with mild mr (20% o jet 50%), 3) ckcs with moderate/ severe mr (jet450%) and no clinical signs of hf, 4) ckcs in hf (hf defined as left atrium to aortic root ratio (la/ao) 41.5, clinical signs of hf and furosemide responsiveness) and 5) non-ckcs in hf. dogs in hf were allowed hf therapy. both hr and hrv were analyzed over a 24-hour period, while hrv were also analysed over a 6-hour nightly period. analyses of variance were performed with hr or hrv as response variables and the explanatory variables dog group and echocardiographic indices of mmvd were included separately. all p-values were bonferroni corrected. minimum-and mean hr were significantly higher in ckcs with moderate/severe mr and in hf compared to ckcs with no/ minimal and mild mr (all p o 0.001). seven out of 26 hrv variables were significantly decreased in ckcs with moderate/ severe mr and in hf compared to ckcs with no/minimal and mild mr (all p o 0.02). another 10 hrv variables showed the same groupwise differences (all p o 0.02), except that the difference between ckcs with mild mr and ckcs with moderate/severe mr did not reach statistical significance. mminimum hr, mean hr and the hrv variables (7 and 10) differing between dog groups, also consistently decreased with increasing mr, la/ao and the proximal isovelocity surface area in ckcs. non-ckcs in hf had a lower minimum hr compared to ckcs in hf (p 5 0.03) and a higher triangular index measured in both periods (all p o 0.04). in conclusion, hr increased and most hrv variables decreased with increasing severity of mmvd in ckcs, even prior to the development of hf. other breeds in hf secondary to mmvd had lower minimum hr, but higher triangular index compared to ckcs in hf. although the cells in the specialized conduction system in the heart are capable of initiating their own impulse, the rate in which those impulses are generated can be influenced by autonomic nervous system. different types of respiratory patterns can stimulate autonomic nervous system in different manners. thus, non-sedated rabbits were studied during forced respiration aiming to evaluate the influence of this breathing pattern on heart rate. twenty male, one-year-old healthy new zealand rabbits were enrolled in the study. animals were set in right lateral recumbency and maintained that way by physical contention. chemical sedation was not used. partial nasal obstruction by digital compression was applied to those rabbits for five seconds, eliciting a forced inhaling and exhaling against semi closed nostrils. heart rate was obtained by measurement of two consecutives rr intervals in the computerized electrocardiography, recorded continuously prior and during the maneuver. heart rate before the intervention was 251 ae 34 bpm (mean ae standard deviation). all rabbits submitted to the maneuver showed a dramatic reduction in this parameter. after nasal partial obstruction, heart rate was 142 ae 32 bpm. data was submitted to statistical analysis by paired student's t test and a significant difference between the heart rate before and after the maneuver was observed (p o 0.0001). although the exactly mechanism involved in this response was not elucidated, the presented data support the applicability of this maneuver as an efficient method for non-pharmacological heart rate reduction in rabbits. obesity can affect cardiac function due to effects on cardiac rhythm, ventricular volume and blood pressure. the purpose of this study was to determine the effects of obesity and overweight on noninvasive systemic blood pressure and doppler echocardiographic parameters in cats without others causes of cardiac hypertrophy. the study groups comprised fifteen obese cats with mean body score index (bsi) of 8,8, seven overweight cats (bsi 5 6,3) and seven cats with ideal bsi (4,9). the blood pressure was measured by doppler method and the doppler echocardiography was performed in conscious animals. the statistical analysis was performed by analysis of variance followed by tukey's test and pearson's correlation. the blood pressure values of the obese cats were superior (159,12 ae 11,22 mmhg, p o 0,0001) than in overweight (134,45 ae 13,81 mmhg) and normal cats (136,90 ae 13,17 mmhg) and 57% of the obese cats had blood pressure higher than 160 mmhg. there were observed differences on the ratio of early (e) and late (a) left ventricular filling velocity (p 5 0,008) of obese animals (e/a 5 1,07 ae 0,39) compared to overweight (1,68 ae 0,37) and normal cats (1,43 ae 0,24). seven obese cats (50%) had inversion of e/a compatible with diastolic dysfunction and there were negative correlation (r 5 à0,453, p 5 0,026) between the e/a ratio and blood pressure values. other differences observed were increases in left ventricular septum in diastole (p 5 0,002) and in free wall in diastole (p 5 0,023) and systole (p 5 0,042) of the obese animals compared to overweight and normal cats. these results demonstrate the possibility of cardiovascular effects related to obesity in cats, such as systemic arterial hypertension and secondary diastolic dysfunction. diuretic therapy reduces preload, and relieves congestion secondary to cardiac dysfunction. torsemide (torasemide) is a loop diuretic with longer duration of action, less diuretic resistance, and adjunctive aldosterone antagonism as compared to furosemide. we hypothesized that torsemide was no less effective than furosemide at diuresis, control of clinical signs, and maintenance of quality of life in dogs with congestive heart failure. a double-blinded, randomized, crossover clinical trial was performed in 7 dogs with stable heart failure receiving bid furosemide and adjunctive medications. dogs were randomized to their current furosemide dose or torsemide (calculated as 1/10 of the daily furosemide dose divided into bid dosing). crossover occurred at day 7 and the study ended on day 14. clinical, laboratory, radiographic, and owner-perceived quality of life variables were evaluated on days 0, 7 and 14. no dog developed recurrent heart failure during the study. average furosemide dose on day 0 was 5.13 mg/kg/day (range, 2.8-9.6). following torsemide treatment, blood urea nitrogen (p 5 0.0028), albumin (p 5 0.0287), and albumin:globulin ratio (p 5 0.0012) were significantly increased, and urine specific gravity (p 5 0.0062) and chloride (p 5 0.0051) were significantly decreased as compared to baseline and/or furosemide dosing (one-way anova with bonferroni correction). no differences in qol were found. results indicate that torsemide is equivalent to furosemide at controlling clinical heart failure in dogs, and might in fact, achieve greater diuresis vs. furosemide. larger clinical trials evaluating furosemide resistance and/or torsemide as a first-line loop diuretic for congestive heart failure in dogs with heart failure are warranted. the purpose of this study was to investigate the feasibility of speckle tracking echocardiography (ste) in healthy cats and to determine whether or not it can detect myocardial dysfunction in cats with diseased heart. radial and circumferential strain and strain rate were measured by ste using left ventricle short-axis view in clinically healthy cats. eighteen cats with hcm whose lv thickness at end-diastole with 6 mm or more were evaluated with ste analysis, and compared with healthy cats. index of left ventricular synchrony (trs-sd) was also assessed in cats with hcm, and compared to healthy subjects. ste resulted in technically adequate images in 100% of the cats. fusions of early and late diastolic (e and a) wave in strain rate were seen in 3 of 16 cats. percent errors in analysis with or without simultaneous ecg monitoring were 5.3-14.7% in all parameters. inter-and intraobserver variability of ste parameters in healthy cats was minimal (4.1-15.6%) except for the systolic circumferential strain rate. sedation using buprenorphine and acepromazine did not affect any ste parameter. e wave in radial and circumferential strain rate of hcm cats was significantly decreased compared with healthy cats. no significant difference was seen in trs-sd. ste analysis was considered clinically feasible to assess cardiac function in cats, and could detect myocardial dysfunction in cats with hcm. further study is warranted to investigate to assess whether or not ste can differentiate the etiology of left ventricular concentric hypertrophy since it is clinically important. carvedilol, a 3rd generation non-selective beta-blocker with ancillary alpha 1 -blocking and antioxidant properties may have therapeutic implications for multiple diseases in cats; however, pharmacokinetics and bioavailability of commercially prepared oral carvedilol has not been determined. hplc for carvedilol measurement in feline plasma was validated and standardization curves created. the pharmacokinetics (pk) of carvedilol was evaluated in 5 apparently healthy male neutered adult cats (average weight of 5 kg) following single dose intravenous (iv) of 0.5 mg/kg and single dose oral administration of 1.6 to 2.0 mg/kg. concentrations of the active parent compound, carvedilol, were detected in plasma using hplc analysis. lower limit of quantification was 5 ng/ml. the mean peak concentration after iv administration of carvedilol was 8639 ng/ml (range, 901 to 8648), elimination half-life was 2.9 hours (range, 2.0 to 5.4), and clearance was 0.35 l/hr/kg. the volume of distribution was 1.18 l/hr. after a single oral administration of carvedilol, the time to peak plasma concentration was 60 minutes (range, 30 to 90 minutes) and the mean residual time was 4.8 hours. the half life was 4.44 hours. maximal concentration 294 ng/ ml and the mean bioavailability was 15.7% with a median of 9.97% (range, 4.7% to 46%). these data demonstrate a low bioavailability of oral carvedilol and a wide variation in cats. all cats tolerated the oral dose of carvedilol with no major adverse effects. also, a mean residual time of 4.8 hours would suggest that a more frequent dosing schedule may be required to maintain therapeutic plasma levels. pharmacodynamic studies investigating beta-adrenergic blockade duration may provide a more accurate dosing interval of carvedilol. abstract c-49 effects of sildenafil citrate on dogs with ei-senmenger's syndrome. k nakamura, m yamasaki, h ohta, m takiguchi. department of veterinary clinical sciences, graduate school of veterinary medicine, hokkaido university, sapporo, hokkaido, japan. sildenafil has shown to be effective for dogs with pulmonary hypertension; however, its efficacy for dogs with eisenmenger's syndrome (es) and secondary erythrocytosis has not yet been determined. the objective of this study is to determine the effect of sildenafil for dogs with eisenmeger's syndrome and secondary erythrocytosis. this was a prospective, single arm, open-label study. five clinical dogs with es and secondary erythrocytosis were included. new york heart association (nyha) functional class, pcv, and pulmonary artery acceleration time to ejection time ratio (pa at : et) were evaluated before and after sildenafil therapy (0.5 mg/kg, bid). nyha functional class was significantly improved after 1 (median 2; range 1-2, p 5 0.031) and 3 months (median 2; range 1-2, p 5 0.031) of sildenafil therapy, compared with the baseline (median 3, range 2-3). pcv was significantly decreased after 1 month (62.4 ae 4.7%, p 5 0.015) and 3 months (59.9 ae 3.6%, p 5 0.01) of therapy, compared with the baseline (68.0 ae 5.6%). at : et was significantly increased after 1 month of therapy (0.39 ae 0.06, p 5 0.013) from the baseline (0.28 ae 0.04). sildenafil resolved the clinical signs and secondary erythrocytosis in dogs with es. sildenafil therapy could be the treatment of choice for dogs with es. sepsis is the number one cause of mortality in neonatal foals. the role of the raas and hpaa in systemic inflammation and response to stress is well documented in critically ill human neonates, but limited information exists in foals. we hypothesized that in septic foals the raas and hpaa will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality. blood samples were collected on admission from 60 septic (sepsis score 4 12), 102 sick non-septic (sns), and 18 healthy foals of o 7 days of age. blood concentrations of corticotropin-releasing hormone (crh), adrenocorticotropin (acth), cortisol, aldosterone, angiotensin-ii (ang-ii), arginine vasopressin (avp) and plasma renin activity were determined by radioimmunoassays. acth, cortisol, aldosterone, ang-ii and avp concentrations were higher while crh was lower in septic and sns foals compared to healthy foals (p o 0.05). septic non-survivor foals had higher concentrations of aldosterone, cortisol, acth and avp and lower concentrations of ang-ii and crh than survivors. avp was associated with ang-ii in septic, and with acth in septic and sns foals (p o 0.05). there was no difference in renin activity and ang-ii concentrations among foal groups. septic foals had a higher acth:aldosterone ratio than healthy foals (p o 0.001). this study shows that in response to sepsis there is raas and hpaa activation in critically ill foals. we propose that in sick foals avp is more important than crh in regulating acth secretion. the increased acth:aldosterone ratio further supports relative adrenal insufficiency in septic foals. this prospective, cohort study aimed to characterize alterations in coagulation and blood-derived inflammatory biomarkers in adult horses that developed diarrhea during hospitalization. physical and hematological parameters were evaluated at times 0 (onset of diarrhea), 6, 12, 24 and 48h, then every 48 h until resolution of diarrhea or death. each hematological analysis included a complete blood count (cbc), thromboelastography (teg), partial-thromboplastin-time (ptt), prothrombin-time (pt), plasma concentrations of lactate, tumor necrosis factor alpha (tnf-a), interleukin (il)-1, il-6, il-10 and nt-proc-type-natriuretic peptide (pcnp). horses were categorized into three groups based on the duration of diarrhea and evidence of systemic inflammation. group 0: diarrhea o 6 h without systemic inflammation (si); group 1 -diarrhea ! 6 h without si; group 2-diarrhea with si. assessment of vital parameters and cbc established a diagnosis of si as previously described (levy, 2003) . descriptive and univariate outcome analyses were based on data normality. 19 horses were enrolled, of which 16 (84.2%) survived to discharge. the mean age was 13.61/-5.3 years. eight horses (42.1%) were categorized as group-0, 2 (10.5%) as group-1 and 9 (47.4%) as group-2. two horses developed thrombophlebitis. based on the results of teg, 6/19 (31.6%) were normocoagulable, 7/19(36.8%) were hypocoagulable and 6/19 (31.6%) were hypercoagulable, at one or more time points. of these, 7/9 (77.8%) group-2 horses were coagulopathic. additionally, group-2 horses had a significantly lower ma than group-0 horses at baseline (43.6 ae 16.7 vs. 61.9 ae 7.2) and 6h (45.9 ae 15.9 vs. 65.5 ae 5.8). biomarker analyses are pending. in conclusion, si was associated with coagulation disorders in horses with hospital acquired diarrhea. clostridium difficile and clostridium perfringens are commonly associated with colitis and diarrhea in equines but asymptomatic carriers exist. reported carrier rates of toxigenic c. difficile and c. perfringens strains in feces range between 0-25% and 0-30% respectively. toxigenic c. difficile has also been isolated from the small intestine of diseased foals and is implicated as etiologic agent of duodenitis/proximal jejunitis in adult horses however scarce information is available on prevalence in gastrointestinal compartments other than feces in healthy horses, and it is unclear whether fecal samples are good predictors of the status of proximal intestinal sites. the objectives of this study were to investigate the presence of c. difficile and c. perfringens in various intestinal compartments of healthy adult horses and to molecularly characterize isolates. intestinal contents were collected from the stomach, duodenum jejunum, ileum, cecum, right dorsal and left ventral colon, small colon and rectum of 10 euthanized horses free of apparent gastrointestinal disease. enrichment culture was performed for c. difficile and c. perfringens and c. difficile isolates were further characterized via toxin gene detection and ribotyping. c. difficile was isolated from 9/90 (10%) samples from 5/10 (50%) horses. between zero and three sites were positive per horse, and multiple sites were positive in three horses. isolates were recovered from duodenum (n 5 1), right dorsal colon (n 5 3), small colon (n 5 1) and rectum (n 5 4). in one horse, the rectal sample was negative but c. difficile was isolated from a proximal site, all other horses were positive on the rectal sample if a more proximal compartment was positive. in three horses multiple compartments were positive however different strains were always present within the same horse (n 5 2). all isolates possessed genes encoding toxins a and b. five isolates were ribotype 078 and also possessed genes encoding the binary toxin. the other isolates were ribotype 001 and were negative for the genes encoding the binary toxin. despite using a method with a detection level as low as 9 cfu/g of feces, no c. perfringens was recovered. rectal samples were a good predictor of overall c. difficile carrier status (4/5 horses), however rectal samples were not always representative for the ribotype carried in more proximal compartments. the presence of variable strains within the same horse suggests transient passage of the bacterium through the gastrointestinal system rather than actual colonization although further study testing multiple colonies per site is needed. the predominance of ribotype 078 is consistent with recent emergence of this strain in this region, as earlier studies found other strains (027, 001) to be more prevalent and a variety of ribotypes were typically recovered from horses. interestingly ribotype 078 has recently emerged as a hypervirulent strain in humans in our area. clostridium difficile, clostridium perfringens and salmonella are important enteric pathogens in horses, however some healthy animals also harbour these pathogens. point prevalence studies have reported these carriage rates, but there are no data regarding longitudinal prevalence of these enteric bacteria, information that would be useful to better understand the epidemiology of these pathogens. additionally, antimicrobial resistance is a pressing concern. commensal e.coli is often used as an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data from horses on farms. the objectives of this study were to longitudinally investigate the above enteric pathogens over the course of one year, molecularly characterize obtained isolates and determine the antibiotic susceptibility profile for e. coli. fecal samples were collected from 25 adult horses from five farms on a monthly basis over the course of one year. selective cultures were performed for c. difficile, c. perfringens, salmonella and e. coli. c. difficile isolates were characterized via toxin gene pcr and ribotyping. broth microdilution was performed to assess antimicrobial susceptibility profiles of e. coli. clostridium difficile was isolated from 15/275 (5.45%) samples from 11/25 (44%) horses. four horses were positive on more than one occasion, three were positive in two consecutive months. different ribotypes were found in two of the latter horses. most isolates were ribotype 078 (n 5 6) with ribotype 001 (n 5 5) and ribotype c (n 5 4) also identified. ribotypes 078 and c possessed genes encoding toxins a, b and binary toxin, while ribotype 001 only possessed toxin a and b genes. despite a detection threshold of 9 cfu/g feces, c. perfringens was not detected in any samples, nor was salmonella. e coli was isolated from 117/225 (52%) samples. resistance to !1 antimicrobial was present in only 19/117 (16.9%) isolates. multidrug resistance (! 3 antibiotics) was present in 5/117 (4%). most commonly, isolates were resistant to sulfisoxazole (17/ 117) and trimethoprim sulfamethoxazole (16/117). the overall detection rate for toxigenic c. difficile in fecal samples of healthy horses was 5.4% which is consistent with previous studies. the cumulative prevalence of 44% was striking but only one horse shed the same strain for more than one month, indicating c. difficile shedding is a transient and dynamic state. the predominant isolation of ribotype 078 is consistent with the suspicion that this strain has emerged and become widely disseminated in this region in recent years. the low prevalence of c. perfringens and salmonella is in agreement with some other studies. the low prevalence of antibiotic resistance in commensal e. coli was encouraging and suggests that healthy horses on pleasure horse farms are not likely a major reservoir of resistance in enteric bacteria. type 1 polysaccharide storage myopathy (pssm) in horses is associated with a dominant missense mutation (r309h) in the skeletal muscle glycogen synthase gene (gys1). since disease severity varies between affected horses, we hypothesised that some clinical variability could be accounted for by the underlying genotype. 107 belgian / percheron horses were genotyped using a validated restriction fragment length polymorphism assay enabling grouping of horses as homozygotes (hh), heterozygotes(hr) or normal (rr). subsequently, semimembranosis muscle samples were biopsied from each of six matched sedentary horses from each group; one sample was formalin-fixed and one fresh frozen. sections were stained using haematoxylin and eosin, periodic acid schiff 1/diastase. anti-dystrophin, nnos and myosin heavy chain immunohistochemistry was performed to examine sarcolemmal intregrity, there were significant differences in resting ck activity (p 5 0.023) (median hh 5 364u/l interquartile range(ir) 332-764; hr 5 301u/l ir222-377; rr 5 260u/l ir216-320) and ast activity (p o 0.001) (ast mean hh 5 502u/l sd116; hr 5 357u/l sd92; rr 5 311u/l sd64) and muscle pathology between the 3 groups, with severity increasing rr o hr o hh. there were significantly more type 2a (p 5 0.04) and fewer type 2x fibres (p 5 0.02) in homozygotes (2a 55% sd 10.2; 2x 32% sd 18) compared with the other groups (hr 2a 38% sd 10.9, 2x 44% sd 10.8; rr: 2a 36.5% sd 12.4 2x 57% sd 11.5). more type 2a fibres contained polysaccharide inclusions in homozygotes (30% sd 11.1) than in heterozygotes (10.6% sd 6.9) (p o 0.001). both dystrophin and nnos expression was normally localised to the sarcolemma in pathologically normal and vacuolated fibres from mutant horses. in conclusion, sedentary homozygotes have more severe skeletal muscle pathology and higher resting plasma ck and ast activities than heterozygotes, and pssm1 is associated with a fibre type shift towards type 2a. although subsarcolemmal vacuolation likely disrupts the contractile apparatus's attachment to the sarcolemma, the latter's integrity appeared intact. the recumbent horse presents a logistic, diagnostic, and therapeutic challenge to the equine practitioner. there is currently very little data available on the prognosis and outcome of horses that are recumbent. therefore, the purpose of this study was to investigate the outcome of hospitalized horses that had been recumbent in the field or in the hospital and the factors affecting their survival. records of horses admitted to the school of veterinary medicine, university of california davis from january of 1995 to december of 2010 with a history of recumbency or horses that became recumbent while hospitalized were evaluated. a horse was defined as recumbent if it was unable to stand on its own. the medical record was examined for the following criteria: history pertaining to the current illness including treatment by the rdvm, breed, age, weight, date of presentation, physical and neurological examination findings, cbc and biochemical profile results, initial drugs administered on arrival, time spent recumbent, time spent in a sling, diagnosis, and hospitalization costs. statistical analysis correlating factors associated with survival was performed using logistic regression. overall there were 112 non survivors and 49 survivors. factors that favored survival included early initiation of treatment in the field by the rdvm, horses that tolerated a sling and spent more time in a sling, increased duration and costs of hospitalization, horses that were recumbent post anesthesia, and those recumbent due to disease of the musculoskeletal system. factors that increased likelihood of non survival included horses that were ataxic on presentation, horses with increased bun, horses that spent more time recumbent, those that did not tolerate a sling, and horses diagnosed with botulism and spinal cord disease. in conclusion, this retrospective study demonstrated that both the cause of recumbency and the ability of horses to tolerate a sling had a direct effect on survival. abstract e-7 plasma peak and trough gentamicin concentra-tions in hospitalized horses receiving once daily gentamicin. jr read 1 , pa wilkins 2 , rd nolen-walston 1 . 1 university of pennsylvania, new bolton center, kennett square, pa. 2 university of illinois, champaign-urbana, il. gentamicin is often used to provide gram negative antimicrobial coverage in horses at 6.6 mg/kg iv every 24 hours. therapeutic drug monitoring in our hospital suggests larger doses are required in many clinical cases to achieve the desired concentration (8-10â minimum inhibitory concentration) for common bacterial isolates (peak target range 32-40 mg/ml). the aim of this study was to determine the correlation between gentamicin dose and plasma concentration in hospitalized horses receiving gentamicin treatment in order to identify an optimum dose range for this population. review of records (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) identified 71 horses ! 3 months old receiving once-daily gentamicin with peak and trough assays performed (n 5 107 sets). spearman rank correlation coefficient analysis revealed a weak (r 5 0.289) but statistically significant correlation (p 5 0.003) between gentamicin dose and peak plasma concentration. horses receiving 7.7-9.7 and 4 9.7 mg/kg gentamicin (groups 2 and 3) had higher median peaks (31 mg/ml) than horses receiving 5.6-7.6 mg/kg (group 1; 25.7 mg/ml). higher doses were more likely to result in peaks 4 32 mg/ml (42 and 41%, groups 2 and 3 respectively) than horses receiving 5.6-7.6 mg/kg (group 1; 16%). all 22 hour post-gentamicin administration trough values were o 2 mg/ml. no correlation was found between dose and change in plasma creatinine during treatment, nor dose and trough level. these data suggest that gentamicin dosage in horses should be individually determined by therapeutic monitoring. additionally, these data support an initial dose of 7.7-9.7 mg/kg iv every 24 hours in order to achieve desired peak concentration and an appropriately low trough concentration. heaves is a common respiratory inflammatory disease, characterized by a pulmonary neutrophilia. this disease is also characterized by an activation of circulating neutrophils after antigen challenge but their specific role in heaves is not well understood. also, there are anecdotal studies concerning heaves-affected horse to be more susceptible to infection. however, to our knowledge, the antibacterial host defense role mediated by circulating neutrophils was not investigated in heaves-affected horses. the objective of this study was to compare phagocytosis activity and bacterial killing by circulating blood neutrophils of heaves-affected and control horses. peripheral neutrophils were isolated from heaves-affected (n 5 6) and control (n 5 6) horses using a density gradient technique. the killing capacity was assessed by incubating neutrophils with streptococcus equi spp equi and spp zooepidemicus. after 1 h of bacterianeutrophil coculture, total viable bacterial cells were measured by quantitative plating. the phagocytosis was evaluated by flow cytometry using fluorescent beads and gfp-transformed streptococcus suis suilysin-negative mutant strain. circulating neutrophils from heaves-affected horses showed a significant decrease in their killing capacity toward s. zooepidemicus (p 5 0.046). a reduced, although not significant (p 5 0.1), killing capacity of s. equi by these neutrophils was also observed. the phagocytosis activity was not different between groups. this impairment of blood neutrophil bactericidal activity in heaves-affected horses could contribute to an increase susceptibility to infection. obesity is a common disorder of the horse, with current prevalence estimated at 30%. in people, obesity is associated with dyslipidemia, insulin resistance, mitochondrial dysfunction and downregulation of lipid and glucose metabolic pathways. in the horse, obesity is similarly associated with insulin resistance and alterations in lipid profiles; however, metabolic regulatory gene expression profiles have not been fully characterized. we hypothesized that obese horses have decreased expression of metabolic regulatory genes and decreased mitochondrial content in skeletal muscle compared with non-obese horses. sixteen light breed horses, 2-27 years of age were included. body condition score (n 5 16) and neck circumference (n 5 9) were recorded. post-mortem biopsy samples of the semi-membranosus muscle were obtained. dna and rna were isolated. relative expression of the metabolic genes, peroxisome proliferator activated receptor g (pparg), pparg coactivator-1a (pgc-1a), fatty acid translocase (fat) and estrogen related receptor a (erra) was determined by quantitative polymerase chain reaction (qpcr). mitochondrial content was assessed by determining mitochondrial dna/nuclear dna ratio by qpcr, using nadh-dehydrogenase subunit 2 and cytochrome oxidase subunit 2 as mitochondrial genes and beta actin as the nuclear reference gene. non-normal data was log transformed for analysis and a pearson coefficient of correlation was calculated for relative gene expression, body condition score and neck circumference. a value of p o 0.05 was considered significant. body condition score was strongly correlated with neck circumference (n 5 9, r 5 0.72, p 5 0.03). relative expression of erra and glut-4 increased with body condition score (erra: n 5 16, r 5 0.66, p 5 0.005; glut-4: n 5 16, r 5 0.50, p 5 0.05). copy number of the mitochondrial genes (nadh-dh and cox-2) was not related to body condition score or metabolic gene expression. expression of glut-4, erra, pparg, and fat were strongly correlated to each other, but not pgc-1a. there was a strong trend towards correlation between pparg and pgc-1a in horses with body condition score 4 6 (n 5 6, r 5 0.77, p 5 0.07). in this study, there was no change in mitochondrial content in obese horses. assessment of mitochondrial function in obese horses and horses with ems is under way. the strong correlation between pparg and pgc-1a observed only in horses with high body condition scores suggests this pathway is activated with obesity. the role of pparg and pgc-1a in equine obesity should be further investigated to determine their potential as therapeutic targets. upregulation of erra and glut-4 in horses with increasing body condition score is unexpected, and may indicate a compensatory response to dysfunction of a downstream pathway. further studies to better define the role of metabolic regulatory gene expression in obese horses and those with ems are ongoing. previously presented at the 7th annual harold hamm diabetes center research retreat, oklahoma city, ok. inflammatory bowel disease is a cause of weight loss, decreased performance, and colic in horses. this condition is difficult to diagnose and clinicians rely upon absorption tests to document malabsorption. the purpose of this study was to compare glucose and xylose absorption tests in normal horses and determine the repeatability of these procedures. eight horses received 500 mg/kg dextrose or d-xylose powder mixed as a 10% solution in water or water alone via nasogastric intubation on three different occasions within the same week for three consecutive weeks (9 tests/horse). a crossover design was employed and the order of treatments was randomized. blood samples were collected at time 5 0, 30, 60, 90, 120, 150, and 180 min. data were analyzed by repeated measures anova and t-tests. results showed that the xylose response over time differed significantly from the glucose response over time (test â time; p o 0.001). mean time to maximum concentration differed (p o 0.001) between tests (glucose 90 min; xylose 60 min). within-horse area under the curve, maximum concentration, and time to maximum concentration values for dextrose and xylose did not differ significantly when tests were repeated. results indicate that glucose and xylose absorption tests are repeatable within the same horse, but plotted curves differ between tests, with peak concentrations occurring at a later time point for the glucose absorption test. we conclude that both tests provide repeatable measures of intestinal absorption, but glucose and xylose appear to differ in their rates of absorption and clearance. the purpose of this study was to examine the records of a population of thoroughbreds with cervical vertebral malformation (cvm) and to determine which factors have an effect on these horses achieving athletic function. this was a retrospective case study of 119 thoroughbreds with cvm treated medically from 2002 to 2010. forty-one were euthanized after diagnosis, while the remaining 78 were discharged for treatment. racing records were reviewed to determine which horses raced after treatment. horses were separated into groups based on whether or not they raced. medical records were reviewed, and results of neurologic examination, radiographic and laboratory findings, treatments, and outcome were assessed and compared between groups. twenty-one of 78 horses treated medically (27%) improved enough to race. median neurologic grade between groups was significantly different (p o 0.0001), with a hind limb grade of 2.0 (range 1-3) for the raced group and 2.5 (range 0.5-4) for the unraced. intravertebral sagittal ratios measured from standing lateral cervical radiographs were equivocal between groups. radiographs of all horses were examined for kyphosis, dorsal over-riding arch, caudal epiphysitis, degenerative joint disease, cystic bone lesions, and cranial stenosis of the vertebral canal. horses with kyphosis (p 5 0.0178), degenerative joint disease (p 5 0.0497), or cranial stenosis (p 5 0.0357) at any site were less likely to return to racing. racing prognosis for horses with cvm treated conservatively is equivalent to that of those treated surgically as reported by rush moore et al (javma, 1993) . radiographic changes and neurologic grade may help serve as indicators for whether a horse will respond to conservative therapy. since pain assessment is vital for management of colic, a valid, reliable and feasible tool for assessing the severity of acute abdominal pain in horses is urgently needed. our aim was to construct and validate a behavior-based pain scale by methodology utilized in construction of pain scales in non-verbal humans. the project consisted of four stages. firstly, behaviors to include in a scale were empirically identified. thirty equine clinicians noted behaviors in each of 23 random film clips of horses with colic using a checklist. nine behaviors (e.g. rolling, pawing, and flank watching) demonstrated good inter-observer agreement without bias (multi-rater kappas: 0.5-0.95). secondly, the clinical judgment of experts was utilized to identify and to weight behaviors. six expert clinicians independently expressed opinions as to which of 46 behaviors to include and the severity of pain they indicate. two contending scales (equine acute abdominal pain scales (eaaps) 1 & 2) were constructed based on both the empirical and the judgmental approaches. each included 12 identical behaviors with a 1-5 point score range; eaaps-2 with gradations to some of the behaviors and eaaps-1 without. in the third stage, blood cortisol and lactate levels and heart rate were shown to only approximate pain since they correlate poorly with degree of pain as assessed by visual analog scale (vas) in 32 horses with colic and 8 controls (spearman rho; lactate 0.359; cortisol 0.214; heart rate 0.261). finally, reliability and validity of the pain scales were evaluated including constructs of pain; face validity, convergent and discriminate validity and extreme groups. thirty of 40 films of horses with colic were randomly presented to 44 expert equine clinicians internationally who were randomly allocated into three groups to score pain; one group by both vas and numerical rating scale (nrs)), and two groups, each by one of the two eaaps scales. inter-observer reliability of both eaaps scales was excellent (intraclass correlation 0.8). intra-observer reliability based on scores given for identical films demonstrated; 87% and 56% agreement, kappa 0.9 and 0.35, and spearman's rho 5 0.97 and 0.58 for eaaps-1 and 2, respectively. both scales varied by 1 score between observations. face validity; each group reported their scale to be valid (67% & 81%). convergent validity; the scales compared favorably with vas/nrs scores (spearman's rho: 0.84-0.89). discriminate validity; correlation to heart rate, lactate and cortisol levels was predictably low (rho 5 0.2-0.5). extreme group validity; colic horses scored significantly higher than control horses; scores of 0.6-0.7 in controls versus 2.6-2.7 in cases. in conclusion, methodology established in human medicine but novel in veterinary medicine was used to construct and validate two clinically feasible equine abdominal pain severity scales that showed excellent reliability and validity. further refinement of the eaaps scale is advised prior to introduction into clinical practice. aortic valve prolapse (avp) is a common echocardiographic finding in horses, but when compared with mitral valve prolapse in dogs, little is known about the natural progression of this condition. previously published data has shown that echocardiographic identification of avp in horses is reliable, diagnostic criteria have been established and that development occurs with training. the aims of this study were to evaluate the different rna and protein expressions of smooth muscle actin (sma), transforming growth factor-b 1 (tgf), nitric oxide synthase (nos) and the concentrations of elastin and collagen in normal, prolapsing and diseased cusps to evaluate what structural changes may predispose them to prolapse. valve cusps were harvested and processed from a group of 176 horses at a commercial abattoir following disease classification using echocardiography. horses were aged 13.7 ae 6.7years, weighing 518 ae 51 kg and with a median body condition score of 4/9. cusps were collected in rnalater s and stored at à801c prior to processing. cdna was produced from half a valve using a standard protocoland qrt-pcr performed to assess relative rna expression of sma, tgfb 1 , endothelial (enos) and inducible nos (inos) and compared with the housekeeping gene 18s. a quarter of cusp was processed using an adapted commercial protocol to evaluate protein expression of sma, tgf b 1 , enos and compared to vimentin. specific antibody binding was assessed with western blotting and protein expression evaluated using dot blots. the remaining quarter cusp was used to measure soluble collagen and elastin concentrations using commercial assays 3 . statistical analyses included one way anova with post-hoc bonferoni, paired student's t-test, linear and logistic regression. there was no effect of gender or age on any of the measurements. valves from animals with avp had lower expression of sma and elastin compared to normal and diseased valves, increased expression of tgfb 1 and enos, whereas inos expression was greater than normal valves (table 1) . collagen content of valves from horses with avp was increased compared to normal but lower than horses with valve disease. prolapsing cusps appear to be a different phenotype from diseased cusps. further studies will help to elucidate the significance of these findings in vivo. a clear association between heart rate (hr) and body mass has been observed across a wide range of mammalian species. furthermore, it is well known that electrocardiographic (ecg) time intervals vary with heart rate and body mass. within the equine species, small breeds are generally thought to have higher heart rates than large breeds. however, despite the large differences in size among different equine breeds, there is little information about normal heart rates and normal ecg time intervals in horses and ponies of different body size. similarly, the relationship between hr and body mass in dogs of various breeds and sizes is still under debate. the goal of this study was to investigate the relationship between heart rate and ecg time intervals to body mass in apparently healthy horses and ponies and to calculate normal ranges for different weight groups. 250 adult horses and ponies at an age of 5.5 (1-30) y [median (range)] and a body weight of 479 (46-1120) kg were included in the study. all animals were considered clinically healthy based on history and physical examination. a standard base-apex ecg was recorded at a speed of 25 (n 5 4) or 50 mm/s (n 5 246) using a multiparameter monitor (datascope passport). during the procedure, the horses were unsedated, standing quiet in a box stall. mean hr over 15 sec was determined for each recording. the following ecg time intervals were measured in triplicate and averaged for further analyses: pq interval, qrs duration, qt interval, and difference between qt and qrs (qt-qrs duration). the relationship between hr, ecg time intervals, and body mass was assessed using linear regression analyses. normal ranges (2.5% to 97.5% percentile) were calculated for 5 different weight groups. the level of significance was p 5 0.05. heart rate was inversely related to body mass (p o 0.0001, r 2 5 0.122). the pq interval (p o 0.0001, r 2 5 0.413), qrs duration (p o 0.0001, r 2 5 0.147), qt interval (p o 0.0001, r 2 5 0.089), and qt-qrs duration (p o 0.0001, r 2 5 0.028) were directly related to body mass. normal ranges for hr, pq, qrs, and qt within the different weight groups were 36-64 bpm, 107-230 ms, 60-127 ms, 360-510 ms (o200 kg); 28-68 bpm, 162-319 ms, 74-155 ms, 362-610 ms (200-399 kg); 28-60 bpm, 211-423 ms, 89-147 ms, 390-581 ms (400-599 kg); 28-54 bpm, 220-380 ms, 87-150 ms, 367-587 ms (600-799 kg); and 24-52 bpm, 240-463 ms, 87-140 ms, 437-533 ms (4 799 kg). we conclude that in healthy horses there is a significant but weak relationship between body mass and hr and between body mass and ecg time intervals, respectively. this study therefore supports the hypothesis that within the equine species, small breeds have faster heart rates and shorter ecg time intervals than large breeds. therefore, body mass has to be considered when comparing hr and ecg time intervals to normal ranges in horses. horses with pituitary pars intermedia dysfunction (ppid) often have elevated plasma acth concentrations. however, ppidaffected horses rarely have resting serum cortisol levels above the reference range or adrenal hyperplasia. we hypothesized that this apparent dissociation between plasma acth levels and adrenal response in horses with ppid is due to the secretion of acth that is less biologically active than that from normal horses. to test our hypothesis, a bioassay to evaluate acth activity was developed. adrenocortical explants were harvested aseptically from normal horses at euthanasia and stimulated with plasma from healthy (n 5 9) and ppid-affected horses (n 5 11). the assay was performed three times with explants obtained from different horses. cortisol secreted by the explants and plasma acth levels were measured with commercially available radioimmunoassays. cortisol secretion stimulated by each sample was standardized to the respective explant protein concentration. cortisol data was normalized for acth concentration in each plasma sample and expressed as a cortisol:protein:acth ratio. ratios from horses with ppid and normal horses were compared by unpaired t-test. horses with ppid had significantly lower cortisol:protein:acth ratios compared to normal horses. (assay 1: 0.06 ae 0.04 vs. 0.33 ae 0.11, p o 0.001; assay 2: 0.06 ae 0.04 vs. 0.30 ae 0.10, p o 0.001; assay 3: 0.07 ae 0.06 vs. 0.20 ae 0.13, p o 0.01). these results suggest that plasma acth from ppid horses is less biologically active than plasma acth from normal horses. our findings give further insight into the pathophysiology of ppid and may aid in the development of novel diagnostic testing protocols. an online survey was conducted to determine perceived needs of potential employers of new acvim-laim diplomates. the survey was designed as the first step in determining what is needed for success in the various sectors of practice employing acvim-laim diplomates. demographic and background data were collected using questions and drop-down menus on the first page. the survey evaluated 189 skills or concepts in 26 areas of veterinary practice. participants answered 4 questions about each skill or concept using drop-down ranked lists. those participants that had completed an acvim-laim training program were asked 3 additional questions about whether they were taught the skill or concept during their own residency. data were collated and descriptive statistics calculated. the mean scores or frequencies of use for each skills or concepts were ranked to determine which of the skills or concepts were most important for an entry-level diplomate. eighty-eight individuals participated in the survey with 86 respondents being acvim diplomates, 1 respondent was not board-certified and 1 respondent was an act diplomate. nineteen respondents were diplomates of acvim and an additional specialty. eighty-three respondents had completed an acvim residency. the majority of respondents were in academia (65%) with 30% being in private practice. equine specialists prevailed (53%) followed by mixed large animal (28%) and then food animal only specialists (13%). the distribution of years post-residency was slightly skewed toward younger diplomates, but overall there was a good distribution of diplomates across years of experience. most respondents stated that they did not make hiring decisions in their practice. competency in disciplines other than internal medicine was expected with ultrasonography and radiology being the most desirable followed by theriogenology and lameness. surgical skills, both equine abdominal (10%) and food animal general surgery (11%) were considered important by some respondents. thirty-six per cent of respondents thought that a new diplomate should expect to make o $50,000 per annum, while only 13% of respondents thought that a new diplomate should expect to make ! $90,000 per annum. not all respondents answered questions on all skills or concepts. the mean number of skills or concepts evaluated was 86 (sd 5 75) with only 18 respondents answering all 189. all skills or concepts evaluated were found to be at least somewhat important, were estimated to be used at least occasionally, were recommended for inclusion in training programs as core or elective, and some level of knowledge was expected. at least some of the respondents were taught each of the skills or concepts during their residency, practiced the skill or concept at least occasionally during their residency, and some degree of competency was expected at the time of completion of their residency. these data will provide a framework for designing future laim residency programs. abstract this study evaluated pharmacokinetics and clinical safety of an oral paste formulation of a commercially available cox1-sparing nsaid in clinically healthy pony foals in a randomized controlled clinical trial. values for complete blood count, serum chemistry profile, urinalysis, pharmacokinetic assay, and gastric endoscopy were evaluated in eighteen shetland pony foals treated with firocoxib (0.1 mg/kg, po, q 24 h) or placebo for 14 days. foals were divided into 3 treatment groups. group 1 and 2 foals received firocoxib while a 3rd group was administered an oral placebo. gastric endoscopy was performed on group 1 and 3 foals prior to treatment and on days 7 and 14 to monitor for the presence of gastric ulcers. group 2 and 3 foals had blood and urine samples taken sequentially for pharmacokinetic analysis, cbc, serum chemistry evaluation, and urinalysis. physical examinations were performed prior to treatment and daily for 17 days. data were analyzed using anova and paired t-tests (p o 0.05). none of the foals presented adverse clinical effects. there were no significant changes in cbc, biochemical profiles within groups, or differences between groups. pretreatment gastric endoscopy scores were not significantly different from evaluations at 7 and 14 days. firocoxib was quickly absorbed with an observed maximum concentration at 2 hr, the first sampling interval, for the majority of animals. firocoxib plasma concentrations decreased in a log-linear manner after reaching the maximum concentration and steady state concentrations were achieved by the 7th dose. based on the sampling times after the final and 14th dose, an average half life of 1.3 days was estimated. administration of firocoxib did not cause any adverse effects on gastrointestinal, or hematological or serum biochemical variables, appears to have been well tolerated, and follows a predictable pharmacokinetic pattern in 4-6 week old foals. equine herpesvirus 1 (ehv-1) is highly prevalent in most horse populations. horses are routinely vaccinated against ehv-1, and neutralizing antibodies have helped to prevent disease. however, the usda has recently classified ehv-1 myeloencephalopathy (ehm) as an emerging disease, in response to the apparent increase in incidence, morbidity, and mortality of ehm that suggests a change in virulence of the virus. it has been reported that cellular immune mechanisms, in particular cytotoxic t-cells (ctls), are important in controlling ehv-1 viremia. interferon-alpha (ifn-a) has a key function in innate immune regulation by inducing the differentiation and maturation of ctls. here, we investigated the influence of abortogenic (racl11, ny03) and neuropathogenic (ab4) ehv-1 virus strains on ifn-a, il-4 and il-10 secretion in equine pbmc. equine pbmc were infected with racl11, ny03 or ab4 ehv-1 strains or kept in medium for 24 hours. ifn-a, il-10 and il-4 secretion was detected in the supernatants by a fluorescent bead-based cytokine assay. the production of ifn-a increased with increasing viral doses and similarly for all three ehv-1 strains. the production of the antiinflammatory cytokine il-10 was significantly decreased after ab4 infection compared to racl11 and ny03 strains at viral infection doses of moi 0.3-1. at high doses (moi 3), il-10 production was suppressed by all three ehv-1 strains. the results suggested that abortogenic and neuropathogenic ehv-1 strains equally induce antiviral ifn-a production in equine pbmc. they also illustrated the differences in the ability of ehv-1 strains to modulate anti-inflammatory il-10. neuropathogenic ab4 strain had an increased potential to down-regulate il-10 production suggesting specific viral mechanisms that interfere with the control of inflammation in the host. the variations in innate il-10 secretion might influence the development of protective immunity and might offer an explanation why neuropathogenic ab4 induces more severe disease, including myeloencephalopathy, than abortogenic ehv-1 strains. previously presented at a conference of research workers in animal disease. rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. treatment protocols are long, expensive and do not always produce good results. rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. recent studies are based on the use of the virulence associated protein a (vapa) which has been described as an important inducer of immunity against r. equi. the present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of s. enterica typhimurium expressing vapa antigen (test group) or s. enterica typhimurium without the vapa gene (control group), previous to and following experimental challenge. two experimental phases were established according to the immunization route: intranasal or oral vaccination up to 12 hrs following birth and at 14 days of age. the experimental and control groups were challenged on day 28 with a virulent stain of r. equi. clinical examination, cbc and image complementary exams were used to evaluate the development of clinical signs. immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation assays, isolation of r. equi and cytological profiles of tbw. clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group (2/3) and was due to r. equi pneumonia. the test group produced a more intense iggb response when compared to controls however no statistical difference was observed. lymphoproliferation and th1 cytokine expression were higher in the test group. in contrast, controls produced an il-4 response. local iga was significantly higher in animals immunized with salmonella carrying vapa. immunization protocols produced no severe toxic effects. the vaccination of neonatal foals with s. enterica typhimurium expressing vapa was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental r.equi infection. this study was conducted to test the hypothesis that the 32 kd protein, myristolated alanine-rich c-kinase substrate (marcks), is involved in equine neutrophil migration and adhesion. in other species, marcks phosphorylation and dephosphorylation causes the protein to cycle between the cell membrane and cytosol, respectively. to investigate marcks phosphorylation in horses, neutrophils were isolated from whole blood and stimulated with platelet activating factor (paf), leukotriene b 4 (ltb 4 ) or phorbol myristate acetate (pma). western blot was performed using specific phospho-marcks and total marcks primary antibodies. these results determined marcks phosphorylation is maximal 30 seconds following stimulation and that dephosphorylation occurs within 3 minutes. to investigate the requirement for marcks in equine neutrophil chemotaxis, isolated neutrophils were pre-treated with mans (a cell permeant peptide identical to the n-terminal 24 amino acids of marcks), rns (a control peptide) or vehicle control (vc) prior to a migration assay toward known neutrophil chemoattractants (ltb 4 or paf). pre-treatment of equine neutrophils with mans significantly inhibited migration while rns pre-treatment had no effect. to investigate marcks requirement in equine neutrophil adhesion, mans, rns or vc treated cells were stimulated to adhere to immulon 2 plates coated with 5% fbs. pre-treatment of equine neutrophils with mans significantly inhibited adhesion while rns pre-treatment had no effect. inhibition of marcks using a cell permeant peptide identical to the protein's n-terminus significantly inhibited equine neutrophil adhesion and migration. these results indicate that marcks is an important regulator of equine neutrophil chemotaxis and represents a potential target for anti-inflammatory therapy. amongst other tests, a thorough neurologic examination of horses may include walking with the head elevated and during blindfolding, in order to help differentiate normal from abnormal and to help with neuroanatomically localising any lesion(s) i.e. in the ataxic horse. consensus amongst equine neurologists suggests that gait abnormalities associated with these specific tests are often exacerbated in horses with underlying proprioceptive deficits however the effect of these tests on temporal gait characteristics in normal horses has not previously been assessed quantitatively. we hypothesized that head elevation or blindfolding, in comparison with walking in a straight line would result in a compensatory decrease in lateral (left front-on to left hind-on and right front-on to right hind-on) and diagonal coupling intervals (left front-on to right hind-on and right front-on to left hind-on) in normal horses. four thoroughbreds without any history or clinical signs suggestive of neurological disease (age range 3 to 5 years) were included in the study. retroreflective markers were applied to the withers, to the sacrum and to left and right tuber coxae; for each limb, lateral fetlock markers and dorsal and lateral hoof wall markers were used. a minimum of 3 trials each with 2-4 walk strides for each task were analysed as horses walked across an 8-force-plate runway i surrounded by a 12-camera kinematic system. ii force-plate data were processed with semi-automated custom written matlab iii scripts. data were analysed with a mixed model using the statistical software r. there was a significant fixed effect of normal walk on a straight line and head elevation on left and right lateral coupling intervals (p o 0.0001) and of the left and right diagonal coupling intervals (p o 0.0001). there was no significant effect of blindfolding on neither lateral nor diagonal coupling intervals. the random effect of horse had no influence on the coupling intervals. the decrease of the lateral coupling intervals indicates a tendency towards a pacing gait during head elevation. we conclude that there is a significant change in temporal gait characteristics of non-neurologic horses when the head is elevated but not during blindfolding compared to normal walking. current results suggest that pacing and increased variation in foot-placement during head elevation should be interpreted with caution however further work is required to determine whether the change differs between horses with neurological disease and non-neurologic disease. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder associated with a mutation in cyclophillin b that leads to impaired collagen folding, aberrant wound repair, and corneal abnormalities. it affects young quarter horses, appaloosa, and paints. herda shows similarities to the human hereditary connective tissue syndrome ehlers danlos (eds). many eds patients suffer from joint pain and osteoarthritis (oa) as adults. the similarity between eds and herda raises the question whether horses suffering from herda develop oa. in oa, excess production of inflammatory mediators such as prostaglandin e2 (pge 2 ) activate enzymes that degrade cartilage as well as impede wound healing. the present study examined articular cartilage from yearling horses afflicted with herda. we hypothesized that chondrocytes from these horses are continually activated to produce inflammatory mediators. to test this hypothesis, articular cartilage from carpal and tarsal joints of herda horses were evaluated using histology. pge 2 production by chondrocyte cultures was measured by elisa and analyzed by one-way anova, tukey post-hoc test, p o 0.05 significance. we also determined the antiinflammatory effects of an avocado/soybean unsaponifiables (asu), glucosamine (glu), and chondroitin sulfate (cs) mixture (ingredients found in cosequin s asu) and phenylbutazone (pbz) on chondrocytes. cosequin s asu and pbz are used alone or in combination for the management of oa. chondrocyte cultures were incubated for 24 hrs with control media alone, a clinically relevant concentration of pbz (4 mg/ml), or the combination of asu (nmx1000 s , 8.3 mg/ml) 1 glu (fchg49 s , 11mg/ml) 1 cs (trh122 s , 20 mg/ml). articular cartilage from joints of five herda-afflicted horses showed gross and histologic evidence of osteoarthritic lesions. chondrocyte cultures from cartilage of horses suffering from herda spontaneously produced greater pge 2 than chondrocytes from normal horses (41000-fold). pbz significantly decreased pge 2 production by $90% (p o 0.001). the combination of asu1-glu1cs also significantly reduced pge 2 production by $60% (p o 0.001). the present study supports anecdotal findings that horses suffering from herda are likely to develop oa. the inhibition of pge 2 synthesis by asu1glu1cs suggests that this combination may be beneficial for the management of oa in herda. research supported by nutramax laboratories, inc. equine inflammatory airway disease (iad) is a common condition often treated empirically with corticosteroids. gene expression analysis in the bronchoalveolar lavage fluid (balf) may help understand the effects of corticosteroids in iad. the first part of the study aimed at identifying reference genes in the balf of iad horses treated with corticosteroids. the second part of the study investigated the effects of dexamethasone and fluticasone propionate treatments on the mrna expression of il-1b, il-4, il-8 and il-17. the expression stability of seven candidate reference genes was determined in balf taken pre-and post-treatment with dexamethasone and fluticasone propionate in horses with iad. primers' efficiencies were calculated using linregpcr. normfinder, genorm and qbaseplus softwares were used to rank the genes according to their stability. gapdh was the most stably expressed gene whereas b2m was the least stable gene. in addition, genorm analysis revealed that the number of genes required for optimal normalization was four (gapdh, sdha, hprt, rpl32). in the second part of the study the mrna expression of il-1b, il-4, il-8 and il-17 was measured in balf samples from seven iad horses treated in a randomized cross-over design with dexamethasone (0.05 mg/kg sid, 15 days) or inhaled fluticasone propionate (3000 mcg bid with aerohippus, 15 days). the balf samples were taken at baseline and after each treatment period. there was no significant effect of the corticosteroids treatment on the mrna expression of il-1b, il-4 and il-8 in the balf. the mrna expression of il-17 was suppressed by dexamethasone and fluticasone propionate treatments. pneumonia is observed in horses after long distance transportation in association with confinement of horses' head position leading to a reduction in tracheal mucociliary clearance time (tmct). we hypothesize that clenbuterol, a beta-2 agonist shown to increase tmct in the horse, will ameliorate the affect of a fixed head position on large airway contamination and inflammation in a long-distance shipping model. six adult horses were enrolled in a cross-over design prospective study. horses were housed with their heads in a fixed position for 48 hours to simulate long distance transport, and treated with clenbuterol (0.8 ug/kg po q12 h) or a placebo starting 12 hours before simulated shipping. tmct was measured using a charcoal clearance technique. data were collected at baseline and 48 hours, and included tmct, tracheal wash cytology and quantitative culture, rectal temperature, cbc, fibrinogen, and serum tnfa, il-10 and il-2 levels. there was a 3-week washout between study arms, and each horse served as its own control. the data was analyzed using regression analysis and wilcoxon rank-sum tests. no statistically significant difference was seen between treatment and placebo groups for any of the variables investigated. tmct did not differ after treatment (1.71 ae 0.64 cm/min) versus placebo (1.55 ae 0.82 cm/ min; p 4 0.10), and intratracheal bacterial counts were similar for treatment (105 â 10 3 ae 42 â 103 cfu; p 4 0.10) and placebo (98 â 10 3 ae 41 â 103 cfu) groups. a reduction of tracheal b hemolytic streptococcus. spp. after clenbuterol versus placebo was also nonsignificant (0% versus 33%; p 4 0.10). in conclusion, treatment with clenbuterol does not appear to combat the deleterious effects of this long-term shipping model. breathing cold air during strenuous exercise is associated with airway inflammation. under these conditions, warming and humidification of inspired air occurs in the lower respiratory tract resulting in mucosal cooling, desiccation, and hyperosmolarity. the purpose of this research was to test the hypothesis that airway hypertonicity causes inflammatory cell migration and alterations in cytokine expression associated with exercise induced airway inflammation. horses (n 5 9) were examined in a randomized crossover design after exposure to hypertonic aerosols (5 minute nebulization with solutions of either isotonic or hypertonic mannitol). airway leukocytes were harvested 5 and 24 hours post aerosol challenge via bronchoaveolar lavage, and were used to determine total and differential nucleated cell count and expression of cytokinespecific mrna. hypertonic aerosol challenge resulted in an increase in total number of cells 5 hr after challenge, characterized by increased macrophage (p 5 0.04) and neutrophil (p 5 0.03) concentrations, but there was no effect on airway leukocyte concentrations 24 hours after nebulization. no significant changes in the relative quantity of mrna for airway cytokines were noted at either time point. these data demonstrate that transient airway hypertonicity can cause airway leukocyte influx and may be responsible for the airway inflammation commonly found in athletes that exercise in cold conditions. however, our data do not support the hypothesis that hypertonicity is the sole initiating cause of changes in cytokine expression secondary to cold weather exercise. it is likely that factors such as airway temperature, shear stress or epithelial damage also play a role in this phenomenon. we studied the importance of abdominal sonograms in neonatal foals suffering from gastrointestinal conditions. we hypothesized that there would be a subgroup of neonates with sonographically detectable pneumatosis intestinalis (pi) as a reflection of a necrotizing component of the disease. records of foals 7 days of age hospitalized between 2005 and 2009 with signs of gastrointestinal disease were evaluated (n 5 89). the association of sonographic, clinical, pathological and clinicopathological signs with outcome and severity of disease was determined. pneumatosis intestinalis was imaged in 19 foals. twenty-seven foals were classified as having necrotizing gastrointestinal disease based on the presence of gastrointestinal signs (colic, diarrhea, gastric reflux or abdominal distension) and pi detected sonographically (19), surgical (2) or pathological (6) evidence of gastrointestinal necrosis. there was a difference between overall survival rate (58%) and survival rate in foals with necrotizing disease (33%, p 5 0.005) or foals with pi detected sonographically (37%, p 5 0.02). pneumatosis intestinalis was the only sonographic finding associated with outcome. sonographic abnormalities in peritoneal fluid, stomach, duodenum, jejunum, cecum, umbilicus or the presence of meconium were associated (p o 0.05) with surrogates of severity of disease (hospitalization cost or days of hospitalization). hypoproteinemia was associated with pi (p 5 0.02). the presence of blood in the feces, reflux and abdominal distension was associated with necrotizing gastrointestinal disease (p o 0.05). abdominal sonograms have prognostic value in neonatal gastrointestinal disease. pneumatosis intestinalis was a common sonographic sign that worsened the prognosis. the therapeutic implications of detecting a necrotizing component of the gastrointestinal disease deserve further study. the interaction of insulin and the microvascular endothelial insulin receptor (irc) plays an important role in the normal and insulin resistant (ir) individual. while endothelial irc signaling is normally vasodilatory, this effect is well-documented to reverse in the ir individual, resulting in vasoconstriction. although vascular dysfunction has been reported in sepsis-associated equine laminitis, the role of the laminar microvasculature in endocrinopathic laminitis remains poorly characterized. the purpose of this study was to characterize the pattern of irc expression in digital laminae in ponies subjected to a dietary carbohydrate challenge that mimicked abrupt exposure to pasture rich in nonstructural carbohydrates (nsc). mixed-breed ponies (body weight 270.9 1/-74.4 kg) received a diet of hay chop (nsc $6% on a dm basis) for 4 weeks prior to initiation of the experimental feeding protocol. following conditioning, ponies either remained on the control diet (n 5 11) or received the same diet supplemented with sweet feed and oligofructose (total diet $42% nsc; n 5 11) for a period of 7 days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including dorsal digital laminae, were collected immediately following euthanasia. the samples were formalin-fixed for 48 hours, transferred to 70% ethanol, and paraffin-embedded. laminar sections were stained immunohistochemically for irc using a commercially-available antibody (abcam); the number of irc (1) cells was quantified in 40x light microscopy fields (n 5 10) for each section. the total number of irc (1) cells was greater in the laminae of challenged ponies than control ponies (p 5 0.0096), and there was a significant correlation between the change in serum basal insulin concentration and number of laminar irc (1) endothelial cells (r 5 0.74; p o 0.05). while the number of irc (1) endothelial cells was significantly greater in the dermal laminae of challenged ponies (p 5 0.0095), there was no difference in the number of interstitial irc (1) cells (p 5 0.82). no epithelial irc (1) cells were observed in any laminar section, and irc (1) cells were conspicuously absent from the deep dermal tissue (including vessels). up-regulation of irc expression in the laminar vasculature occurs acutely in response to dietary carbohydrate challenge and accompanies hyperinsulinemia in ponies. the dramatic increase in endothelial irc expression in the laminar microvasculature in nutritionally challenged ponies, with no apparent epithelial irc present, suggests that hyperinsulinemia associated with exposure to increased dietary nsc may induce laminar injury by causing a similar vasoconstriction in ir equids as described in the microvasculature of ir humans. glucose transport from the blood stream into cells, the limiting step in whole-body glucose utilization, is regulated by a family of glucose transporter (glut) proteins in insulin-sensitive (i.e., muscle and adipose) tissues. we previously demonstrated that glut4, the major isoform, is a key factor in the pathogenesis of equine insulin resistance (ir). while it has been recently demonstrated that glut12 (a newly discovered isoform) increases insulin-stimulated glucose transport in human muscle, its role in other tissues, particularly in the setting of ir, is not well characterized in any species. in addition, as160 has recently emerged as a key downstream signaling molecule regulating translocation of glut to the cell surface, the rate-limiting step in glucose uptake. we hypothesized that glut12 content would be differentially expressed across tissues and that ir would induce alteration in glucose transport by affecting active cell surface glut12. biopsies of skeletal muscle, and subcutaneous and visceral adipose tissue were collected from light-breed horses, characterized as either insulin sensitive or compensated ir, based on the results of an insulin-modified frequently-sampled intravenous glucose tolerance test (n 5 5/group). we specifically quantified active cell-surface glut12 in these biopsies, using an innovative exofacial bismannose photolabeled assay, which has not been previously applied to glut12. total glut12 protein expression was measured by western blots, as well as total and phosphorylated (indicating activation of) as160. glut12 was expressed in all the depots with a significant regional effect. total glut12 protein content was increased (p o 0.05) in visceral (omental and mesenteric) compared to subcutaneous (nuchal ligament and tailhead) adipose tissue and skeletal muscle of healthy horses. ir did not induce alterations in active cell-surface and total glut12 content nor in total and phosphorylated as160 in any of the tissues evaluated. our data suggests that glut12 is abundant in visceral adipose tissue and is therefore likely to play a substantial role in the regulation of glucose transport. however, neither glut12 translocation nor as160 activation are impaired in insulin-sensitive tissues of ir horses. it is concluded that, in contrast with glut4, glut12 does not appear to contribute to glucose transport alterations during naturally-occurring equine ir. insulin resistance (ir), characterized by exaggerated glycemic or insulinemic responses to glucose challenge, is a key metabolic disturbance in horses that develop obesity-associated laminitis. in addition to obesity, diet and age have been demonstrated to affect tissue sensitivity to insulin in other species but these factors have received limited investigation in horses. we hypothesized that there would be greater glycemic and insulinemic responses to a sweet feed meal in aged horses, as compared to adult horses, as well as in horses adapted to a forage-only diet. three diets, grass hay only, grass hay plus sweet feed (starch and sugar-rich, ss), and grass hay plus a fat and fiber (ff) feed, were fed to 17 mares, 8 adult (5-12 yr) and 9 aged (4 19 yr), for a 6-week adaptation period in a randomized design. glycemic and insulinemic responses to a standardized meal of sweet feed (4 g/kg bw offered for 1 hour) were determined for 6 hours from the onset of feeding. peak glucose and insulin concentrations and areas under the glucose or insulin vs. time curves (auc-g, mg/ dl/360 min, and auc-i, mu/ml/360 min) were determined and data were analyzed by one-and two-factor repeated measures anova. there were no differences between age groups in glycemic responses to any of the diets. however, in aged horses peak glucose concentration (p o 0.03) and auc-g (p o 0.01) were greater after adaptation to the forage-only diet, as compared to the other two diets. in contrast, aged horses had a greater peak insulin concentration (p o 0.05) and auc-i (p o 0.03) than adult horses on all diets but no differences in peak insulin concentration or auc-i was found between diets within age groups. as hypothesized, the insulin response, but not the glycemic response, to a sweet feed meal was greater in aged horses, regardless of background diet. further, the glycemic response was greatest after adaptation to a forage-only diet, but this finding was only significant in aged horses. morbidity, mortality, and economic loss to the equine industry. in obese humans and rodent models of nutritional obesity, systemic insulin resistance and hyperinsulinemia are followed temporally in a majority of individuals by decreased glucose tolerance, pancreatic bcell failure, and type ii diabetes mellitus. in stark contrast to humans, obese horses and ponies chronically remain in what is termed a ''prediabetic'' state in human ir, characterized by hyperinsulinemic euglycemia. few data exist describing the biology of the equine endocrine pancreas in the chronically ir animal that may both: 1) explain this unique equine endocrine physiology and 2) characterize the animal at-risk for hyperinsulinemia-associated laminitis. the purpose of the study reported here was to characterize the morphology and physiology of the equine endocrine pancreas in response to a dietary carbohydrate challenge. twenty-two mixedbreed ponies (body weight 266.6 ae 170.5 kg) were conditioned to a diet of chopped hay (nsc $6% on dm basis) for 4 weeks; following conditioning, ponies either remained on the control diet (n 5 11), or received the same hay supplemented with sweet feed and oligofructose (total diet $42% nsc; n 5 11) for 7 days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including pancreas, were collected immediately following euthanasia. the samples were formalin-fixed for 48 hours, transferred to 70% ethanol, and paraffin-embedded. immunohistochemistry was performed on pancreas sections using a commerciallyavailable anti-insulin antibody (abcam), and measurements of islet surface area and b-cell surface area were performed (n 5 10 islets per tissue section) using a commercially-available computer software program (image j). there was a trend for greater total islet surface area in pancreatic tissue from ponies fed the high nsc diet when compared to the ponies on the hay diet (p 5 0.068); however, no difference was noted in b-cell surface area between diet treatments (p 5 0.12). the change in serum insulin concentration was significantly greater in the high nsc-fed ponies than in controls (403.8 1/-317.1 miu/l vs. 1.00 1/à 4.03 miu/l; p 5 0.002); however, this variable was not correlated with total islet surface area (r 5 0.32; p 5 0.17) or b-cell surface area (r 5 0.25; p 5 0.3). due to the relatively modest changes in pancreatic islet surface area that accompany marked increases in serum insulin concentrations in ponies fed a high nsc diet, it is important to assess both b-cell function and insulin clearance mechanisms in future studies to delineate the mechanism(s) of hyperinsulinemia in this model. humans that suffer from obesity show exaggerated inflammatory responses and this may be relevant to the association between increased adiposity and laminitis in horses with equine metabolic syndrome (ems). this study was performed to test the hypothesis that inflammatory responses to endotoxemia differ between healthy horses and those affected by ems. six healthy adult mares and 6 horses with ems received an intravenous infusion of lipopolysaccharide (lps; 20 ng/kg in 60 ml sterile saline) or saline alone. a crossover design was employed with a 7-day washout period. physical examinations were performed hourly for 9h and whole blood was collected at 30, 60, 90, 120, 180, and 240 min for assessment of inflammatory cytokine gene expression. a liver biopsy was performed between 240 and 360 min postinfusion. data were analyzed using mixed model anova. mean rectal temperature, heart rate, and respiratory rate increased following lps infusion (treatment â time; p o 0.001), with higher heart (group â treatment; p 5 0.087) and respiratory rates (group; p 5 0.017) detected in ems horses. lipopolysaccharide infusion significantly increased whole blood gene expression of tumor necrosis factor a (tnfa), interleukin (il)-1b (p o 0.001), il-6 (p o 0.001), il-8 (p o 0.001), and il-10 (p 5 0.002), and hepatic gene expression of il-6 (p o 0.001), il-8 (p o 0.001), and il-10 (p 5 0.016). inflammatory gene expression did not differ significantly between groups, so our hypothesis was not supported. heart rates tended to be higher when lps was administered to horses with ems. elevated serum concentration of cardiac troponin i (ctni) is a biomarker for myocardial damage in horses. preferred times to test blood for ctni levels following athletic performance or other events that may cause myocardial injury are not yet established and would be affected by time of release from the myocytes, location of release within the myocytes, duration of release and half-life of ctni in the horse. this information would be necessary to more accurately and reliably test horses for myocardial injury. the aim of this study was to determine the elimination half-life (t1/2) of equine ctni. to establish the t1/2 of equine ctni in horses, ctni was recombinantly expressed in e.coli. two healthy ponies received intravenous injections of recombinant equine ctni and plasma ctni concentrations were measured with a point-of-care ctni analyzer at multiple time points after injection. standard pharmacokinetic analysis was performed to establish the elimination half-life of ctni. for comparative purposes, data were subjected to pharmacokinetic models describing a single versus biphasic elimination profile. elimination of recombinant equine ctni following intravenous administration exhibits a short half-life. establishing the t1/2 of troponin provides critical information in understanding the clinical application of this cardiac biomarker in clinical practice. this study describes a true biological ctni t1/2, which has not been documented in any species thus far. stall-side assessment of this cardiac biomarker in horses should enhance the ability of clinicians to detect myocardial damage and aid in the management and treatment of horses with cardiac disease. the objective of the study was to evaluate the between-pony, within-pony, between-analyser and within-analyser variation of flow-mediated vasodilation (fmd) measurement in healthy ponies, to investigate the hypothesis that fmd occurs in healthy ponies. six healthy, native breed, unrelated pony mares of varying weight (236-406 kg), body condition score (3/9-7/9) and age (14-25 years) were used. the median artery was occluded for 5 minutes. twodimensional (2d) ultrasonographic images of the artery were recorded for 30 seconds prior to and for 2 minutes after occlusion. the peak luminal diameter was compared to baseline diameter to calculate the relative percentage increase in luminal size (fmd). images were obtained from six ponies on one occasion and from one pony on six occasions. analysis of images was performed by two independent analysers and by one analyser twice. the mean (sd) fmd in 6 ponies was 12.57% (4.28%) and in 1 pony (6 occasions) was 7.30% (2.11%). coefficients of variation were 34.09% and 28.84% respectively. agreement between analysers was fair (icc 5 0.47) and within analyser was poor (icc 5 0.30). fmd is used to assess endothelial function in humans and has recently been assessed for its use in canine subjects. fmd occurs and measurement is feasible in ponies. fmd could be used to assess endothelial function, in the context of laminitis or other cardiovascular diseases. current state-of-the-art technique for measuring blood pressure (bp) in the horse is invasive and involves cannulation of the facial artery. indirect techniques, such as oscillometry, have proven useful in the anaesthetised horse, but have not become routine in the standing horse. monitoring bp can be indicated for the diagnosis and treatment of the hypotensive patient (ie. caused by endotoxemia, hypovolemia, systemic inflammatory response syndrome and cardiac failure) or the hypertensive patient (ie. due to equine metabolic syndrome or pain). the objective of this study was therefore to a) describe the methodology for application of oscillometric bp using a cuff applied to the tail in the standing horse and b) and to determine accuracy and precision of this method applied to the normotensive standing horse. the oscillometric method is simple to apply in a clinical setting. a pneumatic cuff is snugly applied to the unclipped tail-base with the cuff bladder centered over the middle coccygeal artery. the tail circumference must match the manufacturers description of the cuffs diameter range. the oscillometric apparatus inflates the cuff and obtain systolic, diastolic and mean arterial bp (sap, dap and map). at least 2 consecutive measurements must be obtained. a correction of 0.7 mmhg/cm vertical distance between cuff and heart level is added to the measurement to correct for hydrostatic pressure difference. for determination of accuracy and precision of indirect sap, dap and map, eight healthy horses (age 3 to 16 years), was equipped with an intra-arterial catheter ii in the facial artery and a commercial tail-cuff oscillometric apparatus. i measurements were recorded every 2 minutes for 20 minutes. the data were analysed with the statistical software r using a mixed model with repeated measurements and a bland-altman analysis corrected for repeated measurements. oscillometric bp was accurate and precise for map (mean bias, lower confidence level, upper confidence level, variation in difference, all mmhg) (à0.3, à18.5, 19.1, 33.2, respectively) in the conscious horse but not for sap (à1.5, à19.3, 16.3, 38.2, respectively) and dap (0.05, 15.9, 16.0, 49, respectively) . there was no significant contribution to the statistical model of either horse or measurement number. all horses tolerated the tail-cuff well and the method was simple to apply. only map could be measured with acceptable accuracy and precision in the normotensive standing horse using the described oscillometric method. reference intervals for thyroid hormone (th) concentrations have not been established for donkeys. therefore, clinicians must use reference ranges from horses, potentially leading to misdiagnosis of thyroid diseases. we hypothesized that th concentrations are different between donkeys and horses. the purposes of this study were: a) to compare th concentrations between donkeys and horses and, b) to determine whether the age may influence th concentrations. thirty-eight healthy donkeys (8.5 ae 0.8 years), mixed breeds, and 20 healthy andalusian horses (6.4 ae 0.5 years) were used. donkeys were divided into three groups: o 5 years (n 5 13), 5-10 years (n 5 12), and 411 years (n 5 13). serum concentrations of total triiodothyronine (tt3), free triiodothyronine (ft3), total thyroxine (tt4), free thyroxine (ft4), reverse triiodothyronine (rt3) and thyroid-stimulating hormone (tsh) were quantified by radioimmunoassay. all blood samples were collected the same day. neither horses nor donkeys had received any treatment for 30 days before sampling and both farms had similar production conditions. total t3, ft3, ft4 and tt4 concentrations were higher (p o 0.01) in donkeys than horses. in contrast, no statistical differences were found for rt3 and tsh concentrations. young donkeys ( o 5 years) had higher ft4, tt4 and rt3 concentrations compared to other donkey groups (p o 0.05). old donkeys (411 years) had lower tt3 and ft3 concentrations than both younger donkeys groups (p o 0.05). this study shows that there are differences in th concentrations between donkeys and horses, raising awareness on the possibility of misdiagnosis of thyroid gland dysfunction when using values from horses, being necessary to determine exclusive reference intervals for donkeys. ovariectomy is associated with alterations of responses to many hormones, not just those associated with reproductive function. in humans and rats, ovariectomy leads to insulin resistance, increased adiposity and altered fat mobilization. the effects of ovariectomy on energy metabolism have not been reported in horses. ovariectomized mares have been shown to respond normally to an acth stimulation test, but the response to suppression of the hypothalamo-pituitary-adrenal axis has not been previously described. the aim of this study was to evaluate the effect of ovariectomy on insulin response in mares and to determine if mares exhibit alterations in response to dexamethasone administration after ovariectomy. six healthy mares underwent an intravenous glucose tolerance test (ivgtt), an insulin sensitivity test (ist) and a dexamethasone suppression test (dst) before and 5 weeks after bilateral ovariectomy. body weight, cortisol values at baseline, 15 and 24 hours after dexamethasone injection and acth values at baseline, 15 and 24 hours after dexamethasone injection, basal insulin/glucose ratio, time to reach a 60% decrease in blood glucose in the ist, time to reach baseline glucose concentration in the ivgtt and area under the curves plotting blood glucose and time to injection of glucose or insulin were compared before and after ovariectomy using a paired t-test or an anova for repeated measures. significance level was p o 0.05. average body weight was decreased after surgery (6kg ). the injection of dexamethasone resulted in a serum cortisol concentration of less than 1 mg/dl in all mares before ovariectomy, whereas after ovariectomy, dexamethasone injection resulted in a serum cortisol concentration of less than 1 mg/dl in 5 out of 6 mares. in all cases, acth concentration was within the reference range (9-35 pg/ml) before and after ovariectomy. however, acth concentrations at t 0 and at t 15 were significantly higher after ovariectomy. each mare had a normal ivgtt, both before and after ovariectomy. additionally, no significant differences were observed in basal blood glucose (84 ae 12 mg/dl before and 85 ae 3 mg/dl after) or in the time to reach glucose baseline (108 ae 66 min before and 99 ae 51 min after). serum basal insulin concentration and insulin/glucose ratio was not significantly different before or after ovariectomy (22.0 ae 7.9 miu/ml and 17.5 ae 8.9 miu/ml and 0.26 ae 0.09 and 0.20 ae 0.10, respectively), nor was the average time to reach a 60% decrease in blood glucose after insulin injection (30 ae 7 min and 25 ae 9 min, respectively). these findings suggest that, as reported in other species, the shortterm effect of ovariectomy may modify dexamethasone response in mares and that, contrary to other species, it may not modify insulin response. equine gastric ulcer syndrome (egus) is a common medical problem in horses. the high prevalence of gastric ulcers, vague clinical signs and negative effect on performance make it a significant clinical and economic problem within the horse industry. current pharmaceutical treatments are expensive and alter the acidic environment of the stomach. berries and pulp from the seabuckthorn plant (hippophae rhamnoides) are a rich source of vitamins, trace minerals, amino acids, antioxidants, and other bioactive substances and have been used successfully to treat stomach ulcers in man and rats. the purpose of this study was to evaluate the efficacy of a commercially sold, liquid extract of seabuckthorn berries (seabuck tm sbt gastro-plus) for treatment and prevention of gastric ulcers in horses. eight thoroughbred and thoroughbred-cross horses (3-10 years of age, 5 geldings & 3 mares, 380-600 kg) were used in a blinded two-period cross-over study. treatments consisted of control (untreated) and treatment (seabuck tm sbt gastro-plus) twice daily mixed with the grain meal. horses were treated for 5 weeks followed by a 1 week alternating feed-deprivation period to induce or worsen existing ulcers. gastroscopies were performed on all horses on day 0, week 5, and week 6 (at the end of the alternating feed-deprivation period). gastric juice was aspirated and ph was measured. during gastroscopy, gastric ulcer scores were assigned to each stomach based on lesion number and severity. horses acted as their own controls, and between each treatment period the horses had a 2-week washout period. data was analyzed by anova for repeated measures via the glm procedure (sas inst. inc., cary, nc). when significant differences (p o 0.05) were observed, a post-hoc tukey's test was used to determine differences. non-glandular gastric ulcer scores significantly increased in all control and sbt-treated horses from week 5 to week 6, after the feed-deprivation phase of the study. there was no significant difference in the non-glandular gastric number (p 5 0.84) and nonglandular gastric severity (p 5 0.51) scores in sbt-treated horses compared to non-treated controls. glandular ulcer number (p 5 0.02) and glandular ulcer severity (p 5 0.02) was significantly lower in the sbt-treated horses compared to the control horses. there was no significant difference in the ph (p 5 0.06) in sbt-treated horses compared to non-treated controls. thus, seabuck tm sbt gastro-plus, mixed in the feed twice daily, may be efficacious in controlling the severity of glandular ulcers in horses during stress, without increasing stomach ph. the availability of rapid and accurate quantitative fibrinogen measurements may be useful for evaluation of hospitalized equine patients. the abaxis vspro analyzer was evaluated for precision using two levels of human fibrinogen controls (300 mg/dl and 150 mg/dl), four different vspro machines, and two different lots of cartridges, assessed over 5 subsequent days. the coefficients of variation of the assay ranged from 4% (300 mg/dl) to 8% (150 mg/ dl). we subsequently evaluated the abaxis vspro fibrinogen assay compared to fibrinogen concentration measured using the beckman coulter acl-1000 in 50 equine samples of varying fibrinogen concentrations obtained from horses with gastrointestinal disease. all samples were measured in citrated plasma. fibrinogen samples measured on the acl-1000 ranged from 226 to 959 mg/dl (median 501 mg/dl). vspro samples were run in duplicate, and the mean compared to the acl values. pearson correlation coefficient analysis generated an r value of 0.949 (p o 0.001). duplicate measurements on the vspro were strongly correlated to each other with an r value of 0.9690 (p o 0.001). bland-altman analysis of these samples for the vspro compared to the acl-1000 noted a bias of à84 ae 57 mg/dl the results of this study indicate that the vspro benchtop fibrinogen analyzer provides accurate and precise fibrinogen data compared to the acl-1000 reference analyzer. the immune response of foals to r. equi is incompletely understood and believed to be responsible for clinical disease caused by this pulmonary pathogen. in a recent study foals receiving a large inoculum exhibited th2 skewing with pneumonia and a small inoculum exhibited th1 skewing without clinical disease. we hypothesized that cytokine/chemokine production by pulmonary alveolar macrophages, in vitro, would increase with the infective dose and that the magnitude of the response would differ between foals and adults. alveolar macrophages were obtained by bronchoalevolar lavage from 7 healthy mares and their 5-week-old foals. macrophage cultures were infected with r. equi (337011 or 33701-) at a multiplicity of infection (moi) of 1 or 100. total rna was harvested 4 and 24 hours post-infection, reverse transcribed and used as template for quantita-tive pcr. the ddct method was used to calculate relative gene transcripts for il-6, il-12p40, tnfa and cxcl10. cellular infections at moi 100 resulted in significantly higher expression of il-6, il-12p40 and tnfa mrna transcripts compared to moi 1. however, the dose-effect was reversed for cxcl10 with significantly lower expression at the higher moi. there was no difference in magnitude of cytokine/chemokine responses by the alveolar macrophages between adults and foals. dose-dependent responses of alveolar macrophages may represent a novel mechanism by which r. equi could modulate immune responses and therefore disease. significant down-regulation of cxcl10 mrna transcripts associated with a higher dose is of particular interest as this chemokine plays a role in development of protective th1 responses. the intent of this study was to develop likelihood ratios (lrs) for infection attributable to corynebacterium pseudotuberculosis in horses based on synergistic hemolysis inhibition (shi) test titers. medical records for horses presented to the uc davis veterinary teaching hospital with serum submitted for shi titer determination were evaluated and 171 cases met study inclusion criteria. these cases were grouped based on evidence of internal and/or external infection attributable to c. pseudotuberculosis and likelihood ratios with 95% confidence intervals determined. results showed increasing lrs indicating increasing odds for any form of active disease as titer increased with all cases considered. lrs for internal infection were 4 1 for titers ! 1280 overall and for titers 4 160 with external abscess cases excluded. no difference from 1 (and therefore no significant change in pre-test to post-test odds) was seen in any lrs for internal disease when only cases with external disease were examined (external and internal disease vs. external only). overall, the shi test results showed usefulness in determining internal c. pseudotuberculosis infection in horses with no evidence of external abscessation. overall, however, higher titers were more indicative of active external or internal disease than internal disease specifically in contrast to previous reports. the shi test was unable to distinguish internal infection when external abscesses were present. salmonella enterica is a zoonotic pathogen that has tremendous impact on many different animal production and management systems. rapid detection of s. enterica in fecal samples may facilitate effective infection control practices. current detection methods require 24-48 hours (polymerase chain reaction or pcr) or 48-72 hours (enriched aerobic culture) to obtain results. alternatives have been developed, lateral flow antigen detection systems (lfads), which are currently marketed for salmonella detection related to food safety microbiology. the objective of this study was to evaluate two commercially available rapid salmonella detection systems in equine feces. fecal samples collected from repeatedly culture-negative horses were inoculated with known concentrations of salmonella enterica serotype typhimurium (five uninoculated control samples, and 5 samples of each 10-fold dilution [1.9 â 10 0 -1.9 â 10 4 cfu/gram of feces]). all samples were aerobically cultured using a standard enrichment technique. in a blinded fashion, samples were tested using two different lfads as well as plated on agar media for confirmatory testing. at 24 hours of incubation, using bacterial culture as the reference method, test 1 was correctly identify 70% of samples ( bacterial contamination of stalls with salmonella sp. is a serious problem in equine hospitals. salmonella sp. exposure to horses in the facility can result in nosocomial infections which results in temporary facility closure, until the organism is eradicated. hospital closure can result in loss of revenue, damage to reputation and interference with patient care. the purpose of this study was to evaluate three stall cleaning methods on eradication of salmonella sp. at an equine veterinary teaching hospital (vth). horses admitted to the vth were assigned to salmonella sp.negative stalls within areas of the vth during the study period (september 2009 -january 2010 . when the horses were discharged stalls were randomly assigned to one of three cleaning methods (pressure-washing only [pw] , pressure washing and hand scrubbing [pws] , or hand scrubbing only [s]) in a single period, non cross-over design. all stalls were stripped of bedding and surfaces sprayed with tap water and cleaned with a disinfectant quaternary-ammonia solution (super hdq neutral, spartan chemical co., inc, maumee, oh). the pressure-washing system (psc cleaning systems, inc., toronto, canada) used, provided a pressure of 3000 psi and a temperature range of 1851-2151f. following cleaning, each stall was allowed to air dry and within 48 hours, stall surfaces were sampled using three 4 00 â 4 00 sponges moistened with sterile saline. the person collecting the samples was masked to the method of cleaning. sponges were submitted to the louisiana animal disease diagnostic laboratory (laddl) for culture of salmonella sp. a chi-squared analysis was used to determine significant differences (limit p o 0.05) between cleaning methods and salmonella sp. isolation. during the study period, 112 stalls (pw [n 5 29]; pws [n 5 50]; s [n 5 33] were included. all stalls had negative environmental salmonella sp. cultures prior to beginning the study. for pw cleaned stalls, 6/23 (20.7%) were salmonella sp.-positive, for pws cleaned stalls, 12/38 (24%) were salmonella sp.-positive, and for s cleaned stalls, 4/29 (12.1%) were salmonella sp.-positive. although, there were fewer salmonella sp.-positive stalls (12.1%) in the handscrubbed stalls, cleaning method did not significantly (p 5 0.4057) affect the isolation of salmonella sp. from the stall environment. in conclusion, power washing alone, power washing and hand scrubbing, and hand scrubbing alone, using a quaternary-ammonia solution did not significantly affect environmental isolation of salmonella sp. from stalls surfaces in the vth during this study. the objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against streptococcus equi subsp. zooepidemicus (s. zooepidemicus) and rhodococcus equi isolates. a single dose of gamithromycin (6 mg/kg of body weight) was administered intramuscularly. concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (pelf), bronchoalveolar lavage (bal) cells, and blood neutrophils were determined using hplc with tandem mass spectrometry detection. the minimum inhibitory concentration of gamithromycin required to inhibit growth of 90% of r. equi and s. zooepidemicus isolates (mic 90 ) was determined. additionally, the activity of gamithromycin against intracellular r. equi was measured. mean peak gamithromycin concentrations were significantly higher in blood neutrophils (8.35 ae 1.77 g/ml) and bal cells (8.91 ae 1.65 g/ml) compared to pelf (2.15 ae 2.78 g/ml) and plasma (0.33 ae 0.12 g/ml). mean terminal half-lives in neutrophils (78.6 h), bal cells (70.3 h), and pelf (63.6 h) were significantly longer than that of plasma (39.1 h). the mic 90 of s. zooepidemicus isolates was 0.125 g/ml. the mic 90 of gamithromycin for macrolide-resistant r. equi isolates (128 g/ml) was significantly higher than that of macrolide-susceptible isolates (1.0g/ ml). the activity of gamithromycin against intracellular r. equi was similar to that of azithromycin and erythromycin. intramuscular administration of gamithromycin at a dosage of 6 mg/kg would maintain pelf concentrations above the mic 90 for s. zooepidemicus and phagocytic cell concentrations above the mic 90 for r. equi for approximately 7 days. eight western stock yearling horses were infected with ehv-1 (ab4) by nasopharyngeal instillation. venous blood samples for collection of plasma were collected in na-citrate tubes on the day prior to infection (d -1) and on d4 through d11. in addition, clinical data, nasal swabs and peripheral blood mononuclear cells (pbmc) for detection of viremia were collected on the day before infection (d -1) and on d1 through d14 post-infection. d-dimer concentrations were determined in citrated plasma samples using a latex agglutination test (minutex d-dimer, biopool, ireland). viral load in pbmc was determined using quantitative pcr. all horses showed bi-phasic fevers typical for ehv-1 infections. one horse developed acute ehm on d10 and was euthanized after samples were collected. in all horses d-dimers were undetectable on d -1 and on d4, 5 and 11. in contrast, all horses had increased ddimer concentrations for 3 to 5 consecutive days starting on day 6 post-infection. d-dimer concentrations in 2 horses increased to 1000 ug/ml and one of these horses was the horse with acute ehm. interestingly, mean increased d-dimer concentrations showed timely overlap with the mean fever curve and, delayed by 1 day, with the mean viremia curve. because plasma samples for d-dimer measurements were not collected during the first 3 days post-infection, which are typically associated with a primary fever, conclusion on the association of d-dimers with fever of viremia await analysis of a second study currently conducted in our laboratory. in conclusion our data indicates that during ehv-1 infection with neuropathogenic strains activation of the coagulation cascade and production of cross-linked fibrin is wide-spread; not limited to horses with clinical signs of ehm, and can be expected between days 6 and 10 post-infection. lawsonia intracellularis is an emerging pathogen in horses and the causative agent in equine proliferative enteropathy (epe). the goal of this study was to evaluate the exposure of pre-weanling foals and broodmares to lawsonia intracelluaris on several farms in louisiana with a history of epe and compare the results to several farms with no known clinical cases of epe in foals. an additional goal of the study was to identify whether a relationship exists between lawsonia intracelluaris and other gastrointestinal pathogens in foals. whole blood and fecal samples were collected from 66 mares and 68 foals from four breeding farms in louisiana. farms a and b had no known clinical cases of epe, while farms c and d had previous know cases of epe in 2009. serum samples were examined for the presence of antibodies against lawsonia intracellularis using an immunoperoxidase monolayer assay (ipma). dna was extracted from fecal samples using a commercial dna kit and molecular detection of lawsonia intracelluaris was assayed using real-time pcr. fecal ova were determined using quantitative sucrose floatation. the presence of fecal clostridium difficile toxin was measured using a commercial enzyme linked immunosorbent assay (elisa). three of the 4 farms examined had foals and mares with exposure to l. intracellularis as evidenced by serum antibodies against the organism. of the total population sampled, 6 foals (8.8%) and 14 mares (21.2%) had evidence of antibodies to l. intracellularis based on serology. three foals (4.4%) tested positive for l. intracellularis organism by fecal pcr, and all of these foals were located on farm c. of these, one of the foals was seronegative, while the other two were seropositive. farm c also had the highest percentage of mares (28.6%) serologically positive for l. intracellularis, while farm a had the highest percentage of foals (14.3%) with antibody titers against l intracellaris. farm c also had the only pairs (n 5 3) of serologically positive mares with seropositive foals. while farm a and b had seropositive mares and/or foals, none of the foals were positive for l. intracellularis fecal shedding by pcr. all serum and fecal samples were negative for evidence of l. intracellaris on farm d. ten foals (14%) had fecal egg counts greater than 200 egg per gram and 2 foals (3%) were positive for c. difficile toxin. this study demonstrated evidence of natural exposure to l intracellularis on farms both with and without a history of epe in louisiana. further, this study failed to establish a relationship between l intracellularis and other gastrointestinal pathogens. the objective of this study was to examine the clinical, hematological, biochemical, and outcome data from equids infected with anaplasma phagocytophilum presented to a primary care field setting in southeastern pennsylvania. computerized medical records from 19 febrile equids with confirmed anaplasma phagocytophilum infection were reviewed. confirmation of anaplasma phagocytophilum was defined by the presence of granular inclusion bodies seen within leukocytes or eosinophils on microscopic blood smear evaluation and/or a positive polymerase chain reaction (pcr) for anaplasma phagocytophilum. 18 horses and 1 donkey presented with a mean fever of 104.41f and mean fever duration of 39 hours. the mean age at presentation was 9.6 years and the mean pack cell volume was 30.8%. 15/19 cases were diagnosed in the months of may to december. equids ages 5 to 15 years had significantly lower platelet counts. 16/18 cases were positive on blood smear for inclusion bodies and 6/6 cases were positive for anaplasma phagocytophilum on pcr. treatments included intravenous oxytetracycline, oral doxycycline, or both. mean treatment duration was 4.8 days and mean treatment cost was $621. 17/19 cases were normothermic within 48 hours. the treatment used in the two remaining cases was changed from oral doxycycline to intravenous oxtetracycline and was successful. this is the first case series of equine granulocytic anaplasmosis in the mid-atlantic states. all cases were examined and treated in the field. in order to make a definitive diagnosis, some cases required pcr. treatment failures were documented with the use of oral doxycycline alone. 100% of the cases survived. a high incidence of clinical and possibly genetic abnormalities has been reported amongst friesian horses including dwarfism, hydrocephalus, dissecting aortic aneurism and esophageal dysfunction. the purpose of the current study was to develop a new electromyography (emg) method to assess neurophysiological function of the esophagus especially for friesian horses. five friesian horses with esophageal dysfunction were included (ranging in age from 0.5-24 years and comprising 4 mares and a stallion) and two friesian control horses (a 10-and 12-year-old gelding). all five horses with esophageal dysfunction had a history of recurrent esophageal obstruction and were examined histopathologically post-mortem. barium contrast radiography was used as the gold standard to distinguish the diseased from the control horses. an endoscopically-guided percutaneous needle emg procedure (viking quest r ; software version 11.0) was performed just caudal to the larynx and just cranial to the thoracic inlet (to monitor striated and smooth muscle, respectively) to visualize esophageal motility. esophageal contractility in both control horses was predominantly reflected by interference patterns associated with longer duration and lower amplitude in smooth muscle compared to striated muscle. mean (ae sd) values were 35.1 ae 19.4 ms and 167.7 ae 96.7 mv (n 5 19 readings) and 10.8 ae 14.3 ms and 305.8 ae 233.7 mv (n 5 24 readings), respectively. in diseased horses, aperistalsis in smooth muscle was the most remarkable finding suggesting a loss of inhibitory neurogenic input resulting in aperistalsis and thus esophageal dysfunction. preliminary findings suggest that endoscopically-guided percutaneous needle emg might become a valuable method in elucidating the pathophysiology of dysfunction of esophageal motility especially in friesian horses. lymphoma affects horses of all ages. unlike in humans, no etiologic agent has been discovered. a 9 year old thoroughbred/warmblood cross mare presented with signs of upper and lower respiratory disease and was subsequently diagnosed with lymphoma and equine multinodular pulmonary fibrosis (empf) and was positive for equine herpes virus 5 (ehv-5) in both the pulmonary tissue and the lymph nodes. retrospective polymerase chain reaction (pcr) testing of six lymphoma cases found that 5 of 6 of the cases were positive on pcr for ehv-5 (83.3%, p 5 0.0045, rr 5.55). electron microscopy was performed on one sample and herpes virus particles were identified. of the samples in which immunohistochemistry was performed (3 of 6), only t-cell rich b-cell lymphoma was identified. samples of mesenteric or submandibular lymph nodes from 20 clinically healthy horses were submitted for ehv-5 pcr analysis; 15% were positive. gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as kaposi's sarcoma and burkitt's lymphoma. equine herpesvirus 5, also a gamma herpesvirus, is found in association with equine lymphoma; although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia remains unknown. pathologic events reported to occur in the digital laminae in early stages of sepsis-related equine laminitis include leukocyte extravasa-tion into the laminar interstitium, pro-inflammatory cytokine expression, and epithelial stress. while these events have been documented early in the disease process at both a developmental stage and at the onset of obel grade 1 (og1) lameness in the carbohydrate overload (cho) model of laminitis, the later events occurring at the onset of obel grade 3 lameness(og3, time point at which structural failure of the laminae usually occurs) have not been determined. we hypothesized that the inflammatory events described above are sustained through og 3 lameness, likely playing an injurious role culminating in laminar failure. our objectives were to determine pro-inflammatory gene expression, leukocyte extravasation, and epithelial stress at og 3 induced using the cho model. archived laminar tissue samples (snap frozen and paraffin embedded sections) were used from a previous cho study at louisiana state university (control group [n 5 6, water], cho group [n 5 7, corn starch]. calprotectin (cp) immunohistochemistry (ihc) was used to assess both laminar myeloid leukocyte numbers and epithelial stress; rt-qpcr was used to assess inflammatory gene expression. minimal inflammatory changes were present at og3 compared to published values at og1 stage in the cho lameness model including decreased mrna concentrations of cytokines (i.e. 20-fold increase in il-6 at og3 vs. 4 2000-fold increase at og1, no increase in il-1b at og3 vs. 11-fold increase at og1), chemokines (no change in mcp-1at og3 vs. 4 30 fold increase at og1, 8-fold increase in il-8 at og3 vs. 95fold increase at og1) and adhesion molecules (no change in e-selectin at og3 vs. 10-fold increase at og1). laminar leukocyte emigration was also decreased at the onset of og 3 lameness compared to previously reported leukocyte infiltration at og 1. interestingly cox-2, underwent a greater increase at og3 (approx. 50-fold) compared to that reported at og1 lameness (35-fold). finally, epithelial stress at og3 evidenced by cp ihc did not follow the uniform widespread distribution reported at og1 lameness, but instead was present in focal areas in which secondary epidermal laminae on either side of a common primary dermal vascular supply demonstrated increased cp signal. overall, laminar inflammation appears to be subsiding at og3 lameness, with epithelial stress possibly more dependent on vascular dysregulation instead of inflammatory events. the sustained increase in cox-2, central to the induced production of vasoactive prostanoids in disease processes, may play a role in vascular dysregulation. this study was conducted to characterize clinical, laboratory and postmortem findings associated with oleander toxicosis in equids and to determine factors predictive of survival in these cases. retrospective analysis of medical records from our veterinary medical teaching hospital from january 1, 1995 to july 15, 2010 was completed. records of equids demonstrating detectable oleandrin in serum, plasma, urine or gastrointestinal fluid samples or detectable serum digoxin in the absence of pharmaceutical cardiac glycoside administration were included. descriptive statistics were used to evaluate the history, physical examination, and laboratory and postmortem data of affected individuals. logistic regression analysis was used to detect physical examination and laboratory factors significantly associated with survival. thirty equids met inclusion criteria of the study. three of 30 subjects were dead on arrival or died immediately upon arrival (10%). of the remaining 27 equids, 85% presented with gastrointestinal abnormalities, 70% were azotemic and 48% had cardiac arrhythmias. mortality was 50% for all subjects and 44% for those treated. the predominant cause for non-survival was cardiac dysfunction. factors significantly associated with survival included relatively decreased hematocrit and serum glucose, relatively increased serum chloride, absence of cardiac arrhythmias, and increased duration of hospitalization. equids with oleander toxicosis frequently present with gastrointestinal upset and may develop cardiac and renal disturbances. patients with cardiac arrhythmias and relatively increased hematocrit and serum glucose and decreased serum chloride are significantly less likely to survive. oleander intoxication is a differential diagnosis for colic in endemic areas, particularly with concurrent azotemia or cardiac dysrhythmia. the quantitative physicochemical approach emphasizes the importance of strong ions (na, k, cl, lactate), pco 2 , and the plasma protein concentrations in determining plasma ph. serum concentrations of strong ions, proteins, and total co 2 are reported on modern biochemical profiles. we hypothesized that the results of serum biochemical analysis can be used for acid-base interpretation in horses. the objective was to determine whether blood ph, anion gap, and strong ion gap could be quantitatively estimated and clinically used based on the results of serum or plasma biochemical analysis. 100 horses (70 adults and 30 foals) presented to the isolation unit of our veterinary teaching hospital for suspected infectious diseases were prospectively enrolled. a venous serum sample was analyzed using a hitachi 911 or copas 6000 c501 automated machine. measured parameters included strong ion difference (sid 5 {na1k}-{cl1lac-tate}), total protein concentration (tp), and total co 2 (tco 2 ), with lactate being measured by blood gas analyzer. a second venous blood sample was collected into a na-heparin blood gas syringe and analyzed for ph (ph m ), pco 2 and concentrations of na, k, cl, and lactate using a radiometer 800 flex blood gas analyzer; sid was calculated from the measured values, and total solids (ts) were estimated using refractometry. serum/ plasma ph (ph calc ) was calculated using stewart's 8 factor equation from the results of serum or plasma biochemical analysis, assuming pco 2 5 40 mmhg for serum and pco 2 accurate for plasma. anion gap (ag) was calculated as: ag 5 (na1k)-(cl1tco 2 ). strong ion gap (sig) was calculated as: sig 5 0.21x[total protein, g/l]/ (1110 {pka-ph} )-ag. linear regression analysis was used to compare ph calc to ph m, as well as ag and sig to blood lactate concentrations. measured ph ranged from 7.03 to 7.48 (7.37 ae 0.07). measured sid from serum biochemistry (sid sb ) ranged from 22.3 to 51.4 meq/l (36.2 ae 4.7 meq/l) and sid from blood gas analyzer (sid bg ) from 14.3 to 46.6 meq/l (33.3 ae 5.4 meq/l; r 2 5 0.59; sid bg 5 0.919 â sid sb ). sid sb and sid bg showed small variability in measurements. tp ranged from 18 to 88 g/l (51.0 ae 13.9 g/l) and ts from 20-84 (55.2 ae 13.8 g/l; r 2 5 0.77; ts 5 1.071 â tp). using sid sb and tco 2 values with constant pco 2 , ph calc was poorly associated with ph m (r 2 5 0.27; ph calc 5 0.48 1 3.89). in contrast, using sid bg with accurate pco 2 , ph calc was closely associated with ph m (r 2 5 0.54; phcalc 5 0.98 1 0.15) and the equation was not different from the line of identity. anion gap and sig (meq/l) calculated were significantly linearly correlated with lactate concentrations (mmol/l); ag 5 0.93 â [lactate] 1 8.3 (r 2 5 0.39), and sig 5 à0.91 â [lactate] 1 0.5 (r 2 5 0.41). we conclude that ph calc using sid sb , tco 2 and constant pco 2 values is not accurate. however, variability of measured biochemical parameters between machines was small, permitting use of serum biochemistry for clinical metabolic acid-base abnormalities interpretations of patients. these results reemphasize the importance of strong electrolytes and proteins in acid-base balance. metalloproteinases (mmps) are critically important in remodeling processes and in wound healing. however, excessive activation of mmps by pro-inflammatory mediators including cytokines, prostaglandin e2, and nitric oxide lead to tissue breakdown. this is observed in osteoarthritis (oa) which is characterized by erosive lesions in articular cartilage. in hereditary equine regional dermal asthenia (herda), afflicted horses exhibit collagen abnormalities and can have associated chronic inflammation and aberrant wound repair. herda affects horses with quarter horse bloodlines and is similar to the human hereditary connective tissue syndrome ehlers danlos (eds). many adult eds patients suffer from joint pain and oa. we hypothesized that chondrocytes from articular cartilage of herda horses have increased activity of mmps. to test this hypothesis, chondrocytes were retrieved from articular cartilage of homozygous herda carpal and hock joints. chondrocytes from normal horses were also obtained for comparison. chondrocytes were seeded at 5 x 10 6 /ml into 6-well plates and incubated at 371c, 5% co 2 for up to seven days. activity of secreted mmps was determined by zymography using equal amounts of proteins for loading. secreted mmps were analyzed by western blot. zymography showed that normal chondrocytes secreted two major bands with gelatinolytic activity observed at 92 and 72 kda suggestive of the latent form of mmp-2 and mmp-9, respectively. less intense bands of gelatinolytic activity were observed at about 82 and 62 kda suggestive of the active form of mmp-2 and mmp-9. another band of activity was also seen at 240 kda which is suggestive of a dimer of mmp-9 that has been reported when mmps are in excess of tissue inhibitors of metalloproteinases (timps). chondrocyte cultures from homozygous herda cartilage showed a similar profile but with decreased activity by 90% at 92 kda and 10-50% increased activity at 72 kda compared to normal chondrocytes. western blot analysis detected mmp-2 and mmp-9 immunoreactivity in chondrocyte culture media of herda-afflicted and normal horses. the present study demonstrates for the first time that horses suffering from herda have increased mmp activity which may predispose them to the development of lesions in articular cartilage. research supported by nutramax laboratories, inc. equine polysaccharide storage myopathy (pssm) type 1 is a dominantly inherited glycogenosis caused by a mutation in the gene coding for skeletal muscle glycogen synthase type 1 (gys-1). the disease has been reported to affect the haflinger breed but so far its prevalence is unknown. aim of this preliminary study was to estimate the occurrence of the gys-1 mutation in austrian haflingers and establish which of the seven haflinger sire lines appear mostly affected. gys-1 genotyping of 50 randomly chosen haflingers was performed with a validated restriction fragment length polymorphism assay. resting and post-exercise muscle enzyme activities (creatine kinase (ck), aspartate aminotransferase (ast), lacate dehydrogenase (ldh)) and blood lactate concentrations were compared between horses with and without the mutation. among the 50 horses 9 were heterozygous (hr) carrier of the mutation. no homozygotes (hh) were identified. all horses with the gys-1 mutation were descendents of the a-or w-sire lines. the estimated hr prevalence was 18% (95% ci: 8.6-32.4%). ck activity after exercise (p 5 0.022) was significantly higher in hr horses compared with horses not carrying the mutation (rr). ast activity was significantly higher in the hr group at rest and after exercise (p o 0.001). there was no statistically significant difference in resting ck, resting and post exercise ldh activity or blood lactate between hr and rr. results suggest that the prevalence of hr in the austrian haflinger population is higher than in the overall quarter horse population and might be as high as 30%, similar to some draft horse breeds. further research is needed to establish the prevalence within the different breeding lines. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder affecting quarter horse lineages. 1 although a mutation in the gene encoding cyclophilin b has been genetically linked to herda, its causal association with the disease is not yet documented. 2 previously, we demonstrated reductions in ultimate tensile strength (uts), modulus of elasticity, and energy to failure (toughness) of skin from many corporal regions of herda animals. 3 given the presumed relationship between her-da and abnormal collagen structure, and the predominance of type i collagen in skin, we hypothesized that altered biomechanical properties would be detected in tendons which are rich in type i collagen. to evaluate this hypothesis we compared the uts, modulus of elasticity, and energy to failure of forelimb deep digital flexor tendons (dft) from six herda horses to six age-matched controls. isolated dft was secured and pulled to failure on an instron s 1011 universal testing instrument using purpose-built cryogenic clamps. analysis of variance was executed using sas 9.2 proc glimmix program (sas institute, 2009). p-values 0.05 were identified as significant. uts and modulus of elasticity were significantly lower in herda dft when compared with controls (p o 0.0001); energy to failure did not differ between groups. these findings document abnormal biomechanics in herda tendon, leading us to postulate that lower uts and modulus of elasticity associated with the herda defect could convey a competitive advantage in the athletic disciplines in which this defect has segregated. (references on request). a proprietary herbal biocontamination product (bios) approved for cosmetic use in france, inhibits proliferation of medically relevant bacteria, mold, and viruses. these properties make bios potentially useful as a topical wound medication, prompting us to compare bios to silver sulfadiazine (ssd) in a distal extremity wound healing model in horses. 1 using general anesthesia, two 6.25 cm 2 wounds were aseptically created on the dorsomedial aspect of all limbs. for the duration of the study, two contralateral limbs were randomly chosen to be bandaged; the other two limbs were un-bandaged -with one limb of each group being treated with 10% bios and the other with ssd. for each limb the most proximal wound served as an untreated control. every 48 hours wounds were evaluated, digitally photographed, and perimeter and area determined using morphometric software (imagej, nih). analysis of variance did not identify significant differences between ssd or bios treatment for wound perimeter (p 5 0.76) or area (p 5 0.95). at individual time points the effect of bandaging was significant when area was evaluated (p 5 0.019) and trended toward significance for perimeter (p 5 0.084) comparisons, substantiating published reports that bandaging modifies wound healing. difference in perimeter and area between control and treatment were highly significant (p o 0.0001), substantiating the importance of topical treatment. over the study duration, effects of bandaging (p o 0.0001) and topical treatment (perimeter p o 0.0001; area p 5 0.0016) continued to be highly significant. bios performance in the equine distal extremity wound model was equivalent to ssd. both bandaging and topical treatment significantly impacted wound healing. this effect was compounded when both variables were evaluated over time. radiolabeled leukocytes are the only scintigraphic method currently available for identifying sites of infection and/or inflammation in horses; however the clinical applicability of this technique is limited by expense and poor efficacy. this pilot study compares the accumulation of 99m tc-labeled igg, peg-liposomes and leukocytes in an equine muscle abscess model. three mixed breed adult horses had 2 â 10 6 cfu s. equi zooepidemicus inoculated into the right semitendonosis to create an abscess. peg-liposomes were prepared via the film hydration method and labeled using 200 mci 99m tc-hexamethyl-propylene-amine-oxime ( 99m tc-hmpao). autologous leukocytes were obtained from 120 ml whole blood and labelled using 200 mci 99m tc-hmpao. commercial equine polyclonal igg was conjugated with the chelator hydrazinonicotinamide (hynic) and labelled with 200 mci 99m tc. radiopharmaceutical administration was initiated 24 hours after inoculation. horses 1 and 2 received 5 mg 99m tc-igg, 2.7 mmol/kg 99m tc-liposomes and 99m tc-leukocytes, with a 48 hour interval between each radiopharmaceutical. horse 3 received only 99m tc-leukocytes. scintigraphic examinations were performed at 8 and 21 hours post injection (p.i.) with each radiopharmaceutical. after the final study, horses were euthanized and tissue samples collected. the percentage of injected dose per kilogram of tissue (%id/kg) was calculated for the region of the abscess, normal muscle and multiple organs. scintigraphic examinations demonstrated increased radiopharmaceutical in the region of the abscess with all three techniques at both time-points. at 8 hours p.i. abscess-to-background ratio was highest using 99m tc-igg (3.7 ae 0.2). at 21 hours p.i. abscess to background ratio was highest using 99m tc-liposomes (5.9 ae 2). tissue biodistribution data revealed abscess to muscle ratios of 36 ( 99m tc-igg), 24 ( 99m tc-liposomes), and 4.1 ( 99m tc-leukocytes). this preliminary data demonstrates that 99m tc-liposomes, 99m tc-igg and 99m tc-leukocytes exhibit long circulating characteristics and accumulate at inflammatory/infectious foci after intravenous injection in horses. 99m tc-igg and 99m tc-liposomes appear to be superior to 99m tc-labelled leukocytes in this model. due to its low cost and ease of preparation, 99m tc-igg has great potential for clinical use where identification of infectious or inflammatory foci is necessary. digital hypothermia is used clinically to decrease the incidence of sepsis-related equine laminitis, a disease causing structural failure of digital laminae resulting in crippling lameness. due to the fact that hypothermia was recently reported to effectively decrease laminar expression of inflammatory molecules including pro-inflammatory cytokines, chemokines and cox-2 in equine laminitis, our laboratory is investigating the effect of hypothermia on central upstream signaling cascades which may induce expression of these diverse inflammatory molecules. the p38 mapk pathway has recently been reported to be a central component of inflammatory signaling in multiple diseases including human sepsis, and is currently being assessed as a therapeutic target. we thus hypothesized that 1) p38 mapk is upregulated and activated in affected laminae in equine laminitis and 2) digital hypothermia inhibits inflammatory mediator expression by blocking p38 mapk phosphorylation (indicator of p38 mapk activation). western hybridizations using both a total p38 mapk and a phospho-p38 mapk antibody were performed on archived pooled laminar samples from black walnut extract (bwe) model (10 control, 2 developmental (dev) groups [1.5h & 3h post bwe administration] and the onset of obel grade 1 lameness (og1) [n 5 5 each]) and carbohydrate overload (cho) models (con [n 5 8], dev [n 5 6], og1[n 5 6]) of laminitis, and individual laminar samples from two groups of horses from a digital hypothermia (dh) study. in the dh study, one forelimb of each horse was kept at approximately 41c in ice water and the other at ambient temperature following administration of 10 g/kg oligofructose (of). dorsal laminae were harvested for snap freezing at either 24 hours after of administration (dev, n 5 7) or at the onset of lameness (og1, n 5 6) using protein extracted from treated and untreated digital laminae of each horse. increased laminar concentrations of phospho-p38 mapk were present in the developmental periods (1.5h and 3h) in the bwe model, and in both the dev and og1 periods in the cho laminitis models. however, digital hypothermia had no effect on laminar phospho-p38 mapk concentrations. thus, p38 mapk is activated in affected laminae in multiple models of laminitis, but does not appear to be the central signaling cascade through which hypothermia works to block the expression of inflammatory molecules. therefore, p38 mapk is not likely to be a viable therapeutic target as a sole source for blocking the multiple inflammatory signaling mechanisms inhibited by local hypothermia. abstract e-60 does cefquinome penetrate the blood brain barrier in the normal horse? hollis ar 1 duggan ve 2 and corley ktt 3 . 1 scott dunn's equine clinic, berkshire, uk; 2 university college dublin, dublin, ireland; 3 anglesey lodge equine hospital, the curragh, ireland. meningitis is a rare but serious condition that occurs in both foals and adult horses. there is currently a restricted choice of antimicrobials that are both safe to use in horses and penetrate the blood brain barrier. cefquinome is a fourth generation cephalosporin that has activity against streptococcus, the most commonly reported causative organism in adult horse meningitis. therefore, if cefquinome were to achieve therapeutic concentrations in cerebrospinal fluid following routine administration, this would be an exciting advance for the treatment of meningitis in the horse. 5 mature, healthy horses were used on 2 separate occasions, seven days apart, in a crossover design. each horse was administered either cefquinome (1 mg/kg) or saline (equivalent volume). cerebrospinal fluid was collected via atlanto-occipital puncture under general anaesthesia 1 and 4 hours after administration of cefquinome or saline placebo. blood samples were collected prior to, and 1 and 4 hours after administration of cefquinome or placebo. all samples were analysed for the presence of cefquinome by a laboratory masked to treatments administered. cefquinome was detectable in the cerebrospinal fluid in all horses 4 hours after intravenous administration, and in 2 horses 1 hour after administration. cefquinome penetrates the blood-brain barrier and it is therefore a potential treatment for equine meningitis. further investigation of the pharmacokinetics and pharmacodynamics of cefquinome in the cerebrospinal fluid is warranted to establish the optimum intravenous dose. the purpose of this study was to determine if enrofloxacin alters the pharmacokinetics of firocoxib in the horse. firocoxib is a coxibclass nonsteroidal anti-inflammatory drug (nsaid) approved for use in horses to control pain and inflammation associated with osteoarthritis. dosages of firocoxib are species dependent, with the recommended dose for horses being 0.1 mg/kg as an oral paste every 24 h. the main elimination pathway of firocoxib is hepatic; however the effects of concurrent administration of drugs that may inhibit its metabolism have not been evaluated. enrofloxacin is a synthetic antibacterial agent from the flouroquinolone group developed for veterinary use. it is primarily used for gastrointestinal, urogenital, skin and respiratory tract infections in various animals. a well acknowledged problem associated with flouroquinolone usage is their effect on the metabolism of other drugs. co-administration of multiple drugs can result in unpredictable therapeutic outcomes. often it is either diminished therapeutic efficacy or increased toxicity of one or more of the administered drugs. various pharmacokinetic interactions between antimicrobials and nsaids have been described. six healthy, adult mares were administered 0.1 mg/kg of firocoxib orally. samples were collected by direct venipuncture of the jugular vein at 0 (control), 15, 30, and 60 min, 2, 4, 8, 12, 16, 24, 48, 72 , and 96 h after administration. after a 20 day washout period the six horses were pretreated 3 days with enrofloxacin 5 mg/kg intravenously every 24 h then on the fourth day given 0.1 mg/kg of firocoxib orally. samples were collected at 0 (control), 15, 30, and 60 min, 2, 4, 8, 12, 16, 24, 48, 72 , and 96 h after administration. all samples were stored at à801c until analysis using a validated hplc method. the t1/2, c max , t max , auc 0-24 and auc 0-f after firocoxib administration were 30. 66 angiotensin converting enzyme (ace) inhibitors improve survival and quality of life in humans and small animals with cardiovascular and renal disease. there is limited information regarding their effects in horses. the purpose of this study was to determine the pharmacokinetics of quinapril and its effects on ace inhibition in horses. six healthy horses were administered quinapril at 120 mg iv, 120 mg po or 240 mg po in a 3-way crossover design. blood was collected at predetermined times for measurement of quinapril and quinaprilat concentrations using high pressure liquid chromatography, as well as ace concentrations using a radioenzymatic assay. normally distributed data were analyzed with one way repeated measures analysis of variance (rm-anova) and non-normally distributed data were analyzed using friedman rm_anova on ranks. significance was set at p o 0.05. no adverse effects were observed during the study period. plasma quinapril concentrations were low and rapidly declined after iv administration. quinaprilat concentrations were below the limit of quantification (0.1 mg/ml). ace activity was significantly decreased from baseline at 0.5 and 1 hour after iv dosing and at all timepoints after oral dosing. maximum % ace inhibition was 72, 53 and 47% with the iv, high and low oral doses, respectively. these results suggest that, despite low plasma concentrations, quinapril has sufficient oral absorption and results in inhibition of ace in healthy horses. controlled studies in clinically affected horses are indicated. this study determined the pharmacokinetic profile of firocoxib in healthy neonatal foals. foals are more sensitive to the side effects of nsaid, primarily due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. firocoxib, a novel, second generation nsaid, is reported to have reduced side effects due to cox-2 selectivity. the pharmacokinetic profile of firocoxib in neonates has not been established. we hypothesized that firocoxib given po to neonatal foals would achieve therapeutic concentrations in plasma. seven healthy foals of mixed gender were administered 0.1 mg/kg firocoxib po q24h for nine consecutive days, commencing at 36h old. blood was collected for firocoxib analysis at 0 (dose #1 only), 0.25, 0.5, 1, 2, 4, 8, 16 and 24h after doses #1, 5 and 9. for all other doses (2, 3, 4, 6, 7 and 8) blood was collected immediately prior to the next dose (24h trough). elimination samples were collected after dose #9. plasma was stored at à801c until analysis. physical examinations were performed on foals daily and body weight obtained every two days during the sampling period. analysis of plasma samples by liquid chromatography-mass spectrometry revealed firocoxib was rapidly absorbed. after the initial dose, a maximum plasma concentration was reached in 30 min, minimal accumulation after repeat dosing occurred and steady state was obtained after approximately four doses. after the final dose, plasma drug concentration decreased in a linear manner with an estimated terminal t1/2 of 11h. seventy-two hours after the final dose, firocoxib was not detectable (o 2ng/ml). erythrocytosis is reportedly a rare finding associated with hepatocellular carcinoma in horses. the purpose of this study was to determine the relative frequency of erythrocytosis and the clinicopathologic abnormalities and hepatic histopathology associated with erythrocytosis in horses with liver disease. ninety-seven horses aged !1 year with clinicopathologic or clinical signs of liver disease, a complete blood count (cbc), and hepatic histopathology were included. information on cbc, biochemical variables, and hepatic histopathology was collected from records. data from horses with erythrocytosis (packed cell volume 4 45%) were compared to those without using the mann-whitney rank sum test with significance set at p o 0.05. there were no differences between groups in white blood cell count, gamma-glutamyl transferase, sorbitol dehydrogenase, aspartate aminotransferase, and alkaline phosphatase activities, total protein, albumin, globulin, blood urea nitrogen, or glucose concentrations. fibrosis (64%), biliary hyperplasia (56%), inflammatory infiltrate (50%), megalocytosis (25%), degeneration (25%), necrosis (25%), cholestasis (25%), anisocytosis and anisokaryosis (11%), and lipidosis (3%) were observed in livers of horses with erythrocytosis. neoplasia (3%) was observed rarely. this study reports a high frequency of erythrocytosis in horses with liver disease. erythrocytosis is associated with higher total bilirubin and serum bile acids concentrations. common histopathologic changes include fibrosis, biliary hyperplasia, and inflammatory infiltrate. hepatic neoplasia was rare. this study was performed to determine if horses diagnosed with equine proliferative enteropathy (epe) from lawsonia intracellularis (li) infection had long term effects from disease based on their sale price as yearlings and race earnings. a retrospective review of medical records of thoroughbred horses that were treated for lawsonia intracellularis infection between january 1, 2002 and january 31, 2008 at hagyard equine medical institute in lexington, kentucky was performed. three criteria were used for inclusion in this study. first, each horse had presumptively been diagnosed with li based on physical examination findings such as ventral edema, diarrhea, lethargy, or poor body condition. second, horses had hypoalbuminemia of less than 2.5 mg/dl (normal reference range: 3.4-4.5 mg/dl). third, each horse had a positive fecal polymerase chain reaction (pcr) for li, a positive serum immunoperoxidase monolayer assay (ipma), or both. an ipma titer greater than or equal to 60 was considered positive for disease. 116 horses met the initial criteria. 36 of the 116 horses sold at public auction as yearlings. the sale price of these horses was compared to the average sale price of all yearlings by the same sire as the affected horse (control group). 30 of the 116 horses raced in the united states. their monetary earnings from racing were compared to the average monetary earnings of all progeny by the same sire as the affected horse (control group). earnings of horses that were between 3 and 7 years of age (23/30 horses) at the conclusion of the study were compared to the lifetime average earnings of the stallion's progeny. earnings from horses that were two years of age (7/30) at the end of the study were compared to the two year old average earnings of the stallion's progeny. monetary earnings from all races prior to december 31, 2008 were included in the study. 12 horses both sold at public auction and raced. as well as being included in the total number of horses that sold and raced, their sale records and monetary earnings were compared to the averages from their respective sire as a separate group. this retrospective study indicated that yearling horses previously infected with li do not sell for as much at public auction as their herdmates, but their monetary earnings from racing are not significantly different from other horses. these results should assist practitioners in guiding owners in determing if treatment of horses with epe is appropriate and it may aid in reassuring owners that despite the poor condition of the horse during and shortly after the course of disease, horse may still have future athletic potential. this abstract was presented at the aaep in december 2009. bronchopneumonia caused by streptococccus equi subsp. zooepidemicus (s. zooepidemicus) is one of the most important causes of morbidity in weanling foals. ceftiofur crystalline free acid (ccfa) is a long acting third-generation cephalosporin antimicrobial recently approved for the treatment of bronchopneumonia associated with s. zooepidemicus in adult horses. the objective of the present study was to determine the disposition of ccfa in plasma and pulmonary epithelial lining fluid (pelf) of weanling foals. six healthy 4-to 5month-old weanling foals were administered a single intramuscular injection of ccfa at a dose of 6.6 mg/kg of body weight. concentrations of desfuroylceftiofur acetamide (dca) and related metabolites were measured by use of ultra-high performance liquid chromatography and tandem mass spectrometry. following im administration, median time to maximum plasma and pelf concentrations was 24 h (12-48 h) . mean (ae sd) peak dca concentration in plasma (1.44 ae 0.46 mg/ml) was significantly higher than that in pelf (0.46 ae 0.03 mg/ml). terminal half-life of dca in plasma (74.8 ae 20.9 h) was not significantly different from that of pelf (58.5 ae 11.4 h). time above the therapeutic target of 0.2 mg/ml was significantly longer in plasma (185 ae 20 h) than in pelf (107 ae 31 h). based on the results of the present study, intramuscular administration of ccfa at a dose of 6.6 mg/kg would be appropriate for the treatment of bronchopneumonia caused by s. zooepidemicus and other susceptible pathogens in weanling foals. fgf-23 is secreted by osteocytes and osteoblasts in response to hyperphosphatemia. fgf-23 enhances phosphaturia and is postulated to have a central role in the development of secondary renal hyperparathyroidism. hyperthyroid cats have elevated plasma phosphate and parathyroid hormone concentrations, which may in part be associated with underlying chronic kidney disease (ckd). the aim of this study was to determine if plasma fgf-23 concentrations were associated with the presence of underlying ckd in hyperthyroid cats, and to investigate the changes in plasma fgf-23 concentrations that occur following treatment of hth. hyperthyroid cats were recruited from two london-based first opinion practices between 1999 and 2009. cats that were azotemic at diagnosis were excluded. hth was treated with anti-thyroid medication alone or in combination with thyroidectomy. cats were included in the study if they had a plasma total thyroxine concentration o 40 nmol/l documented for a six month period following commencement of treatment. cats were classified as having azotemic ckd if they developed renal azotemia within six months of establishment of euthyroidism. otherwise cats were deemed to have normal renal function. stored edta plasma samples were assayed for fgf-23 using a recently validated elisa. the mann-whitney u test and the wilcoxon signed rank test were used to compare between the groups and assess the response to treatment respectively. results are reported as median [25 th , 75 th percentiles]. correlations were made using spearman's correlation coefficient. thirty one cats with hth (14 azotemic and 17 non-azotemic) were included in the study. plasma phosphate concentrations decreased following treatment in cats that did not develop azotemia (4.84 [3.91, 5.64] mg/dl vs. 3.91 [3.38, 4.37] mg/dl; n 5 13, p 5 0.01) whereas plasma phosphate concentrations did not change significantly following treatment in cats that did develop azotemia (4.28 [3.81, 5.64] mg/ dl vs. 4.22 [3.10, 5.33] mg/dl; n 5 14, p 5 0.158). plasma fgf-23 concentrations were significantly higher in cats that developed azotemia than cats that did not at both pre treatment (211.7 [176.4, 356 .3] pg/ml vs. 148.3 [118.8, 274 .9] pg/ml; p 5 0.039) and post treatment (514.0 [250.2, 800.0] pg/ml vs. 195.1 [160.7, 287 .3] pg/ml; p 5 0.001) timepoints. plasma fgf-23 concentrations increased following treatment in both azotemic (p 5 0.004) and non-azotemic groups (p 5 0.025). plasma fgf-23 concentrations and plasma phosphate concentrations were not correlated at baseline (r s 5 0.189, p 5 0.335) or following treatment (r s 5 0.136, p 5 0.472). plasma fgf-23 concentrations were higher in pre-azotemic cats than non-azotemic cats and increased following treatment of hth. the reason that fgf-23 concentrations increased following treatment, particularly in the face of decreasing plasma phosphate concentrations in cats that remain non-azotemic, is unclear but may be related to the decline in glomerular filtration rate. hyperthyroidism is a disorder resulting from the excessive production and secretion of t4 and t3 by the thyroid gland. although the disorder and its pathological lesions have been well studied and described the cause remains illusive. whole blood and solid tissue samples from non-diseased, severe disease and mild disease cats based on t4 levels and thyroid histology were used in this study. whole blood samples from 29 non-disease cats, 28 severe disease cats and 17 mild disease cats as well as solid thyroid tissue samples from 30 non-disease cats, 31 severe disease cats and 27 mild disease cats were collected and processed. the resulting total rna samples were used for genechip analysis using our custom feline gene chip designed by affymetrix. data analysis was performed using the partek s gs software for gene expression data. the robust multichip average algorithm was used for background adjustment, normalization, and probe-level summarization of the raw data. anova analysis was performed to find significant differentially expressed genes with a minimal false discovery rate control of 0.1 and a fold change of 1.3 in each direction. during the mild disease state, pathways associated with dna damage and apoptosis are most prominent. at later stages when the histopathological disease is more severe in addition to the aforementioned pathways others associated with tgf-beta signaling, cell adhesion and extracellular matrix remodeling take more prominence. the analysis of this unique data set generated from the use of our proprietary genechip revealed molecular mechanisms that are associated with the transition from non-disease, to mild disease to severe disease, in the thyroid tissue as well as the blood. these mechanisms could provide insights into the causes of the disease and identify potential new therapeutic and diagnostic targets. although it is well established that concurrent chronic kidney disease (ckd) develops in about 30% of hyperthyroid cats, no one has reported the use of the iris staging system for ckd before and after treatment of these hyperthyroid cats. the purpose of this study was to compare the effects of treatment in hyperthyroid cats with known stage 1 and 2 ckd in order to determine the effects of restoring euthyroidism or inducing hypothyroidism has on the iris stage in these cats. we evaluated 36 hyperthyroid cats (median age, 14 years) in this study. one day prior to treatment, serum t 4 concentration, serum chemistry analysis, complete urinalysis, and urine protein-to-creatinine ratio (upc) were measured. all cats were again evaluated with the same parameters again 3 months after treatment with 131 i. prior to treatment, 26 (72%) of the 36 cats had no evidence of azotemia (serum creatinine o 1.6 mg/dl), whereas 10 cats (28%) had stage 2 ckd (serum creatinine, 1.6-2.8 mg/dl). in the 36 cats, iris staging revealed proteinuria in 33 cats (92%), 21 with borderline proteinuria (upc, 0.2-0.4) and 12 with overt proteinuria (upc 4 0.4). hyperthyroidism was cured in all 36 cats (median post-t4, 1.3 mg/dl). all cats had a good response to treatment; there were no signs of ckd except for polyuria and polydipsia in some cats. a significant (p o 0.001) increase in median values for both serum urea nitrogen (26 mg/dl to 31 mg/dl)) and creatinine (1.1 to 1.7 mg/dl) occurred after treatment. nine of the 26 cats (34.6%) classified as nonazotemic or iris stage 1 prior to 131 i progressed to stage 2 ckd after 131 i. all 10 cats with stage 2 ckd before treatment remained azotemic after 131 i, with 5 cats remaining in stage 2 ckd, and 4 cats progressing to stage 3 ckd (serum creatinine, 3.1-3.7 mg/dl). there was a significant inverse relationship (p 5 0.002) between pretreatment urine specific gravity (usg) and post-treatment serum creatinine in the 36 cats. of the 19 cats with post-treatment serum creatinine values 4 1.5 mg/dl (stage 2 to 3 ckd), 15 (79%) had pretreatment usg of o 1.040. in contrast, in the 17 cats with post-treatment serum creatinine values o 1.5 mg/dl, only 3 (18%) had pretreatment usg of o 1.040. a significant (p o 0.001) decrease in median upc from 0.3 to 0.1 occurred after treatment, but there was no relationship between degree of proteinuria and iris stage in these cats. two cats developed iatrogenic hypothyroidism after 131 i, diagnosed by finding low serum t 4 and high ctsh concentrations. both hypothyroid cats had progressed from stage 1 before treatment to stage 2 and 3 ckd, respectively, after 131 i; after thyroxine replacement, serum creatinine decreased to near pretreatment concentrations in both cats. conclusions: 1) iris stage 2 ckd is common in untreated hyperthyroid cats. 2) progression to next higher iris stage is common after treatment, but most cats with remain relatively asymptomatic for ckd. 3) usg may be helpful in predicting which cat's iris stage will progress after 131 i. 4) iatrogenic hypothyroidism worsens azotemia, an effect that appears reversible with replacement therapy. home blood glucose monitoring (hbgm) of diabetic pets is likely to result in superior glycaemic control, minimizing episodes and impact of dangerous hypoglycaemia and reducing costs. nevertheless, it has proven difficult to objectively establish a clear benefit of hbgm using biological parameters (clinical signs, blood glucose, fructosamine). the current study aimed to assess the impact of hbgm on owner perceived quality of life (qol) aspects of diabetes mellitus (dm) treatment, using the recently validated psychometric tool diaqol-pet. owners of insulin treated diabetic cats were recruited to complete the 29-item tool, evaluating areas affecting the cat's and owner's qol, including: worry about pet's dm, hypoglycaemia, costs, owner's desire for autonomous control over the pet's dm, etc. item-weighted-impact-scores (iwis), reflecting frequency and importance ratings of each item, were calculated, as well as averageweighted-impact-scores (awis; average iwis of all items), as an overall measure of diabetes dependent qol. frequencies, iwis and awis were compared between owners practising hbgm and those who did not using mann whitney u test (significance p o 0.05). two hundred and eleven owners of insulin treated diabetic cats completed the diaqol-pet; 161 owners practised hbgm, whereas the remaining 50 did not practise any form of home monitoring (including urine glucose). iwis for 'excessive drinking' and 'owner wanting more control' were significantly different between the hbgm-group (mean1/-standard deviation: à2.011/à2.4 and à5.071/à4.8) and the non-hbgm-group (à3.361/à3.8 and à1.861/à3.2). there was no significant difference between the groups with regards to the iwis for other items, including 'worry about hypoglycaemia' or 'worry about pet's dm'. polydipsia was reported significantly more frequently in the non-hbgm-group and this was the reason for the difference between groups in this item's iwis as it was considered of equal importance. frequency and iwis of reported occurrence of hypoglycaemia signs were not significantly different. awis for both groups was not significantly different (hbgm: à1.851/à1.3; non-hbgm: à1.871/à1.1). the current study suggests that hbgm is predominantly practised by owners who desire more autonomous control over their cat's dm. the frequency of polydipsia was lower in the hbgm-group perhaps suggesting superior control. however, hbgm did not detectably affect the impact of the majority of qol-items, nor the frequency of hypoglycaemic episodes. overall diabetes dependent qol of diabetic cat and owner, as measured per diaqol-pet, was unaffected by hbgm. these data argue for the use of hbgm in selected pet-owner combinations rather than as part of a practice's standard dm management protocol, although further studies are indicated. insulin resistance is associated with impaired activation of the insulin signaling pathway in peripheral tissues such as skeletal muscle, visceral and subcutaneous (sc) adipose tissue. high plasma glucose, fatty acid and endotoxin levels are three major causes of insulin resistance in feline and human obesity and in type 2 diabetes mellitus. however, the mechanisms by which these factors influence insulin action are still unclear. therefore, our aim was to investigate the tissue-specific expression of crucial mediators of insulin action such as the insulin-receptor substrate 1 (irs1), the serine/threonine protein kinase b (pkb/akt) and of the principal insulin-dependent glucose transporter protein (glut4) in feline models of hyperglycemia, hyperlipidemia and subacute endotoxemia. healthy cats were infused through the jugular vein with glucose (n 5 5), lipids (n 5 6) or lipopolysaccharide (lps; n 5 5) for 10 days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: 25-30 mmol/l; triglycerides: 3-7 mmol/l) or to induce a systemic low-grade inflammation (lps; rectal temperature: 39.2-40.51c), respectively. healthy control cats were infused with saline (n 5 10). on day 10, specimens were collected from skeletal muscles, visceral and sc fat and processed for irs1 mrna expression, total and phosphorylated pkb/akt and glut4 protein expression. gene transcripts of irs1 were not different between the groups. compared to controls, skeletal muscle pkb/akt phosphorylation was 91% lower in cats infused with glucose (p o 0.01); lipid-infused cats showed a trend for a decrease in pkb/akt phosphorylation (31% lower than saline) and had decreased glut4 expression (p o 0.05) in muscle. total (p o 0.01) and phosphorylated (p o 0.05) pkb/akt protein expression were decreased in the sc adipose tissue of lps-infused cats compared to controls. in these cats, phosphorylation of pkb/akt protein was also decreased in visceral fat (p o 0.01). sustained hyperglycemia and, to a lesser extent, hyperlipidemia impaired insulin signaling and glucose transport pathways primarily in skeletal muscle; endotoxemia reduced insulin sensitivity mainly in adipose tissues. thus, the development of insulin resistance in response to hyperglycemia, hyperlipidemia or endotoxemia might be affected by tissue-specific mechanisms in cats. separately used, single photon emission computed tomography (spect) and computed tomography (ct) both lack sensitivity and are additionally hampered by a poor anatomical location capacity and a lack of specificity, respectively. these drawbacks suggest an interest in the fusion of images obtained by the 2 techniques. the aim of this study is to test spect/ct fusion performance in dogs with insulinoma. inclusion criteria were: 1/ a biological diagnosis of insulinoma; 2/ an examination by high resolution ct scan and 111 in-pentetreotide spect followed by spect/ct fusion; 3/ a surgical or post mortem examination completed by histopathological analysis. spect examination showing abnormal foci and ct scan showing pancreas, lymph nodes (ln) or liver abnormalities were considered positive. in case of double positivity, presence (imp1) or absence (imp-) of superimposition of abnormal images was noted. ten dogs were included. in 2/10 dogs, superimposition of abnormalities couldn't be tested. ct scan detected 3 abnormal images [2 pancreatic nodules (pn), 1 enlarged ln (eln)] while spect failed to show any abnormal uptake. both dogs became euglycemic after removal of pn and ln designed by ct scan. in 6/ 10, all abnormal images were classified as imp1 [6 pn, 1 eln and 1 diffuse hepatic infiltration (dhi)]. surgery performed on 5/6 resulted in euglycemia in 4; 1 dog remained hypoglycemic after partial removal of 1 pn. pn localization and dhi were confirmed after necropsy in the 6 th dog. in 2/10 dogs imp1 and imp-images were both recorded. in 1 dog, a dhi was classified as imp1 but pn localization was imp-: localized in the left lobe by ct scan and in the corpus by spect, the latest localization being confirmed after necropsy. in the other dog pn localization was imp1 but a diffuse spect signal superimposing to the liver considered as normal on ct scan was noted. hepatic biopsy confirmed spect results. this study confirms an imperfect sensitivity of both ct scan and spect. it confirms that ct scan can be associated with unspecific abnormal images. subject to a confirmation on a larger cohort of dogs, it indicates that imp1 images provide specific detection and accurate localization of canine insulinomas' primary lesions and metastasis. the majority of dogs with primary hypoadrenocorticism (ph) reveal clinical and laboratory abnormalities of gluco-and mineralocorticoid deficiency. in some of them sodium and potassium levels are normal, a phenomenon currently called atypical addison's. it has been postulated that in those cases adrenal destruction is confined to the zona fasciculata/reticularis, resulting in isolated glucocorticoid deficiency. however, there are no histological studies confirming a normal zona glomerulosa and in most reported cases diagnosis was based solely on low post-acth cortisol levels. the aim of the study was to evaluate aldosterone (aldo) levels in dogs with ph with and without electrolyte abnormalities. seventy dogs with newly diagnosed ph were included. aldo concentrations (ria, coat-a-count s , siemens) were measured before and 60 min after administration of 250 mg synthetic acth (synacthen s , novartis) iv. results were compared to those of 19 healthy dogs and 17 dogs with diseases mimicking ph. to confirm that peak concentrations were not missed aldo was additionally measured 15, 30 and 45 min after acth in 19 dogs (5 with ph, 14 with ph mimicking diseases). results were analysed by means of non-parametric statistical methods (p o 0.05). post-acth aldo was significantly lower in dogs with ph (0-253 pg/ml, median 0 pg/ml) than in healthy dogs (46-602 pg/ml, median 187 pg/ml) and in dogs with ph mimicking diseases (0-639 pg/ml, median 155 pg/ml). low post-acth aldo was found in 67/70 dogs with ph, in 64/67 of them levels were below the detection limit of the assay. normal sodium and potassium levels were found in 5/67 dogs (7%), 6/67 dogs (9%) had hyponatremia and normal potassium, 56/67 dogs (84%) had hyponatremia and hyperkalemia. electrolyte abnormalities ranged from mild to severe. there was no correlation between post-acth aldo and sodium and a weak correlation between post-acth aldo and potassium (r 5 à0.28). aldo concentrations were not different 30, 45 and 60 min after acth. the results demonstrate that aldo levels are low in most dogs with ph independent of the degree of electrolyte abnormalities. this implicates that all three zones of the adrenal cortex are compromised and that there are mechanisms which allow maintenance of a normal electrolyte balance without aldo. definitive diagnosis of canine hypoadrenocorticism (ha) is based on inadequate cortisol secretion following adrenocorticotropic hormone (acth) administration. an abnormal serum sodium to potassium (na:k) ratio can be used to determine whether an acth stimulation test is warranted. the aim of this study was to examine the utility of combining the na:k ratio with white blood cell counts to determine whether an acth stimulation test is warranted. a retrospective review of medical records of dogs examined between 2005 and 2009 was performed. 53 dogs diagnosed with ha and 110 control dogs, in which a diagnosis of ha was excluded during the study period, were included. inclusion criteria for all 163 dogs were hospitalization with intravenous fluid therapy, a complete blood count, and serum na and k measurements at the time of initial examination. dogs were included in the ha group if they also had pre and post acth stimulation serum cortisol concentrations 1.0 mg/dl. dogs were included in the control group if they had resting or post acth stimulation serum cortisol concentration 4 2.0 mg/dl. exclusion criteria were recent administration of glucocorticoids, prior treatment of hyperadrenocorticism, or serum cortisol concentration 4 1.0 mg/dl but 2.0 mg/dl. continuous variables were compared between groups using the mann-whitney u test. receiver operating characteristic (roc) curves were produced to assess the sensitivity and specificity of detecting ha with various cutoffs for each variable. data is presented with 95% confidence intervals (ci) and statistical significance was defined as p o 0.05. the na:k ratio, neutrophil count and neutrophil:lymphocyte ratio were significantly lower in dogs with ha than in dogs without ha (p o 0.01 for each). lymphocyte and eosinophil counts were significantly higher in dogs with ha compared to dogs without ha (p o 0.001 for each). the areas under the curve by roc analysis were largest for na:k ratio (0.87, ci:0.81-0.92) and lymphocyte count (0.85, ci:0.78-0.90). a na:k ratio 39.6 was 100% sensitive (ci:93-100%) but only 15% specific (ci:9-23%) for detecting ha. a lymphocyte count ! 0.77 x10 3 cells/ml was 100% sensitive (ci:93-100%) and 35% specific (ci:27-45%). conversely a na:k ratio 20.1 was 51% sensitive (ci:37-65%) but 100% specific (ci:97-100%) and a lymphocyte count ! 6.1 â 10 3 cells/ml was 9% sensitive (ci:3-20%) but 100% specific (ci:97-100%). a na:k ratio 35.5 was 96% sensitive (ci:87-100%) and 35% specific (ci:26-44%) for detection of ha and a lymphocyte count ! 0.79 â 10 3 cells/ml was 98% sensitive (ci:90-100%) and 36% specific (ci:27-46%) for detection of ha. a combination of this na:k ratio ( 35.5) and lymphocyte count (! 0.79 â 10 3 cells/ml) was 96% sensitive (ci:87-100%) and 55% specific (ci:47-66%) for detection of ha. these results indicate that the combination of lymphocyte count and na:k ratio results in a better screening test for ha than the use of the na:k ratio alone. pheochromocytoma is a malignant, catecholamine-producing, adrenomedullary tumor. clinical signs resulting from excessive catecholamine secretion are typically non-specific, making differentiation from other adrenal tumors a challenge. elevated plasma concentrations of the catecholamine breakdown products metanephrine (mn) and normetanephrine (nmn) are used to identify pheochromocytoma in humans. this study tested the hypothesis that plasma metanephrine concentrations are greater in dogs with pheochromocytoma than in dogs with other adrenal neoplasms, healthy dogs and dogs with non-adrenal illness. edta plasma was collected from healthy dogs and unwell, hospitalized dogs with non-adrenal illness, pheochromocytoma and cortical tumors between april 2007 and october 2010. samples were stored at à801c before measurement of free mn and nmn concentrations using high pressure liquid chromatography at the central laboratory for clinical chemistry at the university of groningen (33 samples) or the mayo clinic, rochester, minnesota (7 samples). kruskal-wallis tests followed by dunn's multiple comparison analysis were used to compare results between groups. significance was set at p o 0.05. results are reported as median [range] . eight dogs with pheochromocytoma, 5 healthy dogs, 15 dogs with non-adrenal illness and 12 dogs with cortical tumors were sampled. pheochromocytoma was diagnosed histologically (7 dogs) or cytologically (1 dog). cortical tumors were diagnosed histologically (9 dogs) or by response to trilostane treatment after obtaining consistent endocrine test results (3 dogs occult hyperadrenocorticism (hac) has been theorized to exist in which excess adrenal sex hormone secretion induces the clinical signs and laboratory changes associated with classic hac. however, the ability of sex hormones to cause such alterations has never been closely evaluated. if sex hormones can cause a syndrome similar to classic hac, they should be able to induce expression of classic glucocorticoid-induced genes. the purpose of the study was to determine if in vitro expression of the gene for corticosteroid-induced alp (cialp) could be induced by clinically relevant concentrations of cortisol and sex hormones believed to cause occult hac. canine hepatocytes were purchased from a commercial source (cellzdirect or invitro) in 6-well plates. upon arrival (3-4 plates per shipment), the cells were allowed to recover in general media per supplier recommendations. after 4 hrs, media was changed to william's e media (-l-glutamine) containing concentrations of cortisol or sex hormones that have been documented in the literature in dogs with hac or with purported occult hac. each plate was treated with a different hormone (cortisol, 17-hydroxyprogesterone [17ohp], progesterone, estradiol or androstenedione), and each well contained a different concentration (starting with no hormone added as a negative control) to evaluate a dose response. media was changed daily. after 5 days of hormone exposure, rna was extracted. reverse transcription was performed and the product used for quantitative pcr for cialp and beta-actin (roche lightcycler) using a gene-specific fluorescent probe for detection. standard curves were created for each gene. all samples and standards were run in duplicate. using the lightcycler software (vers 4.0), cialp expression was normalized to that of beta-actin. fold change in expression was determined relative to the negative control. each sex hormone was used to treat 3 plates; one plate in each shipment was treated with cortisol as a positive control. for cortisol, a dose response was seen in expression of the cialp gene. compared to no cortisol, 10, 50, 100, 150, 250 and 500 nmol cortisol increased expression 2.8, 4.6, 7.1. 9.3, 9.9 and 9.8 fold, respectively. a 2-fold increase is considered significant (j.vandesompele et al genome biol 2002). expression of cialp was not significantly induced in response to any concentration of 17ohp (10 nm maximum), progesterone (5 nm maximum), estradiol (max 400 pm maximum) or androstenedione (100 nm maximum). we conclude that in vitro these sex hormones do not induce expression of the cialp gene which is classically induced by cortisol in vivo; indeed, elevated serum cialp activity is a hallmark of classic hac. thus, the ability of the sex hormones to induce the gene in vivo must be questioned and evaluated. measurement of sex hormones has been advocated as an adjunct means for diagnosing typical hyperadrenocorticism (hac), i.e. disease due to excess cortisol secretion, as well as for diagnosis of atypical hac, i.e. disease due to excess adrenal sex hormone secretion. however, measurements in either setting have not been widely studied. therefore, our objectives were: 1. to determine the sensitivity of 17-hydroxy-progesterone (17ohp) and estradiol concentrations pre-and post-acth for diagnosis of typical hac. 2. to determine the specificity of 17ohp and estradiol concentrations preand post-acth for diagnosis of occult hac. dogs that had pdh (n 5 12), dogs that were suspected to have hac but proven not to (had non-adrenal illness [nai, n 5 89]) or dogs that were healthy (n 5 20, used to establish reference ranges [rr]) were enrolled. acth stimulation tests were performed (5 mcg/kg cosyntropin iv); blood samples were drawn pre and 60min post; 17ohp and estradiol were measured by previously validated radioimmunoassays. a kruskal-wallis rank sum test was used to compare values between the groups. significance was set at p o 0.05. for basal and acth-stimulated 17ohp concentrations, the rr were determined to be 0.03-0.6 ng/ml (mean ae 2 s.d.; range 0.03-0.55) and 0.3-2.2 ng/ml (range 0.31-1.95), respectively. in pdh dogs, 3 and 7 had basal and post-acth 17ohp concentrations above the rr, respectively; in the nai group, 23 and 25 dogs had concentrations above the rr, respectively. thus, the sensitivity of basal and post-acth 17ohp measurement for diagnosis of hac is 25% and 58%, respectively. specificity of diagnosis is 72% and 74%, respectively. post-acth 17ohp concentration was significantly different between groups. for basal and stimulated estradiol concentrations, the rr were determined to be 81-180 pg/ml (range 89-195) and 71-170 pg/ml (range 74-151), respectively. for both basal and stimulated estradiol, 4 pdh dogs (n 5 9) had concentrations above the rr; for those with nai (n 5 80), 14 and 15 had concentrations above the rr, respectively. thus, the sensitivity of estradiol measurement for diagnosis of hac is 44% for both pre-and post-acth. specificity of estradiol for diagnosis for hac is 83% and 81% for pre-and post-acth, respectively. overall, 23 dogs with nai had at least one elevated estradiol concentration (total specificity 70%). post-acth estradiol concentration was not significantly different between groups. we conclude that use of 17ohp and estradiol concentrations for diagnosis of hac can be problematic. sensitivity and specificity are relatively low, potentially leading to misdiagnoses. diabetes mellitus is one of the most common feline endocrinopathies and is considered to have a similar pathophysiological basis to human type 2 diabetes. several studies have identified risk factors for development of diabetes mellitus in cats, which include age, obesity, inappropriate diet and physical inactivity. however, to date, no specific genetic risk factors have been identified. genome-wide association studies in humans have identified several genes that predispose to obesity and/or diabetes mellitus, one of which is the melanocortin receptor 4 (mc4r) gene. the aim of the current study was to identify polymorphisms (snps) in the feline mc4r gene and to use these to perform a case:control study to determine whether these candidate gene snps were associated with diabetes mellitus in cats. genomic dna from 10 cats (6 domestic short hair [dsh], 4 burmese) was initially analysed by pcr and direct sequencing using felmc4r-specific primers, which identified a missense mutation (mc4r:c.92 c 4 t) in the region encoding the extracellular domain of the receptor protein in dsh cats only. one hundred and nineteen dsh cats were subsequently recruited into the case:control study. fifty nine cats were obese diabetic (29 male, 30 female), mean age 11.8 years (range 6-18y); mean weight 6.68 kg (range 5.15-10 kg). sixty lean cats were used as controls (30 male, 30 female), mean age 13.81 years (range 9-19y), mean weight 3.99 kg (range 2.56-5.68 kg). the t to c base change alters a restriction site in the sequence recognized by the enzyme bstoi, such that dna from cats with the mutant (c) allele can be cut, whereas that from the wild-type (t) allele cannot. primers were designed that flanked the mutation to allow pcr amplification of this region of mc4r from genomic dna obtained from edta blood. the pcr products were purified and subject to restriction fragment length polymorphism (rflp) analysis. bstoi digestion products were then analysed by agarose gel electrophoresis. of the 59 diabetic cats, 32 (54%) were homozygous for the mutation (cc), compared to 21 (35%) of 60 control cats. statistical analysis (two tailed fisher's square test) revealed that this difference between groups was statistically significant (p 5 0.0431). in conclusion, this pilot study has identified a missense mutation in the coding sequence of mc4r. this could be an important predisposing factor for development of diabetes and/or obesity in dsh cats. polymorphisms in a similar region of human mc4r predispose to obesity, which in turn is a major risk factor for type 2 diabetes. hyperadrenocorticism (hac) is one of the most common endocrine disorders of dogs. the two most effective medical treatments are trilostane (vetoryl s ) and mitotane (lysodren s ). previous studies evaluating the effect of treatment on aldosterone secretion measured the hormone at 60 min post-acth administration. however, the optimal sampling time would be at the time of maximal secretion, which occurs 30 minutes after the 5 mg/kg dose commonly used for the test (carlson et al, jvim, 2010). thus, the true effect of either medication on aldosterone secretory capacity is unknown. our objectives were: 1) to assess and compare the effect of treatment with trilostane and mitotane in dogs with pituitarydependent hac (pdh) on aldosterone secretory reserve at 30 min post-acth stimulation and 2) to determine if changes in aldosterone concentration at that time correlate with changes in serum sodium and potassium concentrations. forty-six dogs being treated for pdh with either mitotane (n 5 30) or trilostane (n 5 16) have been enrolled. the dogs could be treated for any length of time. all had acth stimulation tests performed (5 mcg/kg cosyntropin iv); blood samples were drawn before and at 30 and 60 min post-acth for monitoring of cortisol and aldosterone concentration using previously validated radioimmunoassays. ten historical normal controls were also included. serum sodium and potassium concentrations were measured in the basal samples. a kruskal-wallis rank sum test was used to compare values between normal dogs and those treated with mitotane or trilostane. linear regression analysis was used to determine if a correlation existed between electrolyte and aldosterone concentrations or between cortisol and aldosterone concentrations. significance was set at the p o 0.05 level. acth-stimulated aldosterone concentrations in mitotane-treated but not trilostane-treated dogs were significantly lower than that in normal dogs at both the 30 and 60 min time points. no difference was detected between aldosterone concentrations at 30 and 60 min after acth injection in either treatment group. a positive correlation existed between the 60-min cortisol and 30-min aldosterone concentrations in the trilostane-treated group (r 5 0.813), i.e. the peak post-acth concentration for each hormone, but not in dogs treated with mitotane. basal serum sodium and potassium concentrations were not correlated with the basal aldosterone concentration in either treatment group. in conclusion, treatment with mitotane resulted in decreased aldosterone secretory reserve, but this did not correlate with hyperkalemia or hyponatremia. measurement of aldosterone concentrations is not predictive of electrolyte concentrations. previously presented at the auburn university phi zeta research emphasis day, november 10, 2010. antioxidant depletion is documented in humans with hyperthyroidism, and is reversible with treatment. in addition, antioxidant depletion has been shown to increase the risk of methimazole toxicity in rats. the primary aim of this study was to determine whether deficiencies in glutathione (gsh), ascorbate (aa), or vitamin e, along with increases in urinary 8-isoprostanes, were present in hyperthyroid cats, and were reversible after radioiodine treatment. a secondary aim was to determine whether antioxidant abnormalities were associated with a prior history of methimazole toxicity. ongoing prospective, controlled, observational study. otherwise healthy client-owned hyperthyroid cats presenting for radioiodine therapy (n 5 26 to date) and healthy age-matched controls (n 5 32 to date) were recruited. all cats were screened with cbc, biochemical panel, urinalysis, and t4, as well as red blood cell (rbc) gsh, plasma aa, plasma vitamin e, and urinary 8-isoprostanes. hyperthyroid cats were re-evaluated 2 months after radioiodine treatment. unlike in humans, median blood antioxidants were not significantly different in hyperthyroid cats (gsh 1.5 mm; aa 11.4 mm, and vitamin e, 19 g/ml) compared to controls (gsh 1.4 mm; aa 12.5 mm, and vitamin e, 17 g/ml). results for urinary isoprostanes are pending, and associations with methimazole toxicity will be investigated after full recruitment. rbc gsh concentrations did increase significantly (to 1.6 mm; p 5 0.019) after radioiodine treatment. however, this modest change is unlikely to be clinically significant. preliminary data do not indicate clinically significant blood gsh, ascorbate, or vitamin e deficiencies in hyperthyroid cats. with appropriate insulin therapy and a low carbohydrate diet, up to 90% of newly diagnosed diabetic cats are eventually able to maintain euglycemia without insulin administration, and these cats are considered to have achieved remission. there are currently no published data reporting the glucose tolerance status of cats classified as being in remission, and it is unknown whether these cats are truly in diabetic remission, or should be classified as non-insulin dependent diabetics, or having impaired glucose tolerance, and/or impaired fasting blood glucose. the aim of this study was to determine fasting blood glucose concentrations and glucose tolerance status of cats in remission. the study was a prospective study in a feline-only clinic. for inclusion, diabetic cats had to have achieved remission through insulin therapy, and insulin withheld for a minimum of two weeks. five diabetic cats in remission and five matched non-diabetic cats were enrolled in the study. blood samples were obtained via the ear vein but where the cat's temperament precluded this, from the jugular.glucose concentration was measured using a meter calibrated for feline blood (abbott alphatrak). a simplified glucose tolerance test was performed after food was withheld for 24 hours. a 22g catheter was placed in a cephalic vein three hours before the gtt was commenced, to minimize the effects of stress on blood glucose concentration. blood glucose concentration was measured at time 0 and then a 1 g/kg dose of glucose was administered slowly via the intravenous catheter. further blood glucose measurements were made at 2 hours and then hourly until glucose had returned to o 117 mg/dl (o 6.5 mmol/l). in the control group, all cats had a fasting blood glucose below 117 mg/dl, and following glucose administration, glucose had returned to o 117 mg/dl by 3 hours. fasting blood glucose in the remission group was o 126 mg/dl (7 mmol/l) in all cats except one, which had fasting blood glucose of 135 mg/dl (7.5 mmol/l). following glucose administration, all five cats in remission had blood glucose above 117 mg/dl (6.5 mmol/l) at three hours, four were o 117 mg/dl at four hours, and one returned to o 117 mg/dl at five hours. the cat with impaired fasting glucose subsequently became diabetic after steroid administration. the results of this study show that these cats, while no longer diabetic, have mildly impaired glucose tolerance compared to nondiabetic cats, and a minority have impaired fasting glucose. the objective of this study was to determine the role of iodine restriction in the nutritional management of cats with naturally occurring hyperthyroidism. five domestic shorthair cats ranging in age from 8-17 years were confirmed to have hyperthyroidism based on persistently increased serum total thyroxine concentrations (tt4), palpable thyroid nodule and weight loss. serum tt4 concentrations ranged from 55-146 nmol/l (reference range 10-55 nmol/l). the cats were then fed a low iodine containing food (0.47 ppm iodine dmb, as measured by epiboron neutron atomic activation). serum tt4 concentrations were measured every 3 weeks. biochemistry parameters were also evaluated at weeks 0, 6 and 9. at 9 weeks, serum tt4 concentrations had decreased in all cats with 4 of 5 cats (80%) being euthyroid (mean 48 nmol/l; range 41-54 nmol/l). the remaining hyperthyroid cat had an initial serum tt4 of 146 nmol/l, which decreased to 83 nmol/l after being fed the iodine-restricted food. mean decrease in tt4 for all 5 cats was 26 nmol/l (range 8-63 nmol/l). renal parameters remained stable in all 5 cats. these 5 cats along with 4 additional newly diagnosed hyperthyroid cats were transitioned to a similar food that contained less iodine (0.28 ppm dmb). baseline serum tt4 concentrations in the 4 new cats ranged from 55-73 nmol/l. serum tt4 and other biochemical parameters were monitored every 3 weeks for 9 weeks, and then every 4 weeks for an additional 8 weeks. with the 0.28 ppm iodine food the four new cats became euthyroid with a mean tt4 concentration of 41 nmol/ (range 29-50nmol/l). the 4 euthyroid cats from the earlier feeding study had a further decrease in tt4 concentration (mean tt4 5 39 nmol/l, range 38-54nmol/l). the single non-euthyroid cat from the first study had a serum tt4 concentration of 61 nmol/l, a decrease from the baseline concentration of 83 nmol/l. the average decrease in serum tt4 for all 9 cats was 20 nmol/l (range 2-35 nmol/l). finally, 8 of the 9 cats were fed a third iodine-restricted food (0.17 ppm dmb) along with one other newly diagnosed hyperthyroid cat (79 nmol/l serum tt4) and evaluated every 4 weeks. all 9 cats in this evaluation were euthyroid (mean tt4 33 nmol/l; range 23-50 nmol/ l). this result included the cat whose serum tt4 remained in the hyperthyroid range in the first two evaluations. the average decrease in tt4 was 13 nmol/l (range 0-43 nmol/l). biochemical features of renal function remained stable and no other biochemical abnormalities were observed. in summary, the results of these three feeding studies demonstrate that feline hyperthyroidism can be managed effectively with dietary iodine restriction. we have shown previously that restriction of dietary iodine (i) is a safe and effective method for decreasing serum thyroxine concentrations (tt4) in cats with hyperthyroidism. the objective of this study was to determine the maximum level of iodine in a nutritionally balanced feline mature adult food required to maintain normal serum tt4 concentrations in hyperthyroid cats currently being controlled on a food containing 0.15 ppm i (dmb) as measured by epiboron neutron atomic activation. all cats were previously diagnosed at least 14 months prior to the start of the study and their tt4 concentrations were maintained in the normal range by dietary iodine restriction for a minimum of 10 months (range 10 months-3 years). serum tt4 concentrations ranged from 9-42 nmol/l (reference range 10-55 nmol/l) at the beginning of the study. the cats were divided into two groups each containing 9 cats. groups were similar in age and gender distribution (mean age 5 13.8 years, range 12-18 years). one group (group a) was placed on a food that was formulated for mature adult cats containing 0.39 ppm i (dmb). the other group (group b) was placed on a similar food that differed only in that it contained 0.47 ppm i (dmb). blood was collected from all cats every three weeks and analyzed for serum tt4 concentration. biochemistry parameters were also evaluated at weeks 0, 6 and 9. all group a cats exhibited increases in serum tt4 concentration (mean increase of 25 nmol/l above baseline, range 5-48 nmol/l). seven of the cats remained in the euthyroid range (mean serum tt4 5 36 nmol/l, range-27-54 nmol/l). two cats exceeded the upper limit of the reference range (59 and 76 nmol/l respectively). the cats in group b also exhibited increases in serum tt4 concentration but to a greater degree than the cats in group a (mean increase 39 nmol/l, range 20-60nmol/l). four cats remained in the euthyroid range (mean serum tt4 5 41, range 29-49 nmol/l). the five remaining cats all exceeded the upper limit of the reference range (mean serum tt4 5 76 nmol/l, range-59-99 nmol/l). all cats returned to a euthyroid state within 1 month of being returned to a diet containing 0.17 ppm i (dmb). it was determined that serum tt4 concentrations are not ideally controlled in the normal range in hyperthyroid cats fed a food containing ! 0.39 ppm i (dmb). hyperthyroidism is a common disease in old cats. excessive production of thyroid hormones is the hallmark of the disease. three main treatments for feline hyperthyroidism include radioactive iodine, thyroidectomy, and antithyroid drugs such as methimazole. previously we have shown that limiting dietary iodine to or below 0.27 ppm induces euthyroidism in cats with hyperthyroidism compared with a similar diet containing 0.42 ppm iodine. the objective of this study was to test whether dietary iodine at 0.32 ppm would induce euthyroidism in cats with naturally occurring hyperthyroidism. fourteen cats with hyperthyroidism confirmed by serum tt 4 and ft 4 measurements were stratified into two groups based on gender and age. one group (control: 4 males and 3 females, age ranged from 11 to 15 years) was given a positive control dry cat food (0.17 ppm iodine) while the other group (test: 3 males and 4 females, age ranged from 12 to 17 years) was fed a commercial dry cat food (1.9 ppm iodine) for at least 6 weeks before the study. afterwards (week 0), the control cats continued to receive the same food while cats in the test group were given a test food (0.32 ppm iodine) for additional 12 weeks. all cats had free access to their food and deionized water during the study. blood samples were collected during weeks 0, 3, 6, and 12 of the study. the control cats maintained euthyroidism during the study. the test food significantly reduced serum tt 4 (72 ae 12, 43 ae 9 ã , 42 ae 9 ã , 40 ae 6 ã nmol/l in weeks 0, 3, 6 and 12, respectively; ã : p o 0.05 compared with week 0, dunnett's t test). it also significantly reduced ft 4 at the end of the study (17 ae 2 vs. 23ae 2 pmol, week 12 vs. week 0; dunnett's t test, p o 0.05). serum ft 4 was within the reference range (10-55 pmol/l) in cats in both groups. serum tt 3 , ft 3 , and tsh were not affected by the test food and were within the reference ranges (tt 3 : 0.6-1.4 nmol/l, ft 3 : 1.5-6 pmol/l, and tsh: 0-21 mu/l) in cats of both groups during the study. this study demonstrates that dietary iodine at or below 0.32 ppm provides an effective and inexpensive therapy for cats with naturally occurring hyperthyroidism. radioactive iodine ( 131 i) is a widely used treatment for feline hyperthyroidism. prior to 131 i administration, many cats receive methimazole therapy. it has been suggested that recent withdrawal of methimazole prior to 131 i may increase the risk of hypothyroidism, inhibit the response to therapy, or have no effect. to further address this question, a retrospective medical records search was performed to identify hyperthyroid cats that received 131 i therapy after methimazole treatment. inclusion criteria included documentation of the time interval between discontinuation of methimazole and 131 i administration, and measurement of thyroxine (t 4 ) at 7-14 days after 131 i. cats were divided into 2 groups: those receiving 131 i within 1 day of stopping methimazole, and those receiving 131 i treatment 5 or more days after stopping methimazole. sixty cats met the inclusion criteria. forty received 131 i within 1 day of stopping methimazole. of those, 20 (50%) had a low t4 (o 1.2 mcg/dl), 17 (42.5%) had a normal t4 (1.2-4.8 mcg/dl), and 3 (7.5%) had an elevated t4 (4 4.8 mcg/dl) at 7-14 days after 131 i therapy. fourteen cats received 131 i 5 or more days after stopping methimazole: 8 (57%) had a low t 4 , 5 (36%) had a normal t 4 , and 1 (7%) had an elevated t 4 at 7-14 days after 131 i therapy. the results were compared with a fisher's exact test and there was no difference between the groups (p 5 0.76). these findings indicate that stopping methimazole therapy within 1 day of 131 i therapy does not inhibit the response to therapy. pharmacokinetic studies evaluating synthetic insulin analogs such as glargine necessitate the ability to measure the blood concentrations of glargine without cross-reactivity to endogenous insulin. although the cross-reactivity between endogenous human insulin assays and synthetic analogs is often known for commerciallyavailable assays, the degree of cross-reactivity of human insulin assays with feline insulin is not. the purpose of this study was to evaluate the cross-reactivity of feline insulin with a commerciallyavailable human insulin elisa with known cross reactivity to several synthetic analogs. pre-and post-prandial blood samples were collected from four healthy cats immediately prior to and approximately 15 minutes following a meal, for a total of 8 samples. dextrose was added to the meals given to two of the cats. blood samples were immediately centrifuged and the serum was collected, aliquoted, and stored at à201c until analysis. serum insulin levels were determined in parallel with commercially-available feline insulin and human insulin elisas. the elisas were run in duplicate and according to the manufacturer's instructions. concentrations of serum insulin measured by the feline insulin elisa ranged from 12.7 ng/l to 4 700 ng/l. despite the wide range of concentrations of feline insulin, all 8 samples evaluated with the human insulin elisa yielded absorbance readings equal to or lower than the absorbance of the negative control, indicating no crossreactivity between the evaluated human insulin assay and feline insulin. since this assay is reported to cross-react significantly with glargine, it is a great candidate for determination of serum glargine concentrations in cats. the aim of this prospective, controlled study was to compare the efficacy of two trilostane protocols for treatment of canine pituitary-dependent hyperadrenocorticism (pdh). among the 28 client-owned dogs diagnosed with pdh, only the dogs weighing o 5 kg were selected (n 5 16). group a (n 5 9; low-dose treatment group) and group b (n 5 7; high-dose treatment group) received 0.8 ae 0.3 mg of trilostane/kg orally every 12 hours and 30 mg of trilostane/ body orally every 24 hours, respectively. all of the dogs were reassessed at 2, 4, 12, and 24 weeks after the initiation of treatment. the improvement in post-acth stimulation serum cortisol concentration, as well as clinical signs in group a, required more time than group b; however, 2 of 7 dogs in group b had clinical signs and abnormal laboratory findings consistent with hypoadrenocorticism after treatment for 20 weeks. twenty-four weeks later, all of the dogs of both groups improved the abnormal clinical findings. the present study suggests that twice daily, low-dose administration of trilostane is effective in the management of canine pdh and may be safe without the potential adverse effects of once daily, high-dose treatment. however, because this study involved only a small number of dogs, a population-based control study will be needed to clarify the efficacy of low-compared to high-dose trilostane treatment. cobalamin is essential for a variety of metabolic processes in many tissues and organs, and has effects on cell growth and peripheral and central nervous system function. chronic distal small intestinal disease in humans, cats, and dogs has been shown to cause cobalamin deficiency. an immunoassay for the measurement of serum cobalamin concentration in these species is being used in routine practice for the diagnosis of cobalamin deficiency. in pigs, the role of cobalamin has not yet been extensively investigated. thus, the aim of this study was to analytically validate an immunoassay, labeled for use in humans, for the measurement of cobalamin in porcine serum samples and secondly to determine serum cobalamin concentrations in weaned pigs. for the analytical validation of the assay, serum cobalamin concentrations were measured using the commercially available immulite s 2000 cobalamin immunoassay (siemens healthcare diagnostics ltd., deerfield, il, usa) in 30 surplus porcine serum samples from a variety of studies. validation of the assay consisted of determination of dilutional parallelism, spiking recovery, and intra-and inter-assay variability. additional surplus serum samples from 27 piglets from four litters at a texas a&m university farm were obtained. each piglet had been bled twice, the first at weaning (21 days of age) and the second one 12 days later. to investigate results in comparison between age groups, serum cobalamin concentrations were compared using a wilcoxon matched pairs test. significance was set at p o 0.05. observed to expected ratios (o/e) for serial dilutions ranged from 87.3 to 124.9% (mean ae sd: 104.2 ae 14.5%) for four different serum samples at dilutions of 1:1, 1:2, and 1:4, and from 96.8 to 118.3% (mean ae sd: 107.6 ae 15.2%) for one serum sample at dilutions of 1:2, 1:4, and 1:8. o/e for spiking recovery ranged from 87.4 to 116.7% (mean ae sd: 102.0 ae 7.5%) for five different porcine serum samples that had been spiked with each other in a 1:1 dilution. intraassay coefficients of variation (%cv) for five different serum samples were 4.3, 5.7, 4.3, 3.7, and 6.1%. inter-assay %cvs for five different serum samples were 4.9, 7.2, 9.6, 3.5, and 7.6%. serum cobalamin concentration was significantly lower in piglets post weaning (median: 242 ng/l) compared to those at the time of weaning (median: 324 ng/l; p 5 0.009). the immulite s 2000 cobalamin immunoassay labeled for use in humans is linear, accurate, precise, and reproducible for measurement of serum cobalamin concentrations in pigs. this study also showed that piglets that differ in age by only 12 days have significantly different serum cobalamin concentrations. further investigations of cobalamin concentrations in both sows and piglets at different stages of weaning are warranted. primigravid dairy heifers can be infected with mastitis pathogens during the periparturient period. the prevalence of intramammary infection (imi) ranges from 30-75% of quarters pre-partum and 12-45% at parturition. some pre-partum infections self-cure before parturition, however a number of these imis persist into early lactation. these imis may impact milk production and quality and may serve as a reservoir for contagious pathogens. no study has specifically investigated the risk of an imi persisting from the prepartum period into early lactation. the objectives of this study were to describe the prevalence of mastitis pathogens in heifers on a grazing dairy before and after parturition and calculate the relative risk (rr) and attributable fraction of population (afp) for the association between a post-partum and pre-partum imi. two-hundred-ninety-four heifers were systematically assigned to 1 of 3 groups: g1) pre-partum secretions from all mammary quarters (n 5 98), g2) no pre-partum secretions collected (n 5 98) and g3) pre-partum secretions from two diagonal quarters (n 5 98). group assignments were designed to assess whether pre-partum sampling increased the likelihood of imi at calving. mammary quarter secretions were collected for bacterial culture approximately 2 weeks prior to expected calving date. quarter milk samples were collected for bacterial culture once weekly during the 1 st 3-weeks of lactation. bacterial isolates were classified as staphylococci, non-agalactiae streptococci and gram-negatives. mammary quarter samples yielding 2 different bacteria were classified as mixed infections and those yielding ! 3 bacterial types were classified as contaminated. bacterial isolates were speciated using gene sequencing methods and strain-typed using pulse-field-gel-electrophorysis to evaluate the relatedness of bacteria isolated from pre-and post-partum samples from the same mammary quarter. relative risk and afp were calculated using 2 â 2 tables. forty-five percent of mammary quarters had a pre-partum imi. during the 1st 3 weeks of lactation the mean prevalence of imi was 23.3% of quarters. staphylococci were most frequently isolated bacteria from pre-partum secretions and milk with s. chromogenes and s. aureus being the most common species. using data from 228 mammary quarters, the rr and afp for the association between a post-partum and pre-partum imi were 11 and 77%, 43 and 86%, and 12 and 68% for all staphylococci, s. aureus only and cns only imis, respectively. mammary quarters sampled pre-partum were no more likely to have a post-partum imi than those not sampled (chisquare, p ! 0.27). these data demonstrate that pre-partum imis persist into early lactation and that pre-partum secretion cultures may be a useful, not only in predicting imi at calving, but also in assessing risk of introducing new contagious mastitis pathogens, e.g., s. aureus, into the lactating herd. despite concerns about antimicrobial resistance and clostridium difficile in food animals, there has been little study of the prevalence or mechanisms of resistance. this study evaluated the impact of tetracycline treatment on c. difficile shedding in veal calves and the impact on resistance. calves arriving on 1 veal farm received oral oxytetracycline for 5 days as per farm protocols. calves were sampled at arrival and 6 days later. selective culture for c. difficile was performed. isolates were ribotyped, and tested for tetracycline susceptibility and the presence of tetracycline resistance genes. multivariable logistic regression models were used to determine the relationship between tetracycline resistance and the presence of tetracycline resistance genes. clostridium difficile was isolated from 32% (56/174) and 51% (88/ 172) calves, at the first and second samples, respectively. the percentage of tetracycline resistant isolates increased from 79% to 93%. isolates from the second sample were 3 times more likely to be tetracycline resistant (p 5 0.016) and 5 times more likely to possess tet(m) (p 5 0.004). tet(m) was detected in 13% (7/53) and 43% (39/ 91), tet(o) in 23% (12/53) and 19% (17/91) and tet(w) in 2% (1/53) and 1% (1/91) of isolates from first and second samples, respectively. tet(l), tet(k) and tet(s) were not detected. 54 resistant isolates were not carrying any of the genes investigated. routine tetracycline use may have had an impact on both the prevalence of c. difficile, as well as the strain distribution and resistance patterns. this is the first report of presence of tet ( the objectives of this study were to 1) estimate the prevalence of antimicrobial resistance in the study population and 2) to investigate the associations between exposures to antimicrobial drugs and antimicrobial resistance in fecal non-type specific e. coli (ntsec) recovered from individual feedlot cattle. two-stage random sampling was used to identify cattle for enrollment at 4 western canadian feedlots. a fecal sample was collected per rectum from each individual at arrival and in the middle of the feeding period when cattle were rehandled as part of standard feedlot protocol. from samples collected at this second time point, a total of 2,133 ntsec isolates were tested for susceptibility to antimicrobial drugs by disk diffusion. parenteral and in-feed exposures to antimicrobial drugs were recorded for each individual enrolled in the study. the least square means estimates and 95% confidence intervals for the prevalence of resistance at each time point were modeled using poisson regression. multivariable logistic regression was used to investigate associations between antimicrobial resistance and exposure to antimicrobial drugs. regression models were adjusted for clustering of observations among individuals and pens. the most common resistances identified in arrival samples were sulfisoxazole (7.5%; 95%ci: 6.1-9.2), streptomycin (7.7%; 95%ci: 6.3-9.5) and tetracycline (20.0%; 95%ci: 17.7-22.6). at the second sampling point, resistance prevalence was 25.6% (95%ci: 23.5-28.0) for sulfisoxazole, 25.0% (95%ci: 22.8-27.3) for streptomycin, and 72.7% (95%ci: 70.5-75.1) for tetracycline. logistic regression modeling identified weak associations of exposures to tetracycline and macrolide classes of drugs with antimicrobial resistance at the second time point. abstract fa-5 premature/dysmature syndrome in cria: a ret-rospective study of 63 cases (1999) (2000) (2001) (2002) (2003) (2004) (2005) . c. gerspach, d. anderson. the ohio state university, columbus oh. prematurity is widely acknowledged as risk factor for subsequent morbidity and mortality in llama and alpaca cria. a review of medical records for premature cria alive at the time of admission to the veterinary teaching hospital between 1999 and 2005 was performed to determine risk factors of prematurity and to report the outcome and related conditions or diseases in affected cria. medical records for 63 premature or dysmature cria were included in this study. of these cria, 51 were alpaca and 12 llama, 36 were female and 27 were male. reasons for referral were prematurity, failure of passive immunity, dyspnoea, weakness and failure to gain weight. cria were presented at a mean age of 1.4 days and were premature by a mean estimated time of 19.5 days. overall survival rate was 82.5%, with all llama cria surviving. a multivariate logistic regression model was used to identify risk factors associated with not surviving. cria receiving camelid colostrum had a significant better outcome than cria receiving no colostrum or colostrum from different species. dyspnea and tachypnea was associated with a poor outcome. all cria that were able to nurse, without assistance prior to referral, survived. clinical pathology parameters most commonly associated with death were hyperphosphatemia and acidosis. enrofloxacin is approved for the treatment of swine respiratory disease, however there are no published studies describing the pharmacokinetics of enrofloxacin at the approved dose and route in pigs (7.5 mg/kg subcutaneously). furthermore no studies have assessed the unbound concentrations of enrofloxacin at its site of action, the extracellular tissue fluid. therefore the objective of this study was to use an in-vivo ultrafiltration method to measure the active fraction of enrofloxacin, and the metabolite ciprofloxacin, at 3 tissue sites relevant to pigs, and to compare these concentrations with plasma concentrations collected at similar time points. six healthy pigs were used in this study. pigs were recently weaned and weighed an average 16.3 kg. on the day before the experiment, pigs were anesthetized for the placement of jugular vein sampling catheters and interstitial fluid collection probes. three ultrafiltration probes were placed in each pig in a subcutaneous site near the right shoulder, an intramuscular site along the epaxial muscles, and in the pleural space of the chest cavity. each pig received an injection of enrofloxacin (baytril 100, bayer animal health) at a dose of 7.5 mg/ kg subcutaneously behind the left ear. plasma and interstitial fluid samples were collected at pre-determined time points, and enrofloxacin and ciprofloxacin concentrations were measured using hplc with fluorescence detection. protein binding was determined with a microcentrifugation system. pharmacokinetic data was analyzed using a one compartment model. the analysis of plasma and isf showed that only a small fraction of ciprofloxacin was produced in these pigs, therefore ciprofloxacin concentrations were not used in pharmacokinetic measurements. the plasma half-life (t 1/2 ), volume of distribution, clearance, and peak concentration (c max ) for enrofloxacin was 25.9 hr (ae 6.2), 6.29 l/kg (ae 1.23), 0.168 l/kg/hr (ae 0.076), and 1.07 mg/ml (ae 0.28), respectively. the concentrations from each of three tissues were not different in each pig. when pharmacokinetic values from all tissues were combined for the isf, the t 1/2 was 23.6 hr (ae 4.1) and the c max was 1.26 mg/ml (ae 0.10). the enrofloxacin plasma protein binding was 31.1% (ae 3.28) and 37.13% (ae 16.54) at a high and low concentration, respectively. this study has demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. the half-life of enrofloxacin is longer in tissues and plasma than has been reported in previous studies. the high tissue concentrations and long half-life produce an auc/mic ratio sufficient for the pathogens that cause respiratory infections in pigs. ceftiofur crystalline free acid (ccfa), a long-acting ceftiofur formulation labeled for use in cattle, pigs, and horses for treatment of respiratory disease has been used for treatment of ovine respiratory infections in clinical practice. pharmacokinetic data, however, do not exist for ccfa administered subcutaneously in sheep. the present pharmacokinetic study evaluated the single dose subcutaneous administration of ccfa in sheep (n 5 9) at 6.6 mg/kg body weight. concentrations of ceftiofur free acid equivalents (cfae) in plasma were measured by high performance liquid chromatography for 14 days following drug administration. pharmacokinetics of subcutaneous ccfa in sheep were best described using a single compartment model with the following average (ae sd) parameters: area under the concentration time curve 0! 1 (206.6 hãug/ml ae 24.8), observed maximum plasma concentration (2.4 ug/ ml ae 0.5), and observed time of maximum plasma concentration (23.1 h ae 10.1). no significant adverse drug reactions were observed. adequate cfae plasma concentrations were attained to effectively treat respiratory tract pathogens associated with pneumonia in sheep. the purpose of this study was to assess, using thoracic ultrasonography, the prevalence of lung lesions in pre-weaned dairy calves. subsequent aims were to describe ultrasonographic changes within the lung, clinical respiratory score, and treatment of respiratory disease. a longitudinal study was performed using female dairy calves from 6 commercial dairy farms in new york state. calves were enrolled based on age. thoracic ultrasound and clinical respiratory scoring were performed on each calf at 2 time points. a standard 5 mhz linear ultrasound probe was utilized to evaluate intercostal spaces 1 through 11 of each hemi-thorax with the calf in lateral recumbency (us1) or standing (us2). lesion appearance, size, and location were recorded. respiratory score (rs) was assigned based on a previously published protocol incorporating fever, nasal discharge, cough, ocular discharge and ear droop, with a higher numerical score corresponding to more severe disease. abnormal lung on ultrasound was defined as one or more areas of !1 cm width or depth of non-aerated lung. farm records were evaluated to identify treated calves. calves were treated for respiratory disease at the farm manager's discretion, not based upon ultrasound findings or rs. non-parametric methods were used to evaluate the data. ninety-one calves were enrolled into the study, with 6 lost to follow-up. an average of 4 minutes was spent performing the rs and ultrasound on each calf. the median ages at first (us1) and second (us2) examination were 13 (interquartile range 12-15) and 46 (interquartile range 44-47) days, respectively. the majority of calves had a low rs (o 5) and only 3.2% of calves had a rs high enough to warrant treatment based on previous recommendations (rs!5). the prevalence of calves that had abnormal lungs on ultrasound but a low rs (o 5) was 5.5% (us1) and 16.5% (us2). the prevalence of calves that had abnormal lungs on ultrasound and a high rs (! 5) was 0% (us1) and 3.5% (us2). of the calves that had abnormal lungs on ultrasound but a low rs, 13% were treated with antimicrobials within 7 days of examination. none of the calves with high rs and abnormal lungs on ultrasound were treated with antibiotics within 7 days of examination. this study demonstrates a high prevalence of abnormal lungs, as detected by thoracic ultrasonography, without significant clinical signs in pre-weaned dairy calves. the relatively low treatment rate in these calves may suggest an area of opportunity for improvement in calf health, welfare, and herd longevity. further studies and follow up are needed to elucidate the significance of these findings and whether or not treatment is indicated. literature regarding diseases causing lameness in beef cattle is limited. this retrospective study was undertaken to examine beef cattle presented for lameness. medical records of beef cattle having a lameness examination done during the period 2007 to 2010 were reviewed and descriptive statistics generated. lameness was classified based on clinical diagnosis. the medical records of 270 beef cattle were reviewed of which 63.2% were male and 36.8% were female. beef cattle presented for lameness most often during the summer months (34%) and least during autumn (18%). causes of lameness were categorized as infectious (44.6%) or non-infectious (55.4%) and infectious lameness subcategorized as either a primary disorder or a secondary infection. all cases of a primary infectious disorder were interdigital phlegmon. secondary infections diseases included sole abscess (25.9%), septic arthritis (11.1%), tenosynovitis (2.8%), and pedal osteitis (1.3%). non-infectious lameness included proximal limb lameness (19.6%), foot trauma (14.6%), hoof horn cracks (9.5%), hoof defects (2.5%), interdigital fibromas (1.9%), overgrown hooves (1.9%), sole bruise (1.3%), subclinical laminitis (1.3%), white line disease (0.9%), osteoarthritis (0.9%), heel erosion (0.3%), sole ulcers (0.3%), and sole hemorrhage (0.3%). the most frequently affected claw was the lateral digit of the hind limb (36.4%), followed by the medial digit of the front limb (27.1%), lateral digit of the front limb (23.6%), and the medial digit of the hind limb (12.9%). the findings of this study suggest significant differences in the frequency of disease causing lameness in beef cattle compared to published reports for dairy cattle. in people, endoscopic ultrasound (eus) has become the technique of choice for assessing pancreatic disease and eus-guided fineneedle aspiration (eus fna) has proven a useful and safe modality for characterizing pancreatic lesions. reported complications include infections, bleeding and acute pancreatitis. in dogs, laparoscopic-assisted pancreatic biopsy has been suggested to be a safe procedure, however eus and eus fna have not been evaluated in dogs so far. thus the aim of the present study was to assess the practicability and safety of eus examination of the abdominal cavity as well as pancreatic eus fna in healthy dogs. this study was approved by the cantonal committee for the authorization of animal experimentation, zurich, switzerland. the study population consisted of 14 healthy beagle dogs with a median bodyweight of 13.4 kg (10.9-15.7). eus was performed with an olympus gf-uc140p-echoendoscope and fna were performed using 19 g needles (cook echotipultra). after completion of the eus-examination of the abdominal cavity from the stomach (liver, gallbladder, bile ducts, kidneys, adrenals, pancreas), the scope was advanced into the duodenum and eus fna of the pancreas was performed. fna tissue acquisition was made applying negative pressure and 6 to 8 needle passes were made. all dogs received 30 mg/kg metimazole im after eus fna and were re-checked ultrasonographically 20 minutes post eus fna. postoperative activity was assessed using a standardized scoring system. a cbc, serum biochemistry, urinalysis and spec cpl s were measured before, as well as 24 and 48 h after eus fna. the eus examination was complete in 13/14 dogs, the pancreas could not be visualized in 1 dog. the pancreas was hypo-(4/13) to isoechoic (9/13) to the surrounding mesenterium in all cases. in 3/13 dogs parts of the pancreas presented hyperechoic. the mean measured thickness was 0.88 cm. the pancreas was aspirated in 12 dogs using a transgastric approach (3) or transduodenal approach (9). duodenal transmural puncture was not accomplished in 1 dog where a re-sterilized needle was used. a minimal amount of peripancreatic fluid was observed in 1/12 dogs after eus fna. all dogs recovered uneventfully and required no further analgesia. all laboratory results including the spec cpl s measurements were within reference ranges on all three time points. cytologically, conglomerates of exocrine pancreatic cells were seen in 8/12 cases, duodenal villous epithelial cells were seen in 11/12 cases. in 1 dog the aspirated pancreatic material was sufficient for a histological assessment. the 8 aspirates with exocrine pancreatic cells on cytology were obtained by transgastric (4) and transduodenal (4) aspirations. in conclusion, (1) eus examination of the abdomen is feasible in medium-sized dogs, (2) the healthy canine pancreas can be difficult to visualize completely, and (3) eus-guided pancreatic fna using a 19 g needle is a safe procedure in healthy dogs. studies evaluating its use in dogs with pancreatic disease are warranted to assess its clinical utility. miniature schnauzers have a high prevalence of idiopathic hyperlipidemia, which is characterized by an increased serum triglyceride (tg) concentration, with or without an increased serum cholesterol (chol) concentration. a common initial therapeutic approach for the management of hyperlipidemia is the use of a low-fat diet. also, it is believed that low-fat diets may be beneficial in the treatment of pancreatitis in dogs. however, the efficacy of this approach has not been evaluated for either condition. the aim of the present study was to evaluate the effect of a commercially available low-fat diet on serum concentrations of tg, chol, and canine pancreatic lipase immunoreactivity (cpli; measured as spec cpl s ) in apparently healthy miniature schnauzers with hypertriglyceridemia. blood samples were collected from 15 apparently healthy miniature schnauzers with hypertriglyceridemia (serum triglyceride concentrations 4 108 mg/dl). common causes of secondary hyperlipidemia were excluded based on historical information, physical examination findings, and the measurement of serum glucose, total t4, and free t4 (by ed) concentrations. the owners of the dogs were asked to switch their dog to the study diet (royal canin gastrointestinal low fat s ; fat content: 18.6 g/1,000 kcal) and have a second blood sample collected 8 weeks after their dog had been on the new diet. all blood samples were collected after food had been withheld for 15 hours. serum tg, chol, and spec cpl concentrations were measured both before and after the diet change. results were compared between the two time-points using the wilcoxon signed rank and fisher's exact tests. serum tg concentrations were significantly higher before (median: 432 mg/dl) than after the diet change (median: 178 mg/dl; p 5 0.003). the proportion of dogs with hypertriglyceridemia was significantly higher before (15/15) than after the diet change (10/15; p 5 0.042). also, the proportion of dogs with serum tg 4 500 mg/dl was significantly higher before (6/15) than after the diet change (0/15; p 5 0.016). serum chol concentrations were significantly higher before (median: 296 mg/dl) than after the diet change (median: 258 mg/dl; p 5 0.004). the proportion of dogs with hypercholesterolemia was significantly higher before (8/15) than after the diet change (0/15; p 5 0.006). finally, the difference in serum spec cpl concentrations before (median: 88 mg/l) and after the diet change (median: 56 mg/l) approached but did not reach significance (p 5 0.052). also, the proportion of dogs with high serum spec cpl concentrations before (4/15) and after the diet change (0/15) was different, but this difference was not significant (p 5 0.099). in summary, a commercially available low-fat diet was effective in reducing serum tg and chol concentrations in miniature schnauzers with hypertriglyceridemia. toll-like receptor 5 (tlr5) is an extracellular pattern recognition receptor which recognizes flagellin present in motile bacteria. we have previously demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the tlr5 gene (g22a, c100t and t1844c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). recently, we have confirmed that two of these tlr5 snps (c100t and t1844c) are significantly associated with ibd in other canine breeds. to further substantiate the role of tlr5 in canine ibd functional analysis of these polymorphisms would be needed. therefore the aim of this study was to determine the functional significance of the tlr5 snps by transfecting wild-type and mutant receptors in to human embryonic kidney cells (hek) and carrying out nuclear factorkappa b (nf-kb) luciferase assay and il-8 elisa. the tlr5 gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) as determined by the case-control analysis in gsds with ibd were cloned into plasmids expressing yellow-fluorescent protein (yfp). these were then stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were then stimulated with various ligands (0.1 mg/ml flagellin, 0.01 mg/ml flagellin, 1 mg/ml lps, 1 mg/ml pam3csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk) according to the manufacturer's recommendations. the supernatants were harvested and used in an il-8 elisa (r&d systems). human tlr5 transfected hek cells (invivogen) served as positive controls in all experiments. independent t-test was used to determine the significance of relative luciferase activity and il-8 concentration between wild-type and mutated tlr5 cells. although there was no significant difference between the wild-type and mutated receptor when they were stimulated with 0.01 mg/ml of flagellin (p 5 0.14), there was a significant increase when the cells with mutated tlr5 were stimulated with 0.1 mg/ml of flagellin compared to the cells expressing wild-type tlr5 (p 5 0.027). similarly, there was a significant increase in il-8 concentration in the supernatants in the cells with the mutated tlr5 receptor when stimulated with 0.1 mg/ml flagellin compared to the wild-type (p 5 0.025-one-tailed, 0.05-two-tailed) but not with 0.01 mg/ml flagellin (p 5 0.26). we show for the first time that polymorphisms associated with ibd are functionally hyper-responsive to flagellin compared to the wild-type receptor. this suggests that tlr5 may play a role in canine ibd and that blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. however, further in-vivo functional analysis of tlr5, especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions. tlr5 has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (ibd). similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the canine tlr5 gene (g22a, c100t and t1844c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). therefore the aim of this study was to determine the functional significance of the tlr5 snps in the breed of gsds. the tlr5 gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) were stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were stimulated with various tlr ligands (0.1 mg/ ml flagellin, 0.01 mg/ml flagellin, 1 mg/ml lps, 1 mg/ml pam3csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk). the supernatants were harvested and used in an il-8 elisa (r&d systems). peripheral whole blood from dogs carrying the wild type and mutant tlr5 genes was cultured and stimulated with tlr ligands as above. canine tnf-alpha was measured in the supernatant by commercially available elisa (r&d systems). t-test was used to determine differences of relative luciferase activity, il-8 concentration and tnf-alpha concentration between wild-type and mutated tlr5 cells. there was a significant increase in nf-kb activity when the cells with mutated tlr5 were stimulated with 0.1 mg/ml of flagellin compared to the cells expressing wild-type tlr5 (p 5 0.027), which correlated with il-8 expression in the supernatant (p 5 0.26). similarly, in the whole blood assay the tlr5 risk haplotype for ibd in gsds (acc) was significantly hyperresponsive to flagellin at a concentration of 0.1 mg/ml compared to the tlr5 wild-type haplotype (gtt) (p 5 0.001). we show for the first time that polymorphisms associated with canine ibd in gsds are functionally hyper-responsive to flagellin compared to the wild-type receptor. blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. proton pump inhibitors (ppi) are widely used in human and also veterinary medicine. side-effects of ppi treatment reported in people are atrophic gastritis, gastric and esophageal cancer, and rebound hyperacidity following cessation of treatment, which has been speculated to be due to a sustained increased in circulating gastrin concentration. moreover, long-term ppi treatment has been associated with an increased risk for osteoporosis in people. little is known about the effect of ppi treatment on serum gastrin concentration or calcium metabolism in dogs. eight healthy adult research dogs (4 males and 4 females) were enrolled into the study. the dogs received an average dose of 1.1 mg/ kg of omeprazole orally twice daily for 15 days. blood samples were collected prior to initiating the treatment and every 3 days during the 15 days of treatment and during the 15 days after discontinuation of treatment for determination of serum gastrin, ionized calcium, pth, and 25 oh vitamin d3. gastric fluid was collected via gastroscopy after an overnight fast for measurement of gastric ph prior to, during, and after the omeprazole treatment period. normally distributed data were compared with a repeated measures anova and post hoc dunnett's test. data that were not normally distributed were compared with a friedman's test and a post-hoc dunn's test. gastric fluid ph was significantly higher (p o 0.01) at the end of the treatment period (median: 7.4; range: 4.2-8.1) when compared to pretreatment values (median: 1.7; range: 1.0-6.8). serum gastrin concentrations increased significantly from a median baseline of 10.0 ng/l (range: 10.0-27.0) to a maximum median of 379.5 ng/l (range: 49.9-566.0) at day 9 of treatment (p o 0.01). serum gastrin remained significantly increased above baseline values from day 6 to day 15 of the treatment, but was not different from pre-treatment values 3 days after the end of the treatment. omeprazole treatment had no effect on ionized calcium or pth for the duration of the study. marginal, but significant changes of 25 oh vitamin d3 were observed at day 15 (end of the treatment period -increased by 13.8%) and day 21 (6 days after the end of the treatment -decreased by 14.7%). this study shows that treatment with omeprazole for 2 weeks results in a profound and sustained increase in serum gastrin concentration in dogs. this effect is rapidly reversible after cessation of the treatment. no effect on calcium metabolism was observed. however, this study documents only the effect of short-term treatment and it is possible that the effects of long-term administration are different. omeprazole treatment has been associated with small intestinal bacterial overgrowth and a higher risk for infectious enteropathies in humans. using a semi-quantitative sequencing approach, we have previously shown that omeprazole treatment may lead to alterations in both duodenal and gastric bacterial populations in healthy dogs (acvim 2010). however, a sequencing approach can only estimate relative proportions of genomic bacterial targets. therefore, significant changes in the total number of bacteria could not be evaluated. the aim of this study was to quantify gastric and duodenal bacterial populations in dogs undergoing omeprazole treatment. eight 9 month-old healthy research dogs (4 males and 4 females) were enrolled. the dogs received an average dose of 1.1 mg/kg of omeprazole orally twice a day for 15 days. endoscopic gastric and duodenal biopsies were harvested 30 and 15 days before starting omeprazole treatment, on the last day of treatment (day 15), and 15 days after the end of treatment (day 30). all biopsies were fixed in 10% formalin for 24 hours, processed, and embedded in paraffin blocks. fluorescent in situ hybridization was used to quantify mucosa-associated bacteria using fluorescently-labeled probes targeting the 16s ribosomal rna. statistical analysis aimed to compare changes in helicobacter spp. in gastric biopsies and total bacteria in both gastric and duodenal biopsies using the glimmix and npar1way procedures in sas s 9.2. bacteria were counted in 1,174 and 989 microscopic fields (63â) obtained from 155 and 132 gastric and duodenal biopsies, respectively. in the stomach, omeprazole treatment led to a decrease in helicobacter spp. (log of average counts ae standard error: 1.98 ae 0.14 at day 15) when compared to the counts 30 (2.43 ae 0.13, p0.0001) and 15 (2.40 ae 0.13, p0.0001) days before treatment. after completion of omeprazole treatment, helicobacter spp. increased and returned to baseline counts (2.45 ae 0.13 at day 30, p0.0001 vs day 15). also, in the stomach, non-helicobacter spp. bacteria were observed more often during omeprazole treatment (median: 3, range: 0-20) than on days 30 (median: 0, range: 0-3) and 15 (median: 1, range: 0-6) before and 15 days after (median: 0, range: 0-2) omeprazole treatment; however, statistical comparison across time points did not reach significance. in the duodenum, while the median number of bacteria for all time points was zero, non-parametric comparison of median scores (number of points above median) revealed significantly higher numbers of bacteria during omeprazole treatment (p 5 0.0063). our results suggest that omeprazole treatment for 2 weeks leads to a lower abundance of helicobacter spp. organisms in the stomach of healthy dogs. also, this transient decrease in helicobacter spp. was accompanied by a higher abundance of other bacteria in both the stomach and the proximal duodenum. the smartpill ph.p s capsule (the smartpill corporation) is a wireless motility capsule that measures ph, pressure, and temperature as it passes through the gastrointestinal (gi) tract. analysis of this data allows the calculation of gastric emptying time (get), small and large bowel transit time (slbtt), and total gi transit time (tgtt). this study evaluated the variability associated with repeated measurement of gi transit times and the effect of oral administration of ranitidine (zantac s ) on gi transit times in dogs using this system. it was hypothesized that ranitidine would reduce gi transit times. six privately owned healthy adult dogs weighing between 25.0 kg and 41.0 kg were used. on 3 occasions each dog was fed a standard meal followed by oral administration of a capsule. data were recorded until the capsule had passed in the dog's feces. on a 4th occasion each dog was given 75 mg of ranitidine po q12 hrs starting 48 hrs prior to testing. the dogs were then fed the test meal and the capsule was administered as above. ranitidine was given until the capsule had passed in the dog's feces. proprietary smartpill software was used to calculate get, slbtt, tgtt, and the median gastric ph (mgph). mean intra-individual and inter-individual coefficients of variation (cv%) were calculated for get, slbtt, and ttt for the first 3 time points. transit times and gastric ph recorded at all 4 time points were compared using a repeated measures anova. where significant differences were identified, post-hoc testing was performed using a bonferroni's multiple comparisons test. significance was set at p o 0.05. a sharp rise in ph indicating exit of the capsule from the stomach was identified in each experiment. mean (ae sd) get, slbtt, and tgtt without ranitidine were 775 ae 144, 1563 ae 614, and 2338 ae 577 min, respectively. mean get, slbtt, and tgtt during treatment with ranitidine were 719 ae 11, 1442 ae 885, and 2155 ae 897 min, respectively. mean intra-individual cv% before ranitidine for get, slbtt, and tgtt were 12.9, 29.7, and 17.8%, respectively. mean inter-individual cv% before treatment with ranitidine for get, slbtt, and ttt were 16.1, 40.5, and 24.7%, respectively. no significant differences in get, slbtt, or tgtt were found at any of the 4 time points. the mean mgph during treatment with ranitidine (ph 2.85) was significantly higher than at all other time points (overall mean ph for the 3 time points: 1.73; p o 0.05). the smartpill system is an easy to use, ambulatory, non-invasive, non-radioactive method for assessing gi transit times in medium to large breed dogs. measurements of gi transit times, especially slbtt, were subject to considerable intra-individual and interindividual variation. no significant effect of oral ranitidine on gi motility was identified in this group of dogs. however, as expected, oral ranitidine caused a significant increase in gastric ph. the intestinal microbiota has been implicated in the pathogenesis of various gastrointestinal disorders in both humans and dogs. recent metagenomic data suggest that specific bacterial groups, including bacteria within the clostridium clusters iv and xiva (i.e., faecalibacterium spp., ruminococcaceae, and lachnospiraceae) and bifidobacterium spp. are decreased, while proteobacteria are increased in dogs with clinical signs of gastrointestinal disease. the objective of this study was to establish quantitative polymerase chain reaction (qpcr) assays for these specific bacterial groups and evaluate their abundance in healthy dogs and dogs with clinical signs of gastrointestinal disease. fecal samples were collected from 21 healthy dogs (14 females and 7 males) and 20 dogs with clinical signs of gastrointestinal disease (11 females and 9 males). novel quantitative pcr assays were established for faecalibacterium spp., ruminococcaceae, and lachnospiraceae by aligning respective group specific sequences against canine specific sequences obtained from 16s rrna gene clone libraries and sequences available from the ribosomal database project. primers for bifidobacterium spp. and proteobacteria were selected from previously published studies. the specificity of the qpcr assays was confirmed by sequencing of obtained qpcr amplicons. the bacterial dna abundance in fecal samples was compared between healthy dogs and dogs with clinical signs of gastrointestinal disease using a mann-whitney u test. significance was set at p o 0.05. a significantly lower abundance of faecalibacterium spp. (p o 0.01) and ruminococcaceae (p 5 0.02) was observed in dogs with clinical signs of gastrointestinal disease when compared to healthy dogs. proteobacteria were more abundant in dogs with clinical signs of gastrointestinal disease, but this difference did not reach statistical significance (p 5 0.07). there was no significant difference in the abundance of lachnospiraceae (p 5 0.23) and bifidobacterium spp. (p 5 0.77) between both groups. in conclusion, we established novel qpcr assays for faecalibacterium spp., ruminococcaceae, and lachnospiraceae. we observed significant decreases in the abundance of faecalibacterium spp. and ruminococcaceae in dogs with clinical signs of gastrointestinal disease. these bacterial groups are considered major short-chain fatty acid producers and studies are warranted to determine if a decrease in these bacterial groups is associated with decreases in short chain fatty acid production. further studies are also needed to determine if these bacterial shifts are associated with specific gastrointestinal disorders. the pathogenesis of chronic enteropathies (ce) in dogs likely involves complex interaction between the mucosal immune system and the intestinal microbiota. while the application of bacterial 16s rdna sequence-based analysis has shown an association between altered microbial composition and duodenal inflammation in dogs, relatively little is known about alterations in non-invasive mucosal and luminal bacteria seen with diseases involving the ileum and colon. the present study sought to evaluate the relationship of enteric bacteria to type and severity of mucosal inflammation affecting the ileum and colon of dogs with ce. eleven client-owned dogs with ce involving both the small and large intestines were prospectively enrolled. ce was diagnosed on the basis of a history of chronic gastrointestinal signs, exclusion of identifiable underlying disorders, and histopathologic evidence of intestinal inflammation. mucosal bacteria were detected in formalinfixed ileal and colonic tissue sections with fluorescence in situ hybridization (fish) using 16s rdna-targeted probes directed against all bacteria, enterobacteriaceae, e. coli, eubacterium rectale-clostridium coccoides group, bacteroides/prevotella, and helicobacter spp. sections were examined by epifluorescence microscopy and the number of bacteria and their spatial distribution (luminal, superficial mucus, epithelial adherent, within mucosa) was determined in ten 60x fields of each section. microbial composition in ce dogs was compared to the ileal/colonic microbiota of 7 healthy control (hc) dogs using a mixed effect anova model. p values o 0.05 were considered significant. the final diagnoses for dogs with ce included ibd (n 5 8) and lymphosarcoma (n 5 3). when compared to hc dogs, dogs with ce showed regional (ileum versus colon) imbalances in microbiota composition characterized by selective enrichment of mucosa-associated populations. evaluation of colonic biopsies in dogs with ce showed that the total number of bacteria (p o 0.04), clostridium (p o 0.005), enterobacteriaceae (p o 0.04) and e. coli (p o 0.03) were increased in the adherent mucus regions of dogs with ibd as compared to hc dogs. total bacteria (p o 0.05) and e. coli (p o 0.02) were also more numerous in dogs with lsa versus hc and ibd dogs (p o 0.04 for e. coli). ileal biopsies from ce dogs similarly showed variable dysbiosis with increased total bacteria (p o 0.05) but decreased helicobacter spp (p o 0.001) and bacteroides (p o 0.02) observed within inflamed intestines as compared to hc tissues. the spatial distribution of these bacteria was also appreciably different from hc dogs, with higher numbers of bacteria generally found within the adherent mucus compartment as compared to other ileal regions. our data demonstrate that dogs with ce affecting the ileum and colon have altered microbiota composition that may be a cause or consequence of mucosal inflammation. recognition of these microbiota imbalances may provide new opportunities for therapeutic intervention. trichomonads have been rarely reported in the feces of dogs and their pathogenicity remains uncertain. although pentatrichomonas hominis (ph) is considered to be a commensal that may overgrow in dogs with other causes of diarrhea, little is known regarding the history, clinical presentation or prevalence of concurrent gi infections in dogs with trichomonosis. the aim of this study was to determine whether dogs with diarrhea and trichomonosis could be distinguished from dogs having diarrhea without trichomonosis on the basis of clinical signs or presence of concurrent enteric infections. fecal samples from 39 dogs were submitted to ncsu from 2007-2010 for trichomonas spp. pcr testing. dna was extracted using a zr fecal dna mini-prep kit and absence of pcr inhibitors verified by amplification of bacterial 16s rdna. pcr for ph and tritrichomonas foetus (tf) was performed as well as real-time pcr assays for 9 possible concurrent enteric infectious agents. obtainable medical records were reviewed. all submitted fecal samples were submitted from dogs with diarrhea that was variably described as soft, mucoid, hemorrhagic, or watery. mean age of the dogs was 2.33 years (median 1.9; range: 2.5-120 months) and represented a total of 24 breeds. ph, tf, or concurrent ph and tf were diagnosed in 18, 1, and 1 dogs respectively (group a). the remaining 19 dogs were negative for ph and tf by pcr no dogs were identified as infected with canine distemper virus or parvovirus. five samples from each group had insufficient quantity or quality of dna for concurrent infectious disease testing. in this large study of canine trichomonosis, no differences in age, clinical signs, or prevalence and identity of concurrent enteric infection between diarrheic dogs with or without ph were identified. thus, these findings do not appear to support a primary pathogenic role for ph as a causative agent of diarrhea in dogs. gastrointestinal motility disorders are a common clinical problem in domestic animals. many of the g.i. motility disorders have been treated previously with 5-ht 4 agonists although limited availability of drugs in this classification have stimulated interest in the use of new (and old) drug therapies. the dopaminergic antagonists are a group of drugs with well-known anti-emetic effects at central dopamine d 2 receptors, and putative gastrointestinal prokinetic effects at peripheral d receptors. domperidone has been shown, for example, to reverse gastric relaxation induced by dopamine infusion in the dog. similar studies have not been reported in the cat or rabbit, two species at risk for distal gastrointestinal motility disorders. our aim was to study the effects, mechanisms, and sites of action of domperidone in feline colonic and rabbit gastrointestinal smooth muscle contraction. portions of stomach (fundus and antrum), intestine (duodenum and ileum), cecum (rabbits only), and colon (ascending and descending) were obtained from healthy cats and rabbits from 9-12 months of age. longitudinal and circular smooth muscle strips from each site were suspended in physiologic (hepes) buffer solution, attached to isometric force transducers, and set to optimal muscle length (l o ) using acetylcholine (ach; 10 à4 m). muscle strips were treated with domperidone (d; 10 à8 to 10 à4 m) in the presence or absence of ach (10 à8 to 10 à4 m), and maximal force output (p max ) was normalized for cross-sectional area (n 5 â 10 4 newtons/m 2 ). domperidone (d) had a minor direct effect of inducing feline and rabbit gastric, cecal, and colonic smooth muscle contraction. direct effects were similar whether in the longitudinal or circular muscle orientation. the direct effect of domperidone was dose-dependent and maximal (feline colon p max 5 0.15-0.22 n; rabbit colon p max 5 0.10-0.19 n) at a dose of 10 à4 m. domperidone had a much greater indirect effect in augmenting cholinergic (ach; 10 à4 m) contractions in feline and rabbit gastric, cecal, and colonic smooth muscle. domperidone-augmented cholinergic contractions were 157-200% (feline colon p max 5 1.54 ae 0.24 n ach only; feline colon p max 5 2.03 ae 0.23n ach 1 d) of baseline cholinergic contractions. domperidone contractions were of a similar magnitude to those induced by cisapride. domperidone effects were similar in mucosaintact and mucosa-dissected preparations. domperidone contractions were unaffected by prazosin (a 1 receptor antagonist), yohimbine (a 2 receptor antagonist), or terbutaline (b 2 receptor agonist), but were somewhat attenuated by dopamine (d 2 receptor agonist) and a non-specific cholinergic antagonist (atropine). in vitro studies show for the first time that domperidone has minor direct and major indirect effects in augmenting cholinergic contractions of feline and rabbit gastrointestinal (stomach, cecum and colon) smooth muscle. as recognition of acute and chronic pain in dogs has increased, so too has the use of non-steroidal anti-inflammatory drugs (nsaids) often in conjunction with tramadol. in people and rats, co-administration increases the risk of perforation and gastric injury over nsaids alone. using an ex vivo model of acid injury in canine gastric mucosa, we examined the effects of indomethacin and tramadol on gastric permeability and concentrations of gastroprotective prostaglandin e 2 (pge 2 ). mucosa from the gastric antrum was harvested from 5 shelter dogs immediately after euthanasia, and mounted on ussing chambers. the tissues were equilibrated for 30-minutes prior to addition of acidic ringer's solution (ph, 1.2). after 45-minutes of injury, the acid was replaced with neutral ringer's and the tissues were treated with indomethacin, tramadol or both. tissues were maintained for 210minutes total, during which time permeability was assessed electrically. prostanoid concentrations were quantified using a commercially available elisa. western blots were performed for cox-1 and à2. recovery of gastric barrier function after acid injury was inhibited by co-administration of tramadol and indomethacin ( figure 1 ) but not by tramadol or indomethacin alone (data not shown). prostaglandin e 2 increased with acid injury. the increase in pge 2 was inhibited by co-administration of indomethacin and tramadol (in pg/ml: acid injury 681.25 ae 324.88, indo 1 tramadol 149.11 ae 35.24). there was no significant effect of treatment on cox-1 or à2 expression. co-administration of tramadol with a non-selective nsaid inhibits the return of gastric mucosal barrier function after acid injury in canine tissue, suggesting that caution is required in prescribing concurrent use of these drugs in dogs at risk for gastric ulcers. these drugs may exert this effect by decreasing levels of gastroprotective prostanoids. further study is needed to understand the mechanism of this drug interaction. an increased intestinal permeability (ip) has been suggested to be both cause and consequence of gastrointestinal (gi) disease, such as inflammatory bowel and celiac disease, in people. a novel tight junction regulator, larazotide acetate (alba therapeutics, baltimore, md) has been shown to significantly decrease ip in rats and in humans with celiac disease. the purpose of this study was to determine if larazotide acetate reduces ip in soft coated wheaten terriers (scwt) and norwegian lundehunds (nl) with chronic gi disease and ameliorates clinical signs. four nl (2 females, 2 males; median age: 2.5 yrs, range: 1.5-4.5 yrs) and 9 scwt (7 females, 2 males; median age: 5.0 yrs, range: 1.5-9.0 yrs) were enrolled based on presence of clinical signs of gi disease and hypoalbuminemia, increased fecal alpha 1proteinase inhibitor (a 1 -pi) concentrations, and/or hypocobalaminemia. scwt with protein-losing nephropathy were excluded. dogs were fed q12 hrs and received 0.5 mg (4 nl and 2 scwt) or 2.0 mg (7 scwt) of larazotide acetate po before each meal for 90 days. prior to start of treatment (day 1) and at the end (day 90), ip was evaluated by calculating the lactulose/rhamnose (l/r)-ratio in serum samples obtained at 30, 60, 90, and 120 min after oral dosing. also, 3 consecutive fecal samples each were collected prior to day 1 and day 90 for n-methylhistamine (nmh) measurement. pre-and post-treatment data were compared using a wilcoxon signed rank test. the 0.5 mg vs. 2.0 mg dose groups were compared using a mann-whitney u test. statistical significance was set at p o 0.05. l/r-ratios (medians) for the 60 min sampling time point were significantly lower on day 90 (0.046) than on day 1 (0.080; p 5 0.018). dogs treated with 2.0 mg q12 hrs had significantly lower 60 min l/r ratios on day 90 than dogs treated with 0.5 mg (0.033 vs. 0.094; p 5 0.014). no difference was found between breeds. fecal nmh concentrations were not different between time points, treatment groups, or breeds. fecal a 1 -pi concentrations were available for 11 of the 13 dogs and were significantly higher on day 90 compared to day 1 (p 5 0.033). no differences were found between pre-and post-treatment serum albumin or cobalamin concentrations. weight gain was seen in all 4 nl. resolution of diarrhea, vomiting, hyporexia, as well as an increased activity was seen in 1 scwt. another scwt had resolution of diarrhea and a decrease in pruritus. no changes in clinical signs were reported in the remaining 7 scwt. this study indicates that larazotide acetate might be able to reduce ip in dogs. this effect may be dose-dependent. however, not all dogs showed an improvement in clinical signs, suggesting that factors other than increased ip might have been responsible for the clinical signs in these dogs. breed-related effects cannot be ruled out, and further studies are warranted to determine the efficacy of larazotide acetate in dogs of other breeds with gi disease. to analyze different biochemical markers, calculate clinical activity scores, and assess survival in dogs with ple and compare them with those in dogs with food-responsive diarrhea (frd) without protein loss. 29 dogs with ple and 18 dogs with frd, referred to the university of bern, ch, were enrolled. selection criteria included a history of chronic diarrhea (4 3 weeks), exclusion of identifiable underlying causes, and histopathologic evidence of intestinal inflammation, but not neoplasia. underlying disorders were excluded based on cbc, chemistry profile, urinalysis, fecal analysis, trypsinlike immunoreactivity, cobalamin, folate, and transabdominal ultrasound. also, canine pancreatic lipase immunoreactivity (spec cpl s ), c-reactive protein (crp), calprotectin and alpha 1 -proteinase inhibitor (a 1 -pi) were measured in serum from 18 dogs and compared with 18 dogs with frd without ple. all dogs were scored using the canine ibd (cibdai) and the canine chronic enteropathy (cce) clinical activity index (ccecai). total protein, albumin (5-28.1 g/l), and total calcium (1.21-2.32 mmol/l) were decreased in all 29 dogs. cobalamin was decreased in all but 3 dogs ( o 100-490 ng/l). spec cpl was mildly increased in 3/18 dogs with ple and normal in 15/18 ple and all frd dogs. crp was normal in 5/18 dogs with ple (16/18 frd), mildly increased in 7/18 (1/18 frd), and moderately increased in 6/18 ple dogs (1/18 frd). calprotectin was slightly higher in dogs with ple, but all ple and frd dogs yielded values in the normal range. serum a 1 -pi was significantly lower in dogs with ple than in those with frd (p o 0.001), with 13/18 ple dogs below the reference range (1/18 frd). cibdai ranged from 4 to 16 and ccecai from 6 to 19. at the end of the study, 12/29 dogs were still alive with survival times between 26 and 2544 days. 17/29 dogs died with a median survival of 96 days (range 2-874 days). dogs with mildly increased crp died earlier than dogs with a normal or moderately increased crp (p 5 0.011), whereas albumin, calcium, spec cpl, calprotectin, cibdai, and ccecai had no significant impact on outcome and survival. in conclusion, dogs with ple have a significantly lower a1-pi in the serum than dogs with frd. furthermore, most dogs with ple have an increased crp and a decreased cobalamin. a mild increase in crp appears to be a poor prognostic factor. while hypoalbuminemia is a common finding associated with chronic enteropathies, its impact on survival in this population is poorly defined. the aim of this study was to compare dogs with chronic enteropathies on the basis of their serum albumin concentration at the time of presentation. we hypothesized that dogs with a protein losing enteropathy (ple) have a significantly shorter survival time compared to dogs with chronic enteropathies which are not hypoalbuminemic (controls). information obtained from the medical records included signalment, duration and characteristics of clinical signs, physical examination findings, clinicopathologic data and survival time. one hundred seventeen cases fit the inclusion criteria; 68 in the ple group and 49 controls. there was no statistical significance between groups for age (p 5 0.12), weight (p 5 0.17), weight loss (p 5 0.59) and body condition score (p 5 0.072). compared to control dogs, ple dogs had decreased serum concentrations of cobalamin (p 5 0.002), total calcium (p o 0.0001), globulin (p o 0.0001), cholesterol (p o 0.0001) and ionized calcium (p o 0.001). survival analysis revealed a significantly decreased survival time for ple dogs (p 5 0.0008); median survival was 701 days for ple dogs and 4 3,500 days for controls. while the ple group did not survive as long, survival was not directly associated with severity of hypoalbuminemia; patients with albumin concentration o 1.3 g/dl survived longer than those with mild hypoalbuminemia (1.6-1.9 g/dl). this study supports the observation that chronic enteropathy patients have decreased survival time when presented with hypoalbuminemia; however this study suggests the severity of hypoalbuminemia is not a reliable indicator of survival. cobalamin (vitamin b 12 ) deficiency in the chinese shar pei (shar pei) is suspected to be hereditary. inherited causes of cobalamin deficiency have been reported in humans and may affect absorption, transport, or cellular processing of cobalamin. based on human and veterinary studies, an increased serum methylmalonic acid (mma) concentration has been suggested to reflect cobalamin deficiency at the cellular level. in this context, it has been shown in humans that mma concentrations are higher in patients with genetic disorders affecting intracellular processing than in patients with genetic defects affecting gastrointestinal processing and extracellular transport of cobalamin. therefore, the aim of this study was to evaluate serum mma concentrations in shar peis and dogs of six other breeds with cobalamin deficiency. from 2008 in conclusion, serum cobalamin deficient shar peis had a 10 times higher median serum mma concentration compared to cobalamin deficient dogs of six other dog breeds. further studies are needed to investigate the intracellular processing of cobalamin in shar peis with cobalamin deficiency. chinese shar peis (shar peis) have a high prevalence of cobalamin deficiency. two other conditions reported frequently in this breed are shar pei fever and cutaneous mucinosis. shar pei fever is an autoimmune disorder causing periodic flare-ups and is associated with increased serum concentrations of c-reactive protein (crp), a nonspecific marker of inflammation. cutaneous mucinosis is characterized by excessive deposition of mucin in the dermis. also, hyaluronic acid (ha), the main component of mucin, was shown to be significantly higher in serum from shar peis with cutaneous mucinosis than in healthy controls. to date, a possible association between shar pei fever and/or cutaneous mucinosis on one side and cobalamin deficiency on the other has not been investigated in shar peis. thus, the aim of this study was to compare serum concentrations of ha (an indicator of cutaneous mucinosis) and inflammatory markers (crp, calprotectin, and s100a12), assumed to be increased in episodes of shar pei fever, in shar peis with and without cobalamin deficiency. serum samples from 40 shar peis, collected from 2008 to 2010, were analyzed. serum ha and crp (reference interval (ri): 0.0-7.6 mg/l) were quantified by using commercial elisa kits (echelon biosciences, salt lake city, ut, usa and tridelta, maynooth, ireland; respectively). serum calgranulin concentrations were measured using an in-house elisa (calprotectin; ri: 0.9-11.9 mg/l) and ria (s100a12; ri: 33.0-233.0 mg/l), respectively. mann-whitney u tests were used to compare serum ha, crp, calprotectin, and s100a12 concentrations between shar peis with and without cobalamin deficiency. significance was set at p o 0.05. fourteen shar peis were severely cobalamin deficient, defined by an undetectable serum cobalamin concentration ( o 150 ng/l). in the remaining 26 dogs, serum cobalamin concentrations were within the reference interval (251-908 ng/l). serum concentrations of ha, crp, calprotectin, and s100a12 were not significantly different between cobalamin deficient shar peis (medians: 649.9 ng/ml, 4. . fifty percent of cobalamin deficient shar peis had serum calprotectin concentrations above the upper limit of the reference interval, and 43% had serum s100a12 concentrations above the suggested upper reference limit. in this study, serum concentrations of ha, crp, and the calgranulins did not differ between cobalamin deficient shar peis and shar peis with a normal serum cobalamin concentration. this finding leads us to speculate that increased ha and/or inflammatory markers are not associated with cobalamin deficiency in shar peis. further studies are needed to investigate serum cobalamin concentrations in patients with shar pei fever or cutaneous mucinosis. cobalamin deficiency (cd) has been associated with gastrointestinal and pancreatic disease in dogs. hereditary cd has been demonstrated in giant schnauzers and single case reports have suggested congenital cd in the border collie (bc) breed. clinicopathologic findings of cd vary and can be unspecific as cobalamin acts as a co-factor for a multitude of enzymatic reactions. the two most important reactions concern the conversion of methylmalonyl-coa to succinyl-coa and the re-methylation of homocysteine (hcy). these two metabolites increase when cobalamin is lacking and act as markers for cobalamin availability on a cellular level. preliminary data from dogs suggested that measurement of methylmalonic acid (mma) may be a better diagnostic test for cd than serum cobalamin concentration. therefore the goals of the study were (1) to establish reference values for serum cobalamin, urine mma and plasma hcy in healthy pet dogs, (2) to screen a larger bc population from switzerland for cd, and (3) to perform genomic analyses on bc with cd. for determination of reference values 35 healthy pet dogs were used. serum cobalamin was measured using an automated chemiluminescence assay (immulite 2000), urine mma was determined using gas chromatography and expressed as a ratio to urine creatinine and plasma hcy was measured using high pressure liquid chromatography and fluorimetric detection. to calculate reference ranges the 10th and 90th percentile were used. data were analyzed using non-parametric tests. reference ranges for cobalamin, hcy, and mma were: cobalamin 279.2-972.8 ng/l; urine mma 2-4.65 mmol/mol creatinine; and plasma hcy 4.73-18.34 mmol/l. the screened bc population comprised 113 purebred dogs and 4 bc (median 11.5 months; range 8-41) suffering from congenital cd could be identified. clinical signs differed and consisted of tiredness (4), stunted growth (4), anemia (3), dysphagia (2) and persistent fever (1). median (ranges) results for healthy bc and bc with cd were: for cobalamin 592 (142-1855) and 72.00 (30-139) ng/l; for urine mma 2 (2-360) and 4148 (1800-6665) mmol/mol creatinine; for hcy 8.5 (2.8-22.4) and 41.00 (40-86.6) mmol/l. strikingly, healthy bc with cobalamin concentrations well within the reference range had significantly higher urine mma concentrations compared to control dogs. under the assumption that the four affected bc are inbred to a single founder animal, first results of genotyping on the 170 k illumina canine_hd snp chip suggest that mutations in the cubn and amn gene can be excluded to cause the observed cd in these dogs. we conclude that cd is a rare familial disease in bc with variable clinical signs. to define the genomic region responsible for cd further genetic analysis is in progress. it remains to be determined why some bc have high urine mma concentrations despite a serum cobalamin concentration within the reference range. calprotectin is a protein complex that plays an important role in the innate immune response. preliminary data suggest that canine calprotectin (ccp) is a useful marker for the detection of inflammation in dogs. recently, a radioimmunoassay for the measurement of ccp has been developed and analytically validated, but this test requires the use of a radioactive tracer. therefore, the aim of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (elisa) for the quantification of ccp in serum and fecal specimens from dogs. canine calprotectin (ccp) was purified, antiserum against purified ccp was raised in rabbits, monospecific antibodies were purified by affinity chromatography, and a sandwich-elisa was developed. purified antibodies were used for capturing and, after coupling with horseradish peroxidase (hrp), for reporting. a hrp substrate was used for color development. the assay was analytically validated by determination of analytical sensitivity and specificity, dilutional parallelism, spiking recovery, and intra-and inter-assay variability. control intervals for serum and fecal ccp were established from 110 and 52 healthy pet dogs, respectively, using the central 95 th percentile. sensitivity of the assay for serum samples assayed in a 1:400 dilution and for fecal extracts assayed in a 1:4,000 dilution was 0.3 mg/l and 3.2 mg/g, respectively. over a wide range of the assay, there was no cross-reactivity with cs100a12, the closest structural analogue of ccp available. observed to expected ratios (o/e) for serial dilutions ranged from 83.2-118.5% (mean ae standard deviation [sd]: 101.3 ae 10.0%) for four different serum samples, and from 81.7-129.1% (mean ae sd: 101.8 ae 14.0%) for five different fecal extracts. o/e for spiking recovery ranged from 87.8-130.4% (mean ae sd: 100.6 ae 6.5%) for four different serum samples and 6 different spiking concentrations, and from 95.9-152.0% (mean ae sd: 104.6 ae 11.6%) for 4 different fecal extracts and 6 different spiking concentrations. intra-assay coefficients of variation (cv) for 4 different serum samples were 7.8, 5.0, 7.4, and 12.7%, and 10.0, 6.1, 6.2, and 7.0% for 4 different fecal extracts. inter-assay cv for 4 different serum samples were 17. 2, 8.1, 9.9, and 12.6%, and 12.3, 8.3, 7.2, and 9 .6% for 4 different fecal extracts. the control intervals for serum and fecal ccp were established as 0.9-11.9 mg/l and 3.2-65.4 mg/g, respectively. we conclude that this new elisa for the measurement of ccp is analytically sensitive, linear, accurate, precise, and reproducible, and does not cross-react with canine s100a12. further studies evaluating the clinical usefulness of measuring serum and/or fecal ccp are currently under way. the syndrome of hemorrhagic gastroenteritis (hge) is characterized by a peracute onset of hemorrhagic diarrhea, vomiting, depression, and anorexia, and can be associated with a high mortality if untreated. the etiology of hge is unknown, but it is speculated that an abnormal response to bacterial endotoxins, bacteria, or dietary components may play a role. hge is characterized by an increased vascular/mucosal permeability, thought to represent a type i-hypersensitivity reaction, whereas inflammation and necrosis appear to be rare. however, markers of gastrointestinal (gi) inflammation and changes in the intestinal microbiota have not been studied extensively in dogs with hge. therefore, the aim of this study was to evaluate fecal canine calprotectin (cp) and s100a12 (a12), a 1 -proteinase inhibitor (a 1 -pi, a marker of gi protein loss), and bacterial groups that have previously been shown to be decreased (i.e., faecalibacterium spp., ruminococcaceae, bifidobacterium spp.) or increased (i.e., proteobacteria) in fecal samples from dogs with hge. fecal samples from 3 consecutive days were collected from 7 dogs with hge. fecal cp, a12, and a 1 -pi concentrations were measured by in-house immunoassays. bacterial dna was extracted from each fecal sample and was analyzed for faecalibacterium spp., proteobacteria, rumino-coccaceae, and bifidobacterium spp. using quantitative pcr assays. concentrations of fecal cp, a12, and a 1 -pi, and the abundance of bacterial dna were compared using a friedman test with dunn's post-hoc tests. significance was set at p o 0.05. at the time of diagnosis (day 1), fecal cp, a12, and a 1 -pi were above the suggested reference intervals in 6, 6, and 5 of the 7 dogs, respectively. until day 3, this number decreased to 2, 1, and 4, respectively. decreases in concentrations were significant between days 2 and 3 for a12 (p 5 0.016), and between days 1 and 3 for a 1 -pi (p 5 0.012), but not for cp despite a trend (p 5 0.085). no differences in the abundance of faecalibacterium spp. (p 5 0.085), bifidobacterium spp. (p 5 0.192), or proteobacteria (p 5 0.305) were observed. however, the abundance of rumino-coccaceae was significantly lower on day 3 when compared to day 2 (p 5 0.008). in this study, fecal markers of inflammation and gi protein loss were increased in dogs with hge. although the number of patients was small, following initiation of treatment, two of the markers decreased significantly. these results suggest a loss of protein into the gi tract at the onset of hge. the lack of significant increases of faecalibacterium spp., bifidobacterium spp., and ruminococcaceae, and decreases in proteobacteria may suggest gi dysbiosis. further longitudinal studies are needed and are currently under way to evaluate gi dysbiosis in canine hge patients. the most recent antiemetic approved for use in dogs is maropitant citrate (cerenia s , pfizer animal health). maropitant is a selective nk1 receptor antagonist that acts by blocking the binding of substance-p within the emetic center and chemoreceptor trigger zone. label dosage recommendations for maropitant citrate are 1 mg/ kg sc or 2 mg/kg orally once daily for up to 5 consecutive days (acute emesis) and 8 mg/kg orally once daily for up to 2 consecutive days (motion sickness). the study objective was to determine when steady-state is reached and the pharmacokinetics of maropitant administered at label oral dosages once daily for 14 consecutive days. two groups of eight healthy beagles were administered maropitant citrate at 2 or 8 mg/kg orally once daily for 14 days. concentrations of maropitant and its metabolite were measured in plasma using a lc-ms/ms assay. pharmacokinetic parameters were estimated using non-compartmental pharmacokinetic techniques and a modeling approach was used to estimate steady-state. the accumulation ratio for maropitant was 2.46 (auc0-24) and 2.03 (cmax) for the 2 mg/kg dose; and 4.81 (auc0-24) and 2.77 (cmax) for the 8 mg/kg dose after 14 days. the model estimate for the number of doses required to reach 90% of steady-state was 4.30 for 2 mg/kg and 8.09 for 8 mg/kg. three dogs experienced a single episode of vomiting. dosing maropitant citrate beyond the label duration was well tolerated by healthy dogs. steady-state was reached after approximately 4 doses for daily 2 mg/kg and 8 doses for daily 8 mg/kg oral dosing. previously presented at the veterinary cancer society, november 2010. cobalamin (vitamin b 12 ) is involved in a variety of metabolic processes. altered serum cobalamin concentrations have been observed in dogs with gastrointestinal disorders, such as exocrine pancreatic insufficiency (epi) or severe and longstanding ileal disease. this study was conducted to identify breeds with a higher proportion of a decreased serum cobalamin concentration that were submitted to the gastrointestinal laboratory. the study was also aimed at investigating serum trypsin-like immunoreactivity (tli) concentrations that were diagnostic for epi in the dogs with a decreased serum cobalamin concentration. except for csp, breeds identified here, have not previously been identified to have a higher rate of a decreased serum cobalamin concentration. also, a possible association between an undetectable serum cobalamin and a decreased serum tli in ai needs to be further investigated. calprotectin (cp) is a widely used marker for the diagnosis and monitoring of gastrointestinal (gi) inflammation in humans. studies in humans usually report fecal cp concentrations based on a single stool sample although considerable day-to-day variability of fecal cp was found in patients with gi disease and in healthy controls. intra-individual variation of canine cp (ccp) was also substantial in a small number of healthy dogs but has not been determined in dogs with chronic gi disease. thus, the aim of this study was to compare the day-to-day variation of fecal ccp in dogs with chronic gi disease before and during treatment to that in healthy dogs. we hypothesized that fecal ccp would be less variable in patients with chronic gi disease than in healthy controls, and thus collection of a single fecal sample would be sufficient. fecal samples from 3 consecutive days were prospectively collected from 15 dogs (group a; median age: 6.1 years) referred for diagnostic work-up of chronic signs of gi disease, from 8 dogs (group b; median age: 5.6 years) with stable gi disease while being treated, and from 44 healthy adult dogs (group c; mean age: 5.4 years). fecal samples were extracted and ccp was measured by an in-house immunoassay. mean ccp, standard deviation, coefficient of variation (cv), and difference between maximum and minimum ccp for the 3-day sample collection period were calculated for each dog and were compared among groups using a kruskal-wallis test. fecal ccp ranged from 2.9-102.7 mg/g (median: 17.6 mg/g) in dogs with gi disease (group a), from 2.9-265.1 mg/g (median: 18.4 mg/g) in dogs of group b, and from 2.9-93.1 mg/g (median: 7.9 mg/g) in healthy controls (group c). cvs were 0-121.3% in group a (median: 20.0%), 2.0-89.4% in group b (median: 47.4%), and 0-145.2% in group c (median: 40.4%), respectively. patients in group a appeared to have less variable fecal ccp than dogs in group b and c, but this difference was not significant (p 5 0.326). the difference between maximum and minimum ccp for the 3-day sample collection ranged from 0-31.1 mg/g in group a (median: 7.2 mg/g), from 0.1-240.0 mg/g in group b (median: 12.5 mg/g), and from 0-57.4 mg/g in group c (median: 14.8 mg/g), and were not significantly different between any of the groups (p 5 0.530). in this study, considerable day-to-day variation of fecal ccp was found in dogs with chronic gi disease (regardless of treatment) and was comparable to that in healthy dogs. results of this study suggest that for evaluating fecal ccp in dogs with clinical signs of gi disease, three consecutive fecal samples rather than a single fecal sample should be analyzed. because we did not intend to evaluate the clinical usefulness of fecal ccp as a marker of gi disease in dogs, disease severity, quality, and location differed among dogs in groups a and b. the diagnostic utility of fecal ccp in dogs with gi disease is currently being investigated. it has been suggested that diagnosis of clostridium perfringens related enteropathy should be based on the detection of the c. perfringens enterotoxin gene (cpe-gene) by pcr and/or c. perfringens enterotoxin (cpe) by elisa in feces. however, the prevalence of the cpe-gene and cpe in dogs and especially cats with gastrointestinal disease has not yet been reported. also, there is limited information about the stability of cpe in fecal samples at various storage conditions. the aim of this study was to evaluate the prevalence of the cpe-gene and cpe and the stability of cpe in fecal samples from dogs and cats. to evaluate the prevalence of the cpe-gene, a total of 481 fecal samples from dogs and cats with clinical signs of gastrointestinal disease (273 dogs and 208 cats) and 109 fecal samples from those without such signs (80 dogs and 29 cats) were examined using pcr. to evaluate the prevalence of cpe, a total of 90 fecal samples from dogs and cats with clinical signs of gastrointestinal disease (31 dogs and 59 cats) and 11 dogs without such signs were evaluated using a commercially available elisa kit (techlab, blacksburg, va). the results were analyzed using a fisher's exact test. significance was set at p o 0.05. to evaluate the stability of cpe, 8 fecal samples from dogs and 2 from cats with clinical signs of gastrointestinal disease that were positive for cpe were examined. also, 5 cpe negative samples from dogs were evaluated as negative controls. each sample was subdivided into 8 aliquots and evaluated on day 0; on days 2, 5, and 10 after being stored at room temperature (rt) or 41c; and on day 10 after being stored at à201c. the prevalence of the cpe-gene was not significantly different between dogs with signs of gastrointestinal disease (99/273; 36.3%) and dogs without (27/80; 33.8%; p 5 0.79). also, the prevalence of the cpe-gene in cats with signs of gastrointestinal disease (80/208; 38.5%) was not significantly different compared to cats without (6/ 29; 20.7%; p 5 0.06). pcr and elisa results were available for 90 samples. of the 65 pcr positive samples, only 6 (9.2%) were elisa positive. of the 35 pcr negative samples, only 1 (2.9%) was elisa positive. the prevalence of cpe was not significantly different between dogs with clinical signs of gastrointestinal disease (3/31; 9.7%) and those without (1/11; 9.0%; p 5 1.0). the prevalence of cpe in cats with signs of gastrointestinal disease was 4/59 (6.8%), but no samples from cats without such signs were available. when evaluating the stability of cpe, results for all aliquots were consistent with the initial result, except for one sample (on day 5, stored at rt, which was initially cpe positive). these results indicate that only a small proportion of samples that are pcr positive for the cpe-gene are also positive for cpe. studies are warranted to further compare the prevalence of cpe among animals with gastrointestinal disease and those without. furthermore, the results indicate that cpe is relatively stable in fecal samples at various storage temperatures. clostridium perfringens has been implicated as a cause of diarrhea in dogs. the main study objective was to compare two culture methods for the identification of c. perfringens. a secondary objective was to evaluate c. perfringens toxin genes a, b, b 2 , e, ı and cpe from canine isolates using a multiplex pcr and determine their prevalence in a group of normal and diarrheic dogs. fecal samples were collected from clinically normal (nd, n 5 105) and diarrheic dogs (dd, n 5 54) at a primary care veterinary facility. isolation of c. perfringens was performed using direct inoculation of feces onto 5% sheep blood agar (sba) as well as enrichment of stool in bhi broth followed by inoculation onto sba. isolates were tested by multiplex pcr for the presence of a, b, b 2 , e, ı and cpe genes. c. perfringens was isolated from 84% (88/105) of nd fecal samples using direct culture and 87.6% (92/105) with bhi enrichment (p 5 0.79). in the dd, corresponding isolation rates were 90.7% and 93.8% (p 5 0.45). all isolates possessed a toxin gene. b, b 2, e, ı and cpe toxin genes were identified in 4.4%, 1.1%, 3.3%, 1.1% and 14.4% of nd isolates, respectively. in the dd group, b and b 2 were identified in 5%, e and ı were not identified and the cpe gene in 16.9% of isolates. bhi enrichment did not significantly increase the yield of c. perfringens compared to sba but increased time and cost involved. c. perfringens (p 5 0.64) and c. perfringens toxin genes were present in equal proportions in nd and dd groups (p ! 0.15). culture of c. perfringens and pcr for toxin genes are of limited diagnostic utility due to the high prevalence of c.perfringens in normal dogs and the lack of apparent difference in toxin gene distribution between normal and diarrheic dogs. endoscopic biopsies are a relatively convenient, non-invasive test for feline infiltrative intestinal disorders. commonly, only the duodenum is examined due to cost, risks and time required to prepare the colon using lavage solutions, cathartics and/or enemas. the purpose of this study was to evaluate the consistency between endoscopic biopsies of the duodenum and ileum in cats. endoscopic biopsies from 70 cats which had duodenal and ileal tissue specimens were evaluated retrospectively. all slides were randomized and reviewed by a single pathologist (jm) for quality, number of biopsies, and diagnosis according to wsava standards. no information regarding history, clinical signs, endoscopic findings, or previous histological diagnosis was made available to the pathologist. statistical comparison of the diagnosis of sc-lsa and ibd by intestinal location was conducted using fisher's exact test (p o 0.05 significant). 18 of 70 cats (25.7%) were diagnosed with sc-lsa in the duodenum and/or ileum. of these 18 cats, 7 (38.9%) were diagnosed with only duodenal sc-lsa, 8 (44.4%) were diagnosed with only ileal sc-lsa, and 3 (16.7%) had sc-lsa in both duodenum and ileum. in 8 cats with only ileal sc-lsa, 3 had severe ibd in duodenal biopsies, possibly consistent with early sc-lsa. 5 of these 8 had duodenal biopsies without evidence of sc-lsa. our results suggest there is a population of cats in which diagnosis of sc-lsa may only be found by evaluating ileal biopsies. clinicians should consider performing both upper and lower gi endoscopic biopsies in cats with suspected infiltrative small bowel disease. periodontitis is one of the most common diseases in cats and is mainly due to the presence of plaque and calculus. in this study, we investigated putative correlations between dental tartar and gingivitis and also between gingivitis and subgingival bacteria in cats. twelve cats (median age: 5 years; range: 1-10 years; 6 dsh and 6 persians; 8 females and 4 males) were enrolled. dental tartar was obtained during scaling for a dental prophylactic procedure. all cats were negative for felv and fiv infection as assessed by a commercial elisa test (snap s fiv/felv combo test). severity of gingivitis (scores: 0-3; 0 5 normal, 1 5 mild, 2 5 moderate, and 3 5 severe) and dental tartar (scores: 0-3) were scored in each cat. endodontic paper points were applied for collecting a bacterial sample from the subgingival area and transferred to thioglycollate transporting media for bacterial culture. the relationship between gingivitis and tartar thickness scores was analyzed by spearman correlation. a student's t-test was used to compare the mean differences (gingivitis and tartar thickness scores) between upper and lower teeth. the association between severity of gingivitis and bacterial type was tested by chi square test. the spearman correlation coefficient for the average gingivitis score and the average tartar thickness score was 0.91 (p o 0.05). interestingly, the average tartar thickness scores from the upper jaw were significantly higher than those from the lower jaw (p o 0.05). the highest scores were found for the molar teeth in all cats. bacterial culture revealed 28.9% anaerobic bacteria species (i.e., bacteroides spp., peptostreptococcus anaerobius, and eubacterium aerofaciens) and 71.1% aerobic bacteria species (i.e., pasteurella multocida, streptococcus spp., enterococcus spp., staphylococcus spp., bacillus cereus, escherichia coli, and pseudomonas aeruginosa). anaerobic bacteria were found mostly in cats with higher gingivitis scores (2-3; chi square: p o 0.05), while pasteurella multocida was found mostly in cats with lower gingivitis scores (0-1; chi square: p o 0.05). antimicrobial sensitivity testing indicated that all of the anaerobic bacteria were sensitive to clindamycin, chloramphenicol, metronidazole, cefoxitin, or tetracycline, 90% were sensitive to erythromycin, and 80% were sensitive to penicillin. the most abundant aerobic bacterial species, pasteurella multocida, was sensitive to cefoxitin in all cases in which it had been cultured. these results suggest that anaerobic bacteria may be associated in the pathogenesis of severe gingivitis. these data warrant further studies of the prophylactic use of antibiotics in cats undergoing dental prophylactic procedures. inflammatory bowel disease is the most common cause of vomiting and diarrhea in dogs. although it can occur in any canine breed, certain breeds are more susceptible. we have previously shown that polymorphisms in the tlr4 and tlr5 gene are significantly associated with inflammatory bowel disease (ibd) in the german shepherd dog (gsd), a breed at risk of developing this disease. it would be useful to determine if these polymorphisms are significant in other canine breeds as this may allow the development of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. therefore the aim of this study was to investigate whether polymorphisms in canine tlr 4 and tlr 5 genes are associated with ibd in other non-gsd canine breeds. four non-synonymous snps in the tlr4 gene; t23c, g1039a, a1572t and g1807a and three non-synonymous snps in the tlr5 gene; g22a, c100t and t1844c previously identified in a mutational analysis in gsds with ibd were evaluated in a case-control study using a snapshot multiplex reaction. sequencing information from 85 unrelated dogs with ibd consisting of 38 different non-gsd breeds from the uk were compared to a breed-matched control group consisting of 162 unrelated dogs from patients treated for noninflammatory disease at the royal veterinary college, london, uk. as in the gsd ibd population the two tlr5 snps; c100t and t1844c were found to be significantly protective for ibd in other breeds included in this study (p 5 0.023 and p 5 0.0195 respectively). this study confirms the protective effects of the two tlr5 snps (c100t and t1884c) in other canine breeds with ibd. this highlights the importance of tlr5 in the pathogenesis of canine ibd and may represent common pathological pathways of ibd in different canine breeds due to the high degree of haplotype sharing seen among breeds. this may allow for the future expansion of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. further functional studies looking at the role of tlr5 in the pathogenesis of canine ibd are needed to confirm these findings. toll-like receptor 5 (tlr5) is an extracellular pattern recognition receptor belonging to the innate immune system. we have recently shown that three non-synonymous single nucleotide polymorphisms (snps) in the tlr5 gene (g22a, c100t and t1844c) are significantly associated with inflammatory bowel disease (ibd) in german shepherd dogs. in addition, we confirmed that two of these tlr5 snps (c100t and t1844c) were significantly associated with ibd in a population consisting of 38 different dog breeds. in order to determine if other novel snps exist in the tlr5 gene in addition to the ones identified in the gsd population, mutational analysis was carried out in seven boxer dogs with ibd. polymerase chain reaction was carried out to amplify the tlr5 coding region in the seven dogs with ibd. sequencing was carried out using sequence based typing with the abi prism sequencing kit (applied biosystems, uk) and analyzed using an abi3100 automated sequencer (pe applied biosystems). sequencing information from seven boxer dogs with ibd from the uk were compared to the reference sequence published on the ensemble webserver (www. ensembl.org/canis_familiaris). in addition to the three snps identified previously in the tlr5 gene, a novel non-synonymous snp; t443c was identified in the boxer dog population with ibd. this snp has never been reported before and was present as the homozygote genotype in three dogs with ibd and in one dog as the heterozygote genotype. using the simple modular architecture research tool (smart) web server (http:// smart.embl.de/) we were able to map the t443c snp to the leucine rich repeat domain of the tlr5 protein. the leucine rich repeat domain is involved with ligand binding and therefore a change in the amino acid in this region may affect function, especially as the t443c snp results in a change in the amino acid from non-polar to polar. our study further confirms the role of tlr5 in the pathogenesis of canine ibd. our results suggest that in addition to shared risk polymorphisms amongst breeds, individual breeds may harbor unique snps arising after breed formation which may further affect their susceptibility to this disease. however, a case-control study would be needed in the boxer dog to confirm the significance of the tlr5 t443c snp and further functional data would be needed to elucidate the exact role of this polymorphism in canine ibd. an automated power driver device (oncontrol, vidacare) has recently become available for bone marrow aspiration (bma) and bone marrow biopsy (bmb) in humans. the purpose of our study was to compare this automated technique to the traditional manual technique for bone marrow sampling in cats. twelve healthy research cats were anesthetized using a standardized protocol on 2 different occasions, 2 days apart, to have bmas and bmbs performed by the same operator. on day 1, half of the cats were randomized to have a bma performed at both the proximal humerus and the iliac crest, and a bmb performed at the iliac wing, using the oncontrol device (15-gauge needle for bma; 11-gauge needle for bmb). the other half of the cats had the same procedures performed using a manual technique (15-gauge illinois needle for bma; 11-gauge jamshidi needle for bmb). on day 3, each cat had bma performed at the opposite humerus and iliac crest, and a bmb performed at the proximal humerus using the opposite technique from day 1. for each procedure, the operator was given a maximum of 3 attempts to successfully collect a sample. the rate of success, as well as the number of attempts were recorded. the ''ease of use'' of the device was rated by the operator on a 5-point scale after each procedure. using previously determined criteria, the macroscopic and microscopic qualities of the bma and bmb samples were assessed by a board-certified pathologist, blinded to the technique used. the level of pain experienced by each cat was evaluated 6, 12, 18, 24, 36 and 48 hours following each set of procedures, using a previously validated pain scoring system. two sample t-tests were used to compare the automated technique to the manual technique and to compare the humerus to the iliac crest site for bmas and the humerus to the iliac wing site for bmbs. for all procedures, at all sites, the ''ease of use'' was better for the automated technique than for the manual technique (p o 0.05). the duration of the procedure and the number of attempts to collect a sample were significantly lower with the automated technique for bma at the proximal humerus (p o 0.05). there was no significant difference in the level of pain at any time point following each set of procedures with either technique. performing bma at the proximal humerus was associated with a higher rate of success (p o 0.05), a lower number of attempts (p o 0.05), a shorter duration of the procedure (p o 0.05), a higher-rated ''ease of use'' of the technique (p o 0.05), and a better quality sample (p o 0.05) when compared to sampling from the iliac crest, in conclusion, we found the automated bone marrow sampling technique suitable for use in adult cats. this technique was easier to use than the manual technique for both bma and bmb, and reduced the duration of the procedure and the number of attempts for successful bma at the proximal humerus. performing bma at the proximal humerus was faster, easier and allowed collection of better quality samples than at the iliac crest, independently of the technique used. the fractious nature of some feline patients sometimes makes sedation or general anesthesia necessary for routine procedures such as blood collection for hematologic analyses. it has been anecdotally reported that sedation or general anesthesia could induce variations in hematologic parameters in cats, making it important for the clinician to be able to anticipate potential changes on hematologic parameters that could result from chemical restraint. this study evaluated the effects of a standardizecd anesthetic protocol using ketamine (10 mg/kg, iv), midazolam (0.5 mg/kg, iv) and buprenorphine (10 mg/kg, im) on the hematologic parameters of 12 healthy adult research cats. each cat had blood samples collected before and after induction of anesthesia on 2 different occasions, 2 days apart. in total, 24 pairs of complete blood counts were obtained. analyses were performed at a certified veterinary laboratory. paired sample t-tests were used to determine whether there were any statistical differences between hematologic parameters before and after induction of general anesthesia, for each cat, on 2 different occasions. compared to preanesthetic values there was a significant decrease in red blood cell count, hemoglobin concentration, hematocrit, lymphocyte count and plasma total protein concentration after induction of anesthesia. there was no significant difference in the segmented or band neutrophil, eosinophil, basophil, monocyte and platelet counts between the samples taken before and after induction of anesthesia. on average, there was a 23.7% decrease in the red blood cell count (9.06 â 10 12 /l to 6.91 â 10 12 /l) (p o 0.0001), a 23% decrease in hemoglobin concentration (133.88 g/l to 103.08 g/ l) (p o 0.0001), a 24.4% decrease in the hematocrit (0.41 l/l to 0.31 l/l) (p o 0.0001), a 25.3% decrease in the lymphocyte count (3.68 â 10 9 /l to 2.77 â 10 9 /l) (p 5 0.0023), and a 12.1% decrease in the plasma total protein concentration (84.79 g/l to 74.54 g/l) (p o 0.0001) when samples taken before and after induction of anesthesia were compared. if only the hematocrit was considered as a marker of anemia, 29% of the samples from these 12 healthy cats, taken while they were under general anesthesia, would have been misinterpreted as belonging to anemic patients (hematocrit o 0.285 l/l), using the reference interval established in our laboratory. none of the cats would have been considered anemic before induction of general anesthesia. in practice, the decrease in lymphocyte count following anesthesia is unlikely to be of clinical relevance, as all the samples except 2 had a lymphocyte count that was within the reference interval for cats established by our laboratory. this study suggests that complete blood counts performed on blood taken under general anesthesia with this combination of anesthetic drugs in cats should be interpreted cautiously in order not to make a false diagnosis of anemia. the mechanism responsible for the decrease in circulating red blood cell mass following anesthesia induction in cats is unknown and requires further investigation. rivaroxaban is an oral inhibitor of activated coagulation factor x (xa). it is expected to have similar coagulation effects as low molecular weight heparin, without the need for injection, making it an attractive alternative for long-term anticoagulant therapy in cats. citrated blood obtained from five healthy adult cats was exposed in vitro to varying concentrations of rivaroxaban, followed by coagulation testing. the rivaroxaban was extracted from commercially available tablets (xarelto s ) and dissolved in dmso prior to addition to the blood. tests performed included kaolin-activated thrombelastography (teg), prothrombin time (pt), dilute pt (dpt), activated partial thromboplastin time (aptt), and anti-factor xa (axa) activity. dose-dependent prolongations were seen in all coagulation parameters. similar to human data, therapeutic axa levels (between 0.5-1.0 axa units) were achieved at in vitro concentrations between 160 and 220 mg/l. at 220 mg/l, dpt measurements were clinically prolonged in all cats (29.2 ae 4 sec vs. 18.5 ae 0.8 sec, p 5 0.148), while aptt values were only mildly prolonged from baseline (21.9 ae 5 sec vs. 15.6 ae 2 sec, p 5 0.07). significant prolongations were seen in dpt at 500 (60.4 ae 42 ec, p 5 0.005). teg r time did not prolong from baseline values until concentrations of 2000 mg/l were reached (16.0 ae 9 min compared to 3.1 ae 0.7 min, p 5 0.006). rivaroxaban has similar coagulation effects in the cat as in other species and may play a role in feline thromboprophylaxis. kaolinactivated teg does not appear to be sensitive to low concentrations of rivaroxaban in the cat. anticoagulated blood is required for platelet function studies. sodium citrate, a calcium chelater, is the most commonly used anticoagulant to measure coagulation parameters including platelet aggregation but it may have a negative effect on platelet responsiveness. dogs are generally considered moderate responders to collagen on platelet aggregation and are notorious for being poor or inconsistent responders to adp-induced platelet aggregation using citrated whole blood. hirudin, a selective thrombin inhibitor, can also be used as an anticoagulant for coagulation assays and is the anticoagulant of choice for certain assays including the multiplate s platelet function analyzer. ten adult healthy dogs were used to compare whole blood platelet aggregation between citrated and hirudinated blood samples. venous blood was collected atraumatically from the external jugular vein directly into tubes containing 3.2% trisodium citrate or hirudin. whole blood platelet aggregation was performed (whole-blood lumi-aggregometer, chrono-log corporation, havertown, pa, usa) with collagen (5 mg/ml) and adp (10 mm) as agonists. maximal platelet aggregation (ohms) was recorded. there was a significant increase in collagen-induced platelet aggregation from the hirudinated samples compared to the citrated samples (31.2 ae 6.1 vs. 17.2 ae 8.6 o, p o 0.0005). there was also a significant increase in adp-induced platelet aggregation from the hirudinated samples compared to the citrated samples (15.6 ae 6.0 vs. 2.6 ae 2.6 o, p 5 0.0001). the results of this study show a significant difference in platelet responsiveness between citrated and hirudinated whole blood using the chrono-log impedance aggregometer. while both collagen and adp-induced platelet aggregation was attenuated from citrated blood samples, this was most notable for adpinduced aggregation where almost all samples had no objective measurable platelet aggregation. it is suggested from this data that future whole blood platelet aggregation studies performed on the chrono-log impedance aggregometer should use hirudinated blood samples although new reference limits would need to be established. low-molecular-weight heparin (lmwh) is now used to prevent thrombotic complications in dogs. a functional assay such as the calibrated automated thrombogram (cat) may provide a new approach for monitoring lmwh therapy. we hypothesized that cat would detect decreased endogenous thrombin potential (etp) in healthy dogs receiving lmwh (fragmin s ). twenty-four healthy adult beagles were included in this study and divided equally in four groups. one dose of 50 u/kg, 100 u/kg or 150 u/kg of lmwh were given subcutaneously to healthy dogs and compared to a control group. platelet poor plasma (ppp) was collected over a 24 hour period. using a repeated-measure linear model, effect of lmwh on etp was time and dose dependent with a significant interaction (p o 0.0001). compared to control dogs, significant differences were obtained for group 50 u/kg at t60 (p 5 0.037), for group 100 u/kg at t15 (p 5 0.013) and between t30-t240 minutes (p o 0.0001) respectively, and for group 150 u/ at t15 (p 5 0.011), between t30-t300 minutes (p o 0.0001) respectively and at t360 (p 5 0.004 the cat assay can be employed to measure the effects of lmwh at different doses in healthy dogs, resulting in significant time and dose-dependent decreases in etp and warrants further investigation as a tool for monitoring lmwh therapy in dogs. the purpose of this study was to determine the effects of prednisone and prednisone plus ultralow-dose aspirin on coagulation parameters in healthy dogs, with an emphasis on thromboelastography (teg). this was a prospective, randomized, blinded study utilizing fourteen dogs determined to be healthy based on normal physical examination, complete blood count, biochemistry, urinalysis, and fecal floatation. dogs were evenly divided into either prednisone plus aspirin (pa) or prednisone plus placebo (pp) groups. baseline values for teg parameters (r, k, angle, ma, ly30, ly60, g, ci) were measured twice two days apart, and thrombin-antithrombin complexes (tat), and traditional coagulation parameters (prothrombin time, activated partial thromboplastin time, d-dimer, antithrombin (at), fibrinogen) were measured once. each dog received 2 mg/kg/ day of prednisone, and either 0.5 mg/kg/day of aspirin (pa group) or placebo (pp group) for 14 days. a complete blood count, biochemistry profile, teg, tat, and traditional coagulation parameters were then repeated on each dog. day to day variation was calculated for the teg parameters using the two baseline measurements. the change from baseline between and within each group were compared using t-tests, or wilcoxon 2 sample test where appropriate, for teg, tat, traditional coagulation parameters, and hematocrit. day to day variation in teg was acceptable ( 10%) for ma, g, and angle, unacceptable (4 10%) for r, k, ly30 and ly60, and not meaningful for ci. within both groups, ma, g, ci and fibrinogen significantly increased from baseline (p o 0.05). within both groups, ly30 and at significantly decreased from baseline (p o 0.05). for the pp group, ly60 significantly decreased from baseline (p 5 0.03), and approached significance for the pa group (p 5 0.0504). all other within group changes from baseline were not statistically significant (p-values 4 0.05). for all parameters, there was no difference between groups for change from baseline (p values 4 0.05). day to day variation in some teg parameters is high and may preclude their clinical utility. prednisone causes hypercoagulability in healthy dogs based on increased g, ma, and ci. the addition of ultra-low dose aspirin to prednisone has no effect on the parameters measured in this study. 'aspirin resistance' has been identified in people and dogs that develop thrombi despite low dose aspirin therapy. variability in platelet cyclooxygenase (cox) isoform expression is one proposed mechanism for aspirin resistance in people. two isoforms (cox-1 and cox-2) have been identified in canine platelets. high (antiinflammatory) dose aspirin inhibits platelet function and alters expression of both cox isoforms in most dogs. this study evaluated the effects of low dose aspirin on platelet function and cox expression in normal dogs. twenty-five healthy client-owned dogs were evaluated before and at two time points (days 3 and 10) during aspirin therapy (1 mg/kg po sid). platelet response to aspirin (siemens pfa-100 s ; collagen/ epinephrine cartridges), was stratified into one of three groups [aspirin responders (9 dogs), non-responders (8 dogs), or inconsistent responders (8 dogs)]. flow cytometry identified platelet cox-1 and cox-2 expression. an elisa was used to measure urine 11-dehydro-thromboxane b 2 (11-dtxb 2 ). there were no significant differences between groups for cox-1, cox-2 or 11-dtxb 2 at any time point. when all dogs were considered as a single group, there was a significant increase (p o 0.0001ã) in cox-1 and cox-2 mean fluorescent intensity (mfi) from baseline to day 10, 70.1% ae 38.0 (mean ae sd) and 70.8% ae 71.2, respectively. there was a significant decrease in mean urine 11-dtxb 2 :creatinine on day 3 and 10 by 23.4% (p 5 0.0044 ã ) and 45% (p o 0.0001 ã ). as with our previous high dose studies, cox-1 expression was increased with aspirin exposure. however, there was a significant increase in cox-2 expression with low dose aspirin in contrast to the decrease seen at higher doses. our study suggests that levels of platelet cox-1 and cox-2 expression do not influence aspirin response in dogs. although thromboxane levels decreased in most (23 of 25) dogs on low dose aspirin, platelet function was consistently affected in only 36% of dogs, suggesting that differences in response to thromboxane may play a role in the variable affects of low dose aspirin on canine platelet function. delayed postoperative bleeding is common in retired racing greyhounds (rrgs), despite normal results of routine hemostasis assays. the excessive postoperative bleeding in the rrgs is not due to primary or secondary hemostatic defects, and may be due to enhanced fibrinolysis or to a clot maintenance dysfunction. providing a method to prevent or minimize the severity of postoperative bleeding in rrgs will not only have major economic impact for owners, but also will markedly decrease the associated complications of minor or major surgeries in the breed. epsilon aminocaproic acid (eaca) is a potent inhibitor of fibrinolysis that also supports clot maintenance due to unknown mechanisms. the objective of this double-blinded, prospective, randomized study was to evaluate the effects of eaca versus placebo on the prevalence of bleeding in rrgs, and to investigate its mechanism of action by using thromboelastography (teg). we compared the effects of eaca and placebo in 100 rrgs that underwent elective ovariohysterectomy or orchiectomy at the veterinary medical center, the ohio state university during 2 years. the main endpoint was bleeding (prevalence and severity); minor endpoints included most teg parameters. thirty percent (15/50) of the rrgs in the placebo group had delayed postoperative bleeding starting 36 to 48 hours after surgery, compared to 10% (5/50) in the eaca group (p 5 0.0124). on the teg parameters, the r time (clot formation time) was significantly different between treatment groups (p 5 0.0321). the postoperative administration of eaca significantly decreased the prevalence of postoperative bleeding in rrgs. thromboembolism associated with protein losing nephropathy (pln) has been long recognized as a serious and unpredictable complication in dogs, however its prevalence remains unknown. in humans, surrogate indicators are frequently used to assess thromboembolic risk. this study aimed to investigate the prevalence of hypercoagulability in pln dogs based on thromboelastography (teg), and to determine whether hypercoagulability in these patients could be predicted by clinical assessments that identify systemic hypertension (systolic blood pressure 4 160 mmhg), hypoalbuminemia (serum albumin o 2.7 mg/dl), antithrombin activity (o 70%), and degree of proteinuria (urine protein:creatinine ratio [upc] ! 2). between march 2009-september 2010, twenty-seven dogs were identified with pln at the animal medical center. the prevalence of hypercoagulability based on a teg g-value 4 9.6 was 83.3%. there was no statistically significant relationship, either categorically or continuously, in univariate as well as multivariate analyses of all variables. univariate logistic regression (odds ratio; lower and upper confidence limit; p value) for hypertension was à1.18; 0.201, 6.99; 0.851; for albumin -1.37; 0.228, 8.26; 0.728; and for antithrombin activity -0.73; 0.12, 4.39; 0.728. thus, in this patient population, in the absence of teg, prediction of hypercoagulability using abnormalities in commonly measured clinicopathologic variables was not helpful. however, given the documented high prevalence of hypercoagulability in patients with pln, early institution of prophylactic anti-platelet or anticoagulant therapies should be considered. thromboelastography (teg) is a test of global hemostasis. due to the effects of extrinsic factors on whole blood coagulation, sample collection method (scm) may influence results. the purpose was to determine if scm influenced teg using kaolin-activated citrated whole blood (wb). healthy dogs with normal platelet counts were prospectively enrolled. three wb samples were obtained from each dog at least 48 hours apart from alternating jugular veins in a randomized order of three methods: 1) vacutainer s into citrated tube (vac), 2) citrated syringe with transfer into plain tube (cit), or 3) plain syringe with transfer into citrated tube (plain). draw time was recorded in seconds. kaolin-activated teg was performed, with measurement of reaction time (r), clot formation time (k), maximum amplitude (ma), and alpha angle. eleven dogs were enrolled. there were no significant differences in teg indices between vac samples and either cit or plain samples. cit samples had a significantly higher k value (p 5 0.004) and a lower alpha angle (p 5 0.004) compared to plain samples. draw times ranged from 2-10 seconds. a longer draw time was significantly correlated (p 5 0.046; r 5 à0.35) with a shorter r time. higher platelet count was significantly correlated (p 5 0.001; r 5 0.575) with a higher ma. scm did not have a significant effect on teg parameters when comparing vac samples to either cit or plain samples. minimizing sample collection time and trauma during venipuncture may be important in minimizing hypercoagulable changes in teg indices. liquid plasma (lp) is defined as either plasma collected and refrigerated immediately after collection or fresh frozen plasma (ffp) that is thawed and stored refrigerated until use. stability studies in people have shown that adequate clotting factor activity is preserved for at least 14 days. lp is transfused in human level i trauma centers to critically ill people requiring rapid infusion of clotting factors as the time required to defrost ffp is considered prohibitive. the use of lp has not been described in veterinary critical care. the purposes of this study were to 1) determine the length of time required in a water bath for a unit of canine ffp to thaw and 2) describe the use of lp in a busy university emergency room (er). for part 1: six units (250 ml) of canine ffp were individually thawed in a 371c water bath. the duration of time (in minutes) to thaw was recorded. for part 2: the transfusion log was reviewed for dogs receiving lp in the last 6 months. the indications and outcome were recorded. the mean time ae sd thaw time was 34.7 ae 1.38 minutes. ten units of lp were transfused to 7 critically ill or injured dogs during the study time. indications for lp transfusion included hypovolemic shock due to intra-abdominal hemorrhage in 6 dogs (2 traumatic, 4 non-traumatic) and rapid correction of hemorrhage following parenteral tissue plasminogen activator administration in 1 dog. lp volume transfused ranged from 11.1 to 50.5 ml/kg. no transfusion reactions were identified. effect on coagulation was not consistently evaluated. time required to thaw a unit of ffp is greater than 30 minutes which could be detrimental in a bleeding, coagulopathic dog. lp was transfused without incident to critically ill and injured dogs and represents a potential new addition to the armamentarium of treatments in a veterinary er setting. further investigation of canine lp is warranted including evaluation of in vitro factor stability and in vivo efficacy in correcting coagulopathy. immune mediated thrombocytopenia (imt) is associated with increased morbidity and mortality. large prospective research studies in dog platelet antibodies and clinical utilization of platelet immunoglobulin assays are limited. potential explanations include limited availability and low specificity due to nonspecific binding. the focus of this study is to evaluate optimized direct and indirect platelet surface associated immunoglobulin (psaig) and staining with anti-cd61 antibodies (cd61ab) for the utilization in classifying thrombocytopenic dogs. one hundred clinically ill and 30 apparently healthy dogs were prospectively evaluated. data collected included a history of hemorrhage, physical examination evidence of bleeding, complete blood count, and measurement of psaig and cd61ab. the psaig assay utilized polyvalent antibodies with correction for non-specific binding by subtraction of background fluorescence with control antiserum. thrombocytopenia was defined as o 164,000/ml and all dogs were clinically classified into 1 of 4 groups (g): g1 imt, n 5 45, g2 thrombocytopenia from non-immune mediated diseases, n 5 37, g3 ill with normal platelet counts, n 5 18, g4 healthy dogs, n 5 30. median platelet counts, by groups, were g1, 20,000; g2, 69,000; g3, 324,500; and g4, 212,000/ml. for the direct and indirect psaigs in dogs with itp (g1), more dogs (n 5 6 and n 5 9) with clinical evidence of bleeding had antibodies compared to those who were not bleeding (n 5 3 and n 5 6). considering only direct psaig the sensitivity and specificity was 13% and 95%, respectively for the diagnosis of imt. for indirect psaig the sensitivity and specificity was 27% and 97%, respectively, for the diagnosis of imt. when considering both direct and indirect psaig together, the sensitivity was 33% with a specificity of 93%. in g1 interference from high control antiserum background staining was noted in 26.7% of dogs and resulted in a negative direct psaig classification. minimal background interference was noted in g2, g3, or g4. the percentage of platelets stained with cd61ab was significantly less in g1 (median 38, p 5 0.0001) vs. g2 (median 74, p 5 0.007) vs. g3 (median 95.1, p 5 0.857) and g4 (median 95.6). these findings indicate the optimized platelet surface associated immunoglobulin assay has a high specificity, however poor sensitivity, for the diagnosis of imt. the decreased cd61 staining in g1 (imt group) may reflect decreased surface gpiiia expression, blocking by anti-gpiiia antibodies or other proteins, clearance by macrophages, or increased non-platelet debris and has potential applications in the diagnosis and treatment of imt. greyhounds have lower serum concentrations of a-globulin than other breeds, explained by negligible levels of haptoglobin (hp) measured using different methods (colorimetric, immunoturbidimetric and protein electrophoresis). the purpose of the present study was to characterize the hp gene in greyhounds. we isolated dna and rna from blood samples of akc-registered and retired racing greyhounds (akcg, rrg), and a german shepherd dog (gsd). we sequenced the hp exons and splice sites, and conducted array comparative genomic hybridization to identify associated dna structural variation (custom 1m agilent oligonucleotide array). additionally, we tested for the presence of one or multiple haplotypes spanning hp in greyhounds using a high density snp array (180k illumina hd). sequencing results of hp in both dna and cdna revealed three synonymous snps in the racing greyhound. we did not identify structural variation overlapping or near the hp gene. notably, we detected that the rrg and akcg do not appear to share a specific haplotype spanning hp. despite having low or undetectable serum concentrations of hp, we did find that rrg hp mrna is expressed and lacks amino acid variation. this suggests that the clinical absence of the hp is attributable to post-transcriptional hp effects or to an unknown physiological interaction. finally, given the existence of distinct rrg and akcg haplotypes spanning hp, it is important to characterize serum levels of hp in akcg in follow on studies. we reported that hemoglobin in retired racing greyhounds (rrg) has higher oxygen carrying properties and affinity than other breeds. surprisingly, very little is known about canine hemoglobin genetics. the purpose of this study was to characterize genetics of canine beta globins. using computational blast analysis of the dog genome, we identified five beta globin genes in a single locus: two human hbelike followed by three hbb-like genes. we isolated dna and rna from blood of rrgs, akc registered greyhounds (akcg), and german shepherd dog (gsd). all beta globin exons and splice sites were sequenced, and the beta globin locus was examined by array comparative genomic hybridization (custom 1m agilent array). additionally, we determined the number of common haplotypes that span this locus in rrgs and akcgs using high density snp array (180k illumina hd). expression and sequence analysis of cdna showed all five beta globin genes are actively expressed in adults. canhbb1 and 2 were created by relatively recent segmental duplication and have identical protein sequence. canhbb1/2 are abundantly expressed in adults; canhbb3 is expressed at greatly reduced levels. sequencing results revealed one rare non-synonymous single nucleotide polymorphism (snp) in hbe1 of rrgs, but no variation that could explain their abnormal hemoglobin. we did not detect structural variation overlapping or near the beta globin locus. notably, rrg and akcg do not share haplotypes spanning the beta globin locus. this is the first characterization of canine hemoglobin genetics, and the first report of canine embryonic hemoglobins and their expression in adults. sampling of the bone marrow in the dog from the costochondral (cc) junction can be performed with minimal to no sedation and readily available equipment but is not in widespread use in the united states. the aim of this study was to compare the number of attempts needed to successfully obtain a sample, the time needed for the procedure, and the sample quality between aspirates obtained from the cc junction and more traditional sites (humerus or femur) in healthy dogs when performed by novice and seasoned practitioners. samples were obtained from healthy anesthetized laboratory reared adult dogs after undergoing terminal endoscopic surgery. paired samples from separate dogs were obtained by each practitioner using either a 22 gauge needle and 6 cc syringe at the cc junction or an 18 gauge rosenthal needle and 12 cc syringe from either the proximal humerus or femur (clinician preference). three small animal veterinary interns, one experienced technician and one boarded internist were monitored for number of attempts to success and length of time needed for success of each procedure. slides were prepared by a single investigator and read by a blinded clinical pathologist. data were compared using the paired t-test for normally distributed data and wilcoxen signed rank test for non-gaussian distributions. five pairs of samples from three dogs were evaluated. two dogs had two pairs drawn from opposite limbs and ribs. mean number of attempts to success for traditional sampling sites (1.4 1/à0.54) and time to success (8.6 minutes 1/à5.2) did not differ significantly from attempts (2.01/-0.70, p 5 0.25) or time (2.7 1/-0.98, 0.06) needed when aspirating from the cc junction. subjectively, samples were of similar quality with regards to cellularity and number of particles present when compared within practitioners. myeloid: erythroid ratio and percentage of lymphocytes were also not significantly different between sites (m:e ratio p 5 0.37, lymphocyte % p 5 0.07) and were within normal limits. while there were no significant differences between the two sites in terms of number of attempts or time to success, it should be noted that the ''seasoned'' practitioners had never performed an aspirate at the cc site and had an increased number of attempts compared to the traditional sites. if the number of attempts needed for success decreases with experience, it is likely the time required would decrease as well. both subjectively and objectively, there were no significant differences in quality or cell populations between the two sampling sites in healthy dogs. based on this data, bone marrow aspiration from the cc junction appears to be equivalent to more traditional sampling sites in healthy dogs. larger studies in clinically ill dogs should be performed before routinely using the site in the clinical setting. recent research on iron homeostasis has elucidated the tightly controlled intestinal uptake of iron. hepcidin, the major hormone limiting iron absorption and release from macrophages, is downregulated by matriptase-2, a transmembrane serine protease (tmprss6) produced by the liver. while iron deficiency is commonly caused by chronic blood loss anemia and rarely dietary deficiency or intestinal disorders in dogs and other species, we report here the clinical to molecular investigations of a dog with iron-refractory iron deficiency anemia (irida) caused by a matriptase-2 deficiency homologous to a recently described autosomal recessive disorder in humans. the proband, a spayed female cocker spaniel without any clinical signs except for recent occasional idiopathic seizures, exhibited a lifelong history of microcytosis and hypochromasia but not anemia. there was no evidence of any blood loss and the dog was receiving an appropriate meat-based diet. mean values of complete blood cell counts, performed from 0.5-5 years of age, were: hematocrit 41% (normal reference range 39-56); rbc count 9.6 â 10 6 /ml (5.8-8.5 x 10 6 ); mcv 43 fl (62-72); mchc 31 g/dl (33-36). serum iron parameters revealed severe iron deficiency with serum iron 34 mg/dl (88-238); total iron binding capacity 378 mg/dl (246-450); serum iron saturation o 9% (15-50%), and ferritin 410 ng/dl (80-800). prolonged courses of oral ferrous sulfate supplementation and several short courses of intramuscular (dextran) injections and intravenous iron infusions did not result in improvement of any red cell or serum iron parameters. however, this dog was never anemic and the partial seizures could not be directly related to the iron deficiency status. no family members were available for further studies. genomic dna was extracted from the proband's edta blood and the exons of the tmprss6 gene were amplified with flanking primers and then sequenced. in comparison to the normal canine tmprss6 sequence and that of a sequenced control dog we found a homozygous missense muation, r723h, toward the c-terminal end of the protein in the proband's gene. in conclusion, the severe microcytosis and hypochromasia, low serum iron parameters and lack of a response to oral and parenteral iron therapy led to the diagnosis of irida. the missense mutation in the matriptase-2 at position 723 from an arginine, which is conserved across all species currently deposited in the genbank, to a histidine is likely the disease-causing mutation. this is the first report of an irida in the dog with features very similar to those observed in humans. dogs with naturally-occurring irida may be helpful in developing and assessing novel therapies. accidental ingestion of copper-coated zinc pennies minted after 1982 is the most common causes of zinc toxicity anemia in the dog. zinc toxicity anemia may also be seen with ingestion of zinc from other sources as ingestion of metallic foreign material other than pennies, medicines containing zinc, and zinc supplements. the purpose of this study was to determine if there is a weight below which dogs are more susceptible to zinc toxicity anemia secondary to metallic foreign body ingestion. records of dogs presented to the internal medicine service at the veterinary medical center of long island for metallic foreign body ingestion were reviewed for signalment, weight, presenting pcv, and type of metallic foreign body ingested. eighteen dogs met the inclusion criteria and were compared. of the 18 dogs, there were 15 cases of coin ingestion (83%), with 11 (73%) involving ingestion of 1 or more pennies. the other 3 cases involved ingestion of a metallic object (1), decorative garland (1), and bb pellets (1).of the 14 dogs exposed to zinc, 13 (93%) were less than 27 pounds (12.3 kgs). of those 13 cases 11 (85%) had ingested one or more pennies. eleven out of the 14 (79%) zinc exposure dogs were anemic at presentation. the average weight of the 11 dogs was 17.5 pounds (8 kg). this study showed that dogs less than 27 pounds appear to be more susceptible to developing anemia secondary to zinc toxicosis, with the majority of cases due to ingestion of pennies minted after 1982. zinc toxicity anemia secondary to penny ingestion is more commonly seen in small dogs. we suspect larger dogs are able to pass pennies through the pyloric sphincter and thus not develop clinical signs. although thrombocytopenia is common in hospitalized dogs, canine cryopreserved platelet concentrate (pc) is used infrequently. data suggest in vitro efficacy of pc and when administered to research dogs, but efficacy is unknown in clinical patients. study objectives were to determine clinical characteristics of dogs receiving pc as well as safety and efficacy of pc in thrombocytopenic dogs. medical records were evaluated retrospectively to identify dogs that received pc. information evaluated included patient characteristics, platelet count, acute transfusion reactions, and survival. twenty six dogs met study criteria. dogs receiving pc ranged in age from 1-13 years (mean 7.8 years) and 17/26 (65.4%) were spayed or intact females. hemorrhage was reported in 20/26 dogs (76.9%) prior to pc transfusion. platelet counts prior to transfusion ranged from 0 to 77 â 10 3 /ul (mean 29.9 1/à43.9 â 10 3 /ul). change in platelet count was measured in 23 dogs and the mean change was17.1 1/à 45.5 â 10 3 /ul. dose of pc administered ranged from 4.8 to 40 ml/kg with a mean of 14.5 1/à 8.8 ml/kg. no acute adverse reactions were reported. there was no correlation between transfusion dosage and platelet count change post transfusion. survival to discharge occurred in 15/26 (57.7%) of dogs. the only variable correlated with survival was age with survivors being younger than non-survivors (6.4 years-old ae 3.9 vs. 10.1 years-old ae 3.1.; p 5 0.02). efficacy of cryopreserved pc transfusions for improving clinical outcome in dogs with thrombocytopenia is yet to be determined; however, pc is well tolerated in clinical patients. fresh frozen plasma (ffp) is used to treat coagulopathies in dogs. current transfusion guidelines recommend that ffp be administered within 4 hours of thawing to avoid decreasing clotting factor function and bacterial contamination. the purpose of this study was to assess clotting factor activity and bacterial contamination of ffp that had been thawed and refrigerated for 5 days. blood was collected from 10 client-owned healthy dogs with no known history of coagulopathy or of administration of drugs affecting coagulation. plasma was separated from whole blood and frozen (à201c) within 30 minutes of collection. thawed plasma was maintained at 41c (1/à21c). aerobic and anaerobic bacterial culture, prothrombin time (pt), activated partial thromboplastin time (ptt), and factor ii, vii, ix, and x analyses were tested at time of whole blood collection, ffp thaw, 24 hours post-thaw, 72 hours post-thaw, and 120 hours post-thaw. there were no statistically significant differences in pt and ptt at any of the measured time points. statistically significant differences occurred between initial measurements of factors ii, vii, ix, and x and subsequent time points, but there was no difference in activity levels of the factors once ffp was thawed. one bacterial colony was grown from each of two samples from post-thaw plasma. thawed plasma protocols do not significantly decrease the function of factors ii, vii, ix, and x or prolong pt and ptt. bacterial contamination of the plasma supply seems unlikely, but strict aseptic technique should be used when obtaining plasma for patient use. erythrocyte pyruvate kinase (pk) deficiency is the first and most common erythroenzymopathy described in dogs, cats, and humans. the pk enzyme plays a crucial role in the erythrocyte energy metabolism and its absence causes severe hemolytic anemia, often misdiagnosed as autoimmune hemolytic anemia. the disease is inherited as an autosomal recessive trait and affected dogs also develop osteosclerosis. in dogs, the enzymatic diagnosis is complicated by the anomalous expression of malfunctioning m 2 -pk expression, but breed-specific r-pk mutation tests have been reported for basenjis, west highland white terriers (whwt), and beagles. we report here on a survey of canine pk deficiency studied at the penngen laboratory. a biased group of samples were received for screening from dog breeds with known mutations as well as from dogs with chronic, prednisone-and antibiotic-resistant hemolytic anemia and their relatives. edta blood samples and/or cheek swabs as well as medical record information were received and genomic dna and/ or enzyme activity testing were performed. among the 237 whwts 7% and 37% were found to be homozygous deficient dogs or carriers, respectively, with a mutant allele frequency of 0.26. the average age at the time of diagnosis was 1.5 years ranging from 2 months to 5 years of age; some samples came from europe and south america. of the 67 beagles studied, 36% were affected and 3% were carriers (mutant allele frequency 0.37). the average age at the time of diagnosis was 2 years ranging from 7 months to 9 years. surprisingly, very few samples from basenjis were received for screening, and none showed the mutant allele. while pk-deficient basenjis lived o 5 years, whwt and beagles often show milder signs and can reach 9 years of age. several dogs from other breeds were also examined because of chronic regenerative anemia and none had any of the known mutations seen in the other breeds. however, based upon pk enzyme activity studies, chihuahua, dachshund, miniature poodle, spitz, eskimo toy, and labrador retriever dogs were found to be affected; they also had osteosclerosis and at least one labrador retriever developed severe hemochromatosis (hepatic iron 37,300 ppm; normal o 1,200 ppm, analyzed on a dry weight basis). moreover, sequencing of the r-pk cdna from a pk-deficient labrador retriever revealed a new nonsense mutation in exon 6. in conclusion, pk deficiency appears to be a common cause for hemolytic anemia in certain breeds, and mutation testing makes screening simple. pk deficiency should also be considered in dogs of other breeds which may require the more cumbersome enzyme testing. studies to identify new mutations will confirm and simplify the diagnosis. supported in part by nih grant rr02512. immune-mediated hemolytic anemia (imha) is a common hematological condition observed in dogs. the diagnosis is based on clinical history, presenting signs and hematological evidence of imha such as regenerative anemia, leucocytosis and presence of spherocytes. the definitive diagnostic procedure is the coomb's test (direct antiglobulin test, dat) which is known to be highly specific but lacks diagnostic sensitivity. direct flow cytometric assay (fca) for igg, igm or c3 coated red blood cells (rbcs) detection might be more sensitive and thus could be introduced as an alternative diagnostic tool. to investigate the usefulness of fca for imha diagnosis, evaluation of igg, igm or c3 coated rbcs was performed from 15 dogs presented at the veterinary hospital at usp that fulfilled clinical and hematological criteria for imha. thirty eight healthy dogs were included as controls. dat was performed with polyvalent and monovalent anti-dog sera with twofold serial dilutions of each one, incubated with 2% rbcs suspension at 371c and 41c. for fca, 2% rbcs from anemic and healthy dogs were incubated with fitc anti-dog igg, anti-dog igm and anti-dog c3 and submitted to flow cytometry evaluation. specific software and mann whitney u test were used for data analysis. five dogs showed positive results for dat with polyvalent coombs reagent at 41c (titer 64 to 4096) and 371c (titer 64 to 2048) but only three of them had agglutination titer for anti-igg at 4 0 c (1024 to 8192) and 371c (512 to 4096). no positive results were observed for anti-igm and anti-c3 dat. by fc, percentage of igg, igm and c3 coated rbcs in normal and anemic dogs were, respectively, 1,18% and 17,11% (p o 0,001); 1,15% and 21,29% (p o 0,001); 0,66% and 6,99% (p o 0,001). igg coated rbcs percentage were higher in dogs showing dat positive results (min. 33,94%; max. 99,96%; median 54,25%). direct flow cytometric erythrocyte immunofluorescence assay is more sensitive than dat for detection of antibodies coated rbc in anemic dogs and may provide quantitative data useful for laboratorial diagnosis of imzha. bone marrow aspirates from cats are typically obtained from the ilium, humerus or femur, but may be difficult to obtain and/or of poor quality. in this study the feasibility, safety, and nature of sternal aspiration in cats was investigated. under general anesthesia, bone marrow aspirates were obtained in a randomized order by a single investigator from the sternum and ilium of 10 healthy cats weighing 3.4-8.4 kg, with body condition scores of 5-9 (on a scale of 1-9). for sternal aspirates, cats were positioned in sternal recumbency and a 1-inch, 22-23 ga hypodermic needle attached to a 12cc syringe was inserted into the cranial manubrium and directed caudally along the long axis of the sternum. aspirates were also obtained from the right iliac crest using an 18 ga illinois needle attached to a 12cc syringe. difficulty of site localization, needle insertion and advancement, and specimen aspiration, were scored from 1 (easiest) to 5 (hardest). bone marrow smears were prepared by one investigator and reviewed by a pathologist blinded to aspiration site and cat. sample quality was scored from 0 (no marrow particles) to 5 (excellent) based on the number of wellsmeared marrow particles on the slide. particle cellularity was scored from 1 (4 75% fat) to 3 (o 25% fat). post-procedure, cats were treated with tramadol (4-5 mg/kg, po, q12h) for 3 days, and assessed for post-biopsy pain (colorado state university feline acute pain scale, range 0 [no pain] -4 [maximum]) and site swelling (range 0 [none] -3 [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. pneumothorax was not identified. it was significantly easier to perform sternal than iliac aspiration, but the quality of the sample was significantly better for iliac than for sternal aspirates. because of limitations due to sample quality, bone marrow morphology in sternal samples could not be compared to iliac samples in all cats. for samples that could be compared, cellularity was identical for sternal and iliac samples from 1 cat but underestimated in the sternal sample from another cat. myeloid:erythroid ratios and lymphocyte numbers were the same for sternal and iliac samples in 2 and 3 cats, respectively. megakaryocyte numbers were the same in one sample, less in sternal samples compared to iliac samples from 2 cats, and overestimated in the sternal sample from 1 cat. bone marrow cell morphology was normal in all acceptable samples. it was concluded that sternal aspiration of bone marrow using a 22-23 ga hypodermic needle is 1) easier to perform than iliac aspiration; 2) safe; but 3) provides samples of lower quality than iliac aspiration in cats. the diameter of 11-13ga jamshidi-type needles makes bone marrow core biopsy difficult in cats. in this study, biopsies of the left humeral head were taken under anesthesia using a 1-inch, 15ga needle (ez-io s intraosseous infusion system, vidacare) from 10 healthy cats weighing 3.4-8.4 kg with body condition scores of 5-9 (on a scale of 1-9). humeral biopsies were compared to biopsies taken from the left iliac crest using a 2-inch, 13ga jamshidi needle. biopsies were performed in randomized order by one investigator. biopsy was repeated to a maximum of 3 attempts until a specimen ! 5 mm long was obtained. difficulty of site localization, needle insertion and needle advancement were scored from 1 (easiest) to 5 (hardest). specimens were wrapped in tissue paper and placed in davidson's fixative for 15 min and then transferred to formalin. biopsy sections were reviewed by a pathologist blinded to biopsy site and cat. biopsy length on the slide was measured, and biopsy quality was scored from 0 (no hematopoietic tissue) to 5 (! 5 intertrabecular spaces free of artifact). post-procedure, cats were treated with tramadol (4-5 mg/kg, po, q12h) for 3 days, and assessed for postbiopsy pain (colorado state university feline acute pain scale, range 0 [no pain] -4 [maximum]) and swelling of biopsy sites (range 0 [none] -3 [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. there were no significant differences between 15ga and 13ga biopsies except for post-biopsy swelling, and there were no significant effects of body weight and body condition. six (60%) of 15 ga and 5 (50%) of 13ga biopsies were considered acceptable specimens for assessment of bone marrow architecture and morphology; all intact spaces in these biopsies had normal hematopoiesis and cell morphology. comparison of acceptable 15 ga to 13 ga biopsy specimens from 4 cats showed no significant differences for cell density and lymphocytes/plasma cells, while cellularity, assessed as high in 2 of the 13 ga biopsies, was assessed as medium in corresponding 15ga biopsies; and megakaryocytes, assessed as 4-9/low-power field in one 13ga biopsy, were assessed as 3/low-power field in the 15 ga biopsy. myeloid:erythroid ratios were greater in 15 ga biopsies compared to 13 ga biopsies in 2 cats, and less in the 15 ga biopsy in one cat. discordant results between biopsies were not related to differences in quality. in conclusion, 15 ga bone marrow biopsy of the humerus was as likely to yield a specimen of acceptable quality as was 13 ga biopsy of the ilium, and resulted in less post-biopsy swelling. reports on canine acute liver failure (alf) include individual or small case series of animals with a specific diagnosis. the aim of this study was to describe the clinical course, outcome and etiology of alf in dogs presenting to a referral hospital. medical records (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) were reviewed for a diagnosis of alf (elevated serum bilirubin or icterus with concurrent coagulopathy or hepatic encephalopathy (he)). fifty cases were identified representing 22 breeds: 7 labradors retrievers, 6 golden retrievers, 3 german shepherds, and 3 cocker spaniels. median age was 6 years (1 m to humerus, 15 ga 5.1 ae 0.9 5.6 ae 0.6 2.1 ae 0.5 6.0 ae 0.7 0 0 ã (2.2-9.3) (3-9) (0-4) (3-10) ilium, 13 ga 6.1 ae 0.6 9.0 ae 0.7 1.9 ae 0.6 7.5 ae 0.8 0.2 ae 0.1 0.9 ae 0.1 ã (4.5-9.5) (6-12) (0-5) (5-12) (0-1) (0-1) 13 yrs). presenting signs included anorexia (31/50), vomiting (26/50), polydipsia (9/50) and neurologic signs (6/50 granulomatous hepatitis (gh) is a histopathological diagnosis characterized by focal aggregations of activated macrophages mixed with other inflammatory cells that is usually part of a systemic disease process (wsava). published case reports describe many potential infectious causes, but only one retrospective study involving nine dogs with gh has detailed clinically relevant findings. the aims of this study were to describe the clinical and clinicopathologic findings in dogs with a histopathological diagnosis of gh, and to identify infectious agents using differential staining techniques, pcr, and fluorescence in-situ hybridization (fish) in archival paraffin-embedded tissues from dogs with gh. medical records of dogs with a histopathological diagnosis of gh (n 5 22) were reviewed and signalment, historical toxin exposure or evidence of other systemic diseases, clinical signs, physical exam findings, clinicopathologic test results, imaging findings, concurrent diagnoses, treatments administered, and case outcome, when available, were extracted and summarized. twelve archival formalin-fixed, paraffin-embedded hepatic tissue samples were available for special staining and molecular diagnostic testing. two of these samples had sufficient tissue for only pcr. the mean age of dogs with gh was 7 years (median 6.5 years; range 2 to 17 years) and included 12 males and 10 females representing 14 different breeds. common presenting complaints included inappetance or anorexia (n 5 12), weight loss (n 5 11), lethargy (n 5 9), fever (n 5 8), and vomiting (n 5 8). high mixed liver enzyme activity (14/22) was the most common clinicopathologic abnormality. leukemia was diagnosed in one dog and copper-associated hepatopathy in 6 dogs. no infectious agents were identified using differential staining techniques. bartonella species dna was not pcr amplified from the extracted archival tissue. furthermore, no bacteria were identified by means of fish using a universal eubacterial probe. these data suggest a possible role for copper accumulation in the genesis of gh in dogs and support further evaluation of dogs with gh for evidence of copper-associated hepatopathy. future studies should include detailed environmental histories, the collection of adequate sample volumes for quantification of hepatic copper content and the examination of frozen tissues using novel molecular diagnostic platforms. hepatocyte copper and iron accumulation contribute to cell loss, inflammation, and fibrosis. the purpose of this study was to compare copper and iron accumulation in feline liver samples with different disease processes. liver biopsies (n 5 104) submitted between july 1, 2007 and june 30, 2009 were evaluated using wsava guidelines and categorized as non-hepatic/normal, congenital, inflammatory/infectious, neoplastic, and other. copper (by rubeanic acid) and iron (by prussian blue) accumulation were graded by increasing amounts (0-3) and location (centrilobular 5 cl, midzonal 5 mz, periportal 5 pp, random 5 r). associations between metal scores and diagnosis category were assessed using the kruskal-wallis test. histologic diagnoses were non-hepatic/normal (n 5 12), congenital (n 5 6), neoplastic (n 5 16), infectious/inflammatory (n 5 39), and other (n 5 31). ninety-two samples were negative for copper; remaining samples were graded 1 (n 5 5), 2 (n 5 6), and 3 (n 5 1). histologic diagnoses (pattern) for positive samples were congenital (1cl), infectious/inflammatory (7: 2cl, 1mz, 2pp, 2r), neoplastic (2pp), and other (2cl). iron staining was negative in 18 samples; remaining samples were graded 1 (n 5 38), 2 (n 5 40), and 3 (n 5 8). distribution was primarily cl (n 5 38) or r (n 5 33), though mz (n 5 13) and pp (n 5 2) distribution occurred. there were no significant differences by kruskal-wallis analysis for amount or location of hepatocellular iron or copper for the different disease categories. in this study, copper accumulation was rare, had variable distribution and occurred primarily in samples with inflammatory/ infectious disease. in contrast, iron accumulation was common and did not correlate with disease category. further prospective evaluation of copper and iron accumulation in feline liver disease and association with outcome may be warranted. chronic hepatitis (ch) in dogs is a progressive condition without clearly defined treatment. glucocorticoids are commonly used to stop progressive inflammation and fibrosis but are associated with significant side effects including a steroid hepatopathy that complicates enzyme monitoring. cyclosporine is proposed as an alternative therapy, but there are no published reports of its use for canine ch. patient records at the csu veterinary teaching hospital were searched for histologically confirmed cases of ch treated with cyclosporine. data were compiled on cyclosporine dosing, concurrent medications, clinical course and biochemical parameters. 13 patients over a 50-month period were identified. serum alanine aminotransferase (alt) decreased by an average of 71% in 12 dogs. the alt normalized completely in 6 of 10 dogs treated for 4 60 days. in 5 of 6 dogs on 4 9 mg/kg/day, the alt also normalized. five of the 6 patients that exhibited clinical signs prior to treatment showed measurable improvement (weight gain, fewer gastrointestinal signs). eight patients had hyperbilirubinemia or ascites prior to treatment; these resolved in 7. post-treatment histopathology, available in one patient, revealed decreased severity of ch. five patients exhibited adverse effects including gastrointestinal signs (3), gingival hyperplasia (1), and papillomatosis (1). cyclosporine was discontinued in 2 dogs with gastrointestinal signs. cyclosporine was an effective therapy for many cases of ch and should be considered for patients who are refractory to or cannot tolerate glucocorticoids. prospective clinical trials with histological documentation are needed to better define cyclosporine's effectiveness in ch. insertion of the veress needle and establishment of pneumoperitoneum is associated with 22 to 57% of all laparoscopic complications in humans. the purpose of this study was to determine the accuracy of interpretation of tissue impedance measurements for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in 20 cadaverous dogs euthanized for reasons unrelated to the study. placement order was randomized. a third individual evaluated impedance measurements using a handheld device (sensormed, knoxville tn) to determine correct versus incorrect needle placement. veress needle locations were marked using contrasting colors of india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified 29/33 correct and 7/ 7 incorrect placements, respectively. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. agreement was moderate (kappa 0.50, p 5 0.01) for placements by operator 1 and very high (kappa 5 0.88, p o 0.01) for placements by operator 2. results for tissue impedance measurement interpretation are superior to published data for currently available tests. impedance measurements accurately detected all incorrect needle placements. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether it increases operator detection of inappropriate veress needle placement and decreases installment phase complication rates. delayed detection of intestinal perforation during veress needle insertion is associated with high mortality. the purpose of this study was to evaluate the accuracy of tissue impedance measurement interpretation for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in 24 cadaverous cats. placement order was randomized. a third individual evaluated impedance measurements (sensormed, knoxville, tn) to determine placement location. needle locations were marked using india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified 36/38 correct and 2/10 incorrect placements. all 8 undetected incorrect placements were located within the retroperitoneal fat pad. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. correlation was absent (kappa à0.15, p 5 0.34) for placements by operator 1 and substantial (kappa 0.78, p o 0.01) for operator 2. there was no association between correct or incorrect placement and operator on chi-squared analysis. failure of impedance measurements to identify placement in the retroperitoneal fat pad resulted in poor accuracy and discordant kappa statistics. small cat size limited the number of appropriate placement sites, perhaps resulting in excessively dorsal placement. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether impedance measurements increase detection of inappropriate veress needle placements or decrease installment phase complication rates. best clinicopathologic tests detecting portosystemic shunting (pss) in dogs remains controversial. this retrospective study examined performance of single random "fasting" and paired serum bile acids (sba; pre-and 2-hr post-feeding) in a large population of non-icteric dogs with confirmed pss (abdominal ultrasound, colorectal scintigraphy, radiographic or spiral-ct portography, laparotomy, or necropsy). sba were measured by enzymatic colorimetric method with normal o 25 mmol/l. dogs meeting inclusion criteria (n 5 568) included 467 portosystemic vascular anomalies (psva; 368 extrahepatic [e-psva], 99 intrahepatic [i-psva]), and 101 acquired pss (apss). signalment and laboratory parameters were recorded. non-parametric statistical analyses were used, two-tailed p o 0.05 applied with bonferroni corrections. median age and weight of 74 breeds were 1.2 (0.2-12) yrs and 6.6 (0.2-48) kg, with equal gender distribution. random "fasting" sba detected 90% psva and 81% apss, whereas post-feeding sba detected 100% psva and 98% apss. low protein-c (o 70% activity) occurred in 96% psva and 74% apss. low mcv and creatinine occurred in 82% and 94% of psva dogs, respectively; other tests were less helpful. in apss, post-feeding sba was superior. compared to apss, psva had significantly (p 0.002) lower mcv, cholesterol, bun, creatinine, glucose, and protein-c. compared to e-psva, i-psva had significantly (p 0.009) lower post-feeding sba, mcv, albumin, urine specific gravity, and protein-c but higher cholesterol and glucose. post-feeding sba reflect physiologically provoked bile acid challenge and should be the preferred sba test in non-icteric dogs for pss detection. protein-c assists in identifying psva but its utility in apss may be complicated by concurrent coagulopathies and inflammation. this study compared outcomes of treatment with adjunctive nonsteroidal anti-inflammatory drugs (nsaids) or anti-inflammatory glucocorticoids in dogs with severe pulmonary blastomycosis. medical records were reviewed for dogs diagnosed with blastomycosis at the university of illinois veterinary teaching hospital between 1992 and 2007. dogs with a presenting pao 2 of 80 mmhg, and clinical or radiographic signs of respiratory blastomycosis were included. all dogs were treated with either itraconazole, fluconazole, amphotericin b, or a combination of these. group 1 (g1) dogs were treated with nsaids and group 2 (g2) dogs were treated with glucocorticoids as anti-inflammatory adjunctive therapy. the following comparisons were made: days of oxygen supplementation, days in hospital, survival to discharge, and long term patient survival. mann-whitney u tests and chi-squared tests were performed on continuous and categorical data, respectively. p o 0.05 was considered significant. sixty-eight dogs fit the inclusion criteria. g1 consisted of 31 dogs and g2 consisted of 37 dogs. the two groups were found to be similar in weight, age, and sex distribution. there was no significant difference between the two groups with regard to duration of oxygen supplementation, duration of hospitalization, survival to discharge, and patient survival. there does not appear to be a difference between the clinical course or patient outcomes between groups of dogs with severe pulmonary blastomycosis treated with nsaids or anti-inflammatory glucocorticoids. further studies need to be performed to fully evaluate the impact these adjunct treatments have on prevention of ards and additional respiratory complications. diagnosis of feline histoplasma capsulatum infection traditionally relies upon identification of organisms in circulating monocytes or affected organs. in recent years, an antigen assay (aa) was developed for the diagnosis of disseminated histoplasmosis in human patients, but there is little information describing this test in cats. the goal of this study was to determine the sensitivity and specificity of h. capsulatum aa in cats with clinical disorders suggestive of histoplasmosis. urine and serum h. capsulatum aa results for feline patients from 3 veterinary hospitals were evaluated. medical records were reviewed for confirmatory evidence of histoplasmosis (based on cytological or histopathological findings) or an appropriately supported alternate diagnosis. aa results were available for 78 cats; initial testing was performed on 72 urine samples, 6 serum samples, and 1 unspecified sample. of these cats, 17/78 had a definitive diagnosis of histoplasmosis based on organism identification, and 10 had a definitive alternate diagnosis (e.g., neoplasia, other infection) based on necropsy findings (n 5 5) or other clinical data (n 5 5). an additional 16 cats had a clinical alternate diagnosis with no cytological or histopathological evidence of histoplasmosis in the affected body system(s). the remaining cats had unverified histoplasmosis (n 5 9) or an open diagnosis (n 5 26). of the 17 cats with confirmed histoplasmosis, 16 were positive on initial urine aa. one cat (with rectal involvement) was negative, indicating a test sensitivity of 94%. one cat was positive on urine aa but negative on serum aa. all of the 26 cats with definitive or clinical alternate diagnoses had negative results on the aa, suggesting an excellent specificity (100%). however, this result should be interpreted with caution, as the possibility of primary or concurrent histoplasmosis was only definitively excluded in the 5 patients who underwent necropsy examination. these findings suggest that the aa for h. capsulatum is a reliable diagnostic tool in this species. a positive result appears to reliably support the presence of infection, but a small percentage of infected cats may be negative on aa. in addition, tests performed on urine may be more sensitive that those performed on serum. the purpose of this study was to evaluate the sensitivity and specificity of an aspergillus galactomannan antigen enzyme immunoassay (ga-eia) for the diagnosis of canine systemic aspergillosis. serum and urine samples were collected from sick dogs at 2 hospitals (ucd and tamu). group 1 dogs were diagnosed with systemic aspergillosis using culture (sterile site) or microscopy and culture (non-sterile site). group 2 dogs had clinical findings suggestive of aspergillosis but an alternate diagnosis was established. group 3 dogs were not suspected to have aspergillosis. samples were tested using the ga-eia and results expressed as a galactomannan index (gmi). gmis 4 0.5 were considered positive. comparisons were performed using the mann-whitney test. there were 10 dogs in group 1, 22 in group 2, and 23 in group 3. serum was collected from all dogs, and urine from 6, 9, and 10 dogs, respectively. serum gmis did not differ from urine gmis across groups. serum gmis of group 1 dogs were higher than those of group 2 and group 3 dogs (p o 0.0001). results from dogs in group 2 did not differ from those in group 3 (p 5 0.43). two dogs in group 1 tested negative, but had localized pulmonary infections. one dog in group 2, which had paecilomycosis, tested positive. two dogs in group 3 tested positive. one was being treated with plasmalyte. the other had a cutaneous opportunistic mycosis. these data support the utility of this assay to aid in the diagnosis of systemic aspergillosis in dogs. anaplasma phagocytophilum, an ixodes tick transmitted rickettsial bacterium has a wide mammalian host range that is not commonly reported in cats. clinical signs in humans, dogs and cats are often vague and include lethargy, anorexia and malaise. the purpose of this retrospective study was to describe the clinical signs, laboratory data and response to treatment in cats that tested positive for a. phagocytophilum on a commercially available pcr of peripheral blood (fastpanel tm ). this study describes and reports the appearance of intracellular morulae in feline neutrophils contributing to the diagnosis of a. phagocytophilum. the a. phagocytophilum real-time pcr (rt pcr) assay consists of four multiplexed primer systems designed to detect a total of three distinct genes. amplicons were confirmed as a. phagocytophilum by dna sequencing. clinicopathologic data was obtained by review of medical records and interview of primary veterinarians. complete blood counts were available from 13/15 cats and 10/13 blood smears were reviewed. the cats included in this study were all positive for a. phagocytophilum by real-time pcr. the cats ranged from 4 months to 13 years of age with an average age of 3.7 years. fifteen of 15 cats had a history of tick exposure and lived in the northeastern region of the us, an ixodes endemic area. all cats presented with lethargy, 13/15 were anorexic and 14/15 had a fever (temperature 4 103 o f). other clinical findings included hepatomegaly, splenomegaly, ataxia and ocular changes of conjunctivitis and elevation of the nictitating membrane. hematologic findings included leukopenia (1/13), neutropenia (1/13) and lymphopenia (4/13). thrombocytopenia was not noted in any case. morulae were seen within neutrophils in 2/13 cases. all cases in this report responded to treatment with doxycycline. this is the first report of the identification of morulae within neutrophils via peripheral blood smear review in cats confirmed by rt pcr to be infected with anaplasma phagocytophilum in north america. infection with anaplasma phagocytophilum should be considered in a clinically ill cat with tick exposure, living in an ixodes endemic area that presents to a veterinarian for lethargy, anorexia and fever. the spectrum of disease manifestations and the accompanying clinicopathological abnormalities indicative of bartonellosis in dogs have not been thoroughly characterized. the objective of this unmatched case-control study was to compare signalment, clinical and pathologic findings in clinically-ill dogs suspected of a tick-borne disease that were negative for bartonella sp. dna (controls) as were the dogs diagnosed with bartonellosis by pcr amplification, dna sequencing and the bapgm (bartonella alpha proteobacteria growth medium) enrichment culture approach. both groups were tested under the same laboratory conditions and in the same time frame. medical records were reviewed for information regarding signalment, medical history, physical examination findings, clinicopathological abnormalities, microbiological data and treatment. the study population consisted of 47 bartonella-infected dogs and 93 non-infected dogs. healthy dogs with no historical illnesses, such as blood donors, were excluded. the following species were amplified: b. henselae (n 5 28, 59.6%), b. vinsonii subsp. berkhoffii (n 5 20, 42.6%), b. koehlerae (n 5 3, 6.4%), b. volans-like (n 5 3, 6.4%), b. bovis (n 5 1, 2.1%). nineteen (40.4%) bartonella-infected dogs were febrile and lethargic and ten (21.3%) had neurological signs. laboratory abnormalities for both groups are summarized below (number of affected dogs provided in parenthesis): multivariate logistic regression using confounding factors was performed to establish potential associations between specific variables and bartonella sp. infection. there were no differences in signalment, age, sex, body weight and duration of clinical signs between the two groups. compared to the control population, infection with the genus bartonella was associated with a diagnosis of endocarditis (p 5 0.0196, or 5 7.95, 95%ci 5 1.39-51.11) and hypoglobulinemia (p 5 0.0057, or 5 5.30, 95% ci 5 1.62-18.55). controls were more likely to have joint effusion (p 5 0.0059, or 5 5.89, 95% ci 5 1.62-27.22) and azotemia (p 5 0.0353, or 5 2.93, 95%ci 5 1.07-8.86) than were the bartonella sp. infected dogs. bartonella was detected in dogs with signs such as fever, anemia, thrombocytopenia, hyperglobulinemia and proteinuria that are typically associated with tick-borne diseases. when endocarditis or hypoglobulinemia are detected, testing for bartonella should be prioritized. likewise, the detection of bartonella should prompt further testing for endocarditis, if not already investigated. surveillance studies in other species depend on detection of antibodies to the highly conserved influenza a nucleoprotein (np); however, no such antibody detection assay is approved for canine use in the u.s. the purpose of this study was to determine the diagnostic accuracy of a commercial blocking elisa used for avian species in detecting influenza a np antibody in dogs. since the blocking elisa is not a species-specific or viral subtype-specific format, we hypothesized that it would detect np antibodies in dogs infected by influenza a virus. serum samples from uninfected dogs (n 5 204) and dogs naturally infected with canine influenza h3n8 (n 5 150) were tested using the idexx flockchek blocking elisa for influenza a np antibody according to manufacturer instructions. the sample/negative control (s/n) absorbance ratios for infected dogs ranged from 0.12 to 0.67 compared to 0.53 to 1.40 for uninfected dogs. a receiver operating characteristic (roc) curve analysis determined optimum diagnostic sensitivity (99.3%) and specificity (99.0%) at a s/n cutoff ratio of 0.647. using this cutoff ratio, the overall diagnostic accuracy was 99.2%. coefficients of variation for intra-assay (4.7%) and inter-assay (6.1%) testing demonstrated good repeatability with canine sera. the excellent diagnostic accuracy of the commercial blocking elisa makes it a suitable tool for large-scale surveillance of influenza a virus exposure in dogs. upper respiratory disease (urd) can affect a majority of cats in shelters and is one of the leading reasons for euthanasia of otherwise adoptable cats. the purpose of this study was to determine prevalence and risk factors for upper respiratory pathogens in four different models for managing unowned cats: short-term animal shelters (shel), long-term sanctuaries (sanc), home-based foster care (fost), and trap-neuter-return (tnr) programs. conjunctival and oropharyngeal swabs were collected from 543 cats, half of which had clinical signs of urd, and tested for feline herpesvirus (fhv), feline calicivirus (fcv), chlamydiophila felis, bordetella bronchiseptica (bb) and mycoplasma felis by real-time pcr. management model, vaccination, sex, age, body condition, and clinical signs were evaluated as risk factors for infection. a majority of cats in all management models carried one or more organisms capable of causing urd. in many cases, prevalence was similar in cats with or without clinical signs. unlike diseases that can be controlled by segregation of symptomatic animals, the lack of strong correlation between the presence of pathogens with the presence of clinical signs suggests that feline urd control should be managed by vaccination before or at the time of intake ,biosecurity protocols that presume all cats may be shedding pathogens, and minimizing stressful conditions that contribute to disease susceptibility. depending on geographical location, sex, age and environment, 2-40% of cats worldwide are infected with the feline immunodeficiency virus (fiv). knowledge of the fiv status of cats is important to limit the spread of disease and to institute appropriate health management. however, like all lentiviruses, fiv is highly variable in nucleotide sequence, and viral load in cats is variable during different disease stages. detection of antibodies is the most widely employed diagnostic approach, but does not distinguish fiv-infected from fiv-vaccinated cats. in this study, samples from 30 fiv-seronegative cats, 30 fivseropositive cats, and 30 fiv-historically seronegative but vaccinated cats, were analyzed by a commercial quantitative pcr (qpcr) assay and virus isolation. replicate blood samples were coded, and then submitted for 1) qpcr (idexx); and 2) mononuclear cell isolation with 7-day culture and viral p24 antigen detection by elisa. for the p24 antigen elisa, cutoff absorbance values were established from analysis of 10 fiv-negative samples. fiv infection status was pre-determined based on 2 antibody-elisa results and vaccination history. results indicated that qpcr had a sensitivity of 76% for samples from fiv-seropositive cats, and a specificity of 100% and 94.1% for samples from fiv-seronegative and fiv-vaccinated cats, respectively. at a cutoff value of 3 standard deviations above the mean absorbance for p24 antigen elisa, results from fiv-negative samples yielded a sensitivity of 69.2% for samples from fiv-seropositive cats, and a specificity of 77.1% and 64.7% for samples from fiv-seronegative and fiv-vaccinated cats, respectively. conclusions from this study are 1) the commercial fiv qpcr assay has high specificity but limited sensitivity for diagnosis of fiv infection; 2) 7-day virus culture has limited sensitivity and specificity. hence, detection of antibodies remains the most reliable test for diagnosis of fiv infection, but qpcr may be suitable to rule out infection. oral disease is an important clinical problem in feline medicine and includes common painful conditions such as oropharyngeal inflammation (formerly known as gingivostomatitis) and tooth resorptive lesions. a number of infectious agents have been associated with private veterinary clinics in the u.s. were recruited to test feline patients presenting with oral disease. presenting cases included cats with plaque, calculus, gingivitis, stomatitis, periodontal disease, tooth resorptive lesions and other oral diseases as defined by the practitioner. all cats were tested using a commercially available point-of-care elisa test (idexx snap combo). confirmatory tests were not performed as part of the study. seroprevalence was calculated as the percentage of positive tests in the study population for each virus. a total of 11,262 cats were tested. seroprevalence for felv was 6.8% and for fiv was 7.1%. of these, 119 cats (1.0%) were infected with both viruses. seroprevalence was higher in cats with inflammatory oral disease than in cats characterized with other types of oral disease. of 7,805 cats with gingivitis, seroprevalence for felv was 7.4% and for fiv was 7.9%, with 1.1% of cats co-infected. of 1,953 cats with stomatitis, seroprevalence for felv was 12.2% and for fiv was 13.9%, with 2.2% of cats co-infected. the seroprevalence for felv and fiv reported in this population of cats with oral disease was higher than in a recent large study where samples from u.s. cats not specifically selected for oral disease were tested (felv 2.3%, fiv 2.5%). results of this study indicate that further investigation of the role of retroviruses in cats with oropharyngeal inflammation is warranted. reliable tests and preventive vaccines and medications for feline retroviral and heartworm (hw) infections are available, but compliance with protocols to reduce transmission is unknown. no largescale longitudinal studies evaluating prevalence over time have been reported. the purpose of this study was to determine the prevalence and risk factors for infection compared with a similar study completed for the first time 5 years previously. veterinary clinics and animal shelters in the us and canada submitted results of testing using a point-of-care elisa for felv antigen, fiv antibody, and hw antigen (idexx snap triple) and risk factor information for cats tested during march-september 2010. bivariable and multivariable analyses were used to evaluate risk factors for infections. a total of 62,301 cats were tested. only 16% of owned cats were prescribed hw preventive. risk of retroviral infections was increased by outdoor access, adulthood, and male gender. the most important risk factor associated with all 3 infections was clinical disease; in particular, respiratory and oral diseases and abscesses or bite wounds. multivariate analysis revealed differences among geodivisions and across infection types. feline retroviral and heartworm infections are easily prevented, but difficult to treat. despite availability of effective management protocols, compliance remains inadequate to reduce the prevalence of these infections. improved use of preventive care and testing to identify and segregate contagious cats, particularly those at high-risk, is required to reduce the morbidity of these preventable infections. infectious disease outbreaks are common in animal shelters and are frequently managed by depopulation when risk-assessment tools are not available. during a canine distemper virus (cdv) and parvovirus (cpv) outbreak in sheltered dogs, we used a cdv/cpv point-of-care antibody titer elisa, a cdv quantitative rt-pcr test, and a cpv fecal antigen test as risk assessment tools to guide release of exposed dogs from quarantine and euthanasia of diseased dogs. serum samples (for antibody titers) and swabs of the conjunctiva and upper respiratory tract (for cdv pcr) were collected from 111 asymptomatic dogs starting on day 4 of the outbreak. dogs with positive cdv pcr tests were retested every 2 weeks until euthanized for progressive disease or released following recovery from infection. dogs with clinical signs of parvoviral infection were tested using a cpv fecal antigen test. for dogs ! 4 months old, protective antibody titers correlated with resistance to clinical disease, but 10% of dogs shed cdv. lack of protective cdv antibody titers correlated with susceptibility to clinical infection, but most dogs recovered. risk assessment and outcome in 60 dogs !4 months of age feline herpesvirus 1 (fhv-1) is a common ocular and respiratory pathogen of cats that can have clinical illness exacerbated by stress. cyclosporine (csa) is commonly used for the treatment of a number of inflammatory diseases in cats and can induce immune suppression. a small number of cats administered csa to block renal transplant rejection have developed clinical signs of upper respiratory tract disease that may have been from activated fhv-1. in this study, young adult cats experimentally inoculated with fhv-1 several months previously were divided into three groups and administered methylprednisolone acetate (8 cats, 5 mg/kg, im, day 0 and day 21), csa (8 cats, 7.0 mg/kg, po, daily for 42 days), or a placebo (7 cats, corn syrup; 0.075 ml/kg, po, daily for 42 days). each cat was assigned a daily individual clinical score by a trained, masked observer using a standardized score sheet during the initial pre-treatment time period (day -14 to day 0) and throughout the 42 day treatment period. each individual clinical score (conjunctivitis, blepharospasm, ocular discharge, sneezing, nasal discharge, nasal congestion, and body temperature scores), the total clinical score (sum of all parameters), the total ocular score (sum of conjunctivitis, blepharospasm, ocular discharge), and total respiratory score (sum of ocular discharge, sneezing, nasal discharge, nasal congestion) were analyzed using sas proc glimmix with 'treatment', 'time', and the two-way interaction 'treatment by time' all as fixed effects. statistical significance was defined as p o 0.05. on day 42 of the study, all of the csa treated cats had detectable concentrations of csa in serum (mean 5 406.1 ng/ml; standard deviation 5 291.8 ng/ml; median 5 388.5 ng/ml). when group mean values for clinical signs were compared over time as described, significant differences in individual clinical score measurements, in total score, total ocular score, or total respiratory score were not detected over time among any of the treatment groups. while clinical signs of activated fhv-1 occurred in some cats administered methylprednisolone or csa, disease was mild and self-limited in most cats and there were no significant csa sideeffects. these results suggest that the csa protocol described here is unlikely to reactivate latent fhv-1 infection and cause significant clinical illness. the purpose of this study was to determine the prevalence and risk factors for enteropathogens in four different models for managing unowned cats: short-term shelter, long-term sanctuary, home-based foster care, and trap-neuter-return (tnr) programs. fecal samples were collected from 482 cats, half with diarrhea (d) and half with normal feces (n), and tested for a panel of feline and zoonotic enteropathogens by polymerase chain reaction, antigen, and fecal flotation. risk factors for infection evaluated include management practices, fecal consistency, and signalment. a majority of cats had at least one enteropathogen of feline or zoonotic importance, regardless of management model or preventive healthcare protocol. for most enteropathogens, the presence or absence of diarrhea did not correlate with infection, the exceptions being t. foetus in sanctuary cats and fcov in foster cats. prevalence of specific enteropathogens varied between management models, reflecting differences in preventive healthcare and housing conditions. management protocols for unowned cats were inadequate for elimination of infections present at the time of intake and for prevention of transmission of enteropathogens among shelter cats. improved compliance with effective vaccination, deworming, sanitation, and housing protocols is needed to reduce zoonotic and feline health risks. several allergic diseases of cats, including atopy and gingivostomatitis, can be resistant to glucocorticoids but responsive to cyclosporine. toxoplasma gondii infection occurs in approximately 30% of cats and the effect cyclosporine therapy has on the t. gondii oocyst shedding period is unknown. the objective of this study was to determine whether administration of cyclosporine before or after t. gondii infection influences the oocyst shedding period. the young adult cats were t. gondii seronegative when administered 1,000 t. gondii tissue cysts orally on day 42. group 1 cats (n 5 10) were never administered cyclosporine; group 2 cats (n 5 10) were administered cyclosporine (7.5 mg/kg, po) daily on days 84-126; and group 3 cats (n 5 10) were administered cyclosporine (7.5 mg/kg, po) daily from days 0-126. available feces from individual cages were collected daily and fecal flotation by sugar centrifugation was performed for 84 days after t. gondii inoculation. group 3 shed oocysts for a significantly shorter period than groups 1 or 2 and had a significantly lower oocyst shedding scores than groups 1 and 2 on days 5-11 after t. gondii inoculation. group 2 cats had completed the oocyst shedding period prior to being administered cyclosporine and repeat oocyst shedding was not detected during administration of the drug. administration of cyclosporine prior to t. gondii infection lessened oocyst shedding which is likely from the anti-t. gondii effects of the drug. administration of cyclosporine using this protocol is unlikely to induce repeat t. gondii oocyst shedding in client-owned cats. ã 5 group with diarrhea significantly different than group with normal feces p o 0.05 known about its metabolic pathways or mechanism of pathogenicity and whole genome sequencing of feline hemoplasmas has not yet been reported. the aim of this study was to completely sequence the genome of m. haemofelis to further characterise this important pathogen. mycoplasma haemofelis genomic dna was purified and subjected to whole shotgun roche 454 sequencing. gaps were closed using targeted pcr and amplicon sequencing. ribosomal genes and potential open reading frames (orfs) were predicted in silico. putative orfs were annotated and orthologous groups identified. analysis showed a circular genome of 1.15 mbp with a gc content of 38.9%. thirty-one transfer rnas (trnas) were identified, accounting for all amino acids, including a tryptophan trna for the opal codon (uga). of the 1,545 putative proteins identified, 328 (21.2%) matched to proteins from other bacterial species. in common with the pneumoniae group of mycoplasmas, the closest phylogenetic relatives of the hemoplasmas, genes involved in carbohydrate metabolism were limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source for m. haemofelis. the majority of the pentose phosphate pathway genes present in other cultivatable mycoplasmas appear to be incomplete or absent in m. haemofelis, suggesting an alternative mechanism for sourcing purine and pyramidine bases such as scavenging from the host. a gene encoding a glyceraldehyde-3-phosphate dehydrogenase homolog of the immunogenic msg1 protein of mycoplasma suis was present. of the uncharacterized hypothetical proteins, 1,115 were arranged in series of orthologous repeats, or comprised fragments there-of, encoding putative proteins of approximately 200 amino acids. the predicted motifs of the majority of these putative proteins were consistent with these proteins being presented on the cell surface; an n' terminal signal peptide or transmembrane region followed by a non-cytoplasmic tail. these data have provided valuable information as to why this pathogen remains highly fastidious; it lacks some of the metabolic pathways found in cultivatable mycoplasmas. we have also identified a homolog of a known m. suis immunogenic protein, and identified a potential mechanism for host immune system evasion by way of highly repetitive, putatively surface-expressed hypothetical proteins with variable sequences. canine leptospirosis has been recognized as a re-emerging disease in the u.s. over the past 20 years, and several serosurveys of the prevalence of leptospiral antibodies in dogs have been published during that time. the role of cats in the epidemiology of leptospirosis has received little attention. serosurveys of cats for exposure to or infection with leptospires have been published from other geographic areas, but none for cats in the u.s. in the past four decades. the new england states have been found to have a high incidence of canine leptospirosis. the purpose of this pilot study was to determine the prevalence of leptospiral antibodies in a population of feral cats in central massachusetts. blood was collected from 63 sexually intact feral cats presented to a spay and neuter program. microagglutination titers to leptospira serovars autumnalis, hardjo, bratislava, icterohaemorrhagiae, canicola, pomona, and grippotyphosa were determined. three of 63 cats (4.8%) had a positive titer to one or more serovars, with autumnalis being the most common. these results are consistent with previously published prevalence rates in feral cats. further studies are required to determine the role of leptosporosis in clinical disease in the domestic cat. since years the rivalta's test is routinely used in several european countries as a tool to diagnose feline infectious peritonitis (fip) in cats with effusion. it is inexpensive and easy to perform in private practice. there is, however, only little information about mode of action or its diagnostic value. the objectives of this study were to evaluate sensitivity, specificity, positive (ppv) and negative predic-tive values of the rivalta's test to diagnosis of fip and to examine if there is a correlation with any effusion or blood parameters. medical records of 782 cats with effusion in which the rivalta's test was performed between 1999 and 2010 were reviewed concerning diagnosis, blood and effusion parameters, and survival time. effusion and blood parameters were compared between rivalta-positive and -negative effusions using the mann whitney u test. prevalence of fip in cats with effusion was 31.7%. the rivalta's test showed a sensitivity of 91.3%, a specificity of 66.6%, a ppv of 55.9%, and a npv of 94.3% for the diagnosis of fip. the ppv improved, when cats with lymphoma or bacterial infection were excluded (ppv 69.2%) and also, when only cats younger than 2 years (ppv 87.5%) or 1 year (ppv 90.8%) of age were included. the most important significantly different parameters between rivalta-positive and -negative effusions were specific gravity as well as cholesterol, triglyceride, and glucose concentration in the effusion. the rivalta's test in general is a useful tool to diagnose fip, but its sensitivity and specificity are not as high as previously assumed. if the rivalta's test, however, is performed in young cats or if certain diseases have been ruled-out, its diagnostic value is high. effusion total protein is not highly correlated with test outcome. therefore, it is still unclear, which components in the effusion of cats with fip lead to a positive rivalta's test. canine parvovirus (cpv) and canine distemper virus (cdv) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. our study aimed to determine the antibody protection status of dogs at the time of admission into an animal shelter (pre-vaccination) and over the following 2 weeks after vaccination. serum samples were obtained from 57 incoming shelter dogs aged 4 months and older with no known history of vaccination. immediately following serum collection, the dogs were vaccinated against cpv and cdv using a modified live vaccine (mlv). cpv and cdv antibody protection status was determined using synbiotics titerchek. dogs with unprotective serum antibody levels against cpv and/or cdv were retested at 6-8 days post-vaccination and again at 13-15 days post-vaccination, if antibody levels were still unprotective against cpv and /or cdv. at the conclusion of the study, stored duplicate sera were submitted for batch 'gold standard' testing to determine canine distemper virus serum neutralization and canine parvovirus hemagglutination inhibition antibody titers. based on the synbiotics titerchek results, 43/57 dogs (75.4%) were protected against cpv and 21/57 (36.8%) were protected against cdv at intake. older incoming dogs were more likely to be protected against cpv (p o 0.0001) and cdv (p 5 0.0174). dogs that were spayed/neutered were more likely to be protected against cpv on intake than intact animals, although this result was not statistically significant (p 5 0.0627). the number of dogs with protective titers against cpv/cdv was increased at 6-8 days post-mlv (cpv -48/56, 85.7%; cdv -31/52, 59.6%) and further increased at 13-15 days post-mlv (cpv -54/54, 100.0%; cdv -47/48, 97.9%). we conclude that incoming shelter dogs often do not have protective antibody titers against cpv and cdv, but older shelter dogs are more likely to be protected against cpv. based on this population, we further conclude that a large percentage of dogs develop protective antibody titers to cpv and cdv within 1 to 2 weeks when vaccinated with a mlv. mycoplasma spp. are common inhabitants of the feline oral cavity and so likely contaminate many cat bite abscesses. mycoplasma spp. are cell-wall deficient and so do not respond to beta-lactam class antibiotics, the class most commonly use for the treatment of cat bite abscesses. the objectives of this study was to determine whether mycoplasma spp. are common contaminants of cat bite abscesses and are associated with beta-lactam resistant clinical disease. privately owned cats with clinical evidence of an acute abscess suspected to be from a cat bite were included in the study. participants were given a free aerobic and anaerobic culture as well as mycoplasma spp. culture and polymerase chain reaction using mycoplasma genus specific primers. mycoplasma spp. amplicons were sequenced to determine the species. all cats were initially treated with appropriate wound management, were administered an antibiotic in the beta lactam class (amoxicillin-clavulanate or cefovicin), and were rechecked in person or by phone 7 days after beginning treatment. of the 26 cats entered into the study to date, mycoplasma spp. were amplified from 4 cats (15.4%). of the 2 positive samples with adequate dna for sequencing, one was consistent with m. felis and the other was consistent with m. equigenitalium. of the 26 cats, 25 responded by day 7 to the initial treatment, including 3 of the 4 mycoplasma spp. positive cats. the cat that failed initial treatment was positive for m. equigenitalium on both day 0 and day 7 and ultimately responded to administration of a fluoroquinolone. the results suggest that while mycoplasma spp. commonly contaminate cat bite abscesses, routine wound management and antibiotic therapy is adequate for control. however, as mycoplasma spp. infections do not respond to beta lactam class antibiotic therapy, these organisms should be on the differential list for cats with abscesses that fail treatment with this antibiotic class. molecular diagnostic assays are frequently used in clinical practice to aid in the diagnosis of suspected infectious respiratory diseases in dogs. however, most currently available assays cannot distinguish strains of the organisms used in vaccines from naturally occurring strains. our prior studies demonstrated that previously immune adult dogs are unlikely to shed nucleic acids of vaccine strains of adenovirus 2, parainfluenza, or bordetella bronchiseptica. however, whether this is true for puppies is unknown. puppies (n 5 8) at a breeding facility were moved into area without other dogs at 6 weeks of age. swabs of the nasal and pharyngeal mucosa were collected prior to vaccination and on days 0, 1, 2, 3, 4, 5, 6, 7, 10, 14, 17, 21, 24, and 28 after vaccination with an intranasal adenovirus 2, parainfluenza, and b. bronchiseptica vaccine (intratrac 3, schering plough). the swabs were shipped on cold packs by overnight express for dna/rna extraction and assay in the fastpanel tm pcr canine respiratory disease profile at antech diagnostics. all puppies were negative for the infectious agents prior to vaccination. after vaccination, positive assay results for parainfluenza and b. bronchiseptica were first detected on day 1 and on day 2 for adenovirus 2. by day 3, dna or rna of the agents were amplified from all puppies from both sample sites and most samples were positive for all 3 agents through day 10. by day 24, only one dog was still positive for b. bronchiseptica. the results indicate that intranasal administration of adenovirus 2, parainfluenza, or bordetella bronchiseptica vaccines commonly leads to positive molecular diagnostic assay results for a short time period after primary vaccination. these findings should be considered when assessing the results of these assays in client-owned puppies with respiratory disease. antimicrobial resistance in escherichia coli is an increasing concern in both human and veterinary hospitals' patients. the choice drug for treatment in dogs is enrofloxacin, a second generation fluoroquinolone (fq) whose activity reflects, in part, ciprofloxacin. among the difficulties in effective e. coli treatment is rapid detection of fq resistance. the purpose of this study was to determine the specificity and sensitivity of a fret based assay for the rapid detection of urinary tract infections caused by fq associated multi-drug resistant e.coli. 306 clinical e. coli isolated from canine urine and 120 clinical veterinary urine samples being examined for e. coli were subjected to susceptibility testing for 14 drugs representing 6 drug classes. pure isolates were designated ndr (no drug resistance), sdr (single drug resistance) and mdr (multi-drug resistant) (n 5 101 mdr, 116 sdr and 89 ndr). minimum inhibitory concentration (mic) for enrofloxacin ranged from 0.03 mg/ml to 512 mg/ml, with high mic generally associated with mdr. extracted dna from culture and from urine were subjected to fret-pcr targeting single nucleotide polymorphisms in gyra. the resulting product was sequenced to detect other polymorphisms. further, to determine the level of detection, microbial free canine urine was inoculated with 10 6 to 10 1 cfu/ml of 7 isolates characterized by variable susceptibility to enrofloxacin (mic enro 5 0.03, 0.06, 0.15, 1, 64, 128, 256 mg/ml). of 306 pure isolates, 50 were confirmed positive for enrofloxacin resistance (mic enro 4 4 mg/ml), 43 of which were positively identified by the fret-pcr assay giving a sensitivity of 86.00%. only 1 isolate that was resistant was not detected (specificity of 96.66%). however, of the isolates expressing high level resistance (mic 4 8 x breakpoint [64 mcg/ml]), and mdr (n 5 34), sensitivity 5 97.06%. of the 120 urine samples 27 contained e. coli 7 of which determined to be fq-resistant by the assay. colony dilutions of e. coli confirmed the assay able to detect enrofloxacin resistance at as low as 101 cfu/ml. the relationship between cfus and the peak of the -(d/dt) fluorescence of the melting curve was r2 5 0.988. these results conclude that the assay is capable of detecting not only the presence of escherichia coli in clinical samples, but also detecting severity of fluroquinolone resistance and infection. the fluoroquinolones (fqs) are a key class of synthetic antimicrobial agents with an established history in both humans and companion animals of efficacy for treatment of urinary tract infections (utis) caused by e. coli, and fluoroquinolones are common therapy. among the commonly used fqs in dogs and cats are the 2nd generation drugs, enrofloxacin, marbofloxacin, orbifloxacin (all veterinary approved) and the human drug ciprofloxacin; no 3rd and 4th generation fq is routinely used. the purpose of this study was to assess the in vitro activity of different generation fqs toward e.coli uropathogens whose phenotype ranges from no resistance to multidrug resistance. a total number of 51 canine uropathogenic canine or feline e.coli isolates had been subjected to susceptibility testing to 6 drugs classes (15 drugs) and phenotyped as to resistance: none (ndr, n 5 12), single (sdr, n 5 15), or multiple, mdr (resistance to 2-6 drug classes; n 5 24). mdr included isolates susceptible (enr s -mdr, n 5 12) or resistant (enr r -mdr) to enrofloxacin. the minimum inhibition concentrations (mics) for 11 quinolones (1-1st generation, 3-2nd generation, 4-3rd generation and 3-4th generation) were determined for these isolates using broth microdilution methods according to clsi guidelines (e. coli atcc s 25922 served as a negative control). mic statistics were generated for each drug among phenotypes. the results showed that companion animal e. coli expressing ndr or sdr are largely susceptible to 2nd to 4th generation fqs. however, isolates expressing resistance to 1st or 2nd generation quinolone also express high level resistance based on the mic 90 to 3rd and 4th generation fqs. the overall potency (mic) for the 11 drugs for isolates not expressing enr resistance (that is, ndr, sdr and enr s -mdr) is gat canine leproid granuloma (clg) was first reported in brazil in 1990. over the past 20 years, 37 cases of clg were diagnosed in sa˜o paulo, brazil, and clinical and epidemiological findings were similar to those reported in australia. all dogs presented with one or more, uni or bilateral, ulcerated or not, papular, nodular or tumoral lesions, mainly observed in the dorsal surface of the ear, site usually more affected. in general, the lesions are painless and confined to the subcutis and skin, and it does not involve regional lymph nodes, nerves or internal organs, and systemic clinical signs frequently are absent. short-coated breeds show a marked predisposition for this disease. the definitive diagnosis of clg was obtained by histological examination of skin biopsies that were stained with acid fast (ziehl-neelsen) and diffquik s . thirty one (83.7%) of the dogs were purebred; in this study the breed pattern comprised 19 (61.3%) boxers, 2 (6.5%) german shepherd and labrador retriever, 1 (3.2%) dobermann, 1 (3.2%) brazilian terrier, 1 (3.2%) golden retriever, 1 (3.2%) bulldog, 1 (2.8%) american pitbull, 1 (3.2%) mastiff, 1 (3.2%) fila brasileiro and 1 (3.2%) cocker spaniel, 6 (16.3%) were of unknown breed. nineteen (51.4%) of the thirty seven dogs were males. twenty (54.1%) dogs were 4-6 years old. in most cases, dogs presented with unilateral or bilateral ear lesions, but rarely thoracic, foot and caudal lesions. the animals were successfully treated by use of rifampicin orally (''the brazilian protocol'') or enrofloxacin orally and topical rifamicin. anaplasma phagocytophilum is being recognized more frequently in dogs in endemic areas. currently, most suspected cases are evaluated for a. phagocytophilum antibodies by immunofluorescence assay (ifa) or elisa. since a. phagocytophilum is an acute disease, detection by antibody measurement may be negative on initial evaluation. it is possible that a. phagocytophilum dna can be amplified from blood or synovial fluid prior to seroconversion. wild caught ixodes scapularis adult ticks from rhode island were allowed to feed on 18 young adult (2-3 years), mixed sex beagles for up to 7 days. blood (weekly for 12 weeks), serum (weekly for 12 weeks), and synovial fluid (radiocarpal joint; alternating arthrocentesis weekly for 6 weeks) were collected prior to tick attachment and then weekly after tick attachment. joint fluid cytology was performed and total dna was extracted from blood and synovial fluid and assayed in a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii. serum was assayed for a. phagocytophilum antibodies by ifa. time to first positive results for serology and pcr were compared by paired student's t test. none of the beagles developed clinical evidence of disease, and no major changes in synovial fluid cytology were detected over time. of the 18 beagles, 15 were positive for a. phagocytophilum dna in blood or synovial fluid or ifa antibodies in at least one sample after tick attachment. antibody titers appeared in 14 of 15 dogs from weeks 2 to 12 (median to 1 st positive 5 3 weeks ae 1). titer magnitude ranged from 1:40 to 1:10,240. anaplasma phagocytophilum dna was amplified from the blood of 14 of 15 dogs with positive test results ranging from 1 to 12 weeks (median to 1 st positive 5 2 weeks ae 1.5). anaplasma phagocytophilum dna was amplified from synovial fluid from 8 of 15 dogs between weeks 1 to 4 (median to 1 st positive 5 4 weeks ae 1). of the 8 dogs, 7 were pcr positive for only one week and 1 dog was pcr positive for two consecutive weeks. of the 15 dogs, 13 were positive for a. phagocytophilum in both blood and joints by dna analysis. anaplasma phagocytophilum dna was amplified from blood more quickly than seroconversion was detected by ifa antibody titer (t 5 à3.4, p o 0.01) or dna was amplified from synovial fluid (t 5 5.4, p o 0.001). anaplasma phagocytophilum dna can be amplified from the blood prior to development of detectable antibody titers by ifa. amplification of a. phagocytophilum dna from synovial fluid does not occur in all dogs, appears to be transient in most dogs, and a negative test result does not preclude a diagnosis of a. phagocytophilum infection. canine granulocytic anaplasmosis and granulocytic ehrlichiosis are tick-transmitted infections caused by anaplasma phagocytophilum (aph) and ehrlichia ewingii (eew), respectively. both organisms induce an acute clinical disease, frequently accompanied by fever, polyarthropathy and thrombocytopenia. however, aph and eew have different tick vectors, i.e. ixodes scapularis and ambylomma americanum, respectively, with different, but overlapping geographic distributions. in addition, infection outcome may be affected by other regional ticktransmitted pathogens, such as borrelia burgdorferi (mn) or ehrlichia chaffeensis (ar). therefore, we compared serology and pcr results derived from dogs examined at two private practices located in highly endemic areas for either aph or eew. serum collected between april-december, 2005 from minnesota dogs (n 5 420) was tested by snap s 4dx s and whole blood was tested by aph pcr. serum collected from arkansas dogs (n 5 708) for 1 year beginning in august 2009 was tested using microtiter plate elisas for antibodies to eew, e. canis, and e. chaffeensis (ech) while whole blood was tested by ehrlichia pcr. comparisons were evaluated using chi square (ã) and binomial (w) tests with an alpha of 5%. the above results indicated that dogs are frequently exposed to both aph and bb in mn, whereas ar dogs are often exposed to eew, but less frequently to ech. antibodies to e. canis peptides were found infrequently in both mn and ar with only 10 seroreactive dogs detected in both locations. active eew infection, as determined by pcr, was four times more frequent in ar pet dog seroreactors as compared to active aph infections among aph seroreactors. although both organisms induce acute disease, the number of aph and eew pcr positive dogs that were also seropositive was relatively high suggesting that both organisms induce persistent infections or that dogs are frequently re-infected, despite the presence of a measurable humoral immune response. additional studies are needed to determine regional infection profiles in other areas that are endemic for these pathogens. anaplasma phagocytophilum and ehrlichia canis are two of the most common vector borne disease agents that infect dogs and cats. while pcr assays that amplify the dna of these agents from blood are currently available, there is minimal information concerning the performance of these assays in different commercial laboratories that utilize different techniques. the purpose of this study was to compare the e. canis and a. phagocytophilum results of two different laboratories on the same samples collected from client-owned animals. veterinarians in 3 states (az, md, ct) were recruited to participate in the study based on high prevalence rates for e. canis or a. phagocytophilum infection. blood in edta was collected from dogs or cats with fever, thrombocytopenia, or clinical evidence of polyarthritis and an equal volume of the same blood sample was simultaneously shipped on cold packs by overnight express to colorado state and to antech diagnostics. standard operating procedures at each laboratory were followed for total dna extraction and amplification of gapdh as the dna control. at colorado state university, a previously published pcr assay that amplifies the dna of ehrlichia spp., anaplasma spp., neorickettsia spp., and wolbachia was performed on each sample with positive amplicons sequenced to determine the species. at antech diagnostics, a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii was performed. in the study to date, samples from 35 animals (30 dogs and 5 cats) have been assayed at both laboratories. dna of a. phagocytophilum (2 cats and 2 dogs) and e. canis (1 dog) were amplified at both laboratories with a percentage agreement between laboratories of 100%. the results to date suggest that the assay results of the two laboratories for a. phagocytophilum and e. canis are comparable. ehrlichiosis and bartonellosis are zoonotic diseases caused by extremely small, obligate intracellular bacteria that require a mammalian reservoir and a blood sucking arthropod vector. human ehrlichiosis is present in peru, with a seroprevalence as high as 23% in the highlands. bartonella species in humans were also identified in peru since 1905 (b. bacilliformis). recently, a new species (b. rochalimae) was isolated from an american woman who became febrile after travelling to peru. dogs can become infected with the same ehrlichia species, and the majority of bartonella species that affect human beings. the role of dogs as reservoirs for human infections has not been clearly established, but exposure and/or infection in dogs has been used to monitor human exposure to tick-borne disease (tbd), since they share the same environment. the objective of this study was to determine the serological and molecular prevalence of anaplasmosis, ehrlichiosis and bartonellosis in rural dogs in the highlands of peru. a total of 122 healthy adult dogs were enrolled in this study from four communities in the central highlands of peru: ondores, pachacayo, san juan de pachayo, and canchayllo. edta-blood samples were collected from 108 dogs, whereas serum samples were available from 110 dogs. serum samples were tested for ehrlichia canis, anaplasma, borrelia burgdorferi and dirofilaria immitis infections using a qualitative dot-elisa (snap s 4dx). the edta-blood samples were screened by conventional pcr for the groel gene of the genus anaplasma and ehrlichia, and for the intergenic transcribed spacer of the genus bartonella. speciation was conducted by nucleotide sequencing. bartonella genus dna was detected from seven of the 108 dogs (6.5%) and ehrlichia canis dna was detected and sequenced from one dog (0.9%). four of the bartonella positive samples were identified by dna sequencing as b. rochalimae (genbank accession numbers hq185696 and hq185695). the other three bartonella positive samples were identified as b. vinsonii subspecies berkhoffii, the causative agent of endocarditis in dogs and humans. no dog was infected with anaplasma species by dna amplification, but one dog was seroreactive for this genus (0.9%). no specific antibodies against ehrlichia canis and borrelia burgdorferi and no antigens of dirofilaria immitis were detected. this study expands the current knowledge about tbd in peru and describes for the first time the infection of b. rochalimae in dogs in peru. the results suggest that dogs may play an important role in the epidemiology of this infection in humans, since they can be asymptomatic but bacteremic. bartonella spp. dna is commonly amplified from the blood of cats exposed to ctenocephalides felis. in previous work, it was shown that cats administered imidacloprid and experimentally exposed to b. henselae infected cats and c. felis did not become pcr positive for b. henselae whereas untreated cats all developed infection. the purpose of this study was to determine if administration of imidacloprid to clientowned cats likely to be exposed to bartonella spp. and c. felis in the field lessens prevalence of bartonella spp. infection. veterinary students in tennessee and florida that owned cats that spent at least 20 days per month outside and that were willing to apply imidacloprid to their cats monthly for six months were recruited for the study. blood for bartonella spp. pcr assay was collected from the cats seven months after starting imidacloprid administration and assayed at colorado state university. to serve as a control group that was unlikely to have been administered flea control products in the previous 6 months, blood was collected from feral cats during tnr programs in each of the two cities and assayed for bartonella spp. dna. the bartonella spp. dna prevalence rates between the groups were compared by chi square analysis with significance defined as p o 0.05. the overall prevalence rates for bartonella spp. dna in the blood of veterinary student cats (7.4%) and the feral cats (39.1%) were significantly different (p o 0.0001). the distribution of results is shown in table 1 . the results suggest that florida feral cats were more commonly exposed to c. felis than tennessee feral cats. while the cats in the groups were not exactly matched, the student cats were allowed outdoors for approximately 20 days per month and lived in the same cities as the feral cats, so c. felis exposure rates were likely similar. as previously shown in experimentally-exposed cats, the use of imidacloprid monthly may influence transmission rates of bartonella spp. amongst naturally-exposed cats. in an endemic area for leishmaniosis and filariosis, coinfection can occur and immunomodulation produced by wolbachia might influence the clinical signs and progression of both diseases. the aims of the present study were 1) to determine the prevalence of wolbachia in dogs infected with dirofilaria immitis (di) and other filarial nematodes, 2) to evaluate the level of coinfection of leishmaniosis and filariosis by molecular assays and 3) to evaluate any associations between leishmania infantum (li) infection, filariosis with or without wolbachia and clinical presentation and outcome. statistical differences between groups were tested for significance by the fisher exact test using spss v.14.0 software (significance: p-value o 0.05). one-hundred and eighteen owned dogs from southeastern spain presenting for clinical evaluation were included in the study. criteria the results of this study highlight the increased sensitivity of pcr for diagnosis of filariosis, confirm the presence of wolbachia in dogs from the mediterranean basin, show the increased severity of hwd when li-filaria coinfection is present and suggest that wolbachia could play a protective role for leishmaniosis. wolbachia antigens can stimulate a th1-type immune response, as has been previously described. however other factors (as treatment with doxycicline) might be responsible for the lower prevalence of wolbachia among filaremic dogs infected with li and further studies must be done to clarify this interaction. the purpose of the present study was investigate the occurrence of leishmaniasis in 200 cats in the municipality of arac¸atuba, sa˜o paulo, brazil, an endemic area for canine visceral leishmaniasis. animals were evaluated by direct parasitological examination of lymphoid organs and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). thirteen (6.5%) out of 200 cats studied were diagnosed with visceral leishmaniasis; eight (4%) by parasitological diagnosis through cytological examination of lymphoid organs, six (3%) were considered positive by elisa and one (0.5%) by ifat. only two (15.38%) out of the thirteen infected cats had clinical signs, characterized by the presence of crusty lesions on the dorsal cervical region and hepatosplenomegaly. regarding age five cats (38.5%) had between six months and two years, being the others older than 2 years (61.5%). only one cat (7.7%) was positive for the three employed methods. pcr confirmed leishmania sp infection in nine (69.2%) cats, of which six were diagnosed previously by cytological examination, two by elisa and one by the three techniques employed. since its first description in 1912 feline leishmaniosis has been reported in several countries. the purpose of this study was to assess the prevalence of leishmania chagasi infection in cats showing dermatologic lesions from an endemic area for visceral leishmaniasis in brazil. animals were evaluated by direct parasitological examination of lymphoid organs, immunohistochemical technique for detection of amastigotes in lesioned skin and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). twenty seven (49.1%) out of the 55 cats studied were diagnosed with visceral leishmaniosis. twelve (44.4%) were positive by parasitological diagnosis; amastigote forms of leishmania sp were identified in lymphoid organs from 10/55 (18.2%) infected cats, and immunohistochemical technique allowed the identification of nine (16.4%) positive animals. the seroprevalence of leishmaniosis was 25.4% (14/55) by elisa and 10.9% (6/55) by ifat. fiv specific antibodies were found in 6/55 cats (10.9%), of which 5/6 (83.3%) had leishmaniosis. real time pcr confirmed leishmania chagasi infection in three cats. based on the evidence of the high occurrence of leishmaniosis in cats in this study, this disease should be included in the differential diagnosis of skin diseases of felines living in endemic areas. blastomyces dermatiditis is a dimorphic fungus that commonly affects large-breed hunting dogs. a recent advancement in diagnosis has come with the advent of a urine antigen screening test that has both high sensitivity and moderately high specificity. therapy for the disease involves use of antifungal agents, usually itraconazole, and length of treatment is based chiefly on resolution of clinical and radiologic signs. with the new urine antigen test, however, a noninvasive route of monitoring treatment progress is available and could be an adjunct device utilized to determine treatment efficacy and may even reveal a need for prolonged treatment. therefore, the purpose of this study was to determine if monitoring the blastomyces urine antigen test and comparing to pulmonary radiographic signs would elucidate the necessity for prolonged antifungal therapy, even after resolution of radiologic signs. to this end, a retrospective case review was performed that identified a series of client-owned animals with naturally occurring blastomycosis. the inclusion criteria were radiographic pulmonary parenchymal signs consistent with fungal disease and urine antigenconfirmed blastomycosis with repeated testing of both radiographs and urine antigen quantification as monitoring parameters until negative results achieved in each. ideally, intervals between testing dates would be between two and five months. radiographs were considered negative if all radiographic changes had resolved or if repeated radiographs separated by at least one month were considered static after documented improvement had occurred from original diagnostic radiographs (suspected scarring). urine antigen testing was considered negative if concentrations were less than 1.0 enzyme immunoassay units, a reference interval set by the testing laboratory. preliminary data analysis reveals resolution of radiographic signs of blastomycosis occurred earlier in many of the cases presented than did attaining a negative urine antigen concentration. ceasing treatment 1 month after radiographic resolution of signs as has been recommended in the past might have resulted in premature discontinuation of therapy in many of the cases. monitoring of urine antigen concentrations may be of additional clinical use for determining when cessation of treatment should occur in cases of blastomycosis. persistent elevation of urine antigen concentrations after radiographic resolution of infection may account for apparent recrudescence of blastomycosis after suspected clinical resolution. giardia spp. and cryptosporidium spp. are both known to cause infections in dogs and humans in the united states. nevertheless, prevalence rates for dual infection in dogs had not been widely reported. in this study, fecal samples from dogs housed in a northern colorado animal shelter (n 5 121), dogs owned by veterinary students in northern colorado (n 5 132), and dogs from the pine ridge reservation in south dakota (n 5 84) were collected. each sample was assayed with a commercially available fluorescent antibody assay that detects giardia spp. cysts and cryptosporidium spp. oocysts. those samples that were positive for giardia spp. or cryptosporidium spp. with adequate dna available for sequencing were genotyped by the glutamate dehydrogenase [gdh] and by the heat shock protein-70 [hsp-70] genes, respectively. overall, 45 (13.3%) of the dogs had current evidence of a protozoal infection ( table 1 ). the dogs from pine ridge reservation had the highest prevalence rates for giardia infection and also for dual infections. from the student dogs, sequencing was successful for the three giardia isolates (assemblage d from 2 dogs; assemblage c from one dog) and one cryptosporidium isolate (c. canis). from the reservation dogs, sequencing was successful for nine giardia isolates (assemblage d from 4 dogs; assemblage c from 5 dogs) and one cryptosporidium isolate (c. canis). cryptosporidium and giardia co-infections are commonly detected in dogs; in this study dual infections were more common than cryptosporidium infections alone. further studies will be required to determine the clinical importance of this finding. although the giardia and cryptosporidium isolates that were sequenced were the dog specific assemblages/genotypes, more samples should be analyzed in order to assess the potential for zoonotic transmission of either parasite. the current study was conducted to determine the prevalence of intestinal parasites in dogs visiting the veterinary teaching hospital, chiang mai university, northern thailand. fecal samples (n 5 301) were collected and submitted by owners between august 2009 to february 2010. demographic and geographic data were recorded. intestinal parasitic infection was diagnosed by both microscopic examination after zinc sulfate centrifugation flotation and commercially available ifa for giardia spp. and cryptosporidium spp. polymerase chain reaction and dna sequencing were performed on all giardia and cryptosporidium positive samples to provide genotyic information. overall prevalence of intestinal parasitic infection in dogs in chiang mai was 38.9%. the most prevalent parasite was giardia spp. (24.9%) followed by ancylostoma spp. (12.0%), cryptosporidium spp. (7.6%), cystoisospora spp. (6.0%), toxocara canis (2.7%), trichuris vulpis (2.0%), coccidian-like (1.7%), toxascaris leonina (0.7%), and strongyloides spp. (0.7%). the prevalence of having at least one parasite in dogs o 1 year, 1-7 years, and 4 7 years were 49.3%, 39.8%, and 27.4%, respectively. of these infected dogs, 59.0%, 34.2%, 5.1%, and 1.7% were infected with one, two, three, and four organisms, respectively. available dna sequences from giardia spp. positive samples were shown to be dog specific. only one adequate dna sequence was available for cryptosporidium spp., which was shown to be c. canis. the findings suggested that intestinal parasitic infection was common in dogs in chiang mai, thailand. dogs could be potential source for zoonotic intestinal parasitic infection since dogs in this area are allowed for free roaming. regular deworming program is indicated to prevent not only transmission among dogs but also to human. a retrospective study was conducted on 135 parasite positive fecal specimens consisting of 90 canine, 29 feline, 14 equine and 2 from other host species, comparing recovery of eggs, protozoan cysts and coccidian oocysts using 2 standardized methods of parasite concentration: the formalin/ethyl acetate (f/ea) sedimentation concentration and the commercial fecalyzer (flotation) kit procedures. specimens were processed by each technique either according to manufacturer's instructions or according to standard laboratory procedures. formalin/ethyl acetate concentrations used at a ratio of 10 ml normal saline to 4 ml ethyl acetate for extraction of lipophilic material from pelleted stool samples, previously fixed in sodium acetate/acetic/acid/formalin (saf) solution. flotations with the fecalyzer kit were performed with concentrated zinc sulfate solution (s. g. 1.22) . the range of parasites recovered from these specimens included flagellate cysts (40 total), coccidian oocysts (28 total), ova and larvae of nematodes (80 total), and ova of trematodes (12 total) , and cestodes (16 total). recovery rates by fecalyzer flotation were good for protozoan cysts, coccidian oocysts and nematode eggs and larvae, but very poor for cestode and trematode eggs. formalin/ethyl acetate concentration showed excellent recovery of all parasites and consistently outperformed fecalyzer in recovery rates. recoveries by f/ea concentrations were higher by 27.5% for giardia, by 10.7% for coccidia and by 10.0% for nematode eggs and larvae. with the exception of coccidian oocysts, based on z-test analyses, recovery rates were significantly higher, at a confidence level of at least 95%, for all parasites, using formalin/ethyl acetate sedimentation concentration. although capc recommends the use of flotation with centrifugation methods for standard fecal ova and parasite examination for veterinary patients, sedimentation concentration methods are widely and effectively used in human diagnostic parasitology laboratories. these results provide good evidence for the use of f/ea concentration as a preferred method to flotation procedures for stool ova and parasite examinations in veterinary laboratories. cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases. cyclosporine inhibits calcineurin-dependent pathways of t-cell activation and the resultant cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. little work has been done comparing the effects of these agents on cytokine production in dogs. our study assessed these effects by measuring t-cell cytokine production using flow cytometry, and cytokine gene expression using quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) in activated canine t-cells treated with cyclosporine and dexamethasone. for flow cytometric assays, peripheral blood mononuclear cells were separated using density gradients and cultured for 12 hours in the presence of cyclosporine (5, 25, or 100 ng/ml), dexamethasone (10 à7 , 10 à6 , 10 à5 m), or cyclosporine plus dexamethasone. for qrt-pcr, whole blood was cultured for 5 hours with the same drugs at the same concentrations, and rna was then extracted from leukocytes. expression of cytokines il-2 and ifn-g was analyzed in pma/ionomycinactivated t-cells by flow cytometry, and gene expression for il-2 and ifn-g in activated t-cell populations was assessed via qrt-pcr. flow cytometry and qrt-pcr both demonstrated inhibition of il-2 and ifn-g that was generally dose-dependent in response to both cyclosporine and dexamethasone. flow cytometry results from the average of samples collected from 3 different dogs are shown in figure a . similar results were achieved using qrt-pcr ( figure b ). suppression of il-2 and ifn-g in activated t-cells has potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine t-cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients. idiopathic eosinophilic diseases are described in several breeds, but are over represented in rottweilers. the immunopathogenesis of idiopathic eosinophilic disorders is poorly characterised. studies in people highlight the importance of cytokines, particularly interleukin-5 (il-5), in mediating eosinophil maturation, differentiation, egress from the bone marrow, migration and polyclonal expansion. eotaxin-2 and eotaxin-3 also appear important for induction of chemotaxis and release of reactive oxygen species from eosinophils. the aim of the current study was to establish whether definable differences in specific cytokines associated with mediation of eosinophil production and survival are present between healthy rottweilers, non-rottweilers and rottweilers with non-parasitic eosinophilia. secondly, by evaluating cytokine profiles the study aimed to improve understanding of the pathophysiology of eosinophilia therefore assisting development of potential molecular treatment options. quantitative real-time reverse transcriptase polymerase chain reaction (qrt-pcr) assays were used to quantify messenger rna (mrna) encoding cytokines il-4, il-5, il-10, il-23p19, il-12p35, il-12p40, il-18, interferon gamma (ifn-g) and chemokines eotaxin-2 and eotaxin-3 from peripheral blood mononuclear cell (pmbc) samples obtained from healthy non-rottweiler dogs with normal eosinophil counts (n 5 5) and rottweilers with normal (n 5 6), mildly increased (n 5 7) and high (n 5 3) eosinophil counts. quantification of serum ifn-g was also performed using a commercially available canine-specific elisa. all samples were positive for housekeeping genes and all cytokines could be quantified with the exception of eotaxin-2 and -3. results were normalised using three stably expressed housekeeper genes (rpl13a, sdha and ywaz) and a relative copy number was calculated for each sample with the sample with the fewest copies given a value of 1. no significant differences were found between groups but there was a tendency for ifn-g mrna expression to be lower in the rottweilers with moderate to severe eosinophilia versus control dogs (p 5 0.062). this trend was not seen in the concentration of serum ifn-g quantified by elisa as there were no significant differences between normal and diseased animals. in conclusion, there were no significant differences in cytokine mrna profiles between normal dogs and rottweilers with varying degrees of eosinophilia. additional studies including larger numbers of affected dogs are warranted before any accurate conclusions can be made. the presence of large amount of antibody on erythrocyte membrane can accelerate red blood cell (rbc) removal process by the mononuclear phagocyte system. an antigenic stimulus such as the one promoted by vaccines, for example, can induce hypersensitivity reactions and may accelerate rbc destruction. the study objective was to evaluate the erythrocytic membrane potential in inducing lymphocyte proliferative response of recently immunized dogs. healthy adult dogs (n 5 17) were immunized with multiple antigens (commercial vaccine with eight antigens: distemper virus, parvovirus, coronavirus, parainfluenza virus, adenovirus, infectious hepatitis virus and leptospire; and anti-rabies). blood samples from each animal were collected into edta tubes in two moments: pre (immediately before vaccination) and pos (28 to 35 days after vaccination). mononuclear cells were separated by gradient, marked with cfse-fitc and cultured. the stimuli for lymphocyte proliferation used were autologous erythrocytic membrane (aem) and concanavalin a (cona). aem was obtained by hypotonic lysis and tested in two concentrations (m1: 0.1ug/100ul; m2: 0.2 ug/100ul). the proliferation assay was evaluated by flow cytometry and analyzed with specific software. the proliferation index (pi) was calculated dividing the fluorescence intensity of the basal sample by the stimulated one. statistical analysis was performed using paired t-test for parametric samples and wilcoxon test for non-parametric samples (a 5 0.05). the for the tested concentrations, autologous erythrocytic membrane does not constitute a stimulus for lymphocyte proliferation in vitro, either before or after vaccination procedure. additionally, there was no evidence of self-reagent lymphocytes to erythrocyte membrane after vaccination. e. coli is a common cause of canine urinary tract infection. current treatment emphasizes eradication of established infection rather than infection prevention but increased antibiotic resistance necessitates strategies to prevent infection. proanthocyanidins found in cranberry juice inhibit e. coli attachment to human uroepithelial cells, impairing bacterial adherence and colonization. we hypothesized that purified cranberry extract (ce) inhibits bacterial adhesion to canine uroepithelial cells. five healthy female dogs received an oral ce supplement (vetoquinol; 100 mg ce/tablet) according to body weight for 30 days. voided urine collected from each dog before (pre) and after (30-day) completion of the protocol was membrane filtered (22 mm) and stored frozen (-20c). bacterial adhesion was determined using an in vitro assay. briefly, urine samples were incubated with an uropathogenic e. coli strain that had been subcultured to promote fimbriae expression. urine samples containing e. coli were next incubated in 96-well plates containing methanol-fixed madin-darby canine kidney (mdck) cells for 1-hr (35c) to permit bacterial attachment. after incubation, plates were washed to remove nonadherent bacteria and fresh media added. plates were incubated (35c) for 4-hr to grow attached bacteria to detection level. bacterial concentration in each well was determined using a spectrophotometer (650 nm). results were analyzed using the chi-square test. ce significantly reduced bacterial adhesion by 30% (n 5 5; p 0.5) in 30-day urine samples compared with pre samples. the results show that ce supplementation can reduce adhesion of uropathogenic e. coli to canine uroepithelium and suggests one mechanism by which ce might improve urinary tract health. the purpose of this study was to determine prevalence of 4 urovirulence factors (uvfs) and antimicrobial resistance in canine uropathogenic e. coli (upec) and to evaluate associations between uvfs and antimicrobial resistance. two hundred and twenty-one upec isolates from samples collected from 184 different canine patients submitted to the university of tennessee microbiology laboratory in 2007 were evaluated. a multiplex pcr assay was used to detect cnf, hlyd, sfa/foc, and papgiii in dna lysate. in vitro susceptibility was evaluated and if the isolate was resistant to any antimicrobial in a class, it was considered resistant to that class. of the 221 samples, the number of uvf expressed per isolate was: 0 5 127/221 (57%), 1 5 27/221 (12%), 2 5 4/221 (2%), 3-22/221 (10%), and 4 5 41/221 (19%). expression of uvf was sfa (33%), hly (24%), cnf (24%), and pap (19%). presence of 4 uvfs was associated with less resistance (p o 0.0001). the combination of hly, cnf, and sfa was associated with less resistance (p o 0.0001). when sfa was present alone, resistance was less (p o 0.0001). average resistance to antimicrobial class by number of uvfexpressed was: 0 uvf 5 4.1 ae 3.3 classes, 1 uvf 5 1.2 ae 1.1 classes, 2 uvf 5 0.0 ae 0.0 classes, 3 uvf 5 0.8 ae 1.6 classes, and 4 uvf 5 0.5 ae 1.2 classes. urovirulence factors were present in a moderate number of upec and correlated negatively with resistance. neither individual nor combinations of uvfs were associated with increased resistance. obesity is associated with several comorbidities in dogs including pancreatitis, osteoarthritis, oral disease, neoplasia, and lower urinary tract disease. investigator observations led to the hypothesis that morbidly obese dogs are more likely to have asymptomatic bacterial urinary tract infections (abuti) than overweight and moderately obese dogs. therefore, a pilot study was conducted to screen for abuti in obese dogs. urinalysis with urine culture and dual energy x-ray absorptiometry (dxa) were performed on fortythree dogs with body fat (bf) percentages ranging from 36 to 56%. following dxa, subjects were categorized as obese (o)(bf 5 35-44%, n 5 17) and morbidly obese (mo)(bf 4 45%, n 5 26). no dogs had owner-reported symptoms indicative of uti. the prevalence of abuti in o dogs was 6% (n 5 1) and 31% (n 5 8) in mo dogs. the dog in the o group with abuti was close to being mo with a bf equaling 44.3%. of the nine dogs with positive cultures, 4 were neutered males and 5 were spayed females. the prevalence ratio of abuti in mo dogs was 7.5, indicating dogs with 45% or greater bf are 7.5 times more likely to have the condition then dogs o 45% bf. the results of this pilot study coincide with other surveillance data describing an increased prevalence of lower urinary tract disease in obese dogs. in conclusion, dogs with body fat percentages greater than 45% are at risk for abuti, and veterinarians should consider screening all morbidly obese patients for urinary tract infections. calcium carbonate (cac) is recommended to decrease phosphate intake in chronic kidney disease. however, its effect is poorly documented in dogs. our objectives were to assess within-day, postprandial and cac effects on phosphatemia variations in healthy dogs. phosphatemia was measured every 2 hours for 24 hours in eight adult healthy beagle dogs in i) fasted condition and ii) a 2 â 2 crossover design. one group received cac mixed with maintenance diet (0.8% phosphorus), while the second group received the diet alone. after a 1-week wash-out period, groups were switched. a general linear model was used to test the period, sequence, treatment, dog and time effects on phosphatemia and the area under the phosphatemia versus time curve (auc 0-24 ). a significant (p o 0.001) circadian variation existed in fasted dogs. the maximum difference (mean: à1.9 mg/dl; 95% c.i.: à2.4 mg/dl; à1.4 mg/dl) was observed between 8 a.m. and midnight. the auc 0-24 with cac (5936 ae 533 mg.min/dl) was mildly but significantly lower (p 5 0.027) than without cac (6239 ae 631 mg.min/dl). however, it was similar to the auc 0-24 in fasted conditions. feeding, with and without cac, has minor effect on phosphatemia. however, circadian variation of fasted phosphatemia might affect its interpretation. gfr measurement permits diagnosis of kidney injury prior to development of azotemia, and is the gold standard for kidney function assessment. accurate and rapid (o 60 min) gfr measurement has been performed in rats by simultaneous transcutaneous assay of two intravascular fluorescently-labeled markers. a recently developed analyzer assays fluorescence via a fiberoptic cable introduced through a peripheral catheter, and thus should also allow rapid gfr determination in larger species. the purpose of this study was to determine correlation and agreement between fluorescent ratiometry (fr) and iohexol plasma clearance (ipc) in dogs over a range of gfrs. acute kidney injury (aki) was induced in 5 female hound-type dogs (10 mg/kg gentamicin iv q8h), and fr and ipc gfr were simulta-neously determined on days 0, 3, 6 and 9. a 9-sample, 5-hr protocol was used for ipc; fr was determined following bolus injection of a dextran conjugate mixture (2-sulfohexamine rhodamine-carboxymethyl 150 kd dextran, 5-aminofluorescein-carboxymethyl 5 kd dextran) with fluorescence measured over 60 min. gfr was calculated using 2-compartment model concentration-vs.-time curves for both techniques. correlation was determined via spearman's rho; agreement was analyzed via bland-altman plots. ipc gfr and serum creatinine confirmed progressive aki in all dogs. correlation between fr and ipc was 0.91 (p o 0.001). bland-altman plots confirmed good agreement between techniques with slight underestimation of gfr by fr across most observed values. these results suggest fr is suitable for gfr determination in dogs with aki. importantly, the portable analyzer allowed for point-of-care gfr determination in o 60 min using a peripheral vein. previously presented at the american society of nephrology renal week (related but not identical abstract). dogs with protein-losing nephropathy (pln) are at risk of thromboembolic disease, but the mechanism of hypercoagulability and the population of dogs at risk are unknown. the purpose of this study was to characterize thromboelastography (teg) in dogs with pln. twenty-eight client-owned dogs with pln (urine protein:creatinine ratio (upc) 4 2.0) and 8 control dogs were enrolled. teg parameters, antithrombin activity, serum biochemical profiles, and upc were measured. teg analyses were run in duplicate with kaolin activation; reaction time (r), clot formation time (k), maximal amplitude (ma), and g (global clot strength) were analyzed. a wilcoxon sum rank test was used to evaluate differences between groups. twelve pln dogs (42.8%) were azotemic. nineteen pln dogs (67.8%) were hypoalbuminemic [serum albumin (salb) o 3.0 g/dl]; 11 had salb o 2.5 g/dl. dogs with pln had higher k (p o 0.01), ma (p o 0.005) and g (p o 0.005) than controls. r was similar between the two groups. pln dogs with salb o 2.5 g/dl had higher g (p o 0.05) values than dogs with salb 4 2.5 g/dl; however, even pln dogs with normal salb (4 3.0 g/dl) had significantly higher g values than controls (p o 0.005). no significant relationship between upc and g, salb and g, antithrombin and g, or salb and antithrombin was noted using linear regression analysis. these results indicate that antithrombin, salb, and upc cannot be used alone to predict hypercoagulability as assessed by teg in dogs with pln. a comprehensive evaluation of the coagulation system in individual patients may be necessary to predict the point at which to initiate anti-thrombotic therapy. cystinuria is a hereditary renal tubular reabsorption defect of cystine, ornithine, lysine and arginine (collectively, cola). the low solubility of cystine in acidic urine predisposes to the formation of uroliths. type i cystinuria in newfoundland and labrador retriever dogs is an autosomal recessive trait caused by mutations in the slc3a1 gene, whereas in other breeds, the cause of cystinuria has not yet been determined. we report here on the clinical, biochemical and molecular features of cystinuria in irish terriers. urine and edta blood were collected from 222 irish terriers from europe and australia. a nitroprusside screening test was used to identify increased cystine in urine. urinary amino acid concentrations were determined by high-pressure liquid chromatography. cystinuric dogs were defined as having cystine calculi, a positive nitroprusside result, urinary cystine (4 179 mmol/g creatinine) and/ or a cola concentration of 4 700 mmol/g creatinine. all 83 females tested nitroprusside negative and had normal urinary cystine (o 150 mmol/g creatinine) and cola (o 500 mmol/g creatinine) concentrations. the 10 intact males that formed calculi as adults exhibited cystine concentrations ranging from 323-1580 and cola from 1029-4302 mmol/g creatinine. an additional 41 males had similarly high cola values with cystine levels from 0-1580 mmol/g creatinine. among the affecteds tested, 75% were nitroprusside positive. the negative nitroprusside results and/or low urinary cystine levels of affecteds may be due to precipitation of cystine in acidic urine. sequencing the coding regions of the slc3a1 and slc7a9 genes from edta blood identified no mutations. the mode of inheritance remains undetermined. however, castration appears to lower the urinary cystine and cola concentrations and to prevent cystine calculi formation, while diet changes have lesser effects. in conclusion, non-type i cystinuria in irish terriers (and several other breeds like mastiffs and scottish deerhounds) is a unique form characterized by increased aminoaciduria only in males, with lower cystine and cola excretion and fewer and later urolith formation compared to type i cystinuria. castrating cystinuric irish terriers lowers their cystine and cola excretion and thus their risk for calculi formation. cats and dogs that are diagnosed with acute kidney injury (aki) and resultant uremia that is not responsive to standard medical therapy are likely to benefit from renal replacement therapies, such as intermittent hemodialysis (ihd). the purpose of this study was to evaluate the long-term outcome of patients with aki treated with ihd, and to establish whether renal function, as determined by serum or plasma creatinine concentrations, is associated with longterm survival. medical records of 20 cats and 35 dogs that were diagnosed with aki, treated with ihd, and survived longer than 30 days following the last ihd treatment were retrospectively analyzed. standard methods of survival analysis using kaplan-meier product limit curves and the log-rank test were performed. for all-cause mortality, the median survival time was 1823 days (95% confidence interval: 841, 4667) for cats and 1049 days (95% confidence interval: 893, 1931) for dogs. when only renal-related causes of death were taken into account, the median survival time was not reached for cats or dogs. survival time for all-cause mortality was inversely associated with the lowest creatinine concentration within the 30 to 90 day period following the last ihd treatment (p o 0.0011 for cats, p o 0.0104 for dogs). this study demonstrates that veterinary patients that are diagnosed with aki, treated with ihd, and survive greater than 30 days after the last ihd treatment have a good longterm prognosis and frequently die from causes that are unrelated to renal impairment. renal fine-needle aspiration (r-fna) is oftentimes attempted during evaluation of dogs and cats with renomegaly, mass lesions, or suspected infiltrative processes. diagnostic utility of fna is dependent upon the organ being sampled; additionally, in some organs, certain diagnostic imaging findings are associated with improved concordance of fna with final diagnosis. objectives of this study were to evaluate the diagnostic utility of r-fna and determine whether concordance with final diagnosis is associated with specific clinicopathologic or diagnostic imaging findings. we hypothesized that r-fna is most useful in patients with diagnostic imaging results suggestive of renal neoplasia (i.e. masses or suspected infiltrative processes). dogs and cats that had undergone r-fna from jan 1, 1998 to dec 31, 2008 were identified by database search. patient signalment, serum creatinine and blood urea nitrogen concentration, urine specific gravity, dipstick protein, r-fna result, and final diagnosis were recorded. patients were excluded if abdominal radiographs or sonographic images were not available for review, or if diagnostic test results were insufficient for determination of final diagnosis. a single coauthor blinded to final diagnoses interpreted all abdominal images using a pre-set list of descriptors and grading criteria. radiographic kidney shape, margin distortion, and ventrodorsal kidney-to-l2 ratio were evaluated. sonographic kidney margin distortion, cortical echogenicity, and corticomedullary junction distinction were described, and presence of nodules or masses, peri-renal effusion, or a peripheral sonolucent rim was noted. concordance of r-fna and final diagnosis was determined, and the chi-squared or fisher's exact test were used to determine association of concordance with the above variables; p o 0.05 was considered significant. 37 dogs and 41 cats (78 animals) met all inclusion criteria. r-fna results were concordant with the final diagnosis in 43 (55.1%) patients, discordant in 12 (15.4%) patients, and inadequate for cytologic interpretation in 23 (29.5%) patients. neoplasia or fip were the final diagnoses in 19 of 43 (44.2%) and 3 of 43 (7.0%) patients with concordant results, respectively. renal lymphoma (p 5 0.196), renal carcinoma (p 5 0.364), and renal neoplasia in general (p 5 0.451) were not associated with a higher likelihood of r-fna and final diagnosis concordance. there was no association noted between likelihood of r-fna and final diagnosis concordance when patients were stratified by species, serum creatinine or blood urea nitrogen concentration, urine specific gravity, dipstick proteinuria, or any diagnostic imaging variables. this study failed to identify concurrent clinicopathologic or diagnostic imaging findings that enhanced the diagnostic utility of r-fna. future studies should use standardized criteria to prospectively identify patients in which r-fna will be performed, evaluate additional variables that may be associated with increased r-fna diagnostic utility, and directly compare the utility of r-fna with that of other diagnostic techniques. feline lower urinary tract disease (flutd) is a disease with increasing prevalence in private practices and veterinary teaching hospitals. although several underlying causes can cause the obstructive form in male cats, the idiopathic form (feline interstitial cystitis) often is diagnosed as underlying reason in cats o 10 years. the goal of this retrospective study was to identify possible predisposing factors in order to optimize the therapy of these patients. as a study group, 40 cats hospitalized with obstructive flutd at the veterinary university of vienna were examined during a 2 year period (2008) (2009) (2010) . as a control group 40 cats presented for other reasons were randomly chosen during the same time period. the data were examined concerning the signalment and history. furthermore, the long-term outcome was evaluated with a questionnaire. based on assumptions a student's t-test or a chi-square test was used. there were no significant differences in age and breed. the body weight was significant higher in the flutd group than in the control group (p o 0.01). we could observe a significant risk for the disease of a weight of 4 5 kg (p o 0.01). there were significant less cat toilets in the flutd group compared to the control group (p o 0.05). furthermore we could observe that in the households of flutd cats there was significant less than one toilet per cat (p o 0.01) and more cats diseased on flutd lived strictly indoor than outdoor (p 5 0.05).there were no significant differences at the time of hospitalization in age, breed, number of cats per household or season of the year between the two groups. in summary, we could observe that cats over 5 kg body weight kept indoor with less than one toilet per cat have a significant higher possibility to be affected by obstructive flutd. further studies with an extensive history of animal husbandry are needed to identify risks predispoing cats to this frequent and cost-intensive disease. although purine uroliths (ammonium urate, sodium urate, xanthine, uric acid, etc.) represent the third most common stone type in cats, purine uroliths have the highest rate of recurrence (13% in 22 months). in dogs, mutation of the urate transporter (slc2a9) and portovascular anomalies are common risk factors. however the underlying cause(s) for purine urolith formation in cats is unknown. the purpose of this study was to test the hypothesis that hyperuricosuria without alterations in liver function is common in cats with urate uroliths. urine concentrations of purine metabolites were measured by high-performance liquid chromatography in 5 cats with ammonium uroliths (cases), 5 clinically healthy, breed and gender matched cats (negative controls), and 2 cats with naturally occurring xanthine uroliths (positive controls). prior to urine collection, all cats were fed a standard maintenance food (protein 5 7 g/100kcal) for 4 weeks. urinary xanthine, uric acid, and allantoin concentrations and concentration to creatinine ratios were calculated and compared between groups. also, serum pre-and post-prandial bile acid concentrations were measured. when compared to control cats, urinary uric acid concentration was significantly higher in case cats (p 5 0.002). xanthine was not detected in the urine of cases or negative controls. a significant difference in fasted and post-prandial serum bile acid concentrations was not detected in cases or controls (p 5 0.197, 0.212).hyperuricosuria without increased concentrations of urinary xanthine or allantoin appears to be a risk factor for ammonium urate urolith formation in cats. an association between portovascular shunts and purine urolithiasis was not observed in this population of cats. studies indicate that proteinuria is predictive, on a population basis, of those cats at risk of developing azotemia. seldi-tof-ms is a sensitive, high-throughput, proteomic technique utilising chromatographic surfaces to facilitate separation and detection of proteins and peptides within biological fluids such as urine. individual low molecular weight (lmw) urinary proteins have been considered as potential biomarkers for renal damage but provide only a limited representation of the urinary proteome; seldi-tof-ms may provide a more global assessment. normotensive, non-azotemic geriatric cats (4 9 years) were recruited prospectively from two first-opinion clinics for routine health screening. at entry cats received a full physical examination, plasma biochemistry, evaluation of total t4 concentration and urinalysis including urine protein to creatinine ratio. re-examination was offered at 6 and 12 months. cats were divided into two groups based on clinical status at the 12 month re-examination (azotemic; creatinine concentration ! 2.0 mg/dl and non-azotemic). optimisation studies were performed to facilitate the automated preparation (biomek 3000) of cm10 (weak cation exchange) arrays for seldi-tof-ms analysis (ciphergen enterprise 4000) of urine samples from cats at entry to the study. results are reported as median [25 th , 75 th percentile]. mann whitney u-test and wilcoxon signed rank test were used to compare variables between groups and between timepoints, respectively. ciphergen express (3.06) software was used to analyse spectral data and a mann whitney u-test was used to identify clusters which differed significantly between groups (p o 0.05) at entry to the study. twenty non-azotemic cats were recruited, of which 10 cats developed azotemia by 12 months. no significant differences in age, body weight, biochemical or urinalysis variables were identified between groups at entry to the study. as might be expected creatinine increased significantly (1.73 mg/dl [1.58, 1.77], 2.17 [2.00, 3.23], p 5 0.002) between study entry and 12 months in the cats that developed azotaemia and there was a commensurate increase in phosphate concentration (3.81 mg/dl [3.60, 4.22], 4.65[3.94, 6 .48], p 5 0.019). creatinine and phosphorus did not change significantly over time in the cats that did not develop azotaemia. seven clusters with m/z values of 2822, 10 033, 10 151, 10 234, 11 635, 11 700 were found to differ significantly between groups at entry to the study. the low protein concentration of feline urine makes the use of proteomic techniques challenging. however, this pilot study indicates that seldi-tof-ms can be utilised to examine the feline urinary proteome and that differences in low molecular weight protein patterns may be useful to differentiate those cats which are at risk of the development of azotemia. further work is necessary to identify these proteins/peptides. fibroblastic growth factor 23 (fgf-23) is a phosphotonin with an important physiological role in the regulation of phosphorous and vitamin d metabolism, and may therefore play a part in the development of renal secondary hyperparathyroidism. previous studies in cats have shown parathyroid hormone (pth) to be elevated prior to the development of azotemia. the study objectives were to explore the hypothesis that fgf-23 is a mediator of the development of renal secondary hyperparathyroidism in the nonazotemic stages of feline ckd. healthy, non-azotemic (plasma creatinine concentrations (cr) o 2.0 mg/dl) geriatric cats were recruited into the study prospectively and followed for 12 months. at the study end point cats were categorised into the following 3 groups: group 1 (n 5 15)-cr 1.58 mg/dl, group 2 (n 5 33)-cr !1.58 mg/dl but did not meet the criteria for group 3 and group 3 (n 5 14)-cr 4 2.0 mg/dl in association with reduced urine concentrating ability (usg o 1.035) or demonstration of persistent azotemia (cr 4 2.0 mg/dl). plasma samples were subjected to routine biochemical analysis, intact pth, calcitriol and intact fgf-23 assay. variables were compared between the 3 groups at the baseline time point. gfr was measured in an additional group of 19 cats (11 non-azotemic, 4 iris stage ii, 4 iris stage iii) using a corrected slope-intercept iohexol clearance method. relationships were explored using linear regression analysis and determining the coefficient of determination (r 2 ). results are presented as median [range] . at the baseline time point fgf-23 concentrations were significantly higher in group 2 (208.1[51.4-814.6 ], p 5 0.001) and group 3 (237.6[127.4-908.1], p 5 0.001) compared to group 1 (126.2[69.4-505.2] ). weak positive relationships were identified between fgf-23 and pth (r 2 5 0.126, p 5 0.005, n 5 62) and fgf-23 and cr (r 2 5 0.077, p 5 0.029, n 5 62). however, the positive relationships between fgf-23 and phosphate (r 2 5 0.016, p 5 0.323, n 5 62) and fgf-23 and calcitriol (r 2 5 0.085, p 5 0.212, n 5 20) were not significant. the additional group of cats in which gfr measurement was performed there was an inverse relationship between fgf-23 and gfr (r 2 5 0.208, p 5 0.040). in conclusion, fgf-23 was elevated in cats prior to the development of azotemia. the role of fgf-23 in the development of feline renal secondary hyperparathyroidism remains to be determined and should be explored through interventional studies. however, considering the relationship between fgf-23 and gfr, it cannot be excluded that the phosphotonin is simply a marker of reduced filtration. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to the progression of this disease. the aim of this study was to evaluate urinary vascular endothelial growth factor (vegf) as a marker of renal hypoxia. cats were recruited through geriatric clinics held at two first opinion london practices. vegf was measured in stored samples using a canine elisa kit validated for use on feline urine and indexed to creatinine concentration to yield a vegf to creatinine ratio (vcr). two studies were undertaken -firstly a cross-sectional analysis of clinical variables associated with vcr in cats with ckd. diagnosis of ckd was based on concurrent findings of plasma creatinine ! 2 mg/dl and usg 1.035, with persistence of azotemia for ! 2 weeks. only patients receiving no medical therapies were included. normotensive and (pre-treatment) hypertensive cats were included, but borderline cases (mean systolic blood pressure 160-170 mmhg on the date of sampling) were not. hyperthyroid cats were also excluded from this cross-sectional study. associations between vcr and clinical data were initially assessed using the spearman's coefficient and mann whitney test. linear regression was then used for multivariate analysis. the second study used samples from a trial in which hypertensive cats that had been treated with amlodipine for at least 3 months were entered into a randomised cross-over study where they received placebo or benazepril (0.5 to 1 mg/kg daily) for 12 weeks in turn. vcr on placebo was compared with that on benazepril using the wilcoxon signed ranks test. cats with well controlled hyperthyroidism were included in this intervention study. results are reported as median [25th, 75th percentile]. vcr was higher (49.5[33.3, 74.1] vs. 36.1[25.6, 42 .1] fg/g, p 5 0.010) in untreated hypertensives (n 5 30) than normotensives (n 5 63). vcr was correlated with pcv (r 5 à0.236, p 5 0.024, n 5 92), upc (r 5 0.444, p o 0.001, n 5 93), plasma phosphate (r 5 0.286, p 5 0.005, n 5 93), and usg (r 5 à0.284, p 5 0.006, n 5 93), but not plasma creatinine concentration. in the best multivariate model, pcv was associated with vcr independently of upc (r 2 5 0.435, n 5 92). vcr was significantly reduced by benazepril therapy (65.3 [43.1, 92 .7] fg/g) compared with placebo (76.0[48.0, 116.7] fg/g; p 5 0.031, n 5 17) with a reduction seen in 76% of cases. these results suggest urinary vegf excretion is associated with proteinuria in cats with ckd and might be a marker of renal hypoxia induced by low pcv. ace inhibitor therapy might reduce urinary vegf excretion because angiotensin ii causes constriction on efferent arterioles resulting in tubular hypoxia. fgf-23 is a phosphaturic hormone. fgf-23 concentrations increase with declining renal function in humans. the objectives of this study were to validate a method for fgf-23 quantification in feline plasma and to assess the association between fgf-23 concentration and plasma creatinine or phosphate concentration in cats with chronic kidney disease (ckd). non-azotemic and azotemic (plasma creatinine concentration (cr) 4 2.0 mg/dl) geriatric (4 9yrs) cats were recruited into the cross-sectional study from two london first opinion practices. cats were excluded from the study if they were fed a phosphate restricted diet, or had evidence of concurrent disease. the cats were categorized, using a modified iris staging system, into the following four groups: group 1 (cr 1.6 mg/dl), group 2 (cr 2.0-2.8 mg/dl), group 3 (cr 2.9-5.0 mg/dl), group 4 (cr 4 5.0 mg/dl). groups 2 and 3 were further subdivided based on the iris targets for plasma phosphate concentration (po 4 ): group 2a (po 4 4.5 mg/dl), group 2b (po 4 4 4.5 mg/dl), group 3a (po 4 5 mg/dl), group 3b (po 4 4 5 mg/dl). fgf-23 concentrations were measured in feline edta plasma using a human intact fgf-23 elisa, validated by intraand inter-assay variability and assessment of dilutional parallelism. comparisons between groups were made using the kruskal-wallis test and mann-whitney u test, with statistical significance defined as p o 0.05. bonferroni correction was applied where appropriate (statistical significance then determined as p o 0.008). results are reported as median [25th, 75th percentiles]. fgf-23 concentrations ! 800pg/ml (upper limit of quantification) were assigned the value of 800pg/ml. intra-and inter-assay variability of fgf-23 measurements were o 10.0% and dilutional parallelism between feline samples and the calibration curve were demonstrated. plasma fgf-23 concentrations increased with increasing creatinine concentrations (group 1: 158 [115, 274] , n 5 20, group 2: 354 [239, 473] , n 5 20, group 3: 800 [425, 800], n 5 23, group 4: 800 [800, 800], n 5 14). fgf-23 measurements were significantly different between all groups (p 5 0.005 to o 0.001) except between groups 2 and 3 (p 5 0.01). fgf-23 concentrations were significantly higher in cats with higher plasma phosphate concentrations (group 2a: 329 [237, 423] , n 5 16 vs. group 2b: 576 [374, 793], n 5 4; p 5 0.047) and (group 3a: 432 [167, 800] , n 5 10 vs. group 3b: 800 [625, 800], n 5 13; p 5 0.028). in conclusion, fgf-23 concentrations were higher in cats with more severe ckd or higher plasma phosphate concentrations as would be predicted from its known biological actions. further work is warranted to explore the role of fgf-23 in the development of renal secondary hyperparathyroidism by measuring parathyroid hormone (pth) and calcitriol in cats at different stages of ckd. progressive non-cardiogenic edema and lung dysfunction are common complications of acute kidney injury (aki) in people. pulmonary abnormalities have not been systematically reviewed in dogs with renal azotemia, but anecdotal reports of dogs with aki and concurrent non-cardiogenic pulmonary edema are suggestive of uremic pneumonopathy (up), a centrally-distributed pulmonary edema syndrome associated with kidney disease in people. we therefore hypothesized that pulmonary-associated clinical signs or thoracic radiograph abnormalities are more common in dogs with renal azotemia than in non-azotemic dogs, and that this association is more likely in dogs with aki than dogs with chronic renal failure (crf). our study objectives were 1) to describe thoracic radiograph and lung histopathologic abnormalities in dogs with renal azotemia, 2) to compare the occurrence of these findings in dogs with aki, crf, or non-systemic illness, and 3) to determine if these abnormalities are associated with shorter survival times. records of dogs with renal azotemia evaluated from 1/1/2000 to 8/20/2010 were reviewed; dogs which could be classified as having aki or crf and which had complete thoracic radiograph studies available for review were included. dogs with primary intracranial disease and normal serum creatinine and a complete thoracic radiograph study were selected as controls. signalment, weight, presence of pulmonary-related clinical signs, azotemia duration and severity at time of radiography, and leptospirosis antibody titer were noted. alveolar, bronchial, interstitial, or nodular lesions were described using a 4-point scale, and lung tissue collected at time of necropsy was reviewed; both the radiologist and pathologist were blinded to final diagnoses. significance was p o 0.05 for all analyses. the final study population included 54 aki, 50 crf, and 63 control dogs. crf dogs were older (p o 0.001) than aki and control dogs. pulmonary-related clinical signs were more commonly diagnosed at first evaluation in aki dogs (29/53 dogs, 54.7%) than in crf (13/50, 26.0%; p 5 0.003) or control dogs (9/63, 14.3%; p o 0.001). presence of an alveolar pattern was the only radiographic finding which differed amongst groups (more common in aki [n 5 8, 14.8%, p 5 0.047] and crf [n 5 8, 16%, p 5 0.028] dogs than in control dogs [n 5 2, 3.2%]). there was no association between presence of an alveolar pattern and any other variable. alveolar mineralization was the most common lesion in aki dogs (5/8 dogs; 62.5%), with concurrent alveolar space concretions or mineralization of vessels or bronchioles noted in 1 dog each. necropsies had not been performed in any of the crf dogs, but mineralization was not seen in lung tissues from any control dogs (n 5 9). neither pulmonary-associated clinical signs nor alveolar pattern were associated with median number of days from discharge until death in dogs with aki (p 5 0.220 and 0.468, respectively) or crf (p 5 0.280 and 0.253, respectively). in this group of dogs, presence and type of radiographic pulmonary abnormalities were associated with renal azotemia but not with median time until death. the association between and clinical relevance of alveolar mineralization in aki dogs were not determined, but both the radiographic and histopathologic abnormalities reported here differ from up in people. chronic kidney disease (ckd) is a common cause of morbidity and mortality in cats. the purpose of this study was to investigate the effects of chinese rhubarb (rheum officinale) supplementation on the progression of feline ckd. cats with stable iris stage ii or iii ckd and without comorbidity were included in the study. cats were divided into 3 treatment groups and administered rhubarb extract (group 1, rubenal s , vetoquinol, 75 mg tablet po q 12 h), benazepril as a positive control (group 2, 0.5 mg/kg po q 24 h), or both (group 3). cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. body weight, laboratory data, and blood pressure were recorded every 3 months for up to 34 months. variables between groups at enrollment and within groups over visits were compared with anova and repeated measures ano-va, respectively. a treatment by visit interaction term was included in all repeated measures models. significance was set at p 0.05. except for body weight there was no significant differences between treatment groups at enrollment. there was no significant change in body weight, hematocrit (hct), upc, or creatinine over time as compared to baseline within any group. there was no significant difference between groups over time in regards to change in weight, hct, upc, or creatinine. the treatment by time interaction was non-significant in all models. although there was no benefit associated with combination treatment, the results for rhubarb treatment alone were not different from benazepril treatment. azodyl, an encapsulated, enteric-coated probiotic/prebiotic nutraceutical, is marketed for reduction of azotemia (bun & creatinine) in dogs and cats. cat owners often sprinkle contents onto cat food to facilitate administration. however, exposure to air and stomach acid are thought to inactivate the lyophilized bacteria within the product. therefore, we examined the ability of foodsprinkled azodyl to reduce azotemia in cats with ckd. 10 cats with ckd were enrolled in the study and randomized receive azodyl or placebo. owners were provided with 3-4 capsules of azodyl prior to enrollment to ensure compliance with administration. 2 baseline blood samples were obtained 1 month apart, and then 1 & 2 months after beginning therapy. clinicians and owners were masked as to medication assignment. we hypothesized that a 30% decrease in bun and/or creat in the azodyl group would be significant, and set a 5 0.2. in order to maximize the probability of detecting a difference, we determined the % change as being the difference between the maximal baseline analyte concentration and minimal therapeutic concentration. we compared the % change between groups by mann-whitney u test. bun and creatinine did not differ between groups. based on these results, azodyl, applied by sprinkling onto food fails to reduce azotemia in cats with ckd. whether intact capsule administration reduces azotemia in cats with ckd remains unknown. lower urinary tract disease (lutd) occurs commonly in cats, and idiopathic cystitis (fic) and urolithiasis account for over 80% of cases in cats less than 10 years of age. although several strategies have been recommended, a common recommendation is to induce dilute urine resulting in more frequent urination and to dilute calculogenic constituents. in addition to conventional therapy using modified diets, traditional chinese and western herbs have been recommended, although only one, chorieto, has published data. we evaluated 3 commonly used herbal treatments recommended for use in cats with lutd including (1) san ren tang, (2) wei ling tang, and (3) alisma. we hypothesized that these 3 chinese herbal preparations would induce increased urine volume and decreased urine saturation for calcium oxalate and struvite. six healthy, spayed female, adult cats were evaluated in a placebocontrolled, randomized, cross-over design study. cats were randomized to 1 of 4 treatments including placebo (p), san ren tang (srt), wei ling tang (wlt), or alisma (a). treatment was for 2 weeks each with a 1 week washout period between treatments. at end of each treatment period, a 24-hour urine sample was collected using modified litter boxes. urine volume and biochemistries were measured, and urine saturation for struvite and calcium oxalate was estimated using equil 1.5b. analysis of variance (anova) was used to analyze data statistically if distributed normally and kruskal-wallis was used to analyze data statistically if data were not distributed normally. a p o 0.05 was considered significant. body weights were not different between treatments. no differences were found in 24-hour urinary analyte excretions, 24-hour urine volume, urine ph, or 24-hour urinary saturation for calcium oxalate or struvite between treatments (table) . urolithiasis is a multifactorial disease, frequent and recurrent in dogs in the worldwide, in which breed, sex, age, diet, some anatomical abnormalities, urinary tract infection, urine ph and some geographical and hereditary features in the populations studied have been implicated as risk factors. the effective long-term management of urolithiasis depends on identification and control of the pathophysiological mechanisms involved, which, in turn, depend on accurate knowledge of the mineral composition of the uroliths. the aim of this study was to determine for first occasion the main epidemiological data of canine urolithiasis in mexico. this study was developed with 491 dogs with urolithiasis from 25 of the 33 states of the country. chemical composition of the uroliths was determined by stereoscopic microscopy, infrared spectroscopy, scanning electron microscopy and x-ray microanalysis. urolithiasis affected nearly the same number of males and females; with ages ranging from two months to 15 years with a median age of 5 years. adult animals were the most affected. breeds more affected were schnauzer miniature, poodle, dalmatian, yorkshire terrier, scottish terrier, chihuahua and bichon frisee´. uroliths were found in the lower urinary tract in 97.74% of the cases. mineral composition of the uroliths was: struvite 49.69%, followed by calcium oxalate 25.46%, purines 7.13%, silicate 6.72%, others 0.20%, mixed 8.15% and compound uroliths 2.44%. struvite uroliths affected females in most cases, whereas calcium oxalate, purines and silicate uroliths, were mainly observed in males. our results are similar to studies developed in other countries and continents, though we found a higher frequency of uroliths containing silicate, either pure, mixed or compounds uroliths (10.79%); in mexico city the frequency reached 15%. this high frequency may be due to high consumption of silicate in home-made food or in the groundwater derived from aquifers. acknowledgments: this work has been partially supported by a project of waltham foundation in mexico and the consejo nacional de ciencia y tecnologı´a (conacyt) of mexico. voiding urohydropropulsion is a non-invasive method for removing small urocystoliths from the dog, most commonly used in females due to the relatively wider and shorter urethra. this procedure is typically performed under general anesthesia to allow complete relaxation of the urethra, however, anesthesia results in longer procedure times and difficult endotracheal tube stabilization due to the vertical positioning of animals, especially in larger dogs. the aim of this study was to devise a novel injectable sedation protocol for urohydropropulsion when cystoscopy was not concurrently required. an intravenous catheter was placed, and a combination of medetomidine (10 to 15 mg/kg iv) and hydromorphone (0.025 to 0.05 mg/kg iv) was administered, with the addition of ketamine (2 mg/ kg iv) in fractious animals; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this protocol was considered in cardiovascularly healthy, non-diabetic dogs without evidence of urinary obstruction. monitoring equipment included electrocardiography, blood pressure measurement, and pulse oximetry, and supplemental flowby oxygen was provided. two dogs received the proposed sedation protocol in order to perform urohydropropulsion. dog one was a 3 year old female spayed shih tzu cross, and dog 2 was a 2 year old female spayed standard poodle. ultrasonography revealed a moderate number of urocystoliths present in both dogs, measuring up to 1 mm in dog 1 and 2.3 mm in dog 2. urohydropropulsion was performed and resulted in retrieval of 15 urocystoliths in dog 1, and approximately 20 urocystoliths in dog 2. repeat ultrasonography revealed no uroliths present after urohydropropulsion in both dogs. the time from administration of sedation to administration of reversal agent was 6 minutes for dog 1, and 8.5 minutes for dog 2. records were obtained from 3 dogs that had traditional general anesthetic protocols for urohydropropulsion with cystoscopy for confirmation of urocystolith removal, performed within the last 2 years, and the average anesthetic time was 64 minutes. subsequent to the use of medetomine-based sedation protocols for the above dogs, cystoscopy was performed in a 9 year old neutered male golden retriever with prostatomegaly. medetomidine (15 ug/kg iv) and butorphanol (0.2 mg/kg iv) were administered; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this sedation allowed adequate immobilization for cystoscopy of the urethra and urinary bladder, and endoscopic biopsying of the prostatic urethra and urinary bladder. the time from administration of sedation to administration of reversal agent was 15 minutes for this dog. in conclusion, a novel sedative protocol for urohydropropulsion is proposed which allows for an appropriate level of sedation along with a short procedure time and rapid recovery. this sedation protocol may also be useful for certain cystoscopic procedures. analysis may be delayed for a variety of reasons, including the need for sample batching within the laboratory or shipping to an outsourced location. therefore, it is important to know how storage of the sample may affect enzyme activity. we hypothesized that urinary nag and ggt activity would be affected differently in samples stored by refrigeration vs. freezing. thirty-four canine urine samples submitted to the clinical pathology laboratory at kansas state university were included. samples were collected from clinical patients with a variety of medical/surgical disorders and were selected based on the day of the week and a minimum volume of 10 ml. a complete urinalysis was performed on each sample; however there were no exclusion criteria based on urinalysis results. nag and ggt activity in the urine supernatant was assessed by colorimetric assay. aliquots of each supernatant were refrigerated for 5 days and frozen at à201c for 5 and 30 days at which time enzyme activity was re-assessed. compared to baseline values, enzyme activity for both nag and ggt were stable after 5 days of refrigeration, however there were significant (p o 0.01) declines in ggt and nag activity when urine supernatants were frozen for 5 and 30 days. treatment for canine urinary tract infections (uti) typically consists of 7-14 days of antimicrobial drugs in primary care veterinary practice. compliance with this drug regimen can be difficult for some clients. enrofloxacin is a veterinary approved fluoroquinolone antimicrobial and is useful for treatment of canine uti. fluoroquinolones are often used in human medicine to treat uncomplicated utis in women and can be prescribed for as little as 3 days. the primary objective of this study was to determine if dogs with naturally occurring uncomplicated uti have equivalent microbiologic cure with a high dose short duration protocol of enrofloxacin, compared to a standard antimicrobial protocol. client-owned adult dogs with naturally occurring, uncomplicated uti were prospectively enrolled in a multi-center clinical trial and assigned to 1 of 2 groups in a randomized blinded manner. group 1 received treatment with 18-20 mg/kg oral enrofloxacin once daily for 3 consecutive days. group 2 dogs were treated with 13.75-25 mg/kg oral amoxicillin-clavulante twice daily for 14 days. both groups had urinalyses and urine cultures submitted on day 0, 10, and 21. at the time of this interim analysis, thirty-six dogs have completed the trial. bacteriological cure was achieved in 15 dogs (83%) treated with enrofloxacin and 14 dogs (78%) treated with amoxicillinclavulante, respectively. these data suggest that the high-dose, short-duration enrofloxacin protocol was equally effective to the standard protocol in treating uncomplicated canine uti in the sample patient population. and may represent a viable alternative therapeutic regimen for similar patients. azotemia is frequent in dogs with dmvd (nicolle et al; jvim 2007; 21:943-949) and could result from renal hemodynamic alterations. renal resistive index (ri) allows assessment of renal vascular resistance. the aim of this prospective study was to assess ri in dogs with different dmvd stages. fifty-five dogs with dvmd were used (isachc class 1 (n 5 28), 2 (n 5 19), and 3 (n 5 8)). physical examination, renal ultrasonography and echo-doppler examinations were performed in awake dogs by trained observers. plasma creatinine, urea and nt-probnp were measured. statistical analyses were performed using a general linear model. whereas ri of renal and arcuate arteries were unaffected by isachc class, left interlobar ri increased (p o 0.001) from 0.62 ae 0.05 (mean ae sd) in class 1 to 0.76 ae 0.08 in class 3. left interlobar ri was also higher (p o 0.001) in azotemic (0.74 ae 0.008) than in non azotemic (0.62 ae 0.005) dogs. similar findings were observed for right interlobar ri. a positive effect of nt-probnp (p 5 0.002), urea (p o 0.001), creatinine (p 5 0.002), urea-to-creatinine ratio (p o 0.001), left atrium-to-aorta ratio (p o 0.001), regurgitation fraction (p 5 0.011), systolic pulmonary arterial pressure (p o 0.001) and shortening fraction (p 5 0.028) on ri was also observed. in conclusion, interlobar ri increases with the severity of dmvd and azotemia. a cause-effect relationship remains however to be established. antibodies against alpha-enolase are associated with immunemediated nephritis in people. it was previously shown that vaccinated cats commonly develop antibodies against alpha-enolase. the purpose of this study was to assess for associations between alphaenolase antibodies and azotemia in privately-owned cats. clinically stable privately owned cats ! 10 years of age, with and without azotemia (creatinine 4 2 mg/dl), and with an available vaccine history for ! 5 years were recruited for the study. sera were assayed for creatinine concentrations and alpha-enolase antibodies by use of previously validated techniques. results from cats with and without azotemia were compared by student's 2-tailed t test or fisher's exact test with significance defined as p o 0.05. median ages were 15 years (range: 10-18) and 12 years (range: 10-15) for cats with (n 5 35) and without azotemia (n 5 27), respectively. there was no significant difference in vaccine events (number, type, or route of administration) between groups. azotemic cats (34.3%) were more likely than normal cats (12.5%) to be positive for antibodies against alpha-enolase (p 5 0.016). in addition, alpha-enolase antibody concentrations were greater (p 5 0.041) in azotemic cats (mean % elisa 5 62.5%) than cats with normal creatinine concentrations (mean %elisa 5 47.2%). results of this study suggest that antibodies against alpha-enolase in cats may be associated with renal disease. additional prospective evaluation in a larger number of cats is indicated. aki is used in human medicine as a predictor of mortality based on the akin (acute kidney injury network) scoring system which utilizes relative increases in creatinine to determine stage. with this scheme, mortality has been shown to increase as the stage of kidney injury (indicated by akin score) increases. accordingly, we hypothesized that this system would improve predicting prognosis in dogs and cats. we retrospectively evaluated 1088 dogs and 856 cats (2008) (2009) ) that had ! 2 creatinine measurements within 7 days, and whose first creatinine was o 1.6 mg/dl. patients were categorized as: level 0 (no aki); level 1 (second creatinine value o 1.6 mg/dl, but creatinine increased ! 0.3 mg/dl); or level 3 (second creatinine 4 1.6 mg/dl with a creatinine increase ! 0.3 mg/dl). thirty and 90 day survival for each level was compared to level 0. adjusted odds ratio (or) in dogs for 30 day survival was 1.3 for level 1 (ci 95%, 0.8-2.2) and 3.2 (ci 95%, 1.8-5.5) for level 2; or for 90 day survival was 1.3 for level 1 (ci 95%, 0.8-2.2) and 3.7 (ci 95%, 2.1-6.5) for level 2. for cats, or at 30 days was 1.5 (ci 95%, 0.5-4.6) for level 1 and 3.1 (ci 95%, 1.5-6.7) for level 2; or for 90 day survival was 0.9 (ci 95%, 0.3-2.8) for level 1 and 4.1 (ci 95%, 1.8-9.3) for level 2. thus, detecting increasing stage of aki helps predict mortality in dogs and cats. abstract n/u-27 feline urate urolithiasis: 143 cases (2000 -2008 . j dear 1 , r shiraki 2 , a ruby 2 , j westropp 3 . 1 william r pritchard veterinary medical teaching hospital, university of california, davis, ca, 2 gerald v. ling urinary stone analysis laboratory, university of california, davis, ca and 3 the department of veterinary medicine and epidemiology, university of california, davis, ca. feline urate urolithiasis accounts for 10% of the feline stones our laboratory analyzes each year; little information is known about this disease, particularly the incidence of those cats with hepatopathies. the objective of the study was to characterize the signalment, clinicopathologic data, and diagnostic imaging of cats with this disease as well as the salts of uric acid present. a retrospective analysis of feline urate uroliths submitted to the stone lab between january 2000-december 2008 were included. from these data, primary veterinarians were solicited to submit records. furthermore, all records from cats with urate uroliths from the vmth were analyzed separately. 143 records were received from the primary care veterinarians. sixteen cases were identified from the vmth. median values for the cbc and chemistry panels available were within the reference ranges provided, with only a few outliers present. of the 78 cats with radiographic reports, 70 (90%) had visible evidence of uroliths. two external cases had confirmed pss; five cases from the vmth had a pss. cats with urate uroliths and pss were younger than cats without a documented hepatopathy (2 years vs. 7 years). the siamese breed was overrepresented. all stones were ammonium hydrogen urate. the pathogensis of urate uroliths in cats is poorly understood. most cats were not completely evaluated for pss, however, there were few clinicopathologic parameters which indicated hepatopathies were present. further studies are warranted to evaluate genetics and purine metabolism in cats with urate uroliths to help tailor proper management and breeding strategies. 3-indoxyl and p-cresyl sulfate (is, and cs, respectively), small protein-bound molecules derived from gastrointestinal protein metabolism, are among the most important uremic solutes affecting morbidity and mortality in human chronic kidney disease (ckd). in the blood stream, these compounds are predominantly bound to protein, but their debilitating effects on prognosis and quality of life in ckd appear to be driven by the free fraction. the objectives of the present study were to assess the normal, physiological levels of is and cs in healthy cats and to evaluate the correlation of the respective free and protein-bound levels. blood samples were taken from 105 clinically healthy adult cats enrolled at five participating veterinary practices in germany. after centrifugation, the serum was deep frozen until transport on dry ice to the analytical laboratory. serum creatinine and urea levels were quantified by vettest s (idexx laboratories, inc.). total and free is and cs, respectively, were quantified by turbulent flow chromatography coupled with a tandem mass spectrometry detector. statistical analysis of the results comprised i) a descriptive report of the median with upper and lower bounds of the 95% confidence interval for reference values of is and cs, ii) a calculation of various pearson correlation coefficients r, also tested with reference to the null hypothesis of no relationship, and iii) wilcoxon-mann-whitney utest for an estimation of the effect of hemolysis on serum is or cs levels. six animals with serum creatinine or urea levels outside the reference range were excluded from the calculation of reference values. median levels of is in cat serum were 1.19 mg/l with upper and lower bound 95% confidence intervals at 1.46 and 0.99 mg/l, respectively. the corresponding median levels of cs were 2.11 mg/l (median) and 2.46 vs 1.57 mg/l (upper vs lower bound levels, respectively). these values showed a low, non-significant correlation with serum creatinine or urea levels. however, is and cs serum levels were moderately correlated (total levels r 5 0.4808, p o 0001). their respective free levels constituted about 5% of the total serum levels (r ! 0.8977, p o 0.001). non-hemolytic samples tended to yield lower values than hemolytic samples. due to the low number of hemolytic samples (n 5 14) , the group difference could, however, not be statistically confirmed. the results indicate that it is sufficient to determine total levels of either is or cs in serum while studying the effects of therapeutic or dietetic interventions on the evolution of these parameters in feline ckd. reference values are provided for orientation towards clinically relevant changes. disrupted urothelial differentiation has been implicated in the pathogenesis of feline idiopathic cystitis (fic). studies of cultured human urothelium have shown that abnormalities in urothelial differentiation and repair may be mediated by persistent 15-hydroxy-prostaglandin dehydrogenase (pgdh) activity and subsequent metabolism of cytoprotective prostaglandins. the goal of this study was to confirm persistent pgdh expression in fic bladders compared to desmoplakin i1ii expression, a marker of urothelial differentiation. urinary bladder biopsy specimens were obtained by cystotomy from 9 symptomatic cats with chronic fic. cats with a history of another major disease, previous cystotomy, or recent treatment with corticosteroids, nsaids, antihistamines, antidepressants, or glycosaminoglycans were excluded. urinary bladder tissue specimens were also obtained from 10 untreated clinically normal specific-pathogen-free cats. tissue specimens were fixed in buffered 10% formalin and embedded in paraffin. tissue sections were deparaffinized and subjected to citrate buffer microwave antigen retrieval. tissues were stained for pgdh using a rabbit anti-pgdh antibody, an isotype negative control or goat anti-desmoplakin i1ii and developed using the avidin-biotin peroxidase complex method. all fic (9/9) and normal (10/10) cat bladder samples showed similar staining of urothelial cytoplasm for pgdh. however, desmoplakin i1ii staining, found on the luminal cell surface in 4/4 normal tissues, was disrupted in 6/6 fic bladder samples. desmoplakin i1ii staining confirmed altered urothelial differentiation in fic cats. however, pgdh expression remained intact in fic samples. we hypothesize that pgdh expression in fic may contribute to its pathophysiology due to breakdown of prostaglandins essential for urothelial healing. additional studies will explore this hypothesis. the university of tennessee college of veterinary medicine's picture archiving and communication system was searched over a 9 month period for cats that had undergone both abdominal radiographs and ultrasound during the same visit. one hundred and three cats were identified (age range o 1 to 18yrs; median 11yrs). kidney size was determined based on radiographic and ultrasound findings. of the included cats, 41.8% had two normal sized kidneys, 18.4% had one small and one normal, 15.5% had one large and one normal, 11.7% had two small, 8.7% had two large, and 3.9% had one small and one large kidney. the presence of mineralization, uroliths and hydronephrosis was also noted. medical records were reviewed for clinical chemistry data and historical information concerning previous urinary disease. no significant differences were found between kidney size and renal function, kidney size and the presence of uroliths, renal mineralization and function or the presence of uroliths and function. the presence of uroliths was significantly associated with hydronephrosis. of the 30 cats with at least one large kidney, 9 (30%) had hydronephrosis. of the 23 cats with current or previously diagnosed uroliths, urinary tract infections or other uropathies, 10 (43.5%) had at least one small kidney. small kidneys were commonly found in older cats, however, this correlation was not statistically significant. based on these findings, small kidneys are more likely to be the result of urinary disease as opposed to being either congenital or due to aging. this study aimed to evaluate ife, which has been advocated for treatment of lipid-soluble drug intoxication, in the treatment of clinically-occurring canine ivermectin toxicosis. one australian shepherd and two miniature australian shepherds were included. all three dogs were homozygous for the mdr-1 gene mutation. two dogs roamed on horse ranches where ivermectin-based deworming products had recently been used. ivermectin was administered to the third dog (165 mg/kg po). all three dogs exhibited tremors, ptyalism, and cns depression, which progressed over several hours to stupor in two dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. a 20% formulation of ife (liposyn ii, hospira) was administered as a bolus (1.5 ml/kg) followed by a slow iv infusion (7.5-15 ml/kg over 30 minutes). no change was observed in the neurologic status of any patient. lipemia visible upon blood sampling persisted for 36 hours in one dog. no other adverse effects were noted. serum ivermectin levels confirmed ivermectin exposure in each case. in this study, ife administration did not result in clinical benefit in cases of ivermectin toxicosis. brain ivermectin concentrations in mdr1 mutant/mutant genotype dogs may be too high to be overcome by ife. additionally, these dogs may lack p-glycoprotein-mediated biliary clearance mechanisms needed for optimal ife function. further investigation is needed to determine the utility and optimal dosing of ife in canine toxicoses, to characterize its safety, and to determine how mdr-1 status may alter the efficacy of ife in treatment of canine ivermectin intoxication. rufinamide is a recently approved antiepileptic drug used for the treatment of seizure disorders in human patients. rufinamide is administered at a dose of 45 mg/kg divided twice daily to achieve therapeutic concentrations of 15 mg/ml. the objective of this study was to determine the pharmacokinetic properties and short-term adverse effects of single-dose oral rufinamide in healthy dogs in preparation for a possible clinical trial evaluating the efficacy of rufinamide in the treatment of canine epilepsy. six healthy adult dogs were included. the pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of 20.0 mg/kg (range 18.6-20.8 mg/kg), extrapolated from the dose used in human patients. dogs were monitored by repeat physical examinations, electrocardiograms and blood pressure assessments during the course of the study. plasma rufinamide concentrations were determined using high-performance liquid chromatography. pharmacokinetic data were analyzed using winnonlin version 1.0. no adverse effects were observed. the mean terminal half-life was 9.86 1/à 4.77 hours. the mean maximum plasma concentration was 19.561/à5.82 mg/ml and the mean time to maximum plasma concentration was 9.33 1/à 4.68 hours. mean clearance was 1.448 1/à 0.703 l/hr. auc inf was 410.72 1/à 175.88 mgãh/ml. results of this study suggest that rufinamide given orally at 20 mg/ kg twice daily in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted. the aims of this study were to investigate the abg for (i) the prevalence of skull abnormalities; (ii) the prevalence of sm; (iii) an association between lateral ventricular size, cerebellar size and sm; and (iv) associations between sm, skull abnormalities, csf pleocytosis and clinical signs. seventy-six abgs, recruited as part of a larger epidemiological and genetic study, underwent brain and spinal mri evaluation (3.0t general electric signa hdx, milwaukee, wi). all dogs were evaluated neurologically, recording deficits and the presence of spinal pain. sequences acquired included t2w, t1w pre-and postcontrast, and t2w flair, sagittal and transverse. cervical spinal cord central canal (cc) and or syrinx size and its percent area of spinal cord was measured using osirix s . the presence of chari-like malformation (cm) was assessed by recording the presence of caudal cerebellar deviation and/or foramenal vermal herniation. lateral ventricle and cerebellar volume was expressed as a percent of the cerebrum and intracranial volume2qa respectively. forty-five dogs underwent atlanto-occipital cerebrospinal fluid tap at the time of mri and the white blood cell (wbc) count was recorded. student's t-tests were used to compare the measured variables between groups with and without skull abnormalities, spinal pain and neurological signs. the mean age of the 30 males (24 intact) and the 46 females (34 intact) was 50.4 months (range 8-135; median 44 months). neurological deficits and neck pain were noted in 21 (27%) and 15 (19.7%) of dogs respectively; 5 dogs (6.57%) exhibited both. cerebellar deviation and vermal herniation were present in 37 (48.68%) and 46 (60.52%) dogs respectively; twenty-three dogs (30.26%) had both. mean height of the cc was 2.3 mm (0-7.2 mm). forty (52.63%) ccs were greater than 2 mm in height; the mean length of these lesions was 2.03 vertebrae (0.5-7). mean csf wbc count was 4.97/ml (0-39). syrinx height and extent were significantly higher in dogs with neurological signs (size p 5 0.01; extent p 5 0.0004). there were no significant differences in syrinx sizes and extent in dogs with or without skull abnormalities or spinal pain. there were no associations of syrinx height or extent with csf wbc count or age of dog. intact females had a significantly lower syrinx extent than intact males (p 5 0.009). there were no significant differences in presence of spinal pain or neurological signs between dogs with or without skull abnormalities. there was a significant negative association of ventricular percentage and cerebellar percentage (p o 0.0001). there was a significant association of ventricular percentage with syrinx percentage (p 5 0.0015) and height (p 5 0.0007). this study suggests that sm and cm are prevalent in abgs. syrinx size and extent are associated with neurological signs and ventriculomegaly is associated with both small cerebellar size and large syrinx size. however, sm may not be associated with cm as defined by cerebellar herniation and deviation and is not associated with csf inflammation. the power tissue resection device (ptrd) is a hand-piece comprised of an outer cannula with motor driven vacuum-assisted inner cutting blade. this device was designed and is marketed for human neurosurgical brain/spinal cord tumor resection. the purpose of this study is to describe the use of the ptrd for intervertebral disc fenestration and to compare the effectiveness of manual fenestration to that of the ptrd. fifteen cadaveric lumbar spines were randomly placed into three study groups: group 1 was the control group on which no fenestrations were performed, group 2 was the manual fenestration group and group 3 was the ptrd fenestration group. the effectiveness of fenestration via both manual and ptrd was assessed by calculating the ratio of remaining nuclear weight post fenestration to total nuclear volume. discs with lower ratios were more effectively fenestrated. results showed a smaller ratio of post fenestration remaining nuclear weight to nuclear volume following fenestration with the ptrd (0.23 ae 0.09) as compared to manual fenestration (0.30 ae 0.10). these results did not show statistical significance. when fenestrated samples were compared to control samples (0.39 ae 0.07), there was a statistically significant reduction in ratios. in conclusion, the ptrd is easy to use and is as effective as the manual technique for canine intervertebral disc fenestration. according to the human who classification gliomatosis cerebri (gc) is a rare astrocytic tumor affecting at least three lobes of the brain with extensive infiltration, but relative preservation of brain architecture. gc has not been reported to occur as a hereditary disease, neither in man nor in animals. here, we report the temporally clustered occurrence of gc in a family of bearded collies. a 7 years old female bearded collie with forebrain signs was presented. differentials included inflammatory/ infectious, metabolic/ toxic, and neoplastic diseases. within a time period of 12 months, 3 offspring of this bitch were presented with similar clinical signs. two dogs were full siblings (2 males). the remaining female dog originated from a match with a different male dog. mri was performed in all 4 dogs and revealed a diffuse and extensive intra-axial lesion with moderate mass effect and midline shift. the ill defined lesion showed mainly a white matter distribution with hyperintense signal in t2-w and flair images and iso-to hypointense signal in t1-w images without contrast enhancement. the lesion was bilateral in all cases, continued along the white matter extending partially into the gray matter with contact to the brain surface. neuropathology revealed a diffuse and extensive infiltration of the brain and spinal cord by a neoplastic glial cell population involving white and gray matter of both hemispheres, thalamus, brainstem and cerebellum in all 4 dogs. based on the cell morphology and immunoexpression of glial fibrillary astrocytic protein by neoplastic cells diagnosis of gc was made. this is the first report of familial occurrence of gc, which is likely the result of a germ-line mutation. several human hereditary cancer syndromes are associated with cns tumors including amongst others the li-fraumeni cancer family syndrome (p53 mutation), neurofibromatosis (type 1 and 2) (neurofibromin, merlin mutation), and tuberous sclerosis (hamartin, tuberin mutation). furthermore, familial clustering of human gliomas unassociated to the known inherited cancer syndromes has been described. in the dog, hereditary cns tumors are not known. the exact mode of inheritance and putative gene mutations of gc in this bearded collie family are currently under investigation. preliminary results are consistent with a monogenic autosomal dominant mode of inheritance, although a recessive inheritance cannot be completely ruled out at this time. mutations in the tp53 gene were not found following amplification and sequencing of exons 5-8 in 2 affected dogs. previously presented at the ecvn annual meeting in cambridge, uk. the gm 2 gangliosidoses are characterized by a deficiency of bhexosaminidase. there are two isoforms: hex a composed of an a and b subunit encoded by hexa and hexb genes respectively and hex b with two b subunits. hex a requires an activator encoded by gm2a. two japanese chin dogs with confirmed gm 2 gangliosidosis showed elevated total hexosaminidase and normal hexosaminidase a activity, a pattern associated with the ab variant in humans and consistent with prior reports in the breed. this study was performed to identify the mutation responsible using resequencing with an applied biosystems 3730xl dna analyzer as previously described (awano 2009). mutations in gm2a cause the ab variant in humans, but resequencing gm2a revealed no mutation that could account for the disease. resequencing hexa and hexb revealed a c.967g 4 a mutation in hexa which was homozygous in both affected dogs. sixty-five normal japanese chin dogs were screened for the mutant allele; 60 were homozygous for the ancestral allele and 5 heterozygous. this mutation predicts a p.323e 4 k substitution affecting one of two primary active-site amino acids that participate in the hydrolysis of gm 2 ganglioside. substitution of a lysine residue at this site is likely to eliminate subunit a enzymatic activity. the apparently normal levels of hexosaminidase a activity in affected dog samples may be a result of b subunit overexpression. human hex b possesses low levels activity against the artificial substrate used to assess hex a activity, but specificity of activity of the canine enzyme is not known. previously presented at the american society for neurochemistry: additional data in this abstract. phenytoin (pht) is the intravenous drug of choice in humans for seizure emergencies following benzodiazapines. iv fosphenytoin (fos) is a pht pro-drug which causes less administration related adverse events. while the short half-life of pht is not suitable for chronic oral therapy in dogs, iv fos has not been studied. two dogs received 15 mg/kg phenytoin equivalent (pe) and two dogs received 25 mg/kg pe of fosphenytoin intravenously at a rate of 50 mg pe/min. blood for plasma levels were drawn at 10 time-points over 12 hours; total and unbound drug levels were measured by hplc. vital signs including ekg, blood pressure, and neurological examination were monitored. the half-life of metabolism of fos to pht was $10 min, with 4 80% of fos metabolized to pht by 30 minutes. eighty to 84% of pht was protein-bound during the first 15 minutes after dosing, compared to 90-95% in humans. the elimination half-life for total pht ranged from 2.8-3.5 hours and for unbound pht ranged from 1.9-5.4 hours. dogs receiving 15 mg/kg pe intravenously achieved unbound pht plasma maximum concentrations of 2.2-2.4ug/ml at 5 minutes, consistent with human loading dose levels. adverse events observed in some dogs included vomiting, mild ataxia, and short lived tremors, the severity of which appeared dose dependent. all dogs were clinically normal within 30 minutes of all doses. a 15 mg/kg pe dose of iv fos appears adequate for production of pht levels predicted to be effective for the treatment of canine seizure emergencies. further studies in clinical canine patients are warranted. acquired myasthenia gravis (mg) is caused by antibodymediated inactivation of the acetylcholine receptor on the neuromuscular endplate causing focal, regional or generalized muscle weakness. many medical treatments have been reported; however, responses to therapy and outcomes are unpredictable and death often results from aspiration pneumonia. therapeutic apheresis is an extracorporeal procedure that separates blood into its components for removal or specific alteration prior to return to the patient. therapeutic plasma exchange (tpe) is an apheresis treatment in which plasma (containing pathologic antibodies) is removed and exchanged with donor plasma. tpe is used routinely to treat mg in human patients with severe disease or disease unresponsive to conventional therapy. we report the successful use of tpe to treat 2 large breed dogs with confirmed mg (aceytlcholine receptor antibody concentration: 3.05 and 3.32 nm/l, respectively; normal concentration: o 0.6 nm/ l) that was severe and not adequately responsive to traditional therapies. both dogs were non-ambulatory, recumbent, and demonstrated megaesophagus and aspiration pneumonia. three tpe treatments (1 plasma exchange each) were performed over 5 and 7 days, respectively, in each dog without complication. both dogs became ambulatory within 3 days of starting tpe treatment with subsequent resolution of regurgitation and megaesophagus. pyridostigmine was continued during tpe sessions and discontinued in both dogs within 3-6 months. both dogs remain asymptomatic and have had no recurrence of mg during 16 and 4 months of follow-up, respectively. tpe is a viable treatment option for dogs with mg that have severe disease, life-threatening complications or that remain unresponsive to traditional therapies. tpe may alleviate clinical signs more rapidly, and improve long-term outcomes when compared to historical experiences in patients with comparable disease. clinical findings, clinicopathologic data, imaging features, and treatment of canine spinal meningiomas have been described in the veterinary literature, but histological characteristics and tumor grading have less commonly been reported. the aims of this retrospective case series were to describe the clinical, imaging, and histologic features of seven canine spinal meningiomas including a cervical spinal cystic meningioma that had imaging and intraoperative features of a subarachnoid cyst. medical records from dogs with a histopathological diagnosis of spinal cord meningioma presented to the veterinary teaching hospital between 2006 and 2010 were reviewed. signalment, presenting clinical signs, physical and neurologic examination, clinicopathologic data, surgery reports and available images were reviewed. all meningiomas were histologically classified and graded following the international who human classification for cns tumors. seven dogs were included, 4 males and 3 females. median age at presentation was 8.7 years (range, 3.5-11.4 years), and median weight was 35 kg (range, 8-45 kg) . median time between onset of clinical signs and diagnosis was 108 days (range, 45 days -1 year). cerebrospinal fluid (csf) analysis was performed in 4 dogs, showing increased protein concentration in 2 cases, and being normal in the other 2. spinal radiographs revealed vertebral canal widening in one case. myelography (4/7) showed intradural/extramedullary lesions in three cases, one of them consistent with a csf-filled subarachnoid cavity, and an extradural lesion in one case. magnetic resonance imaging (mri) was performed in all cases and revealed mild to marked hyperintensity on t2w and precontrast t1w images and homogeneous contrast enhancing (ce) intradural/extramedullary masses (4 cervical and 2 thoracic) in six cases, with one of these showing an additional intramedullary ce pattern. a dural tail was identified in two dogs. one dog had a fluid-filled subarachnoid enlargement located dorsally to the spinal cord. this lesion was hyperintense on t2w, hypointense on t1w and flair images, and did not enhance. it was diagnosed as a spinal subarachnoid cyst, but the histopathological study of the surgically resected mass revealed a grade i cystic meningioma. five other cases underwent cytoreductive surgery, two transitional meningiomas (grade i) that survived 3 (alive at the time of writing) and 7 months; and three anaplastic meningiomas (grade iii) that survived 10-16.6 months before neurological deterioration and euthanasia. another anaplastic meningioma was euthanized right after diagnosis. there are few reports grading canine spinal meningiomas, with most being grade i or ii. of the few grade iii tumors reported, only one had been treated surgically and was euthanized 90 days later because of neurological deterioration. we report four grade iii (anaplastic) meningiomas, three of which surgically treated and with longer survival times. finally, cystic meningioma should be considered in the differential diagnosis of cases with imaging features consistent with arachnoid cyst because of their similar appearance, making histopathological analysis essential for a definitive diagnosis. head trauma is a common veterinary emergency, but few prognostic indicators have been studied in dogs, making it challenging for clinicians to counsel clients about the odds of recovery. a recent meta-analysis showed that higher plasma glucose, lower plasma ph and lower hemoglobin at admission were associated with increased risk of death in human head trauma. the goal of this retrospective study was to investigate the association between admission point of care blood gas parameters and survival to discharge in dogs with head trauma. fifty one dogs presenting to the cornell university hospital for animals with head trauma from 2007 to 2010 that had a blood gas analysis done within 1 hour of presentation were eligible for inclusion. parameters assessed included glucose, base excess (be), anion gap (ag), ph, hemoglobin, and sodium. biochemical data were found to be normally distributed using the kolmogorov-smirnov test. t-tests or welch tests were used to compare parameters between survivors (s,n 5 42) and non-survivors (ns, n 5 9). of glucose, be, ag, ph, hemoglobin, and sodium, only mean glucose (s 5 131 mg/dl, ns 5 171.4 mg/dl, p 5 0.029) was significantly different between groups, although there was a trend for a difference in mean be (s 5 à3.6, ns 5 à8,5, p 5 0.055). logistic regression analysis showed that of the parameters, only be was independently associated with outcome (odds ratio 0.79, 95% ci 5 0.63-0.98, p 5 0.036). these results suggest that two easily measured biochemical parameters (glucose and be) may yield useful prognostic information in dogs with head trauma, but further studies are needed to further elucidate these findings. type i intervertebral disc disease (ivdd) commonly affects chondrodystrophic dogs. neurological recovery and outcome following surgical decompression may be unpredictable due to suspected ischemic neuronal injury. hyperlactatemia has been associated with spinal cord injury in humans and experimental animals. the purpose of the study was 1) to determine the relationship between serum and csf lactate levels and 2) to compare lactate levels with neurological outcome following decompressive surgery in dogs with ivdd. healthy, chondrodystrophic dogs diagnosed with ivdd localized to the t3-l3 spinal cord were included. serum lactate levels were obtained at: anaesthetic induction, skin incision, muscle dissection, and extubation. in patients with hyperlacatemia at extubation, additional samples were obtained. csf was analyzed for lactate concentration. neurological status was recorded at presentation and multiple times during the recovery period. 31 dogs were included in the study (3-12 years old). 22/31 dogs had normal lactate levels throughout the study. 9/31 dogs had serum hyperlactatemia prior to anaesthetic induction; 6/9 dogs returned to normal during anaesthesia and 3/9 dogs had continued hyperlactatemia until the end of the observation period. neurological status of the dogs varied similarly between all groups. in 12/14 dogs where csf lactate levels were measured, initial serum levels were lower than csf lactate levels; in 5/14 dogs where csf and serum were collected simultaneously, serum lactate concentration was consistently lower than csf lactate. no association between presenting neurological status or neurological outcome and serum or csf lactate concentration was made. neither serum nor csf lactate concentration is useful for predicting neurological outcome in dogs with ivdd. chiari-like malformation (cm) has been associated with syringomyelia (sm) in cavalier king charles spaniel (ckcs) and is postulated to result from a mismatch between the volume of the caudal cranial fossa and the brain parenchyma contained within. the objective of this study was to assess the role of cerebellar volume in caudal cranial fossa overcrowding and syringomyelia. three dimensional models were created using t2-weighted transverse magnetic resonance images in the commercial software package mimics s . volumes of cerebellar parenchyma were analyzed as percentages of caudal cranial fossa volume (cerebellar caudal cranial fossa percentage) and total brain parenchyma volume (cerebellar brain percentage). data was assessed for normality and the appropriate statistical test was used to compare means/medians between groups. forty-five small breed dogs (sb), 58 ckcs and 31 labradors (ld) were compared. as sm is thought to be a late onset disease process, two subgroups were formed for comparison: 21 ckcs younger than 2 years with sm (group 1) and 13 ckcs older than 5 years without sm (group 2). ckcs had a larger cerebellar caudal cranial fossa percentage than the other groups .76%] vs. sb 47.81% [40.36-62.91%] and ld 41.32% [32.59-52.95%]; p o 0.001). the cerebellar brain percentage was also larger in ckcs compared to the other groups (ckcs 8.90% [6.62-11.46%] vs. sb 7.37% [5.25-11.34%] and ld 7.23% [6.36-9.54%]; p o 0.001). group 1 had a significantly larger cerebellar caudal cranial fossa percentage than group 2 (53.71% ae 1.27 vs. 49.31% ae 2.35, p 5 0.001) and a significantly larger cerebellar brain percentage (9.45% ae 0.43 vs. 8.58% ae 0.55, p 5 0.021). our findings show that the ckcs has a relatively larger cerebellum than small breed dogs and labradors and there is an association between increased cerebellar volume and sm in ckcs. chiari-like malformation (cm) is nearly omnipresent in the cavalier king charles spaniel (ckcs) breed. the mis-match of the caudal cranial fossa and the parenchyma within is thought to lead to syringomyelia (sm). there is currently a lack of information if the morphological changes seen in ckcs with cm are progressive or non-progressive. in this retrospective study we used established measurements of cerebral volumes, foramen magnum height and cerebellar herniation length to assess if there is a significant difference between subsequent magnetic resonance (mr) imaging of the brain of the same dog. electronic patient records were reviewed for ckcs with cm which had two separate mri scans, which were a minimum of 3 months apart. ckcs with diseases affecting measurements were excluded. for the volumetric measurements three-dimensional models were created using t2-weighted transverse mr images in the medical imaging software (mimics v12.0, materialise n.v, 2008) . volumes of the caudal cranial fossa parenchyma were analyzed as percentages of caudal cranial fossa volume and caudal cranial fossa volume was analyzed as a percentage of total cranial cavity volume. the volume of the ventricular system was recorded as a percentage of total parenchymal volume. data was assessed for normality and the appropriate statistical test was used to compare means/medians. twelve ckcs were included with a median scan interval of 9.5 months (3-83 months). the size of the foramen magnum increased significantly between the first and second scan (1.52 ae 0.08cm vs. 1.59 ae 0.09cm; p 5 0.03), as did the length of cerebellar herniation (0.17 ae 0.05cm vs. 0.22 ae 0.09cm; p 5 0.02) and the caudal cranial fossa percentage (13.44% [9.9-15.28%] vs. 13.96% [9.9-15.48%]; p 5 0.02). there was no significant difference noted between the two time points in any of the other volumetric measurements ( this work could suggest that overcrowding of the caudal cranial fossa in conjunction with the movements of cerebrospinal fluid and cerebellar tissue secondary to pulse pressures created during the cardiac cycle causes pressures on the occipital bone. this leads to a resorption of the bone and therefore an increase in caudal cranial fossa and foramen magnum size allowing cerebellar herniation length to increase. the cord dorsum potential (cdp) is a stationary potential arising in dorsal horn interneurons after stimulation of sensory nerves. cdps have been recorded in normal anesthetized dogs previously, and normal latency values have been determined for tibial and radial nerves. this study was undertaken to determine whether cdps could be reliably recorded from the caudal nerves in normal dogs, thus allowing electrophysiological assessment of the cauda equina, and whether neuromuscular blockade improved recording quality. ten adult dogs weighing from 23.2 to 32.0 kg were anesthetized and cord dorsum recordings were compared before and after administration of atracurium. recording needles were placed onto the dorsal lamina at intervertebral sites from l7/s1 to l2/3. stimulations were made on the lateral aspect of the caudal vertebrae approximately 5-8 cm from the tail base. recordings from 500 stimulations were averaged. cdps were recorded successfully in all dogs. onset latency varied from 2.2 to 4.7 ms. the cdp was largest when recorded closest to the site of entry of the stimulated nerve into the cord, as determined by post-mortem examination immediately after testing in 6 dogs. administration of atracurium did decrease muscle artifact, and in some cases helped isolate the origin of the cdp. these data show that cdps can be readily assessed from the caudal nerves of anesthetized dogs, with or without atracurium. cord dorsum potentials from caudal nerves may add important information about the integrity of the cauda equina in dogs with suspected degenerative lumbosacral stenosis. canine intracranial glial tumors and many human brain tumors express heat shock proteins (hsps) associated with their degree of malignancy. the up-regulation of hsps during tumor cell growth helps keep tumor proteins stable and therefore makes them a reasonable target for therapy. ki67 expression and ec have been strong indicators of cell proliferation and dedifferentiation, respectively.the aims of this study were to determine (i) if canine meningiomas express hsp 27 and/or hsp 72; (ii) whether the expression of the hsps was associated with ki67 and/or e-cadherin (ec) expression; and (iii) whether peritumoral edema was associated with hsp, ki67 and/or ec expression. forty-one formalin-fixed, paraffin-embedded canine intracranial meningiomas underwent immunohistochemical staining using anti-hsp 27, or 72 antibodies. these tumor samples were also immunohistochemically stained for ki67 and ec expression. canine mammary carcinoma and squamous cell carcinoma tissues served as the control samples, as both have previously been shown to express hsps. skin was used as control for ki67 and ec. four non-overlapping high power fields of each stained sample were selected and cell staining was analyzed using a semi-quantitative method for hsps and ki67; a qualitative assessment was used for ec. all analyses were performed using sas v 9.2 (cary, nc). descriptive statistics of staining percentages were calculated for all tumors tested. simple pearson's correlation was used to test for correlations of ec area with hsp areas and ki-67 percent positive cells and of ec intensity with hsp intensities and ki-67 percent positive cells. all hypothesis tests were 2sided and the significance level was a 5 0.05. thirteen meningiomas had mr images quantitatively evaluated for peritumoral edema using t2flair sequences. the edema index (ei) was evaluated for an association with hsp 27, hsp 72, ec and ki67 expression. hsp 27 was expressed in 36% (mean 8.7% of cells; range 0-58%), hsp 72 in 52% (mean 5.2% of cells; range 0-26%) and ec in 68% of meningiomas. there was no association demonstrated between either hsp expression variable and ec or ki-67 expression. there was also no association between the ec expression variables and ki-67. however, there was a significant negative association between hsp 72 extent (p 5 0.03) and area (p 5 0.04) with ei. in conclusion, hsp 27 and 72 expression was demonstrated in canine intracranial meningiomas but was not associated with ki-67 or ec expression. this study suggests that hsps may not have a significant role in the maintenance of canine meningiomas and so do not represent a novel treatment target for this group of tumors unlike canine glial cell tumors. however, hsp 72 may be involved in the pathogenesis of peritumoral edema in meningiomas and warrants further investigation. an extended release (xr) formulation of levetiracetam, a second generation antiepileptic drug, was recently approved for human use on a once daily basis. although levetiracetam is clinically effective for seizure control in dogs, it requires a three times daily administration. the potential benefits of the xr formulation include reduced daily dosing leading to improved compliance and relatively constant plasma concentrations. the aim of this study was to compare the pharmacokinetics of levetiracetam xr tablets with immediate release (ir) tablets following single dosing in dogs. five clinically and neurologically normal mixed breed dogs were used in a cross-over design. all dogs (mean body weight 25.9kg; range 23.2-30.5) had normal hematology, serum chemistry and urinalyses. following a 12 hour fast, each dog was administered oral ir levetiracetam (500 mg; mean dose 19.3 mg/kg; range 16.4-21.6). heparinized blood for drug analysis was taken from each dog prior to administration and 0.25, 0.5, 0.75, 1, 2, 4 and 8 hours after. blood was immediately centrifuged and supernatant plasma was stored at à801c until analysis. after a 4 day wash-out period, each dog was administered 500 mg oral xr levetiracetam and blood samples were taken at identical timings. plasma samples were thawed at room temperature before preparation by solid phase extraction for hplc analysis. reverse phase chromatographic separation was performed. levetiracetam and an internal standard were detected using ultraviolet spectroscopy at 205 nm. concentrations of levetiracetam were determined by peak area comparison to the internal standard. mean data were fit to a one compartment pharmacokinetic model with first order elimination and absorption and included a lag-phase for xr formulation. no adverse clinical effects were noted in any of the dogs. the auc associated with xr was 230 hr ug/ml, a 5.14 fold increase over that with ir (44.8 hr ug/ml). the absorption half-life was 3.2 hr with xr and 0.41 hr with ir, a 7.75 fold difference. the elimination halflife was 3.13 hr with xr and 2.19hr with ir, a 1.43 fold difference. the tmax associated with xr 5.01 hr and 1.22 hr with ir, a 4.21 fold difference. the cmax associated with xr was 18.5 mg/ml and 9.62 mg/ml with ir, a 1.92 fold difference. the plasma concentration of ir levetiracetam was not detectable at 8hr after administration whereas it was greater than 10 mg/ml at 8hr after xr administration. based on the auc data, there is an approximately 5 fold increase in bioavailability of the xr compared to the ir formulation. the cmax was approximately 2 times greater following xr administration and a high plasma level in excess of the suggested canine therapeutic concentration (5 mg/ml) for at least 8 hours. although specific dosing recommendations cannot be made from this data, the favorable pharmacokinetics of xr over ir suggests that single, daily administration could be efficacious. thoracic and lumbar vertebrae are frequently affected by fractures and or luxations in dogs following trauma. surgical repair is part of the emergency treatment described for this disorder but does not guarantee improvement of the associated clinical signs. multiple surgical repair techniques have been described but have not been compared in terms of their success and the factors associated with a positive outcome. the aims of this study were to retrospectively evaluate the effect of 3 different types of vertebral repair, injury type and injury location on outcome in dogs with thoracolumbar (tl) and lumbosacral (ls) spinal trauma. medical records were searched for dogs with radiographic evidence of a tl or ls vertebral fracture and or luxation (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) ; signalment, body weight and duration of disease were recorded. dogs were retrospectively scored neurologically (0-5; normal to plegic with absent pain perception) on admission and at re-evaluation following surgery. lesion location was classed as t3-l3 and l4-s3; dogs were evaluated as one group and as two separate groups with respect to outcome. a subset of lesions were classed as cord compression or not based on advanced imaging. three repair techniques were evaluated (i) pins and polymethylmethacrylate (pmma); (ii) screws and pmma; and (iii) spinal stapling. regression analysis was applied to test for an association between the type of surgery and a successful outcome (non-painful and ambulatory). simple bivariate analyses were performed to investigate for variables predictive of a successful outcome. fifty-nine dogs were included. twenty-eight dogs were classed as t3-l3 and 31 were l4-s3. there were 55 dogs with fractures and 43 with luxations; 23 dogs had both. thirty-one of 35 dogs evaluated had spinal cord compression. ten dogs were repaired with im pins and pmma, 18 dogs with screws and pmma and 31 dogs with spinal stapling. overall, there was a 78.7% success rate; there was no significant difference in outcome between the anatomic sites (p 5 0.5). all dogs initially graded as 1-3 pre-operation were classed as a successful outcome after at least one week following surgery; 79% of dogs initially graded as 4 (plegic with pain perception) were classed as successful recovery. one dog (12.5%) initially as graded as 5 (plegic with no pain perception) had a successful outcome. a low admission score was statistically predictive of a successful outcome (p o 0.0001). surgery type was not associated with a successful recovery (p 5 0.13). signalment, body weight, location of injury, injury type (fracture, luxation or both), presence of compression, and duration of disease did not predict outcome. from this study, the successful recovery of dogs following surgical fixation is high and is only dependent on the neurological score at the time of admission. the choice of surgical technique does not seem to influence outcome although a prospective study comparing two surgery types is warranted to further investigate this issue the results of which can be confounded by surgeon experience and variable follow-up. cranial thoracic intervertebral disc disease (ivdd) is extremely rare due to the presence of the intercapital ligament, although anecdotic data suggest german shepherd dogs (gsd) can share some predisposition for this disorder. the objective of the study was to retrospectively evaluate through mri if cranial thoracic ivdd is significantly more common in gsd compare to other large breed dogs. a search was done through database of the ontario veterinary college. any gsd were a spinal mri including t1-t10 spine was performed was recruited. a group of large-breed non-gsd was used as a control. in the midsaggital t2wi plane, three variables were assessed and graded for each intervertebral disc space t1-t9: spinal cord compression (scc), disc degeneration (dd), and herniation. wilcoxon sign rank test was used to assess if scores were different between groups. exact conditional logistic regression was used to determine whether any intervertebral disc space was a risk factor. 22 gsd and 46 large breed non-gsds were recruited. the gsd group had significantly higher scores than the non-gsd for scc, and herniation. regarding the individual intervertebral discs, in the gsd group t2-3, t3-4, t4-5 discs had significantly an increased risk for scc, and t3-4 for herniation. the results of this study show that gsd have a higher risk of cranial thoracic disc ivdd than other large breed dogs. that risk was higher in discs t2-t3, t3-4, and t4-5, particularly in t3-4. genetic and/or conformational factors, such as weakness of the intercapital ligament, may predispose gsd to this lesion. diskospondylitis is a common disease of the canine spine; however, few reports of mr imaging findings in dogs are available. the purpose of this study was to describe the signalment, clinical and mr imaging features in affected dogs. twenty-three dogs with a diagnosis of diskospondylitis based on clinical signs, mr imaging, and urine, blood, csf and/or intervertebral disk cultures were included. large breed dogs (4 25kg) accounted for 21of the cases. the mean age was 6.8 years with males and females equally represented. most dogs (15/23) were ambulatory with varying degrees of pain and paresis. mr imaging characteristics of 27 sites were reviewed. on t2w images, vertebral endplates were of mixed signal intensity (16/27) while the vertebral body was hypointense (11/27). the intervertebral disk space was hyperintense on t2w (16/27) and stir (14/15) images and mixed signal intensity (7/13) on t1w images. paravertebral soft tissue hyperintensities were noted on 10/27 t2w and 12/16 stir images. contrast enhancement occurred at 12/19 endplates and 15/19 intervertebral disk spaces. only 4/18 vertebral bodies and 7/18 parvertebral soft tissues contrast enhanced. intramedullary spinal cord t2w hyperintensity was noted at 10/23 sites. spinal cord or cauda equina compression occurred at 22/27 sites. based on the spearman correlation coefficient, a significant direct correlation was found between the degree of spinal cord or cauda equina compression and the patient's neurologic status (p 5 0.0011). the incidence and severity of spinal cord compression in canine diskospondylitis may have prognostic value and may have been previously underestimated using other imaging modalities. hemilaminectomy and pediculectomy are both well described and commonly utilized techniques to access the spinal canal. these procedures are most often performed to approach a compressive lesion, such as intervertebral disc disease and neoplasia, the goal being adequate visualization of the spinal canal and access to the offending lesion. a proposed benefit of pediculectomy is preservation of the articular facets and thus better maintaining stability of the vertebral column, but at the cost of reduced access to the spinal canal. the purpose of this study was to describe standardized anatomical limits of each technique and report any observed differences that could be considered during presurgical planning. ten canine cadavers had both procedures performed on opposite sides to access the t11-12, t13-l1, and l2-3 spinal canal. measurements were obtained after performing a computed tomography study of the spine and recorded from the transverse slice most representative of the defect. the surgical technique, vertebral site, and side of vertebral column were compared with the mean spinal canal and defect height using a covariate model. dorsal and ventral remnant lamina heights were also compared. the height of the defect relative to the spinal canal was 76-87% with hemilaminectomy and 58-75% with pediculectomy. the observed difference in defect height of 12-18% (p o 0.0001) and varied with spinal canal height. dorsal remnant lamina height was 0.9-2.4% of spinal canal height with hemilaminectomy and 7-19% with pediculectomy. ventral remnant lamina height ranged from 1-2% and 0.6-2.3%, respectively, though the difference was not statistically significant. while a larger defect is expected with a hemilaminectomy procedure, our results demonstrate that this difference increases with increasing spinal canal height. interestingly, the proportion of exposed spinal canal decreases with increasing canal height for both procedures. the difference in defect height between techniques was due to greater removal of the dorsal spinal canal, possibly making the hemilaminectomy technique better suited for more dorsal lesions, while no statistically difference in access to the ventral canal is observed. no effect of vertebral site was detected. of note was the involvement of articular facets in half of the pediculectomy defects, involving an average of 22% of the articular facet height. this result questions the suggested benefit for the vertebral stability, but further biomechanical studies would be required. low level laser therapy (lllt) is a treatment used in human and veterinary medicine for a variety of clinical syndromes. some uses in human medicine include acute pain associated with osteoarthritis, rheumatoid arthritis, tendonitis, tmj disorders, chronic joint disorders, and wound healing. research is currently on-going to determine the adequate wavelengths to promote effective treatment results with lllt in these conditions. it is purported that lllt acts via the mitochondria to increase cellular metabolism promoting wound healing and a decrease in pain and inflammation. in this study, we hypothesized that dogs treated with lllt in conjunction with hemilaminectomy would display quicker recovery times regardless of the presence or absence of deep pain sensation. seventeen dogs (9 dachshunds, 2 chihuahuas, 2 french bulldogs, 2 lhasa ahpsos, and 1 each of a pembroke welsch corgi, and a miniature poodle) were selected and divided into two groups. the dogs ranged in age from 2 to 11 years old, weighed between 7 and 33 pounds, and underwent hemilaminectamies after acute onset of paraplegia secondary to intervertebral disc disease (surgically confirmed). one group received laser treatments on days 1 through 4 of hospitalization. the second group did not receive lllt, but followed the same peri-operative medication protocol. the laser used in this study was an erchonia laser model pl5000 (635nm). the hertz setting was similar for each patient using the previously established protocol for intervertebral disc disease (ivdd) with pulse rate ranging from 9hz to 1151 hz. all dogs received advanced imaging pre-operatively with myelogram or mri. results of the study revealed that treatment with lllt of 635nm wavelength did not shorten or improve recovery times for dogs with acute onset paraplegia secondary to ivdd after hemilaminectomy procedures. dogs that showed recovery to ambulation at the two week recheck were consistently dogs that were deep pain positive on presentation. a lengthened recovery time or no recovery was seen in the majority of those dogs with absent deep pain on presentation as has been revealed historically in past studies. lllt did not appear to have an effect on this result. however, there are few data describing normal glucose uptake of the canine brain for comparison with suspected or confirmed disease. thus the purpose of this study was to assess the normal distribution of fdg uptake of canine brain structures using a high-resolution research tomography-pet and 7 t-magnetic resonance imaging (mri) fusion system. fdg-pet and t2-weighted mr imaging of the brain were performed on 4 healthy laboratory beagle dogs. acquired pet and mr images were automatically co-registered by the image analysis software. on mr images, regions of interest (roi) were manually drawn over 48 intracranial structures, including 6 gross structures (whole brain, telencephalon, diencephalon, mesencephalon, dorsal metencephalon, ventral metencephalon and myelencephalon). a standard uptake value (suv) and relative suv ratio (rsuv 5 suv of roi/suv of whole brain) were calculated for each roi. 7 t-mr images compensated the low anatomical resolution of pet qj;by proving good spatial and contrast resolution for the identification of the clinically relevant brain anatomy. among gross structures, mesencephalon and ventral metencephalon had the highest (suv: 4.17 ae 0.23; rsuv: 1.12 ae 0.03) and the lowest (suv: 3.34 ae 0.35; rsuv: 0.90 ae 0.06) fdg uptake respectively. when suvs were calculated on 42 detailed regions, rostral colliculus and corpus callosum had the highest (suv: 6.00 ae 0.16; rsuv: 1.62 ae 0.05) and the lowest (suv: 2.81 ae 0.12; rsuv: 0.76 ae 0.06) value respectively. these data acquired from normal dog brain will be used in clinical neurology to investigate various intracranial diseases such as inflammation, neoplasm and behavioral disorders. degenerative lumbosacral stenosis (dlss) is a multifactorial condition affecting predominantly large breed dogs. the combination of stenosis and compressive neuropathy cause lumbar pain, lameness and neurologic dysfunction. previous reports describe urinary and fecal incontinence in severely affected dogs. the objectives of this retrospective case series were to describe the clinical signs associated with dysuria and eventual diagnosis of dlss in dogs, and to describe factors associated with regained micturition following prompt diagnosis and treatment. medical records from the university of georgia and the university of missouri between 1995 and 2009 of 11 dogs were reviewed. inclusion required observation of dysuria, urine retention, absence of structural lower urinary tract disease and concurrent presumptive diagnosis of dlss. dysuria was defined as inability to initiate or sustain a urine stream. urine residual volume was evaluated postvoiding. dysuria was further evaluated using urethral contrast studies, urodynamic testing (urethral profilometry (4) and cystometry (4)), ultrasonography (5), and urine culture (8). presumptive diagnosis of dlss was based on imaging using plain radiography and epidurography (8), computed tomography (1) or magnetic resonance imaging (2). breeds represented included the german shepherd dog (n 5 3), golden retriever (n 5 2), burnese mountain dog (n 5 2), and 1 each labrador retriever, weimaraner, rottweiler and mixed-breed. all dogs were male. 8 were intact at onset of clinical signs. median body weight was 38.5 kg (range 29.5-46) and median age was 5 years (range 2-10). median duration of clinical signs prior to admission was 2 months (range 0.25-12). other pertinent presenting clinical signs included dyschezia (2), fecal incontinence (4), general proprioceptive ataxia (2), weakness (2), and difficulty rising (1). physical examination findings included pelvic limb muscle atrophy (2) and prostatomegaly (1). abnormal neurologic examination findings included postural reaction deficits (6), hyporeflexia (4), decreased tail tone (3) and lumbosacral hyperesthesia (6). neurologic examination was normal in 3 dogs. dorsal laminectomy was performed and diagnosis confirmed in 9 dogs; recovery was monitored for a median of 5.5 months (range 0.25-9). three of the 9 dogs (33%) regained normal micturition within 0.25-1.5 months of surgery. though not statistically significant, dogs that regained micturition tended to have a shorter duration of clinical signs (median 0.25 months, range 0.25-2) versus dogs that remained dysuric (median 5 months, range 2-12). two of the 3 dogs that regained micturition were neutered at the onset of clinical signs, but only 1of 6 dogs that remained dysuric was neutered. signs improved in all dogs with postural reaction deficits and decreased tail tone. hyperesthesia resolved in 5 of 6 dogs (83%) and fecal continence returned in 2 of 4 dogs (50%). these findings suggest that following prompt diagnosis and surgical decompression, normal micturition could be regained in dlss affected dogs presenting with signs of dysuria. glycogen storage disease type ia (gsdia; von gierke disease) is an inherited metabolic disorder resulting from a deficiency of glucose 6-phosphatase-a (g6pase). previous reports indicate that clinical manifestations of gsdia occur only in individuals with homozygous expression of a p.i121l mutation. heterozygote dogs (het) have been previously reported to exhibit an overall normal outward phenotype. the purpose of this report is to briefly describe some differences that have been observed between het and homozygous wild type (wt) dogs. a colony of dogs at the university of florida contains a mix of affected, wt, and het individuals. in the course of studies designed to determine the effectiveness of gene therapy for correction of gsdia in dogs, both wt and het dogs have been utilized as controls. available information about body weights, clinical pathology tests, fasting studies, and liver biopsies was retrieved from records for both wt and het dogs and compared. although birth weights are similar, het dogs have a slower average rate of weight gain than wt dogs and this difference is especially prominent during the first few months of life (figure 1) . in contrast to affected dogs, both wt and het dogs are able to maintain normal blood glucose concentrations for up to 10-12 hours of fasting, however, after longer fasts of 12-17 hours, het dogs have lower glucose and higher lactate concentrations (table 2 ). in addition, liver biopsy samples from het dogs had greater apparent levels of glycogen suggested by pas staining than did samples from wt dogs, and this correlated with the results of proton magnetic resonance spectroscopy which demonstrated 2.9 times greater glycogen content in a liver biopsy sample from a het dog compared to a sample from a wt dog. together, these findings suggest that the level of g6pase activity in heterozygote dogs does not provide a completely normal physiological, biochemical, or histological phenotype as previously reported. the glucokinase gene (gck) encodes an enzyme involved in cellular glucose-sensing mechanisms in pancreatic beta cells and hepatocytes. gck mrna is present in feline pancreas but the gene is not expressed in feline liver. hepatic gck expression is abundant in omnivores so its absence may reflect an evolutionary adaptation of strict carnivores, like feline species. we hypothesized speciesspecific features in the gck hepatic promoter may underlie the gene expression pattern observed in cats. the putative feline gck (fgck) promoter region was located using bioinformatic software to identify homology with human gck (hgck). genomic dna from a dsh cat was subjected to direct sequencing using a series of pcr reactions with speciesspecific primers. dna clones thus obtained were aligned to generate the feline sequence. direct sequencing yielded 8.9 kb of genomic dna sequence with high homology with sequences (acbe01359231, acbe01359221) archived in the feline genome project. the feline sequence had six regions homologous with non-coding regions of hgck; four of these conserved regions are upstream of the putative fgck start. a 0.8 kb segment immediately upstream of feline hepatic exon 1 is not present in hgck. the 0.8 kb insert is the reverse complement of a conserved sequence located downstream of exon 1 in feline and human sequences. in conclusion, the putative hepatic promoter of fgck shares extensive homology with the hgck promoter but contains a 0.8 kb insert not found in hgck. functional studies are needed to confirm the role of the unique insert in regulation of fgck gene expression. deuterium oxide (d 2 o) dilution has been proposed for quantifying body water content, but remains difficult to perform routinely. the objective of this study was to assess if the volume of distribution (vd) of creatinine could be proposed as an alternative in dogs for such a measurement. creatinine and d 2 o vd were measured before (c) and after induction (o) of obesity (by giving an hypercaloric diet (6100 kcal/ kg) for 6 months) in six healthy adult beagle dogs. creatinine (40 mg/kg) and d 2 o (200 mg/kg) were simultaneously injected by bolus iv. blood was collected before administration and then at 5, 10, 30, 60, 120, 180, 240, 360, and 480 min post-injection (creatinine), and 10, 30, 60, 90, 120, 150, 180 and 240 min (d 2 o) . plasma concentrations of both markers were determined. vd was calculated using pharmacokinetic equations. the body weight increased from 10.7 ae 0.6 (c) (mean ae sd) to 15.8 ae 1.0 kg (o). d 2 o vd decreased from 636 ae 18 (c) to 468 ae 19 (o) ml/kg. similarly, creatinine vd decreased from 624 ae 31 (c) to 420 ae 25 (o) ml/kg. the individual difference between creatinine and d 2 o vd (expressed in % of d 2 o vd) ranged from à9.5 to 1.0% (c) and from à17.3 to à7.1% (o). in conclusion, creatinine vd provides a good estimate of d 2 o vd in both normal and obese conditions. a 16 wk double blinded study was conducted comparing the affect of two foods on mobility in dogs. all work was approved by an iacuc. 53 healthy beagle dogs (7-15 years old, mixed gender) were used. 33 affected (a) and 20 non-affected (na) dogs were identified based on orthopedic examination and radiography as having or not having evidence of naturally occurring joint pathology (presence of osteophytes, dysplasia, effusion, pain on manipulation etc) in one or more joints. a and na dogs were evenly distributed between two locations. foods had nutrient profiles adequate for maintenance according to the 2009 aafco official publication. the test food contained greater amounts of methionine, manganese, carnitine, vit. e,, vit. c, alpha linolenic acid (ala), and eicosapentaenoic (epa) acid: the food provided 388 mg n3 fatty acids and 847 mg n6 fatty acids per 100kcal. all dogs were fed the control food for 4 wks followed by a 12 wk feeding period where 17 a and 10 na dogs consumed the test food and 16 a and 10 na dogs the control. blood and urine were collected at weeks 0, 4, 8 and 12 and analyzed for serum fatty acids and urine thromboxane:creatinine ratios were determined. evaluators in this study were different than those making the original diagnosis and so were blinded as to treatment and diagnosis. orthopedic exams were performed by two veterinary surgeons at each site on weeks 0, 4, 8 and 12. the same two evaluators examined the same dogs throughout. the data was evaluated for the difference between a and na dogs and between foods with age, gender and location as covariates. body weight, disease status, age and gender were blocked. analysis included anova repeated measures mixed procedure (sas version 9.0) to determine treatment effects over time.serum epa was greater and arachidonic acid lower at weeks 4, 8 and 12 in the test food fed dogs (p o 0.05). urine thromboxane:creatinine ratios were decreased in the a dogs fed the test food compared to the a dogs fed the control food at 12 wks (p o 0.05). lameness score was significantly improved (p o 0.05) within and between groups of dogs fed the test food. a significantly greater proportion of a dogs fed the test food had improvement in total het (n511) blood glucose (mg/dl) 78 (ae17) 64 (ae13) blood lactate (mmol/l) 1.1 (ae0.5) 1.7 (ae1.1) joint score, lameness, functional disability and overall assessment score at 12 wks compared to a dogs fed the control food. 69% of a dogs had an improved overall assessment score on the test food after 4 wks and at 12 wks compared to 40% at 4 wks and 27% at 12 wks of a dogs consuming the control food. this study shows that a food with moderate amounts of added linolenic acid and epa can have a positive impact on systemic inflammation and mobility in 4-12 weeks. a similar abstract will be presented at the orthopedic research society meeting in january 2011 to an audience largely of orthopedic researchers interested in human orthopedics. fat is an important dietary component, serving both as a source of energy and as a supplier of essential fatty acids (fa). medium-chain triglycerides (mct) contain intermediate length fa that do not rely on l-carnitine for transport across the inner mitochondrial membrane, bypassing this rate-limiting step in fa oxidation. longchain (n-3) polyunsaturated fatty acids (pufa) from fish oil (fo), and in particular eicosanoids derived from eicosapentaenoic acid (epa), may protect against excessive inflammatory reactions, which may be exacerbated by eicosanoids derived from (n-6) arachidonic acid (aa). this study investigated the effects of adding mct:fo and l-carnitine to a control diet (prescription diet s k/d s ) on lean body mass, and serum fa and metabolites. forty healthy beagles (3.1 to 14.8 y) were fed one of three foods (n 5 13 to 14 dogs each) for 6 mo. the study protocol was reviewed and approved by iacuc, hill's pet nutrition, inc. all foods were complete, balanced, and sufficient for maintenance of adult dogs; and had similar concentrations of moisture, protein, and fat (approx. 7.4%, 14.0%, 18.1%, respectively). composition of serum fa was determined by gas chromatography of fa methyl esters. metabolomic profiles of serum samples were determined from extracted supernatants that were split and run on gc/ms and lc/ ms/ms platforms, for identification and relative quantification of small metabolites. body composition was determined by dual energy x-ray absorptiometry. serum concentrations of lauric and myristic fa increased; epa and dha increased in a dose-dependent manner; and aa decreased in dogs fed treatment food 3 (proc-mixed procedure in sas; all p 0.05) when compared to dogs fed treatment foods 1 or 2. serum concentrations of acetylcarnitine and succinylcarnitine increased, indicating lcarnitine incorporation, in dogs fed treatment foods 2 and 3. thus, a diet enriched with mct:fo significantly altered serum fa composition, enriching (n-3) pufa and lowering aa concentrations. there was no change in lean body mass for any of the diets compared to baseline values, and no difference between treatments, showing that all three treatment foods met protein requirements. ten owned dogs, obese for more than 12 months (body condition score [bcs] of 9; fat mass [fm] 5 45.7 ae 1.51%) were studied. these dogs had their weight reduced by 20% (bcs 5 8; fm 5 33.5 ae 1.92%; p o 0.001) being designated weight reduced (wr) group and then were fed to maintain constant body weight during 150 days (bcs 5 8.2; p 5 0.6), designated maintenance (main) group. a control (ct) group of 10 beagles was also included (bcs 5 4.5; fm 5 18.3 ae 1.38%; p o 0.01). in all groups the glucose postprandial response test was performed after 12 hours of fasting. blood samples were taken prefeeding and after 5, 10, 15, 30, 45, 60, 120, 180, 240, 300 and 360 minutes of the consumption of cooked rice enough to the ingestion of 6g of starch/kg body weight. tnf-a and il-6 were dosed in milliplex tm map panel, insulin and leptin by radioimmunoassay. statistical analysis included paired or non-paired t-tests and wilcoxon (p o 0.05). the regimen normalized meal glucose response, the area under the curve (auc) of glucose for wr was lower than for obese (p o 0.05) and similar to main and ct (p 4 0.05). insulin secretion did not normalize immediately, as obese and wr exhibited similar auc of insulin and higher values than for ct (p o 0.05). main, however, presented similar auc of insulin than ct, with lower values than obese and wr (p o 0.01), suggesting that dogs require some time to adapt their metabolism. leptin, tnf-a, and il-6 presented significant reductions after weight loss (p o 0.05), without differences between wr, main and ct (p 4 0.05), suggesting an improvement of the pro-inflammatory state consequent to obesity. studying food base excess (be) modification, methionine intoxication was described. in a basal kibble dog diet (be 5 305 meq/kg; 1.98 g/kg of s) two dosages of ammonium sulphate and methionine was added, resulting in diets with be of 135 meq/kg (4.26 g/kg of s) and à51 meq/kg (6.65 g/kg of s), or be of 142 meq/kg (4.68 g/kg of s) and 4 meq/kg (7.49 g/kg of s), respectively. a 2 â 2 factorial plus a control diet design, resulting in five treatments, and 32 adult health beagle dogs were used, in a completed randomized design with six dogs per diet. a 5-d adaptation phase followed 3-d of total urine collection (in bottles with 100 mg of thymol). urine were pooled by dog and analyzed for density, volume and ph. food macroelements were determined by standards methods (aoac, 1995) and used for be calculation. dog's acid-basic status was studied by blood gas analysis of venous blood, at 8:00h (pre feeding) and 6 hours after meal. a dose-dependent reduction of urinary ph was verified for both compounds (p o 0.01). blood bicarbonate (r 5 0.98; p o 0.01), and blood base excess (r 5 0.75; p o 0.01) were highly correlated with food be. acidemia and reduced blood be were verified in diets with be close to zero (higher dose of both compounds, or 4 6.6 g/kg of s), resulting in daily or each other day vomiting episodes in the dogs. ataxia, seizures, and vomiting were previously describe in dogs fed 47 g/kg of methionine, but our results suggest that a much lower value (27.7 g/kg) was toxic and that the safe upper limit should be between this value and 15.7 g/kg (the lower evaluated dose). in people with chronic kidney disease and heart failure, obesity is associated with longer survival times. this association, called the "obesity paradox," also has been recognized in dogs and cats with heart failure. excess weight appears to modulate the serious deleterious effects of muscle loss in these diseases. the purpose of this study was to determine the effects of body condition and body weight changes in dogs with naturally-occurring chronic kidney disease (ckd). dogs diagnosed with ! iris stage ii ckd between 2008 and 2009 at iowa state university and tufts cummings school of veterinary medicine were eligible for the study. dogs o 1 year of age and those with acute renal failure or suspected congenital renal diseases were excluded. medical records were reviewed using a standardized data form, and data were collected for initial body weight and body condition score (bcs, 1-9 scale), clinicopathologic values, changes in body weight and bcs, comorbidities, and treatments. dogs were classified as underweight (bcs 5 1-3), moderate weight (bcs 5 4-6), or overweight (bcs 5 7-9). a change in body weight was defined as 4 0.2 kg. survival times were determined for all dogs that were discharged from the hospital and lived 4 1 day. associations between survival and bcs or body weight changes were analyzed using cox proportional hazards models. one hundred two dogs were enrolled in the study. at the time of diagnosis, 21 dogs were classified as iris stage ii, 57 dogs were stage iii and 24 dogs were stage iv. median body weight at baseline was 18.7 kg (range, 2.4-50.1 kg). for dogs with body condition scores recorded (n 5 74), 13 were underweight (18%), 51 were moderate (69%), and 10 were overweight (14%). for dogs that had at least two body weights recorded over the course of their disease, 22 gained weight, 46 lost weight, and 10 had no change in weight. changes in body weight were not associated with survival; however, bcs at the time of diagnosis was significantly associated with survival. dogs classified as underweight had a significantly shorter survival time compared to both moderate (p o 0.001) and overweight dogs (p o 0.001). these results suggest that body condition is an important consideration in dogs with acquired chronic kidney disease. further studies are warranted to evaluate the relationship between obesity and longer survival in dogs with ckd. protein restriction is the cornerstone of dietary management of kidney disease. the national research council recommends 20% crude protein and the american association of feed control officials (aafco) recommends a minimum of 26% crude protein for maintenance for healthy adult cats. protein requirement is unknown for adult cats with kidney disease. most commercially produced cat foods for adult maintenance contains 30% or more crude protein on a dry matter basis. a typical therapeutic food for cats with kidney disease contains about 28% crude protein. the objective of the present study was to investigate whether dietary crude protein at 28.5% would be adequate for the maintenance for adult cats with impaired kidney function. seven adult cats, 3 female and 4 male, with age ranging from 5 to 13.7 years old (mean: 8.1 years) were used in the study. all cats had elevated serum creatinine concentration (4 1.6 mg/dl, range: 1.61-2.10 mg/dl) and reduced glomerula filtration rate (mean: 32% reduction; range: 12-60% reduction) during the study. they did not have other systematic diseases, e.g., hyperthyroidism, at the beginning of the study. cats were fed an expanded dry food made with ingredients commonly used in commercial dry cat foods. the food contained 28.5% crude protein (chemical analysis) and 4366 kcal/kg (calculated) on a dry matter basis, or 65.3 g protein/1000 kcal. each essential amino acid in the food was at least 130% of that recommended by aafco. other nutrients in the food also exceeded aafco's recommendations for maintenance for adult cats. cats were fed the food for 30 weeks. lean body mass (dual x-ray absorptionmetry; hologic, hologic, inc, ma) and serum albumin concentration were measured periodically to monitor protein status of cats. the average lean body mass (mean ae sd) was 3.63 ae 0.82 kg, 3.72 ae 0.85 kg, 3.74 ae 0.84 kg, and 3.75 ae 0.89 kg in weeks 3, 11, 17, and 30 of the study, respectively. paired t-test did not detect statistical difference (p 4 0.05) when comparing the lean body mass in weeks 3 versus weeks 11, 17, and 30, respectively. serum albumin concentration were within the normal reference range during the study (mean ae sd: 3.04 ae 0.35%, 2.80 ae 0.35%, 3.04 ae 0.32%, and 3.07 ae 0.40% in weeks 3, 11, 17, and 30, respectively) . these data show that 28.5% dietary crude protein in a dry food with 4366 kcal/kg on a dry matter basis, or 65.3 g protein/1000 kcal, is adequate for maintenance for cats with impaired kidney function. in humans, several disease conditions exist that involve abnormal patterns of polyunsaturated fatty acids and similar abnormalities may be present in companion animals. indeed there have been reports of decreased plasma arachidonic acid and reduced delta-6 desaturase activities in dogs with atopy and other skin disorders. the present study investigated serum fatty acid profiles in dogs and cats presented to the texas a&m university veterinary teaching hospital, clinical pathology laboratory over the past one year period. results were compared with normative data generated among dogs and cats from earlier feeding studies. sera used were residual samples submitted to the laboratory for other diagnostic procedures and stored frozen for no more than 2 months after collection. the samples were grouped according to presenting disorders involving liver, kidney, digestive, and cardiac diseases. total lipids were extracted using chloroform:methanol (2:1 v/v) and fatty acid methyl esters were prepared for capillary gas chromatography. relative percentage distribution of individual serum fatty acids for each animal were then compared with average normative serum phospholipid fatty acid values (dogs, n 5 44; cats, n 5 29) by calculating the ratio of the value in the diseased individual to the normal mean value and used as an index of normalcy. normalcy ratios were then plotted on a logarithmic scale with normal at 1.0. the ratio was then compared to changes greater than 3, 2 and 1 standard deviations of the normal mean values. in this way a graphical presentation of resultant values was obtained. although the animals had been fed various commercial diets and some home-prepared foods, a number of noteworthy patterns emerged from this analysis. dogs showed increased linoleic acid, decreased arachidonic acid, increased total monounsaturated and decreased saturated fatty acids at p o 0.001; oleic acid was increased at p o 0.01. remarkably, these findings were similar for all canine disease categories evaluated (n 5 30, heart; n 5 17, kidney; n 5 28, liver, and n 5 28 digestive disorders). in cats, a slight decrease in arachidonic acid and large decrease in 22:0 was observed but only in heart disorders. by contrast, modest elevations of arachidonate were observed in kidney, liver, and digestive disease groups but at p o 0.01. sample sizes of the feline sera were considerably smaller (range of 4-13 per group). a limitation of this analysis is that variability of normal data may exist depending on diet fed making comparisons less reliable however, these preliminary data suggest that metabolic diseases of dogs may depress plasma arachidonic acid independent of diet fed suggesting either reduced conversion from linoleic acid or increased utilization of arachidonate for eicosanoid production during times of metabolic stress. conversely, in cats, increases in arachidonic acid may be associated with diet arachidonate or other mechanisms. additional studies to verify these findings are warranted. the objective of this study was to determine whether or not lalanyl-l-glutamine (ala-gln) supplementation in dogs with parvoviral enteritis improves the survival rate and ameliorates clinical signs without side effects. this randomized, double-blinded, placebo-controlled clinical trial included 39 client-owned dogs. the dogs were randomly assigned into two groups and administered ala-gln solution (dipeptiven; 0.4 g/kg) or an equivalent volume of placebo orally twice a day. all of the dogs (ala-gln group [n 5 20] and placebo group [n 5 19]) received standard treatment while hospitalized and were monitored daily according to a clinical scoring system and diagnostic evaluation for 11 days. among the 39 dogs, 17 (ala-gln-treated group [n 5 8] and placebo group [n 5 9]) were vaccinated and 22 (ala-gln-treated group [n 5 12] and placebo group [n 5 10]) were not vaccinated. the population consisted of 29 purebreds and 10 mixed breed dogs, with a mean age of 12.2 ae 2.2 weeks. the survival data were compared statistically by means of a log-rank test for the kaplan-meier survival curves. the clinical scores of ala-gln-treated dogs improved significantly relative to the placebo group. there was a significant difference between the two groups in the survival distribution (p 5 0.038); specifically, 3 of the ala-gln-treated dogs (15.0%) died, whereas 8 of the dogs in the placebo group (42.1%) died. no side effects were associated with the administration of ala-gln. these results suggest that the oral administration of ala-gln is effective in improving clinical signs and survival rate in dogs with parvoviral enteritis. bleeding disorders, thrombocytopenia and alterations in platelet function have been documented in humans receiving lipid-containing parenteral nutrition formulations. despite a lack of evidence in the veterinary literature, it is believed that parenteral lipids are contraindicated in critical illness when the development of bleeding disorders is likely. the objective of this study was to determine if there is an in vitro effect on platelet function and thromboelastography (teg) in normal dogs with varying concentrations of a 20% soybean oil emulsion (intralipid s ). twelve clinically healthy dogs were used for this study. whole blood platelet aggregation, using adp and collagen agonists, was measured using multiple electrode aggregometry in hirudinated blood with final lipid concentrations of 0, 1, 10, and 30 mg/ml. the teg parameters r, k, a-angle, and maximum amplitude (ma) were evaluated from citrated whole blood with equivalent final lipid concentrations as platelet aggregation. there was no significant difference between groups with collageninduced platelet aggregation. there was a significant increase in the area under the curve (auc) with adp-induced aggregation at a lipid concentration of 30 mg/ml (p 5 0.027). the ma was significantly reduced at both the 10 mg/ml (p o 0.001) and 30 mg/ml (p o 0.001) lipid concentration. there was no statistical difference between groups evaluating the other teg parameters. while platelet aggregation appeared enhanced at the highest concentration evaluated, this concentration is not clinically relevant. the reduction in ma seems discordant but both fibrinogen and platelets contribute to the ma. therefore the higher lipid concentrations may be interfering with fibrinogen kinetics or fibrinogenplatelet interaction. in vivo studies are indicated to determine if any of these changes are clinically significant. rosiglitazone is a peroxisome proliferator-activated receptor gamma (pparg) agonist and an fda-approved anti-diabetic agent in humans that has been investigated for its ability to reduce tumor cell growth. specifically, the combination of rosiglitazone and carboplatin has demonstrated enhanced tumor control. the purpose of this study was to determine the peak plasma concentrations and side effect profile of rosiglitazone after oral administration in dogs with spontaneously occurring cancer. all dogs received carboplatin intravenously concurrently with oral rosiglitazone. ten cancer-bearing dogs with normal pre-treatment hepatic and renal function were enrolled. complete pre-treatment hematological and biochemical parameters were available in ten dogs and post-treatment parameters in nine dogs. peak plasma concentrations varied with dose and ranged from 150.3-959.4 ng/ml and occurred between 30 minutes and 4 hours post administration and rapidly declined after the peak. the dose limiting toxicity was hepatic at a dose of 9 mg/m 2 . there was one grade iii, two grade i alt, and one grade iii ast elevations noted. no changes in total bilirubin, alkaline phosphatase, or ggt values were noted. blood glucose values remained within normal limits. mild, self-limiting gastrointestinal and hematologic toxicities were observed when rosiglitazone was administered in combination with carboplatin. based on this study, the recommended dose of rosiglitazone in cancer-bearing dogs with normal hepatic function is 6 mg/m 2 orally once daily. side effects of the combination appear similar to side effects noted with carboplatin alone. further study is needed to determine efficacy of this combination and if more frequent dosing is required to maintain plasma concentrations. carboplatin has shown little activity as a single agent for the treatment of canine transitional cell carcinoma (tcc). however, gemcitabine has shown synergism with carboplatin in human cell lines. the purpose of this study was to evaluate the activity of gemcitabine against canine tcc cell lines alone or in combination with carboplatin. we hypothesized that gemcitabine in combination with carboplatin would have synergistic effects in vitro. the results of this study could provide a rationale for treatment of canine tcc with the combination of these drugs. tcc cell lines tcc-kiss, tcc-knapp-js, tcc-axa, tcc-hxc, and tcc-sh were treated with gemcitabine, carboplatin, or the combination. cell proliferation was assessed using cyquant assay, cell cycle was evaluated using propidium iodide staining, and apoptosis was assessed by measuring caspase-3/7 activation. synergy was quantified by combination index analysis using compusyn software. treatment of canine tcc cell lines with carboplatin or gemcitabine decreased cell proliferation, induced cell cycle arrest, and apoptosis. when tcc cell lines were treated with gemcitabine and carboplatin in combination at a therapeutically relevant concentration (gemcitabine o 100um, carboplatin o 250um), a significant decrease in cell proliferation was observed compared to gemcitabine or carboplatin alone, and the drug combination was synergistic in 3 of 5 cell lines, and additive in the remaining 2 lines. gemcitabine exhibits biologic activity against canine tcc cell lines and carboplatin combined with gemcitabine exhibits synergistic activity at biologically relevant concentrations. our results support further evaluation of these drugs in dogs with tcc to determine the clinical efficacy of this combination. metronomic chemotherapy has been shown in murine models and humans to improve tumor control by inhibiting tumor angiogenesis and suppressing regulatory t cells (treg). treg are a subset of t lymphocytes demonstrated to be increased in humans and dogs with cancer and are thought to suppress cellular immune responses against tumors. the purpose of this study was to determine whether metronomic cyclophosphamide therapy depletes treg and/or exhibits antiangiogenic activity in dogs with soft tissue sarcoma. client owned dogs with histologically confirmed grade i or ii soft tissue sarcoma were administered cyclophosphamide at 12.5 mg/m 2 or 15 mg/m 2 orally once daily for 28 days. whole blood and tumor biopsies were obtained on days 0, 14, and 28. flow cytometric analysis of blood was performed to assess changes in t lymphocyte subsets, including cd4 1 and cd8 1 cells as well as cd4 1 foxp3 1 treg. tumor microvessel density (mvd) was assessed by performing immunohistochemistry for cd146. five dogs were enrolled in the 12.5 mg/m 2 /day dose cohort and six dogs were enrolled in the 15.0 mg/m 2 /day dose cohort. in patients that received cyclophosphamide at 12.5 mg/m 2 /day, the mean number of treg decreased from day 0 to 28 but there was no change in the mean percentage of treg or mvd. for patients that received 15.0 mg/m 2 /day, both the mean number and percent of treg as well as mvd decreased over the 28 day time period. cyclophosphamide at 15.0 mg/m 2 /day or greater selectively depletes treg and inhibits angiogenesis in dogs with soft tissue sarcoma. arsenic trioxide (ato) is used to treat leukemias, multiple myeloma, and relapsed lymphoid malignancies in humans; its use has not been explored in veterinary oncology. prior therapy with glucocorticoids decreases likelihood and duration of remission for dogs with lymphoma treated with chemotherapy. we hypothesized that ato will re-sensitize glucocorticoid-resistant canine lymphoma cells to glucocorticoid-induced apoptotic death. the osw canine lymphoma cell line was cultured with 200 um dexamethasone. remaining viable dividing cells were considered resistant. resistant cells were exposed to 0.01 um and 0.02 um of ato without dexamethasone, after which cells were washed and re-exposed to 200 um dexamethasone. after 24, 48 and 72 hours of dexamethasone exposure, cells were counted using trypan blue stain. apoptosis was assessed by tunel assays on cytospin preparations collected at 24, 48, and 72 hours from ato-exposed and control groups. statistical analysis was performed using 1 way anova and tukey's test. the proportion of dead cells increased over time in both 0.01 um and 0.02 um ato exposed groups. the proportion of dead cells was greater for 0.01 um ato (p o 0.001) and 0.02 um (p o 0.001) groups compared to control. apoptosis increased with increasing ato concentration and duration of dexamethasone exposure compared to control. these results support the effectiveness of ato at re-sensitizing glucocorticoid-resistant canine lymphoma cells to apoptotic death following re-exposure to glucocorticoids. ongoing gene expression studies aim to elucidate this mechanism. additional studies to determine if this effect is seen with other chemotherapeutic agents are warranted. lymphoma is the most common hematopoietic tumor of dogs. protein disturbances may be associated with this disease including monoclonal gammopathies in a low percentage of cases. serum protein electrophoresis (spe) is routinely used to aid diagnosis of various canine diseases including lymphoma when total protein concentration is elevated. the purpose of this study was to compare spe changes in lymphoma patients without elevated total proteins with a population of healthy dogs. agarose gel electrophoresis was performed on residual serum from 17 healthy control dogs and 21 untreated dogs with multicentric lymphoma (stage iii -v) after measuring total protein (tp) using the biuret method. densitometric traces of the protein bands were obtained using computer software (totallab 100) and the albumin, alpha-1, alpha-2, beta-2 and gamma globulin subfractions were identified by visual inspection. the total protein concentration, the number of subfractions and the relative and absolute protein subfraction concentrations were then compared statistically between the two populations. in lymphoma dogs, tp, absolute albumin, beta-2 and gamma globulin concentrations and both relative and absolute concentrations of the alpha-1 globulins were significantly lower however relative and absolute alpha-2 globulin concentrations were significantly elevated. no monoclonal gammopathies were identified in any of the dogs and not every patient with lymphoma had the above changes in their electrophoretogram. this study has demonstrated that significant changes occur in the albumin and globulin fractions of canine lymphoma patients despite no obvious increase in tp. further investigation is required to identify the proteins responsible for these changes. it is well known that immunophenotype has a prognostic value for the outcome of canine lymphoma, with t-cell lymphomas having a worse prognosis than b-cell lymphomas. the recent advent of flowcytometric techniques allowed easy detection of many different markers on lymphoma cells and therefore, not only distinguish between t and b cells, but also estimate possible aberration on immunophenotype. in human oncology, although some controversy persists, it seems that non-hodgkins lymphoma and acute leukemia carrying aberrations have a worse prognosis. the aim of this study was to evaluate the role of immunophenotype aberration in canine high-grade lymphoma considering outcome and time span to achieve complete response under chemotherapy. samples of bone marrow, blood and lymph node suspensions from twentythree dogs were evaluated with flow-cytometry. eleven dogs had aberrant expression of neoplastic lymphocytes and twelve were non-aberrant. the most common aberrations found were: positivity to cd34, biphenotypes, double expression of tantigens (cd41, cd81), diminished expression of cd45. all dogs were treated with a chop-based protocol. there was a significant difference for the time to achieve response to chemotherapy (partial or complete). 12/12 non aberrant lymphomas went into cr or pr after the first treatment (l-asparaginase), while aberrant lympho-mas needed more than 2 treatment to reach cr or pr. there was a trend for a prolonged disease free interval with non-aberrant versus aberrant, although it was not statistically significant. aberration of immunophenotype may be a prognostic factor for canine lymphomas, but further studies with larger groups are needed. class ii major histocompatibility expression is a significant and independent predictor of prognosis in human b cell lymphoma. low class ii mhc is consistently associated with poorer outcome. the mechanism underlying this relationship is not clear, but one hypothesis is that high class ii mhc allows for better antigen presentation and tumor-specific immune responses. in the this study, we investigated whether that class ii mhc expression in canine b cell lymphoma was associated with remission and survival times. a total of 160 patients were categorized by level of class ii mhc,expression of cd34 and cell size for on neoplastic b cells. multivariable cox-proportional hazard analysis was used investigate this research question using a randomly selected subset of the data, and the predictive ability of this model was validated on the remaining 1/3 of patient data. results suggested that low class ii mhc expression was associated with decreased times to relapse and death as is seen in human b cell lymphoma, and that large neoplastic cells were associated with decreased survival time. cd34 expression was not associated with patient outcomes. these findings have implications for the use of dogs to model human lymphomas, for the study of tumor vaccines, and for prediction of mortality in dogs with b cell lymphoma with a high level of specificity. one of the reasons for the failure of canine lymphoma treatment is related to the resistance of tumor cells against chemotherapy drugs. the major form of this resistance is provide by multidrug resistance abc transporters. abc transporters proteins comprise a large superfamily of transmembrane proteins, atp-dependent, that extrude a large variety of drugs from the cells. multidrug resistance phenotype in cancer cells is associated with overexpression of these transmembrane proteins. abcg2, also known as bcrp, is a 655 residue half-transporter protein that protect hematopoetic stem cells against toxic compounds. the aim of this study was to investigate the expression of bcrp (abcg2) in canine multicentric lymphoma. samples were collected by fine needle aspiration of an enlarged lymph nodes, from 25 dogs with multicentric lymphoma (stage iii to v) at diagnosis, and 8 normal lymph nodes (control). dogs that were previously treated with prednisone or chemotherapy were excluded from the study. quantitative rt-pcr was used to measure the mrna expression level of bcrp and flnb expression as a endogenous reference canine gene. a widely range expression value for abcg2 expression was found for canine multicentric lymphoma. high gene expression was observed in 52% (13/25) canine lymphoma, but 48% of dogs had a lower expression when compared with normal lymph node. gene expression was not associated with clinical staging, complete or partial remission, relapse and survival time. in conclusion, abcg2 was expressed in canine lymph node and canine multicentric lymphoma at the diagnosis, and it was not correlated with clinical response. osteosarcoma (osa), the most frequent primary malignant bone tumor of dogs, is both locally aggressive and highly metastatic. prognostic factors for canine osa include tumor location, distant metastatic disease, and serum alkaline phosphatase (alp) concentration. an increased serum alp concentration is associated with poor prognosis; however the mechanisms underlying this phenomenon are currently unclear. during normal bone development alp may be used as a marker for osteoblasts. additionally, alp is a downstream target of activated canonical wnt/b-catenin signaling. therefore, we hypothesized that increased serum alp would be associated with increased expression of b-catenin in canine osa. the goals of this study were: (1) characterize and compare cellular alp expression in osa tissue from patients with normal and high serum alp; and (2) assess b-catenin expression in those same patient populations. we used frozen osa samples collected from patients with either high alp (n 5 3) or normal alp (n 5 3). total rna was isolated from the frozen tissue, converted to cdna, and analyzed using quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) with either target gene alp (aim 1), or target gene b-catenin (aim 2). additionally, b-catenin expression was analyzed by western blot. qpcr data for bcatenin and alp expression were normalized to 18s, and relative expression was calculated by the ddct method. the relative expression of cellular alp was higher in high serum alp samples compared to normal serum alp samples: 44.74 ae 22.04 (mean relative expression ae standard deviation; p o 0.001). further, the relative expression of b-catenin was also increased; b-catenin expression of high serum alp samples relative to low serum alp samples was 24.57 ae 11.41 (p o 0.001), which is also seen by western blot. this study begins to clarify the mechanism behind high serum alp in canine osa, and suggests the wnt signaling pathway may be active in this population of patients. further work will focus on elucidating the role active wnt signaling plays in the biology of osa. in the future, serum alp status of osa patients may help identify patients that would benefit from therapies targeting this pathway. accurate assessment of abdominal lymph node status is of vital importance for appropriate treatment planning and determining prognosis in dogs with apocrine gland adenocarcinoma of the anal sac (agaas). pretreatment knowledge of lymph node status is helpful for determining prognosis and planning the optimal extent of lymphadenectomy. in addition, pretreatment knowledge of lymph node status may help in selecting patients who might benefit from adjuvant chemotherapy and radiation therapy. abdominal ultrasound is currently the most commonly employed test to screen for abdominal lymphadenopathy in dogs with agaas. imaging studies in people indicate that magnetic resonance imaging ( to determine and compare the plasma concentration of cyclophosphamide and its metabolite 4-ohcp, within the plasma of lymphoma bearing dogs being treated with either oral or intravenous cyclophosphamide. in this prospective study, patients were randomly assigned to either receive oral or intravenous cyclophosphamide, at a dose of 250 mg/m2. based on a priori power calculation eight patients per treatment group were enrolled. plasma was obtained at times 0, 15, 30, 60 minutes, and then at 2, 4, 6, 8, 24 hours post administration for evaluation of 4-ohcp concentrations by liquid chromatography-dual mass spectrometry (lc/ms/ms). average values were obtained for both cyclophosphamide and 4-ohcp concentrations within the plasma of both groups. the following values were obtained, half life (hl), time to maximum concentration (tmax), maximum concentration (cmax), and area under the curve (auc). the mann-whitney statistical test was used to compare the groups. the auc for cyclophosphamide was statistically significant (p o 0.05) when compared between the two groups. the auc for 4-ohcp was not statistically significant between the groups. the difference between cmax for cyclophosphamide and 4-ohcp was statistically significantly (p o 0.05) between the groups. although the auc for cyclophosphamide was statistically significant between the two groups, the auc for the active metabolite 4-ohcp was not different when administered intravenously or orally. thus drug exposure to the active metabolite of cyclophosphamide is the same when administered intravenously or orally. previously the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g3 power driver by a wide spectrum of novice participants in feline cadavers were evaluated. novice users' mean io catheter insertion time using the ez-io g3 driver was also compared to the mean iv catheter insertion time in normovolemic feline and canine patients presented to the western college of veterinary medicine (wcvm) small animal hospital. novice users included 40 wcvm personnel (8 technicians, 11 veterinary students, 5 interns, 8 residents, 8 clinicians). after watching a 5-minute ez-io g3 training video, each participant inserted 3 io catheters using the ez-io g3 driver. site (proximal humerus or trochanteric fossa of the femur) and side of cat (right or left) were randomized for each attempt for each participant. a 15 gauge x 15 mm long needle and a 15 gauge x 25 mm needle were used for io catheter insertion in the humerus and femur, respectively. participants then graded the ''ease of use'' of the ez-io g3 device on a visual analog scale (vas) that was converted to a 5-point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by wcvm small animal hospital personnel (6 technicians, 16 veterinary students, 1 intern, 1 resident, 2 clinicians) were then timed and compared to the mean io catheter insertion time in feline cadavers by study participants using the ez-io g3 device. the io catheter was inserted correctly on every attempt by 70% (28/40) of participants. no difference was found between participant groups for mean io catheter placement confirmation percentage (p 5 0.72). percentage of io catheter ''slippage off the bone'' at the time of placement did not vary across participant groups (p 5 0.12). mean io catheter insertion times were all less than 10 seconds and did not differ significantly as a function of attempt number (p 5 0.93) or as a function of participant group (p 5 0.78). participants rated the ez-io g3's ''ease of use'' favorably and subjective scores did not differ across participant groups with varying levels of clinical experience (p 4 0.2). compared to the mean insertion time for iv catheterization (188 sec), mean io catheter insertion by participants using the ez-io g3 (8 sec) was significantly faster (p 5 0.001). regardless of their level of clinical experience, participants rated the ez-io g3 device favorably in terms of its ''ease of use'' and their willingness to use the device in the future. regardless of their level of clinical experience, study participants successfully placed io catheters using the ez-io g3 device and did so significantly faster than the reported iv catheter insertion time in normovolemic feline and canine patients in the wcvm small animal hospital. intraosseous catheterization using the ez-io g3 has the potential to provide very rapid vascular access and is a skill that can be easily learned. previously presented at the western college of veterinary medicine undergraduate poster competition. multicavitary effusion is a common cause of presentation for dogs to emergency medical centers. the goal of this study was to identify common underlying causes of multicavitary effusion as well as determine their relative importance. a retrospective analysis of 43 cases of multicavitary effusion admitted to the icu of a tertiary referral center (ontario veterinary college) from 2007 to 2010 was performed. twenty-three different breeds, with golden and labrador retrievers (25.6% and 14%, respectively) being most commonly seen, were included in the study. ages ranged from 2 to 14 years with a median age of 9 years and a mean of 8.1 years. 69.8% of cases were males (30/43 cases). most common presenting signs included lethargy (48.8%), anorexia (32.6%), vomiting (21%) and dyspnea (21%). cavitary effusion was detected by either ultrasonography (pericardial, pleural or abdominal) or radiographs (pleural). bicavitary effusion was present in 28 cases (65.1%) whereas 15 cases (34.9%) had tricavitary effusion. neoplasia was found to be the most common underlying cause overall (51.2%), with hemangiosarcoma being the leading type (40.9% of neoplasia cases), followed by congestive heart failure (9.3%), gastrointestinal lymphangectasia (4.7%), peritonitis/pancreatitis (4.7%), cirrhotic liver disease (2.3%) and acute renal failure (2.3%). in 11 cases (25.6%), no underlying cause could be found. of these, 4 (9.3% of all cases) were diagnosed as having idiopathic pericardial effusion. taken together, these findings suggest a strong association between multicavitary effusion and diseases carrying a guarded prognosis in dogs. infection control practices in veterinary clinics and hospitals are becoming increasingly important, with rising client expectations, growing concern about the spread of antimicrobial-resistant pathogens, and the potential for zoonotic transmission of disease. surgical patients are at increased risk of developing infections, and can serve as sources of these pathogens for other animals and people with whom they have contact within and outside the clinic. taking all reasonable precautions to reduce the risk of surgical site infections, beginning with preoperative preparation of the surgeon and patient, is therefore an important part of any infection control program. while guidelines are available for preoperative preparation procedures, there has been no objective investigation of compliance with these guidelines in veterinary practices. the objectives of this pilot study were to describe a range of preoperative hand scrub and surgical site preparation practices in veterinary clinics, and to determine if there were any areas that consistently require improvement. observation of preparation practices was performed in each of ten clinics over 9-14 days using 2-3 small wireless surveillance cameras. data was coded for 148 surgical patients, and 31 surgeons performing a total of 190 hand scrubs. patient hair removal was most commonly performed after induction of the animal (129/140, 92%) and using clippers (114/137, 83%) . steps in surgical site aseptic preparation ranged from 1-4. contact time with soap ranged from 18-369s (mean 82s, median 53s), and with alcohol from 3-220s (mean 41s, median 30.5s). application of alcohol or antiseptic using a ''cleanest to dirtiest'' pattern was infrequent (29/66 (44%) and 11/83 (13%), respectively). potential contamination of the surgical site occurred most frequently when the animal was moved to the surgery table after initial preparation (40/58, 69%). preoperative alcohol hand rub was used in 2/10 facilities, but soap and water hand scrub was still more commonly used even at these clinics. proximal-to-distal scrubbing was noted in 95/142 (67%) of soap and water scrubs. contact time during surgeon hand preparation ranged from 7-529s (mean 144s, median 124s) for soap and water and from 4-123s (mean 34s, median 25s) for alcohol-based hand rub. approximately 75% of the variation in contact time was due to inter-surgeon variation. no significant changes in practices were identified over the course of the observation period. some preoperative preparation practices were fairly consistent between clinics in this study, while others varied considerably. contact times with preparatory solutions were often far shorter than recommended, and there was a high frequency of non-sterile contact with the surgical site during movement of patients to the surgical suite. the camera system used to perform this study did not have a significant time-dependent effect on the behavior of participants, and could be useful for performing similar field-based observational studies in the future. this prospective randomized study compared the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g3 power driver to manual io catheterization in feline cadavers. the io catheter insertion time in cadavers using the ez-io g3 device was also compared to iv catheter insertion time in normovolemic feline and canine patients. after a purposely limited training period, a preclinical veterinary student was timed and video-recorded as she performed 20 io catheter placements in feline cadavers (10 io insertions by placing an illinois needle manually and 10 io insertions using the ez-io g3). order of technique (manual or ez-io g3), site of io placement (proximal humerus or trochanteric fossa of the femur), and side of cat (right or left) were randomized for each attempt. when using the ez-io g3, a 15 gauge x 15 mm long needle and a 15 gauge x 25 mm needle were used for io catheter insertion in the humerus and femur, respectively. after each attempt, the student graded the ''ease of use'' of each technique on a visual analog scale (vas) that was converted to a 5-point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by western college of veterinary medicine (wcvm) small animal hospital personnel (6 technicians, 16 veterinary students, 1 intern, 1 resident, 2 clinicians) were then timed and compared to the student's mean io catheter insertion time using the ez-io g3.median io catheter insertion times for the 2 techniques were significantly different (manual io technique 24 sec; ez-io g3 5 sec) (p o 0.001); the manual method took 48 seconds longer (95% confidence interval of 28 to 67 seconds) than the ez-io g3 method. insertion time was more variable for the manual technique than for the ez-io g3. percentage of catheter ''slippage off the bone'' and extravasation around the inserted catheter were significantly higher for placement of the manual io catheter compared with placement of the ez-io g3 catheter (p o 0.001). student's subjective ratings were more favorable and more consistent for the ez-io g3 technique compared to the manual technique for io catheter insertion. compared to the mean insertion time for iv catheterization in the wcvm small animal hospital, io catheter insertion by the student using the ez-io g3 was significantly faster (iv catheter 188 sec; ez-io g3 io catheter 7 sec) (p o 0.001). intraosseous catheter insertion using the ez-io g3 can be said to be significantly faster, less traumatic, more user-friendly, and as effective as io catheter placement using the manual technique. vascular access via io catheter insertion using the ez-io g3 device may be suggested to be faster than iv catheter insertion. previously presented at the western college of veterinary medicine undergraduate poster competition. computed tomography (ct) has been widely investigated and applied as a means for non-invasive quantitative bone mineral determination in human medicine. the aim of this study was to assess age-related changes and anatomic variation in bone mineral density (bmd) using quantitative ct in normal cats. seventeen normal cats were included in this study and divided into the following 3 age groups: o 1 year (n 5 4); 2-5 years (n 5 10); and 4 6 years (n 5 3). a computed tomographic scan of each vertebra from the 12 th thoracic to the 7 th lumbar spine, and the pelvis, was performed with a bone-density phantom (50, 100, and 150 mg/cm 3 , calcium hydroxyapatite, cirs phantom s ). on the central transverse section, the elliptical region of interest (roi) was drawn to measure the mean hounsfield unit value. those values were converted to equivalent bmd by use of the bone-density phantom and linear regression analysis (r 2 4 0.95). the mean bmd value of the thoracic vertebrae (651.3 ae 100.4 mg/cm 3 ) was significantly higher than of the lumbar vertebrae (520 ae 119.5 mg/cm 3 ). the maximum bmd occurred at the t12, t13, and l1 levels in all age groups. there was a statistically significant difference in the mean bmd value among the 3 age groups at the t12 (p o 0.001), t13 (p o 0.001), and l4 levels (p 5 0.013), respectively. in addition, there was no significant difference between the mean bmd value of the left and right iliac bodies (485.1 ae 140.4 mg/cm 3 and 482.1 ae 148.2 mg/cm 3 , respectively). the present study suggests that age-related changes and anatomic variation in bmd values should be considered when assessing bmd using quantitative ct in cats with bone disorders. dynamic contrast-enhanced computed tomography (dce-ct) is a rapid and widely available method of cerebral perfusion imaging. however, there is no established reference value of cerebral blood flow (cbf) measured by dce-ct according to a dog's age. the purpose of this study was to identify the correlation between regional cbf and aging in clinically normal dogs using dce-ct. fourteen dogs with no evidence of hemodynamic disorders and central nervous system dysfunction were included in this study. dogs were assigned to the following 3 age groups: o 1 year (group 1); 3-6 years (group 2); and o 10 years (group 3). dce-ct scans were performed at the level of the third ventricle and mesencephalic aqueduct. cbf in the gray and white matter was calculated using stroketool-ct s software. the overall mean ae standard deviation quantitative estimate for regional cbf in clinically normal dogs was 67.8 ae 12.9 ml/min/ 100 g, 44.7 ae 4.1 ml/min/100 g, and 31.0 ae 3.4 ml/min/100 g in groups 1, 2, and 3, respectively. there was no significant regional cbf difference between the right and left sides of the brain in each group. also, a statistically significant difference in the regional cbf was observed between groups 2 and 3 (p o 0.001). thus, aging affects the regional cbf in normal dogs and the values should be considered assessing the results of dce-ct. according to several clinical behavior guidelines, ''toileting'' type inappropriate urination (i.e. large amounts of urine deposited in horizontal surfaces) can arise in cats suffering from a medical problem (typically lower urinary tract disease). by contrast, ''spraying'' type behaviour (i.e. possibly smaller amounts of urine deposited on vertical areas) is more typically associated with anxiety brought about by a threat to local resources, arising from either a change in the physical environment or threat to these resources from another cat. however, there is some evidence that ''sprayers'' may also be presented with a medical problem, which might be linked to the disease (e.g. painful voiding associated with crystalluria may lead to a standing posture being adopted and small amounts eliminated at a given time). this might be associated with an apprehensive state or simply a co-morbid state. as part of a larger research project aimed at investigating behavioral and physical aspects of cats presented with inappropriate urination, owners of 14 ''spraying'' and 18 ''toileting'' cats with appropriate control subjects from the same households were recruited throughout local media coverage and the internet. the case-control dyads were brought by the owners to the veterinary hospital of the university of sa˜o paulo, at the same time, for a medical work-up (i.e. physical examination, complete blood count, biochemical profile, urinalysis, urine culture and abdominal ultrasound). no significant differences between the ''sprayers'' and ''toileters'' regarding the occurrence of medical problems were found. both groups had a similar proportion of cats affected by medical illnesses (sprayers: 35.7%, toileters: 44.4%; chi 2 , p 5 0.618), directly or indirectly relating to the urinary system (e.g. diabetes, chronic kidney disease). in both groups, control cats also had a relatively high occurrence of medical concerns (21.4% and 27.8%, respectively for each control group). these results emphasize the importance of careful medical evaluation of cats presented for a urinary housesoiling problem. the relatively high prevalence of medical concerns among apparently healthy cats in multi-cat households may have arisen, at least in part, as a result of an inability/failure of owners to monitor individuals, thus allowing some early signs to pass unnoticed. the way in which medical and behavioral elements are linked (if at all) remain unknown but deserve further investigation. considered as a semi-social species, domestic cats appear to be highly sensitive to the effects of social stress, especially when living in high density populations. cats are capable of adapting to live ingroup; nonetheless, they do not appreciate living in close proximity with others as result of an environment lacking of great opportunities of escaping and hiding. this study aimed at testing the following hypotheses: (a) owners' perceived quality of life affects cats' global levels of stress; (b) cats' global levels of stress are influenced by cats' personality; (c) cats' living style (single housing versus large group housing) does affect stress levels in cats. to our knowledge, this is the first study investigating stress levels of domestic owned cats, under natural conditions, throughout measurement of faecal glucocorticoids metabolites concentration, and taking into consideration cat personality, cat living style and owner's subjective life quality. in this study, adrenocortical activity, as a valuable physiological indicator of emotional stress, was evaluated throughout the measurement of faecal glucocorticoids metabolites in fourteen single and sixteen in-group housed cats. cat personality as well as owners life quality was evaluated by self reported questionnaires given to the owners to answer. significant differences in mean glucocorticoids metabolites concentrations (mgcm) between the two populations (i.e. single versus in-group cats) were not detected (random effect model, p 5 0.178). however, when mgcm were taken as a function of cat personality, there were differences regarding single catstimid cats showed higher levels in comparison to easy-going (random effect model, p 5 0.018) and bossy (random effect model, p 5 0.020) cats. as to owner subjective life quality, a direct association between the scores given by the owners to the social dimension and mgcm was found for single cats only (i.e. the better the owner felt itself social wise the higher the mgcm of the cat; random effect model, p 5 0.002). social stratification may compensate the stress resulting from spatial restriction in large in-group living cats. other underexplored factors such as feline personality and owner life style seem to play an equally important role in domestic cats' day to day levels of stress, especially in the cats kept as single pets. in dogs, raas activation is a major feature of congestive heart failure (chf). benazepril (fortekor s ) is a potent ace inhibitor with well-documented effectiveness in canine chf. although ace activity (ace a ) has been used in preclinical studies as a surrogate marker of efficacy, some authors have reported a poor correlation between plasma ace a and changes in angiotensin ii (aii) or aldosterone (al). the purpose of this study was to investigate the effect of benazepril on canine plasma renin activity/concentration (pra/prc), angiotensin i (ai), aii, al, and fractional excretion of potassium (ufek), sodium (ufena) and aldosterone (ufeal). sixteen beagle dogs were fed a low-sodium diet and dosed with placebo or benazepril tablets (10 mg po, q24h) for 5 days. blood and urine samples were collected on day 1 (d1) and day 5 (d5) over 24-hour periods. data were analyzed by repeated measures anova of baseline corrected values, and anova of auc 24hours . compared with placebo, benazepril induced a significant increase in pra and ai at d1 (p-value [pra] :0.001, p-value [ai] :0.002) and d5 (p-value [pra] :0.001, p-value [ai] o 0.001). no differences in prc were noticed. based on auc 24hours, aii levels were 34% lower in the benazepril group at d5 (p-value [aii] :0.01). ufeal and al decreased by up to 27% and 24% at d1 and d5, respectively, though differences did not reach statistical significance. benazepril markedly influences raas dynamics in dogs. decreased exposure to aii and al are likely to be the key events required to counteract pathological remodeling of the heart in chf. this study compared two intravenous anesthetic agents, alfaxalone (alf) (alfaxan s , jurox pty. ltd.) and propofol (ppf) (rapinovet s , schering plough animal health) and their effects on spontaneous ventilation after induction of anesthesia in dogs at various doses. this randomized, crossover, dose-escalation study used six dogs in weight and gender-matched pairs (3m-3f). for each drug, each dog was dosed incrementally at 1, 2, 5, 10 and 20 times the labeled anesthetic induction dose rate (alf 2 mg/kg, ppf 6.5 mg/kg) or until a dose was reached that rendered the dog apneic. a minimum of three days was allowed between doses. for each dose administration, the entire calculated dose was delivered constantly over 1 min. the primary variable was apnea, defined as an absence of spontaneous ventilation for 1 minute. apneic dogs were manually ventilated with oxygen until they resumed adequate spontaneous ventilation. once the apneic dose was determined for an individual dog for one drug, the dog began incremental doses with the alternate drug. for each anesthetic episode times were recorded from completion of induction dose to; removal of endotracheal tube, dog lifting head, dog attaining sternal recumbency and dog standing. pulse rate, respiratory rate, spo 2 and etco 2 were each measured every 5 min. within-dog comparisons were made using the paired student's t-test. for both alf and ppf all 6 dogs respired voluntarily at the labeled (1 x) dose. for ppf at 2 and 5 x doses, 4 and 0 dogs respired voluntarily respectively. for alf at 2, 5 and 10 x doses, all 6, 4 and 1 dog respired voluntarily respectively. for all six dogs to become apneic required 5 x dose of ppf and 20 x dose of alf. the mean no observable adverse effect dose (noael) expressed as a multiple of the labeled dose was higher for alf (4.8 x) than for ppf (1.7 x) (p 5 0.035). there were no significant differences between times to extubation, head lift or attaining sternal recumbency after alf and ppf at 1, 2 and 5 x doses. at the 2 x dose, dogs took longer to stand after alf (29.0 ae 7.0 min) than ppf (25.0 ae 8.3 min). we concluded that based on anaesthetic duration, the manufacturer's labeled dose rates of 2 mg/kg for alf and 6.5 mg/kg for ppf were equivalent. however, based on the dose escalation, the number of dogs becoming apneic at each dose-multiple is consistent with ppf having a narrower safety margin, i.e., ppf caused more respiratory depression than alf. parenteral levetiracetam (lev) has been shown to rapidly attain therapeutic levels in dogs when given iv or im, and has been used offlabel for the treatment of seizure emergencies. the purpose of this study was to determine the safety and pharmacokinetics of subcutaneously administered levetiracetam in healthy dogs. potential application of these results would be use of sq lev instead of or in addition to rectal diazepam for the treatment of cluster seizures at home. lev was administered sq between the shoulder blades to 4 healthy, purpose-bred hound dogs, at a dose of 60 mg/kg (undiluted). blood samples were collected at 15, 120 and 420 minutes after lev administration via jugular venipuncture. plasma lev concentrations were measured by high pressure liquid chromatography. none of the dogs became sedated, nor was there pain evident on palpation of the injection site. mean (standard deviation) lev concentration was 65.2 (29.5), 114.5 (10.5) and 84.9 (20.6) mg/ml at 15, 120 and 420 minutes, respectively. administration of sq lev was well tolerated, and exceeded the suggested therapeutic range (5-45 mg/ml) within 15 minutes of administration, and remained above the range for at least 7 hours. these data indicate that sq lev administration may be an alternative for the at-home treatment of cluster seizures in dogs, and prospective studies in epileptic dogs are warranted. the purpose of this study was to assess the effects of cyp inhibitors (ketoconazole, chloramphenicol, fluoxetine, trimethoprim, cimetidine, and medetomidine) in varying combinations on the bioavailability of oral methadone in healthy greyhound dogs. the iacuc approved this study. cyp inhibitors were administered po for 48 hours prior to methadone administration. methadone hydrochloride was administered po at a targeted dose of 1 mg/kg. blood was obtained for the determination of methadone plasma concentrations by mass spectrometry. the area under the curve (auc) of methadone for each treatment group was compared statistically to the auc of methadone administered without inhibitors using the mann-whitney rank sum test. significant increases (p o 0.001) in the methadone auc occurred in all treatment groups which included chloramphenicol, including chloramphenicol as the only inhibitor. the magnitude of increase was at least 50 fold. mean concentrations of methadone exceeded 10 ng/ml for at least 10 hours in all groups administered concurrent chloramphenicol. no significant increases in the auc occurred in any of the groups which did not include chloramphenicol. in conclusion, chloramphenicol significantly inhibits the metabolism of methadone in greyhound dogs. as a result, the oral bioavailability of methadone is significantly increased and plasma concentrations are achieved that are reported to be effective in humans for 10-36 hours after a single oral administration. doxycycline hyclate is used frequently in small animals, horses and exotic animals for treatment of a wide variety of infections. because doxycycline hyclate tablets may not be suitable for oral administration in some animals, particularly horses and cats, it has been compounded into liquid suspensions. the commercially available doxycycline calcium 10 mg/ml oral suspension, vibramycin s (pfizer) is not suitable for use in animals due to its low concentration and flavoring that animals find unpalatable. because of the known inherent instability of doxycline in aqueous vehicles under storage, this study was conducted to determine the potency of two formulations stored in dark and light conditions. a high pressure liquid chromatography (hplc) assay with uv absorption at 350 nm was developed for analyzing doxycycline in formulations, in comparison to a reference standard from the united states pharmacopeia (usp). doxycycline hyclate 100 mg tablets were first tested for potency. the tablets were then crushed and mixed with a pharmaceutical vehicle to make two concentrations: 33.3 mg/ml and 166.7 mg/ml. the vehicle used was a 50:50 mixture of a vehicle for oral solution (ora-sweet, usp-nf) and vehicle for oral suspension (ora-plus, usp-nf). the suspensions were prepared in replicates of 3. each replicate was divided, with one aliquot stored at room temperature in lighted conditions, and the other aliquot stored at room temperature in the dark. doxycycline was extracted from the formulations and measured by hplc at day 0, 1, 4, 7, 14, 21, and 28 . each replicate was tested and the potency reported as the percent doxycycline relative to the usp reference standard. on day 0, 1, 4, and 7, the potency of each formulation was within 90-110% of the reference standard (range 93.4-109%). this value is within the accepted range cited in usp o 795 4 on pharmaceutical compounding-non-sterile preparations. however, starting at day 14, the potency declined dramatically and remained low for the tests performed on day 21 and 28. the potency on day 14, 21, and 28 was below 20% of the reference standard (range 14-18%). there was also a noticeable change in the quality of the formulation starting on day 14, and a change in the color of the formulation to a dark brown. these results indicate that when doxycycline hyclate tablets are compounded as a suspension in an aqueous vehicle as described in this study, at 33.3 and 166.7 mg/ml under the storage conditions used in this study, potency of the formulation cannot be assured beyond 7 days. we recommend a beyond-use-day (bud) of 7 days for formulations prepared and stored at room temperature in light or dark conditions. therapeutic options for multidrug resistance (mdr) escherichia coli urinary tract infections (uti) are limited. fosfomycin (fos) tromethamine is an oral, broad-spectrum, cell-wall active, bactericidal drug approved for treatment of uncomplicated uti in humans. the purpose of this study was to determine time dependency of fos and the disposition of fos tromethamine in dogs. using a randomized, double crossover design, 12 client-owned dogs received fos sodium iv (40 mg/kg) and fos tromethamine (po, 80 mg/kg) either with (n 5 6) or without food (n 5 6). serum and urine were collected for 24 hr; fos was quantitated with a bioassay (atcc e. coli 25922, serum or atcc proteus vulgaris 13315, urine). in-vitro killing curves (cell counts through 24 hours) were performed at 0 (control),0.5,1,2,8,16 and 32 x mic for mdr e. coli canine fos susceptible (e-test s ) uropathogens. killing curves indicated fos to be time dependent. after iv administration, clearance (mlãkg/hr), volume of distribution (l/kg), elimination half-life (hl; hr) and mean residence time (mrt, hr) were (mean ae sd): 210 ae 104, 0.23 ae 0.15, 0.36 ae 0.19, 1.14 ae 0.35 and 1.7 ae 0.4, respectively. for po, c max , hl and mrt were 66 ae 21, 2.5 ae 1.09 and 5.1 ae 1.7, respectively. serum fos exceeded the mic 90 reported for multidrug resistant (mdr) e. coli (1.5 mg/ml) for 7hr (iv; 2.5 mg/ml) and 12hr (po, 9 mg/ml diminazene is an aromatic diamidine, anti-protozoal drug that has shown promise in a small number of cases of cytauxzoonosis. in a noncontrolled case series, 5 of 6 cats with clinical cytauxzoonosis given 3 mg/ kg of diminazene aceturate survived infection. dosage frequency was two intramuscular injections given one week apart. commercial formulations contain the diminazene diaceturate salt. the active base is diminazene with the salt consisting of two aceturate molecules. currently there is no data available on the pharmacokinetics of either diminazene compound in cats. the objective of this study was to determine the pharmacokinetics of diminazene diaceturate in healthy cats. four purpose bred cats with normal physical examination, cbc, chemistry and urinalysis were used. a powdered commercial drug formulation (veriben s , ceva sanet animale) was freshly reconstituted with sterile water to a concentration of 7 mg/ml prior to administration and sterile filtered solution. heparinized blood samples were collected just before (hour 0) or 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, 120 , and 168 hours after intramuscular administration of 3 mg/kg (1.68 mg/kg of diminazene base) diminazene diaceturate. the plasma was separated by centrifugation within 30 minutes of collection and frozen (à801c) until analysis. concentrations of diminazene were measured by hplc analysis using uv absorption and ion-pairing conditions. the pharmacokinetic profile was analyzed using a simple one-compartment model. in these cats, diminazene had a mean terminal half life (t 1/2 ) of 1.70 (1/-0.29) hrs and mean peak plasma concentration (c max ) 0.51 (1/à 0.11) mg/ml. the mean residence time (mrt) of diminazene was 2.45 hrs (1/à0.42). systemic clearance (cl/f) was 1.38 (1/à 0.26) l/kg/hr. the volume of distribution per fraction absorbed (vd/f) was 3.36 (1/à 0.72) l/kg. a single intramuscular dose of diminazene diaceturate was well tolerated by all 4 cats. without knowing the concentration required to inhibit or kill cytauxzoon felis, it is not yet possible to make suggestions regarding optimum dosing schedules for this drug. additional toxicology data and studies to assess clinical efficacy for the treatment of cytauxzoonosis are indicated before routine clinical use can be considered. meloxicam has been shown to accumulate in areas of inflammation in both the rat and human. the objective of this study was to investigate the concentration of meloxicam in synovial fluid of inflamed joints versus that of non-inflamed joints in dogs. eight male dogs were treated with 0.2 mg/kg of meloxicam on day one and 0.1 mg/kg of meloxicam on day two. all treatments were administered orally. on day three reversible acute synovitis was induced in one stifle by aseptic, intra-articular administration of 1 ml sodium urate crystal suspension (10 mg/ml). in four dogs synovitis was induced in the l stifle and in four dogs the same procedure was used in the r stifle. in each dog the stifle without induction of synovitis served as the ''normal'' joint sample. a synovial fluid sample was collected from both the r and l stifle of each dog. sample collection occurred eight hours after administration of sodium urate and twenty four hours after the last administration of meloxicam. synovial meloxicam concentration was analysed using high performance liquid chromatography-mass spectrometry (hplc/ ms-ms). the concentration of meloxicam in the inflamed versus non-inflamed joint in each dog was compared using the paired t-test. the results indicate that meloxicam preferentially accumulates in inflamed joints in the dog as meloxicam concentrations are statistically significantly higher in inflamed joints than in non-inflamed joints. no national surveillance system exists for monitoring emergent resistance in companion animals. however, e. coli resistance is an increasing therapeutic and public health concern in these in dogs and cats. the purpose of this study was to describe current resistance patterns of canine and feline pathogenic e. coli throughout the united states and identify risk factors of antimicrobial resistance. isolates (n 5 1512) of clinical e. coli collected from dogs or cats from may 2008 through may 2010 located in 6 different regions. susceptibility was determined to 15 drugs (6 drug classes) by broth microdilution methods. pharmacodyamaic statistics were described regionally. phenotypes were determined and type of resistance was based on the number of drug classes to which resistance was expressed: none (ndr), single (sdr) and multi (mdr). the majority of isolates were from urinary tract (71.5%) and dogs (75.7%). the proportion of resistance type for each drug was: ndr (17.5%), sdr (56.4%) and mdr (26.12%). the proportion of mdr was greatest in the southwest (25.29%) and least in the northwest (9.19%) (p o 0.05). for all regions, the proportion of resistance was: cephalothin (cph, 65.2%) 4 amoxicillin-clavulanic acid (amx, 59.4%), ampicillin (amp, 52.9%), tricarcillin-clavulanic acid (tcx, 21.8%), doxycyline (dxy, 15.9%) 4 cefoxitin (cfx, 15.2%), cefpodoxime (cpx, 13.7%), chloramphenicol (chp, 13.2%), enrofloxacin (enr, 13.0%) , ciprofloxacin (cif,12.3%), trimethoprim-sulfamethoxazole (tmx, 10.7%), ceftazidime(cfz, 10.0%), gentamicin (gtm, 10.0%), cefotaxime (cft, 9.2%) 4 meropenem (1.1%) (p o 0.05). the mic 90 exceeded the resistant breakpoint for amp, amx, cpx, cph, cif, cfx, dxy and enr whereas mic 50 did not surpass the susceptible breakpoint. beta-lactams (96.37%) was the most and aminoglycosides the least (0.12%) sdr. the drug class most frequently involved in mdr was beta-lactams (97.72%) and least, gen (30.13%). resistance differs regionally, being greatest in the southwest. cph is the most and meropenam is the drug least associated with resistance; these patterns are consistent with current drugs used by veterinarians. the fluoroquinolones (fqs) are common choices for treatment of e. coli urinary tract infections (utis) in animals and humans. 2nd generation drugs approved in animals include enrofloxacin (enr), marbofloxacin (mar), orbifloxacin (orb); human drugs include ciprofloxacin (cip). 3rd and 4th generation fq for humans include moxifloxacin (mox), gatifloxacin (gat) and ofloxacin, (ofl]), its lisoform levofloxacin (lev). for animals, pradofloxacin (pra) is approved for use in europe. the purpose of this study was to assess the in vitro activity of 1st (naladixic acid [nal] through 4th generation fqs (n 5 11) toward dog or cat e.coli uropathogens (n 5 51). isolates were subjected to susceptibility testing to 6 drugs classes (15 drugs). isolate phenotypes included no (ndr; n 5 12), single (sdr; n 5 15) or multidrug (to more than 2 drug classes; mdr; n 5 24) resistance (including enr resistant [enr r -mdr; n 5 12] or enr susceptible (enr s -mdr, n 5 12). the minimum inhibition concentrations (mics) were determined for each isolates using broth microdilution (e. coli atcc s 25922 served as a negative control). mic statistics were generated for each drug among phenotypes. the overall potency (mic 90 ) for all enr susceptible isolates (ndr, sdr and enr s -mdr) was gat 4 pra, mox, mar, lev, cip 4 sar, orb, ofl 4 enr 4 nal. each e. coli isolate expressing ndr or sdr was susceptible to all fq. however, isolates expressing resistance to 1st or 2nd generation fq were also resistance to later generation drugs. glucocorticoids (gc) are standard therapy for allergic asthma but do not reverse the underlying type i hypersensitivity. allergenspecific immunotherapy (asit), a process of ''desensitization'', is potentially curative but requires identification of offending allergens. the purpose of this study was to determine if oral or inhaled gc administered at routinely used dosages would interfere with allergen identification. we hypothesized that oral but not inhaled gc would interfere with accurate identification of allergen-specific ige using skin and serum testing in experimentally asthmatic cats. asthma was induced in eighteen cats using bermuda grass allergen (bga). cats (n 5 6/group) were randomized to receive oral gc (10 mg prednisolone q 24 hr po), inhaled gc (600 ug budesonide q 24hr) or placebo (gelatin capsule q 24hr po) for one month. intradermal skin testing (idst) and bga-specific ige amounts were measured prior to, during (weeks one and four) and every two weeks after treatment until both tests were positive. a paired t test was used to compare serum ige among groups pre-and post-treatment (p o 0.05 significant). idst reactivity was eliminated in 4/6 cats on oral gc, 3/6 on inhaled gc, and 1/6 placebo-treated cats. within two weeks after stopping treatment, idst was again positive in all cats. contrary to our hypothesis, serum ige reactivity to bga was not significantly diminished by any treatment. in conclusion, a two week withdrawal from gcs is adequate for idst identification of allergen but no withdrawal is required prior to serum ige testing to identify the sensitizing allergens. previously in people, increasing severity of asthma is associated with low serum concentrations of 25-hydroxyvitamin d (25-oh-d). 25-oh-d is thought to ameliorate lower airway inflammation primarily by decreasing the production of pro-inflammatory mediators, and by increasing the production of the anti-inflammatory cytokine il-10. in people, serum 25-oh-d concentration is associated with sunlight exposure as well as dietary intake. cats do not rely on sunlight for vitamin d synthesis; all vitamin d comes from dietary intake. cats have a naturally occurring lower airway disease syndrome (lad) that shares many features with human asthma. the goal of this study was to evaluate serum 25-oh-d concentrations in cats with lad. cats with naturally developing lad were enrolled. criteria for a diagnosis of lad included a history of cough, wheeze or respiratory distress, radiographic evidence of a bronchial pattern and hyperinflation, negative heartworm antigen and antibody test, and a resolution of clinical signs in response to glucocorticoids. dietary history was obtained. 25-oh-d concentrations were determined on serum samples by a commercial laboratory. twelve cats with lad were enrolled. all cats ate commercial cat food. the median 25-oh-d concentration was 112 nmol/l with a range of 65-176 nmol/l which is within the reported reference range of 65-170 nmol/l. in contrast to human asthma, lower airway disease in cats is not associated with low serum concentrations of 25-oh-d. interstitial lung diseases (ild) are uncommon in dogs, with the most commonly recognized ild idiopathic pulmonary fibrosis (''westie fibrosis''). in human medicine, ild represent a large umbrella of pulmonary diseases, with ipf only a subset. other, more treatable, ilds are also identified, and may respond to either the removal of a stimulus (hypersensitivity) or steroid therapy. the goal of this report is to describe the clinical course, including outcome, computed tomography and histopathology of dogs affected with an ild. the computed tomography (ct) log was reviewed for dogs that underwent thoracic ct scanning for evaluation of respiratory signs, and had changes consistent with ild as the primary abnormality, including the presence of diffuse disease in all lobes, and at least 2 of the following: reticulation, ground glass opacity, consolidation, or traction bronchiectasis. survival time from ct date was calculated. the presence of moderate pulmonary hypertension [phtn] (4 50 mmhg) as estimated by tricuspid regurgitant jet, was also reported and survival times were compared with a mann-whitney rank sum with p o 0.05 considered significant. thirteen dogs were identified. terriers and chihuahuas were the most commonly affected breeds. two dogs were adolescents, the remaining dogs ranged from 7-16 years, with a median of 11 years. histopathology results (n 5 5), including moderate to severe interstitial fibrosis (3) alveolar proteinosis with fibrosis (1), and interstitial eosinophilic pneumonia (1). one had suspected cryptogenic organizing pneumonia and had a good response to glucocorticoids. eight dogs died of respiratory failure, with a median post ct survival time of 42 days (range 2-365), two dogs died of non-pulmonary disease, 2 dogs had severe lower respiratory infections as puppies with persistent respiratory signs, and both are still alive at 4 2 years since diagnosis, 1 terrier is alive at 19 months and 1 was lost to follow up. 5 dogs had phtn, with a median survival of 60 days (42-590), while the 5 dogs without had a survival of 180 days (range 21-730), [p 5 0.3]. interstitial lung disease in dogs is not just idiopathic pulmonary fibrosis. following respiratory infection, young dogs may develop an ild with a relatively indolent course and rare ild is steroid responsive. ct is useful to identify ild but further research correlated with echocardiography and histopathology is advised to use it to prognosticate. idiopathic pulmonary fibrosis (ipf) is an interstitial pulmonary disease, mainly described in west highland white terriers (whwt). identification of molecular pathways important in the pathogenesis of ipf would improve our understanding of this disease and may help identify therapeutic targets. the aim of the present study was to investigate gene expression in lungs of whwt with ipf using oligonucleotide microarray. total rna was extracted from post-mortem pulmonary samples from five whwt with ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. genes with a minimum of a two-fold difference in expression between the two groups were selected for further analysis. the most significant biological functions for these genes were identified using ingenuity pathways analysis. more than 1000 genes were identified as having greater than twofold difference in expression. the significant biological functions associated with these genes were related to cellular movement, cellular proliferation and apoptosis. most notable among these were genes encoding the leukocyte chemotactic proteins: ccl2 (fold change 14.93), ccl17 (12.74) and il8 (14.32); the proteins involved in fibroblast migration; and the matrix metalloproteinases (mmps) involved in matrix degradation: mmp7 (à17.55), mmp9 (-3.42), mmp1 (à2.76). this study has identified genes which may be important in pathogenesis of ipf, e.g. proteins involved in leukocytes chemotaxis, fibroblast recruitment and activation, regulation of apoptosis, and extracellular-matrix turn-over. however, real-time quantitative rt-pcr studies are needed to confirm these results before any definitive conclusions can be drawn. idiopathic pulmonary fibrosis (ipf) is an interstitial disease, mainly described in west highland white terriers (whwt). defini-tive diagnosis ultimately relies on lung histopathology. identification of specific biomarkers would be very helpful. expression microarray is a powerful screening tool to study local gene expression in a disease state. the aim of the present study was to measure gene expression profiles in lungs of whwt with ipf to identify potential blood or bronchoalveolar lavage fluid (balf) biomarkers. total rna was extracted from post-mortem pulmonary samples from five whwt with histopathologically confirmed ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. ipa-biomarkers analysis (ingenuity system) was used to filter and prioritize biomarkers candidates using the three following criteria: a minimum of a two-fold difference in expression between ipf and ctrl; expression of the gene in lung tissue; possible detection of the protein in blood or in balf. fifty-four molecules met all the criteria. based on difference in expression, promising proteins included ccl7 (fold change 17.8), a3-actinin (15.7), ccl2 (14.9), serum amyloid a1 (14.4), il8 (14.3), plunc (à25.1), mmp7 (à17.6). some are well-known biomarkers of ipf in humans either for diagnosis (mmp7, il8) or prognosis (ccl2). these results provide novel potential biomarkers of canine ipf. measurement of these proteins in blood and balf of healthy dogs, dogs with ipf and with other respiratory diseases is needed to assess their use as biomarkers of canine ipf. heliox is a mixture of helium and oxygen that has been used therapeutically in human medicine for treatment of airway obstruction. helium's low density and other physical properties have been shown to reduce the work of breathing by limiting turbulence. the purpose of this study was, therefore, to evaluate respiratory parameters in response to inhaled heliox in dogs with meso-and brachycephalic conformation. eleven healthy dogs were recruited, five were mesocephalic and six were brachycephalic. flow-volume loops were collected using commercial software (buxcor) while breathing 70:30 helium: oxygen (heliox) and 70:30 nitrogen:oxygen (nitrox) in a randomized order via a low dead-space face mask. due to the intrinsic gas properties, gas flow rates and volumes were corrected in-vitro by a conversion factor for the effect of helium on the pneumotachograph. respiratory rate, tidal volume (ml), minute ventilation (l), inspiratory time (ti), expiratory time (te), peak inspiratory flow (pif) and peak expiratory flow (pef) were recorded while breathing heliox or nitrox. values were compared using a paired sample t-test, with p o 0.05 considered significant. all dogs cooperated with testing. there was no significant difference in respiratory rate, tidal volume, minute ventilation, inspiratory or expiratory times, or peak inspiratory flow. peak expiratory flow was significantly higher (p 5 0.01) while breathing heliox than when breathing nitrox in brachycephalics but not in mesocephalics (p 5 0.22). heliox is well-tolerated in healthy dogs and results in an increased expiratory flow rate in brachycephalic dogs. further investigation of heliox is warranted in dogs with airway obstruction. of this prospective multicentric study is to assess the effects that surgical correction has on the severity of clinical signs and levels of acute phase proteins (c-reactive protein [crp] , haptoglobin [hp]) and cardiac troponin i (ctni). thirty three brachycephalic dogs with boas were included and evaluated before and, approximately two months, after surgical correction. the most common components of boas found were elongated soft palate (33/33; 100%), stenotic nares (31/33; 94%) and everted laryngeal saccules (13/33; 39.4%). staphylectomy was performed by means of two different surgical techniques: laser (n 5 12) or electrical scalpel (n 5 21). there were significant differences between dogs depending on the surgical technique used, with a higher reduction of respiratory signs (p o 0.002) and a better postsurgical improvement (p o 0.015) with the use of laser. the levels of crp, hp and ctni were categorized into normal or elevated. before surgical treatment three (9.1%), six (18.2%) and thirteen (39.4%) dogs had elevated values of crp, hp and ctni, respectively. two months after surgical correction, five (15.1%), eleven (33.3%) and fourteen (42.4%) dogs had elevated values of crp, hp and ctni, respectively. there were no statistical differences between values of crp and ctni before and after surgical correction but the levels of hp increased significantly after surgical treatment (p o 0.008), probably due to postsurgical treatment with corticosteroids. as previously suggested by others, there was a statistically significant reduction of respiratory and gastrointestinal signs in dogs with boas submitted to surgical correction (p o 0,001). according to the results obtained in the present study, the determination of crp, hp and ctni before and two months after surgical treatment do not have a prognostic value in dogs with boas. even though, near half of the dogs studied had elevated levels of ctni (39.4%) that persisted after surgical treatment (42.4%), suggesting some degree of myocardial damage is present. further studies are needed considering the influence of breed and age. to the authors' knowledge, this is the first description of crp, hp and ctni determination in dogs with boas. overweight and obesity are common conditions that lead to alterations in respiratory mechanics, airway resistance, pattern of breathing and gas exchange in humans. the objective of the present study was to investigate if there are significant differences on respiratory parameters and arterial gas analysis of obese and overweight cats, in conscious state and under general anesthesia. twenty nine adult cats were arranged in three groups: obese (n 5 15), overweight (n 5 7) and with ideal body score index (bsi) (n 5 7). mean of bsi in the groups were: 8,8 (obese), 6,3 (overweight) and 4,9 (ideal bsi). cats did not had respiratory, cardiac or others systemic diseases. the respiratory parameters were evaluated with a ventilometer equipment coupled to facemasks in conscious cats and directly to the endotracheal tube in anesthetized cats under spontaneous respiration. the anesthesia was performed with propofol (3 ae 1,2 ml/kg) and the cats were maintained in the same anesthetic plan. the three groups were compared by analysis of variance followed by tukey's test and conscious and anesthetized cats were compared by student's t test, with a 5% significance level. there were not observed differences on the respiratory parameters evaluated on ventilometry (tidal volume, expiratory and inspiratory times and peak pressures, respiratory rate and partial pressure of end tidal co 2 (petco 2 )) and on arterial gas parameters (pao 2 e paco 2 ) in the three groups. the pao 2 of cats with ideal bsi was 88,1 ae 13,5 mmhg, although was not significantly different (p 5 0,06) from overweight (72,0 ae 11,9 mmhg) and obese cats (72,6 ae 14,6 mmhg). comparison of anesthetized to conscious cats, it was detected decreases in tidal volume, expiratory and inspiratory times and peak pressures and increase in petco 2 in respiratory rate in the anesthetized cats. only petco 2 , inspiratory time and respiratory rate in overweight cats did not differ in anesthetized cats. these results suggest that obesity and overweight did not result in impairment of respiratory function in cats and propofol induced respiratory depression. osteosarcoma (osa) is the most common bone tumor in dogs, however, little is known regarding the mechanisms underlying malignant transformation in these tumors. breeds such as rottweilers and greyhounds are at higher risk for developing osa, suggesting that heritable factors play a role in this disease. mirnas have tumor/tissue specific roles in regulating gene expression and dysregulated mirna expression is found frequently in cancer. we hypothesize that canine osa is characterized by a unique mirna expression profile(s) with dysregulation of some mirnas being associated with specific breeds. mirna expression profiling of primary osa tumors from 6 greyhounds and 6 rottweilers was performed using the nanostring technologies ncounter mirna expression assay kit, interrogating the mirna expression profile of 651 human mirnas, 168 of whose mature sequences are 100% conserved between human and dog. 17 mirnas were differentially expressed in greyhound versus rottweiler tumors (p o 0.05), suggesting that breed-specific dysregulation of mirnas may contribute to the development and progression of spontaneous osa. hierarchical clustering revealed distinct mirna expression signatures in greyhound osa tumors as compared to rottweilers. based on these preliminary results, we are evaluating a larger cohort of osa tumor samples including greyhounds, rottweilers, golden retrievers, and a mixed population of other breeds. statistical analysis will be performed to determine the association of mirna transcript levels with specific breeds and overall outcome. characterization of mirna expression in canine osa will facilitate our understanding the biology of this disease and has the potential to identify targets for therapeutic intervention. originally combination therapies using drugs with documented single-agent activity and lack of overlapping toxicities could potentially improve outcome. the hypothesis intended to be tested is that palladia s can be safely administered concurrently with a standard weekly protocol of vinblastine (vbl), at dosages known to have activity against mast cell tumors. dogs with histologically confirmed measurable mast cell tumors were evaluated for eligibility to enter a standard phase i dose-finding trial (313 cohort), at a starting dose of 2.3 mg/m2 iv vbl (weekly for a total of 4 treatments) and 2.25 mg/kg po palladia s eod, concurrently. dose escalation of palladia s was scheduled in 0.25 mg/kg increments until mtd was established or fda label dose completed (3.25 mg/kg). safety evaluation was performed weekly throughout the 4 week study period. dose-limiting toxicities were described following established vcog-ctcae(v1.0) criteria. while antitumor response is not a primary endpoint of phase i trials, activity was documented prior to vbl treatments 2-4, and monthly thereafter, based on recist criteria. nine dogs have been enrolled; cohort 3 is filled and approaching completion of the evaluation period. hematologic dose limiting toxicity led to 2 de-escalations of vbl. the current safe combination appears to include vbl at 1.6 mg/m2 every other week and palladia s at 2.25 mg/kg eod. response was seen in all but one dog. without head to head trials comparing efficacy of bi-weekly vbl combined with palladia s and vbl alone, choice of therapy should remain at the clinician's discretion. originally prostate specific membrane antigen (psma) is a transmembrane protein expressed by tumor-associated neovasculature, but not normal blood vessels. based upon its selective expression in endothelial cells associated with cancer, psma may serve as a conserved angiogenic target shared by macroscopic solid tumors of various histologies. to investigate the feasibility of targeting a homogenous population of psma-expressing endothelial cells as a novel anticancer strategy, we have investigated psma expression in several canine hemangiosarcoma (chsa) cell lines, and subsequently developed self-assembling nanoparticles containing diagnostic (near infrared dyes) and therapeutic (doxorubicin) cargo which selectively bind to psma by means of the a10 aptamer, a commercially-available oligonucleotide. the expression of psma by chsa cells was confirmed transcriptionally and translationally by real time pcr and immunohistochemistry, respectively. selective binding and endocytosis of a10 decorated nanoparticles was studied by fluorescent microscopy. the ability of a10 decorated nanoparticles encapsulating doxorubicin to exert in vitro cytotoxic effects in chsa cells was assessed by colony forming assays. using a chsa xenograft murine tumor model, clinically-relevant anticancer effects of a10 decorated nanoparticles encapsulating doxorubicin were tested. all chsa cell lines expressed psma mrna and protein. a10 decorated nanoparticles were selectively endocytosed by psma-expressing cells, and when these nanoparticles encapsulated doxorubicin, significant cytotoxic effects were exerted in vitro. finally, a10 decorated nanoparticles encapsulating doxorubicin significantly reduced the size of macroscopic chsa tumor burdens in transplanted mice. diagnostic and therapeutic nanoparticles can be targeted to psma-expressing endothelial cells, and chsa provides a comparative model for the future study of nanoparticle therapeutics. canine transitional cell carcinoma (tcc) is the most common tumor of the urinary tract, and is similar to human invasive tcc in histopathologic characteristics, molecular features, sites of metastasis, and response to medical therapy. prevalence is increasing, and novel therapies and strategies are needed to effectively treat this aggressive form of cancer in both species. personalized medicine techniques intend to improve treatment outcome by using patient tumor profiling to identify potential and individualized therapeutic targets. a genomic algorithm has been developed termed ''coexpression extrapolation'', or coxen, that aims to use expression microarray data to predict drug activity in patient tcc samples. the utility of this predictive methodology has been established in other types of cancer in vitro, however its clinical utility has not yet been determined. validation studies of coxen in 10 canine tcc cell lines were conducted. the goal was to determine the value of coxen in predicting baseline sensitivity of canine tcc to 5 chemotherapy agents (gemcitabine, mitoxantrone, carboplatin, vinblastine and cisplatin) that would then be used in a proposed clinical trial. additionally, expression data from 25 canine treatment-naı¨ve primary tumor samples were generated on an affymetrix array platform (canine genome v 2.0). both the expression data and tcc cell line data (antiproliferative effects, 50% growth inhibition or gc 50 ) were used to establish a canine specific predictive coxen algorithm. coxen scores for canine tcc cell-line drug activity were then analyzed. scores predicted the activity of cisplatin, gemcitibine, and mitoxantrone in all 10 cell lines, and of carboplatin in 6 cell lines. because all of the cell lines were sensitive to vinblastine (gi 50 o0.05 mm), the coxen score was not predictive of its potency. interestingly, coxen fails to predict vinblastine response in human tcc cell line data as well. in concurrent work, comparative genomic studies to define and compare the gene expression signatures of tcc in dogs and humans provides further evidence that canine tcc is a valuable genomic model of the human disease. current studies involve testing the chemo-predictivity of this derived canine coxen algorithm in additional canine tcc cell lines. canine tcc offers an excellent model for in vitro and in vivo studies of the coxen approach. this preclinical work will be used to guide the feasibility of future coxen clinical trials in dogs and humans with tcc. a small molecule complex (aminoact) isolated from bovine milk is a natural peptide mixture with multi-kinase inhibitory effects against epidermal growth factor receptor (egfr) and insulin-like growth factor receptor-1 (igfr-1). ingestion of aminoact in people with cancer results in lower serum tnf-alpha, an increase in antioxidant superoxide dismutase (sod) enzyme activity, and subjects' blood serum causes apopsotis in cancer cell lines. this study was designed to first assess safety and secondly the efficacy of three dosage levels of ax-3 in sustaining progression free survival (pfs) for dogs with refractory advanced and/or metastatic cancer. the prospective, open label study included dogs of different breeds with naturally occurring histologically confirmed malignancies. the first 13 dogs received aminoact at 1g/m 2 ; the second group of 7 dogs subsequently received the same dosage 1 350 mg of aminoact; and the third group of dogs subsequently received 2g/ m 2 . each dog was treated orally daily for six weeks along with 550 mg betaine hcl, that aids in peptide absorption. all patients were evaluated for toxicity using the vcog-ctcae and efficacy using the recist criteria via assessment of clinical parameters, blood work and client questionnaires. no toxicity other than mild, transient (grade i) nausea was noted, nor were there any changes in hemograms or biochemical profiles in any patient. dogs with tumors that were confirmed as responders (4 50% reduction in size) include pulmonary adenocarcinoma, mast cell tumor, trichoepithelioma and soft tissue sarcoma. it appears in limited studies that the response rate may be more durable at higher dosages. the response to aminoact is dose dependent and only transient mild toxicity was observed, which suggest the maximum effective dosage has not been reached. further clinical studies will be valuable in determining the effective dosage and response duration. treating cancer in dogs with aminoact offers a unique opportunity as a model for human cancer biology and translational cancer therapeutics. stereotactic radiation therapy (srt) combines patient immobilization, image guidance, and intensity modulated delivery to achieve ablative radiation doses within the tumor, while preferentially sparing surrounding normal tissues. the purpose of this study was to evaluate the efficacy of srt as a means of achieving local tumor control for canine nasal tumors. retrospective analysis was performed on dogs with a nasal tumor confirmed by histopathology and computed tomography, no previous surgical or radiation therapy, at least six months of follow-up, and completion of three fractions of srt at csu.srt was administered via the varian trilogy linear accelerator once daily for three consecutive days. the varian eclipse treatment planwas reviewed to determine the planned target volume (ptv) and dose to 95% of the ptv. kaplan-meir survival analysis was performed for disease free interval (dfi) and overall survival (os). sixteen patients with nasal tumors (8 adenocarcinoma/carcinomas, 2 squamous cell carcinomas, 3 chondrosarcomas, 2 osteosarcomas, and 1 undifferentiated sarcoma) were treated with srt. a median dose of 29.1gy was administered to 95% ptv with a median ptv of 75.3cc. srt was well tolerated by the normal tissues with minimal, manageable side effects. to date, the median dfi is 270 days, while the median os is 367 days. based upon the initial clinical experience, stereotactic radiation therapy is an emerging modality in the management of canine nasal tumors. canine leptospirosis can vary from subclinical infection to illness that ranges from mild to severe, including death, depending on the susceptibility of the dog, virulence of the organism, and route and degree of infection. the objective of this study was to evaluate the ability of a canine leptospira bacterin to prevent infection and disease following challenge with virulent leptospira canicola, l. pomona, l. grippotyphosa, or l. icterohaemorrhagiae. groups of 8week-old beagles were vaccinated (day 0) and boosted (day 21) with placebo (n 5 10) or the 4-way bacterin (n ! 20) and subsequently challenged with each serovar. the results demonstrated that blood and various tissue samples from placebo-recipients became reliably infected, and the dogs developed typical clinical signs of leptospirosis including loss of appetite, ocular congestion, depression, dehydration, jaundice, hematuria, melena, vomiting, petechiae, and death. in addition, placebo-recipients developed kidney and liver dysfunction. in contrast, some vaccine-recipients became infected, but the organisms were cleared quickly from the blood. vaccinated dogs failed to develop severe clinical disease requiring medical intervention, and no animals died (p ! 0.001). a few of the vaccinated dogs developed clinical abnormalities, but the clinical signs remained mild and were self-limiting (p o 0.0001 for each serovar). administration of the bacterin also prevented thrombocytopenia ( ciprofloxacin, a synthetic fluoroquinolone antimicrobic, is not fda-approved for veterinary use. however, due to recent availability of less expensive generic formulations, extra-label use of ciprofloxacin by veterinarians appears more common. although ciprofloxacin crystalluria and uroliths have been reported in humans, we are unaware of any published reports in dogs. this is surprising since mean urine ciprofloxacin concentration (0.36 mg/ml) in dogs following a modest iv dose (13 mg/kg) was 3 times higher than the solubility of ciprofloxacin in water (0.12 mg/ml). to identify the occurrence of ciprofloxacin uroliths in dogs, records from the minnesota urolith center were reviewed. between january 2001 and december 2009, ciprofloxacin was identified in uroliths from 58 dogs; uroliths were composed of 100% ciprofloxacin in 10, mixed uroliths containing ciprofloxacin were identified in 6, a shell of ciprofloxacin was observed in 21, and ciprofloxacin surface crystals were identified in 21. based on an experimental study in which 83% of human volunteers consuming 1000 mg of ciprofloxacin with nahco3 exhibited ciprofloxacin crystalluria (urine ph 47.3), while no volunteers consuming 1000 mg of ciprofloxacin and nh4cl to acidify urine formed crystals; we postulated that ciprofloxacin uroliths could be dissolved in acidic urine. to test this hypothesis, canine uroliths composed of 100% ciprofloxacin from a single source (6-yr-old male, english bulldog receiving 24 mg/kg of ciprofloxacin po, q12 hr to manage superficial pyoderma; turbulent flow chromatography/tandem mass spectrometry detected 517 mg of ciprofloxacin/g of urolith) were incubated in urine at selected ph's and monitored for dissolution. urine obtained from multiple dogs not receiving fluoroquinolones, was pooled and divided into 5 aliquots. aliquots were adjusted with hcl or naoh to a ph of 3, 5, 6, 7, or 8. aliquots were capped and preserved by refrigeration; ph was monitored and readjusted weekly. ten uroliths of approximately equal weight were randomly assigned to individual flasks containing 10 mls of urine. flasks were constantly agitated and maintained at 381c. every 24 hours, urine was discarded and replaced with 10 mls of urine of identical ph until stone dissolution was complete. ciprofloxacin urolith dissolution times at each urine ph are reported below. ciprofloxacin uroliths are a newly recognized disease and a potential adverse effect of ciprofloxacin administration in dogs. in vitro dissolution of ciprofloxacin uroliths was achieved in canine urine, supporting the premise that in vivo dissolution is possible. urolith dissolution times were shortest at lower and higher ph's, which is consistent with the pka (6.0 and 8.8) of this amphiprotic antimicrobic (more soluble at ph below the acidic pka and above the alkaline pka). foods designed to promote struvite urolith dissolution may be designed for short term feeding facilitating rapid dissolution or may be formulated with a more moderate target urine ph to allow for dissolution and then life-long maintenance feeding minimizing recurrence. the purpose of this study was to compare the efficacy and rate of dissolution of a maintenance food with a struvite dissolution food. sixteen client-owned adult cats (13 fs, 3 mc) with naturally occurring struvite urocystoliths (mineral composition based on history, radiographs, urinalysis, urine culture and physical examination) were randomized to either a dry maintenance food (test) or a dry food known to dissolve struvite uroliths (control). the clinical care team and owner were blinded to treatment assignment. the test food was formulated to provide 0.06% mg (dm), 0.65% p, 35% protein, and a calculated target urine ph value (uph) of 6.2-6.4. the control food was formulated to provide 0.06% mg (dm), 0.77% p, 35% protein, and a targeted urine ph of 5.9-6.1. owners were advised to feed the assigned diet exclusively in an amount to maintain body condition. after diet assignment radiographs were performed at eight weekly intervals until there was no evidence of uroliths or until there was evidence that the uroliths were the same size or larger. a physical examination, complete blood count, serum chemistry profile, urinalysis and urine culture were repeated at the conclusion of the study. statistical analysis was by anova. all uroliths dissolved in all cats and both foods were palatable. radiographs of cats fed the control food indicated the uroliths dissolved in a significantly shorter time (mean ae std dev of 1.6 ae 0.7 weeks) compared to cats consuming the test food (mean 3.9 ae 2 weeks; po0.05).). cats in the control group finished the study at 1 (n 5 4), 2 (n 5 3) and 3 weeks. cats in the test group finished the study 1, 2, 3 (n 5 2), 4, 5, 6, and 7 weeks. all the minnesota urolith center occasionally receives uroliths for analysis that are immersed in formalin. results of quantitative analysis of these uroliths revealed that some submitted in formalin consisted of newberyite (magnesium hydrogen phosphate trihydrate). because newberyite is uncommonly found in uroliths formed by cats and dogs, we hypothesized that this mineral was an in vitro artifact caused by exposure of struvite (magnesium ammonium phosphate hexahydrate) to formalin. the purpose of this study was to determine if formalin alters the mineral composition of uroliths. urolith submissions containing stones of either 100% struvite (n 5 5 dogs and 5 cats), 100% calcium oxalate (n 5 5 dogs and 5 cats), 100% calcium phosphate apatite (n 5 5 dogs and 5 cats), 100% cystine (n 5 5 dogs and 5 cats), 100% ammonium urate (n 5 5 dogs and 5 cats), and 100% silica (n 5 5 dogs) preserved by only air drying were tested. one urolith from each submission was quantitatively analyzed by polarized light microscopy or infrared spectroscopy. a subsequent urolith from the same submission was immersed in 1 ml of 10% buffered formalin for 48 hours at room temperature. uroliths were then air dried for 30 minutes and the analysis was repeated. after exposure to formalin, portions of all 10 struvite uroliths were transformed into newberyite. three (1 dog and 2 cats) of 10 ammonium urate uroliths were completely dissolved. newberyite was not detected in any of the remaining uroliths. likewise quantitative mineral analysis of non-struvite uroliths remained unchanged. to avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis. we previously reported that transfusion to normal dogs of autologous erythrocyte concentrates (prbcs) that had been stored for 21 days causes a profound inflammatory response (2x increase in leucocyte count and fibrinogen, 60x increase in c-reactive protein). we speculated that inflammation was due to cytokines produced during the storage period, and hypothesized that transfusion of fresh (f) prbcs would elicit less inflammation than would stored (s) prbcs. a whole blood unit was collected from healthy dogs (n 5 10) for prbcs on day 0, then again on day 32. on day 35 dogs received an autologous transfusion of prbcs stored for either 35 days (s, n 5 5) or 3 days (f, n 5 5). cbcs and in-tem thromboelastometry (ct:coagulation time, cft:clot formation time, a:alpha, mcf:maximum clot firmness) were evaluated on blood samples collected at 0 (pre) and 5,9,24,48, and 72 hours after transfusion. fresh prbcs did not elicit any change in leucocytes, platelets, or thromboelastometry. stored prbcs elicited a degenerative left shift (5 hr) followed by a regenerative left shift (9-48 hr), thrombocytopenia (36% decrease at 5 hr), and marked hypocoagulability characterized by prolonged ct (5,9,24 hr) and cft (5,9 hr), and decreased a (5,9 hr) and mcf (5,9, 24 hr). data are mean(sd). a: p o 0.05 between groups f and s by t test. b: p o 0.05 compared to ''0'' by rm anova. transfusion of autologous stored prbcs elicits a greater inflammatory response than fresh prbcs, and results in hypocoagulability on thromboelastometry. clopidogrel is a potent antiplatelet drug that is gaining popularity in veterinary medicine for antithrombotic therapy. the parent molecule is an inactive prodrug that must be converted by hepatic isozymes to an active metabolite. the majority of the parent molecule is directed to the formation of inactive metabolites with only an extremely small proportion of parent molecule directed to the formation of the active metabolite. there are multiple hepatic isozymes responsible for the formation of the active metabolite. a non-specific hepatic isozyme inducer such as rifampin could increase the formation of the active metabolite of clopidogrel thereby increasing the pharmacodynamic response which may allow a reduced drug dose to achieve a clinical effect. we have previously presented data supporting the increased pharmacodynamic response of clopdiogrel after rifampin therapy. the goal of this study was to demonstrate an increased pharmacokinetic response of clopidogrel after rifampin induction of hepatic isozymes. six healthy, purpose-bred dogs were used for this study. the pharmacokinetics of clopidogrel were determined by measuring the parent molecule, primary inactive metabolite and active metabolite through lc/ms/ms. the pharmacodynamics of clopidogrel were determined by measuring collagen-induced whole blood aggregation. blood samples were collected prior to clopidogrel administration (baseline), after 7 days of 2 mg/kg clopidogrel po q 24 hrs, and after 7 days of 2 mg/kg clopidogrel po q 24 hrs 1 10 mg/ kg po q 12 hrs rifampin. given the absence of a known standard for the active metabolite, only a semi-quantitative assessment of active metabolite concentration can be made. there was no identifiable active metabolite peak noted at baseline or after clopidogrel treatment. however, with clopidogrel and rifampin combined administration there was an active metabolite peak identified in all dogs with a mean area of 41.7 ae 23.5. the development of the active metabolite peak was associated with an increase in the pharmacodyamic response of clopidogrel in the dogs. this is the first study in any species to document the increased formation of the active metabolite of clopidogrel in response to a strong, non-specific hepatic isozyme inducer. this increased pharmacokinetic response was associated with an increased pharmacodynamic response of clopidogrel. this data provides supportive evidence to develop therapeutic protocols to improve the pharmacodynamic response to clopidogrel in dogs that may reduce dosing requirements or correct subtherapeutic pharmacodynamic response. critical illness-related corticosteroid insufficiency (circi) has been identified in humans, foals, dogs and cats with lower-thanexpected circulating cortisol concentrations, and/or by a blunted cortisol response to acth stimulation. our purpose was to determine if circi exists in critically ill horses. endogenous plasma acth and serum cortisol concentrations, and cortisol at t 5 0 and t 5 30 min after 0.1 mg/kg cosyntropin, were measured by radioimmunoassay from horses with colic or systemic illness on admission, and days 2, 4 and 6 of hospitilization. horses were divided into mild, moderate, or severe illness groups based on clinicopathologic data. inappropriately low cortisol was defined as endogenous cortisol o mean-1sd achieved after administration of 0.1 mg/kg cosyntropin to normal horses ( o 269 nmol/l). inadequate delta cortisol was defined as o mean delta cortisol in normal horses after 0.1 mg/kg cosyntropin ( o 159 nmol/l). cortisol, acth and delta cortisol were compared using anova between groups, with p o 0.05 considered significant. fifty-eight horses classified as having mild (11), moderate (30) and severe (17) disease at admission had survival rates of 100%, 97% and 35% respectively. admission acth and cortisol concentrations were highest in severely ill horses (93 ae 198pg/ml, 361 ae 137 nmol/l) compared to moderate (31 ae 36, 279 ae 137) and mildly ill horses (18.0 ae 12.0, 237 ae 133). admission cortisol concentrations were higher overall in severely ill horses (p 5 0.016), but were low in 24% (4/17). admission delta cortisol was low in 85% (11/13) of severely ill horses, and was associated with marked adrenal hemorrhage in non-survivors. severely ill horses have high cortisol and acth, but low cortisol and delta cortisol may indicate circi secondary to adrenal hemorrhage. equine pituitary pars intermedia dysfunction (ppid) is a common endocrinopathy of aged horses that results from neurodegeneration of the dopaminergic periventricular neurons that innervate the intermediate lobe of the pituitary. factors that initiate spontaneous dopaminergic neurodegenerative disease remain elusive, however accumulation of misfolded a-synuclein protein and dysfunctional protein clearance have been implicated. misfolded protein accumulation occurs due to increased protein production or decreased clearance of damaged macromolecules through the process of autophagy. while have previously demonstrated that horses with ppid have increased asynuclein in the periventricular neurons compared to controls, it remains unknown whether the protein accumulates due to increased production or decreased clearance. we hypothesized that autophagy is decreased in the pituitary neurointermediate lobe from horses with ppid compared to controls. neurointermediate lobe pituitary tissue was from collected from horses with ppid (n 5 12) and healthy horses (n 5 37, 2-35 years). realtime pcr was used to determine the relative expression of autophagy genes (mtor, beclin1, atg12, atg7, atg5, pink, lamp2) and a-synuclein relative gene expression from horses with ppid were compared to healthy horses by t-test following log transformation. a pearson coefficient of correlation was calculated comparing a-synuclein expression with autophagy gene expression. the expression of a-synuclein, autophagy-related genes (atg12, beclin, lamp2), and mtor was greater in horses with ppid than in healthy horses. age was not correlated to a-synuclein or autophagy gene expression. there was a significant positive correlation between expression of a-synuclein and beclin1, atg12, atg7, atg5, and pink, but not mtor expression. accumulation of a-synuclein protein in horses with ppid may result from increased a-synuclein expression. autophagy genes are upregulated in horses with ppid, suggesting a compensatory response, although these findings need to be confirmed by demonstrating an increased functional response. asynuclein expression was positively correlated to expression of autophagy genes except mtor, suggesting a-synuclein may stimulate autophagy in an mtor independent manner. acvim forum session 86a efficacy of delayed antiviral therapy against ehv-1 challenge. lk maxwell 1 , ll gilliam 1 , n pusterla 2 , r carmichael 1 , rw eberle 1 , jw ritchey 1 , tc holbrook 1 , t gull 1 , gb rezabek 1 , d mcfarlane 1 , cg macallister 1 . 1 oklahoma state university, stillwater, ok. 2 university of california, davis, ca. equine herpes virus type-1 (ehv-1) outbreaks are often not recognized until exposed horses are at immediate risk for developing equine herpes myeloencephalopathy (ehm). the objective of this study was to determine whether delayed therapy with the antiviral drugs valacyclovir or ganciclovir could protect those horses most at risk for ehm. eighteen aged ( 4 20 years) mares were randomized to treatment: no therapy (control), oral valacyclovir therapy, or intravenous ganciclovir therapy. drug administration was initiated at the onset of the second febrile phase, between days 4-6 after ehv-1 inoculation (pi), and continued for one week. neurological examinations were performed prior to the study and for three weeks pi. one horse was excluded from the study for failure to become febrile. body temperature was significantly lower in the ganciclovirtherapy horses as compared to control horses on days 6-8 pi (p o 0.05), whereas valacyclovir-therapy horses did not differ from control horses. viremia in whole blood, as determined by pcr, was also lower in the ganciclovir-therapy horses on days 7-10 pi and on day 7 pi in the valacyclovir-therapy horses (p o 0.05). although antiviral drug administration did not reduce the risk of ataxia (p 5 0.06) or nasal shedding, ganciclovir therapy did decrease the severity of ataxia (p o 0.05) as compared to valacyclovir-therapy and control horses, where 0/6, 2/5, and 4/6 horses, respectively, developed at least a two grade change in ataxia. in summary, ganciclovir administration provided better protection against ehm than did valacyclovir when therapy was initiated just prior to the onset of neurological disease. equine vaccination is amongst the most important method of prophylaxis against equine influenza virus (eiv), a pathogen in which continuous antigenic drift can lead to vaccine failure. a 6month duration of immunity (doi) challenge infection study was conducted using commercial inactivated vaccines containing different strains of a/equine/2/influenza virus's, including innovator tm , containing kentucky/97 (pfizer animal health, new york, ny), and calvenza, containing a combination of ohio/2003, kentucky/ 95, and newmarket93 (boehringer ingelheim vetmedica, st. joseph, ms) . the challenge virus strain was colorado/07, the most contemporary challenge strain currently in use. the study design was a blinded, randomized challenge trial. three groups of 10 yearling ponies, with no history or serological evidence of eiv infection were established. each group received one of three treatments: vaccination with innovator tm ; vaccination with calvenza tm ; or injection with a saline placebo. each treatment was administered 3 times, at intervals of 1 month between the first two treatments, and 3 months between the second and third treatments. all ponies were challenged by nasal nebulization of 5x10 7 eid 50 influenza virus a/eq/2/colorado/07 6 months after the third treatment. clinical signs of disease, including rectal temperature, nasal discharge, anorexia, coughing, and depression, were recorded daily for 2 days prior to challenge infection, and 14 days post-challenge. nasal shedding of eiv was measured on the same days, using a realtime pcr test procedure. eiv-specific antibody responses were measured by elisa. differences between groups were analyzed by non-parametric repeated measures anova, and differences were declared significant when p o 0.05. all control group ponies demonstrated clinical signs of disease consistent with eiv infection post-challenge infection, including pyrexia, nasal discharge, inappetance and partial anorexia. these signs were significantly lower in both vaccine groups; mean body temperature was elevated ( 4101.51f) for 8 days in controls, but only 2 days in vaccine groups. nasal shedding of eiv was detected in all ponies in all groups: over the duration of the study the calvenza group shed significantly less virus than innovator and control. over time antibody titers were significantly higher in the calvenza than the innovator group, and both were significantly greater than controls. this study demonstrated that both current commercial inactivated eiv vaccines have a duration of clinical protection of at least 6 months after a highly pathogenic challenge with a recent eiv isolate. both antibody responses and virological protection differed between the vaccines. formulation difference between the vaccines, including the eiv antigens employed, may have contributed to this performance difference. degenerative myelopathy (dm) may be homologous to a form of amyotrophic lateral sclerosis in humans which has excitotoxic and immunologic pathogeneses described. the aims of this study were to determine (i) presence or absence of abnormalities in concentrations of csf amino acid (aa) neurotransmitters (glutamate, glycine and gàaminobutyric acid (gaba)) and cytokines in dogs with dm and if present (ii) investigate associations with disease severity. twenty-two dogs histopathologically confirmed for dm and 21 dogs with suspected dm based on thorough diagnostic investigations and 42 clinically normal age-matched control dogs were included in the study. the neurological severity of the dm dogs was graded (1-4) using an established scale. csf was evaluated for presence of glutamate, glycine and gaba by high performance liquid chromatography and for gm-csf, ifn-g, il-2, il-4, il-6, il-7, il-8, il-10, il-15, il-18, ip-10, kc (keratinocyte chemoattractant), mcp-1 (monocyte chemotactic protein-1) and tnf-a using a commercially available, canine multiplex immunoassay (millipore, billerica, ma). all data analyses were performed using sas v 9.2 (cary, nc). analyte levels were compared between dm confirmed, dm suspected and control dogs by an analysis of variance (anova). spearman correlation was used to test for correlations of analyte levels and neurological grades. all hypothesis tests were 2-sided with a 5 0.05. there were no significant differences between individual csf analytes in dm confirmed and dm suspected dogs. glutamate levels were not significantly different between dm affected (mean 5 0.09 mg/ ml; range 5 0.01-0.29; sd 5 0.069) and control dogs (mean 5 0.07 mg/ ml; range 5 0.01-0.29; sd 5 0.056). control dogs (mean 5 0.86 mg/ml; range 5 0.08-1.16; sd 5 0.213) had significantly higher levels of gaba (p o 0.0001) than dm dogs (mean 5 0.12 mg/ml; range 5 0.07-0.79; sd 5 0.12). control dogs (mean 5 2.14 mg/ml; range 5 0.01-3.83; sd 5 0.76) also had significantly higher glycine concentrations (p o 0.0001) than dm dogs (mean 5 0.45 mg/ml; range 5 0.01-0.98; sd 5 0.24). dm-affected dogs also had significantly higher levels of il-2 (p 5 0.03), kc (p o 0.0001) and mcp-1 (p 5 0.005) than control dogs. neurotransmitter levels were not significantly associated with neurological grade. kc levels were significantly higher in the least affected dogs (p 5 0.0015). there were no associations with disease severity and analyte concentrations. dm affected dogs have an imbalance of csf aa concentrations creating a relatively excitotoxic environment. reports in human als confirm an imbalance between csf excitatory and inhibitory aas suggesting a pathogenic role for excitotoxicity in als. it also appears that dm affected dogs have increases in csf cytokines and chemokines suggestive of an immunologic component to the pathogenesis as is similar to als. further prospective analysis of dm is warranted to evaluate the role of treatment on csf variables. the pathogenesis of neuropathic pain (np) and syringomyelia (sm) in association with chiari-like malformation (clm) in dogs has focused on the anatomical anomalies and secondary cerebrospinal fluid (csf) flow abnormalities. neuropathic pain in humans has been associated with abnormalities of neurotransmitters such as glutamate and serotonin as well as immunologic mechanisms. the aim of this study was to investigate the csf neurotransmitter and cytokine levels in brussels griffon dogs (bgs) with clm, sm and np. as part of an mri study investigating the prevalence of sm in bgs, atlanto-occipital csf was acquired from 46 dogs and stored at -80c until analysis. all dogs underwent a neurologic exam prior to mri; osirix s software was used to measure sm and the presence of cerebellar herniation and deviation were recorded. deproteinized csf samples were analysed for presence of serotonin (ng/ml), glutamate, glycine and gaba (mg/ml) by high performance liquid chromatography. all csf samples were evaluated simultaneously for gm-csf, ifn-g, il-2, il-4, il-6, il-7, il-8, il-10, il-15, il-18, ip-10, kc, mcp-1 and tnf-a. a commercially available, canine multiplex immunoassay (millipore, billerica, ma) was used for the cytokine analysis (pg/ml). student's t-tests were used to compare the means of neurotransmitter and cytokine values between groups with and without skull abnormalities or spinal pain. simple pearson's correlation was used to test for correlations of neurotransmitter and cytokine values with syrinx dimensions and correlations of neurotransmitter with cytokine values. all hypothesis tests were 2-sided and the significance level was a 5 0.05. np was detected in 8 dogs (17%); sm was present 24 dogs (52%); and cm was detected in 24 dogs (52%). ifn-g levels were significantly lower in dogs with np than without (p 5 0.036). there were significant positive correlations between syrinx size and il-8 (p 5 0.017), kc (p 5 0.025) and mcp-1 (p 5 0.003). there were significant negative correlations between ifn-g and syrinx height (p 5 0.025) and extent (p 5 0.042). there was a significant negative correlation between il-2 and syrinx height (p 5 0.042). neurotransmitter levels were not associated with skull abnormalities or spinal pain, but there was a positive correlation of glycine with il-2 (p 5 0.004) and mcp-1 with glutamate (p 5 0.0147) and serotonin (p 5 0.0059). the size of the syrinx in bgs with sm is associated with several cytokine elevations but only a decrease of ifn-g was associated with np. based on this study it does not appear that excitotoxicity plays a role in either sm development or np. further work is justified on the role of the immune system in cm, sm and np. current knowledge about the conservative management of disk associated cervical spondylomyelopathy (da-csm) is rather limited and mainly based on retrospectively retrieved data. the goals of this study were to prospectively evaluate the evolution of clinical signs in dogs treated conservatively for da-csm. additionally, several potential prognostic parameters and the correlation of initial clinical signs with magnetic resonance imaging (mri) and transcranial magnetic stimulation (tms) were investigated. twenty-one dogs were included. after neurological evaluation, neurological status was graded from 0 (5 normal) to 6 (5 tetraplegia). all animals underwent low-field mri and tms with measurement of onset latencies and peak-to-peak amplitudes from the extensor carpi radialis and cranial tibial muscles. from the mr images, the following dimensions were calculated: remaining spinal cord area; compression ratio; vertebral occupying ratio of the spinal cord; canal height to body height ratio (cbr); canal height to body length ratio (cblr); and the canal compromise ratio. intraparenchymal intensity (isi) changes were graded from 0 to 3. all dogs were reevaluated by the same person after 1, 3, 6, 12, and 24 months. eight of 21 dogs (38%) experienced a positive clinical evolution with improvement of clinical signs or stabilization of mild clinical signs. all dogs with a negative clinical evolution 1 month after diagnosis experienced a further progression of clinical signs resulting in a poor outcome. the opposite was true for all dogs with a positive clinical evolution after 1 month. outcome was further significantly associated by the remaining spinal cord area and the vertebral canal compromise ratio. prognosis was not significantly affected by clinical presentation or tms. progression of clinical signs, in unsuccessfully treated dogs, was generally characterized by a rapid and dramatic deterioration of neurological status. there were no significant correlations between clinical presentation, mri and tms. two dogs underwent necropsy and histopathological examination. this revealed in both cases chronic wallerian degeneration and segmental myelomalacia. the results of this study suggest that conservative treatment of da-csm is associated with a rather guarded prognosis. clinical evolution 1 month after diagnosis and selected mri parameters can be considered as prognostic indicators. the lack of correlation between clinical presentation and outcome, medical imaging and electrophysiological evaluation is disturbing and warrants further investigation. a mri-guided stereotactic brain biopsy system has not been clinically evaluated in dogs. the purpose of this study was to determine the ability of the brainsight tm system to obtain histologically diagnostic samples and access the impact of this procedure on neurologic status for 72 hours after the biopsy. five dogs with mri definable lesions in the brain have been enrolled. breeds included a pitbull mix, pembroke welsh corgi, french bulldog, border terrier and west highland white terrier. age ranged from 5-11 years. weight ranged from 8.5-18.8 kg.dogs presented with seizures (n 5 5), ambulatory paresis(n 5 4), unilateral blindness(n 5 2) and head tilt(n 5 1). one dog had a normal neurologic exam. lesions chosen for biopsy were in the olfactory and/or frontal lobes (n 5 3), parietal lobe(n 5 1), and pyriform lobe(n 5 1). lesions were between 14-22 mm in diameter. all lesions were well-circumscribed and contrast enhancing except for one. histologic diagnosis of meningioma(n 5 3) and granulomatous meningoencephalitis(n 5 1) were made. the poorly-circumscribed, non-contrast enhancing frontal mass yielded non-specific necrosis. following biopsy, three dogs returned to pre-biopsy neurologic status within 12 hours. the french bulldog took 48 hours to return to previous neurologic status due to brachycephalic syndrome that required oxygen support. one dog had acute respiratory arrest 16 hours post-biopsy. necropsy is pending. these results suggest that this mri-guided biopsy system can provide an accurate histologic diagnosis of brain lesions. biopsies of poorly-circumscribed and non-contrast enhancing brain lesions may be less diagnostic. further evaluation is on-going to determine the true diagnostic yield and complication rate of this procedure. concurrent malformations of the craniocervical junction are commonly identified in humans with chiari type i malformation. recent evidence suggests such craniocervical junction abnormalities (cjas) also occur in dogs suspected of having chiari-like malformation (clm). the purpose of this study was to objectively describe morphometric features of the craniocervical junction region of dogs with suspected clm and to investigate for associations between these features and the occurrence of other malformations in this region. magnetic resonance (mr) and computed tomographic (ct) images from 274 dogs with clm were evaluated. three regions of neural tissue compression were assessed: cerebellar compression (cc); ventral compression at the c1/c2 articulation, termed ''medullary kinking'' (mk); and dorsal compression (dc) at the c1/c2 articulation. a compression index (ci) was calculated for all abnormal regions for each dog. multiple logistic regression analysis was performed (p o 0.05) to ascertain whether ci values for the different regions of compression were associated with the incidence of other craniocervical junction abnormalities. 68% of dogs had mk and 38% of dogs had dc. 28% of dogs also had evidence of atlanto-occipital overlapping (aoo medical infrared imaging (mii) is a non-invasive diagnostic imaging technique that measures skin surface temperature and generates thermal pattern maps based on predetermined color scales. because skin temperature, dependent on regional perfusion, is under direct control of the sympathetic nervous system, mii provides information about the function of the autonomic nervous system. because of recent advances in technology and lack of sedation needed to image patients, mii has potential use as a screening test for a variety of conditions that may result in autonomic dysregulation like chiari-like malformation in dogs (clm). the purposes of this study were to establish a mii protocol for dogs suspected of having clm, to identify thermal imaging patterns for various regions of interest (roi), to evaluate changes in thermal patterns and compare the results to those of mri findings, considered the standard for diagnosing clm in dogs. one hundred and five cavalier king charles spaniel dogs with clinical signs attributable to clm and confirmed clm with mri were evaluated with a complete blood count and chemistry profile, examination by a board certified surgeon/neurologist, multidetector ct scan of the craniocervical junction, whole body mri and mii. the protocol for thermal imaging included cranial and caudal views of the body, full lateral right and left body views, dorsal views of the head and body, and right and left lateral views of the head. thermal patterns were assessed with custom image recognition software. after each dog was imaged awake, general anesthesia was administered and the dogs re-imaged using the same protocol. mri findings in dogs with severe or moderate cerebellar compression and cerebellar herniation were compared with mii results. the top of head and front of head roi were 89.2% and 97.3% successful in identifying dogs with clm. based on these preliminary findings, mii may be a viable screening tool to detect clm in dogs. medical infrared imaging (mii) is an imaging technique that measures skin surface temperature derived from cutaneous perfusion and generates thermal pattern maps based on color scales. mii has been used as a test for a variety of conditions that cause autonomic dysregulation resulting in altered cutaneous perfusion. acute thoracolumbar intervertebral disk disease (tlivdd) is common in dogs. the purpose of this study was to: 1) determine the success of mii in identifying dogs with tlivdd, 2) compare the mii localization with mri results and surgical findings 3) determine if the mii pattern returns to that of normal dogs following decompression surgery. 72 small breed chondodystrophic dogs with tlivdd confirmed with mri and 14 dogs with no tlivdd were evaluated with mri and mii. regions correlating with the intervertebral disk spaces were analyzed for average temperatures and thermographic patterns. thermal patterns were assessed with computer recognition pattern analysis (crpa) software. 21 dogs were re-evaluated 8 weeks after surgery using the same protocol. when analyzing temperature averages over a region, no significant difference was found between control and affected dogs. crpa was 90% successful in differentiating normal from affected dogs. crpa was 97% successful in identifying the intervertebral disk space when compared with mri and surgical findings. based on these findings, mii may be a viable screening tool to detect tlivdd in dogs. microglia physiologically shows regional topographical differences in immunophenotype and function within the central nervous system indicating the endowment for a prompt response to pathological stimuli such as trauma. spinal cord injuries (sci) consist of a primary injury encompassing the mechanical impact and the ''secondary wave'' of injury occurring minutes to weeks later and comprising various consecutive effects such as increased production of free radicals, excessive release of excitatory neurotransmitters and inflammatory reactions. activated microglia has the potential to perform some of these reactions, their contribution to the secondary wave is therefore controversially discussed. it has to be considered a double-edged sword as both, beneficial and deleterious effects have been attributed to these cells. the purpose of the presented study was to assess microglial involvement, particularly in the ''secondary wave'' following sci. microglia from 15 dogs with sci was isolated and characterized ex vivo in terms of morphology, immunophenotype, and function by flow cytometry. the results were compared to region-specific findings obtained from healthy control dogs (n 5 30). the histopathological exam confirmed the diagnosis of sci in the cervical (n 5 5) and thoracolumbar (n 5 10) spinal cord, and revealed a significant activation of microglia/ macrophages and upregulation of myelinophagia in dogs with sci 5 days or longer prior to euthanasia. microglial ex vivo examination showed significantly increased expressions of b7-1, b7-2, mhc ii, cd1c, icam-1, cd14, cd44, and cd45, and significantly enhanced phagocytosis and generation of reactive oxygen species (ros) in sci compared to healthy controls. microglial cells seem to be highly activated following sci with an immunophenotype indicating their active role in co-stimulation of t cells, in leukocyte adhesion and aggregation, and in lipid and glycolipid presentation. microglial phagocytosis might play a pivotal role in removal of injured or damaged cells and initialize subsequent healing processes. however, as ros can be directly neurotoxic an enhanced microglial generation might lead to bystander damage of the traumatized spinal cord and might therefore add to the deleterious effects of the secondary wave. modulating the microglial response in sci might be a valuable novel therapeutic strategy alleviating further damage to the spinal cord. thymidine kinase (tk) is a soluble biomarker present in s-phase of a salvage pathway for dna synthesis, and can be measured in serum. tk activity correlates with stage, prognosis, and relapse in canine and human lymphoma. we previously reported the results of a pilot study evaluating tk activity in archived canine osteosarcoma, transitional cell carcinoma, and hemangiosarcoma (hsa) sera, and found elevated tk activity in 80% of canine hsa sera evaluated. the purpose of this study was to prospectively evaluate serum tk activity in a large number of dogs presenting to emergency clinics with hemoabdomen and a splenic mass, to determine if tk activity could be used as a noninvasive means to distinguish hsa versus benign conditions in this population. dogs presenting with hemoabdomen and a splenic mass identified on ultrasound examination were studied. serum was collected prior to anesthesia, euthanasia or surgical intervention and frozen until batch analysis. tissue from all patients was evaluated histologically by a single pathologist. sera from age-matched normal dogs comprised a control population. an elisa using azidothymidine as a tk1 substrate was used. comparisons between groups were made using 2-tailed student t-tests, and receiver-operator characteristic (roc) curves were generated. sixty-two patients and 39 normal controls were studied. there were 35 dogs with hsa, 10 dogs with other splenic neoplasia, and 17 dogs with benign diseases. using a training set of 24 normal dogs, a cutoff of 6.55 u/l was established from the roc curve. tk activity was significantly higher (p o 0.0001) in dogs with hsa than in the validation set of 15 normal dogs (mean1/àsd 5 17.711/à4.5 and 2.011/à0.6 respectively), but not between dogs with hsa and benign splenic disease (mean1/àsd 5 7.021/à3.7, p 5 0.13). using a cutoff of 6.55 u/l, tk activity demonstrated a sensitivity of 0.54, specificity of 0.76, positive predictive value of 0.83 and negative predictive value of 0.45 for distinguishing hsa versus benign splenic disease. when interval thresholds of o 1.55 and 47.95 u/l were used together, diagnostic utility was markedly increased for distinguishing both hsa versus normal and hsa versus benign disease. in conclusion, serum tk evaluation may assist in detection of canine hsa, and may also discriminate between benign disease and hsa in dogs with hemoabdomen and a splenic mass. t cell chronic lymphocytic leukemia (cll) is a heterogeneous disease that affects a number of dog breeds. cll patients have variable disease outcomes. the objectives of this study were to use gene expression profiling of cd8 t cell leukemias with variable outcomes in order to identify markers that can be used in routine diagnostic tests to distinguish good from poor prognosis disease, and to identify potential targets for novel therapy. gene expression profiling of 12 cd8 t cell leukemias (7 good, 5 poor prognosis) was conducted. samples from 6 normal dogs were also profiled. several differentially expressed genes were found including cd9, cd94, and cd 25. these were selected for further study using flow cytometry to determine expression of protein on the cell surface. seventy nine cases of cd81 t cell leukemia were screened for cd 9 expression. forty seven had associated outcome information. based on analysis to date, cd9 expression as assessed by flow cytometry does not appear to provide prognostic information. a monoclonal antibody to cd25 was recently made available. to date 33 patients with cd8 t cell leukemia have been profiled. cd25 is variably expressed on t cell leukemias compared to normal cd8 t cells. cd25 is the receptor for interleukin 2. cyclosporin, a commonly used immunosuppressive drug, inhibits il-2 production, and has been used to treat a subset of t cell leukemias in people. thus, the finding that cd25 is up regulated on t cell leukemias compared with normal t cells suggests a possible new therapeutic avenue. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of dogs. there is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (ibd). similarly, compositional changes of the intestinal bacterial ecosystem have been associated with ibd in humans. the aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders using a next generation sequencing technique. fecal samples were obtained from healthy dogs (n 5 31), dogs with acute uncomplicated diarrhea (n 5 7), dogs with acute hemorrhagic diarrhea (ahd; n 5 13), and dogs with active (n 5 8) and therapeutically controlled ibd (n 5 10). the bacterial composition was analyzed by massive parallel 16s rrna gene 454-pyrosequencing. differences between groups were analyzed using mann-whitney u tests and kruskal-wallis tests followed by dunn's multiple comparison tests. statistical significance was set at p o 0.05. significant differences in the proportions of several bacterial groups were identified between healthy and diseased dogs. dogs with gastrointestinal disease had significantly higher proportions of proteobacteria (p o 0.01). proportions of firmicutes were lower in diseased dogs, but this difference did not reach significance (p 5 0.06). within the firmicutes the most notable findings were decreases in bacterial groups belonging to clostridium clusters iv and xiva (i.e., ruminococcus, dorea, and faecalibacterium spp.; p o 0.01 for all). dogs with ahd had the most profound changes of the microbiota, followed by dogs with acute uncomplicated diarrhea, and dogs with active ibd. faecalibacterium spp. was the bacterial group most prominently depleted in dogs with active ibd, but was not significantly different between healthy dogs and dogs with therapeutically controlled ibd (p 5 0.66). results of this study revealed bacterial dysbiosis in fecal samples of dogs with various gi disorders. bacterial changes were more profound in dogs with severe disease, but were not identified in dogs with therapeutically controlled ibd, suggesting that the microbiota is stable in non-active disease. the bacterial groups identified are considered to be important short chain fatty acid producers and may serve as candidates for the diagnosis or therapeutic monitoring of gi disease. future studies are necessary to determine if these microbial changes correlate with functional changes in the intestinal microbiota. ciprofloxacin oral tablets, available in a generic formulation for people, are widely used for treatment in dogs. oral absorption data for ciprofloxacin in dogs has been variable, and too limited to guide accurate dosing. subsequently, published doses for dogs in veterinary formularies have varied from 5 to 25 mg/kg. this study was undertaken to explore the factors that may affect oral absorption of generic ciprofloxacin in dogs, and to derive a pharmacokinetic-based dose for treating susceptible bacteria. six healthy adult beagle dogs were used for the study (11.2 kg mean weight). after placing jugular vein catheters for collecting blood samples, these dogs were administered either a single oral dose of ciprofloxacin (250 mg tablet; mean dose 23 mg/kg), or an intravenous (iv) dose (10 mg/kg; 2 mg/ml solution). a randomized crossover design was used with a washout time between treatments. blood was collected for plasma drug analysis for 24 hours. ciprofloxacin concentration in plasma was analyzed using high pressure liquid chromatography (hplc) and pharmacokinetics analyzed using a computer program. oral absorption was also evaluated via deconvolution analysis. the oral dose was well-tolerated, but the iv dose produced transient vomiting and depression in some dogs. after the oral dose, the peak plasma concentration (c max ) was 4.4 mg/ ml (cv 55.9%), terminal half-life (t1/2) 2.6 hr (cv 10.8%), auc 22.5 mg á hr/ml (cv 62.3%), and systemic absorption (f) 63.7% (cv 41.6%). after the iv dose, the t1/2 was 3.7 hr (cv 52.3%), systemic clearance 0.587 l/kg/hr (cv 33.9%), and volume of distribution 2.39 l/kg (cv 23.7%). after examining the pharmacokinetic results from the oral dose, it was apparent that oral ciprofloxacin was absorbed well in some dogs (approximately 80%), but poorly in others (approximately 30%). to explore the factors that may have affected oral absorption, two high absorbers and two low absorbers were administered an additional oral dose as a 10 mg/ml solution (250 mg total dose) via gastric tube. after administration of the oral solution, the plasma concentrations were more uniform and consistent among dogs. absorption of the oral solution of ciprofloxacin was 71.0% (cv 7%) with a t1/2 of 3.1 (cv 18.6%) hr and c max of 4.67 mg/ml (cv 17.6%). therefore, it appears that inconsistent oral absorption of ciprofloxacin in some dogs may be formulation-dependent, and affected by tablet dissolution in the canine small intestine. doses were calculated using the data for oral tablets in these dogs. the pharmacokinetic-pharmacodynamic (pk-pd) target was an auc/ mic ratio of 100. because of the wide range in oral absorption of tablets, a dose to reach the pk-pd target ranged from canine distemper (cd) is a highly contagious, acute or subacute systemic viral disease of dogs and other carnivores which can be controlled efficiently by the use of modified live-virus (mlv) vaccines. however, mlv strains do cross-react with molecular diagnostic tests and cause significant confusion for clinicians. the purpose of this study was to use quantitative real-time pcr viral load information to differentiate between vaccine virus used in mlv vaccines and wildtype infections in dogs. a real-time pcr test for cd virus (cdv) based on the p gene for phosphoprotein was used to determine viral loads in vaccinated and wildtype infected animals. a total of 158 respiratory mucosal swab samples from mlv vaccinated and asymptomatic dogs were obtained within the first 3 weeks after mlv vaccination. based on the viral load in vaccinated animals, a cutoff value was established for the differentiation of dogs with clinical signs of respiratory distress and presumably infected with a wildtype strain of cdv. two hundred clinical cases with known clinical and vaccination histories were analyzed to validate the cutoff value. the cdv real-time pcr proved to be of high analytical and diagnostic sensitivity: a standard curve was established using known numbers of cdv molecules to allow absolute quantitative cdv viral load data. the limit of detection was in the single molecule range while the limit of quantitation was established at around 10 molecules per pcr reaction. a comparison to ifa showed real-time pcr to be 30% more sensitive. the cdv viral load in vaccinated animals averaged 26,738 viral particles per swab. a cutoff value of 107,903 viral particles was calculated by adding 3 standard deviations to the average value. this cutoff value correctly detected 95.2% of the vaccinated samples. acutely infected dogs with cdv compatible clinical signs have high viral loads normally several logs higher than the cutoff value. in dogs with clinical distress, recent cdv mlv vaccination but viral loads below the cutoff value, other infectious agents were detected by using a panel of real-time pcr tests. testing additional infectious agents in clinical settings is important in order to explain clinical signs when viral loads below cutoff values indicate that cdv is not the cause of clinical signs. in conclusion, quantitative real-time pcr is a sensitive, rapid and reliable test regardless of recent vaccination. the use of a cutoff value will be of significant help to discriminate between vaccine interference and wildtype infection in clinical settings. feline ureteral obstructions are a common urinary dilemma and traditional therapy is associated with substantial morbidity/mortality. feline nephrostomy tubes are reported as being effective when pelvic drainage is required. the biggest limitation is externalized drainage, requiring careful management to prevent infection/dislodgement. the development of an indwelling ureteral bypass using a combination locking-loop nephrostomy/cystostomy tube was modified from humans, resulting in permanent indwelling drainage, reduced complications, and improved quality of life. the objective is to describe the technical and clinical outcome using a novel device called a subcutaneous ureteral bypass (sub) in cats with ureteral obstructions. fifteen cats (16 kidneys) had a sub placed for: ureterolithiasis (9), ureteral stricture (1/à stones) (6), and ureteral stent rejection (2). the median pre-and post-procedure creatinine was 6 mg/dl (range: 2.8-13) and 2.5 mg/dl (range: 2.4-9), respectively. the median pelvis diameter pre and post-procedure were 15 (range: 7-28) and 5 mm (range 3.4-6), respectively. six french tubes were placed in 14, and 5 fr. in 2. the bypass remained indwelling for a median of 4240 days (range 64-4450). there were 3 major complications resulting in nephrostomy tube dislodgement (2) and port leakage (1) 5 days after surgery. one patient with severe coagulopathy developed a clot which resolved with tpa infusion through the port. no sub got occluded/obstructed long-term. overall, the use of a sub for cats with ureteral obstructions can be considered a functional option when other therapies have failed or are contraindicated, but shtime. oxidative stress is considered central to the pathogenesis of many systemic diseases. in humans, biomarkers of oxidative stress, antioxidant depletion and lipid peroxidation, have been correlated with disease severity and associated with poor clinical outcomes. therapeutic antioxidant supplementation with nac in glutathione (gsh)-deficient patients has shown clinical benefits, including repletion of intracellular gsh levels. we have shown that clinically ill dogs are gsh-deficient, and that gsh deficiency correlates with mortality, but it is not clear whether there are direct benefits of antioxidant intervention in these patients. the purpose of this randomized, investigator-blinded, placebo-controlled, prospective study was to evaluate the effect of nac to normalize blood antioxidants (rbc reduced gsh (rbc gsh), plasma cysteine (cys), serum vitamin e (vit e), and whole blood selenium (se)), reduce lipid peroxidation (urine isoprostane/creatinine ratio (u i/ c)), and improve illness scores (spi2) and outcome (survival to discharge) in clinically ill dogs. clinically ill client-owned dogs, admitted to the uw veterinary medical teaching hospital that did not receive blood transfusions, tpn, vitamins, or antioxidants were eligible for the study. dogs enrolled in the study were randomized to receive iv infusions q. 6 h. of either nac (1 â 140 mg/kg and 6 â 70 mg/kg) or equal volumes of 5% dextrose (placebo) over 48 hours. at the time of enrollment, and 2 hours following the final 48 hour infusion, blood and urine were collected to quantify rbc gsh, cys, vit e, and se concentrations; u i/c ratios; and calculate spi2 scores. rbc gsh and cys concentrations were quantified by hplc. commercially available hplc, atomic absorption spectroscopy, and eia were used to quantify vit e, se, and u i/c ratios, respectively. nonparametric statistical analyses were used, with results reported as medians and p o 0.05 considered significant. sixty-one ill dogs were randomized to either nac (n 5 30) or placebo (n 5 31). overall this group of ill dogs had significantly decreased rbc gsh (1.50 vs. 1.91 mm; p 5 0.0013), vit e (27 vs. 56 mg/ml; p 5 0.0002), and se (0.37 vs. 0.55 mg/ml; p 5 0.0308) levels and elevated u i/c ratios (969 vs. 398 pg/mg; p 5 0.0005) in comparison to healthy control dogs. dogs in the placebo group showed a significant further decrease in rbc gsh over the next 48 hours (1.48 to 1.42; p 5 0.035). nac supplementation significantly increased plasma cys levels (9.1 to 15.1 mm; p o 0.0001), and prevented a further decline in rbc gsh (1.55 to 1.58 mm; p 5 0.174). however, serum vit e (30 vs. 28 mg/ml), se (0.40 vs. 0.35 mg/ ml), u i/c ratios (946 vs. 806 pg/mg), spi2 scores (0.74 vs. 0.75), and outcome (81% vs. 76%) were not significantly different between the nac and placebo groups after treatment. the results of this study further support that clinically ill dogs experience oxidative stress, and suggest that antioxidant supplementation with nac within the first 48 hours of hospitalization prevents further rbc gsh depletion. further studies are necessary to investigate whether longer duration or combined antioxidant supplementation normalizes the redox state and impacts long-term outcome. diabetes mellitus in cats is very similar to type ii diabetes in humans, preceded by a period of insulin resistance. evaluating insulin resistance in a cat is a time consuming, expensive, and difficult procedure. there is a need for a simple biomarker based test predictive of insulin resistance. there is a biomarker based assay predicative of insulin resistance in humans. the purpose of this study was to evaluate the utility of this assay in overweight cats and show improvement in insulin sensitivity following weight loss and weight maintenance. the insulin resistance assay is based on the quantitative analysis of 5 metabolites (2-hydroxybuterate, creatine, palmitate, decanoylcarnitine, and oleoyl-lpc). a proprietary algorithm (metabolon, inc, durham nc) was used to generate a predictive rd (rate of disposal) value (normal range in cats 6.5-10.5). individuals with an rd value less than 6 will have a greater than 50% chance of being insulin resistant and an rd value less than 3 will have a greater than 90% chance of being insulin resistant. initial studies demonstrated that the rd values indicating insulin resistance in cats correlated with age, obesity and severity of diabetes as determined by histopathology and blood glucose levels. in a feeding study of 40 cats (4 39% vs. o 25% body fat) rd values improved from 6.19 ae 1.23 to 6.8411.38 (p 5 0.029). during weight maintenance, 19% body fat for 4 months, further improvement was observed (rd, 8.30 1 1.08 (p 5 9.2e-12)). these results demonstrate that long term weight maintenance following weight loss is critical for increasing insulin sensitivity in cats. the use of monoclonal antibodies and antibody fragments to directly target tumor antigens and neutralize their growth factors has shown promising results in human clinical trials. however, these targeted approaches have not been possible in dogs since specific tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses. to overcome these obstacles, we have generated canine antibody phage display libraries from canine splenocytes. these libraries consist of single chain variable fragments (scfv) comprised of canine variable heavy (vh) and variable light (vl) immunoglobulin chains displayed on the surface of bacteriophage (fig. 1) . the antigen specificity within these libraries is diverse and recapitulates the antigen-experienced immunoglobulin repertoire of the dog. we can now use simple panning techniques to isolate scfv of canine origin that bind to either known targets or unknown targets which can then be identified using standard molecular techniques. canine hsa is a highly aggressive malignancy of vascular endothelial cells that affects large breed dogs. although there are no confirmed immunological targets for hsa, serum levels of vascular endothelial growth factor (vegf) are elevated in these patients and, as in many human cancers, vegf may represent an important therapeutic target for neutralization. we used simple panning techniques to screen canine scfv libraries generated from the spleens of dogs with hsa against canine vegf and successfully isolated 3 scfv clones that bind and neutralize canine vegf in vitro. these scfvs are now being taken into a murine model of canine hsa to determine whether they can inhibit tumor growth and metastases. in addition, we have panned the same antigen-experienced scfv phage display libraries against allogeneic primary canine hsa cells of low passage number to isolate canine-derived antibody fragments that can target malignant endothelial cell surface molecules. early results demonstrate enrichment of scfv phage libraries for malignant endothelial cell binders. these scfv can be readily linked to chemotherapeutic agents or other toxins and used to deliver high doses directly to the malignant cell. this novel approach aims to reduce side effects of systemic chemotherapy and augment therapeutic response. calcitriol, (vitamin d 3 ), has antineoplastic activity and acts synergistically to potentiate the antitumor activity of a diverse array of chemotherapeutics. ccnu, vinblastine, corticosteroids, and tyrosine kinase inhibitors, are used to treat canine mast cell tumors (mct). vitamin d receptor is expressed in the majority of canine mcts, suggesting a role for calcitriol in the management of dogs with these tumors. the purpose of our study was to examine the in vitro effects of calcitriol in combination with ccnu, vinblastine, imatinib, or toceranib on canine mastocytoma c2 cells. also, we evaluated the antitumor activity of dn101, a highly concentrated oral formulation of calcitriol, as single-agent treatment in dogs with naturally occurring mcts. c2 cells were incubated with serial dilutions of calcitriol (0.1-25 nm). twenty-four hours later, cells were then treated with vehicle control or serial dilutions of ccnu (2.5-20 um), vinblastine (1.25-10 nm), imatinib (1.67-12.5 nm), or toceranib (3.13-25 nm). cell viability was assessed with an mtt assay after 48 hours and data was used to derive a combination index (ci: values o 1, 1, 41 indicate synergism, additivity, antagonism, respectively). in the phase ii clinical trial, dogs were eligible if they had at least 1 measurable, histologically confirmed, mct. calcitriol was administered orally. recist criteria were used to assess tumor response. calcitriol, ccnu, vinblastine, imatinib, and toceranib each suppressed c2 cell viability in a dose-dependent manner. ci values o 1 were obtained for calcitriol (0.1-6.25 nm) combined with ccnu (5 and 10um), vinblastine (2.5 and 5 nm), imatinib (1.67-12.5 nm) and toceranib (0.1-12.5 nm). due to the occurrence of toxicity (vomiting, anorexia, hypercalcemia), the phase ii trial was terminated early; only 10 of 20 planned patients were treated. one dog with a metastatic muzzle mct had a complete response that lasted 89 days. three dogs achieved partial response lasting from 74-90 days. in summary, our in vitro data demonstrate that calcitriol combined with ccnu, vinblastine, imatinib or toceranib has synergistic effects on c2 mastocytoma cells. antitumor responses were observed in dogs with spontaneously occurring mcts treated orally with single-agent calcitriol, but the frequency of adverse effects was high. together these results suggest calcitriol combination therapies might have significant clinical utility in the treatment of canine mcts but refinement of the calcitriol-dosing regimen must be done. cyclosporine is a potent immunosuppressive agent used to treat many canine inflammatory and immune-mediated diseases. cyclosporine has gained popularity as an immunosuppressive agent because of a favorable toxicity profile compared to many other immunosuppressive agents. optimal dosing regimens for cyclosporine in the dog remain unclear, primarily because standard methods that monitor effectiveness of immunosuppression have not been established. pharmacokinetic testing is currently used during treatment with oral cyclosporine to adjust doses based on measurement of blood drug levels. individual patients, however, often demonstrate marked variations in blood drug levels while on similar oral doses of cyclosporine, and can also demonstrate different clinical responses even at comparable drug levels, making correlation of blood cyclosporine levels and degree of disease control extremely difficult. pharmacodynamic testing offers an alternative method for regulating cyclosporine dosing by objectively measuring the effects of cyclosporine on t-cells, the drug's main cellular target in the body. our acvim foundation-funded research has focused on developing and evaluating a comprehensive panel of biomarkers of immunosuppression that can be utilized for pharmacodynamic monitoring during treatment with cyclosporine and other immunosuppressive agents that affect t-cell function. we have completed several studies using flow cytometry to evaluate activated t-cell expression of surface molecules (cd25 & cd95) and cytokines (il-2, ifn-g & il-4) as potential biomarkers. our first study was an in vitro study evaluating expression of surface molecules and cytokines in canine t-cells exposed to varying concentrations of cyclosporine. this study established consistent drug-associated suppression of the cytokines il-2, ifn-g and il-4. our second study was an in vivo study in normal dogs evaluating the effects of two doses of oral cyclosporine, a high dose considered to be reliably immunosuppressive (starting dose 10 mg/kg bid, titrated upwards as needed to attain trough drug blood levels of at least 600 ng/ml) and a lower dose used to treat atopy (5 mg/kg sid), on t-cell expression of these three cytokines. significant suppression of il-2 and ifn-g expression was seen at the high cyclosporine dose, while at the lower dose only ifn-g expression was suppressed. because tcell expression of il-4 was not significantly suppressed at the high cyclosporine dose, il-4 was not evaluated at the lower drug dose. because of specialized sample handling requirements, flow cytometry is not as practitioner friendly as other assays (such as pcr) for routine use in pharmacodynamic testing. we have therefore conducted an in vitro study comparing the effects of cyclosporine on activated t-cell expression of il-2 and ifn-g using flow cytometry and qrt-pcr, and demonstrated dose dependent and comparable suppression of il-2 and ifn-g using either methodology. we are currently evaluating, using qrt-pcr, the effects of oral cyclosporine on t-cell expression of il-2 and ifn-g in normal dogs prior to moving on to pharmacodynamic trials in our clinic patients. effect of hypothyroidism on reproduction in bitches. dl panciera 1 , bj purswell 2 , ka kolster 2 , sr werre 3 . departments of 1 small animal clinical sciences, 2 large animal clinical sciences, and 3 laboratory for study design and data analysis, virginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va. numerous reproductive abnormalities, including irregular interestrous period, anestrus, and infertility have been attributed to hypothyroidism. we previously documented reduced fertility and lower birth weight and increased periparturient mortality in pups born to bitches with experimentally-induced hypothyroidism for a median duration of 56 weeks. the purpose of this study was to evaluate reproductive function in these same bitches after hypothyroidism was treated with a replacement dose of levothyroxine. twelve multiparous bitches were studied. hypothyroidism was induced in 6 dogs by administration of 1 mci/kg 131 i. hypothyroidism was confirmed by finding serum t4 concentrations before and 4 hours after iv administration of human recombinant tsh that were o 5 nmol/l. levothyroxine (0.02 mg/kd q 24 h) was administered to all hypothyroid bitches. six bitches served as euthyroid, untreated controls. dogs were evaluated daily for signs of estrus and were bred by 1 of 2 males when serum progesterone was !5 ng/ml. interestrous interval, gestation length, strength and duration of contractions during whelping, time between pups, number of live pups and stillbirths, viability of pups at birth, weight of pups, and periparturient mortality were recorded. the student's t-test and anova were used to compare differences between control and hypothyroid bitches for continuous, normally distributed data. the wilcoxon rank sum test was used to analyze data between groups that was not normally distributed. the mean duration of hypothyroidism prior to levothyroxine administration was 102 ae 8.1 weeks. breeding took place after levothyroxine treatment for 46 ae 12.6 weeks in the hypothyroid group. all 6 dogs in the hypothyroid group and 5/6 control dogs were pregnant, while 4/8 hypothyroid and all 6 control bitches became pregnant prior to levothyroxine administration. no difference in interestrus interval or gestation length was noted between groups. during whelping, no difference in strength of contractions, contraction duration, interval between pups, or viability scores of pups was found between groups. litter size, birth weight and peirparturient mortality were similar between groups. levothyroxine administration reverses the detrimental effects of hypothyroidism on fertility and neonatal health. racing sled dogs have a high prevalence of exercise-induced gastric erosions/ulcers, with reports ranging from 50-67% of dogs running at least 100 miles in a day or less. omeprazole reduces the severity of, but does not completely prevent, gastritis under racing conditions, and can be difficult to administer under these conditions. famotidine can be administered in food, but has only demonstrated efficacy under less intense training conditions. the purpose of these studies was to evaluate different acid suppression strategies under racing conditions for the prevention of exercise-induced gastritis. experiment #1 was a randomized placebo-controlled study using 36 sled dogs (3-8 years) competing in a 330 mile race over 50-60 h. treatment groups were famotidine (approx 1 mg/kg qd) or no treatment, beginning 2 days prior to the start of the race and proceeding until gastroscopy was performed 24h after the race. experiment #2 was a randomized positive-control study using 52 sled dogs (2-8 years) running a mock race of 300 miles in 50h. dogs were divided into omeprazole (approx 1 mg/kg qd, administered 30 min prior to a meal) or famotidine (approx 2 mg/kg bid) groups beginning 2 days prior to the exercise challenge and continuing for 24h after completion. gastroscopy was performed immediately prior to the start of dosing and 24h after completion of the exercise. in all cases, mucosal appearance during gastroscopy was blindly scored using previously described scoring system. famotidine (1 mg/kg qd) reduced the prevalence of clinicallyrelevant, exercise-induced gastric lesions compared to no treatment (7/16 vs 11/16, p 5 0.031). compared to famotidine at 2 mg/kg bid, omeprazole significantly decreased the severity (0.4 vs 1.2, p 5 0.0002) and prevalence (2/23 vs 7/21, p 5 0.049) of gastric lesions. although famotidine provides some benefit in the prevention of exercise-induced gastric lesions, neither the recommended dose nor the higher dose were considered acceptable in the prevention of exerciseinduced gastritis as between 33-44% of the dogs receiving famotidine had clinically significant lesions. a previous study examining omeprazole under racing conditions, but without careful administration on an empty stomach, resulted in a 22% prevalence of clinically significant gastric lesions. however, the bioavailability of omeprazole is reduced in the presence of food, and when the daily administration of the drug is carefully scheduled to coincide with an empty stomach, the resulting prevalence of clinically significant lesions induced by racing-intensity exercise is reduced to just over 10%. the conclusions of these studies are that omeprazole is superior to famotidine in preventing gastritis in racing sled dogs during competition. routine administration of omeprazole is recommended to prevent stress-associated gastric disease in exercising and racing alaskan sled dogs. mares may be an important source of environmental contamination with rhodococcus equi on breeding farms. attempts to reduce fecal shedding of r equi by the mare and the effects of the mare's fecal r equi concentration on airborne concentrations in the foaling stall have not been previously reported. twenty-one arabian mares were treated daily with either oral gallium nitrate or placebo in a randomized double-blind study. fecal samples were collected at day 320 of gestation (time 1), the week before foaling (time 2), and the week after foaling (time 3). airborne concentration of r equi were measured in the stall within 6 hours post foaling using a microbial air sampling system into which standard (100-mm) culture plates with a media selective for r. equi have been loaded. concentration of total r equi were determined by morphological characteristics. the concentration of virulent r equi was determined using a modified colony immunoblot method. concentrations of total and virulent r equi were compared among mares to examine effects of treatment, time, and treatment by time interaction. there were significant (p o 0.05) effects of treatment that depended on time of sample collection. at sample times 1 and 2 there were no significant differences between groups in the fecal concentration of virulent r equi. at time 3 concentrations of virulent r equi were significantly lower among mares in the treatment group (p o 0.05) compared to control. effects of time depended significantly on groups: for the control group, there were no significant effects of time. for the treatment group, concentrations tended to decrease over time, and concentrations at time 3 were significantly (p o 0.05) lower than those at time 1. no other differences among times for concentrations in the treatment group were statistically significant. there were no significant effects of treatment, sample time, or their interaction on the concentration of total r equi between groups; however, the pattern for these data was similar to that observed for the virulent isolates. no significant differences were determined between treatment groups for airborne concentrations of virulent or total r equi. treatment of mares with oral gallium nitrate significantly reduced the fecal concentrations of virulent r equi over time, but had no impact on the airborne concentration of r equi shortly after foaling. the purpose of this study was to evaluate the protein profile of bronchoalveolar lavage fluid (balf) in horses affected with recurrent airway obstruction (rao) and in control horses using proteomics and western blot techniques. rao-affected (n 5 5) and control horses (n 5 6) were subjected to an experimental exposure trial; when the rao-affected horses showed clinical signs of disease, balf was collected from all horses. balf was also collected from client-owned rao-affected horses (n 5 15) with naturally-occurring clinical signs of disease and client-owned control horses (n 5 12) from the same environments. the balf from the experimental exposure trial horses was subjected to trypsin digestion and proteomics analysis with mass spectrometry (ms). peaks detected with ms were identified using tandem ms analysis and database searches. western blot was used to confirm the identity and expression levels of two proteins identified using proteomics techniques in the balf of all horses. data from ms experiments were analyzed with the student's t-test to compare peak intensity between rao-affected and control horses. western blot band density data was analyzed with the kruskal-wallis anova for comparison between groups of horses. significance level was set at p o 0.05. with ms proteomic analysis of the balf from the experimental exposure trial horses, 2049 total peaks (peptides) were identified. of these peaks, 100 were differentially expressed between the rao-affected (24 over-expressed) and control horses (76 over-expressed). identifications were made for 250 balf proteins. transferrin and secretoglobin were chosen for validation with western blot. proteomics indicated that secretoglobin was not differentially expressed between the experimental exposure trial group; this was confirmed with western blot analysis. western blot also showed that clientowned rao-affected horses had lower secretoglobin expression than client-owned control horses and control horses before experimental exposure. according to the proteomics data, transferrin was over-expressed in control horses after experimental exposure compared to rao-affected horses. while the western blot analysis did not show a statistically significant difference in this comparison, transferrin was significantly over-expressed in control horses before experimental exposure compared to client-owned rao-affected horses. in addition, both secretoglobin and transferrin band densities on western blot were negatively correlated with airway obstruction and neutrophilic pulmonary inflammation. this study demonstrates that proteomics techniques can be used in the investigation of equine balf proteins. the proteins identified as differentially expressed between rao-affected and control horses in this study including, but not limited to, secretoglobin and transferrin should undergo further evaluation for their use as biomarkers of rao, and as potential targets of new therapeutic agents for rao. cardiotoxic effects of rattlesnake venom in the horse are not well defined. the first aim of this study was to document cardiac damage in naturally envenomated horses. twenty horses with clinical diagnosis of snake bite were included. a snake venom elisa was utilized to confirm envenomation when possible. serum and plasma were collected at selected intervals. plasma was assayed for cardiac troponin i (ctni) using a flurometric assay (stratus cs s , dade behring). holter monitors (zymed s , philips) were placed at admission, 1 week and 1 month post presentation. echocardiography was performed on available horses 5-7 months after envenomation. the second aim of this study was to investigate potential mechanisms of the cardiac damage. serum samples were assayed for tnfalpha using a commercial assay (endogen). antibody titers to crotalus atrox venom were measured at admission, 1 week and 1 month after natural envenomation and compared to titers in vaccinated horses (crotalus atrox toxoid, red rock biologics). a significant number of horses showed elevations in ctni (p o 0.05) at one or more time point indicating myocardial damage. holter readings revealed the presence of arrhythmias or persistent tachycardia in 19 horses. five of twenty horses were available for echocardiography; no abnormalities were noted. horses with increased ctni tended to have greater tnfalpha concentrations compared with horses without increased ctni. peak venom titers in bitten horses were significantly higher than peak titers in vaccinated horses (p o 0.05). rattlesnake envenomation was associated with evidence of cardiac damage in a significant proportion of bitten horses. further studies are needed to determine the cause as well as mechanisms to treat and/or prevent its occurrence. little is known about the gastric mucosal flora in healthy horses and its role in gastric disease has not been critically examined. our laboratory previously reported that a diverse microbial flora with a predominance of streptococcus spp. and lactobacillus spp. exists in healthy horses using fluorescence in situ hybridization (fish). the present study sought to further characterize the gastric mucosal flora of healthy horses using massive parallel 16srrna bacterial tag encoded flx-titanium amplicon pyrosequencing (btefap). biopsies of the squamous, glandular, antral and any ulcerated mucosa were obtained from 4 healthy horses via gastroscopy after a 12-hour fast and 2 horses immediately post-mortem. dna was extracted from the mucosal biopsies and btefap and data processing was performed. hierarchical cluster analysis based on relative abundance data on the genus level were performed to look for trends in bacterial diversity among the individual horses. pyrosequencing yielded between 4,500 and 13,000 reads per horse with 9238, 16587, 16587 reads in the antrum, squamous and glandular regions, respectively. the microbiome segregated into two distinct clusters: cluster 1 comprised of 2 horses that were stabled, fed hay and sampled at post-mortem and cluster 2 consisted of 4 horses that were pastured on grass, fed hay and biopsied gastroscopically after a 12-hour fast. samples from different antomic regions clustered by horse rather than region. despite being very similar at the higher taxonomic level (phyla) differences in the distribution of bacteria were seen at the genus and species level. the dominant bacteria in cluster 1 horses were firmicutes (483% reads/sample) consisting of mainly streptococcus spp., lactobacillus jensenii, l. fornicalis and sarcina maxima. cluster 2 had more diversity with a predominance of proteobacteria, bacteroidetes and firmicutes and 51 genera identified such as streptococcus spp., moraxella spp., actinobacillus spp., and others. though the relative abundance of the individual taxonomic groups was significantly different between individual horses, no significant differences in the overall diversity could be found (as assesed by shanon weaver, ace and choa i diversity indices). helicobacter spp. sequences were not identified in any sample (out of 58,891 reads). the ulcerated mucosa from horse 3 (group 1) had lower diversity and higher numbers of bacteria predominated by lactobacillus equigenerosi. this data shows that the equine gastric mucosa harbors an abundant and diverse microbiome which is unique to each individual and differs by sampling method, fasting prior to sampling and diet. seasonal pasture myopathy (spm; atypical myopathy [am] in europe), typified by nonexertional rhabdomyolysis, occurs in pastured horses during autumn or spring. clinical signs rapidly progress from muscular weakness to recumbency and frequently death. extensive myonecrosis and intramyofiber lipid storage occur in highly oxidative respiratory and postural muscles. recently, a defect of lipid metabolism called madd has been identified in european horses with am. this report documents the first cases of equine madd in the united states. six midwestern us horses suspected of having spm in the spring or fall of 2009 were evaluated for madd by urine organic acids, plasma acylcarnitines and/or muscle carnitine and histopathology. five horses had clinical signs and clinicopathologic data consistent with severe rhabdomyolysis. one horse was found dead on pasture after 2 days of rear limb stiffness and inappetance. urinary organic acid profiles revealed markedly elevated ethylmalonic and methylsuccinic acids, butyrylglycine, isovalerylglycine, and hexanoylglycine, consistent with equine madd. plasma acylcarnitine profiles from 2 horses had marked elevations of short chain acylcarnitines, while the third horse and only survivor had minor elevations of short chain acylcarnitines. affected muscle showed extensive degeneration with intramyofiber lipid accumulation, a marked decrease in free carnitine, and high levels of carnitine esters. spm appears to be a highly fatal emerging disease of pastured horses in the us characterized by weakness, colic-like signs and myoglobinuria. the disease is associated with a defect in muscular lipid metabolism that can be diagnosed by performing lipid staining of muscle samples and urine organic acid profiles. candidatus mycoplasma haemolamae (cmhl) is a common red blood cell parasite of new world camelids. the high degree of parasitemia that develops in an infected splenectomized animal allows for the efficient collection of parasitic dna. this dna can then be used in the development of genetically-derived tools such as pcr and in-situ hybridization. thus, one splenectomized animal can replace many immunologically intact animals within a research setting. the purpose of this study was to track the natural progression of cmhl parasitemia and associated clinical signs in a splenectomized alpaca. an intact, 9-month-old, 39.1 kg male alpaca was used in this study. he had tested positive via pcr for cmhl on three different occasions, although no organisms were seen on peripheral blood smears. the alpaca was placed under general anesthesia and a ventral midline incision was made. the spleen was located, the vessels ligated, and the organ removed. buprenorphine and flunixin meglumine were given for 2 and 4 days after surgery respectively. body weight, attitude, rectal temperature, blood glucose, and pcv were recorded daily. in addition, a peripheral blood smear was examined daily and the percent of red blood cells that were infected with mycoplasma organisms was determined. the alpaca was not parasitemic prior to surgery. one percent of the rbc's contained mycoplasma on days 2 and 3 after splenectomy. parasitic bloom developed on day 4 with 85% of the red blood cells infected, and over 70% containing 3 or more organisms. the alpaca was treated with 20 mg/kg oxytetracycline i.v. on day 4. on postoperative day 5 no parasites were seen in the peripheral blood. the peripheral blood remained free of parasites for 11 days. on the morning of the 12 th day, 3% of the peripheral red blood cells contained mycoplasma. by late that afternoon, 75% of the observed rbc's contained 3-4 organisms. the alpaca again received oxytetracycline. there were no more parasites observed from that time until the alpaca was euthanized 5 days later. the alpaca lost 3.7 kg between days à1 and 12 after surgery. his weight fluctuated between 34.9 and 35.4 kg for the remainder of the study period. blood glucose ranged between 87 and 163 mg/dl there was no major change in pcv (range 21-29%), a finding that was expected as the spleen was not available to remove infected red blood cells. body temperature ranged between 38.1 and 39 degrees celsius except for days 4 and 16 when more than 70% of red blood cells contained parasites. on those days rectal temperature reached 39.4 and 39.1 degrees respectively. this study confirmed that a non-parasitemic, yet pcr positive alpaca did indeed harbor cmhl. the time from splenectomy to parasitic bloom was shorter, and the length of oxytetracycline suppression longer than has been observed in other species. gastro-intestinal (gi) disease frequently results in increased wall thickness in many species. identification of changes in gi wall thickness using ultrasound has proved to be a useful diagnostic tool and is widely used in human patients, small animals and horses. although gi motility has been evaluated in cattle, normal reference ranges for wall thickness has not been reported in ruminants. the aims of this study were to report normal values for wall thickness of various gi structures and to assess the repeatability of this technique in adult dairy cows, sheep and goats. eight healthy adult holstein friesian (hf) cattle (656 ae 11 kg), eight jersey (j) cattle (458 ae 56 kg), thirteen adult sheep (79 ae 11 kg) and eleven adult goats (43.5 ae 8 kg) were recruited and examined on three consecutive days. ultrasonographic images were optimised for the structure of interest. structures were identified based upon appearance and anatomical position. a minimum of three cineloops were obtained of the abdominal organs per intercostal space (ics) and three along the ventral midline in each ics; images were analysed offline. data were analysed using anova and post-hoc bonferoni, student's ttest and intra-class correlation coefficients. each structure was measured per ics per species; if no differences were noted for structures in different ics, then measurements were pooled. no differences were noted between hf and j cattle so data were pooled. data are displayed in table 1. good repeatability (icc40.91) was obtained for all measurements and no differences were noted between animals of the same species or between days. these measurements for assessment of normal gi thickness are repeatable and may allow valuable additional information to be gained from ruminants with gi disease. ocular infections with the infectious bovine keratoconjunctivitis (ibk) agent moraxella bovis (m. bovis) are associated with significant economic loss in the cattle industry.although antibiotic therapy is the treatment of choice for ibk, treatment failures are common and current vaccines are not optimally effective mainly due to antigenic variation. as a result, our laboratory has been actively investigating the therapeutic potential of bdellovibrio bacteriovorus 109j (b. bacteriovorus); a predatory bacterium capable of attacking and inducing lysis of gram-negative bacteria, as a new treatment for ibk. we have previously shown that b. bacteriovorus can reduce the number of m. bovis attached to bovine epithelial cells in an in vitro model of ibk and that b. bacteriovorus can be trained to kill m. bovis as effectively as e. coli using serial passages. in this study, we hypothesized that b. bacteriovorus can remain viable in bovine tears without its prey for up to 24 hours. this hypothesis was addressed by incubating inocula of active b. bacteriovorus in its preferred media peptone yeast extract (pye) and comparing b. bacteriovorus viability in bovine tears or phosphate buffered saline (pbs) at time 0, 2, 4, 12, and 24 hours. using a plaque assay to quantify the mean amount of plaque forming units (pfus) of b. bacteriovorus exposed to each treatment, it was determined that viability of b. bacteriovorus over time was comparable between treatment groups. overall, the results supported that b. bacteriovorus can remain viable in tears for up to 24 hours in the absence of prey bacteria. further studies are needed to determine the therapeutic potential of b. bacteriovorus in an in vivo model of ibk. correction of the measured ionized calcium concentration (cca 21 ) to a ph 5 7.40 is routinely applied in experimental studies in order to assist in the interpretation of measured values relative to a reference range. the equation most commonly used for ph correction in bovine plasma is: cca 21 ph 5 7.40 5 cca 21 â10 (-0.24â{7.40 -ph}) . the validity of this equation for bovine plasma is unknown. accordingly, our first objective was to characterize the in vitro relationship between cca 21 and ph for bovine plasma. feeding rations with a low dietary cation-anion difference (dcad) during late gestation mitigates periparturient hypocalcemia in dairy cows, particularly when chloride containing acidodgenic salts are fed. the mechanism for this beneficial effect remains unclear. our second objective was to determine whether hyperchloremia displaces calcium from binding sites to albumin, thereby increasing cca 21 . the in vitro relationship between plasma log(cca 21 ) and ph in was investigated using lithium heparin anticoagulated blood from 10 healthy holstein-friesian calves. plasma was harvested and tonometered with co 2 at 371c over a ph range of 7.10-7.70. plasma chloride concentration (ccl -) was altered by equivolume dilution of plasma with 3 electrolyte solutions of varying ccl -(97 ae 1, 110 ae 1, and 123 ae 1 meq/l; mean ae sd). the slope of the linear regression equation relating log(cca 21 ) to ph for 112 tonometered plasma samples from the 10 calves was -0.24 ae 0.05 at normal values for cca 21 (2.63 ae 0.09 meq/l), albumin concentration (30.9 ae 2.0 g/l), and ccl -(99.1 ae 2.2 meq/l). the experimentally-determined value for the slope for bovine plasma was identical to that determined previously for human plasma. the formula for correcting cca 21 in bovine plasma for change in ph from 7.40 is therefore: cca 21 ph 5 7.40 5 cca 21 â 10 (à 0.24 â {7.40-ph}) . this equation is only valid at normal concentrations of albumin and chloride in plasma. equivolume dilution of plasma by electrolyte solutions of varying cclindicated that cca 21 ph 5 7.40 increased by 0.007 meq for every 1 meq/l increase in ccl -. in other words, plasma cca 21 at a given ph increases directly in response to an increase in plasma ccl -, presumably because the additional chloride displaces calcium that is electrostatically bound to albumin. furthermore, the increase in cca 21 is independent to the change in plasma ph induced by an increase in ccland decrease in plasma strong ion difference. our finding that hyperchloremia directly increases plasma cca 21 provides an additional mechanism by which ingestion of high chloride (acidogenic) rations prevents the clinical signs of periparturient paresis. our finding is consistent with the results of other studies that indicate acidogenic salts that contain chloride as the predominant anion (ie, nh 4 cl, cacl 2 ) are more effective in increasing cca 21 than equimolar quantities of acidogenic salts such as mgso 4 . coagulase negative staphylococci (cns) are among the most common bacteria isolated from the bovine mammary gland. historically, these bacteria were lumped together as minor mastitis pathogens. modern molecular techniques have allowed accurate speciation and fingerprinting of the cns species. these methodologies have recently been applied to the study of cns in bovine mastitis. the aim of the studies presented here was to evaluate the role of individual cns species on milk somatic cell count (scc) and duration of intramammary infection (imi). in the first study, mammary quarter foremilk samples were aseptically collected from all lactating cattle ($180 head) at the university of missouri dairy research center once monthly for 17 months for bacterial culture and milk scc. staphylococcal isolates were speciated by sequencing the rpob gene and strain-typed using pulsed-field gel electrophoresis (pfge). using species and fingerprint data along with published definitions for staphylococcal imi, 91 cns imis were identified. overall, 11 species of cns were identified with staphylococcus chromogenes, s. cohnii, s. epidermidis, and s. simulans being most prevalent. duration of imi and scc data were analyzed using regression models accounting for repeated measures. mean milk scc and duration of imi were found to differ between cns species (p o 0.05). although most imis were of short duration (1 month), staphylococcus capitis and s. chromogenes imis had longer mean durations of infection than 3 or more of the other species isolated. mean sccs were under 350,000 cells/ml in most cases. however, staphylococcus simulans and s. xylosus imis were more inflammatory (mean 4 600,000 cells/ml) and had a higher mean scc than s. cohnii, s. epidermidis, and s. haemolyticus. to examine the relationship between cns imi and milk scc in a larger population of cattle, cns isolates from the canadian bovine mastitis research network (cbmrn) culture collection were obtained for speciation. speciation and fingerprinting were performed as above. isolates were from subclinical imi from before and after the dry period and from subclinical imi during lactation. data associated with each isolate were obtained from the cbmrn database. nine-hundred-thirty-eight isolates from 696 mammary quarters in 89 herds were successfully speciated. twenty-two different species of cns were identified. staphylococcus chromogenes was the most frequent species identified accounting for 40% of the infections. three species, s. chromogenes, s. xylosus, and s. simulans accounted for 475% of all infections. data were analyzed using a linear hierarchical repeated measures mixed model. differences in mean scc were found between some cns species and culture negative control quarters and also between different species of cns (p o 0.05). overall, our data demonstrate potential differences in pathogenicity between strains of cns that cause bovine mastitis. passive transfer of maternally derived antibodies via ingestion of good-quality colostrum within the first 24 hours of life is crucial for the health and future productivity of dairy calves. however, infectious diseases can be transmitted via colostrum feeding, which may require use of a colostrum replacement product or pasteurization to decrease disease transmission. while pasteurization of colostrum is effective for sterilization, heating during pasteurization can alter the viscosity of colostrum, destroys important nutritional biomolecules, and has been shown to decrease colostral igg concentrations. the purpose of this study was to investigate the effect of high pressure processing (hpp) on the viscosity, igg concentration, and bacterial contamination of bovine colostrum. first milking colostrum samples were collected from 18 cows from 3 different farms, and 50 ml aliquots of each sample were pooled for analysis. pooled colostrum was processed in triplicate using an isostatic press at 400 mpa (60,000 psig) for 0, 5, 15, 30, and 45 minutes. samples were tested for the effects of hpp on the viscosity, bacterial load (cfu/ml), and igg concentration. there was a significant decrease (p o 0.05) in bacterial load at each time point when compared to time 0. no significant difference in igg concentration was found between any time points. subjectively, the colostrum viscosity appeared to increase with the processing time, though the rheologic assessment has not been completed at this time. hpp appears to be an effective method to decrease bacterial contamination of colostrum while maintaining appropriate igg concentrations. minimizing the processing time or pressure may be necessary to maintain an acceptable viscosity of the colostrum. based on these results, additional studies are justified in order to determine the optimum combination of processing time and pressure and the effect of hpp on specific bovine pathogens. the heme-associated iron-binding apoprotein lactoferrin (lf) is known for its, anti-inflammatory, anti-parasitic, antimicrobial and bactericidal effects. lactoferrin demonstrates ubiquity throughout mammalian host biological fluids: saliva; tears; mammary secretions, as well as at mucosal surfaces. it is also released from immune cells under pathogenic stimulation. the purpose of this study which has been approved by western university's institutional animal care and use committee is to further characterize the mechanisms through which lf modulates inflammation in the face of bacterial endotoxin. it was hypothesized that lf would inhibit p38 phosphorylation. numerous studies speak to the ability of lf to alter leukocyte function, inhibit cytokine production, and bind lipopolysaccharide (lps); mechanisms through which it is believed to achieve its anti-inflammatory effects. recently, investigators demonstrated its ability to interact with host dna while others describe regulation of granulocyte adhesion and motility; elucidating its roles in the apoptotic signaling. in earlier studies, dawes me, et al. demonstrated lactoferrin's ability to limit the expression of inducible cyclooxygenase-2 and the gelatinase, matrix metalloproteinase -9 by lps-induced macrophages. the generation of these inflammatory mediators is modulated by pro-inflammatory cytokines such as interleukin-1 b (il-1 b) and tumor necrosis factor-alpha (tnf-a), the production of both being dependent on signaling through the p38 mitogenactivated protein kinase (mapk) pathway. peripheral mononuclear cells (5 â 10 6 )isolated from buffy coat cells of healthy neonatal to 4-month old holstein calves were cultured in the presence and absence of lf (200 ng/ml), lps (1 mg/ml), anisomycin (25 mg/ml), a known p38 activator -the positive control, and 50 mm of sb203580, a known p38 inhibitor -the negative control. sample lysates obtained post culture was subjected to immunoprecipitation and kinase reactions. reactions were terminated under reducing conditions and evaluated using western immunoblotting. phosphorylation of activated transcription factor-2 (atf-2) by phosphorylated p38 served as the marker of investigation. immunologically reactive atf-2 expression by lps and anisomycin-treated cells was compatible with a prominent band at 40 kd. evidence of lf-induced inhibition of lps-induced p-38 activation was observed in lanes representative of co-cultures of lf 1 lps; lf 1 anisomycin; and anisomycin 1 sb203580, which was demonstrated by decreased immunological reactivity at 40kd. the findings here, suggest that lf interferes with lps-induced p-38 activation of transcription factor atf-2, in vitro. this serves as additional proof of its potential use in attenuating the systemic effects of lps. six (6) clinically normal, purpose-bred cats of similar age and body condition were imaged with [ 18 f] fluorodeoxyglucose ([ 18 f]fdg) and [ 18 f]ftha by using dynamic cardiac-gated fused pet/ct for kinetic assessment of myocardial glucose and fatty acid uptake and metabolism, respectively. kinetic tracer uptake within the myocardium was achieved by initiating image data acquisition simultaneously with tracer injection. pet data were acquired over a 1 hour period with the heart in the center of the scanner field of view. regions of interest were drawn in the left ventricular wall and thoracic aorta for the purpose of measuring the kinetics of tracer redistribution. serial blood samples were also taken during pet imaging for comparison with image data. the equilibrium biodistribution of both tracers was documented 1 hour post-injection in a whole body pet/ct image. standard echocardiographic examination of cardiac structures was also performed. both radiotracers remained in the plasma fraction; however, [ 18 f]ftha was cleared from the more rapidly than [ 18 f]fdg (t 1/2 $ 2 and $ 20 min, respectively). the tracers were readily visualized within the feline myocardium in dynamic pet images and analysis of the blood pool clearance from the kinetic image data agreed with blood sampling data. myocardial uptake of each tracer was best described by a double exponential analysis and was rapid but variable among animals (range 1-30 bq/cc/min), although blood glucose levels were similar in all cats during image acquisition. physiologic [ 18 f]fdg was observed in the brain, salivary tissue, gastrointestinal tract, renal pelves and urinary bladder, with [ 18 f]ftha seen in the myocardium, liver and renal cortex. all cats were normotensive with normal echocardiographic parameters. this study demonstrates the utility of kinetic imaging using the left ventricle (lv) shape has been suggested to change from elliptical to more globular in response to chronic volume overload. real-time three-dimensional echocardiography (rt3de) offers new modalities for lv assessment. the aim of the study was to investigate left ventricular changes in shape and volume occurring in response to different severities of naturally acquired myxomatous mitral valve disease (mmvd) in dogs using rt3de. privately owned dogs were classified by standard echocardiography into: healthy (20), mild (20), moderate (8) and severe mmvd (17). a lv cast was obtained using semi-automated endocardial border tracking from rt3de dataset, from which global and regional (automatically acquired basal, mid, and apical segments based on lv long-axis dimension) end-diastolic (edv) and endsystolic volumes (esv), lv long-axis dimension and rt3de sphericity index, were derived. global and regional edv and esv increased significantly with increasing mmvd severity, assessed by mmvd group-wise comparisons and linear regression analyses using left atrial to aortic root ratio, and lv end-diastolic and end-systolic dimensions. all three segments contributed to the overall increased global volumes, but the mid edv segment was strongest associated with increasing lv end diastolic dimension (p 5 0.048). furthermore, lv long axis distance and lv sphericity index increased with increasing mmvd severity. the basal and apical edv segments were strongest associated with sphericity index (p o 0.0001). in conclusion, this rt3de study showed that increased lv edv, primarily in the mid segment, leads to rounding of lv apical and basal segments in response to increasing mmvd severity in dogs. 84 dogs from shelters in florida with naturally acquired di infection were euthanized and necropsied. all adult di in each dog were sexed using morphological features. total worm burdens and numbers of males and females were recorded. no other information was available for any dog. all data, raw and transformed, were examined visually and descriptively. raw numerical data were further examined by a paired t-test; log-odds transformed data were examined by logistic regression. we also conducted a binomial distribution goodness of fit analysis assuming a null hypothesis of a m:f 5 1.0. worm intensities ranged from 1 to 143 di per dog. eight dogs had unisex infections: 7/8 had all-female infections. dogs with lowintensity dual-sex infections were more likely to have greater numbers of female di. overall, sex ratios were equal (paired t-test, p 5 0.7). however, logistic regression demonstrated that the probability of being female is strongly affected by the total worm intensity, with lower intensities increasing the probability of having a predominance of female worms. our data show that di sex ratios in naturally-infected dogs equal 1 when examining the entire dog population, but deviate to favor female worms at low worm intensities. these data could impact adulticide treatment strategies. the reasons for sex ratio distortion in di are unknown. we evaluated cardiac reverse remodeling after mitral valve repair under cardiopulmonary bypass (cpb) for mitral regurgitation in small breed dogs. fifty dogs (body weight 1.8-9.3 kg, age 5-14 years) with mitral regurgitation were treated between august 2006 and november 2009. the cardiac murmur was grade 4/6-6/6. the preoperative chest x-rays showed cardiac enlargement (vertebral heart scale (vhs) 11.0-13.1). echocardiography showed severe mitral regurgitation and left atrium enlargement (la/ao 2.0-4.2). after inducing anesthesia, a thoracotomy was performed in the fifth intercostal space. cpb was started by using a cpb circuit connected to carotid artery and jugular vein catheters. after inducing cardiac arrest, the left atrium was sectioned and chordae tendineae rupture confirmed. the chordae tendineae were replaced with expanded polytetrafluoroethylene. a mitral annulus plasty was also done, and the left atrium was closed. after de-clamping for restarting the heart, the chest was closed. heart rate decreased from 118-164 bpm to 75-138 bpm. the grade of cardiac murmur was reduced to 0/6-3/6 three months postoperatively, and the heart shadow was reduced (vhs 9.8-11.5) in the chest x-rays. echocardiography confirmed the marked reduction in mitral regurgitation and the left atrial dimensions (la/ao 1.2-2.2). mitral valve repair reduced enlarged cardiac size by reduction of regurgitant rate. pulmonary arterial hypertension (pah) is a well recognized condition in dogs leading to considerable morbidity and mortality. the majority of therapeutics has focused on endothelial dysfunction causing reduced production of vasodilators, such as nitric oxide and prostacyclin, coupled with overproduction of vasoconstrictors, such as endothelin-1. more recently, it has been shown that the mitochondria play an important role in the development of pah as oxygen sensors and regulators of cellular proliferation. in pah, pulmonary artery smooth muscle cells undergo a metabolic shift from oxidative phosphorylation in the mitochondria to glycolysis in the cytoplasm as the major energy source and this leads to suppression of apoptosis and increased proliferation. dichloroacetate (dca) inhibits pyruvate dehydrogenase kinase to activate pyruvate dehydrogenase which catalyzes the rate limiting step for entry of pyruvate into the krebs cycle, thus increasing mitochondrial respiration. in three different rat models of pah, dca has been shown prevent and reverse pah by normalizing molecular pathology, stimulating apoptosis of pulmonary artery smooth muscle cells, and reducing pulmonary artery hypertrophy. dca has known toxic effects, including reversible hepatotoxicity and peripheral neuropathy, and has not been studied in any species with naturally occurring pah. the objective of this open label pilot study is to evaluate the therapeutic and toxic effects of dca in naturally occurring canine pah. three dogs with pah diagnosed by doppler echocardiography and no correctable underlying cause are enrolled in the study. dogs are orally administered 25 mg/kg of dca divided daily for 2 weeks, and then 12.5 mg/kg of dca divided daily for the remainder of the study. at baseline, 2, 4, 8, and 12 weeks, an echocardiogram, cbc, serum chemistry profile, urinalysis, nt-probnp, blood uric acid, blood lactate, noninvasive blood pressure, nerve conduction study, and trough dca level (12 hr post-dose) are obtained. the measured echocardiographic parameters include peak and mean tricuspid regurgitant flow velocity and pressure gradient, peak and enddiastolic pulmonary regurgitant flow velocity and pressure gradient, pulmonary valve flow velocity acceleration time and ejection time, pulmonary valve flow velocity time integral, right ventricular myocardial performance index, tricuspid annular plane systolic excursion, and systolic tricuspid annular tissue velocity. variables are inspected for normalcy and equality of variances and a two-sided paired t-test is used to compare the variables before and after treatment at each evaluation time. the basis for the role of the mitochondria in pah and the results of this pilot study will be presented to determine if dca warrants further study as a therapy for dogs with pah. study produced the strongest associations between the ncl phenotype and cfa2 markers. all 19 ncl-affected tibetan terriers were homozygous for the same haplotype which extended for 22 consecutive snps spanning 0.95 mb. none of the 20 annotated genes within this target region had previously been associated with human or rodent ncl. we used dna from ncl-affected tibetan terriers to resequence the coding regions and intron-exon borders of several genes harbored within the target region and found a single base pair deletion, c.1623delg, in exon 16 of positional candidate atp13a2. this deletion produces a frame shift and a predicted premature termination codon. we genotyped all 454 tibetan terrier dna samples in our collection and found all 45 ncl-affected tibetan terriers to be homozygous for the c.1623delg allele. eleven additional c.1623delg homozygotes were either less than 5 years old, or lost to follow up. there were no known cases of ncl in the remaining 398 tibetan terriers which were either heterozygous (n 5 149) or homozygous for the ancestral allele (n 5 249). atp13a2 is a member of group of ion transport genes and has been associated with lysosomes. mutations in human atp13a2 cause kufor-rakeb syndrome (krs), a rare neurodegenerative disorder with clinical features that include parkinsonism plus spasticity, supranuclear upgaze paresis, and dementia. post-mortem findings in krs have not been reported. we conclude that ncl in tibetan terriers is caused by a mutation in atp13a2. our results suggest that krs may be a form of adult onset ncl in humans. niemann-pick type c (npc) disease is a progressive neurological disorder characterized by dementia and ataxia, hepatic and pulmonary disease, and death typically within the first or second decade. despite the identification of causative mutations, the pathogenesis is not clear and therapies to successfully treat npc disease have been ineffective to date. the recent use of intravenously administered 2-hydroxypropylbeta-cyclodextrin (hpbcd), an fda-designated orphan drug (may 2010), in a small number of children with npc disease is based on favorable treatment outcome data in subcutaneously treated mouse and cat models. to rigorously evaluate the mechanistic, pharmacologic, and toxicity issues associated with hpbcd therapy in npc disease, we have utilized the spontaneous feline npc model harboring a missense mutation in npc1 (pc955s), orthologous to the most common mutation in juvenile-onset patients. the feline npc model has clinical, neuropathological and biochemical abnormalities similar to those present in juvenile-onset patients making this model homologous to the most common disease form seen in human patients. we identified that intrathecal administration of hpbcd ameliorated all clinical aspects of neurological disease at least up to 24 weeks of age (an age when untreated cats die) but had no effect on hepatic disease. we identified that while subcutaneous therapy with hpbcd at all doses ameliorated liver disease, only 8000 mg/kg substantially affected neurological disease but also resulted in early death due to pulmonary toxicity. finally, we identified a dose-related toxic effect of hpbcd on hearing function that had not been described in any other species. leukodystrophies are disorders of myelin synthesis and maintenance that affect cns myelin. they are subdivided as leukodystrophies, hypomyelinating disorders and spongy degenerations. although infrequently seen, several forms have been described in various dog breeds. we present a novel form of complex leukodystrophy consisting of hypomyelination and spongy degeneration that presents primarily with hind end tremors in border terrier puppies. three border terriers from two different litters (and lineages) are described here that presented with a history of shaking movements. the youngest dog was a 3-week old male. it was the only dog affected in the litter. the other two dogs were 6-week old female littermates. there were two unaffected males in the same litter. physical examination revealed no abnormalities. on neurological examination, the affected dogs displayed severe hind end tremors, with a characteristic swinging side-to-side movement (best described as ''rumpshaker''). the tremors also involved the head and thoracic limbs but to a lesser degree, and disappeared when the dogs were asleep or at rest. severe cerebellar ataxia was observed when the dogs ambulated. proprioceptive positioning was delayed in the pelvic limbs of all 3 dogs. spinal reflexes and nociception appeared normal. necropsy was performed in all 3 puppies. no macroscopic changes were observed. histologic evaluation of the cns revealed spongy degeneration and hypomyelination in all funiculi of the cervical and thoracic spinal cord. white matter of the frontal, temporal and parietal cortices had mild multifocal spongy degeneration and hypomyelination, whereas white matter of the cerebellum, medulla and pons showed severe diffuse spongy degeneration and hypomyelination with gliosis. the combination of reduced myelin formation combined with spongiform white matter changes in the absence of microglial responses suggest a complex pathogenesis affecting both oligodendrocytes' capacity to synthesize myelin and the stability of the myelin that was formed. the number of oligodendrocytes and axons appeared subjectively normal indicating a primarily hypomyelinating process. the clinical and pathological features of this disease have not been described in any other canine leukodystrophy. the primary and most striking clinical feature is the presence of severe tremors in the hind end, causing the ''rumpshaker'' pheynotype. genetic studies are underway to determine if the disease is inherited and the inheritance mode. a syndrome of border collie collapse (bcc) appears to be common in dogs used for working stock. this syndrome has also been called malignant hyperthermia, heat intolerance, exerciseinduced collapse and ''wobbles''. a presumptive diagnosis of bcc can only be made by eliminating other causes of exercise intolerance and weakness. the purpose of this study was to describe the clinical features of collapse in affected dogs and determine if there were characteristic clinical or laboratory features at rest or after exercise that could aid in diagnosis. seven adult border collies with a history of collapse during sheep herding (affected) and 5 adult border collies regularly used for sheep herding but showing no signs of exercise intolerance (normal) were evaluated before and after participating in a videotaped 10 minute exercise protocol consisting ofa series of continuous short outruns and fetches of three sheep in an outdoor pen. exercise was halted at 10 minutes or earlier if there were signs of gait or mentation abnormalities. pre-exercise evaluation included physical examination, orthopedic and neurological exam. pre and immediate post exercise rectal temperature, pulse and respiration, patellar reflexes, ecg, cbc, serum biochemistry profile, cortisol, arterial blood gas and plasma lactate and pyruvate concentrations were measured. clinical parameters (gait, temperature, reflexes) and lactate and pyruvate concentrations were evaluated at intervals up to 120 minutes after exercise. additional testing in affected dogs included measurement of acetylcholine receptor antibodies (achrab) and dna testing for dynamin-associated exercise induced collapse (deic) and the ryanodine receptor mutation associated with canine malignant hyperthermia(mh). one week after exercise affected dogs had thoracic radiographs and echocardiography performed and were anesthetized for emg and muscle biopsies. there were no significant differences in temperature, pulse, respiration, or any laboratory parameter at any time point between normal and affected dogs. no arrhythmias were detected. affected dogs were negative for the dna mutations tested and for achr ab. thoracic radiographs, echocardiograms, emgs and muscle biopsies were normal. the 5 normal dogs had no alterations in mentation or gait during or after exercise. three of the affected dogs had exercise halted early (6 min-9 min) because of altered gait or mentation. all 7 of the affected dogs were abnormal in the 15 minutes following exercise. abnormalities seen in all dogs included disorientation, dull mentation, swaying, falling to the side, exaggerated lifting of limbs each step, choppy gait, delayed limb protraction, scuffing of rear and/or forelegs, and crossing legs when turning. all dogs returned to normal by 30 minutes. bcc appears to be an episodic nervous system disorder that can be triggered by exercise. genetic testing excluded deic and the described canine mh mutation. common causes of exercise intolerance were eliminated, but the cause of collapse in bcc was not determined and no clinical or biochemical marker to aid diagnosis was established. equine cushing's disease (ecd) is common in older horses. the purpose of this study was to determine the frequency of diagnosis, identify prognostic factors and assess owner satisfaction with treatment. the study was a retrospective cohort design evaluating equine accessions reported to the veterinary medical data base (vmdb) and the ohio state university from 1993-2004. proportional accessions, annual incidence and demographic characteristics of horses with ecd were compared with all accessions in the vmdb. medical records for a subset of horses were extracted and owners contacted to obtain long-term follow up information. two hundred seventeen new cases of ecd were reported to the vmdb. incidence increased from 0.25/1,000 in 1993 to 3.72/1,000 in 2002. eighty-one percent of horses were ! 15 years of age. average delay from onset of signs to diagnosis was 180 days (range 1 to 1,824 days). hirsutism (84%) and laminitis (50%) were the most common clinical signs. improvement in one or more signs 2 months after diagnosis was reported by 9/22 (41%) of horse owners. none of the clinical or laboratory data were associated with survival and, 50% of horses were alive, 4.6 years after diagnosis. 17/20 (85%) of horses were euthanatized and 13/17 (76%) were euthanatized due to conditions associated with ecd. twenty-eight of 29 (97%) of horse owners said they would treat a second horse for ecd. ecd is becoming a more frequent diagnosis. fifty percent of horses survived 4.5 years after diagnosis and owners were satisfied with the horse's quality of life. supported by centers of excellence in livestock diseases and human health, college of veterinary medicine, university of tennessee. the role of the hypothalamic-pituitary-adrenal (hpa) axis in sepsis has been a subject of a great deal of research. the role that the somatogenic axis plays in sepsis is less well understood and how these two axes interact during critical illness is not clear. the purpose of this study was to assess inter-relationships of adrenocorticotropin (acth), cortisol, and insulin-like growth factor-i (igf-i), in septic and non-septic term foals. blood samples were obtained from term septic foals less than 7 days of age (n 5 20) admitted to texas a&m university veterinary medical teaching hospital or mid-atlantic equine hospital. the foals were classified as septic by a sepsis score ! 11 and/ or a positive blood culture. non-septic term foals less than 7 days of age (n 5 8) and having a sepsis score o 11 and a negative blood culture, were obtained from texas a&m university veterinary medical teaching hospital and mid-atlantic equine hospital. plasma and serum were processed from whole blood collected by jugular venipuncture upon admission, at 24 hours post admission and at 5 days post admission or at the time of discharge. plasma concentrations of acth, and serum concentrations of cortisol and igf-i were determined by specific rias. data were analyzed using linear mixed-effects modeling with foal modeled as a random effect and day of admission modeled as an ordered categorical variable; post-hoc testing of pair-wise comparisons was made using the method of sidak. significance was set at p o 0.05, and analyses were performed using s-plus software (tibco, inc., seattle, wa). plasma concentrations of acth were not significantly different between septic and non-septic foals whereas septic foals had greater serum cortisol (37 ae 8 ng/ml vs 25 ae 7 ng/ml) but lower serum igf-i (116 ae 14 ng/ml vs 152 ae 15 ng/ml) relative to non-septic foals pooled overall sampling times. the positive association of the peripheral blood concentrations of acth and cortisol depended on disease status of the foals. specifically, cortisol and acth were positively correlated for the septic foals (p 5 0.027) but not significantly correlated in the non-septic foals. peripheral concentrations of acth and igf-i were not significantly correlated whether data were pooled overall or stratified by sepsis status. however, peripheral concentrations of cortisol and igf-i were negatively associated (p 5 0.019); disease status did not influence this association, although it appeared to be a stronger association for the septic than the non-septic foals. the negative correlation between serum concentrations of the adrenal axis steroid cortisol and the somatogenic axis peptide igf-i may reflect interactions of these homeorhetic hormones. further studies of these and other metabolic hormones in a greater number of foals are warranted to better understand how these factors contribute to survival or non-survival of critically ill foals. botulism is a potentially fatal paralytic disorder which definitive diagnosis is difficult. the purpose of this study was to investigate if repetitive stimulation of the common peroneal nerve will aid in the diagnosis of suspected botulism in foals. four healthy foals were used for its comparison with 3 foals with suspected botulism. controls were anesthetized and affected foals were sedated to avoid risks of anesthesia. the common peroneal nerve was chosen for its superficial location and easy access. stimulating electrodes were placed along the common peroneal nerve. for recording, the active and reference electrodes were positioned over the midpoint and distal end of the extensor digitorum longus muscle, respectively. repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies (1 to 50 hz). amplitude, area under the curve and percentages of decrement or increment for each m wave over subsequent potentials for each set of stimuli were analyzed. baseline m waves were decreased in affected foals compared to controls. a decremental response was seen at all frequencies in control foals. decremental responses were also observed in affected foals at low frequencies. however, an incremental response in amplitude and area under the curve was seen in all affected foals at 50 hz. reduced baseline m waves with incremental responses at high rates are supportive of a presynaptic neuromuscular disorder which botulism was the most likely cause in these foals. repetitive nerve stimulation is a safe, simple, fast, and non-invasive technique that can aid in the diagnosis of suspected botulism in foals. this study examined the frequency with which dogs are exposed to e. chaffeensis and e. ewingii relative to e. canis, which is transmitted by the more ubiquitously distributed brown dog tick (rhipicephalus sanguineus). a total of 6,512 canine serum samples, ranging from 182 to 614 from each of the 14 participating institutions, collected at random from clinical accessions, diagnostic laboratories and/or shelters were evaluated. all serum samples were tested by three microtiter plate elisas using species-specific peptides for antibodies to e. canis, e. chaffeensis and e. ewingii. zip code information for sample origin was provided by the collaborator and was used to assess seroprevalence by region. comparisons were evaluated using the chi-square test. seroreactivity for at least 2 of 3 ehrlichia spp was found in samples from every institution both mississippi and oklahoma had greater than a 7% samples from ohio had the lowest aggregate seroprevalence (1.0%) with only 4 dogs e. canis seropositive, one e. ewingii seropositive and no e. chaffeensis seroreactors. the geospatial pattern of e. chaffeensis and e. ewingii seropositive samples was similar to that previously reported based on modeling seroreactivity to e. chaffeensis in white-tailed deer as well as the distribution of human monocytic ehrlichiosis (hme) cases reported by the cdc. this study provides the first large scale regional documentation of canine exposure to these three ehrlichia spp., highlighting where infections most commonly occur and thus identifying areas where heightened awareness about these emerging vector urinary incontinence (ui) occurs in approximately 20% of spayed female dogs. the most common cause is urethral sphincter mechanism incompetency (usmi). pharmacological agents are effective, however, not all dogs respond, and dogs may become refractory to treatment over time. urethral bulking, where a compound is injected submucosally in the urethra, has been used in women and in female dogs with urinary incontinence. new synthetic compounds have been used in human medicine; the most promising is polydimethylsiloxane (pdms), which has been shown to be more effective than glutaraldehyde cross-linked collagen. the purpose of this descriptive clinical trial is to evaluate the safety and effectiveness of pdms urethral bulking agent (pdms uba) in client-owned, spayed female dogs with naturally-occurring ui due to usmi.twenty-two, spayed female dogs were included. dogs had a median age of 6 years (2 to 11 years). eighteen dog breeds were represented, and dogs weighed a median of 29.9 kg (7.3 to 62.7 kg). average length of time of ui was 2.5 ae 2.3 years; 18/22 dogs had been treated medically, of which 2/18 were continent, 14/18 were improved, and 2/18 had no improvement. dogs were deemed healthy based on results of physical examination, complete blood cell counts, plasma biochemical analysis, and urinalysis; urine cultures were negative.dogs were anesthetized, positioned in dorsal recumbency, and cystoscopy performed using a 2.7 mm, 0-or 30-degree, 18 cm rigid cystoscope. urethral bulking was performed with pdms uba. on average, 2.5 ae 0.9 ml were injected in 3 to 5 locations approximately 1 to 1.5 cm distal to the trigone submucosally in the proximal urethra. good coaptation was achieved in all dogs. the procedure took on average 15.9 ae 4.3 minutes. one dog experienced urethral obstruction after the procedure; a foley catheter was inserted for approximately 12 hours and removed at which time she urinated normally and was continent. three dogs experienced an acute allergic reaction characterized by blepharedema and urticaria treated successfully with diphenhydramine. dogs were discharged on day of procedure except for the one dog that experienced urethral obstruction. all dogs were treated with meloxicam (0.1 mg/kg po q24h for 3 days).owners were contacted on day after discharge and 21/22 dogs were continent; 1/22 dogs was improved. dogs were re-evaluated 1 week after discharge and 21/22 dogs were continent and 1/22 dogs polyneuropathy in large breed dogs is a relatively common clinical problem for which the genetic basis is generally unknown. the first cases of polyneuropathy in the leonberger breed (leonberger polyneuropathy or lpn) were identified in 1999 by one of the authors (gds) and a report published in 2003 (musclenerve 27:471-477) . in this report a spontaneous, distal and symmetrical polyneuropathy with onset between 1 to 9 years of age was described and characterized clinically, electrophysiologically, histologically and morphometrically. there were striking similarities between lpn and the charcot-marie-tooth group of human inherited sensory and motor polyneuropathies, which have many known genetic mutations.a genome-wide case-control association study for lpn was performed with 53 cases and 42 controls on high-density 170k canine snp arrays and revealed a significantly associated region on cfa 16 (p raw 5 2.36 â 10 à10 p genome 5 9.99 â 10 à5 ). a clear association of an approximately 1 mb cfa16 haplotype with cases (p 5 1.71 â 10 à8 ) was observed, particularly with those cases that were affected more severely and at a younger age (p 5 2.55 â 10 à11 ). a positional candidate gene, arhgef10, which has previously been associated with peripheral nerve abnormalities in humans, was sequenced, revealing a deletion that results in a frame shift and premature stop codon. of all leonbergers with young onset lpn (before 4 years), 48.5% (32 of 66) have two copies of this deletion, and, of all young onset leonbergers that are nerve biopsy positive for lpn, 59.4% (19 of 32) have two copies of this deletion. importantly, nearly all dogs carrying two copies of the deletion (32 of 34 or 94.1%) are affected with lpn by the age of 4 years.the leonberger breed was generated from crossing several breeds, including the st. bernard, and a polyneuropathy clinically and histologically similar to lpn occurs in this breed. to determine if the arhgef10 mutation was associated with polyneuropathy in the st. bernard, dna was extracted from archived frozen muscle biopsy specimens from clinical cases (n 5 3). the identical arhgef10 startle disease or hyperekplexia is caused by defects in mammalian glycinergic neurotransmission resulting in an exaggerated startle reflex and extensor hypertonia triggered by noise or touch. in humans and animals, startle disease is typically caused by mutations in one of three genes (glra1, glrb, and slc6a5) encoding postsynaptic glycine receptor subunits (a1 and b) or a presynaptic glycine transporter (glyt2). a litter of seven irish wolfhounds was recently identified in which two puppies developed muscle stiffness and tremor beginning at 5-7 days of age post-partum. signs were dramatic when the puppies were handled and resolved when the puppies were relaxed or sleeping. both puppies were euthanized due to ongoing stiffness, tremor and breathing difficulties. necropsies were performed, but no microscopic pathological abnormalities were identified in the peripheral or central nervous system.based on the clinical signs, exons from the three candidate genes were amplified from genomic dna isolated using pcr and directly sequenced. no deleterious polymorphisms were identified in either glra1 or glrb. however, difficulties were experienced in amplifying slc6a5 exons 2 and 3 from affected animals, although control samples were positive, suggesting that the pcr primer designs and conditions were not at fault. further pcrs revealed that the reason for this anomaly was the presence of a homozygous 4.2 kb deletion encompassing exons 2 and 3 of the glyt2 gene in both affected animals. this deletion is predicted to result in the loss of part of the large cytoplasmic n-terminus that is vital for trafficking of glyt2 to synaptic sites, and a loss of all subsequent transmembrane domains via a frameshift. this genetic lesion was confirmed by defining the deletion breakpoint, southern blotting and multiplex ligationdependent probe amplification (mlpa). this analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, suggesting recessive inheritance of this disorder. wider testing of related animals has identified a total of 18 carriers of the slc6a5 deletion and enabled the identification of non-carrier animals to guide future breeding strategies. insulin resistance (ir), obesity, and type 2 diabetes affect glucagon-like peptide 1 (glp-1) concentrations in humans and rodents, but this incretin hormone has not been examined in horses. we therefore hypothesized that glp-1 concentrations would change in horses as obesity and ir were induced or exacerbated by overfeeding. six horses previously diagnosed with equine metabolic syndrome were provided with twice the amount of digestible energy required for maintenance as sweet feed and hay for 8 weeks. intravenous and oral glucose tolerance tests (ogtts) were performed at 0 and 8 weeks. effects of time and period (0 and 8 weeks) were assessed by repeated measures anova.mean body weight increased from 438 ae 61 kg (range, 381 to 533 kg) to 464 ae 61 kg (range, 394 to 550 kg) over 8 weeks, with individual horse weight gain varying from 2 to 10%. mean body condition score increased (p 5 0.006) from 6 ae 2 (range, 4 to 8.5) to 8 ae 1 (range, 7 to 9). three horses developed mild laminitis. glucagon-like peptide 1 concentrations increased over time during ogtts (p 5 0.023), but the period â time effect was not significant (p 5 0.141). area under the glp-1 curve remained unaffected by weight gain, whereas area under the insulin curve increased (p 5 0.003) over time, indicating a reduction in insulin sensitivity. obesity and ir were induced or exacerbated when horses previously diagnosed with ems were overfed, but glp-1 concentrations did not change as a result. hypertonic saline solution (7.2%) (hss) is an intravenous fluid used for the emergency treatment of intravascular volume deficits. the use of this fluid in horses with severe dehydration is controversial. the purpose of this study was to compare the use of hss and isotonic saline solution (0.9%) (iss) for the emergency treatment of endurance horses.endurance horses eliminated from competition and requiring intravenous fluid therapy were eligible for enrollment in the study. twenty-two horses were randomly assigned to receive 4 ml/kg of either hss or iss along with 5 l lactate ringer's solution (lrs). following this bolus, all horses were treated with an additional 10l of lrs. blood and urine samples were collected before, during and after treatment. data was compared using two-way anova with repeated measures.as compared to iss, hss horses showed a greater decrease in pcv (p 5 0.04), total protein (p 5 0.01), albumin (p 5 0.01), and globulin (p 5 0.02). hss horses showed a greater increase in sodium and chloride (p o 0.001) as compared to iss horses. horses receiving hss had a shorter time to urination (p 5 0.03) and lower specific gravity (p o 0.001) than those receiving iss.results of this study indicate that hss may provide faster restoration of intravascular volume deficits than iss in endurance horses receiving emergency medical treatment. more profound electrolyte changes should be expected with hss however. b 2 -adrenergic receptor agonists have been shown to increase erythrocyte carbonic anhydrase activity, which may stimulate the jacobs-stewart cycle and increase pulmonary circulation transvascular fluid fluxes during exercise. increase in pulmonary transvascular fluid fluxes (j v-a ) and consequent increase in the pulmonary interstitial fluid would be detrimental for alveolar o 2 exchange during the fast erythrocyte transition time across the pulmonary capillaries. therefore, we hypothesised that treatment with inhaled b 2 -adrenergic receptor agonist will increase j v-a and the alveolar-arterial po 2 difference (aado 2 ) during exercise.six stb horses were exercised on a high-speed treadmill at 80% vo 2 peak until fatigue. horses were randomly assigned to treatment with salbutamol (sal: 500mcg) or placebo (control: con) inhalation via aeromask ã 60 min prior to exercise, with cross over treatment used at the repeated exercise test (8 days later). arterial and mixedvenous blood, as well as co 2 elimination and o 2 uptake, were sampled simultaneously at rest, during exercise at 60 sec intervals until fatigue, and into recovery. blood gases were analyzed. aado 2 was calculated using the inspired po 2 (149 mmhg), and blood partial pressure of o 2 and co 2 . blood volume (%) changes across the lung were calculated from changes in hemoglobin and hematocrit values in venous and arterial blood. cardiac output (q) was calculated using the fick equation. j v-a was calculated using q and blood volume changes across the lung. variables were analyzed using two-way repeated-measures anova (p o 0.05).the duration of exercise to fatigue was 4.3 ae 0.3 min and 4.4 ae 0.4 min in both con and sal, respectively. at rest sal had no effect on j v-a , oxygen consumption (vo 2 ), blood oxygen saturation (so 2 ) or aado 2 (p40.05). at the onset of exercise j v-a increased in con and sal (p o 0.0001) and at fatigue reached 10.0 ae 2.4 l/min and 10.0 ae 1.6 l/min, respectively. treatment with sal had no effect on j v-a during exercise (p 5 0.9). at the onset of exercise so 2 and vo 2 increased in con and sal (p o 0.0001). treatment with sal had no effect on so 2 or vo 2 during exercise (p40.05). aado 2 increased during exercise in con and sal (p o 0.0001) and at fatigue reached 19.4 ae 2.3 mmhg and 18.1 ae 2.4 mmhg, respectively. treatment with sal had no effect on aado 2 during exercise (p 5 0.3).inhaled b 2 -adrenergic receptor agonist salbutamol at the dose of 500mcg given 60 min before exercise did not affect the duration of exercise to fatigue, j v-a , vo 2 , so 2 or aado 2 . therefore, it had no detrimental effect on alveolar-capillary diffusion distance and the ventilation/perfusion mismatch in exercising horses. inflammatory airway disease (iad) and recurrent airway obstruction (rao) represent two classes of equine lung inflammatory diseases that may share some similar immunologic mechanisms. there is evidence that th2 cytokines and il-17 play some role in rao. iad is a common condition in horses, but its pathophysiology is still not understood. the aim of the present study was therefore to determine the mrna expression of th1, th2 and th17 inflammatory cytokines, to understand the immunological mechanisms of iad.the mrna expression of ten inflammatory cytokines and chemokines was measured in the bronchoalveolar fluid (balf) of seventeen horses with iad and compared with ten control horses. the horses were selected based on 1-their clinical signs, 2-the inflammatory cells count in the balf, 3-their physical examination and 4-their medical history. the mrna expression of il-5, il-1b, il-6, il-8 and il-10 was significantly up-regulated in balf from horses with iad.furthermore, the balf samples were subdivided in two groups based on the differential cells count 1-balf with increased mast cells (iad-mast) and 2-balf with increased neutrophils (iad-neutro). il-4 was significantly down-regulated in the iad-neutro group compared to the iad-mast group. il-17, il-5 and il-8 were significantly up-regulated in the iad-neutro group compared to the iad-mast group.the present study shows that iad in horses is characterized by a th2 and a th17 mrna inflammatory expression profile and that different immunological mechanisms are involved in mast cells or neutrophils accumulation in the balf of horses with iad. b 2 -adrenoreceptor (b 2 -ar) agonists are a class of medications that promote smooth muscle relaxation and bronchodilation in horses and humans with airway disease. activated human peripheral blood lymphocytes (pbls) also respond to b 2 -ar agonist stimulation by attenuating the production of cytokines associated with the pathogenesis of asthma and recurrent airway obstruction (rao). the aim of this study was to develop an in vitro technique for measuring the response of equine pbls to stimulation with salbutamol, a b 2 -ar agonist. this method was then used to compare the response of pbls from rao-affected and non-affected horses to b 2 -ar agonist stimulation. pbls from 4 rao and 4 nonaffected horses were cultured (4x10 6 /ml) in rpmi complete media with concanavalin a (cona, 2ug/10 6 cells) for 0, 1, or 2 days then stimulated with salbutamol (30 minutes). using flow cytometric techniques, response was measured by detecting protein kinase a phosphorylation of vasodilator stimulated phosphoprotein (vasp). results were verified by western blot analysis. activated pbls were then incubated with cona for one day were pre-incubated with b 2 or b-adrenoreceptor antagonist (ici 118,551, sigma s ; atenolol, sigma s ) for 15 minutes, followed by 30 minutes salbutamol (500 nm) stimulation. results were analyzed by anova or ancova and differences were considered significant when p o 0.05.response to b-antagonist was only observed in activated pbls (pre-cultured with con a) and was greater in cells from rao horses as compared to cells from non-affected horses. the addition of b-antagonist attenuated the response of pbls to salbutamol while the addition of a b 1 -antagonist had no effect. these findings indicate that activated pbls from rao-affected horses have a greater response to salbutamol as compared to pbls from non-affected horses, and this response is mediated mainly through the b 2 -ar.human b 2 -ar are known be polymorphic and this polymorphism results in a variable response to b 2 -agonist binding that affects long term outcome in human asthmatics. further studies are required to determine if the difference in response of pbls from rao affected as compared to non-affected horses is due to genetic polymorphism in the equine b 2 -ar, and whether this difference is associated with a propensity for horses to develop equine rao. key: cord-010092-uftc8inx authors: nan title: abstract of 29th regional congress of the isbt date: 2019-06-07 journal: vox sang doi: 10.1111/vox.12792 sha: doc_id: 10092 cord_uid: uftc8inx nan in the fin de siecle was heavily concentrated in vienna. freud, boltzmann, schr€ odinger and mach might be the first names to find, whenever one cites austrian scientists. but more related to transfusion are the noble prize winners max perutz and karl landsteiner. landsteiner 0 s fate illustrates the brain drain beginning in the early 30 s escalating in 1939 with the "anschluss", which lead to the forced emigration of many scientists. a loss which was not regenerated in the post war years and was further aggravated by dubious and often undisclosed relations and scandals in the nazi-era. all together this leads to a severe loss of credibility and productivity of universities across decades. opening university access in the early 80 s and intensive historical work-up of scandals transformed the austrian universities to open and effective scientific institutions driving innovation in the country. austria has achieved a great economic deal in recent decades, which was accelerated by the eu membership in 1995. as a result of strong long-term economic performance, the country's gross domestic product (gdp) per capita is the eighth highest among oecd countries and fourth in the eu28. levels of poverty and income inequality are both below the oecd average. investment in research and development (r&d) increased since the eu accession, when austria's r&d intensity (aggregate r&d expenditure as a percentage of gdp) was well below the oecd average and significantly far lower than switzerland -a country to which austria prefers comparison. the eu target of 3% r&d intensity was first met in 2014 and is 2018 the sixth highest among oecd countries and the second highest in the eu28. austria showed the second highest increase in r&d intensity of all oecd countries, exceeded only by korea. the rapid expansion was matched by a similar increase in human resources and scientific output of universities. austrian science in quantum mechanics, quantum communication and information is world renown. vienna is a major biotech hub, as is linz in mathematics and mechatronics and graz in automotive and production technologies. austria has been a net resource recipient in the horizon 2020 and the preceding 7th framework programme. small and mediumsized enterprises show a high propensity to co-operate with universities and other research organisations and more and more included in scientific grant schemes. vienna is the largest student city in the german-speaking world and consistently ranks among the top cities in the world on quality-of-life indices. as austria possesses globally recognised cultural attractions ranging from famed salzburg festival to the vienna new year concert its inhabitants are not aware of the progress made in r&d and how thriving innovation is going on in their country. they still love to show their cultural heritage and events and impress the world with some kind of eternal sound of music. patients with refractory b-cell malignancies as non-hodgkin lymphomas (nhl) resistant to standard therapies have a dismal prognosis. the outcome is even poorer in patients relapsing after autologous stem cell transplantation. most of these patients do not qualify for an allogeneic hematopoietic cell transplantation (hct) due to refractory disease, lack of a suitable allogeneic donor, higher age or cumulative toxicity of previous chemotherapy. despite patients undergoing allogeneic hct normally profit from a graft-versus -lymphoma effect, overall survival in patients with nhl after hct remains short. a similar situation can be observed for patients with acute lymphoblastic leukemia (all). therefore novel treatment modalities are urgently needed. chimeric antigen receptor (car)-t cells, a new class of cellular immunotherapy involving ex vivo genetic modification of t cells to incorporate an engineered car have been used in clinical trials. in the majority of studies b-cell malignancies treated with cd19 targeting car-t cells have been analyzed. austria had the advantage to participate in two international trials in the past and is currently involved in further car-t studies. recently, results from cd19 directed car-t cell trials with an increased follow-up of patients led to fda (food and drug administration) and ema (european medicines agency) approval of tisagenlecleucel and axicabtagene ciloleucel. common adverse events (aes) include cytokine release syndrome and neurological toxicity, which may require admission to an intensive care unit, b cell aplasia and hemophagocytic lymphohistiocytosis. these aes are manageable when treated by an appropriately trained team following established algorithm. in this presentation, results of four large phase ii cd19car-t cell trials for patients with nhl and all and focus on aes is summarized. preoperative anemia is a known risk factor for increased perioperative morbidity and mortality in patients undergoing major surgery. previous studies have not only shown higher in-hospital mortality, but also an increased hospital length of stay, greater postoperative admission rates to intensive care and prolonged use of mechanical ventilation and intensive care resources in patients with anemia compared to those with normal preoperative hemoglobin concentrations. about 30% of patients scheduled for major surgery suffer from preoperative anemia. this figure is even higher in patients requiring orthotopic liver transplantation, where up to 75% of all patients are diagnosed with anemia prior to surgery. transfusion of packed red blood cells (prbcs) is commonly used to correct anemic hemoglobin values. however, transfusion of prbcs has been associated with increased morbidity and mortality in patients undergoing cardiac, orthopedic, and abdominal surgery. additionally, transfusion of prbcs is associated with a greater incidence of postoperative acute kidney injury in patients undergoing orthotopic liver transplantation. as preoperative anemia might increase the perioperative use of prbcs, negative effects observed after prbc transfusions might even be augmented. data on the influence of preoperative anemia on morbidity and mortality after orthotopic liver transplantation are limited. thus, we retrospectively analyzed the association of preoperative anemia and mortality in adult patients undergoing orthotopic liver transplantation at our institution. in addition, we examined the influence of anemia on perioperative parameters such as transfusion requirements, surgical complications, early allograft dysfunction, acute kidney injury, and the need for renal replacement therapy. based on the results obtained in the retrospective analysis, an ongoing prospective randomized clinical trial was initiated. the two suspensive treatments in sickle cell disease (scd) are hydroxycarbamide, inducing the production of the functional hbf, normally repressed at birth, and red blood cell (rbc) transfusion, a critical component of scd management. however, rbc transfusion is not without risk. repeat exposure to allogeneic rbcs can result in the development of rbc alloantibodies which can make it difficult to find compatible rbcs for future transfusions. however, the main concern of alloimmunization is the development of hemolytic transfusion reaction, with in the most severe cases, hyperhemolysis, leading to multi organ failure and death in 4% of the cases. the prevention of this life threatening condition must be based on risk factors. however, although some risk factors, such as alloimmunization, have been identified, much of the mechanism underlying dhtr remains a mystery, particularly in severe cases presenting hyperhemolysis. here we will describe the current and future development to prevent and treat this severe syndrome in order to decrease exposure to transfusion in scd but also improve red blood cell quality, some new products are developed. oxidative damage is one of the parameter that could be diminished. some work is also ongoing to prevent filter blockage during leucodepletion of precious rbcs units from afro-caribbean donors carrying the sickle cell trait. finally, in countries with higher risks of transmission of infectious disease, treatment of red blood cell units against infectious agents can be discussed. the only current curative treatment of scd is hematopoietic stem cell transplantation (hcs). however, the occurrence, frequency, and effects of immune hematologic complications in hcs remain and will be discussed. finally, gene therapy is a real hope as a definitive curative treatment. clinical trials are ongoing in france and will be discussed as well as the remaining place of transfusion in this therapeutic. in the context of the chronic myeloid leukemia (cml), we have hypothesized that quiescent leukemic hematopoietic stem cells (hsc) compartment, escaping to the current tyrosine kinase inhibitors (tkis) treatment, in part associated in the molecular relapse, may be targeted by cart-cells immunotherapy. gene expression profiling studies have established that a cell surface biomarker il-1rap is expressed by the leukemic but not by the normal cd34 + /cd38-hsc. this talk will focus of the whole process of development of a cart-cells starting from recombinant il-1rap protein mice immunization to produce a specific monoclonal antibody (mab), to the proof of concept demonstration, before moving into the clinic. we produced and selected a specific anti-il-1rap mab (#a3c3 clone, diaclone sa, besanc ßon, france). after molecular characterization of antigen-binding domain, nucleotide sequences were fused with 3rd generation t cell activation coding sequences and cloned as a single chain into a lentiviral backbone comprising a safety switch suicide gene icasp9 (inducible caspase 9) and a monitoring/selection cell surface marker δcd19. we demonstrated in-vitro and in an in-vivo xenograft murine model that il-1rap car t cells can be activated in the presence of il-1rap+ cell lines or primary cml cells, secrete pro-inflammatory cytokines, degranulate and specifically killing them. we also demonstrated that multi-tkis treatment over a 4-year period does not affect transduction efficiency of cml patient t-cells by il-1rap car vector and that autologous cart-cells are able to target il-1rap+ leukemic primary hsc. "off-tumor-on target" toxicity prediction, by studying il-1rap expression on a tissue macroarray comprising 30 normal human tissues (3 donors) , with #a3c3, detected various il-1rap intensity staining in only few tissues. regarding the healthy hematopoietic system, #a3c3 flow cytometry staining did not detect hematopoietic cells, except monocytes that express poorly il-1rap. as expected, monocyte subpopulation is targeted by autologous il-1rap cart cells (ratio e: t = 1:1), but at a lower level that il-1rap cml cell line. in-vivo investigation of specific toxicities of autologous il-1rap cart-cells against hsc and/or immune cells on a human-cd34 + cord blood cell engrafted/nog murine model, but also by an in-vitro cd34 + colony forming unit assay didn't reveal any significant toxicities in immunocompetent cell subpopulations, suggesting that healthy cd34 + hsc are not affected. finally, to overcome potential toxicity, functionality of the icasp9/ rimiducid â safety switch was demonstrated in-vitro but also in-vivo in a nsg tumor xenograft model, showing that, when activate, the system is able to eliminate more than 90% of cart-cells, after exposure to ap1903. in conclusion, based on cml model, we demonstrated that il-1rap is an interesting target for cart-cell immunotherapy, with a limited "on target, off tumor" predictable toxicity. next step will be the up-scaling of the process in order to match with the use in human regarding also the regulatory requirements. this strategy may be applied, in the future, in other hematological malignancies. mortality ranges from 20 to 30% for trauma victims with severe bleeding and is largely dependent on the transfusion therapy from which they can benefit. the nature of this therapy has an impact on prognosis with a halving of mortality when the plasma/prbc ratio is greater than ½ and a decrease of about 20% when the proportion of platelets transfused is close to that of whole blood. the speed with which such therapy is actually administered has a major impact as well with an increase in mortality of 5% for each minute of delay in making the entire therapy available. this can be explained mainly by the fact that the probability of death of these patients is greater within minutes of their admission to hospital with a median time to death of 2 h after admission. to allow plasma, platelets and prbc to be made available in a timely manner, north american trauma centers have mandated that trauma centers have massive transfusion packs at the patient's bedside within 15 min. to further simplify and speed up logistics from distribution to transfusion, several trauma centers now use whole blood stored at 4°c. this return of an "old" product is largely inspired by military experience where whole blood is mainly used "warm" immediately after collection with compelling evidence of its effectiveness. its return to civilian practice requires the ability to deleukocyte it while preserving platelets and to store it while maintaining their hemostatic functions. good quality data shows this is achievable and several clinical studies are planned to begin in the coming months. in france, the french blood establishment and the french army are cooperating to initiate the prospective randomized non-inferiority storhm trial (sang total pour la r eanimation des h emorragies massives) which will be comparing whole blood to separate blood components in an 1/1/1 ratio in severely bleeding trauma patients. the primary endpoint will be a thromboelastographic parameter (maximum amplitude) assessed at the 6th hour after admission. secondary endpoints will include early and overall mortality, lactate clearance (reflection of the effectiveness of resuscitation) and 24 h organ failure. this trial will be recruiting 200 patients in 6 french trauma centers and is planned to be initiated second half of 2019. local/neighbours day: innovation in germany 1c-03-01 mesenchymal stromal cells for regenerative therapy bm-msc / asc obtained from these protocols have been characterized in detail in preclinical evaluations. manufacturing licenses for msc and asc and a platelet-derived growth factor concentrate have been obtained and they have been explored in several clinical trials for treatment of bone defects (ortho-ct1: eudract number: 2011-005441-13; ortho-ct2: eudract number: 2012-002010-39; maxillo1: eudract number: 2012-003139-50) . we will summarize results of completed clinical trials which confirmed feasibility and safety of autologous msc /asc treatment and provided evidence for efficacy (gjerde et al, stem cell res. 2018 introduction: in vitro produced megakaryocytes (mks) may serve as source to produce platelets (plts) ex vivo or in vivo. we have established a strategy to differentiate mks from induced pluripotent stem cells (ipscs) in bioreactors. this study aimed at the large-scale production of mks using microcarriers to increase the mk yield and to characterize their phenotype and functionality after irradiation as a method to decrease possible safety concerns associated to the ipsc origin. methods: ipscs were cultured in an aggregate form in presence or absence of microcarriers using 50 ml stirred flasks. cells were differentiated into mks using tpo, scf and il-3 in apel2 medium for a period of 22 days. non-irradiated or irradiated ipsc-derived mks were analysed for polyploidy, phenotype and proplt production using flow cytometry and fluorescence microscopy. also, plt-production was investigated in vivo. non-irradiated or irradiated mks were transfused to nod/ scid/il-2rcc-/-mice and blood was analyzed for human plts. results: differentiation of mks in presence of microcarriers resulted in an 8-fold increase of mks per ipsc in comparison to only aggregates. this resulted in mean of total mk harvest of 18.7 ae 6.8 9 10 7 in microcarrier-assisted bioreactors in comparison to 4.9 ae 1.3 9 10 7 mks collected from bioreactors containing only aggregates. interestingly, mks produced in microcarrier-assisted bioreactors showed higher proplt formation capacity than mks derived from only aggregates bioreactors. mk phenotype and dna content was comparable between mks derived from both types of bioreactors. irradiation of mks did not affect their phenotype and capability to form proplts or plts after transfusion into nod/scid/il-2rcc -/mice. conclusion: microcarriers showed to significantly increase the yield of ipsc-derived mks in stirred bioreactors to clinically relevant numbers. this may facilitate the use of ipsc-derived mk for ex vivo production of plts, direct transfusion or for innovative mk-based regenerative therapies. although the rosetta stone, found by the troops of napoleon in egypt near the city of rosetta (rashid) contains only a small amount of text in three languages it was key in deciphering hieroglyphs. the rosetta mission tried to achieve something similar: by looking at a tiny body its goal was to decipher the origin of the solar system, planets including earth and life. after more than 12 years the rosetta spacecraft softly crashlanded on comet churyumov-gerasimenko on september 30, 2016 . it has travelled billions of kilometers, just to study a small (4 km diameter), black boulder named 67p/ churyumov-gerasimenko. the results of this mission now seem to fully justify the time and money spent in the last decades on this endeavor. where are we from? where are we going? are we alone in the universe? these are some of the big questions. in this talk i will show which answers we got from rosetta and comet chury. we follow the pathway of the material which makes up our solar system from a dark cloud to the solar nebula and finally to planets and life. i will show that indeed we are the result of stardust and that what happened here may happen elsewhere in the universe. cells, tissues and entire organs can collectively be seen as "living drugs". genetically unaltered cells are routinely used in clinical practice to treat diseases as diverse as anemia, bleeding disorders, leukemia and organ failure. ground-breaking advances in genetic and genome engineering technologies are propelling cell therapies to the frontline of medical research and practice. the hematopoietic system is particularly amenable to genetic engineering because specific cell types can be purified based on the expression of specific surface proteins and the ability to culture and expand cells ex vivo. the recent unprecedented clinical success of killer t cells reprogrammed by chimeric antigen receptors (cars) to attack cd19 expressing tumor cells demonstrates the power of immunotherapy with genetically engineered immune cells. however, given the rapid development of novel genome engineering and synthetic biology tools we are likely only at the beginning of a new era of engineered cellular therapies. i will present recent progress in immune cell reprogramming, gene correction, safety aspects and remaining challenges such as manufacturing. 1d-04-03 cell free nucleic acids (cfna) circulate in the plasma of all individuals and are thought to be released by host and foreign cells into the circulation. after fractionation by centrifugation, cfnas can be extracted from the supernatant of whole blood samples or manufactured blood products. these dna or rna sequences can be of human, bacterial, viral or fungal origin. most of them are human double stranded dnas. research on cfnas is increasing, thanks to technological advancements in molecular biology. some of their results are already implemented in clinical practice in the areas of prenatal diagnosis, oncology and infectious diseases. the latter investigation focuses on the exploration of non-human cfnas, the field of metagenomics. high throughput sequencing associated with bioinformatics, the so-called new generation sequencing (ngs), has sped up the investigations of non-human cfnas. this tool provides the opportunity to classify cfnas into a human or non-human category, and then to identify them. it is thus possible to explore simultaneously the whole landscape of bacterial, viral and fungal populations. presently, ngs of human blood has already proven its feasibility and its value in identifying emerging viruses or investigating clinical cases of fever of unknown etiology. ngs of cfnas is also particularly effective in analyzing the different genotypes of a virus in case of a co-infection (e.g. hepatitis c virus). studying cfnas with the new molecular technologies is therefore of great importance in transfusion medicine, especially regarding security and clinical transfusion reactions. first, transfusion transmitted infections are the most feared adverse complications. second, febrile non-hemolytic transfusion reactions are also the most frequently reported adverse events in hemovigilance systems and their physiological mechanism -if only one-remains not clearly elucidated. investigating cfnas could thus improve our understanding and strategy aiming at reducing those two clinical adverse events. surveying comprehensively the composition of circulating infectious agents in a blood product by ngs technology could be very interesting for investigating a severe febrile transfusion reaction. moreover, when the costs of analysis will be reduced, it might be possible to screen prospectively and regularly the whole metagenomics of asymptomatic blood donors, in addition to the classical epidemiological surveillance. for instance, in a study testing a ngs method on manufactured fresh frozen plasmas, an astrovirus (mlb2) has been identified. finally, it is the responsibility of transfusion physicians implicated in the manufacturing of blood products to ensure that cfnas within a blood product do not have a clinical impact on the innate immunity of the recipients. according to recent research in vitro, cfnas purified from blood products can induce the transcription of inflammatory cytokines by mononuclear cells. as nonhuman cfna have an effect on toll-like receptors (tlr-linked inflammatory pathways), it would be also relevant to insure that donor's cfnas have no significant effect on the immune system of the recipient. in conclusion, cfnas are very diverse molecules contaminating blood products. technological progress makes now their investigation more available. besides being useful markers of infection in asymptomatic donors, their impact on the recipients' immunity should be further investigated. an active life and regular training is part of a healthy life style and for many this includes participation in endurance exercise competition at different levels. thus, it is highly relevant to know how a blood donation affects exercise performance and how close this can done to an endurance competition. endurance exercise performance is determined by many factors, but three of the primary are maximal oxygen uptake, the relative load that an individual can sustain over time and finally the efficiency of movement in the given discipline. over the years, a number of studies have sampled blood volumes ranging from 450-1200 ml and applied different methodological approaches to measure maximal oxygen uptake over a recovery period ranging between 3-28 days. overall, the general finding is a reduction in blood haemoglobin and an attenuated maximal oxygen uptake as well endurance performance after blood donation. in normal to well-trained men maximal oxygen uptake and performance was normalized after two weeks in one study after a normal blood donation (450/470 ml), but remained attenuated after four weeks in another study, despite the change in blood haemoglobin concentration was similar and the design and methodology also similar in the two studies. in addition to maximal oxygen uptake the relative load that can be tolerated during exercise is probably also attenuated, through a decreased arterio-venous oxygen extraction, but the available data is very limited. the first part of this talk will highlight the major findings and discuss some of the methodological issues that complicate interpretation and conclusions. there are sex differences in circulating blood volume, haemoglobin concentration, haematocrit and hormone levels and thus it is entirely possible that there is a sex difference in the effect of blood donation on physical performance and the recovery after blood donation. in addition to the basic physiological sex differences, there is also a higher prevalence of iron deficiency in premenopausal women, physically active women and women donating blood. therefore, we studied the influence of a standard 450 ml blood donation on maximal oxygen uptake and endurance performance and the subsequent recovery in physically active women. we observed that in iron sufficient women blood haemoglobin concentration and maximal oxygen uptake were back to baseline 28 days after blood donation, but endurance performance was normalized already after 14 days. the second part of this talk will discuss the sex differences in the effect of blood donation on maximal oxygen uptake and endurance performance. overall, the available data suggest that, with a careful conservative approach, 4 -5 weeks are needed after a normal blood donation to be fully recovered to participate in endurance exercise competitions. more than one in ten attempts to donate blood result in a temporary deferral, due to concerns about the impact of the donation on the donor or recipient. there is well established evidence that temporary deferrals impact negatively on donors, with a large proportion of those deferred failing to return at the end of the deferral period. this presentation provides an overview of deferrals from the donor perspective, describing the likelihood of receiving a deferral for different donor subgroups. the impact of temporary deferrals on the future donation of donors, considering both short-term and longer-term donation patterns, will also be reviewed, outlining which donors are at highest risk of non-return following a deferral, and what is known about the accumulative impact of multiple deferrals on donors. several hypotheses have been proposed to account for the strong negative impact of deferrals on donor behaviour, and there is preliminary evidence of psychological factors, such as emotional reactions, predicting intention to return. research is also beginning to emerge on the effectiveness of tailored interventions to mitigate the impact of deferrals on donor behaviour. the evidence for these preventative interventions, and for strategies to reactive donors once they have lapsed post-deferral, will be reviewed. recommendations for blood centres will be made, as well as suggestions for future research to address continuing gaps in knowledge. in his influential study "the gift relationship" (1970) , richard titmuss coined the idea of voluntary, non-paid, blood donation being the gift of life for a fellow citizen. this metaphor has been powerful in mobilizing donors (busby 2004) . it conveys a direct relationship between blood donation and patients' vitality, as well as a difference between gains and costs. as the gift of life, blood donation is seen to symbolize pure altruism and promoting solidarity between strangers. but can we apply the metaphor as successfully into donating blood for research? we asked a group of finnish blood donors what they would think if the frc blood service invited them to give a blood sample and personal information for research. the blood donors were usually willing to contribute to research for the public benefit, because they saw great potential in science to create solutions to help patients in the future. however, based on our interview data and previous research, we suggest that the analogy between gift of life and donation for research did not work all the way. the metaphor fails to address donors' questions on new types of relationships, interests and risks related to the use of personal data for research. left unanswered these could discourage donating for research. hence, we argue that the gift of life metaphor is not applicable to donor recruitment at the research context. in this presentation we wish to look for a better metaphor for donation for research that blood services collecting research data could apply. academy day: transfusion challenges in patients with sickle cell disease 2a-02-01 immunohaematological features of patients with sickle cell disease (lfa) should be considered as polymorphic antigens in the african population and these lfa are not present in most commercial panels. the situation is even more complicated when recipients lack high-frequency antigens, the most common ones being hr-, hr b -, sec-, u neg , u+ var , js(b-), (hy-), and jo(a-). finally, there is a high rh diversity among people of african descent. because they harbor variant alleles and/or partial rh antigens, they are at risk of developing alloantibodies. in this setting, screening for partial rh antigens makes sense. the figures illustrating this diversity vary with the approach used. one of them is to take into consideration rhd or rhce*ce variant alleles. in several american studies, their prevalence was estimated to be 29-36% and 53-72%, respectively. other teams take into consideration d, c and e partial antigens. their prevalence was estimated to be 8.4-14%, 12.5-27.7%, 3.3-3.5%, respectively, and the alloimmunization rates were 17.6%, 14.3-30%, 7.1%, respectively. as a result of these phenotype discrepancies, scd patients are more likely to be alloimmunized. an overall immunization rate of 2-6% is commonly admitted in the general population. depending on the unit selection policy and/or the study design, the immunization rate in scd patients varies from 7% to 76%, the highest figures being established when an abo/rh1-only matching policy is implemented. in a meta-analysis of 24 publications, the overall alloimmunization rates were around 20%. alloimmunization is thought to be enhanced by an inflammatory state, which is often present in scd patients. they are more prone to develop a new alloantibody. using a stochastic modeling of alloimmunization, they have a 61% increased risk of producing additional antibodies versus 30% in the general population. autoantibodies have been identified as a risk factor of alloimmunization. as a result, scd patients often have complex mixtures of allo and autoantibodies. rh antibodies and those considered as irregular natural antibodies are present in a significant proportion. another characteristic of the antibodies in scd patients is their evanescence; up to 70% of alloantibodies become undetectable within a few years of their initial development. relatedly, about a third of dhtrs are reported to happen in patients with no previous history of immunization. in addition, a third of patients will not develop an antibody after a dhtr. identifying patients at risk of developing a dhtr is key to managing them properly. alloimmunization is a serious risk of red blood cell transfusion in patients with sickle cell disease (scd) and can result in severe (delayed) haemolytic transfusion reactions, exacerbation of clinical symptoms and life-threatening hyperhaemolysis. once alloimmunized, the presence of alloantibodies in the patients' blood further complicates pretransfusion testing and hampers the selection of compatible blood products. numerous studies have shown that scd patients have a relatively high risk of alloimmunization as compared to the 'general' population. this is not only explained by the large number of transfusions given but also by the increased exposure to foreign antigens as a result of differences in the antigen make-up of the scd recipients and the blood donor population. other factors involved in the immune response such as age at first transfusion, inflammatory state, hla typing are under investigation and are starting to unravel. because blood transfusion is still one of the main treatment modalities for scd and some patients have a life-long transfusion dependency it is important to minimize the alloimmunization rate. theoretically, complete matching for all relevant blood group antigens would prevent alloimmunization. this however, is only possible when all donors are comprehensively. matching strategies should be developed to minimize alloimmunization while balancing patients' need and donor availability and is cost effective. to develop a (preventive) matching strategy some factors need to be established; 1) which antibody specificities are clinically relevant 2) which antigens are most immunogenic 3) what is the availability of specific antigen typings in the donor population 4) how should recipients (and donors) be typed, phenotypically and/or genotypically and to what extent. the latter is especially important in scd patients since they are of african descent and the prevalence of genetic variations in this population is relatively high. rhd and rhce variants are common and can remain undetected when serological typing is used but can be discovered with high resolution molecular typing. patients with partial rh phenotypes are at risk for alloimmunization. apart from special rh phenotypes in individuals from african descent, the fy(a-b-) phenotype related to the gata-box mutation in the fyb allele and the u-or u var phenotype resulting from genetic variations in the mns alleles are also common. several studies have shown that in scd patients antibodies directed against rhd, rhe, rhc and k are most frequently found when unmatched transfusions are given. preventive matching for these antigens has proven successful in reducing alloimmunisation. extended matching for all rh antigens fy(a), jk(a) and jk(b) can further decrease the alloimmunisation rate. currently, different countries have preventive matching strategies in place for this vulnerable patient group. as genotyping is more and more available and within reach, optimal antigen typing approaches for patients and donors, combining serology and genetics are being developed. in this lecture several aspects of antigen typing approaches and preventive matching strategies that will most benefit scd patients of will be discussed. artificial intelligence has become a buzzword that will appear about anywhere in the media. we can forget that ai, or the subfield in this computer science field machine learning, has been around for over 50 years. improvements in computing power, abundance of data, progress in computer science, and the arrival of affordable cloud solutions have now brought it to our daily lives. also in health care news about ai has become omnipresent. and some landmark papers have come out on algorithms outperforming (teams of) physicians in the diagnosis of all kinds of skin disease, eye disease from retina scans, and detect cancer in ct scans. however, little of these solutions have actually shown up in our clinical practice yet. in anaesthesia, we worked with the first algorithm to come to anaesthesia practice; hypotension during surgery is associated strongly with poor outcome like myocardial ischemia, surgical complications, renal failure and even mortality. we worked on a machine-learning trained algorithm that predicts hypotensive events using the arterial blood pressure curve up to 15-30 min before the actual event. to get fda and ce approval, however, mere mathematic validation is required. this can be achieved on retrospective datasets. in reality, we need more before we can use these algorithms to support our decision-making; after internal (retrospective) and external (prospective but passive use) validation steps, clinical (i.e. rct validation is needed. moreover, we will need to assess the economic impact too. ultimately this tool has now reached clinical practice and is starting to help us go from reactive to more proactive hemodynamic management. like this, we have started to work on machine-learning tools to predict the incidence of specific types of patients coming into a&e and predicting infections after surgery. we will discuss our approach, essentials to start with machine learning, practical learnings. we will also discuss a first project design to use machine learning in managing bleeding patients to get the best therapy advice for blood product use like plasma, fibrinogen et cetera. how can we start using this tool in unison with our existing tools to improve science and clinical practice in our respective (bio)medical fields? thrombocytopenia is a very common hematological abnormality found in newborns, especially in preterm neonates. two subgroups can be distinguished: early thrombocytopenia, occurring within the first 72 h of life, and late thrombocytopenia, occurring after the first 72 h of life. early thrombocytopenia is associated with intrauterine growth restriction, whereas late thrombocytopenia is caused mainly by sepsis and necrotizing enterocolitis. platelet transfusions are the hallmark of the treatment of neonatal thrombocytopenia. most of these transfusions are prophylactic, which means they are given in the absence of bleeding. however, the efficacy of these transfusions in preventing bleeding has never been proven. in addition, risks of platelet transfusion seem to be more pronounced in preterm neonates. because of lack of data, platelet transfusion guidelines differ widely between countries. in a recent randomized controlled trial (planet-2/matisse study) among preterm infants with severe thrombocytopenia, we found that those randomly assigned to receive platelet transfusions at a platelet-count threshold of 50 9 10 9 /l had a significantly higher rate of death or major bleeding within 28 days after randomization than those who received platelet transfusions at a platelet-count threshold of 25 9 10 9 /l. this presentation summarizes the current understanding of etiology and management of neonatal thrombocytopenia. transfusion-associated circulatory overload (taco) is a severe transfusion adverse reaction that is associated with increased mortality and morbidity. the incidence of taco in adults varies from 1% to 8%, but is probably underdiagnosed and underreported. the incidence in the pediatric population is undetermined. taco usually occurs in patients who receive a large volume of blood product over a short period of time. it is more common in patients with known risk factors such as cardiovascular disease, renal failure, and older or younger age (> 60 years or < 3 years). hospitalised patients and intensive care patients are also more at risk. the typical presentation of taco is respiratory distress (dyspnea, tachypnea) occurring within 12 h of a blood transfusion. associated signs and symptoms are hypoxia, hypertension, tachycardia, positive fluid balance, high central venous pressure, and acute or worsening pulmonary edema on chest x-ray. echocardiography and measurement of brain natriuretic peptide (bnp) or its n-terminal prohormone (nt-probnp) is helpful for diagnosis. several definition criteria have been proposed for taco, but none are adapted for children, particularly critically-ill children who are more at risk. this is probably the main reason why taco is even more underdiagnosed and underreported in the pediatric population. in a recent study, we compared the incidence of taco in a pediatric intensive care unit using the international society of blood transfusion (isbt) criteria, with two different ways of defining abnormal values: 1) using normal pediatric values published in the nelson textbook of pediatrics; and 2) using patients as their own controls and comparing pre-and post-transfusion values with either a 10% or 20% difference threshold. we monitored for taco up to 24 h post-transfusion. a total of 136 patients were included. taco incidence varied from 1.5% to 76%, depending on the definition used. with such wide variability, we conclude that a more operational definition of taco is needed in pediatrics, particularly for critically-ill children. differential diagnosis from other dyspnea-associated transfusion adverse reactions (e.g. transfusion-associated lung injury, anaphylaxis) is important because treatment differs, as do guidelines to the blood bank. treatment for taco is similar to that of any other cardiogenic pulmonary edema: oxygen, diuresis, ventilatory support. prevention is possible by avoiding unnecessary transfusions, transfusing only the necessary amount of blood product, avoiding rapid transfusions, and using diuretics. background: the risk and importance of transfusion-transmitted hepatitis e virus (tt-hev) infections by contaminated blood is currently a controversial discussed topic in transfusion medicine. in particular, the infectious dose is a not finally determined quantity. the different countries have chosen different approaches to deal with this pathogen. one central question is the need of individual nat screening (id) versus minipool nat screening (mp) approaches to identify all relevant viremias in blood donors. aims: comparison and evaluation of the available screening strategies in relation to the infectious dose to minimize the risk of tt-hev infections. methods: we systematically reviewed the presently known cases of tt-hev infections and available routine nat-screening assays. furthermore, blood donation screening strategies for hev ehev in effect in the european union were compared. we also describe our own experiences of hev screening utilising an id-nat-based donor screening algorithm compared to mp-nat in pools of 96 samples. from november 2017 to january 2018, a total of 10,141 blood donations were screened for the presence of hev rna using a mp-nat (in house, realstar hev rt-pcr kit) and an id-nat (cobas 6800 platform). results: the review of the literature revealed a significant variation regarding the infectious dose causing hepatitis e. in the systematic case review, all components with a viral load (vl) greater than 5.00e+04 iu caused infection (definitive infectious dose (difd) . the lowest infectious dose resulting in tt-hev infection observed in general was 7.05e+03 iu (minimal infectious dose (mifd). the infectious dose of the different blood products is mainly influenced by the remaining plasma content. our data comparing the two different hev screening algorithms revealed eight hev rna positive donations using a mp-nat (incidence 1:1,268) , whereas 17 hev rna positive donations were identified by id-nat (incidence 1:597); all id-nat only positive donations had vl < 25 iu/ml. summary/conclusions: taken into account the current knowledge on the required mifd, the difd, and the analytical sensitivities of the screening methods, we extrapolated the detection probability of hev-rna positive blood donors using different test strategies (nat assay, id vs. minipool with different pool sizes). we also considered the amount of plasma in the different blood products and calculated the infectious doses needed to be detected. only id testing would be sufficient to detect the minimum vl in the donor to avoid tt-hev infections based on the currently known mifd, but a highly sensitive mp-nat should be adequate as a routine screening assay to identify high viremic donors and avoid tt-hev infections based on the difd. we have also determined that the incidence of hev infection was approximately 50 % higher if id-nat was used. however, vl were below 25 iu/ml and will most likely not result in tt-hev infection taken into account the currently known mifd or difd. the clinical relevance of and need of identification of these low level hev positive donors still require further investigation. in the last 25 years several pathogen inactivation (pi) technologies have been developed to be applied to blood components. technologies for inactivating pathogens in plasma and platelets are available in the european union, and some others are currently under development. the first pi technology introduced in the market was for plasma, and was based on the addition of methylene blue and the illumination with light (theraflex mb-plasma, macopharma and gr ıfols). for platelets and plasma two technologies are licensed, one is based on the addition of amotosalen and the illumination with ultraviolet light (uv) (intercept â , cerus) and the other one combines the addition of riboflavin (vitamin b 2 ) and the illumination with uv light (mirasol â , terumo bct). currently another technology for platelet inactivation, based on the illumination with uvc light and strong agitation is under development (theraflex, macopharma). for red blood cells one technology based on the addition of one molecule (amustaline, cerus) is being developed. the mechanism of action, and the spectrum and level of inactivation of pathogens varies among the different technologies. in addition, the number of studies with clinically relevant endpoints and the number of patients included in the studies is not homogeneous. there is published evidence for most of them that show that the treated blood components are safe and efficacious for the patients although, for treated platelet concentrates some decrease in the posttransfusion recovery and survival of the transfused platelets occur, with differences between the different technologies. however, cumulative experience on the use in routine, for some of the technologies for almost 20 years, support the concept of the safety and efficacy of the blood components treated with pathogen inactivation technologies without a significant increase in utilization. the use of pathogen inactivation for blood components is not widespread. differences in epidemiology between countries, infectious risk perception, concerns about potential adverse effects associated with its use and economical considerations might explain the differences observed in its implementation. the history of the p1 and p k antigens is complicated and sometimes confusing because of several changes to the nomenclature. the association between the antigens and their genetic home has raised many questions as well as the longstanding enigma regarding the molecular mechanism underlying the common p 1 and p 2 phenotypes. the system (isbt no. 003) currently includes three different antigens, p1, p k and nor. the p1 antigen was discovered already in 1927 by landsteiner and levine while p k and nor were described in 1951 and 1982, respectively. as for the abo system, naturally-occurring antibodies of igm and/or igg classes can be formed against the missing p1/p k carbohydrate structures. anti-p1 is usually a weak and cold-reactive antibody very rarely implicated in hemolytic transfusion reaction (htr) or hemolytic disease of the fetus and newborn (hdfn). however, some antibodies against p1 have been reported to react at 37°c, bind complement and cause both immediate and delayed htrs. the p k antibodies can cause htr and anti-nor is regarded as a polyagglutinin with unknown clinical significance. a higher frequency of miscarriage is seen in women with the rare phenotypes p and p 1 k /p 2 k . the rbc of the fetus as well as of the newborn express low amounts of p1, p and p k antigens but the placenta shows high expression and is consequently a possible target of the antibodies and the cause of the miscarriages. the p k and p1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. furthermore, altered expression of p k antigen has been described in several cancer forms. a longstanding question has been why individuals with p phenotype not only lack p k and p expression but also p1. recently it was clarified that the same a4galtencoded galactosyltransferase synthesizes both the p1, p k and nor antigens and in addition the p 1 and p 2 phenotypes was confirmed to be caused by transcriptional regulation. transcription factors bind selectively to the p 1 allele in the 5'-regulatory region of a4galt, which enhance transcription of the gene. it has been debated whether the p k and p1 antigens exist on glycoproteins in the human rbc membrane or if glycolipids are the only membrane components carrying these epitopes. a recent publication shows that the p1 antigen can be detected on human rbc glycoproteins and thus glycosphingolipids can no longer be considered as the sole carriers of the antigens. the blood group system which started out with one antigen, p1, has now gained two more members namely p k and nor. step by step the biochemical and genetic basis underlying the antigens expressed in this system has been revealed but still many questions remain to be solved. neither gata1 nor klf1 represent a blood group system but mutations in the genes encoding these transcription factors (tfs) have been shown to result in simultaneous altered expression of blood group antigens in certain rare blood group phenotypes. in particular, mutations in the klf1 gene are responsible for the dominantly inherited in(lu) phenotype, commonly referred to as lu(a-b-) because of the gross reduction in lutheran antigens expression. red cells from in(lu) individuals, though, have also weakened expression of other blood group antigens, like the high-incidence antigen anwj, the antigens of the indian blood group system (cd44) and p1, among others. since the first description of klf1 variants associated with the in(lu) phenotype, many other variants of this gene have been reported with an impact on blood group antigen expression and they are listed on the klf1 table of blood group alleles. other than klf1, a mutated gata1 gene has also been found associated with the x-linked form of the lutheran-mod phenotype and has likewise been registered in the gata1 allele table. besides the effect of tf variants on blood group antigen expression, there are transcription factor-binding site polymorphisms in regulatory regions of blood group genes, which also have an impact on the expression of the encoded antigens in red cells. the first example of such type of polymorphisms was described in 1995, when the disruption of a gata motif in the ackr1 gene promoter was found to abolish erythroid gene expression in fy(a-b-) individuals of african descent. the impact of mutations affecting gata1 binding sites has also been described in some abo subgroups, like the am and bm phenotypes. a regulatory element with gata binding sites in the first intron of the abo gene has been found to be altered in individuals with these phenotypes, either by deletion or by a point mutation disrupting the gata motif. recent findings have also revealed that xga expression on red cells is dependent on gata1 binding to a control element located 3.7 kb upstream of the xg gene. a single nucleotide polymorphism (snp) within this region was shown to correlate very well with the expected distribution of the xga negative phenotype in different populations. further work has demonstrated that this g>c snp disrupts a gata1 binding site and consequently abolishes erythrocyte xg expression. overall, these investigations have allowed to elucidate the underlying genetic basis for xga expression and have made xga genotyping possible. similar to xga, the p1 antigen has been known for a long time to be determined by the a4galt gene but the molecular basis underlying the common p1/p2 phenotypes has remained elusive till recently. several cis-regulatory snps had been identified in non-coding sequences around exon 2a, which showed a very good correlation with p1 antigen expression. interestingly, potential binding sites for several hematopoietic tfs were identified in the same region. finally, recent investigations have demonstrated the role of the runx1 tf in the expression of p1 antigen, by selective binding to a regulatory site present in p1 but not in p2 alleles. to summarize, variation in blood group antigen expression may result from mutations or polymorphisms in the regulatory region of blood group genes. recent reports have unravelled the molecular mechanisms underlying the expression of p1 and xga blood group antigens, which involves tf binding to allele-specific regulatory elements. similar mechanisms may also regulate antigen expression in other blood group systems. 2c-06-03 clinical immunology, copenhagen university hospital, copenhagen, denmark since the discovery of cell-free fetal dna (cffdna) in pregnant women's blood, the development of noninvasive prenatal testing (nipt) has provided new diagnostic applications in prenatal care. in transfusion medicine and clinical immunology, cffdna is extracted from maternal plasma to predict fetal blood groups with the purpose of 1) guiding targeted rh prophylaxis in non-immunized rhd negative women and 2) assessing the risk of hemolytic disease of the fetus and newborn (hdfn) in immunized women. i will give an overview of noninvasive prenatal testing of fetal blood groups. based on the literature, i will summarize the current experience with noninvasive prenatal testing of fetal rhd and other blood groups. for rhd negative pregnant women, routine clinical testing is available in several countries world-wide to assess the risk of hdfn in d immunized women, and routine testing to guide rh prophylaxis is now implemented as nationwide service in 6-7 european countries. noninvasive prenatal testing for fetal rhd is highly accurate with sensitivities of 99.9%, as reported from clinical programs. in general, the sensitivity is challenged be low quantities of cffdna, especially in early pregnancy. the specificity is challenged by the polymorphic rh blood group system, where careful attention is needed to navigate among the many rhd variants. rhd variants may complicate cffdna analysis and interpretation of results, especially in populations with mixed ethnicities. despite these challenges, fetal rhd testing is very feasible when implemented with careful attention to these issues. for blood groups that are determined by snps, such as kel or rhc, the main challenge has been interference from the maternal dna when analyzing the fetal dna which has resulted in low accuracy and lower sensitivity, when using qpcr. with the application of more novel techniques such as next generation sequencing and droplet digital pcr, accurate noninvasive prediction of these fetal blood groups has been demonstrated. the success of predicting fetal rhd and its successful clinical implementation into national programs should encourage wide-spread use of cell-free dna based analysis. future work on noninvasive prenatal testing of fetal blood groups determined by snps may consolidate the application for cell-free dna testing for such targets, including human platelet antigens. at isbt, the newly formed cfdna subgroup of the red cell immunogenetics and blood group terminology working party will work to facilitate clinical applications, implementation and evaluation of cell-free dna testing. blood banks in most of the nordic countries all share a vein-to-vein approach which in short means that the collection of blood, the preparation of blood components, testing/release and storing is served by a single actor. on top of that recipient and donor blood grouping, crossmatching, delivery, registration of transfusion and of any complications is usually handled in a single blood banking information system (bbis). this means that blood banks in the nordics are traditionally operated by a single vendor. the needs for process control in a single vendor bbis, the present solutions, unsolved challenges and untapped possibilities of streamlining processes have been scrutinized with the intention to describe separate processes and to acquire best of breed or best of suite it-systems. the aim for integrations, rather than building an integrated it-system, to support the need for a vein-to-vein process is a precondition in the nordic countries. with multiple it-systems supporting isolated processes, we intend to facilitate development in these and furthermore increase the flexibility in the whole process. we set out to reveal any existing knowledge in the literature on it vendor strategies for blood banks, but we didn't succeed in identifying any relevant literature. however, a systematic literature study on vendor strategies when choosing health it was based on the prisma method, and identified 837 studies, but only 10 was eligible for full text review and 5 met the inclusion criteria. even this broader literature study reveals very little evidence. two studies find single vendor strategies poor and conclude "best of suite" solutions to be optimal. one study was not able to correlate vendor strategies to the investigated productivity, but concludes that best of suite and best of breed strategies requires larger organizational changes than a single vendor strategy. in summary, the existing research is contradictory. this paper adds basic knowledge for breaking down the process control of blood banking in smaller processes. this adds the possibility for identifying best of breed or best of suite vendors, instead of relying on single vendor it solutions. furthermore it is a call for more research in the field of vendor selection strategies which this study didn't succeed in identifying. 2d-07-02 applying drones to supply blood to remote areas: rwanda's experience biomedical services, rwanda-ministry of health-national blood services, kigali, rwanda background: in rwanda, blood transfusion services started in 1976. during the 1994 genocide against the tutsis almost all the socioeconomic fabric of rwanda was destroyed as well as its health infrastructure. the healthcare system was suffering in its aftermath, and there were health inequalities between urban and rural areas, including access to blood for transfusion. from 1995, the government started to rebuild all courses of life including the health system and the blood service in particular. the national center for blood transfusion (ncbt) was then mandated to provide safe, effective and adequate blood and blood components to all patients in need. this was pivotal in achieving health related mdgs 4, 5 and 6. today, rwanda has an ambitious vision to put all 12 million citizens within 30 minutes of any essential medical product. while every second matters in emergency management, the use of drones was the perfect solution to many of the last mile challenges that have been traditionally difficult to overcome. it is impossible to forecast accurately down to the need of a single patient. the government has provided an easy solution by centralizing supply and providing on-demand, emergency medical deliveries by drone. doctors are now empowered to provide the quality care with all the supplies on hand, patients can now be treated close to home, and we eliminate waste from potential overstocking when health workers know that they have a quick and reliable source of supply. description: in 2016, the government of rwanda started to operate the world's first national drone delivery program for blood and other lifesaving medical products. these drones can carry two to six units of blood at a time and deliver in 15 -45 minutes depending on a hospital's location. the average duration was between 2 -4 hours round trip with the vehicle system, before the use of drones. drones currently deliver blood to 25 health facilities throughout the country and are set to reach 90% of transfusing health facilities outside kigali by the end of the year. within the first year, healthcare workers saved an average of 3.1 hours per delivery and a total of 10,115 hours of lost time on road pick up they could instead dedicate to patient care. by march 2019, over 11,000 deliveries have been made, with 30% of those being emergency deliveries. a total of more than 19,500 blood units have been delivered. in february 2018, zipline obtained the highest rating from the health facilities being served in a performance evaluation conducted by the national center for blood transfusion. when a doctor or medical staffer needs blood, they place an order through the hemovigilance order portal. they are then sent a confirmation message saying a drone is on its way. the drone flies to the health facility at up to 100 km/h. when it is within five minutes of the destination, the medical staffer receives a notification. the drone then drops the package, attached to a parachute, into a special drop zone. conclusion: supply is not a developing country problem, it is a global issue. rwanda was just the first one to recognize the potential of this technology and decided to do something about it first and fast, to ensure access to universal access to all blood products. 2d-07-03 scottish national blood transfusion service, edinburgh, united kingdom supporting the provision of viable transfusion services in remote/rural areas is more than just a geographical challenge. limited qualified blood bank resource; small throughput volumes; increased regulation are only three of the additional factors combining to threaten safe and sustainable transfusion service delivery. inventory management and out of hours service provision were identified as essential areas where it was thought that technology, in the form of a remotely controlled blood fridge, could provide a key element of the overall solution. a radio frequency identification (rfid) fridge racking system was installed in a standard blood bank fridge and connected to the laboratory information management system (lims) common to both the remote and central blood bank. the central blood bank was enabled to test patient samples from the remote laboratory, identify components located in the remote fridge suitable for the patient and allow correct component issue, even when qualified staff were unavailable in the remote laboratory. testing has concluded that installation of remote fridge management can play a major role in helping to maintain a remote inventory permitting patient compatible components to be issued. by sustaining transfusion services in remote communities we can avoid transportation of patients who require transfusion support to locations miles from home. antibodies to hna-1b, fcgriiib and hna-2 have been reported, too. neonatal alloimmune neutropenia results from maternal antibodies transferred transplacentally to the fetus and is caused by all known hna-antibody specificities, i.e. hna-1a, -1b, -1c, -1d, hna-2, hna-3a, -3b, hna-4a, -4b and hna-5a specificity. hna and hla antibodies can induce mild febrile transfusion reactions and trali. since the introduction of the male only plasma strategy, in many countries the trali incidence decreased but it is still one of the most common causes of severe transfusion reactions. especially hna-3a antibody containing plasma from female donors is responsible for severe or even fatal reactions. but also hna-1a, -1b, hna-2 and hla class i and class ii antibodies were reported. the latter activate monocytes to secrete soluble factors that act on the primed neutrophils in the narrow lung capillaries. laboratory testing: laboratory work-up requires the knowledge of the patient's clinical condition and the methods that are appropriate to detect the relevant antibodies. the classical granulocyte agglutination test (gat) in combination with the granulocyte indirect immunofluorescence test (gift) can detect nearly all relevant antibodies. hna-1, -2, -4, -5 and hla class i antibodies are clearly detectable in the gift while hna-3 antibodies strongly agglutinate neutrophils in the gat. the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) test detects all hnaantibodies except for hna-3 with high glycoprotein specificity and sensitivity but is time consuming and requires highly skilled personnel. for trali diagnostics laboratory testing is completed by methods like the indirect lymphocyte immunofluorescence test (lift) or elisa for hla class i antibodies and hla class ii specific elisas. since several years fluorescent bead based assays (luminex) enable faster and more automated hna antibody detection but to date not all specificities, especially hna-3, can be reliably detected so that still the classical gift and gat have to complete the methodological spectrum. serological typing today is mostly reduced to the determination of hna-2 in the gift because the molecular reason for the hna-2-null phenotype is not completely understood. establishing only one pcr-asp reaction for the main cd177*787a>t polymorphism would comprise the risk to miss other molecular causes. however, for all other hna allelotyping by pcr methods is the first choice. summary/conclusions: granulocyte serology still today is widely based on a variety of manual methods and will be reserved to specialized laboratories as it requires experienced laboratory staff and profound knowledge of granulocyte immunobiology. 2d-08-02 norwegian national unit of platelet immunology, laboratory medicine, university hospital of north norway, tromso, norway maternal alloantibodies against antigens on human platelets can cause severe thrombocytopenia and bleeding in fetus or newborn, identified as fetal/neonatal alloimmune thrombocytopenia (fnait) . although most cases the thrombocytopenia is selfresolving within the two first weeks of life, some infants present bleeding symptoms and thus require platelet transfusion. a set of laboratory analyses are required to confirm the fnait diagnosis. in addition to guiding compatible platelets to the affected newborn, the correct diagnosis will be valuable to assess the risk of fnait in subsequent pregnancies. in addition, platelet alloantibodies may also complicate platelet transfusions by immune-mediated platelet refractoriness, and require proper identification of the patient's antibody specificities prior to selection of donor platelets. the algorithm for laboratory investigations include both serological and molecular assays, and depend on the objective and timing: whether there is an urgent need for platelet transfusion, follow-up of a pregnancy with known risk, or to do full-scale laboratory testing to confirm diagnosis. molecular genotyping should include all hpa systems relevant for the local population (in caucasians hpa-1, -2, -3, -5 and -15), preferably with optional extended panels for systems for low frequency populations due to immigration/mobility and for less frequently seen alloantibodies (hpa-4, -6 to -11 are most commonly included). serological testing of antibody binding to platelets is often initially tested by flow cytometry analysis (direct test and/or cross-match). however, the detection of platelet-specific antibodies is often complicated by the presence of anti-hla class i antibodies and thus require sensitive platelet glycoprotein-specific assays. serological testing for platelet-specific antibodies includes as a minimum panels of antigens on gpiib/iiia, gpib/ix, gpia/iia and cd109 and preferably additional targets for populations with asian/african origin. several methods are available; i.e. bead-based assays and elisa based methods. however, most reference laboratories perform variants of the monoclonal antibody immobilization of platelet antigens assay (maipa), as reported by the 19th international platelet immunology workshop of isbt (2018) . the investigations also include measurement of the anti-hpa-1a by quantitative maipa if present, as this is reported to potentially predict the severity of fnait. for pregnancies with known risk of fnait, there are methods available to perform non-invasive prenatal typing from maternal plasma. the most feasible and so far appropriate for routine testing is fetal hpa-1 typing with quantitative pcr or by melting curve analysis. other sophisticated, yet resource-demanding techniques have also recently been reported -importantly also for typing of other hpa-systems. 2d-08-03 molecular basis of hna-2 expression justus liebig university, institute for clinical immunology and transfusion medicine, giessen, germany human neutrophil antigen 2 (hna-2) is a neutrophil-specific antigen located on gpi-anchored glycoprotein cd177 (also known as nb1). hna-2 is absent on the neutrophil surface of 3-5% of the healthy individuals that divided the population to hna-2 positive and hna-2 null individuals. exposure of hna-2 null individuals to hna-2 positive neutrophils during pregnancy, after transfusion or transplantation, induces immunization against hna-2 and consequently the production of iso-antibodies. the hna-2 iso-antibodies are involved in the mechanism of neonatal alloimmune neutropenia (nain), transfusion-related acute lung injury (trali) and graft failure following bone marrow transplantation. presence of cd177 on a neutrophil surface of hna-2 positive individuals follows a bimodal expression that categorizes the circulating neutrophils to hna-2 positive and negative subsets. the cd177 gene contains 9 exons encoding a protein of 437 amino acids. the lack of hna-2 (in hna-2 null individuals) is associated with the presence of a missense mutation, cd177*c.787a>t in exon 7 of cd177 gene inducing a premature stop codon in codon 263. this mutation alone or in combination with cd177*c.997delg has been introduced as the main reason for the absence of cd177 in hna-2 null individuals. a pseudogene (cd177p1) highly homologous to exons 4-9 of cd177 gene is located downstream of the cd177 gene. conversion of exon 7 of cd177p1 into cd177 gene is responsible for the generation of cd177*c.787a>t missense mutation. in addition, the heterozygosity or homozygosity of cd177*c.787a>t is accounted for regulation of hna-2 negative and positive neutrophils subpopulations. genotyping has revealed the hna-2 null individuals, heterozygous for cd177*c.787a>t mutation without cd177*c.997delg, indicating the presence of a complementary mechanism regulating cd177 expression. newly in hna-2 null individuals and individuals with atypical cd177 expression a cd177*1291g>a polymorphism in combination with cd177*787a>t is described. altogether these data indicate a complex compound mechanism(s) for regulation of cd177 expression on the neutrophil surface. this presentation will summarize recent findings on cd177 expression and highlights the potential genotyping methods for genetic assessment cd177 expression of donors and patients. blood products ordered, transfusion start and end times, whether patients experienced a reaction to the transfusion as well as vitals measurements taken before and during the transfusion, including temperature, oxygen level, blood pressure and heart rate. for the validation process, 100 transfusion nursing notes were sampled and reviewers assessed the accuracy of the information regarding 1) blood product ordered, 2) whether the patient experienced a reaction, and 3) the start and end times of the transfusion. for each of these fields across all 100 sampled notes, the claritynlp tool reproduced these data points with 100 percent accuracy. in addition, the tool supplied transfusion end times for numerous structured records that were missing this key data point. summary/conclusions: claritynlp can very efficiently digest a large number of transfusion nursing notes simultaneously and also does an excellent job of extracting the main characteristics of a transfusion, which can be used in partnership with structured data to produce a more accurate and more complete picture of patient transfusions. immunohematology and genomics, new york blood center, new york, united states antibody-based typing, with a positive result reflected in agglutination of the red cells (rbcs), has served the profession for nearly a century enabling safe and effective transfusion therapy. the power of rbc typing by serologic methods lies in the availability of standardized antibody reagents which target many of the specificities of significance for transfusion, and the ability to directly detect antigen expression on the rbcs. hemagglutination has historically been relatively inexpensive, particularly for abo and rhd as the most important blood groups in most populations. serologic rbc typing is reliable, requires no sophisticated equipment, is generally straightforward to perform, and is fast requiring less than 1 h to results. hence, antibody-based testing has been considered the "gold standard" for blood group typing. with the age of genomics, dna-based genotyping is increasingly being used as an alternative to antibody-based methods. most antigens are associated with single nucleotide changes (snps) in the respective genes. genotyping has been validated by comparison with antibody-based typing and has been shown to be highly correlated. the power of genotyping of rbcs lies in the ability to test for antigens for which there are no serologic reagents, and to type numerous antigens in a single assay using automated dna-arrays. this increases accuracy and weak antigen expression can be revealed. fresh rbc samples are not required for dna extraction, and there is no interference from transfused rbcs or igg bound to the patient's rbcs. dnabased typing is economical in that it provides much more information, but testing requires special equipment, training, and 24-h turn around. what then is the best approach to use? will serologic typing be replaced by dnabased typing? indeed, genotyping will increasingly be used in the practice of transfusion medicine, especially with the growth of whole genome sequencing (wgs). however, because serologic typing for abo and rhd is fast and accurate and often relied on for sample identification, genotyping will not be the sole means for routine typing. genomic sequencing approaches will certainly reveal unrecognized changes and genetic variability in rbc membrane proteins, but not all variation will be immunogenic. a genetic polymorphism must be associated with antibody production to be considered a blood group antigen. the importance of an antibody, and antibody reactivity, then will continue to be the central defining principal in transfusion. as two sides of a coin, both are key to safe and effective transfusion therapy. since the mid-1980s, research in the molecular basis of structural and functional aspects of proteins carrying and producing the antigens has led to an upgraded and modern understanding of blood group variation. most commonly, single nucleotide polymorphism (snp) based basic molecular typing techniques were utilized to test new findings on a smaller subset of samples, and resulted in concordant sero-and genotyping results in general. however, quite commonly a small number of all samples delivered discrepant results, triggering consecutive rounds of analysis and resolution, finally resulting in a better knowledge with respect to the underlying blood group variation. such rounds of repetitions represented the synergistic incremental process of learning, learning for serologists and molecular biologists. inheritance of public, presence of high frequency, or low frequency, or partial antigens and notice of weakly expressed, or almost undetectable antigens marked the path of incremental learning and may best be exemplified by discoveries within the blood group systems abo, rhd and kell. naming for pheno-and genotypes coevolved alongside the permanent discovery of new antigens. at present, antigens and their antithetic counterparts (if tested and if existent), are more commonly reported independently, as exemplarily shown for the following kell phenotype consisting of three antithetic antigen-couples: kk, kp (a+b+), js(a+b+). alternatively, the same phenotype could be stated as: kel:-1,2,3,4, (5),6,7. genotypes, on the other hand, rather mirror the actual biological background, e.g. display the two parental alleles (or "haplotypes") present in an individuals' sample. genotype of the above mentioned example would read: kel*02.03/ 02.06 (italicized). in an idealized diagnostic environment and for most blood group systems encoded by proteins, every single blood group allele would be defined by its full genomic sequence, derived from one parental chromosome (including "some" 5'and 3'-untranslated regions). thereby, every such "ideal allele" would fully declare presence or absence of all its public, low-and high-frequency antigens and possess its "ideal name". by trend, biallelic snps and their immediate relation to antithetic antigen couples might have distracted from the originally intended meaning of "blood group alleles", more recently. finally, genotypes only dependent on (ideal) allele names, and considering mendelian inheritance patterns (dominant, recessive), would allow for fully comprehensive phenotype predictions. more recently, blood group serology, e.g. the "second side of the coin", seems to gain momentum. since the advent of whole genome sequencing and access to many more than 1000 human genomes, it seems that dozens of new blood group alleles are discovered, almost on a daily basis. beside the challenge of naming this multitude of alleles, respective discoveries are frequently made in samples lacking any phenotypic blood group pre-values. clear procedures will be needed to address naming and analyzing the phenotypes resulting from previously unknown alleles. as a consequence, questions asked 30 years ago have changed: today, molecular biologists looking at hundreds of newly discovered blood group alleles find themselves not being asked by serologists any more: "can you confirm my serology?", but instead, pose their question to the experts for the blood group phenotype: "can you confirm my genotype?" 3a-s02-03 background: the screening of blood donors and returning travelers from active transmission areas have highlighted the importance of diagnosis of acute arboviral infections. in the context of co-infections and similar clinical signs in endemic zones, the differential diagnosis of arboviruses is essential to discriminate the causative agent. the detection of viral nucleic acids in serum or plasma provides a definitive diagnosis, however, in most instances, viremia is transient within less than two weeks after the onset of clinical illness. in addition, the cross reactivity due to the high degree of structural and sequence homology between zikv and other flaviviruses is a significant concern. the combination of molecular (identification of viral genomes) and immunological assays (detection of the immune response) is a key challenge to follow the natural history of these infections and to improve the patient management and the epidemiological surveillance. aims: in this context, we have developed an innovative platform based on agglutination of superparamagnetic nanoparticles (npmag) covalently grafted either with nucleic or proteic probes to face the continuing emergence of arboviruses. methods: dengue (denv) and zika (zikv) viruses are selected as models in this study. a pan-flavivirus rt-pcr is used for the molecular assay to amplify the viral genomes. then, biotinylated viral amplicons are captured specifically on complementary original polythiolated probes coated on npmag. for the immunological assay, npmag are grafted with viral ns1 proteins to capture anti-denv or anti-zikv antibodies potentially present in the plasma samples. both tests are performed in disposable cuvettes in a homogeneous format. a magnetic field generated by an electromagnet is applied to the reaction medium to align the npmag into chains to enhance the capture of the targets between two npmag. aggregates formed are detected when the field is turned off. the optical density is measured in real-time at 650 nm during several cycles of magnetization / relaxation. results: in this study, molecular analytical performances were evaluated on human samples from blood donors with no history of infections as negative controls, on viral standards and on clinical samples. using viral references standards, we have observed sensitivities of 10 -10 2 tcid 50 /ml for zikv and denv (serotypes 1/2/3/4) after a detection phase of around 5 min. the first results obtained on 16 zikv (+) clinical samples previously tested by commercial real-time pcr (ct < 36, altona) showed an 88 % correlation between the two detection methods. no false positive results or cross reactions were observed. concerning immunological assays, commercial human plasma from donors tested positive for denv or zikv antibodies were detected positive with our innovative approach in less than 10 min (sampling + detection) instead of 2 h with classical elisas. further assays on clinical samples are planned to confirm these preliminary results. summary/conclusions: this innovative strategy combining molecular and immunoassays on the same analytical platform offers new opportunities for rapid blood testing to improve the surveillance and the prevention of arboviral infections. background: zika virus (zikv) caused a dramatic epidemic in puerto rico (pr) during 2016, with up to~2% of blood donors reactive for zikv rna in id-nat testing at the peak in june 2016. aims: perform a serosurvey for anti-zikv igg using six panels of 500 donor specimens each collected in march 2015, at the beginning, peak and end of the 2016 epidemic, and from march 2017 and april 2018. methods: we employed a commercially available zikv igg elisa antibody (ab) assay based on the zikv ns1 antigen from bio-techne to characterize zikv seroprevalence in the 6 9 500 cross-sectional sample sets (anonymized with selected demographic information). results: 500 pr donor samples collected in april 2015 were initially evaluated using the manufacturer supplied cut-off to confirm that the zikv ab results were largely negative (3 positive, 3 equivocal) despite the high dengue virus seroprevalence (>90%) in pr that could potentially lead to false positive zikv ab results. we then used this dataset, together with known positives collected 1-3 months postdetection from zikv nat yield donors, to set a population-specific cut-off based on receiver operating characteristic (roc) curve analysis. this cut-off yielded sensitivity and specificity values of > 99%, and an area under the curve (auc) of 0.999, demonstrating a highly accurate assay. we used this new cut-off to calculate final rates of seroreactivity in the additional 5 sample sets (2500 samples) and estimate seasonal incidence. rates of reactivity, together with mean net od for only the reactives (shown in parentheses), were calculated for each sample set: background: sex hormone intake in blood donors occurs in three demographic groups: premenopausal women who take contraceptive drugs (progestins with or without estrogens), postmenopausal women who receive estrogen replacement therapies that may be combined with progestins, and testosterone therapies in men. we hypothesized that sex hormone therapies may modulate the quality of red blood cell (rbc) products via alterations of rbc function and predisposition to hemolysis during cold storage. aims: the objectives of this study were to evaluate the association between sex hormone intake and rbc measurements of hemolysis, and to examine possible mechanisms by which sex hormones interact with rbcs. methods: self-reported sex hormone intake and menstrual status were evaluated in 6,636 female blood donors from the national heart, lung and blood institute's rbc-omics study. the associations between hormone intake and donor scores of spontaneous storage, osmotic, or oxidative hemolysis were determined in all women and by menstrual state. the interactions between sex hormones and rbcs were determined by sex hormone (progesterone, 17b-estradiol, or testosterone) potency to inhibit calcium influx or hemolysis during incubations or cold storage. the calcium fluorophore, fluo-3am, was used to define rbc calcium influx in response to treatments with sex hormones or drugs that modulate transient receptor potential cation (trpc) channel activity including hyp9 (a selective trpc6 activator). results: sex hormone intake by menstrual status was higher in premenopausal women (25.3%) than in postmenopausal women (7.4%). female hormone intake was significantly (all p < 0.0001) associated with reduced storage hemolysis in all females (0.32 ae 0.16% versus 0.35 ae 0.29% in controls), enhanced susceptibility to oxidative hemolysis (37.9 ae 9.3% versus 35.8 ae 9.9% in controls), and reduced osmotic hemolysis in postmenopausal women (23.1 ae 10.2% versus 26.8 ae 12.0% in controls). in vitro, supraphysiological levels of progesterone (10 or 20 lmol/l), but not 17b-estradiol or testosterone, inhibited spontaneous or hyp9-induced calcium influx into rbcs, and were associated with lower spontaneous hemolysis after 30 day cold storage (0.95 ae 0.18% versus 1.85 ae 0.35%, progesterone 10 lmol/l versus solvent control (dimethyl sulfoxide, 0.1%), p < 0.0001). co-incubations (2.5 h, 37°c) of rbcs in the presence of progesterone and a trpc6 activator (hyp9, 25 lmol/l) suggested that progesterone protected against hyp9-induced hemolysis (1.45 ae 0.13% and 1.01 ae 0.09% versus 2.63 ae 0.19%; hyp9 + progesterone at 10 or 20 lmol/l versus hyp9 alone, p < 0.05 by one-way anova). summary/conclusions: this study revealed that sex hormone intake in blood donors is capable of modulating rbc predisposition to hemolysis and led us to propose new mechanistic pathways by which progesterone regulates calcium influx and hemolysis in human rbcs. pre-and postmenopausal women respond differently to hormone intake and its effects on rbc responses to osmotic or oxidative stress. progesterone modulates calcium influx into rbcs via a mechanism that may involve interactions with membrane trpc6 channels, activation of which is associated with pre-hemolytic events such as senescence and eryptosis. 3a-s05-01 international cooperation, swiss red cross, wabern, switzerland background: red cross and red crescent societies were playing an important role in setting up blood transfusion establishments in many low resource countries. by the mid-1970s, the red cross was active in the national blood programs in approximately 95% of countriesmostly in blood donor recruitment and education. today, major organizational developments in blood transfusion services were made in high income settings, where nearly 50% of all worldwide donations take place (home to only 17% of the population). who data shows that the median annual blood donation rate in high-income countries is 3.21% of the population compared to 0.46 % in low-income countries. the factors for this low turnout are multilayered, but it is well-known that most resource poor settings suffer from a low rate of regular donors and challenges to set-up and financially sustain a national blood donor program. the red cross and red crescent (rc) societies assume a key role by reaching and retaining donors from the communities and contribute significantly to better safety and availability of blood. partnerships and international collaboration, such as the swiss red cross (src) program, are aiming to strengthen national structures to improve blood safety and to face today's epidemiological, demographical, and technological challenges. aims: the present work aims to review the role, the mandate and the impact of rc societies in improving blood safety through systematic "voluntary non-remunerated regular blood donor" (vnrbd) programming and international partnerships. methods: data and evidence is drawn from the src international cooperation projects over the last 30 years, more specifically partnering with three rc societies, and the data from the global advisory panel (gap) of the ifrc including their 2018 global mapping. results: the promotion of vnrbd has been a specific objective in all src supported programs. through the engagement of the rc society, the training of volunteers and partnering with the health authorities, the projects significantly increased the blood donor rate by recruiting and retaining donors from the communities. for example, the rc societies increased total donations by 25 % in lebanon; vnrbds by 71% annually in kirgizstan, and from practically zero to 3'588 in south sudan. the importance of rc societies was also underlined in the 2018 published global mapping of gap, which showed that 36 (19%) of them provide level a (full blood service), 75 (39%) are level b (systematic blood donor recruitment) and 47 (25%) are level c (vnrbd blood promotion) blood services. gap has also commenced a new three year vnrbd support program aimed at establishing tools and materials for national societies. summary/conclusions: the red cross / red crescent movement has a unique mandate and position in improving global blood safety at all levels; with its huge network of volunteers, even blood donors in remote communities can be reached and retained. rc societies in low resource settings with a level b or level c role should further capitalize on partnerships with local and international actors to leverage technical assistance and funding for their activities. background: it is essential to motivate and encourage the public to donate blood and be eager to help saving lives, in order to maintain safe and adequate national blood supply. aims: "technical assistance for recruitment of future blood donors (europeaid/ 132420/d/ser/tr)" project aimed to avoid problems in supplying the safest blood to contribute to the improvement of public health by (i) increasing the knowledge of primary and secondary school students regarding blood donation, (ii) creating sensitivity in school principals and teachers regarding voluntary non-remunerated blood donation (vnrd), (iii) motivating family members of the students for blood donation and (iv) creating public awareness through media. methods: an effective coordination is established between ministry of health (moh), ministry of national education (mone) and turkish red crescent (trc). the existing curricula and textbooks of primary and secondary schools were reviewed and revised, and corresponding materials on the importance of blood donation were created. the human resources capacity of moh, mone and trc to support raising awareness on blood donation were developed. to raise public awareness on blood donation nationwide, education and recruitment campaigns on were organized in 500 pilot schools. additionally, media and public relation campaigns on blood donation were organized throughout the country. results: (1) existing curricula and textbooks relevant to promoting blood donation were reviewed, revised and reported to the board of education of mone. (2) corresponding educational materials for students and teachers were developed and distributed. (3) blood donation clubs were established in 500 pilot schools. (4) trainings were conducted for 688 personnel of moh and trc on blood donation regarding their responsibilities. (5) cascade trainings were conducted for 3218 personnel of transfusion centers and 4399 school principals in 81 provinces. (6) information seminars were delivered to 251.475 students and 15.739 teachers and family members of students during school campaigns. (7) four animation films on blood donation were produced and broadcasted on the national tv channel (trt). (8) three different computer games targeting different age groups were developed and uploaded to the web portal of the project and distributed to the pilot schools. (9) media spots were produced and broadcasted 3.851 times in 33 different tv and radio channels. (10) billboard posters and brochures were prepared and distributed to 81 provinces for raising public awareness. (11) advertisements about the project and the importance of vnrd were displayed 386 times on national and local newspapers, 1.117 times on online news, and broadcasted on 6 national tv channels. (12) during the campaigns, 28.310 units of whole blood were collected in pilot schools. (13) visibility kits to recruit future blood donors are prepared and distributed throughout the project activities. (14) awareness and knowledge level of students and their teachers/parents on the importance of blood donation are increased to 32.07 % and 35.99 % respectively, assessed through pretest and posttest. voluntary non-remunerated donation rate of national demand increased from 73.66% to 82.54 in two years. summary/conclusions: training and campaign programmes successfully increased the knowledge on blood donation. to achieve national self-sufficient safe blood supply, efforts for recruitment should be continued. background: despite 70% of pakistan's population being under 29 years, only 10% of blood supplies come from voluntary donors while remaining blood is collected from 'family replacement donors'. in pakistan the system has outsourced the mobilization of blood donors to the patient families. as a result many people reach out to their networks including on facebook to locate blood donors. there are thousands of posts each month in pakistan seeking blood donors on facebook. to facilitate needy families, the global social media giant facebook launched a special blood donation feature for pakistan in collaboration with sbtp, pakistan. the feature makes it easier for people to sign up to become blood donors and helps connect these voluntary donors with people and organizations in need of blood. similar features have been launched by facebook in india, bangladesh and brazil to address the problem of blood shortages in those countries. however, among these four countries pakistan has unique position because of the existence of a national counterpart, sbtp which can facilitate facebook in promoting its feature and provide the feedback on the impact of this innovative effort for continuous improvement of the feature. aims: to promote voluntary blood donations and blood safety in pakistan through facebook. methods: the facebook and sbtp teams launched a pilot to study the impact and effectiveness of the facebook blood donation feature as a tool of community engagement. a six months plan has been chalked out to measure the impact of this tool in five selected blood centres. a checklist called "p0 checklist" was shared with these blood centres to fulfill some basic requirements for an official blood bank page including a display picture, cover photo, contact information, directions, etc. regular skype meetings are held between the teams of sbtp, facebook (san francisco and singapore) and the blood centres to monitor the progress of the pilot and generate feedback. results: the facebook blood donation feature has recorded remarkable success with over one million signups within few months in pakistan. the blood centres participating in the pakistan study have experienced enhancement in the voluntary blood donations trend with 3-10 walk-in donors and an average of more than 20 telephonic queries regarding voluntary blood donation per month in each center. the trend is gradually surging as the feature is being refined on the basis of feedback received. the pilot will end in june 2019. the statistics generated since january 2019 are very encouraging and underscore the importance of social media in reaching out to the untapped potential blood donors. the study will be used to plan an effective nationwide strategy to increase donor mobilization, recruitment and retention. background: in our region, an increasing number of patients of african or asian origin with sickle cell disease (scd) or transfusion dependent thalassemia (tdt) require red blood cell (rbc) transfusions, and many have rbc alloantibodies. selecting optimally matched rbc units for these patients is essential for preventing not only acute hemolysis but also further alloimmunisation. beside antigen-matching for abo, rh d, c, c, e, e and k, patients with scd and tdt should ideally receive rbc units matched also for m, s, s, fya, fyb, jka and jkb (extended phenotype). this is the policy at our center, which currently provides rbc products to 31 patients with hemoglobinopathies. because the vast majority of our blood donors are caucasians, the selection of matched rbc units for patients of different ethnic origin can be difficult. therefore, expanding the number of available african and asian blood donors is becoming increasingly necessary. aims: hereby the recruitment strategy of non-caucasian blood donors introduced at our center is described and the results obtained during six years are reported. methods: since 01.01.2013, whenever a first-time blood donor of non-caucasian origin is registered, an alert is entered in the donor file to trigger the determination of the extended rbc phenotype along with routine testing. rbc antigen determination is performed in our laboratory with serologic methods. in selected cases (i.e. suspected rhd or rhce variant), samples are sent for molecular analysis (ssp pcr). rare rbc phenotypes relative to ethnicity are, among others, fy(a-b-), s-s-, lu(b-) and those with uncommon rh phenotypes. if a rare rbc phenotype is detected, a coded comment is entered in the donor data and the donor is listed in the national rare donor file. results: from 01.01.2013 until 01.03.2019, an extended determination of rbc antigens was performed in 352 subjects presenting for blood donation. twenty-nine rare donors (8%) were identified and included in the rare donor file: 25 fy(a-b-), 1 lu (a-b-), 1 lu(b-), 1 fy(a-b-) and s-, 1 ccddee (r'r'). overall, these 29 donors provided 105 rbc units (range . to date, all donors are still active and 14 are reserved for dedicated donations. the internal price of rbc antigen testing per donor is approximately 100. -chf, resulting in a total financial effort of around 35,200.-chf in the time since the project was started. summary/conclusions: in our experience, a "passive" recruitment of non-caucasian blood donors based on ethnicity has an overall low efficiency from a logistic and financial point of view. moreover, african and asian blood donors may require investigations for hemoglobin variants and serology for malaria in addition to routine testing. nevertheless, a targeted determination of extended rbc antigen phenotype does allow the identification of persons with rare phenotypes. currently, measures for the active recruitment of potentially rare blood donors are being implemented at our center. after a pilot phase, a project for a nationwide recruitment strategy will be elaborated. a further goal is to build a national registry of patients with hemoglobinopathies requiring transfusions. blood transfusion is an essential treatment. transfusion safety consists of several components. although all are important, ion richer countries the order of priority is typically: 1.) avoidance of transfusion transmittable infections; 2.) quality of the blood product with a strong focus on component therapy; 3.) prevention of severe transfusion reactions; 4.) avoidance of clerical errors; 3.) sufficient availability of blood. the keynote of this lecture will be that the order of priorities on transfusion safety should probably be different in resource limited environments. 1.) sufficient availability of blood and proper utilisation; 2.) avoidance of transfusion transmittable infections; 3.) avoidance of clerical errors; 4.) prevention of severe transfusion reactions; 5.) quality of the blood product. most important, in regions with limited resources patients suffer from under-transfusion because not enough blood is available. all efforts should be made to reduce wastage of the available blood either by inappropriate storage, handling, or nonindicated transfusions. in addition, prevalence and number of pathogens transmittable by transfusion is much higher than in the richer parts of the world. this is aggravated by the fact that rejection of blood donors based on their history is problematic when the blood bank is empty. the aim to develop centralized national blood services with few sites manufacturing cost effective (low personnel costs) high-quality blood products, which are distributed to regional hospitals is not matching the reality of infrastructure, governmental support, and functionality. in healthcare systems with limited resources usually available personnel (hands) is not a problem, while reagents, equipment and even electricity are precious resources. the lecture will propose to focus on staff training and education, establishing local hospital-based transfusion services, which provide the blood products for the region, based on donor recruitment campaigns adjusted to the available technology, local culture, including replacement donor programs, with retention of safe donors as highest priority. fractionation of blood into components had been standard in transfusion medicine, but recently whole blood has experienced revival for patients with acute blood loss. given main transfusion indications such as postpartum hemorrhage, or severe trauma, in regions with limited infrastructure, whole blood might be the more appropriate product. most patients requiring transfusion in these regions are younger and volume overload by whole blood is not a major issue. in addition, frequent electricity failures do not allow prolonged storage of plasma at -20°c (this is therefore mostly wasted), although this issue can be overcome by solar powered freezers. ideally whole blood should be pathogen inactivated for which two methods are currently available. to reduce frequencies of acute hemolytic transfusion reactions again education and training to minimize clerical errors in the transfusion process are most important. extended testing for other rhesus antigens and k beside abo and rh-d in transfusion dependent hemoglobinopathy patients may help to reduce delayed hemolytic transfusion reactions. currently a leukodepleted pathogen-inactivated whole blood product might mostly serve the needs for blood transfusion in regions with limited infrastructure . the developed world should invest research efforts to develop such a product available at affordable costs. background: in modern transfusion medicine, serological investigations for blood cell antigens are complemented by genotyping arrays and pcr assays. whilst these platforms are informative in the majority of reference investigations, they are limited in their ability to define nucleotide variants associated with rare antigens and unable to detect novel variants potentially affecting antigenicity. next-generation sequencing is increasingly being employed in reference settings, providing information that cannot be obtained through these methods. whole genome and whole exome sequencing have been successfully employed in many investigations of novel and rare antigens, however concerns remain regarding the collection of genomic data unrelated to reference investigations, and reporting clinically significant incidental findings. these concerns can be addressed through the use of targeted sequencing panels. we report on the design, testing and efficacy of a panel providing a comprehensive genotyping profile for red cell, platelet and neutrophil antigens in a single test. aims: design a customised targeted exome sequencing panel for red cell, platelet and neutrophil antigen genes, and benchmark the efficacy against a commercial medical sequencing panel (illumina trusight one -tso). -test the panel and in-house genotype prediction script on sequence outputs from 51 samples with known red cell, platelet and neutrophil genotypes and phenotypes and determine whether predictions are concordant. methods: the panel was designed with 2654 probes covering exons of 64 genes associated with red cell, platelet and neutrophil antigens. using illumina nextera rapid capture technology, 51 samples were tested over five sequencing runs, on standard and micro chemistries, to determine optimal sample plexity per standard run, and the efficacy of smaller flow cells for lower throughput applications. an in-house python script was used to predict star-allele genotypes based on variants listed in isbt and embl databases. these predictions were compared to results from serology, snp array and previous tso data. results: coverage consistently averaged > 2509, with 94% of target at a quality of q30. optimal sample plexity for a standard run was determined to be 14 samples, allowing for sufficient coverage of all clinically significant variants. for red cell samples with previous typing data (excluding rh structural variants), the script correctly predicted 99.5% of snp based red cell genotypes. script predictions were 100% concordant for platelet genotypes, and four of five neutrophil antigen genotypes. hna2 genotypes defined by cd177 could not be reliably determined. the increased target coverage of the panel allowed for detection of a clinically significant heterozygous variant in scianna system, previously undetected by the tso panel due to extremely low coverage. additionally, a variant defining a potentially novel null allele was detected in the p1pk system. summary/conclusions: the panel demonstrates considerably higher coverage, quality and throughput compared to the tso and allows for detection of variants previously overlooked due to low sequencing coverage. up to 14 samples can be reliably sequenced in a single run. our script correctly predicts over 99% of snp based alleles; however, rh structural variants require further manual analysis. background: to ensure the safety of a transfusion it is critical to identify the blood type of both donor and recipient. serological methods for typing abo, rh and kel use monoclonal antibodies, however, typing reagents for rare blood groups are expensive, unavailable or unreliable. dna-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. however, to date, the cost per sample has prevented the universal application of dna-based donor typing aims: to achieve universal adoption of dna based donor typing, the blood transfusion genomics consortium (bgc) set out to develop an affordable dna based platform, capable of typing all red cell antigens, hla class i and ii and human platelet antigens. methods: the uk biobank axiom array, previously used to type 600,000 uk citizens, was redesigned for donor typing using three approaches: i) mining transfusion medicine knowledge, e.g. isbt allele tables; ii) inclusion of loci associated with donor health; iii) extraction of all coding variants in relevant genes with a frequency > 1:20,000 identified in large-scale sequencing data. samples from nhsbt and sanquin blood donors (n = 7,871) were used for performance assessment. red cell and platelet antigens for each donor were inferred from genotypes using the bloodtyper algorithm and concordance with clinical serological typing results assessed. results: concordance between genotypic and serological typing results was 99.9% for 95,022 comparisons; 29 of the 89 discrepancies were serologically negative and genotypically positive for a given antigen (k/k, fy[a/b], lu[a/b]). in all cases dna variants known to modify or weaken antigen expression were detected, displaying the power of genotyping to detect variant 'weak-antigen' expression. across 48 antigens for which serology was available, genotyping provided a 3.6-fold increase in the number of typing results available per donor (47.9 vs 13.2). furthermore, genotyping provided data on an additional 224 clinically relevant antigens, allowing identification of antigen-negative donors and blood group identification for which antibodies are not commercially available. the power of a genotyped panel of donors to support patient management was demonstrated by a retrospective analysis of clinical cases referred to nhsbt. from 3,146 patient referrals with > 3 alloantibodies between 2014 and 2018, 772 unique alloantibody profiles were identified. we found that there was a 2.6-fold greater likelihood of finding o negative compatible donors for these patients when using genotyping data from the 4,721 nhsbt donors. importantly, the number of alloantibody combinations for which no compatible antigen-negative donor could be identified fell from 293 to 246, representing an additional 164 patients that could be provided with directly compatible blood using the same donors. summary/conclusions: through the bgc efforts an affordable fully automated genotyping platform, including the processes for quality assurance and data analysis, has been developed. furthermore, we have demonstrated the real-world benefits more extensive donor characterisation can provide when selecting blood for patients with multiple antibodies. the results of this international collaboration provide opportunities to introduce fully-automated genotype-based donor typing in a safe and cost-efficient manner in blood supply organisations. 3c-s06-04 biobank performs whole-genome sequencing (wgs) from individuals nation-wide. these data are suitable for allele frequency analysis to demonstrate gene expression and genetic profile in our population, also to estimate the significance of each antigen in transfusion practice. aims: we aim to provide and verify population-based blood group antigen profile using wgs and dna samples from taiwan biobank. methods: a near 1500 wgs and demographic data were analyzed. annotations of blood group antigen were performed according to variants from isbt allele tables, including 2 transcription factors; variants for the lewis system were obtained from previous studies. annotations of blood group variants were verified by 4 dna samples with targeted sequencing on illumina miseq, and specific variants were verified by 40 dna samples with the commercial genotyping kit or sanger sequencing. allele frequencies from wgs analysis were compared with population serology data using two-proportion z test. results: population-wide blood group antigens were analyzed, revealed in-depth antigen expression profiles in all systems (except ch/rg). the antigen frequencies from wgs were similar compared with published serology data, except for the antigens and possible explanations listed as follow, 1) m, n: insufficient sequencing reads, 2) c, c: identical rhce exon 2 with rhd exon 2 for c allele, 3) mur: insufficient read length/depth for gypa/gypb hybridization calling and individuals from high prevalence of mur antigen in aboriginal tribes were not enrolled. blood group antigen predictions and variants from wgs were accord to dna verification. furthermore, 13 systems shown no genetic variations, predicting uniform antigen expression in our population, and we can manage transfusion with minimum concerns for antigen mismatch in these systems. moreover, we found weak and null alleles in our population for blood group systems that we had previously no knowledge of, such as lan, jr, and vel. these variants were helped to identify a patient with anti-jr a carrying homozygous jr a null alleles. summary/conclusions: taiwan biobank wgs is suitable for full blood group antigen profile determination with few adjustments required for specific antigens. the population antigen allele frequency provides valuable insight to antigen significance in transfusion practice, matching strategy for our patients, and estimation of the likelihood to obtain for specific antigen negative blood from mass population. also, the genetic variants revealed in this study can help us to locate rare donors, and to integrate variations into routine donor blood group screening to provide suitable blood at a low cost efficiently. background: providing adequate amounts of safe, appropriately matched blood to meet the demands of an expanding and aging patient population presents a challenging global problem. for these reasons, sustainable in vitro sources of red cells may offer a desirable alternative to reliance on donor blood. the first stable immortalized early adult erythroblast cell line, bel-a2, has been shown to differentiate efficiently into mature, functional reticulocytes (trakarnsanga et al., nat commun. 8:14750, 2017) and consequently could provide a readily available tool for diagnostic use and proof of principle for future therapeutic use. aims: at ibgrl, next generation whole exome sequencing (wes) has been used previously to accurately predict blood group phenotype in a number of blood group systems, including abo, rh and mns (tilley & thornton, transfusion medicine 27 (suppl.2) :42, 2017). here we have used it to analyse and document bel-a2 blood group-related genotypes and predict blood group phenotypes. additional genes involved in cell-growth and enucleation were also analysed in order to further elucidate the characteristics of the bel-a2 cell-line. methods: bel-a2 cells (day 196) were cultured in expansion medium and genomic dna (gdna) was isolated from 1 9 10 6 cells on day 5. for wes, gdna libraries were prepared using nextera â rapid capture exome enrichment and sequenced on illumina â miseq. sequence alignments for 41 genes encoding all 36 known blood group systems and 12 further genes encoding transcription factors and cell enucleation-associated proteins were visualised using integrative genomics viewer, whilst illumina â variant studio was used to identify observed mutations. mutations in coding regions were used to determine bel-a2 genotype and predicted phenotype. results: good coverage of most of the selected genes was achieved. alignment of homologous blood group genes including rhd/rhce, gypa/gypb and c4a/c4b was problematic and additional analysis of coverage of these genes was required for accurate interpretation. despite a number of polymorphisms observed across the tested genes, bel-a2 did not express any novel or rare blood group antigens. genotyping results predicted a common antigenic profile, in agreement with previous serological and genotyping results where available. although a number of missense single nucleotide variations were detected in analysed genes, including cr1, cdan1 and tmx4, these were common polymorphic variants and unlikely to be of any functional significance. summary/conclusions: wes was used to determine bel-a2 genotype in relation to blood group genes and selected genes encoding transcription factors and proteins associated with cell enucleation. wes allowed accurate prediction of blood group phenotypes, showing full concordance with available serological data (trakarnsanga et al, 2017) . a small number of mutations were identified which are of unknown significance and require further work to determine any potential phenotypic effects. this complete record of the bel-a2 blood group-related exome will enable reliable gene editing strategies for future diagnostic and therapeutic purposes. additionally, knowledge of the full cell line exome will allow analysis of any emerging genes of interest and provide better insight into the mechanisms of erythroid differentiation and enucleation. background: emerging evidence, especially in neonates, has shown potential harm associated with liberal platelet transfusion strategies. very little evidence exists regarding optimal platelet transfusion thresholds in critically ill children. randomized controlled trials may be difficult due to lack of equipoise from providers. if regional variation in practice exists, comparative effectiveness studies may be an alternative approach. aims: to describe regional variation in platelet transfusion practices in critically ill children. methods: secondary analysis of a prospective, observational study. subjects were grouped according to region (north america, europe, middle east, asia and oceania) and nation. transfusions were analyzed as prophylactic (given to prevent bleeding) or therapeutic (given to treat bleeding). the primary outcome was the total platelet count (tpc) prior to transfusion. sub-groups analyses were performed in children with an underlying oncologic diagnosis and those supported by extracorporeal life support (ecls). the dosing and processing of the platelet transfusions were analyzed as secondary outcomes. results: five hundred and forty-nine children from 16 countries were enrolled (67% in north america, 17% in europe, 7% in oceania, 5% in asia, and 4% in the middle east). overall, the median (iqr) tpc prior to prophylactic transfusions (n = 360) differed significantly on a regional basis (p = 0.04) and ranged from 12 (8-41) x10 9 cells/l in the middle east to 45 (20-66) x10 9 cells/l in asia. the median tpc prior to prophylactic transfusions did not significantly differ between countries (p = 0.08), nor did the tpc prior to therapeutic transfusions (n = 189) differ on either a regional (p = 0.16) or national (p = 0.57) basis. for children supported by ecls (n = 90), there were no regional (p = 0.06) or national (p = 0.40) differences for prophylactic transfusions. however, significant differences in the tpc prior to therapeutic transfusions were observed on both a regional (p = 0.02) and national (0.04) basis with the middle east, in particular israel, transfusing at the lowest median (iqr) tpc [28 (16-81) x10 9 cells/l]. for children with an underlying oncologic diagnosis (n = 233), no differences were seen in the tpc for prophylactic transfusions (n = 175) on a regional (p = 0.19) or national (p = 0.20) basis. nor were differences seen in the tpc prior to therapeutic transfusions on a regional (0.86) or national (p = 0.33) basis. there was significant variability in the dosing of platelet transfusions on both a regional (p < 0.001) and national basis (p < 0.001). the median (iqr) dose based on volume ranged from 8.4 (5.0-11.2) ml/kg in north america to 12.6 (9.8-16.0) ml/kg in europe. the vast majority of transfusions were leukoreduced and irradiated but significant variation exists in storage duration on both a regional (p < 0.001) and national (p < 0.001) basis. summary/conclusions: regional and national variation exists in platelet transfusion practices among critically ill children, especially in those given therapeutic transfusions while supported by ecls. considering this variation, comparative effectiveness studies may be an appropriate approach to gain evidence to optimize platelet transfusion thresholds. background: the optimal threshold for prophylactic platelet (plts) transfusion in pediatric patients with cancer is still controversial and current clinical practice comes from studies on adults and on inpatient setting. the international guidelines (icmtg, 2015) recommend, for all age patients, a prophylactic platelet transfusion when plts count is ≤ 10 9 10 11 /l and a platelet dose of 1.1 9 10 11 per square meter (sm) of body-surface area (bsa) in inpatient and 2.2 9 10 11 /sm in outpatient setting. aims: in january 2018 we started in our children's hospital a prospective protocol in order to evaluate the impact on bleeding risk of current clinical practice of prophylactic platelet transfusion in inpatients and outpatients onco-haematological patients. methods: bsa was calculated from age-standardized weight. inpatients received a dose per transfusion of 1.1 9 10 11 /sm and outpatients a dose per transfusion of 2.2 9 10 11 /sm. platelets were transfused when the count was ≤ 10 9 10 11 /l or in presence of bleeding signs; pediatric aliquots were obtained from buffy coat derived pooled platelet concentrates or apheresis platelet concentrates, according disponibility. results: from january 2018 to december 2018 a total of 9839 platelet pediatric aliquots were transfused: 5534 (56.2%) were obtained from apheresis platelet concentrates and 4305 (43.8%) from buffy-coat-derived pooled platelet concentrates. the majority of platelets pediatric aliquots (7505-76.3%) were transfused to onco-hematological patients undergoing hematopoietic stem cells transplant (hsct) or conventional chemotherapy. among them, 6365 aliquots were transfused in inpatient setting: 1675 (17%) in the hematology unit, 2772 (28.2%) in the oncology unit and 1918 (19.5%) in hsct unit. a total of 1140 (11.5%) aliquots were transfused in outpatient setting: 840 (8.5%) to patients affected by hematological malignancies and 300 (3%) to patients with solid tumors. five major bleeding events (who grade ≥ 3) were observed during the study period and all of them occurred in hospitalized patients. two patients with solid neoplasm developed a who grade 3 bleeding event. two patients with hematologic malignancies and a patient with neuroblastoma (n = 3, 1.2%) developed intracranial bleeding (who grade 4). the platelet count at the time of the event was 22 9 10 9 /l, 25 9 10 9 /l and 8 9 10 9 /l, respectively. summary/conclusions: our results showed the efficacy, in onco-hematological pediatric patients, of a prophylactic platelet transfusion protocol based on international guidelines: a very low incidence of who grade 4 bleeding has been observed in inpatients setting only (1.2% vs 1% of plado trial, sj slichter, nejm, 2010) , while in outpatients setting the double platelet dose prevents the major bleeding event (who grade ≥ 3) occurrence. background: the problem of blood-borne infections remains relevant in transfusion medicine. pathogen reduction technologies (prt) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. to date, prt widely used for platelet concentrates and blood plasma, however, the use of these technologies for the treatment of red blood cell-containing blood products undergo research. aims: the aim of our study was to evaluate the safety and efficacy of transfusions of pathogen-reduced (test group) red blood cell suspensions (rbcs) and compare these data with gamma-irradiated rbcs (control group). methods: the technology based on the combined action of riboflavin and ultraviolet (mirasol prt, terumo bct, belgium) was used to reduce pathogens in whole blood. subsequently, the rbcs of the test group were derived from pathogenreduced whole blood. the control rbcs were irradiated at the gammacell 3000 elite (best theratronics, canada) at a dose of 25 gray. all rbcs were used for transfusion for 14 days from the harvest day. 70 pediatric patients with various oncological and hematological diseases were randomized to 2 groups of 35 members in each group. the test group of patients received transfusions of a pathogen-reduced rbcs; the control group received transfusions of a gamma-irradiated rbcs. the next day after transfusion were assessed hemoglobin and hematocrit increment, the level of potassium and haptoglobin in the patients' serum, the frequency and severity of transfusion reactions. 3-5 days after the transfusion, the direct antiglobulin test (dat) was performed and after 14-21 days the indirect antiglobulin test (iat) was performed. the interval to the next need for transfusion was also evaluated. results: the increase in hemoglobin and hematocrit (p = 0.2), as well as the concentration of potassium (p = 0.44) and haptoglobin (p = 0.25) in the patients' serum after the transfusion did not differ between groups. none of the patients in both groups had hyperkalemia after transfusion. in each group, two patients had febrile non-hemolytic transfusion reactions of comparable severity (p = 1). all dat and iat tests were negative in both groups. the interval between transfusions were not significantly different between groups (p = 0.39). only in the test group was found the correlation between the increase in the hemoglobin and hematocrit values with the volume of transfusion, with the dose and the adjusted dose of hemoglobin obtained for the transfusion on body weight. and in this group was found inverse correlation between the hemoglobin and hematocrit increment with the level of hemolysis in the rbcs. summary/conclusions: we found that the clinical efficacy and safety of rbcs of the compared groups did not differ. there was no evidence of immune elimination and allo-sensitization caused by pathogen-reduced rbcs. according to our data, the spectrum of efficiency and safety indicators of pathogen-reduced rbcs is no worse than that of gamma-irradiated rbcs, provided that rbcs is used for 14 days of storage. the founded correlation suggests that the efficiency of pathogen-reduced rbcs transfusions is more dependent on the characteristics of the rbcs. background: patient blood management (pbm) programs are expanding at an international level. a recent nationally representative study from united states observed pediatric age group as the only age group showing lack of objective evidence of pbm initiatives (goel et al, jama 2018) . aims: this study aims to identify trends in peri-operative blood utilization in children undergoing elective and non-elective surgeries over 5 years duration from 2012 to 2016. methods: using 5 years data (2012) (2013) (2014) (2015) (2016) perioperative transfusions decreased steadily per year from 6.4% in 2012 to 5.5% (14% cumulative decline) in 2016 for children of all ages (or 0.966; 95% ci 0.957-0.976; p trend < 0.001). the cumulative change in elective procedures was 9.2% versus 27.2% decrease in urgent/emergent procedures (p trend < 0.001). summary/conclusions: in this large prospective registry study of > 350,000 children undergoing elective/non-elective surgeries, a statistically significant decrease in utilization of peri-operative rbc transfusions was seen across 5 years from 2012 through 2016 with more significant decrease in urgent/emergent procedures than elective procedures while these findings need evaluation for non-surgical indications of transfusion, these results may provide first evidence of peri-operative pediatric patient blood management strategies being implemented to optimize transfusions in pediatric population. adverse events -tti, immune interactions and risk 3c-s08-01 transfusion-transmitted infections (tti) are a long-standing and well recognized concern in medicine, which is tackled on the highest level to guarantee the safety of the transfusion procedure for all stake holders. these include the recipient patients, the donating volunteers, the health care workers involved, and their respective contact persons. accordingly, current national and international guidelines including expert societies and the who provide medical, technical, and legal frameworks, which are the basis for the standard operating procedures. nevertheless, there are important challenges, which render tti a "moving target", and reflect the dynamics in three main areas. first, a change in the type and number of recipient patients with past or ongoing immunomodulatory / immunodeficiency component (examples being hiv/aids, sot, allogenic hct, monoclonal antibody therapies, small molecule inhibitors). second, changing exposure to known agents in donors due to global travel, migration and displacement, as well as environmental/climate change. third, discovery and diagnostics of old and new agents with their known or presumed impact as tti. these aspects will require careful review of data and studies, and judicious discussion of the potential action such as selection versus close monitoring to keep tti rates as low as possible, to deliver maximal safety of patients and stakeholders. background: the implementation of nucleic acid testing (nat) and the development of sensitive and specific serologic assays to detect hbsag and anti-hbc antibodies significantly reduced the risk of hbv transfusion-transmission. the apparent redundant testing for two direct viral markers prompted debates on maintaining hbsag screening, particularly in low endemic countries where blood donations are screened for anti-hbc. however, frequencies of 2-20% of hbsag-confirmed positive/nat negative donations have been reported depending on the sensitivity limit of the molecular assays used. the nature of this discrepancy between hbsag and dna remains largely unknown and it is essential to evaluate any potential negative impact on blood safety before considering removing hbsag testing. aims: the prevalence in blood donors and the molecular mechanisms responsible for a persistent undetectable or barely detectable level of viral replication in the presence of a sustained hbsag production were investigated in a collaborative study including five laboratories/blood centers in europe and south africa. discrepancy between viral dna and hbsag levels suggested the presence of mutations that may negatively affect hbv replication and/or infectious viral particle production. methods: donor samples from france, south africa, poland, and croatia were selected for having hbsag levels ≥ 100 iu/ml and being id-nat (procleix-ultrio plus tm [95% lod: 3 iu/ml]) non-reactive/non-repeatable reactive (nr/nrr) with undetectable viral load (vl) or < 6 iu/ml (n = 44) or nat repeat reactive (rr) with vl < 6 iu/ml (n = 32). french samples initially tested nat nr/nrr with procleix-ultrio (lod 95%: 11 iu/ml) were retested with ultrio plus prior inclusion in the study. hbv dna load was quantified (cobas taqman hbv [loq: 6 iu/ml]). hbv dna was purified from 5 to 12 ml of plasma after ultracentrifugation. the whole hbv genome, pre-s/s, precore/core and bcp regions were amplified and sequenced. results: following viral concentration, hbv dna presence was confirmed in 79% of all samples with undetectable or vl < 6 iu/ml. hbv genotypes were a1 (33.3%), a2 (18.4%), a3 (3.3%), b (3.3%), c (1.7%), d (25%), and e (15%). all samples were anti-hbc positive and 73% of ultrio-negative samples tested positive with ultrio plus. unusual 1-2 nt insertions/deletions identified in bcp regulatory elements (tata boxes, pginr, epsilon domain) suggest altered viral replication. amino acid substitutions (n = 16) or deletions (n = 4) at positions reported involved in nucleocapsid formation, particle envelopment and virion formation were observed in the core protein of 30 samples. the replicative properties of the bcp and core variants are currently evaluated in vitro as a surrogate model for direct infectivity testing. preliminary results indicate that the variants tested so far have replicative capabilities similar to those of control viruses. analysis of pol, s, and hbx proteins is ongoing. summary/conclusions: these data confirmed the presence of extremely low level of circulating dna-containing viral particles in id-nat non-reactive or nonrepeated reactive blood donations with concomitant high hbsag levels and anti-hbc reactivity. despite the presence of mutations in the viral genomes potentially affecting virion production, preliminary data indicate that some of the viruses in plasma retain the ability to replicate in vitro and to constitute a potential infectious risk. 3c-s08-03 background: in switzerland highly sensitive nucleic acid screening in an individual donation format for hepatitis b virus (hbv id-nat) and hepatitis b surface antigen (hbsag) detection is mandatorily performed (guidelines of swiss transfusion src, switzerland). since 1998, hbv (hb) vaccination is recommended in switzerland for children and adolescents until the age of 15 and for adults belonging to known risk groups. aims: to highlight that low anti-hbs titers several years following hbv vaccination still confer protection and enable the host immune system to clear hbv dna without development of serologic markers of disease. methods: a retrospective donor interview was conducted to complete information not covered by the questions included in the standard donor questionnaire. routine hbv serological donor screening was performed on a quadriga system (diasorin, former siemens) with the enzygnost hbsag assay (diasorin, former siemens). further hbv tests were performed on the abbott architect i1000 analyser (hbsag neutralisation, hbeag, anti-hbc igg/igm, anti-hbc igm, anti-hbe and anti-hbs). routine id-nat screening for hiv/hcv/hbv was performed with the roche cobas mpx test on a roche cobas 8800 platform. hbv id-nat positive samples were confirmed with a quantitative hbv nat assay (abbott). background: hepatitis b core-related antigen (hbcrag) is a structural antigen of hbv, consisting in hbcag, hbeag and the p22cr precore protein. quantitative hbcrag measurement is a sensitive marker of viral replication reflecting the cccdna content and persistence of disease. hbcrag positivity was found to be a significant risk factor of hbv reactivation in hbsag-, anti-hbc+, hbv dna-patients (occult hbv infection, obi) undergoing immunosuppressive therapy. aims: no data about hbcrag status in apparently healthy subject with obi are available. the aim of this study was to analyse this marker in our cohort of obi blood donors. methods: hbcrag was measured in 69 blood donors confirmed to be carriers of obi (hbsag-, hbv dna+). of them, 59/69 (85.5%) donors were anti-hbc positive, and 10 (14.5%) negative. 18 donors had both anti-hbc and anti-hbe reactivities. a group of 11 young blood donors vaccinated for hbv infection (hbsag-, hbv dna-, anti-hbc-), and 9 patients with chronic hbv infection (hbsag+, hbv dna+) were used as negative and positive controls group, respectively. serum hbcrag was measured using a chemiluminescent enzyme immunoassay on the lumipulse g600 automated analyzer (fujirebio, tokyo, japan). the lower limit of detection (lod) of the quantitative assay is 2 logu/ml and the lower limit of quantification (loq) is > 3 logu/ml, due to nonlinearity results between 2 and 3 logu/ml. levels of hbcrag were tested in the three groups and analysed in comparison to the presence of anti-hbc and anti-hbe. statistical analysis was performed by the ibm statistics spss 19.0.0. results: all donors in the negative control group had undetectable hbcrag levels, whereas all patients in the positive control group have detectable hbcrag (mean value: 3.0 logu/ml, range 2.0-4.9), confirming that individuals without prior exposure to hbv would not have detectable hbcrag. hbcrag was detectable in 34/69 obi donors (49.3%), with a mean value of 2.21 logu/ml (range 2.0-3.20). hbcrag could be measured only in 2 obi donors (3.0 and 3.2 logu/ml), being below the loq of the test in the majority of obi (32/34). considering the presence of anti-hbc, hbcrag was detected in 29/59 (49.1%) anti-hbc+ and in 5/10 (50%) anti-hbc-obi, with no significant difference in their mean levels (2.21 ae 0.32 vs 2.14 ae 0.26; p = 0.44). interestingly, the presence of anti-hbe (18/62) was independently associated with higher hbcrag levels (2.38 ae 0.42 vs 2.14 ae 0.22; p = 0.03). summary/conclusions: identification of donors with obi is critical to prevent the risk of hbv transfusion-transmission. being hbcrag associated with the cccdna content and replication, our results suggest that the presence of hbcrag, even if not quantifiable, could be useful marker to confirm the occult infection status, even in anti-hbc negative donors. the association between hbcrag, anti-hbc and anti-hbe could also be a useful marker to identify obi donors with a higher risk of hbv reactivation. 3c-s08-05 hc group. human peripheral blood mononuclear cells (pbmcs) from blood donors were stimulated with hbv polypeptides pool in vitro. t cell proliferation assays (cfse) was used to detecting t cell proliferation, enzyme-linked immunospot assay (elispot) was used to detecting the frequency of hbv-specific ifn-c secreted t cells. spss 20.0 statistical analysis software was used for statistical analysis. the measurement data of normal distribution were tested by two independent samples t test; and the comparison between multiple groups was analyzed by one-way anova. mann-whitney u test was used for comparison between non-normal data sets. p < 0.05 was considered statistically significant. results: 1. proliferation characteristics of t cells. the proliferation of cd4 + t lymphocytes was mainly stimulated by specific hbv polypeptide pool, and the proliferation rates of obi group and chb group were significantly higher than those of hc group (3.0%, 3.3% vs. 1.7%), with significant difference (3.0% vs. 1.7%, p = 0.016, 3.3% vs 1.7%, p < 0.001). 2. the frequency of specific ifn-c secreted t cells. the response intensity of the obi group (25 sfc/10 6 pbmcs) and chb group (25 sfc/10 6 pbmcs) was higher than that of the hc group (5 sfc/10 6 pbmcs) under the stimulation of hbv polypeptide pool, and the positive rate of t cell response to the stimulation of hbv polypeptide pool was the highest in the obi group (64.0%). summary/conclusions: both obi and chb had higher rates of hbv-specific t effector cell proliferation and ifn-c secretion than the healthy control group. compared with the chb group, obi group had a higher positive rate of t cell response, which may be one of the causes of host immunity resulting in obi. further studies on other immune factors are required. background: western blood transfusion practices are currently changing due to various drivers such as blood management policies, ongoing technological developments, and new therapeutic options. in the netherlands, as in many high-income countries, these have resulted in a diminishing trend of red blood cells. therefore, it is important for blood bank management to anticipate the future demand of blood products for the sake of medium and long term decision making. to support this decision making, we have employed scenario development, which is used in many other sectors (such as finance and transportation) and can also be applied to blood transfusion. building upon a prior literature review and semi-structured interviews of international experts, we gathered experts together for scenario sessions to assess the opportunities and threats for sanquin's medium-term (15-20 years) strategy using an online platform and face-to-face discussions. aims: to assess for opportunities, threats, and the organizational implications thereof for the medium-term future of sanquin, the dutch national blood bank. methods: twenty-one multidisciplinary experts in blood transfusion agreed to participate and were separated into two groups for half-day interactive sessions. using an iterative process through an online platform, experts brainstormed opportunities and threats for sanquin, which were categorized into themes. these themes were ranked according to importance and certainty, and through consensus, experts chose two themes with high impact and high uncertainty. for these chosen themes, specific actions for the blood bank were listed to mitigate and/or enhance the threat or opportunity. discussions were ample throughout. results: with regards to opportunities and threats for sanquin's medium term strategy, experts brainstormed many ideas and categorized them under 10 themes: political context/ changing legislation, novel products and alternative applications, donors, international markets, commercialization, digitalization, change in perceptions, research, demand, and organizational structure. after ranking for importance and certainty, six themes were chosen: change in perceptions, international markets, political context (opportunities), demand (opportunities), research (vulnerabilities), and donor (vulnerabilities). for each of these themes experts provided specific actions for the organizations to mitigate threats or stimulate opportunities accordingly. these actions included increased transparency and improved communication with the (donor) public, lobbying in political spheres, increased activities in educational institutes and large funding organizations, and creating and collaborating on novel blood products on an international level, to name a few. summary/conclusions: these results show that mapping and assessing a blood bank's future using a multi-disciplinary group of experts is conducive as an effective means of collection a diverse range of opportunities and threats. this provides an opportunity for blood bank management to become proactive towards these potential opportunities and threats and possibly evolve future strategies for the organization. showed that iron-deficient female blood donors were more likely to have depressive symptoms than non-iron deficient female blood donors. among participants with depressive symptoms, females with low plasma ferritin levels had significantly increased odds for reporting a "feeling of lacking energy and strength" (or = 2.11; 95% ci: 1.03-4.31). as it is known that blood donors are at an increased risk of iron deficiency, it is important to determine whether those genetically predisposed to lower plasma ferritin levels have a higher risk of experiencing the tiredness/lack of energy symptom. aims: to investigate whether there is an association between polygenic risk scores (prss) based on plasma ferritin levels and the tiredness/lack of energy symptom in blood donors. methods: the dbds is an ongoing nationwide blood donor cohort, of which genome-wide genotype data are available for 72,000 participants. genotyping was performed using the infinium global screening array (illumina â ) and imputation was achieved based on a scandinavian reference genome. ferritin prss, based on an icelandic ferritin gwas (n = 150,000), were calculated for all dbds participants. 12,903 female donors were available for the analysis. data on depressive symptoms were obtained using the validated major depression inventory scale (mdi), a selfreport mood questionnaire, which assesses the presence of 10 depressive symptoms. a donor was classified as "tired" if they responded "all the time" or "most of the time" to the question "how often do you feel that you lacked energy and strength?". logistic regression analysis was performed, adjusting for age. for generating the quantile plots, the participants were distributed evenly into six quantiles based on their prs, whereby quantile 1 contained the donors with the lowest prss (genetically predisposed to lower ferritin levels) and was set as the reference quantile with or = 1 from the age-adjusted regression analysis (tiredness~quantile). results: prss in females ranged between -0.42 and 0.50 (mean 0.01). a total of 12,523 female donors were classified as "not tired" and 380 (2.9%) were classified as "tired". no significant difference in ferritin prs was found between "tired" and "not tired" female donors (tired mean prs: 0.00; not tired mean prs: 0.01). an age-adjusted logistic regression model found this to be insignificant (or: 0.74, 95% ci: 0.33-1.66), p = 0.47). to visualise the lack of association, a quantile plot was created, separating the female donors into six equal quantiles based on their prs. no clear trend was observed; donors with the highest prss (in quantile 6) had or = 1.02 (p = 0.93) of being tired when compared to those in quantile 1 (or set as 1). summary/conclusions: no significant association was found between the ferritin prss of female blood donors and the tiredness/lack of energy symptom. further studies are needed to understand the effect of blood donation versus genetic constitution on tiredness among female iron-deficient blood donors. background: antiretroviral therapy (art) is critical for the control of clinical progression of human immunodeficiency virus (hiv) infections. however, the outcome of art could be limited by drug resistance-associated mutations (drms), even lead to the transmission of drug-resistant hiv to treatment na€ ıve patients such as blood donors, which is a huge concern to art. drms surveillance in hiv infected groups is strongly recommended by world health organization. characteristics of genetic diversity and drms of hiv among blood donors may provide comprehensive data to monitor viral evolution and optimize art, play important roles in blood safety. aims: limited data concerning the epidemic of hiv-1 subtypes and drms of blood donors is available in china. this study is to investigate genetic characteristics and drms of hiv-1 infected blood donors. methods: from 2016-2018, 177 blood donations collected from 24 blood centers, covering almost the whole of china, were confirmed as hiv-1 positive by national centers for clinical laboratories using abbott realtime hiv-1 assay or cobas taq-man hiv-1 test, version 2.0. then hiv-1 gag (973 bp, hxb2: 1074-2044), pol genes (1454 bp, hxb2: 2068-3521) (encoding the whole protease (pr) and a part of reverse transcriptase (rt)) was sequenced after viral rna extraction and amplification. hiv-1 subtype based on gag and pr-rt regions was determined by comprehensive analyses of los alamos hiv blast tool, rega hiv-1 subtyping tool, phylogenetic trees and online jphmm program. drms analysis was performed in the stanford hiv drug resistance database. results: among 177 donations, gag and pr-rt regions of 160 samples were sequenced successfully. the distribution of hiv-1 genotype was as follows: crf_07bc = 66 (38.8%), crf_01ae = 58 (36.3%), b = 9 (5.6%), crf_08bc = 2 (1.3%), crf55_01b = 4 (2.5%), crf59_01b = 1 (0.6%), crf65_cpx = 1 (0.6%), crf67_01b = 2 (1.3%), crf79_0107 = 1 (0.6 %), crf85_bc = 1 (0.6 %), urf_0107 = 6 (3.8%) and urf = 9 (5.6%). 26 of 160 hiv-1 isolates were identified to have drms. there were 4 (15.4%, 4/26) protease inhibitors (pi) accessory drms, 3 pi major drms and 20 (76.9%, 20/26) non-nucleoside reverse transcriptase inhibitors (nnrti) drms. most of blood donors with drms were crf01_ae and crf07_bc (61.5%, 16/26). 3 of 4 pi accessory drms were q58e. the pi major drms included m46l, m46i and n88s. n88s could result in hlr to atazanavir (atv) and nfv, llr to indinavir (idv) and saquinavir (sqv). v179d/e is main nnrti drm (70.0%, 14/20). a combination of v179d and k103r among two samples acted synergistically to reduce efavirenz (efv) and nevirapine (nvp) susceptibility. furthermore, two blood donors with k103n mutation in reverse transcriptase gene had high level-resistance to efv and nvp. summary/conclusions: overall, the most prevalent subtypes among blood donors in the study were crf07_bc (38.8%), crf01_ae (36.3%). besides, other rare crfs and several urf_0107 and urfs were also found in these hiv-1 isolates, which suggested the epidemic of hiv has been shifted from high risk populations into general populations, including blood donors in china. drms were observed in 16.3% donors in the study, which may result in resistance to pis and nnrtis, especially the hiv-1 variants with n88s mutation in pr gene and k103n mutation in rt gene. in summary, our findings indicate that increasing diversity of hiv-1 in blood donors and remind us the necessity of timely genotypic drug resistance monitoring and molecular epidemiology surveillance of hiv-1 among blood donors. background: labeling of platelets is required to measure the recovery and survival of transfused platelets in vivo. currently a radioactive method is used to label platelets. however, its' application is limited, due to safety issues and the inability to isolate transfused platelets out of the circulation. biotin-labeling of platelets is an attractive non-radioactive option, however, no validated protocol to biotinylate platelets is currently available for clinical purposes. aims: the aim of this study is to develop a simple, standardized, reproducible method to label platelets with biotin as a non-radioactive alternative to trace transfused platelets in vivo. methods: six pooled buffy coats derived platelet concentrates (pcs) stored in 100% plasma were biotinylated at day 1 and day 7 of storage. to distinguish the effect of the processing steps from the effects of biotin incubation, 'sham' samples were processed. for the biotinylation procedure, 50 ml of pcs was washed twice and incubated with 5 mg/l biotin, dissolved in phosphate buffered saline-pas-e (1:9), for 30 min. stability of the biotin labeled platelets after irradiation was tested. annexin v and cd62p expression were assessed as measures of platelet activation. applicability of this method to other platelet products was assessed in three pooled pcs stored in 65% pas-e and three single donor apheresis pcs. results: the method was reproducible performed in a closed system. after biotinylation, 98.4% ae 0.9% of platelets were labeled. platelet counts, ph and 'swirling' were within the range accepted by the dutch blood bank for standard platelet products. the number of annexin v positive cells was not significantly altered by the biotinylation procedure in both fresh and stored platelets. in contrast, cd62p expression was increased in biotinylated platelets 48.4% iqr(41.7-56.2%) compared to the control samples 12.3% iqr(9.5-12.7%) on day 1 of storage. however, biotinylated platelets were not more activated compared to sham samples 50% iqr(41.7-56.2%). thus only the procedural steps led to increased cd62p expression and not the biotin label itself. all samples showed maximal response to thrombin receptor-activating peptide. for platelets labeled at day 7, a similar pattern was observed. irradiation of biotin labeled platelets did not alter the stability of the biotin label nor cell quality. furthermore this method is also applicable to pooled pcs stored in pas-e and apheresis pcs, with similar patterns in annexin v and cd62p expression. summary/conclusions: we developed a standardized and reproducible protocol according to good practice guidelines (gpg) standards, for biotin-labeling of platelets for clinical purposes. the procedural steps, which are similar to the steps used for production of hyperconcentrated platelet products, led to an increased cd62p expression, but did not alter the annexin v expression. this method can be applied as non-radioactive alternative to trace and recover transfused platelets in vivo. blocking activity over the prototypic chs4 insulator in cell lines and substantially reducing genotoxicity in a c-retroviral vector-mediated carcinogenesis mouse model. in contrast to chs4, these insulators are small-sized (119-284 bp vs 1.2 kb) and can be easily accommodated in gt vectors without detrimentally affecting vector titers. aims: we aimed to test whether a1, one of the newly discovered cis, could reduce vector-mediated genotoxicity in the challenging context of sin-lvs, by insulating a therapeutic globin-vector. methods: we tested the genotoxicity effect in the il-3-dependent 32d cells, which upon transduction with oncogenic vectors become il-3-independent, leading to transformation. 32d cells were transduced with sin-lvs: the b-globin-τνs9.3.55-, the insulated b-globin-a1-tns9.3.55and the oncogenic sffv-gfp-vector. transduced cells were expanded in 10% il-3 and transduction efficiency was determined by vector copy number (vcn). transduced 32d cells were seeded in methylcellulose with 10% or 0-1% il-3 to detect the il-3-independent and potentially transformed clones. the il-3-independent clones were further expanded in 10% il-3 and infused in partially myeloablated and il-3-treated c3h/hej mice. wbc analysis, blood smears and bone marrow(bm) cytospins were performed. results: the a1 insulator did not negatively affect vector titers (τνs9.3.55, a1-tns9.3.55, sffv-gfp: 2.8, 1.8, 2.5x10^8 iu/ml, respectively). 32d cells were successfully transduced with all vectors (%vcn positive colonies: 40-100%) and expanded up to 400-fold. the a1-insulator decreased the number of il-3-independent colonies by 78-93% over the uninsulated vectors. the uninsulated vector-transduced, il-3-independent colonies, were greatly expanded in culture with 10% il-3 over the a1-transduced colonies (sffv, τνs9.3.55, a1-tns9.3.55: 48, 40, 10 fold change, respectively). il-3 independence as a transformation event was confirmed in vivo by the development of overt leukemia (hyperleukocytosis, splenomegaly, bm-and extramedullary site-infiltration) in mice transplanted with the il-3-independent and expanded colonies. summary/conclusions: under forced oncogenic conditions, the a1 insulator effectively protected a therapeutic vector from vector-mediated genotoxicity. a1 may serve as a safety feature in the construction of globin-sin-lvs. background: novel rare nucleotide substitutions are frequently identified in rhd, the gene encoding the immunogenic d antigen of the clinically-relevant rh blood group system, resulting in d variant phenotype. so far, it has been commonly accepted that substitutions of amino acids located either in a transmembrane or intracellular domain of the rhd protein induce weak d phenotype, i.e. reduced d antigen density at the surface of red blood cells. recently we showed by functional analysis using a "minigene splicing assay" (msa) that a decrease in d antigen expression may be due also to alteration of cellular splicing. aims: here we pay attention to the general disruption of this mechanism and the related phenotypic consequences in novel and previously reported single-nucleotide variations in rhd. we then sought to characterize functionally by msa novel candidate splicing variants in rhd. then we extended the project by studying prospectively all single-nucleotide variations reported in rhd exons, in order to assess globally the correlation between in silico prediction and functional analysis and to gain insights into the reliability of bioinformatics tools in line with the available phenotypic and/or clinical data. methods: seventeen novel or uncharacterized rhd variations, including missense, synonymous and intronic substitutions, were selected for functional analysis by msa in human cell models. a second set, including 46 missense variants reported in rhd exons 6 and 7, was further analyzed. functional data were compared with an algorithm derived from the quepasa method and tools available in the alamut suite. a published 3d protein model was used to visualize the location of missense amino acid substitutions and to assess potentially their respective phenotypic consequence. results: a novel "universal" minigene was validated and used successfully to characterize eleven novel splicing variants. those variants include six intronic and four missense substitutions close to the consensus dinucleotide splice sites, as well as the c.1065c>t synonymous variation associated with a weak d phenotype, which creates a de novo splice site. very interestingly, c.1154g>t (gly385val; d-negative) disrupts totally normal splicing, while c.1154g>c (gly385ala; weak d) and c.1154g>a (gly385asp; d-negative) only partially alter the mechanism. further visualization of amino acid changes in a 3d model suggests that gly385asp, but not gly385ala, dramatically impair rhd protein structure/folding. subsequently the global analysis of mutations in rhd exons 6 and 7 by msa showed that inclusion of whole exon sequence in the mature transcript is significantly reduced in 15/46 (32.6%) variants, which correlates well with the quepasa-like prediction (sensibility = 0.93, specificity = 0.94). additionally, while normal exon inclusion is affected by c.1012c>g (weak d type 70), the associated leu338val substitution does not seem to be deleterious to the protein. summary/conclusions: on the basis of our functional data, this work shows that splicing disruption in the presence of rhd variants is a common and general mechanism that may act independently or synergistically with alteration of protein structure through amino acid substitutions, resulting in a weak d phenotype. it also illustrates the potency of combining functional tests and in silico tools towards the phenotypic/clinical interpretation of rare variants. background/aims: monetary and non-monetary incentives may support blood services in recruiting blood donors but have also been criticized for violating ethical principles and threatening blood safety by attracting donors with a high risk for infectious diseases. although incentives for blood donors have been discussed extensively over the past decades, empirical research on this topic remains limited. the aim of this study was to describe attitudes towards incentives for blood donors in europe and show donor return rates of compensated and non-compensated blood donors in south-west germany. methods: first, we present results of a secondary analysis of the eurobarometer, a nationally representative survey in all member states of the european union. in 2014, participants were asked to evaluate eight potential incentives for blood donations as acceptable or unacceptable. these incentives were refreshments (e.g. coffee), physical check-ups (e.g. blood pressure), free (testing) laboratory parameters, free medical treatment, complimentary items (e.g. first aid kits), monetary travel reimbursements, additional cash reimbursements, and release from work. second, we conducted a retrospective analysis of donor return patterns of 6.210 compensated and 68.205 non-compensated donors who started donating blood at mobile and fixed donation sites. compensated donors received either 25 eur as a regular reimbursement for their expenses (at a fixed donation site), in accordance with the german transfusion law, or a singular free entrance for an amusement park (at a mobile donation site). these compensated donors were compared with noncompensated donors who started either at a fixed or mobile donation site. chisquare statistics were used to test for differences in regular donor status after 24, 36, 48 and 72 months between compensated and non-compensated first-time donors. results: among german participants of the eurobarometer, physical check-ups (51.9%), refreshments (50.0%) and free (testing) laboratory parameters (38.3%) showed the highest acceptance as an incentive for blood donors. travel reimbursements and free medical treatment were rated as acceptable by 28.6% and 25.3%, respectively. the lowest acceptance was for release from work (17.8%), complementary items (13.9%) and additional cash reimbursement (11.8%). interestingly, the acceptance of potential incentives varies considerably across europe. in south-west germany, donor return of first-time donors differed significantly by type of compensation. among compensated first-time donors, who received 25 eur as a monetary reimbursement, the proportion of regular donors after 48 months (19.9%) was significantly higher than among comparable non-compensated donors (9.2%). however, a non-monetary compensation (free entrance) did not increase donor return rates. conclusion: the eurobarometer survey indicates that in most european countries monetary incentives are only accepted by a small minority. refreshments, checkups, free (testing) laboratory parameters and free medical treatment were most popular as incentives for blood donors. however, results of our four non-randomized donor samples from south-west germany suggest that monetary compensation may increase the likelihood of donors returning to fixed donation sites. regular monetary reward may therefore help to recruit regular donors especially in urban settings. incidentally, non-monetary compensation by a free entrance, however, may not affect donor return. background: previous research showed that whole blood (wb) donors that are temporarily deferred on-site are at higher risk of lapsing, yet very little studies have focused on differentiating the effects that different deferral reasons (e.g., travel, hemoglobin [hb]) may have on donor lapse. in addition, donor experience (i.e., firsttime or repeat donor) has also previously been found to affect donor lapse, yet novice (1-5 prior donations) and reactivated donors (returning after years of not donating) may respond differently. finally, it is currently unclear how and why different deferral reasons and donor experience interact in influencing donor lapse. aims: our aims were to understand 1) how deferral reasons and donor experience jointly affect donor lapse, and 2) why donors may lapse after temporary deferral. methods: a mixed methods approach was used. first, we used sanquin's donor database for a quantitative analysis of return behavior of all dutch wb donors between 2013 and 2015 (n = 343,564). the first wb donation for each donor was identified as the target donation. lapse was defined as non-return within a followup period of two years after the target donation. target donations included 25% new donors, 41% novice donors, 30% experienced donors, and 4% reactivated donors. deferral reasons included travel, hb, medical short-term (<28 days duration), medical long-term (>28 days duration), and miscellaneous. next, we interviewed 31 temporarily deferred donors to understand the deferral process from their perspective. semi-structured interviews were used to understand how these donors cognitively and emotionally experienced on-site temporary deferral. we analyzed the interviews (using the framework approach, cf. hillgrove et al., bmc public health, 2012) to identify key topics and underlying themes. results: of target donations, 11% were deferred, mostly for travel (40%), medical short-term (23%), and hb (19%). survival and time-to-events methods showed that the different deferral reasons and donor experience levels differentially impacted donor return or lapse. importantly, experience and deferral interacted in influencing return (rate). for instance, deferred new donors were more likely to lapse than eligible or experienced donors (ors < 0.75, p's<0.001). even though deferral also affected return of experienced donors, this effect was smaller or even non-existent for certain deferral reasons (e.g., travel-and hb-related deferrals). qualitative results showed that almost all donors experienced temporary deferral as disappointing, particularly when it was unexpected (e.g., first-time deferral). not all donors (fully) understood the aims of deferral or how to prevent on-site deferral. donor beliefs about why deferral would lead to lapse were related to recurring deferrals, (mistakenly) interpreting deferral as permanent, or feeling all the effort did not pay off. summary/conclusions: reasons for temporary deferral differently impact risks of donor lapse at different levels of donor experience. for new donors all reasons for deferral are related to higher risks of lapse, whereas some reasons for deferral seem not to affect lapse among more experienced donors. unexpected or recurring deferrals may explain why donors lapse after temporary deferral. blood banks may tackle disappointment after deferral by explicitly showing that the donor is still valued, for instance by using personalized communication or offering an alternative good deed. background: blood donors experience a temporary reduction in their hemoglobin (hb) value after whole blood donation. in the netherlands, the hb value is measured before each donation, and a too low hb value (cut-off values: 8.4 mmol/l (135 g/l) for men and 7.8 mmol/l (125 g/l) for women) leads to a deferral for donation, in order to prevent iron deficiency and anaemia. the minimum interval between two donations is internationally set at 8 weeks, but over time donors exhibit iron deficiency so that blood donors are temporarily deferred from donation each year. in the us 35% -75% of deferrals are due to low hb, especially in women (editorial, transfusion, 2012) . due to the recovery process after each donation and the unobserved heterogeneity of donors, advanced statistical methods are needed to model the longitudinal data of hb values of blood donors. aims: to estimate the shape and duration of the recovery process of hb until the hb value has returned to its pre-donation level, to assess whether one can distinguish between donors with fast and/or slow recovery of their hb level and to predict future hb values. methods: the study is based on data of the donor insight study, which was a prospective cohort study performed by sanquin in the netherlands from 2007 to 2016. we employed three statistical models for the hb value: (i) a mixed-effects models, (ii) a latent-class mixed effects model, and (iii) a latent-class mixed-effects transition model. in each model, a flexible function was used to model the recovery process after donation. the latent classes identify groups of donors with fast or slow recovery times, and donors whose recovery time increases with the number of donations. the transition effect accounts for possible state dependence in the observed data. all models were estimated in a bayesian way, using data of a sample of 1000 new entrant donors (500 males and 500 females). prior information from the clinical literature (boulton, vox sanguinis 2004) about the recovery process three days after blood donation was incorporated into the analysis since these values were not identified in the observed data. results: the results show that the latent-class mixed-effects transition model fits the data best. we also found that the recovery process shows a concave process (initially fast followed by slower recovery). the estimated recovery time is much longer than the current minimum interval of 58 days between donations. namely, depending on the subgroup that the donor belonged to, males showed a recovery time of 100 to 419 days, while the estimated recovery time for females varies between 54 to 503 days. these results suggest that an increase of this interval may be warranted. summary/conclusions: the analysis shows the usefulness of the sophisticated statistical models that make use of historical information to model complex processes in time, in this case the hb trajectory over time across repeated donations. in addition, our results suggest a (much) longer time lag between subsequent donations to avoid anemia. background: complications of blood donation are known to reduce donors' return for future donation. the episode study (experience success in donation) showed that water drinking shortly before donation had an effect of 23% reduction of selfreported vasovagal reactions (vvr) in younger novice whole blood donors (wiersum-osselton, transfusion, 2019) . aims: in this study we analysed the return for a subsequent donation of the donors participating in the episode study. this was a predefined secondary outcome of the episode study. methods: the episode study was conducted in young (<30 years) whole blood donors making their first, second, third or fourth donation in geographically selected collection centres. the study interventions were: 330 ml water drink, 500 ml water drink or squeezing a ball (placebo intervention) during the wait after the screening interview and before phlebotomy, and a control group without intervention. participating donors were sent an online questionnaire about their experience within a week following their donation attempt. in the netherlands donors are usually invited for blood donation in accordance with hospitals' needs; the aim is to invite eligible donors at least once a year. donors were included in the return analysis if they had received at least one invitation within 400 days after the index donation and we analysed their return for a donation attempt within 421 days. associations with the interventions and donors' donation status, gender and reported symptoms at their index donation were analysed by calculating return percentage of eligible donors and by binomial logistic regression. results: out of the 8199 episode participants who had received an invitation, 6538 (79.7%) returned within the study period. there was no difference in donor return between the two water groups. the likelihood of return was significantly increased in both water and placebo intervention donors compared to the questionnaire group (or 1.2, 95% ci 1.06-1.4 and 1.3, 1.12-1.5 respectively). return was slightly lower in women (or 0.88, ) and lower in first-time donors (or 0.86, 0.77-0.96) than after a 2 nd -4 th donation. a staff-recorded or self-reported vvr at the index donation reduced donor return (or 0.47, 95% ci 0.37-0.60 and or 0.53, 0.46-0.61 respectively). other symptoms following donation were also associated with a lower return percentage. summary/conclusions: in this cohort of younger new and novice blood donors, 79.7% returned for a subsequent donation. a vvr (either staff-recorded or selfreported) reduced donor return. donors who received a study intervention, either water or placebo, were more likely to return, whether or not they had suffered a vvr. it is conceivable that the mere fact of study participation could also have increased donor return, even in de questionnaire group; this will be examined in the total population of target group donors. background: the contribution of older blood donors to the blood supply is substantial. in australia, donors aged > 50 years contributed 41% of all donations made in 2017. however, with ageing, the general health status of older donors changes relatively faster, thus progressively affecting their ability to donate. an indepth understanding of the relationship between older donors' health status, future donation patterns, and risk of iron-deficiency could be of a great value to inform the blood service to predict the number of future donations, and manage the risk of iron-deficiency. aims: to understand the relationship between self-reported health, blood donation patterns, and the management of identified iron-deficiency in older blood donors. methods: we linked the sax institute's 45 and up study baseline data collected between 2006 and 2009 to the blood service donation records, inpatient records, and medicare records*. the data-linkage was conducted by centre for health record linkage. using these linked data, we examined the relationship between health, donation patterns, and iron-deficiency and its management. results: we followed up 22,058 active whole blood donors for 200,403 eligible person-years (average age at recruitment 56.4 years, 56.3% female, average follow up 9.1 years per-person). after adjusting for the effect of age, sex, body-mass index, education, non-english language spoken at home, country of birth, smoking, physical activity, regular use of multivitamins, alcohol consumption at enrolment, and total number of whole blood donations in the 2 years prior to enrolment, participants with better self-reported health at recruitment showed significantly higher rates of donation. excellent, very good, good, and fair/poor health status donors made 1066 (95% ci 1056-1075), 987 (981-993), 900 (891-909), and 710 (690-730) donations per 1000 person-years, respectively. iron-deficiency was identified in 8.9% of donors in the study (n = 1964, 95% ci 8.5-9.3) . sixty percent of those with iron deficiency (n = 1,175, 95% ci 57.6-62.0) visited their general practitioner (gp) within 60 days of the identification of irondeficiency, and 48.4% (95% ci 45.5-51.3) of those visiting gp underwent further iron status examination and monitoring. after adjusting for several potential confounders including the total number of donations made during the follow-up period, excellent self-reported health status was independently associated with lower risk of iron-deficiency (p for trend = 0.004). summary/conclusions: information on self-reported health status can be an effective indicator to estimate the future donation yield of an older blood donor panel, and risk of developing iron-deficiency. donors with better self-reported health had a higher number of future whole blood donations and a lower risk of iron-deficiency. donors referred to gps for management of their iron status utilised the health services as expected, however there is an opportunity to improve their contact with their gps. * medicare records was provided by australian government department of human services. anaemia is a major public health issue, affecting 25% of the population worldwide according to the world health organization. iron deficiency is responsible for approximately half of all cases globally, with other causes including anaemia of chronic disease, other nutritional deficiencies, haemoglobinopathies, renal impairment, malignancy and bone marrow disease. in the elderly, where anaemia is even more common, the cause is frequently multifactorial. anaemia is associated with increased mortality, decreased cognitive and physical function, depressive symptoms and fatigue, particularly in older adults. poor outcomes have also been reported in anaemic patients with underlying comorbidities such as cardiac and renal disease, and cancer. within a hospital setting, anaemia is highly prevalent. preoperative anaemia, affecting up to 30% of patients, is associated with poor clinical outcomes including higher in-hospital mortality, longer length of stay and higher icu admission rates. anaemia management requires a proactive and multi-faceted approach, typically involving a multi-disciplinary team in which the transfusion practitioner plays a vital role. this includes screening of high-risk patients and pre-admission clinics to identify and manage patients at high risk of peri-operative anaemia. implementation of patient blood management (pbm) guideline recommendations has been shown to be effective to prevent and optimally manage anaemia within the community and hospital settings. the transfusion practitioner has key roles in the coordination, monitoring and auditing of pbm programs. active patient involvement and engagement of all members of the multidisciplinary team, including primary care clinicians, are also key to enhance the success of such programs. tp01-02 the role of the transfusion practitioner in anaemia assessment and management: processes, tips and resources for creating background: patient blood management (pbm) is an evidenced based integrated multi-disciplinary approach aimed to improve clinical outcomes by effectively managing and conserving the patient's own blood, thus reducing unnecessary exposure to transfusion. pbm has the patient as the central focus with the aim being to improve their outcomes and include them in the process. pbm includes three pillars: 1) optimising the patient's own blood, 2) minimising blood loss and 3) optimising a patient's physiological tolerance of anaemia. delayed assessment/management of anaemia contributes to increased health costs and unnecessary blood transfusions, and transfusion has been recognised to be associated with increase morbidity and mortality. the term transfusion practitioner (tp) includes those known as transfusion nurses, transfusion safety officers, haemovigilance officers, or patient blood management (pbm) coordinators. a key aspect of the role is driving and influencing clinical blood management activities to help align practice to internationally recognised guidelines and standards, including pbm. aim: to demonstrate the tp role in anaemia assessment & management and discuss strategies, processes, tips and resources for creating organisational and cultural change to implement pbm. context: literature outlines the importance of a multidisciplinary team to implement pbm related changes, and tps play a fundamental role within these teams to support 'buy in'. tps are seen as enablers, pulling resources together, engaging with those involved, providing education and facilitating change. they are often the ones to conduct audits, collating data and evaluating outcomes. approaches to implement pbm should be tailored to suit individual organisations. the authors will outline different approaches, highlighting where the tp can support or lead activities. one approach to anaemia assessment is to undertake an audit, examples of available tools will be shown. with this data, the tp along with the pbm team can explore options for corrective action. these could include interventions such as developing a pathway where all or a specific group of patients are assessed and or treated either at a preoperative clinic, or with their local general practitioner; through to more complicated strategies such as establishing anaemia clinics. the skills of the tp are a valuable asset to analyse clinical specialties/patient mix who should be targeted to achieve best outcomes, they know the organisation and as such are well placed to help develop a process/concept that will suit, and they can provide education and support to promote and embed these practices. conclusion: appropriate assessment and management of anaemia requires a multidisciplinary approach. the tp plays an active and crucial role in this team. examples of processes, tips and resources to support change and embed a pbm culture across the clinical spectrum will be shared. 3d-s11-01 department of hematology and central hematology laboratory, inselspital bern, bern, switzerland immune haemolytic anaemia (iha) is characterized by an increased breakdown of red blood cells (rbcs) due to allo-and/or autoantibodies directed to rbc antigens with or without complement activation. clinical and laboratory signs of haemolysis in concert with the presence of a positive direct antiglobulin test characterize iha. alloantibodies formed during pregnancy and/or after prior transfusions may cause acute or delayed haemolytic transfusion reaction after transfusion of a rbc product incompatible with the specificity of the alloantibody. autoantibodies to rbcs reduce the survival of endogenous and hamper the recovery of donor rbcs after transfusion. lymphoproliferative disease, autoimmune disease, infection or drugs often cause autoantibodies to rbc, but frequently no obvious cause can be identified. besides the antigen specificity, the isotype critically determines the biological activity of rbc antibodies in vivo. the isotype defines the affinity to fc-gamma receptors on cells of the reticuloendothelial system as well as the capacity to activate the classical pathway of complement, igm being the most effective. antibody-mediated complement activation results in the opsonisation of rbc with c3bc/c3d with subsequent complement receptor-mediated removal by phagocytes (extravascular haemolysis). occasionally, complement activation proceeds via the activation of c5 to the formation and insertion of the membrane attack complex resulting in intravascular haemolysis. there is growing evidence that the innate immune system plays an important role in the pathogenesis of iha. the process of complement-mediated haemolysis results in systemic inflammation, which contributes to morbidity and mortality of patients suffering from iha. complement activation results in the release of anaphylatoxins, which are strongly vasoactive and mediate chemotaxis, inflammation and formation of radical oxygen species. release of cell-free haemoglobin and cell-free haeme upon haemolysis induces endothelial cell activation, no-depletion, cytotoxicity, ros formation and neutrophil activation. natural plasma scavengers, such as haptoglobin and hemopexin complex with their target molecules, cell-free haemoglobin and haeme, with subsequent removal of the complexes via cd163 and cd91-mediated phagocytosis. although being positive acute phase proteins due to consumption the plasma scavengers become exhausted during chronic haemolysis thereby failing to prevent the adverse biological effects of cell-free haemoglobin and haeme in the circulation. inducible haeme oxygenase-1 (ho-1) is an efficient cellular scavenger by breaking down haeme into biliverdin with subsequent formation of bilirubin, co and ferrous iron with subsequent oxidation to ferric iron and storage by the ferritin h chain. ho-1 has an established role in the systemic protection from systemic inflammation induced by haemolytic and non-haemolytic diseases. the lecture will emphasise the role of innate immunity with a special focus on different plasma-and cellular systems involved in the pathogenesis of systemic inflammation in patients suffering from iha. 3d-s11-02 understanding erythrocyte clearance c roussel, p amireault, p ndour and p buffet research and teaching, institut national de la transfusion sanguine, paris, france the clearance of erythrocytes is essential in physiology, disease and transfusion. elimination of erythrocytes altered because of senescence or pathological processes is expected to protect the microcirculation from obstruction by adhesive or rigid erythrocytes. it also contributes to the harmful consequences of anemia and hemolysis in hereditary and acquired red blood cells diseases as well as in conditions associated with auto-or allo-immunization. immunobiology has explored in great details antibody-mediated clearance of erythrocytes but conventional approaches may not be fully operational to explain delayed hemolytic transfusion reactions. some important clearance processes are independent from the recognition of molecules or antigens on the erythrocyte surface. increased erythrocyte stiffness triggers their clearance in hereditary spherocytosis, malaria and possibly also in the context of autoimmune anemia. knowns and unknowns on the mechanisms and sites of erythrocyte clearance will be presented based on a critical review of old and recent contributions. 3d-s11-03 cardiovascular and endocrine-metabolic diseases and aging, istituto superiore di sanit a, rome, italy existing literature indicates that red blood cells (rbcs), beyond gas transport, exert a complex role in human physiology, being involved in many functions essential to maintain ion, metabolic and immunological homeostasis. rbcs display an immunomodulatory activity on adaptive immune cells by promoting t cell growth and survival and inhibiting activation-induced cell death. the balance between cell death and survival controls t cell homeostasis and anomalies in this balance account for diseases linked to excessive or faulty t cell growth. rbcs are able to modulate innate immunity by binding endogenous molecules such as chemokines and mitochondria-derived dna, as well as external agents such as pathogens. rbcs can also directly modulate innate immune cell activation or tolerance by controlling the maturation of the circulating pro-inflammatory subset of dendritic cells (dcs). these cells are potent inducers of primary antigen-specific t cell responses, produce tnf-a when stimulated by lps and are the principal il-12p70-producing cells among leukocytes. the pro-inflammatory capacity of circulating dcs is controlled by rbcs that are able to inhibit their maturation and il-12 production. in diseases characterized by local th1 inflammatory response such as psoriasis vulgaris and rheumatoid arthritis, pro-inflammatory dcs play a role in the induction and perpetuation of inflammation. collectively, literature data indicate that rbcs exert important modulatory functions that may result in immune activation or quiescence, depending on the environmental conditions. when rbcs encounter a microenvironment characterized by an intense production of ros, the rbc defenses get overwhelmed or are unable to counteract the new pro-oxidant status and become themselves a source of ros, which cause the generation of senescent signals on rbcs. the major feature of oxidized rbcs is the clustering and/or the breakdown of band 3. other features are the complexation of hb with spectrin, the loss of glycophorin a, the externalization of phosphatidylserine and the reduction of the "marker of self" integrin-associated protein cd47. a similar senescence phenotype has been documented in rbcs during the storage period. oxidized, senescent or stored rbcs, due to surface antigen modification and to the release of pro-inflammatory molecules, fail to control immune cell homeostasis thus contributing to the perpetuation of inflammation and to the pathogenesis of immune-mediated diseases associated to oxidative stress, such as autoimmune diseases and atherosclerosis. our research group demonstrated that rbcs from patients with carotid atherosclerosis presented a senescent phenotype similar to that acquired by rbcs from healthy subjects following to in vitro oxidation. oxidized erythrocytes fail to control t lymphocytes apoptosis and lipopolysaccharide-induced monocyte-derived dc maturation, thus representing dangerous signals for adaptive and innate immunity and contributing to the pathogenesis of atherosclerosis. in conclusion, the crosstalk between rbcs and the immune system represents a mechanism to maintain immunological homeostasis. however, in high oxidative stress conditions, that can take place during a prolonged storage period or in particular diseases, rbcs can acquire a pro-oxidant behaviour and lose their functional and homeostatic features. by interfering in immune system homeostasis, rbcs become a potential tool that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of adaptive and innate immunity. transfusion therapy remains an important treatment modality for patients with sickle cell disease (scd). transfusions are given to lower the percentage of circulating sickle rbcs, and to decrease blood viscosity and have been shown in clinical trials to reduce the risk of stroke by 90%. however, many indications for transfusion in scd remain controversial partly due to insufficient randomized clinical trials data and in part because of our limited understanding of the complex pathologic networks leading to diverse disease complications in scd despite the common single mutation. similarly, we have incomplete mechanistic understanding of why chronic transfusion protocols must be continued for those indications supported by clinical data. the beneficial effects of transfusion therapy in scd need to also be weighed against potential transfusion risks including alloimmunization associated with lifethreatening delayed transfusion reactions, increased iron stores associated with increased oxidative stress and exposure to infectious agents. we believe that a deeper understanding of the benefits as well as harmful effects of transfusions is crucial to optimize our current transfusion therapy protocols in scd. this knowledge may provide highly needed guidance, which is currently lacking, for expansion or limiting existing indications for chronic transfusions in scd. 3d-s12-02 treatment of thalassaemia department of pediatric hematology, ege university, faculty of medicine, bornova/ izmir, turkey thalassaemia is a devastating blood disease with a significant worldwide burden. annually, 60,000 children are born with a major thalassemia. life-time rbcc transfusions and iron chelation remain standard of care treatment in thalassaemia. transfusion therapy still account for significant iron overload related morbidity and mortality despite chelation therapy which is associated with poor adherence, safety concerns and varied efficacy. higher risk for transfusion transmitted infections (ttis) exists for thalassemia patients whose transfusion exposure sustains lifelong. although, the risk of transmission for traditional viruses is exceedingly rare in the modern era, emerging infectious diseases continue to be recognized as potential threats to transfusion safety. the inadequacy of blood safety points to the necessity for an additional layer of security for the blood supply in the developing world. pathogen reduction technologies for rbcc may imply a proactive, more generalized approach against new and re-emerging pathogens in the developed world and may be an ultimate safeguard for transfusion safety in the developing countries. rbc alloimmunization may become a major challenge in thalassaemia management. prevention is the key reducing the burden of alloimmunization. while the recommendation is to transfuse thalassaemic patients with c/c,e/e,kell compatible blood, it is not universally practiced. extended molecular rbc typing may be an appropriate adjunctive test in addition to serological typing before embarking on transfusion therapy. if a complete rbc antigen profile has not yet been performed in an alloimmunized patient, genotyping is the only option for accurate detection of rbc antigens that may guide the antibody identification. allogeneic stem cell transplantation (a-sct) is the only available curative therapy in children with hla matched sibling which is available to approximately 20% patients. in the absence of msd, mud transplant with high compatibility criteria has still limited experience. mismatch related, cord blood and haploidentical donor scts are considered experimental. a-sct carries a substantial risk of saes and mortality, both increasing with recipient age and disease severity. dfs is 88% in paediatric and 65% in adults. gene therapy for correction of the a-globin chain imbalance overcomes the problems of donor availability and immunologic complications associated with a-sct. multicenter clinical studies on gene addition therapy by using self-inactivating lentiviral vector are currently underway. recently, gene editing by either gene disruption or gene correction emerged as a potential alternative to gene addition therapy in beta-thalassaemia. a new era of novel therapeutics is unfolding in thalassemia management. several targets have been identified that can improve alpha/beta chains imbalance, ineffective erythropoiesis, or iron dysregulation and a number of those now have agents in preclinical and clinical development. hydroxyurea may improve globin chain imbalance and be beneficial for reducing or omitting transfusion requirement in selected group of patients. ruxolitinib has shown the limited effect on pretransfusion haemoglobin and reduction in transfusion needs, but allowed steady decrease in spleen volume that may serve for avoiding splenectomy in beta thalassaemia. luspatercept may restore normal erythroid differentiation and improves anaemia and hepcidin mimetics or tmprss6 inhibitors may modulates ineffective erythropoiesis by iron restriction and improves anaemia and organ iron loading. background: thalassaemia major (particularly b-type) and sickle cell disease (scd) are the commonest clinically important haemoglobinopathies, representing major sources of morbidity. recommended therapy is regular transfusion of safe, good quality blood, and monitoring of related complications. thalassaemia international federation (tif) guidelines, in place since 1987, include strategies for precautionary measures and use of scientific progress in detection, inactivation and elimination of transfusion transmissible pathogens. antigen-matching strategies to avoid alloimmunization against rbc antigens and other measures including haemovigilance are key components for safe blood, alongside voluntary, non-remunerated blood donation and laboratory quality assurance programmes. aims: we present the contribution of tif and the greek experience in ensuring safety and availability of blood for thalassaemia patients applying internationally accepted standards and recommendations. methods: tif -a non-profit, patient-driven organization with 204 national thalassemia associations in 62 countries -promotes national control programmes for prevention and management contributing to the achievement of final cure. the main working methods are provision of education, expert support, networking, communications and projects to support improvements in the quality of health, social and other care. in greece, technical standards for blood donor selection and testing are applied in compliance with directive 2004/33/ec as well as haemovigilance programmes and traceability procedures for recording adverse reactions and events associated with the transfusion of rbcs (directive 2005/61/ec). pre-transfusion and transfusion measures recommended by the council of europe are applied. in particular, measures for transfusion of "the right blood at the right time for the right patient", leucodepletion, rbc washing and accurate cross-matching and antigen and antibody screening for an extended matching policy are practised. fresh (up to 15 days old) rbcs are used. molecular testing for abo and rh d is performed in cases with blood group discrepancies. haemovigilance in greece covers 95% of total blood supply. data on ttis in 3,067 patients with thalassaemia and scd-thalassaemia in 2010-2017 are analysed. results: tti prevalence in thalassaemia syndromes was: hbv 1.8% (occult type 1.3%), hcv 54%, hiv 0.3%, htlv 0.8%, wnv 0.5% and hev 3%. most frequent adverse reactions in 2015-17 were allergic (incidence 1:854), non-haemolytic febrile reactions 1:2,631, "other" 1:5,883, alloimmunisation 1:6,667, taco 1:100,013, tad 1:50,006, tt-hev 1:100,013. hyperhaemolysis was diagnosed in two scd patients, delayed haemolytic transfusion reaction in one thalassaemia intermedia patient. trends in 2010-2017 show reduced incidence of alloimmunisation against rbcs. rates of allergic and pyrexial ars remained stable. no major abo incompatibility case was reported and no fatal transfusion reaction of transfusion has been recorded. summary/conclusions: blood safety in transfusion has significantly improved in high and upper-middle income but unfortunately not in lower and low income countries. blood shortages and lack of stringent protective measures for thalassaemia patients is the reality for many developing countries. tif focuses particular attention on the provision of support and the promotion of initiatives promoting the safety and adequacy of blooda key component of the lifelong management of patients with transfusion-dependent thalassaemia. background: b thalassemia is the most common group of hereditary hemoglobinopathy diseases. affected people with major thalassemia are dependent on regular blood transfusion which leads to iron overload. hepcidin is a peptide and an important regulator of iron homeostasis. expression of this hormone is influenced by polymorphisms within the hepcidin gene, hamp. aims: this study aimed to analyze the association of three polymorphisms in promoter of hamp, rs10421768, rs117345431, and rs142126068 with iron overload in major b thalassemia patients who do not respond to iron chelating therapy. materials and methods: a total of 102 samples from major b thalassemia patients were collected. genomic dna was extracted and sequenced for snps rs10421768, rs117345431, and rs142126068. statistical analysis was performed on ibm*spss* statistic 23 using independent t test and fisher test. results: our analysis revealed statistically significantly difference between the level of cardiac iron concentration and c.-582a>g variant (p = 0.02). for rs117345431 statistical analysis was on the edge of significant relationship between minor allele and serum ferritin (p = 0.058). all samples were homozygous for allele t of rs142126068. summary/conclusions: different factors affect iron overload in thalassemia. our findings and others emphasize the role of hepcidin polymorphism as a key component in iron homeostasis. ten to twenty years ago, countries in south eastern africa faced the peak of the devasting hiv/aids epidemic leading to an up to 20 years drop in general life expectancy. with the burden of hiv/aids falling mainly on the economically active population of young and medium-aged adults, the epidemic endangered social and economic stability in nations most heavily affected. today, despite aids still being a major cause of death in south eastern africa, the epidemic has become an example of public health gains that can be achieved through programmatic, evidencebased approaches that are endorsed by globally aligned policy and funding strategies. based on his work from lesotho, where one out of four among adults is infected by hiv, niklaus labhardt will take the auditors through the history of hiv programs in south eastern africa and show how innovative, pragmatic and evidence based implementation brought the region to a stage where the goal to end the aids epidemic by 2030 might be in reach. background: in france, the deferral for men who have sex with men (msm) was reduced from permanent to 12 months in july 2016. since this change has not impacted the residual risk (rr) of undetected hiv among blood donations, the ministry of health is considering a greater access of blood donation to msm. two scenarios have been studied: s1. deferral of msm during the 4 months preceding the donation; s2. deferral of msm who have had more than one sexual partner in the 4 months preceding the donation, similarly to all other blood donors in france. aims: to assess the impact of these two scenarios on the hiv rr estimated over the period july 2016-december 2017 which is the baseline rr with the current 12month deferral for msm. methods: baseline hiv rr was calculated with the classical incidence-window-period method, where hiv incidence was derived from a detuned assay (eia-ri) detecting recent infections (≤180 days) since all hiv-1 antibodies positive blood donations are tested with this test. the assessment of the impact of both scenarios on the baseline hiv rr was based on (i) data obtained from surveys among msm in the general population and in blood donors (compliance survey), to estimate the number of additional msm who would give blood in each scenario, and on (ii) hiv incidence estimate among these additional donors. this incidence was estimated: for s1, from msm blood donors with the current deferral policy (12 months) and for s2, from monogamous msm of the general population. results: from july 2016 to december 2017, 8/28 (29%) hiv-1 positive blood donors tested with the eia-ri were identified as recently infected, allowing to estimate the baseline hiv rr at 0.16 in 1 million donations [95% ci: 0.04-0.34], or 1 in 6,380,000 donations. for s1, the number of additional msm donors was estimated at 733 and the number of additional hiv positive donations at 0.09, resulting in an hiv rr of 0.16 in 1 million donations [95% ci: 0.04-0.35] or 1 in 6,300,000 donations. for s2, the number of additional msm donors was estimated at 3,103 and the number of additional hiv positive donations at 4.92, resulting in an hiv rr of 0.23 in 1 million donations [95% ci: 0.05-0.56] or 1 in 4,300,000 donations. sensitivity analysis shows that if both the number of msm and the hiv incidence were multiplied by 1.5, the risk would be 1 in 6,225,000 donations for s1, and 1 in 3,000,000 for s2. summary/conclusions: for both scenarios, the hiv rr remains very low. for s1 (4-month deferral), the risk is identical to the baseline rr and is very robust to variations in the model parameters. for s2 (no more than one sexual partner, 4 months), the risk is 1.5 higher than the point estimate of the baseline rr and sensitivity analysis shows that this estimate is less robust than for s1, since the risk could be 2 times higher than the baseline rr. for both scenarios, there was a modest increase in eligible msm donating. 3d-s13-03 background: recruiting safe blood donors amongst the largest hiv-positive population in the world is a major challenge for south african blood transfusion services. south african donor deferral criteria and deferral periods for perceived high risk activities have evolved over time, but current risk factors for infection have not been formally assessed. in addition, most studies have reported risk factors for prevalent hiv infection whereas risk behaviours for incident infection are more informative as donations with these infections could occur during the window periods of available screening assays. aims: to identify the demographic and behavioural risk factors associated with incident hiv infection among blood donors in south africa. methods: we conducted a case-control study with incident hiv-infected blood donors compared to infectious marker negative controls. incident hiv cases and controls seronegative for hiv, hepatitis b and c viruses and syphilis were accrued from a donor pool covering 8 of 9 provinces in south africa. controls were frequency matched at a 3:1 ratio to cases on race, age and geography. incident hivinfections were hiv rna positive by individual donation nucleic acid amplification testing (id-nat; procleix, grifols) but antibody (ab) negative (prism, abbott) as well as those rna+/ab+ donors with recently-acquired hiv based on limiting antigen avidity (lag) assay results with normalized optical density values of < 1.5. eligible cases and controls completed a confidential audio computer assisted structured interview (acasi) on motivations for blood donation and behavioural factors, including behaviours in the 6 months before donation. frequencies and measures of statistical association for risk behaviours comparing cases and controls are reported after adjusting for multiple comparisons. results: from november 2014 to january 2018, we enrolled 323 incident hiv cases and 877 controls; 202 (62.5%) cases and 544 (62%) controls were ≤ 29 years old. there were significantly more female cases 230 (71.2%) than female controls 439 (50.1%) (p < 0.0001). significant hiv risk factors (all p < 0.0001) reported within the 6-months before donation included: having a primary sex partner who is male; reporting increasing numbers of male sexual partners for both females and males; frequency of vaginal sex; frequency of vaginal sex without condoms; use of methods to clean, dry, or tighten one's anus before sex; and having visited a traditional healer for medical care. lack of medical aid (private health insurance) and reports of injury or accident with blood loss were also associated with an incident hiv infection. summary/conclusions: our study has identified a set of novel, putative risk factors for incident hiv infection among south african blood donors while confirming a number of previously known sexual risk behaviours. not having private health insurance and being injured may be markers of socio-economic context that place individuals at higher risk rather than behaviours that directly increase hiv transmission risk. the detection of risk behaviours by acasi in donors who passed predonation questionnaires and interviews suggests that acasi has the potential to improve risk behaviour identification. background: in france from 2000 to 2016, among male blood donors (mbds) found hiv-1 positive at blood donation screening, 31% did not disclose any risk factor for hiv infection during post-donation interviews, while 38% reported having sex with men (msm), and 24% and 7 % reported heterosexual sex (hts) and other risk factors, respectively. aims: in order to gain new insights into the risk factors for hiv-1 infection in mbds, we performed an hiv-1 genetic network analysis, including hiv-1 positive mbds and patients included in the french primary hiv infection anrs co6 primo cohort (pc). methods: 284 mbds, who donated blood between 2000 and 2016, and 1340 pcs, included between 1998 and 2014, were studied. epidemiological data were collected by the french blood service (efs) upon blood donation or post-donation interviews for mbds, and upon inclusion for cps. viral strains were sequenced and genotyped in pol gene, and a recent infection assay was performed to date infection in mbds (recent: <6 months). a partial transmission network was computed based on tamura-nei 93 nucleotidic distance (threshold for hiv-1 s/t b = 1.5%; for non-b s/ t = 0.8%) and assortative mixing was evaluated for mbds epidemiological data, including risk factors for hiv infection (msm, hts, others and unknown). selfreported data were then compared to assortativity-enhanced data. results: hiv-1 strains from 81 mbds and 126 pcs were linked into 54 clusters including at least one mbd. primo-only clusters were excluded from the analysis. compared to mbds who did not cluster, those found linked to the network were younger (30 vs. 36 year-old; p < 0.01) and were more likely to have a recent infection (48% vs. 34%; p = 0.03). assortative mixing indexes showed that paired individuals were more likely to live in the same area (p < 0.001) and to have the same risk factor for hiv infection (p < 0.001) compared to a random distribution. imputing msm risk factor to non-msm individuals paired with msm changed the distribution of risk factors as follows: msm: 38% vs. 49%, hts: 24% vs. 21%, other: 7% vs. 6% and unknown: 31 vs. 24%. summary/conclusions: after validating the assortativity of risk factors between paired individuals, and imputing msm risk factor to individuals self-reported as non-msm (including those with no identified risk factor), up to 18% (31/176) of mbds could be reclassified as msm. this is a worst-case scenario, as the network analysis does not exclude the possibility of one or several persons between two paired individuals (missing link). altogether, these results could help reevaluate the hiv residual risk linked to msm mbds, especially in the frame of the evolution of blood donor deferral criteria. background: although most individuals remain asymptomatic, htlv infection can lead to adult t-cell leukaemia/lymphoma (atll) and htlv-1 associated myelopathy (ham). the serious nature of these diseases, evidence of transmission via non-leucodepleted blood, and concern about a high prevalence among donors originating from endemic areas led to the uk blood services introducing universal blood donation screening in 2002. monitoring through routine surveillance commenced and htlvinfected donors were invited to participate in the htlv national register cohort study to assess disease progression. these data together with evidence from lookback to previously untested donations and cost-effectiveness analysis were reviewed by an expert working group in 2013 and 2015. aims: to describe the epidemiology of htlv among uk blood donors and evidence of disease progression from long term follow up of asymptomatic donors. methods: uk blood donations screened, and infected donors identified are reported to a national surveillance scheme. these donors are contacted, their results explained and information about clinical history and possible sources of infection are collected. where appropriate, htlv-infected donors are consented to the register, with participants completing a baseline questionnaire about their health, flagged in registries for cancer or death, and followed up about every 2-3 years. results: in the uk 2002 -2017, 254 htlv-infected donors were identified. prevalence among new donors was steady around 5/100 000 donations. prevalence among repeat donors peaked in 2002 (2.7/100 000 donations), with most in previously untested. from 2004 to 2016, prevalence of 0.07 per 100,000 donations (average of one positive/year) was recorded. in 2017, prevalence among new donors increased to 9.9/100,000 donations (17 positives), with increased numbers associated with asian ethnicity and coinciding with an increase in collections from bame groups. overall, most were women (182/254, 72%), uk-born (125/254;49%) and htlv-1 infections (228/254;90%). mean age was 43 years. almost all positive donations were from previously untested donors (240/254), with seroconversion within a year of previous donation confirmed for only 5 of the 14 previously tested donors. typically, infections were associated with endemic countries (including caribbean region, west africa, iran, india and japan), acquired through breast feeding or from their heterosexual partner originating from these countries. interestingly, three were thought to have been infected through self-flagellation. a total of 114 htlv-positive asymptomatic blood donors have already been recruited to the htlv national register, and during over 800-person years follow-up, none had developed atll or ham. summary/conclusions: over 16 years of testing, few seroconverters were identified, suggesting very little ongoing transmission among uk blood donors. the lack of disease among the cohort study was also reassuring, although it is likely too early to detect associated symptoms of a slow progressing disease. recruitment to this unique dataset continues, also outside of the blood donation setting. as a result of these surveillance data, evidence from lookback, and cost-effective analysis, in 2017 nhsbt ceased to test donations from previously tested donors unless the donation was being used to manufacture a non-leucodepleted component. finland lies in northern europe between the 60°and 70°n latitude. the length of the country is 1150 km and width 550 km. by surface area it is the fifth largest country in eu. the population of the country is 5.5 million resulting in the lowest population density in eu (15.8 inhabitants/km3). the whole country is inhabited, although most of the population is packed in the south. the climate of finland is influenced mainly by its latitude, but the warm waters of the gulf stream and the north atlantic drift current also play a role. due to finland's northern location, winter is the longest season. the southern portions of the country are snow-covered about three or four months of the year, and the northern regions for about seven months. long distances, low population density and the extreme climate give logistical challenges. it is estimated that these logistical costs can be as much as 10-20% of gdp in finland. the finnish red cross blood service (frc bs) has been the nationwide blood service provider in finland since 1948. frc bs collects annually about 200 000 whole blood units of which 50 % are collected in 10 fixed sites and 50 % in mobile sessions around the country. central activities (donor recruitment, medical support, production, testing, supply chain management, digital services and administration) are located in helsinki. management of transfusion is highly dependent on the logistical arrangements from blood donation sites to the central facilities and from the central inventory to the hospitals. the logistics is outsourced to three major partners all of whom have their roots in nationwide public transportation and logistics services. posti ltd is a state owned company having its roots in the national postal and telecom office. today it is the leading postal and logistics service company having the widest network coverage in finland. blood units collected at different fixed sites and mobile sessions are transported overnight by posti ltd to the frc bs central facilities by 8 am on the day following the blood donation. posti ltd is also used for the regular deliveries of blood products to the hospitals. the other important partner is matkahuolto ltd, which was founded in the 1930s to maintain bus stations and to serve as a common marketing company for the bus and coach services in finland. it maintains a nation-wide package delivery system based on the scheduled bus route network. matkahuolto ltd is used to transport donor testing samples from the donation sites to the central laboratory. by this arrangement it is possible to obtain most of the donor samples to the laboratory around midnight, which significantly speeds up the completion of laboratory results. the third logistics partner is jetpak finland ltd, which operates the air freight for the national flight company finnair. blood transfusion services can be managed centrally in a large sparsely populated country in a manner that is of high quality, safe and cost effective. however, the supply chain has to be planned carefully. background: elearning is a divisive topic. it is often criticised as an inferior form of education while simultaneously being promoted as a means to provide education to large numbers of people in a consistent, cost-effective manner. bloodsafe elearning australia (bea) is a government-funded blood transfusion education program that commenced in 2007 and provides courses in clinical transfusion practice and patient blood management (pbm) including: -clinical transfusion practice (4 courses) -pbm: general (7 courses) -pbm: medical (6 courses) -pbm: acute care and surgical (4 courses) -pbm: obstetrics and maternity (3 courses) -pbm: neonates and paediatrics (7 courses) aims: to determine the engagement, outcomes and impact of learning of bloodsafe elearning australia courses. methods: a retrospective analysis of user registrations, course completion records, course evaluation data and red cell usage in australia to determine learner demographics, and the impact on acquisition of knowledge and application to clinical practice. results: in the period from 1 july 2007 to 31 january 2019: -489,600 people registered as learners -1,072,299 courses were completed -these learners came from 182 countries, with 11,141 (2.3%) of them from outside of australia. analysis by profession shows that: -82.4% are nurses and/or midwives -11.2% are medical -6.4% are laboratory, anaesthetic technicians or other. analysis of user evaluation data (n = 2,885) from 1 april 2015 to 31 january 2018 shows that these courses have a positive impact, with 88.7% of respondents stating they gained additional knowledge, 65.2% able to make changes to clinical practice, and 88.2% reporting that these changes will improve patient safety and outcomes. analysis of international participants shows greater benefits with 93.5% gaining knowledge, 76.4% able to change their clinical practice and 91.9% believing this will improve patient outcomes. analysis of red cell usage in australia shows that since 2012 there has been a 21.1% reduction in red cells issued. this has been achieved through a number of pbm activities including development of guidelines, research and audits, education, waste reduction strategies, and promotional campaigns. bloodsafe elearning australia courses on pbm were released in 2012 and are one part of this pbm activity, and it is notable that these courses have the widest reach as they are undertaken by a large proportion of doctors, nurses and midwives in australia who are not directly involved with the blood sector. stakeholder feedback shows that the program provides credible, consistent education that is cost-effective, reduces duplication, is 'best-practice' elearning, is readily accessible, and allows institutions to focus on the development of practical transfusion skills. summary/conclusions: this analysis shows that elearning is a well-accepted, wellutilised form of education for healthcare workers to learn about clinical transfusion practice and patient blood management, and learners gain knowledge that can change their clinical practice and improve patient outcomes. it is also likely that these courses have contributed to better utilisation of a scarce, freely-donated resource. this approach has global reach and availability, and is a cost-effective model for improving transfusion practice in the developing world by providing education for millions of healthcare workers. 3d-s14-03 prospective platelet auditing: analysis of trainee compliance with guidelines pathology, columbia university, new york, united states background: apheresis platelets are a component product with high cost and limited supply. furthermore, there is a potential for severe transfusion reactions associated with this product such as transfusion related lung injury (trali), and sepsis due to bacterial contamination. therefore, transfusion guideline compliance is closely monitored by many centers. this quality assurance analysis describes our experience with prospective platelet auditing performed by physicians in pathology residency training. aims: this study aims to evaluate the ability of physicians in training to perform prospective auditing and compare policy compliance for different levels of experience. methods: this is a quality assurance analysis of a prospective platelet audit program for a 12-month period (january 2018-december 2018). the blood bank paged the on call physician any time an order was placed for a patient with a platelet count of > 50,000/ll, ≥2 doses of platelets with no interim repeat count, or an unknown platelet count. audit records created by physician trainees in their first post graduate year (pgy1) were compared to subsequent years (pgy > 1). information collected included the total number of doses requiring approval, number of products approved, training year for the approving physician, and transfusion indication. cost analyses assumed $500 for a dose of platelets. descriptive statistics and comparative analysis using a pearson's chi-square were used with a difference of p < 0.05 considered statistically significant. results: there were 1446 platelet doses requiring approval with 670 (46%) routed to the pgy1 group and 776 (54%) to the pgy > 1 group. there were 847 (59%) ordered doses that were in compliance with hospital transfusion policy and 599 (41%) that were not in compliance with hospital policy. of the 847 appropriately ordered doses, the pgy1 group declined release of 7 necessitating the clinical team to insist upon release without approval, and there were zero such instances in the pgy > 1 group. when paged by the blood bank, pgy1 physicians approved product release not in compliance with policy for 191/670 (29%) doses while pgy > 1 physicians approved not indicated products for 79/776 (10%) of doses (p < 0.01). products not indicated by hospital policy were held from release by pgy1 physicians for 113/670 (17%) doses and 216/776 (28%) doses by pgy > 1 physicians (p < 0.01). the ordered doses not in compliance with hospital policy had an estimated cost of $299,500. of this cost, there was a calculated $164,500 savings of products not released due to prospective auditing. there was an additional potential savings of $135,000 for products not indicated but released ($95,500 from the pgy1 and $39,500 from the pgy > 1 group). summary/conclusions: despite a higher number of requests being routed to the more senior pgy > 1 group, there were a disproportionately higher number of out of compliance platelet orders being released by the pgy1 group in addition to withholding needed products on several occasions. potential mitigation strategies for this could include a closer level of oversight for pgy1 physicians, and the potential monetary savings could justify a hiring a dedicated patient blood management team or quality assurance manager to monitor compliance and provide feedback to clinicians. 3d-s14-04 what can we learn from how adverse events are detected? norwegian directorate of health, oslo, norway background: the primary aim of reporting systems, such as haemovigilance systems, should be learning and improvement and to identify risk areas, not simply counting errors. to understand and learn from adverse events the description of how, where and why they occur, and how they are detected, is important. to support our understanding, we use a predetermined classification that is required for reporting to eu, supplemented by classification suggested by ihn, who and ourselves. in 2015 we started asking the blood establishments what steps they would take to prevent recurrence of the event, and we added a simple classification to tell how the adverse event had been detected. aims: this study aims to analyze how different types of adverse events reported to the haemovigilance system were detected, whether the current quality management systems used in norwegian blood establishments had effective barriers and whether new barriers should be considered. methods: adverse events reported to the norwegian haemovigilance system in 2017 and 2018 were analyzed with focus on how the adverse event had been detected. in all cases classification had been performed by the reporter of adverse events and were confirmed, or reclassified if necessary, by the haemovigilance team before analysis. for analysis based on classification we used powerbi (microsoft). results: a total of 188 adverse events were reported from norwegian blood establishments. all had been classified according to how the adverse event had been detected. twenty (10.6 %) adverse events were detected because of alarms or warnings from it-systems or equipment. routine checks by blood establishment staff detected 39 (20.7 %) events and formal internal or external reviews detected one event. seven (3.7 %) events were detected because the donor became ill shortly after donation, but the illness was not caused by the donation. sixty-four percent of events were detected in a way that did not fit our present classification and hence were classified as "other". twelve out of 16 wrong blood in tube were detected by an alarm from the it-system or routine check, as were six of 22 events related to blood ordering, two of seven errors in testing, six of 17 events where incorrect blood had been transfused, and eight of 64 events related to donor selection. in 80 reports human error was listed as the cause of the event and 27 of these were detected by alarms or routine checks. summary/conclusions: detection of adverse events by alarms or routine checks are highly efficient when the blood establishment has historic data to check against, as exemplified with wrong blood in tube or a patient require irradiated blood components. when no historical data exists or when the quality management systems do not require routine checks, events are usually detected by chance. further analysis is needed to see if and where the quality management systems should be improved. the wide variety of adverse events can make it difficult to select which area to prioritize in the improvement work. results: the hb measurements from the finger prick were on average 1.2 g/l (0.9%) higher than from the venous blood samples. the range of the difference was -21 -+22 g/l. these results were used in order to add novel information to determine the measuring uncertainty of hb measurement in frcbs. in 1.4 % (12/845) of the donors in this study the venous hemoglobin measurements were below the cut-off point of donor eligibility. in those measurements the difference of the finger prick and venous hemoglobin measurement was at most + 9 g/l. 92 % of the hemoglobin results from the finger prick were in the range ae10 g/l compared to the venous hemoglobin results. 63 % of the results from the finger prick were between ae5 g/l (the precision of the device) compared to venous hemoglobin results. in 13 cases the difference between finger prick and venous measurements was outside 2 standard deviations from the mean i.e. 2.5 % from the bottom (n = 9) or top (n = 4) of distribution. systematic errors were seen in some nurse's results both towards too low or too high hb result in the finger prick measurement and some nurses had random errors in both directions. the batch of cuvettes, donors' age, gender or the time of sampling were not detected to have an impact on the difference between finger prick and venous hb measurements in this study. summary/conclusions: the results of the poc measurements compared to the cell counter were in agreement with published data and with manufacturers' information on the device. the practical skill test is a workable way to develop competence and operations to measure the hemoglobin from the finger prick. it offers an opportunity to give personal feedback to nurses concerning their personal performance in the use of the current hb measurement technic. it also provided data on the accuracy of the poc method in the everyday donor selection process. background: whole blood donation has frequently been related to iron deficiency. a blood donor loses per donation about 8% (men) to 81% (menstruating women) of iron stores. to replenish the iron lost by blood donation in a donation interval of 56 days, a donor needs to absorb 4.5 mg iron per day. this amount exceeds the reported maximal amount of absorbed iron of 3-4 mg/day, eventually leading to iron deficiency, with consequences such as donor deferral and possibly iron deficiency-related symptoms (decreased physical endurance, fatigue, pica, restless legs syndrome, and cognitive functions). since hb levels do not reflect donors' true iron status, measuring ferritin is a better way to detect low iron stores in whole blood donors. studies from usa and denmark showed that on the introduction of ferritin measurement with either extension of donation intervals or iron supplementation in case of low iron stores, deferral percentages for low hb declined in both male and female donors. aims: to gain more insight in iron status of whole blood donors during their donor career, how this affects donor health and which measures may prevent low iron stores in donors. methods: in the netherlands, sanquin blood bank is currently implementing a policy with ferritin-guided donation intervals. in brief, ferritin levels are measured in all new donors and in repeat donors every 5th donation or in case of an hb below the deferral threshold. donation intervals are extended if ferritin levels are < 15 lg/ l, or ≥ 15 and ≤ 30 lg/l (for 12 and 6 months respectively). we anticipate that routine ferritin measurement will ultimately result in a lower prevalence of iron deficiency, less hb deferrals and improved donor retention. this will be further evaluated in a stepped wedge cluster-randomized trial 'find'em', which may also identify subgroups of donors prone to develop (symptoms of) iron deficiency. in addition, implementing ferritin screening may lead to a decreased donor availability. for this purpose, we modeled the impact of the implementation of our ferritin deferral policy on donor availability over time, which provides insight for both the expected size of the impact of the ferritin deferral policy and the time and rate at which this impact is expected to occur. this allows the blood bank to timely plan actions to counterbalance possible donor shortage and ensure an adequate blood supply. lastly, iron supplementation can be an alternative measure instead of donation deferral. as the used and recommended dosage of iron supplementation varies widely across blood services, sanquin is planning to start a new study in whole blood donors to gain evidence on the dosage and frequency of iron supplementation and its effect on ferritin and hemoglobin levels and donor health. results: the before-mentioned studies are ongoing and results will be expected from 2020 onwards. summary/conclusions: iron deficiency is a frequent side effect of whole blood donation. to prevent iron deficiency and its consequences, like donation deferral and health issues, more evidence-based insight in iron management of whole blood donors is being generated. 3d-s15-02 superdonors -genetic risk profile and risk of low hemoglobin deferral background: no reliable method exists for stratifying new blood donors into those who can maintain sufficient hemoglobin (hb) levels and those who will be deferred because of a low hb (<7.8 mmol/l [<12.57 g/dl] for women and < 8.4 mmol/l [<13.54 g/dl] for men). polygenic risk scores (prss) have shown great promise in predicting complex disease risk. prss could also prove useful for identification of donors genetically predisposed to low hb levels, and, thus, to an increased risk of deferral. aims: the objective of the study was to evaluate the association between prs (modelled to predict hb level as a quantitative trait) and risk of deferral as a binary outcome. methods: the danish blood donor study (dbds) is an ongoing nationwide blood donor cohort since 2010 with more than 110,000 participants. extensive genotyping has been performed on approximately 72,000 dbds participants using the infinium global screening array (illumina â ) and extended by use of imputing based on the pan-scandinavian reference genome. based on hb and genetic data on more than 150,000 icelandic individuals (an independent discovery cohort), we constructed 9 different weighted prss for individuals from dbds. information on the donors' whole blood donations following inclusion into dbds unto end of 2017 was obtained from a nationwide donation database, scandat. the best predictor of hb among the nine prss was chosen and used in all subsequent analyses. we performed multilevel mixed-effects linear regression analysis with hb as outcome, and prs as factorized explanatory variable with cutoffs at 5, 10, 25, 40, 60, 75, 90 , and 95 th percentiles, respectively. moreover, the models had a two-level clustering on donor id and donation site and an id-specific random intercept; and further adjusted for: sex(binary), age(continuous), year of donation(factorized), and time since last donation (continuous). lastly, risk of deferral was evaluated in random effects logit models with similar covariables and clustering structure. results: mean number of donations per donor after dbds inclusion was 6.9 donations. generally, we observed a statistically significant positive association between prs(hb) and current hb levels. compared with donors in the 40-60 prs percentile group, donors below the 5 th percentile had lower (-0.23 hb mmol/l (95% ci: -0.25; -0.21)) and donors above the 95 th percentile higher (+0.19 hb mmol/l (95% ci: 0.18; 0.21) hb levels. in the random effects logit models we observed a marked increase in deferral risk with decreasing prs percentile strata. with the 40-60 prs percentile stratum as reference, donors below the 5 th percentile and donors above the 95 th percentile had odds ratios of deferral of or = 2.58 (95% ci: 2.27; 2.93) and or = 0.46 (95% ci: 0.39; 0.54), respectively. summary/conclusions: we found a statistically significant positive association between prs(hb) and hb levels and a markedly increased risk of deferral with decreasing prs(hb). from a scientific point of view, it is unsurprising that a genetic score for hb from an independent cohort is associated with hb in another cohort. however, from a practical perspective, prss may be the first step in a personalized donation approach to donors and their risk of deferral. background: individually calibrated inter-donation intervals for repeat blood donors have the potential to minimize the risk of iron related adverse outcomes (e.g., hemoglobin deferral or collecting a donation from a donor with low or absent iron stores) without unduly impacting the donated blood supply. machine learning has shown promise for personalized clinical risk assessment. aims: our aim is to use machine learning to develop donor-specific, personalized inter-donation intervals that minimize the risk of adverse outcomes while maintaining or improving the adequacy of the donated blood supply. methods: using a public use dataset from the reds-ii donor iron status evaluation (rise) study (cable, transfusion, 2012) we defined donor profiles with physiological measures including hemoglobin, ferritin and soluble transferrin receptor along with questionnaire responses regarding diet, reproductive health indicators, and demographics. we used these profiles (58 features, 3,162 donations from 1,025 repeat donors) and the time until the next donation attempt to predict iron-related outcomes of the next donation attempt. possible outcomes were no adverse outcome, hemoglobin deferral, low-iron donation (ferritin < 20 ng/ml for women and < 30 ng/ml for men), or absent-iron donation (ferritin < 12 ng/ml for men and women). we trained multiple machine learning models on 2,543 of the donations and selected the model with the best performance (lowest cross-entropy loss in cross validation). we assessed the best model's performance on a hold-out test set of 620 donations, which were not used to train or select the model. we then used our model to generate risk estimates for these 620 test donors as a function of days since their last donation, which varied from 56 days to 250 days. to show individual variation, we generated graphical representations of individual donors' risk over time. results: ferritin, log ferritin, body iron, and time since last donation were most useful for predicting iron-related adverse outcomes at the next donation attempt. the estimated risk of adverse outcomes at the next donation attempt varied considerably across donors. as expected, the risk of adverse outcomes 250 days after the last donation was lower than the risk 56 days after the last donation for most donors (risk of hemoglobin deferral decreased for 84% of donors; risk for low-iron donation decreased for 61%; and risk for absent-iron donation decreased for 94%). summary/conclusions: the risk of iron-related adverse outcomes as a function of time since last donation varies considerably between donors. machine learning models trained on relevant donor profiles can effectively estimate how an individual's risk will change over time. individual risk estimates could allow blood centers to protect highrisk repeat donors while continuing to allow more frequent collections from low-risk donors. further study is needed to ensure this approach works well for donor classes that are not well-represented by the rise dataset, to assess risk prediction outside of the physiological measures collected in the rise study, and to determine the viability of assigning an optimal inter-donation interval to a first-time donor using this approach. background: iron depletion is common among repeat blood donors, who contribute a large proportion of the blood supply in many countries. exogenous iron from multivitamins with iron or iron-only supplements helps prevent donation-induced iron depletion, but whether dietary iron protects against iron depletion in repeat donors has not been rigorously evaluated. available data from the reds-ii rise study in the us (cable, transfusion, 2011) and from the danish blood donor study (rigas, transfusion, 2014) suggest minor impact of dietary iron consumption on blood donor iron status in multivariable regression models. both studies, however, analyzed food items singly, such as beef or fish, rather than in aggregate, so precision was limited. aims: to evaluate whether a composite measure of dietary heme iron consumption, weighted for frequency and iron content, was associated with incident iron depletion among repeat blood donors. methods: a re-analysis of the rise cohort was undertaken to test the hypothesis that reported levels of animal protein consumption was associated with lower risk for incident iron depletion among repeat blood donors. the six blood centers participating in rise enrolled first-time and frequent donors for 15-24 month follow-up of donation frequency and iron status. a brief checklist of 8 food categories was administered at baseline to assess frequency of consumption of several categories of animal protein that are rich in heme iron, the biochemical form of iron most readily absorbed. an iron composite score (ics) weighted for frequency and heme iron content was derived and subjects were grouped into tertiles (thirds) of ics. iron status was assayed at enrollment and study completion and at roughly one-third of donation visits in between. modified poisson regression with generalized estimating equations was used to generate risk ratios controlling for donation frequency and other covariates. results: of 2425 enrolled donors, 1406 were iron replete at baseline and completed the food checklist. the median value of the ics for each tertile (lowest to highest) was 7. 2, 13.1, and 22 .0 mg of heme iron weekly. these values are equivalent to approximately 3, 6, and 9 servings of beef per week, or alternately twice as many servings of chicken or pork. across 2236 follow-up visits with iron outcomes assayed, almost 33% of donor visits were associated with intermediate iron depletion (serum ferritin < 26 ng/ml) and 8.5% with complete depletion of iron stores, representing serum ferritin < 12 ng/ml. after controlling for demographic factors and donation frequency, the lowest tertile of ics was associated with a greater than 2fold higher risk for complete iron depletion during all follow-up visits (rr 2.40, 95% ci 1.51, 3.81, compared to the highest tertile). summary/conclusions: in this longitudinal evaluation of dietary iron and iron status, blood donors with low intake of heme iron had an elevated risk for developing advanced iron depletion. these results suggest that blood centers should continue to recommend iron-rich diets to repeat blood donors. background: blood donors lose approximately 250 mg of iron with every blood donation. as a result, frequent blood donors are at risk of iron deficiency and low hemoglobin (hb) levels, which may affect their health and eligibility to donate. lifestyle behaviors such as dietary iron intake and physical activity, may influence iron stores and thereby hb levels. gaining insight into associations between lifestyle behaviors and hb levels is valuable for blood supply organizations, as lifestyle behaviors can potentially be considered to prevent hb deferrals. examining the mediating role of ferritin, a measure reflecting iron stores, in these associations will help to gain insight into whether iron stores could be the limiting or enabling factor that links lifestyle behaviors to hb recovery after donation. aims: to investigate associations between lifestyle behaviors (dietary heme and non-heme iron intake and physical activity) and hb levels, and whether ferritin mediates these associations. methods: donor insight-iii (dis-iii) is a dutch cohort study of blood and plasma donors and included 2,552 donors. participants who were pregnant, had hemochromatosis, used iron supplements/medication, got a hysterectomy or bilateral oophorectomy were excluded (n = 292). hb levels were measured in edta whole blood samples using a hematology analyzer (xt-2000, sysmex, japan) and ferritin was measured in plasma from lithium heparin tubes (architect ci8200, abbott laboratories, u.s.a.). dietary heme and non-heme iron intake (grams/day) were assessed using a food frequency questionnaire adapted to measure iron intake. moderate-tovigorous physical activity (mvpa, minutes/day) was assessed using the international physical activity questionnaire (ipaq)-short form. results: in total, 2,260 (1,186 female) participants were included. donors with higher intakes of heme iron had significantly higher hb levels (regression coefficient (b) (95% confidence interval (95% ci)) in men and women respectively: 0.147 (0.069 to 0.225) and 0.094 (0.013 to 0.175) mmol/l), independent of age, smoking, menstruation, number of donations in previous two years, donation interval, sedentary behavior, the other lifestyle variable (i.e. (non-)heme iron intake or mvpa), and initial hb level. non-heme iron intake was negatively associated with hb levels (-0.015 (-0.026 to -0.004) and -0.018 (-0.032 to -0.005) mmol/l for men and women respectively). ferritin mediated associations between dietary iron intake and hb levels (indirect effect in men and women respectively: 0.073 (0.046 to 0.107) and 0.065 (0.031 to 0.100) lg/l for heme and -0.003 (-0.008 to 0.000) and -0.007 (-0.013 to -0.002) for non-heme). more mvpa was negatively associated with hb levels in men only (-0.004 (-0.008 to -0.001)), which was not mediated by ferritin. summary/conclusions: in conclusion, higher heme and lower non-heme iron consumption are associated with higher hb levels in donors via higher ferritin levels, indicating that donors with high heme iron consumption may be more capable of maintaining iron stores to recover hb levels after blood donation. more mvpa was associated with lower hb levels, although effect sizes were small, independent of ferritin. taking a donor's lifestyle behaviors into account may be useful in preventing low hb levels in blood donors. immune thrombocytopenia (itp) is still diagnosed by exclusion of other causes for thrombocytopenia. sensitive and specific detection of platelet autoantibodies may support the clinical diagnosis and prevent misdiagnosis of itp. for example, the direct monoclonal antibody immobilization of platelet antigens (maipa) assay, performed with in vivo sensitized patient platelets, offers platelet glycoprotein specific autoantibody detection with high accuracy. a drawback is that low platelet counts demand a large blood sample to have sufficient patient platelets available for analysis. circulating platelet autoantibodies are more difficult to detect by maipa; and may demand more sensitive detection platforms, such as those using surface plasmon resonance. in general, the presence of anti-gpiib/iiia, anti-gpib/ix and anti-gpv platelet autoantibodies is investigated. all these antibody specificities have been found in patients with itp. in itp, platelet autoantibody-mediated destruction via the spleen has been proposed; but also other mechanisms leading to low platelet counts in itp may play a role. inhibition of megakaryocytopoiesis by autoantibodies or by t cells has been suggested. in mice, gpib-directed antibodies induce loss of platelet-sugar epitopes, inducing hepatocyte-medicated platelet destruction. platelet autoantibodies can cause complement activation, which may contribute to platelet autoantibodymediated destruction. interestingly, we recently found that lack of detectable platelet autoantibodies is correlated with non-responsiveness to rituximab (cd20 moab) treatment in itp patients. in children with newly diagnosed and often transient itp, platelet autoantibodies of igg class or not often found, but of igm class are present for short duration. in conclusion, testing for platelet autoantibody characteristics and their pathologic effect may be helpful in establishing the diagnosis of itp and in choosing the best individualized therapy for itp patients. 4a-s16-02 thrombopoietin receptor agonist (tpo-ra) treatment raises platelet counts and induces immunomodulation in immune thrombocytopenia (itp) jw semple 1 , r aslam 2 , e speck 2 , j rebetz 1 and r kapur 1 1 lund university, lund, sweden 2 st. michael's hospital, toronto, canada background: itp is an autoimmune bleeding disorder in which autoantibodies and/ or autoreactive t cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. several therapeutic options e.g. corticosteroids, intravenous immunoglobulins (ivig), rituximab and splenectomy are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. for the last 10 years, tpo-ra e.g. romiplostim and eltrombopag have made a substantial contribution to the treatment of itp patient's refractory to first-line treatments. of interest, approximately 30% of patients that are tapered from tpo-ra therapy show a sustained response (e.g. a stable higher platelet count than before treatment). the mechanism of how tpo-ra induce these sustained responses is unknown. aims: to analyze the efficacy and immunomodulatory properties of a murine tpo-ra (amp4, amgen) in a well-established murine model of itp that demonstrates both antibody-and t cell-mediated thrombocytopenia (chow l et al., blood 2010) . methods: platelet glycoprotein (gp) iiia (cd61) knockout (ko) mice were immunized with cd61 + platelets and itp was initiated by the transfer of their splenocytes into mice with severe combined immunodeficiency (scid). the scid mice were treated with either placebo or tpo-ra weekly and platelet counts and serum anti-platelet antibodies were measured weekly. results: in an initial pilot dose escalation study, control na€ ıve scid mice treated with a single subcutaneous bolus of different concentrations of murine tpo-ra (1, 10 and 20 ug/kg) had significantly higher platelet counts by 72 h post infusion. in addition, compared with untreated mice, bone marrow histology revealed significantly increased numbers of megakaryocytes. maximal platelet count increases were observed with the highest tpo-ra dose and this dose was chosen to treat scid mice suffering from itp. when scid mice were treated with weekly injections of tpo-ra, platelet counts began to increase after 2 weeks and were fully rescued to control levels after 3 weeks post splenocyte transfer. of interest, compared with non-treated itp mice, serum igg anti-platelet antibody production in the tpo-treated mice was significantly reduced starting from two weeks post splenocyte infusion. summary/conclusions: these results suggest that murine tpo-ra is not only an efficacious therapy for murine itp but also induces immunomodulation indicative of immunosuppression. thus, this model may be able to elucidate the mechanism of how tpo-ra's induced immunosuppression in patients with itp. background: desialylation, the loss of sialic acid content on platelets (plts) glycoproteins (gps) was recently identified to contribute in immune thrombocytopenia (itp). however, the potential impact of autoantibodies (aabs) on megakaryocyte sialylation remains unclear. aims: to investigate the effect of itp aabs on plts and megakaryocytes (mks) sialylation and the subsequent impact on plt survival. methods: aabs from well-characterised itp patients induced gp-modifications were tested using a lectin binding assay. after incubation of mks or plts with itp or control sera, glycan changes were analysed by flow cytometry (fc). to investigate the impact of desialylation on plts life-span, the nod/scid mouse model was used. results: 112 itp sera were investigated in this study. 35 (31%) sera induced a significant increase in rca signal on plt surface compared to control sera from healthy donors (rca-mean fold increase (rca-fi): 3.23, range: 1.76-13.61, p = 0.0001). in addition, 23 (21%) sera caused higher ecl binding to test plts (ecl-fi: 2.31, range: 1.54-5.7, p = 0.0001). injection of desialylating aabs resulted in accelerated clearance of human plts from the circulation of the nod/scid mice which was significantly reduced by a specific neuraminidase inhibitor that prevents background: autoimmune hemolytic anemia (aiha) is a rare autoimmune disease characterised by hemolysis associated with the presence of immunoglobulins (igg, igm, or iga) and/or components of complement system on red blood cells (rbcs), which is usually demonstrated by a positive direct antiglobulin test (dat). depending on the presence of an underlying disorder, aiha can be subdivided into primary and secondary and, by the temperature at which autoantibodies bind optimally to rbcs, into warm antibody aiha (waiha), mixed aiha (including both warm igg and cold igm antibodies), cold agglutinin disease (cad), paroxysmal cold hemoglobinuria (pch) and dat negative aiha. a frequent finding in immunohematology is the presence autoantibodies on rbcs without clinical symptoms of hemolysis that may later develop. aims: the aim of this study was to analyse serologic findings and transfusion support in patients with aiha and also to analyse dat positive patients without clinical symptoms. methods: we included data for all adult patients with aiha and dat positive patients without clinical symptoms diagnosed and/or treated at the university hospital centre (uhc) zagreb, croatia in the period between 2014 and 2018. the diagnosis of aiha was defined by anemia with features of hemolysis (elevated bilirubin and/ or elevated lactate dehydrogenase and/or low haptoglobin level) and a positive dat. results: the data from 64 patients (52% women) meeting the inclusion criteria was analysed. the mean age at the time of aiha was 63 years (range 22-89 years). the mean hg level at diagnosis was 68.60 g/l. dat results were positive mostly with igg+c3d (59%) or igg (31%). most patients had warm aiha (70%). other types of aiha diagnosed were mixed aiha (15%), cad (11%), pch (1.5%) and dat negative aiha (1.5%). in 6 cases alloantibodies were detected with autoantibodies in the patient's plasma. patients were treated with corticosteroids as 1st line therapy and some with intravenous immunoglobulins (ivig). in severe or refractory patients rituximab and/or splenectomy was applied. a total of 80% of patients were transfused at a mean hemoglobin level of 67.88 g/l. during this period we detected 136 dat positive patients without clinical symptoms. summary/conclusions: most patients from our study were diagnosed with warm type of aiha, followed by mixed type aiha and cad. on the other hand, pch and dat negative aiha were very rare, which is in concordance with relevant literature. most patients were transfused despite therapy used, which is not desirable in patients with aiha and should be better controlled, especially in moderate cases of anemias, where this is rarely necessary. a significant number of patients that were dat positive without clinical symptoms may later develop aiha and should be closely monitored. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to diagnosis, investigation and management of patients with aiha in english nhs trusts. methods: a survey of diagnostic and management practice was designed, piloted and disseminated to clinical transfusion leads in all english acute nhs trusts from november 2017 to march 2018. completion was by a consultant haematologist treating patients with aiha but a response that represented a departmental consensus was encouraged. results: responses represented 42% (58/137) of english acute trusts. median number of adults with aiha diagnosed annually was 4-6. in the preceding 5 years, 31% (18/58) recalled at least one patient who had died due to aiha. although 7% (4/57) undertook a bone marrow biopsy in all patients, 93% required additional features, mainly: neoplasia, age over 60 or being treatment-refractory. for patients with suspected drug-induced immune haemolysis, 59% (34/58) would not organise confirmatory tests, either because it was considered unnecessary (29/34), or because clinicians were unsure how to access tests (5/34). when determining aiha subtype, 29% (17/58) indicated there were no circumstances in which they would undertake cold antibody testing (antibody titre and/or thermal amplitude), with 12 considering this unnecessary and 5 unsure how to access tests. in 4 clinical scenarios of patients with aiha and dat positive to c3d ae igg ae cold associated symptoms, up to 87% (47/54) of respondents would not test for cold antibodies. for first line treatment of primary warm aiha, mean duration of prednisolone 1 mg/kg given before judging the patient refractory and reducing the dose was 3.5 weeks (sd 1.70, range 1-19 weeks). second line treatment of choice was rituximab for 82% (45/55) of respondents and splenectomy for 5%. intravenous immunoglobulin and splenectomy were the most cited rescue therapies. for primary cold haemagglutinin disease (chad), first line treatment was rituximab-based for 88% (49/56) but single agent steroid for 9%. we also explored the potential for future audit and research. 64% (37/58) of respondents were able to identify patients with aiha who previously required transfusion. 96% (55/57) of respondents would consider supporting a registry of patients with aiha requiring transfusion. the key questions that respondents thought a registry should address were: morbidity and mortality, treatment response, and differences in the diagnosis and treatment of aiha subtypes. there was uncertainty over access to cold and drug-induced antibody tests and clinicians do not always conduct bshrecommended cold antibody tests for aiha with c3d positive dat. initial treatment of primary warm aiha and chad broadly matched bsh guidelines although 44% (25/57) would continue prednisolone at 1 mg/kg beyond the recommended 21 days before starting a taper, with greater toxicity risk. summary/conclusions: the findings support the need for a range of research initiatives, including creation of an aiha registry. preoperative anemia is common and is associated with adverse outcomes in the peri-operative period. preoperative anemia also increases the risk of allogeneic blood transfusions, which may lead to increased perioperative mortality, increased hospital length of stay and infections. diagnosis and treatment of anemia is one of the tenets of patient blood management (pbm), along with reduction in unnecessary transfusions and diagnostic phlebotomy, as well as use of hemostatic agents to reduce bleeding among many others. effective pbm is multi-disciplinary, multi-modal, timely, individualized and patient-centered. early referral to pbm and multi-modal pbm interventions are associated with greater improvement in pre-operative hemoglobin. pbm has been shown to reduce transfusions and cost, while system-wide, multi-modal programs may also be associated with improvement in mortality. using examples from our local research and practice, i will discuss three aspects of pbm. iron and erythropoiesis stimulating agents (esa) are effective, safe and used extensively in management of pre-operative anemia. previous studies have questioned whether esa leads to increased risk of thrombosis, however, recent systematic reviews do not support these concerns. another pbm approach is to reduce bleeding during surgery by using hemostatic agents such as tranexamic acid (txa). txa reduces transfusion requirements in knee and hip arthroplasty, and is safe, widely available and relatively cheap. txa is effective in both anemic and non-anemic patients, making it an attractive universal pbm strategy. finally, recommendations and evidence-based guidelines on pbm exist, including the most recent international guidelines developed by the pbm international consensus conference. however, knowledge translation in pbm has been a problem and a number of barriers to its implementation have been identified. these include perceived or actual lack of expertise, time, and resources, as well as lack of physician and patient engagement. one way to address patient engagement is education through character driven animation and we are currently trying this approach. 4a-s17-02 low vs . high hemoglobin trigger for transfusion in vascular surgery (tv): a randomized clinical feasibility trial (the tv trial) background: current guidelines advocate to limit red-cell transfusion during surgery, but the feasibility and safety of such strategy remains unclear as the majority of evidence is based on postoperative stable patients. aims: we assessed the effects of a protocol aiming to restrict red-cell transfusion during elective vascular surgery. methods: fifty-eight patients scheduled for lower limb-bypass or open surgery of abdominal aortic aneurysm were randomized to a low-trigger (hemoglobin < 8.0 g/ dl, 5 mmol/l) vs. high-trigger (hemoglobin < 9.7 g/dl, 6 mmol/l) for red-cell transfusion throughout hospitalization. intraoperative change in cerebral-and muscle tissue oxygenation was assessed by near-infrared spectroscopy. we used a nationwide registry to collect data on death and major cardiovascular events, which encompassed (1) severe adverse transfusion reaction, (2) acute myocardial infarction, (3) stroke, (4) new-onset renal replacement therapy, (5) vascular reoperation, and (6) amputation of the lower limb. results: the primary outcome, mean hemoglobin within 15 days of surgery, was significantly lower in the low-trigger group: 9.46 g/dl vs. 10.33 g/dl in the hightrigger group (mean difference 0.87 g/dl; p = 0.022, longitudinal analysis) as were units of red-cells transfused ( background: controlled non-hemato-oncological studies have consistently demonstrated a single-unit red blood cell (rbc) transfusion policy as well as a stringent hemoglobin (hb) rbc transfusion threshold to be safe and reduce blood product utilization. yet, it is unclear whether these conclusions also apply to the hemato-oncological patient population. aims: to quantify reduction of rbc blood product utilization by the introduction of a restrictive single-unit hb-triggered rbc transfusion policy among the inpatient hemato-oncological population. methods: under the liberal transfusion protocol, applied up till november 1, 2017, standard double-unit rbc transfusion was indicated with a hb threshold ≤ 8.1 g/dl and/or anemia-related symptoms. following this date, the restrictive transfusion protocol was introduced involving a lowering of threshold to 7.3 g/dl and single-unit transfusion. for patients with an asa-score of ii-iii and iv, a hb threshold of respectively ≤ 8.1 g/dl and ≤ 9.7 g/dl applied. we evaluated rbc blood product utilization over a 6 month period starting december 1, 2016 (liberal protocol) and december 1, 2017 (restrictive protocol) in all hemato-oncological patients admitted for chemotherapeutic treatment including hematopoietic stem cell transplantation (hsct) with an expected duration of neutropenia of ≥ 7 days. analysis of categorical and continuous data was performed using the chi-square and mann-whitney test, respectively. results: during both observational periods, 137 patients were admitted who in total received 164 therapy cycles, including 56 acute myeloid leukemia (aml) induction cycles and 69 autologous hscts. distribution of indications of admittance, median age, duration of hospitalization and duration of neutropenia did not differ between both periods. during the restrictive period, in 303/402 (75.4%) of transfusions the assigned hb trigger was adhered to. the percentage of single-unit transfusion episodes increased from 29/112 (29.0%) to 81/111(73.0%) with the introduction of the restrictive protocol. overall, rbc blood product utilization per admittance did not reduce under the restrictive protocol (cumulative number of transfused rbc units 4.0 (interquartile range (iqr) 2.0-8.0) during the liberal versus 2.5 (iqr 0.0-9.0) during the restrictive period (p = 0.36)). however, rbc blood product utilization per neutropenic day demonstrated a trend towards reduction: 0.25 (iqr 0.11-0.33) versus 0.15 (iqr 0.00-0.32) units per day during the liberal versus restrictive period, respectively (p = 0.06). this reduction was mainly attributed to autologous hscts during which rbc blood product utilization decreased from 2.0 (iqr 0.0-2.0) to 0.0 (iqr 0.0-1.5) units (p = 0.06), corresponding to a reduction from 0.13 (iqr 0.00-0.21) to 0.0 (iqr 0.0-0.13) (p = 0.01) units per neutropenic day. moreover, 14/38 (36.8%) patients during the liberal versus 21/31 (67.7%) during the restrictive period did not require rbc transfusion during admittance. consequently, stringent hb thresholds as compared to single-unit transfusions seem to more strongly impact rbc blood product utilization. summary/conclusions: a hb-triggered single-unit transfusion policy results in a strong reduction of rbc blood product utilization in the setting of autologous hsct. no utilization reduction was observed among other hemato-oncological inpatient populations receiving intensive chemotherapy. further improvement of protocol adherence rates could potentially increase the benefit of this blood saving strategy. 4a-s17-04 assessment of hb content of packed red cells (prbc): is it time to label each unit with hb content? r jain 1 , n marwaha 2 and s sachdev 2 1 transfusion medicine, aiims, new delhi 2 transfusion medicine, pgimer, chandigarh, india background: in the current era of evidence based medicine and individualized care of patients, rbc transfusion continues to be administered on the basis of conventional wisdom and the notion of an average benefit per unit. the existing blood transfusion practice based on the "number of units transfused" ignores the fact that the total hb varies markedly among the individual rbc units. aims: the present study was aimed at estimating the hb content in packed red cell unit prepared by three different protocols from 350 ml and 450 ml whole blood collection in three types of blood donors: replacement blood donor (rd), first time voluntary donor (ftvd) and regular voluntary blood donor (rtvd). methods: a total of 900 prospective blood donors were included in this study. three hundred whole blood collections were performed in each of the three groups of donors (rd, ftvd, and rtvd). within each group 100 collections were done in double 350 ml, triple 450 ml and quadruple 450 ml blood bags respectively. a pre-donation venous sample was drawn from sample collection pouch for analysis in hematology analyzer as reference method for hb concentration of donor. the hb content of packed red cell units were estimated after collection of representative sample from the blood unit. volume of prbc unit was estimated by the formula of weight of blood in prbc divided by specific gravity. the hb content in unit was estimated by the formula: hb content in unit = hb value of the prbc unit (g/dl) 9 volume of prbc unit (dl). results: in this study the hb concentration (g/dl) was comparable among three types of blood donors except that rtvd had lower hb values when compared to rd (p = 0.007). hemoglobin concentration of prbc ranged from 14.2-29.6 g/dl; mean hb was 21.02 ae 2.90 g/dl. net hb content of prbc bag was lower in prbc prepared from rd as compared to ftvd (p = 0.0001) and rtvd (p = 0.008). the hb content of prbc units prepared from 450 ml collection ranged from 35.19-87.36 g and from 350 ml collection ranged from 30.77-65.78 g. we observed a wide range of net hb content in the prbc units and the correlation coefficient showed the strongest association of net hb content of the prbc unit with the overall volume of prbc (r = 0.730, p = 0.0001).higher volume prbcs have more hb content. volume of prbc bags in the study ranged from 155 ml to 370 ml (including both 350 and 450 ml collections). summary/conclusions: the present study shows that labelling hb content of the prbc unit help in better inventory management for patients. the hb content may help in decision making for release of units for paediatric/low weight versus adults/ higher weight patients. adopting a policy of optimizing dosage of rbc transfusion could have the potential to significantly improve rbc utilization and decrease patient exposure to allogenic blood. this would help further in the clinical transfusion practices based on evidence. 4a-s17-05 nv more, p desai, s rajadhyaksha, a navkudkar and n deshpande transfusion medicine, tata memorial centre, homi bhabha national institute, mumbai, india background: red blood cell (rbc) transfusion is an important medical therapy benefiting the patient in a wide spectrum of clinical setting. critically ill intensive care unit patients in particular, as well as medical and hemato-oncology patients, are among the largest group of the user of rbc. periodic review of blood components usage is essential to assess the blood utilization pattern in any hospital or health care set up. our institute is a 639 bedded tertiary care oncology centre with approximately 18,000 to 20,000 rbc transfusions annually. these transfusions are required in various stages of patient treatment like chemotherapy, radiotherapy, surgical and palliative care and there are established guidelines by the institute to be followed by clinicians. aims: to study clinical practices of rbc transfusions based on indications and to evaluate appropriateness of rbc utilization practices at the institute. methods: this was a prospective observational study, started after approval from institutional ethics committee. total of 4413 rbc transfusion events in adult patients over a period of four months were included and analyzed as per institutional guidelines for their appropriateness. details of transfusion events in form of pre transfusion hemoglobin, indication of transfusion, type of request, number of unit requested and issued, time of issue, site of transfusion and adverse reactions, etc were obtained from department of transfusion medicine records. overall statistical analysis was descriptive using spss software. chi-square test in cross tables was applied to see the relationship between different variables and considered significant if p-value was < 0.05. results: total 4413 rbc transfusion events for 2012 patients were analyzed. there were 1877 (43%) events in 628 patients of medical oncology and 2536 (57%) in 1384 patients of surgical oncology. maximum transfusions were received by patients in age group of 41 to 60 years (47%). total 83% of transfusion events were appropriate as per institutional guidelines. all transfusions administered in operation theatre were found to be appropriate with p value < 0.05. inappropriateness was more 53%(396/735) and significant in daycare setup (p < 0.05). anemia was the most common indication of rbc transfusion observed in 90% of events (3971/4413). total 62% rbc transfusions were given as planned and 38% as urgent transfusions. most common adverse transfusion event observed was allergic reaction in 0.3% of total transfusion reactions. summary/conclusions: clinical practice of rbc transfusions in our hospital was largely found to be appropriate and rational with adherence to institutional guidelines. blood utilization audits should be conducted regularly by transfusion services and results should be discussed with clinician for ensuring judicious use of the scarce resource. the concept of transfusion safety officer (tso) can be introduced for better coordination between clinicians and blood transfusion services to improve practices. 4a-s18-01 paul-ehrlich-institut, langen, germany on a global scale, blood services are quite diverse in regard to aspects like organisational structure, regulatory background, donor populations, donation rates or pathogen epidemiology. the world health organization (who) recognizes blood and blood products as essential medicines and provides guidance to member states for various aspects like blood regulation, best practices in blood collection and transfusion, or screening parameters. more recently a who guideline on residual risk of transfusion associated infections has been established which may facilitate decision-making for the most appropriate screening algorithms. it emphasizes the need for regional evaluation of screening assays and regulatory control of blood-associated ivds. background: babesia, a protozoan parasite that infects red blood cells, is a leading infectious cause of mortality in u.s. transfusion recipients. babesia is usually transmitted through the bite of an infected tick but may be transfusion transmitted (tt) or transmitted from mother to child during pregnancy. babesiosis is a world-wide disease; the ticks that carry babesia have a global distribution. babesiosis has been reported throughout europe and in canada, korea, india, and japan. prospective testing of blood donations in endemic areas of the u.s. revealed 0.38% of donors were positive for babesia dna or antibodies (moritz, nejm, 2016) aims: -to report results of ongoing babesia clinical trial -to explain significance of babesia as a tt infection methods: in cobas â babesia for use on the cobas â 6800/8800 systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (wb) donor samples the 4 babesia species that cause human disease: b. microti, b. duncani, b. divergens, and b. venatorum. testing began in october 2017 under a u.s. fda-approved investigational new drug application. wb was collected into a proprietary medium that lysed red blood cells and stabilized babesia rna and dna. donations were collected in states with high, low, and no babesia endemicity and screened as individual blood donor (idt) samples. reactive index donations were retested in simulated minipools of 6 (mp6), plus 3 idt replicates with cobas â babesia. reactive index donations were also tested with 2 validated alternate babesia nat and for b. microti igm and igg antibodies. donors with reactive results were invited to enroll in a follow-up study to test for additional evidence of infection. results: to date, 256,802 valid donations have been screened with cobas â babesia, and 15 (0.006%) were reactive. 13 of 15 (87%) initially-reactive donations were confirmed to be positive for babesia with a positive alternate nat or serology result. 1 of 13 (8%) confirmed-positive donations was collected in a state with low babesia endemicity (pennsylvania), and 1 (8%) was collected in a state where babesia is not considered endemic (iowa). 11 of 13 (85%) confirmed positive donations were collected in states with high endemicity. 8 of 13 (62%) confirmed babesia-positive donations were detected in late fall or winter. all 13 (100%) confirmed babesia-positive donations were reactive in mp6. serology results are available for 12 of 13 confirmed-positive donations: at index, 6 of 12 confirmed babesia-positive donations were only igg-positive, while none were only igm-positive; 3 were positive for both igg and igm. 3 of the confirmed-babesia positive donations were negative for both igg and igm antibodies. cobas â babesia showed an overall specificity of 99.999% (256,787/256,789; 95% exact ci: 99.997%>100%). summary/conclusions: the cobas â babesia test successfully identified 13 babesiapositive donations, including 3 confirmed-positive donations with no igm or igg reactivity. 2 donations were collected in states considered low-or non-endemic for babesia. 8 confirmed-positive donations were collected outside of the summer babesia season, when most clinical cases occur. screening with cobas â babesia continues in several laboratories. cobas â babesia is not fda licensed or available commercially. background: babesiosis in humans is caused by the erythrocytic protozoan parasite, babesia microti which is transmitted by tick bites, but is also transfusion transmitted. although frequently asymptomatic or presenting with flu-like symptoms in a normal host, if immunocompromised infection can lead to severe complications and death. b. microti is endemic in the north eastern/upper midwest united states where partial testing of donations has been implemented. in canada, a 2013 study of~14,000 donors did not identify any b. microti antibody-positive samples, suggesting low risk at that time, but risk should be monitored. aims: to evaluate the prevalence of b. microti-positive donations in potentially atrisk areas in canada. methods: between july and november 2018, 50,752 blood donor samples were selected from sites near the us border. minipools were tested for b. microti nucleic acid by transcription mediated amplification (tma) using the procleix â babesia assay on the panther â system with individual testing on reactive pools. reactive donations were also tested by b. microti-specific: american red cross (arc) igg immunofluorescence assay [ifa] and imugen ifa/pcr. a subset of 14,758 tma-negative samples, primarily from the province of manitoba and eastwards to nova scotia, were tested for b. microti antibody using the arc ifa and if positive, the imugen ifa/pcr. donor age, sex, donation status, residential location and collection site location were recorded. donors who tested reactive/positive were informed, deferred and asked about risk factors (possible tick exposure and travel within canada, the usa and elsewhere, history of symptoms) and a follow-up sample was requested for supplemental testing (tma, arc ifa). reactive donations were removed from inventory. results: the 50,752 donor samples were proportional to collections in target geographic regions. age group, sex and donation status were also similar to the donor base in the collection areas. one sample from winnipeg, manitoba was tma reactive and antibody positive on supplementary testing. the donor did not remember symptoms or spending time in wooded areas. he visited the city of fargo, north dakota, usa. the subset of 14,758 samples tested for antibody were also proportional to collections in the targeted areas. four antibody-positive samples were identified from mid-september to october 1, all in south western ontario near lake erie. none were tma reactive. three were interviewed and none remembered any symptoms, any likely tick exposure, or relevant travel within canada or the usa. summary/conclusions: this is the largest b. microti prevalence study in canada. the results indicate very low prevalence with only 1 tma-confirmed-positive donation of 50,752 tested. the donor was from the only region in canada where one autochthonous human case has been reported and active tick surveillance identified b. microti positive tick populations. seropositive donations in south western ontario may suggest low prevalence in that region, but interpretation is less certain due to lack of corroborating supplementary results or case history. given the close proximity to the us border, forgotten us travel should not be ruled out. 4a-s18-04 background: the protozoan parasite toxoplasma gondii is prevalent in animals and humans worldwide. wild and domestic felids are the definitive hosts, and homoeothermic animals serve as the intermediate ones. after primary infection, the parasite persists lifelong within latent tissue cysts. transmission is by ingestion of undercooked or raw meat infected with cysts, by ingestion of food or water contaminated with oocysts, or transplacentally. however, it can also be acquired by blood transfusion and organ transplantation. toxoplasmosis can be a severe disease in immunosuppressed people and neonates whose mothers have acquired primary infection during pregnancy. aims: there is no information about the specific epidemiology of t. gondii infection in blood donors in portugal. therefore, we sought to determine the seroprevalence of t. gondii and associated risk factors in the population of blood donors in portugal. methods: between september 2015 and july 2017, 520 blood donors who attended the portuguese blood and transplantation institute blood banks located in oporto, coimbra and lisbon, and also at regional blood collection meetings, were invited to participate in the study. a written informed consent was obtained and a questionnaire about socio-demographic and behavioural variables was answered. sera were assessed for igg antibodies to t. gondii by a modified agglutination test (mat) commercial kit (toxo-screen da â biom erieux, lyon, france). results: of the 520 blood donors (mean age 38.55 ae 11.14; range 18-65 years old), 38.1% were positive for antibodies to t. gondii. when questioned about toxoplasmosis, almost half the blood donors did not have any knowledge about the disease. the centre of portugal had the highest seroprevalence (55.1%) followed by the north (37.2%) and the south (25.3%). blood donors living in rural areas had a significantly higher seroprevalence (p = 0.001) than those living in urban areas. seroprevalence increased with age, with the highest seroprevalence (60.2%) found in the age group of 46-55 years old (multiple logistic regression [mlr]: or = 7.68; ci: 4.08-14.46; p < 0.001), and decreased with educational level (p < 0.001). engaging in soil-related activities (gardening or agriculture) was significantly related to t. gondii seropositivity (p = 0.02). regarding water consumption, untreated sources (even though including mineral and tap water) was confirmed as a risk factor (mlr: or = 2.72; ci: 1.27-5.86; p = 0.001). other behavioural and eating characteristics, including cats in the household, eating raw or undercooked meat, processed pork products, or not washing raw fruit and vegetables before eating, were not associated with t. gondii infection. summary/conclusions: the risk of t. gondii transmission through blood transfusion is low, and serologic testing of antibodies, with exclusion of blood donors, appears not to be feasible. immunosuppressed individuals, organ transplant patients and pregnant women, should receive t. gondii antibody-negative blood components for transfusion. this study explored the epidemiology of t. gondii in portugal thus providing useful information on the seroprevalence and potential risk factors for t. gondii transmission. information regarding toxoplasmosis and its prevention could be promoted by medical and public health authorities among blood donors, and also the general population, when addressing policies, and designing screening programs, for monitoring and controlling infection and disease in portugal. 4a-s18-05 who is syphilis testing excluding? c reynolds 1 , c pearson 2 , k davison 2 and s brailsford 1 1 nhsbt/phe epidemiology unit, nhs blood and transplant 2 nhsbt/phe epidemiology unit, public health england, london, united kingdom background: screening for treponemal antibodies to detect syphilis in blood donors has been in place in england since the 1940s. there have been no reported syphilis transfusion transmissions in england since records began in part due to sensitivity of the organism to cold storage. since we have specific tests in place for other sexually transmitted infections such as hiv and hepatitis b virus (hbv), the utility of syphilis screening is often questioned. however, it may be a useful proxy for higher risk behaviours particularly following shortening of deferrals for higher risk sexual behaviours from 12 to 3 months in november 2017 and against a background of increasing infectious syphilis in the general population. aims: here we describe the epidemiology of recently-acquired syphilis in blood donors in england compared with hiv and acute hbv infection between 2009 and 2018. methods: monthly donation testing results are collected from the nhs blood and transplant (nhsbt) screening centres and reference laboratory. the demographics, possible sources of infection, and compliance to donor selection in confirmed positive donors are collected by proforma at post-test discussion with the nhsbt clinical team. recent syphilis is classified as igm positive and/or recent history including a negative donation within 12 months for regular donors. results: between 2009 and 2018 there were 153 recent syphilis cases, 121 hiv and 32 acute hbv infections identified by donation screening. recent syphilis rates per 100,000 donations increased from 0.66 to 1.64 whereas hiv decreased from 1.04 to 0.19 with less than 5 positive donations in 2018. acute hbv rates rose slightly from 0.28 to 0.38 in 2018. males outweighed females accounting for 71.9%, 63.6% and 59.4% of cases of recent syphilis, hiv and acute hbv respectively. nearly a quarter of cases of recent syphilis and hiv were seen in donors below 25 years old. of the male donors with recent syphilis, 58.2% reported sex between men and women (sbmw), 19.1% sex between men (sbm) and 22.7% did not report a risk. this contrasted with hiv where 45.5% of male donors reported sbm, just 2.6% not reporting a risk. overall 19, 32 and 3 males with recent syphilis, hiv or acute hbv respectively were non-compliant to the sbm deferral in place at the time of donation. in 2018, 26 donors with recent syphilis aged 23-65 years (median 36 years) were excluded from the donor pool, including 3 non-compliant to the sbm deferral. there were fewer than 5 hiv cases identified in 2018, all over 40 years old, all compliant, reporting sbmw. of the 6 hbv acute cases in 2018, 5 were male, all but one in the 45 and over age-group. summary/conclusions: over the 10 year period demographics of recent syphilis cases appeared similar to hiv with highest rates in young males, albeit lower proportions reporting sbm. following the switch to a 3 month deferral, hiv case detection continued at low level, while syphilis screening continued to exclude higher numbers spanning all age-groups, potentially at risk of other sexually transmitted infections, including non-compliant donors. background: globally, an estimated 113 million blood donations are given annually. in the blood service we are obliged to monitor donor health and ensure that blood donation is safe. in recent years, large-scale blood donor cohort studies in several countries have increased our knowledge on health effects of blood donation. health concerns relate both to immediate side effects like fainting and to possible long-term health issues related to repeated blood or plasma donation. the studies have provided us with data that can now help us introduce an evidence-based individualised donor care -a parallel to personalised medicine. individualised donor care in the management of iron depletion: studies have shown that a large percentage of our frequent whole blood donors, especially young women, are iron depleted. iron depletion is a strong predictor of deferral for low haemoglobin but has also been associated with e.g. restless legs syndrome and lower birth weight in children of frequent donors. the risk of iron deficiency can be mitigated by ferritin-guided prolongation of interdonation intervals or by iron supplementation. prolongation of interdonation intervals can challenge our inventories. iron supplementation, on the other hand, may give gastrointestinal side effects and other effects have been proposed as well, e.g. the masking of malignant disease and increased iron availability with subsequent risk of infection. in a large study we found that iron supplementation is not associated with increased risk of infection. what is the optimal balance between iron supplementation and prolongation of interdonation intervals? a growing number of blood services have implemented various flavours of iron management regimens generating more results. moreover, genetic studies in e.g. the uk, us, holland, and denmark can help us to find donors at high risk of iron depletion or low haemoglobin. we can use all these data in a big data approach in the pursuit of an individualised risk assessment model. other risks for blood donors: the presentation will also cover other risks associated with donation. new studies identify predictors of fainting after blood donation and also new interventions to prevent fainting. the global demand for plasma derived medicinal products has increased severalfold the last 10 years. plasma donors are bled up to 104 times per year in the us. very little is known about the health effects of frequent plasma donation. we know that immunoglobulin levels decrease with frequent donation but how does this affect health? summary/conclusions: the precautionary principle mitigates risk through early intervention prior to evidence. we tolerate next to no risk of transfusion-transmitted infectious diseases. the health of the blood donors, however, has not been protected similarly. we owe to our whole blood and plasma donors to investigate health effects of blood donation and ensure their safety. while the first attempts may not be perfect, we now have the tools to construct models for individualised donor care. background: in 2014, the isbt, aabb, ihn and eba jointly issued the standard for surveillance of complications related to blood donation which categorized donor adverse events (dae) into 6 categories (16 subcategories) defined by specific criteria. severity and imputability were briefly described but were optional. subsequent validation of these categories demonstrated consistency in categorizing reactions, but wide variation in assignment of severity. in 2018, with international input, the aabb donor biovigilance committee developed a severity grading tool (sgt) using a recognized medical adverse event grading system in which neutral grades replace subjective terms (mild, moderate, severe). aims: a large us blood collection establishment (bce) applied the draft sgt to assess its use in real cases of dae. methods: we performed retrospective analysis of all allogeneic and apheresis needle-in donations between 1/1/2017 to 9/30/2018. severity grading was assigned based on criteria defined by the sgt. database review of dae was performed, and each event was assigned a grade based on the type of outside medical care (omc), and on specific key search terms. search terms for omc included emergency room, emergency medical response, urgent care, healthcare professional, and hospital admission. additional specific key search terms included fracture, concussion, laceration, dental injury, surgery, and hospitalization. since duration and activities of daily living (adl) limitations were not captured in our dae database, cases in our dae claims' database were reviewed. case files of events classified as grade 2 or higher were individually evaluated by a physician for grading accuracy. results: in 1,511,758 needle-in collections, 31,320 dae were graded for severity. the majority (16,143, 51.5%) were vasovagal reactions (vvr), followed by 8,570 apheresis-related (27.4%), 6,572 needle-related (21.0%) and 35 allergic (0.1%) events. the majority of dae were grade 1 accounting for 98.6% of all dae, followed by grade 2 (1.2%), and grade 3 (0.1%). there were 2 grade 4 and no grade 5 dae. among the vvr, 98.1%, 1.6%, 0.2% and 0.01% were grade 1, 2, 3, and 4 respectively. grade 3 vvrs included 14 concussions, 11 fractures, 1 dental injury, and 2 pre-faint and 8 fainting events requiring hospitalization for work-up. two grade 4 vvrs involved falls resulting in intracranial hemorrhage requiring immediate medical intervention. for allergic and apheresis dae, there were only 6 and 5 grade 2 reactions respectively, and no grade 3 or 4 events. needle-related dae included 98.3% grade 1, 1.6% grade 2, 0.1% grade 3, and no grade 4 events. of the six grade 3 needle-related dae, 4 were nerve irritations lasting > 6 months, and 2 were dvts requiring hospitalization. summary/conclusions: the sgt provided consistent assignment of severity for the majority of dae, based on outside medical care and specific key search terms. assignment of severity based on impact on activities of daily living or on duration of injury/condition requires tracking over time making such assignments more difficult; modification of our dae tracking database and claims database to capture adl and duration should improve severity assignment for such cases. background: the international haemovigilance network (ihn) has collected aggregate data on complications of whole blood and apheresis donations from member national haemovigilance systems (hvs) since 2006. aims: we analysed the data collected in 2006-2016 in order to learn from the data and consider future improvement of data collection. methods: national hvs entered annual data on donor complications in the passwordprotected "istare" (international surveillance of transfusion adverse reactions and events) online database. from 2008 the donor complication spreadsheet allowed entry of separate data for whole blood donation (wbd) and apheresis, but also provided an option for entering data for all donation types. annual numbers of whole blood and apheresis donations were also collected. the harmonised international standard definitions were implemented in 2015. reactions were captured according to severity level (mild, moderate, severe) but without distinction between donor sex or first time vs repeat donation. extracted data were used to calculate national and aggregate donor complication rates (generally per 1000 donations). results: twenty-four hvs provided figures for donations and donor complications for one or more years (median years per country was 7, iqr 2-8). the total number of country years (cy) was 138, covering 155 million donations. the overall complication rate was 6.3/1000 donations and the median country rate was 3.2 complications/1000 donations (iqr 1.1-10.1). rates were generally consistent within a hvs from year to year but showed considerable variation between hvs; this was also the case for reactions classed as severe. not all countries differentiated between mild and moderate reactions and some reported all reactions under a single severity level. vasovagal reactions were the most commonly reported complication: overall 4.6/ 1000 donations, median country rate 3.1/1000 donations (iqr 0.6-7.7). rare and apheresis-related types of complications such as generalized allergic reaction (0.10 per 100,000, 40 cy), and major blood vessel injury (category available since 2015; overall 0.12 per 100,000, 6 cy) were only reported occasionally. eighteen of the hvs provided separate data for complications of whole blood and apheresis donations in one or more years (total 89 cy, 101.6 million wbd and 26.3 million aphereses, total 128 million donations). for these hvs the median rate of vasovagal reactions was 3.4/1000 wbd (iqr 1.0-9.1) and 1.5/1000 apheresis procedures (1.0-4.2) . reported haematoma rates were higher for apheresis than for wbd: the median per hvs was 0.39/1000 wbd (iqr 0.31-1.2) vs 4.2/1000 aphereses (0.69-5.6); rates of arm pain and/or nerve injury (not separated in 2006-2013) also tended to be higher: median 0.09/1000 with wbd, iqr 0.03-0.34, vs 0.39/1000 with apheresis, 0.05-0.57. summary/conclusions: international reporting allows hvs to study rates of blood donation complications, to distinguish between wbd and apheresis complication rates and capture information about very rare events. variability of reporting and of severity assessment between countries impairs the feasibility of comparisons between hvs. work is needed to improve harmonisation of classification of donation complications and severity assessment for data comparison and research. background: to prevent iron related hb loss, screening with ferritin testing was implemented in stockholm county (approx. 52000 registered blood donors) during a two-year roll-out. iron supplementation is offered to blood donors but has not prevented hb deferrals resulting in 8000 control visits per year. ferritin testing is hypothesized to increase iron compliance. aims: implementation of ferritin testing for surveillance of iron levels for the entire blood donor population with specific attention to new donors, women returning after pregnancy, donors with low hb and at return visit after low hb. yearly testing of plasma and platelet donors. methods: ferritin testing, following a staff education program, was implemented for applicant donors, donors with low hb, women after pregnancy, apheresis donors, followed by screening of registered blood donors per donation site. after initial screening, donors will be tested at each 4 th (women) or 8 th (men) donation, and with yearly testing of young adult blood donors below 25 years. six nurses were educated to process ferritin and blood count results. donors with aberrant ferritin were contacted by letter. results: establishment of cut-off levels and algorithms for ferritin testing and iron treatment was evidence based but met practical limitations such as number of analyses and results that could be processed per week, limitations in liss set-up, blood demand contra preferred cut-offs, iron supplementation compliance. for applicant donors, hb testing show that 20% of female and 9% of male applicants cannot be registered because of low hb (125 and 135 mg/l respectively). adding ferritin testing, a preferred cut-off level of 30 lg/ml (male reference level), would result in additionally 24% female and 1.3% male applicant donor loss. as this would threat the blood demand, cut-off was set to 15 lg/ml for women, above the female reference 10 lg/ml, with an acceptable 6% loss of female applicant donors. for registered blood donors, 4000 mg of extra iron tablets were offered at low ferritin (10-29 lg/ml). this was sometimes combined with prolonged intervals and often repeated before ferritin was restored above 30 lg/ml. donors with ferritin below 10 lg/ml (in 0.6% applicant donors, 0.8% registered donors) or above 600 lg/ml (0.5% applicant donors, 0.1% registered donors) were deferred and recommended to see their physician. for hb deferral, the interval was prolonged from 3 to 5 months, irrespective of ferritin levels. this, together with iron supplementation, resulted in an increase from 50% to 70% approved hb at return. the team of nurses processing ferritin and blood count results (1½ nurse fulltime weekdays) reacted to approximately 40 donor results daily, representing 20% of test results. summary/conclusions: many female donor applicants have suboptimal ferritin levels although they meet required hb for donation. iron treatment was added to retain donors with low ferritin as only prolonged intervals may danger the blood supply. for implementation of ferritin testing, it is necessary to have a well-functioning and agile organization to create and apply algorithms for testing, extension of intervals and iron treatment. background: since november 2017 a new donor screening regime is introduced in the netherlands where serum ferritin levels in whole blood donors are measured periodically to further control potential iron deficiency in donors. donor deferral thresholds are set at 30 and 15 ng/ml, and donors are deferred for six and twelve months respectively if ferritin levels are below these values. as limited information is available on ferritin recovery in whole-blood donors, the policy is introduced in parts such that adaptations to the implementation may be considered based on intermediate results and the impact of the measure on donor well-being can be evaluated. aims: to assess the effect of donor deferral on donor ferritin levels. methods: ferritin levels are measured in new donors and at every fifth donation in repeat donors. donors with ferritin levels below the indicated thresholds are deferred and ferritin is re-evaluated at their return for donation after six or twelve months. the policy allows estimating long term trends in ferritin levels post donation in repeat donors. as ferritin levels are measured in all new donors a reference distribution of ferritin levels in healthy individuals is obtained as well. results: among repeat donors 46% (44% of 16,433 male donors, and 48% of 13,525 female donors) have ferritin levels below 30 ng/ml and are deferred for their next donation. furthermore, the distributions of ferritin levels in repeat male and female donors are similar and each has an average ferritin level of 43 ng/ml. in contrast, we found that only 27% of new female donors (n = 13,283) and 1.7% of new male donors (n = 6,334) have a ferritin levels below 30 ng/ml. the average ferritin level in new donors was 148 ng/ml for males and 60 ng/ml for females. comparing the ferritin levels in new and repeat donors, a reduction in average ferritin levels between 1.5 and 2.0 was observed in female donors and between 3.2 and 4.4 in male donors. both ratios increased with donor age. at the end of december 2018 2884 donors with low ferritin levels returned for donation after six or twelve months deferral. repeat ferritin measurements show that on average the ferritin levels in female donors increased by 13 ng/ml per year whereas average ferritin levels in male donors increased by 34 ng/ml per year. summary/conclusions: in line with earlier findings in literature our results show that repeat donations substantially reduce ferritin levels in repeat donors. these range from 1.5 to 2.0 in female and from 3.2 to 4.4 in male donors, who generally have higher ferritin levels. deferral of donors with low ferritin levels seems to be effective in increasing ferritin levels in donors, however, further monitoring of follow-up in repeat donors is warranted to see whether the proposed scheme allows for sufficient donor recovery over time. there are~7000 different rare diseases and the genes for half have been identified. approximately 3.5 million uk citizens experience premature ill-health because of a rare disease. a conclusive diagnosis is generally not reached and on average the diagnostic odyssey lasts 2.2 years. the main aims of the 100,000 genomes project are to reduce the diagnostic delay by embedding whole genome sequencing (wgs) to accredited standards in the care path of patients with undiagnosed rare diseases. the project started in 2013 and dna samples from 100,000 nhs patients and their close relatives have been analysed by wgs. here we review the results from the nihr bioresource pilot study for the 100,000 genomes project comprising phenotype and genotype data from 13,037 individuals recruited at 83 hospitals using approved eligibility criteria for 15 rare disease domains. we determined the population structure including ethnicity and relatedness estimation, high level phenotypes collected using human phenotype ontology (hpo) terms and quality control and summary metrics for samples and variants. the sequence resource contains over 165 million unique variants in the 10,258 genetically independent samples, with 47% of variants previously unobserved in other large scale publicly available genome datasets. we summarise the curation of gene lists and pertinent findings in 2,000 unique diagnostic-grade genes for the 15 domains. over 1,000 reports assigning pathogenic or likely pathogenic causal variants have been issued with diagnostic yields varying between domains from 0.5% to 55%, while the proportion of novel causal variants ranged between 25% and 73%. we show the power of the bayesian association test, bevimed, to recapitulate decades of clinical genetics discoveries and by identifying >30 novel genes and novel diseasecausing variants in the non-coding space of the genome. we show how typing data for all red cell, hpa and hla class antigens can be extracted from wgs data. we mined the data from the 100,000 genomes project and similar sequence resources to re-version the probe content of the uk biobank axiom array. we genotyped 7588 donors from england and the netherlands with this new array and observed a 99.92% concordance when comparing 92,387 blood centredetermined antigen typing results with genotype-determined ones. for the 48 red cell and hpa antigens that were available for 7,473 donors, the array typing provided a 3.6-fold increase in typing results per donor (13.2 vs 47.9) and 38 rare donors were identified. using the genotyping data we identified 2.6 times more compatible units among this cohort of donors when blood demand was modelled using referral data from 3,146 english patients with more than three red cell alloantibodies. in conclusion the 100,000 genomes project has shown the feasibility of using wgs across a universal healthcare system to deliver a diagnosis for patients with rare diseases. based on these results the nhs has commissioned the analysis of another 500,000 dna samples from patients with cancer and rare disease. with analysis of dna by wgs and arrays becoming part of routine clinical care, blood services must develop competencies to extract transfusion and transplant relevant information from clinical-grade genotyping data. next-generation sequencing (ngs) enables the sequencing of thousands of genes, the exomes, and even entire genomes by single experiments at a reasonable price. there have also been advances in cytometry: use of antibodies with different fluorescence tags enables simultaneous monitoring of the expression of dozens of antigens. however, immunological methods cannot detect every variant discovered by ngs. genome sequencing reveals not only the exome but also the regulatory elements of transcription/translation, such as promoters and enhancers. rna sequencing determines which genes and spliced transcripts are expressed. it is amazing to realize how much this novel technology has been contributing to the better understanding of various biological phenomena. since the initial cloning of the human blood group a transferase cdnas in the early 1990s, we have been studying the abo genes, a and b glycosyltransferases, and a and b oligosaccharide antigens. various scientific disciplines including genetics, immunohematology, biochemistry, enzymology, and glycobiology have been applied to their study. we have made several important scientific contributions. we demonstrated the central dogma of abo: the a and b alleles at the abo genetic locus encode a and b transferases, which synthesize a and b antigens, respectively. we elucidated the allelic basis of the abo system. we found 4 amino acid substitutions between a and b transferases and inactivating mutations in o alleles. we became the first who succeeded in the abo genotyping, discriminating the aa and ao genotypes, as well as the bb and bo, which was impossible by the immunological approach. we have taken a simple experimental strategy: preparation of eukaryotic expression constructs of a/b transferases and their derivatives, dna transfection to human hela cells or their sublines, and immunological detection of the a/b antigen and/or biochemical examination of the enzymatic activity. we used this to show that the codons 266 and 268 are crucial in determining the sugar specificities of galnac/galactose of a/b transferases. we also identified mutations in several subgroup alleles causing restricted substrate use and diminished transferase activity. we also showed that cis-ab and b(a) alleles specifying the expression of both a and b antigens by single alleles encode a-b transferase chimeras. since then, other scientists have characterized more than 250 abo alleles. recent human genome sequencings have identified many more single nucleotide polymorphism variations. the genome sequences of many species are also available. taking advantage of those sequences and associated information, we have expanded our research to include evolutionarily related a1,3-gal(nac) transferases and their genes and scaled it up from the genetic to genomic level. in this talk, i would like to present the followings. 1: our elucidation of the molecular genetic basis of the abo blood group system (as requested by the organizer); 2: identification of novel abo alleles by others; 3: more snp data from genome sequences and potential problems for abo genotyping; 4: findings obtained from analysis of abo genes from other species; bacteria, vertebrates, to primates; 5: a1,3-gal(nac) transferases and their genes and the crosstalk between a transferase and forssman glycolipid synthase (fs); and 6: the potential causes of generation of abo polymorphism and of species variations of the gbgt1 gene specifying the fors polymorphism. in recent years, there has been a concerted effort to improve our understanding of the quality and effectiveness of transfused blood components. the expanding use of large datasets built from electronic health records allows the investigation of potential benefits or adverse outcomes associated with transfusion therapy. together with data collected on blood donors and components, these datasets permit an evaluation of the effect of donor and blood component factors on transfusion recipient outcomes. large linked donor-component recipient datasets provide the power to study exposures relevant to transfusion efficacy and safety, many of which may not otherwise be amenable to study for practicality or sample size reasons. analysis of these large blood banking-transfusion medicine datasets allow for characterization of the populations under study and provide an evidence base for future clinical studies. knowledge generated from linked analyses has the potential to change the way donors are selected and how components are processed, stored and allocated. however, unrecognized confounding and biased statistical methods continue to be limitations in the study of transfusion exposures and patient outcomes. results of observational studies of blood donor demographics, storage age, and transfusion practice have been conflicting. this review will summarize statistical and methodological challenges in the analysis of linked blood donor, component, and transfusion recipient outcomes. 4c-s20-01 a large deletion spanning xg xg and gyg2 gyg2 constitutes a genetic basis of the xg null phenotype, underlying anti-xg a production background: the xg blood group system comprises the homologous antigens xg a and cd99. the cd99 gene resides within pseudoautosomal region 1 on the short arms of the sex chromosomes and thus mimics autosomal inheritance. xg, on the other hand, is x-linked and straddles the pseudoautosomal boundary; a truncated pseudogene composed of only the first 3 exons remains on the y chromosome and therefore males carry a sole full-length copy of xg. this phenomenon manifests as asymmetric frequencies of the xg(a+) phenotype between the sexes: roughly 11% of women and 34% of men are xg(aà). also, whilst xg a immunization is rare, the vast majority of all anti-xg a makers reported are men. recently, we reported that the rs311103c variant disrupts a gata motif between xg and cd99. this abolishes erythroid xg a expression and causes the common xg(a-) red cell phenotype. however, rare individuals who produce anti-xg a cannot be accounted for by this finding. we hypothesized that a structural defect in the xg coding region causes the true xg null phenotype underlying anti-xg a production. aims: we undertook to determine a genetic explanation for anti-xg a production. methods: genomic dna (gdna) was extracted from two whole blood samples and cell-free dna (cfdna) from 13 archived plasma samples from donors producing anti-xg a ; one cfdna sample was from a female donor and the rest from males. polymerase chain reaction (pcr) experiments, sanger sequencing, and database searches were performed to identify and confirm the deletion. aliquots of gdna from four males reported to carry a similar deletion in the 1000 genomes project were also tested. results: in one gdna sample, exon-specific pcr identified a deletion involving part of xg and the downstream gene gyg2. database searches indicated that the most likely deletion was the infrequent genomic structural variant esv2662319 reported in the 1000 genomes project. further analyses with a short (714 bp) and a long (3555 bp) pcr amplicon across the suspected breakpoint determined that this deletion was approximately 114 kb and corresponded well with esv2662319. this finding was confirmed in the second gdna sample. given the rarity of anti-xg a producers, we decided to test for the same deletion in cfdna extracted from old archived plasma samples. of the 13 cfdna samples, poor quality in four samples prevented amplification even from control reactions and one was contaminated with bacterial dna. in the remaining nine samples, eight could be amplified for the deletion-specific 714-bp short amplicon while one was negative for the deletion. sanger sequencing of the amplicons revealed a heterogeneous repetitive dna element, ltr6b, hinting at a previously-reported recombination event. this deletion was not detected in the samples from the 1000 genomes project which reiterates the previously identified deficiency in data interpretation and reporting for deletions. summary/conclusions: a large deletion disrupting the xg and gyg2 genes accounts for the xg null phenotype underlying the majority (10 of 11) of anti-xg a makers. one sample remained unexplained, indicating further heterogeneity to be explored. our data help to explain why anti-xg a production is rare and has primarily been reported in men. background: s and s antigens encoded by gypb differ by one nucleotide (nt), c.143c>t, p.thr48met. two different genetic backgrounds are associated with silencing of s antigen and a u+ w phenotype. these include the nt change c.230c>t (p.thr77met) causing partial exon skipping and designated gybp*03n.01 (gypb*ny) and c.270 + 5g>t, an intron change causing complete skipping of exon 5, designated gypb*03n.03 (gypb*p2). aims: samples from three individuals, a previously transfused african american sickle cell patient (p1), a blood donor of unknown ethnicity (p2), and an african american patient (p3) (lapadat r. 2014 aabb abstract) were investigated for discrepant serologic and molecular results when determining s and s phenotype. methods: standard methods were used for rbc typing with licensed s and s reagents and rbcs from donor p2 were also tested with monoclonal and polyclonal anti-s and anti-s. dna was isolated from wbcs and hea precisetype performed on p1 and p2. p1 was also tested by gypb*s/s as-pcr, exon 5 pcr-rflp for c.270 + 5g>t and as-pcr for c.230c>t. p3 was tested for gypb*s/s and c.230 c>t and c.270 + 5g>t changes by a real-time pcr-fluorogenic 5' nuclease taqman chemistry. for all, gypb exons 1-6 were amplified and sanger sequenced and aligned to consensus using clustal x. results: rbcs of all three probands typed s-and strongly s+ while dna testing indicated c.143t/c (gypb*s/s). assay for the two common gypb*s silenced alleles, c.230c>t and c.270 + 5g>t, indicated all three samples had both silencing mutations previously reported to be independently associated with a sàu+ w phenotype. hea precisetype could not interpret this novel allele combination and indicated gypb*s as pv (possible variant). samples were confirmed to be heterozygous for c.143c/t, c.230c/t and c.270 + 5g/t by exon specific sequencing and as-pcr, pcr-rflp and real-time pcr. by long range sequencing of gypb, all three were heterozygous c.59t/g and c.60a/g (p.20leu/trp), c.67a/t (p.23thr/ser), c.71a/g and c.72g/t (p.24glu/gly), c.143c/t (s/s), c.208g/t (p.70val/leu), c.230c/t (p.77thr/met), and c.270 + 5g/t. all samples were also c.251g/g (p.84ser) and heterozygous for several previously recognized silent changes in exon 2, c.87t/c, c.96t/c and c.102a/g. summary/conclusions: we report a novel silenced gypb*s allele that can confound gypb genotyping interpretation. the allele was found in three probands associated with a sàs+ phenotype. in these samples, two changes previously reported to be inherited independently and both associated with silencing of s antigen are carried on the same allele. dna-based testing could not rule out that c.230t or c.270 + 5t are separate and that gypb*s was also silenced. robust s+ rbc typing indicates both changes are on gypb*s. gene sequencing confirms the c.270 + 5t change is on a gypb*03n.02 [gyp*he(ny)] background. c.230c>t (rs79492560) and c.270 + 5g>t (rs139511876) have a frequency of 0.0053 respectively 0.032 in the african population (exac). although we identified 3 samples, the frequency of this novel allele is unknown. background: the lutheran blood group system currently consists of 25 antigens. these antigens are of low immunogenicity and may cause mild-to-moderate transfusion reactions and hemolytic disease of the fetus and newborn. the activation of lu-glycoprotein/bcam on red blood cells (rbcs) and its interaction with laminin-5a is thought to play a role in vaso-occlusion in sickle cell disease and other hematological disorders. the two glycoprotein isoforms lu-glycoprotein and bcam are encoded by the bcam gene which consists of 15 exons located on chromosome 19q13.2. a number of rare lutheran phenotypes have been previously recorded in israel, including lu:-6,9 observed among iranian jews, lu:-20 in one thalassemia patient and one case of lu:-21. in this report, a previously transfused pregnant arab patient with b-thalassemia intermedia was investigated because she presented with an antibody to an unknown high frequency antigen (hfa), potentially related to the lutheran system. aims: to characterize a novel lutheran antigen through serological and molecular investigation of a patient with a lutheran related antibody. methods: initially, the red cell phenotype and the presence of a lutheran related antibody in the serum of the patient were detected by standard serological techniques, utilizing enzyme treated and chemically modified cells and rare cells and sera from the nbgrl collection. further serological investigations were carried out using standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were performed using soluble recombinant lu protein (srlu). eluates were prepared using acid elution method (gamma elu-kit ii). genomic dna was isolated from whole blood and all exons of the bcam gene were amplified by pcr and directly sequenced by sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: the patient's plasma reacted with all cells tested, except for three examples of in(lu) cells and cells treated with 2-aminoethylisothiouronium bromide, trypsin and a-chymotrypsin. inhibition studies with srlu protein showed complete inhibition of the antibody, thereby confirming the antibody to be directed toward an epitope on the lu-glycoprotein. in addition, testing of inhibited plasma revealed the presence of underlying anti-e and anti-fy a . an eluate was prepared to isolate the patient's lu-related antibody and this eluate was found to be incompatible with examples of lu:-5, lu:-6, lu:-8, lu:-13, lu:-21, lu:-22, and lu:-23 cells, whereas in(lu) were compatible. results of serological typing of the patient's cells, for lu system hfas, could not be conclusively determined due to the patient having been recently transfused. however, results suggested (through absence of mixed field reactivity) the patient's cells to be lu: -1,2,8,12,13,-19,21 . bcam sequence analysis confirmed the patient to be lu*02, lu*18 and revealed a novel homozygous mutation c.1351a>c in exon 11, encoding p.lys451gln in the lutheran glycoprotein. summary/conclusions: a novel homozygous mutation c.1351a>c (p.lys451gln) in exon 11 of bcam was identified in a patient with an antibody to a lutheran hfa. serological and genetic evidence presented here indicates discovery of a novel antigen of the lutheran blood group system, which we propose to name lura. background: lutheran glycoprotein and basal cell adhesion molecule antigen b-cam are two isoforms of a type i membrane glycoprotein residing on red cell surfaces. both isoforms are adhesion molecules with the main function of laminin binding, and both carry antigens of the lutheran blood group system (lu). the system currently comprises 25 antigens, all encoded by mutations in the alternatively spliced single gene bcam located on chromosome 19. currently, isbt lists 20 high incidence antigens in the system. aims: we report a case study of an individual with an unidentified alloantibody to high incidence antigen present in her plasma. samples from the patient and her family were investigated. we provide here serological and molecular evidence for a novel high incidence antigen of the lutheran blood group system. methods: serological investigations were performed by standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were completed with soluble recombinant lu (srlu) protein. genomic dna was isolated from whole blood of the patient and her family members; all the exons of the bcam gene were amplified by pcr and analysed by direct sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: presence of a lu-related antibody in the patient's plasma was confirmed, reacting moderate strength by liss iat with untreated and papain treated cells. cells from the patient's mother, father and two siblings were all incompatible with her plasma, though weaker than panel cells, reflecting dosage. only in(lu) cells were compatible with patient's plasma. the antibody was successfully inhibited with srlu protein, thereby confirming the epitope recognised by the antibody resides on the lutheran glycoprotein. the patient's cells were found to be lu: -1, 2, 3, 5, 6, 8, 13, 20, 21 . bcam sequencing revealed a novel homozygous mutation c.121g>a in exon 2, encoding p.val41met in the lu glycoprotein. the c.121g>a change appears to be an extremely rare mutation, listed in gnomad database with a frequency of 3.98 9 10 -6 and with no known homozygous examples. homology model of the novel lutheran glycoprotein was subjected to all-atom molecular dynamics calculations to analyse potential conformational changes. summary/conclusions: we report serological and genetic evidence for a novel antigen of the lutheran system, which we propose to name lunu. the evidence will be submitted to the isbt red cell immunogenetics and blood group terminology working party for consideration for allocation of antigen status. the absence of this high incidence antigen arises from a rare single amino acid change p.val41met in the lutheran glycoprotein and the presence of anti-lunu in the patients' plasma was presumed to have been made in response to previous pregnancy. on native, papain-treated (diagast) and trypsin-treated (sigma) rbcs. genomic dna was extracted from peripheral blood cells by an automated method, amplified by sema7a exon-specific primers and sequenced. results: the proband was a 32-year-old female patient of moroccan origin, group a, d+c+e-c+e+, k-, without transfusion history. she was hospitalized at 27 weeks gestation for a blighted ovum requiring a manual vacuum aspiration, with a significant hemorrhage risk. a rbc antibody screening was performed by a first laboratory. the antibody reacted 1 + by iat on all native reagent rbcs, with negative autocontrols, but was nonreactive on papain-and trypsin-treated cells. an anti-ge2 was initially suspected, due to the pattern of reactivity and ethnic background. new blood samples were referred to our national immunohematology reference laboratory. the antibody showed the same profile. anti-ge2 and anti-ch1 could be ruled out. the serum was nonreactive with two jmh:-1 and positive with two jmh:-3 samples. the patient was found to be jmh1 positive. in addition, a soluble recombinant jmh protein (jmh imusyn/inno-train) fully abolished the reactivity of the panagglutinating antibody. the antibody was an igg4. overall, these results were consistent with a probable jmh variant and prompted us to perform sema7a sequencing. three nucleotide changes were found, in homozygous state: a rare nonsynonymous change in exon 7, c.709g>a (p.asp237asn, rs140707085, maf < 0.01, sift score = 1); a common synonymous change in exon 12, c.1545a>g (p.gln515gln, rs741761, maf = 0.5); a rare non-synonymous change in exon 14, c.1865g>a (p.arg622his, rs140128092, maf < 0.01, sift score = 0.36). the analysis of surface accessibility of asp 237 and arg 622 using the 3d structure of sema7a (rcsb pdb-3nvq https://www.rcsb.org/structure/3nvq) showed that only arg 622 was predicted to be an exposed-epitope. interestingly, all other reported jmh variant phenotypes correspond to an arginine substitution. of note, we retrospectively found another individual of algerian ancestry (pregnant woman) with a pan-agglutinating igg4 antibody showing a similar pattern of reactivity, and with the same three changes in sema7a. we unfortunately could not perform a cross-compatibility testing with the proband (no material left and unsuccessful contact). summary/conclusions: serological and molecular studies allowed us to provide evidence for a novel high-prevalence antigen in the jmh blood group system, very likely encoded by the p.arg622his substitution in sema7a. we propose to provisionally assign the name jmh7 for this antigen. interestingly, our two unrelated jmh:-7 individuals were from north african ancestry. background: the abo system was discovered almost 120 years ago and the underlying structures later elucidated as carbohydrates carried by glycoproteins and glycolipids. the terminal trisaccharides galnaca3(fuca2)gal and gala3(fuca2)gal constitute the clinically important a and b epitopes, respectively. clausen et al. (pnas, 1985) showed that the a antigen could be extended to a repetitive glycolipid a epitope, galnaca3(fuca2)galb3galnaca3(fuca2)galb4glcnac-r. however, extended forms of b antigen have not been described. we encountered two related situations with unexplained serological reactivity. firstly, enzyme-conversion to group o treatment of group b (b-eco) red blood cells (rbcs) with a3-specific gh110 family exogalactosidase (bzyme) abolishes b antigens as detected by hemagglutination and flow cytometry with all monoclonal anti-b tested. despite this, 40% of group o plasmas have been reported to give positive crossmatch results with b-eco rbcs. secondly, plasmas from ab and b individuals of the globoside-deficient p k phenotype contain anti-p and anti-px2 but react stronger with bpp-rbc than with app/opp-rbc. based on these findings, we hypothesized the presence of a bzyme-resistant, b-related glycolipid. aims: to identify the molecular basis of the enigmatic serological observations outlined above. methods: plasma and eluates from an a 1 b individual with the p1 k phenotype were investigated by hemagglutination and flow cytometry, as were eluates from b p 1 k and o plasma. rbc membrane glycolipids were extracted from two batches of pooled, expired group b-rbc units (frozen 500-litre reference preparation and confirmatory preparation from 24 freshly collected units). native or enzyme-treated glycolipid fractions were analysed by liquid chromatography electrospray ionizationmass spectrometry (lc-esi/ms) and immunostaining of thin layer chromatography (tlc) plates. antigen expression in the h+bà human erythroleukemia (hel) cell line was analysed by flow cytometry following overexpression of selected glycosyltransferases. results: anti-p-depleted eluates made from a 1 b p 1 k plasma contained anti-px2 and antibodies of unknown specificity that reacted stronger with native or papaintreated bpp-rbcs compared to app/opp-rbcs. anti-px2 was removed by adsorption onto opp-rbcs but reactivity (here designated anti-extb) remained against b/bpp/b-eco rbcs. lc-esi/ms of glycolipid fractions from group b units revealed an unknown hexnac-hex-(fuc-)hex-4hexnac-hex-4hex heptasaccharide. upon b-nacetylhexosaminidase treatment of this candidate structure, a group b type 2 hexasaccharide was produced, demonstrating that the terminal hexnac of the hexnac-gala3(fuca2)galb4glcnacb3galb4glc heptasaccharide was b-linked. since the discovery of the anti-platelet effects of aspirin platelets have been a major therapeutic target for pharmaceutical companies and also a very profitable target. however, the effectiveness of aspirin has also been a challenge as it is an inexpensive drug and any new agent needs to show clear benefit over aspirin. furthermore the risk of bleeding from anti-platelet agents, especially cerebral bleeds, has also presented challenges. in the 1990's orally active gpiib/iiia antagonists were considered to be the 'super aspirin' but clinical trials showed increased mortality and ultimately this class of drugs was relegated to iv use only in high-risk patients. gpib/ix/v antagonists were also a promising drug target but no agent made it to market. the real breakthrough was the discovery of the p2y12 antagonist clopidogrel which, in conjunction with aspirin, proved to be very effective at preventing thrombotic events and as a result it became the biggest selling drug in the world at the time. with clopidogrel now offpatent the combination of aspirin and clopidogrel is a formidable challenge to any new agent both in efficacy terms and pharmacoeonomic terms. so is there a future for new anti-platelet agents? with the growing awareness of the role of platelets in inflammation and an understanding of how the immune activation of platelets differs from the classical haemostatic activation of platelets it is now possible to develop novel anti-platelet agents that target inflammation without compromising haemostasis. it is here that we should look for the next generation of anti-platelet agent. 4c-s21-02 university hospitals of geneva, geneva, switzerland platelet function defects, either congenital or acquired, are associated with increased bleeding risk, particularly in a perioperative setting. the use of platelet function assays is therefore tempting in order to tailor transfusion and limit platelet transfusion to those bleeding patients with impaired platelet function, as assessed by those assays. however, the current guidelines provide only weak recommendations supporting the routine use of these assays. indeed, there are numerous platelet function assays on the market that differ in their method of evaluation of platelet function and agreement between their results is at best moderate. the threshold values beyond which procedure-associated bleeding risk becomes worrisome is not standardized. moreover, observational studies addressing the predictive value of platelet function testing in perioperative or spontaneous bleeding are not consistent. finally, management trials with randomized patients assessing the benefit of platelet function testing are scarce. more recent data identified selected situations where platelet function testing may be useful though. i will review the different platelet function assays as well as selected clinical studies addressing the impact of platelet function testing to improve bleeding and transfusion-related outcomes. the latest recommendation will be addressed too. background: platelet refractoriness complicates the provision of platelet transfusions in management of thrombocytopenia in oncology patients. platelet refractoriness poses challenge due to alloimmunization to hla and human platelet antigens and is associated with adverse clinical outcomes. aims: a prospective study was undertaken to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory oncology patients. pulmonary complication after blood transfusion is the leading cause of transfusionrelated morbidity and mortality, with an incidence reported between 0.05 -15% of all transfused patients. the most important transfusion related pulmonary complications are transfusion associated circulatory overload (taco), transfusion related acute lung injury (trali) and transfusion associated dyspnea (tad). in this presentation the recent changes in the international definitions will be presented and discussed. furthermore, insights in the different underlying pathophysiologic mechanisms will be highlighted. in the past decades only for trali prevention strategies have successfully been designed and implemented. currently no evidencebased treatment strategy is available for any of these life-threatening syndromes. insight in the pathogenesis of pulmonary complications after transfusion should pave the way for future prevention and treatment studies. the issue of the impact of iron overload / toxicity on the hematopoietic stem transplantation (hct) outcome has been firstly addressed in the field of transfusion dependent thalassemia. today the concept has been extended to other diseases characterized by periods of variable duration of transfusion dependence such as myelodysplastic syndrome (mds) and myeloproliferative diseases. patients requiring regular blood transfusions certainly develop iron overload leading to tissues and organ damage. iron burden before transplant significantly impacts outcome and long-life posttransplant. it is well known that iron overload is deleterious for organs such as liver, heart and endocrine glands and it has been postulated could also increases the risk of infections and severe graft versus host disease early after hct. recent preclinical data has shown how increased production of reactive oxygen species (ros) resulting under iron overload condition, could impair the stem cells clonality capacity, proliferation and maturation. also, microenvironment cells could be affected through this mechanism. for this reason, iron overload is becoming an important issue also in the engraftment period early post-transplant. high baseline ferritin levels before hct have been shown to negatively influence clinical outcome, but nowadays, ferritin is considered a steady and not biologically active form of iron, while free iron forms as non -transferrin bound iron (ntbi) and labile plasma iron (lpi) are considered the main trigger of cell damage more representative of the dynamic tissue damage. the scientific community is moving the iron disease from a "bulky" disease, such as classically in thalassemia (based on quantitative iron parameters as ferritin, red blood cell transfusion number, mri) to a "toxic" disease (based on active and dynamic biological markers as ntbi/lpi). at this time in all the studies published on hct setting, only the correlation between direct or indirect estimates of iron overload (mainly serum ferritin) and outcome parameters has been explored, while the duration of exposure to toxic iron species has not been taken into account. the first study that explored the lpi role in relationship with outcome was published by wermke and colleagues in malignancies. they investigated the predictive value of both stored (mri-derived liver iron content) and non-transferrin-bound-iron, defined as enhanced labile plasma iron (elpi) on post-transplantation outcomes in patients with acute myeloid leukemia or mds. their prospective, observational all-ive study showed that patients who had raised elpi concentration at baseline, also had significantly increased incidence of non-relapse mortality at day 100 (33%) compared with those who had normal elpi at baseline (7%) (p = 0.00034). reinterpreting transplant predictive factors in the light of the current advances in understanding iron homeostasis further supports the concept that the key to successful transplantation is regular and life-long chelation therapy to consistently suppress tissue reactive iron species and prevent tissue damage in the years before hct. in transfusion medicine, the role of donor sex was long considered to be limited to the increased risk of trali observed after transfusions from female donors. this risk has been shown to be limited to female donors with a history of pregnancy and to plasma rich products (i.e. excluding red blood cell products, typically containing < 50 ml plasma). until, in 2011, we found that sex-mismatched red blood cell transfusions were associated with increased recipient mortality. since then, several other studies have confirmed these findings, but some studies also did not find an association. all of these studies relied on the analyses of routinely collected health care data, which was not primarily intended to be used for research. as a result, analyses are complex and often difficult to properly appraise based on published descriptions. therefore, the discussion about possible reasons for these discordant findings has largely focused on the methodological approaches of the different studies. other potential explanations include differences in donor or patient populations, production methods, or storage time of blood products. the different potential explanations are expected to be associated with different underlying biological mechanisms. therefore, further delineating which donor, patient, and product characteristics modify the observed association could provide more insight into the underlying mechanism. in 2017, we observed that only transfusions from female donors with previous pregnancies were associated with increased mortality and only in male recipients under 50 years. this leads us to postulate that pregnancy induced long term changes in the female immune system are transferred during red cell transfusion, with negative consequences for young male recipients. the low amount of plasma present in red cell products further lead us to assume a cellular component, like passenger leukocytes, to be involved. it has been shown that micro-chimerism of passenger leukocytes can persist for decades after transfusion, even of leuko-reduced blood products, suggesting long term immune-modulation could play a role. we hypothesized that passenger leukocytes would die during storage of blood products and the negative effect of ever-pregnant female donors, on the survival of young male red cell recipients, would therefore be attenuated by increased storage time. however, our data seem to indicate the opposite. the risk of death was increased over three-fold for young male recipients of old (>24 days storage) red cells from ever-pregnant donors, compared to for young male recipients of fresh (<10 days storage) red cells from ever-pregnant donors (3-year cumulative incidence of death 15.4% versus 4.8%). the negative control group (i.e. young male recipients of red cells from male donors) showed a much weaker association of mortality with storage time (i.e. 7.2% versus 4.7%). these findings seem to falsify our hypothesis that mortality could be caused by passenger leukocytes, establishing long term immune-modulatory effects. another potential mechanism that has been suggested could be the presence of cellfree dna in transfused blood products. this cell-free dna increases during storage. however, more research is needed both to establish if cell-free dna can also be linked to previously pregnant blood donors and by which mechanism it could negatively affect young male transfusion recipients. clinical trials (cts), the gems in clinical research for generating robust evidence in medicine and public health, are costly and complicated undertakings. in resource limited setting like sub-saharan africa (ssa) where the health systems are sub-optimal and where capacity for research is limited, the conducting of cts can be a daunting challenge. the challenges of undertaking cts in rls may be categorized based on the occurrence of the bottleneck(s) in relation to the ethics and regulatory approval process: pre-approval: protocol development: in order to develop a context-specific protocol which is subsequently subjected to an ethics and regulatory approval process, investigators need to review and ensure that the protocol is pragmatic and feasible with respect to implementation. this results into a time-consuming reiterative process of reality-checking the protocol. site selection: in light of the limited research infrastructure, investigators in rls and their developed world partners spend considerable time reviewing and selecting suitable sites for participation in the anticipated protocol for the cts. suitable sites are usually very few and with competing on-going studies. approval: institutional review board (irb) approval: the irb approval process can be quite lengthy (6-9 months) with considerable unpredictability in the periods between the initial and subsequent irb reviews. national regulatory approval: the requirements by national regulators are unusually innumerable with limited flexibility to accommodate specific cts. post-approval: the key post-approval challenges for cts implementation in rls are attaining appropriate participant enrolment and maintaining high retention rates. specifically, for participant enrollment, the challenge may be unforeseen competing cts targeting the same participant pool or community perspectives that may discourage participants from getting screened for the cts. retention may also be a challenge particularly where participants view enrollment as a chance to access healthcare services may therefore not have any incentive to keep in a study after the initial study visits. in conclusion, cts are complex undertakings wherever they are conducted but are doubly challenging in rls like sub-saharan africa. the bottlenecks at the preapproval, approval and post-approval stages are considerable. nevertheless, it is rewarding to perform ctus in rls given that the data generated therein is highly valued by national regulators and may hasten the registration process for medical products. background: interest in an appropriate and effective whole blood (wb) pathogen reduction technology (prt) is growing, especially in sub-saharan africa where the residual risk of transfusion-transmitted infections (ttis) remains unacceptably high and wb is still frequently used. cerus corporation, manufacturer of the intercept tm blood system, and swiss transfusion src are collaborating on a clinical development program to adapt intercept prt using amustaline (s-303) and glutathione (gsh) for red blood cells (rbcs) into an appropriate prt for wb in resource-limited settings in africa. treatment with amustaline/gsh has been shown to inactivate a broad spectrum of transfusion-transmissible pathogens in rbcs. studies with amustaline/gsh in wb have shown effectiveness against a duck hepatitis b virus (>5.3 log reduction) and plasmodium falciparum (>7.5 log reduction), with future studies planned. a wb prt system with amustaline/gsh also has the potential benefit of minimal electricity requirements. aims: to describe the safety and clinical objectives for a phase 1 clinical trial using the amustaline/gsh prt system for wb in africa, and describe research and development efforts to adapt the intercept prt system for rbcs into a robust and appropriate wb system for settings with high burdens of tti and limited resources. methods: the protocol for a phase 1 clinical trial using pathogen-reduced wb treated with amustaline/gsh in an african country is presented, as are current research and development activities related to the development of a prt system for wb. results: in the planned phase 1 clinical trial in africa, 20 clinically stable patients with anemia who require wb transfusion will be randomized into two study arms at a large medical center in a sub-saharan african country. enrolled patients will receive one unit of non-leucocyte-reduced wb treated with amustaline/gsh, or a unit of untreated control wb or rbcs. the primary safety endpoint will be the incidence of high-imputability transfusion reactions (swissmedic ≥grade 2) within the first 24 hours of transfusion. data will also be collected on all adverse events and transfusion reactions (all grades) and the development of treatment-emergent antibodies to pathogen-reduced wb or auto-antibodies within 59 (ae3) days of the study transfusion. clinical efficacy will be characterized by hemoglobin increment 24 hours after transfusion adjusted to hemoglobin dose and body weight. summary/conclusions: a prt system for wb is being developed based on the intercept prt for rbcs that is in advanced development in europe and the united states. intercept-treated rbcs have met efficacy and safety endpoints in phase 3 clinical trials. the amustaline/gsh prt system used to treat intercept rbcs has demonstrated effective inactivation against a broad spectrum of agents that may result in ttis. a phase 1 clinical trial using an adapted prt system for wb in africa is the first step in a clinical development program that includes additional pathogen inactivation efficacy studies and improvements to the wb prt implementation process. together, these developments and evaluations represent progress toward a realistic and appropriate prt for wb in africa and other resource-limited settings. background: in australia, demand for plasma-derived products has increased dramatically, and there is a need to increase plasma collections. first-time donor retention, including the rate at which first-time donors return, is a pressing issue. a quick return is optimal as this increases the overall plasma yield and is associated with long-term retention. however, we lack evidence of effective interventions to encourage first-time donors, particularly those donating plasma, to return and to establish a higher frequency donation routine. working from schultz's (2014) framework, this intervention study was based upon insights from interviews with first-time plasmapheresis donors. participants identified barriers such as time and lack of knowledge about plasmapheresis. facilitators included being able to help more people and to donate more frequently than allowed with whole blood. participants generally favoured donating at a frequency of every 4 weeks. aims: the aim of this study was to test the effectiveness of three intervention conditions compared with the business-as-usual (bau) procedure on the proportion of donors returning to donate plasma and the number of plasma donations. we report on the data from 2 months post-donation. methods: donors were randomly assigned to one of four study conditions. in conditions 1 and 2, donors received an email one day after their initial donation. in the first condition, donors received the bau 'thankyou' email. donors in the second condition received an alternative email with content derived from the interview study. donors in the remaining conditions received either the bau email (condition 3) or the revised email (condition 4) coupled with a telephone call. the phone call was scripted to provide additional information about plasma, including how often plasma can be donated, a suggestion to donate every 4 weeks, and a prompt to forward-book appointments. results: the final sample (n = 6788) comprised 3859 women (57%) and 2929 men (43%) aged 18-70 (mean = 32). after two months 37.2% of donors returned to donate plasma at least once. after controlling for gender, age, and blood group, donors in each of the intervention conditions were more likely to return to donate plasma than were donors in the bau condition. the greatest effect was found between donors randomized to condition 4 (revised email + phone call), or = 1.305, ci = 1.128-1.510, and bau. donors assigned to the two telephone conditions (condition 3 and 4) donated plasma at a higher frequency than bau. summary/conclusions: this study tested the effectiveness of interventions designed to encourage first-time plasma donors to return to donate plasma and to establish a routine of donation. early indicators suggest that the evidence-based email and phone call elements are more effective than bau in bringing donors back to donate plasma, and the revised email combined with a phone call had the greatest positive effect on short-term plasma yield. background: healthy individuals with hereditary hemochromatosis (hh defined as hyperferritinemia and homozygous p.c282y mutation), but also carriers of other hfe mutations (p.c282y/p.h63d or homozygous h63d) with elevated serum ferritin (sf) are accepted as blood donors, if allowed by local regulations and if eligibility is fulfilled. generally, blood components are released for transfusion at normal sf levels (< 200 ng/ml in females, < 300 ng/ml in males). aims: prospective, two-center, randomized study comparing the efficacy and tolerability of double-erythrocyte apheresis (2rbcaph) and whole blood phlebotomy (wbph) for iron depletion in asymptomatic subjects with hh or hyperferritinemia and other hfe mutations in the setting of routine blood donation. methods: eligibility criteria included age ≥ 18-60 years, total blood volume ≥ 5 l, bmi < 35 kg/m 2 , hb ≥ 140 g/l, elevated sf levels and no end organ damage due to iron overload. 2rbcaph (360 ml rbc) were scheduled every 14 days and wbph (450 ml) every 7 days until sf was < 100 ng/ml. a complete blood count and sf were measured at baseline, at every visit and at follow up 8 weeks after completion of the study. adverse events were systematically recorded. the treatment effect was tested by poisson regression, with gender, hfe mutation, bmi and baseline sf as covariates. results: 30 subjects (5 females; mean age 47 years) were randomized to wbph (n = 16; 1 female) or 2rbcaph (n = 14; 4 females). hfe mutations were p.c282/ p.c282y in 17 subjects, p.c282y/p.h63d in 9, and p.h63d/p.h63d in 4. at baseline, mean hb was 149 g/l (sd 7.8) and median sf was 504 ng/ml (iqr 406-620 ng/ml). 222 procedures (wbph n = 146, 2rbcaph n = 76) were completed; 9 were interrupted (local hematoma, insufficient flow); 35 (16 wbph, 19 2rbcaph) were postponed because of low hb and 15 for non medical reasons. there were 2 drop-outs in the wbph arm due to depression and poor compliance, respectively. anemia (hb < 130 g/l in males, <120 g/l in females) occurred after 15 visits in 8 wbph subjects and after 5 visits in 3 2rbcaph subjects. fatigue was reported after 37 phlebotomies and 31 aphereses. only 5 participants (17%) completed the study per protocol. 136 blood components (94 rbc concentrates and 42 plasma units) for transfusion were obtained. overall, a median of 7.5 wbph (iqr 6.2-9.8) was needed to reach sf < 100 ng/ml, corresponding to 1.8 times of 2rbcaph (median 4.0, iqr 3.0-5.8) (p = 0.0001). analyzing separately p.c282/p.c282y and p.c282y/p.h63d carriers, the relation wbph to 2rbcaph was 1.6 and 1.8, respectively. treatment arm and hfe mutation were the covariates with significant effect on the primary endpoint (p = 0.0001 and 0.007, respectively). summary/conclusions: 2rbcaph is more efficient than wbph for iron depletion in healthy subjects with hh or other hfe mutations and moderate hyperferritinemia. intensive treatment schedules, generally recommended for hh, are difficult to keep because of hb drop and compliance. less intensive treatment in asymptomatic individuals with hh and their inclusion in blood donation would avoid negative effects on quality of life and benefit blood collection centers in the long term. background: serum ferritin (sf) measurements in whole blood (wb) donors demonstrated that female sex and intensity of donation are major risk factors for iron deficiency. approximately 200 ml red blood cells (rbc) and 200-240 mg iron are lost with wb donation. double unit rbc (2rbc) collections of 360 ml (ca. 40 ml less than the rbc amount of two wb donations) lead to a loss of about 400 mg iron. in switzerland, the maximal allowed donation frequency for male donors is once every 6 months for 2rbc and once every 3 months for wb donation. aims: to describe and compare the course of hemoglobin (hb) and sf in male subjects donating wb and 2rbc at our institution. methods: we included 294 wb and 151 2rbc donors (n = 445) who donated with the maximal allowed donation frequency over 48 months between 2008 and 2013, yielding 4,704 wb and 1,208 2rbc donations. we excluded subjects with hyperferritinemia and known hfe mutations. hb limits were 135 g/l for wb and 140 g/l for 2rbc donation. with 2rbc apheresis 360 ml rbc were collected. sf was measured on a predonation serum sample; hb was determined from finger prick samples. the donors received no iron substitution. we used generalized estimating equation models for hb and sf trajectories. results: mean age at the first blood donation was 53 (wb) and 48 years (2rbc), respectively. at the first donation, mean hb was 153 g/l (sd 13) in wb and 159 g/l (sd 8) in 2rbc donors; mean sf was 44 (sd 52) and 73 lg/l (sd 56), respectively. on average, hb and sf were higher in 2rbc donors (5.1 g/l and 26 lg/l, respectively; p < 0.001). there were 137 subjects with sf < 30 lg/l in wb and 19 in 2rbc group, and 85 with sf < 50 lg/l (but > 30 lg/l) and 40, respectively. in 2rbc donors, between the first and the last donation, mean hb declined from 159 g/l to 157 g/l (p < 0.05) and mean sf from 73 lg/l to 66 lg/l (ns). in wb donors, mean hb dropped from 153 g/l to 152 g/l (p < 0.05) and sf from 44 lg/l to 35 lg/l (p < 0.001). similar results were found when adjusting for age and season. hb values dropped from baseline until the 11 th donation for wb donors and until the 4 th donation for 2rbc donors with an upward trend thereafter. in both groups, no hb value below the limits of blood donation and no anemia were observed. sf reached a nadir at the 4 th donation in both wb and 2rbc donors (37 lg/l and 60 lg/l) and increased thereafter in 2rbc donors. in wb donors, sf followed a parabolic trend that peaked at the 10 th donation, and then declined until the last donation. summary/conclusions: the maximal allowed blood donation frequency for wb and 2rbc male donors in switzerland is not only protective for the development of anemia, but also for deferral of blood donors because of low hb. this was observed even in subjects with low sf at baseline. background: granulocyte concentrate transfusion is a potentially lifesaving option for patients without functional neutrophils. however, recent studies have failed to demonstrate the anticipated clinical effectiveness of this procedure. granulocyte concentrates are manufactured using sedimentation agents to separate granulocytes from red blood cells and enhance granulocyte collection efficiency. high-molecularweight hydroxyethyl starch (hes) is most commonly used for this. however, authorities recently restricted the use of hes due to its unfavorable risk-benefit-profile. modified fluid gelatin (mfg) is an already used alternative sedimentation agent. as the granulocyte product contains these substances, any impact of the sedimentation agent on granulocyte function may affect the clinical effectiveness of granulocyte transfusion. aims: we tested the hypothesis that mfg is not inferior to hes in terms of the functionality and viability of granulocytes. methods: granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15% or 30% mfg (gelafundin 4%, b. braun melsungen ag) or hes (hespan 6%/450/0.7, b. braun medical inc.), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ros) production, neutrophil extracellular trap formation (netosis), antigen expression of cd11b, cd62l and cd66b, and viability were subsequently investigated in vitro. testing was performed using live cell imaging of the cells embedded into a collagen i matrix for parallel testing of migration, ros production and netosis. in addition, flow cytometric (facs) analysis was utilized for surface marker expression, viability and respiratory burst measurement. results: granulocyte migration decreased in a dose-dependent manner in response to hes and mfg. relative to the controls, all three concentrations of hes lowered migration distances (p < 0.001 respectively), whereas only the higher concentrations (15% and 30%) of mfg showed lower relative migration distances (p < 0.001 respectively). track straightness was reduced with both sedimentation agents at 15% and 30% to the same extent (p < 0.001 respectively). hes resulted in lower cd11b expression (p = 0.028) and higher cd62l expression (p = 0.007) compared to the controls, whereas the differences for cd66b did not reach statistical significance. mfg did not affect the expression of any investigated surface antigen mediating endothelial adhesion and transmigration in comparison to the controls. no significant differences in the timing of ros production or netosis, or in neutrophil viability or respiratory burst were observed. summary/conclusions: these results indicate that mfg is not inferior to hes in terms of granulocyte phenotype and function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with hes. background: plateletpheresis donation leads to a well-known transient decrease of donor's platelets. the question of long-term effects raised with the development of regular donations by some donors in order to satisfy a growing demand. a seminal work (lazarus, transfusion, 2001) stated that there is a sustained thrombopenia in frequent plateletpheresis donors, correlated with the total number of donations. aims: french regulation authorizes up to 12 plateletpheresis donations per year, with a minimum 4 weeks interval between them. we tried to evaluate the risk of sustained thrombopenia under these conditions. methods: we retrieved all plateletpheresis donations occurring between 01/01/2014 and 08/31/2018 from the french civilian blood donors' base and then selected a cohort of donors with at least 24 donations during that period. in order to minimize measurement errors, platelet counts analysed were means of three consecutive donations, i.e. 8 measures for each donor. results: the cohort includes 2,186 donors (384 women and 1,802 men). mean platelet counts fluctuate between 276.4 and 278.6 platelets/ml. analysis of variance does not show any statistically significant difference (f = 0.462), even taking donor's sex or age in consideration. there is no difference if we consider the total duration of the 24 donations, either. donors with the lowest first counts show a significant rise in subsequent measures and donors with the highest counts show a decrease trend, exhibiting a classical regression toward the mean. summary/conclusions: plateletpheresis french regulation does not seem to be at risk of sustained donor thrombopenia. this conclusion is in agreement with recent literature data. the primary biological role of the human leukocyte antigen (hla) system is the regulation of the immune response to foreign antigens. because of this role, hla genes and molecules have an important role in transplantation, etiology of many autoimmune, non-autoimmune and infection diseases, but also in transfusion medicine. an increasing probability of an hla non-compatible blood products, tissues or organs exists due to the extremely high polymorphism of hla genes, with more than 20,000 described alleles to date, and their different frequency distribution in various worldwide populations. the hla system, originally discovered as a result of a transfusion reaction in the 1950s, can cause detrimental immune reactions in transfusion therapy. hla antibodies present in the patient are responsible for some of these reactions, while in other cases hla antibodies or hla reactive cells present in the transfused product are accountable for the immunoreactivity. hla antibodies form as a result of exposure to foreign hla antigens during pregnancy, transplantations and blood transfusions and can cause platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury, and transfusion associated graft versus host disease. in order to avoid or reduce the development of these transfusion-related events, hla antibody negative or compatible products should be used. almost all existing methods presently used for molecular typing of hla polymorphisms are based on polymerase chain reaction, but with different resolution levels (low resolution -two digits or high resolution -four digits). in addition to providing a more precise detection of polymorphisms at hla classical loci (e.g. hla-a, -b, -c, -drb1, -dqb1), molecular methods can also determine polymorphisms at hla loci which previously could not be typed by serology (e.g. hla-drb3, -drb4, -drb5, -dqa1, -dpa1). the most commonly used method for the detection of hla antibodies was until recently complement-dependent cytotoxicity (cdc) technique, but it is increasingly being replaced by a more sensitive, solid phase based method (luminex technology). in conclusion, an accurate and precise determination of both hla gene polymorphism and hla antibodies presence is essential for the safe and efficient administration of transfusion products. background: in only a minority of pregnancies complicated with anti-hpa1a antibodies serious fetal/neonatal disease develops. the difficulty in predicting which mothers should be treated with ivig hampers implementation of fnait screening. we found that fc-core fucosylation and galactosylation are highly variable in anti-hpa1a igg, and that these glycan features strongly affect binding to fccriiia receptor. the level of fc-core fucosylation of anti-hpa 1a alloantibodies was found to correlate with platelet count and outcome of the newborn, suggesting that antibodyspecific fucosylation might serve as a biomarker in fnait screening. however, at present the fc-glycosylation pattern can only be determined by complicated methods involving purification of the antigen-specific igg, and analyzing trypticly released -igg-derived-glycopeptides by tandem liquid chromatography-mass-spectrometry (ms) techniques. these methods, although powerful, are not yet suited for high throughput clinical screening. aims: our aim was to provide a simplified method to quantify the biological activity of anti-hpa-1a antibodies, and possibly other alloantibodies against blood cells. methods: here we explored if cellular surface plasmon resonance (spr) imaging can replace ms, resulting in less complicated handling of patient sera and donorantigen-bearing cells. the strength of the binding of platelets to fccr on spr sensor was monitored under flow. the spr sensor was equipped with both wt fccriiia (sensitive to fc-glycosylation status) and mutant fccriiia-n162a (insensitive to fcglycosylation status). in addition, the biosensor was prepared with anti-platelet cd61 (c17) and anti-igg to calibrate the number of injected platelet as well as to quantify igg-opsonization. the quality of the anti-hpa 1a glycosylation was monitored as the ratio of the binding of opsonized platelets to the wt and the mutant n162a-fccriiia. platelets opsonized with recombinant glycoengineered anti-platelet antibodies with different levels of fc-fucosylation were used as standards. for validation, 166 plasma samples with anti-hpa1a antibodies, already analyzed by mass spectrometry and with known clinical outcome were tested (sonneveld, bjh, 2016) . results: we found that the ratio between the binding to the wt fccriiia and to the mutant n162a-fccriiia correlated with the level of fucosylation of the hpa1a antibodies, as measured by mass-spectrometry (r = à0.524; p < 0.0001). overall, a similar predictive value for disease severity was obtained as we previously reported for this retrospective cohort. in addition, quantitative information on antibody concentration can also be extracted using the fccriiia-n162a receptor as sensor on the chip, while anti-igg gave aspecific signals, presumably because it recognized cytophilic platelet-fccriia-bound antibodies as well. summary/conclusions: in conclusion, the combined use of wt and mutant fccriiia in a label free spr assay provides both quantitative and qualitative information of platelet bound anti-hpa 1a antibodies, which circumvents the need for purification of specific antibodies and laborious mass spectrometric analysis. this approach might be generally applicable to determine the biological activity of cell bound antibodies not only for anti-hpa1a in fnait, but also for anti-rhd alloantibodies in hdfn or anti-platelet antibodies in itp. background: immunization against the human platelet hpa-1a alloantigen is the most common cause of severe fetal and neonatal alloimmune thrombocytopenia (fnait) in otherwise healthy term newborns. the screening for hpa-1a antigen in pregnant women is an important tool for identification of pregnant women at risk of having a fetus/neonate with fnait. any targeted intervention depends on efficient screening methods as well as sensitive and specific methods for detection of anti-hpa-1a. within the framework of the polish-norwegian project (prevfnait) we have performed hpa-1a screening program in poland. aims: our aim was to assess the frequency anti-hpa-1a antibody detection and the clinical outcome of newborns identified through the study. women who joined the program due to the fnait in the previous child or in the current newborn are not analyzed in this study. methods: hpa-1a screening of 24 244 pregnant women in 8-40 gestational weeks was performed by facs phenotyping or rq-pcr genotyping at ihtm in warsaw. hpa-1a negative/hpa-1b/1b women were tested for hla drb3*01:01 and for anti-hpa-1a antibodies by maipa (followed up at week 17-20, 28, 32, 38-40 and 6 weeks after delivery). if anti-hpa-1a were detected, quantitative maipa was performed. all hpa-1a negative women were contacted for information concerning the newborn. if the baby had thrombocytopenia and anti-hpa-1a were not detected by maipa, the look back samples were tested retrospectively by paklx test (immucor). results: 554 hpa-1a negative women were identified (2.3%). anti-hpa-1a was antibodies were detected by maipa in 44 women (two delivered tweens). in addition, anti-hpa-1a antibodies were later detected by paklx in further 3 women who delivered baby with severe thrombocytopenia and/or ich. total number of immunized mothers was 47 (8.5%). they delivered 49 babies; 35 were boys. three women were treated by ivig: two by 4 and 8 injections since 33 th and 26 th gw respectively. the anti-hpa-1a concentration in the 1 st one was 0.4; 0.1; 5.88 iu/ml in 17, 28, 32 gw respectively and in the 2 nd <0.05 iu/ml in all examined samples. the decision on treatment was based on the low plt count~50 g/l in the fetus in cordocentesis. their newborns (one delivered tweens) were healthy. the 3 rd treated woman entered the program in 35 gw (anti-hpa-1a concentration was high 22.64 iu/ml). she obtained one injection of ivig. her baby was born with mild thrombocytopenia with no ich. severe fnait occurred in 5/49 newborns: in 3 with anti-hpa-1a detected in paklx only and in 2 with antibody concentration in maipa -1 st : 6.86/12.91/ 23.36 at 28/32/38 th gw respectively; 2 nd : 8.85/17.58 at 32/38 th gw respectively. ich was observed in all of them; plt count was < 50x10 9 in four, 56 9 10 9 / in one. summary/conclusions: 1/ the severe thrombocytopenia due to anti-hpa-1a alloimmunisation in our prospective study occurred in 2/10 000 pregnancies 2/ the paklx could improve anti-hpa detection in the screening program and should be considered as an additional diagnostic test, if maipa result is negative 3/ the hpa-1a alloimmunisation frequency is higher in pregnancies with male than female fetus. background: foeto-maternal platelet alloimmunization (fmpai) is mainly characterized by foetal and / or neonatal thrombocytopenia (fnait), sometimes revealed by intracranial hemorrhage (ich) or even by foetal death in utero (fdiu). the experience of the pnil milwaukee (usa) reported in 2014 that the diagnosis of alloimmunization was carried in only 33% of neonatal thrombocytopenia cases with a clinical symptomatology highly suggestive of an alloimmune etiology. aims: the aim of this two-year study was i) to determine the frequency of platelet incompatibilities in fnait, ich and fdiu and ii) to evaluate the frequency of detectable platelet alloantibodies (alloab) and their specificity in cases of incompatibility. methods: platelet genotyping was performed by hpa beadchip genotyping kit (bioarray solutions, immucor, warren, nj). serology investigation was carried out by 3 different methods: complete maipa kit (apdia bvba, turnhout, belgium), pack lxtm assay (immucor gti diagnostics, waukesha, wi) and « in house » maipa. all 2017 and 2018 data were collected using the laboratory information management system. results: 544 patient files were analyzed. no incompatibility is demonstrated in hpa-1 to -9, -11 and -15 systems in 19.3% (n = 105). hpa-1 and / or 3 and / or 5 incompatibilities were found in 271 cases (49.8%), hpa-2 and / or 15 in 87 cases (16%). platelet alloimmunization was globally confirmed in only 10.6% of the cases. 58 platelet alloabs were identified regardless of clinical manifestations: 24 anti-hpa-1a (41.4%), 20 anti-hpa-5b (34.5%), 8 anti-cd109 (13.8%), 2 anti-hpa-5a and anti-hpa-15b (3.4% respectively) and 1 anti-hpa-1b and anti-cd36 (1.7% respectively). 40 alloabs were found in the context of neonatal thrombocytopenia, 6 in ich and 10 in fdiu, and 1 in a follow-up of pregnancy. even if no anti-hpa-3 alloab could be identified, the incompatibility in this system was highly associated with fnait, ich and fdiu (n = 55, n = 32 and n = 17 on 114 cases). summary/conclusions: this study strongly confirmed the known immunogenicity of some hpa systems and highlighted overall the severity of hpa-3 and hpa-5 incompatibilities. the definite diagnosis of fmpai is difficult to make due to the present technical difficulties in the detection of antibodies against the hpa-3 and hpa-15 systems. however, our results suggest that special attention should be paid to the management of pregnancies with these incompatibilities due to the frequency of severe foetal/neonatal adverse events. background: fetal and neonatal alloimmune thrombocytopenia (fnait) is a potentially life threatening disease caused by maternal alloantibody formation against fetal human platelet antigens (hpas), of which anti-hpa-1a is accountable for the fast majority of the cases. population-based screening for fnait has been topic of debate for over decades. logistically as well as financially, the major challenge of such a screening is the typing of pregnant women to recognize the 2% hpa-1a negative women. at present, hpa-1a typing is mostly done by genotyping. for costeffective implementation of anti-hpa-1a screening there is need for a high-throughput, quick and low-cost phenotyping assay. aims: the aim was to develop a high-throughput, quick and low-cost phenotyping assay in order to identify hpa-1a negative pregnant women. methods: an automated sandwich elisa was developed to perform hpa-1a phenotyping using a murine monoclonal anti-gpiiia as coating antibody and horseradishperoxidase-conjugated recombinant igg1 anti-hpa-1a as detecting antibody. to ensure the applicability for high-throughput testing in a potential screening setting, 20 ll of the uppermost plasma of 3 -6 days-old stored edta anticoagulated blood tubes was used, without first swirling or spinning them. in two phases, samples of pregnant women were tested and compared to an allelic discrimination polymerase chain reaction assay as golden standard. in the first phase, samples from unselected consecutive pregnant women were tested. the second phase was part of a prospective screening study in pregnancy and confirmatory genotyping was restricted to samples with an arbitrary set od < 0.500 in the hpa-1a elisa. the developed elisa was optimized to require no additional handling (swirling or spinning) of stored tubes. during phase i, 506 consecutive samples were tested. in phase ii, the hpa-1a elisa was performed in another 62,171 consecutive samples, with confirmatory q-pcr in 1,825. the two phases combined, samples from in total 1,585 hpa-1a negative and 823 hpa-1a positive pregnant women were genotyped. the assay reached a 100% sensitivity with a cut-off od between 0.075 and 0.200, leading to a specificity of 99.6%. summary/conclusions: a quick, low-cost and reliable assay for hpa-1a phenotyping was developed that can be used in a population-based screening setting to select samples that has to be tested for the presence of anti-hpa1a antibodies. because plasma from non-mixed or spinned tubes of three to six day-old samples can be used, this assay is applicable to settings with suboptimal conditions. background: cytomegalovirus (cmv) sero-prevalence in ireland is lower than that which is reported in many other european countries. a study of 1047 pregnant women in 2002 found that 30.4% of irish women were cmv seropositive in comparison to 56% from western europe and 92% eastern europe and 97% from africa. an internal study carried out by the irish blood transfusion service (ibts) in 2010 indicated the rate of cmv seropositivity in irish blood donors was 21.97%. therefore a significant proportion of the irish donor and recipient population are susceptible to primary cmv. this is of particular concern for patients for certain at-risk groups such as very-low birthweight cmv seronegative neonates, cmv seronegative patients undergoing transplantation and other cmv seronegative immunocompromised patients. this results in a demand for the provision of cmv sero-negative blood components. in 2018 the ibts evaluated the abbott alinity s cmv igg assay as a replacement for the cmv mastazyme eia (total ab eia). aims: to assess the performance of the abbott alinity s cmv igg screening assay in comparison to the cmv mastazyme eia (total ab eia). methods: diagnostic sensitivity was determined by testing 48 confirmed cmv igg positive donors from an external laboratory. sensitivity was assessed using three seroconversion panels (n = 54). analytical sensitivity was calculated using linear regression analysis of the who first international standard for anti-cmv igg. diagnostic specificity was determined by testing 6127 donors. further evaluation of discordant results was carried out using the architect anti-cmv igg and igm assays and vidas anti-cmv igg and igm assays. results: the diagnostic sensitivity of the alinity s anti-cmv igg assay was determined to be 100%. the seroconversion sensitivity reported 42 out of 54 samples reactive. the analytical sensitivity of the alinity s cmv igg assay was determined to be 1.12 iu/ml. the validation reported 65 discordant results from 6127 donor samples tested with both the alinity s cmv igg assay and the current mastazyme total assay. 60 discordant results were observed (alinity s anti-cmv igg positive/mastazyme total negative). further testing of these samples classified 27 discordant results as positive, 12 as negative and 21 as indeterminate. 5 discordant results were observed (alinity s anti-cmv igg negative/mastazyme total positive). further testing classified these samples as negative. overall the diagnostic specificity was determined to be 99.80%. summary/conclusions: both the seroconversion and analytical sensitivities are comparable between the alinity s cmv igg assay, the cmv mastazyme total ab assay, the architect cmv igg assay and the vidas igg assay. the slight variations can be attributed to the individual assay cut-off definitions, which can vary greatly between cmv assays. it must be noted that the determination of the diagnostic specificity (99.80%) does not include indeterminate discordant results. further testing will be carried out to try to characterize all discordant samples in collaboration with abbott. this evaluation did not identify any donors with isolated confirmed cmv igm antibodies in a pool of 6127 donors. based on this evaluation the abbott alinity s cmv igg assay is a suitable replacement to the mastazyme total ab assay for blood donor screening. background: africa has a unique set of challenges regarding safe blood transfusion. two of the largest contributing factors are: 1) the most common disease states in sub-saharan africa (ssa) require large amounts of blood as lifesaving interventions e.g. malaria, 2) the highest burden of infectious diseases transmissible through transfusion (tapko, toure, & sambo, 2014) is found in ssa. this has often led to the binary donor base that exists in ssa, consisting of voluntary non-remunerated blood donors (vnbd) and family or replacement donors (frd) as transfusion centres are unable to supply the demand when relying only on vnbd. voluntary non-remunerated donors are the safest blood donors as they have no incentive (other than altruistic motives) and are not under social pressure to donate, both factors that may induce individuals knowing or suspecting themselves to be infected with a blood-borne agent to donate blood. nucleic acid testing (nat) in conjunction with serological testing is the gold standard for testing, however, the vast distances and high temperatures of africa makes transport of traditional plasma samples a logistical challenge. many publications evaluating the stability, suitability, and ease of use of dried blood spots (dbs) for nat have been published. generally, results have been shown to be comparable to traditional plasma samples. dbs is being used successfully in the early infant diagnosis (eid) programs for hiv by means of pcr testing, especially in africa. aims: 1. to demonstrate that dbs and/or dried plasma spot (dps) testing is suitable for blood donor screening and can make nat testing more widely available in africa 2. to determine the diagnostic sensitivity and specificity of testing dps and dbs samples, in comparison to testing of plasma samples. methods: 900 negative new donor samples and 100 confirmed positive donor samples, as defined by routine blood safety screening done at western cape blood service, were screened using a dried blood spot kit. after routine testing was completed, one dbs sample and one dps sample for each blood donor were prepared and analysed with the ultrio elite assay on the panther analyser. summary/conclusions: dbs/dps can be used as a sample for screening blood donors as the invalid rate was 0.56%, and only found on dbs samples. logistically dbs/dps is well suited for the resource-poor countries as samples are: -easy to obtain (fingerpick samples could be used.) -transport is simplified as samples will not leak or haemolyse due to high temperatures. -samples can be stored at room temperature dbs/dps demonstrated acceptable specificity. the ultrio elite performed well with regards to hiv and hcv sensitivity. sensitivity with regard to hbv was not as high but this could be due to very low and erratic viral loads. background: sanquin blood supply is responsible for the blood transfusion services in the netherlands. at the national screening laboratory sanquin (nss) annually more than 750.000 blood and plasma donations are tested, on average 3.000 samples per day. for more than 10 years, infection serology testing was performed using the prism (abbott diagnostics), but since mid of july 2018, serological testing for the hbsag, hiv ag/ab, anti-hcv and anti-hbc is done with abbott's alinity s system. aims: to compare the numbers of initially and repeatedly reactive results of whole blood and plasma donation samples and the rate of non-specific results leading to deferral of donations and donors for prism and alinity s assays using data from 6 months before and 5 months after implementation of the alinity s systems at nss. methods: initial and repeat reactive rate of the assays run by either prism (hbsag, hiv o plus, hcv) or alinity s (hbsag, hiv ag/ab combo, anti-hcv,) were calculated for january to june 2018 (prism) and august to december 2018 (alinity s). due to the lack of a true confirmatory method for anti-hbc, we only compared the rate of repeatedly reactive results for prism hbc and alinity s anti-hbc. results: the rate of repeat reactive results for prism (p) and alinity s (a) assays were as follows: 1) hbsag p 0.01 % (42/390.736) versus a 0.03 % (87/322.394); 2) hiv p 0.07 % (262/390.735) versus a 0.06 % (201/322.470); 3) anti-hcv p 0.11 % (427/390.737) versus a 0.03 % (109/322.476). the rate of anti-hbc reactive samples was not significantly different between prism (0.39 %) and alinity s (0.42%). over the study period, the rate of initially reactive samples for the three main screening assays (hbsag, hiv, hcv) was also comparable between alinity s (0.39 %) and prism assays (0.34 %), mainly attributable to a rather high number of initially reactive alinity s hiv ag/ab results. this was due to initial issues with blood collection tubes that were resolved. as a result in december, the rate of initially reactive samples decreased to 0.16 %, which was significantly lower than for the three prism assays (0.33%). summary/conclusions: the introduction of the alinity s assays lead to a decrease of the average repeat reactive test results (hbsag, hiv, hcv) by 0.17 % as compared to the prism, mainly due to a lower false reactive rate of the alinity s anti-hcv assay. this will be further investigated for first time and multiple time donors. with the implementation of the alinity s at sanquin we aimed to improve not only the operational efficiency but also to further minimize unjustified disapproval of donors. these first data show that the low initial and repeat reactive rates of the alinity s assays indeed have a positive impact on unnecessary deferrals of donations and donors. background: in blood banks, testing all blood donations for markers of infectious diseases plays an important role in maintaining the safety of blood transfusions. mandatory serological testing in switzerland is performed for anti-hcv, hiv ag/ab, hbsag and syphilis. highly specific and sensitive tests with corresponding automation are essential for this purpose. aims: a comparative study was carried out to evaluate the usability of the newly launched alinity s system (abbott) and the specificity of the infectious disease parameters hbsag, anti-hcv, hiv combo and syphilis (abbott) with the currently used elisa methods on the quadriga befree system (all diasorin, formerly siemens healthcare diagnostics). methods: the study took place at the interregional blood transfusion service in berne, switzerland. the specificity of the parameters was studied on 2,748 blood donor sera from both first time and repeat donors. the samples were tested first on the quadriga be free system with enzygnost hbsag 6.0, enzygnost anti-hcv 4.0, and enzygnost hiv integral 4 assays and on the pk7300 with the newbio-pk tpha assay (newmarket biomedical). all samples were retested on the same day with hbsag, anti-hcv, hiv combo and syphilis on the alinity s. initial reactive samples were repeated in duplicate. discriminatory tests were carried out for repeatedly reactive samples using alternative screening tests and neutralisation (for hbsag) on an abbott architect i1000 system and immunoblots (hiv-, hcv-, syphilis-inno-lia, fujirebio). for all samples, results from our routine individual donation nucleic acid testing (hcv, hiv, hbv, roche cobas 8800 system) were available. results: based on the results from testing 2,748 blood donations, the observed specificities of alinity s assays (a) and enzygnost assays ( summary/conclusions: the alinity s system was easy to use by the operators after a very short introductory training and provides good operational efficiency such as high throughput even when selective testing for samples is needed. the observed specificity of abbott alinity s versus siemens enzygnost assays is comparable in a blood donor screening setting. unfortunately, we were not able to analyse statistically the specificity data due to the insufficient number of donor samples tested in parallel. it is worth mentioning that around 90% of the samples included in the study derived from repeat donors who had been previously tested with the enzygnost assays but were "first time donors" for the alinity s assays. all four assays from both systems exhibit a very good specificity and are highly suitable and practicable for routine blood donor screening. background: effective screening for transfusion-transmissible infections is essential to ensure safe blood transfusions. the world health organization recommends mandatory serological testing of blood donations for human immunodeficiency virus (hiv), hepatitis b (hbv)/c (hcv), and syphilis. due to increasing demands on clinical laboratories, there is a need for reliable and accurate automated blood screening tests. the fully automated cobas e 801 analyser can be used with elecsys â infectious disease parameters to screen donor blood samples. aims: to compare the performance of elecsys â infectious disease parameters on the cobas e 801 analyser (roche diagnostics) with other commercially available assays for routine first-time blood donor screening. methods: we provide results from etablissement franc ßais du sang (montpellier), a blood bank which participated in a large, multicentre study of the cobas e 801 analyser. the following infectious disease marker assays were compared: hiv, elecsys â hiv duo versus prism hiv o plus; hcv, elecsys â anti-hcv ii versus prism hcv; hbv surface antigen (hbsag), elecsys â hbsag ii versus prism hbsag; hbv core antigen antibodies (anti-hbc), elecsys â anti-hbc ii versus prism hbcore; syphilis, elecsys â syphilis versus newbio pk tpha assay. specificity was tested using residual fresh serum samples from unselected first-time blood donors, and calculated according to assay package inserts and site-specific cutoffs. samples were tested using comparator assays, then retested the same day using elecsys â assays. initially reactive samples were repeated in duplicate; confirmatory tests were conducted on repeatedly reactive samples. confirmatory tests: hiv, nucleic acid testing (nat), architect hiv ag/ab and inno-lia â hiv i/ii score assays; hcv, nat, archi-tect hcv and inno-lia â hcv score assays; hbsag, nat, architect hbsag and elecsys â /prism hbsag confirmatory assays; anti-hbc, nat, hbsag, anti-hbs, and architect anti-hbc assays; syphilis, architect syphilis tp and inno-lia â syphilis score assays. sensitivity was tested using 30 preselected, anonymised, positive, citrate-phosphate-dextrose-plasma samples (plasmatec laboratory products) and compared with archived data for comparator assays. sensitivity was calculated according to the final nat result. results: across all infectious disease markers, specificity to detect repeatedly reactive samples using elecsys â versus comparator assays was similar (99.81-100.00% versus 99.79-99.98%; n ≥ 5195). in specificity analyses, there were 14 discrepant results for hiv testing, 27 for hcv, two for hbsag, eight for anti-hbc, and five for syphilis. sensitivity of the elecsys â hiv duo assay (83.33%; 95% ci 65. 28-94.36) was higher than the prism hiv o plus assay (76.67%; 95% ci 57.72-90.07), but the difference was not statistically significant. sensitivities of elecsys â and comparator assays were the same for hcv (85.19%; 95% ci 67.33-95.97), hbsag (70.00%; 95% ci 50.60-85.27), anti-hbc (100.00%; 95% ci 85.75-100.00), and syphilis (100.00%; 95% ci 88.43-100.00); three hcv and six anti-hbc samples were classified negative/ indeterminate and excluded from the analyses. in sensitivity analyses, there were two discrepant results for hiv testing, three for hcv, and five for anti-hbc. summary/conclusions: elecsys â infectious disease parameters on the cobas e 801 analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays. background: individual plasma and serum specimens from whole blood or plasmapheresis donors are tested for absence of infectious agents by serological assays prior to use for transfusion or production of blood derived therapeutics. the department of plasma analytics (pa), takeda (austria), and haema ag, grifols (germany), both labs with high throughput and a high level of automation, were seeking for alternatives to replace their current serological test systems (abbott prism next). aims: to allow a direct comparison of the two final candidate analyzers alinity s (abbott) and cobas e801 (roche diagnostics gmbh), a side by side evaluation was carried out by the pa and haema with support from abbott and roche (provision of instruments and reagents). the aim was to compare assay specificities as well as handling and performance of the instruments. the outcome should be used to better understand potential specificity differences and practical handling aspects (throughput, etc.) of a next generation serological analyzer. methods: the two candidate instruments were installed in the pa. from march to june 2018, close to 10,000 aliquots from routine preselected repeat donors, provided by haema, were run on both study instruments in parallel. plasma samples were tested for hbs antigen (ag), hcv antibody (ab), hiv ag/ab, and partially for syphilis ab. serum samples were additionally tested on hbc ab. samples with repeat reactive results ("rr", two reactive results out of three tests) not confirmed by confirmatory tests were counted as false reactive. the necessary sample size was calculated based on a one-sided comparison of proportions with the aim to detect potential specificity differences (a = 10%) in the size of those specified by the manufacturers' instructions. two different lots were tested for the three main assays. results: out of 7,389 plasma and 2,242 serum samples, 41 test results representing 37 individual donations were found rr on one or both instruments. two samples were confirmed positive (1 9 hbsag, 1 9 hcv), two others were indeterminate. the sample containing low level antibodies against hcv was pcr negative and only detected by the roche system. the percentage of false reactive results for the five assays on the two systems were (alinity s/e801): hbs ag: 0.04/0.03 % in a total of 9590/9589 samples tested; hcv ab: 0.01/0.09 % in 9620/9585, p < 10%; hiv ag/ab: 0.03/0.09% in 9362/9329, p < 10%; syphilis ab: 0.1/0.07% in 2971/2987; hbc: 0/0% in 1531/1549. no significant difference was found between the calculated specificities in our study and the manufacturers' data. a potential influence of sample matrix and kit lots was assessed. a trend towards more false reactive results in serum vs plasma was found for nearly all assays. no clear-cut statistical difference was seen between lots. summary/conclusions: the study results are in line with the manufacturers' specificity data, showing that the alinity s hcv ab and hiv ag/ab assay show a slightly higher specificity in a population of plasma and serum samples from repeat donors prescreened by prism. a possible influence on the test specificity by the sample matrix was detected but needs further investigation. the possibility to edit complex genomes in a targeted fashion has not only revolutionized basic research but biotechnological and therapeutic applications as well. with the rapid development of genome editing tools, in particular zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and the crispr-cas system, a wide range of therapeutic options have beenand will bedeveloped at an unprecedented speed. therapeutic genome editing in hematopoietic cells enable new interventions in the blood and immune system, including novel approaches to treat immunological disorders, infectious diseases, and cancer. we have developed gmp-compliant protocols to manufacture gene edited cd34 + hematopoietic stem and precursor cells (hspcs) as well as chimeric antigen receptor (car) t cells, with the final goal to provide novel cell therapies for patients suffering from primary immunodeficiencies, chronic infection with human immunodeficiency virus type 1 (hiv-1), and some tumor entities. despite great success in improving their specificity, engineered designer nucleases can induce genotoxic side effects by introducing mutations or chromosomal aberrations. we have established novel genome-wide assays that enable us to detect chromosomal aberrations induced not only by off-target activity but also by on-target activity, such as micro-aberrations and translocations, with unparalleled sensitivity. in toto, our developed protocols allow us to achieve genome editing in hematopoietic cells with high efficiency and to assess the genotoxic risk associated with the expression of crispr-cas nucleases and talens in clinically relevant human cells, so forming the basis for planned phase i/ii clinical studies. adoptive t cell therapy (act) has proven a potent means to treat blood-borne tumors and solid tumors. adoptive cell therapies include t cells that are genetically engineered with tumor specific t cell receptors (tcrs), or with chimeric antigen receptors (cars). in addition, tumor infiltrating cells (tils) can be isolated from tumor lesions, which are then expanded and reprogrammed in vitro prior to transfusion into the patient. the anti-tumoral efficacy of act products depends on several parameters, including the capacity of cd8 + t cells to produce cytokines, chemokines and granzymes, a feature that is critical for effective anti-tumoral responses. here i will discuss our efforts to develop and improve act products for future clinical use. i will present pre-clinical work on developing til therapy for non-small cell lung cancers. in addition, i will show that human cd8 + t cells can be divided into different subsets, and that only one of those subsets is highly cytotoxic. this finding may help improve the quality of genetically engineered t cell products, like tcr and car t cell products. background: the baltic states -estonia, latvia and lithuania have a lot in common. we are located side by side, share the baltic sea as a gate to the west, and more importantly, a common history. we were members of the ussr and suffered 50 years of soviet occupation. we held hands in a 600 km long human . . .chain" across the three states to express our mutual support, and later on, even joined the european union on the very same day -june 1 st , 2004. the three differ a bit in size, population and more in the languages spoken in each one, but that does not explain why the path towards voluntary unpaid donation varies as it does. aim: the aim is to describe the journey towards voluntary non-remunerated blood donation in the baltic states after regaining independence from the soviet union. methods: the information was collected from published and unpublished memories, annual reports and written interviews with latvian and lithuanian colleagues. results: in soviet times, all orders came from moscow and quality control was conducted from the capital city of latvia, riga. donors were mostly paid and given an extra vacation day. big factories were the best places to collect blood and people were queuing to donate. in 1991, the soviet union fell apart and the baltic states suddenly got the freedom and responsibility to decide. in estonia the first edition of "guidelines for the preparation, use and quality assurance of blood components" was taken as guidance in 1992. a lot of advice came from finnish colleagues. in 1997, it was decided to move towards non-paid voluntary donations. the process took 6 years. the first couple of years were economically difficult for the reborn state, as money had less value than food. instead of cash, donors were given rapeseed oil, sugar and pasta, for example. as the situation improved, food items were replaced by small symbolic gifts that carry sentimental value. it has been this way for more than 15 years by now. in lithuania, the process started later, the first program for developing a framework for voluntary non-remunerated donations being carried out in 2006-2015. it resulted in 51% of the donations being unpaid. the second program initiated in 2016 is still ongoing, aiming towards 100% non-remunerated donations by 2020. by the end of 2018, they had reached 99.2%. in the beginning, the main obstacle was a private blood center creating unfair market conditions. in latvia, monetary compensation for blood donations still exists, but the younger generation has been encouraged to donate blood for free and some results can already be seen. summary/conclusions: a common starting point does not guarantee the same results, at least not at the exact same time. examining the circumstances leading to the different outcomes could benefit countries yet to start moving towards non-remunerated donations as well as those considering the opposite. haemoglobin (hb) was as expected significantly different between women and men (meanaesem: 13.8 ae 0.01 vs 15.5 ae 0.01 g/dl; p < 0.000). percentage of females with low hb < 12.5 g/dl were 7.1%, 4.9%, 5.2%, 6.7% and 4.2%, percentage of males with hb < 13.5 g/dl were 0.5%, 0.6%, 0.7%, 1.7% and 1.2% for the age groups 1-5 respectively. ferritin values were higher in males compared to females (median; 25 th -75 th %>tile: 51;31-79 vs 157;106-231 lg/l; p < 0.000) and in older age groups compared to younger age groups (median; range in age groups 1-5 in females: 40;3-702, 52;6-619, 60;6-480, 60;8-725, 98; 10-604 and in males: 99;8-661, 161;18-1080, 206;15-1090, 220;26-1430, 221;33-981 respectively) . percentage of females with ferritin ≤ 15 lg/l were 8.6%, 3.8%, 3.7%, 6.7% and 2.0%, while percentage of males with ferritin ≤ 15 lg/l were 0.2%, 0.0%, 0.1%, 0.0% and 0.0% for the age groups 1-5 respectively. white blood cell counts (wbc) were slightly higher in females compared to males (meanaesem: 7.2 ae 0.02 vs 6.6 ae 0.03; p < 0.000). percentage of females with wbc > 12x10 9 /l were 1.8%, 1.3%, 1.7%, 1.4% and 0.3%, while percentage of males with wbc > 12x10 9 /l were 1.4%, 0.9%, 0.5%, 1.2% and 0.9% for the age groups 1-5 respectively. none had wbc < 2x10 9 /l. platelet counts (plt) were higher in females compared to males (meanaesem: 257 ae 0.6 vs 222 ae 0.6; p < 0.000).percentage of females with plt < 150x10 9 /l were 1.0%, 1.5%, 2.2%, 2.4% and 1.0%, while percentage of males with plt < 150x10 9 /l were 2.6%, 3.6%, 2.7%, 2.4% and 1.6% for the age groups 1-5 respectively. among the low plt counts most were caused by edta-dependent pseudothrombocytopenia. extreme deviations from normality were seldom and referred to gps for further investigations. summary/conclusions: first time donors are young with 75% younger than 30 years of age and the female/male ratio was 58/42. of the 16 583 first time donors with data on ferritin available, 14% had low ferritin (≤ 30 lg/l). the typical male first time donors neither had low hb nor low ferritin, even with a significantly lower ferritin in younger donors. in female first time donors the prevalence of low hb (6%<12.5 g/dl) and low iron stores (23%≤30 lg/l) is high. in all, while all first time donors are highly appreciated, campaigns could target the male population to even out the gender imbalance. blood centers must be aware of the higher prevalence of low iron stores in the youngest donors. background: the aim of assessing suitability of prospective blood donors is protection of their health and the safety of transfused patients. selection process is not always effective in obtaining all relevant information from blood donors in a timely manner. for several reasons, some risks remain undetected or they are disclosed at a future donation(s). therefore, recording and management of post-donation information (pdi) are of great importance for improvement of transfusion safety, donor counselling and education as well as overall improvement of the selection process. aims: the aim of the study was to present results of pdi management at croatian institute of transfusion medicine (citm) and the effect of education activities on their trends. methods: we have analyzed reports on pdi recorded in two-year period (2017-2018), according to the types of information obtained, age and sex of blood donors, total number of their donations preceding pdi, and the time of receiving the information. the effect of an information leaflet on pdi launched in november 2017 was assessed by comparing results in two study years. results: a total of 491 pdi were recorded: 266 in 2017 (1/396 donations) and 225 in 2018 (1/452 donations) with the following distribution: nonsexual risk as tattoo and piercing (17.5%), surgical procedures (17.1%), travel history (16.1%), infections/ contact (15.3%), other medical reasons (13.4%), endoscopy/invasive diagnostic procedures (12.2%), malignancy (4.3%), autoimmune diseases (3.7%) and sexual risks (0.4%). majority (76.4%) were late pdi, revealed on the future donation(s): 58.7% on the first next donation, 28.9% on the second and 12.3% after more than 2 subsequent donations. the mean age of blood donors associated with pdi was 40 ae 12 years (median 39 years), while the mean age of all donors in 2017/2018 was 38 years (median 37 years). of all pdi, 81.1% were related to male donors (84% in total pool of citm donors). using chi-square test there were no significant difference between female and male donors in total pdi frequency and in their distribution to early and late pdi (p > 0.05). the median number of all donations preceding pdi was 6 for female donors and 19 for male donors. implementation of education leaflet for blood donors resulted in 15.4% reduction of pdi in 2018 compared with 2017 (p > 0.05). the effect is more pronounced (p < 0.05) when comparing second and first half of 2018 (-25.6%). reduction is observed in all types of pdi with the exception of infections/contact (because they are mostly early pdi) and malignant diseases. the share of early pdi increased from 21.1% in 2017 to 26.7% in 2018, which may suggest better awareness of blood donors on the importance to inform blood bank on changes in their health status. summary/conclusions: our study points to the importance of systematic recording and management of pdi, including education of blood donors about the need of providing all relevant facts related to their health and the safety of donated blood in a timely manner. we are planning further improvements by providing information on this topic on posters and screens on donation sites. background: currently, the transfer of data between organizations and/or computer systems is very limited, and where present is typically proprietary. in the absence of a standardized reference format individual organizations and vendors attempting to integrate disparate databases must develop unique solutions. aggregation of information from multiple sources is complex and costly, constituting a significant barrier to effective analysis of data to improve practice and inform policy. aims: to standardize the definitions and facilitate integration of key data items used in blood donation and transfusion. we report here on an initial effort to map internationally harmonized critical steps in the blood collection/donation process in order to test the approach. methods: through a collaborative process of serial conference calls and correspondence, an informal multi-national consortium of experts across the transfusion industry are attempting to create a vocabulary with sufficiently precise definitions to be usable by automated systems and that can be the foundation of a blood collection/transfusion medicine common data model (cdm), using the following steps: -define the scope of activity to be addressed and segment into key processes. -identify the set of data elements in each segment that are common to all systems. -review and consider existing standards and definitions for each data element. -develop draft definitions for each data element. -release draft to public domain for critical review and refinement with long-term goal of gaining widespread endorsement. results: a standardized approach to blood donation was mapped through identification of common pathways and core mappable data elements. denominator data associated with donor characteristics and blood collection was selected as the first segment to address. a dictionary (or vocabulary) of common terms has been created and will be presented for international comment. summary/conclusions: developing an international consensus on the core elements and their definitions across the transfusion chain is critical for data integration and automation efforts. the expected benefits of this endeavor include that it allows the establishment of algorithms to automate reporting and thus reduce hands-on staff time; reduces time and resources needed to integrate new databases; allows systems to continue to use existing concepts and definitions internally while also providing data output in a standardized format; supports the ability to consistently analyse, interpret and present information regardless of the data source; establishes data definitions against which new systems can be developed; helps to improve comparability of results by providing a common data model for researchers and policy makers; improves confidence in data integrity and reliability of the derived information as a © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 basis for rational decision making; and reduces data gathering effort and cost thus improving opportunities for more efficient/complex data analysis. standardizing the transfusion medicine dataset is the first step in achieving the automation of data transfer and analysis needed globally to drive patient safety, research innovation, and best business practices. further steps must address the precise methods of data exchange, identification of responsible entities for maintenance and further development, and engagement of computer system developers. red blood cell (rbc) alloantibodies develop in a subset of individuals following exposure to non-self rbcs through transfusion, pregnancy, or other activities; these antibodies can lead to difficulty locating compatible rbcs, acute or delayed hemolytic transfusion reactions, or hemolytic disease of the newborn. alloimmunization is underestimated due in part to antibody evanescence, the random nature of posttransfusion antibody screens, fragmented medical care, and the lack of widespread antibody registries. factors that influence who will develop detectable alloantibodies are not well understood. transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many rbc units (and many non-self abo blood group antigens). individuals with sickle cell disease (scd) and myelodysplastic syndrome (mds) are more likely to form rbc alloantibodies than most other patient populations. individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher than average risk of forming rbc alloantibodies. inflammation, in a broad sense, is one common thread among these diagnoses associated with high prevalence rates of rbc alloimmunization. reductionist murine models support some types of inflammation (including viral-like stimuli) around the time of rbc exposure as being associated with an increased likelihood of alloantibody formation. strategies other than transfusion avoidance or extended antigen matching beyond abo/ rh would be beneficial to prevent new rbc alloantibody formation, especially in patients at highest risk. background: the unique genetic makeup of the omani population makes them rich in the genetic blood disorder. 45 % of omani populations are àa/àa gene carriers, 44% àa/aa, and 11% of the population are aa/aa. around 10 % of omani nationals carry the gene for hbs, and 2 -3 % carry the gene for b-thalassaemia. recent statistics show that there are around 400 patients with thalassaemia major and 3000 with scd in oman. the other rbc abnormality that is common in oman is g6pd deficiency which is found in 28 % of males and 12 % of females. omanis are known to have the highest frequency of a thalassaemia and g6pd reported so far in any race. although blood transfusion is one of the supporting treatments of scd, it can cause some serious complications for the patients. alloimmunization of red blood cells is one of the consequences of blood transfusion. alloimmunisation of the rbcs can cause haemolytic transfusion reactions and may trigger hyperhaemolysis, in which transfused and patient's own rbcs are destroyed. alloantibodies can cause delay in the process of transfusion, it can be costly and time consuming. high number of patients developing alloantibodies may indicate a major difference in the patient and donor population. it may also indicate lack of a controlled, generalised sickle patients management policy. in oman the decision of transfusing scd patient is left to physicians attending the patient. aims: this study is aimed to highlight the increasing number of alloimmunised sickle cell patients. in the royal hospital we get 40 new cases of sickle patient with alloantibodies each year. the acknowledgement of these cases may help in is assessing the current practice of transfusing scd patients, or will help to define the donor and patient population difference. methods: 418 patients were recruited in the royal hospital for this study. edta blood samples were taken for antibody screening test and in the positive cases antibody was identified, all tests done by capture technique using immucor neo machine. results: of the 418 scd patients, 49% of the patients were male and 51% female, mean age was 17 years, in the range of 1-72 years. 38 % of the scd cases were positive for the alloantibodies, 72% were female and 28% were male, the age range was from 6-68 years. 78% of the positive were scd, 19% s trait and 3% were s/ bthal. most of the patients developed one antibody, however cases of multiples antibodies were also detected. 51% of the patients were with single alloantibody, 30 % of them with two antibodies, 10 % with three antibodies, 7 % with four antibodies and 2 % with five antibodies. the majority of the cases were igg against rh antigens anti-e is being the majority 23%, followed by anti-d 13%, anti-k 17%, anti-c 14%, anti-c 8%, anti-jk a 5%, anti-jk b 5%, anti-fy a 5%, anti-e 3%, anti-s 3%, antis 1%, anti-kp a 0.7%, anti-fy b 0.3% and igm being 2%. summary/conclusions: rbc alloimmunisation rate is high in oman majority of the patient affected are female. interestingly sickle trait patients were also transfused and 19% of them developed alloantibodies. the practice of transfusing rh and kell matching blood unit is implemented four years ago and still high alloimmunization percentage is achieved. background: in ghana, routine pre-transfusion investigations for patients with sickle cell disease (scd) involve only abo-d typing and immediate spin crossmatch, without screening for irregular rbc antibodies aims: determine the prevalence and specificities of and risk factors for rbc alloantibodies in multi-transfused patients with scd methods: in 2018, a cross-sectional study in multi-transfused patients with scd, from two tertiary hospitals in ghana was performed. participants' data on demography, transfusion and medical history were recorded. antibody screening and identification tests were done at sanquin, the netherlands, with standard serology using liss as enhancer and with papain treated rbc panel cells ('enzyme only'). characterization of rhd genes was done by multiplex ligase amplification assay. logistic regression was used to determine the association of patient characteristics, i.e. sex, age at enrollment (continuous), age at first transfusion (categorized as ≤ 1, 2-5, 6-9 and ≥10), previous pregnancy, number of transfused units (2, 3-5 and 6-10 and > 10), and years after last transfusion (<1, 1-2, 2-5, >5y) with presence of alloantibodies results: 226 patients (100 males and 126 females, median age 17 years, range 1.7-66) were included. the median number of transfusions was 3 (range 2-40). the median years after last transfusion was 2 (range 2 weeks-55.5 years). in 56 patients, anti-rbc antibodies were detected. in 14 of them the antibodies were weakly reactive with enzyme treated cells only or pan-reactive, possibly some of them representing autoantibodies or antibodies against high frequency antigens. in seven patients enzyme-only anti-le a was demonstrated, likely naturally occurring antibodies. thus, in at least 34 patients (15.0%) alloimmunization was demonstrated or suspected; in 11 patients the alloantibodies were 'enzyme only'. besides, the 36 alloantibodies of known specificity (8 anti-d, 2 anti-d+c, 16 anti-e, 1 anti-c, 3 anti-e, 1 anti-k, 1 anti-s, 1 anti-le a , 1 anti-go a ), three antibodies reactive only with fy(a-b-) cells and two antibodies of yet unidentified specificity were detected. in six d-patients (3 had been pregnant) anti-d (together with anti-c in two patients) was found. in three out of four d+ patients with anti-d, an rhd variant gene was demonstrated (2 dau-alleles and 1 diii type 4 or diva-2). logistic regression revealed that none of the risk factors analysed was associated with the presence of antibodies in the 34 patients. immunobiology -red cell alloimmunity 65 fifty-eight patients, had experienced an adverse reaction during or shortly after transfusion (12 patients had dark urine). adverse reactions were associated with the number of units received (or 1.71 (95% ci, 1.25-2.33; p = 0.001), but not with the presence of antibodies (p > 0.7) summary/conclusions: in at least 15% of multi-transfused patients with scd alloimmunization could be demonstrated, mainly (80%) directed against rh antigens. the enzyme only reactivity, coupled with absence of antibodies in seven of 12 patients with probable haemolytic reaction and known evanescence of especially non rh antibodies suggest possible low titre and disappearance of some clinically relevant antibodies. given the high immunization rate together with the high frequency of adverse transfusion reactions, pre-transfusion screening for rbc antibodies should be considered for patients with scd. background: rh blood group system and mainly antigen d is one of the most immunogenic, diverse and clinically important protein-based blood group. antibody anti-d may induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. anti-d prophylaxes become ineffective if an anti-d immunization has occurred. approximately 1% of the d+ population carries rhd alleles associated with reduced d antigen expression. qualitative variants, in which some epitopes are lacking and can produce anti-d antibody, are usually termed partial d. by contrast, d weak is commonly defined as a quantitative variant that have all d epitopes and should not make anti-d. del is a very weak form of d antigen and cannot be detected by routine serological tests. because some of del individuals have already developed an anti-d antibody whereas others did not this group contains both qualitative and quantitative changes. aims: investigation was prompted by finding discrepant results in typing of d antigen in a pregnant woman /3 rd pregnancy, 1 st delivery, 2 abortions in 1 st trimester/. routine serological techniques detected d negativity and the presence of antibody allo-anti-d in clinically significant titre. the non-invasive testing of d status of the foetus from maternal peripheral blood was indicated, but this was not applicable due to presence of the rhd gene in the woman's dna sample isolated from buccal swab. our aim was to investigate the discrepancy and determine the underlying rhd genotype. methods: blood samples, dna from peripheral blood and buccal swab of the pregnant woman were investigated. routine blood grouping and antibody testing were performed by column agglutination. two anti-d sera (id-diaclon anti-d igg (cell line esd1) by biorad and anti-d duo igm+igg, clone: th28 + ms26 by immucor) were used for adsorption/elution test for identification of del phenotype. initial rhd genotyping was performed by rt-pcr (exons 5,7,10) with the dna from buccal swab; further resolution was performed using pcr-ssp (fluogene; inno-train diagnostik gmbh); sequencing was performed by sanger analysis (inno-train diagnostik gmbh). results: genotype was identified as rhd positive by ce-certified pcr-ssp kits (fluogene). sanger sequencing of rhd from exon 1 to 9 revealed presence of a nucleotide deletion in position c.147dela, which is specific for allele rhd*01el.04. this nucleotide change results in the amino acid change p.val50leufs*5 causing the del phenotype. presence of antigen d was proved by adsorption/elution technique. titre of the anti-d was rising during the pregnancy to the 2000 level two weeks before the delivery. the newborn was delivered by s.c. without a sign of hemolytic disease. blood grouping of the newborn revealed blood group a, d negative, dat negative, testing for del was not performed. summary/conclusions: the case reported here shows that females with rhd*01el.04 allele are able develop strong anti-d immunization, so this type of del phenotype belongs to the "partial del subgroup". presence of variant rhd gene in mother disabling antenatal fetal genotyping from maternal blood by current methods requires a more attentive approach to care for such pregnancies. supported by mh cz-dro uhkt 00023736 and rvo-vfn 64165. 5a-s29-05 ea scharberg 1 , s rothenberger 1 , a st€ urtzel 1 , n gillhuber 1 , s seyboth 1 , e richter 1 , g rink 2 and p bugert 2 1 institute for transfusion medicine and immunohematology, drk-bsd ba-w€ u-he, baden-baden 2 institute for transfusion medicine and immunology, heidelberg university, medical faculty mannheim, mannheim, germany background: rb a (di6) is a low prevalence antigen of the diego blood group system. it has been found in few families only. the clinical significance of anti-rb a is unknown so far. the slc4a1*c.1643c>t (p.pro548leu; isbt allele name: di*02.06) allele is the molecular basis of the rb a antigen. in the gnomad database this gene variant was found in only one of 125,742 sequenced genomes (allele frequency: 0.000004). aims: to prove the frequency of the allele in our population and gain an rb a positive donor we performed a molecular screening for di6 in 1,700 blood donors. after our antibody screening test accidentally contained an rb a positive test cell we found out that anti-rb a is a very common antibody specificity. the frequency of the antibody in patients and blood donors was proved. methods: for the molecular screening of the blood donors we developed a pcr-ssp method. the antibody screening test in 3,652 patients and in 964 blood donors was performed in the gel technique (biorad ahg id-cards) using a 3 cell screening panel (drk-bsd src) including an rb a positive test cell. positive reactions with the rb a positive cell were confirmed by an additional rb a positive test cell of different source. additional antibodies were excluded or identified in the same method using an antibody identification panel (drk-bsd irc). results: the molecular screening for the di*02.06 allele in 1,700 blood donors revealed no single positive individual. within the first 2 weeks of usage of our antibody screening test which accidentally contained the rb a positive test cell 84 patients with anti-rb a were found. it was 2.3% of 3,652 patients tested in 21 laboratories in different parts of germany. some laboratories stopped using the rb a positive lot to avoid expensive and time consuming identification and conformation tests. in 18 of 964 randomly tested blood donors (1.9%) anti-anti-rb a was also present. summary/conclusions: despite the very low frequency of the di*02.06 allele, anti-rb a is a very frequent unexpected antibody in patients and blood donors in germany. it is obviously naturally occurring and is even more frequent than anti-wr a and anti-vw we found in previous studies in around 1% of patients and donors. 5a-s30-01 national blood center, ministry of health and sports, yangon, myanmar hemovigilance which detects every event not only for patient' reactions and donor's complications but also incidents and near misses definitely improve quality of blood transfusion services especially for those situations where implementation of all the standards in one time is not possible. healthcare system in myanmar is still in the stage of requiring priority for clinical professions and has limited resources for supportive roles. supportive services including transfusion service are still not a center of interest from prioritization of health care system. blood transfusion service has been practiced in myanmar since 1935. real essence of transfusion service is hidden behind laboratory practice and transfusion is regarded as part of laboratory investigation. hospital laboratories take care of testing of blood donated by replacement donors. this kind of transfusion services under laboratory umbrella is still being practiced in myanmar except national blood center (nbc) which was established in 2003 in accordance with blood and blood product law. this law was formulated cohesively with who strategies of blood safety. in 2002, who global data-based study sent questionnaires for assessment of safety status of transfusion service. nbc noticed that there was no data which can support corrective actions for safety. from that time onward, active retrospective review of existing data and introduction of records, prospective finding of process errors and any events from hospital blood banks were recorded and taking into actions at local level. cost of every unit of blood is supported by government. in 2017, national blood and blood product committee was established. the steering committee is working hard to get cooperation from every service by aiming to prevent those undesirable events before establishment of national level policy, standards and guidelines for sustainable service quality. in conclusion, by using essence of hemovigilance as a tool, quality of transfusion service can be improved step by step to fulfil the gap in spite of limited resources. the system started in local, extends to regional level by getting agreement of importance from hemovigilance results and is finally approaching to national level endorsement. background: erroneous transfusion of abo-incompatible(aboi) blood almost always reflects a preventable breakdown in transfusion protocols and standard operating procedures and can have disastrous consequences, with significant morbidity and mortality. these incidents need to be investigated in a systematic manner to identify system vulnerabilities to mitigate risks and improve patient safety. since 2016, reporters to shot have been asked to score(0-10) the extent to which the cause of incidents can be attributed to key factors: staff, environmental, organisational and government/regulatory which helps recognise the key factors identified whilst investigating these incidents. aims: to understand why unintentional transfusion of aboi blood components continue to happen despite standard procedures and national guidance available. methods: retrospective analysis of unintentional transfusion of aboi blood components reported to shot between 2010-2017(inclusive) was done to identify common themes and recognise areas of improvement. information provided using the shot human factors investigation tool (hfit) between 2016-2017 was reviewed to understand more about why the errors occurred. results: sixty-seven unintentional aboi transfusions were reported between 2010-2017; majority (56/67, 83.6%) were red cell transfusions but aboi plasma (9/67) and platelet transfusions (2/67) were also seen. most errors occurred in the clinical area (45/67, 67%), and could have been detected at point of administration. in 21(31%) cases, the error could not have be detected at the point of administration with a primary laboratory error in 10/21(48%) incidents. reviewing data from hfit for cases in 2016-2017 (13 aboi cases), the total score for staff culpability was 100, compared to a total score of 99 for all the other three organisational and system factors. this disparity is most obvious for the 4 aboi red cell cases, all of which scored the maximum 10 for staff culpability, i.e. 40/40 compared to 8/120 as the combined total score given to the other factors. in the preceding years (2010 to 2015), there were no hf scores available; however, the emphasis on staff-related culpability is demonstrated by 37 cases that included an outcome of the local case review and 14 (37.8%) mentioned staff-related retraining or disciplinary procedures. the risk of haemolysis and serious harm is more likely with aboi red cells than with other components with 2/56(4%) that resulted in death, 14/ 56(25%) major morbidity and 40/56(71%) no or minor adverse reaction. of these cases, one resulted in conviction for manslaughter and at least two staff dismissals. summary/conclusions: transfusion never events continue to occur, and it is evident that investigations into such incidents focus mainly on staff failings and do not consistently identify system wide changes that need to be incorporated to address prevalent issues. national recommendations and a safety alert to 'use a bedside checklist' immediately prior to administration were issued between 2015-2018 to support prevention of such errors but never events continue to persist. current approach is ineffective because it often leads to apportioning blame, rather than understanding the often-complicated and multidimensional factors contributing to the error. this must be replaced by a holistic approach which addresses local work pressures and embraces advances in automated technology like electronic prescribing and barcode scanning. of the confirmed trars, n = 27 were possibly related to treatment, n = 2 trars were probable, and n = 2 were definitely related to treatment; n = 37 trars were grade 1, n = 4 were grade 2, and none were grade 4. in recipients of conventional wb, there were n = 13 (1.12%) ars, n = 32 (2.75%) fnhtrs, n = 1 (0.09%) taco, n = 0 trali, and n = 16 (1.38%) unclassified transfusion reactions. of the confirmed trars, n = 55 were possibly related to treatment, n = 1 trar was probable, and n = 8 were definitely related to treatment; n = 54 trars were grade 1, n = 1 was grade 2 and n = 1 was grade 4. there were 21 mirasoltreated wb transfusions in pregnant women and 2 trars (9.5%), both grade 1 and probably related. there were 80 transfusions of mirasol-treated wb and 84 transfusions of conventional wb in patients < 18 years old resulting in n = 7 (8.33%) trars in recipients of mirasol-treated wb and n = 10 (11.90%) in recipients of conventional wb. summary/conclusions: timely data reporting of trars and expanding the hv infrastructure has helped to improve the hv system in ghana. of 2181 wb transfusions in routine use in ghana, there were 4.02% trars in recipients of mirasol-treated wb and 5.59% in recipients of conventional wb. additionally, mirasol-treated wb was safely transfused in pregnant women and pediatric patients. haematology, monash health, melbourne, australia background: transfusion-associated graft-versus-host disease (ta-gvhd) is rare and usually fatal. it can be prevented by provision of irradiated blood products to at-risk individuals, such as those receiving nucleoside analogues, alemtuzumab, bendamustine or with hodgkin lymphoma (hl). duration of risk is uncertain, so ensuring these individuals correctly receive lifelong irradiated blood components, as currently recommended by anzsbt and bsh guidelines, is challenging. in australia, platelets are routinely irradiated, but red blood cells (rbc) are not. aims: to determine whether patients receiving fludarabine, cladribine, bendamustine, alemtuzumab, or dacarbazine (for hl), appropriately received irradiated rbcs. secondary outcomes included rates of ta-gvhd after unintended exposure to non-irradiated components, factors influencing correct issue of irradiated rbcs such as transfusion management plans, and provision of adequate clinical information on blood requests. methods: we performed a retrospective audit to identify patients receiving therapies indicating risk for ta-gvhd using pharmacy dispensing records from january 2008 to october 2018 at monash health, a multi-campus university hospital in melbourne, australia. diagnosis, treatment dates, group and hold (g&h) requests, rbc transfusions, and follow-up information were sourced from laboratory and medical records. results: we identified 310 patients who received fludarabine (n = 52, 17%), bendamustine (n = 29, 9%), cladribine (n = 17, 5%), dacarbazine for hl (n = 164, 53%) and alemtuzumab (n = 48, 15%). the median age of patients was 46 years (range 8-92) and 171 (55%) were male. median follow-up was 30 months (range 0-132). post-exposure, 42 patients (14%) received transfusions with 33% correctly receiving irradiated rbcs. the remaining 28, all from haematology/oncology, received a total of 192 unirradiated rbcs. in 8 patients, this was rectified on subsequent transfusions. there were no cases of ta-gvhd at median follow-up of 14.5 months (range 0-75) from first rbc transfusion. after medication administration, 99 patients had g&h requests after a median of 3 months (range 0-129). only 23% of requests had sufficient clinical information to prompt irradiation, such as hl or medication details, and only 20% asked for irradiated components. preventive strategies have now been employed. transfusion management plans for haematology patients were implemented in march 2017. for audited patients, these were written from 38 days prior to 104 days after medication exposure. two were written following inadvertent unirradiated rbc transfusion. patients identified in this audit will have a laboratory flag generated and prospectively, pharmacy dispensing records will be sent to blood bank to identify at-risk patients. our hospital is transitioning to electronic medical records (emr). an alert will be generated in emr when ordering transfusions if there has been exposure to these medications. however, clinical awareness and documentation remain vital. additional measures include patient education, alert cards, and ongoing collaboration with medical staff to encourage transfusion planning. summary/conclusions: recognition of patients at risk for ta-gvhd remains low, even among haematology units. we are making progress on ensuring provision of lifelong irradiated blood components in patients exposed to nucleoside analogues or alemtuzumab, as well as hl patients. implementation of an emr and additional strategies in this domain is important to prevent ta-gvhd. background: blood transfusion is considered an essential element in the management of patients globally. it might be risky and transfusion related adverse reactions may occur with the less adherence of transfusion policies. standard guidelines regarding the screening of blood for infectious disease, genuine need of transfusion and abo compatibility are followed and monitored drastically. however, patient assessment during transfusion especially at patient bedside and post transfusion is also equally important. aims: we are a newly established hospital and are working towards the best possible management of patients. in this regard to minimize the transfusion errors and to highlight if any lacking being practiced during transfusion, we conducted this study to observe the compliance rate of documentation of transfusion form by the healthcare staff and also to observe the compliance of line of action taken in case of occurrence of transfusion reactions. methods: this was a observational study conducted at nibd and bmt, pechs campus from february 2018 to february 2019. ethical approval was obtained prior to the study. transfusion form for each transfusion was filled. the form provided information on documentation of blood product receiver name, employee identity number, date and time of receiving blood product, patient name, medical record number on units, on patient's wrist band and on transfusion form. abo compatibility on the unit and on form, medical record number from wrist band, name and employee identity number of two healthcare staff started transfusion, transfusion start and completion time. time, temperature, blood pressure, pulse and initials of staff at the time of order, onset, after 15 minutes and at the completion of transfusion were also included. transfusion reaction form was also filled by the healthcare staff. data was analyzed by using spss version 23.0. results: a total of 500 transfusions forms were analyzed. over all compliance rate was 18%. out of 500, 115(23%) forms were available in source notes and of 115, 88 (76%) were partially and completely filled. higher compliance was seen in the initial months of hospital establishment than later months (p-value = 0.000). highest non compliance was seen in documentation of initials of duty doctors on transfusion form at the completion of transfusion(3%) and highest compliance was seen in documentation of name by healthcare nursing staff at the start of transfusion(89%). a total of 02(0.4%) adverse events were reported from red blood cells and platelets. mean time of start of symptoms was 2 hours and 30 minutes for red blood cells and for platelets it was 1 hour and 13 minutes. transfusion was instantly stopped as the symptoms appeared with no delay of time and actions were taken to resolve the reactions. time of appearance of symptoms and time of start of medication were documented and error free. all blood bags were returned to the blood bank and discarded after 6 hours as per the policy of hospital. summary/conclusions: the study was conducted to highlight the scarce practices that are being implemented by healthcare staff in context of documentation and reporting of transfusion reactions at our hospital. stringent actions should be taken for the adherence of compliance by healthcare staff to avoid morbidities and mortalities. we believe that it will also be helpful to provide baseline information in the process of preparation of a national guidelines and protocol on blood transfusion procedures. 5a-s31-01 as buser, a holbro and l infanti regional blood transfusion service, swiss red cross, basel, basel, switzerland to make blood supply safer, pathogen inactivation (pi) technologies have been developed. they are based on photochemical (amotosalen/uva or riboflavin/ uv) or uv-c light treatment to reduce potential pathogens in blood components. this gain of safety might however be offset by "off target" effects of these technologies. in virtually all clinical platelet transfusion trials, it has been demonstrated that post transfusion increments with pi platelet (plt) components are lower as compared to conventional components, indicating different biological behaviour such as survival/ clearance of nontreated and treated plt. published studies have also suggested shorter survival of platelets in vivo in animal studies. additionally, data of the rates of alloimmunization and refractoriness after transfusion of pi platelets are show discrepant results. animal studies suggest a reduction of the rates of alloimmunization when transfusing (leukoreduced) pi plt as compare to conventional plt. in the clinical setting, published data, including very recent reports, showed different rates of hla class i and ii alloimmunization with the two currently available photochemical based pi technologies. while pi of plt components surely benefit patients regarding pathogen safety, the impact of potential off target effects possibly impairing efficacy of pi plt transfusions need more investigation. background: brucellosis is an endemic disease and still a major health problem in saudi arabia. ministry of health in saudi arabia listed brucellosis as a notifiable disease due to its endemicity. in the last ten years, the incidence has decreased significantly to approximately 15 cases per 100,000 but is still higher than that in developed countries. human-to human transmission is extremely rare including breast feeding, transplacental, sexually and blood transfusion. five cases of brucellosis through blood transfusion have been reported in the literature. brucella transmission through blood transfusion is likely underreporting due to the long incubation time of 2-4 weeks (range, 5 days to 5 months),vagueness of clinical presentation and lack of hemovigilance systems in endemic areas. (allohsct) and 130 (4.6%) autologous (autohsct) hsct patients, with mean corrected count increments (cci) of 8.7 9 10 3 , 8.0 9 10 3 and 7.2 9 10 3 , respectively. mean cci decreased in a linear fashion between day ≤ 2 and day 7 pcs (9.0 9 10 3 , 9.1 9 10 3 and 8.5 9 10 3 at ≤ 2 days; 6.7 9 10 3 , 5.8 9 10 3 and 4.9 9 10 3 at 7 days, respectively), although the number of pc transfused on day 7 to autohsct patients was small (n = 71). background: nipah virus (niv) is a paramyxovirus (genus henipavirus) that emerged in the late 1990s in malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in bangladesh and india. niv infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of niv contaminated foods. nipah virus (niv) belongs to the list of pathogens identified by the who to have the potential for a global pandemic. aims: this study aimed to investigate the efficacy of the theraflex uv-platelets system to inactivate niv in platelet concentrates (pcs). the theraflex uv-platelets system (macopharma) uses uvc light without the need of any additional photoactive compound. methods: plasma reduced pcs from 4 bcs (35% plasma in additive solution ssp+) were spiked with virus suspension (10% v/v). pcs (n = 2, 375 ml) were then uvcirradiated on the macotronic uv machine (macopharma) and samples were taken after spiking (load and hold sample) and after illumination with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) j/cm 2 )). the titre of the niv (malaysia) was determined as tissue culture infective dose (tcid 50 ) by endpoint titration in microtitre plate assays on vero 76 cells (atcc â crl-1587 tm ). the results of the infectivity assay demonstrated that uvc irradiation dosedependently inactivated niv. after spiking a niv titer of 6.2 (bag no. 1) and 6.5 (bag no. 2) log 10 tcid 50 /ml was received in the pcs. at a uvc dose of 0.10 j/cm 2 and higher niv was inactivated down to the detection limit of the system (1.9 log 10 tcid 50 / ml), resulting in log 10 reduction factors of ≥4.3 (bag no. 1) and ≥4.6 (bag no. 2). summary/conclusions: our results demonstrate that the theraflex uv-platelets procedure is an effective technology to inactivate niv in contaminated pcs. vs. 364 ae 20.6 9 10e11 platelets/unit, p < 0.001), whereas the platelet content of apheresis pc did not change (305 ae 9.9 vs. 300, ae16.1, p = 0.57). summary/conclusions: pathogen reduction resulted in the transfusion of older pc on average, but without altering the number of pc ordered or the use of pc per patient. pathogen reduction has improved pc stock management without an increase in platelet demand, despite lower platelet content of buffy coat pc after pr implementation. donors and donation -donor adherence -are we doing the right thing? the transfusion procedure is the last step in a multi-process supply chain. the task of matching supply with demand requires donor managers to consider average consumption rates on a weekly or monthly basis, but to also have insight into variability in order distribution and possible attribute (blood groups) requirements. since hospitals and blood banks are usually not deeply interwoven and often only ex-post data is available, forecasting methods should be implemented. a thorough analysis of order pattern to set weekly target inventories and safety levels is required to close the information gap. a collection plan needs to identify possible bottlenecks which can be prevented through the planning of inter-shipping, changes in message urgency and building of reserve donor pools. constant analysis of collection and mobilization kpis allows donor managers to implement the rolling-wave planning approach and continually adapt to changing requirements, unexpected events and overall systematic variability. the variability happens on the demand side, as order quantities and their attributes, such as blood group distribution, are subject to change. however, also the supply is subject to significant variation, as donor response rates, attrition, deferrals and overall availability of donors are not constant. the data was collected with the face-to-face interview method right after the donation. 1478 first-time donors has attended to the study in 18 regional blood centres in 29 cities in turkey. the survey included items in accordance with the standard tpb predictors of attitude, self-efficacy, and intention. self-identity, anticipated regret, donation anxiety, paraphernalia anxiety, personal moral norm, descriptive norm, satisfaction, motivation also assessed for the first-time donors. the relation between the predictors and intention confirmed with correlation analyses. the predictors' distribution analysed by multiple linear regression. a number of goodness-of-fit indices were calculated and examined for each tested models (ibm, amos spss). the results of goodnessof-fit tests for proposed model provided a better fit to the data than these models (cmin/df = 14). moreover, this result indicated that the fit between the proposed model and the data could be improved with further modifications with the inclusion of paths between motivation and attitude, self-identity and intention. moreover, inclusion the paths between donation anxiety and intention and between self-efficacy and attitude, on contrary to recent analyses suggesting opposite paths. evaluation of goodness-of-fit tests showed good result for revised model with a value of cmin/df = 3.55, close to perfect fit. the revised model revealed that attitude was the strongest positive direct predictor of intention followed by personal moral norm, self-identity, motivation and anticipated regret (path coefficients: 0.47, 0.28. 0.19, 0.17, and 0.5, respectively). donation anxiety was the negative direct predictor of intention (-0.05). satisfaction was the strongest positive indirect predictor of intention via attitude and followed by self-efficacy (0.90 and 0.08). paraphernalia anxiety was the negative indirect predictor of intention (-0.03). descriptive norm did not show any significance. our model accounted for 75.3% of the variance in intention. summary/conclusions: these findings suggest several potential avenues for enhancing donor retention. the results obtained with this study provide important data from the standpoint of donor retention, which should be, implemented in the future strategies of turkish red crescent. background: transpose-transfusion and transplantation: protection and selection of donors, is a european consortium project, including partners from 16 countries, reviewing donor selection and protection policies for substances of human origin (soho).one of the main issues in the current donor selection system, which transpose aims to tackle, is that for many, if not most criteria, is not evidence based. the transpose consortium therefore tries to re-assess selection criteria, revised them where needed and provide recommendations as evidence-based as possible. transpose additionally adds to the current european directorate for the quality of medicines & healthcare (edqm) guidelines by emphasizing donor safety. aims: the aim is to compare existing donor eligibility criteria throughout europe, and to compile a list of risks to consider, with evidence-or consensus-based deferral criteria to provide more uniform donor screening criteria. methods: there are three horizontal work-packages (wps); wp1 coordination, wp2 dissemination, and wp3 evaluation of the project, and four technical ones with specific deliverables and milestones to be regularly produced: -wp4 inventory of donor selection & protection practices; -wp5 development of risk-based guidelines for donor selection and protection; -wp6 development of a standard donor health questionnaire (dhq); -wp7 training course/workshop on the use of the guiding principles, guidelines and the dhq. the transpose project launched in september 2017 and will complete in spring 2020. wp4 has completed its work in october, wp5 will complete its work in june 2019, and wp6 and wp7 have recently commenced. results: with the use of the deliverables created by wp4, we have created an indepth inventory of current practices in donor selection and protection, including overview of similarities and differences across european countries and across soho types. there is an agreement amongst experts that existing guidelines are often based on the precautionary principle rather than on risk assessment. consequently, in the development of wp5's guidelines for donor selection and protection, we now make an effort to also emphasize donor safety, in a more evidence-based way via the use of risk-based assessments. this will result in a standardized dhq with a common trunk and more in -depth questions per soho. summary/conclusions: the impact of the outcomes of transpose will be threefold. first, outcomes are expected to be of help in revising donor selection and protection related eu directives. second, the set of guiding principles and donor selection & protection guidelines will facilitate eu member states to take a next step in implementing donor selection and protection policies in a consistent and clear-cut way to the benefit of both donors and recipients of soho. third, a standard donor health questionnaire with carefully guided local/regional/national adjustments will become available per soho which can be used widely and will consequently enable comparisons of the prevalence of certain risks and risky behaviours throughout europe. background: transpose-transfusion and transplantation protection and selection of donors is a european consortium project, including partners from 16 countries, that reviews donor selection and protection policies for blood, plasma, tissues, assisted reproductive technology (art) and stem cells (together soho). donor selection criteria (dsc) in europe are based on eu-directives, guidelines and countries' own additional criteria. literature shows that particular criteria are outdated or not risk-based, often leading to unnecessary donor deferral or an underestimation of risks for donors. aims: to 1) provide a comprehensive inventory of current systems for selection and protection of donors and donations, 2) critically review them and 3) recommend an over-arching donor health questionnaire (dhq) including all necessary criteria currently used by different eu-member states (eu-ms). methods: in-depth semi-structured interviews with key stakeholders in blood collection were conducted to identify main topics for improvement in the current dsc. these formed the basis for a survey sent to professionals from collection institutions of all soho to get feedback on current systems from as many eu-ms organisations as possible. questionnaires were sent to a total of 163 experts (40 blood; 40 plasma; 27 tissues; 47 stem cells; 9 art) and 39 (24%) completed questionnaires were received. where information was lacking, additional experts were asked to recommend upon dsc. results: for blood and plasma donation four main areas of concern in dsc were identified: risk-based selection, adaptability, flexibility and consistency. the stakeholders agreed that dsc are often outdated and lack evidence, hence leading to unnecessary deferral of donors and underestimated risks for donors. they suggested to base dsc on group risk-assessment (risk-based selection) and on conducting more research to achieve standardized risk perceptions and evidence-based deferrals, either for safety of recipient or donors. criteria could be made more detailed to fit specific groups to defer less donors (adaptability). furthermore, implementing criteria was considered easy, but abolish criteria when not regarded as a risk anymore seems almost impossible (flexibility). additionally, deferral periods are perceived too long, seen as both negative, i.e. jeopardizing donor return intention and positive, i.e. no risk for safety (consistency). changing legislation into guidance was an often-mentioned suggestion to improve dsc. specific feedback on plasma donations revealed that many whole blood topics are not applicable to plasma-only donors, e.g. parasite infections such as malaria (no deferral needed); travel history (no deferral needed), and recent bacterial and viral infections (deferral periods currently too long). a clear need for more research on plasma collection-related issues was identified. summary/conclusions: dsc are perceived redundant on a substantial number of aspects by most stakeholders. besides achieving the goal of save and sufficient soho for patients, many regulations could be improved to diminish deferrals and decrease donor risks. transpose will add to reviewing, improving and harmonising these regulations and criteria. furthermore, transpose will provide suggestions to improve directives and guidelines and a dhq, focusing on both donor health protection and safety of donations, but also removing deferral criteria that are not relevant (anymore), and offer a future research agenda to make dsc more evidence-based. background: transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of 24 stakeholders from both not-for-profit and private blood collecting organizations as well as researchers and officials. the project aims to create new evidencebased donor selection criteria as well as guiding principles for risk assessment of threats to the safety of all substances of human origin (soho) except solid organs. as part of this, an inventory of current donation-related risks was performed, including an investigation of both type and number of adverse events reported. aims: we here aim to present an overview of reported adverse events in plasma and whole blood donation in europe and to compare this to the anticipated risks rated by transpose stakeholders. methods: national or local data on adverse reactions from the years 2014-2017, both serious and mild, in whole blood and plasma donors was collected from the relevant stakeholders (eighteen and nineteen respectively). stakeholders were also asked to grade the most important anticipated donor risks according to severity, level of evidence and prevalence. we then compared the relevant risk categories as evaluated by the stakeholders with the categories of the provided data, as well as the heterogeneity of category numbers. results: thirteen stakeholders provided data on adverse events during whole blood donation in a given year, including in total thirty-three different categories of adverse events, ranging from only one unspecified reaction to seventeen different categories, with an average of nine categories per stakeholder. the most frequently used categories were hematoma (included by 77%), arterial puncture (77%) and nerve damage (54%). vasovagal reactions were also frequently included (75%); however, this was being done variably as vasovagal reactions unspecified, and acute and/or delayed vasovagal reactions. only one stakeholder reported iron deficiency. for plasma donation, seven stakeholders provided data on adverse events. a total of twenty-seven different categories were reported, ranging from one to seventeen per stakeholder, with an average of nine. the most frequently reported adverse events were hematoma (86%), citrate reactions (86%) and arm pains and nerve damage (both 57%, respectively). anticipated risks in blood donation were rated by nine stakeholders rating iron deficiency, vasovagal reactions and hematomas the greatest risks to donors. for plasmapheresis, six stakeholders rated vasovagal reactions, hematomas and citrate reactions as highest risk. summary/conclusions: as shown, categories used to describe adverse events in blood donation vary tremendously across europe, with some countries only being able to provide total numbers of adverse events without further specification. furthermore, there is a gap between perceived high donor risks and reported adverse reaction categories in donor vigilance for whole blood, as reports on iron deficiency are virtually absent despite being considered the most significant risk. our findings show the need for international collaboration on creating an international standardized donor vigilance system, to gather more insight into donor risks to protect the health of donors. plenary session -a glimpse of the future pl-03-01 modern transplantation medicine has made significant progress within the last decades due to a better immunological understanding of rejection and advances in immunosuppression. however, the severe side effects of long-term, typically lifelong, immunosuppression and the shortage of donor organs remain the major restrictions in transplantation. the idea behind all research to improve transplant outcome has always been the modification of the recipient's immune system to ideally induce a specific tolerance towards the donor's graft. in fact, the immunological blindness of the recipient towards the donor's graft is achieved by a general reduction of the immune system's competence and represents a major burden for transplant patients. the idea of invisible organs is an entirely different approach to solve the problem: instead of inducing an immunological blindness of the recipient's immune system an immunological invisibility of the donor's organ is created. this is achieved by genetically engineering the transplant to eliminate the organ's immunogenicity defined by the gene products of the major histocompatibility complex (mhc) and minor histocompatibility antigens. in addition to manipulating the expression of mhc genes required for immune recognition, immune cloaking strategies are used to evade immune rejection. these approaches take advantage of creating an immunosuppressive environment and expressing immune suppressive molecules by immunomodulatory transgenes. mhc engineering and immune cloaking in an entire organ is achieved during ex vivo perfusion by lentiviral transduction of gene expression modifiers and transgenes to induce a permanent immunological invisibility of the organ. importantly, mhc engineering also prevents the presentation of minor histocompatibility antigens, which usually are not possible to match between donor and recipient, but which trigger potent immune responses and graft rejection. eliminating the targets of cellular and humoral rejection as well as creating an allograft-specific immune environment through immune cloaking camouflages the organ and equips it with a powerful set of defense weaponry. immune-engineering of transplants achieved during the inevitable ex vivo period of the allograft after explantation without the need to accept off-target effects allows keeping the recipient's immune system fully functional and capable to combat infections and cancer. in pre-clinical in vivo studies from rodents to minipigs a clear survival advantage of ex vivo engineered transplants could be demonstrated. this approach has the potential of eliminating the burden of organ rejection and immunosuppression, thereby sustainably increasing transplant survival, organ availability and quality of life. gene editing for sickle cell disease: re-expression of the fetal c-globin genes (hbg1/2) could be a universal strategy to ameliorate the severe b-globin disorders sickle cell disease (scd) and b-thalassemia by induction of fetal hemoglobin (hbf, a 2 c 2 ). we have previously identified bcl11a erythroid enhancer sequences, marked by hbf-associated common genetic variants, that are required for repression of hbf in adult-stage erythroid cells but dispensable in non-erythroid cells. recently we have optimized conditions for selection-free on-target crispr-cas9 editing in human hscs as a nearly complete reaction without detectable genotoxicity or deleterious impact on stem cell function. we demonstrate that cas9:sgrna ribonucleoprotein (rnp) mediated cleavage at core sequences of the + 58 bcl11a erythroid enhancer results in highly penetrant disruption of gata1 binding motif, reduction of bcl11a expression, and induction of fetal c-globin. erythroid progeny of edited engrafting scd hscs express therapeutic levels of hbf and resist sickling, while those from b-thalassemia patients show restored globin chain balance. moreover we find that hscs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. nhej-based bcl11a enhancer editing approaching complete allelic disruption in hscs appears to be a feasible therapeutic strategy to produce durable hbf induction. in this presentation, i will compare and contrast bcl11a enhancer editing to other autologous curative gene therapy and gene editing approaches at various stages of clinical and pre-clinical evaluation. oxygen is vital for life. without oxygen death is assured for aerobic organisms. although everybody knows this fact a lot of medical acts forget to take care of it, leading to a lot of potential troubles. indeed, during cell respiration the glucose oxidation by oxygen gives carbon dioxide, water and energy. this energy also called atp is necessary for cellular metabolism and consequently for life. we have identified an extracellular hemoglobin coming from a marine worm, called arenicola marina, which is able to deliver oxygen to this animal living in the intertidal areas on the atlantic coast in france between the north sea and biarritz. this molecule called m101 was developed in the medical device named hemo 2 life â . we have showed that this product was very efficient to protect organs before transplantation. a multi centers clinical trial performed under the supervision of pr. le meur from the chu of brest, on 60 patients waiting kidney grafts showed a delay graft function reduced roughly by three between the two kidneys harvested on the same donor with and without hemo 2 life â and grafted on recipients. in 2018, a world first was realized in france by the pr. lantieri to georges pompidou hospital in paris, france. indeed, it was the first time that a patient received a second graft face. this surgery was realized with hemo 2 life â and showed a very nice result according the pr lantieri, the anastomosis were very easy and no edema was observed. furthermore, we have developed dressing incorporating m101 making a product called hemhealing â . preclinical data on diabetic mice showed an increase of healing process. hemoxycarrier â , a therapeutical oxygen carrier, is also in progress of development in order to address ischemic diseases such as the sickle cells disease, myocardiac infraction and stroke. this universal oxygen carrier without blood typing, which is the ancestor of our red blood cell containing hemoglobin showed that it is able to deliver oxygen at different biological levels, cellular, tissues and organs and could address a multitude of medical applications. background: main goal of transfusion is saving life and/or improve the health status of human by "safe blood" which needs regular, voluntary, unpaid blood donors. donor recruitment is being more sensitive and challenging part of the blood supply system in actual global socio-economic conditions. achievement to enough voluntary non-remunerated blood donation (vnrbd) can be established by an efficient donor recruitment. efficiency of the donor recruitment has still close relation with blood donor recruiter although there are so many new tools. occupational specifications, rights and responsibilities of blood donor recruiter have wide range differences between countries which cannot be explained completely by the specific conditions of each country. also, a concrete document which has an international consensus was not existing on this subject. turkish blood foundation (tbf) has been organizing an international workshop since 2012; anatolian blood days (abd). "who is a blood donor recruiter?" was the topic of abd-vii at 9-11 march 2018. aims: main aim of the workshop was to check and evaluate the existing systems of the participant countries. than create a model for clearly defining occupational specifications, responsibilities and rights of blood donor recruiter. methods: experts from 25 countries participated in the workshop. those countries are albania, algeria, bosnia-herzegovina, estonia, france, germany, hungary, india, kazakhstan, lithuania, macedonia, montenegro, oman, portugal, qatar, romania, russia, saudi arabia, serbia, slovenia, sri lanka, tajikistan, turkey, uganda, uzbekistan. these countries reflect almost all religious, ethnical, social, cultural and economic situations of the world. a questionnaire which was analyzing existing systems at participant countries sent before the workshop. after country presentations 4 different discussion groups were organized. below listed topics were announced at final declaration. results: donor recruiter: 1. should have university degree preferably in marketing and business administration field. 2. should have a certificate and/or professional experience in public relation 3. should have efficient skill in conversation, sociability, independence, self-confidence, reliability, resilience and conscientiousness as well as to work in a team 4. should get a special training which includes not only social topics such as public relation, marketing, etc. and medical topics related bb&tm before practicing alone as a donor recruiter 5. should be a permanent staff 6. should have basic salary and performance bonus might be given 7. is eligible to monitor and modify mobile team working period at blood drive 8. should participate the mobile blood drive which he/she has organized 9. should participate the group who will create promotional materials for national blood service 10. number at each blood establishment should be defined based on annual blood collection such as 5 staffs for 50,000 whole blood collection annually in germany. summary/conclusions: in conclusion; both donor recruitment and retention are not easy tasks to undergo while public are aging, and birth rates are decreasing all around the world. dedicated blood donor recruiter whose occupational specifications, rights and responsibilities are clearly defined will be the corner stone of the success for providing enough safe blood for transfusion. ct smit sibinga 1 and j emmanuel 2 background: africa is a large continent with 55 independent states and a total population of 1,275,710,034 (february 2018) . healthcare policies and strategies are developed through who's advocacy, guidance, and support from hq in geneva and the 2 who regional offices; eastern mediterranean regional office (emro) supporting 8 arabic speaking countries and the african regional office (afro) responsible for 47 sub-saharan countries. population distribution is approximately 40.6% urban. there are a large number of different local dialects and languages spoken. the main languages spoken are english, french, portuguese, spanish and arabic. countries are mainly classified by undp as being of low and medium human development index the africa society for blood transfusion (afsbt) has members in most countries, advocates for the development of sustainable and effective blood services, and has developed a stepwise 3 level accreditation program. in 2016 emro held a consensus meeting developing a "strategic framework for blood safety and availability for 2016-2025" with a set of priority interventions focusing on leadership and governance, cooperation and collaboration, provision of safe blood and blood products, appropriate clinical use of blood, and quality system management. in 2001 all 54 member states of the african union (au) countries, in abuja, nigeria, pledged that national budget for health should be at least 15% of the national fiscal budget. in 2013 ministers of health of who member states endorsed that blood and blood products be included in the essential medicines list; these endorsements and who's universal health coverage (uhc), have yet to be fully implemented. aims: to analyze (gap-analysis) to what extend countries in africa implement the world health assembly resolution wha63.12 on availability, safety and quality of blood products, which urges governments to ensure safe, accessible, affordable and available supplies of blood and components from voluntary non-remunerated blood donors, which meet clinical transfusion requirements and achieve national self-sufficiency, following who guidelines and recommendations. methods: to provide an overview of the current status of the blood supply in africa strengths and weaknesses, data from who's 2016 global status report on blood safety and availability were analyzed and used. the study has been descriptive and explorative. results: the 2016 report identified a number of areas requiring attention; principle amongst these were -inadequate funding; -lack of governance and leadership; -ineffective public education on blood donation; -absence of capacity building for clinicians on rational use of blood; -lack of haemovigilance and implementation of quality management systems; -the need for regulatory or oversight mechanisms. summary/conclusions: national authorities should address areas requiring attention if progress towards ensuring a sustainable safe and sufficient supply of blood products is to be achieved. key is the commitment and support of national governments, which should implement resolutions and recommendations agreed by ministers of health at wha and african union. background: the core function of the blood donation testing (bdt) laboratory is to screen every unit of blood collected from a donor for blood group type and infectious disease markers to ensure safety of the national blood supply prior to transfusion. the lab operates daily on two work shifts, comprising of 4 staff on the morning (am) shift (from 8:00 to 17:30) and 5 staff on the afternoon (pm) shift (from 13:00 to 22:00) on weekdays and 3 staff on the am shift and 5 staff on the pm shift on weekends. bdt lab has a staff strength of 15 to be rostered for the 2 work shifts. each staff is on a five-day work week and has to work 11 pm shift and 9 am shift per month on average. the higher number of pm shift leads to staff feedback that they do not get sufficient time in the evenings for family and social or leisure activities. a lean six sigma project was initiated to review the work rostering to improve the work-life balance of the staff. aims: the project aims to reduce the number of staff working on the pm shift without affecting the downstream processes and continues to meet the timely release of blood supply to the hospitals. methods: lean six sigma tools were used to study the bdt lab workflow process and to identify factors that contributes to the higher number of pm shifts that the staff has to take on. data on the turn-around time and the man-effort required for each screening tests performed was analyzed. a survey was also conducted to understand the preference of the staff on the acceptable number of pm shifts per month. results: the main contributing factor for more staff required to perform the pm shift is due to majority of the daily donation samples being received only in the evening. as this factor is beyond the control of the bdt lab, redeploying work from the pm shift to the am shift was eventually selected as the solution to reduce the number of staff needed for the pm shift. the screening test that was shifted was determined based on the test system that has the shortest turn-around-time and is able to allow continuous release of results. at the same time, most of the staff must be trained for that test system. a trial on the new roster involving 5 staff on am shift and 4 staff on pm shift was conducted. the total number of pm shift per month was reduced from 124 to 112 using the re-defined process. the 10% reduction translates to fewer number of pm shifts that the staff has to undertake and was able to meet the staff's expectation. summary/conclusions: with the adoption of the new process workflow, bdt lab was able to reduce the number of pm shifts that the staff needs to be rostered using evidence based process improvement method. most importantly, the lab has a satisfied team of staff with better work-life balance. background: preparedness of blood transfusion services for emergencies and crisis situations is an important issue concerned with patient and transfusion safety. aims: having an experience of delay in blood component supply in an emergency situation due to partial interruption of hospital information system (his), it is aimed to create a crisis kit and constitute an alternative work flow for emergency in crisis situations. methods: it is stipulated that the failure of his which is normally conducts all process for transfusion would be disabled in a disaster or crisis situation. a brain storm was made on possible challenges associated with disability of his during transfusion emergencies. according to the scenarios a kit was developed for the process management of transfusion emergencies. results: a flow chart was designed in proper with transfusion emergency definitions of who and instructions were written to explain the flow chart. all forms categorized with different colour codes are designed to fill with handwriting. the kit consists of flow chart and instructions, analysis request forms (blue coloured), blood component request forms (pink coloured), proceeding forms (green coloured), pens and blood sample tubes with edta were put into a plastic folder labelled as transfusion emergency disaster & crisis (tedc) kit. additionally, the kit is placed in a sealed clear plastic bag and delivered to all inpatient and intensive care units of pediatrics and pediatric surgery. a training programme concerned with transfusion emergency situations and usage of the tedc kit was developed for health care workers involved in blood transfusion process. pre and post-assessment tests were developed for the evaluation of effectiveness of the training programme. summary/conclusions: it's challenging to improve the response capacity of blood transfusion services during emergencies and crisis situations. abstract withdrawn. background: india is a developing country having 2760 licensed blood banks majority have manual documentation which causes inaccuracies and errors in blood bank activities. monitoring is a herculean task. computerisation is the need of the hour but this goal involves many hurdles and challenges aims: the aim of this study is to discuss the challenges faced during computerisation of blood bank activities and the remedial solutions for it methods: department of transfusion medicine, king george medical university, lucknow is one of the biggest blood bank of the country with annual collection of 70,000 blood units. two years ago, the blood bank worked on totally manual system. computerisation involved challenges associated with hardware and software installation and personnel training. hardware was installed in two phases. initially hp system but later shifted to apple imac due to frequent breakdowns. with hp server. software installation (easy software) involved erratic internet connectivity hence changed to lan. customisation involved radical changes according to our needs. at times we had to change our way of working to suit the software. biometrics linking, medical registration, cash id generation, donation, serological crossmatch, automated blood grouping, labelling, chemiluminescence & nat testing, blood component preparation and camps were all included with challenges at every level. remedial actions were taken from small to big. training of the staff was the most essential part of the implementation of computerisation who initially showed considerable resistance and at time faking ignorance due to apprehension that their mistakes will be highlighted and they will be penalised for it. it was a herculean task in creating their password protected identity and enforcing them to use it. gradually the staff realised that computerisation made their task easier as it cut down on paper work and repetition and also prevented serious mistakes from happening. hard copies at certain essential areas were still maintained to continue work in the event of major breakdown of computer results: computerisation aided us in regulating the movement of the donor which at times was repetitive due to manual entry. transfer of data ensured a safe supply and the mistakes could be retraced very easily. implementation which included installation, training and enforcement took a period of 6 months. after overcoming all the challenges we minimised hard copies to 6 registers and started taking printouts of the other necessary details. the turnover time for the employees due to computerisation decreased by 20%. waiting time for attendant decreased by 10%. traceability of all the units became 100%.supervision of the activities being carried out was 90% accurate. identification of the donors was easy due to biometrics which included thumb impression and iris scanning. the decision making time for donors decreased by 50% thus making the system more efficient. summary/conclusions: manual to computerisation involves involvement from source to supply and it is essential to anticipate the challenges and be prepared for solutions in order to make its implementation successful p-007 ct smit sibinga 1 , y abdella 2 and f konings 2 1 iqm consulting, zuidhorn, netherlands 2 who eastern mediterranean office, cairo, egypt background: who defined essential medicines (ems) as medicinal products that satisfy health-care needs of the majority of a population. they should be available at all times, in adequate amounts, in appropriate dosage forms, with assured quality and affordability. in 2013 blood and blood products (whole blood, red cells, platelets, plasma, and plasma-derived products) were added to the who model list of ems. appropriate and effective regulatory framework (legislations, regulations, etc.) and a functioning regulatory authority (ra) is crucial for management of blood products as ems. however, particularly in the less developed world, these prerequisites have barely been implemented. aims: to analyze and advise on existing legislation and regulations. existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. methods: existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. results: various formal legislative documents of only 9/22 countries are put in force by governments [1960 (egypt) till 2017 (pakistan -sindh)]. most are detailed descriptions of ra, operational establishments, and specific requirements. however, none of these legislations complies with who and eu recommended formats and contents, and will not support effective regulatory oversight to promote and enhance quality, safety and availability of these ems. summary/conclusions: government should provide effective leadership and governance in developing a national blood system (nbs, fully integrated into the national health-care system. essential functions of a nbs include an appropriate regulatory framework with legislations, regulations and other non-legislative instruments administered by a ra. these documents should spell out principles and cadres, standard setting, and organization of the blood system to ensure an adequate supply of blood and blood products and safe clinical transfusion for which a model was designed. the structure of nbs will depend on organization and level of development of the health-care system. however, all critical activities within nbs should be coordinated nationally to promote uniform standards, economies-of-scale, consistency in staff competency, quality and safety of these ems, and best transfusion practices. key is formulation of an appropriate regulatory framework administered by a ra responsible for regulating the vein-to-vein chain in the preparation and use of these ems. background: the capacity of blood transfusion service to provide adequate supplies of blood components is the issue of concern for health providers worldwide; longer term observations of trends in this respect are therefore of crucial value. aims: the study aim was analysis of some basic activities of the polish blood transfusion service in 2008-2017 including organizational changes, numbers of donors, donations and blood components as well as activities directed at increasing their safety. methods: retrospective analysis of data supplied by the regional blood transfusion centers (btcs). results: in the discussed period, blood and blood components were collected in 21 regional btcs and local collection sites as well as during mobile collections. although the number of local collection sites decreased from 170 in 2008 to 133 in 2017 in favor of mobile collections, which increased from 8,672 to 13,189, the former is still the number one location for donating blood. on average 47.36% of all donations were performed in local collection sites. the total number of blood donors both at the beginning and the end of the discussed period was similar (583,908 in 2008 and 588,184 in 2017); over 99% of all donors were non-remunerated. however, the number of first-time donors dropped significantly (from 237,658 in 2008 to 143,038 in 2017). the total number of donations increased from 1,076,655 in 2008 to 1,249,655 in 2017; most frequent were whole blood collections (from 1,016,411 in 2008 to 1,171,302 in 2017) . some blood components (mostly plasma and platelet concentrates) were also collected by apheresis. most frequently prepared blood components were red blood cell concentrates -rbcs (996,678-1,154,239 units per year), fresh frozen plasma -ffp (1,090,369-1,289,021 units) and platelet concentrates -pcs (81,692-129,143 units, with significant increasing tendency). additional processing methods such as leukocyte depletion and irradiation were more frequently applied to pcs (52-57.6% in respective years irradiated, 75.18-92.07% leukocyte-depleted), than to rbcs (4.46-8.46% irradiated, 7.65-20.7% leukocyte-depleted). in 2010, the pathogen reduction technologies in plasma and the pcs were implemented. up to date however the use of these technologies is limited in most btcs. in 2017 approximately 11.5% of pcs and 8% ffp units issued for transfusion were subjected to pathogen reduction technologies. summary/conclusions: our study data may contribute to the assessment of some long-term tendencies observed in polish blood transfusion service and may serve practical-benchmarking. this in turn may prove beneficial to the transfusion community as a whole. background: in poland 80% of hospitals depend on blood for the treatment of patients; over 1.5 mln units of blood components are annually transfused. it is therefore purposeful to expand the knowledge on factors impacting on blood transfusion service (bts). the institute of hematology and transfusion medicine (ihtm) as competent authority is responsible for collection of data related to the activity of all polish blood transfusion centers (bcts). this data is exploited to a much lesser degree than the recently available statistical methods and data processing tools would allow. moreover, survey of research in the field of public health indicates a negligible share of issues related to bts. it seemed therefore necessary to "fill in the gap" with true assessment of performance of the polish bcts for improvement of bts activity. 1 st stage of our investigation refers to collection, merging of data from different sources, their unification and preparation (big data) for further analysis to be performed using multidimensional statistical analysis and data mining methods. aims: assessment of the activity of the polish btcs over the 20 year-period in two stages. goals at 1 st stage: 1. data digitalization; scanning of paper documents. 2. development of a uniform template for collecting digital data from various sources. 3. standardization, unification and quality improvement of available data: filling in missing data, elimination of errors, duplicate records etc, that may distort the outcome of analyzes. 4. selection of data for analysis. methods: digitalization and big data methods for processing various types of data: a) stored in paper form (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) , b) digital stored in two file types (.doc and .xls, for the years 2005-2010 and 2011-2017, respectively). for each data-type, a separate excel file model was created. the models were then merged into one analytical table with data processing methods. results: 1. 1344 pages of paper documents were scanned. 2. models developed for data from 3 different sources: a. paper-data were rewritten and ascribed to its model; outcome -3 tables, 272 columns, 10,400 rows. b. .doc and .xls. filesdata were ascribed to 2 other models; outcome -6 tables, 1656 columns, 788 rows. 3. the 3 models were merged into 1 analytical table to create a 588 mb database (comparable to approx. 784 min of music). 4. the data was subjected to standardization, unification and quality improvement: filling in missing data, elimination of errors and duplicate records that may distort the results of analyzes. 5. selection of data for analysis at 2 nd stage. summary/conclusions: the 1 st stage provided a set of selected data for analysis in the 2 nd stage which will rely on multidimensional statistical analysis and data mining methods. the outcome of such analysis will contribute to optimal realization of objectives: a) gaining in-depth knowledge about the fundamental phenomena that shape polish bts, b) identification of potential changes bcts, c) development of overall guidelines for change management. aimed to touch untouched or less touched topics of bb&tm. so far 8 workshops were organized and each year around 30 countries were participated from almost all religious, ethnical, social, cultural and economic situations of the world. 6. supported realization of major changes in bb&tm in turkey; a convincing medical individuals and agencies mainly moh to give the deserved consideration to bb&tm b encouraging the recognition and establishment of national blood program c issuing a new blood act and numerous necessary bylaws, etc. d creating an appropriate standard donor questionnaire form e changing blood transfusion practice from %96 whole blood (at 1996) to 5%. f changing donated blood screening criteria; while anti-hcv screening became obligatory malaria, screening cancelled g preparing national guidelines h promoting haemovigilance nurse post i promoting patient blood management 7. around 11,000 blood bankers attended national courses, 7,000 attended national congresses, 18,000 attended nationwide symposiums. summary/conclusions: bbtst can be accepted as a sample how a scientific nongovernmental organization may give a very positive impact on developing and progressing bb&tm activities with close collaboration moh and other related organizations abstract withdrawn. background: globally there is growing investment in information technology (it) in business. this similar trend has been observed in blood establishment computerized systems (becs). the it investment can be high hence it decisions need to be properly informed. the africa society for blood transfusion (afsbt) encourages the use of its in african blood services as this optimize quality blood services, thereby improving patient's outcomes. afsbt established in 2016 an it working group (afsbt itwg) with the support of the swiss red cross (src) to spearhead the it standards among member blood services. a number of priority it thematic areas were identified. these includes it governance which focuses on creation (strategic alignment) and preservation (risk management) of business value. there is absence of published literature on how a structured it governance framework can be implemented in a resource constrained setting. a review of the national blood service zimbabwe (nbsz) it governance was done based on published it governance framework. aims: to explore how a structured it governance can be developed, implemented and monitored in a resource constrained setting. methods: a published mit-cisr framework which has six components was used to assess the strength, gaps and opportunities of the it governance. results: nbsz has been implementing an evolving structured it governance system. in terms of service strategy and organization there is a well-established it function which is reflected in the nbsz strategic plans. this ensures it annual budgetary support, which averages 4.3% of the total budget. the it governance arrangements are such that decision rights are assigned to different it staff (executives, it specialist, and users). a range of it solutions have been embedded within the nbsz operations such as becs, financial, donor mobile application, social media, temperature monitoring, and human resources. the business performance goals are defined and are congruent across the various business units. it organization and desirable behaviors are documented in the ict policies and procedures and were needed remedial actions are available through the code of conduct. the it metrics are included within the nbsz monitoring and evaluation system which use a four colored traffic lighting reporting system. it was noted that the it accountabilities are undesirably tilted to the it specialist only hence some ict projects tend to have delayed deliverables. the it governance mechanisms are supported with tools such as service level agreements and established communication approaches. simple excel based solutions are used to track critical performance metrics such as on the interactive blood supply management status, which averages 122.8% (2018) based on a 5-day projected stocking and supply levels. nbsz need to properly document the return on investments on all these ict initiatives, which is estimated (2016/2017) to be at 3.2% of annual savings. summary/conclusions: blood services in resource constrained settings can implement a properly structured it governance and this will ensure maximum return on it investments. the nbsz approach will be shared and further developed in the afsbt itwg to support other blood services in improving their it governance. haematology/blood transfusion, alfred health, melbourne, australia background: in october 2018, an integrated electronic medical record (emr) was implemented at an australian metropolitan multi-campus heath service using cerner millennium tm , aiming to achieve himss (healthcare information and management systems society) level 6. prior to implementation, large numbers of blood specimens were collected from patients unnecessarily and sent to pathology without a test request attached (no blood test requested -ntr). these specimens required additional processing in the laboratory. electronic specimen collection using cerner specimen collect tm allowed streamlining of specimen processing by eliminating paper requests. as part of the new workflow, individual specimen labels are printed with the specified blood test and correct tube type. this helps prevent the practice of collecting additional specimens due to uncertainty of the collection requirements. aims: • to quantify the expected reduction in ntr specimens following introduction of electronic specimen collection, and outline the benefits • to determine the impact on collection errors and wrong blood in tube (wbit) events methods: data was obtained directly from cerner millennium tm using a ccl (cerner command language) query which is run monthly by pathology it staff. this data includes all specimens registered for the month with indication of rejected specimens, wbit & ntr samples. 'rejected specimens' includes incomplete specimen and/or request certification, unlabelled specimens/requests and mismatched specimens. further information about wbit events was collated from riskman reports and staff interviews. results: data from the 12 months prior to emr implementation was compared with 3 months post. ntr numbers reduced from 4220/month to 2019/month (52% reduction), freeing up more storage space in fridges. rejected specimens due to inadequate patient request labelling reduced from a mean of 27/mth to 6/mth. wbit numbers have increased slightly: before having median 1 (range 0-2), after with median 2 (range 0-3). although it was hoped that wbit incidence may reduce with the new emr, 4 of the post implementation 6 wbits involved electronic specimen collection. departure from planned protocols involving a lack of working printers, causing staff to print patient labels away from the patient's bedside, as well as multiple patient labels printing on individual printers appear to be a main cause of the emr wbits. summary/conclusions: emr implementation has led to a reduction in ntr, and rejected specimens due to inadequate request labelling, as well as increased storage space in laboratory refrigerators. associated benefits include: • decreased financial costs of the wasted equipment • decreased staff time collecting and processing unusable specimens • decreased environmental impact of manufacture and disposal of unused specimens • decreased potential of iatrogenic anaemia work in preventing the occurrence of further wbits is ongoing, by ensuring that label printers are in working order, are in plentiful supply and easily accessible to staff; and also ensuring positive patient identification and blood collection by the patient's bedside remains a priority. jm mustaffa 1 , k teo 2 , s tsai 1 , p heng 1 , r sagun 1 and m wong 1 1 laboratory medicine 2 khoo teck puat hospital, singapore, singapore background: khoo teck puat hospital (ktph) is a 761-bed general and acute care hospital, opened in 2010, serving more than 800,000 people living in the northern sector of singapore. the blood bank of ktph department of laboratory medicine provides specimen testing and blood transfusion services for ktph as well as the neighbouring yishun community hospital (ych), one of the largest community hospitals in singapore providing intermediate care for recuperating patients including rehabilitative services. the process of ordering transfusion-related test requests in both hospitals is through printed forms. aims: in line with the hospital directive to move towards electronic patient management, the ktph blood bank intended to implement an electronic type and screen (e-t&s) system. the goal of the project is to ensure zero patient identification errors and maintain full traceability and accountability for the blood collection process in all transfusion-related testing. another aim of the system is to reduce repeated venepunctures when specimens are rejected due to missing essential patient information on the printed forms by implementing mandatory fields in the e-t&s form. methods: the e-t&s was implemented in phases. phase 1: an online version of the printed form was signed electronically by the ordering doctor and a witness within the electronic medical record system, sunrise clinical manager (scm) system with the doctor counter-checking by signing on the specimen label to ensure correct patient identification. phase 2: the ordering doctor is not required to sign on the specimen label since fingerprint biometrics are required for the electronic signin. phase 3: elimination of the witness step for blood collection. specimen collection and rejection data from 2016 to 2018 was analysed. specimen rejection rate was presented as percentage of rejected specimens (mislabelled, unlabelled and clerical errors) over total specimen count for each month. results: between january 2016 and march 2017, before the implementation of the e-t&s phase 1, the average rejection rate for blood bank specimens was 0.16% and 1.14% for identification and clerical errors respectively. during phases 1 and 2 of implementation, rejection rate increased due to unfamiliarity to the new work processes. by february 2018, with the implementation of the final phase of the e-t&s system the specimen rejection rate was 0.38% and 0.12% for identification and clerical errors respectively. rejected specimens were mostly from the few locations that had to use paper requisition due to workflow or infrastructure limitations. summary/conclusions: the e-t&s system was implemented successfully in ktph. full traceability and accountability of the blood collection process was maintained with the fully electronic system. the adoption of electronic documentation has also reduced the number of preventable repeated venepunctures that were due to incomplete order information on the printed forms. future developments in technology and full implementation of e-t&s system in all hospital locations may make zero patient identification error achievable and ensure transfusion safety in all patients in the near future. background: blood component administration represents a critical phase due to the possible occurrence of errors during the different steps from the identification of the patient to the infusion of the product. error occurrence can be reduced by the implementation of validated information systems. we tested the scweb â system at the bedside in a transfusion outpatient clinic. aims: the aim of the study is to validate a system designed to assist and to control blood administration step by step using electronic devices to ensure traceability and documentation of the process methods: the scweb â system is based on it monitored checklists which guide the personnel to follow the procedure, according to best practices; the system must initially be activated by the operator which is recognized by an auto-signing system based on bluetooth low energy which avoids the operator having to identify himself/herself beforehand. appropriate privacy protection is provided. thereafter the system takes up the task to give instructions and to verify the adherence, by asking an active confirmation of the proper fulfillment of the activities; a continuous registration and documentation is made by the system. standards and specifications for each step of the procedure have been configured on scweb â system to track in detail operator and patient identification, presence of informed consent to transfusion, blood pressure, pulse and temperature recording, vein access, verification of the blood unit. an alarm has been set after 15 min, to ensure the control of patient's conditions. for each step, an active confirmation of the action is required and nurse and doctor direct involvement must be actively confirmed on the device by both operators. the system has been tested at the bedside on 30 patients admitted to the outpatient clinic for 45 red cell concentrate transfusion; compliance of the personnel and organizational impact has been recorded. results: the system required a very short training: ease of scweb â system allows its implementation without negative impact on organization of transfusion outpatient clinic and without difficulties by operators (nurses and doctors), who appreciated the help given by the it check system. the registration of the electronic check list offered a reliable tool for the traceability of the transfusion procedure, also granting a paperless and timely available documentation of the entire process through a registration in electronic format of all the operator's action in every single phase of the transfusion process. when prescribed, confirmation of the checklist was only possible in the presence and with the active confirmation of two operators (doctor and nurse). summary/conclusions: the scweb â system is useful as a barrier against the mismatch of transfusion (preventive measure), as a traceability and documentation measure and as a tool for training of personnel in blood transfusion administration; it avoids paper registration during the transfusion process, due to the timely registration of the activities performed by operators recognized by the system thanks to the bluetooth low energy auto-signing device. the scweb â system will be connected to the transfusion data management system, to monitor all the process from the arrival of the unit from the blood bank. background: he blood banks aims at reducing cost and increasing customer satisfaction by providing quality in service. the quality in service can be attained by streamlining the processes and restructuring the supply chain of the organization by implementing it tools. aims: aim is to understand the complex flow of information and processes within the supply chain of the blood bank. the requirement of such a study is a part of the integrated erp modeling for the integrated functioning of a blood bank. methods: he approach used to understand and map the sequence of processes, and the work responsibilities of each process and the operational decisions involved at each step is process mapping and data flow diagrams for front end system modeling and analysis. the processes are mapped and represented in a schematic diagram. dfd (data flow diagram) are constructed for representing the system. a context diagram is also constructed for understanding the entities interacting with the system. the emr systems aim at replacing (or supporting) the paper based medical records. the whole model of the system is divided into two parts-front end and back end. the front end design and analysis is done using epc (event-driven process chains), resource views, data flow diagram for data view. reporting was on donor selection, finance and collection of blood bag, blood collection process, component preparation, blood testing and blood distribution results: process mapping using event driven process chain generated a whole view of the processes involved. the resource view gave an organizational structure and the personnel involved. the data view using context diagram and data flow diagram gives a flow of data and amount of data involved. this framework can be used for business process reengineering for the blood banks by conducting a time study and removing non value added activities. data view helps analyze redundant data in each process. it also helps in staff training and orientation within the department. summary/conclusions: a systematic overview presented in this paper facilitates in removal of non value added processes, duplication of data, bottlenecks, reduction of cycle time and thereby improving service quality in blood banks. background: the transfusion of blood components, one of the most prevalent interventions in clinical practice is a major expenditure item in healthcare services which tend to increase in recent years. aims: it is intended to investigate the impact of transfusion associated costs to hospital costs in pediatric intensive care unit (picu) patients. methods: during a year period (january 2017-december 2017) 76 patients, 40 females and 36 males receiving transfusion with blood components along the stay in picu were included in the study. transfusion associated costs and total costs for healthcare services for children treated in picu was collected by using hospital information system (his). statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). mann-whitney u test and kruskal-wallis test was performed for comparison of independent categoric variables and numeric data; chi-square analysis was performed for comparison of two numeric variables and spearman correlation analysis was performed for associations. results: the median age of patients was 12.0 months (interquantile range-iqr 26). the median length of stay was 16 days (iqr 30). in total 400 blood components were transfused in which of 227 red blood cell concentrates, 114 apheresis platelet concentrates, 6 granulocyte concentrates, 51 fresh frozen plasmas, and 1 cryoprecipitate and 1 whole blood. the ratios of transfusion associated expenditures to hospital costs were categorized in intervals of percentages as < 5%, 5-10%, 11-15% and > 15%. most of the patients (63.2%) were ranked in the lowest interval. the medians for hospital cost and transfusion associated cost were 5478.76 euros (iqr = 11280.02) and 130.57 euros (iqr = 354.86), respectively. a significant strong positive correlation between numbers of transfusions and hospitalization cost of picu was detected (r: 0.674, p < 0.01). while it was found a significant weak positive correlation between transfusion associated cost and hospital cost (r: 0.247, p = 0.032) there was also a significant weak positive correlation between the age and transfusion associated cost (p = 0.048, r: 0.227). a significant difference was found between the patients with and without hematological malignancies (p < 0.01) for transfusion associated cost. the reason why pediatric dosages are mostly prefer is that the hospital provides healthcare for only children and splitting of the blood components was common in the hospital. but unexpectedly a significant increase on the transfusion associated costs which is related to split blood components was detected (p < 0.05). summary/conclusions: studies on the economics of blood transfusion have been conducted mostly in patients who require chronic or multiple transfusions. picus, specialized facilities that provide care for patients with severe life-threatening diseases are major departments often necessitate multiple transfusions. there are many variables to evaluate the impact of transfusion associated cost to hospital cost in picu patients, but the major factors are underlying conditions, admitting diagnoses and transfusion strategies. although there are unexpected data in our study demonstrated the increasing impact on transfusion associated cost originated from blood components split for pediatric usage no significant relationship was determined to explain this situation. further studies on the economics of blood transfusions have to be carried out to clarify the variables of transfusion associated costs. background: approximately 55.7% of the transfused blood component is packed red cell (prc). over ordering of prc unit is a common practice and excessive pretransfusion testing was being wasteful of resources and have adverse consequences on cost. high crossmatch to transfusion (c/t) ratio as quality indicator of blood bank implies that crossmatches were performed unnecessarily. aims: the aims of this study were to evaluate the cost effectiveness of strategies for limiting the number of pretransfusion testing of ordering prc. methods: all prc units who ordered from dr. hasan sadikin hospital from january 2016 to december 2018 were collected in this retrospective study. number of ordering prc unit, completed pretransfusion testing of ordering prc units, and prc units that were transfused were recorded. restrictive pretransfusion testing strategies were done based on the hemoglobin level and diagnosis as transfusion indication criteria. cost effectiveness was measured by multiplying the unit cost of pretransfusion testing and number of prc unit. results: out of total 177,370 ordered prc unit, 166,910 (94.1%) were subjected to pretransfusion testing and 63.1% (105,260) of ordering prc unit which are pretransfusion testing were transfused. this means that 5.9% (10,460) of ordering prc unit were not subjected to pretransfusion test. this showed savings of 1,098,300,000 rupiah. c/t ratio was 1.6 which demonstrate a good ordering pattern. however, 16.4% (27,451) of completed pretransfusion testing of ordering prc unit were not transfused, leading to blood bank loss of 2,882,355,000 rupiah. summary/conclusions: strategies for limiting the number of pretransfusion testing on the good c/t ratio was still associated with saving cost effective background: blood is a precious resource for saving patient lives. the purpose of blood and blood component therapy is to provide suitable and safe blood products to achieve best clinical outcomes. nurses have an important role in ensuring safe blood transfusion. it is crucial for nurses to have sufficient knowledge about blood donation and collection, storage, component preparation, possible adverse effects of blood transfusion and necessary management and care. aims: the aim of this study was to assess the impact of an educational intervention on knowledge and awareness of nurses regarding blood transfusion services and practices. methods: the baseline study to assess the knowledge and awareness regarding blood transfusion services and practices of the nurses posted at various areas of the hospital including wards, operation theatres and critical care areas was carried out at our institute hospital which is a tertiary care teaching centre. the nurses were then sensitized and educated regarding blood transfusion services and practices during their day to day activities by referring them to the blood transfusion guidelines of the institute. subsequently, a self-developed questionnaire which was used for the baseline assessment of knowledge and awareness of the nurses was again used to reassess them. a total of 19 questions were included in the questionnaire pertaining to: general awareness (two questions), blood donation (two questions), testing and blood component preparation related (two questions), storage of blood/blood components (two questions) and pre-transfusion checks and bed side transfusion practices (eleven questions). fifty nurses each were included for both the baseline as well as post-sensitization assessment. for different category of questions, the correct response rates were compared with those obtained in the baseline study using mann-whitney test. the entire study duration was spread over a period of three months (december, 2014 to february, 2015 . results: the overall mean percentage of 'correct' responses for 19 questions in the baseline study was 61.75%, whereas post sensitization it was 77.21%. the mean percentage increase in general awareness related questions was 21.49%, 13.95% for storage of blood/blood components related questions, 17.37% for pre-transfusion checks and bedside transfusion practices related questions, 21.33% for testing and blood component preparation related questions and 27.27% for blood donation related questions. the percentage increase in correct response was found to be statically significant for each of the five categories of questions. the overall mean percentage increase in correct response rate was also statistically significant (p < 0.001). summary/conclusions: this study revealed that after sensitization and educational intervention there was a significant improvement in the knowledge and awareness of nurses regarding the blood transfusion services and practices. abstract withdrawn. background: tact, introduced in the uk in 2014 to support managers, provides resource-saving, continual, 'real-time' monitoring of knowledge-based competency of staff in transfusion laboratories. tact is available online 24/7, complementing existing practical competency schemes and external quality assessment. multiple variations on a standard pre-transfusion testing scenario are generated using constrained randomisation; logic rules for automatic assessment of sample acceptance, abo/d, antibody screen and identification (as/id), and component issue are based on bsh guidance. during 2018, tact was offered internationally to transfusion laboratory managers to trial, and saw uptake in five countries. the core tact programme, based upon uk guidelines, is under review for programming conversion, to be customisable for the international community. aims: to assess the feasibility of tact programming conversion to meet the requirements of country-specific pre-transfusion testing guidelines, and to direct future programming in line with feedback from international users. methods: guidelines from 3/5 international users were obtained and translated where necessary. these were compared against the core assessment elements of current tact programming. international users were approached for their feedback on the current version of tact, as it compared to their local policies and practices. results: the following criteria were cross-referenced: specification of transfusion request forms, sample label acceptance criteria, reagents used for abo/d and as/id, resolution of grouping anomalies, alloantibody confirmation/exclusion, and selection criteria of blood components for transfusion-dependent patients and women of child-bearing potential. apparent differences included:-australia:--selection of red cells for patients with immune anti-d. greece:--inclusion of the name of the patient's father on the transfusion request. italy:--testing of all new patients with an anti-a,b reagent and two different monoclonal anti-d reagents. international users in the same three countries supplied feedback. this included suggestions for:-greater complexity of cases presented, provision of patient history, inclusion of follow-on tests e.g. phenotyping and cells for antibody confirmation/exclusion, broader range of reaction strength grading, and official professional cpd credits. the following differences were noted:-nomenclature used, the format and content of the request form, use of english abbreviations of patient clinical details, and the availability, provision and specification of blood components. summary/conclusions: this analysis has shown very few instances where the current tact iteration differs from the guidelines reviewed, and that it is feasible to expand the use of tact on a more international basis. the current iteration of tact has been developed to represent an abbreviated scope of pre-transfusion testing practices, which can be applied to laboratory practice outside of the uk without difficulty. further work is required to enable international users to configure tact such that the system represents all laboratory practice on an international basis. aims: these courses provide education to clinicians on patient blood management and safe transfusion in neonatal and paediatric settings in order to improve patient outcomes and increase awareness of the national patient blood management guidelines. this analysis aimed to investigate the uptake, practical use, and perceived value of the courses by learners. methods: a retrospective analysis of course completion statistics and course evaluation data. results: there have been 2,376 paediatric and neonatal courses completed from 6 march 2018 to 28 february 2019 with 89.6% of learners being nurses and/or midwives. analysis of course evaluation data (n = 33) showed that these courses: -provide knowledge (96.9%) -improve patient safety and outcomes (84.9%) -result in change to clinical practice (69.9%) -are relevant to clinical practice (70.9%) -are easy to use (67.7%) -are readily accessible (58.1%). examples that learners provided of how they can apply this learning to their clinical practice include: -"[i am now] more aware of special requirements for neonatal blood transfusion" -"[i] feel more confident especially when talking with parents" -"[i will now be] checking the patient's blood results and will speak up for unnecessary blood sampling" -"[it's good that] when there is ambiguity in clinical practice [this is] very well shown by explanation from experts in the field" -"we don't do a lot of transfusions [and this is] a reminder that transfusions are not always the first answer to the baby's clinical picture". summary/conclusions: analysis of course and user evaluation data demonstrates that these courses are being used by nurses and doctors working in the neonatal and paediatric settings and that they provide knowledge of pbm that can be applied to clinical practice, thereby contributing to improved patient care. background: blood transfusion is a high-risk clinical activity that must be mastered both theoretically and practically in order to guarantee the required result without any incident or complications. the mastery of transfusion knowledge among nurses represents a very important link in the transfusion chain. the objective of this work is to compare the theoretical and practical knowledge of transfusion among two groups of nurses divided according to their seniority. aims: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. methods: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. results: the participation rate in the survey was 100%. the 2 nd group participants had an average seniority of 9 years . more than half of them (64%) had seniority of less than 10 years. only 12% had more than 20 years of experience. the rate of correct answers for all items combined was 44.4% for students versus 42.5% for practicing nurses. the theoretical knowledge part was more mastered in the 1 st group than that of practicing nurses (44.8% vs 33.4% of correct answers). on the other hand, the control of the transfusion act was better in 2 nd group (44% vs 50.5%). the overall "dangerous" response rate was 47% for students and 41.7% for practicing nurses. false practical knowledge was more common in group 1 (59.5% vs. 41.5%). summary/conclusions: the theoretical as well as the practical knowledge remains not well mastered by the care staff. our study highlighted the best theoretical mastery for young students and practical for practicing nurses. this could be explained by the freshness of knowledge in the first group and the daily practice in the second group. background: the european commission (ec) directive 2004/33/ec on blood donor selection criteria is 15 years old. in the meantime, knowledge on risks related to blood donor selection has progressed and challenged several obligatory rules. transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of more than 24 stakeholders from both not-for-profit and private organizations providing substances of human origin (soho). the project aims to provide evidence-based donor selection criteria and guiding principles for risk assessment of threats to the safety of soho. as part of this work, an inventory of current blood donor selection criteria in europe and an evaluation of the evidence behind current practice was performed by experts working on this project. aims: to identify the gap between the ec directive 2004/33/ec on whole blood donor selection criteria and a current evaluation of the clinical relevance of the criteria based on scientific literature by a panel of european experts within the transpose project. methods: in 2018, we performed an inventory of blood donor and transfusion recipient risks in participating european countries. project members were asked to provide the existing donor selection criteria related to these risks and to carry out a risk-based evaluation for each of them. the evaluation was based on the available scientific literature and on a risk assessment template based on the abo risk-based decision-making framework, developed by transpose. all risks with divergent assessments within the panel were resolved through discussion; in all cases an expert consensus was established. subsequently we compared the results with the content of the ec directive 2004/33/ec for every risk, thereby identifying discrepancies and missing items in the directive. results: the panel identified 64 risks considered to be significant, distributed between donors and recipients. for 35/64 (55%) of them the expert evaluation deviated from the content of the ec directive, or the ec directive provided no information about the decision making. in particular, a discrepancy was observed for 9/20 criteria concerning general health and medication, 12/22 for transfusion transmissible infections, 10/13 for high-risk behaviour and travel, and 4/9 for other diseases. summary/conclusions: our results highlight a significant gap between the whole blood donor selection criteria stated in the ec directive 2004/33/ec and the scientific evaluation performed by a panel of transpose participating experts. this gap includes both new risks not addressed in the ec directive and addressed risks that are however evaluated differently. this involves both blood donor and transfusion recipient safety, and various medical and epidemiological topics covering several aspects of the blood donation criteria. we strongly recommend a change in the european legislation, allowing a procedure to guarantee that blood donor selection criteria are updated regularly within the framework of the european institutions, to keep aligned with scientific progress, epidemiology and changes in medical practice, in order to enable an updated risk-and evidence-based framework for donor selection criteria. the risk-assessment method elaborated in the transpose project is a valuable instrument for this purpose. background: the brazilian health regulatory agency -anvisa has developed the method for assessment of potential risk in hemotherapy services (marpsh) which is based on the data collected during the inspections of blood services carried out by regulatory authorities. using marpsh any blood service can be classified in one of 5 possible potential risk categories: high, medium-high, medium, medium-low and low risk. each category represents a different potential risk level, according to the proportion of compliance with the established regulatory requirements. marpsh has been used since 2007, showing a trend of risk reduction on blood services evaluated all over the country. aims: this work aims to describe the utilization of marpsh as a tool for an integrated risk management model. also, it shows the main results obtained after 10 years of data monitoring and coordination of regulatory actions and policies by anvisa, targeting quality and safety of blood products. methods: the utilization of marpsh follows a network risk management model since the inspections are carried out by decentralized organs in all 27 states and some municipalities. the inspectors fulfill a standardized inspection guide containing the regulatory requirements, where each item is associated with a level of risk, varying from i to iii as the risk increases. at the end of the inspection, after a statistical calculation, the service is categorized. this classification gives an estimate of its quality profile, guiding the adoption of suitable measures for risk management by local authorities and services. these data are send to the states (if realized by municipalities) and to anvisa that perform consolidation in a national level. either states or anvisa use data to coordinate risk management measures in a broader spectrum. data are continuously monitored by anvisa as part of its strategical panel of indicators. anvisa follows up specially blood services in high and medium-high risk with the aim of helping or complementing local authorities' actions. additionally, anvisa periodically sends this information to the brazilian ministry of health and local governmental organs from brazilian national blood system that also support actions to improve quality in their blood services networks. results: since 2007, when the assessment covered 109 blood services, marpsh reached 1218 blood services in 2017 (57% of the blood services registered) what corresponded to almost 100% of the inspection cover in this year. over this period (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) it is possible to notice a dramatic decreasing in trend for proportion of blood services classified as high and medium-high risk, varying from 26% to 9%. summary/conclusions: marpsh generates data necessary to the categorization of blood services into five levels of potential risk. as a result, the regulatory actions are applied by local organs with the purpose of reducing or eliminating risks involved in the production and use of blood components. data have shown a significant risk reduction over 10 years of marpsh's utilization. additionally, monitoring of this data at a national level has been permitting appropriate planning and prioritization of integrated strategies directed to risk management and strengthening of blood services in brazil. background: sub-saharan africa has the highest need of blood transfusion in the world, mainly for childbearing women and children suffering from malaria. meeting basic quality and operational requirements to provide patients with safe blood remains a challenge in these settings. aims: the aim was to identify and prioritize potential hazards for patients in blood bank practices in the democratic republic of the congo (drc). we focused on two subsets: (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products, using failure mode effect analysis (fmea). methods: two risk analysis workshops were organized at the national blood transfusion centre in kinshasa, the democratic republic of the congo. in both workshops, a multidisciplinary team was invited to represent different hospitals and profiles in transfusion management in drc: quality coordinators (n = 2), training coordinator (n = 1), medical doctor for donor selection (n = 2), hemovigilance officer (n = 1), laboratory technicians performing donor sampling, blood qualification and production (n = 7), biomedical scientists (n = 3), microbiologist (n = 1), clinical biologist (n = 1), nurse (n = 3). the principle of fmea was applied, which implies identification of possible hazards (failures) in a process flow, followed by scoring each hazard according to their impact, probability, detection and feasibility. in the both workshops, participants were guided by an external facilitator who guaranteed common understanding of the methodology. main focus of the risk analysis was potential harm to the transfused patient in the process of (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products. all ideas were written on coloured cards and mapped on a chart according to their impact, probability, detection and feasibility score. hazards were ranked according to their final risk score by multiplying these four scores. results: in the process of sensibilisation and selection of donors, the three hazards with the highest final score for impact, probability, detection and feasibility were: (i) a paid donor is recruited after sensibilisation by family members of the patient, (ii) donor selection staff approves a non-eligible donor for blood donation because of personal/financial motivation, (iii) blood donors are not correctly informed about blood donation during sensibilisation by family members of patients. in the flow of qualification of blood products, highest scores were made for: (i) no double check for validation and sorting of qualified blood products, (ii) stock-out of reagents, (iii) no check for match between registered test result and tested blood tube. regarding production of blood products, the top three consisted of: (i) transport time between blood collection and processing is >8 h, (ii) storage of qualified and quarantined blood products in the same fridge (some sites only), (iii) power cuts. summary/conclusions: the risk analysis resulted in three prioritized hazards in the process of donor selection/sensibilisation and blood product qualification/production. some are very specific to the sub-saharan african setting and have been described before (power cut, family and paid donors, stock rupture,. . .). an action plan needs to be put in place to reduce their final risk score. the risk analysis needs to be continued for the remaining blood transfusion flows. background: 19.3 million of germany's population, so just under a quarter of residents, have a migration background. the majority of these has roots in regions where the population has a distribution pattern of blood group and hla-antigens that differs considerably from the predominant one in the german population. sufficient supply of these individuals with red blood cell (rbc) and platelet concentrates (tc) will continue to be a major challenge in the future, as blood donors with compatible blood group antigens are dramatically underrepresented in the local donor pools. many migrants suffer from severe hematological disorders such as b-thalassemia or sickle cell disease and will not only need compatible blood transfusions, but an allogeneic stem cell transplantation in the foreseeable future. as healthy family donors often are not available, at present suitable stem cell donors with a similar genetic background can only be found in international donor registries. aims: this project was initiated to recruit new donors with a migration background for blood donation and to increase the number of blood stem cell donors among this group. methods: serological extended blood group phenotyping was performed by automated gel card technique (fa. grifols, erytra) and included ab0, rh (ccdee), kk, fy (ab), jk(ab), lu(ab), m, n, s, s. hla typing for hla-a, -b, -c, -dr, -dq, and -dp was performed by next generation sequencing. allele frequencies were analysed using genepop version 4.2; the rare and very rare alleles were defined according to the allele frequency database (www.allelefrequencies.net) rbc genotyping using next generation sequencing is currently being established and will include additional antigens with the most frequent distribution pattern differences between migrant and resident populations according to literature. results: so far, more than 8800 blood donors with a migration background have been recruited for a blood donation in this project. amongst this group, over 1000 blood donors from more than 20 non-european countries enrolled as potential stem cell donors. an initial evaluation of the data revealed a very similar distribution of blood groups compared to the current blood donor population in north rhine-westphalia. of 600 migrant donors, ten fy(a-b-) donors were identified, which corresponds to a percentage of 1.6%. amongst 509 hla-typed potential stem cell donors, we found 28 (5.5%) with rare and very rare alleles. summary/conclusions: blood donors with rare blood group and hla phenotypes (e.g. null types such as fy(a-b-)), are in demand for adequate medical care of people with a migration background. the technological development of blood group determination by next generation sequencing will significantly improve the supply for all blood transfusion recipients in germany. this project is funded by the european development fund 2014-2020 (erdf) and the european union. background: mortality due to uncontrolled haemorrhage following trauma is the most important cause of potentially preventable deaths. trauma care systems in low and middle income countries like india, are still in developing phase. also, the role of blood component therapy in improving patient outcomes has been mostly derived from combat settings. application of these protocols in an urban setup has not been well established and marked variation in practice exists. hence this study aims to identify the key components of transfusion practices to optimize the transfusion protocol in trauma settings. aims: to study the current transfusion practices in severely injured trauma patients, admitted to the red area/resuscitation bay after initial triage in ed methods: this prospective observational study was conducted over a period of 1 year starting from june 2017 to may 2018 at the department of transfusion medicine in collaboration with emergency department at jpnatc, aiims, new delhi. the study included severely injured patients (iss ≥ 16) that were admitted within 24 h to the red area/resuscitation bay after triage. data collected included the demographics, injury, laboratory and transfusion details for these patients results: during the study period 885 patients (83.5% males) were enrolled. mean iss scores was 21.89 . mean time to hospital admission after injury was 9:03 (iqr 3.38-13:48) hours. mean time to first rbc transfusion following admission was 2:09 (iqr 0:27-2:45) hours. approximately 49.3% (436) patients were in shock (sbp < 90 mm hg &/or pulse rate > 110/min). whereas, 160 (18.1%) patients were coagulopathic (pt ≥ 1.5 times of normal). during initial 24 h of admission, these patients were transfused with 2929 (69.7%) rbc, 1986 (51.8%) ffp, 2327 (42.9%) rdp and 384 (81.5%) cryoprecipitate of total blood components utilized for these patients. massive transfusion (defined as transfusion of ≥ 5 units/4 h) was given to 190 (21.4%) patients. summary/conclusions: significant quantity of blood components were required during initial resuscitation in severely injured patients. pre-hospital transfusion can significantly reduce the time to transfusion. further studies are needed to assess utility of pre hospital transfusion in severely injured patients. background: allogenic stem cell transplantation recipients are known to be the main consumers of platelet concentrates (pc). the geneva university hospital is one of the three allogeneic hematopoietic stem cell transplantation (hsct) centers in switzerland. since the blood center is also part of the hospital, data of pc consumption are easily available. as needs rose steadily since several years, with an average increase of 9% per year, pc supply is a serious concern for our center. aims: in this study we tried to evaluate if any pre-transplant indicator could help to forecast the number of pc needed after an allogeneic hematopoietic stem cell transplantation. methods: this observational retrospective study was conducted in geneva hospital on 78 patients suffering from various inherited or acquired disorders of the hematopoietic system who were treated by hsct in 2016. pc consumption was examined from january 2016 to december 2018. the five indicators were: gender, stem cell source (bone marrow (bm) vs peripheral blood stem cell (pbsc)), donor type (hla matched (10-8/10) vs haploidentical), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient. results: data for a total of 78 patients aged from 3 to 74 years were analyzed; 48 (62%) were male and 30 (38%) female; 35 (45%) were cmv-negative and 43 (55%) were cmv-positive. out of a total of 78 transplants, 14 (17.9%) were haploidentical and 64 (82.1%) hla-matched. according to the stem cell source, bm was transplanted in 22 cases (28.2%), and pbsc in 56 cases (71.8%). two patients also received a cd34 + stem cell boost. our analysis showed that, with a mean follow-up of 652 days, the number of pc transfused to our patients treated by hsct ranged from 0 to 383 units, with an average of 37 and a median of 15, illustrating a high variability. the results indicated that gender, stem cell source (bm vs pbsc), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient do not have any statistical impact on platelet consumption. however, we observed a tendency of an increased need for platelet transfusion when patients were cmv positive. our results also showed a statistically significant (p = 0.034) higher number of pc transfused for patients treated with a haploidentical (89) versus hla-matched (26) transplant. summary/conclusions: this study points out the high variability of platelet consumption after hsct, which limits the forecast of platelet production needed to support allogeneic hsct recipients. a larger cohort would be required to confirm a potentially higher platelet consumption in cmv positive patients, and to consolidate our results showing a higher pc consumption for patients treated with haploidentical transplant. abstract withdrawn. background: historically at our institution, a minimum of four red blood cell (rbc) units were crossmatched for all cardiac surgery cases regardless of surgical case-type or patient characteristics. two rbc units were packed in validated blood product coolers and brought to the operating room (or); the balance of crossmatched units remained in the blood bank. a retrospective review revealed that very few rbcs were transfused (2016: 20% (371/1813), 2017: 19% (322/1742)). moreover, approximately 8 products were wasted each month as a direct result of this practice. thus, we recognized an opportunity to improve inventory management in terms of personnel activities and blood component utilization. aims: the goal of this study was to reduce advance preparation of coolers in cardiac surgery cases without compromising patient care and safety. we limited our intervention to those patients who were eligible for electronic crossmatch. we maintained the aforementioned historical practice for those patients with history of and/ or those who currently demonstrated clinically significant red blood cell alloantibodies. methods: a multidisciplinary group consisting of representatives from the blood bank, cardiac surgery, cardiac nursing, cardiac anesthesia and surgery quality department was assembled in october 2017 to determine whether a modification of practice was reasonable and safe. group members evaluated site specific society of thoracic surgery (sts) cardiac surgical data between july 2014 and december 2016 to establish intraoperative red cell transfusion rates classified by type and urgency of surgery. the group's main goal was to discontinue preparation of default coolers for patients eligible for electronic crossmatch who were scheduled for all types of non-emergency cardiac surgery cases in which ≤ 25% of historical cases required at least one red cell transfusion. additionally, team members simulated the multiple protocols by which red blood cells could be prepared and delivered to the or and estimated the time for each scenario. results: review of sts data showed that the following cases met the criteria of ≤ 25%: elective primary coronary artery bypass graft (cabg), urgent primary cabg, elective mitral valve repairs, and elective aortic valve replacements. simulation showed that, in patients eligible for electronic crossmatch, preparation from receipt of order to completion of unit packing for delivery took 2.5 min using the pneumatic tube system (maximum of 2 units per tube) and 4.5 min using delivery of a cooler using a human courier. summary/conclusions: based on the simulation results, and with consensus agreement from the multidisciplinary group, default cooler preparation for elective primary cabg, urgent primary cabg, elective mvr, and elective avr was discontinued in december 2017. one year following implementation of the change in policy 940 rbc units were issued to the or (a 54% reduction); 38% (361) were transfused, compared to 19% in 2017. wastage rates decreased from 8 products a month to 1 per month on average. summary/conclusions: the most obvious drawback of pabd is the higher cost in running the program in comparison with collection of allogeneic blood in the areas of additional patient attention and clerical input in labeling, separate storage and so on. in this audit, 34% of the autologous blood components were not transfused into the intended recipients and wasted; in this context, the pabd program could not be considered as a cost-effective approach in protecting blood safety. background: the national blood service zimbabwe (nbsz)'s blood supply management status (bsms) is an integral process of ensuring the availability of a safe and sufficient blood supply provision. nbsz introduced a new daily blood bank statement with improved metrics from 1 may 2018. the new analytics approach focuses on three interactive components of the blood bank statement; the available stock, quarantine stock (as per the desired 5-days stocks level), and the demand versus supply. it is imperative to have a closely monitored blood supply chain because blood has limited shelf life with uncertainties in both supply and demand. the 'blood-for-free' proclamation by the government of zimbabwe in july 2018 set more pressure on the blood demand. these metric-based analytics seek to assess if the nbsz's improved blood bank statement is a realistic model for the bsms. aims: to assess the use of the interactive metrics in monitoring the blood supply management status. methods: a prospective cross-sectional study was conducted. a total of 704 daily blood bank statements which were submitted between may and december 2018 from each of the five branches were analyzed. the bsms which is calculated as the average of the three interactive measures of quarantine stock, available stock and demand versus supply was determined. sub-analysis of branches was done to determine individual branch performance. analysis by month was done to assess seasonal variations. findings and recommendations were shared among key stakeholders to validate the bsms methodology. results: overall the quarantine stock average was 130.4% (sd +/-101.6), the available stock was 142.3%: (sd +/-118.8) and the demand versus supply was at 95.6% (sd +/-11.3).the overall bsms was 122.8%; (sd +/-77.2) for the study period. gweru and masvingo nearly supplied all the demanded blood with 99.2%, overall bsms of 118.1% and 99.7%, overall bsms of 144.3% respectively. bulawayo supplied 98.3% of the blood demanded with an overall bsms of 99.1%. mutare supplied 97.8% with a bsms of 180.1% and harare 85.6% and a bsms of 78.0%. there were monthly variations but the service could supply above 90% of the blood demand. in the month of may the service met 91.6% of the demand and a bsms of 87.8%. in november and december it supplied 92.6%, bsms of 100.8% and 92.8%, bsms 131.8% respectively. august also had a below average supply of 93%, bsms -98.2%. june, october and september recorded above the average values; 97.3%, bsms of 126.8% and 98.7%, with a bsms of 117.2% respectively. summary/conclusions: the overall bsms performance was satisfactory and it was noted that branches capacitated according to demand. the new interactive analytics approach is appropriate for showing the blood bank status and assessing the performance of the branches. this new approach has optimized the decision-making process in blood supply management. the metrics are tracked using excel based model hence this approach is suitable for resource constrained settings with limited ict infrastructure . st vincent's hospital melbourne (svhm), a tertiary hospital supporting medicine, surgery and non-major trauma emergency and itu services implemented a mtp in 2008. subsequent mtp reassessment has led to implementation of regular multi-disciplinary review of all mts to identify areas for improvement in transfusion and other aspects of support for critically bleeding patients. aims: to implement a systematic service-wide stakeholder review of mt events at svhm aiming to identify deficiencies and implement improvements in mt management. methods: a multi-disciplinary mt review team was established as a subcommittee of the hospital transfusion committee (tc) to update the organisational mtp in 2016 and subsequently continued to meet quarterly as the mt review subcommittee (mtrs) of the tc, systematically reviewing all aspects of mts at svhm. instances where 4 or more red cell units are transfused in <4 h are identified from the laboratory information system and reviewed by the mtrs which includes representatives from accident and emergency, intensive care, operating suite (os) and transfusion laboratory staff; the head of the patient's treating unit is also invited to contribute. reviews include: demographics, clinical details, comorbidities, time from patient arrival to pre-transfusion specimen collection/receipt, time from blood request to release/transfusion, regularity of full blood examination (fbe)/coagulation (coag) testing, timing of blood component transfusion, total component provision/ratios, component waste, patient outcome, and communication between various clinical areas and also the laboratory. a discussion summary with actions/ recommendations is provided to the tc and some cases referred to the hospital mortality/clinical review committee. results: cases reviewed: 65 from 10 treating units including cardiothoracic surgery (18) hepatobiliary/gastrointestinal/colorectal surgery (24), vascular surgery (6), neurosurgery (4), orthopaedic surgery (3), endocrine (2) and "other" (encompassing general surgery, urology, general medicine and oncology -8). areas for monitoring/improvement identified: transfusion documentation, regularity of fbe/coag specimen submission, reducing time between patient arrival and specimen collection, reducing specimen transport time, interfacing point of care bloodgas analysers to the central pathology result management system as well as component management/waste reduction and the introduction of viscoelastometry assessment in the os. 18 of 65 reviewed cases involved the transfusion of emergency uncrossmatched o rhd negative red cell units. the appropriateness of the use of this precious resource is also reviewed by the mtrs. summary/conclusions: the svhm mtrs meets regularly to review mt events and formalise multidisciplinary collaboration in identifying possible improvements to support these often critically ill patients. matters highlighted include communication issues, delays in specimen delivery and blood component waste minimisation. areas for further work include minimising delay between mt events and review, and formalisation of key performance indicators for mts. background: the use of radio frequency identification (rfid) technology to manage the blood supply chain is recognized as a major enhancement to the operations of blood banks and hospital transfusion services. to facilitate optimal blood supply management, it is crucial to guarantee the integrity of rfid tags throughout the transfusion chain. since rfid tags can be affixed to blood products very early in the process, these tags undergo the same process-steps as the blood products themselves (e.g. centrifugation, label printing, shock-freezing and irradiation). aims: the goal of this study was to validate the mechanical and functional resistance of biolog-id rfid tags through different blood related processes: centrifugation, label printing, shock-freezing, intensive reading at à40°c, and irradiation. biolog-id tags are passive hf (13.56 mhz) tags. they are compliant with is0 15693, iso 18000-3 and follow the guidelines for the use of rfid technology in transfusion medicine (vox sanguinis, 2010). methods: biolog-id tags were evaluated using a series of rfid encoding and reading tests. before each of the processing steps, each tag was encoded with donation number, site id, product code, blood group and expiry date. the data was encoded using the isbt 128 format. the different processing steps and conditions tested were: -centrifugation: quintuple whole blood bags, filled with 450 ml water. centrifugation at 4,500 rpm for 10 min. 270 tags processed, 15 tags per kit affixed at different positions. -shock-freezing at à80°c: shock-freezer (angelantoni, sf40), 50 units processed, reading immediately after removal from shock freezer. water, 30 tags irradiated at 30 gy and 30 tags at 50 gy results: all biolog-id tags were encoded and read with a 100% success rate in all series of tests. summary/conclusions: biolog-id rfid tags can be encoded and read through common processes used throughout the blood transfusion chain. their mechanical and functional integrity is not affected by centrifugation, shock-freezing, intensive reading at à40°c, printing, eto sterilization and irradiation. background: the provisioning of compatible red blood cells by international cooperation is presented. the units were meant for an 18-year old female, with homozygous sickle cell disease (scd) and multiple complications. patients' blood group was a positive with anti-c, -e, -wr a and an antibody to a high prevalence antigen in the rh system, anti-hr b possibly combined with anti-hr b (rh34). the antibody was not reactive with rh null , -d-or hr b negative cells. the donor center put out an international request for group a or o, rh null or -d-units lacking wr a and possibly k, fy a , jk a , wr a , do a and s (the latter antigens for prophylactic matching). the patient sample had been genotyped for rhd and rhce using mlpa and sanger sequencing and the patient was found to carry rhd*01/rhd*03n.01 and rhce*cevs.01/ rhce*cevs.03. aims: the request was sent to the american rare donor program (ardp). the ardp working with the american red cross national molecular laboratory, used the rh genotype information to identify donors carrying the same or similar rh variant alleles using the rh allele matching approach described previously (keller et al. transfusion 2013 53(2s):174a). methods: a recent blood sample was used to confirm anti-hr b ; no anti-hr b was detected. the patient rhd and rhce alleles were used to build punnett squares for both genes with donors carrying the same and similar alleles that would be predicted to be compatible. tier 1 donors are those predicted to carry the same combination of rhd and rhce alleles as the patient. tier 2 donors are those predicted to be homozygous for one of the allele combinations carried by the patient. tier 3 donors are those predicted to carry alleles similar (but not identical) to those carried by the patient, with similar predicted phenotype. the database of donors in the ardp carrying rh variant alleles was queried against the alleles in the patient-specific punnett square. results: donors of group a or o and matched for rh alleles were identified as follows: 102 tier 1, 357 tier 2 and 100 tier 3 donors. after the clinical team agreed to drop one or more of the prophylactic antigen matches, one tier 2 unit lacking s and jk a was identified at the american red cross. while the request was being processed, the patient experienced a sickle cell crisis, red cell aplasia and recurrent aiha and her hemoglobin level dropped from 7 to 1.9 g/dl. at that time, she was transfused the only compatible units available -2 of the rare -dphenotype and her hb increased to 3.8 g/dl and eventually to 7 g/dl. the tier 2 rh allele matched unit was shipped to amsterdam where it was frozen, and reserved for the transfusion care of this patient. summary/conclusions: this case illustrates how rh allele matched blood can be found for a highly rh alloimmunized patient, and can avoid use of the exquisitely rare -d-or rh null blood. background: blood transfusion has been a complicated and high-risky clinical procedure. any error could cause serious injuries to patients. to better assure the procedure safety. aims: we enhanced and built a blood transfusion database platform and develop inventory management strategies to better guarantee the patient transfusion safety. methods: we designed six new features of the platform (1) assuring the patient identification with barcode techniques; (2) designing a structured order entry; (3) proactively reminding the physicians with patient's previous blood transfusion reaction with related precautions including the use of leukoreduction filter; (4) automatically reminding physicians the happening of reaction and suggesting relevant test; (5) building a complete traceability log system; and (6) supporting data analysis. the blood transfusion safety team includes medical technologists, nurses, physicians, system analysts, and blood transporter and the whole process is electronic management. the new blood transfusion platform integrated the workflow, reduced the incidence of abnormal blood samples collected (0% after implementation, p < 0.01), reduced the time of call for medical technologists with blood component preparation and improved the achievement rate of emergency 30-min blood crossmatch (98.1% after implementation, p < 0.05). the barcode correctly identified patients and monitored the entire transfusion process to reduce the error rate of blood component supply (0% after implementation, p < 0.01). summary/conclusions: after the transdisciplinary team approach with e-monitoring and a better design of clinical decision support module with barcode technology, blood transfusion database platform improve the blood supply efficiency and assure blood transfusion safety. background: in the modern world, terrorist acts are characterized by a multiplicity of combined injuries to a large number of victims. qualified medical care is urgently required for a large number of patients in one locality at the same time. it leads to increase in emergency demand for blood components, mostly red blood cells. the desire to donate blood to the victims is a natural manifestation of society's solidarity in response to tragic events. however, donor activity and patient needs do not always correlate. aims: to analyze the donor activity during the terrorist attacks. methods: a retrospective analysis of donation activity in periods of terrorist attacks in moscow (2004 moscow ( -2011 . the average daily blood donations' number (dbdn) before ta compared with the number of donations in day after ta and with the dbdn during 7 days after ta. also the number of delivered rbc units (d-rbcu) daily before ta and daily in 7 days after were compared. results: in 2004-2011, 5 terrible ta occurred in moscow: 141 people died and more than 629 were injured. with the explosion in subway in 02/2004 42 people died, 250 were injured. the number of d-rbcus increased by 10% on ta-day, and by 30% during next 7 days. dbdn in the 1st day after ta increased 6,7 times, and in the next 7 days -1,6 times. second explosion in subway in 08/2004 resulted in 10 died, 50 injured. the number of d-rbcus increased by 80% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 1,1 times, and in the next 7 days -2,2 times. in 2006 (explosion on market) resulted in 14 died, 61 injured. d-rbcus delivery increased by 20% on ta-day, and by 10% during 7 days. dbdn in the 1st day increased 2,0 times, but decreased to 0,6 times during the next week. with subway explosion in 2010 40 people died, 88 were injured. the number of d-rbcus increased by 10% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 6,5 times, and in the next 7 days -1,6 times. with the explosion in airport in 2011 35 people died, 180 were injured. rbcus delivery increased by 50% on ta-day, and by 40% during next 7 days. dbdn in the 1st day after ta increased 6,1 times, and in the next 7 days -2,0 times. summary/conclusions: an increase in donor activity is observed already the next day after ta and usually lasts for 7 days, but does not correlate with the number of victims. the rbcs' delivery from blood bank increases in all cases on the day of the ta. therefore, the guarantee for patients is the maintenance of rbcs' stock, including cryopreserved ones. it is also necessary to promptly send excess of red blood cells harvested at the peak of activity to the cryobank. background: rh system is the major blood group system besides abo system. even after proper blood grouping and cross matching there is a possibility of alloimmunisation in recipients against the rh or minor blood group antigens like kell, mnss, duffy etc. in medical colleges which cannot bear the financial burden of complete phenotyping of patient and donor, implementation of rh & kell phenotypes match blood transfusion can play a major role in preventing alloimmunisation and adverse events in multitransfusion patients aims: to evaluate the efficacy of rh & kell phenotyping as a cost effective measure instead of extended phenotyping in multitransfused patients methods: study was carried out in the department of transfusion medicine, one of the biggest blood bank of the country with annual collection of 70,000 blood units. 2000 patients of thalassemia, aplastic anemia and leukemia were taken who required multiple transfusions. complete phenotyping was done initially of all the patients before transfusion. 1000 patients were taken as control and the other 1000 were taken as cases. 2482 blood units of healthy donors were chosen (2442 were males and 40 were females). in all the donor units, identification of rh & kell phenotyping was done by the antigen antibody agglutination test by the erythrocyte magnetize technology on fully automated immunohaematology analyzer qwalys. these blood units were transfused to 1000 patients who had been selected as cases. in the control group, patients were transfused blood units which were not phenotyped for rh & kell but gel crossmatching was done. follow-up was done on these patients for transfusion reactions and at the end of six months they were evaluated for any alloimmunisation. results: at the end of 6 months, no reactions were reported in cases receiving rh & kell phenotype blood and no alloimmunisation was seen on repeat phenotyping. the control group on the other hand reported reactions in 5 cases (0.5%) and phenotype at the end of three months showed alloimmunisation with 'e' antibody. the phenotypic frequencies of rh & kell blood groups in the population were comparable with other published studies. amongst the rh antigens (e) was the most common (99.44%) followed by d (95.45%), c (89.65%), c (53.1%) and e (17.65%). thus 'e' was the most common and e was the least common of all the rh types. background: the prevalence of a particular blood group has an uneven distribution in different geographic areas and is largely determined by the national composition of the population. moscow is one of the largest city of europe with population of 12.5 million. the understanding of prevalence of red blood cells antigens (rbc-ag) among the population has great importance for blood banking planning. aims: to determine frequency and distribution patterns of transfusion-significant rbc-ag among donors in the moscow region. methods: the results of immunohematological studies on ab0, rhesus and kell systems were analyzed retrospectively in 352362 blood donors for 14 years (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) in moscow. data collection and processing was carried out using the regional information system for transfusiology. rbc-ag detection (ab0, rh, kell) systems was performed using microplate method (automatic immunohematological analyzer "galileo neo" (immucor, inc., usa)) and "ih-1000" (bio-rad laboratories, usa) with diagnostic cards. results: the most frequent blood group is a (ii) 35.8%, 0 (i) blood group 34.3%, b (iii) 21.3%, ab (iv) 8.6% (n = 352362). rh(d+) was established as positive in the presence of antigen d and as rh(d-) negative in its absence. donors with weak variants of antigen d (du) were determined as rh (d+) positive. the ratio of rh (d+) and rh (d-) was 82.8% and 17.2%, respectively. donor's phenotype detection was routinely conducted from the 2013 year, therefore the number of donors was 152883. the most common phenotype among donors ccdee (32.61%), the second in frequency ccdee (19.33%), the third in frequency rhesus negative phenotype ccddee (15.42%) in the studied population. the ccdee and ccdee phenotypes were 14.04% and 12.17%, respectively. the most rare are ccdee (2.65%), ccdee (1.86%), ccddee (1.54%). other options: ccddee, ccdee, ccdee, ccddee, ccddee, ccdee, ccddee were detected in single cases and amounted to a total of 0.37% (n = 152883). cw antigen was tested in 104230 donors and was detected in 5.28%. cw is most commonly found in donors with ccddee phenotypes (2.36%), ccdee (2.03%) and ccdee (0.79%), with other variants of the data phenotype, the antigen was detected in 0.1% of the examined (n = 104230). antigen k was detected in 6.8% of donors, in 93.2% of this antigen is absent (n = 352362). summary/conclusions: the study of transfusion-relevant antigens distribution in population is necessary for building of effective and flexible model for blood service managing. a differentiated approach in choosing a strategy to form a long-term bank for storing blood components, taking into account the frequency of various antigen variants, contributes to improving the quality, accessibility and safety of medical care. of dislocated division of our blood establishment in orthopaedic hospital valdoltra (obv) in 2014 the number of outdated units at the hospital side dropped considerably. results: since 2008 when the issued number of red blood cell units (rbc) was 5170 the amount of issued units rose to 5678 in 2011 and then dropped more or less steadily to 4850 in 2018. in this period the hospitals' programmes rose for 10% in all areas. number of donated units declined from 6330 in 2011 to 4820 in 2018. after reorganization in 2009 the number of outdated units fell from 3% of stocked units to 1.5%. after setting a dislocated unit of ctdiz on obv location the number of discarded rbc fell from 397 to 41 in 2018. for transfusion specialist who is constantly in contact with the clinician in the hospital the most important day routine is when the stock availability is displayed. it happens 4 times a day; at 10 a.m. when the previews' day collection is released and another three times a day when the updates occur. the central base is led in ljubljana (the capital) and all centres are able to control and order the stock for the blood banking. blood wastage remained low and the traceability of the blood usage in south-western region remains high (99%). though it is not supported by an informational system the traceability form the blood bank to the patient is done on paper. this issue demands a big effort by the staff in blood bank and in hospitals. summary/conclusions: reorganization enabled better stock utilization and traceability of issued units. sometimes it is impossible to predict the peak demand of rbc especially during the summer season when the population of the area doubles and car accidents as well. transfusion specialist's effort in assuring the optimal blood stock represents the crucial daily routine. blood bank, grande international hospital, kathmandu, nepal background: voluntary non-remunerated blood donor consists of 78% blood donor's population in nepal. therefore demographic about the distribution of blood donors according to the age group is important to achieve 100% voluntary nonremunerated blood donors in nepal. aims: to explore the demographic distribution of the blood donor in different age group in the kathmandu nepal. methods: this is retrospective study conducted at nepal red cross society central blood transfusion service. data from january 2013 to january 2017 were collected from donor management software. the data includes socio demographic data. data has been process with spss version -17 results: during 4 years study period, total of 276,290 blood donation happened from both mobile blood collection and in-house blood collection. out of 276,290 collection, 48351 (17.5%) are from 18-24 age group; 106924 (38.7%) are from 25-31 age group 53324 (19.3%) are from 32-38 age group; 34536 (12.5%) are from group 39-45 age group; 23761 (8.6%) are from age group 46-52 and 9394 (3.4%) from age group above 53 respectively. summary/conclusions: the distribution of abo blood group varies regionally and from one population to another. in kathmandu, nepal 18-38 years age group is the most common age group encountered donating blood. the data generated in the present study and several other studies of different geographical region of india will be useful to health planners and future health challenges in the region. background: the information system on hemovigilance sihevi-ins©, coordinated by the national health institute was available in 2018 to all blood banks in the country. this software allows to centralize and record the identification data of a donor, its infectious and immunohematological tests, as well as the fractionation and final destination of each blood component obtained from a donor. aims: to describe the abo and rhd typing discrepancies in blood donors found in the blood group variables registered by each blood bank to sihevi-ins©. methods: retrospective analysis of the information registered by 80 of the 81 blood banks authorized nationwide between january and december 2018. results: sihevi-ins© received information of 854,462 accepted donors, 33% of them with more than one donation in the same blood bank in a period of 12 months. a total of 72 abo or rhd discrepancies were identified in 69 people, who made donations in 20 blood banks (estimated risk: one discrepancy per 11,876 accepted donors). five of the blood banks implicated in these discrepancies are hospital-based (annual average collection of 6,086 ae 3,785 units, representing 3.6% of the national collect). the remaining blood banks are distributors (average collection: 31,389 ae 25,704 units per year, representing 44% of the national collect). 75% of blood group typing discrepancies (n = 36) were related to the abo group. the most common discrepancy was between a typing group and ab typing group (67%) . in 25% of the cases, the same blood bank initially registered in the same donor, an o blood type donation and later an a blood type (n = 10) or b type (n = 4). rhd typing discrepancies account for 25% (n = 18) of the total. additionally, in three donors, a simultaneous discrepancy between abo and rhd typing was detected in the same blood bank. the results could be due to: a) failure in the warning mechanism before the release of the blood component; b) errors in typing the information of the donor registered in the system or c) failures in the identification of the donors at the time of selection. the above shows risk in the process of control of blood components release, which can impact patient safety unless abo and rhd typing blood groups are systematically verified before transfusion. summary/conclusions: despite blood banks have a verification and validation process through software to release blood components, flaws were detected. although sihevi-ins© is not a software to validate the information before the release of blood components, it was through this program that abo and rhd typing discrepancies were identified in donors who attended the same blood bank multiple times. this finding implies increasing the controls that should be used in each blood bank, to avoid lose traceability of the processes and to put at risk the life of the recipients. blood donor testing department, blood transfusion institute of nis, nis, serbia background: the ability to automate blood grouping and antibody detection procedures is a requirement for blood donor testing laboratories. mistakes in the sample identification and testing procedures could be prevented by testing on automated immuno-hematology systems. irregular antibody screening and abo/rhd grouping of blood donors are tests performed routinely in blood transfusion institute of nis. neo iris (immucor, usa) is a fully automated instrument for the abo and rh d grouping using microplate hemagglutination technique and antibody screening and identification using solid phase red cell adherence (sprca). aims: evaluation of the automated neo iris system for abo and d grouping and irregular antibody screening of blood donors in blood transfusion institute of nis. methods: during the evaluation period a total of 4417 edta-anticoagulated samples for abo and d forward and reverse grouping using microplate anti-s, 1 anti-s, 1 anti-jka and 6 anti-k. in one case ih-1000 failed to identify anti-c antibody in very low titer in sample with anti c+d antibody presence. in two samples (0.045%) false-positive result were observed both on ih-1000 system and neo iris and in two cases (0.09%)only on neo iris due to nonspecific reasons. summary/conclusions: abo/rhd grouping results obtained on neo iris system, using microplate method, have a good correlation with results on ih-1000 system as our routine column agglutination method. for antibody screening and identification neo iris showed high sensitivity for detection of clinically significant antibodies which is important step for increasing blood transfusion safety. background: continuing improvement of laboratory quality to provide accuracy test results for precise diagnosis and treatment is the mission of advanced laboratory. immunogenetic testing for histocompatibility including human leukocyte antigen (hla) typing, hla antibody detection and cytotoxicity test is critical for diagnosis and evaluation of transplantation and prognosis monitoring. in order to improve the quality of experiment competency, an external quality assurance schemes with review and education per year program was established and performed during the period from 2017 to 2018 in taiwan. aims: the proficiency testing (pt) held semiannually from 2017 to 2018 were reviewed to investigate the outcome of competency improvement of laboratories participated in the program. methods: the test items in the exercises were classified into 4 groups, hla genotyping (including pharmacogenetics hla typing), cytotoxicity test, hla and platelet antibody. the methods of hla genotyping include ssp (sequence specific primer), sso (sequence specific oligonucleotide), sbt (sequence based typing) and either ssp+sso or ssp+sbt were used, the methods of hla antibody including elisa, flow cytometry and luminex were used and the methods of platelet antibody including sprca and elisa were used. there are four shipments of exercise materials in two years and each shipment include two positive and one negative samples for antibody detection, two each of whole blood and serum for cytotoxicity of t and b cell and three whole blood for hla genotyping. aims: this study aims to survey transfusion related laboratory tests for the quality improvement of hospital's blood bank management. methods: we analyzed survey results of 11 kinds of routine work categories of 841 blood banks that were registered on korean association of external quality assessment service. blood bank worker voluntarily replied this electronic survey. the 11 categories were as follows: 1. characteristics of institution 2. the equipment of blood bank 3. the kinds of tube in blood bank 4. the present kinds of blood bank tests 5. abo and rh type tests 6. the cross-match tests 7. the irregular antibody tests 8. hemovigilance system 9. other blood bank tests 10. massive transfusion protocol 11. quality control issues we analyzed and compared each category data according to considering characteristics of hospitals. results: there were consensus and some differences of current blood bank tests. we presents the result of a pilot survey. especially the cross-match tests were divided by saline phase method added with irregular antibody tests or completion of 3rd step anti-human globulin phase according to institutional environment. automated typing machines or automated irregular antibody test devices were more increased in large-scale hospitals than small-scale hospitals. different kinds of tubes were used such as edta tube for abo and rh typing, plain tube for cross-match test. the retention segments of rbc were reserved for minimum 7 days. most blood bank were registered and regularly listed up transfusion events to korean hemovigilance system for safety transfusion. also, a lot of institution have none or underdeveloped massive transfusion protocol. more specific survey results will be analyzed in further poster presentation. summary/conclusions: this survey will show the current status of transfusion related blood bank test. this institutional blood bank comparison will be helpful to assess the currency of individual blood bank environments. abstract withdrawn. background: we know that quality management is a continuous process, involving implementation, maintenance and improvement. aims: our purpose is to show our experience in implementing the quality management system in the whole institution and our first steps in achieving the jacie accreditation in the stem cell collection facility in order to provide our patients and donors the best possible care. methods: the institute for transfusion medicine of the republic of macedonia (itm) is the main institution in charge of blood transfusion service (bts) in the whole country, which is the national unified system. the stem cell collection facility is a part of the itm. this facility is operational since 2001 year with 844 collections of stem cells (686 in patients and 159 collections in sibling donors) till now. we are obtaining the implementation and maintenance of qms through the establishing of the iso standardization for the whole institution (itm), as well as of implementing jacie standards in the stem cell collection facility. the two of our colleagues became the jacie inspectors and the standard operating procedures (sops) were developed, followed by regular meetings, trainings and self-evaluation of the personnel. we asked for the orientation visit from the independent jacie inspector in order to come one step closer to the jacie accreditation and to improve our overall qms. results: the institute for transfusion medicine of rm was a part of the ipa project "strengthening the blood supply system". this project aimed to ultimately bring the blood transfusion service to european union standards allowing the exchange of blood components and all other types of collaboration with other european union countries in future. the project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel. although a lot of strengths were found during the orientation visit from jacie inspector, there are still a lot of areas for improvement. our strengths are motivated team and supportive institutional leadership including macedonian ministry of health. areas for improvement are: labeling of cellular therapy products and lack of laboratory for quality control. there is a national regulatory framework in place and who and world bank initiatives in macedonia which support quality in health care and accreditation. summary/conclusions: our institution has in plan to implement isbt 128 standards for labeling of cellular therapy products and to establish a laboratory for quality control of cellular therapy, as well as to meet all the requirements to become jacie accredited facility. working by standards, following the rules and regular self-evaluations will help us to maintain the strong quality management system. every institution will benefit from a quality management system that brings you into line with international standards. ensuring the quality of our services and products is essential to keep safe and strong blood transfusion service. background: implementation of robust quality assurance program is key to high performing blood establishments. quality control and quality assurance systems together constitute the key quality systems and are parts of quality management. effective and efficient quality control policies not only provide guidance that help to increase the reliability of results but also maintains the laboratory's consistence performance overtime. aims: therefore, we established a set of qc limit using historical data which can timely identify unexpected variation in the testing systems and trigger a review of test processes in blood screening laboratories as part of quality assurance system. methods: last two consecutive years (jan, 2017 to dec, 2018) qc data from archi-tect i2000sr (abbott laboratories, chicago) was extracted using abbottlink for philippines red cross tower national blood center total of 6532 data points (3523 data points in 2017 & 3009 data points in 2018) were obtained for 10 different qc levels for four serological blood screening assays (hiv combo, hbsag, anti-hcv, syphilis). the data was sorted for each assay/lot and qc level combination by year. qc limits were calculated using simple mean, standard deviation (sd) and coefficient of variation (cv%) and were validated and compared with manufacturer's recommendation. results: all the six positive quality control levels cv% (5.70-12.05) were within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2018. five out of six positive quality control levels cv% (5.72-15.80) showed within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2017 except syphilis tp positive control (15.8%). all four negative quality control levels showed the sd values within 0.009-0.41 in 2017 and 0.009-0.41 in 2018 respectively. summary/conclusions: excellent qc performance was observed in philippines red cross tower national blood center blood screening laboratory based on historical data and evidence-based laboratory qc limit for blood screening assays were established using historical data which takes into account total variation expected in a test system and offers a more robust and meaningful mechanism for setting control limits, for the first time. background: quality indicators (qi) in transfusion medicine (tm) are 'critically important aspects of transfusion medicine practice that are measured and utilized to gain insight for continuous quality improvement, into the degree to which the tm is capable of providing quality tm care, products or services for the aspect of practice measured following comparison of the measurement against acceptable local or international reference thresholds, benchmarks, standards, or practice guidelines'. the critical control point (ccp) selected for this study is 'administration techniques and monitoring of key elements'. this has been selected since the clinical fraternity plays a larger role in ensuring quality services in administration of blood components. there was a need to follow up compliance to standard protocol for bedside transfusion practices hence was decided to study the same with four selected quality indicators and introduce corrective measures if necessary. aims: 1. to assess the existing transfusion practices in the institute with specific quality indicators 2. to introduce corrective reforms to improve the existing practice 3. to assess the transfusion practices after interventions using the same quality indicators methods: to assess the existing transfusion practices in our centre, 295 transfusions were prospectively followed up with a structured checklist. the quality indicators used were (i) verification of blood components prior to transfusion (ii)initiation of transfusion within 30 min of release from the blood bank (iii) close observation of transfusions for the first 15 min (iv)completion of transfusion within the right time frame for each component. as a corrective measure, a transfusion monitoring format was designed which was distributed in every ward and the nursing officers were informed to monitor and document transfusions using that. in addition, the blood bank staff was made to call up the wards and ensure that the transfusions of every component had been initiated within 30 min of issue. transfusion practices were once again monitored by following up 306 transfusions using the same quality indicators. results: there was significant difference in all the four variables between the two phases. 57.3% transfusions were verified in phase i while 73.6% were verified in phase ii (p < 0.001). 69.5% transfusions were started within half an hour of issue while in the second phase, it rose to 83.1% (p < 0.001). 47.8% transfusions were observed in the first 15 min in phase i and 88.6% were observed in the second phase (p < 0.001). in phase i, 54.6% transfusions were completed within right time while the same in phase ii was 73.9% (p < 0.001). summary/conclusions: we recommend the following as quality indicators for bedside transfusion practices: background: antibody titration consists in performing antibody detection with selected red cells of different sample dilutions. the titer is reported as the reciprocal of the highest dilution that induces macroscopic agglutination. the usual applications of titration are prenatal studies and complex antibodies identification. some publications have demonstrated that more variation in antibody titer and titration score are noted upon repeat testing of the same sample when testing was performed in tubes as compared to repeat testing in gel. aims: to evaluate the efficacy of automated antibody titration versus manual method by using gel microcolumn technology. methods: edta-anticoagulated whole blood donors' and plasma frozen samples containing a known irregular (rh, kidd, duffy, mns, etc.) and regular (a1 & b) antibodies were selected. the titers of 126 samples were determined in parallel by using grifols analyzers (erytra and erytra eflexis) and compared versus grifols gel manual method by using grifols gel microcolumn technology and grifols red blood cell reagents. sixty of these also processed in parallel in erytra and erytra eflexis analyzers for comparison. for the precision study, 12 of these samples were tested in the 2 automated systems for 5 times (120 datapoints for each analyzer) on different testing days. the hands-on (manual intervention) average time required to complete a titration was measured (3 expert technicians) in 2 different sample workload (1 and 10 samples testing). these results were compared with the same number of independent titrations performed in grifols analyzers. for the walk-away time, 2 different sample workload (1 and 10 samples testing) were assessed in manual method (3 expert technicians) and compared to timings obtained when reproduced in grifols analyzers. results provided by analyzers were reviewed and compared to manual method. results: titer obtained by erytra or erytra eflexis was equivalent to the titer obtained manually (differences ≤ 1 titer: 73% ≤0.5 titer). the results proved that both instruments were equivalent in performing titration (differences ≤ 1 titer; 85% ≤0.5 titer). the precision results showed no difference between titers obtained through the 100% of the runs performed with the grifols analyzers (differences ≤ 1 titer: 75% ≤0.5 titer). the manual hands-on in automated system was reduced in a 15% compared to manual method for 1 sample. when the number of samples was increased (10 samples), the difference in hands-on in was even more reduced (65%). in addition, the walk-away was 46% higher in automated system compared to manual method. furthermore, when the number of samples was increased (10 samples), the walk-away difference was increased even more (78%). finally, automated system software demonstrated to increase the standardization of the test as all samples, results and reagents traceability were automatically managed. summary/conclusions: grifols gel system including erytra and erytra eflexis analyzers provided a scalable and efficient solution to perform standardized titrations in the immunohematology lab. the study proved that using grifols gel system, titrations can be run in an automated reliable way (less than one-fold differences versus manual gel), thus reducing at least 15% the hands-on, increasing at least 46% the walk-away, rising the standardization and automating all testing traceability. , and fourth case of use (results) scenarios, tasks were considered "very easy" by 30%>50% of users and "easy" and by 50-80% of users; 90%>100% of the users considered "sufficient" the design to ease the interaction; and 60%>80% of users never founding any situation of not knowing how to proceed. for the fifth case of use (user roles), 40% of users considered tasks "very easy" or "easy"; 40% of users considered "sufficient" the design to ease the interaction; and 40% of users never found any situation of not knowing how to proceed. for the sixth case of use (maintenance plan), 100% of users considered tasks considered "very easy" or "easy"; 90% of users never found any situation of not knowing how to proceed; and 100% of users considered the maintenance plan similar or better than other instruments. reliability analysis ( background: quality control procedures in blood group serology for reagents, techniques, personnel working and automated equipment are essential for the accuracy of the laboratory results. the observation of high number of uninterpreted results during blood donor grouping was a motive for investigation and possible targeting the problem. aims: to identify blood group interpretation problems by analyzing the testing results obtained with the commercial quality control samples routinely used during blood grouping. methods: a microplate (mp) system for performing abo and rhd, as well as rh phenotype and kell blood group determination with two automated analyzers techno (1 and 2) and correspondent two mp-readers lyra (1 and 2) using maestro software from diamed is currently in use for blood donor typing. three types of mp are being used such as: a, b, ab, dvi-, dvi+, ctl/a1, b profile for first time donors, then a, b, d ctl for repeat donors and finally, the c, c, e, e, k, ctl profile. the accuracy and safety of the blood grouping results is ensured by using the diamed q.c. system which consists of 4 + 2 tubes of whole blood and 2 tubes containing serum with known specific antibodies. we analyzed and compared the interpretation of the q.c. whole blood samples' results from both of the analyzers after a new optic camera was installed on the techno 1/lyra 1 system. ward to ward. methods: a prospective observational pilot study was done for around 140 prbc unit issues which were followed in real time for understanding the tat within blood bank & from ward to ward. as per the definitions, the areas where the times are documented perfectly are understood and considered for calculations. based on the conclusions of pilot study a monitoring form has been designed and utilised to monitor the tat within bb & wtw. the data is analysed monthly and an avg tat for bb & wtw is calculated. the common causes of delay in providing the blood components were analysed and strengthened to both reduce & control the tat. results: in pilot study, total wtw tat averaged to 90 min, with highest time taken 795 min, where there were additional processings like leukodepletion, irradiation, saline washing of red cells and holding the transfusion. lowest wtw tat was found to be 10 min where there was a prior information for crossmatch. after the surveillance form has been started, the average time taken for wtw tat came down to 70 min, maximum being 310 min (jan 2019), the areas where delay happened were identified as internal courier delays, technician delays, billing & other logistics delay. the concerned staff are put on regular training to maintain the tat. summary/conclusions: although ethically all the staff work for providing better care for patients, there will be few areas that delay the life supporting blood transfusion. monitoring using tat surveillance forms help in avoiding the delays and hence provide better & timely transfusion support. blood donation -blood donor recruitment p-059 hematological and physiological characteristics of regular blood donors with beta-thalassemia traits background: according to recent evidence, the physiological variability observed in the hematological characteristics of regular blood donors (linked -in certain caseswith genetic factors or the donor's lifestyle) may affect red blood cell (rbc) storage lesion. beta-thalassemia heterozygous (b-thal-het) blood donors represent a group of particular interest because of a) the high frequency of thalassemia mutations in specific geographical areas b) the physiology of the b-thal-het rbcs, which predisposes towards more effective management of storage-associated stress. aims: the goal of the present study was the comparative examination of the hematological and rbc physiological features of regular blood donors with or without beta-thalassemia traits before blood processing for transfusion purposes. methods: 204 healthy blood donors of greek origin (18-24 years old), who met the blood donation criteria were recruited in this study. plasma/serum (uric acid, electrolytes, extracellular hemoglobin, antioxidant capacity), cellular (rbc indices) and biological parameters (corpuscular fragility, proteasomal activity etc) were measured. the results were statistically analyzed and topologically represented in biological networks for both donor groups (+/-b-thal-het). significance was accepted at p < 0.05. results: b-thal-het represented 9% of the donor cohort. no differences in lifestyle (smoking, alcohol consumption, physical exercise) were observed between the two groups. nevertheless, regardless of sex and sex-dependent parameters (e.g. hct, hb concentration), b-thal-het demonstrated: a) reduced hct, mcv and mch (11% p = 0.039, 26% p = 0.008 and 30% p = 0.006, respectively) and b) increased rbc count (20%, p = 0.042) compared to the average donors. moreover, mpv platelet index was found slightly elevated (p = 0.053) and serum total protein concentration slightly reduced (p = 0.054) in the same group. a trend for higher plasma antioxidant capacity (p = 0.052) was evident in the group of b-thal-het, in addition to statistically significant lower levels of osmotic fragility (by 13%, p = 0.022) and hemolysis (by 41%, p = 0.001) compared to controls. finally, analysis of the three proteasome-associated enzymatic activities (n = 10 per group) in the rbc cytosol and the membrane, revealed similar levels in the two groups (p > 0.05). the b-thalhet and control biological networks showed insignificant variations in respect to the amount of connections and their hub profiles. however, differences were observed regarding the number or type of connections, or even their topology in the network, in the cluster of lipids (triglycerides, ldl etc), nitric oxide, clusterin, carbonylated plasma proteins and rbc osmotic fragility (correlated with the concentration of electrolytes selectively in b-thal-het donors) between the two groups. summary/conclusions: b-thal-het who meet the criteria for blood donation are a non-negligible sub-group of the total donor population in greece. they exhibit several similarities to the general cohort, but differ in fine characteristics of rbc physiology, including resistance to hemolysis and extracellular antioxidant capacity. the differential network profile of hematological and redox parameters may be important in respect to the subsequent blood processing and storage of b-thal-het erythrocytes for transfusion purposes. background: blood service in poland is based on voluntary and non-remunerated donations. regional blood donor centre in poznan as well as other regional centres (total of 23) are the only entities authorized to collect, process, store and distribute blood and its components to hospitals in the region of their activity but they are also responsible to provide sufficient amounts of blood and its components. regional blood donor centre in poznan is one of the largest blood centers in poland with the total number of donations exceeding 100,000 per year. in the recent years we have observed a growing popularity of tattoos among various age groups as well as among people registering to donate blood (first time and repeat donors) hence, it is critical to introduce suitable measures to ensure the safety of blood and its components. aims: the aim was to analyse the correlation between the increasing number of donors deferred from donating blood due to having tattoos made and the number of recorded confirmed hcv infections and the effect it may have on the safety of blood and its components. methods: the analysis was made using the data for the years 2010-2017 obtained from the computer system 'blood bank' which is in operation in regional blood centre in poznan, poland. we have analysed the total number of deferrals of donors due to recently acquired tattoo and the total number of recorded confirmed hepatitis c infections. we must note that the category of temporary deferrals due to tattoos is a broad one: it includes so called regular 'artistic' tattoos, permanent make-up procedures as well as medical tattoos. results: we have recorded a significant increase in number of deferrals due to tattoos from 400 in 2010 to 716 in 2017 (+79%). in the group of male donors this trend remained rather stable with a slight decrease: from 181 in 2011 to 151 in 2017 (à16.6%). in the group of female donors the growth was more prominent: from 219 in 2010 to 565 in 2017 (+158%). in terms of the recorded confirmed hcv infections a downward trend can be observed: from 63 in 2010 to 16 in 2017 (à74.6%). in the group of male donors from 43 in 2019 to 12 in 2017 (à72%), in the group of female donors from 20 in 2010 to 4 in 2017 (à80%). summary/conclusions: as we can conclude from the analysis the applied policy of temporary deferrals of donors with recently acquired tattoos (in the last 6 months) proves to be a reliable method of increasing the safety of blood and its components. nevertheless, the current conduct of the qualification of the donors which requires a 6 month deferral following the new tattoo must be complemented by various and numerous educational activities regarding the means of hcv transmission (and other bloodborne viruses such as hbv, hiv) and ways of protection from possible infections. special emphasis must be put on the group of female donors as the growth of deferrals was more prominent among them. at the same time it is vital to ensure for constant availability for all donors of well designed, concise educational materials (hard copies on the premises, articles, infographics, downloadables etc. on the website). background: a temporary deferral has a negative impact on donor retention, with many donors failing to return at the end of their deferral period. anecdotal evidence collected by the australian red cross blood service suggested that many donors do not know when they are eligible to return to donate, suggesting that a reminder message may be effective at promoting donor return once the deferral has ended. aims: the aim of this study is to evaluate the effectiveness of a reminder message on the return rates of deferred donors at the end of their deferral period. this reminder message notified donors that their deferral period was ending and encouraged them to make an appointment to donate. this study also aimed to determine the most effective time to send the message, message content, and mode of communication (sms vs email) in optimising donor retention post deferral. methods: three separate randomised controlled trials were conducted to answer these questions. data on donors' attempted return behaviour and subsequent deferrals, appointments and donations made one month after the deferral end date were collected and analysed. results: overall, 18.3% of donors who received a reminder message attempted to return compared to 12.8% of donors in the control group (p < 0.05). looking at each time point, donors who received the message 1 week before their deferral ended were 64% more likely to attempt to return compared to the control group (p < 0.05). the 1 week prior reminder message was particularly effective with males, with 30.3% attempting to return to donate, compared with 25.2% of females (p < 0.05). there were no significant differences in the return rates of donors who received the recipient versus non-recipient focused message, or donors who received the message via email or sms. summary/conclusions: a reminder message sent to deferred donors at the end of their deferral period is a simple, cost-effective way to promote donor retention, providing clear information regarding the date on which the donors can return to donate as well as a prompt to make an appointment background: our challenge is to provide 100% voluntary donation for safe blood, thus taking into account the current history of family donation, promotion of blood donation, level of awareness and voluntary donations from various institutions, the opinion of 1000 interviewees will give us a clearer idea of what we want to achieve and what needs to be improved in the future. aims: 1. provide 100% voluntary donation for safe blood. 2. establishing a special department within the national blood transfusion center responsible for marketing and promotion of voluntary blood donation. methods: this study was conducted as a combination of qualitative and quantitative methods. the study was a combination and identification of existing data, direct interviews with persons of different age groups, preparation and dissemination of questionnaires and analytical processing of the collected information. the study questionnaire with 60 questions in total was divided into 6 sections out of which 14 questions on blood practices were answered by all 1000 interviewees. 416 people answered 9 questions on the blood transfusion service. 5 questions on blood knowledge were answered by 270 people. 5 questions on the knowledge of the blood transfusion were answered by 220 people, 17 questions on blood donation were answered by 420 people and 10 questions on the communication channels were answered by 500 people. results: out of 1000 interviewees, 19% have never donated and did not intend to donate, due to the fact that most of them were afraid of needles and infections, while the smallest part didn't donate blood because it was not allowed by the religion, 40% did not donate, but expressed the readiness to donate in the future, 10% have donated voluntarily only once, 24% were family donors, 3% regular volunteer donors, and 4% have donated voluntarily several times and did not want to donate anymore. from those who have donated, 59% have donated for one of their relatives, 28% have donated for thalassemic children, 10% have donated to benefit free check-up and 3% have donated because it was valuable for their health. the question as to whether they would voluntarily donate again, 57% have answered yes, 22% no and 21% were still not sure. this means that donation of those who have donated once did not leave a positive impression, did not increase the desire to repeat the donation once again, rather it has restrained or made it unsafe for them to repeat donation. among the causes mentioned by the interviewees were bad conditions in the donation facilities, staff behavior, inadequate treatment, they did not feel good after donation and had hematoma at the venipuncture. summary/conclusions: based on the results obtained from the study, the national blood transfusion center needs the establishment of a genuine promotion department where there is a need for a transfusion doctor who should be an active part of it. the national blood transfusion center should build up and implement a rigorous retention policy for voluntary blood donors, as the study found out that around 60% of donors who have donated once would like to donate again. their attraction through a donor retention policy will surely lead to self-sufficiency with safe blood. the safe blood is a public good and for this reason it is the duty of all state instances, the media and non-governmental organizations to give their support in the promotion of voluntary blood donation. background: smoking, unhealthy diet, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders, including obesity. a balanced and equilibrate nutrition may prevent the negative consequences associated to the status of obesity. in italy, overweight and obesity is increasing with 3 adults of 10 overweight and 1 of 10 obese in 2017 with a higher frequency in the south. blood centers can play a public health role in obesity surveillance and interventions. aims: since the quality of life, self-reported by the patient, related to health and adequate quali-quantitative nutrition, are becoming necessary and relevant in the field of nutrition, we conducted a demographic study to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. methods: a descriptive cross-sectional face-to-face questionnaire was developed. it included a 41 item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. normal weight was established with bmi < 25 kg/m 2 , overweight with a bmi ≥ 25 and < 30 kg/m 2 , and obesity with bmi ≥ 30 kg/m 2 . obesity prevalence was standardized by sex. donors were repeat blood donors, who had made at least 3 donations in the last 2 years, and were eligible to donate. results: of the 2468 blood donors enrolled between july 2017 and january 2018, 1390 were regular repeat donors, 1102 did not wish or chose not to respond at survey for several reasons (i.e. lack of time or privacy) and 288 accepted, of which 83 were deferred from blood donation and were excluded from the analysis. among the 205 included participants 68.3% (n = 140) were male, age ranged from 19-61 years with a mean age of 39.8 ae 11.1 sd and 31.7% (n = 65) were female age ranged from 20-62 years with a mean age of 37.7 ae 11.0 sd. data showed that donors followed mainly a mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. the prevalence of overweight was found 50.7% in men and 16.9% in women. our survey showed that 84.4% of the participants evaluated their health as "good", without gender difference (men, 86.4% vs women, 80.0%). besides, 14.6% reported their health as "very good". summary/conclusions: overweight and obesity are common among regular blood donors and it is more frequent in men than women. our preliminary data showed that women have a better knowledge of the nutritional properties of food and consequently adopt a more balanced and proper diet. furthermore, it is clear that they are aware about the relationship between lifestyle and health putting into practice their information. unfortunately, the survey structure, of observational nature, does not make it possible to establish whether women are more alert to health to participate more in donation programs or if, on the contrary, the status of regular donor could help the improvement of knowledge and healthy lifestyle. background: donor recruitment pose an ongoing challenge to blood banks worldwide. one approach to improve the effectiveness of donor recruitment is to target influencing factors. a yearly league is conducted at the sultan qaboos university (squ) to encourage university students and faculty to donate blood. during this, the colleges are evaluated based on different measures including the number of donors recruited from each college and the efforts made by the students in increasing the awareness of blood donation in the colleges and in the society via different means including the utilization of the social media. the whole competition is organized and ran by an independent group of students. aims: this study aims at studying the impact of the yearly squ college competition on the perception of blood donation among squ students. methods: a comprehensive anonymous voluntary survey was developed and used to assess perception of students aged 18-25 attending squ and other universities (non-squ) over a two years' period. analysis was performed using ibm spss statistics 22.0. categorized variables were presented in numbers with percentages and associations between the groups were analyzed using chi-square test. a p-value of < 0.05 was considered statistically significant. results: a total of 600 students were surveyed (300 squ, 300 non-squ). there was no statistical difference between squ and non-squ students with regard to past history of blood donation and the number of donations made. when comparing between both cohorts, 73% of the squ and 25% of non-squ students reported the university as the main source for information (p < 0.001), while 56% of squ and 45% of non-squ students reported that the social media was the main source respectively (p = 0.048). there was no statistical difference between male and female donors on their perception of level of self-knowledge on blood donation (p = 0.868). about 84% of the youth agreed that blood donation is one of the duties toward the community. squ students reported higher rates of respond to specific requests for blood donation (44.3% vs 29.7%, p < 0.001). squ students reported greater influence of peers (84% vs 60.7%, p < 0.001), personal knowledge (82% vs 74.7%, p = 0.029) and personal experience (79.3% vs 69%, p = 0.005) when compared to non-squ students. they also reported more feeling of commitment to the society (90.3% vs 78%, p < 0.001). squ students reported lower influence of parents (53% vs 65%, p = 0.005), lower rates of fear from needles (18% vs 32%, p < 0.001) and lower rates of fear from blood (16% vs 29%, p < 0.001). there was no difference between male and female genders in any of the discouraging factors. summary/conclusions: these results highlighted the positive impact and important rule of the youth in the promoting blood donations among themselves through this yearly college competition; in recruiting blood donors and in the dissemination of the knowledge of blood donation. distinct promotion strategies should be adopted to increased first time and repeated blood donation among the youth. we advocate for similar initiatives in encouraging blood donation and disseminate knowledge among individuals in the community. dubai blood donation center, dubai health authority, dubai, united arab emirates background: dubai is multicultural city in united arab emirates. only about 15% of the population consists of uae nationals with the rest comprising expatriates from various countries all over the world. approximately 85% of the expatriate population (and 71% of the emirate's total population) are asian, chiefly indian (51%) and pakistani (16%). dubai blood donation centre is the only centre providing blood donation services in dubai. arabic is the national and official language and english is used as a second language. in order to have good quality screening, it is important that blood donors understand the educational material and questionnaire properly. aims: dubai blood donation centre receives donors (nationals, residents and gcc card holders) from various countries. the aim of this study is to analyze the multinational profile of donors and to find out the need to add any third language to meet the customer needs and expectations. methods: a cross-sectional study of blood donors was conducted in dubai blood donation centre in 2019. the donors were asked about their country of origin, languages which they can read & understand and about the preferred mode of communication. results: a total of 1080 donors were surveyed and asked about the languages which they can read and understand and 1860 responses were obtained. the most common languages which can be read and understood by blood donors in dbdc are english (n = 760; 41%), arabic (n = 272; 14.6%), hindi (n = 268; 14.4%) and malayalam (n = 233; 12.3%). the donors come from different countries, most common 591 (54.7%) donors are indian and 73 (6.8%) are from uae. it was found that 45% donors can read and understand only one language. majority 884 (81.9%) donors can read and understand either of the official languages arabic or english. however, 196 (18.1%) donors can't read and understand these two official languages, the other common languages being hindi and malayalam. the donors were asked about the preferred mode of communication, 1290 responses were obtained. the most common mode of communication were sms and telephone (85% together). summary/conclusions: based on the above findings, it can be concluded that the blood donor profile in our centre is multinational which is a unique and almost similar to the population profile of dubai. as 18.1% donors can't read and understand arabic and english, so it has been decided that the educational material and questionnaire need to be prepared in one more language. hindi has been decided as the third language in the centre and donor questionnaire and educational materials in hindi will also be made available to the donors. further,the donors will be communicated through sms for routine messaging and disease notification while telephonic calls will be done only when the blood is urgently required. background: metabolic disorders (metds), including hypertension, dyslipidemia, hyperglycemia, and central obesity, are tightly associated with cardiovascular diseases and type 2 diabetes mellitus. due to the sedentary lifestyle and increased consumption of high-calorie diet in modern society, metds have become serious health problems worldwide. to have a better understanding and possible improvement on blood donors' health condition, we conducted a survey of the prevalence of metds among blood donors in a blood donation station located in the hsinchu science park in taiwan. participants with metds will be provided with health education materials about metabolic risk reduction, in order to prevent the development of future complications. aims: the aims of this study were to determine the prevalence of metabolic disorders among blood donors, and to calculate how much money would be paid to identify a case of hyperglycemia, hyperlipidemia, or undiagnosed diabetes. methods: this study was approved by the institutional review board of taiwan blood services foundation (tbsf). the body weight, body height, waist circumference (wc) and blood pressure (bp) of participants were measured. blood samples were obtained to determine the values of hemoglobin a1c (hba1c) background: the law on blood donation supports the development of the blood service and guarantees the protection of the donor's rights and the maintenance of health during blood donation in the russian federation. national criteria for donor selection for blood donation are used in the activities of blood service establishments and are aimed at ensuring the blood products safety. the study of the characteristics of blood donors allows to predict the development of blood service and to plan the volume of blood products for transfusion and plasma fractionation. aims: the aim of this work was to study the characteristics of whole blood and apheresis donors in the blood service in the russian federation. methods: indicators of activity in the blood service establishments in the russian federation in sectoral statistical observations over the period 2015-2017 and the calculation of indices characterizing the whole blood and apheresis donors were analyzed. data are presented according to the administrative division of russian federation into federal districts (fd). results: the proportion of whole blood donors was 83.7%, plasmapheresis donors -13.2%, blood cell apheresis, including plateletapheresis, donors -3.1%. for the period 2015-2017, the percentage of repeated and regular whole blood and apheresis donors increased from 66.2% to 72.2%. the percentage of first-time donors ranged from 27.8% to 33.8%. the largest proportion of plasmapheresis donors was observed in the volga fd (22.0%). about 28.9% of the total plasma was collected by apheresis from donors. the percentage of plateletapheresis donors increased from 1.9% to 2.3%. the largest percentage of plateletapheresis donors was observed in the central fd (2.9%), where a significant part of medical centers of cardiac surgery, hematology and bone marrow transplantation are located. the proportion of platelet concentrate collected by apheresis increased to 69.9% in 2017. actions to recruit young donors for blood donation and its components were regularly carried out in all federal districts. summary/conclusions: in the russian federation, the structure of donation is characterized by an increase in the proportion of plateletapheresis donors, stabilization of the percentage of plasmapheresis donors and an increase in the proportion of repeated and regular whole blood and apheresis donors. there are significant regional variations of donor's characteristics in the federal districts. background: shortage of blood supply despite continuous blood donation campaigns especially during local festive seasons has been a major issue in our country. thus, our faculty initiated blood donation drives in collaboration with national blood centre in order meet the demand for the blood requirements. however, the pre-donation deferral rate was relatively high among our young blood donors leading to loss of valuable blood units. understanding the causes of donor deferral provides direction on strategies for young donor recruitment and retention of future blood donation. aims: the aim of this study is to evaluate the young donor deferral pattern and to identify factors which could help in minimizing the preventable deferrals. methods: this is a retrospective study of voluntary young blood donors age between 20 to 23 years old recruited during mobile blood donation in faculty of medicine, universiti teknologi mara, malaysia. the study was conducted between january 2016 to december 2018. the data were retrieved from the official reports of each mobile blood donation. results: a total of 828 young blood donors had attended mobile blood donation during the study period. the overall pre-donation deferral rate is 25.6%. the main causes of deferral are low haemoglobin (hb) level (20.8%) followed by low blood pressure (16.0%), upper respiratory tract infection (11.3%) and sleep less than 5 h (9.4%). summary/conclusions: low haemoglobin and low blood pressure are the two common reasons for blood donation deferral among our young blood donors. in our study a significant proportion of deferrals are due to sleep less than 5 h whereby this could be prevented if the donors are aware of the donor selection criteria. strategies to mitigate preventable deferrals and improve blood donor retention particularly young blood donors as source of motivation for future blood donation are urged to avoid additional stress on the blood supply. background: in the modern world, donating blood has become a humane manner for saving of patients life. but there are barriers to blood donation which are designed to ensure both donor and blood recipients' safety. anemia is one of the most common health problems in the world. based on the who estimation, nearly a quarter of the world's population are suffering from anemia, its prevalence varies among the populations and age groups. the prevalence of anemia among men is 12.7% and in non-pregnant women is 30.2%. aims: the aim of this study was to determine the status of hemoglobin in volunteer blood donors referring to fars province blood transfusion service and to determine the demographic status of them during the last two years. our study included blood donors for all blood donors during the last two years. methods: the study is descriptive cross-sectional and our sampling was non-random and simple sampling method. all parameters related to the donors, including age, sex and type of donation were investigated and analyzed in spss software. results: the total number of referrals for blood donation was 454913. repeated blood donors was 44.8% of total population and had the highest number of referrals, followed by first and lapsed donors with 31.2% and 24% respectively. in terms of gender distribution, 6.2% were female and 93.8% were male. the highest rate of hemoglobin level less than 12.5 g/dl was found in first-time donors with 51.6% and the lowest prevalence was observed in lapsed donors, followed by repeated donors with 24.7%. 44.8% of the repeated blood donors have hemoglobin higher than 16.5. there was a significant difference between blood donation type and hemoglobin level. summary/conclusions: according to our findings, low hemoglobin levels are more common among first-time and female donors, and this requires a special training among these groups. because the high share of first time donors in blood supply and the positive impact of female donors on the blood safety, corrective action for that groups is recommended. finnish blood donor biobank j partanen, t wahlfors, m arvas, j clancy, k l€ ahteenm€ aki, e palokangas and n nikiforow background: the increasing need for large, well-characterized cohorts of healthy individuals for modern biomedical research, such as genomics or phenomics studies typically including tens or even hundreds of thousands of subjects, has posed the possibility of using blood services as an option for collecting samples and related data. the possibility to re-contact blood donors for repeated sampling or asking for additional data has further increased interest in collecting large biobanks from blood donors. there is also a need to study more thoroughly the effects of blood donation on donor health. aims: the first-phase goal is to recruit 50,000 blood donors with broad biobank consent for the finngen (https://www.finngen.fi/) project, a large publicprivate effort aiming to collect genome and health-related registry data of 10% (500,000) of the finnish population. (11.58%), dental examination 10(3.32%) and medication history 6(1.98%). permanent deferral namely, risk factor involving transfusion transmitted infections and chronic disease were 5(1.65%) and 8(2.64%) respectively. the prime cause of permanent deferral was risk factor involving transfusion transmitted infections while the temporary deferral was bed side hypertension. gender wise, the leading cause of donor deferral in male was bed side hypertension and anaemia was the major cause in female. summary/conclusions: the findings of the survey aid to evaluate the significant causes of blood donor deferral. this study suggests that the restrictive criteria can be used for blood donor selection. this will in turn increase the blood supply of tertiary care hospital. background: donor selection is the first step towards safe blood but retaining blood donors is also very important for the blood supply. donor questionnaire and the medical interview should provide optimal doctor deferral. aims: to evaluate deferral rate in blood donors in order to identify the main reasons and to target eventual corrective activities. methods: we analysed the data concerning blood donors who were registered in the period of three years (2016-2018). we used data from the information system e-delphyn. background: iron deficiency (id) in blood donors is an underestimated issue in many countries and may cause symptoms to blood donors even without anemia. id prevention is mainly based on the prevention of anemia in whole blood donors, which is done by deferring donors whose haemoglobin level is under defined threshold (120 g/l in women, 130 g/l in men in france). efs (french blood establishment) studies has observed that the rate of deferral for anemia is significantly higher in women than in men, either in french west indies (15.9% versus 3.4%) or in continental france (2.9% and 0.8%). assessing the prevalence of id is of great interest since strategies to counteract it must deal with donor health and self-supply. however, data on id are missing in france. aims: to estimate the prevalence of id in french whole blood donors and to identify risk factors associated with id. methods: this non-interventional, cross-sectional, multicenter study is performed in blood donors of efs and ctsa (blood center of the french military health service). all whole blood donors who met selection criteria are potentially included. donors coming for bloodletting and donors who refuse to participate to the study are excluded. no additional sample is taken for the study, ferritin is tested after blood qualification on surplus amount. samples are selected at random within all the geographical areas and all mobile blood drives and blood centers between march 11 and march 28, 2019. results: this study ferridon has been approved by ethical research committee. nine thousand (9000) whole blood donors will be included in efs centers in continental france. to have information on donors of afro-caribbean origin and comoros origin, 150 donations should be included in the french west indies and 120 in reunion island. additionally, 1500 whole blood donors will be included in ctsa centers. in this study, id is defined by ferritin lower than 26 ng/ml and iron overload is defined by ferritin higher than 300 ng/ml. all donors with iron deficiency or overload will received a letter advising to consult their general practitioner. weights will be calibrated on age, sex and geographical area so the sample will be representative of the french whole blood donors. estimation of id prevalence will take into account the weights and logistic regression model will be used to analyze risk factors associated with id. data will be analyzed during april and may 2019 to get result at the end of may. summary/conclusions: ferridon will be the first study on id in the french blood donors. considering the french health care system and diet, it will be interesting to compare those results to results from other countries. mostly this study will allow to consider various strategies dealing both with donor safety and self-supply. background: in portugal, with an aging population of around 10 million people, only 3.8% are blood donors. the country has a national center of blood supply and some central hospitals with a blood donation center. despite the growing practice of the excellent concepts of patient blood management, it is imperious to attract new donors. this need has been our inspiration to use new approaches towards people, in a constant work of promotion. aims: reach the majority of our local population using radio and telecommunication as well as social networks in an attempt to raise the number of new blood donors in a central hospital of the north of portugal. methods: active communication with the population of our reference area, via the social networks facebook tm and instagram tm , through educational digital posters and messenger service to answer any kind of questions. establish contact with radio and television stations as well as with the mayor of the city, journalists, schools, town hall deputies and celebrities, through email and telephone calls. design posters, flyers and public advertising to distribute in the city. results: through the social networks it has been possible to reach a population of dozens of thousands in our city, in a daily basis. the national and world donor days were celebrated with success, in our health facility, with city mayor and journalists, and also in three television stations with national broadcast, reaching millions of people. celebrities (sport, television, music, stand-up comedy, journalists and a magician) have accepted our challenge through videos or donating blood, appealing to blood donation and sponsoring our cause. these projects and continuous availability to innovate have given our hospital a self-sufficiency of 95% in 2018, instead of 80% in 2016, which implied receiving less blood unities from the national center of blood supply. our most recent project involves high schools, in an attempt to educate our next generation of donors, with meetings in the town hall with deputies and district school delegates. summary/conclusions: the aging population and the low percentage of blood donors are an important issue concerning public health. nevertheless, the good will and continuous advertising and educative work towards the population, appealing to the ethical and civil responsibility since young ages have shown to improve our capacity of response as a central hospital, increasing the auto-sufficiency of blood unities and the interest of younger donors. it is of the utmost importance to understand that this is a continuous and a hard work of the professional team of our hospital, involving countless calls, emails and hours to obtain some positive response, in an endless job. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . the mean interval between donations is shorter for former regular donors (4.9 months, p < 0.01) whereas donors with an interval of 9 to 12 months are more likely to be regular (aor 16; 95% ci 1.6-164). summary/conclusions: at the provincial blood transfusion centre of bukavu, the percentage of regular donors is low and there is a substantial loss of former regular donors. some factors identified to be linked to fidelity are unique to our study: female gender and a longer interdonation interval. other factors that are similar to those found elsewhere have a particular significance in our donor population which consists mainly of young people and people without income. efforts must be undertaken to ensure a supply from voluntary donations; recruitment strategies and target groups must be refined. future qualitative studies are needed to explain the various associated factors and better understand the motivations of regular and non-regular donors to improve donor retention. results: in kazakhstan, the proportion of donors is higher, especially primary. the number of blood and especially plasma donations is higher, which can be explained by the presence of several albumin and immunoglobulin production sites. increased evidence of blood transfusion rules, the development of a patient's blood management in combination with an increase in the quality of blood components cause a reduction in clinical need for red blood cells and plasma for transfusion. at the same time, the need for platelets is growing. it is difficult to assess the correctness of comparing the amount of banked whole blood. it is equally difficult to compare the number of received and distributed donor red blood cells and plasma: in russia they are measured in liters, and in kazakhstan in doses. with a certain degree of conditionality platelet extraction can be compared. in russia, they are counted in equivalent doses isolated from a dose of whole blood (at least 60 9 10 9 cells per dose), and in kazakhstanin therapeutic doses (at least 200 9 10 9 cells per dose). in 2017, the estimated consumption of platelets in kazakhstan exceeded the russian indicator by 28.0%. 86% of platelets in kazakhstan and 69.9% in russia are harvested by the apheresis method. inactivation of pathogens is performed in 92% of platelets in kazakhstan and in 15.4% in russia. pathogen inactivation with amotosalen allows us to abandon the examination of donors for cytomegalovirus and irradiation of platelet concentrate. the main modern trend in the use of cryoprecipitate is using it as a source of fibrinogen, a blood coagulation factor that is first depleted in coagulopathy associated with injury and massive bleeding. its production is growing in both countries, endowment in kazakhstan in 2017 exceeded the russian indicator by 141.4%. a significant plasma percentage in both countries does not pass quarantine due to repeated non-appearance of the donor and is subject to destruction. inactivation of pathogens is performed in 10% of plasma in kazakhstan and in 3.6% in russia. despite the instruction and selection of donors, laboratory screening of infection markers remains effective: russia more often identifies hiv and viral hepatitis c from potential donors, and viral hepatitis b and syphilis are detected in kazakhstan. 0.7% of donors in kazakhstan are exempted due to the results of multiplex screening of nucleic acids of three hemotransmissive viruses. summary/conclusions: the blood services of russia and kazakhstan perform tasks to provide medical organizations with effective blood components. in conditions of decreasing demand for red blood cells and plasma, it is advisable to focus on the efficiency of resource use and improving the quality of blood components produced. blood collection including apheresis p-091 finnish red cross blood service, helsinki, finland background: skin disinfectant must effectively reduce microbes from the arm of the donor. as a result of poor disinfecting microbes may be transferred from skin via venepuncture to the collected blood and contaminate the blood components. aims: to ensure the efficiency of the skin disinfectant used for donor arm disinfecting by validation. the validation has two criteria which the post-disinfection samples must achieve: 1. no bacteria growth near the puncture spot (result 0 cfu) in ≥ 90% of the samples. 2. total amount of bacteria on average is at most 2 cfu/25 cm 2 . at most 10% of samples are allowed to have 2-10 cfu/25 cm 2 . methods: microbiological samples were taken with contact plates from 30 voluntary persons' elbow folds before and after skin disinfection. the disinfecting was performed according to normal procedure by five nurses altogether with ethanol based disinfectant used in blood donation. on the sample plates an x was marked and this was directed to the puncture spot pointed by nurse. post-disinfection sample was taken at the moment the skin would be punctured with needle. results: the amount of bacteria varied from 20 to above 500 cfu/25 cm 2 in the pre-disinfection samples. disinfection reduced bacteria very well; the critical puncture spot was totally clean (0 cfu/25 cm 2 ) in 93.3% of the samples and 86.7% of the samples had 0 or only 1 cfu/25 cm 2 . the average number of bacteria after disinfection was 0,6 cfu/25 cm 2 and the maximum number was 4 cfu/25 cm 2 . most of the remaining bacteria were single colonies at the edges of the plates. summary/conclusions: both main criteria are fulfilled. the sub criteria of the second main criteria is also full filled if the not so critical colonies at the edges of the plates are not taken into account. the skin disinfectant in question is shown to be effective and can still be used in blood donation paying attention to thorough procedure performance according to the instructions and sufficient drying time of the disinfectant. background: research questions involving blood donation and recipient data often require advanced statistical methodologies. while such methodologies may appear in other medical research areas, specific tailor-made statistical tools and approaches are required for the analysis of blood-related data. these toolkits, which often require collaboration, are not always readily available to blood services, especially so in resource-limited settings. an international network of statisticians, epidemiologists and clinical researchers has been established for this purpose, which started with an invited session at the 2018 meetings of the international biometric society. aims: to exchange ideas, experience and knowledge to further improve the quality of blood sector research. methods: currently our network covers four major blood services and members from five different countries. the network has monthly conference calls about past and current research topics. we wish to extend this network further, and establish a subcommittee on statistical and epidemiological methodology with regular face-toface meetings at an international organization such as isbt. results: the monthly meetings have already demonstrated that the members share common problems and interests. for example, we are discussing techniques to analyze data with repeated measurements, e.g. eligibility haemoglobin tests, ways to assess the healthy donor effect, e.g. determining appropriate controls groups, and predictive models of blood supply and demand, e.g. stochastic processes and queuing models. the network also aims to organize training sessions in methodology either on site and/or by developing web lectures. summary/conclusions: an international network on statistical methodology for the analysis of blood donation and recipient data will improve the quality of research in the field of transfusion medicine research. expanding the network to include countries and blood services in research limited settings needs to be actively pursued. background: blood donor hemoglobin concentration (hb) is commonly measured from a skin-prick sample at the donation site, and low hb is the most common reason for temporary donor deferral. while a proportion of the deferrals do reflect true low hb, the skin-prick sample is prone to preanalytical error and variation resulting in false deferrals. aims: we assessed the applicability of a venous blood sample for second-line hb screening in blood donors failing the initial skin-prick test. methods: initial hb was measured from a skin-prick sample with the hemocue hb201 + (hemocue ab) point-of-care (poc) device. donors with hb < 125 g/l for females or < 135 g/l for males or with a decrease > 20 g/l from latest donation were included in the study. in the study group, a venous blood sample was collected for hb measurement with the poc device at the donation site. donation eligibility was based on this hb result. venous hb was also determined with a hematology analyzer (sysmex xn, sysmex co.). the blood service's current workflow served as the control group: two more skin-prick samples were collected and the donor's final hb and donation eligibility assessed with an algorithm based on all three skin-prick hb results. results: in the study (n = 305) and control (n = 331) groups, the proportion of male donors (27% and 26%) and the mean initial skin-prick hb (122 g/l and 123 g/l) were similar. significantly less donors were deferred from donation in the study group (40%) than in the control group (51%; chi-square test p = 0.004). the mean difference in venous hb with the poc device versus the hematology analyzer was à3 g/l (range à12 to + 6 g/l). two donors were incorrectly accepted based on venous sample poc result; however, in both, hb measured with the hematology analyzer was only 1 g/l below the limit of donation eligibility (124 g/l for a female and 134 g/l for a male). interestingly, a further 33 donors (27% of all deferred in the study group) would have been eligible for donation based on the hematology analyzer result. summary/conclusions: utilizing a venous blood sample for second-line screening of donors failing the initial skin-prick hb test significantly decreased low hb deferrals without compromising donor health. blood donors' and blood service nurses' reactions to the new workflow have been favorable. we conclude that valuable donations can be recovered and donor satisfaction increased by implementing a second-line hb screening model utilizing venous sample analysis at the donation site. background: there is a paucity of literature on haemoglobin (hb) reference values for adults above 60 years of age. this age group has been reported to use up to 50% the blood supply. some studies report a decline of mean hb with age, but others have found no change with age. conflicting findings of hb levels in the healthy elderly population may be associated with challenges in accessing data from healthy older adults, small sample sizes, selection bias and recent health population data. to donate blood, each individual is assessed as 'healthy' and must meet the minimum hb criteria. however, the hb criteria across countries vary and many blood collection services have an upper age limit for donors. as many populations around world are aging, restricting the upper age limit for blood donation may potentially affect the size of the donor pool and consequently the nation's blood supply. aims: to explore the hb levels of healthy older adults, through a multi-centre retrospective observational study of blood donors aged 60 years or older. methods: over a one-year period, hb values were collected from blood donors aged ≥ 60 years from blood centres of four countries. the estimated proportion of blood donors aged ≥ 60 years old for each country was 0.81% in south korea (sk); 2.57% in hong kong (hk), <3.18% indonesia (indo) and 9% in japan (jap). the minimum hb criteria varied between each country and ranged from 11.5-12.5 g/dl for women and 12.5-13.0 g/dl for men. hb levels were determined using point of care testing (hemocue, compolab, hemcontrol) or the xe-2100d sysmex dependant on the country of origin. statistical analysis of the mean, standard deviation and cumulative distribution of hb were determined by gender and age. background: medication usage is assessed to determine donor eligibility from the perspective of both recipient and donor safety. different time frames since last taken apply to different medications. assessment of medication use varies by jurisdiction, but most european centres use multiple questions. these often include a general question about recent medication use whereas the usa does not. at canadian blood services there are 6 medication questions on the donor history questionnaire (dhq), including any medication use in the last 3 days, vaccination and specific medications over different time frames (high teratogenicity medications). the name of each medication taken and reason for use are documented by staff at each donation attempt. assessment of the frequency with which this process occurs is the first step in improving efficiency of this aspect of donor screening. aims: to determine the percentages donors answering yes to medication questions by demographic variables. methods: all whole blood donors who completed the dhq (full length or abbreviated) in 2017 were included in the analysis. donors' answers to each of the 6 medication questions were extracted from the national epidemiology donor database, as well as sex and age. the number and percentage of donation attempts in which a donor answered yes to each medication question were calculated. donors who answered yes to any medication question were sorted by sex and by age group, the totals and percentages calculated. results: there were 975,067 donation attempts with a completed dhq. overall, 34% of donors answered yes to medications in the last 3 days, 8% to vaccination, and less than 0.1% to others (38% any). slightly more were female (42 vs 36%) of those who answered yes to any medication question, as well as by individual question. the percentage of donors answering yes to any medication question increased progressively in each age group from 24% of 17-19 year olds to 57% aged 60 + (p < 0.0001 for trend). summary/conclusions: more than one third of all donation attempts answer yes to a medication question and require further questioning and documentation. this is more common in older donors and follows a similar trend to general population medication use. comparison of ways of assessing medication use in different countries may help identify effective but more efficient approaches. in addition, the contribution to donor and recipient safety of assessing all medications should be assessed. blood center experience with trima accel 7 and tomes software j schreier 1 , a davison 1 , j gambarte 2 , y l opez 2 , c calonge 2 and e herranz 2 1 terumo bct, lakewood, united states 2 centro de transfusi on de la comunidad de madrid, madrid, spain background: in the madrid community, more than 4000 apheresis platelet collections were completed in 2018, of which almost 3000 were completed in the blood transfusion center and the remainder in several hospitals in the region. trima accel 7 was implemented to meet the productivity needs of the blood transfusion center while improving the donation experience. tomes (terumo operational medical equipment software), which enables bidirectional communication with trima accel devices, was used to connect and centrally manage all trima accel 7 devices with automated data capture and reporting. aims: the aim of this study was to evaluate operational improvements using trima accel 7 with tomes compared to trima accel version 6. methods: this was a retrospective study analyzing 1372 apheresis procedures on trima accel version 6 during the control period from 2 january 2018 to 10 september 2018 compared to 541 apheresis procedures on trima accel 7 during the test period from 11 september 2018 to 28 december 2018. this was not a paired study. operator interventions, and completed procedure rate comparisons, were analyzed using a 2-proportions test, whereas donor demographic data were analyzed using a 2-sample t-test. results: trima accel was used to collect single and double platelet products stored in platelet additive solution. operators selected either a single (target platelet yield = 3.0 or 3.5 9 10 11 ) or double (target platelet yield = 6.0 9 10 11 ) platelet donation based on desired procedure time not the maximum number of products that could have been collected per donor. no statistically significant differences were observed for donors in the test arm compared to donors in the control arm for total blood volume (control = 5148 ml, test = 5124 ml, p = 0.545), hematocrit (control = 43.7%, test = 43.6%, p = 0.233), or platelet pre-count (control = 249 9 10 3 / ll, test = 253 9 10 3 /ll, p = 0.091). females represented 21% of donors in the control arm compared to 23% of donors in the test arm. platelet split rate (platelet products per procedure) increased from 1.15 with trima accel version 6 to 1.18 with trima accel 7; procedure time decreased from 55.7 min to 53.3 min for single collections and from 63.0 min to 60.9 min for double collections with trima accel 7 (these differences were not statistically significant). the percentage of procedures that completed with no operator interventions due to access alerts increased from 62.4% to 84.8% (p < 0.001) and the rate of completed apheresis procedures increased from 89.8% to 92.4% (p = 0.083) with trima accel 7. manual transcription of data during the procedure was discontinued with the implementation of trima accel 7 with tomes. tomes captured procedural data and operator steps with barcode scanning and tracking of configured events. this information was transferred to tomes post procedure and printed as a final report. summary/conclusions: trima accel 7 significantly decreased operator interventions, and automated data capture with tomes eliminated manual transcription of data. both outcomes freed operators to complete other tasks and focus on donor well-being. background: the european committee (partial agreement) on blood transfusion (cd-p-ts) of the council of europe (coe) has appointed a working group (wg) to focus on issues with plasma supply management (psm). the task of the wg is, among others, to collect and analyze data in order to fill knowledge gaps concerning donor safety in plasmapheresis. in doing so, the working group will gather evidence base data to support the upcoming revision of the 19 th edition of coe's "guide to the preparation, use and quality assurance of blood components", the blood guide. an international survey was conducted sept-dec 2017, distributed to blood establishments (bes) by the cd-p-ts representatives to coe's member and observer states. the questionnaire included sections covering collection practices (volume and frequency), management of red cell loss, donor panel demographics and data on donor adverse events. aims: the aim of this study was to investigate whether collection practices following the recommendations published in the blood guide for maximal collection volumes and number of donations per year were indeed associated with higher levels of donor safety and improved donor base sustainability. methods: from the total of 36 respondents, 24 bes collected plasma for fractionation (pff) by apheresis and the study had a dataset covering 2,888,390 plasma donations in the latest fiscal year (lfy). the parameter used as marker of donor safety was the rate of immediate vasovagal reactions with loss of consciousness (vvr with loc) per 10,000 plasma collections. the parameter used as marker of donor base sustainability was the retention rate of donors, ie % donors active in the previous year returning to make a donation in the lfy. results: in the blood guide, the collection volume per apheresis is limited to 16% of the estimated total blood volume but maximally 750 ml, including anticoagulant. respondents had differing practices and scale of collection program 15 be were aligned or lower, and 9 be had higher collection volumes. altogether 14 reported the immediate vvr with loc rate, which mainly was lower than 10/10000 collections. there was a small trend towards reduced rate with larger collection volumes than allowed by the current blood guide. saline compensation during or after collection did not affect the rate of vvr with loc. no correlation was observed between the annual donor retention rate and the rate of vvr with loc or saline compensation practices, as reported by 16 respondents. the retention rate banded in the range of 50%>80% (mean = 60%, min = 25%, interquartile range = 14%, max = 90%). the association between maximum allowed yearly plasma collection (25 l) appears to be reasonably constant and showed no clear association with the donor retention rate. summary/conclusions: restricting the maximum collection limit according to the current blood guide was not associated with either lower vvr with loc or with higher donor retention rate. this study supports reassessment of current blood guide 0 s limits for collection volume of maximum of 750 ml per donation and 25 l per year per donor. methods: serum ferritin concentrations were established from sera stored at à30°c from repetitive platelet donors between 2014 and 2016, using architect â ferritin assay chemiluminescent microparticle immunoassay (cmia). the hematimetric parameters were evaluated in a total blood sample using the celldyn 1800 â . sixteen samples were obtained from women (age: 35.3 ae 12.4 years, range: 18-61) and 35 samples from men (age: 36.2 ae 9.4 years, range 20-61), corresponding to 55.2% and 37.2% of the total female and male repetitive donors of platelets by apheresis using trima accel â terumo-bct and amicus tm fresenius-kabi. the difference in the concentration of serum ferritin between the last and first donation was established, as well as the change in the predonation platelet count between the last and first event. results: in the study population, 43.8% of women and 40% of men performed repetitive donations of platelets by apheresis with an interval of less than three months. the change in ferritin concentration was evaluated according to the interval between donations. in women ferritin delta was à38.4 ae 52.2 ng/ml when the donations had an interval less than three months, vs 3.3 ae 13.0 ng/ml when the time between donations was higher (p = 0.046). in men the change in the ferritin levels was à38.5 ae 61.2 ng/ml with donation times less than three months vs © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 à3.3 ae 38.9 ng/ml with prolonged donation times (p = 0.042). in women, the change in platelet count was à2 ae 46.2 9 10 3 /ul, when the donations had an interval less than three months vs à22 ae 45.3 9 10 3 /ul when the time between donations was greater (p = 0.43). in men, the delta of the platelet count was à12 ae 37.0 9 10 3 /ul in donation times less than three months vs à13 ae 26.6 with higher donation times (p = 0.93). no correlation was found between the concentrations of serum ferritin and the platelet count (r = 0.05, q = 0.75 for males, and r = 0.34, q = 0.22 for females). summary/conclusions: the data obtained suggest that repetitive donation of platelets by apheresis with intervals between donations of less than three months, significantly reduce serum ferritin concentrations in women and men, although normal levels were maintained in both groups. there was no correlation with platelet count. therefore, it is proposed to develop prospective studies to establish the minimum time interval safety for platelet apheresis donor procedures. background: the demand for platelets concentrates is increasing continuously and becomes a challenge for the blood establishments. apheresis platelet collections may be a solution for this challenge. improving apheresis collection efficiency while maintaining blood donor safety is an important goal for the service du sang of the belgian red cross. aims: our establishment evaluated the improvements of the trima accel automated blood collection system version 7 (ta7) by comparing its routine performance with that of the previous software version 6.06 (ta6). methods: prospective, multi-site, controlled, non-randomized trial. apheresis collections were performed in three sfs sites: liege, mons and namur using the two trima software versions ta6 and ta7 sequentially. data were collected from december 2017 to april 2018 on ta6 and from june to july 2018 on ta7. simple and double doses of platelets (respectively 5.0 9 10 11 , 5.5 9 10 11 and 8.0 9 10 11 , 9.0 9 10 11 ) were collected in platelet additive solution (ssp+, macopharma) with concurrent plasma from the same cohort of donors in accordance to donor's eligibility and preferences. in order to maintain the same final platelets content in platelets concentrates, the trima accel's tool yield scaling factor (ysf) was subsequently adjusted from 1.06 (ta6) to 1 (ta7). platelet yield, duration of procedure, number of alarms requiring operators' interventions were recorded and evaluated. donor's hypocalcemia was avoided by giving preventively oral or intravenous calcium which was documented by the operators. results: five hundred ninety (590) collections with ta6 and 607 with ta7 were recorded, with 92% and 95% complete procedures respectively. mean duration of procedures was 70 min on ta6 against 72 min on ta7, p < 0.05. the mean alerts number per procedure on ta6 was 4.1 against 0.5 on ta7, p < 0.05 whereas the maximum alerts number per procedure was 57 and 12 respectively. on ta7, 76% procedures did not require operator's intervention against 40% on ta6,. with ta7 the inlet flowrate was automatically adjusted in 97.2% procedures. the inlet flowrate was increased in response to access pressure in 45.6% of procedures, for 3% of the procedures the inlet flowrate was decreased and for 48.6% of the procedures the inlet flowrate was increase and decreased on the same procedure by the ta7 autoflow system. summary/conclusions: ta7 with its autoflow function improves apheresis donors experience while decreasing operator' interventions through a significant reduction of access draw alerts. as expected from the trima accel platform, post-donation safety remains high. a weak increase in procedure duration was observed for the same platelet yield which may be resolved with further adjustments. background: trima accel system is an apheresis platform relying on continuous flow centrifugation to collect from a donor platelets, plasma or rbcs based on donor qualification. the latest software version -trima accel 7 (ta7) introduced the autoflow feature which allows for automated flow rate adjustments. moreover, ta7 leverages the mobilization capacity of the spleen increasing potential platelet productivity while maintaining high post-donation safety standards characteristic of trima accel. aims: the objective of this evaluation was to assess the impact of ta7 software by retrospective comparison of procedure data and potential for increased productivity with those of trima accel version 6 (ta6) in the same cohort of platelet donors. methods: eight hundred twenty one procedures, started on ta6 from 4th january 2016 to 10th october 2017 were compared to 995 procedures, started on ta7 from 18th october 2017 to 31 st december 2018. procedural data from the trima devices were captured using the cadence system (terumo bct, lakewood co). parameters investigated were the number of machine access pressure alerts per procedure, the potential for higher platelet yield collections and the actual collected yields within the same cohort of platelet donors. results: both donor populations (ta6 vs. ta7 respectively) were comparable and were characterized by: tbv -5207 vs. 5344 ml; platelet count pre-procedure -252 9 10³/ll vs. 252 9 10³/ll; hematocrit pre-procedure -44% vs. 44%. gender distribution was 11% female with ta6 vs. 2% with ta7. venous access pressure alerts were significantly improved by ta7 with an average of 0.2 alerts per procedure as compared to 2.0 with ta6, i.e. 92% decrease. this decrease went down to 91% if only male procedures were analyzed. the maximum number of pressure alerts went down by 84% from 90 alerts in one particular run in the ta6 cohort to 14 alerts in one ta7 procedure. procedure time for single platelet products was reduced from 51 to 49 min and for double platelet products from 59 to 54 min (ta6 and ta7 respectively). donor qualification possible was 52% of procedures yielding single products and 48% of procedures yielding double products with ta6. the percentage of procedures qualifying for doubles increased to 91% with ta7. in terms of split rates, i.e. how many platelet doses could be produced per apheresis collection, potential split rates increased from 1.48 to 1.91 from ta6 to ta7, respectively. in fact, the observed split rate rose modestly from 1.01 to 1.05, as shorter procedures were generally selected according to donors' preferences. summary/conclusions: in comparable donor populations, implementation of ta7 decreased the number of access pressure alerts significantly compared to previous trima versions. the average procedure duration was also found to be slightly reduced. implementation of ta7 has the potential to increase productivity significantly. the observed modest actual rise in split rate suggested that factors related to donor and inventory management will determine at which extent the potential of the new software will be used. donor compared experience on trima accel 7 to trima accel version 6 august 11 2017 to october 17 2017 or trima accel 7 during the test period from november 28 2017 to january 9 2018. this was not a paired study. donors completed the survey while recovering from the apheresis procedure in the cantina. results from the paper survey were transcribed into excel for analysis. results: 63 donors completed the survey during the control period whereas 86 donors completed the survey during the test period. the mean number of previous donations for the control period was 3.0 (min 1 max 6) and for the test period was 3.2 (min 0 max 7). there were no first time donors during the control period and 20 first time donors during the test period. 91% of donors rated their overall donation experience as good on trima accel 7 compared to 90% on trima accel version 6. zero (0) donor rated their experience on either trima accel device as poor. 100% of donors who responded to the question said they would donate on trima accel 7 again. summary/conclusions: no significant difference was observed in donor experience between trima accel version 6 and trima accel 7 as both versions receive high marks. background: the trend on growth of query for donor platelet concentrates is observed in russia for past few years. as reported by edqm in 2014, a higher number of the platelets was consumed compare to 2011 by 7.8%. patients' with hematological malignancies treatment requires platelet concentrates transfusions during chemotherapy, immunotherapy and hematopoietic stem cells transplantation. according to the data collected in national research center for hematology (nrch) in 2018, 635 (8.6%) of the 7,371 patients, treated within facility, received platelet transfusions as transfusion therapy. the total number transfused units is 14,292, which is 668 higher (by 4.9%) comparing to 2017. platelet concentrates production can be performed either by apheresis process or by pooling individual units recovered from the whole blood. taking into account that the nrch produces blood units for its own needs, the pooling is not suitable method for production because its implementation doesn't cover require for platelets and overproduces rbcs. that is why platelet concentrates in nrch are obtained by apheresis only. in summary, the growth on requirement for platelet concentrates and their safeness explains the need for a comparative study for effectiveness of platelet production using various apheresis systems. aims: the aim of the study is to compare effectiveness of platelet concentrate production using mcs + 9000 (haemonetics), upp and trima accel (terumo bct) version 6.0 protocols. methods: the data for 4868 protocols of platelet donations performed in 2018 were analyzed: 2773 on trima accel and 2095 on msc + 9000. all donors were voluntary and non-paid donors with previous experience of blood donations. the choice of the platelet collection device was random; analysis of the main characteristics of donors did not reveal any significant differences between the groups. the median age of the donor was 32 years old, height -175 cm, weight -76 kg, platelet count before donation -254 9 10 9 /l, hematocrit -42%. detailed data are presented in table 1 . student's t-test for unrelated sets was used for statistical analysis of the data. a value p of less than 0.05 was considered as significant. results: the data obtained showed significant difference (p < 0.01) between average number of platelets collected on trima accel (6.3 ae 1.06 9 10 11 /l) and on mcs + 9000 (5.09 ae 0.78 9 10 11 /l). while cost of consumables are comparable, trima accel demonstrated 23.7% higher efficiency. procedure duration also was comparable and averaged within 94 min for both devices. detailed data are presented in table 2 . it is crucial to mention that proportion of trima accel's donations was significantly increased in nrch during 2018 and reached 56, 7% in total (2017-19.4%). a flexible usage of trima accel's consumables for different procedures (regular platelet collection and collection in pas) allowed to change the pas/regular platelets collection ratio from 7.64% up to 43.7%. summary/conclusions: obtained results proved the effectiveness of the trima accel's use for platelets concentrates production. it allowed to increase the average count of platelets obtained for one procedure by 23.7% compared to mcs + 9000 while the cost of consumables and procedure duration are comparable. the donor's comfort during procedure did not affect either. in long terms increasing of number of platelets collected is reducing the cost of platelet concentrates production. abstract withdrawn. background: apheresis collected platelet concentrate is preferable in terms of reducing the risks of adverse reactions in platelet transfusion when compared to random donor platelet concentrates. aims: the aim of our study is to present our experience in collection donation of single donor platelets with apheresis. methods: this is a retrospective study performed in the institute for transfusion medicine from 2010 till 2019. all donors were fully informed on the donation procedure and signed an informed consent for donation. the optimal platelet count that we want to achieve was ≥ 3.0 9 10 11 equal to 12 random donor platelet doses. minimum preapheresis platelet count in donors requested to start the apheresis collection was 150.000/ll. platelet collection was performed using flow cell separators haemonetics mcs+ and trima accel. acid citrate dextrose formula a was used for anticoagulation. median precollection platelet count of donors was 273.000/ll, with range from 182.000/ll to 397.000/ll. male were 77% of the donors and females were 23%. the single procedure usually took 45-70 min. the median platelet count collected was 4.0 9 10 11 , range 2-6.5 9 10 11 . the median processed blood volume was 3215 ml and median used acd-a was 352 ml. mean total volume of collected product was 312 ml. the adverse effects included vein perforation and the numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and was very mild. summary/conclusions: the collected platelet count was more than the wanted optimum platelet count. the number of apheresis donors is increasing and we are working on expanding our voluntary platelet donors registry and increasing the number of typed donors in the registry. background: to determine value of hemoglobin in blood donors, there are some tools or methods used, such as: cyanmethemoglobin method that can detect of hemoglobin quantitative and methods cupric sulfate solutions (cuso4) can detect of hemoglobin qualitative. according to who (world health organization) to determine the level of hemoglobin in blood donors enough used cuso4 solutions with specific weight (y) 1.053 can to detect value of hemoglobin above or same with 12.5 gr/dl. but, cuso4 solution specific weight 1.053 can not to detect and elimination value of hemoglobin above 17 gr/dl or polycythemia sick. because it, central blood transfusion unit (utdp) as the central of blood service in indonesia to manufacture cupric sulfate solution (cuso4) with a specific weight (y) 1062 to detect value of hemoglobin below 17gr/ dl and determine value of hemoglobin above 17 gr/dl. because it, do the testing the accuracy of the solution cupric sulfate in detecting and eliminating donor with value of hemoglobin above 17gr/dl with the test of 35 samples. aims: to determine accuracy and effectiveness by blood donors unit in indonesia red cross to use cuso4 solution with specific weight 1.062 in to detect and elimination value of hemoglobin donors above 17 gr/dl. methods: used the method cyanmethemoglobin and cuso4 (y) 1.062 determination value of hemoglobin donor. test results were analyzed with spss software version 23 using nonparametric analysis wilcoxon test. results: this research testing the accuracy and effectiveness of using a cuso4 solution with specific weight (y): 1.062 in detecting and eliminating hemoglobin value donors above 17gr/dl. from data processing using spss with the wilcoxon test p value 0.02. summary/conclusions: it was found that the cuso4 solution (y): 1062 can detect hemoglobins value above 17gr/dl and more effective in checking the hemoglobin in blood donors. it can be seen from the data processing with spss version 23 with the wilcoxon test p value < 0.05. it is important to monitor the precise course by which repeated blood donation affects hb and the probability of low hb deferral. zinc protoporphyrin (zpp) is a functional indicator of body iron levels and is hypothesized to predict hb levels among blood donors. advanced statistical methods are necessary to properly analyze the longitudinal associations between zpp and hb in data with repeated donations per donor. aims: to determine whether predictions of future hb levels using current hb levels can be improved by taking zpp levels into account, and to illustrate the use of statistical models for repeated measurements of blood donors. methods: we used data from the zpp and iron in the netherlands cohort (zinc) study. we identified previous zpp levels (log-transformed) as the main predictor and adjusted for previous hb level, age, day and time of donation, donation history, bmi, blood volume and blood pressure. we used linear mixed models, which take into account missing data in the outcome and associations between repeated measurements, to investigate the longitudinal association between previous zpp and current hb levels. the longitudinal analysis with linear mixed models was contrasted with a simpler analysis based on the area under the receiver-operating-characteristic (roc) curve for the probability of low hb deferral. results: in total, 8194 whole blood donors (20,864 whole-blood donations) were included in the zinc study, 63% being female donors. previous zpp showed a statistically significant association (p < 0.001) with hb levels in females, but the size of the association was quite small (regression coefficient, b = à0.17, 95% confidence interval à0.22 to à0.11). the same was true for males, but the size of the association was even smaller. blood volume and age for women were significant secondary predictor variables; blood volume, age and donation interval for men. by comparison, the roc analyses showed relatively larger, but less statistically significant predictive effects of zpp on hb. summary/conclusions: zpp is a statistically significant predictor of hb levels, but the size of the effect after adjustment for previous hb and other variables is small. the results cast doubt on whether zpp is an effective predictive marker for hb level and low hb deferral, and suggest that zpp should not be included in prediction models for hb levels. by properly adjusting for associations between repeated measurements and by using all available data, longitudinal models provide less biased and more precise estimates than simpler cross-sectional analyses. background: finnish red cross blood service (frcbs) is a national blood service and is responsible for all blood collection and component production in finland. the highest age for blood donors was 66 years until the end of year 2013 and since beginning of 2014 donors between 66 and 70 years have been able to donate blood. blood donation after the age 66 is possible if the donor has donated within the last 24 months. the upper age limit was raised up based on adverse event data from frcbs donors up to 66 years and on published data from other blood establishments. aims: the aim of this study is to find out if the new policy from 2013 with upper age limit of 71 years is safe. therefore donor adverse event data was analyzed in order to evaluate if the blood donors older than 66 years have more adverse events compared to other donors. the most common donor adverse event for donors over 66 years, haematoma, was registered 211 times (0.05%). in the other age groups haematoma was registered 4408 times (0.56%) and the difference between the oldest age group compared to all other donors was statistically not significant (chi2, p = 0.012). vvrs with loc were registered 21 times (0.05%), vvrs without loc 25 times (0.06), and the total number of all daes was 293 (0.65%) in the age group 66 years or older. the respective numbers in the other age group were: 1152, 0.15%; 11055, 1.41%; 17550 (2.23%). the number of vvrs with and without loc, and total number of all daes in the age group 66 years and older was smaller than in the other groups and the difference between these groups was statistical significant (chi2, p < 0.0001). summary/conclusions: donors over the age of 66 years have less donor adverse events than other age groups. decision to raise the age limit from 66 to 71 seems proven to be right as the older age group has even less donor adverse events than other donors. background: deep vein thrombosis (dvt) of the donor's phlebotomy arm is a rare, but serious complication of blood donation that needs to be recognised and managed appropriately in a timely manner. post donation dvt will be classed as a 'serious adverse events of donation' (saeds)these are events that either result in donor death, hospitalisation, intervention or significant symptoms persisting for more than one-year post donation. aims: to review cases of dvt post donation reported in uk in 2016-2018 years and identify any common themes for improving practice methods: all data relating to saeds from the four uk blood services reported to shot in the last 3 years (2016-2018 inclusive) were reviewed to look for reports of dvt post donation results: a total of 135 saeds were reported in uk from approximately 5.8 million donations (whole blood and apheresis) collected during this period. three cases of upper limb dvt were reported during this time accounting for 2% of the saeds reported and rate of dvt of 1 in 1.9 million donations collected. -case 1: a regular male whole blood donor in his early 30s reported worsening arm pain 12 days following blood donation, had a painful venepuncture and a small bruise at site of donation. he was diagnosed to have an upper limb dvt extending to the subclavian and brachiocephalic vein and started on oral anticoagulants. no other contributory factor was obvious -case 2: a female donor in her mid-40s gave her sixth whole blood donation without event. 11 days after donation, she developed worsening arm pain in the donation arm and was diagnosed with an upper limb dvt and commenced oral anticoagulation. there was no other identifiable risk factor for the thrombosis -case 3: a female donor in her 20s developed pain, swelling, redness and itchiness in her donation arm and chest wall two days after donation. she also described prominent veins on the affected side compared to her other arm. she contacted the transfusion service one week after donation; by this time she was also breathless on minimal exertion. she was admitted to hospital and commenced on anticoagulant therapy. a diagnosis of dvt and associated pulmonary embolus was confirmed. the donor's only risk factor for thrombosis was use of the oral contraceptive pill. summary/conclusions: rare complications of blood donation, like dvt, can occur. superficial venous thrombosis may occasionally progress into the deeper veins of the donor's arm but dvt can also occur without signs and symptoms of superficial thrombosis. none of our patients had any overt evidence of superficial thrombosis. one patient in our series reported using oral contraceptive pill. no other risk factors for thrombosis was forthcoming. transfusion services should encourage donors to make early contact with the blood service if they experience arm complications so that they can be investigated and managed in a timely manner. staff dealing with such donors must recognise the possibility of this rare complication, explore other additional contributory factors and initiate prompt and appropriate management. background: voluntary blood donation is widely considered to be safe with very minimum chance of adverse reaction, which may occur during or after the end of phlebotomy procedure. aims: to find the adverse blood donor reaction among voluntary blood donors in tertiary care hospital in kathmandu methods: this is a prospective study done among voluntary blood donors at grande international hospital, kathmandu, nepal from february 2013 to march 2015. the outlines of reported and communicated adverse donor reaction were also collected after the blood donation from voluntary blood donors in different locations including outdoor and in-house blood donation drive results: in the present study 6,955 whole blood donors were included, during the period of 2 years, 105 (1.50%) adverse donor reactions were reported. majority 89 (84.76%) of adverse donor reactions were mild in nature such as, sweating; 27 (25.72%), light headedness; 19(18.09%), nausea and vomiting; 15(14.28), allergy and bruises; 11(10.47%), sore arm; 9(8.58%) and hematoma; 8(7.62%) while 16 (15.24%) were severe adverse reactions similarly, anaphylaxis; 11(10.49%), loss of consciousness; 3(2.85%) and convulsive syncope; 2(1.90%). markers of the adverse donor reaction were age, sex, pulse, weight, blood pressure and donation status. age and first time status were related with significantly higher risk of adverse reaction with 18-23 years old at higher risk compared to 24-55 years old. first time donors were at higher risk compared to repeated volunteer donors. summary/conclusions: the results of the study are helpful to identify and understand the complication of adverse donor reactions though the incidence of reactions in the blood donors is lower than in other studies. donor age and donation status were strong possibilities of complications. background: blood donors with pollen-induced allergy and asthma must often refrain from donation in pollen season despite medication, because of symptom severity or similarity to airways infection. extracts of the medicinal mushroom agaricus blazei murill (abm) given orally have been found to reduce ige anti-ovalbumin levels and ameliorate the skewed th1/th2 cytokine balance in mice sensitized to ovalbumin (takimoto, immunopharm immunotox, 2008; ellertsen & hetland, clin mol allergy, 2009). aims: the objective was now to examine whether supplementation with the abmbased extract that we used in the mouse model for allergy, could alleviate allergy and asthma in blood donors by reducing specific ige levels and basophil sensitization. methods: sixty donors at oslo blood bank with self-reported birch pollen allergy and/or asthma were recruited and randomized in a double-blinded, placebo-controlled study with oral supplementation for 7 weeks before the birch pollen season with the abm-based extract andosan tm (immunopharma, oslo, norway). this is a water extract of the bacidomycetes mushrooms abm (82%), hericeum erinaceus and grifola frondosa. the participants filled in questionnaires for allergic conjunctivitis & rhinitis, asthma and medication. serum ige (immunocap â , immunodiagnostics, sweden) and bet v 1-induced basophil activation in whole blood determined by cd63 expression in a flow cytometer (flow cast â , b€ uhlmann lab ag, switzerland), were analyzed before and after the pollen season. (trial record: nct03198455, clinical.trials.gov). results: there was significant reduction in allover allergy-related ailments and types of allergy medication used in the abm extract compared with placebo group during the pollen season and no side effects. also, abm treated asthmatics had fewer symptoms and used less medication than controls. in the abm group, serum levels of specific ige anti-bet v 1 and anti-t3, were significantly reduced during the pollen season as compared with levels in the placebo group. whereas the maximal allergen concentrations needed for eliciting basophil activation before the season changed significantly to lower concentrations (i.e. enhanced sensitization) after the season in the placebo group, these concentrations remained similar in the group given the mushroom extract. summary/conclusions: oral pre-seasonal supplementation with an abm-based extract for 2 months reduced general allergy ailments, asthma symptoms and medication in blood donors with birch pollen-induced allergy and asthma during the pollen season. this was due to reduced specific ige levels and basophils rendered less sensitive to allergen activation. the study suggests that supplementation with abm mushroom extract can have prophylactic effect on aeroallergen-induced allergy and asthma in blood donors. it may therefore reduce such ailments in affected blood donors and impact blood donations in the pollen season. results: dhv started in 20/9/2014. the data presented in this abstract is till 28/2/ 2019. data is collected from total of 114475 blood donors (86.67% male donors and 13.33% female donors). repeat donors accounted for 59.34% against 40.66% of first time donors. of the total number of donor adverse events recorded, 2.73% (2633) reported for male donors and 6.97% (1063) for female donors when the donor adverse events stratified age-wise, the highest incidence reported in age group 18-24 years (male 5.45% and female 13%). among age group 25-40 years, (male 2.4% and female 4.02%), whereas in age group 41-65 years, (male 1.52% and female 2.11%) data analysis of total 6226 reported and registered donor adverse events, are categorized as hyperventilation (864), sweating (1458), dizziness (pre-syncopal 3160), loss of consciousness (416), vomiting (108), convulsions (220), hematomas with re-bleed (255), nerve irritation (8) and off-site reactions (333). many donors showed multiple forms of reactions. summary/conclusions: evaluation of donor side effects helps to improve donation process and donor compliance. most frequently recorded reaction remains dizziness (pre-syncopal). our donor vigilance data show reactions occurred more frequently in younger age, female and first time donors. repeat donation and age are predictors for low rates of adverse events. participation in dhv implies an effort to improve donor care and safety infrastructure and a desire for national and international comparisons to determine best practices and also to look into effectiveness of risk reduction strategies and follow-up trends. pre-donation hydration was implemented as an interventional tool to test the effects of hydration on pre-syncopal reactions to blood donation, specifically targeting those at highest risk such as female, first-time, high school donors. the results are awaited. background: descriptions of deferral categories and a knowledge in the percentage of deferrals in each category are of value in formulating recruitment and retention strategies. this can also help in planning more efficient recruitment strategies and thereby assist in reducing the shortage in blood supply. aims: the aim of the study is to categorize all donors who were deferred during medical checkup and find out the donor deferral rate in dubai blood donation center from january 1 st 2016 to december 31 st 2018 and also to find out whether there is any yearly or seasonal trend in any of the categories of deferral criteria's which can aid in forecasting and managing donor pool. methods: a retrospective study of donors deferred during last three years from january 1 st 2016 to december 31 st 2018 was done in dubai blood donation centre. the donors deferred during pre-donation medical check-up were categorized into 8 categories including low hemoglobin, high and low bp, intake of antibiotic, fever and flu, taking other medications, travel history etc. the deferrals were analyzed monthly and yearly and then were compiled to find any yearly trend or seasonal trend in the donor deferral rate in any of the categories. the data were analyzed using the spss software and a p value of < 0.05 was considered significant. the assessment of donor suitability is in accordance with aabb standards and is consistently applied in every blood donation setting on each occasion of donation to all blood donors. results: during this study, 193,228 donors were registered from january 1 st 2016 to december 31 st 2018 and 41,037 (21.24%) donors were deferred. the common reasons of deferral were low hb, high bp, travel history, intake of antibiotics and cough/flu symptoms. there was a significant decrease in deferral rate from 24.07% (15302/63563) in 2016 to 20.79% (13180/63394)in 2017 and further to 18.74% (12419/66271) in 2018 (p < 0.05). the specific deferral rate due to low hb also significantly (p decreased during these three years (86/1000 in 2016, 68/1000 in 2017, 58/1000 in 2018), though no change was seen in the deferral due to other reasons. the reduction in the rate of deferral due to low hemoglobin may-be linked to the change in the staff performing the hemoglobin testing in dbdc (nurses instead of phlebotomist were assigned to perform hb estimation of donors). there was a seasonal variation in the deferral rate in all the three years-lowest in june (6/1000) and then increasing with a peak in october (19/1000) and plateauing till january. this pattern of deferral corroborated with the rate of deferral due to flu/fever and cough and antibiotics with an average of 4/1000 in june and increasing to 10/1000 in october (p < 0.05). summary/conclusions: staff competency is pertinent in accurately deferring donors. there is also a significant seasonal pattern in flu/fever and intake of antibiotics deferral rate that is reflected in the total donor deferral pattern. seasonal variation of specific category of donor deferral should be taken into account for donor recruitment and retention efforts. background: west nile virus (wnv) is a mosquito transmissible flavivirus. it has been shown (vogels 2017 thesis) that the common mosquito in the netherlands can transmit wnv in laboratory circumstances but presently does not lead to effective transmission. however, the number of outbreaks of wnv is increasing and moving from the eastern and southern european borders towards the traditionally more colder western and northern parts of europe. in order to prevent wnv transmission to blood transfusion recipients, dutch donors that travelled to regions with wnv risk are deferred for a period of 28 days for whole blood, platelet donations and quarantine plasma in order to exclude potentially infected asymptomatic donors. aims: to assess numbers of dutch donors who are deferred for travelling to wnv risk areas within europe, and the return after onsite and offsite deferrals of donors. methods: data from 2015 to 2018 on donation attempts and deferral were retrieved from eprogesa, the blood bank information system. onsite deferral is defined as a donation that was attempted in the deferral period or in the 7 days prior to deferral, all other deferrals are considered as offsite. a generalized estimating equation model was used to assess the association between onsite versus offsite wnv risk deferrals in 2015-2016 and subsequent return rates within two years (after which a donor is inactive according to domaine). results: in 2015-2018, 16,400 donation attempts led to onsite deferral for wnv risk; 87% at whole blood donation attempts, 13% at new donor examinations. in total 19,624 offsite deferrals could not be traced directly to a donation, but based on the next donation more than 90% were probably whole blood donors. the number of deferrals peaks each year during august, the major holiday period in the netherlands, and increased from 920 in august 2015 to 1711 in august 2018. this increase is probably caused by the expansion of wnv risk regions. the return rate of wnv deferred whole blood donors is slightly lower than for donors who are not deferred (92% versus 95%); for wnv deferred new donors the return rate is 75% (versus 87% for no deferral). thus wnv deferral resulted in approximately 400-500 extra lapsing donors during these 2 years. however wnv deferred donors, that are older (odds ratio (or) 1.03; 95% confidence interval (95% ci) 1.02-1.03), of male sex (or 1.16; 95% ci 1.03-1.31) and whole blood donors as opposed to new donors (or 2.01; 95% ci 1.70-2.38) were more likely to return to donate. there was no difference in return rate by offsite and onsite deferral. summary/conclusions: travel-related wnv deferrals are increasing with expanding risk regions, especially in the holiday season where the availability of donors is already low. although the numbers of donors who are permanently lost after wnv deferral are limited, the increasing numbers of lost donations make it important to consider alternatives to donor deferral such as wnv nat testing. background: low haemoglobin due to iron deficiency is increasingly recognized as a serious problem in many blood centers. donor education, iron supplementation, ferritin monitoring, and lengthening of inter-donation interval are currently the main mitigation measures. however, a number of factors in particular donor knowledge could impact their success. locally, iron supplementation programme was implemented since 2012 with target group of donors who have given blood within the last six months. aims: here we look at an online donor survey to gain insight on their view of the programme and knowledge. methods: donors with successful blood donation in the past six months would be given 14 days of one tablet of iron supplementation (100 mg elemental iron) since 2012. an electronic questionnaire was sent to blood donors in 2017 to assess their view on the programme and knowledge which focused on iron store and absorption, compliance and any side effects occurred. results: 3212 donors (male to female was 1:1.9) replied to the questionnaire. of them, 594 received iron supplementation (male to female was 1:1.6). most of the respondents (94%) had one or more donations in the preceding 12 months. of the 594 donors received iron tablets, only 246 (41%) took all; 69 (12%) took more than 50% but not all; 130 (22%) took some but less than 50% and 149 (25%) did not take any. gastrointestinal upset was reported in 113 (25%) donors and constipation seen in 193 (43%) among those who took at least some of the iron supplementation. most respondents answered correctly to the questions on the knowledge on iron store and absorption. when comparing those with better compliance (took more than 50%) to those who did not (took less than 50%), significantly more donors in the former knew vitamin c could enhance iron absorption (p < 0.05). on the other hand, no difference was seen when they were asked if 1) iron can only be absorbed from meat; 2) tea and coffee consumed during meal can enhance iron absorption; 3) everyone can take iron supplementation on their own; and 4) iron store in male is always more than female. summary/conclusions: the results suggested that there is definitely more room to enhance the blood donors' knowledge on iron store and absorption in order to improve the effectiveness of iron supplementation programme. besides, the side effects reported by the donors could be an important limiting factor that better alternatives should be explored and considered. background: vasovagal reactions (vvr) are a well-established deterrent to donor return. however, the correspondence between vvr experience and donor lapse is not perfect. in australia, for example, vvrs only reduce two-year return rates by 27% for whole blood donors and 22% for plasma donors. the elements of a vvr and the donor's interpretation of this event that protect against or encourage lapse have not yet been identified. aims: in this study we explored the views of donors on donating following a vvr, with a particular interest in their emotional reaction to the vvr, their understanding of what caused the reaction, and their intentions to return. methods: semi-structured telephone interviews were conducted with 36 whole blood and plasma donors who had a recent vvr experience. data were analysed using the framework approach. results: donors are generally motivated to give blood to help others and to positively impact on those in their communities. they anticipate feeling good after their donation but in contrast, for many, a vvr leaves them feeling anxious, embarrassed, and disappointed. for donors, the experience of a vvr negatively influences their perceived ability to donate successfully, and many fear it will happen again. however, this effect appears minimised among donors who at least partially attributed their reaction to their own behaviour, such as poor hydration. for donors already juggling multiple demands, a vvr may tip the balance with donating becoming too much of an effort and perceived risk. however, donors appeared more confident to return if they felt supported by staff or if they could donate with family or friends. summary/conclusions: this study provides valuable insight into the vvr experience, which will aid in the improvement of donor safety and retention. the findings highlight the need to improve communication at the time of and following a vvr, to educate donors on how to reduce their vvr risk, and to intervene to help donors maintain their perceived ability to give blood in order to maximise retention following a vvr. background: frequent blood donation depletes the iron stores of blood donors. iron depletion might have negative effects on the health of the general population, but its effect on the blood donor population is not well known. aims: to investigate the iron status of finnish blood donor population and how it relates to donor health, the finnish red cross blood service set up the findonor 10,000 study in 2015. we investigated whether there were changes in donors' selfrated health and if these possible changes could be associated with differences in iron biomarkers (ferritin and soluble transferrin receptor -stfr) or hemoglobin levels during the first study visit. methods: participants were recruited in three donation sites in the capital region of finland between may 2015 and december 2017. participants filled out an electronic questionnaire about their health and lifestyle at the donation site during their enrollment visit. participants were asked by letter to fill out the same questionnaire electronically during the summer 2018. we included the 1435 participants (596 men and 480 premenopausal and 359 postmenopausal women) who completed both health questionnaires. to evaluate self-rated health we used the well-validated single question: "how would you rate your health in general?". participants were able to evaluate their health status on a five-point scale: excellent, very good, good, moderate, and poor. iron biomarkers and venous hemoglobin were measured from blood samples collected at the first study visit. we first computed the odds-ratios of reporting poorer health depending on demographic group. we then compared iron biomarker and hemoglobin levels between donors who reported improved, similar or poorer health rating. results: donors who rated their health in the first questionnaire as moderate (n = 31), good (n = 483), very good (n = 684) or excellent (n = 237) health tended to report improved (58%), similar (63%), similar (55%) or poorer (55%) health ratings respectively in the second questionnaire. pre-menopausal women reported their health poorer in the second questionnaire compared to the first questionnaire more often than post-menopausal women (pre-menopausal 51%, post-menopausal women 38%), or = 1.37 95% ci 1.02-1.85). there were no differences between other groups. there were no significant differences in iron biomarkers levels (ferritin and stfr) or hemoglobin levels between donors whose health ratings were improved, similar or poorer. summary/conclusions: in this cohort, pre-menopausal women rated their health poorer at the end than at the beginning of the study more often than post-menopausal women. no association was found between changes in self-rated health and iron levels (ferritin, stfr) or hemoglobin levels. further studies about the factors relating to blood donors' self-rated health need to be carried out. background: in recent years, the blood donation business has made great achievements, but it still cannot avoid the occurrence of adverse reactions to blood donation which not only brings certain obstacles to the blood donation work, but also affects the enthusiasm of blood donors. aims: to understood the causes and other relevant factors of adverse reaction among blood donors, the information of blood donors at dai autonomous prefecture of xishuangbanna were analyzed in 2018. methods: the data of volunteers from january to december 2018 were analyzed. the causes of adverse reactions were classified, and the incidence of adverse reactions was compared in terms of gender, frequency, age and blood type of blood donors. results: there were 9081 blood donors in 2018, 69 (0.76%) of whom had adverse reactions and 9 causes were induced, among which mental stress was the most common factor that accounted for 78.26% (54 cases). there was no significant difference in the incidence of adverse reactions between men and female (p > 0.05). from the frequency of blood donation, the incidence in the first donor was significantly higher than that in the second donor (p < 0.01). when it comes to age, the incidence was different and the 18-25 age group was the highest (1.61%). among different blood group donations, there was no significant difference (p > 0.05). summary/conclusions: adverse reactions of blood donation is closely related to the psychological state and age of the blood donors. the staff of the blood center should further optimize the service, strengthen the communication and publicize the knowledge of blood donation. the ultimate goal is to increase the blood donation rate on the basis of reducing adverse reactions. background: blood loss due to repeated blood donation can lead to iron deficiency or anemia, but currently there is no management plan for the prevention of iron deficiency in korean blood donors. female and male donors are required to wait at least 8 weeks between blood donations in korea, which is the shortest period among all northeast asian countries. female and male donors are allowed to donate whole blood up to five times per year and platelets up to 24 times per year (if spaced more than 14 days apart for the latter) due to the chronic blood supply shortage. these facts induce concern about the impact of blood donations on the donors' iron status. aims: this study aimed to evaluate the effect of oral iron supplementation in repeat donors based solely on donation history. methods: the high-risk group included male donors with ≥4 whole blood donations or 16 plasmapheresis or plateletpheresis donations, and female donors with ≥3 whole blood donations or 12 component donations, both within the previous year. the control group consisted of first-time or reactivated (ft-ra) donors who had no history of blood donation in the past 2 years. the hemoglobin (hb) level, ferritin level, total iron binding capacity (tibc), transferrin saturation, and soluble transferrin receptor (stfr) of repeat donors at high risk for iron deficiency were compared to those of ft-ra donors. iron deficient erythropoiesis (ide) is defined as present if the log of the ratio of soluble transferrin receptor to ferritin was ≥ 2.07. the repeat donors took iron supplements for 4 weeks and the same tests were repeated after 2 and 4 weeks to evaluate their effects and the side effect and compliance was assessed. results: a total of 53 male and 57 female repeat donors were recruited, and 30 each male and female ft-ra donors were recruited to the control group. after 4week iron supplementation, among male donors, the prevalence of: low hb level (<13.0 g/dl) decreased from 24.5% to 1.9%; low ferritin level (<15.0 ng/ml) decreased from 58.5% to 3.8%; high tibc level (>380 lg/dl) decreased from 66.0% to 37.5%; low transferrin saturation (<20.0%) decreased from 58.5% to 35.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 28.3% to 2.3%. among female donors, the percentage of: low hb level (<12.0 g/dl) decreased from 43.9% to 9.8%; low ferritin level (<15.0 ng/ml) decreased from 78.9% to 11.8%; high tibc level (>380 lg/dl) decreased from 68.4% to 24.5%; low transferrin saturation level (<20.0%) decreased from 70.2% to 22.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 94.7% to 44.4%. in total, 15 male (28.3%) and 29 female (56.9%) blood donors reported undesirable side effects related to iron supplementation. a total of 38 male (71.7%) and 36 female (70.8%) blood donors were administered iron supplementations for 28 days. 89 participants (85.6%) answered that they were willing to take a complimentary iron supplementation. summary/conclusions: ferritin level, considered a reliable indicator of iron status, increased and ide decreased significantly after iron supplementation in female donor group, but not in male donor group, compared to the ferritin levels and ide of control donors. iron supplementation in repeat donors at a high risk of iron deficiency was shown to reduce their risk of iron deficiency or anemia irrespective of gender; however, 4-week oral iron supplement was not enough to restore iron storage level in the male donor group. background: c-reactive protein (crp) is an acute-phase protein and a non-specific maker of inflammation and tissue damage produced by the liver. several prospective epidemiologic studies have demonstrated that high-sensitivity c-reactive protein (hs-crp) is a predictor of future coronary events among apparently healthy men and women, hs-crp level greater than 3 mg/l has been independently associated with a 60% excess risk in incident of coronary heart disease (chd) as compared with levels less than 1 mg/l. frequent blood donation has been associated with a lower incidence of coronary artery disease (cad); however, there is a dearth of information on serum levels of crp in the nigerian donor population. aims: to investigate whether regular blood donation is associated with lower serum hs-crp level in nigerian blood donors. methods: a descriptive cross-sectional study carried out to measure serum levels of high sensitive c-reactive protein (hs-crp) and ferritin among blood donors attending the donors' clinic in lagos university teaching hospital (luth). subjects who did not meet criteria for blood donation were excluded. additional data on sociodemographic characteristics was collected using interviewer-administered questionnaire. serum ferritin was analysed using chemiluminescent microparticle immunoassay performed on the abbott architect ci4100 (abbott laboratories, abbott park, il, usa). serum concentration of hscrp was estimated by immunoturbidimetry method using analytical kits from erba diagnostics mannheim gmbh in semi-autoanalyzer (xl 180, erba mannheim). data was analysed using stata version 15 (stata corp) statistical software. results: in total of 313 blood donors, 278(90.8%) were males and 28(9.2%) were females, the mean age was 32.3 ae 9.3 years. two hundred and thirty four (74.8%) were first time donors and 79(25.2%) were regular donors, serum levels of hs-crp was slightly higher in regular donors compared to first time donors (0.95 ae 3.7 vs 0.35 ae 1.7 mg/l, p = 0.06) though the difference was not significant. serum levels of ferritin was significantly higher in first time donors compared to regular donors (87.56 ae 23.9 vs 46.44 ae 31.4 ng/ml, p = 0.02). interestingly, levels of serum hs-crp were significantly higher in male than female population (0.52 ae 2.4 vs 0.17 ae 0.2 mg/l, p < 0.03) and smokers than non-smokers (1.4 ae 4.6 mg/l vs 0.4 ae 1.9 mg/l, p = 0.02). correlation analysis showed no correlation between serum hscrp and serum ferritin levels in both categories of donors while there was a weak positive correlation between hs-crp levels and white blood cells among the first time donors. summary/conclusions: this present study did not reveal any decrease in baseline levels of serum hs-crp with regular blood donation; smoking status and gender were however associated with an increase in baseline hscrp. this finding suggests that hs-crp level might not be a useful marker of future coronary events in healthy blood donors in nigeria. background: because the blood donation removes 250 mg of iron from the donor, iron deficiency, frequently occurs in regular blood donors leading at a long term to the anemia. aims: to determine the effect of blood donations on ferritin levels in regular blood donors. methods: all prospective donors have been submitted to a physical examination and a health history assessment intended to ensure that the prospective donor is in a good general health and eligible to donate blood. the acceptance criteria are: • hemoglobin > or = 13.5 g/dl for male and > or = 12.5 g/dl for female • inter donation interval = 56 days • 5 donations/year for male and 3/year for female all eligible donors and deferred donors for all reasons except for low hemoglobin who accepted to enroll in this study and signed a consent. in addition to the medical exam, two samples have been collected one for cbc and another for ferritin. donation history, sex, age and weight have been documented. results: 1056 first time and regular donors accepted to enroll in this study. only 38 female donors (3.6%) participated to this study. 11.3% of the participants were first time donor. 21% of male and 26% of female frequent donors are iron deficient 173 out of 1018 male blood donors were iron deficient (17%) with serum ferritin < 30 ng/ml. 98.8% were repeat donors. 7 out of 38 female donors were iron deficient (18.4%) with serum ferritin < 13 ng/ ml, all were repeat donors. 19.2% of repeat donors were iron deficient 3/180 of the deplete donors were first time donors summary/conclusions: frequent blood donors have higher prevalence of iron deficiency than first time donors. female donors have a slightly higher prevalence of iron deficiency than male donors. prevalence of iron deficiency in abu dhabi donor population is lower than the published data. changes need to be done on: increase inter donation interval or restrict the total number of allowable donations in a 12-month period for whole blood and red cells modifying donor hemoglobin requirements testing for serum ferritin iron supplementation donor education abstract withdrawn. background: haemovigilance procedures aim to guarantee not only the safety of the recipients of blood and its components but the safety of the donors as well. every adverse reaction that occurs during the donation of blood or its components can potentially be a threat to the health of the donor which can subsequently lead to the decision of the donor to resign from donating blood. aims: the aim is to analyse the type and the frequency of occurrence of adverse reactions among the donors donating blood or its components independently of the method of the donation. methods: we have analysed the number of collected donations and the number of adverse reactions in the years 2014-2018 in the group donors of aged 18-24. we have specified following adverse reactions: vasovagal response without fainting, vasovagal response with fainting, vascular reactions (bruises) and other (e.g. allergic reaction to the anticoagulant, loss of blood pressure due to hypovolemia). the analysis was made using data obtained from computer system blood bank which is in operation in blood center in pozna n, poland. results: in years 2014-2018 the total number of 1904 adverse reactions among the donors was recorded which is 0.39% of the total number of collected donations. 50% of the adverse reactions occurred in the group of donors aged 18-24. vasovagal response without fainting was the most common adverse reaction in the total number of reactions and totalled 50.2% of all adverse reactions. in the group of donors ages 18-24 it totalled 27% of all adverse reactions. the second most common type of adverse reactions was vasovagal syncope that totalled 29.8%, in the analysed group of donors 15.96%. vascular reactions (bruises) totalled 10.2% of all adverse reaction, in the analysed group 5.6%. the remaining adverse reactions totalled 9.8%. summary/conclusions: 1. vasovagal reactions (with and without fainting) were proved to be 2 most common adverse reactions in the group of donors aged 18-24 i.e. in the groups of donors just starting to donate blood. it seems reasonable to continue with further research into the reasons for the occurrence of this psychosomatic reactions. 2. it seems beneficial to provide constant educational activities of young donors regarding the preparation for the process of donation of blood and its components (proper nourishment, hydration as well as planning the time for scheduled donation long enough for a safe and pleasant procedure. 3. it seems beneficial to provide constant training for the medical staff involved in the process of donation regarding active observation of donors, proper conduct in the situation when the adverse reactions occur during the blood donation, ways to minimize the fear of donors, effective communication with the donors (explaining the process of blood donation, proper behaviour after the donation e.g. avoiding physical exercise or straining the arm). blood products -blood processing, storage and release background: the accumulation of microvesicles (mvs) in rbc concentrates during storage may be responsible for clinical symptoms such as inflammation, coagulation, and immunization. aims: our aims was to determine whether any of cd molecules responsible for important functions are present on the microvesicles, and if their expression level is dependent on the storage period of rbc units. additionally, by using cytometric analysis and phagocytosis visualization in a confocal microscope, we examined the interactions of donor monocytes with erythrocyte microvesicles, depending on their time of storage. methods: erythrocyte microvesicles were isolated from "fresh" (2 nd day) and "old" (42 nd day) stored rbc units. qualitative and quantitative cytometric analysis of these membrane structures was performed using the annexin v-fitc, anti-cd235a-pe antibody, and calibrated beads. the microvesicles were also visualized under a confocal microscope. the expression of the molecules cd235a, cd44, cd47, cd55, cd59, and of phosphatidylserine was analysed using flow cytometry. measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. results: the analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0.5 lm in the "fresh" and "old" blood samples. we observed a statistically significant increase in the number of microvesicles in the "old" units (201 ae 139 mvs per 1 ll), as compared to the microvesicles in the "fresh" (67 ae 53 mvs per 1 ll). at day 2, the microvesicles had elevated expression levels of cd47 and reduced expression levels of phosphatidylserine. significant changes were also observed in the case of cd55 and cd59 molecules. the expression of these molecules of vesicles isolated from "fresh" rbcs was lower than in the case of 42-day vesicles. the phagocytosis index was significantly higher (28.44%) for the microvesicles isolated from 42-day stored rbcs than for microvesicles from the 2background: platelet concentrates (pcs) are conventionally stored at room temperature with a limited shelf-life of 5-7 days. alternative storage methods, such as cold storage and cryopreservation are attractive options due to the potential for extended storage, reduced bacterial growth and improved hemostatic function. cryopreservation of human pcs has been associated with formation of more microparticles and elevated procoagulant activity compared to liquid-stored (room temperature-and cold-stored) pcs. microparticles are submicron plasma membrane particles that have been postulated as potential mediators of adverse transfusion outcomes. similarities in the size and storage-related changes up to 7 days suggest that sheep may be a suitable model in which to investigate the effects of pc transfusion. previous research has established that room temperature stored sheep pcs contain fewer microparticles than human pcs. however, nothing is known of the effect of other storage conditions. aims: this study aimed to determine whether cold storage and cryopreservation contribute to variation in concentration and size of sheep platelet derived microparticles compared to conventionally stored sheep pcs. methods: sheep buffy coat derived pcs in 30% plasma/70% ssp+ were prepared with minor modifications to standard procedures for preparation of human pcs. sheep pcs were split into 3 units (n = 6 of each) on day 2 and stored either at room temperature (rt; 20-24°c with agitation) for 5 days, cold stored for 21 days (2-6°c no agitation) or cryopreserved (à80°c with the addition of 5-6% dimethyl sulfoxide) for 33-109 days and sampled post-thaw. platelet supernatant, prepared by double centrifugation, was stored at à80°c. the mean size and concentration of microparticles were measured using nanosight ns300 nanoparticle tracking analysis system (malvern instrument). results are mean ae standard deviation. storage associated changes overtime were determined using a one-way analysis of variance with bonferroni's post-test. paired t-tests were applied to determine the effect of cryopreservation. a p-value of < 0.05 was considered significant. results: at day 2, sheep pcs had a microparticle concentration of 3.77 9 10 10 ae0.84 9 10 10 microparticles/ml with a mean size of 156.6 ae16.7 nm. storage duration at rt sheep pcs was not associated with significant changes to microparticle concentration or size. cryopreservation of sheep pcs significantly increased the concentration (4.39 9 10 10 ae0.81 9 10 10 microparticles/ ml; p = 0.0087) and the mean size (170.9ae 20.5 nm; p = 0.0008) of microparticles post-thaw. the mean size and concentration of microparticles in the cold-stored pcs at day 21 was comparable to room temperature pcs stored for 5 days (165.4 ae17.5 nm vs. 163.6 ae20.9 nm; p = 0.4081 and 4.11 9 10 10 ae 0.54 9 10 10 microparticles/ml vs. 3.82 9 10 10 ae0.71 9 10 10 microparticles/ml; p = 0.16 respectively). summary/conclusions: cold storage of sheep pcs did not impact formation of microparticles over the 21 days storage period; however, cryopreservation increased microparticle concentration and the size post-thaw. further investigation is required to determine whether these findings are influence hemostatic function. a pre-clinical sheep model of cold-stored and cryopreserved pc transfusions can facilitate mechanistic studies and complement clinical trials. background: during storage, the properties of rbc in storage solution change ("storage lesion"). for instance, ph, atp and 2,3-dpg concentrations decrease upon prolonged storage. these changes can affect oxygen delivery by the cells. the capacity to deliver oxygen is defined as p50: the oxygen tension (po2) at which 50% of the hemoglobin is saturated with o2. an oxygen dissociation curve (odc) represents the non-linear relationship between saturated hemoglobin and po2. this relationship is dependent on temperature, ph, pco2 and 2,3-dpg. due to changes in these factors, the curve will shift along the x-axis. in whole blood, p50 is at a po2 of about 26 mm hg. not much is known about p50 of rbcs in storage solution, and the changes during storage. aims: to determine the oxygen dissociation of rbcs stored in standard red cell additive solution sagm and in pagggm (an experimental red cell additive solution, transfusion. 2010;50:2386-92). methods: rbcs were prepared in sagm (n = 3) or pagggm (n = 3). pagggm is designed to better maintain both atp and 2,3-dpg during storage. rbcs were stored at 2-6°c and sampled on day 1, 35 and 56 for (internal) ph, atp, 2,3-dpg and p50. p50 was determined by hemox analyzer (tcs scientific corp.). the principle of the hemox is based on the measurement of spectrophotometric properties of hemoglobin at different oxygen pressure. rbc samples were brought from oxygen-rich environment to oxygen-poor environment (2%) using n2 gas. p50 was determined from the obtained odc. results: the whole storage period, ph i of pagggm-rbcs was higher compared to sagm-rbcs. 2,3-dpg content of sagm-rbcs decreased during storage and was below the detection limit after day 14. 2,3-dpg content of the pagggm-rbcs increased the first days of storage and slowly decreased from day 7 on. at day 56, pagggm-rbcs still contained 2.3-dpg (2.2 lmol/g hb). p50 values decreased during storage from 26 mmhg at day 1 to 20 mmhg at day 56 for sagm-rbc and from 31 mmhg to 26 mmhg for pagggm-rbc. p50 values of pagggm-rbcs were higher during the entire storage period. summary/conclusions: during storage, the p50 decreased in all rbcs. the p50 was higher for the pagggm-rbcs during the whole storage period. the higher p50 in pagggm-rbcs seems to correlate with the higher 2,3-dpg content in these cells. background: in belgium 100% of the platelets are pathogen inactivated (pi) and legislation requires a minimum platelet content of 3.0 9 10 11 per platelet concentrates (pc). therefore routine pools are produced with 6 buffy-coats (bc). facing increased demand of pc and stable to slightly declining red blood cells (rbc) demand, production of whole blood (wb) derived platelets must be adapted to switch flexibly from 6 to 5 bc per pool. this dual pooling strategy should allow alignment between wb collection forecast, pc inventory, pc demand and pc production. aims: first develop a pooling procedure with 5 bc and 250 ml platelets additive solution (pas) instead of 280 ml for 6 bc, without changing the settings of our wb separators and platelets separators. maintain a content of ≥ 3.0 9 10 11 platelets with a ratio plasma/pas between 32 to 47% required for pi. after validation, deploy a dual pooling strategy (5 or 6 bc/pool). methods: wb is collected with top and bottom kit (composelect; fresenius kabi) and separated (macopress; macopharma) to produce 44 ml bc with 40% haematocrit (htc) and > 90% platelets recovery with average platelets content of 1 9 10 11 . 5 random bc are pooled with 250 ml or 6 bc are pooled with 280 ml of pas-e, platelets are then extracted on tacsi pl (terumo bct) and pc are treated for pi (intercept blood system; cerus). each pc is sampled and platelet content is determined (abx pentra xl 80; horiba). results: during the study 45594 bc were processed into 8225 pools (3756 (45.7%) with 5 bc and 4469 (54.3%) with 6 bc). before tacsi separation, bc mixture with pas-e had volumes of 421 ae 9 ml (5 bc) and 491 ae 8 ml (6 bc) with respectively htc of 19 ae 1% and 20 ae 2%. the plasma/ pas ratio was 37 ae 1% in both cases. tacsi separation was performed with one same program for both types of pools. after pi, platelets content of the pools was 3.8 9 10 11 ae 0.5 with 5 bc and 4.7 9 10 11 ae 0.5 with 6 bc (average ae standard deviation). pools below the limit of < 3.0 9 10 11 were 139/3756 (3.7%) with 5 bc and 1/4469 (0.02%) with 6 bc. the platelets concentrations (10 3 /ll) were 1395 ae 167 (5 bc) and 1491 ae 155 (6 bc). platelets recovery was 85% ae5 for 5 bc and 86% ae 6 for 6 bc. summary/conclusions: 45594 bc could theoretically produce 7599 pools of 6 bc or 9118 pools of 5 bc. this means a maximum potential gain of + 20% pc. in practice during shortage periods we switched from 6 to 5 bc when dictated by the actual inventory levels and hospital needs. the advantage of this dual pooling strategy was a gain in production capacity to cover these shortage periods (626 pc, +8%). the disadvantage of pooling randomly 5 bc is that 139 pools contained less than 3.0 9 10 11 platelets per pool potentially limiting their usage to low weight or paediatric patients. a preselection of the bc based on platelet count could optimize the 5 bc pooling procedure. background: apheresis-derived platelet concentrates (apcs) is a standard medical therapy indispensable to contrast bleeding or hemorrhage. however, bacterial infection caused by storage at room temperature (rt) still remains the major drawback. recently, we showed that cold-stored apcs are associated with better plt functionality but with accelerated clearance (haematologica 2018, pmid: 30115655). cold-induced apoptosis was identified as a potential mechanism of the shorter plt survival aims: to investigate the protective effect of apoptotic inhibitors during cold storage of apcs methods: apcs were collected and stored at rt and 4°c in the presence or in the absence of caspase-3 inhibitor. the phosphatidylserine exposure and the mitochondrial membrane potential (mmp) (tetramethylrhodamine ethyl ester perchlorate [tmre ] staining) were measured using flow cytometry. the protein expression was quantified by western blot results: a higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored apc compared to rt (% apoptotic events meanaesem: 13 ae 1% vs. 5 ae 1% p = 0.018). to verify if the apoptotic signal, observed with phosphatidylserine, specifically involved the intrinsic pathway, the mmp was analyzed as a marker of alive cells. interestingly, after cold storage a decrease of the mmp was observed compared to rt indicating the activation of the intrinsic pathway (mean fluorescence intensity tmre meanaesem: 6.13 ae 1.89 vs. 18.53 ae 3.64, p = 0.038). accordingly, a decrease of the procaspase-3 level after cold storage was detected by western blot analysis. however, when plts were stored in the presence of caspase-3 inhibitor a significant rescue of the cold-stored cells viability was observed (tmre staining: % alive cells meanaesem: 74 ae 3% vs. 22 ae 3%, caspase 3 inhibitor vs. ionomycin, p = 0.005). this indicates that the activation of the apoptotic pathway, induced during cold storage, can be prevented using caspase inhibitor summary/conclusions: our results show that the reduction of cold-stored plt viability can be prevented by a specific caspase inhibitor. consequently, cold storage, associated with a better plt functionality, may become an efficient strategy for apc storage in combination with apoptotic inhibitors background: gamma-irradiation is used to treat red blood cell (rbc) concentrates (rccs) for patients who are immunosuppressed. this treatment is known to damage rbcs and to increase storage lesions. one of the causes of the storage lesions is the presence of oxygen. several studies have shown, based on different strategies to reduce o 2 , a reduction of storage lesions related to metabolism, protein modifications and cell morphology. aims: the present research work investigated the effect of gamma-irradiation on rccs stored under normal condition and hypoxia/hypocapnia. methods: saturation of o 2 (so 2 )-and abo-matched rccs from whole blood donations, leukoreduced and prepared in paggsm (macopharma, france) were pooled and split in two identical rccs within 24 h post-donation. one bag (treated) was submitted to oxygen and carbon dioxide adsorption (oxygen reduction bag, hemanext, usa) for 3 h on an orbital shaker (50 rpm) at 22°cae2 and then transferred to a storage bag impermeable to gas. the other one (control) was left as it is. the two bags were then stored at 4°c. a g-irradiation treatment (25 gy, gammacell 3000 elan, theratronics) was applied at day 2 or 14 and the rccs (expiry dates at day 16 or day 28, respectively) were stored until day 43. hematological parameters, glycolytic metabolites, extracellular potassium level, antioxidant power, morphology and deformability were measured. results: starting so 2 values were of 63.7%ae18.4 (n = 12) in control and of 20.8%ae9.8 (n = 12) in treated bags, and reached 90.8%ae9.1 and 6.6%ae5.9 at day 43, respectively. as expected, an increase in glycolysis rate was observed during deoxygenation without any influence from the irradiation. potassium levels were identical in treated and control, and reached around 70 mm at expiry with an irradiation-dependent kinetic release. antioxidant power and deformability were identical in both conditions. no difference in hemolysis was observed after irradiation on day 2 and the values stayed equivalent through end of storage (at day 16, hemolysis (control) = 0.37%ae0.24, hemolysis (treated) = 0.34%ae 0.15, p-value > 0.9999). when irradiated at day 14, hemolysis was lower (p-value = 0.033) in treated rccs at the end of storage (day 28, 0.67%ae0.16) compared to control (1.06%ae0.33). seven days post-irradiation, two-third of the control rccs were above the limit of 0.8% whereas all the treated rccs remain below the limit. quantification of microvesicles and morphological analysis confirmed these data. summary/conclusions: the storage under hypoxia has a beneficial effect on rbc storage thanks to a decrease in o 2 content and to an improvement of metabolism. this benefit provided equivalent storage when rccs were irradiated at day 2 and was an advantage when irradiated at day 14. importantly, the results show that combining irradiation with hypoxia/hypocapnia retained the improved hemolysis profile of o 2 depleted rbc. in summary, the reduction of o 2 level in rccs enables a better storage of rcc when a late irradiation is applied. background: in vitro blood circuit machines require a constant monitoring of blood flow rate which have to be maintained at a constant value. also, measuring the hematocrit of flowing blood in such machines is essential for performing real-time diagnostics. recently, acoustophoresis has emerged has a promising blood separation technology capable of replacing centrifugation for the preparation of platelet concentrate. to avoid damaging blood cells, the technique is used without infusing pumps thus increasing the need of flow monitoring. however, acoustophoresis chips performs at low flow rates, outside the range of available commercial flow meters. in addition, hematocrit measurement is of a particular interest for acoustophoresis since it is a direct indicator of the separation efficiency. aims: in this study, we present a straightforward doppler ultrasound system designed for measuring blood flow rate and hematocrit in an acoustophoresis chip [bohec et al, platelets, 2017] . we show that the stability of the in vitro environment can be used to obtain high level of accuracy of the doppler method using a basic and low-cost experimental set-up. this improvement allows a precise measurement of flow rates as low as 0.5 ml/min in sub-millimeter tubing. furthermore we evaluate the capability of the system to measure hematocrit of human blood samples coming from different donors. methods: the experimental set-up was constituted of an ultrasonic continuous wave doppler probe mounted on a 3d printed support. the accuracy of flow rate measurements between 0.5 ml/min and 1.5 ml/min was evaluated as well as the optimal measurement time. for 4 different blood bags, the relationship linking the total energy of doppler signals and hematocrit was derived. hematocrit in a range under 8% was estimated from doppler signals for each blood bag. results: the system is able to acquire exploitable doppler signals for the whole flow rate and hematocrit range. flow rate estimation from the signals shows a high accuracy with a mean measurement error under 3% for a measurement time of 2s. the mean error is still under 5% for a measurement time of 0.5s. hematocrit estimation from doppler signals shows a good linear correlation with reference measurements for bags 2, 3 and 4. hematocrit estimation for bag 1 diverges from reference for values above 6%. summary/conclusions: the proposed doppler ultrasound system is capable of measuring low blood flow rate in narrow medical tubing with a high accuracy. it is particularly suited for an acoustophoresis device but the versatility of the system makes it easily applicable to any in vitro blood circuit. we furthermore demonstrated that the system can be used for measuring hematocrit under 8% without additional developments. this finds interesting applications in blood sorting technologies but also demonstrates that doppler ultrasound is a potential simple and low cost method for measuring hematocrit of flowing blood in vitro. background: hereditary hemochromatosis (hh) is the most common genetic disorder in populations of northern european descent manifesting with high levels of storage iron (ferritin) in blood and tissues. the standard treatment is serial therapeutic phlebotomy to decrease iron overload. the collected blood is frequently discarded but some blood banks allow "healthy" hh patients to donate blood for patient use. red cell concentrates from hh donors have been reported safe for transfusion, but little or no data is available on platelet concentrates from hh donors, including the potential contribution of surplus iron to the "platelet storage lesion". aims: the aim of this study was to compare platelet quality, activation and aggregation over seven-day storage in platelet-rich plasma from patients with newly diagnosed hh and from healthy controls. methods: whole blood (450 ml) was drawn into compoflow blood bags containing cpd and sag-m from 10 healthy controls and 10 newly diagnosed hh patients. platelet-rich plasma (prp) was prepared from whole blood and split into four compo-flex bags each containing 20 ml prp (range 270-320 9 10 9 platelets/l). platelet quality tests were performed on days 0, 1, 3, 5 and 7 of storage. platelet aggregation was tested using a chrono-log aggregometer and four agonists (adp, arachidonic acid, collagen, and epinephrine). platelet expression of cd41, cd42b, and cd62p was measured with flow cytometry while ph and metabolites were measured with a blood gas analyzer. scd40l and scd62p in the supernatant were quantified using enzyme-linked immunosorbent assays. results: both hh and control groups included 7 males and 3 females. the mean age was significantly lower in the control group, 35 years (22-55 years), than in the hh group, 55.7 years (29-77 years) (p = 0.004) while ferritin levels were significantly higher in hh patients (median 847.5, range 498-1889) than in controls (median 45.8 ng/ml, range 9.28-97 ng/ml) (p < 0.001). in the hh group, 5 each had the c282y/c282y and c282y/h63d genotypes. results of prp quality control tests were comparable between the two study groups over seven days of storage (p < 0.05) with the exception of glucose (higher in hh patients on all time points, p < 0.05). platelet aggregation and the expression of activation markers (cd62p and cd42b) on platelets and in the supernatant (scd62p and cd40l) were comparable between hh and control prp units over all seven days of storage. the analysis revealed comparable and expected alterations in metabolic and platelet activation markers over seven-day storage in both groups. ph increased, glucose decreased, and lactate increased over time while cd42b expression decreased and cd62p increased. platelet aggregation responses decreased during storage but to a varying degree depending on the agonist, however, the decrease was comparable in cases and controls. summary/conclusions: these results suggest that high iron stores in hh do not adversely affect the quality of platelet units produced from hh patients. furthermore, the data also suggest that blood from hh patients, including platelets, can be donated for patient use. background: platelets are often shipped over long distances from collection centres to blood processing centres and subsequently to hospitals. platelet agitation facilitates oxygen transfer, thus promoting aerobic metabolism, and maintaining platelet ph. during shipment, platelets cannot be agitated continuously, which may promote anaerobic metabolism. previous studies have examined the effects of prolonged periods without agitation on apheresis platelets collected in plasma, but not platelets in platelet additive solution (pas). it is therefore important to determine whether platelet quality and function are maintained during prolonged transport or hold time in a shipper. aims: the aim of this study was to evaluate the effects of prolonged storage without agitation on the in vitro quality of apheresis platelets in pas. methods: triple dose apheresis platelets (n = 16) were collected using a trima accel platform in 40% plasma/60% pas (ssp+). after resting for 1 h, platelets were split equally into three components, packed into a shipper and transported immediately to the blood centre. upon arrival, one of the platelet components was removed (<6 h; t0), and the others remained within the shipper, without agitation. the second component was removed at 6 h post-collection (t6), and the third was removed at 23.5 h post-collection (rounded up to 24 h; t24). platelets were tested on day 1, 5 and 7 post-collection and in vitro quality and function were monitored. data were analysed using a two-way repeated measures anova, where a p-value of < 0.01 was considered significant. results: platelets held without agitation for 24 h consumed significantly more glucose than those removed at 6 h or immediately upon arrival (p < 0.0001), even on day 1 post-collection. this was accompanied by increased lactate production (p < 0.0001), indicating increased anaerobic glycolysis. consequently, the ph was significantly lower in t24 platelets (p < 0.0001), and on average it was 0.4 ph units lower than in platelets held in the shipper for 6 h or less. however, the ph remained above 6.9 in all components. mean platelet volume was also reduced in t24 platelets (p < 0.0001), suggesting acceleration of the platelet storage lesion. phosphatidylserine exposure, surface expression of cd62p and microparticle generation were significantly higher in the t24 platelets throughout the storage period (all p < 0.0001), suggesting platelet activation. release of scd62p was also increased in t24 platelets (p = 0.0006), whereas extended storage in a shipper did not affect release of rantes (p = 0.4488). adp-induced activation of glycoprotein iib/iiia, measured by pac-1 binding, was decreased in t24 platelets (p < 0.0001), indicating reduced platelet responsiveness to agonist stimulation. additionally aggregation in response to collagen (p = 0.0001) and adp (p = 0.003) were significantly lower in t24 platelets, suggesting a decrement in platelet function after prolonged storage without agitation. summary/conclusions: significant in vitro changes were observed in platelets held without agitation for 24 h. these results suggest that the length of time that platelets are held in a shipper should be minimised where possible. background: the shelf-life for platelet products has been restricted to 5 days. this very limited window of time is intended to sustain the quality of platelet and to reduce the risk of bacterial growth. we have recently demonstrated that in suitable platelet bags, the platelet product quality remains high after 5 days of storage. this was proved by examining in vitro, the quality parameters of platelets such as platelet concentration, glucose, ldh, and ph (alexopoulos k. et al., haema, 9, 2018) . our new target is to extend this research in 2 extra days of storage. we also want to determine if there is any bacterial development in this period. aims: the goal is to investigate the capability of storage period for platelet units, from 5 to 7 days. methods: in this study, platelets were collected from 11 normal blood donors in the blood bank department of general hospital of patras "agios andreas". a total of 450 ae 10 ml of whole blood was drawn into triple cpd/sag-m top-top bags blood container systems, lmb technologie (gmbh). the platelet concentrates were prepared by platelet rich plasma (prp) method and then they were placed in a platelet incubator with agitator (helmer pc 1200). samples were drawn aseptically with a needless access coupler (cair-lgl) on days 1, 5, and 7. platelet count was done by ceeldyn ruby (abbott all data shown are reported as mean ae standard deviation (sd). the swirling effect remained positive (+) during the seven days storage period. the bacterial screening was found negative. summary/conclusions: platelet concentration in the bag remained constant between day 5 and day 7, maintaining platelet yield. the decrease in glucose and increase in lactate, along with the decreased ph, show that the platelets remain metabolically active between days 5 and 7 of storage. the ph remained well within the acceptable range. no bacterial contamination was reported. thus, we conclude that platelet concentrates in these specific bags may be used with an extended shelf life of 7 days. further studies are needed with other platelet bags to confirm our hypothesis. abstract withdrawn. aims: we introduce rt-dc as a fast, robust and unbiased quality control tool for pc, rcc and hpsc. utilizing the interdependency between cell deformation and the molecular state of the cytoskeleton, we demonstrate that rt-dc is capable to assess the quality of blood products. methods: by rt-dc we assessed: i) platelets after storage at 4°c or room temperature (rt) over 10 days for 4 apheresis pcs in addition to standard in vitro platelet function assays; ii). red blood cells before and after gamma irradiation in addition to hemolysis; and iii) hpsc after cryopreservation with 5% or 10% dmso in addition to cell count, and in vitro viability. in addition we compared the regeneration time of patients' platelets and leukocytes after transplantation of hpsc products containing either 5% or 10% dmso. results: for pcs standard quality assurance tests did not show a major difference between 4°c and room temperature storage while rt-dc showed a highly significant difference between both start conditions (day 1-7, p < 0.001 and day 10, p < 0.02). for red cells, we found by rt-dc no impact of gamma irradiation with 30 gy over the entire storage period of 42 days assessing 3 different rcc. for hpsc, rt-dc showed that cryopreservation in liquid nitrogen resulted in a significant increase in deformation (0.052 for 5% dmso versus 0.034 for the control without dmso; p < 0.00004). however, this did not differ to high extent whether 5% or 10% dmso were used for cryopreservation (0.035 and 0.041, respectively; p < 0.02). hpsc viability was lower after cryopreservation using 10% dmso in comparison to using 5% dmso. overall, blood cell regeneration is comparable between 5% and 10% dmso. summary/conclusions: studying platelet and red blood cell concentrates as well as hematopoietic stem cells under different, clinically relevant, storage conditions our results demonstrate that intrinsic material properties reveal insights into cell function and allow to predict cellular state in a robust way and using small sample volumes. in order to offer more flexibility to the production process, the storage of bcs overnight (16 h) has been validated in our blood center. aims: the aim of the study was to assess the platelet quality in platelet concentrates derived from overnight stored buffy coats. methods: whole blood collected at day 0 was separated into plasma, bc and red cell concentrates either at day 0 or at day 1. bcs were then stored until the pooling step at 20°c without agitation and pcs were prepared at day 1 by pooling 5 isogroup bcs. seven "16 h-pcs" were prepared from bcs stored for 16 h (whole blood separation at day 0) and six "90 min-pcs" from bcs stored for 90 min (whole blood separation at day 1). standard quality control measurements were performed during the process and the storage. in addition, the quality of the platelets into the prepared pcs was assessed throughout the period of storage by measuring the hypotonic shock response (hsr) and by measuring by flow cytometry the proportions of platelets in apoptosis (marked with annexin v), of functional platelets (marked with cd42) and of activated platelets (marked with cd62 the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment (amotosalen and uva) with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the ipp pooling and leukodepletion set developed by kansuk. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. methods: 32 dd-bc-pc were prepared with 8 bc and 280 ml of pas (intersol, fresenius kabi (germany) are sterile docked to the octopus harness and combined into a 600 ml pooling bag. the pool is centrifuged and the pc supernatant expressed through a bioflex cs leukodepletion filter into a temporary platelet storage container. the obtained dd-bc-pc were tested within 1 h of preparation and after storage for 8 h in the platelet storage container for volume, platelet content, residual leukocytes (wbc), plasma ratio and biological parameters, ph, po 2 , pco 2 , glucose, lactate, mpv, ldh, p-selectin and swirling. results: the platelet content of dd-bc-pc (n = 32) was on average 6.9 ae 0.3.10 11 in a volume of 393 ae 6 ml. the mean of plasma ratio was 42% [min: 39.3max: 43.9]. all pc contain <1.10 6 wbc [min: <lq; max: 0.38]. po 2 decreased from 166 ae 12 mmhg after separation (1 h) to 44 ae 18 mmhg after 8 h, ph (+22°c) and pco 2 was stable during the same time period. glucose decreased from 9.0 ae 0.3 mmol/l (1 h) to 8.7 ae 0.3 mmol/l (8 h) while lactate increased from 6.07 ae 0.46 mmol/l (1 h) to 6.57 ae 0.54 mmol/l (8 h). the vpm was stable with 7.6 ae 0.2 at 1 h and 8 h. ldh increased from 120.4 ae 8.6 u/l at 1 h and 125.8 ae 10.3 u/l at 8 h as p-selectin 45.7 ae 7.6 ng/ml at 1 h and 57.7 ae 7.0 ng/ ml. all units have maintained swirling. the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the platelet pooling and leukodepletion set developed by fresenius. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. background: the irish blood transfusion service (ibts) is the statutory body with the responsibility for ireland's national blood supply. in 2018 the ibts collected 130,183 whole blood units and performed 9,654 platelet apheresis procedures. blood is collected in three fixed and six mobile sites and is returned overnight in temperature controlled vans to a single processing site. in september 2017, the ibts implemented the tacsi automated system for the preparation of buffy-coat derived platelets as an alternative to the orbisac system. aims: the aim of this study is to compare operational aspects and quality data of buffy-coat derived platelets prepared by the tacsi (t-pcs) and orbisac (o-pcs) automated systems. methods: qc data of t-pcs and o-pcs from years 2016 to 2018 were analyzed. in 2016, the ibts prepared 5,609 orbisac platelet pools of which 5,477 were qc tested. in 2017 5,031 orbisac and 1,625 tacsi platelet pools were prepared and tested. in 2018 6,783 tacsi platelet pools were prepared of which 4671 were qc tested. qc data comprised pc volume, platelet content (10 9 /unit), residual leucocyte content (10 6 /unit) and ph at expiry. feedback from the processing laboratory operators was collected comparing operational aspects of preparation of t-pcs and o-pcs. results: from the 10,508 o-pcs tested in 2016 and 2017, mean pc volume was 292 ae 9 ml. initially the t-pcs mean volume was 273 ae 13 ml but more recently the volumes were increased by addition of a larger volume of additive solution to 303 ae 13 ml. platelet content (10 9 /unit) was significantly improved in t-pcs (384 ae 47) compared to o-pcs (356 ae 50) [p < 0.01] as well as residual leucocyte content (10 6 /unit) which was lower in t-pcs (0.05 ae 0.04) compared to o-pcs (0.1 ae 0.2) [p < 0.01]. ph at expiry was lower in t-pcs (7.32 ae 0.07) compared to 0-pcs (7.38 ae 0.06) but remained within the quality requirements. from an operational perspective with two tacsi devices, five operators produce approximately 48 t-pcs per hour compared to a lower throughput of 20 o-pcs per hour with five orbisac devices. the tacsi system is more compatible with batch processing which aligns with our staffing arrangements in routine production and has resulted in greater productivity since its introduction. with tacsi implementation, we reverted to manual pooling of buffy coats and new staff had to undergo training to achieve optimal platelet recovery. once staff were trained during validation, we observed a 10% increase in platelet recovery. processing laboratory staff indicated that tacsi devices were easier and simpler to maintain in comparison to orbisac, but highlighted that some improvements to tacsi system boxes could be made to make the system more robust. summary/conclusions: pcs prepared with tacsi and orbisac automated systems both met national and european quality requirements, however we observed a significant improvement in quality markers of pcs prepared with tacsi. processing staff reported easier maintenance and processing with tacsi compared to orbisac. throughput is higher with tacsi and there is greater additional capacity to increase production in the future if required. a perez aliaga 1 , f puente 1 , a aranda 1 , j domingo 1 , l call en 1 and a bah 2 1 banco de sangre y tejidos de arag on, aragon, spain 2 terumo bct europe n.v., zaventem, belgium background: the blood bank and tissues of arag on (bsta) is responsible for the collection, processing and distribution of blood in the region of aragon in spain. with five fixed and five mobile sites, the bsta collects and processes 44,000 whole blood units per year. to improve quality of processed units and working conditions of personnel, the bsta decided to implement the reveos automated system. validation studies were carried out in november 2012 and routine use started in july 2013. in parallel, the bsta initiated pathogen inactivation of platelet concentrates (pc) in november 2012 to improve transfusion safety. in november 2015, the mirasol prt system was implemented due to simplicity of this platform. aims: the aim of this abstract is to (i) describe the results of the validation studies carried out during reveos implementation (ii) present routine use data of products processed with reveos and (iii) provide information on mirasol implementation. methods: in 2012, 369 whole blood units were processed using reveos. quality parameters for red cell concentrates (rcc), such as: hemoglobin (hb), hematocrit (hct) and volume; plasma: volume and residual platelet content; and pcs: volume and yield, were analyzed. a control group consisting of units processed with a semiautomated platform was used as comparison. a survey was sent to the collection and processing staff for feedback on operational aspects of the use of reveos. from 2013 to 2018, qc data of rccs (n = 3025), plasma (n = 660) and pcs (n = 660) processed with reveos were collected. number of mirasol treated pcs and hemovigilance data from 2015 to 2018 were collected. results: volume, hb and hct of rccs processed with reveos were significantly higher compared to control rccs (volume: 296 ae 21 ml vs 251 ae 19 ml; hb: 59.9 ae 6.2 g/ unit vs 49.2 ae 7.8 g/unit; hct: 59.5 ae 2.7% vs 56.1 ae 6.3%). control plasma units had higher volume (280 ae 17 ml) and lower residual platelet content (9 ae 7 10 9 /l) compared to reveos plasma units (volume: 249 ae 23 ml; residual platelet content 19 ae 13 10 9 /l). for pcs, volume was higher for units processed with reveos compared to control (350 ae 20 ml vs 310 ae 10), but no statistical difference was observed in platelet content (reveos: 361 ae 60 9 10 9 /unit; control: 386 ae 80.10 9 /unit). we observed either an increase or stabilization in qc parameters of rccs, plasma and pcs during the 5 years routine use of reveos. for example, percentage of rccs units hemolyzed dropped from 4% to 0% between 2013 and 2018 and platelet content in pcs increased from 370 9 10 9 /unit (first six months in routine) to 390 9 10 9 /unit. plasma volume increased from 249 ml during validation to 267 ml in 2018. survey results from staff highlighted the user friendliness of the reveos device. number of mirasol treated pcs increased from 10% in 2015 to 59.7% in 2018 with no pathogen transmission nor serious adverse events reported following transfusion of these products. summary/conclusions: with implementation of two user-friendly systems, the reveos whole blood automation system and mirasol prt system, we observed an increased standardization of our processes enabling us to provide higher quality and safer blood products to hospitals and the patients they serve. background: white blood cells (wbc) may be regarded as contaminants in blood components and potentially reduce transfusion safety. there is currently enough evidence showing that wbc reduction through pre-storage filtration reduces the incidence of three major complications of allogeneic blood transfusions: febrile nonhemolytic transfusion reactions (fnhtr), refractoriness to random-donor platelet transfusions and transmission of cmv. leukodepletion is now mandatory in canada and several european countries. aims: the aim of this study was to evaluate leukoreduction performance and the quality of the resulting red blood cell concentrate (rbc) produced with new quadruple system top & bottom imuflex crc with an integrated red blood cells filter which was used in whole blood fractionation under routine conditions of san paolo asl blood bank methods: twenty-seven whole blood donations (450 ml) were collected with using quadruple blood bags top & bottom system with an integrated rbc filter (imuflex crc, terumo bct). centrifugation and processing (using t-ace ii + -terumo bct) of whole blood units followed the blood bank sop to obtain rbcs, plasma and buffy coat. rbcs were diluted with sagm (100 ml) and then filtered by gravity in average 2-3 h after blood collection to obtain leukoreduced rbc. average temperature during filtration was 26°c. samples were collected before and after leukoreduction for calculation of hemoglobin loss through the filtration process. filtration time was recorded. final hb, hematocrit (hct), residual platelets and white blood cells were determined after filtration using standard laboratory methods (blood cell counter) results: wb donations (n = 27) were performed. rbc characteristics post-filtration and separation were as follows: volume (266.8 ae 17.1 ml), hematocrit (57.8 ae 2.1%) hemoglobin (19.8 ae 1.1 g/dl) and hemoglobin per unit (53.0 ae 5.4 g); residual platelets (11.7 ae 2.2 x10³/ll); residual wbcs (0 à 0.004 9 10³/ll). the mean filtration time was 19 ae 3 min and no filter blocks were observed summary/conclusions: the usability and performance of the new quadruple system top & bottom imuflex â crc with integrated rbc filter was evaluated. all quality parameters complied with italian and european guidelines and requirements. in conclusion, the set was found to be a reliable and efficient collection and separation system that performs consistently background: novel devices for automated whole blood processing (reveos tm terumo bct) have been installed in regional center for blood donation and blood treatment in katowice to modernize and automate production of blood components. this system allows to separation of four whole blood units into four buffy coat depleted red blood cell concentrates (rbc), four units of plasma and four interim platelet concentrate (ipu) units in one automatic cycle. an ipu is the starting blood component for the production of pooled, leucoreduced platelet concentrates (lpc). residual leukocytes (wbc) are obtained as a byproduct and discarded. the use of this system shortens production time because it combines otherwise two separate steps: centrifugation and blood separation in press. aims: to evaluate the quality of the blood components obtained with the newly developed reveos nlr kits, which are a whole blood collection kits containing no leucocyte reduction filter for the rbcs. methods: blood [wb] was collected into nlr reveos set (terumo bct), and processed between 2 and 8 h from collection using the 3c protocol. quality control was performed on rbc, plasma and ipu. rbc were tested for hemoglobin (hb) content, hematocrit (ht), residual white blood cells (wbc), volume and hemolysis level on the last day (42 nd day) of storage. the content of wbc and platelet count (plt), protein, factor viii on the day of production and after one month of storage were determined in plasma. for ipu, platelet and leucocyte counts, volume and average platelet yield index (pyi) were displayed at the device after processing was recorded. furthermore, a comparison between fully automated and semi-automated whole blood processing was done, by comparing the amount of time necessary to fractionate 4 whole blood units using the two different processes. background: direct, single step automatic blood separation devices (reveos tm terumo bct) was installed in regional center in katowice in 2018. the system allows for full automation of whole blood fractionation. one of produced blood components is an interim platelet unit (ipu), containing most of the platelets and limited of white blood cells (wbcs). it is then pooled through a filter to form a therapeutic unit of leucoreduced platelet concentrate (lpc). a rough estimate of the platelet yield in the ipu, the so-called platelet yield index (pyi) is calculated and displayed by the device after the end of the separation phase. it is a useful tool to optimize selection of ipu for pools to produce lpc. aims: to determine the quality of polled lpc obtained on the basis of ipu from whole blood processed up to 8 h after collection and the usefulness of this method in daily practice. methods: ipus used for production of lpc were obtained using fully automated whole blood processing single step system -reveos tm (terumo bct). from the beginning of the application of the method, 1005 therapeutic units of lpcs were produced after overnight hold: 38 pools of 4 ipus and 966 of 5 ipus were made using platelet pooling set (terumo bct), using 250 ml ssp+ solution (macopharma). results: 42 therapeutic units of lpc were analyzed. the mean volume of lpc was 387 ae 13 ml, platelet content was 3.67 ae 0.46 x10 11 , and white blood cells (wbc) count was 0.15 ae 0.13 x10 6 , 100% all lpcs had wbc below 1 9 10 6 . ph measurements were made on the fifth day of storage, the ph was determined in 15 units of lpc and was 7.2 ae 0.04. summary/conclusions: the use of interim platelet units in daily practice increases the possibility of producing high quality blood components for patients and is a great alternative to the laborintensive manual methods of lpc's production. background: the development of cold-stored platelets has been hampered by the rapid removal of blood after transfusion by apoptosis and desialylation due to platelet storage lesions caused by refrigeration. previous studies have shown that antioxidants inhibited the activation and desialylation of cold-stored platelets for 5 days and also inhibited rapid elimination after transfusion in animal models. aims: it was tried whether in long-term (10 days or longer) storage conditions, antioxidants showed similar effects inhibiting the activation and desialylation of cold-stored platelets. methods: platelet-rich plasma (about 250 ml) were obtained from a healthy blood donors of type o blood group and divided into a 30 ml platelet storage bag manufactured by the manufacturer, and the conditions including room temperature and refrigerated condition and various kinds of antioxidants were prepared, (ph, glucose, lactic acid) and activation index (cd62p, gpiba (cd42b), annexin v), degree of reactive oxygen species and sialidase activity on the 7th, 10th and 12th day, and phagocytosis using the hepg2 cell line and platelet recovery after in vivo transfusion using scid mice were compared and compared. results: cold-stored platelets showed autoaggregation on the 2nd day of storage, and apparent aggregation on the fifth day of visual observation but not in the antioxidanttreated group. as expected, the ph, glucose and lactate levels of the cold platelets were maintained and the platelets at room temperature changed significantly. nac (n-acetylcysteine) -treated platelets were less active on day 12 and sialidase activity was maintained lower than that of control group on day 12. the degree of phagocytosis by the hepg2 cell line remained at the same level during the storage period, and the in vivo recovery rate in the animal model was superior to that of the antioxidanttreated cold platelets for 2 h and 24 h after transfusion. summary/conclusions: cold platelets could be developed as clinically effective agents for up to two weeks if appropriate preservatives were added, and antioxidants, especially n-acetylcysteine, were effective. background: the reveos 3c procedure produces the following three blood components for transfusion: plasma, rbcs, and a single interim platelet unit (ipu). whole blood is collected into the reveos blood bag set and held at room temperature a minimum of 2 h before processing on the device. after processing on the reveos device, the plasma product is ready to freeze. the rbc product is ready to combine with sag-m additive solution and leukoreduce using the integrated leukoreduction filter. the ipus are rested and agitated for the recommended time. for example, when whole blood is processed within 8 h of collection, the ipu is agitated overnight before pooling. because the ipu is made from one unit of whole blood, approximately 4 to 6 ipus are pooled together and leukoreduced by filtration prior to storage in a platelet storage bag. this leukoreduced platelet pool (4 units) can be supplied to the demand of hospitals like a platelet apheresis product. aims: to assess the usability of the reveos automated blood processing system to collect units of whole blood and to process the units on the reveos device, producing blood components that meet product quality specifications. methods: this study will evaluate approximately 19 units of whole blood using the reveos system and approximately 4 units of pooled platelet products. a total of 19 units of whole blood (wb) have been collected into the reveos blood bag set and then held at room temperature without the use of an active cooling system (or temperature control system), such as cooling plates, or in this case, ice packs. of the 19 units, 10 units have been processed on the reveos device using the fresh procedure (within 8 h of collection). nine units have been processed using the overnight wbhold procedure. all units have processed successfully without alarms, resulting in three components (rbcs, plasma, platelets). we also evaluated four platelet pools created from eighteen interim platelet units (ipus). one ipu has been discarded due to a 22-min whole blood collection time. the leukocyte count on all component using the automatic sysmex-xn 2000 cell blood counter. results: the red blood cells meet the criteria for hematocrit, hemoglobin and residual wbc counts are less than 1 million per unit. the plasma units meet the criteria for displayed volume accuracy compared to measured volume and also for factor viii levels. the average residual platelets in plasma levels meet the criteria. the platelet pools meet the acceptance criteria for platelet yield and volume. limited data (n = 4) suggests that this criteria will be easily met, provided that wb hold conditions are suitable for platelets. summary/conclusions: final results indicate that products produced from the reveos system meet or exceed product quality expectations. staff report that the reveos system is very easy to use. abstract withdrawn. background: thorough manual mixing of overnight-held whole blood (wb) units prior to centrifugation to separate red cell (rc) from platelet-rich-plasma (prp) is currently practised at the hong kong red cross blood transfusion service (hkrcbts). the mixing step is crucial to maximise platelet yield but laborious, and may cause elbow and wrist injuries in operators. a laboratory shaker, reax 20 (heidolph instruments, schwabach, germany), originally designed for mixing solvents, was modified to allow mixing source wb units by the supplier. to enhance the well-being of the operators in the context of occupation health and safety (oh&s) management, feasibility of the application of such blood mixer to replace the manual mixing steps in blood component preparation was explored. aims: to evaluate the in vitro quality control (qc) parameters of the blood components prepared with the use of the modified mixer. methods: fifty-eight wb collections, including 40 units in 450 ml quadruple bags (450q) with/without integrated leucofilter and 18 units in 350 ml quadruple bags (350q), were processed in accordance with hkrcbts' prevailing component processing protocols except using the motor-driven mixer in lieu of manual mixing. wb collections were divided into 2 groups, mixed at 2 revolutions per minute (rpm) for 2 min (n = 30) and 30 min (n = 28), then processed into rc, prp platelet and plasma components. in vitro qc assessments including haematocrit (hct), haemoglobulin (hb) and haemolysis at expiry for rc; absolute platelet count and ph for platelet concentrates (plt); volume and residual platelet count for plasma units were evaluated and compared. results: all components derived from both 450q and 350q processed with the blood mixer fulfilled the qc requirements stipulated by aabb standards and council of europe (coe) guidelines. there were no significant differences for all the qc parameters in rc and plasma components produced when mixed for 2 or 30 min. summary/conclusions: the overall performance of blood components processed with the motor-driven mixer met the acceptance criteria at the hkrcbts in accordance with the aabb and coe standards. higher platelet yield was observed with prolonged mixing for 30 min. however, prolonged mixing will much delay routine component processing. further evaluation on intermediate mixing time such as 3, 5 or 10 min may help determine an optimal mixing duration to maximise platelet yield without compromising processing workflow. to conclude, using the modified blood mixer to replace laborious manual mixing in components preparation is promising to improve the oh&s for blood component preparation operators. background: leukocyte reduction is widely used to reduce the risk of non-hemolytic febrile transfusion reactions and alloimmunization by multiple blood transfusions. around and after year 2000, universal leukocyte reduction was adopted in many developed countries. there are mainly two types of filter, one to filter whole blood and the other to filter red cell concentrate (rcc) used during pre-storage blood processing. aims: in this study, we conducted the filtration tests with sepacell tm rs2 for rcc in comparison with sepacell tm r-s11 and sepacell tm pure rc widely used in the world. background: the cardarelli hospital blood bank processes over 40,000 whole blood units yearly. in order to constantly cope with the huge demand for platelets from oncohematology and intensive care departments, a progressive review of the processing protocols for platelet concentrates (pc) from buffy-coat (bc) pools has recently been implemented to reduce the number of units assembled, from 5 (routine in italy) to 4. the 4-bc pc units have slightly lower volume and plasma content, factors that further limit the transfusion risk. in addition, the 4-bc assembly allows to facilitate the production of the pc units obtained from the same blood type, in order to minimize both the adverse reactions related to the abo/rh incompatibility and the amount of bcs not used. finally, the described innovation also impacts on the workload, with reduction of assembly, centrifugation and separation time. aims: the aim of this work is to define an optimized protocol for the preparation of pc units from 4-bc pools, so that the platelet yield is always higher than the minimum value required by current regulatory (2.0 9 10 11 cells/unit). background: blood is a basic component for human life and hence blood transfusion is an integral intervention in the treatment of patients. the need of blood transfusion is increasing with decreasing blood supply due to limited blood donations. conversely wastage of blood products is also a topic for discussion. to ensure the availability of blood products when they are needed, wastage of blood products should be controlled and minimized. aims: the study was designed to investigate the quantity and reasons of wastage of blood products at our hospital. methods: this was an observational study based on the statistical data available from blood bank department of nibd and bmt, pechs campus, karachi, pakistan. the study period was from february 2018 to february 2019. study was approved from institutional review board. for all the blood products wastage and reasons of wastage were evaluated. frequencies were calculated by using spss version 23.0 results: a total of 2880 blood products were available at our institute including 960 each of platelets, pack red cells and fresh frozen plasma(ffp). the overall wastage rate was 3.5%. pack red cells and platelets were fully consumed yet shortage of supply was observed. however, highest wastage was observed in ffp in our blood bank i.e. 102(10%). results of investigating the common causes of blood product wastage at the hospital revealed that it was highly associated with 02 common causes, including the expiry of unused products 60(59%) followed by broken bags 30(29%). summary/conclusions: this study showed over all diminutive wastage rate. ffp wastage was highest among all the blood products. wastage of blood products is a genuine issue in a hospital setup, strategies and plan of action should be discussed and implemented including enhanced collaboration with other centres to outsource the blood products or even donate at times, education and training of staff for better clinical use of blood products and ensure the availability when and where they are needed most. background: in the early 2000s, the blood service of the republic of kazakhstan functioned along the lines of the soviet period: donation of blood was approached to the place of its use (carried out at hospitals), screening of donor blood was carried out at one stage (enzyme immunoassay) on analyzers of semi-automatic type, there was no mechanism calculating the cost of blood components, the activity of blood centers was funded at the rate of 1 liter of whole blood. donation was not popular in society, household fears of infection were common when donated. aims: research objective is to study the main stages of reforming of blood services of the republic of kazakhstan (rok). the research methods are based on the system analysis of the condition of the rok's blood services based on the indicators of production activity for the period of 2010-2017. results: in order to effectively reform the blood services, the following steps were taken. the blood supply was centralized and optimized. blood collection at hospitals was terminated, more than 200 blood collection subjects were closed. the result was a more efficient use of technical, human and financial resources, as well as ensuring the high quality of the blood components produced. today, there are 18 blood centers in the republicone for each of the 18 administrative territorial units of the republic. the screening of donor blood for transfusion infection markers has been transferred into a two-step principleimmunological analysis and nat testing. a fully automated analytical equipment of a closed type is used for screening, unified for all blood centers of the republic. unification of the equipment choice allowed to provide centralized service maintenance of complex equipment and its uninterrupted operation. since 2012, the national reference laboratory has been operating in the blood service, which conducts an external assessment of the quality of laboratory research in blood centers, as well as research in controversial and complex cases. introduced pathogen inactivation of platelet concentrates in 100% of cases of platelets. erythrocytes are used only in the weighing solution and only after leukoreduction. the use of resource-saving blood harvesting technologies, as well as methods to further enhance the infectious and immunological safety of components, are expanding annually. the foundations for the development of applied scientific research in the field of transfusion medicine were laid, and an own school of transfusiologists was created. as a result of reforming the blood services, the mechanisms of reimbursement of blood services costs from the budget of the republic have been changed, uniform tariffs have been defined by type of blood components for all blood components suppliers in the country. summary/conclusions: the transformations made it possible to create a blood service in kazakhstan that corresponds to the best international practice and ensures the quality, efficacy and safety of transfusion therapy. further development of the blood service is aimed at deepening reforms, studying and developing cellular technologies, new properties of plasma blood components, and improving the clinical practice of using donor blood components. background: reconstituted platelet concentrate (rpc) is a component that contains platelets (thrombocytes) resuspended in plasma that is blood group compatible with the recipient. it is used when transfusion of platelet concentrate is necessary but the fresh platelet concentrates of the specific blood group are not available. group 0 rh-(negative) platelets resuspended in the ab group plasma are of universal use as such components can be used for all recipients independently of their blood group. in the area of activity of regional blood center in poznan rpc is used most often for patients after stem cell transplant incompatible with their blood group. aims: the aim of the analysis is to evaluate the loss in the number of platelets in the reconstituted platelet concentrates in order to determine their quality and usage. methods: we used for the analysis 30 units of pooled platelet concentrate derived from buffy coats and 30 units of pooled platelet concentrate derived from the apheresis. for the reconstitution group ab plasma was used. we investigated the number of platelets prior to and after the reconstitution. following measures were calculated: number of platelets in pooled platelet concentrates derived from buffy coats, number of platelets in pooled platelet concentrates derived from apheresis; average values and the standard deviation as well as the loss in number of platelets due to the reconstitution. the number of platelets was calculated with impedance method using horiba pentra xl 80 analyser. 1. pooled platelet concentrates derived from buffy coats the number of platelets before the reconstitution: 4.12 9 10 11 /unit ae 0.50 the number of platelets after the reconstitution: 3.40 9 10 11 /unit ae 0.50 the loss in the number of platelets: 17.5% 2. platelet concentrates derived from apheresis the number of platelets before the reconstitution: 3.68 9 10 11 /unit ae 0.55 the number of platelets after the reconstitution: 2.91 9 10 11 /unit ae 0.49 the loss in the number of platelets: 20.7% summary/conclusions: 1. reconstitution results in the loss of 17.5% of platelets in comparison to the initial value of pooled platelet concentrate derived from buffy coats. 2. the average number of platelets after the reconstitution is 3.4 9 10 11 /unit, which fulfils the quality requirements. 3. reconstitution results in the loss of 20.7% of platelets in comparison to the initial value of pooled platelet concentrate derived from apheresis. 4. the average number of platelets after the reconstitution is 2.9 9 10 11 /unit, which slightly deviates from the quality requirements. 5. the results that we obtained show that the process of reconstitution always results in the loss of number of platelets and that the quality of platelet concentrates that have not been reprocessed is always higher. because of that we recommend that the reconstitution should only be performed in special cases such as for patients after stem cell transplant incompatible with their blood group and that hospitals should be supplied with platelet concentrates derived from buffy coats and apheresis. methods: pf4 was extracted from human pcs by using immunoaffinity chromatography and specific anti-pf4 antibody on the column. hereafter, pf4 was purified with concentration tubes having 5 kda molecular weight cut-off and dialyzed via dialysis tubing with 3.5 kda cut-off. pf4 concentration was measured by bradford method. specific enzyme-linked immunosorbent assay (elisa) and dot blot analysis were used to determine the pf4 specificity. molecular weight of obtained pf4 was determined by polyacrylamide gel electrophoresis (page). results: our data confirmed clearly that separated pf4 had 30 kda molecular weight which is corresponding with a tetramer pf4. in addition, elisa showed that the pf4 qualified true antigenicity. dot blot analysis helped us to assure the specificity of pf4. summary/conclusions: we used and optimized a homemade protocol to isolate and purify the pf4. nevertheless, there was no any scientific report among the databases that utilized the similar purification method. recombinant pf4 is more feasible and ready-to-use, but its price may discourage the researchers to study the biology of pf4. unused human pcs and specific anti-pf4 antibody can considered as an alternative to separate the pf4 particularly in labs with the shortage of resources. background: whole blood is increasingly used in civilian hospitals as an alternative to blood components in trauma resuscitation with massive bleeding. however, the hemostatic effect of whole blood depends on the initial platelet content and decreases rapidly within the first 2 weeks of storage at 4°c. consequently the problem arises of how to provide wb to trauma centers without losing valuable low titer group o units if the units are not used within the expiry date of use. aims: to assess the in-vitro quality of red cell concentrates (rcc) produced from 7 days cold stored leucodepleted whole blood units filtered with a platelet-sparing whole blood filtration filter in order to save the rcc once the wb units has reached the expiry date. methods: 34 units of whole blood were leucodepleted within 24 h using a platelet sparing filter (imuflex wb-sp/terumo bct) and stored for 7 days at 4 ae 2°c. on the 8 th day of storage, units are centrifuged and the rcc is supplemented with sag-m additive solution for an additional storage of 35 days at 4 ae 2°c. in vitro parameters such as hemoglobin, hematocrit, residual leucocytes, platelet content, hemolysis, glucose, lactate, atp, 2.3 dpg were tested during storage. results: the imuflex wb-sp provided satisfying leucodepletion of the wb units (0.23 ae 0.2.10e6/u)) while maintaining 80% of the initial platelet content (85 ae 20.10e9/u). mean volume (464 ae 24 ml), hemoglobin content (54 ae 6 g/u) and hematocrit (36 ae 3%) was within conformance range. after 7 days of storage, 50% of the initial platelet content at collection is maintained and mean hemolysis is 0.27 ae 0.15%. rcc processing at day 8 was uneventful, without loss of hemoglobin or noticeable hemolysis. mean volume (266 ae 20 ml), hematocrit (56 ae 3%), hemolysis (0.18 ae 0.19%) were in conformance with mandatory standards at day 8. after 7 days of storage (i.e. day 14 after collection), rcc quality parameters were maintained with however an increase in hemolysis (0.34 ae 0.36%) which was above usual values routinely observed with rcc produced from day 1 processed units. 3 rcc out of 34 were above the maximum limit of 0.8% at day 14 after collection such increased hemolysis was more pronounced at day 28 after collection (0.70 ae 0.45%) with a proportion of rcc with a non-conforming hemolysis ratio above the accepted threshold (20% of the tested units). summary/conclusions: rcc units can be readily obtained from leucodepleted wb units with spared platelet contents after 7 days of cold storage. in vitro quality attributes of such rcc are within conforming ranges for hemoglobin, hematocrit, volume and hemolysis. hemolysis increases more rapidly than in standard rcc, thus limiting the possibility of use within 21 days after collection. additional studies are being initiated to reduce hemolysis during storage. g hetland 1,2 , f mahmood 3 , l nissen-meyer 1 and i nentwich 1 1 immunology and transfusion medicine, oslo university hospital 2 immunology, university of oslo, oslo 3 immunology and transfusion medicine, akershus university hospital, lørenskog, norway background: allergen-specific ige in donors' plasma can be transferred from blood donors to recipients via plasma-containing products and consequently sensitize recipient's effector cells bearing receptors for ige (mast cells, basophils, thrombocytes etc.). these cells can then degranulate after exposure to allergen. this can cause allergy symptoms of various degrees (johansson, allergy, 2005) . food allergen-specific ige antibodies, although causing mild or no symptoms in blood donors, can be of clinical importance to plasma recipients due to varying sensitization and activation grade of recipients' effector cells. aims: the aims of the study were 1) to assess sensitization profiles against an array of 55 allergens in 100 randomly selected blood donors and 2) to identify donors with high concentrations of food-specific ige antibodies that could bear risk of clinically important sensitization of plasma product recipients. methods: we tested serum samples from 100 blood donors randomly selected on one donation day outside the pollen season. we used 300 ll serum per sample in a multiplex ige line-blot enzymoimmunoassay for 55 analytes (euroline atopy screen, euroimmun, germany), which comprised 28 food allergen extracts and single allergens, 24 inhalant allergen extracts, 2 insect venom extracts and one carbohydrate allergen ccd. this high-throughput method enables scanning of 44 serum samples for ige against 55 allergens in less than 3 h. results are expressed as east (enzymoimmunoassay) classes: class 0 (no sensitization), 1-2 (weak sensitization), 3 (moderate sensitization), 4-5 (strong and very strong sensitization). results: east class, allergen, number of samples. food allergens: class 5none detected. class 4, sesame seed: 1. class 3, apple 1; hazelnut 2. class 2, apple 1; almond 2; hazelnut 1; sesame seed 1; soybean 1; cow milk alpha-lactalbumin 3. class 1: 26 donors weakly sensitized to one or more allergens as kiwi, apple, almond, hazelnut, sesame, soybean, wheat, mutton, beef, cow milk beta-lactoglobulin and alpha-lactalbumin. we did not find any donors sensitized to apricot, peanut, rice, rye, yeast, cow milk casein and carbohydrate allergen ccd. insect venoms: class 5, 4 and 3 none, class 2, wasp venom 7; honey bee 2. lower classes (0-1) not reported in this abstract. summary/conclusions: we found considerable sensitization to inhalant allergens but such sensitization probably represents low risk of clinical allergies due to passive transfer of antibodies. sensitization to insect venoms was negligible. we disclosed only one blood donor (1%) with high ige to sesame seed with a potential to transfer clinically relevant ige antibodies via plasma products. the need for a broad screening of donors for ige sensitization remains still questionable. background: the platelet storage lesion (psl) is one of the key factors that limit the platelet shelf-life to 5 days. the mechanisms mediating the psl are not well understood, but it is becoming increasingly evident that platelets from some donors do not store as well as others. similarly, assessment of many in vitro quality parameters has been used to predict transfusion outcomes, with varying degrees of success. donor attributes such as age, and body mass index (bmi) may contribute to these differences. aims: to characterise the variability in the quality and function of apheresis platelet donations and to examine associations between donor attributes and platelet characteristics. methods: apheresis platelets (n = 53) were collected, stored at 22°c with agitation, and tested at day 1, day 5 and 7 post-collection. vwf and fibrinogen binding (indicators of platelet adhesion; ae ristocetin stimulation), phosphatidylserine (ps) exposure (annexin v binding) and microparticles (cd61 + /annexin-v + ) were measured by flow cytometry. platelet aggregation was initiated with collagen, thrombin or arachidonic acid. thromboxane a 2 (txa 2 ) was measured by elisa. the proportion of procoagulant platelets (% coated platelets) was assessed by fibrinogen binding following collagen/thrombin stimulation. platelet-attached glycans were measured using flow cytometry and lectins ricinus communis agglutinin-1 (rca-1; detecting galactose-residues) and wheat germ agglutinin (wga; detecting sialic acid and nacetyl-d-glucosamine, glcnac-residues) ae stimulation with ristocetin. donation time, donor age, blood pressure (bp) and bmi were recorded. linear regression was performed using graphpad prism software. results: for many of the parameters measured, there was high inter-donor variability. platelets from subsets of donors showed up to 4-fold higher vwf and fibrinogen binding, formation of coated platelets and microparticle numbers. binding of lectins was also highly variable between the donors, indicating variability in sugar cleavage. following ristocetin stimulation, binding of all lectins increased, and again there was high variability in the responses, with up to 4-fold higher binding in platelets from a subset of donors. of particular interest, aggregation in response to arachidonic acid was observed in only 27% of platelet donations tested; although platelets from non-responders aggregated in response to the collagen and thrombin. txa 2 was also higher in the arachidonic acid responders (p = 0.027). there were few associations between platelet parameters and donor attributes, and these associations were typically weak. ph at day 5 was weakly associated with donor age (p = 0.009, r 2 =0.2613) but not bmi (p = 0.9143, r 2 <0.001), with a trend towards lower ph in younger donors. summary/conclusions: these data indicate that apheresis platelet products are highly variable. whilst donor characteristics may have some influence on the quality of the donated platelet product, there is a high level of inherent inter-donor biological variability. a much larger sample size is required to confirm these findings, and determine whether they have clinical implications. background: the frequency of beta thalassemia mutations, and thus, of beta thalassemia heterozygotes (b-thal-het) is particularly high in certain geographical regions. it has been reported that a significant proportion of donors (occasionally more than 10%) are b-thal-het that meet the criteria for blood donation (hb > 13.5 g/ dl). red blood cells (rbcs) of b-thal-het donors are characterized, in vivo, by particular geometry and redox status. despite sporadic indications that the rbc storage lesion may be milder in b-thal-het, targeted research on this donor group is still missing. aims: the aim of this study was to investigate whether b-thal-het rbcs storage at blood banks leads to a distinctive hemolytic, physiological and redox profile, thus, making b-thal-het a unique blood donor group. methods: blood samples from 10 healthy non-smoker donors (5 b-thal-het carriers and 5 controls) were analyzed before and after preparation and storage of leukoreduced packed rbc units in cpd/sagm at various time intervals. susceptibility in hemolysis (in the presence/absence of oxidative, mechanic and osmotic stimuli), redox status (lipid peroxidation, reactive oxygen species (ros) accumulation, antioxidant capacity), intracellular ca 2+ and proteasomal activities were determined. for statistical analysis, significance was accepted at p < 0.05. samples from the red cell units were collected aseptically, processed (dual centrifugation at 3,000 g for 15 min) and stored at à80°c. processed samples were thawed, and then analysed using the nanosight ns300 nanoparticle tracking analysis system (malvern instruments). samples from all time-points from each unit were analysed on the same day. data were analysed by one-way anova with bonferroni's multiple comparisons test. results: at d2, red cell units contained an average of 8.1 9 10 9 ae 4.0 9 10 9 mvs/ ml. the mean size of these mvs was 112.8 ae 10.5 nm and the mode size was 79.9 ae 10.3 nm. the concentration of mvs increased gradually throughout storage (p = 0.0276), reaching a maximum at d42 of 32.4 9 10 9 ae 1.8 9 10 9 mvs/ml. both the mean (p < 0.0001) and mode (p < 0.0001) size of the mvs increased during storage; however, this size increase primarily occurred in the first week of storage (d2 vs. d7: p < 0.0001 for both mean and mode). by d42, mean and mode size of mvs was 176.1 ae 4.8 nm and 171.8 ae 5.6 nm summary/conclusions: nanoparticle tracking analysis demonstrated the presence of mvs smaller than 400 nm in red cell units. both the concentration and size of mvs present in red cell units increased during the 42 days of routine storage. the concentration of these mvs was approximately 100-fold higher than we had previously detected using flow cytometry (aung, pathology, 2017) indicating the advantages of more sensitive techniques in characterisation of mvs. background: the lack of availability of sterile saline in a format suitable for use in blood centers for manual washing has led to an urgent need for blood services to consider alternative methods. for operational flexibility it would be desirable to be able to produce a washed rbc unit that had a shelf life longer than 24 h. aims: the aim of this study was to validate the manual method for washing rbcs using sagm solution both as wash and storage solution and to ascertain whether an extended storage period for washed rbcs may be feasible. methods: six 14 day old leukocyte depleted red blood cells (ld-rbc) and six 4 day old ld-rbcs were manually washed and stored in sagm, and half of the units were pre-stored irradiated (25 gy). a volume of 350 ml wash solution (sagm) was added to the ld-rbss by sterile connection. after mixing the units were centrifuged for 6.5 min at 2888 9 g at 4°c (hettich roto silenta 630 rs) before removing the supernatant using compomat g5 extractor. wash procedure was repeated twice using 450 ml sagm solution, and after removal of the last supernatant, 100 ml of sagm solution was added. all units were immediately measured for volume, haematocrit, albumin, iga, potassium, haemolysis, haemoglobin, ph, glucose and lactate and tested again after 24 h, 7 days and 14 days storage at 4 ae 2°c. results: all washed ld-rbcs met european specification for haematocrit (0.50-0.70) and all but one for hb content (≥ 40 g/unit). hemolysis increased during storage. the rate of hemolysis in irradiated ld-rbcs was greater over time than in nonirradiated units. all units, both irradiated and nonirradiated, met european specification for hemolysis (less than 0.8%) 14 days after washing. after wash, potassium levels were low and then increased during storage; increase was greater in irradiated than nonirradiated units. potassium concentration 14 days after washing and irradiation did not exceed those levels found at the end of shelf life (day 35) of standard ld-rbcs. ph decreased during storage due to the metabolic activity of red blood cells converting glucose to lactate. the ph level of the supernatant depends on the age of the unit and not on the irradiation. the glucose concentration of the supernatant after washing is high due to sagm solution. the concentration of glucose decreased and lactate increased due to the metabolic activity of red blood cells. there is currently no specification in europe or finland for iga in washed rbcs. aabb and american red cross rare donor program stipulate that level of iga should be less than 0.05 mg/dl (0.5 mg/l). our iga method 0 s lower limit for detection is 0.05 mg/l and all results were below this level. total albumin were well below finnish specification (<250 mg/unit). background: room temperature has been the standard storage condition for platelets since the 1970s, when it was shown that this improved in vivo survival compared to when stored at 4°c. however, storage at room temperature has several disadvantages, including risk for bacterial contamination and short outdating. recently, the interest in cold-stored platelets increased, especially for patients with a hemostatic need. using extensive analysis techniques, we evaluated the in vitro quality of cold stored platelets in additive solution. aims: investigation of the in vitro quality of platelets stored at 4-9°c in pas-e. methods: three experiments were performed, in which two platelet concentrates, prepared from 5 buffy coats and 300 ml of pas-e (pcs) were pooled and split in equal pcs. pcs were stored for 23 days at 4-9°c, one of each pair with agitation on a flatbed shaker and the other without agitation. various parameters were analyzed to study the in vitro quality during storage and compared to routine room temperature storage. results: during cold storage, the swirling phenomenon disappeared within one day. due to the lower temperature, metabolism of the platelets was lower as compared to room temperature storage. the metabolic conditions were acceptable with ph d1-d16: 7.05-6.77 with platelet count 400 9 10 9 /u and glucose still at 2 mm at least until 16 days of storage. platelet activation maintained acceptable levels with cd62p expression < 30%, while ps exposure increased rapidly; >20% after 6 days of storage. aggregation tests showed functional platelets until 13 days of storage. agitation during storage had no effect on any of the tested parameters. summary/conclusions: during storage of platelets at 4-9°c, the hematological parameters and ph met routine requirements, while swirling phenomenon disappeared already at the first day. the functionality of the platelets did not decrease during cold storage, indicating that the swirling phenomenon is not a good surrogate marker under these conditions. the strong increase of ps exposure might be involved in the observed short survival of cold-stored platelets. platelet concentrates stored at 4-9°c are potentially suitable as a hemostatic agent for patients with a bleeding in need of platelets, but more studies are needed. aims: the goal of the study is the reinforcement of platelet reserves for case of emergency events and increasing their availability for treatment, preferentially in patients with massive bleeding. methods: we performed a comparative study with cpp and fp in vitro. buffy coatderived pooled leukoreduced platelets 0 rhd negative were frozen in 5-6% dmso and stored at à80°c for 6 months. cpp were thawed at 37°c, then reconstituted in platelet additive solution ssp+ and compared to fp. we measured these parameters: platelet content, platelet concentration, platelet loss during preparation process, coagulation properties, volume, ph, dmso concentration, titres of anti-a and anti-b antibodies. results: the average platelet loss after the process of freezing and reconstitution was 24%. the amount of platelets and platelet concentration in unit was lower in cpp compared to fp, but high enough (amount 194 9 10 9 /unit, concentration 0.73 9 10 9 /unit). both types of plts (either pcc or fp) maintained an acceptable ph during storage. swirl was on value 3 in fp and on value 1 in cpp. the average plasma content in fp was 31% compare to 3.22% in cpp after reconstitution. measured titres of igm anti-a and anti-b antibodies were very low (0-1:2). cpp had faster clot initiation (rotem clotting time (ct) in cpp 69.2 s, fp 81.8 s). cpp contributes to a sufficient clot (rotem maximum clot firmness (mcf) in cpp 39.8 mm, fp 53.2 mm). summary/conclusions: our results shows, that cpp have higher procoagulation activity and simultaneously lower clot firmness. thawing and reconstitution of platelets are easy and fast processes if platelet additive solution is used. this method helps to increase the availability of platelets in emergency medicine. low plasma content in cpp enables their use as washed platelet product in specific groups of patients. methods: after donation, the whole blood was stored in room temperature overnight before separating next morning by reveos â system. seven abo compatible ipus, each with a target volume of 24 ml, were selected and then they were connected to the pooling set provided by terumo bct. prior to the pooling of ipus, 250 ml of additive solution (t-pas+ provided by terumo bct) was added and distributed evenly between the ipu bags. the pooling set was then kept 1 h on bench in room temperature followed by 1 h on agitator at 22 ae 2°c. after filtration, the pool might be manually adjusted if its volume exceeded the maximum of 420 ml to meet the requirements by the intercept tm blood system. the final products were two pathogen-reduced platelet units with a shelf life of 7 days. results: during validation of the method, 12 pathogen-reduced platelet units were controlled, in addition to the platelet count, for ph, glucose, po2, pco2 and lactate on day 2, 5 and 7 of storage. the platelet count was 2.27 ae 15 9 10 11 per unit on day 2. the ph value was 6.9 on day 2, 7.1 on day 5, and 6.9 on day 7. the glucose concentration decreased from 5.8 to 3.5 and 1.6 mmol/l on day 2, 5 and 7, respectively. the mean po2 level was 25.3, 25.0 and 28.7 kpa while the mean pco2 was 3.8, 1.7 and 1.8 kpa and the lactate concentration was 5.9, 9.6 and 12.9 mmol/l on day 2, 5 and 7, respectively. since routine implementation of the method in april 2017, regular quality controls showed an average of platelet count of 2.63 ae 34 9 10 11 (n = 161) with a volume of 204 ae 7 ml per unit. summary/conclusions: the validation of the method and the following two years of experience in routine shows that the pooling of 7 ipus processed in reveos â system meet the requirements needed for intercept tm ds processing set for pathogen reduction of platelets. the results from the quality controls of the final platelet units were in accordance with the local and eu guidelines. methods: data was analyzed from 11 published and unpublished clinical studies that performed both primary and secondary testing of platelets using the bta system. the studies included apheresis and whole blood derived buffy coat platelets and tested 4-10 ml sample volume per culture bottle. the 11 studies classified results based on aabb bulletin 04-07 definitions with some modifications. the following assumptions were made including: • data was summarized as total number of positive tests, observed by the total number of tests performed on each day post collection; • it was assumed that one test was performed per platelet unit; • all units eligible for secondary testing were negative by the primary test the data needed to demonstrate a benefit for the use of the bta 3d systems for detecting contamination that was not revealed by previous bacterial testing as well as clinical specificity. results: a total of 217,932 platelet units from the studies where secondary testing of platelets was performed were analyzed. platelets were tested on day 3, 4, or ≥ 6, and represented 8.9%, 44%, and 47% of the units tested, respectively. true positives were detected in 174 platelet units representing 0.08% of the total platelets tested. the majority were reported from platelets tested on day ≥ 6 with a total of 128. data showed the bta 3d system used for secondary testing detects the most prevalent contaminates reported, staphylococcus spp., in ≤27 h with the majority detected in ≤24 h after incubation, allowing for interdiction of the units prior to transfusion. instrument specificity was reported in 5 of the 11 studies for platelets tested at 3 days and ≥ 6 days with a total false positive rate of 0.27% (range of 0-1.1%). instrument sensitivity when used for secondary testing could not be determined since subculture of negative bottles is not performed during routine use. during previous validation testing of lrap and lrwbpc, 1,136 culture bottles were confirmed true negatives by subculture. summary/conclusions: data from the studies that tested platelets at 3 to 8 days post collection provided evidence that the bta 3d with bpa & bpn detects contaminants missed in previously tested platelet units. the data supports that the bact/alert 3d system is an effective safety measure for secondary testing of platelet products to extend platelet dating beyond day 3 and up to day 7 when testing is performed using the test parameters described in the bpa and bpn bottle ifus and according to the fda draft guidance. background: magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (hsa). however, the optimal hsa density coated on particles and the uptake mechanism of single particles in platelets remain unclear. aims: we characterize the interaction between single particles and platelets and determine the optimal hsa amount required to coat particles. methods: ferucarbotran iron oxide nanoparticles were coated with hsa in different amounts (0.5-2 mg/ml) and we confirmed successful hsa coating by addition of a crosslinking hsa antibody (dynamic light scattering). we labeled platelets from pooled platelet concentrates with 2 mm ferucarbotran coated nanoparticles and analyzed labeled platelets for iron content (atomic absorption spectroscopy) and particle localization (transmission electron microscopy). single-molecule force spectroscopy was used to determine binding forces of nanoparticles to platelet compartments. we applied hsa-particles via linkers of different length (i.e. short~2 nm, medium 30 nm and long~100 nm) on the cantilever tip and let them interact with a platelet provided on a collagen surface. after interaction we determined the rupture force required for platelet retrieval. results: the iron content per platelet reached a maximum at 0.5-1.0 mg/ml hsa coated particles with 0.57 ae 0.36 and 0.54 ae 0.39 pg/platelet, respectively. however, the 1.0 mg/ml hsa coating resulted in~100-fold higher binding affinity to platelets than particles coated with 0.5 mg/ml hsa. depending on peg length between tip and particle, particles interacted differently with platelets as shown by one, two or three force distributions of 80, 220, and 360 pn, which correspond up to three different binding pathways, respectively. the results indicate that a particle can interact with three targets including platelet membrane, open canalicular system, and platelet granules. summary/conclusions: our results reveal mechanism of platelet-particle interaction on a single particle level and provide an optimal hsa concentration coated on particles to gain maximal platelet labeling efficiency. labelling of platelets by magnetic nanoparticles may substitute radioactive labeling. results: the activation/lesions on total platelets and small and medium-sized platelets platelet population was detected on storage day 3, by the increased expression of cd36. the percentage of cd36-positive cells among the population of large platelets did not change during storage. on the day 3, increased expression of cd42b and cd62p was detected, but only on large platelets. small and medium-sized platelets had increased cd62p expression only on day 5. the expression of cd42a on total platelets increased significantly on day 3, and stayed unchanged until day 5. the same pattern of cd42a expression was detected for small and medium-sized platelets, whereas on large platelets the expression continued to increase until the end of storage. a decreased percentage of cd41-positive cells was detected among the total platelets and populations of medium-sized and large platelets. the storage induced externalization of phosphatidylserine on total platelets or on any of the platelet populations was not detected. the levels of tgf-b and p-selectin in the pc-bc supernatants were unchanged during the 5-day storage period. increased annexin and pf4 concentrations were detected on day 5. the concentration of b-tg increased on day 3 of storage, and continued to rise until the end of storage. summary/conclusions: the evaluation of expression of activation markers on different platelet populations could be used as an additional analysis in quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation i.e. for testing new additive solutions, cryoconservation protocols, and cryoprotectants. background: the primary goal of autologous blood transfusion is to reduce the risks related to allogeneic blood transfusion, including transfusion-associated graft-versus-host disease (ta-gvhd). although downward trends in rates of autologous blood transfusion have been reported in europe and the americas, it still plays a role in eliminating risks related to allogeneic blood transfusion in japan, especially ta-gvhd. since february 2009, the transfusion service in our hospital has managed 3 autologous blood conservation techniques and helped to prevent mistransfusion by employing a bar code-based electronic identification system. aims: the objective of this study was to determine the use of 3 types of autologous blood components in a single institution over an approximately 9-year period. methods: between february 2009 and december 2017, we retrospectively analyzed autologous blood transfusion, including perioperative autologous cell salvage (pacs), pre-operative autologous blood donation (pad), and acute normovolemic hemodilution (anh). we investigated the use of autologous blood components and the rate of complying with electronic pre-transfusion check at the bedside in the operating rooms. we also determined the adverse reactions to autologous blood transfusion, which were categorized according to the definitions proposed by the international society of blood transfusion (isbt) working party on haemovigilance. results: a total of 9,193 patients (9% of whom received operations) received blood transfusion, of which 5,080 (55%) received autologous blood transfusion alone, 2,101 (23%) both autologous and allogeneic blood transfusions, and 2,012 (22%) allogeneic blood transfusion alone. the rate of autologous blood transfusion among patients who received blood transfusion was 78%. pacs units were transfused to 5,830 patients (60%), pad units to 2,845 patients (30%), and anh units to 982 patients (10%), and multiple blood conservation techniques were used for one patient. the overall compliance rate with electronic pre-transfusion check at the bedside in autologous blood components was 98.6%. adverse reactions were observed only with pad transfusion and not pacs nor anh. the number and rate of adverse reactions to pad transfusion were 12 and 0.1%, respectively, of which the most common was febrile non-hemolytic transfusion reaction at 9 (75%), followed by allergic reactions at 2 (17%), and hypotensive transfusion reaction at 1 (8%). the severity of adverse reactions to pad transfusion was grade 1 (non-severe) in all cases, and no serious adverse reactions were observed. among pad units, the rate of adverse reactions to whole blood pad units was 0.55%, that to frozen pad units was 0.05%, and that to autologous ffp units was 0.10%. summary/conclusions: our observations indicate that the rate of autologous blood transfusion among patients who received blood transfusion was 78%, when all types of autologous blood conservation techniques were included. to accurately determine the rate of autologous blood transfusion in a hospital, it may be better for the transfusion service to manage all types of autologous blood conservation techniques. they are now clinically available as a blood product. the residual plasma level of this product, which is prepared using the automated cell processor acp215 (haemonetics corp.), is approximately 1%. recently, a retrospective multicenter study was reported that this product was effective and safety for prevention of recurrent or severe transfusion reactions. plt products are generally stored with continuous agitation to maintain their quality. the interrupted agitation of plt suspended in additive solution (plasma carryover: 3-40%) for up to 24 h was previously found to have only a slight impact on in vitro plt properties. however, in some small hospitals with no agitator, if the initiation of transfusion is delayed by an emergent change in a patient's condition, the interruption of agitation may be prolonged. aims: the aim of this study was to evaluate the effects the interruption of agitation for 48 h (shelf life of wpc in japan) on the in vitro qualities of plt. methods: leukoreduced apheresis platelet concentrates in 100% plasma were washed on day one after blood collection using the automated closed-system cell processor acp215 (n = 6). wpc, which were rested 1 h after preparing, were divided equally into control and test units using polyolefin containers on day one. control units were agitated from days one to seven. test units were stored without agitation from days one to three, and agitation was subsequently performed until day seven. both units were stored at 20-24°c. in vitro plt quality was examined on days one, three, four and seven. results: the plt concentration of prepared wpc was 110.9 ae 1.9 (910 10 /l) and the volumes of the control and test units after the division were 120 ae 11 and 123 ae 12 (ml). the ph values of the test units on day three were lower than those of the control units; however, the ph of both units were maintained at higher than 6.85 during the seven-day storage period. swirling was well maintained and no clumping was visible in both units during storage period. no significant differences were observed in plts concentrates, mpv, hsr, aggregation response. the pco 2 , po 2 , bicarbonate, glucose and lactate mean values of test units were slightly lower or higher than those of the control units on days three or four. the levels of cd62p expression were significantly higher in the test units than in the control units on days three (52.6% ae 6.0 vs 40.9 ae 5.7, p < 0.01); however, this difference decreased in a time-dependent manner after agitation resumed. the levels of cd42b expression of test units were relatively lower than those of the control units until day seven, but no significant difference between the two units. background: monitoring residual white blood cells (rwbcs) is a requirement for quality monitoring (qm) the production of leucocyte depleted blood components. although flow cytometry is widely used for monitoring rwbc, there are no widely accepted methods to accurately and consistently measure rrbcs in blood components. sysmex have developed a novel algorithm, termed the blood bank (bb) mode for their xn-series of haematology analysers which is specifically designed to quantitate the levels of rwbcs and rrbcs in blood components. aims: we have previously assessed the linearity, accuracy and reproducibility of the bb mode on spiked samples in an r+d lab. we sought to further assess the performance of the bb software in a routine, high throughput blood component manufacturing department. methods: units of plasma, platelets (pcs) and red cell concentrates (rccs) were produced according to standard uk specifications within nhs blood and transplant (nhsbt). qm of residual cells was tested using the bb mode whilst rwbc was additionally analysed by flow cytometry using bd leucocount kit. results: during a 2-month field trial over 10,000 data points were collected representing all types of manufactured component. for some pcs, bb mode results from some sample tubes that did not contain edta gave very high rwbc values, indicating a potential large number of ld failures. the results were significantly different from those obtained from pcs using edta samples (p < 0.0001) which did not show the same high values. for rccs or plasma, the range of results from plain and edta tubes were not significantly different (p ≥ 0.3246). the analyses of ld platelet and plasma concentrates by either bb mode or flow cytometry both show more than > 90% of ld components have less than 1 9 10 6 rwbc/unit. for ld failures (n = 14) there was a good correlation (r 2 =0.9848) between flow cytometry and bb mode measurements. spiking studies suggested that the limits of detection and quantitation of rrbc were around 6 and 8 rrbc/ll respectively. residual red cell counts from manufactured components showed a wide variation in their numbers between units. as expected platelet production methods also showed a significant difference (p < 0.0001) in rrbc contamination, with lower levels in apheresis platelets (median = 15 rbc/ll, n = 1820) compared to those produced from buffy coats (median = 783 rbc/ll, n = 77) in our hands, although the time taken to analyse samples is similar for flow cytometry and bb mode, considerable time can be saved on manual handling and the processing of samples for flow cytometry (approximately 2-3 h for 60-90 samples). summary/conclusions: we have been able to embed the sysmex bb mode into a routine production environment and confirm that its performance in spiked samples is mirrored in routine use. for platelets, sample collection in edta is essential. the bb mode offers an opportunity to reduce operator time compared to flow cytometry whilst gaining additional information on rrbc. abstract withdrawn. results: in our experiment, the typical size of a spectrin matrix section (l) was 60 to 220 nm (without oxidation). the heights (h) of dips were 4 to 10 nm. due to oxidation, the junctional complexes between spectrin and membrane proteins can rupture. only 10% to 15% of the spectrin surface has the same structure as in the control group. the values of l and h vary significantly depending on the intensity and time of exposure. we observed significant changes in the spectrin matrix after exposure to uv radiation in a model experiment. the local topological defects in the membrane arise from the action of oxidizing agents on the red blood cells. the mechanism of their appearance is connected mainly with the distortion of the spectrin matrix. as a result of oxidation processes, the spectrin molecules can be damaged. there is a transformation of tetramers to dimers. additionally, it can be easily seen with the afm, that spectrin network structure was essentially destroyed. most parts of the spectrin matrix have damaged structures with mesh breaks and dips after uv irradiation. also the results of network distortions in response to temperature changes were obtained. there are presented preliminary results of spectrin matrix change during long-term storage of prbc. summary/conclusions: atomic force microscopy in direct biophysics experiment allows to observe and to quantitatively measure the disturbances in the spectrin matrix nanostructure in response to oxidation processes in rbcs. these studies are important for the fundamental research of interactions of rbcs on the molecular level during redox processes and the consequences to their structure and function on the cellular level. this is important for the advancement of transfusion medicine, intensive care medicine, and molecular and radiation biology. methods: blood samples were taken from 8 donors during a prophylactic examination and collected with edta-filled microvettes (sarstedt ag and co., germany). all experiments were carried out in accordance with the institute guidelines and regulations. the polylysine-coated glass was used to perform the sedimentation method for formation of native rbc smears. it is important that any fixatives weren't used. the stiffness of rbc membranes was studied in native rbcs (control) and native cells after the application of modifiers: glutaraldehyde, hemin, zn 2+ (heavy metal ions). local stiffness was studied by atomic force spectroscopy (afs) (ntegra prima, (nt-mdt, russian federation). results: experimental kinetic curves i(z) were measured. nonlinear fitting method was used to determine the young's modulus. the experimental dependences of membrane bending were approximated by the hertz model to a depth up to 600 nm. the young's modulus e = 10 ae 5 kpa for control rbc. it was shown that some natural oxidants (hemin), membrane fixatives (glutaraldehyde) modifiers, heavy metal ions (zn 2+ ) significantly increased the absolute value of the young's modulus of rbc membranes up 2-3 times. the biophysical parameter hertz depth (h hz ) was determined for each curve. under the influence of modifiers the hertz depth h hz was changed from 200 nm to 600 nm. there are presented preliminary results during long-term storage of blood. summary/conclusions: the blood rheology is determined by rbc deformability, associated with membrane stiffness. the young's modulus can be used as a quantitative criterion to estimate the membrane state of a native cell. the results of the work can be used in clinical practice, in assessment of the quality of donor blood during storage before transfusion, in biophysical studies of rbc state. abstract withdrawn. immunohemotherapy, centro hospitalar universit ario são joão, epe, porto, portugal background: transfusion of blood and blood components is an essential resource in modern medicine. a proper use of human blood, being an irreplaceable resource, is necessary in order to achieve minimal wastage. blood wastage may occur for a number of reasons, like expiry date, haemolysis, seroreactivity or low volume. monitoring wastage of blood product during collection, testing and processing of blood is used as a quality indicator. aims: to determine the annual rate of discarded blood components due to expiry date in a portuguese university hospital blood bank (bb) from january 2016 to december 2018, in order to implement appropriate measures to minimize the number of discarded blood to a reasonable rate. methods: we retrospectively analysed the rates of blood components discarded after meeting their expiry date of a portuguese university hospital blood bank from january 1st 2016 to december 31st 2018. results: a total of 54,599 whole blood units and 3,166 apheresis platelets were collected during the study period. of the 66,289 blood components (packed red cells, whole blood pooled platelets and apheresis platelets) prepared during the study period, a total of 2,891 (4.4%) blood components were discarded, of those 75.4% due to expiry date. the rate of discarded packed red cells, according to this component production, decreased considerably over the years, in 2016 was 5.6%, in 2017 was 3.8% and 0.2% in 2018. similar tendency was shown in the pooled platelets for 2 consecutive years with 4.8% (2016) and 3.8% (2017), but with an increase in 2018 (7.7%). the rate of apheresis platelets had a more variable behaviour from 2016 to 2018 with rates of 1.3%, 0.6%, and 0.9% respectively. summary/conclusions: blood transfusion is an essential part of patient care. for this reason, the implementation of a quality system and continuous evaluation of all activities of the bb can help to achieve maximum quantity and quality of safe blood. we identified that date expiry was the main reason of discarded blood components, although there was a significant decrease in the rate of discarded packed red cells over these three years. properly implemented blood transfusion policies, donor screening and training staff as well as implementation of automation also helps to improve the process, reducing the discarding rates of blood and blood component. background: storage performance of platelets (plt) is associated with age of the donor. the risk for plt with poor storage performance, characterized by high lactate production and rapid acidification of a plt concentrate (pc), shows a positive correlation with age. we wished to explore whether high lactate production was associated with donor health issues. aims: to investigate high lactate production by stored platelets in relationship to donor health. methods: single-donor pc were collected by apheresis or prepared from 1 buffy coat and donors were evaluated who could be marked as 'rapid acidifiers'. in total, 31 apheresis pcs and 66 pcs from whole blood were included in four studies. information about donor health was obtained either from the blood bank information system or using questionnaires. in some donors, the lipid profile was measured from plasma, and the diabetes marker hba1c from red cells. triglyceride levels > 2.5 mmol/l and hba1c levels > 42 mmol/mol were defined as high. results: twenty two percent (21/97) of the donors were marked as 'rapid acidifiers' and 71% (15/21) of these donors had health issues. 'rapid acidifiers' were of age 57, 31-69 (median, range) years. three groups of donors can be distinguished: a) donors affected by metabolic syndrome, prediabetes and type 2 diabetes, indicated by high cholesterol and/or triglycerides, high hba1c and/or the use of medication to treat diabetes. b) donors affected by vascular diseases who reported or used medication to treat high blood pressure. c) "other" donors who used other medication to treat various other conditions. the remaining 'rapid acidifiers' (29%) did not have high triglyceride or hba1c levels and did not report health issues. summary/conclusions: pcs with rapid acidification by high lactate production are mainly collected from older donors with health issues. we postulate that high lactate production by stored plts is associated with health issues, and we will combine detailed donor information (health and behavior) with in vitro quality for a significant number of donations. background: the development of applied biotechnologies requires a search and creation of new methods of cells' functional completeness analysis. the instrumental assessment of platelets quality for the selection of the most effective donors, quality assessment of platelet concentrates for short and long-term storage and for the selection of platelets for cryopreservation is in demand in blood service. assessment of platelets morphofunctional status is possible using morphological studies of various platelet granules fractions (makarov, med. alfavit, 2012). among the biologically complete platelets there is a special population of cells, the so-called granule-rich platelets (grp). these cells contain the largest number of cytoplasmic granules (more than 10 visually distinct granules). it is established that grp have increased viability and functional activity. earlier we found a correlation between the grp level in blood plasma and shift of the redox potential value in blood plasma after cryodestruction of platelets (tsivadze et al., doklady physical chemistry, 2016). it was suggested that the shift of the redox potential may be partly due to the release of the low molecular weight antioxidants contained in functionally complete platelets outside the cell. in turn, the concentration of low molecular weight antioxidants can be estimated using the cyclic voltammetry. aims: the aim of the study was to estimate of cyclic voltammetry method possibilities for quality assessment of platelets. methods: the functionality of platelets was examined in platelet concentrate (pc) obtained by apheresis (1246.8 ae 90.1/ll). voltammetric analysis in pc before and after the platelets cryodestruction was carried out on platinum electrode in the potential range from à600 mv to +1100 mv using a potentiostat ipc pro l and saturated ag/agcl electrode as reference. for the morphofunctional analysis platelets were vitally stained with fluorochrome stains trypaflavin and acridine orange. microscopic examination of platelets was carried out using confocal microscope nikon eclipse 80i. the following parameters were evaluated: concentration and percentage of platelets with granules and concentration and percentage of grp. results: voltammetric studies in pc show that there are two oxidation peaks of low molecular weight antioxidants on voltammogram at potentials + 500 mv and + 800 mv. analysis of pc before and after the cells cryodestruction showed that changes in the height of oxidation peaks occur, indicating an increase of antioxidant content in blood plasma. at the same time a correlation between the changes in the height of oxidation peaks and the grp content in the sample was found. in samples with reduced initial grp content (less than 10%) after the cells cryodestruction significant changes in the height of oxidation peaks were not observed, regardless of the total number of cells in the pc. summary/conclusions: in conclusion, voltammetric analysis allows to indirectly estimate the population of functionally active platelets that in combination with other methods of analysis can serve to assess the quality of platelet products. background: determination of hemoglobin derivatives in blood is one of the most important studies in clinical laboratory diagnostics, especially during the storage of donor blood and its transfusion. concentration of hemoglobin derivatives can be changed during redox process. aims: to show the possibility of using non-linear fitting method to calculate concentrations of hemoglobin derivatives during reduction-oxidation processes. methods: for this we performed model biophysical experiment, in vitro. blood samples were collected into edta microvettes from 4 healthy donors (sarstedt ag and co., germany) during prophylactic examinations. all the donors gave their consent to participate in the study. a suspension of erythrocytes was prepared in pbs buffer with ph 7.4. we used ultraviolet (uv) irradiation of blood or nano 2 as oxidizing agent. the drug cytoflavin (stpf "polisan", russian federation) was used as an antioxidant. in our study we used digital spectrophotometer (unico 2800, usa) to measure the absorption and scattering of light (0.5 nm step). the method of nonlinear fitting was used to find the concentrations of hemoglobin derivatives. the empirical spectrum d l (k) exp was approximated by the theoretical curve d l (k l ) theor , which fits the experimental curve in the best way. under approximation the light absorption by different hemoglobin derivatives was considered in model. simultaneously effects of rayleigh light scattering on structures with size d<< k (coefficient s) and light scattering on particles with size d≥ k (coefficient k) were taken into account: d l (k l ) theor = e hbo2,l c hbo2 l+ e hb,l c hb l+ e methb,l c methb l+ e hbno,l c hbno l+ e methbno2 -,l c methbno2 -l+ e methbno,l c methbno l+k+s/k 4 l (1), where e h,l is the molar absorption coefficient for each hb h derivative at given wavelengths k l , c h is the concentration of the derivatives hb h , l is the thickness of the solution layer, d l (k l ) is the optical density of the substance, k and s are the parameters of the model. results: we determined the concentrations of hemoglobin derivatives without any additional chemicals in blood. there were measured experimental spectra for different agents action on blood. it was shown that concentration of methb increased after uv irradiation and nano 2 action (up to 90%). there were calculated c h for each hb h derivative. it was established that theoretical curves coincide with experimental data with good accuracy (r 2 = 0.98). incubation of rbcs with cytoflavin leads to reduction of methb to hbo 2 . summary/conclusions: the determination of hemoglobin derivative concentrations by the method of nonlinear fitting (without adding special chemical agents to blood) can be used for measurement of carboxyhemoglobin in blood during toxic state of organism. also it is important for assessment of rbcs quality before blood transfusion. background: the use of in line leukoreduction filters have been highly expanded in iranian blood transfusion centers within the last decade in order to provide sufficient leukoreduced blood fractions from healthy safe frequent blood donations to be supplied to the leukocyte sensitive patients. leukoflex lcr5, the dominant brand of such filters procured by iranian blood transfusion organization, is the most updated generation of the filters used around the world. aims: in this study, it is tried to recover the trapped leukocytes from this novel filter by different buffering systems and having optimized the elution mode, the cell differential of the viable recovered white blood cells were determined by flow cytometry. methods: having passed the routine virological tests, eight leukoflex lcr5 leukoreduction filters freshly used in tehran blood transfusion center were daily collected and each were back flushed by a self-designed mechanical system (a peristaltic pump, a triple junction with regulator part and an air pump) using various conditions and additives for pbs buffer at different phs in order to find the highest recovery yield for leukocytes. the optimized elute was characterized by flow cytometry for subcellular profile to be determined. results: it was illustrated that a system consisting of pbs (without cacl2 and mgcl2) in ph 7.2 containing 2 mm edta and 4%(w/w) dextran 40 without additive amounts of triton x100 was the most optimized buffering system for lcr5 filter back flushing. total cell content was also determined as 6.28*10 8 granulocytes, 4.27*10 8 lymphocytes and 0.8*10 8 monocytes using auto hemoanalysis and flow cytometric methods. summary/conclusions: in addition to partly compensating of the overhead expenses inflicted by application of leukoreduction filters on healthcare system, the results will assist blood organization system to be more classified in rational profile design, future cell therapy strategies and exceptional blood management. also, the recovered cells could be of significance in stem cell science, cellular interaction studies as well as novel molecular developments in drug discovery. vox sanguinis (2019) results: three lines of strategy are in place to pursue self-sufficiency of the largest number of pdmps. first strategy line: maximizing the yield of driving proteins, represented by immunoglobulins (ig) and albumin; this was assured by csl behring with a yield of 5.2 g ig (70% intravenous -privigenand 30% subcutaneous -hizentra) and 26 g albumin (alburex) per kg plasma fractionated, corresponding to 998.000 g ig and 4.992.000 g albumin; based on present demand, this represents 97% and 100% self-sufficiency, respectively, for naip regions. second strategy line: ensuring other products from plasma fractionation; the fractionator granted 6.000 g fibrinogen (riastap) and 6.000.000 iu vwf/fviii (haemate p) per year, which corresponds to the present demand of naip regions for both products, but it is under the full potential of plasma, thus providing a high margin of safety in case of increased demand (now the case of fibrinogen). third strategy line: exchanging cryoprecipitate, fibrinogen and vwf with italian regions whose plasma is fractionated by other companies to obtain prothrombin complex concentrates (pcc -kedcom) and antithrombin (atked) as to satisfy naip regions demand; this strategy allowed a supply of 5.000.000 iu at and 3.000.000 iu pcc, capable of ensuring self-sufficiency for naip regions until 2020. summary/conclusions: in italy, differentiation of plasma contract manufacturing among companies with different portfolios allowed naip regions to obtain a significant contribution to self-sufficiency from vnrd plasma for a variety of pdmps by different and complementary strategies consisting in maximizing the yield and the portfolio of proteins from the fractionator and exchanging products among regions for other pdmps at high demand but not included in the portfolio of a single fractionator. plasma check system: a valuable tool for plasma freezing validation and monitoring. background: the validation of plasma freezing processes may result problematic in the monitoring/control of critical process parameters (cpp). in 2017 in italy 923,944 litres of plasma were produced and frozen. aims: in order to assist plasma freezing validation and cpp monitoring, 151 of the 176 italian bes performing plasma freezing utilize the plasma check system (pcs), a system able to record, store and certify the temperature (t) detected at the core of "surrogate" bags during the entire freezing session, consistently with gmp requirements. pcs is patented and commercialized by expertmed srl, verona, italy (http://www.expertmed.it). methods: pcs consists of 3 parts: a) "surrogate" bags (check-bags) of 250 and 700 ml corresponding to the average standard volumes of the real products, containing a fluid validated to simulate the thermal behaviour of plasma; b) a mobile probe (cryo-med) positionable at the core of the check-bags; c) a dedicated software (memo-track). plasma freezing session data are tracked via barcode/rfid and can be consulted by the pcs that associates blast freezer code, operator code, cryo-med and check-bag. data on plasma freezing are stored in a shared folder and transferred to the be information system. the pcs can also be used to check and monitor the out-of-storage variations of core t of frozen plasma unit, i.e. during labelling and packaging procedures, thus allowing to establish optimal timeframes and operations and suitably validate these procedures. in the period 2016-2018, at the pievesestina be of emilia romagna region 210,871 plasma units were frozen so as to allow complete freezing within 60 0 to a temperature below à30°c, in 8,349 freezing sessions, using the pcs both for process validation, change control and for the systematic monitoring of core t at each freezing session. furthermore, at the bologna be 108 tests on the out-of-storage conditions of plasma units were carried out to revalidate the procedures of labelling and packaging. results: out of 8,349 freezing sessions carried out at the pievesestina be, 27 (0.3%) were detected to fail to reach à30°c at the core of the check-bags within 60 0 . of the latter, in most cases (70%) a technical error in the activation of the cryo-med was identified. in addition, the pcs was systematically utilized for periodical revalidation of the freezing procedures. the tests performed at the bologna be to validate the out-of-storage procedures of frozen plasma labelling and packaging allowed to modify the operating procedures in place so as to establish optimal timeframes and operations. this prompted corrective actions regarding: i) number of units to be taken out of storage sites at each labelling session (<24 units), ii) labelling time (<8 0 ), iii) optimal storage t (à40°c vs. à30°c), iv) optimal time between two openings of storage sites (>30 0 ). summary/conclusions: the pcs is a valuable system for plasma freezing validation and monitoring, as well as to perform monitoring and control of the whole pathway of frozen plasma in the be. it is a technologically advanced, easy-to-use and costeffective tool that can efficiently replace other traditional methods commonly used for the above-mentioned purposes. assessment of blood group matching quality using six sigma metrics background: six-sigma metrics provides a general methodology to evaluate a process performance on a sigma scale. implementation of six-sigma for quality assurance can benefit the health care sectors. one of the most important health care sectors is blood transfusion service. for that reason, maintaining a high quality in blood transfusion service is required. pathogen in activated plasma is one of the main products that are provided by the blood transfusion service. the process of producing pathogen inactivated plasma involves blood group matching step. the quality of this blood group matching is extremely significant for the delivery of plasma that satisfies the recipient need. aims: the aim of this study is to assess the quality of blood group matching of pooled plasma units using six sigma metrics, and to clarify the potential implementation of six sigma metrics as a quality management tool. methods: this retrospective study was conducted in the component preparation lab of kuwait central blood bank. the twelve months (january 2018 -december 2018) data of pooled fresh frozen plasma units were recruited and examined. the data was separated to data without double check (6 months) and data with double check (6 months). data statistics and analysis were conducted by the use of six sigma metrics. results: in a sample size of 6818 from the first six months a 38 mismatch was found which equals 5573 dpmo and 4.1 sigma metric. and in a sample size of 5854 from the second six months a 25 mismatch was found which equals 4277 dpmo and 4.2 sigma metric. out of the whole 12672 pooled units 63 were found to be mismatched. some of which were found to be discarded as abo discrepancy, broken, or expired. other was still available in the system, while the rest of the mismatched units were issued. summary/conclusions: using the six sigma principle the study presents a successful assessment of blood group matching quality. as a 4.1 sigma metrics obtained from the first 6 months, were shifted to a sigma metrics of 4.2 in the second 6 months, after the addition of a double-checking step to the blood group matching of pooled plasma process. the implementation of these metrics in our laboratory quality management has been shown to be very beneficial. in which six-sigma metrics were able to clarify the reduction in blood group matching errors. although six-sigma benefits in major quality improvements and helps to reach an error free laboratory services, yet it presents a new challenge to laboratory practitioners. currently, the hemophilia a patients treated with factor viii concentrated as the first line of therapy but it is more expensive and the supply is not sufficient so for now they have not used factor viii concentrate as prophylaxis therapy. for some cases, hemophilia patients in indonesia depend on subsidy from the world federation of hemophilia. the first handicapped concentrated case is just for therapy not for prophylaxis. big blood centers in indonesia produce routinely fresh frozen plasma (ffp) and cryoprecipitate-anti hemophilic factor (ahf) as replacement therapy for hemophilia a, but its content and safety of factor viii from 150 ml ffp need to be improved. nowadays, there is an available kit for producing minipool cryoprecipitate (mc) that has better safety and quality but it is available as liquid products, stored in very strict and specific temperature (à20°c). prophylaxis therapy for hemophilia patients needs a stable product, easy to use and convenient treatment for patients. aims: to analyze the content and safety of f viii with minipool cryoprecipitate (mc) and lyophilized mc for home therapy. methods: we produced 7 mc; 1 mc as the control, 3 mc were lyophilized with excipient and 3 mc without excipient. we analyzed the number of factor viii, the safety, and stability. we count the erythrocyte, leukocyte and platelet residual in mc using flow cytometry. we also measure the ph, osmolality, solubility to learn its stability after storage at 30 days at room temperature (30-32°c) and blood bank refrigerator temperature (2-8°c) at central blood transfusion services (cbts). results: we found the content f viii with excipient is higher (9.8 iu/ml) than without excipient (4.5 iu/ml) and the storage at blood bank refrigerator (2-8°c) is better than at room temperature (30-32°c) . in both group, there were no residual cells and bacterial found in mc. no significant difference in the ph, osmolality and solubility in both groups. summary/conclusions: the lyophilized mc with excipient stored at blood bank temperature (2-8°c) is better than room temperature. this experiment will be continued to know its stability in extended storage time. background: peptic ulcer disease (pud) is a multifactorial and complex disease, and it affects a wide range of people in the world. however, a perfect therapy for pud has not yet been available at present. therefore, we provided a novel therapeutic approach for pud patients and observed its effect in this study. aims: we provided a novel therapeutic approach for pud patients and observed its effect in this study. methods: in this randomized controlled trial, pud patients residing in chongqing were enrolled from 2016 to 2017. they were randomly assigned to two groups: (a) a control group used only rabeprazole, and (b) a platelet-rich plasma (prp) group that received a combined therapy of autologous platelet-rich plasma (aprp) and rabeprazole. the aggregation rate of aprp was measured via aggregation remote analyzer module. the therapeutic effect was assessed via the ulcer size and the symptom score. all data were recorded and analyzed statistically using spss. results: a total of 27 patients were included (12 patients as control group) and (15 patients as prp group) in the analysis. we found that the aggregation rate of aprp is not affected in ph 2.5 after treatment with pepsin. our results showed that there were no significant differences between the prp group and control group before the treatment, and there was also no significant difference in healing time between the two groups in different variables. however, regression analysis revealed that the healing time was 6.99 d less in the prp group than in the control group, and the patients with higher symptom scores in the initial examination need more time to heal in treatment. summary/conclusions: this study showed an encouraging preliminary result that aprp has a positive result in the peptic ulcer patients, and it seems to be a better choice for refractory pud patients. despite the further follow-up studies are needed to determine the duration of efficacy of aprp, the approach will be helpful for improving the pud treatment in clinical. background: the croatian institute of transfusion medicine (citm) collects, produces and distributes blood components in an area of 4.2 million habitants. annually, it collects about 100,000 whole blood and 3,500 apheresis donations. platelet concentrates (pcs) are more inclined to bacterial contamination due to storage conditions that favor bacterial replication. the citm decided to evaluate the mirasol pathogen reduction technology (prt) system as it offers the possibility to work with a non-toxic, non-mutagenic compound that upon uv illumination induce nucleic acid damage, reducing the risk of septic transfusion. aims: the study objective was to evaluate quality of pcs treated with the mirasol prt system for platelets and stored in tpas+ for 7 days at 22°c on a platelet shaker. methods: pcs were produced according to the citm's s.o.p., either through pooling of 4 bc with tpas+, "wbd", or through apheresis collection using two devices: the fresenius amicus, "ad" and haemonetics mcs+ system, "mcd". pcs were stored in 33% plasma and 65% pas and mcd were subsequently evaluated also in 38% of plasma and 62% pas. identical pcs were produced with a pool-split protocol to be prt-treated or serve as untreated control. pcs were treated with the mirasol system according to manufacturer's instructions. qc parameters, such as yield, ph and swirl were measured at days 1, 5 and 7. bacteria sterility test was performed at day 7 for a sample of all treated platelets. protein content of pcs produced routinely at the citm was determined to assess accuracy of plasma carry-over calculations for all processed pcs. results: mirasol-treated wbd (n = 10) and ad pcs (n = 8) stored in 33% plasma showed at day 7 an average ph ≥ 6.9; swirl ≥ 2.5 and yield = 3.3 9 10 11 . their untreated counterparts showed average values for ph ≥ 7:0, swirl ≥ 2.9 and yield 3.3-3.4 9 10 11 . mcd stored in 33% plasma (n = 6) that underwent prt showed at day 7 average values for ph = 6.7, swirl = 1.2 and yield = 3.05. control mcd showed average values for ph = 7.0, swirl = 2.8 and yield = 3.1 9 10 11 . mcd stored in 38% plasma (n = 8) that underwent prt showed average values for ph = 6.6., swirl = 1 and yield = 3.1. their untreated counterparts had average ph = 7.1, swirl = 2.9 and yield = 3.2. total protein content in pcs derived from wbd (n = 12), ad (n = 15) and mcd (n = 27) was 22 g/l, 20 g/l and 20 g/l, respectively. while the coefficient of variation of wbd and ad ranged from 3% to 4%, plasma products 129 respectively, the one of mcd reached 14%. all prt-pcs were negative for bacterial growth at day 7. summary/conclusions: mirasol treated wbd and ad produced according to citm current s.o.p. were quite similar to untreated controls at expiry, on day 7 and passed the requirements of the eu guidelines (19 th edition). quality of mcd units met eu criteria at day 5; swirl decreased significantly at day 7 which might be explained by the variability in plasma content of mcs+ -derived platelets, challenging the accurate calculation of illumination index for the mirasol treatment. all mirasol treated pcs showed minimal platelet loss at the end of storage. as the implementation of pr had to be cost-neutral it could only be implemented for~25% of the annual produced buffy coat platelet concentrates (bcp) (~40.000 bcp/year) and required a change in the bcp production method. the primary aim of the implementation was to offer increased blood safety to our most vulnerable patients. the secondary aim was to ensure that we built-up enough routine experience with pr to enable us to quickly ramp-up the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. aims: to verify if we could produce~25% pr-bcp without increasing the overall production cost (opc) for bcp. also evaluate the impact of pr on overall scrap rates of bcp, outdate rates and usage of other safety measures. methods: we compared opc for bcp between the pre-pr period (2017) . this cost was offset by substituting a semi-automated production method for bcp, which was used in 2017 to produce 28.7% of bcp-units. a manual double dose buffy coat production method (dd-bcp) in combination with pr enabled us to reduce the bcp-disposables cost by 89.2%. despite the moves from a semi-automated to a manual production method the overall scrap rates during production decreased in 2018 by 0.19%. the extension of max. storage time from 5 to 7 days for 24% of the bcp-units that were pr resulted in decreasing our overall outdating rates by 29% (versus 2017). this reduction in outdating rates reduced our opc in 2018 by 2.2%. in 2018 we gamma-irradiated 25.9% fewer bcp-units, but this had only a minimal impact on the opc. summary/conclusions: results of this study confirmed that we reached our initial objectives of producing~25% pr-bcp without increasing the overall production cost (opc) of bcp. it enabled us to offer increased blood safety to the most vulnerable patients. we built-up enough routine experience with pr so we could quickly rampup the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. background: irradiation of red cell units is undertaken to prevent transfusion-associated-graft-versus-host-disease (ta-gvhd) in immuno-compromised patients. while irradiators using radioactive c-ray sources are primarily found in blood establishments, they require regular recalibration and supplementary safety measures. xirradiation has been shown to have similar biological effectiveness to c-irradiation and does not require a radioactive source. there is international interest in moving away from gamma sources to reduce vulnerability to terrorism. although damaging, impacts of irradiation on red cells are well recognised. only a limited number of studies have compared red cell component quality following cand x-irradiation for both standard volume red cell concentrates (rcc) and neonatal red cell splits (rcs). aims: to compare the in vitro quality of rcc and rcs when subjected to cor xirradiation on day 14 of storage then stored for a further 14 days. rcs were also irradiated on day 5 of storage as that is most common practice in nhs blood and transplant (nhsbt). methods: four rcc were pooled and split into 4 arms on day 1 of storage, with 10 units in each arm. all units received an irradiation dose of 25.9-48.3 gy. two arms remained as standard volume rcc and were either cor x-irradiated on day 14 of storage. the other two arms were both split into 6 rcs on day 4 of storage before being irradiated on day 5 (early arm) or day 14 (late arm) of storage. for each replicate in these arms, 3 splits were c-irradiated and 3 splits x-irradiated. all arms were tested a day prior to irradiation and 1, 7 and 14 days post-irradiation for red cell quality parameters: haemolysis, intracellular atp and 2,3 dpg, supernatant potassium, glucose and lactate, ph and red cell microvesicle release. the rcc arms were sampled over storage; while for the rcs arms, 1 split was tested on each testing day post-irradiation. a 2-way anova was used to detect statistical differences over storage between cand x-irradiation for the same components. results: all components produced were within nhsbt specification for volume, haemoglobin and haematocrit. there were no significant differences in red cell in vitro quality parameters studied over storage between cand x-irradiated units, for standard volume arms or neonatal arms and whether rcs were irradiated early or late in storage. moreover, all arms were within haemolysis specification for the end of storage (>75% of units with < 0.8% haemolysis) and 100% of units had atp levels above the recommended minimum for acceptable post-transfusion survival (2.3 lmol/ghb). both haemolysis and potassium levels at the end of storage for the standard c-irradiated rcc were comparable to our laboratory's historic data for the same component. summary/conclusions: in summary, the storage quality of rcc and rcs post-xirradiation did not differ from c-irradiation in this study, providing reassurance that either method could be used in routine manufacturing. a pajares herraiz 1 , c coello de portugal 2 , m morales 3 , f solano 4 , c perez parrillas 5 , a rodriguez hidalgo 5 , t diaz rueda 5 and m flores 5 1 direccion, regional transfusion center toledo-guadalajara 2 transfusion service, toledo hospital complex, toledo 3 transfusion service, general university hospital of guadalajara, guadalajara 4 transfusion service, hospital nuestra señora del prado de talavera de la reina, talavera 5 regional transfusion center, regional transfusion center toledo-guadalajara, toledo, spain background: the regional transfusion center of toledo-guadalajara (rtc) manages the collection, processing and distribution of blood components for the hemotherapy area of castilla la mancha (spain) that serves 3 general hospitals (hospital complex of toledo (hct), university general hospital of guadalajara (ughg) and hospital nuestra señora del prado de talavera (nspt)) and the needs of 943,000 inhabitants. by also managing the hct transfusion service, it facilitates the handling of stocks. since 2008, rtc has initiated pathogen inactivation (pi) for a part of its platelet components(pc) with the intercept blood system (cerus) using a photochemical treatment with amotosalen and ultraviolet-a. this system allows the inactivation of a broad panel of pathogens and leukocytes, extending the shelf-life of the cp from 5 to 7 days. this affects the expiry and discards of this blood component, allows a better management of the inventory and has an influence on production costs. aims: the objective was to evaluate the influence of pi in the production of cp at rtc and the expiry in the hemotherapy area during the last 8 years divided into four periods ( results: pc were predominantly obtained from whole blood collections with 85% of bc platelets/15% of apheresis platelets. 77% of the available bc were used in production for period a and 88% for periods b, c and d. after wastes of approximately 1.9%, the distribution of pc was stable for the 4 periods studied. 7075 pc were distributed for period a, 7047 pc for b, 7467 pc for c and 7083 pc for d. the % of pi platelets with 7-day shelf life available in the 3 hospitals was limited to 13% during period a. it was then increased to 14.1%, 20.9% and 26% for periods b, c and d respectively. the percentage of wastes was stable at 0.2-0.3% but the discards due to expiry went down from 24.24% (period a) to stabilize at 10.9% in periods b and c and 10.3% in period d. in the 3 general hospitals the expiry went down from 20% to 5.89%(hct), 29.4% to 14.5% (ughg) and 33.36% to 17.68%(nspt) respectively. summary/conclusions: greater control of pc stocks through historical analysis and consumption projection, together with it tools and the use of pi pc with 7-day shelf life allowed reducing discards for expiry from 24.24% to 10.3% in the last period analyzed at rtc and the 3 major hospitals of the hemotherapy area. this has a great value in cost-reduction and improves inventory management and the efficiency of the processes. background: blood centers are faced with many challenges including availability of concentrate platelets as well as ensuring highest quality of the product. overcoming the shortage of platelet apheresis by using pooled platelet derived from whole blood units separated using automated standardized system, which can assist blood banks to meet the increase demand in platelets. the pathogen inactivation (pi) technology can improve the quality of the product by mitigating the risk of transfusion-transmitted diseases (ttd) and residual white cells, resulting in minimizing non -hemolytic transfusion reactions. however, the pathogen inactivation treatment must not impact the platelet quality and functionality significantly, as well as the patient safety. aims: evaluate the quality of pooled platelets derived from whole blood (five interim platelet units), separated using reveos automated blood processing system (terumo bct), pooled in 100% donor plasma and pathogen inactivated by amotosalen/uva technology. methods: five interim platelet units (ipus) produced with reveos device (terumo bct) from single whole blood donations, were pooled with a platelet pooling set (terumo bct) and leucodepleted with a lrf-xl filter (haemonetics). thirty pools have been included in this study, the units were treated using a large volume cerus intercept processing set for platelets according to the manufacturer's instructions and stored until day 5. the swirling was determined by visual inspection. the volume and yield content were assessed preinactivation and after treatment by pathogen inactivation with a cell counter (dxh-800, beckman coulter), rbc contamination was also measured preinactivation with a cell counter (beckman coulter), bacterial contamination was assessed by automated blood culture with a bact/ alert system (biomerieux). the ph of the platelet units was assessed with a phmeter (jenway), and residual amotosalen levels were assessed by an hplc assay. results: the impact of amotosalen/uva pathogen-inactivated pool platelet products quality were assessed. the pre and post-inactivation of the units showed a swirling score of 2-3. the average volume per unit of the pre-inactivation was 288 ml (278-302 ml) and post inactivation was 269 ml (254-284 ml), with average volume loss during inactivation was 36 ml (24-43 ml), corresponding to 13% (8-15%). the average platelet yield per unit pre-inactivation was 3.7 9 10 11 (3.0-4.4 9 10 11 ) and post inactivation 3.2 9 10 11 (2.8-3.9 9 10 11 ) with an average platelet loss of 13% (3-20%) . the average rbc contamination per unit (0.01-0.02 9 10 9 rbc/ml). the culture tests were negative, the average ph at day 1 was 7.4 (7.1-7.6), average ph at day 4/5 was 7.3 (7. 2-7.4 ). the average residual amotosalen concentration post treatment was 0.30 lm (0.23-0.35 lm). summary/conclusions: the quality of pathogen-inactivated pool platelets tested, met the criteria set by aabb guidelines. the volume and platelet loss were in acceptable range, in alignment with previously published data. a residual amotosalen concentration below 2 lm is considered safe and acceptable by french and german authorities. the evaluated data support the reasonable assurance of quality and effectiveness of the device when used in accordance with indication for use. background: the implementation of a pathogen inactivation process (pi) allows the redesign of processes to obtaining safe blood components by reducing the need for additional testing for pathogens detection, minimizing the residual risks (such as the infectious window period for those pathogens that are detected as usual), eliminates the need for selective tests (eg cytomegalovirus serology test) and complements gamma irradiation given its ability to inactivate white blood cells. in addition, the routine implementation of pi reduces the incidence of bacterial infection in recipients of blood components and allows blood services to proactively protect the blood supply against future emerging infections. aims: to verify the functional integrity and viability of platelet concentrates after being inactivated of any pathogenic agent, to be used as safe and functional components for transfusions. methods: a total of 106 independent platelet concentrates were studied. platelets are donated through a process called plateletpheresis according to the established norms, after the process, platelet concentrates were submitted to pi on the intercept blood system tm platform with uv-a illuminator; an immediate sampling of each donation of platelet concentrates was carried out taking a sample of 2 ml pre-inactivation and another sample post-inactivation (16 h after pi). the platelet viability of each sample was evaluated by demonstrating the cd62p expression marker by flow cytometry. once processed, platelet concentrates were released as safe components for donation. compiled the experimental data of the platelet count with platelet activation marker with respect to the total platelet, a comparative, nonparametric test of wilcoxon was carried out between two measurements (pre vs post) and the platelet viability after pi was determined. results: a total of 106 independent platelet concentrates were studied, where the average percentage of pre-inactivated platelets with expression of the cd62p marker, was 18%, while the percentage of functional platelets post inactivation was 19%, this result only shows that the functionality of the platelets is not being altered after the inactivation process. the wilcoxon test confirms that there is no significant difference between platelet activity pre-and post-inactivation, with a 95% confidence level. summary/conclusions: the process of photochemical treatment with amotosalen hydrochloride and long-wavelength ultraviolet light (uva) applied to platelet concentrates provides functional products without alterations in platelet function to be transfused. background: treatment of platelet concentrates (pcs) with pathogen reduction technologies is widely implemented in blood establishments to reduce the risk of bacterial contamination and to face the presence of new emerging agents in blood components. aims: the reduction of antioxidant power (aop) could be a quality control test to prove the complete viro-inactivation treatment. this evaluation has the goal to study the feasibility of the method from "abonnenc et al., transfusion, 2016" in another blood service, assessing the aop of platelet units treated by intercept technology. methods: the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid. repeatability, intermediate precision and accuracy were determined. linearity was evaluated using the linear regression and the calculation of pearson's coefficient (r²). limit of quantification (loq) was determined by measuring aop using nacl samples to define the background. roc curves were used to determine a threshold to discriminate pcs before and after treatment. a distinction was realized between men and women and between apheresis (a) pc and buffy coats (bc) pcs. a one-year evaluation was assessed on pcs before and after treatment on the routine production. results: the coefficient of variation for the repeatability was less than 10%. for the intermediate precision, the coefficient of variation was less than 15%, but for the pcs after treatment, this result rose up to 21%. the r² value for the linearity was 97.7%. the detection limit corresponded to a result of 6 edel and the loq (equal to 10xsd) is 19 edel. concerning roc curves, the men apcs threshold was 58.5 edel compared to women apcs with 62.5 edel. the threshold for bcpc was 54 edel. all of these results had 100% of specificity. below this threshold, intercept treatment was considered to be executed. about the one-year experience on routine pcs production, 404 apcs (182 women and 222 men) and 263 bcpcs were tested. all of the bcpcs and women apcs were under the threshold after treatment. concerning men apcs, 14.0% of the pcs after treatment were not under the threshold. summary/conclusions: the device validation was satisfied. for the one-year evaluation and concerning men group apcs, the threshold found by abonnenc et al. was 89 edel. our study showed a threshold with 100% specificity and 90% sensitivity at 58.5 edel which is much lower. specificity was favored compared to sensitivity but the analysis should be revised to adapt the threshold to get higher sensitivity. this can lead to reduce the non-conformity and allows measuring the aop only after treatment. for women, our threshold was found at 62.5 edel compared to 66.5 edel for abonnenc et al. concerning sex in apcs, results were statistically lower in women group than men group before and after treatment. and for bcpcs, the two populations (before and after treatment) were very distinguishable and our threshold (54 edel) was lower than abonnenc threshold which was at 59.8 edel. in conclusion, edel threshold enables the segregation and depends on the preparation process adapted in each blood service. aims: this study has the goal of measuring antioxidant power (aop) level in plasma units treated by mb technology. the aim is to use such a test as a quality control assay for documenting the execution of pathogen inactivation treatments during the preparation of plasma units. methods: aop measurements were performed using a potentiostat electrochemical analyzer. a 3-ll volume of sample is deposited over the electrodes on a single-use microship. the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid and reflects the redox status of the plasma units. different protocols were established to understand the role of mb, the illumination and the filtration on the aop variation measure: 1) complete treatment, 2) plasmas with mb without illumination, 3) plasmas without mb with illumination and 4) plasmas without mb without illumination. ten dosages on men donor samples, except for protocol 1 where n = 20, were realized during the viro-inactivation process, t1 corresponds to a dosage of plasmas before treatment, t2 the plasma after the mb dry tablet passage, t3 is the time after illumination and t4 corresponds to the final product (after filtration). results: in each protocol with mb, an increase was observed after addition of mb before illumination. after illumination, the edel values decreased for about less than 50%, which was expected because of the degradation of mb in its photoproducts during the illumination. in the series 1 and 3, the illumination seemed to have an effect by itself, with or without mb because the aop increased. the final filtration has the goal to eliminate the residual mb and its photoproducts. after this step, the aop values fell down. the series 2 was a confirmation of the efficacy of the filter to remove the mb as shown by the decreased aop in t4 (238 ae 16 edel at t3 and 209 ae 17 edel at t4). however, in the absence of mb (series 3 and 4), the results at t1 and t4 were not statistically different. summary/conclusions: the filtration decreases the aop rate, except when there was no mb. the results of non-complete viro-inactivation treatment allow concluding that the measure of aop rate may not indicate that the treatment was completed or not since significant differences before and after treatments were found in the non-complete treatment series. vox sanguinis (2019) background: the intercept blood system (ibs), a photochemical treatment with amotosalen and uva, is used to inactivate pathogens and leukocytes in plasma. the intercept tm plasma processing set (cerus bv, netherlands) was modified to incorporate plastic containers in non-pvc materials sourced from alternate suppliers and connecting parts and accessories in non-dehp pvc formulations, making the system dehp-free. the final storage container was modified with a higher contact surface with plasma to limit the thawing time. proportion of units with a fibrinogen concentration ≥ 2.00 g/l was 94% (>70% required). mean recovery fviii fibrinogen after ibs treatment and frozen storage were 75% and 90%, respectively. residual platelets were < 25 9 10 9 /l, leucocytes < 1 9 10 4 /l and red blood cells < 4 9 10 9 /l. all units had a protein content > 50 g/l. residual amotosalen was below 2 lm in all post-cad samples. the concentration of tat complexes was slightly reduced after treatment and frozen storage. concentrations of c3a and c5a were significantly reduced with the cad treatment. the plasma thawing time in a water bath at 37°c was consistently short (6-7 min). summary/conclusions: pathogen inactivated plasma units (ffp-a-ibs and ffp-wb-ibs) prepared with dehp free intercept processing sets retained in vitro characteristics which meet the quality standards for therapeutic plasma. the process did not activate coagulation or complement. reducing ffp thawing time from routine 8-10 to 6-7 min is an important benefit for emergency use. background: plasma coagulation factor concentrations usually differ for individual donors, therefore pooling of whole-blood derived plasma units moderates high or low coagulation factor concentrations and ensures transfusion of more standardized blood components. moreover, pooling contributes to dilution of reactive antibodies and may reduce the risk of non-hemolytic transfusion reactions and trali. additionally pathogen inactivation reduces the risk of transfusion-transmitted infections, and non-hemolytic transfusion reactions as well as gvhd through inactivation of residual lymphocytes. aims: assessment of the impact of plasma pooling and pathogen inactivation on the standardization of blood components and plasma quality. methods: the study included 5 experiments. for each experiment 5 male-donor, abo-compatible whole-blood derived plasma units (≥ 270 ml) were collected from different donors and pooled using the donopack optipool plasma pooling set (cerus europe b.v.). each of the 5-unit pools were split into 2 equal minipools which were subsequently treated with the in intercept blood system (cerus europe b.v.). then, each minipool was split into 3 (≥200 ml) therapeutic units. samples were collected before and after pooling as well as after inactivation to assess the coagulation factor content (fviii, fix, fibrinogen, vwf antigen using elisa) and coagulation time (aptt, pt). the study-analysis included samples from five pools from 5 single plasma units respectively ( background: biotin (bio) is an alternative to radioactive red blood cell (rbc) tracers which allows one to concurrently track in vivo multiple cell populations labeled at different bio densities. in american clinical trials, multi-labeled biorbc have been transfused in man to assess their survival (mock et al, transfusion, 2018) . in these studies, the different biorbc populations were monitored by ex vivo flow cytometry analysis using streptavidin. so far, the biotinylation reagents biosulfonhs was not complying with good manufacturing practices (gmp). moreover biorbc, with bio ≥ 18 lg/ml, have induced immunization of the recipient, in rare cases (schmidt et al, transfusion, 2017) . this represents an obstacle regarding the regulatory european authorities. aims: the aim of this study is to describe a procedure of biotinylation of rbc intended for clinical trials while refining the levels of bio ≤ 18 lg/ml. methods: sterile status is met throughout the process. rbc are taken from standard rbc concentrates and treated with biosulfonhs of gmp-grade (0 to 70 lg/ml) recently commercialized. washing buffer is of injectable-grade. biotinylation efficacy is controlled by flow cytometry with streptavidin conjugated to 2 different fluorochromes: phycoerythrin (pe) or brilliant violet (bv421). results: labeling with biosulfonhs of gmp-grade or non gmp-grade is comparable and 4 populations of rbc could be easily distinguished between themselves and from unlabeled blood cells. biosulfonhs (lg/ml): 0 (mfi 0.4), 4 (gmp mfi 12; non gmp mfi 9.5), 18 (gmp mfi 44; non gmp mfi 42), 70 (gmp mfi 174; non gmp mfi 180). streptavidin-bv421 brighter than streptavidin-pe is a promising tool because it amplifies by 1.7 the signal of fluorescence and allows a good differentiation of the 4 populations of rbc treated with only 0, 1, 4 and 18 lg/ml biosulfonhs. summary/conclusions: this preliminary study explores the feasibility of multilabeled biorbc production for clinical trials. the benefits of this approach are to overcome the need for non-radioactive tracers, to follow simultaneously various populations of rbc and consequently to limit the number of volunteers, and to reduce the risk of immunization using bio ≤ 18 lg/ml. background: rejuvenation is aiming to revert ageing-related disease development. heterochronic parabiosis studies revealed eotaxin in young and old murine blood as a regulator of brain aging and neurogenesis. umbilical cord blood (ucb)-borne factors including tissue inhibitor of metalloproteinases 2 (timp2) and neonatal immune cells also contributed to rejuvenation in animal models. human platelet lysate (hpl) is commonly used by us and others for highly efficient cell propagation in vitro (burnouf et al., biomaterials, 2016) . published data indicate only limited differences between adult and ucb-derived hpl, partly questioning enigmatic rejuvenation effects. aims: to verify candidate regenerative factors in neonatal blood products we compared protein contents of neonatal and adult plasma and platelets, respectively. methods: heparinized ucb samples (n = 9) were centrifuged within 3 h to collect neonatal platelet rich plasma. aliquots from apheresis platelet concentrates (n = 9) were used as adult counterpart. platelet concentration was adjusted to 7-10 9 10 11 / l. plasma supernatants and platelets were obtained by centrifugation and platelet pellets were re-suspended in saline. after two freeze/thaw cycles at à30°c/37°c for platelet lysis (npl; apl) the platelet fragments were removed by centrifugation. the protein content was analyzed with a proteome profiler tm array. nine samples of each group were pooled to avoid individual donor variations. a threshold of 50,000 au spot density was defined as cut-off. data were analyzed by graphpad prism 8 using two-way anova. results: semi-quantitative evaluation of 105 analytes per array revealed significant differences. in plasma samples and platelets 63 and 48 analytes were detected above cut-off, respectively. in neonatal plasma we found more highly prevalent proteins (>200,000 au spot density) compared to adult plasma (6/63 vs. 3/63). thirteen proteins were significantly elevated in neonatal plasma including growth/differentiation factor 15 (gdf 15), platelet derived growth factor aa (pdgf-aa) and serpin e1 (p < 0.0001). more highly prevalent proteins were detected in npl (10/48) compared to apl (1/48), and 20 proteins were significantly elevated including vascular cell adhesion molecule-1 (vcam-1), platelet factor 4 (pf4/cxcl4), epidermal growth factor and lipocalin-2 (p < 0.0001). in adult samples only 10 proteins were significantly higher in plasma and three proteins in apl compared to the neonatal groups (p < 0.05 to p < 0.0001). summary/conclusions: we detected significant differences in regenerative growth factor and cytokine contents of neonatal and adult plasma and platelet samples, respectively. additional experiments are underway to further characterize their impact in distinct functional readouts. background: the production and storage conditions of platelet (pl) products intended for transfusion are constantly evolving and need sometimes in vivo evaluations in clinical trials to ascertain whether the platelets have retained their ability to survive in the circulation. this requires that the transfused platelets can be distinguished from the recipient's circulating platelets. labeling of platelets with biotin (bio) affords to track in vivo and concurrently, multiple cell populations covered with various biotin densities as already described for red blood cells (mock, transfusion, 2018) . surprisingly, there is only one study describing the transfusion of human biopl (stohlawetz, transfusion, 1999) . so far, the biotinylation reagent bio-sulfonhs was not complying with good manufacturing practices (gmp), which represents an obstacle regarding the regulatory authorities. aims: the aims of this study are 1) to describe a procedure to label injectable human platelets with 2 densities of biotin, 2) to evaluate the impact of biotinylation on platelet functions, 3) to track human biopl in the circulation of the mouse. methods: platelets are taken from standard platelets concentrates and treated with 1.2 and 10 lg/ml biosulfonhs of gmp-grade, recently commercialized. main platelet functions are assessed in vitro. human biopl survival is evaluated in immunodeficient nsg-mice treated with liposome-clodronate to eliminate macrophages and to prevent rejection. circulating human biopl are detected ex vivo by flow cytometry with streptavidin phycoerythrin. results: using trap (60 lm), p-selectin externalization reveals a normal capacity of secretion for all biopl. gpiba and gpiibiiia expression is not affected by the biotinylation process. biopl have the ability to aggregate: using arachidonic acid (1 mm), amplitude of aggregation is 81.7 ae 2.5% (bio 0); 82.6 ae 2.3% (bio 1.2 lg/ ml); 79.2 ae 1.6% (bio 10 lg/ml). using collagen (2.5 lg/ml), amplitude of aggregation is 65.6 ae 4.2% (bio 0); 61.4 ae 4.0% (bio 1.2 lg/ml) 40.8 ae 6.6% (bio 10 lg/ml). the 2 biopl populations could be easily distinguished between themselves and from unlabeled blood cells in the mouse circulation during more than 48 h. after 4 h, the mean fluorescence intensities are 0.13 ae 0.02 for unlabeled circulating mouse platelets, 1.01 ae 0.03 and 8.2 ae 0.15 for circulating human biopl covered respectively with 1.2 and 10 lg/ml biotin. summary/conclusions: this labeling approach should be helpful to evaluate new platelet products in vivo and represents an alternative to radioactive tracers. it allows to follow simultaneously different platelet populations and consequently limits the number of volunteers in clinical trials. background: severe ocular surface diseases, dry eye syndrome, persistent and recurrent corneal epithelial defects and diabetic or neurotrophic keratopathy are mainly successfully cured by standard treatment protocols. however, not rarely does refractory to these usual treatments appear, especially with serious forms of disease. in military medical academy, autologous serum eye drops -auto seds and autologous platelet lysate -apl eye drops have been being applied in the treatment of ophthalmological patients in these categories, who were previously resistant to standard therapy. aims: to show the achieved results of therapeutic use of autologous blood products (auto seds and apl) in the treatment of ophthalmological patients who previously had not responded to conventional therapysingle center experience. methods: auto seds are prepared by taking autologous blood into tubes (bd vacutainer, cat, 10 ml) and apl in tubes with anticoagulants (greiner bio-one, acd-a, 9 ml). control on tti of every patient and sterility of every series has been conducted. before and after the treatment, subjective ocular discomfort (ocular surface disease index -osdi), objective parameters of the tear film (schirmer's test, rose bengal, tear breakup time -tbut) and measuring of epithelialization zone were analyzed. apl, obtained from platelet-rich plasma which had been frozen, unfrozen and diluted with nacl solution, up to 30%. auto seds were administered in the form of 20% eye drops. results: auto seds have been applied to 17 ophthalmological patients (10 men and 7 women), previously resistant to standard therapy. in total 44 treatments were performed (each lasted 20 days). for successful curing, one or two treatments per patient, in average, were applied. apl has been used multiple times to one patient with sj€ ogren syndrome and severe multiple tropical corneal changes. all ophthalmological patients had subjective improvements (the average pre and post treatment osdi scores were 71.3 and 19.6 respectively). also, objective progress was present in 83% of all patients (p < 0.001). summary/conclusions: the use of auto seds and apl in the treatment of ophthalmological patients, previously resistant to standard therapy, is in constant increase, because of its simplicity and low expenses. apl has turned out to be better than auto seds for patients with severe trophic changes, because apl contains larger amounts of the nerve growth factor, tgf-b, vegf and platelet derived growth factor. however, a larger number of clinical cases is needed for future conclusions. background: whole blood (wb) has recently regained favor in treatment of massively bleeding patients in military and civilian settings. 1 platelets (plts) are a vital component in clot formation. as a component of wb, it is critical that they maintain functionality throughout storage. red blood cells (rbcs) stored in hypoxic/ hypocapnic conditions preserve high level of 2,3-dpg while reducing storage lesions stemming from oxidative stress. 2, 3 on the other hand, effects of steady hypoxia (pco2~10-20 mmhg) on plts contained in leukoreduced wb is poorly characterized. aims: examine the effects of hypoxic conditions on plt function and microvesicle (mv) formation in wb stored hypoxically (h) and conventionally (c) for 3-week storage at 1-6°c. methods: 11 units of wb were collected at mayo clinic rochester blood donor center from normal healthy volunteers into 70 ml cp2d. wb was leukoreduced using plt-sparing filter (terumo wb-s) then split into control (c) and hypoxic (h). h-wb was processed by the oxygen-reduction bag (hemanext, lexington ma) and unit was stored in o2-free bag. 5 ml of wb were collected from each unit at day 1, weeks 1, 2, 3. plt counts, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation, nonactivated and agonists activated plt surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding), and microvesicles (mv) were measured by coulter counter and digital flow cytometer. paired student t-tests were used to analyzed differences in degradation rates; significance: p < 0.05. results: h-plt counts declined to~60% by the o2-reduction process, while similar decline was observed after 1 week in c, and thereafter remained steady. plt activation (ps) increased over time (h >> c after processing; c increasing more rapidly during storage). p-selectin increased over time (h < c), while pac-1 showed large increase after 1 week, then remained steady (h << c). plt activation by trap or adp declined modestly over 3 weeks (~15%) while h-plt showed additional~10% reduction for all time points. collagen activation for c-plt increased after 1 week (74%) and gradually increased to 100% after 3 weeks (~20% reduction with h compared to c). plt-derived mv (cd61 and cd61/annexin v) increased~4-fold over storage time; day 0 mv levers were significantly higher for h, but subsequent increase rates were similar or lower. total number of plt-derived mv (cd42a) in wb supernatant increased 17-fold after 3 weeks for c, while h suppressed increase to 7-fold. (majority of the trends described above showed significant differences between h and c.) summary/conclusions: plts were activated over 3-week period when stored at 1-6°c in leukoreduced wb, accompanied by a modest loss of agonist-induced activation. oxygen reduction treatment initially activated h-plts, while subsequent increase in activation rates were suppressed compared to c-plts. wb plts retained activatability, and hypoxic condition showed only modest further reduction on the activatability. hypoxic wb may provide higher quality wb for trauma patients if the levels of initial plt activation can improved during oxygen reduction procedure. methods: after informed consent, eligible patients were randomized to either first receive autologous followed by allogeneic seds or first receive allogeneic followed by autologous seds. each sed treatment phase was one month, separated by one month of patient's standard treatment (wash out period) between sed treatment phases. the patients each donated 500 ml whole blood from which the autologous seds were prepared. allogeneic seds were prepared from blood from never-transfused male donors with blood group ab. all serum was diluted 1:1 by adding saline, and aliquoted in an eye drop dispensing system (meise, schalksm€ uhle, germany). at each visit, the osdi was determined using a validated questionnaire, with higher scores reflecting poorer outcomes. the results were analyzed intention-to-treat, and a random effects linear mixed model for cross-over design was used. results: in total, 19 patients were enrolled, 4 of whom were excluded because they failed the autologous blood donation. background: the following blood components for non-transfusional use (bcntu) are produced in our transfusional center (tc): 1) allogeneic platelet gel (pg), derived from buffy-coats (bc) and human cord blood platelet gel (cbpg); 2) autologous serum eye drops (sed). the creation of both types of platelet gel started in 2014 but only in 2017 we confirmed the process for daily production: these blood components are used to treat pediatric patients with epidermolysis bullosa. the sed, produced from 2018, is dedicated to treat patients with dry eye syndrome. aims: production and storage bcntu. methods: the whole process production of bcntu is traced on the transfusional informatic system (emonet-insielmercato), under the same conditions of another blood transfusional components. the process takes place in closed circuit using the laminar flow hood. 1) pg production starts from the bc resuspended in plasma that are not used for daily platelet concentrates, instead the cbpg is produced using cord blood units that are not used for hematopoietic transplant. both have a platelet concentration between 800-1200 10 3 /ll and negative blood cultures, required by the italian law; the units are frozen at à80°c and last 5-year. pg and cbpg must be activated with calcium gluconate or batroxobin to be used. 2) the ophthalmologist's patients, with dry eye syndrome, donate 120 ml of autologous blood; the serum is separated and after the dilution with a balanced saline solution (30%) are divided in 2 boxes containing 30 single-dose vials each: they are stored at à80°c and they last one year. negative blood culture was evaluated. results : background: candida albicans is the most common pathogen detected in fungal infections. aims: in this study, we aimed to evaluate the in vitro antifungal activity of volunteerderived platelet rich plasma (prp) against c. albicans atcc 10231 strain and the possible effects of certain chemokines, kinocidins that might play a role in this activity. methods: prp from nine volunteers were derived by using magellan prp â kit. 10% calcium gluconate was used to obtain autologous thrombin. c. albicans isolates with a final yeast concentration of 1 9 10 3 cfu/ml and 1 9 10 4 cfu/ml were inoculated on sabouraud dextrose agar at the 1 st , 2 nd , 4 th , 8 th and 16 th hours of incubation to reveal the antifungal activity of autologous thrombin-activated prp. the colonies were counted after 18-24 h of incubation at 30°c. chemokines and kinocidins (platelet factor-4, interleukin-8 and thymosin-b4) were also measured simultaneously by elisa method. results: compared with the pbs-control group, the prp-10 3 group showed that the antifungal activity was still going on at the 8 th hour. the difference in colony production between the two groups at 8 th hour was statistically significant (p < 0.05). it was observed that the antifungal activity continued at the 4 th hour, decreased at the 8 th hour in the group prp-10 4 group. although the same amount of prp was used and the same amount of chemokine and kinocidins were released in both groups, the concentration of c. albicans was considered to be important in the detection of more effective prp-10 3 group. although there was an increase in il-8 levels by hours in the two prp groups by elisa method, no antifungal effect was detected against c. albicans. it was observed that decrease in tmsb4 values results from the antifungal activity on the advancing hours in the prp groups. whereas pf-4 did not act an antifungal activity on prp-10 3 and prp-10 4 . summary/conclusions: even in our study group where the highest platelet counts were obtained at the lowest concentration, c. albicans reproduction could not completely eliminated as mentioned in the literature. repeated doses of prp applications, such as drugs used in patients, may have longer duration of action and even complete repression of reproductive outcomes. background: generally, blood is available in developed countries for transfusion. sometimes, transfused or previously pregnant patients form alloantibodies to red cell antigens and rarely, to antigens of high prevalence. this case focuses on a twoyear-old girl, of pakistani descent, diagnosed with neuroblastoma stage iv with anti-in b and -e. although the publications indicate that 4% of the pakistani, indian or iranian populations are in(b-), it was discovered that this blood type is exceedingly rare. an international search was required to ensure blood product availability for chemotherapy and autologous hematopoietic progenitor cell transplants aims: illustrate the response of the public to a powerful story of a child needing rare blood for treatment and international collaboration for provision of very rare units. methods case report: a two-year-old patient's sample was referred for antibody identification. the patient had received four transfusions (883 ml of red cells) in the preceding 10-day period. hgb level fluctuations were consistent with decreased transfused red cell survival. following the last transfusion of 225 ml, the hemoglobin decreased from 8.6 to 5.8. anti-in b , and a ficin-only reactive anti-e was identified in the serum and anti-in b in the eluate. the monocyte monolayer assay predicted the anti-in b to be clinically significant (68% reactivity). transfusion of antigen neg units once obtained, resulted in a stable transfusion response. although it was expected that in(b-) blood would be more easily sourced, only two donors in the usa were in (b-) e-. as 7-10 units of blood were requested for the post-transplant period, a national and international search was initiated, as was a robust media appeal to donors resulting in many donors for an intense domestic screening effort in the usa. the search of the who international rare donor panel by the international blood group reference laboratory revealed three known in(b-) e-donors; two british and one australian. they were contacted, recruited, collected and shipped to the usa with the work of the american rare donor program (ardp) staff and the isbt working party on rare donors (isbt wprd) members in each of the countries. results: the intense media coverage of oneblood (the florida blood center collaborating on treatment with the hospital) included online news outlets (youtube, facebook) resulted in over 27,000 responses from national and international potential donors to be tested for in b . isbt wprd members were sent the web information of potential donors identified in their countries by the ardp. over 3,500 samples from 75 blood centers and associated laboratories tested with anti-in b by oneblood. two new in(b-) donors were discovered (0.06%); but both typed e+, thus were not a match for the child. summary/conclusions: this intense media coverage and the overwhelming donor response was unprecedented in our experience. the coordination and cooperation among the numerous blood centers reflect the deep dedication of the blood banking community to the well-being of special patients in need. this case illustrates the response potential that a powerful story and a medical appeal for exquisitely rare blood utilizing social media and other online news outlets can generate. background: blood platelet units are generally stored in blood banks for 3-5 days, afterwards they are discarded. prepared infusible platelet membrane (ipm) from fresh or outdated human platelets correct the prolonged bleeding times in thrombocytopenic animals such as rabbits. infusible platelet membrane (ipm) as a platelet substitute may be the most feasible approach to reach the target market. our previous experiments have shown that ipm has a hemostatic efficacy to shorten bleeding time without any adverse effects in rabbits. aims: abnormal toxicity is the european pharmacopoeia standard for assessment of biological products which the test material is administered to the mice. in this study, abnormal toxicity of ipm was evaluated in experimental animal model such as mice to assure the safety of ipm without any evidence of serious toxicity. methods: in this experimental study, infusible platelet membrane (ipm) was prepared from outdated platelet concentrates. platelet concentrates were pooled, disrupted by freeze-thaw procedure, pasteurized for 20 h to inactivate the possible viral or bacterial contaminants with a sodium caprylate stabilizer, formulated by sucrose and human serum albumin and finally lyophilized. at first, the test for sterility is carried out under aseptic conditions for ipm vials and then we injected 0.5 ml of ipm (2 mg/kg) intravenously between 15 to 30 seconds into each 5 health mice, weighing 17-22 grams. these tests were performed according to eu pharmacopeia monographs. results: in the sterility test no evidence of microbial growth in our product is found. the abnormal toxicity test will be passed if none of animals die during 24 h after injection. if more than one animal dies, the preparation fails the test. if one of the animals just dies, the test is repeated. in our experiment all five mice were alive after 24 h of ipm injection. summary/conclusions: in this research the results showed that ipm as a platelet substitute is free of abnormal toxicity with adequate safety and it may be used in human clinical trial studies as a feasible approach to develop a platelet substitute in the future. however, further studies are required to confirm the different aspects of its safety as well. the success of such investigations may affect patients' care in transfusion medicine in the future. a substantial number of infants, especially premature infants, are unable to receive adequate amounts of their mothers' milk for a variety of reasons. the world health organization recommends that infants, especially preterm and ill infants are fed with quality-controlled donor milk if they cannot be fed with their own mother 0 s milk. due to the possible transmission of the human immunodeficiency virus many human milk banks closed in the 1980s, therefore the availability of donor milk has decreased. aims: we analyzed the processing of donor milk and the required laboratory tests to establish a human milk bank within our blood donation service in cooperation with the department of neonatology at the frankfurt university hospital. methods: based on the recommendations for promoting human milk banks in germany, austria, and switzerland (efcni) we evaluated the manufacturing steps and the quality controls require to establish a human milk bank. background: for patients suffering from severe ocular surface disorders treatment with blood derived serum eye drops (sed) is a highly effective therapy. autologous sed, prepared from the patient's own blood, is used preferably. for this approach we have more than 6 years of experience. if auto-sed cannot be manufactured due to medical reasons allogeneic sed present an alternative. since 2 years, the allogeneic approach is well established in our center. aims: retrospectively evaluation of our experience with allo-sed. methods: in germany manufacturing of allo-sed is only possible as an "individual healing attempt". for each patient experienced regular ab0-identical male donors without blood borne disease, who never received blood products and not taking any kind of medication are selected. additionally, donors must pass a questionnaire excluding any form of dry eye syndrome. allo-sed are manufactured directed for each individual patient according to the process for auto-sed in a closed system. patient files of our serum eye drops donors were screened for patients receiving an allogeneic treatment. data concerning indication for allo-sed, contraindication for phlebotomy, problems with donor selection and manufacturing, as well as serological and microbiological testing results were obtained. clinical results were evaluated © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 by ocular surface disease index (osdi) and patient's questionnaire, asking for subjective benefit, symptom reduction, possible side effects, consumption and comparison with artificial eye drops or, if applicable, with auto-sed. furthermore patients are undergoing regular ophthalmologic examination within a special consultation for dry eye syndrome at our hospital. results: 31 patients were identified receiving allogeneic sed, 15 patients had been treated autologous previously. in total, allogeneic sed have been produced 54 times since june 2017. indications were ocular gvhd (n = 15.48%), neurotrophic keratopathy (n = 9.29%), mucous membrane pemphigoid (n = 2.7%), sj€ ogren syndrome (n = 1.3%) and secondary keratoconjunctivitis sicca by virtue of chemotherapy, meige syndrome, rosacea, morbus bruton (n = 4.13%). contraindications for autologous donation were underlying disease (n = 19.61%), poor venous access (n = 12.38%), low haemoglobin (n = 8.25%), low body weight (n = 8.25%), very young age (n = 7.22%), circulatory disturbances (n = 3.10%) and lack of response to auto-sed (n = 2.7%). some patients presented more than one contraindication. manufacturing problems were: lipemic donor plasma (n = 2.4%), high donor haemoglobin (n = 2.4%) and unspecific positive serological findings (anti-hbs n = 2.4%). microbiological testing was sterile every time. as side effects one case of allergic reaction, suspected as serum protein allergy, appeared. clinical outcome can be considered equivalent to ased. subjectively, all patients benefited from the therapy and reported an alleviation of their symptoms. for some indications (highly active gvhd) allo-sed might even be the better option. summary/conclusions: considering our previous experience, allo-sed seem to be a safe and equally effective alternative to auto-sed for patients unable to donate blood. in case of urgent indication, timely supply can sometimes be difficult. to overcome this disadvantage licensing allo-sed as a new blood product with the possibility of production and storage in advance would be a desirable goal. in addition supply would become even safer by preparing allo-sed according to a quarantine principle like ffp. abstract withdrawn. background: vernal keratoconjunctivitis is a chronic, recurrent bilateral inflammation of the outer ocular layer. mostly affected are children and young people and the condition is more common in boys. the disease presents with eye pruritus (itching eye), photophobia (sensitivity to bright light), excessive tearing and foreign eye syndrome. severe cases manifest with diffusion of overgrown papillae usually of the upper eyelid, bursting of the connective tissue barriers and appearance of giant papillae that press on the cornea. corneal ulceration is a severe complication of vernal keratoconjunctivitis that may induce scarring, corneal neovascularization and occasionally perforation. treatment of keratoconjunctivitis mainly relies on steroids, mast cell stabilizers, antihistamines, immunosuppressive drugs (cyclosporine), artificial tears, contact lensdressing, cryotherapy and surgical papillae removal. we present the case of a 8 year-old girl with corneal ulceration who was applied artificial tears after traditional methods of treatment proved unsuccessful. aims: the aim was to share our experience on artificial tears therapy applied in ophthalmic disorders. methods: autologous blood (20 ml) was collected into disposable, sterile transfer bags used for routine blood component preparation (no anticoagulant) and incubated for 1 h at 37°c. the clot was then removed by centrifugation and the serum containing erythrocytes was press extracted. centrifugation was applied again to obtain serum free of cellular components. the serum was then divided into 0.3 ml segments (capsules)and the artificial tears applied to the left eye 8 9 daily. results: ulcer healing was reported after 4 weeks of therapy with artificial tears. the dosage was reduced to 4 9 daily. no recurrence of corneal ulceration was observed after subsequent 8 weeks. summary/conclusions: artificial tears are a safe and effective therapy for ophthalmic disorders in children. background: arv non-disclosure among hiv-positive donors who tested hiv antibody (ab) positive but rna negative (ab+/rna-), so-called false elite controllers, was previously described by our group in south africa, with > 80% of ab+/rnadonations since 2016 testing arv positive. the extent of undisclosed arv use at time of donation represents a significant risk to blood safety in a country with a growing treated hiv population. aims: to establish the prevalence of arv non-disclosure among four subgroups of hiv-positive donors in south africa along with demographic correlates of non-disclosure. methods: south african blood donors are screened by a self-administered questionnaire, which includes questions on current hiv status and arv use, followed by a semi-structured personal interview. specimens for hiv, hepatitis b and c testing are collected at time of donation. based on id-nat (procleix, grifols) and antibody (prism, abbott; western blot) testing, hiv-positive blood donations were classified as acute (ab-/rna+), recent (ab+/rna+, limiting antigen avidity [lag] odn ≤ 1.5), longstanding (ab+/rna+, lag odn > 1.5) and potential elite controller (ab+/rna-) cases. stored plasma from these donations were tested for four arv drugs using qualitative liquid chromatography-tandem mass spectrometry (detection limit 0.02 lg/ml). chi-square tests were used to assess associations of hiv case type, gender, ethnicity, age, donor type, and donor clinic (fixed, mobile) type with arv non-disclosure. results: during 2017, 1671 donors tested hiv-positive of whom 1315 had samples available that were tested for arvs. the overall prevalence of undisclosed arv use was 9.3% (n = 122) with efavirenz most frequently detected (115), followed by lopinavir (5) and nevirapine (2) . potential elite controller cases had the highest proportion of detectable arv (68/80; 85%) (p < 0.0001) followed by longstanding (37/741; 4.9%) and recent (17/366; 4.6%) infections. none of 82 acute hiv cases tested positive for arvs. there were no associations between arv use and gender or ethnicity. however, older (35 to 64 years) hiv-positive donors (49/305; 16.1%) were significantly more likely to test positive for arv than younger (15 to 34 years) donors (73/1010; 7.2%) (p < 0.0001). arv use was more frequent among first time (101/ 716; 14.1%) than in lapsed (13/277; 4.7%) or repeat (8/322; 2.5%) donors (p < 0.0001). donors at mobile clinics had significantly higher arv non-disclosure than donors at fixed sites (10.4% vs 5.0%; p = 0.0051). summary/conclusions: the 9.3% prevalence of undisclosed infection and arv use among hiv-positive south african blood donors is alarming. higher rates of nondisclosure among first-time donors was expected, but non-disclosure among repeat and lapsed donors suggests failure in donor education and assessment. the 4.7% prevalence among concordant ab+/rna+ cases may suggest sub-optimal viral suppression. lack of detection of arvs in acute cases should be qualified because the samples were not tested for tenofovir, the most common drug used in pre-exposure prophylaxis. donor motivation for non-disclosure of known hiv infection and arv use needs further investigation, since early arv initiation or infection while on prep could lead to low ab and rna levels, failure to detect hiv-infected donations and transfusion-transmission of hiv. blood bank, rotary blood bank, new delhi, india background: voluntary blood donation ensures safe blood transfusion. careful blood donor selection is of importance to provide safe blood to patients, although new methodologies have also been adopted by blood centers for blood safety and to minimize risk of transmitting infections through blood transfusion. the quality and the availability of blood components depend on the willingness to donate and reliability of the information given by the donors about their own health, including risk behaviour. blood donor history questionnaire is designed to evaluate donor's history in accordance with the guidelines laid down by the fda. donors, once deferred by the blood bank, will be less motivated to return for donation if he is not counseled effectively. it is important to reduce the number of deferrals by good donor comprehension and the centre should have a mechanism to recall temporarily deferred donors aims: the aim of the study is to analyse donor history and test results of those who donated blood with past history of jaundice. based on their history which suggested the type of viral infection they had, these donors were accepted or deferred. data was collected from voluntary blood donors who were screened for blood donation in the year 2018. methods: in this study, donor history was analysed with reference to history of jaundice. jaundice in donors after the age of 11 yrs, history of surgery, blood transfusion, body tattoos and acupuncture treatment within past one year of donation, history of multiple sex partners and related history and intravenous drug abuse history was taken into consideration. donors who revealed past history of jaundice were asked in detail about their illness and recovery. blood was donated by donors from whom the history of jaundice was elicited and it was understood that the type of virus which caused jaundice was not hepatitis b or c. those who could not give the correct history or were not sure of the cause of hepatitis, those individuals were deferred. aims: to assess the performance of this follow-up program in terms of donor participations, successful confirmed positivity rates, and potential reentry rates. methods: eligible donors were tested for hbsag, hcvab, hivab/ag, and tpab with two eias for each marker. samples reactive with at least one assay were tested further with electro-chemiluminescence assay (eca) and reactive samples were considered repeated reactive (rr). tpab reactive donations were re-tested with particle agglutination assay (tppa). samples eca or tppa non-reactive were considered non-repeatable reactive (nrr background: the blood donation service in suhl processes more than 160.000 samples annually from whole blood and apheresis donations, testing on average around 600 samples per day. for the last 5 years, serology screening was performed on the architect instruments (abbott) (arc), but will be changed to the alinity s system (aly) by middle of 2019. although the design of the aly assays is based on those of the arc assays, we undertook a thorough evaluation of the four mandatory screening assays detecting hbsag, hiv ag/ab, anti-hcv and anti-hbc. aims: to validate the 4 mandatory screening assays on the new aly system in our lab in terms of sensitivity and specificity, also including samples with known falsereactive results. determine the rate of false reactive results for hbsag, anti-hcv and anti-hiv that may lead to deferrals of donations and donors. methods: for sensitivity, we used known positive samples confirmed by immunoblot or nat. known unspecific positive samples for arc not confirmed by immunoblot or nat were testes for aly also. close to 2.000 unselected samples (edta plasma) from routine blood and apheresis donors were tested in parallel on both systems, arc and aly to determine the rate of initial and repeat reactive results. results: all known confirmed positive samples were identical detected by aly. samples with known unspecific reactive results were retested by aly with the following results: 19/33 anti-hcv, 19/21 hiv ag/ab and 04/22 hbsag were found reactive by aly to. one donation from an acute hiv infection in the early seroconversion period was detected by both methods in routine testing. there are no reactive results for aly not already known for arc. the specificity for the screening assays on aly versus arc assays were as follows: 1) hbsag aly 100. 00% (1994 00% ( / 1994 00% ( ) vs arc 99.95% (1993 00% ( /1994 ; 2) hiv aly and arc 99.95% (1992/1993); 3) anti-hcv aly 99. 90% (1994 90% ( /1996 90% ( ) vs arc 99.75% (1991 90% ( /1996 . the number of anti-hbc reactive samples did not differ between aly and arc. summary/conclusions: while the switch to the new system is mainly driven by operational efficiency, obviously, the high specificity of the alinity s assay will reduce unnecessary deferrals of donations and donors. abstract withdrawn. background: blood donor selection is the cornerstone for blood transfusion safety, designed to safeguard the health of both donors and recipients. donor safety is targeted by reducing the risk of complications associated with blood donation and transfusion safety by reducing the risk of transfusion-transmitted infections (tti) and other preventable transfusion reactions. there is always a compromise on blood donor safety as well as blood safety during outdoor mega blood donation drives due to various reasons, mainly due to more number of donations within a stipulated time. aims: to compare the blood donor selection patterns between in house blood donations and donations at mega blood donation drives and its influence on donor safety and blood safety in a tertiary care hospital in india. methods: a retro prospective study was done to audit and compare blood donor safety and blood safety over a period of 2 years from january 2016 to december 2017. blood donor safety was analyzed by two indicators: donor health questionnaire (dhq) monitoring and blood donor reaction rates and blood safety through tti positivity rates. (78) during mega blood donation drive. summary/conclusions: a good donor selection is a lengthy process which involves pre-donation information and advice: this is usually provided in a leaflet, especially about transfusion-transmitted infections (and the associated risk factors) and the potential risks of donation, filling of dhqs by the donor himself, donor interview: conducted by a qualified medical specialist trained in donor selection process and health assessment at the end of the interview to declare if the donor is eligible to give blood or deferred temporarily or permanently. it was observed that seroprevalence rates, number of donor reactions and incompletely filled dhqs were more among blood donations at mega blood donation drives when compared to blood donations during in house collections. this is mainly due huge number of blood donations with in a stipulated time where there is limited time spent on proper donor selection. stringent implementation of who strategy: "safe donor safe blood" is the only way for blood donor and transfusion safety. background: safety of blood transfusion is a great concern especially in crisis countries and during humanitarian emergencies. transfusion transmitted infections (ttis) are one of the major health problem in yemen that are associated with blood transfusion complications. aims: the aim of this study is to determine the prevalence of ttis among blood donors at national blood transfusion and researcher center (nbtrc this contributed to an additional reactivity of 0.09%, thereby total reactivity being 2.39%. 55% (22/40) of these were hcv reactive & 45% (18/40) for hbv. the nat yield was 1 in 1086 and the viral loads of nat reactives ranged from 1-9 x10 4 iu/ml for hcv & all the hbv yields had an extremely viral load of < 06 iu/ml. 12/40 nat reactive showed sero-conversion after 4-8 months with follow-up eclia screening, and 7 of these were hcv reactive and 4 hbv reactives. summary/conclusions: incidence rate indicate that the current risk of transfusion transmitted viral infections attributable to blood donation is relatively high in our country. parallel use of both serology and nat screening of donated blood in countries that have high seroprevalence can improve the blood safety. at our centre, by using best in class serology and nat technologies, we were would add an extra layer of safety to blood supply by interdicting samples from donor with recent infections. abstract withdrawn. abstract withdrawn. (1/190,298) . the both hiv-rna and hcv-rna detected donors by nat were identified in the window period. summary/conclusions: in this study, we found that nat could detect 283 infected cases with hbv-dna, hiv-rna and hcv-rna which were forgotten by serological methods therefore, nat is a sensitive screening method to detect low viral load and shorten the window period of the virus infection to ensure the safety of blood transfusions. service du sang, croix rouge de belgique, namur, belgium background: due to enhancement of kits specificity and machines throughput, roche elecsys â technology is a potential partner for blood donations screening laboratories. aims: the aim of the study was to assess the performance of the elecsys serology assays on a cobas e801 equipment for clinical specificity, analytical sensitivity and reproducibility. background: deceased donors are the primary source of organs and tissues for transplantation but the risk of infectious complications in the recipient is high and is the main cause of morbidity and mortality after transplantation. to minimize the risk of infections by organ or tissue transplantation, donors should be tested for anti-hiv-1/2, hbsag, anti-hbc, anti-hcv, and syphilis. further laboratory tests may be required depending on the history of the donor and on the tissue properties. certain grafts can be donated after circulatory death of the donor; however, the absence of the heartbeat may change dramatically the blood composition by e.g., haemolysis and proteolysis. this may have an impact on test performance and lead to false results. therefore, an assay validation is needed for testing of cadaveric samples. aims: a validation study was performed to demonstrate the suitability of elecsys hbsag ii, anti-hbc ii, anti-hcv ii, hiv combi pt, hiv duo, syphilis, htlv-i/ii, and chagas for the use in cadaveric samples from non-heart beating donors. methods: as the basis for validation, we followed the recommendations of the paul-ehrlich-institut (pei) "proposal for the validation of anti-hiv-1/2 or hiv ag/ ab combination assays, anti-hcv assays, hbsag and anti-hbc assays for use with cadaveric samples". comparison of spiked samples from living donors and cadaveric donors was used to demonstrate accuracy. to determine precision, two cadaveric specimens were tested in several replicates. acceptance criteria were implemented according to the pei recommendations. results: results were found to be within specifications requested by the pei recommendations for all tested assays summary/conclusions: the evaluated results support the extension of the use of these assays with cadaveric specimens. background: in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. aims: in this context, metagenomic analyses of viral content in blood donations collected in geographical zones recognized as "hotspot" for viral emergence represents a suitable approach without any a priori for the identification of a potential emerging viral risk that may compromise blood safety. methods: in the framework of a viral discovery program founded by the french national agency for medicine security (ansm in french), more than 900 plasma samples collected in 7 sub-saharan africa countries (2011) (2012) and the amazon region of brazil (2016) have already been analysed by metagenomics. results: although no viral sequence could be described as novel (i.e. new species or even a new genus), we unexpectedly identified a feline bocavirus in two donors from mauritania. a large diversity of known viruses that are not part of the regularly monitored agents were also observed, among which anelloviruses, hpgv-1 (formerly known as gbv-c), papillomaviruses, herpes viruses, parvovirus b19, chikungunya virus, enterovirus, and various small circular viruses (circo-, cycloand gemycircularviruses). while no significative differences was observed in the higher classification of detected virus (above families/genera) between africa and brazil, we observed variations at the sequence level allowing better resolution of the genetic diversity for several viruses (for example characterization of hpgv-1 genotypes). summary/conclusions: overall, the absence of novel viruses in blood samples collected across countries of two distant continents is reassuring regarding threats emergence. however, continuous monitoring of prospective blood banks should be continued. summary/conclusions: after the high peak observed in 2014 during the first period, this study shows that the decrease in the seroprevalence of viral markers is continuous over the next five years. the second period is marked by an irregular evolution of seroprevalence but with lower levels than the first period. the recruitment of new donors allows a quantitative increase in donations. however, improving the quality of blood products essential condition of transfusion safety is achieved through retention of recruited blood donors. background: in blood screening laboratories, samples may be transferred between automated serological and molecular instruments, and the potential for sample contamination is a serious risk to the integrity of nucleic acid testing (nat) results. the sensitive limit of detection (lod) for hiv and hcv nat assays combined with the high viral titers encountered in specimens from patients with acute infections presents a challenge for maintaining the sample integrity of negative specimens. at additional cost per test, this risk can be reduced with single-use filter pipette tips. aims: we evaluate the efficacy of applying induction heated washes to a non-disposable pipettor on serology instruments-alinity s, alinity i, and architect i2000sr (abbott diagnostics)-to preserve the integrity of samples transferred to a downstream molecular instrument, the m2000 realtime (abbott molecular diagnostics), which amplifies viral nucleic acid targets exponentially. methods: in this application of induction heating, the metallic pipettor warms under its own resistance to coil-induced electrical currents. by sweeping the pipettor through an induction coil, temperatures on the pipettor are elevated throughout its length. single donor high viral titer hiv genotypes a (5.78 log iu/ml), b (5.90 log iu/ml), c (5.62 log iu/ml), crf01 (5.61 log iu/ml), crf02 (6.19 log iu/ml), and urf (6.33 log iu/ml), as well as single donor high viral titer hcv genotypes 1a (6.84 log iu/ml), 1b (6.73 log iu/ml), 3a (6.64 log iu/ml), 2 (6.10 log iu/ml), 2q (6.65 log iu/ ml), and 4t (6.07 log iu/ml) were used as potential sources of contamination; these genotypes account for the majority of hiv and hcv infections worldwide. on serology instruments, one high viral titer hiv or hcv specimen and three consecutive susceptible negative samples (hiv/hcv rna negative human plasma, abbott molecular diagnostics) were tested on an hiv ag/ab combo or anti-hcv immunoassay (abbott diagnostics), and this schema was repeated four times per positive specimen. induction heated washes occurred between all samples processed on the serology instruments. the first susceptible negative from each testing block, with approximately 1 ml of residual sample volume, was then tested using the 0.6 ml abbott realtime hiv assay (lod 40 copies/ml) or 0.5 ml abbott realtime hcv assay (lod 12 iu/ml) and an hcv ag immunoassay (lod 1.24 fmol/l; abbott diagnostics). study acceptance criteria required that any susceptible negative sample had no detectable level of hiv or hcv rna. results: all first susceptible negative samples (n = 24 per platform per virus schema) run on alinity s, alinity i, and architect i2000sr using induction heated washes after a high viral titer hiv specimen or hcv specimen were hiv ag/ab combo nonreactive (< 0.20 s/co) and reported no detectable level of the hiv rna target, or were anti-hcv nonreactive (< 0.20 s/co) and reported no detectable level of the hcv rna or core antigen targets. summary/conclusions: while precautions should continue to be taken for samples run on molecular instruments, the integrity of samples originally tested on the alinity s, alinity i, and architect i2000sr was preserved for downstream molecular testing through the use of induction heated washes. aims: increasing the safety of blood and blood products -motivating the blood donors to be regular donors methods: national reporting system showed the high prevalence of ttis among first blood donors in compares with the regular donors. in 2015 per 2.083.914 donations, 54% % of confirmed positive hiv, 87% of hcv, and 93% of hbv cases has been reported among first blood donors. in the end of 2015 a national program named "pre-donation screening tests "has been developed and has been implemented in high prevalence provinces in whole country. based on this program, all first blood donors who accept in donation sites, if after donor selection process are eligible to donate blood, they refer to give just a blood sample for screening ttis tests. after 3 months, the invitation letters and smss send to the donors who have negative results for all screening ttis tests, and they can be eligible to donate blood after another donor selection process. in 2015, about 0.156% of all donations have been rejected because of at least one of hiv, hcv, or hbv confirmed positive results, while this reject rate in 2017 was 0.082%, which shows a significant decreasing the ttis prevalence among blood donation from 2015 to 2017. the prevalence of hiv, hcv, and hbv among donations has been decreased significantly in 2017 compared with the 2015. prevalence of hiv among donations reduce from 0.0032% in 2015 to 0.0024% in 2017, for hcv and hbv the same results have been experienced, respectively from 0.040% and 0.113% in 2015 reduce to 0.026% and 0.053% in 2017. it seems this applied study could effectively scale up the safety of national blood supplies. in addition this intervention could support iranian blood transfusion service to increase the proportion of regular blood donors from 80.19% in 2015 to 86.97% in 2017. it means that with increasing the regular blood donor population sizes, the safety of iranian blood and blood products will be more and more scaled up. summary/conclusions: evidence based reports show there is a high rate of prevalence of transfusion transmitted infections (ttis) among first blood donors. so an effective intervention which can reduce the risk of unsafe first blood donation can effectively increase the safety of blood and blood products. pre donation screening tests program in iran can support the national program to decrease the rate of ttis among blood donations from 0.0156% in 2015 to .082% in 20117. abstract withdrawn. abstract withdrawn. background: despite the universal application of viral inactivation and elimination technologies during the preparation of plasma-derived products, the exclusion of infectious donations before any other procedure remains the first essential step as well as the major determinant for the safety of untreated labile blood products. current selection and screening techniques have reduced the risk of viral transmission to very low levels, but there is still a very low but quantifiable risk of transmission through donations beyond routine detection, particularly during the " seroconversion window". "of an infection in a blood donor that is to say during the period when the recently infected donor has not yet developed a serological response. the level of residual risk, which must be as low as possible, is mainly conditioned by the rates of the infections concerned (hiv and hepatitis b virus (hbv) and c (hcv)) to blood donors. summary/conclusions: the evolution of serologic markers is generally satisfactory with continued regression, which has improved particularly for hiv. on the other hand, hepatitis b is still a concern because of its still high rate among new donors. it is desirable to initiate a regular donor vaccination program to protect against hepatitis b. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hcvab, havab igm and havab igg essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 61 samples were tested (19 for hcvab, 24 for havab igm and 18 for havab igg) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hcvab ranged from 0 to 4.474%. 5 samples were tested for havab igm in a total of 55 essays and the %cv ranged from 0 to 11.475%. havab igg was tested in 3 samples during 33 essays and the %cv ranged from 0 to 4.861%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hcvab, havab igm and havab igg. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hbsag, hbsab, hbcab, hbeag and hbeab essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 85 samples were tested (10 for hbsag, 20 for hbsab, 18 for hbcab, 14 for hbeag and 23 for hbeab) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 30 essays we found the %cv hbsag ranged from 0-4.375%. 3 samples were tested for hbsab in a total of 33 essays and the % cv ranged from 0-5.196%. hbcab was tested in 3 samples during 32 essays and the %cv ranged from 0-2.811%. using 3 samples and a total of 28 essays we found the %cv hbeag ranged from 4.176-5.147%. 4 samples were tested for hbeab in a total of 29 essays and the %cv ranged from 0-4.444%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hbsag, hbsab, hbcab, hbeag and hbeab. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hivag/ab, syphilis and htlv i/ii essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 38 samples were tested (12 for hivag/ab, 14 for syphilis and 12 for htlv i/ii) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hivag/ab ranged from 0 to 3.05%. 2 samples were tested for syphilis in a total of 22 essays and the %cv ranged from 0 to 1.481%. htlv i/ii was tested in 3 samples during 28 essays and the %cv ranged from 3.342 to 7.5%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hivag/ab, syphilis and htlv i/ii. , human immunodeficiency (hiv) and hepatitis c (hcv) viruses' infection in blood donors were 1.27%, 0.20% and 0.50% respectively. consecutive positive results for hbv were 9.20% (63/685), for hcv were 6.0% (16/266) and nil for hiv. there was no sample carry over in this. out of 79 consecutive reactive donors 69 were donated for same patients and 32 were related with infected patient which were statistically significant (p < 0.0001). summary/conclusions: among all tti reactive donors 7.4% (79/1061) were consecutive reactive. the reason for the same may be process related like sample carry over or reagent carry over or donor related. donor related reasons may be, one of the close relative is reactive for virus and that is transmitted to other family members. in our study 32 reactive donors either had close contacts with persons with history of infective disease or were their first degree family relatives. these findings were found statistically significant (p < 0.0001). this study recommends that in analysis of consecutive positive results in elisa along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patient. background: screening for transfusion-transmitted infections (ttis) is critical in ensuring safety of blood products. transmission of infections through transfusion remains a major source of viral hepatitis especially hbv and hcv. the effectiveness of rapid immunochromatographic test (ict) devices for screening of blood is a concern and needs validation through advanced methods like chemiluminescence immunoassay (clia) and polymerase chain reaction (pcr). aims: the current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with clia and pcr. methods: this single centre, cross sectional study was conducted at the department of blood transfusion services, shaheed zulfiqar ali bhutto medical university, islamabad, from january -june 2018. a total of ten commercially available ict devices and one clia kit (liaisonr xl) were tested for their sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy using 100 positive and 100 negative samples each for hbv and hcv respectively, in comparison with the values determined by pcr. the ict kits included hightop, rightsign, wondfo, accu-chek, fastep, abon, immumed, insta-answer, biocheck and ctk. results: the sensitivities and specificities of ict kits for hbsag were 65% and 70% (hightop), 67% and 85% (rightsign), 62% and 73% (wondfo), 70% and 80% (accu-chek), 68% and 77% (fastep), 73% and 85% (abon), 77% and 83% (immumed), 80% and 90% (insta-answer), 67% and 81% (biocheck) and 72% and 83% (ctk) respectively. similarly the sensitivities and specificities of different ict kits for hcv were 69% and 80% (hightop), 76% and 83% (rightsign), 69% and 81% (wondfo), 78% and 79% (accu check), 68% and 68% (fastep), 63% and 73% (abon), 71% and 70% (immu-med), 79% and 68% (insta answer), 62% and 66% (biochek) and 69% and 78% (ctk) respectively. the sensitivity and specificity of diasorin liaison murex assay for both hbv and hcv were found to be 100%, when compared with pcr. the ppv, npv and accuracy were determined accordingly. summary/conclusions: rapid testing ict devices for both hbv and hcv available in pakistan were found to have a variable degree of sensitivity and specificity, when compared with pcr. comparatively expensive but quality methods are more reliable as compared to rapid devices. the data generated will help policy makers to prepare future plan of action and introduce the concept of quality control in blood centres. the analysis has shown that the population of blood donors also included people infected with syphilis. in reference to the number of the tested samples this number is quite significant. the analysis proved the increase in the number of syphilis infections among the blood donors which is consistent with the general trend in the population. summary/conclusions: we have proved that testing blood donors for the treponema pallidum infection increases the safety of recipients of blood and its components and that obligatory testing of donors is fully justified. , 1940 and 1945 , the use of third or fourth generation serological assays is mandatory for screening of blood donor units for hbv and hcv infection before transfusion. routinely, blood banks in india screen the units by the elisa testing. nat is not very common due to cost constraints. aims: the aim of this study is to determine the frequency and load of hbv dna and hcv rna in hbs and hcv reactive blood donors respectively, and hence it was intended to contribute to determining whether routine hbs and hcv screening of blood donors, using elisa method alone, provides any concrete benefits with regard to hbv and hcv risk reduction or whether the implementation of nat will be of great benefit to low-resource countries like india, which has high prevalence of hbv and hcv. abstract withdrawn. ,906 donors were routinely tested for hbv dna by using cobastaqscreen mpx-1 and mpx-2 (roche) or procleix ultrio and ultrio plus id (grifols) assays. obi was confirmed by repeat dna testing and by performing additional serological and molecular investigations on index and follow-up samples. anti-hbs concentrations were determined and anti-hbc antibodies were tested with three distinct commercial clia assays (anti-hbc elecsys roche, architect anti-hbc ii abbott, and hiscl anti-hbc assay sysmex). hbv pre-s/s, precore/core and bcp regions were pcramplified after viral particle concentration and viral amplicons were sequenced. results: 348 hbsag-/dna+ donors (1:1,462) including 294 confirmed obi were identified (1:1,731 prevalence). among obi donors, 160 (54.5%) tested anti-hbc+/anti-hbs-, 108 (36.7%) were anti-hbc+/anti-hbs+, 25 (8.5%) were anti-hbc-/anti-hbs+, and 1 anti-hbc-/anti-hbs-primary obi (0.3%). anti-hbc-/anti-hbs+ obi donors were significantly younger (mean: 21 years [range: 18-57 years]) than those with anti-hbc+/anti-hbs+ (mean: 44 years [range: 19-60 years]) and anti-hbc+/anti-hbs-(median: 45 years [range: 21-55 years]) profiles (p < 0.0001). hbv vaccination was documented for 18 (72%) of these donors and was reported in one donor but without definitive evidence. extremely low hbv dna loads (range: <10-155 iu/ml) were transiently detected in seven donors during follow up. genotypes identified were genotype b (n = 1/20) and genotype c (n = 19/20). preliminary analysis of core protein (n = 16) and bcp (n = 10) sequences showed no particular genetic feature that could be associated with altered antigenicity or core gene expression. follow-up was available for 15/ 25 anti-hbc-/anti-hbs+ donors (2-6 samples/donor; range: 2-56 months). anti-hbc remained undetectable with all clia assays in these donors except one. low transiently detectable levels of hbv dna were observed overtime with anti-hbs levels fluctuating between 12 and 1,000 iu/l. no significant difference in hla-a, -b (except hla-b*46 more frequently detected in anti-hbc negative obi), and -drb1*. summary/conclusions: overall, the 8.5% prevalence of anti-hbs-only in hbv dna positive obi carriers (1:20,355 of total donors) in dalian blood donors confirmed previous reports from south east asia. this phenomenon was not related to core antigenic variations but was significantly associated with younger age of carriers. a particular route and natural history of the infection may be considered. the hypothesis of acute-phase vaccine breakthrough was ruled out in 15/25 donors by the over 50 months stability of this serological profile. breakthrough in immunized donors may still be suspected. further studies are needed to evaluate the potential infectivity of anti-hbs-only/hbv dna+ obi carriers, and to characterize the potential viral and immunological mechanisms responsible for this unusual hbv infection profile. confirmatory laboratory, hungarian national blood transfusion service (hnbts), budapest, hungary background: vaccination against hepatitis b virus (hbv) is an effective tool to avoid the infection. in hungary, population born after 1986 is considered to be immunized, because inoculation has been mandatory for children as campaign vaccination since 2000. hbv vaccine is strongly recommended for healthcare workers, moreover trips to endemic countries and awareness of individuals could also be reasons of vaccination in immunologically na€ ıve age-groups. since the hbv vaccine contains surface antigen, a recent inoculation can cause reactivity of hbsag screening assays and positivity of confirmatory tests for several days resulting in deferral of donors from blood donation. the former regulation of hnbts, which was valid until december 31, 2018, allowed the re-entry of donors whose immunization records and negative hbv confirmation of the second blood samples proved that the previous vaccination had resulted in the hbsag confirmed positivity. aims: the aim of this study was to strengthen that vaccination against hbv before blood donation had resulted in the reactivity of hbsag screening and confirmatory assays between 2012 and 2018. background: in brazil, the introduction of nucleic acid tests (nat) for hbv-dna detection in the routine screening at public blood banks is relatively recent. at fundac ßão pr o-sangue/hemocentro de são paulo (fps-sp), about 130,000 blood donors are submitted to serological screening tests (hbv, hcv, hiv-1/2, chagas disease, syphilis and htlv-1/2) and nat for hiv, hcv and hbv per year. approximately 2% of the blood donations are discarded due to some reactive result; of these, the hbv discard rate was 0.65% in 2016. aims: our study aims to determine the potential infectious cases among samples that had one or more hbv-reactive screening results (anti-hbc, hbsag and mp-nat-hbv) and verify the different categories of hbv infection (acute, chronic, occult hepatitis b infection (obi) and immunological window). furthermore, to characterize the distribution of hbv genotypes, drug resistance and escape mutations and analyze the risk factors. methods: we carried out a cross-sectional study of roughly 74,000 donations from may 2016 to december 2016. hbv antibodies and antigen screening were performed using cmia kits architect â -abbott/hbsag and architect â -abbott/anti-hbc. nat screening was performed in minipools (mp) of six samples using kit nat hiv/hcv/ hbv -bio-manguinhos (sensitivity 95% lod 300 iu/ml for hbv). reagent samples (n = 407) that presented one or more hbv-reactive screening results (anti-hbc, hbsag and/or mp-nat-hbv), were submitted to individual nucleic acid extraction and "in house" quantitative real-time pcr-hbv (id-pcr-hbv) targeting the hbsag region (sensitivity 10-20 ui/ml). the hbv genotypes and mutation analyses were determined by direct sequencing of the hbv pol-gene/surface-gene and use of the online analysis tool geno2pheno [hbv] 2.0. socio-demographic and epidemiological data were also analyzed. financial support: fapesp 2017/16658-6. results: among the 407 hbv reactive samples, 377 were reactive for anti-hbc only (92.6%), 10 for hbsag only (2.5%) and 20 were reactive for both markers and hbv dna (4.9%). routine testing and id-pcr-hbv identified 20 (0.027%) samples of active infections that had all hbv reactive/positive tests results. no hbv dnayield samples or hbsagyield or obi were observed. viral loads for active infections samples ranged from 1.08e+01 to 2.45e+09 cop/ml (median, 2.12e+08cop/ml). hbv sub-genotypes a1, a2, c1, d3 and f1 were found in 70%, 5%, 5%, 15%, and 5% of the donors, respectively. no reverse transcriptase inhibitor-resistant variants were detected. escape mutations in small hbsag protein shb region were detected in 30% (6/20), with the following main substitutions 100c (3x), 120r, 123n and 144g. the mean age of donors with active hbv infection was 40 years, mostly donors were males (80%), mixed (40%) or white (40%) and had concluded high school (45%). summary/conclusions: discard rate due to isolated anti-hbc is high but no obi was found in the blood donor population studied. in addition, no case of immunological window for hbv or hbsagyield was detected. there was a predominance of subgenotype a1 and mutations associated with escape were found in 30% of hbv-dnapositive samples. continuous research and surveillance about hbv prevalence among blood donors are needed to maintain and evenly increase blood safety in brazil. background: screening for anti-hbcore antibodies in blood donors is considered to contribute significantly to blood safety, since it reveals donors with occult or probable occult hepatitis b, with variable results in molecular screening, due to very low viral load. however, universal anti-hbcore testing in blood donors, might exclude a considerable number of blood donors in countries with high hbv prevalence and even in countries with low to moderate prevalence, like greece. aims: the aim was to investigate the percentage of blood donors with natural hbv infection (confirmed positive anti-hbcore) or hbv immunization due to vaccination (anti-hbs+ only, due to vaccination) and predict the impact of generalized anti-hbcore testing. methods: during the period 15-30 november 2018, all blood donors were asked to give their consensus for additional screening for hepatitis b anti-hbcore and anti-hbs antibodies, besides the obligatory serological and molecular screening, the samples of few donors who disagreed, were not examined. all samples with repeated positive anti-hbcore results, were further examined for anti-hbcore igm and anti-hbe antibodies. furthermore, a new donor sample was requested, to confirm reactivity. the serology results were recorded in an excel spreadsheet. additional data, including age, sex, nationality, number of previous blood donations, abo blood group, family history of hbv infection, hbv vaccination, were also recorded and statistically evaluated. donors were informed of the positive results. results: a total of 620 edta samples were tested using the architect anti-hbcore, anti-hbs, nti-hbcore-m and anti-hbe assays (chemiluminescent microparticle immunoassay (cmia). repeated anti-hbcore(+) occurred in 24 (3.87%) samples, among which 7 (19%) were also anti-hbe(+), while anti-hbs was found > 200 m iu/ml in 15 (62.5%), between 100 and 200 miu/ml in 3 (12.5%), and < 100 miu/ml in 6 (25%). among anti-hbcore positive donors, 4/24 were foreigners (16.6%) and 20 were greeks, while foreigners consisted 6,29% (39/620) of donors examined. so, anti-hbcore was found positive in 10,25% of foreigners (4/39, all from countries with high prevalence for hepatitis b infection) and in 3,44% of greeks (20/581). in total, 285 (45.96%) samples had anti-hbs > 10 miu/ml (considered seroprotective for the donor). summary/conclusions: almost half of our blood donors (45.96%) were immunized, 261 by vaccination and 24 (3.87%) by natural infection. the incidence of natural infection was significantly higher in foreigners (10.25% versus 3,44%). if not all anti-hbcore+ donors, 25% with anti-hbs < 100 iu/ml, might be potentially infectious, especially for immunocompromised patients. if we choose to screen all blood donors for anti-hbcore and reject those with positive results, regardless of the anti-hbs levels, we would probably lose a significant number of donors and jeopardize blood sufficiency. alternatively, we could reject only those with anti-hbs < 100 or < 200, or choose to selectively screen pre-donation blood donors from countries with high prevalence of hbv infection. following this pilot study, the prevalence of immunization against hbv in large numbers of blood donors from various parts of greece, must be investigated, in order to decide whether to introduce such screening. aims: the aim of this study was to perform phylogenetic analysis of the donor samples with hcv found in the neighbouring villages to determine the nature of transmission. methods: altogether, approximately 2 million blood donor samples were screened with anti-hcv immunoassay (architect anti-hcv, abbott gmbh, wiesbaden, germany) and reactive results were confirmed with anti-hcv line-immunoassays (inno-lia hcv score, fujirebio europe, gent, belgium). based on lia positivity, in 20 samples an association of hcv infection was supposed, because the residence places of donors were in three neighbouring villages situated less than 20 km to each other. pcr was positive in 15 samples. from these samples, hcv sequencing and phylogenetic analysis were performed. fourteen hcv infected samples of general population and 11 of ivd users were also included into the study. results: phylogenetic analysis detected genetic relationship among the hcv virus sequences in donor samples. the most abundant was the 1a subtype, and it formed two different groups on the phylogenetic tree. according to their genetic distance, a more distant mutual ancestor could be supposed. two samples with 1b subtype originated from the same village, and their difference was only 2 nucleotides. three hcv from the ivd user control group showed close genetic relationship with the viruses detected in the donor samples. summary/conclusions: based on our phylogenetic analysis, hcv transmission in blood donors could be the consequence of the ivd use and the origin might be related to 2 or 3 primary human sources. during 2011 and 2014, a significant increase in the hcv seroprevalence among the ivd users was observed, which was approximately threefold in the rural areas of hungary. our recent findings highlight the importance of the proper donor selection, which can identify the typical signs of the ivd use. moreover, enhancing awareness of blood donors with education is a further significant issue in order to reduce the risk of transfusion transmitted infections. abstract withdrawn. background: in china, the residual risk of transfusion-transmitted hcv has been declining since screening of blood donors for anti-hcv and/or hcv nat from 1993. however, many high sensitivity reagent, using to test blood donors' samples, lead to false-positive results and donors loss. aims: this study intended to establish a donor reentry procedure for hcv screening reactive donors in china. methods: from march 2014 to december 2017, there were 2083 blood donor samples which were screened reactive or belonged to "grey zone" by elisa and/or reactive by nucleic acid test(nat) at the local blood centers were collected from 15 chinese blood centers. all these samples were sent to institute of blood transfusion (ibt) national reference laboratory where anti-hcv and hcv individual nucleic acid test (id-nat) were conducted. if the results were reactive for anti-hcv, then the samples were tested with a recombinant immunoblot assay (riba). results: based on this study, 1178 of 2083 donors in the study who could be classified into two categories for hcv status: 201(17.1%) true positive and 974 (82.9%) false positive. a total of 905 of 2083 donors lost to follow-up, their hcv status cannot be determined with certainty. based on these data, a reentry procedure for hcv screening reactive donors was proposed. summary/conclusions: based on our proposed donor reentry procedure for hcv screening reactive donors, a majority of screening false-positive donors (82.9%) can re-entry safely. abstract withdrawn. background: providing safe blood for transfusion in sub-saharan africa (ssa) is a particular challenge due to a combination of factors; limited resources and infrastructure, suboptimal diagnostics and a high prevalence of the major transfusion-transmissible infections (ttis). average seroprevalence data estimates from the ugandan and kenyan blood transfusion services (bts) for hepatitis c (hcv) currently stand at 6.6% and 0.9% respectively. between january and december 2016, in mbale (eastern uganda) the hcv prevalence amongst blood donors was an alarming 8.1%. with no provision or funds for confirmatory testing, the bts are unable to confirm or refute a diagnosis of active hcv. this results in large quantities of blood wastage, unnecessary anxiety in potential donors and high donor deferral rates limiting the donor pool. aims: we aim to determine the true prevalence of active hcv infection amongst seropositive donors in bts in uganda and kenya. in addition, we aim to compare the performance of locally used serodiagnostics and best available alternative tests and to examine the feasibility of cost-effective additional or alternative tests to help provide accurate results on the infectious status of blood. methods: 235 hcv seropositive blood samples from 3 bts study sites (kampala, mbale, mombasa) will be re-tested using the local serology screening test (abbott architect anti-hcv), an alternative who pre-qualified rapid antibody test (sd bioline) and a confirmatory test (hcv core antigen test). where there is discrepancy in the results or need for clarification, samples will be tested on the cepheid xpert platform by reverse-transcriptase pcr to obtain a quantitative rna result. s/co (specimen to cut-off) values for false positive samples (by screening serology) will be analysed and presented. pre-analytical factors (centrifugation speed, haemolysis check, time delay between collection and testing) will be controlled for and documented. results: pilot data from re-testing quarantined hcv seropositive donor blood (mbale bts) in uganda demonstrated that 45/50 seropositive blood (90%) was rna pcr negative. in december 2018, 156/249 (63%) of seropositive samples (by screening anti-hcv serology) in kampala bts had s/co values between 1.00-1.99 (1.00 is the cut-off indicating a positive sample). data from the re-testing of 235 seropositive samples as true representation of active hcv will be demonstrated and s/co values for the study period concomitantly with a retrospective analysis of january to december 2018. preanalytical factors, cost analysis comparisons of the diagnostic platforms coupled with costs of the donor deferral process in false positive cases will be presented. summary/conclusions: for the bts in ssa there are significant resource and financial implications, as repeat testing and donor deferral counselling is required. evaluating and introducing new and appropriate diagnostics and algorithms in the screening of hcv is crucial in improving the supply of safe blood transfusion services in east africa. background: in november 2017, the blood services of england, scotland and wales reduced donor deferral to three-months for commercial sex workers and individuals with higher risk sexual partners, including sex between men. the change was recommended after a detailed review by an external expert committee (sabto) which recommended that a shortened deferral of 3 months would allow detection of recently acquired infection and maintain residual risk (rr) at a tolerable level. recommendations were accepted by government but with a government commitment to explore a more individualised approach. aims: to assess the impact of a 3-month deferral on blood safety in terms of epidemiology of infections in blood donors and compliance with donor selection criteria, and to explore evidence required to develop a more individualised approach to donor selection policy methods: routine uk blood donation surveillance data for 2010-2018 (2018: preliminary) were reviewed. annual prevalence and incidence of hbv and hiv infection were estimated, with a poisson regression models to test for trends. incidence was calculated from donors seroconverting within 12-months, and/or microbiological and clinical evidence of recent infection. for donors positive in 2018, compliance with the 3-month deferral was determined. uk hemovigilance data were scrutinised for evidence of transfusion transmitted infections (tti) associated with newly eligible donors. results: from 2010 to 2018 among new donors, annual hiv prevalence decreased significantly by an average of 11.3% each year (p = 0.02) to 1.49/100,000 donations in 2018; no significant trend was observed for hbv. annual hiv incidence among repeat donors also decreased significantly by an average of 17.3% each year (p = 0.03) to 0.23/100,000-person years (pyrs) in 2018 (based on 2 seroconverters). there was no significant trend in hbv incidence over the study period, however between 2017 and 2018 incidence increased from 0.35/100,000 pyrs to 0.84/ 100,000/pyrs (based on 3 and 7 seroconverters respectively). with the information available to date, none of the 2018 seroconverting donors were non-compliant, and there was no reported confirmed ttis associated with the policy change. summary/conclusions: hiv prevalence and incidence has continued to decline. hbv incidence in repeat donors increased in 2018 although initial analysis suggests this is not associated with the policy change. monitoring continues, and residual risks will be re-estimated as data post-change accumulate. these data are reassuring, and therefore it is appropriate to scope the evidence for, and feasibility of, a more individualised approach to selection policy. a multidisciplinary steering group has been convened including representation from patient and stakeholder groups. gaps in knowledge are being defined, and a package of work is in development under the project of fair (for the assessment of individualised risk), using the abo rdf for guidance. background: permanent deferral of men who have sex with men (msm), established in the 1980s, primarily to minimise the risk of hiv transfusion-transmitted infections is increasingly challenged. accordingly, blood services in many countries have changed to time-based deferral. in canada, a 5-year deferral was implemented in 2013, reduced to 12-months in 2016; a 3-month deferral is now being considered. aims: to estimate the risk of undetected hiv among screened blood donations under a 3-month deferral since last sex between men. methods: the applied model combines features of previously published english and canadian models to estimate hiv risk under a 12-month deferral. three scenarios varying hiv incidence, prevalence and non-compliance under a 3-month deferral were modelled. assumed constants were the hiv nucleic acid window period, testing procedure error rate and assay sensitivity. model inputs were incidence under the current 12-month deferral, calculated as hiv positive donors with a previous negative within 12 months divided by number of person years, numbers of hiv positive donations, hiv positive msm, hiv msm incident cases and newly eligible msm donors (from donor surveillance and compliance surveys). the risk with a 3-month deferral was estimated for three scenarios, one determined "most likely", where msm donor non-compliance, hiv incidence and hiv positive donations do not change and msm newly eligible to donate are estimated from compliance surveys. this scenario is based on data from two sequential policy changes in canada. an "optimistic" scenario where non-compliance halves and a "pessimistic" scenario where msm hiv incidence, hiv positive donations, non-compliance and new msm donors double were also used. the median hiv residual risk was used as the final estimate. the uncertainty in this estimate was assessed with the 2.5th and 97.5th percentiles over the simulation (95% ci). results: incidence, per 100,000 donations, was estimated to be 0.15, 0.13 and 0.27 for the "most likely" "optimistic" and "pessimistic" scenarios, respectively. for the 3month deferral "most likely" scenario, hiv residual risk was predicted to be 1 in 32.6 million donations (95% ci: 1 in 217,692 million to 1 in 8.3 million). for the "optimistic" scenario, hiv residual risk was estimated to be 1 in 34.3 million donations (95% ci: 1 in 257,692 million to 1 in 9.1 million). finally, for the "pessimistic" scenario, hiv residual risk was estimated to be 1 in 16.0 million donations (95% ci: 1 in 34,091 million to 1 in 5.7 million). with these residual risk estimates, based on the number of donations in canada, one hiv infectious donation would be in inventory every 30 years for the "most likely" scenario, every 32 years for the "optimistic" scenario and every 15 years for the "pessimistic" scenario. summary/conclusions: the risks of hiv entering the blood supply in canada for a 3-month msm deferral are predicted to be very low for all modelled scenarios, including a "pessimistic" doubling of hiv incidence post change. background: safety of blood and blood products is a major concern in pakistan. the prevalence of transfusion transmitted infections among multi-transfused thalassaemia patients is high (above 25%). the hiv epidemic in pakistan is following the asian epidemic model where after establishment among the high risk groups, its transmission to general public is rapid. fear, stigma and ignorance have contributed heavily to hiv transmission in pakistan. the hiv detection among blood donors is on the rise and reports occur in media repeatedly. aims: to investigate the possible transmission of hiv through blood transfusion in punjab, pakistan and to highlight the steps being taken to reduce further transmission of infections methods: in september 2018, a report of hiv transmission through blood transfusion was reported in the media where a mother and her newborn acquired hiv after blood transfusion from a hiv positive donor (confirmed later). the case was referred to and investigated by the punjab blood transfusion authority (pbta). the pbta team took blood samples of both recipients (mother and her newborn) and the blood donor who was a family relative. the samples were tested by highly sensitive chemiluminescence immunoassay (clia). the clia results confirmed the presence of hiv in both recipients and the blood donor. due to maternal hiv antibodies transfer through the placenta, the infection status of the newborn was not re-confirmed as he died within two weeks. the donor informed that he had donated 22 times in the past few years. the pbta was able to trace only one earlier donation three months ago. the recipient (a female) was found, tested by clia and was found to be hiv positive. all these cases occurred in unlicensed private blood banks that were screening for hiv on rapid manual devices. the blood banks were sealed by the authority and infected cases were registered by the provincial aids control programme and are being treated. summary/conclusions: the main reasons for hiv spread through blood transfusion is the use of sub-standard rapid screening devices which are not evaluated and validated at a national level. in addition, the existing system relies on the family/replacement donors. the national safe blood transfusion programme, is implementing blood safety system reforms as recommended by who. under the reform agenda, the blood transfusion authorities have been made functional and grant licenses to only those blood banks with proper systems to ensure quality and safety of blood products. the programme is developing a national system for the evaluation, selection and validation of all assays used for screening of blood in close coordination with the drug regulatory authority of pakistan. to promote the culture of voluntary blood donations, the programme has taken concrete steps initiating with the formulation of a national blood donor policy, interaction with celebrities, celebration of world blood donor day and more recently the launch of blood donation feature through 'facebook'. the promotion of voluntary blood donation concept along with regulation of blood sector will reduce the risk of hiv transmission through blood transfusions in pakistan. mianyang blood center, mianyang 9 urumqi blood center, urumqi, china 10 rti international, rockville 11 national heart, lung, and blood institute, bethesda 12 stanford university, stanford, united states background: the incidence of hiv infections has increased substantially over the past decade in china, especially among young people, who represent nearly half of the chinese blood donor population. this upward trend in hiv infections underscores the importance of monitoring hiv prevalence and incidence in chinese blood donors. aims: to estimate hiv prevalence and incidence rate (ir) among chinese blood donors using blood donation data from five geographically-disperse blood centers in 2013-2016 participating in the recipient epidemiology and donor evaluation study-iii (reds-iii) china program. methods: western blot confirmatory testing was done on samples of blood donations reactive for hiv-1/2 on one or both rounds of routine elisa tests or positive by nucleic acid amplification testing (nat). multiple imputation was used for samples with missing confirmatory test results. hiv prevalence was calculated among first-time donors. to estimate hiv ir in first-time donors, single-well lag-avidity eia testing was conducted with first-time hiv recent (incident) infections defined as being infected within approximately 129 days based on avidity of hiv antibodies. a novel model was derived to estimate hiv ir among infrequent repeat donors who had provided only one donation in the 2013-2016 estimation interval. to derive an overall hiv ir for repeat donors, this estimate was combined with the classical-model ir estimated for repeat donors who had given at least 2 donations in the estimation interval. multivariable logistic regression model was used to examine factors associated with hiv infection. results: a total of 1,276,544 whole blood and apheresis platelet donations with postdonation screening results were collected at the five blood centers between 2013 and 2016, including 648,607 donations from first-time donors and 627, 937 donations from repeat donors. hiv prevalence among first-time donors was 68.04 per 100,000 donors (95% ci, 61.68-74.40). hiv ir was estimated to be 37.93 per 100,000 person-years (95% ci, 30.62-46.97) among first-time donors and 20.55 per 100,000 person-years (95% ci, 16.95-24.91) among repeat donors. hiv prevalence and ir varied across regions with an increasing trend observed at some blood centers. among first-time donors, being male, older than 25 years, minority ethnicity, less than college education, and certain occupations (commercial services, factory workers, retired, unemployed, or self-employed) were associated with positive hiv confirmatory testing results. summary/conclusions: although hiv prevalence and incidence remain low among chinese blood donors, it is important to monitor hiv epidemiology in blood donors on a continuous basis, especially among populations and regions of higher risk. further donor screening and education strategies need to be developed and evaluated to reduce these risks. the ir methods used in this study for first time donors as well as repeat donors who donate very infrequently is readily applicable to other countries who have similar donation patterns. background: in thailand, the national blood centre is responsible for blood donation service which includes follow-up and blood donor counseling in order to indicate the infection status, especially hiv-positive blood donors. currently, although the epidemic of hiv infection in thailand is in decline, the hiv-positive cases still have been found in blood donors screening. thus, monitoring of hiv infection status in blood donors and post-blood donor counseling are important for providing the hiv-positive infected donors lead to access the hiv treatment immediately. aims: to study the hiv follow-up cases on serological testing over 10 years for assessment of the hiv infection in thai blood donors. the retrospective analysis of hiv follow-up cases on serological testing (cmia, ics and western blot) was conducted during 2009 to 2018 at thai national blood centre. results: a total of 6,408,024 voluntary blood donations over 10 years, the repeated reactive results on hiv serological screening were 5,978 (0.093%) cases and only half of these hiv reactive donors returned to follow-up testing for ascertaining their hiv status. for hiv follow-up process, the hiv reactive screening donors must be followed for 6 months and tested by using the different three principles of hiv serological testing. a total of 5,978 hiv reactive results were separated to three patterns including hiv positive results, inconclusive results and negative results which the number of each group was 1,130 (35.7%) cases, 742 (23.5%) cases and 1,292 (40.8%) cases respectively. out of 1,130 hiv positive results, we found that 1,105 (97.8%) cases were positive with all hiv serological testing for the first-time follow-up and 25 (2.2%) cases were tested and become to positive results after follow-up more than one time. in 742 cases of inconclusive results, 539 (72.6%) cases were reactive only 1 or 2 testing(s) which these donors did not return to confirm again leading to temporarily deferred donors in blood donor system. in addition, 203 (27.4%) cases of inconclusive results could not conclude the hiv result although they were repeated several times. for the last pattern, 1,292 negative results cases showed 762 (59.0%) cases were negative results after follow-up over 6 months while 530 (41.0%) cases were inconclusive results before changing to negative results which almost cases of this group were reentry as blood donor after deferral period is over. summary/conclusions: the number of repeated reactive results on hiv screening was constant over 10 years of which returned to follow-up only half of hiv reactive donors leading to accumulation of temporarily deferred donors in blood donation system. hiv follow-up positive cases were informed and counseled immediately then referring to anonymous clinic for treatment. the problem and challenges of hiv follow-up were inconclusive results that were unclear and some of these did not return to retest lead to loss of re-entry donor who might be changed to negative result afterward. hence, the effective counseling and follow-up system need to be taken urgently to encourage the temporarily deferral donors returned to retest for reducing stigma of deferred donors in hiv follow-up cases. . we only analyzed the information that had non-reactive results for infectious markers reported by blood banks to sihevi-ins©, because they represent a risk for blood recipients. results: when loading the information of sivigila in sihevi-ins©, 241 donors were found (80% men); of these people 256 donations were obtained (97% whole blood). 166 donors (69%) had a reactive result for hiv being subsequently reported in sivigila. in addition, five of them were reactive simultaneously for hbv in blood banks and took on average 174 ae 83 days to be reported in sivigila. 25 donors (10.4%) had an hiv reactive result notified by sivigila and subsequently they were reactive in blood banks. this behavior may suggest an attempt to spread the disease. 20 donors (60% men) despite being initially reported in the sivigila database, presented a non-reactive result in a blood bank for hiv; one of them was reactive for syphilis and hbv and only one for hbv. this pattern may suggest false positive or negative results in one of the two databases analyzed. fourteen donors had negative test in blood banks for hiv and in a range of up to 6 months they were reactive by sivigila (93% of them donate whole blood). this conduct may suggest that accepted donations were in a window period and therefore warrant further investigation. considering that two blood components could be obtained on average from each donor, a potential risk is estimated for 28 recipients. summary/conclusions: the donors reported first in the blood banks through sihevi-ins© and later in sivigila allow to estimate an adequate orientation to the health services. the information from general epidemiological surveillance programs could improve the selection of donors and transfusion safety. background: it is assumed that bacterial contamination of blood products most often takes place during the donation process. the number of bacteria at this time point is estimated to be around 10-100 cfu per bag. little is known about the growth behavior of different bacteria species in whole blood (wb) units during storage and the distribution of bacteria to the different blood products. aims: aim of the current study was to determine the growth of different bacteria species in contaminated wb units and to study the distribution of the bacteria to the different blood components. methods: whole blood (n = 4-12 per species) was inoculated with approximately 10 cfu of different bacteria species (escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, staphylococcus aureus, staphylococcus epidermidis, streptococcus dysgalactiae, streptococcus pyogenes) and stored for 22 to 24 h at room temperature before centrifugation and separation into red blood cells (rbc), buffy coat (bc) and plasma. bcs from spiked wb were each pooled with 3 random bcs to prepare plasmareduced platelet concentrates (pc). samples were taken from wb after storage and from the blood products (rbc, bc, plasma and pc) right after preparation, and the bacterial titer was determined. sterility of pcs was tested by bact/alert after seven days of storage. results: bacterial growth in wb varied remarkably between donations and bacteria species. the highest titers in wb were detected for the streptococcus species, whereas staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas fluorescens did not multiply. bacteria preferably accumulated in the bcs during separation, reaching titers of up to 3.5 9 10 3 cfu/ml in bcs and up to 0.9 9 10 3 cfu/ml in the corresponding pcs right after preparation. in total, 33 out of 48 pcs tested positive for bacteria at the end of storage. the results were dependent on the species used: e.g., 6/6 pcs tested positive after spiking with streptococcus pyogenes, while only 1/8 pcs tested positive after spiking with escherichia coli. bacterial contamination of rbc and plasma units was much less frequent and associated with higher bacterial titers in the parental wb units. summary/conclusions: the growth and distribution of bacteria during processing of wb into the different blood products is species-dependent and remarkably varies between donations. results: both patients were male (69 yo and 65 yo) with a history of acute myelogenous leukemia status-post haploidentical stem cell transplant. the patients were thrombocytopenic and underwent simultaneous transfusion of irradiated, non-pr, day 4 platelets stored in platelet additive solution, from a single apheresis collection. the blood supplier's primary pre-release bacterial cultures were negative, and the on-site point of release secondary safety measure pan genera detection (pgd) testing was negative for both gram positive (gp) and gram negative (gn) organisms. both apheresis units also passed visual inspection prior to release from the blood bank. during transfusion, both patients displayed signs of septic transfusion reaction including rigors, fever, hypoxemia, tachypnea, tachycardia, and hypotension. transfusion reaction evaluations were initiated, and both patients were admitted to the medical intensive care unit and started on broad-spectrum antibiotics. gram stain of one platelet unit demonstrated gram negative rods (gnr) and gram positive cocci (gpc) in clusters, and the second platelet unit demonstrated gnr only. repeat secondary safety measure pgd testing of both units was negative for both gp and gn organisms. direct bacterial cultures of both platelet units grew both gnr and gpc identified as a. baumanii and s. saprophyticus after 18 h of incubation. colonies on the initial bacterial plates were too numerous to count (tntc), and subsequent re-plating of the platelet units showed: unit 1: a. baumanii tntc and s. saprophyticus with 8.5 9 10 4 cfu/ml unit 2: a. baumanii 6.6 9 10 7 cfu/ml and s. saprophyticus 2.2 9 10 6 cfu/ml blood cultures collected from both patients became positive within 8 h with gnr on gram stain, and both blood cultures ultimately grew both a. baumanii and s. saprophyticus. the primary pre-release cultures at the blood supplier remained negative. after days on antibiotics and pressors, both patients stabilized and were discharged home. the blood donor was interviewed, and he was well. no cultures were collected. summary/conclusions: this case documents failure of both primary pre-release bacterial testing and secondary on-site point of release pgd testing to identify two pathogenic organisms. a. baumanii and s. saprophyticus are unusual causes of septic transfusion reactions, and it is possible that these organisms have different limits of detection in the pgd assay compared to other organisms. rapid attention to clinical signs during transfusion and prompt initiation of antibiotics is critical for the management of septic transfusion reactions. we are currently evaluating ways to further reduce septic transfusion reactions, including increasing the utilization of pathogen reduced platelets. background: transfusion-associated infections due to the transmission of bacteria still represents a major risk in developed countries nowadays. despite the refrigerated storage of red blood cells (rbc), fatal reactions of patients receiving contaminated rbc units are repeatedly reported. in order to further increase the safety of blood transfusions, new strategies and measures have to be developed. in this context, transfusion-relevant bacteria reference strains can serve as a valuable tool for the validation, comparison and interpretation of these new developments. aims: conducting a collaborative study to establish the first repository for red blood cell transfusion-relevant bacteria reference strains. methods: six bacterial strains (serratia liquefaciens, serratia marcescens, pseudomonas fluorescens, listeria monocytogenes, yersinia enterocolitica a-105 and yersinia enterocolitica a-176) were distributed from the paul-ehrlich-institut to 17 laboratories in 10 countries for enumeration, identification and growth measurement in a 7-day interval for a total of 42 days after low-count spiking of rbc bags (10-25 colony-forming units (cfu)/rbc bag). results: except for s. marcescens, all other strains showed good-to-excellent growth in rbc. s. liquefaciens, p. fluorescens, y. enterocolitica a-105 and y. enterocolitica a-176 achieved >10 6 cfu/ml at day 14 and 10 9 cfu/ml at day 21. growth of l. monocytogenes was lower reaching a maximum concentration of >10 6 cfu/ml at day 35. in 9 out of 17 laboratories, s. marcescens showed no growth at all. summary/conclusions: five of the six tested strains showed robust growth in rbc independent of donor variability and are promising candidates to be adopted as official rbc transfusion-relevant reference strains by the world health organization. background: the samplok sampling kit (ssk), itl biomedical, is used by nhs blood and transplant (nhsbt) for sampling of platelet components (pc) for bacterial screening using the bact/alert 3d system. inoculation of bact/alert bottles is performed immediately after sampling. validation of delayed inoculation, with retention of the sample within the ssk devices, would allow a contingency for transport to other screening sites in the event of an incident that prevented testing at the sampling site. ssk maintain a closed system for sampling of pc and are compatible with standard blood collection bags. a graduated chamber (10 or 16 ml) ensures that only the required sample volume is collected and an integrated needle allows inoculation into bact/alert bottles. aims: the national bacteriology laboratory, nhsbt, evaluated the impact of prolonged storage of pc samples in ssk devices with regard to bacterial viability and detection. methods: four reference species were assessed: staphylococcus aureus (atcc 29523), streptococcus agalactiae (atcc 13813), escherichia coli (atcc 25922), clostridium perfringens (atcc 13124). a pool and split method was used with apheresis pc suspended in plasma. units were screened using bact/alert 3d prior to spiking to prove the absence of contamination. pc were spiked with a single species (range 4 9 100-2.8 9 103 cfu/ml) and tested on bact/alert with a 1 ml inoculation into anaerobic and aerobic bottles (positive control). enumeration was performed to confirm the bacterial concentration. each unit was sampled using two 10 ml ssk, which were held for a period of 6 h at 20-25°c. the process was repeated with a 24-h period. once elapsed, 8 ml of each ssk was inoculated into an aerobic and anaerobic bact/alert bottle, one ssk per atmosphere per species and the remaining sample was enumerated. all bottles were incubated on the bact/ alert system for a maximum of 7 days (36ae0.5°c) and subcultured if positive. results: positive controls had detectable growth by bact/alert, excluding aerobic bottles with c. perfringens. this was expected as it is an anaerobic organism. after the storage periods, all bottles had detectable growth by bact/alert. s. aureus showed an increase of 0.6-log10 after 6 h (2.0 9 102 to 8.9 9 102 cfu/ml) and 2.0-log10 after 24 h (3.1 9 101 cfu/ml to 3.4 9 103 cfu/ml). s. agalactiae increased by 0.7-log10 after 6 h (2.1 9 102 cfu/ml to 1.1 9 103 cfu/ml) and 0.09-log10 after 24 h (1.0 9 102 cfu/ml to 1.2 9 102 cfu/ml). c perfringens increased by 0.6-log10 after 6 h (1.3 9 102 cfu/ml to 5.0 9 102 cfu/ml) and 2.7-log10 after 24 h (4.0 9 100 cfu/ml and 2.2 9 103 cfu/ml). for e. coli, the concentration after 6 h was reduced by 0.28-log10 (2.8 9 103 cfu/ml and 1.5 9 103 cfu/ml), however this was possibly a result of inherent counting errors. at 24 h, an increase in growth by 2.7-log10 (1.5 9 102 to 7.9 9 104 cfu/ml) was obtained. summary/conclusions: storage of pc samples in ssk devices for up to 24 h does not have a negative effect on bacterial viability and detection using the bact/alert 3d system. background: the intercept tm blood system for platelets efficiently inactivates pathogens and leukocytes in platelet concentrates (pc). the system utilizes amotosalen and uva light and is available for the treatment of apheresis and whole blood (wb) derived platelets (mostly buffy coat pools) in europe in plasma or platelet additive solution (pas), and the treatment of apheresis platelets in the us (trima tm in 100% plasma or amicus tm for 65% intersol pas/35% plasma). acinetobacter baumanii and staphylococcus saprophyticus strains were isolated from a saline flush taken 7 h after successful and complete transfusion of an apheresis intercept-treated pc in 65% pas/35% plasma, from a patient with a suspected septic reaction that occurred 2 h post transfusion. bacterial isolates, and a sample of a gram stain-negative and culture-negative sister split pc were submitted for evaluation. we report here the in vitro inactivation of the fast growing, gram negative bacterium a. baumanii and slower growing gram positive s. saprophyticus. the sister unit was assessed for amotosalen break down products as an indication of successful inter-cept treatment. aims: the objective of the study was to evaluate bacterial inactivation of a. baumanii and s. saprophyticus in apheresis platelets, assessed immediately after treatment and with re-culture at the end of a 7 day shelf-life. methods: a double apheresis pc collected in 65% pas/35% plasma was split into three equal components, yielding approximately 250 ml of platelets/pc. a. baumanii and s. saprophyticus were grown in lb broth and each pc unit was inoculated with either bacterial strain or the combination of both strains, each at~6 log colony forming units/ml (cfu/ml). after inoculation, the contaminated units were treated in small volume (sv) intercept kits. samples were taken: pre and post-inactivation treatment, and at 3, 5 and 7 days of storage. the samples were analyzed by plating on lb plates (100 ll-10 ml). residual amotosalen levels were assessed by hplc. results: initial bacterial titers were 5.9-6.0 cfu/ml. following the inactivation treatment, no viable bacteria were detected by plate culture. no bacteria were detected after 3, 5 and 7 days of storage, corresponding to > 5.9 log 10 inactivation of both bacterial strains. performance of the intercept treatment process was confirmed in the sister pc unit as evidenced by levels of amotosalen and its byproducts characteristic of intercept treatment, as well as by review of the process documentation. summary/conclusions: amotosalen/uva effectively inactivated a. baumanii and s. saprophyticus individually and together below the limit of detection after 7 days storage. no bacteria in the sister pc by gram stain and culture, and the presence of amotosalen byproducts suggested that the pc collection involved in the septic reaction was sterile at the time of intercept treatment and was successfully illuminated. the possibility of only one-of-two split pc being contaminated due to biofilm formation is minimized in the intercept system which decants platelets into virgin storage bags at the end of treatment. contamination of the source pc container likely occurred after intercept treatment, possibly at the time of spiking for transfusion. background: studies in sub-saharan africa have documented bacterial contamination of blood products for transfusion varying between 0,3%>17,5%, up to 5000 times higher than in the north. published data from central africa are lacking. aims: the aim of this study was to determine the proportion of blood products contaminated with bacteria in three hospitals in the democratic republic of the congo (drc). to assure aseptic sampling, we used a closed system of sampling bags. in addition to the presence of contamination, we assessed semi-quantitative colony counts. methods: from july to december 2018, a total of 2411 blood products were sampled, of which 979 in hôpital provincial g en eral de r ef erence, kinshasa (hpgrk), 662 in hôpital p ediatrique kalembe lembe, kinshasa (hpkll) and 770 in hôpital saint-luc, kisantu (hslk). after compatibilization of blood products, 16 ml of blood was transferred from the primary blood bag to an attached sampling bag. sampling bags were sealed off, stored in the fridge and transported once daily to the bacteriology laboratory. using the adapter connected to the sampling bag, 4 ml of blood was inoculated in a blood culture (bactalertpf, biom erieux) and incubated at 35°c for 7 days. cultures were checked daily for signs of growth. in addition, 2 ml of blood was equally distributed on the cled and macconkey agar-coated sides of a dipslide (meus s.r.l.). dipslides were incubated 48 h for semi-quantitative culture, expressed as colony-formingunits (cfu) per ml. in case of blood culture growth, bacteria were identified and a second blood culture was inoculated to exclude contamination during processing. bacteria grown on semi-quantitative culture were also identified. results: a total of 1.6% (39/2411) of whole blood and red cell concentrates were contaminated with bacteria. in hpgrk, 2.7% (26/979) of blood products were contaminated, in hpkll 1.5% (10/662) and in hslk 0.4% (3/770) . the proportion of contaminated blood products was significantly higher in hpgrk compared to hslk (p = 0.00047). there was no significant difference between the other sites (p = 0.051 and p = 0.12). majority of isolated bacterial species were coagulase-negative staphylococcus spp./micrococcus spp. (46.7%) and bacillus spp. (33.3%). the remaining 20% of bacterial isolates were identified as non-fermentative gram-negative rods, klebsiella pneumoniae, staphylococcus aureus, mould, listeria spp., corynebacterium spp./coryneform bacteria. the concentration of all isolated bacteria was lower than 10³ cfu/ml, except for one coagulase-negative staphylococcus spp. found in hpgrk at 10³ cfu/ml. summary/conclusions: to our knowledge, we were the first to reach a sample size of more than 2000 blood products for bacterial culture and the first to use a closed system of sampling bags in sub-saharan africa, which guarantees aseptic sampling of blood cultures. this might explain the low bacterial contamination rate (1.6%) of blood products in three hospitals in drc compared to previous studies in other sub-saharan african countries. moreover, bacterial concentrations in the contaminated blood products were low (<10³ cfu/ml). the different proportions of contamination between study sites suggest that different environments and practices play a role in the risk for bacterial contamination. background: although the screening of the treponema pallidum (tp) is mandatory in blood donations, its necessity is controversial, because there have been no transmissions by blood products documented in the developed countries in the last few decades. aims: based on laboratory markers, active and early tp infected donors (aeid) were determined. the demographics of aeid and the frequency measures of cases were compared with that of early infected syphilis cases (syc) notified from the 18 to 65-year-old general population registered at the nphc. methods: altogether, 474,017 18 to 65-year-old donors were screened with anti-tp immunoassay (architect syphilis tp, abbott, wiesbaden, germany) between 2015 and 2017. reactive results were confirmed with immunoblots (viramed biotech ag, planegg, germany), which discriminated both specific anti-tp (igg, igm) and non-specific vdrl antibodies in five dilutions. meeting the criteria of anti-tp igg positivity with a vdrl titer of ≥ 1:8 and anti-tp igm positivity, donors were considered as aeid. they were stratified by age, gender and residence regions. the proportion of aeid (paeid) and syc (psyc) were calculated in first time (ft), and repeat tested (rt) donors and in the 18 to 65-year-old general population, respectively, in each year studied. the period prevalence (pp) of aeid and syc was estimated in the populations at risk 1,2 across 2015-2017. statistics: weighted proportions and one-way anova with tukey post-hoc test and z score test were applied. statistical significance was defined as p < 0.05. results: anti-tp reactivity was confirmed in 167 blood donors. aeid was proved in 85 cases with 36 ft and 49 rt donors. in that period, 1696 syc were notified. both in aeid and syc, the age group of 25-34 years with approximately 35% and 36% of individuals was the dominant. the proportion of men was 74% and 77% (p = 0.5) in the aeid and syc, respectively. paeid estimated in ft donors was significantly higher (0.020%; 0.023%; 0.032%, p < 0.0001) than that of rt donors (0.007%; 0.009%; 0.009%) and the proportion of syc (0.008%; 0.009%; 0.010%) in the general population. pp of aeid showed a roughly homogenous distribution in the 7 regions (0.011%-0.025%), however, pp of syc had a significant (0.058%; p < 0.0001) central hungary dominance in relation to the other regions (0.008%-0.016%). comparing the pp of aeid to syc, central hungary indicated a significant difference (0.025% vs. 0.058%, p < 0.0001), however, other regions showed no substantial differences. summary/conclusions: donors with anti-tp confirmed positivity are referred to the healthcare system. based on the laboratory markers tested, aeid could be separated and their demographical characteristics are pretty similar to that of syc notified from the general hungarian population. the proportion of early and active infection in ft donors is significantly higher than that of rt donors and the proportion of syc in the general population. given the considerable number of tp infection in background: quality assurance and safety of hematopoietic stem cells (hsc) with emphasis on prevention of bacterial and fungal contamination are the prerequisites for any transplantation procedure. bacterial contamination is of particular significance as it occurs relatively more frequently and bacteria are gradually gaining more antimicrobial resistance. aims: the aim was to determine the incidence rate of bacterial and fungal contamination during processing of transplantation material at the institute of hematology and transfusion medicine (ihtm) taking into account the hsc sourceperipheral blood (pbsc), bone marrow (bm) or cord blood (cb). methods: analysis involved both autologous and allogenic components collected at ihtm and other hospitals and dedicated for ihtm patients. in all, the analysis comprised 4135 donations, including 112 bm (2.70%), 3787 pbsc (91.60%) and 236 cb (5.70%) donations processed in our laboratory in the years 1996-2016. bm was collected in operating theatre-conditions, pbsc with cell separators -cs-3000 (baxter), cobe spectra (gambro) and trima accel (terumo bct) and cb was acquired from umbilical vein at obstetrics and gynaecology wards. aerobic and anaerobic bacteria contamination was determined at various incubation temperatures (room temperature and 35°c) using a variety of culture media. pbsc and bm were tested using 2 samples with trypcase-soy broth (tsb-t) and 1 with schaedler + vit k3 (biomerieux). cb was tested using bactec peds plus/f and bactec lytic/10/anaerobic/f (becton-dickinson). results: in the 1996-2016 period 42 contaminated products were found: 25 pbsc (0.66% of all tested pbsc units) and 17 cb (7.20% of all tested cb units). no infected bm products were determined. the overall percentage of contaminated products was estimated at 0,1%. in 2010, three (3) products were found contaminated with staphylococcus epidermidis; all came from one patient with central venous catheter and were collected on consecutive days. other products were contaminated mostly with staphylococcus epidermidis (61.36%). detailed results to be presented on the poster. summary/conclusions: according to ihtm policy no contaminated product is admitted to clinical use. the outcome of our study identifies processing experience of the staff as the main indicator of product quality. important is also proper assessment of donor health and condition of the injection site as products are usually collected from central venous catheter. the closed system is an additional safeguard against contamination during processing. the sample collecting procedure should help to avoid false positive results. background: syphilis is considered a global public health problem. the world health organization (who) estimates that there are annually around 12 million new cases of syphilis in the world, more than 90% occurring in developing countries. despite significant decrease in syphilis transfusion transmission. the recent increase in worldwide incidence associated with the risk of transmission through platelet concentrates (cp), which are stored at room temperature, have called attention to the potential residual risk of syphilis transmission by transfusion. between 2015 and 2017 we observed in our institution a significant increase of 24% in positivity of syphilis among blood donors from 0.62% in 2015 to 0.73% in 2016 and 0.77% in 2017 (p < 0.0000001). aims: to determine the prevalence of active syphilis in blood donors and characterize the serological profile of syphilis positive donors. methods: each positive sample in a treponemic chemiluminescence assay (cmia, abbott architect) performed during blood donor screening in 2017 was submitted to a treponemic elisa anti-treponema pallidum igm (euroimmun) and a non-treponemic test (antigen-omega diagnostics). samples with positive results for one or two of these tests (indicating recent syphilis) were submitted to a real-time pcr for syphilis. the inno-lia syphilis-fujirebio immunoblot test was also performed for samples that presented a positive result for elisa-igm alone. financial support: fapesp 2017/23028-9. results: among 123,851 samples screened in 2017, 958 (0.77%) presented a positive result for cmia -syphilis. of these, 626 (65.4%) were included in the study. a total of 106 samples (17%) showed vdrl+/igm+; 96 (15%) vdrl+/igmand 28 (4.5%) vdrl -/elisa igm+. the inno-lia syphilis test was performed as a confirmatory test in 28 (4.5%) samples that presented positive results for elisa igm and vdrl negative with 21 (3.35%) positive results, 1 (0.15%) undetermined and 6 (0.95%) negative. none of the 626 samples showed the presence of treponema dna by real-time pcr. the prevalence was 0.77%, the incidence was 0.1% in the year 2017, and the incidence in relation to the total positivity was 20.4%. both, prevalence and incidence were higher in men, white, not married, aging 18-29 years and high school educational level. we observed a 6% a-hbc seroprevalence in the elisa igm-syphilis positive samples and a prevalence of 1.5% htlv coinfection. summary/conclusions: we observed a significant increase in prevalence of syphilis in 2017 (0.77%) with an incidence of 20.4% of the total of cases initially positive in the cmia test. according to our data, we could identify a risk of syphilis transfusion transmission in blood banks that exclusively use the vdrl for donor screening, once we found 22 (3.5%) cases with negative vdrl and elisa igm and inno-lia positive. continuous monitoring of the profile of donors infected with syphilis at this time of reemergence of the disease is useful and important not just for blood banks, as it reflects the epidemiological situation of disease in community, and can contribute to the definition of health policies. background: transfusion related sepsis is a serious concern limiting platelet storage time to 5 days at room temperature. while most units are screened for bacterial contamination when collected, bacterial monitoring methods can take up to 7 days to detect contamination. thus, cold storage of platelets represents an attractive alternative for improving platelet safety. in this study, we assessed bacterial growth in platelets stored either at room-temperature (rt; 22°c) or refrigerated (cs; 4°c). aims: the aims of this study were to 1) assess the effect of storage temperature on platelet function and bacterial growth in "contaminated" platelet units, and 2) identify factors contributing to bacterial growth during rt storage. methods: apheresis platelets in plasma (plt) were obtained from healthy donors using the terumo trima accel automated blood collection system (terumo bct). fresh plasma (fp) was collected similarly. aliquots of plt or fp were transferred to ph safe minibags (blood cell storage, inc) and "contaminated" with acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, or pbs (uninfected control). minibags stored at rt were agitated using an orbital shaker set to 60 rpm while cs aliquots were stored under static conditions. bacterial growth was monitored daily through dilution plating. lactate levels were assessed by istat (abbott) cg4 + test cartridges. plasma glucose levels were assessed using blood glucose testing strips (germaine laboratories). platelet activation and aggregation were assessed on days 0, 1, 3, and 5 by flow cytometry and multiplate platelet aggregometry, respectively. results: bacterial growth progressed rapidly over the first 3-4 days post-collection in all plt aliquots stored at rt except those challenged with s. epidermidis. significant growth of s. epidermidis was not detected until day 4. bacterial numbers remained unchanged in refrigerated aliquots through day 5. rt storage resulted in significantly (p < 0.05) decreased platelet aggregation over time which was exacerbated by bacterial challenge. plt function was largely preserved with refrigeration regardless of challenge. bacterial growth was significantly reduced, or at least delayed, in fp. fp challenged with gram-negative pathogens exhibited a significant (p < 0.05) delay in bacterial growth at day 1. while growth of e. coli and p. aeruginosa recovered by day 2, growth of a. baumannii was significantly (p < 0.05) inhibited throughout. fp challenged with gram-positive pathogens exhibited significant (p < 0.05) reduction in bacterial growth relative to plt aliquots. bacterial growth correlated with plt lactate production. lactate levels in plts challenged with e. coli showed diminished significantly after day 3, indicative of lactate utilization. with exception of fp challenged with s. aureus, bacterial growth was restored in fp supplemented with lactic acid in all challenge groups. summary/conclusions: refrigeration preserved platelet function while both inhibiting bacterial growth and lactate production. conversely, the opposite was observed with rt storage. these data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and rt storage may potentiate growth of certain bacterial strains through accelerated plt metabolism. background: bacterial contamination of peripheral blood progenitor cell (pbpc) for transfusion has been the cause of serious sepsis and life-threatening infections. however, a standard procedure or choice of test sample(s) has not been established to screen pbpc products for microbial contamination, because these products are not large enough to facilitate inoculation of the recommended volume for the automated blood culture systems. samples taken from by-product plasma and low volume pbpc product were cultured in routine sterility test. aims: to evaluate the residual risk of microbial contamination in pbpc products for transplantation, we cultured sufficient post-thaw inoculation volumes from pbpc products which were discarded for various reasons in our blood center. methods: in routine sterility test, a 20-ml sample of by-product plasma collected with pbpc product was inoculated into bact/alert bpa and bpn culture bottle (10 ml each) within 48 h after collection. the bottles were then placed in the bact/ alert system and incubated for at least 7 days or when a positive reaction was indicated by the automated liquid-media culture system. moreover, a 2-ml postthaw sample would be cultured before transplantation performed. in the residual risk investigation, discarded pbpc products were thawed, and then a 2-ml and a 20-ml aliquot were taken and cultured with the same method. all positive bottles were subcultured for bacterial isolation and identification. results: in september 2008 and march 2018, after maintaining in liquid nitrogen for 1 to 17 years, 205 pbpc products collected from 45 patients, which was preserved in a volume between 35 and 60 ml, were discarded. all of these products had been cultured negative in routine sterility tests with plasma samples. these 205 final products were thawed and cultured with both the 20-ml and the 2-ml aliquot. one of these pbpc products had the positive culture result with the 20-ml retested samples. nevertheless, the same pbpc product had the negative result with the 2-ml post-thaw pbpc sample and the 20-ml by-product plasma sample. propionibacterium acnes was isolated from the bpn positive bottle. summary/conclusions: the residual risk of microbial contamination in pbpc postthaw products still exist after routine sterility test with the plasma sample and the 2-ml volume of pbpc sample. the bacterium isolated from pbpc product was normal skin flora bacterium. an optimal screening method of pbpc products merits further study to increase the safety of the blood supply. background: hospital hygiene tools that serve as a proxy for assessment of microbial contamination are increasingly used. they include adenosine triphosphate (atp) bioluminescence and air particle counting. however, their use for microbial monitoring of blood banks remains underexplored. they could be of particular interest in a sub-saharan african setting (temperatures, dust) to circumvent bacterial culture and provide direct results usable for monitoring over time. aims: the aim of this study was (i) to quantify environmental bacteria in the air and on surfaces that are regularly in contact with blood products, and (ii) to evaluate atp bioluminescence techniques and particle counts as a predictor for bacterial contamination, in three blood banks in the democratic republic of the congo (drc). methods: samples were taken in three blood banks in the democratic republic of the congo: hôpital p ediatrique de kalembelembe (hpkll) (10 surfaces, 1 air), hôpital provincial g en eral de r ef erence (hpgrk) (24 surfaces, 2 air) and the national blood transfusion centre (cnts) (20 surfaces, 2 air). surfaces that are regularly in contact with blood products were selected (sealer, fridge, donor chair,. . ..). regular surfaces were sampled using rodac contact plates (23.7 cm²) containing cled and macconkey agar, irregular surfaces using swabs (nrsii, medicalwire). atp was measured on the same surface (pd30, kikkoman), expressed as relative light units (rlu) per 100 cm². air was sampled by active sampling (500 liter; spinair, iul) on cled and macconkey medium. in parallel, particles > 0.5 lm and > 5 lm were counted using a particle counter (14,15 liter; metone 227a). culture media were incubated for 48 h at 35°c before counting colony forming units (cfu). results: for regular surfaces, the median (range) viable bacterial count was 27 (6-60) cfu/rodac, 69 (6-103) cfu/rodac, 82 (3-132) cfu/rodac for hpkll, cnts and hpgrk, respectively. at hpkll, highest viable counts were found in the sink (plain growth) and cool boxes (43 and 60 cfu/rodac). in cnts the blood processing bench, the donor chair arm support and washing basin showed the highest counts (plain growth). whereas in hpgrk, most bacteria were found in a fridge (plain growth), blood bag trolley (plain growth) and manual separator (132 cfu/ rodac). gram-negative bacilli were isolated from water basins and sink in cnts and hpkll, but also surfaces close to donor chairs at hpgrk. the median (range) of atp per 100 cm² was 2.906 (778-26.497) rlu at hpkll, ) rlu at cnts and 35.235 (1.754-348 .052) at hpgrk. atp results and total viable count were not correlated (n = 49, p = 0.52). median (range) bacterial count in the air was 258 (210-375) cfu/500l for all sites together. there was no correlation found between total bacterial count and particles > 0.5 lm or > 5 lm (r = 0.7 and r = 0.6 respectively; p < 0.05; n = 5) summary/conclusions: total viable bacterial count of surfaces varies over blood bank sites. according to our results, atp and particle counts did not correlate with bacterial counts on surfaces and in the air, respectively. bacterial isolates from blood bank environments in drc need to be identified and seasonal variations need to be evaluated. background: the risk of transfusion-transmitted hepatitis e virus (tt-hev) infections in line with the question of the necessity of hev-nat screening of blood products is currently subject to an ongoing debate on the importance of timely introduction of hev screening of blood donors and the impact of blood safety. different countries have chosen different regulatory approaches. just recently, the german federal authorities have introduced mandatory testing of all therapeutic blood products beginning from january 1st 2020. however, we already decided to voluntarily test all our blood products since january 2015. aims: in this study, we present our results of a 100% screening of therapeutic blood products for hev rna including four years of active surveillance of hepatitis e infection among blood donors in germany. methods: from january 2015 to december 2018, a total of 386,307 allogenic blood donations from 69,956 individual german blood donors were screened in a minipool format of 96 samples of for the presence of hev rna (realstar hev rt-pcr kit, altona diagnostic technologies (adt), hamburg, germany). nucleic acids were extracted from 4.8 ml plasma using the chemagen msm-i extractor (viral 5k, perkin elmer chemagen gmbh). the 95% lod of the assay was determined to 4.66 iu/ml (447 iu/ml per single donation). the presence of hev-specific igm and igg antibodies was determined using the anti-hev igm/igg elisa (euroimmun, luebeck). hev rna concentrations were quantified using the first who international standard for hepatitis e virus rna for nat-based assays. all hev rna positive donors were deferred from donation for 3 months. follow-up samples were tested for the presence of hev rna and hev-specific antibodies. genotyping was performed by sequencing of the hypervariable region (hvr) and orf1. results: in total, 274 hev rna positive donors were identified. of these, 274 hev rna-positive donors, 216 were nat-only positive donations (78.83%, negative for anti-hev igm and anti-hev igg), three donors had a positive igm titer (1.09%), 30 donors showed reactive igm and igg titers (10.95%), 25 donors already had isolated igg titers (9.12%). median values of viral loads were approximately twice as high in index donations that were antibody negative. merely 62 donors showed elevated alt levels (22.63%), mostly within a double increase within the reference range (16.06%), only 6.57% of donors had even further elevated alt levels. significantly higher alt values were found in donors with a viral load > 1,000 iu/ml compared to the group with viral loads between 100 and 1000 iu/ml. available follow-up samples confirmed igg seroconversion for all donors, however we also observed long-term igm positivity in some donors. genotyping revealed genotype 3 in all cases. the month-dependent incidence ranges from 1:719 to 1:3,781 blood donations with a peak in june and july. summary/conclusions: the high number of identified hev rna positive donors emphasizes the need for hev-nat screening to increase the safety of blood products. this study further confirmed that hev infection is common in german blood donors. background: zika virus (zikv) is a mosquito-borne virus that has caused outbreaks in central and south america in february 2016, and has threatened the safety of blood transfusion globally. there is a high risk of zikv transmission by whole blood and blood components transfusion. it was reported that, zikv rna in infected patients plasma can only be detected within 1 to 2 weeks. however, in whole blood, zikv rna might present positive up to day 101 after the symptoms appear in some patients, even if the clinical symptoms disappeared with zikv rna negative in plasma. this phenomenon suggested that the presence of zika is associated with red blood cells (rbcs). moreover, another report showed that viral load in whole blood of type a west nile virus (wnv) patients was higher than type o, implying that the binding of virus to rbcs may be related to blood group glycoprotein. both of zikv and wnv are member of the flavivirus genus. the study is intended to explore whether zikv have the same adherence mechanism to rbcs as wnv. aims: to investigate the distribution of zikv in blood components and adherence of zikv to different blood types of rbcs in whole blood. methods: five units for each blood type of a, b, o and ab whole blood were randomly selected. each unit of 200 ml whole blood was divided into two half-unit. zikv was added to one half-unit in a certain proportion, and incubated at 37°c for 3 days. each component of whole blood was collected for viral load detection. in the other half-unit,rbcs were suspended in the same type pools of plasma with equal volume after the plasma removed from the whole blood after centrifugation. zikv was added with the same certain proportion, and then incubated at 37°c for 3 days. the whole blood samples and the upper plasma by centrifugation were collected detected for zikv rna. meanwhile, rbcs were washed and resuspended with normal saline followed by viral load detection. results: zika rna of these samples which extracted from whole blood, rbcs, and plasma were determined in a quantitative reverse transcription pcr, and viral rna of each component was all up to 10 9 copies/ml. although, zikv rna loads did not show significant difference in distribution between rbcs and their corresponding plasma components, zikv rna quantification were significantly higher than those in plasma (p < 0.05) in type o rbcs and lower than those in plasma (p < 0.05) in type ab rbcs. summary/conclusions: in our study, we detected high viral rna loads in rbcs. it was demonstrated that zikv adheres to erythrocyte in whole blood, and the blood type may have influence on the adherence. background: hong kong is not endemic for dengue virus (denv) with most of the documented cases being imported. the presence of sufficient number of mosquito vectors, aedes albopictus, in the territory has led to two self-limiting indigenous outbreaks affecting 20 residents from 2000 to 2014. during 14 august to 4 september 2018, another outbreak of 29 confirmed cases of autochthonous dengue fever were reported to the department of health, linked to two epidemiological clusters, one in lion rock park near wong tai sin (wts) district (19 cases) and the other in cheung chau, an outlying island (10 cases). aims: we assessed the risk of dengue transmission from blood donors during the 2018 outbreak using a simplified version of the probabilistic model developed by biggerstaff and petersen (b-p) and the european up-front risk assessment tool (eufrat) model (oei, transfusion, 2013) . methods: patient demographic and general population data were obtained from the centre for health protection and the department of census and statistics of the hong kong government for the number of 15-to 64-year-old patients in the 2018 outbreak and residents of the same age range in hong kong and wts district as at mid-2018 respectively (16-65 years old being the eligible age range for first time donation). to apply the b-p model, we estimated denv incidence among donors in hong kong territory and in wts with confirmed denv infection during 12 august to 8 september 2018 after correction for clinical:subclinical infections ratio, the mean length of asymptomatic viraemia and the probability of collecting blood from asymptomatic donors as described previously (seed, transfusion, 2009 ). to estimate the risk using eufrat model, outbreak and blood donation variables were entered into eufrat's web-based interface (https://eufrattool.ecdc.europa.eu/), which provided automatic calculation of risk-related output parameters. results: while using the b-p model, the estimated risk of collecting a denv viraemic donation was one in 778,000 (266,000-1,822,000) territory-wide for the 28-day study period but increased to one in 147,000 (50,000-345,000) in wts. similarly while applying the eufrat model, the risk of encountering a viraemic donor was 1 in 279,000 (118,000-752,000) territory-wide and 1 in 52,000 (21,000-188,000) in wts during the same period. the eufrat also predicted a territory-wide issue of 0.08 unit of denv-contaminated labile blood component during the outbreak period. summary/conclusions: like many mosquito-borne infections such as denv, the risk is characteristically localised and varies geographically and seasonally during outbreaks. the average predicted risk of collecting a denv-viraemic donation territory-wide is low at 1 in 778,000 during the 2018 outbreak based on the b-p model, which was generally considered as tolerable. however, the risk increased by 4 folds when blood donations were collected from wts residents, who had higher chances of paying visits to lion rock park in close proximity. it was then justifiable to institute risk mitigation policies such as geographically-based deferral and/or fresh component restriction, enhanced post-donation reporting, etc. to protect against blood safety. background: hev is a developing threat to blood safety following the reporting of several cases of transfusion transmission hev (tt-hev). transfusion-related hev infection has been reported in several countries but its true frequency is probably underestimated because it is often asymptomatic and testing of blood donors is infrequent. pakistan is classified as a highly endemic region; with sporadic cases of hev occurring throughout the year, mainly affecting the adult population. to the best of our knowledge, no studies have been reported from pakistan on the epidemiology of hev in blood donors. aims: to assess the epidemiology of the hev specific antibodies and serum alt levels in blood donors of capital twin cities of pakistan. methods: this cross sectional study was conducted from july 2017 to december 2017 at three blood banks in the capital twin cities (rawalpindi and islamabad) of pakistan. the blood donors were equally distributed across the three blood banks. only donors who tested negative for hiv, hbv and hcv were included in the study. serum alt levels were analyzed by using automated clinical chemistry analyzer (selctra pro m) using merck kits. all samples were tested for hev-specific antibodies (igm and igg) by using enzyme linked immunosorbent assay (elisa) kits (adaltis, italy). statistical analyses were performed using spss software version 21.0 (ibm). results: in our study population there were 445 (98.9%) males and 5 (1.1%) females. the mean age of recruited blood donors was 28.38 (sd ae 7.34), with a range of 18-55. younger donors were more common with a frequency of 18-27 year olds of 249 (55.3%). we found an overall hev igg prevalence of 17.5% and an hev igm prevalence of 9.1%. there were 10 (2.2%) blood donors who were positive for both igg and igm antibodies. our results revealed that the hev specific antibodies (igg, igm) prevalence increased with age. the mean value of serum alt was 33.8 (sd ae 41.0) with a range of 4-554 iu/l. the serum alt levels were elevated (>45 iu/l) in 73 (16.3%) blood donors. there was significant correlation (p=<0.001) between serum alt level and hev specific antibodies for igg and igm. summary/conclusions: this study shows that a significant proportion of blood donors at our blood centers have been infected with hev and may be able to cause tt-hev. as we have not yet measured hev rna, we have used igm antibodies as a proxy for donors who have active infection. hev is generally asymptomatic, so it is debatable whether mandatory hev screening in blood donors should be required. results of this pilot study show that there is a need to conduct a larger study at national level with highly sensitive assays before making screening for hev mandatory in pakistan. background: hepatitis e virus (hev) is a zoonotic virus. who estimates that there are 20 million hev infections, 3 million acute hev cases and 56 thousands hevrelated deaths worldwide each year. in recent years, the prevalence of hev in european and american countries has increased significantly. the survey results show that the positive rate of hev igg in blood donors is respectively 4.0% in new zealand, 13.5% in britain, 20.6% in denmark, 16% in the united states and 27.0% in the netherlands. hev has become a global public health concern. in addition to the food route of infection, several cases have been reported that hev can be transmitted via blood products. aims: to investigate the prevalence of hepatitis e virus (hev) infection among voluntary blood donors and potential impact on blood safety in guangzhou china. methods: blood samples from 5552 blood donors were collected from april 2017 to april 2008 at the guangzhou blood center and were tested for anti-hev igg antibody (hev igg), anti-hev igm antibody (hev igm) and hev antigen (hev ag)by enzyme linked immunosorbent assay (elisa). hev rna detection was performed on hev antigen positive samples by rt-pcr. the association of age, gender, ethnicity, occupation and alt with hev igg and igm were analyzed by chi-square test. multivariate logistic regression analysis was applied to identify the independent risk factors of hev infection. results: the positive rates of hev igg, igm and hev ag were 20.05% (1113/5552), 0.76% (42/5552) and 0.04% (2/5552), respectively. no positive hev rna was detected. age and ethnicity were independent risk factors for hev igg and hev igm. the rate of hev antibody increased significantly with age (igg or = 1.089, p < 0.001; igm or = 1.055, p < 0.001). donors who were zhuang minority (32.69%, 7.69%) showed higher anti-hev than those who were han ethnicity (19.89%, 0.70%), and the difference was statistically significant (igg or = 2.052, p = 0.023; igm or = 12.029, p < 0.001). in addition, we found that occupation was an independent risk factor for hev infection, where students showed the lowest anti-hev rate. summary/conclusions: the results indicate that the positive rate of hev antibody among blood donors in guangzhou is high, and the infection status differs in different populations. our study provides basic data for the estimation of risk of transfusion-transmitted hev. background: human cytomegalovirus (hcmv) belongs to the viral family of herpesviridae. it is an enveloped double-stranded dna virus, widely distributed in the human population (60-100% seropositive subjects worldwide) and cause of severe disease in immunocompromised patients and upon infection of the foetus. in normally healthy subjects, hcmv persists lifelong without clinical manifestation undergoing alternating phases of active viral replication and latency. since hcmv can be readily detected in blood, as free virus as well as associated to neutrophils and monocytes, hcmv transmission is a complication of blood transfusion. even though leukoreduction of blood products has been shown to significantly reduce the risk of hcmv transmission, higher inactivation standards may be required for high-risk, immunocompromised groups of patients. aims: in this study, murine macrophages infected with murine cytomegalovirus (mcmv) were used as a model to study the inactivation cell-associated cmv in human plasma using the theraflex mb-plasma system (macopharma). methods: mcmv expressing the green fluorescent protein was used to infect murine macrophages. infected macrophages were harvested 20 h after infection, washed and used for spiking of plasma. plasma units (n = 2, 290 ml) were spiked with infected cell suspension (10% v/v) and treated with the theraflex mb-plasma system according to the manufacturer's protocol using the macotronic-b2 illumination device (macopharma). samples were taken after spiking (load and hold sample), after illumination with different light doses (0 after addition of mb, 30, 60, 90 and 120 [standard] j/cm 2 ) and after blueflex filtration. mcmv titers were determined by endpoint titration and large volume plating on murine fibroblasts. infectious virus, which expressed gfp in infected cells, was detected using a fluorescence microscope. results: the results of infectivity assay showed that the treatment of human plasma by the theraflex mb-plasma system inactivated cell-associated mcmv in a dosedependent manner. after spiking with mcmv infected macrophages a mcmv titer of 4.2 (bag no. 1) and 4.5 (bag no. 2) log 10 tcid 50 /ml was achieved in the plasma units. in hold samples, a mcmv titer of 3.8 (bag no. 1 and bag no. 2) log 10 tcid 50 /ml was determined. the illumination step of the theraflex mb-plasma treatment procedure efficiently inactivated mcmv. already three-fourths of the standard light dose decreased infectivity of cell associated and remaining cell-free mcmv to infectivity levels below the limit of detection (≥ 2.9 log). further investigations would be needed to evaluate the log reduction capacity of the blueflex filtration step for cell-associated mcmv. summary/conclusions: the results with the murine model virus suggest that the theraflex mb-plasma system is an effective technology to inactivate cell-associated cmv in human plasma units. background: the use of pathogen inactivation (pi) technologies for platelet concentrates and plasma is slowly but steadily increasing. methods for treatment of red blood cells (rbcs), the most commonly used blood component, are still under development. aims: a novel approach for pi in rbc units employing uvc light was developed. methods: pi treatment was applied to full-scale rbc units after leukodepletion. the pi capacity of the uvc-based method was evaluated by bacteria and virus infectivity assays. a panel of in vitro assays to measure quality, metabolism, functional, morphologic, and blood group serology variables was applied to a pool-and-split approach in which pathogen-reduced rbcs were investigated in comparison to untreated rbcs. results: uvc treatment caused dose-dependent inactivation of bacteria and enveloped and non-enveloped viruses in rbc units. at a full dose, the mean log 10 reduction factors ranged from 4.2 (bacillus cereus) to 6.1 (serratia liquefaciens) for the tested bacteria, and from 3.1 (emcv) to ≥ 4.9 (vsv) for the tested viruses. uvc treatment did not alter rbc blood group antigen expression. quality of uvc-treated rbcs was maintained during storage, e.g. hemolysis in uvc-treated and untreated rbcs were well below 0.8% until day 35 of storage. summary/conclusions: the data obtained until now show that uvc irradiation is a potential new method for pi in rbcs and justify further development of this process. background: histo-blood abh antigens are the mayor allogeneic antigens in human and they are widely distributed in almost all tissues. the expression of a-1,2-fucosyltransferase (fuct2), encoded by fut2 gene, determines the secretor status of an individual. about 80% of caucasian population have a functional copy of fut2 (secretor gene) expressing abh blood group soluble antigens in organic fluids such as saliva and seminal plasma. this individuals are known as "secretors". soluble abh blood group antigens have been associated with several metabolic and infectious diseases as well as reproductive failures. the incidence of infertility related of both male and female factors continues to rise despite many advances in reproductive technologies. it is well known that abo antigens are expressed on sperm membrane and in seminal fluid of secretors as well as abo antibodies are present in cervical mucus. in previous studies we observed significant loss in progressive motility of spermatozoa of non-secretors compared to secretor ones caused by specific cervical mucus antibodies in abo-incompatible couples. in addition, sperm cells are haploid cells, so that a heterozygous individual has two sperm subpopulations, each expressing the corresponding allele. the specific antibody of cervical mucus will attack only its complementary sperm. aims: to evaluate the prevalence of secretor character in men belonging to fertile and infertile couples in order to investigate a possible association with reproductive success. methods: 126 samples of semen, 68 from infertile men and 58 from fertile controls were studied. comprehensive infertility evaluation was performed in all patients according to who 2010 criteria. secretor phenotype was evaluated in seminal plasma by inhibition of hemagglutination assay using saline erythrocyte suspensions, monoclonal antibodies anti-a, anti-b and lectin from ulex europaeus (anti-h). to distinguish between abo genes, genomic dna was extracted by an enzymatic digestion method. pcr was designed with two sets of oligonucleotides that allow to amplificate two different regions of the transferases without use of restriction enzymes. by comparison of bands of the pcr products, the individual genotype was determine. cervical mucus antibodies of their female partners were titrated with the corresponding red blood cells. results: results were analysed in both groups. in infertile couples with abo incompatibility, the frequency of non-secretor phenotype of male partners (76.9%) were significantly higher than those from fertile couples (21.6%) (p < 0.03) the results obtained by pcr in sperm cells correlated 100% with red cells phenotypes. summary/conclusions: incidence of infertility continues to increase. several factors have a negative impact on men's reproductive health. immunological implications are now being studied and considered as a cause of failure in sperm-egg interaction, even among normal gametes. secretor phenotype in male partners could help reproductive success by blocking cervical abo antibodies. furthermore, if the male is heterozygous, cervical mucus antibodies will only affect the corresponding sperm. we propose to evaluate abh antigen expression on sperm membrane and seminal plasma as well as abo antibodies in cervical mucus to contribute to the diagnosis and treatment of human infertility. background: the h blood group contains one antigen, the h antigen, which is present on virtually all red blood cells (rbc) and is the acceptor substrate of both a and b gene-specified glycosyltransferases. in blood group o the h antigen remains unmodified and therefore its rbcs show the highest and the rbcs of blood type ab the least amounts of h antigen. individuals with the so called bombay phenotype carry homozygous h null alleles (h | h) and do not produce any h antigen. the para-bombay phenotype retains some h antigen on rbcs either induced by a weakly active (h+ w | h+ w ) or completely silenced fut1 gene (h | h), mandatory linked with an active fut2 gene. aims: in this study, we aimed to develop an adapted flow cytometric method to quantify the relative amount of h substance present on rbcs in order to distinguish different abo phenotypes in routine diagnostics as well as to capture rare h-deficient phenotypes. methods: analyses were performed on a flow cytometer (facs canto ii, bd biosciences, ch) and measured with identical instrument settings. list mode data were evaluated and visualised using bd facsdiva software. rbcs were incubated with increasing concentrations of monoclonal anti-h antibodies (bric231-pe and a 1:1 mixture of bric231-pe/bric231, ibgrl, uk). after rinsing the cells with pbs, micro-aggregates were mechanically dissolved. rbcs from 29 blood donors with different abo phenotypes (o (5), a 1 (5), a 2 (5), b (5), a 1 b (5), a 2 b (4)) and 2 patients with genetically confirmed bombay and para-bombay phenotype were assessed. results: saturation of h antigen binding sites on type o rbcs was achieved only upon use of a 1:1 antibody mixture (bric231-pe/bric231) covering approx. half of the h-binding sites by unconjugated bric231. in contrast, non-o type rbcs reached saturation of h-binding sites using pure bric231-pe. rbcs coated with bric231-pe at saturation revealed a distinct pattern of mfi (mean fluorescence intensity) depending on the abo phenotype. in addition, mfis of rbcs upon staining with bric231-pe did discriminate bombay-and para-bombay type rbcs, respectively. summary/conclusions: adapted flow cytometry is able to distinguish variant expressions of rbcs h antigen. thus, our flow cytometric method may complement traditional serological and genetic analyses in routine abo phenotype cases or, more intriguing, when the bombay or para-bombay phenotype is suspected. it will be of interest to further prove this method by investigating additional rare h-deficient phenotype cases. s chen 1,2 , x xu 1,2 , x hong 1,2 , k ma 1,2 , j he 1,2 , j chen 1,2 and f zhu 1,2 1 blood centre of zhejiang province 2 zhejiang provincial key laboratory of blood safety research, hangzhou, china background: weakened a and b antigen expression results in abo typing discrepancies. h gene controls the development of h substance from which a and b antigens develop. depressed a and b antigen expression and strengthened h antigen expression are always simultaneously observed in abo subgroups. there are other possibilities for weak antigen expression of abo system such as leukemic change and pregnancy. it is undiscovered whether abnormal expressions of a, b and h antigen stand for abo subgroups in hemopathic patients. aims: the aim of this study is to explore the role of enhanced reactions with anti-h in direction to abo subgroups of hemopathic patients. methods: 109 samples from blood donors and hemopathic patients with nonconcordant abo typing by serological tests were collected after consent information. the agglutination strength of these rbcs with anti-h reagent was recorded. enhanced reactions were determined by comparison with the results from normal abo groups. the genomic dnas of 109 samples were extracted and genotyped for abo system. this work was sponsored by the medical science research foundation of zhejiang province (2018rc029). results: 69 samples in 80 blood donors showed increased expression of h antigen, of which 47 were identified as abo subgroups. there were 22 enhanced reactions in 29 hemopathic patients. however, 19 were finally confirmed as normal abo genotypes. no statistical significance (86.3% vs 75.9%, p > 0.05) in the frequency of strengthened h antigen expressions was observed between donors and hemopathic patients. the total number of subgroups is 50 and 3 respectively in blood donors and hemopathic patients. extremely significant statistical differences (68.1% vs 13.6%, p < 0.005) existed in the frequency of subgroups with enhanced h antigen, which meant the possibility of subgroups in hemopathic patients samples was less. summary/conclusions: the expression of h antigen is comparably enhanced in subgroups and hemopathies. but most of hemopathic patients with strengthened h antigen expression present normal abo genotypes. as a result, the enhanced reaction with anti-h is necessary but not sufficient for serological identification of abo subgroups in hemopathic patients. background: although the use of automated blood bank analyzer with the advantages of speed and efficiency has recently increased, the abo discrepancies in automated blood bank analyzer have caused the reporting delays of the results and increase of the task. aims: we analyzed the causes of abo discrepancies in automated blood bank analyzer and suggested a solution strategy based on the causes. methods: from november 2018 to january 2019, 55 cases (0.6%) of abo discrepancies among 9,550 abo blood type tests performed using the 15-min reaction mode of ih-500 in chonbuk national university hospital blood bank were identified. we compared the test results of 15-min mode with results of immediate mode using different red cell reagents, and analyzed the causes of discrepancies by performing additional tests such as microscopy, auto-control, antibody screening and identification, anti-a1 and abo genotyping. results: in the immediate reaction using different red cell reagents, 45 cases (81.8%) of discrepancies disappeared and 10 cases (18.2%) remained discrepancies. all abo discrepancies observed in the 15-min reaction were due to serum side causes, and one case (1.8%) was due to both of serum and red cells side cause. nonspecific response (28 cases, 50.9%), cold antibody (20 cases, 36.4%), rouleaux formation (3 cases, 5.5%), cis-ab (3 cases, 5.5%), and abo subtype (1 case, 1.8%) were analyzed as causes of discrepancies. one discrepancy due to cis-ab was disappeared in the immediate reaction using different red cell reagents, abo subtype was changed to totally different blood group, a. on the other hand, in cases of the discrepancy corrected by the immediate reaction using different red cell reagents, the intensity of the positive results still observed in immediate reaction was not different from the 15-min reaction. summary/conclusions: ih-500, an automated blood bank analyzer, was considered useful for automation of abo blood typing, and some observable abo discrepancies are expected to be mostly addressed by reexamining with immediate reaction mode using different red cell reagents. abstract withdrawn. background: abo blood group antigens mainly expressed on red blood cells, but along with that they also present on many organs and tissues like epithelia, platelets, vascular endothelia and neurons etc. the importance of abo antigens extends beyond transfusion medicine by association with various systemic diseases like cardiovascular diseases, gastric diseases, cancers, infectious diseases etc has been proven previously. previous researchers also tried to find out the involvement of abo antigens in neurological diseases like alzheimer's disease, parkinson diseases etc. but association with neurological tumours is less explored. aims: this study aimed to analyse the association of abo blood group antigens with neurological tumours. methods: a retrospective study in a tertiary care institute in india analysed the 2 years data from jan 2017 to dec 2018. the carcinoma patient's admitted in neurosurgical department during study period were included in our study. their diagnosis and abo blood groups were collected from hospital information system. data were analysed into microsoft excel 2016 and spss (version 22). results: during study period a total of 1970 patients with neurological tumours were admitted in our hospital. the blood group frequency of these patients were 20.91%, 37.51%, 32.74%, 8.83% for a, b, o and ab respectively. the common neurological tumours found in our study were glioma (33.55%) followed by pituitary adenoma (20.05%), meningioma (18.58%), schwannoma (8.98%), cavernoma (2.54%), neuroma (2.23%) and space occupying lesions (14.06%). the prevalence of abo antigens was almost similar in all neurological tumours except in neuroma. neuroma was found in 47.73% o group patients as compared to other blood groups which was found statistically significant (p < 0.05). summary/conclusions: in this study we tried to analyse the association of neurological tumours with abo blood groups antigens. we found there is no association of neurological tumours with abo blood groups because the prevalence on abo group in general population is almost similar in patient with neurological tumours except neuroma. neuroma group of tumours like neurofibroma, neuroblastoma, nerve tumours etc. were more common in o group of patients while in our population frequency of b blood group antigen (38.2%) is more common as compared to o blood group(33.4%). background: rhd and rhce represent homologous genes in head-to-head position on chromosome 1 (chr1, p36.11). they encode for the proteins rhd resp. rhce which compose together with rhesus associated glycoprotein (rhag), band 3 and ankyrin the ankyrin complex (ac) linking the red blood cell (rbc) membrane to aspectrin of rbc cytoskeleton (s.e. lux, blood, 2016). cooperatively, the proteins of ac are important for maturation and physiologic properties of rbcs. many proteins of the rbc membrane express blood group antigens on their extracellular surface and are therefore of concern in transfusion medicine. cepellini et al. described weakened hemagglutination reactions of rhd+ rbcs in the presence of an rhc+ antigen (cepellini et al, pnas, 1955) . we attempted to further elucidate the expression of rhd/rhag proteins in various rhce/rhce pheno-/genotypes using a sophisticated flow cytometry approach. aims: in this study, we investigated a flow cytometric method for measurement of the antigen-density of various rhce-phenotypes. methods: analysis was performed on a flow cytometer (facscanto ii, becton dickinson (bd)) using bd facsdiva software and identical instrument settings for all samples. optimized number of rbcs was incubated with saturating concentration of pe-conjugated anti-rhd antibodies brad-3/brad-5/fog-1 (ibgrl, bristol, uk). debris was excluded by rbc gating in fsc/ssc plot. quantibrite-pe beats (bd) were applied according to manufacturer's instruction to quantify the relative expression of rhd epitopes. in addition a representative number of samples from common phenotypes were assessed for expression of rhag using bric-69pe (ibgrl). results: a total of 146 samples from healthy blood donors with serologically defined rhcde phenotypes were included into this study (rr(21), r1r(20), r1r1(23), r2r(18), r0r(15), r1r2(27), r2r2 (22)). variant expression of rhd by different rhce phenotypes using brad-3-pe was shown. rhd was weakly expressed in presence of rhc antigen (cepellini effect). effect of rhd gene dose on rhd protein expression is mitigated by rhc/c genotypes. when only samples with molecularly confirmed phenotypes were assessed, the rhdce genotype predicts consistently the strength of rhd protein expression. outlier samples (3) were retrospectively genotyped and revealed rhdce genotypes as expected from the strength of rhd expression falsifying serological rhcde phenotypes. in contrast, rhe/e polymorphic site does not correlate with rhd expression. in addition, rhag protein is equally present across all rhcde phenotypes. similar results were obtained by using alternative anti-d antibodies such as brad-5-pe and fog-1-pe, although different antibody's avidity precludes quantitative comparison of antigen expression on rbcs. summary/conclusions: sophisticated facs methods reveal different expression of rhd on rbcs according to rhce/rhce phenotype/genotype. rhc/c polymorphic sites (c.48g>c, c.201a>g, c.203a>g of exon 1, exon 2 resp. and intron 2) are in linkage with rhd expression, confirming the observation by cepellini et al. in contrast, rhe/e (c.676c>g, exon 5) is not in linkage with rhd expression. based on epigenomic signature it is conceivable that altered transcription factor binding sites (tbs) of rhd mirrored by homologous rhc/c may cause variant rhd expression. rhe/e snp mirroring the homologous sequence of rhd in exon 5 is not recognised as tbs. in addition, although ac comprises all three rh proteins (rhd, rhce, rhag), their transcriptional regulations seem to be distinct. red cell reference laboratory, australian red cross blood service, perth, australia background: the rh26 antigen was first described when an antisera thought to contain a potent anti-c did not react with all c+ cells. these non-reacting c+ cells were classified as c+, rh:-26, and the antibody specificity anti-rh26. most polyclonal anti-c contain anti-c and anti-rh26. previous studies have shown 2 of 10 monoclonal anti-c reagents are actually anti-rh26. these reagents will not detect the c antigen where the red cells are rh:-26. aims: the australian red cross blood service investigated a phenotype discrepancy in a blood donor. the donor's historic phenotype c+ (r 1 r) was inconsistent with the current donation phenotype c-(r 1 r 1 ). we aimed to investigate the cause of the discrepancy so the donor could be assigned the correct phenotype, identify the root cause of the discrepancy and implement any corrective actions. methods: the donor's red cells were phenotyped with all available anti-c reagents as per the manufacturers product insert across both manual and automated testing platforms. following variable results and weaker reactions with some reagents, dna was extracted from the edta sample and was genotyped using immucor bioarray tm hea precise and rhce beadchip tm . targeted dna sequencing of rhd and rhce was also performed using the trusight tm one sequencing panel. a review of the historical phenotype results, including the testing platform and reagents used at the time was also performed. results: on the current sample the donor's red cells gave a 2 + reaction by tube with bio-rad seraclone â (2) [clone ms35] and immulab epiclone tm [clone anti-c reagents. the sample tested negative on the beckman coulter pk7300 using beckman coulter anti-c [clone 951] blood grouping reagent and tested positive (4) reaction on the immucor neo using immuclone â (1) anti-c [clone . immucor bioarray tm hea precise beadchip tm predicted a c+ phenotype and no variants were detected with the bioarray tm rhce beadchip tm . the trusight tm one sequencing panel genotyped the donor as rhd*01/*01n.01 and rhce*01.15/*02 with a predicted phenotype of c+, c+ w , d+, e-, e+, rh:55 (locr+), rh:-26. a review of the donor's historical records indicated the donor tested as c+ on the pk7200, which at the time was being used with an in-house bromelain preparation (sigma-aldrich) and diagast olymp pheno anti-c reagent [clone ms33]. summary/conclusions: results indicated the phenotype discrepancy was caused by the c+ rh:-26 variant associated with the rhce*01.15 allele. reagents containing clones ms-33 and ms35 correctly phenotyped the donor as c+, with the manual tube reagents showing a weaker reaction which may alert the operator to a possible variant which is important in the patient setting. the beckman coulter pk7300 and associated anti-c [clone 951] failed to detect the c antigen. this reagent appears not to detect the c antigen where it is associated with the rh:-26 phenotype, which is in contrast to the previous report by faas et al, transfusion, 1997 where it was demonstrated that clone 951 reacted with c+ rh:-26 bromelain treated red cells. abstract withdrawn. background: although serological rhd typing has always been challenging due to variation of techniques and variable sensitivity of anti-d reagents, most individuals are unequivocally typed as either rhd positive or rhd negative. however, variants of d (weak d and partial d phenotypes) may present typing difficulties. individuals with partial d (missing epitopes of the d antigen) must be typed as rhd negative as blood receivers, but as rhd positive, as blood donors. aims: the aim of our study was to evaluate the algorithm used since 2017 at ahepa university blood center, to resolve rhd typing problems among first time donors. methods: since automatic analyzers may type variants of rhd as rhd+, our practice is to routinely perform two different typing methods in first time donors: an automated microplate method on the neo analyzer (immucor) and the slide test, using a potent reagent (anti-d blend-immunodiagnostika). in case of negative, weak, slow or mixed-field reaction, further testing with an automated microplate weak d method [immucor-modified indirect antiglobulin (anti-igg) test] follows. the next step of the protocol consists of testing with the commercial id-partial rhd typing kit (bio-rad) comprising a panel of 6 monoclonal anti-d reagents, in an indirect coombs gel test assay. the patterns obtained with this kit can distinguish between d weak and partial d and can also differentiate between categories ii, iv, v, vi, vii dfr, dbt and dhar. the last step of our algorithm consists of molecular testing (immucor bioarray rhce and rhd beadchip assays) at the hellenic national blood transfusion center, in case of remaining uncertainty. results: we applied the above algorithm in 32 samples: a) by using the partial d kit, 23 samples were typed: four samples were characterized as "partial d" (3 dfr, 1diii) and 19 as "weak d". four of the weak d samples (all from women of reproductive age) were confirmed by molecular typing ("weak d type 1" three samples, "weak d type 4.0 or 4.3" one sample). b) the nine (9) remaining samples that showed atypical serological pattern, were sent for molecular testing, which characterized 2 samples as "weak d type 1", one sample as "weak d type 3" and another as "weak d type 11". results are pending for 5 samples. summary/conclusions: in our experience some partial rhds may be mistyped as rhd+ if the technologist does not inspect the pattern of the reactions and only takes into account the assignment by the automatic analyzer as d+ or d-. by use of our algorithm, serological characterization was sufficient to distinguish between weak d and partial d in 68,75% of cases. molecular typing was necessary in the rest. the integration of molecular techniques improves the quality and accuracy of d typing of blood donors. if applied to patients, it also allows administration of d positive blood without compromising safety to those carrying prevalent weak d types that have not been reported to produce anti-d. furthermore, it permits withholding rhig in case of pregnant women carrying such weak d types. background: rhd antigen is one of the most clinically significant blood group antigens. except d positive and d negative phenotypes, there are over 200 rhd variants, which represent as serologic weak d phenotypes (swd). patients with certain swd can make anti-d alloantibodies. by serology testing it is not possible to clearly distinguish among different swd. in croatian institute of transfusion medicine (citm) patients and pregnant females with swd are mostly reported as rhd negative and generally did not refer for confirmation, because molecular testing was not part of the algorithm. that remains the risk of shortages of rhd negative blood and overuse of anti-d immunoprophylaxis for pregnant females. according to uk guidelines patients with swd who are likely to require chronic transfusion support and females ≤ 50 years are treated as d negative and refer for confirmation of d type. people who are rhd genotyped as weak d type 1, 2 or 3 are not susceptible for rhd alloimmunisation. one study showed that in croatian population the most frequent variants are weak d type 1, 2 and 3. aims: the aim of this study is to estimate the prevalence of swd in patients and pregnant females and to find out serologic and molecular characteristics of swd referred for confirmation. methods: from 2013/01/01 to 2018/10/01 we analysed 119.845 samples of patients and pregnant females. rhd typing was performed by anti-d igm monoclonal reagents in direct agglutination micromethod on tango (bs232, bs226) (biorad, dreieich, germany), swing maestro [lmh 59/20 (ldm3) + 175-2 and th-28 + rum-1 + ldm1] and ih-1000 [lmh 59/20 (ldm3) + 175-2] (id-card, biorad, cressier, switzerland). cut-off value for tango was determined as ++ and for gel microtyping as +++. the samples with results below the cut-off were reported and treated as rhd negative, all except those which gave discrepant results at current testing or with historical data. these were sent to rhd genotyping for confirmation. dna extraction was done by qiaamp blood mini kit (qiagen, hilden, germany) and rhd genotyping by pcr-ssp kits ready geneweak d and ready genecde (inno-train, kronberg im taunus, germany). results: from 119.845 samples 300 (0,25%) were swd. 71/300 (24%) were referred to rhd genotyping. 55/71 (77%) samples were weak d type 1, 2 or 3, while 16/71 (23%) were weak d type 14 and partial d variants vii and va. serologic reactions with monoclonal igm anti-d reagents showed different pattern for weak d types 1, 2 and 3. clearly negative serologic reactions were given in 27/29 samples with bs 226 and bs 232, in 30/33 samples with lmh 59/20 (ldm3) + 175-2 and in 18/39 samples with th-28 + rum-1 + ldm1. summary/conclusions: the prevalence of swd in this study is rather low (0,25%). after rhd genotyping 77% of referred samples were finally reported as d positive. serologic determination of d variants is inconsistent and only rhd genotyping can resolve rhd status in swd. to define the permanent rhd status of swd female of childbearing potential and patients who are likely to be chronically transfused we will introduce rhd genotyping in the new algorithm. background: among all blood group systems, the antigens of the abo system are by far the most clinically significant. comes second in importance is the antigens of the rh system, which comprise d, c, e, c, and e antigens. another clinically relevant antigen is the k of the kell blood group system, which is known to be involved in both htr and hdfn. the distribution of the major blood group antigens, such as rh, and kell, is well-studied among populations of developing countries. in contrary, a relatively few studies have addressed their frequencies in saudi arabian population this is also the case in jazan province, where only two published studies have analysed the prevalence of abo and d antigens, while the frequency of other clinically important antigens, such as rh and kell antigens, is yet to be explored. aims: to determine the frequency of the following clinically relevant blood group antigens; rh(d, c, e, c, e) and k among saudi blood donors in king fahd central hospital in jazan province. methods: a retrospective, cross-sectional study was carried out in the blood bank of king fahd central hospital in jazan province. the red cell phenotyping records for blood donation of 3243 randomly selected saudi donors, who donated blood between january and june 2018, were reviewed to identify the prevalence for the following antigens: d, c, e, c, e and k. the hospital blood bank routinely performs rh/k1 phenotyping for all blood donation using either bio-rad or ortho diagnostic column agglutination technology (cat) platforms. phenotype frequencies were expressed as percentages. results: this study included a total of 3243 saudi voluntary as well as family replacement blood donors. the d antigen was found to be positive in 93.7%, while k antigen was positive in 11.1%. among other studied rh antigens, e was the most common (98.1%) followed by c (78.2%), c (70.3%) and e(26.9%). dce/dce (34.2%) and dce/dce (5.3%) were the most common phenotypes amongst d-positive and dnegative donors, respectively. surprisingly, dce/dce phenotype was significantly prevalent (11.9%) with almost 5 times higher frequency compared that reported in caucasians (2.0%). the rare phenotype dce/dce was found in 3 donors (0.09%), while dce/dce and dce/dce phenotypes were found in only one donor each. summary/conclusions: this study is the first to determine the frequency of rh and k antigens in saudi blood donors in jazan province. determination of the frequency of these clinically significant antigens in our geographical area will facilitate the selection of antigen-matched red cell units for transfusion in recipients with multiple alloantibodies. it will also help in the management of blood donation processes and planning the estimated need of blood stock of different blood group phenotypes to meet the patient's needs. abstract withdrawn. background: the gerbich (ge) blood group system includes several high-frequency antigens located on glycophorin c and d. with only few reports published on the clinical significance of antibodies directed against these antigens, it is unclear whether blood transfusions have to be antigen negative in the presence of an anti-ge antibody. the monocyte monolayer assay (mma) is an in-vitro method used to estimate the clinical significance of alloantibodies. aims: to illustrate the role of the monocyte monolayer assay (mma) in the transfusion management of a patient with an anti-ge alloantibody. methods: the clinical and transfusion history was retrospectively retrieved from the patient's medical records. serological investigations were performed by indirect antihuman globulin test. papain and trypsin treated cells were also used. the clinical significance of the antibody was assessed by mma. genomic dna was isolated from whole blood and the samples were further characterized by pcr. results: a 58-year-old male patient with lung cancer without previous transfusions was admitted (06/2009) for surgery. his hemoglobin was 13.2 g/l. an anti-ge antibody was detected and it was decided to transfuse ge-positive packed red blood cells (prbcs). however, no blood transfusion was needed. in july 2017, the patient was admitted for colon cancer surgery with a hemoglobin of 11,0 g/dl. the anti-ge alloantibody was still detectable and a ssp-pcr revealed the genotype ge*01.-03. an mma performed on the pre-transfusion sample revealed a monocyte index (mi) of 0.35% and the antibody was considered not to be clinically relevant. the mi was interpreted as following: 0-3% not significant; 3-5% inconclusive; >5% clinical significant. however, due to the clinical background of the patient it was decided to transfuse ge-negative prbcs, which were obtained from etablissement francais du sang (efs), paris, france. two days after surgery, the patient received 2 units of ge:-2,-3 prbcs without any transfusion reaction. one and a half year later (11/2018), peritoneal carcinomatosis, as a complication of colon cancer, was diagnosed. the patient's hemoglobin was 87 g/l and he had a passage disorder, symptoms of deterioration and an adynamia. based on the mma results from july 2017 indicating no clinical significance of the antibody, it was decided to transfuse ge-positive prbcs. in the following 16 days the patient received a total of 5 units of gepositive prbcs no immediate or delayed transfusion reaction were observed following these transfusions. two further mma's, performed on samples drawn on december 12 th and 16 th (12 days after transfusion of a total of three and two days after two further prbcs respectively), showed a mi of 0.8% und 1% respectively and the anti-ge antibody was considered still not to be clinically significant. summary/conclusions: we report the case of a patient with an anti-ge antibody transfused with ge-positive prbc. as ge-negative prbc are not available in switzerland and not easy to obtain internationally the mma can help in the decision on how to transfuse. in this case, the clinical course confirmed the mma-based prediction. transfusions of ge-positive prbcs were tolerated without signs or symptoms of immediate or delayed transfusion reactions. background: abo grouping discrepancies occur when the results of forward grouping are not corroborative to those of the reverse grouping. these may be due to weak subgroups of a and b, missing or weak abo antibodies or red cell alloantibodies. determination of correct abo blood group of a donor is essential for preventing abo incompatible transfusions and to avoid hemolytic transfusion reactions in the recipient. aims: to determine the frequency of abo discrepancies and their resolution to correctly identify the blood group of the donors. we also determined the frequency of 'weak d' positivity in rhd negative donors. methods: this was a retrospective study on donor samples collected from 1 st april, 2013 to 30 th september, 2015 (two and a half years). for discrepant samples, the abo and rhd grouping was repeated using tube technique using commercial antisera {anti-a, anti-b, anti-ab and anti-d (igm), anti-d blend (igm+igg), anti-h and anti-a1 lectins}. adsorption-elution testing was done for detecting weak subgroups of a and b. antibody screen (3-cell) and identification (11-cell) was done by gel technique (bio-rad, switzerland). 'weak d' testing in rhd negative donors was also performed by gel technique. antibody titration was done using tube technique. the donor details including name, age and the registration/unit number of the donation were also checked for all the discrepancies to avoid repetition while data analysis. results: we detected 104 (0.072%) abo discrepancies out of the total 144279 donor samples tested during the study period. out of these, 135043 (93.6%) were rhd positive. the most common cause of abo discrepancies was weak anti-b antibody (33/104; 31.73%), followed by weak anti-a antibody and weak subgroups of a (24/104 each; 23.07% each) and weak subgroups of b (5/104; 4.8%). the remaining 17.3% (18/104) discrepancies were due to agglutination with o cells in reverse grouping. the overall frequency of weak subgroups of a and b collectively was 0.02% ( background: detection of unexpected red blood cell (rbc) antibodies before transfusion is critical for prevention of hemolytic transfusion reaction. ideally, unexpected rbc antibody detection is carried out within 3 days after receiving a patient's sample. however, in some cases, retests could be performed after more than 3 days for evaluation of any transfusion reaction, quality control or research. therefore, it is necessary to determine the stability of antibodies after refrigeration or freezing for a certain period of time. aims: we carried out antibody identification test with fresh, refrigerated and frozen samples using automated analyzer ih-500 and manual tube methods to evaluate the stability of antibodies after storage and compare the results between the two methods methods: antibody identification tests were performed using ih-500 (bio-rad, 1785 cressier fr, switzerland) and manual tube methods. fifty samples showing positive results in antibody screening test by both methods were divided into three and tested immediately, 1 week after storage at 4°c and 1 month after storage at à20°c. the specificities and reactivities of antibodies at each storage state were recorded and compared between the two methods. results: specificities of antibodies identified were concordant between ih-500 and manual tube methods irrespective of the storage state. the results were as follows: anti-e/e+c, 15; anti-le a , 4; anti-di a , 4; anti-c+e, 3; anti-m, 3; anti-d, 2; anti-c, 1; anti-k, 1; anti-jk a , 1: anti-xg a , 1; unidentified antibody, 13; autoantibody, 2 cases. with regard to the changes in reactivity owing to storage, 26 (52%) samples (anti-e+c, 12; anti-m, 3; anti-di a , 3; anti-d, 2; anti-c+e, 2; anti-le a , 1; anti-c, 1: autoantibody, 1; unidentified antibody, 1) showed identical reactivities after 1 week and 1 month storage by both ih-500 and tube methods. however, 19 (38%) samples, comprising 12 unidentified antibodies, 3 anti-le a , 1 anti-c+e, 1 anti-e, 1 anti-e+c, and 1 autoantibody, showed decreased reactivities after storage in both methods. three samples, comprising anti-di a , anti-e+c and anti-k antibodies, showed increased reactivities after storage. one sample with anti-jk a showed increased reactivity only after 1 month storage, while one sample with anti-xg a showed decreased reactivity only after 1 month storage. higher reactivities were observed in all samples detected using the ih-500 analyzer than manual tube methods (p < 0.005, wilcoxon rank sum test). summary/conclusions: the specificities of unexpected antibodies detected by ih-500 and tube methods were the same in all storage states; however, reactivities were higher in ih-500 than in the tube method. twenty-six (52%) of 50 samples showed identical reactivities after 1 week refrigeration and 1 month freezing. nineteen (38%) samples showed decreased reactivities after storage; however, 12 (12/19, 63%) of them were nonspecific antibodies, unable to identify using commercial id panels. therefore, it is suggested that retests for evaluation of transfusion reaction, quality control or research could be reliably performed after more than 3 days, if stored appropriately in refrigerated or frozen states. abstract withdrawn. t gleich-nagel 1 , d huber-marcantonio 1 , n rufer 1 , g canellini 1 and c niederhauser 2 1 unit of transfusion medicine, interregional blood transfusion src, lausanne 2 laboratory diagnostics, interregional blood transfusion src, bern, switzerland background: a positive direct antiglobulin test (dat) is mainly found in patients with warm/cold autoantibodies or alloantibodies directed against transfused erythrocytes. the identification of antibodies fixed on red cells is important for the clinician, allowing the further evaluation of a patient's clinical situation including their current medication. in immunohematology the elution of a positive dat remains a tedious and expensive procedure. the blood transfusion service src (bts src) has derived a flow chart that indicates in which situation an elution of dat positive samples should be performed. in order to follow the bts src guidelines, it is mandatory to obtain additional data related to the patient's condition, such as haemolytic parameters and recent transfusion history. currently, our laboratory is not always able to apply the recommended flowchart, since information is often unavailable. aims: here, we performed a comparative study between the algorithm provided by bts src and our in-house strategy, which is based on the qualitative changes of a positive dat, without the need for additional patient and biological information. methods: details of dat positivity and the patient's transfusion history was taken from the software eprogesa (mak-system) and analysed in excel. we analysed a total of 3'753 dats and evaluated them for their positivity, whether an elution was performed or whether antibodies were detectable in the eluate. furthermore, we performed an additional analysis on those samples, that were derived from recently transfused patients (<14 days). results: a positive dat was found for igg and c3d in 421 out of 3'753 (11.2%) samples, a level similar to previous reports of positive dats for hospitalized patients. among these positive samples, 244 (57%) were eluted because of a qualitative change in their positivity according to our in-house algorithm. identification of warm autoantibodies or alloantibodies occurred in only 10.7% (26/244) of the cases. from the 161 patients transfused within the last 14 days and having a positive dat, 60 (14%) were eluted according to our in-house algorithm. the same samples would have been analysed if the swiss transfusion guidelines had been applied. however, this comparative study reveals a significant discrepancy in regards to overall sample numbers that should have been eluted according to the two algorithms (244 versus 60 samples). this is mainly due to the fact that the swiss transfusion based algorithm does not recommend an elution of positive dats from patients who did not receive a transfusion within the last 14-days, except if there is a significant clinical suspicion (e.g. haemolysis). summary/conclusions: this comparative study indicates that our elution-based algorithm was performed on all clinically relevant samples as recommended by the bts src guidelines. qualitative changes in the dat positivity represent our main parameter for selecting those samples to eluate. besides ensuring that no clinically relevant samples were missed, this strategy also led to a large number of unnecessary elution analyses. in conclusion, a significant reduction in the laboratory workload and economical savings arises if the relevant clinical information and patients history is known prior to laboratory analysis. background: novel anti-cd38 monoclonal antibodies, such as daratumumab (dara) and isatuximab, used in treatment of multiple myeloma, interfere with routine blood bank serologic tests. as part of the strategies to manage these patients, it is recommended to perform extended phenotyping to provide matched units (aabb association bulletin #16-02). many investigations have focused on the interference with iat for the screening and identification of underlying alloantibodies and how to overcome them, but less has been published on the potential interference with extended phenotyping techniques. aims: the purpose of this study is to compare different technologies to type the most important antigens in myeloma patients before and during the treatment with therapeutic anti-cd38 antibodies. vox sanguinis (2019) 114 (suppl. 1), 5-240 methods: edta-anticoagulated whole blood samples coming from 30 patients in different stages of treatment with daratumumab and 5 with isatuximab have been typed in parallel with dg gel microcolumn (grifols) and mdmulticard technology (grifols). the results are also compared with genotyping results obtained with id core xt (grifols). direct coombs, autocontrol and antibody screening has also been performed as complementary tests. results: the study provides that four patients had positive dat and/or ac before therapeutic cd38 antibodies treatment. in these cases, 6 of 7 negative antigens (fy / jk and/or s) turn to positive in gel technology but mdmulticard showed 100% agreement with genotype id core xt results. focusing in the data obtained during the treatment, 8 negative antigens were type as positive in gel technology (12% of the tests). mdmulticard agreed with genotype in 100% of the analyzed antigens. as complementary data, 13 of 66 patient-treated samples had dat or ac positive and 59 showed panagglutination. summary/conclusions: the results demonstrated that mdmulticard is an effective method to type cd38-directed cytolytic antibodies treated samples in addition to dat and or autocontrol positive samples. background: antibody titration is a semi-quantitative method to estimate the strength and concentration of antibodies present in plasma or serum sample. titration methodology should be validated together with clinical data to evaluate the relevance of the titer value in each application. the titer of an antibody depends on different parameters: the antibody concentration in the sample, the density of the corresponding antigen expressed on the red blood cells used, the affinity constant of the antibody-antigen and other parameters regarding the technique used (e.g. gel cards or tube test). gel cards technology reduces the intra and inter-laboratory variation in titration studies comparing with the tube technique. aims: to evaluate the suitability of dg gel coombs, dg gel anti-igg and dg gel neutral (grifols) for titrations using two sample volumes 25 ll and 50 ll. methods: twenty frozen plasma samples containing unexpected antibodies from different specificities (anti-jk a , -fy a , -k, -d, -e and -c) were titrated in dg gel coombs and dg gel anti-igg cards and 20 donor fresh plasmas with natural occurring antibodies (anti-a and -b) were titrated in dg gel coombs and dg gel neutral (saline technique). the titer of the antibodies was determined by testing two-fold dilutions of the plasma with selected red blood cells depending on the antibody tested. plasma samples were diluted in dg gel sol. selected red blood cells serascan diana, serigrup diana or donor blood were added into the card (50 ll at 0.8%). further, sample dilutions were dispensed into the card (25 ll or 50 ll). subsequently, cards were incubated 15 min, 37°c (coombs technique) and 15 min, 18-25°c (saline technique), centrifuged in dg spin and the results read. agglutination intensity was graded visually according to the instructions for use of dg gel cards. the reciprocal of the highest plasma dilution that gives macroscopic agglutination was interpreted as the titer. results: titers obtained with dg gel coombs and anti-igg (n = 80 titrations, titer ranged 0-256) were compared for each sample with unexpected antibodies. no differences were found between gel cards types (differences were ≤ 0.5 titer in the 98% of the cases). differences between dg gel coombs and neutral (saline technique) (n = 80 titrations, titer ranged 2-512) were observed when anti-a and -b antibodies were titrated using the same sample. the titer was similar or higher in coombs in comparison to the saline technique. coombs titers may be a mix of igm antibodies reacting at 37°c and igg antibodies. differences were > 1 titer in 35% of the comparisons and ≤ 1 titer in the rest of the cases (65%). comparing sample volumes of 25 ll and 50 ll in all cards (n = 160 titrations), higher titers were observed using 50 ll, as expected. differences were 1 titer in the 51% of the comparisons, <1 titer in 44% and > 1 titer in the 5% of the cases. background: autoimmune haemolytic anemias (aiha) are characterized by production of antibodies directed against red blood cells and destruction by the mononuclear phagocytic system or complement system. aiha observed in paediatrics is usually self-limiting and often precipitated by viral infections. in some, the condition is secondary to autoimmune diseases, drugs, infections or underlying primary immune deficiencies. appropriate immuno hematological evaluation to characterise the underlying autoantibody can help identify the type of aiha to aid in diagnosis & treatment of these cases. aims: retrospective analysis of immune-hematological evaluation, treatment and outcome of aiha in paediatrics. methods: patients aged 0-18 years, diagnosed with aiha, between april 2017-december 2018 (21 months) were included in this analysis. aiha was defined as positive direct coombs' test (dct) with anemia associated with corroborative evidence of haemolysis in the form of raised indirect hyperbilirubinemia, raised ldh, raised reticulocyte counts or red cell agglutination on peripheral smear. further monospecific dct and evaluation for the specificity of autoantibody was done for all patients using biorad gel cards and panel cells. steroids were given as first line in all; second line agents included cyclosporine and rituximab. red cell transfusion was given in those with severe anemia with cardiac decompensation. results: 10 patients were diagnosed during the study period with autoimmune haemolytic anemia. haemoglobin at presentation ranged from 2.5 to 9 grams/dl. the initial presentation was severe anemia in 7 children and mild-moderate anemia with thrombocytopenia (evan's syndrome) in 3. the trigger for haemolysis was infection in 4 children. rheumatological evaluation was performed for 5 children out of whom 2 were diagnosed as evolving lupus. primary immune deficiency evaluation was advised for 4 and one child was diagnosed as suffering from combined immunodeficiency. dat was positive in 9 out of 10 aiha patients as one of the infant had dat negative iga mediated aiha secondary to viral infection. two out of 9 dat positive cases had igg & c3d positivity on monoclonal dat testing whereas rest 7 had only igg coating the red cells. dat titration was more than 1:300 in 4 patients; where only 1 of these 4 patients had both igg1 and igg3 coating and rest 3 had only igg1. alloantibody screen was negative in all. specificity of autoantibody was found only in one case, which was against rh blood group antigen (anti e). all patients received prednisolone as the primary treatment. three children attained remission following a 4-6 weeks of steroids. in those who were steroid dependent, cyclosporine was used as the second line agent in 2 and rituximab was used in 3. out of these children 5 children are in sustained remission and off any medication, whereas the rest are on low dose steroids with cyclosporine. summary/conclusions: aiha is not an uncommon problem in children and can vary in its clinical severity. the proper diagnosis and management involves efficient immuno-hematological evaluation, as detailed characterization of the autoantibody coating the red cell is very important in guiding the clinician for management and prognosis. abstract withdrawn. background: drug-induced immune hemolytic anemia (diiha) is rare and has only been described once with dexchlorpheniramine (polaramine tm), an antihistaminic agent widely used in the treatment of a variety of allergic reactions. we report a case of diiha complicated with acute renal failure associated with antibodies to dexchlorpheniramine. a 64-year-old woman with no history of transfusion, was treated semimonthly with a combination of chemotherapy and targeted therapy for metastatic colorectal adenocarcinoma. her chemotherapy regimen consisted of oxaliplatin and 5-fu with leucovorin rescue (folfox). panitumumab (monoclonal antibody anti-egfr) was used as targeted therapy. premedication with dexchlorpheniramine iv was systematically given at the beginning of each cycle of treatment to reduce the risk of perfusion reactions mainly associated with panitumumab. the patient developed chills and febrile agranulocytosis during the first and second infusion respectively. the third infusion was not performed due to pyrexia, chills, general discomfort experienced by the patient at the beginning of chemotherapy. probabilistic antibiotherapy was administered and the patient recovered rapidly. during the next infusion (day 1), following premedication with dexchlorpheniramine, a more "impressive" reaction including all the above mentioned symptoms occured along with back pain and dark colored urine. the infusion was halted and no chemotherapy was delivered. bacterial infection at the implantable port was first thought to be the cause of this adverse event but was not confirmed. additional laboratory findings revealed biological signs of inflammation associated with iha and acute renal failure. the patient was treated with hemodialysis (day 5), two units of rbcs (day 6) and was discharged one week later in stable condition. dexchlorpheniramine was then suspected and samples collected on day 24 were sent for a diiha laboratory workup. aims: the aim of this study was to support a clinical diagnosis of diiha. methods: laboratory workup included direct and indirect antiglobulin tests (dat and iat). drug antibodies investigation was performed by incubating patient's serum and eluate from patient's rbcs in the presence of drug against normal donor rbcs that had not been previously treated with the drug (i.e., by the so-called "immune complex" method). control tests were performed in parallel. drug was diluted in pbs and tested at 1 and 5 mg/ml. results: dat was positive (anti-igg 2 + , anti-c3d 2 + ) and no unexpected rbcs antibodies were detected by iat in patient's serum and eluate without the in vitro addition of the drug. an antibody directed against untreated (titer 2) and enzymetreated (titer 8) normal donor rbcs was demonstrated only in patient's serum in the presence of the drug tested at 5 mg/ml by the gel method. the pool of normal sera did not react in the presence of the drug. summary/conclusions: the multi-drug treated patient described in this study was demonstrated to have dexchlorpheniramine dependent antibody detected by the "immune complex" method. the key to the diagnosis was the observation of positive dat with negative eluate tests which prompted a reexamination of the medications administered in temporal relationship with the hemolytic event. although rare, this case report should alert physicians to the need to investigate the possibility of dexchlorpheniramine induced hemolytic anemia in any patient who develop unexpected anemia after hematologic or oncologic procedures p-331 singapore, singapore, singapore background: daratumumab is a monoclonal antibody against cd38 used in the treatment of multiple myeloma and has been known to bind to cd38 on rbc's and interfere with indirect antiglobulin based serologic tests such as red cell antibody screens and crossmatch compatibility testing. in order to negate the interference of daratumumab, our reference laboratory follows the daratumumab protocol recommended by the aabb which uses dithiothreitol (dtt) treated reagent red cells in red cell antibody screening and identification test in patients known to have received daratumumab. aims: the objective of this study is to determine the impact of daratumumab in the turnaround time (tat) for red cell antibody screening and identification. methods: a retrospective review of the tat for red cell antibody screening and identification samples of patients known to be treated with daratumumab from october 2016 to december 2018 was performed. turnaround time is defined as the time the sample is received up the time the results were reported. the tat for routine red cell antibody screen and identifications were also reviewed during the same period and was compared with the tat of samples from patients treated with daratumumab. results: a total of 55 patients on daratumumab had samples sent to our reference laboratory for red cell antibody screen and identification during the study period. information on daratumumab treatment was not provided to the reference lab prior to the start of testing in 23 of the 55 patients while the use daratumumab was mentioned in the serology request form of the other 32 patients. the median tat for red cell antibody screen and identification is 212 min (range: 47-877) if information on daratumumab was provided prior to start of testing and 301 min (range: 133-1417) if information was not provided prior to testing. the median tat for routine testing is 198 min (range: 15-2245). using wilcoxon rank-sum test, turn-around time for antibody screening and identification for daratumumab treated patients was observed as statistically not significant when compared to routine samples (p value 0.72634). however, tat for serologic tests requests with appropriate medical history compared to the testing requests without relevant information was also observed to be significantly difference (p value 0.00694). summary/conclusions: there is no significant impact in the tat of red cell antibody screen and identification in patients known to receive daratumumab as compared to routine testing. however, there is a significant difference in the tat if information on daratumumab treatment is not provided prior to testing. this highlights the importance of providing the relevant medication information in the request form in order to prevent delays in testing and provision of blood to patients on daratumumab, which can result in improved organizational efficiency and have positive impact on cost and resource savings. background: daratumumab, an anti-cd38 monoclonal antibody, has been shown to be highly efficacious in the treatment of multiple myeloma (mm). cd38 is a glycoprotein highly expressed on plasma cells and, to a less extend, on the surface of red blood cells (rbc). when bound to cd38 on rbc, daratumumab interferes with the pretransfusion tests, with positive antibody screening and crossmatch. anti-cd38 interference is an important challenge as many mm patients will require blood transfusions during their treatment. dithiothreitol is a reducing reagent with multiple applications in blood bank testing. treatment of rbc with dithiothreitol irreversibly removes cell surface cd38 tertiary structure, avoiding the binding and testing interference by the anti-cd38 daratumumab. aims: to demonstrate the efficacy, safety and celerity of the protocol between the blood bank (bb) and haemato-oncology of our institutions, using just the crossmatch. methods: a retrospective research was used for the evaluation of the results obtained from the implemented protocol. this comprehends a previous contact by haemato-oncology that leads to a study of the patient before the beginning of daratumumab treatment, and consists in: abo/rhd grouping; rh and kell phenotyping, and other clinically significant antigens; antibody screening; and direct antiglobulin test. genotyping may be required for some patients who received previous blood transfusions. before the beginning of the therapy, a blood sample of the patient is sent to the bb to perform laboratory tests and frozen after. this frozen sample is used for crossmatching in patients that already started therapy, did not have a blood transfusion in between, and have a positive antibody screening and/or crossmatch. in further transfusions, in case of positive tests, the dithiothreitol-treated donor rbc is applied. the donor rbc antigens are always selected accordingly to patients negative clinically significant antigens, when transfusional support is needed. the laboratory tests are executed in gel column agglutination technique. results: since 2016, 32 patients were studied, from which 16 were transfused with 108 blood units, according to the protocol. there were no immunizations or adverse reactions to transfusion registered within the transfused patients, neither delay on the availability of blood units. patient blood sample collected and frozen prior to the beginning of the treatment, has shown to be a good strategy by reducing significantly the waiting time for the blood unit in the first transfusion. summary/conclusions: this protocol, which defines the communication among the involved professionals, has shown to be a secure and effective way of reducing interferences caused by daratumumab. it ensures the previous study of the patients and their transfusion with rbc respecting the patients negative clinically significant antigens. if not adopted, the mitigation measures described in this protocol, delays in the availability of the rbc requested and alloimmunizations, may and will possibly occur. a good communication between the bb and the haemato-oncology is crucial for a good time management when a transfusion is requested for these patients. three methods were used to resolve this dara interference. reagent rbc's were treated with dtt, which know to denature cd38 and then tested with patient plasma. allo-adsorption study was performed using a certain ratio of red cells to plasma. in addition, a selection of phenotyped cord cells were used as an antibody screening panel. results: dtt treatment of reagent red cells was successful at eliminating dara interference and allowing for the presence of underlying antibodies to be identified. in this case, underlying antibodies were not detected by using reagent dtt treated red cells or phenotyped cord cells. adsorption technique was ineffective at elimination the reactivity. summary/conclusions: dara is the first commercial fda-approved therapeutic monoclonal antibody used in treating multiple myeloma patients. • since cd38 is weakly expressed on normal red blood cells, dara attachment to red blood cells can interfere with pre-transfusion iat testing. • dtt treatment of reagent red blood cells and cord cells can abolish the interference of dara to test for the presence of underlying alloantibodies. • to prevent delays in issuing red blood cell units to patients, hospitals should send patient samples to be tested before receiving dara treatment to ensure that clinically significant alloantibodies are not being masked. background: antibody screening (as)is considered superior to antihuman globulin (ahg) cross match during pretransfusion compatibility testing. in spite of knowing the utility and superiority of as, it has not been adopted uniformly in india. therefore, scarce data is available from this subcontinent in terms of optimisation of red cell antibody detection during pretransfusion testing in form of "type and screen" aims: the main objective was to study the benefits of performing simultaneous antibody screening along with the blood grouping during the first hospital visit to the hospital. other objectives were to study the prevalence of clinically significant antibody among the indian population and to follow up the patients who were transfused antibody screen negative but cross match incompatible blood. we also studied some other relevant quality indicators related to efficiency of blood transfusion services methods: this prospective study was carried out at a tertiary healthcare centre in india between july 2014 and dec 2018 (54 months). the study protocol was submitted to institutional review board and permission was granted. blood grouping and as were done during patients' first hospital visit, which we called "type and screen". when the patient got admitted to the hospital and required blood transfusion, a blood request form was generated by the user and sent to blood bank. depending upon the results of antibody detection, further course of action was chosen. if patient was found to have no antibody, immediate spin test (ist) cross match compatible blood was issued and transfused. in such cases the procedure of ahg crossmatch testing was continued even after issue of blood. cases where ahg cross match test was found negative no further follow-up of the patient was done whereas when ahg cross match was found positive, patients were followed after the transfusion results: a total of 22888 patients were "type and screened". majority were from hemato-oncology, bmt, liver transplant, paediatric cardiac surgery, and medical icu units. clinically significant allo-antibody was detected in 145 patients and autoantibody was detected in 53 patients. alloantibody was detected mainly against rh and kell blood group systems. in diagnosed aiha cases, majority were in the form of warm aiha (58%) and 20% of aiha cases were having hidden single or multiple alloantibody. significantly higher proportion of patients in as positive group required blood transfusion than as negative group (45% vs 34%, p < 0.05). in both the groups, in planned cases, most of the time blood was issued immediately within the defined turnaround time except in 21 where either transfusion was delayed or surgery was postponed. it happened only in trauma or surgical bleed cases. expiry of blood was decreased significantly due to no usage of blood (1.2% vs. 5%, p < 0.05). during the period of study we obtained 10 cases where the ist cross match was compatible but the ahg cross match was incompatible. during follow up none of the cases demonstrated any sign of hemolysis summary/conclusions: in developing countries like us, optimization of as during pretransfusion testing increases operational efficiency and significantly decreases the expiry of blood. results: during the period when absc was performed on pk7300, 250,599 donation samples were tested and 4,895 (1.95%) were found absc positive. antibodies to red cells were identified in 250 donations out of 4,895 (5.11%) absc positive samples and in the rest, no irregular antibodies were detected. the prevalence rate for atypical antibody was 0.10%. the top 5 most frequent antibody specificities were: nonspecific cold antibodies (28.8%), anti-e (26.8%), anti-mi a (19.6%), anti-m (16.8%) and anti-le a (4.0%). a total of 225,124 donations were screened for atypical antibodies by ih-1000 and 2,275 (1.01%) were screened positive. among these, anti-red cell antibodies were identified in 1,239 samples (54.46%), which was significantly higher than those identified in pk7300 screened positive samples (p < 0.00001). the prevalence rate for atypical antibody as screened positive by ih-1000 and with confirmed red cell specificities was 0.55%, which was also significantly higher (p < 0.00001). the top 5 most frequent antibody specificities were: anti-mi a (55.3%), anti-m (21.1%), anti-le a (10.2%), anti-e (6.5%) and non-specific cold antibodies (3.6%). anti-fy b was detected in 7 cases, which would be missed detection by enzyme treated reagent cells on pk7300 system. summary/conclusions: the performance of the ih-1000 system using a 3-cell screening panel including one cell with mi(a+) expression and column agglutination technology with iat phase was superior in comparison with that of pk7300 in the context of higher sensitivity in detecting more true positive results and higher specificity in detecting more true negative and less false positive results. this has translated into the advantages of reduction in workload of reference laboratory in performing less antibody identifications in those false positive samples as well as enhanced transfusion safety by removing more irregular red cell antibody positive plasma-containing components from the issuable inventory, which may potentially lead to haemolytic transfusion reactions. the prevalence of irregular red cell antibodies of 0.55% in healthy blood donors in hong kong reflects more the true statistical figure. background: chronic red blood cell (rbc) transfusion is the upfront therapy for thalassaemia patients, however this therapy is featured by several adverse events including rbc alloimmunization. phenotype matched products transfusion policy can prevent alloantibody formation, but it makes routine transfusion more difficult for both the donor center and the transfusion service. a recent systematic review (franchini et al, blood transfus 2019) reported a rbcalloimmunization prevalence of 11.4%, with a higher incidence against rh and kell systems in thalassemia intermedia patients. aims: the aim of our retrospective study is to evaluate the rbc alloimmunization prevalence in thalassemia patients transfused in a single center over a 18 years period with limited phenotype matched rbc (rh and kell system antigens) units. methods: from 1990 to 2018 thalassaemia patients, with a minimum follow up of 1 year and transfused with more than > 10 rbc units, were included in our study. patients were studied for: blood group and rh / k phenotype determination, direct antiglobulin test (dat), irregular antibodies research (abirr). cross-match and detection of alloantibodies were performed using the indirect antiglobulin test by the column agglutination method. six-monthly dat and antibody screening were performed using the indirect antiglobulin test and enzymatic papain-treated rbc test. results: overall 57 patients (38 females, 19 males) were included in our retrospective analysis: 54 patients were affected by thalassaemia major and 3 by thalassaemia intermedia. rbc alloimmunization prevalence was 12.3% (7 patients): 3 patients were found to be positive for rbc alloantibodies, four with alloantibodies and autoantibodies. eleven alloantibodies were detected (1 anti-h, 1 anti-cw, 4 anti-e, 1 kpa, 1 anti-jka, 1 anti-jkb, 1 anti-m and 1 anti-lua). in 2 out of 3 alloimmunized patients we found an anti-e antibody reactive in enzymatic papain-treated rbc test only, in the third alloimmunized patient anti-kpa and anti-lua antibodies were detected, while in the remaining 4 patients, in which auto and alloantibodies were detected, a severe autoimmune hemolytic anaemia (aea) requiring therapy was diagnosed. in these cases the appearance of alloantibodies is concomitant with the presence of autoantibodies. among the 7 patients positive for alloantibodies, 6 were affected by major thalassemia and one by intermedia thalassaemia summary/conclusions: in our experience a limited phenotype matched rbc transfusion policy showed a rbc alloimmunization prevalence similar to literature data: 12.3% vs 11.4%; we didn't find higher alloimmunization prevalence in thalassemia intermedia patients may be due to the low patients number. we believe that introduction, in our department, of an extended-phenotype matched transfusion, including antigens of the main group systems (fy, jk, mns) and the main rare antigens (cw, kp, lu), could reduce the risk of red blood cell alloimmunization in thalassemia patients. abstract withdrawn. background: undoubtedly, preventing alloimmunization has an advantage over overcoming its consequences. however, the high cost of technical and organizational aspects of preventive measures requires their scientific substantiation confirmed by clinical and laboratory data. selection of donors of the rhesus (d, c) and kell (k) antigens for the red blood cell transfusions to hematological patients has been regulated in the russian federation since 1998. recipients with the phenotype c+c-transfuse red blood cell only with the same antigenic combination. for transfusions red blood cell obtained from k-negative donors are used. the compatibility of the donor and recipient with the antigens c, e, e, c w (rhesus system) and k (kell system) is additionally taken into account from april 2013. that is, transfuse red blood cell that do not contain antigens in the phenotype that are not in the recipient's phenotype. aims: to evaluate the efficiency of red blood cell donor selection using antigens of rhesus (c, c, e, e, c w ) and kell (k, k) systems for the prevention of the recipient alloimmunization. methods: immunohaematological studies using equipment and reagents of biorad (usa) were performed in 3642 patients of the hematology clinic. non-hodgkin lymphoma was diagnosed in 759 patients, acute leukemia in 600, multiple myeloma in 390, chronic lymphatic leukemia in 319, chronic myeloid leukemia in 218, aplastic anemia in 147, hemophilia in 105, myelodysplastic syndrome in 73, and other hematological diseases in 1031. the frequency of detection of antibodies to antigen c (0.25% vs 0.06%) and to antigen e (0.46% vs 0.12%) decreased four times. the frequency of detection of antibodies to the c w antigen has not changed significantly (0.10% vs 0.06%, respectively). selection of antigens c (rhesus) and k (kell) has been carried out in the clinic since 1998, therefore the immunization index for these antigens remained unchanged and amounted to 0.10% vs 0.12% for anti-c antibodies; 0.36% vs 0.36%for anti-k antibodies. alloantibodies to the antigens e (rhesus) and k (kell) were not detected for the entire observation period. summary/conclusions: research verified the effectiveness of alloimmunization prevention of recipients by selecting red blood cell for antigens c, c, e of the rhesus system and k (kell). the study concluded that selection of red blood cells for the antigens c w , e (rhesus) and k (kell) does not affect the level of alloimmunization of patients and is not clinically justified. background: in the russian federation, there is an order according to which patients requiring multiple transfusions, who are at high risk of immunological complications are to typed for red blood cell antigens: abo, d, c, c, e, e, cw, k, k. selection of erythrocyte-containing blood components is carried out taking into account the donorrecipient compatibility according to all the listed antigens. aims: analysis of results of immunological evaluation of patients of hematological clinic. methods: the study included 466 first time patients of hematology clinic in 2017-2018. typing of antigens of abo, rhesus, kell systems, screening and identification of antibodies were carried out using equipment and reagents from bio rad (usa). results: interpretation of results of immunohematological screening was complicated in 84 (18.0%) patients. the total number of complex cases was 96. the double population of red blood cell was most often determined in antigens of the rhesus system (10.9% of the total number of patients) as a result of previous transfusion therapy. of those, chimera for the antigen e was detected in 31 cases (60.8% of patients with the chimera for rhesus and kell antigens), cin 23 (45.1%), sin 15 (29.4%), e -5 (9.8%), cw -8 (15.7%), k -5 (9.8%). in such cases, donor red blood cells were chosen not carrying chimeric antigen for transfusions, in the presence of chimeras in both paired antigensred blood cell transfusion with the cc phenotype and / ee. chimera for abo antigens was detected in 0.6% of the examined individuals. the discrepancy between the direct and reverse blood grouping of the abo system in patients (1.1%) was due to a decrease in the production of antibodies -4 cases and the appearance of extra agglutinins -1 case. autoantibodies were detected in 3.9% of all patients, including 0.6% of patients, when they caused panagglutination phenomenon. upon detection of autoantibodies that complicate the individual selection of donors, transfused red blood cells that are compatible with antigens of abo, rhesus, kell, duffy, kidd, mns systems. alloantibodies were detected in 2.8% of patients, including specific anti-din 4 (0.86%), anti-dcin 2 (0.43%), anti-kin 1 (0.21%); antibodies of unidentified specificityin 2 (0.43%), polyspecificin 4 (0.86%). summary/conclusions: the complexity of interpreting immuno-hematological tests in hematological patients is due to intensive transfusion therapy, changes in red blood cell antigens and appearance of nonspecific antibodies due to underlying disease. red blood cell for transfusion in these patients should be selected taking into account the expanded red blood cell antigen profile. abstract withdrawn. background: blood transfusion is an essential part of therapy for many patients. although life-saving for many patients, blood transfusion is not without risk. the main goal of blood transfusion services is that transfused blood should be compatible with the patient. the clinical and serologic evaluation, which allows for the transfusion of the most compatible (or "least incompatible") blood, requires a joint effort between the clinician and the transfusion medicine physician. aims: root cause analysis of incompatible cross matches in patients. methods: in this prospective study, total of 3,49,497 crossmatches were performed over period of last four & half years, out of which 867 units were found incompatible by column agglutination method-cat in polyspecific (anti-igg+ c3d) gel media. a root cause analysis protocol was formulated to resolve incompatibility to ensure safe transfusion. results: on the evaluation of 3,49,497 crossmatches, only 867 units were found to be incompatible (0.14%). the major cause for incompatibility found in patients was aiha-(32.87%). other causes of incompatibility were infections (27.44%), multiple transfusions (17.41%), trauma (11.23%), evan's syndrome (4.15%), rh negative mother (3.57%), sca (2.99%) & incompatibility due to dat positive packed red cells (0.34%).the most common antibody found were anti-'c', anti-'s' & anti-'m'. summary/conclusions: the rca protocol involves a thorough evaluation of the patient's clinical condition and underlying pathology to identify the cause. a logical stepwise approach will enable provision of safe transfusion to the patient. background: antibodies to high-frequency antigens (hfas) are a transfusion hazard, as compatible blood is often very difficult to obtain. other clinically significant alloantibodies represent an additional transfusion risk. in patients treated with allogeneic bone marrow transplantation (bmt) recipient red cell alloantibodies may cause acute or delayed haemolysis of donor red blood cells (rbc) and contribute to morbidity and mortality. aims: the aim is to present the case of a patient with myelodysplastic syndrome (mds), multiple "common" alloantibodies and an additional alloantibody to a highfrequency antigen, treated with allogeneic bmt. methods: a forty-one-year-old caucasian patient with mds (raeb-1) was admitted to our hospital in january 2017 for unrelated allogeneic bmt. she previously received myeloablative conditioning therapy according to the flu / bu4 / atg protocol (5 days of 250 mg iv. fludarabine, 4 days of busulfan 792 mg iv, 2 days of 300 mg iv. antithymocyte globulin). the indirect antiglobulin test (iat), done in august and december of 2016, was negative. according to anamnestic data, the patient had two pregnancies. she received red cell transfusions during childbirth and platelets in december 2016. results: the patient's blood group was o rhd positive, iat positive. the donor blood group was a rhd positive, iat negative. phenotype of the recipient's rbcs, as well as the donor rbcs, was also determined. anti-e and -c w were found in the patient's plasma, but an additional alloantibody was suspected. the autocontrol was negative. column agglutination technology (cat) and tube technology were used to identify rbc antibodies. plasma was tested with pheno-matched rbcs, papain-and 0.2 m dithiothreitol-treated rbcs, as well as cord and autologous rbcs. adsorption and elution tests were done, excluding other "usual" clinically significant alloantibodies, and the patient received three incompatible (xm in iat, cat) yt(a+), e-, c w -, k-red cell units. the sample was urgently sent for an antibody investigation at the international blood group reference laboratory (bristol, uk). in the reference laboratory, anti-e, -c w and an alloantibody to a high-frequency antigen were confirmed, whose specificity was determined to be anti-yt a . anti-jk b was also suspected and later confirmed. before the patient was discharged from the hospital, she received eight more red cell units (yt(a+), e-, c w -, jk(b-)), during which she was serologically closely monitored. summary/conclusions: the results of the antibody investigation in this case study indicate the presence of multiple alloantibodies in a patient who has previously received immunosuppressive myeloablative conditioning therapy. in addition to the "common" alloantibodies (anti-e, -c w , -jk b ), an alloantibody to a high-frequency antigen (anti-yt a ) was detected in the patient. this patient was transfused with incompatible red cell units (yt(a+)) in an emergency, with no ill effects. although anti-yt a is rarely a clinically significant antibody, according to literature, it can cause immediate haemolytic transfusion reaction. additional risk were "common" clinically significant alloantibodies, especially anti-jk b , which was in this case extremely difficult to detect and had further complicated the selection of blood. background: the identification of an antibody against a high-incidence antigen always introduces a challenge due to the difficulty in finding compatible units of red blood cells (rbcs). in patients needing surgery it is important to minimize their transfusional needs by implementing patient blood management programs (pbm). tests that predict the clinical significance of antibodies, such as monocyte monolayer assay (mma) are also useful in guiding clinical decisions. kell blood group system contains highly immunogenic antigens. antibodies against these antigens are immunoglobulin g, and can cause severe hemolytic transfusion reactions and fetal anemia. results: case report we report the case of a 75-year-old female, with non-hodgkin lymphoma, chronic anemia and scoliosis with severe neurological compromise, proposed for lumbar spinal stabilization surgery. she had a total hip replacement surgery in 2004, with unknown transfusion history. her obstetric history was g6p4a2. the patient had no history of thromboembolic or hemorrhagic events. during pre-transfusional tests, she was typed as a 0 rr and had a positive antibody screening test. the identification studies were suggestive of an antibody against a highincidence antigen, so the surgery was delayed until clarification of these results. she was also referred to a pbm appointment where her hemoglobin was improved from 9.0 g/dl to 12.0 g/dl by administration of ferric carboxymaltose iv and darbepoetin sc. the patient was phenotyped as kp(a+b-) with anti-kpb, an antibody against a highincidence antigen (>99% prevalence worldwide). it is a rare antibody with variable reactivity, causing from none to moderate/delayed transfusion reactions. to access the clinical significance of this antibody, a mma was performed, resulting in a reactivity of 1.2%, suggesting no clinical relevance, however it could be altered after transfusion of kpb+ blood. in order to find compatible rbc's, several family members were phenotyped, however they were all positive to the kpb antigen. in portugal there were no 0 rr kp(b-) blood donors, as it is extremely rare, so we searched in the international rare donor panel (irdp) and two donors were found in spain. two units of compatible rbc's were requested prior to the surgery, which was performed successfully four months later without transfusional support. summary/conclusions: anti-kpb is a rare antibody that in some cases can cause hemolysis of the transfused kp(b+) red blood cells. the combination of kp(b-) and o rr, an extremely rare phenotype, presented a challenge in finding compatible rbcs. this case illustrates not only the complex transfusional and logistic problems that an antibody against a high-incidence antigen can pose, but also the importance of an efficient pbm programme to mitigate the transfusional needs in these patients. background: blood transfusion is an integral part of the supportive care for patients with sickle cell disease (scd). allo-immunization is a recognized complication to red blood cells transfusion (rbc) in those patients. this may result in difficulties in providing compatible blood, and may be associated with the risk of acute of delayed hemolytic transfusion reactions. aims: to describe transfusion management in a patient with scd who has multiple alloantibodies with difficulty in obtaining compatible blood, in order to highlight the importance and clinical consequences of this complication and suggest a possible management approach methods: an 18-years-old female patient with scd presented to our hospital with hemoglobin level of 4 g/dl secondary to acute splenic sequestration. she had a history of multiple previous admissions and many previous rbc transfusions. blood grouping and pre-transfusion compatibility testing were performed in addition to phenotyping of the patient's red cells. screening was done using column agglutination technique by automated machine (ortho; usa) and antibody identification was performed manually using commercial 11 cells identification panel. phenotyping for the patient was done using haemagglutination technique with mono-specific anti sera (bio-rad; switzerland). results: the patient was of group o rhd (positive). antibody screening was positive and antibody identification revealed probable anti-e and anti-fya with possible development of anti-k allo-antibodies, in addition to recent development of autoantibodies; giving pan-positive reactivity with the identification panels. phenotyping of the patient's rbcs was found to be r1r and k-negative. other masked allo-antibodies of undefined specificities were suspected and no compatible blood was found. the clinical condition warranted a blood transfusion, so least incompatible phenotypically matched rbc unit was released. the patient developed acute hemolytic transfusion reaction with drop of the hb level to 2.8 g/dl. despite screening hundreds of rbc units, no compatible units were identified, and no transfusion was given. the patient was managed conservatively using hydration, analgesics, hydroxyurea, erythropoietin, intravenous immune globulin (ivig), steroids, and rituximab. hb level increased to 8 g/dl in 2 weeks, and the patient was discharged from the hospital. the sample of the patient was sent to a reference lab (institut fur klinische chemie und laboratoriumsmedizin-regensburg -germany) for further investigations, clarifications and advice for compatible transfusion in case of need. the report of the reference lab revealed the development of additional anti-m and anti-s with confirmation of the presence of anti-fya, anti-k and warm auto-antibodies. phenotyping of rbcs was confirmed by molecular diagnostic testing done in the reference lab; as r1r, k-neg. summary/conclusions: finding compatible blood may be extremely difficult in patients with scd who develop multiple alloantibodies. it is therefore essential to perform an initial extended red cell phenotyping for the patients at diagnosis and to have on shelf ready phenotyped blood units for issuing to the patients, to minimize allo-immunization. transfusion may occasionally be avoided in allo-immunized patients, utilizing alternative options of treatment and reducing the risk of serious complications such as hemolytic transfusion reactions. background: red blood cell (rbc) antigens that are present on less than 1% of most populations are known as low incidence antigens and those present on more than 90% are known has high incidence antigens. the mns blood group system consists of 49 antigens carried on glycophorin a (gpa), glycophorin b (gpb) or on hybrids of these glycophorins. there are 35 low incidence and 10 high incidence antigens in the mns blood group system. an individual that is homozygous for gp.mur will be negative for the high incidence jenu (mns49) antigen. anti-jenu was first described in a thai patient with thalassemia where only 2 compatible units were found following screening of 3600 units. the jenu negative phenotype is a rare phenotype with an estimated frequency of 0.06%. a male patient with a history of previous transfusion presented with an anti-e and a weak auto antibody with no apparent specificity. a donor unit selected for cross match (group o rhd positive, c+e-c-e+, k-) was incompatible with a reaction grade of 4 + by column agglutination technology. the patient's sample and donor unit were referred to the red cell reference laboratory for investigation for a possible antibody to a low incidence antigen. aims: we aim to characterize the phenotype of the incompatible donor unit. methods: standard serological procedures were used to identify the antibody specificities in the patient's sample. blood group phenotyping of the patient and donor was performed by standard serological procedures. genotyping and zygosity testing was performed using polymerase chain reaction (pcr) high-resolution melting (hrm) assay. gp.mur is a gp(b-a-b) hybrid glycophorin resulting from a gene conversion event between gypa and gypb . this disruption to gpb impacts s expression. the donor was negative with anti-s moab (albaclone), positive with anti-s polyclonal (immulab) and negative with anti-s monoclonal antibody (immulab). this s and s phenotype was consistent with the previously reported examples of gp.mur homozygote jenu negative individuals. molecular testing was consistent with serology supporting gp.mur homozygosity and jenu negative phenotype. summary/conclusions: this donor has been added to our rare donor panel and their red cell donations are cryopreserved for future use in our rare donor frozen inventory. there is limited anti-jenu antiserum available to confirm the jenu negative phenotype. we currently rely on the serological profile of red cells presenting with the gp.mur phenotype, s negative and the discrepant s phenotyping to identify jenu negative donors. this case has highlighted the importance of following up unexplained serological incompatibilities. the development of a monoclonal antibody directed against jenu antigen would provide an opportunity to screen for suitable donors for this rare phenotype. background: molecules expressed on tumor cells are a target of interest for drug development by the use of monoclonal antibodies or blocking proteins. however these drugs have the potential to interfere in pretransfusion testing when the target molecule such as cd47 is also expressed on red blood cells (rbcs). recently, many drugs targeting cd47 have been developed but appropriate mitigation strategies and approach to selecting rbcs for safe transfusion is still an obstacle. aims: we describe a case of delayed hemolytic transfusion reaction (dhtr) by anti-jk a in a patient treated previously with cd47 targeted high affinity sirpa fusion protein alx148. methods: a 35-year-old woman diagnosed with nasal cavity squamous cell carcinoma was enrolled in an alx148 clinical trial. her blood type was group ab, rhd positive, and the antibody screening test was negative for the past 8 months. she had no previous transfusion history during the past two years. after two infusions of alx148, two units of apheresis platelets were requested for transfusion. the blood bank noticed that the antibody screening was positive and further investigation was proceeded. results: antibody screening showed trace positivity in both panel cells (i & ii) at room temperature (rt) and 37°c albumin phase, and 2 + at anti-human globulin (ahg) phase by tube method. the auto control was negative at rt and 37°c albumin phase, but 2 + at ahg phase. antibody screening (2 cells) and identification (11 cells) all showed 3 + at ahg phase using gel cards. direct antiglobulin test was 4 + for anti-igg and 3 + for anti-c3d using gel cards. two units of rbcs were requested for transfusion after hemoglobin decrease to 7.8 g/dl. rbc genotyping was unavailable at the moment. as her previous antibody screening was negative (anti-jk a not detectable), e-, c-, fy b -rbcs were given as a second best option, considering the phenotype distribution of major blood groups in the korean population. the hemoglobin level was well sustained between 11.1-12.0 g/dl but it decreased again to 9.2 g/dl twenty days after rbc transfusion. further laboratory investigation was consistent with a dhtr. the patient was no longer being given alx148, and antibody screening and dat decreased to 0-1+ reactivity. we presumed that antigen typing results would be reliable after chloroquine dissociation and cell washing using antisera that did not require ahg for testing. serologic phenotyping showed that the patient's cells were c+, e+, c+, e+, jk a -, jk b +, fy a +, fy b -, s-, s+, m+, n + . antibody identification using papainized panel cells revealed anti-jk a antibody. we concluded that the dhtr was due to anti-jk a , and jk a -, fy b -, s-rbcs were issued for further transfusion requests. the patient's hemoglobin level recovered to 13.6 g/ dl. the patient's genotype was later identified to be the same as serologic typing. summary/conclusions: communication with the physician and blood bank to perform adequate pretransfusion testing before administration of drugs targeted to cd47 is important to achieve safe transfusion for patients. serologic phenotyping using antisera which do not require ahg for testing can be used as a second option when genotyping is unavailable in a timely manner. background: transfusion is still a key treatment for sickle cell disease (scd) patients. as a result, these patients are much more exposed to transfusions' risks, the most feared one being a delayed hemolytic transfusion reaction (dhtr). we investigated a female scd pediatric patient with no known antibody, who was referred to us for a suspicion of two dhtrs. three transfusion episodes were reported (a total of four units collected from four donors). for the last transfusion, a premedication with rituximab was done. the patient was planned to undergo a bone marrow transplant with her brother as her donor. aims: to describe the molecular and serological workups needed to investigate a dhtr in a scd patient. methods: antibody identification and crossmatches were performed by iat gel testing with red blood cells/panels, which were used raw, papain-treated and trypsintreated. rbcs' phenotypes were determined by conventional techniques. semi-quantitative phenotypes were conducted by serial dilutions with a monoclonal anti-jk a (ms15/seraclone â ). dna was extracted using the magna pure compact instrument (roche). sequencing of jk exons 4-11 was carried out by in-house techniques. results: the antibody identification showed a very weak anti-jk a , which was only reactive on papain-treated rbcs. autologous control was also only positive in this technique. dat and the eluate were negative. as the patient had recently been transfused (less than four months earlier), on this first sample we were neither able to perform autologous adsorptions, nor verify her jk a /jk b phenotypes. in order to rule out the imputability of an anti-lfa in the dhtr outcome, crossmatches with her donors' rbcs were undertaken. three out of the four donors were tested. apart from the anti-jk a reactivity, none of them was reactive. because the patient had previously been phenotyped as jk(a+b+), her jk gene was sequenced. her genotype was determined as jk*01(28a,226a,303a,588g)/jk*02. to confirm this jk a variant allele, a family study was conducted. all her siblings were found to harbor the same genotype. her mother's and father's genotypes were jk*01(28a,226a,303a,588g)/ jk*01 and jk*02/jk*02, respectively. subsequently, autologous adsorptions were performed, which proved the anti-jk a to be an autoantibody. considering the weakness of this antibody, internal controls were used, in order to evaluate a possible dilution effect of this technique. finally, serial dilutions with the anti-jk a reagent showed a weakened jk a expression encoded by the jk*01(28a,226a,303a,588g) variant allele. this finding is consistent with the fact that the crossmatches between the proband's serum and her brother's rbcs were weaker than those performed with (jka+b+) rbcs. summary/conclusions: about a third of dhtrs are reported to happen in patients with no previous history of immunization. performing sensitive serological techniques in order to identify antibodies is necessary to select the most appropriate units. molecular work and extra serological testing can be useful to determine whether an antibody is an allo or autoantibody. even though in this case the anti-jk a was the only antibody identified, because it was proven to be an autoantibody, it is difficult to conclude if it was the cause of the dhtr. nevertheless, jk(a-b+) blood was issued, and no adverse events have been reported. luckily, the patient's bone marrow donor harbors the same variant allele. background: according to the aabb, a pre-transfusion sample must be obtained within 3 days of transfusion if a patient has been transfused or pregnant in the preceding 3 months. despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this 3 day window. to avoid issuing incompatible red blood cells (rbcs) to these patients, a new antibody screen (abs) sample should be drawn and tested shortly before anticipated transfusion. aims: we report a case of a 60 y/o man who presented to the ed (hospital day 0, hd0) with a post-fall intracranial hemorrhage and multiple fractures. anti-e and anti-jka were identified after admission on a new specimen prior to current specimen expiration (<3 days). methods: specimen #1 (s1) was sent on hd0 for type & abs (t&s) and crossmatch (xm) of 2 rbcs. abs and immediate spin xm were negative; there was no patient history. by hd9, he had 4 negative t&s specimens (hd0: s1; hd2: s2&3; hd6: s4) and had been transfused 4 rbcs (hd2: 2; hd5: 2) via electronic xm (exm). at 1730 hr on hd9, 2 rbcs were requested and could have been issued via exm since s4 was not expiring until midnight. however, given recent transfusions, bb staff first called the patient's nurse to review history. patient was uncommunicative, but had scars suggesting past trauma or surgery. s5 was requested and received at 1801 hr. results: s5 showed anti-e and anti-jk a in plasma and eluate. his hemoglobin/hematocrit (h/h) decreased from 10.2 (14.0-17.5 g/dl)/30.1 (41.5-50.4%) on hd6 to 6.9/ 20.6 on hd9. during this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. two e-jk(a-) rbcs were transfused on hd9, which he tolerated well with an increase of hemoglobin from 6.9 g/dl to 8.6 g/ dl. he did well post transfusion with stable h/h between 8.1/24.2.0 to 8.5/25.3. he was discharged on hd19. repeat abs on s4 was negative. of the 4 rbcs transfused before s5, one was e+ and four jk(a+). the family reported that he was injured 20 years prior and had been admitted to 3 hospitals, but was unaware of transfusion. hospital #1 (h1) reported admissions 20 years ago (2 rbcs transfused) and 4 years ago; all abs were negative. h2 admission was 5 years ago with positive abs and inconclusive workup. h3 admission 4 years ago showed negative abs. summary/conclusions: the patient developed a significant antibody response in less than 3 days from the specimen collection, likely a secondary immune response to sensitization from a transfusion 20 years earlier. a new specimen was requested prior to transfusion even though the existing sample (which was abs negative) had not expired. this approach identified new antibodies, preventing transfusion of incompatible rbcs, and a potentially serious hemolytic transfusion reaction. this case suggests that for high-risk patients, abs more frequently than every 3 days may be beneficial. it is important to increase clinicians' and laboratorians' awareness of this issue. background: red cells with partial d antigen have historically been classified as such, based on the fact that the red cells type as d positive, but individuals make anti-d antibody when exposed to conventional d antigen. a definitive confirmation of the variant of d antigen is obtained after the rh d genotyping. aims: to present a case study of the patient's alloimmunisation with the present d partial antigen type dnb, most likely on previously received transfusions. methods: the patient's pretransfusion testing included the determination of the abo blood group and rhd type (id card diaclon abo/d dv+, dv-, reverse grouping, monoclonal antibodies, gel method), antiglobulin crossmatch, additional phenotyping (gel and tube methods), antibody screening, identification of the specificity of irregular anti-erythrocyte antibodies by commercially available red cell panels (id dia-panel bio rad gel method, panocell immucor, tube method) through an indirect antiglobulin test (iat) and enzymes. after routine rhd typing we continued further characterisation of the rhd antigen by serologic assay (bio-rad id-partial rhd typing),and finally by rhd antigen molecular genotyping (fluogene method on fluo vista machine). results: our patient is a 75 year old woman with a diagnosis of tu mammae who was preparing for total mastectomy surgery. she had a history of blood transfusions twenty years ago, and she also had two births. the blood group typing was: o, ccdee, k-, fy (a-b +), jk (a+b +), ss, mn, le (a+b -). the agglutination reactions that we tested with anti d serums were strong (4+). the compatibility test with rhd positive donated blood units was positive. the presence of anti-d and anti-fya antibodies in the serum of the patient was determined. we prepared one compatible blood unit, rhd negative and fya negative, for a surgery. interpretation of the id-partial rh d typing set indicated that this is a diii category of d partial antigen. a sample of blood of our patient was sent to the blood transfusion institute of serbia, where molecular typing of d antigen was performed and the presence of partial form of antigen d, dnb type, was found. summary/conclusions: rhd positive patients or donors with anti-d antibody presents in their serum should be tested for d genotyping. the recommendation for further transfusions of our patient with dnb d partial and her alloimmunisation is to prepare d negative, fya negative erythrocytic blood components, and as a possible blood donor it would be labeled as rh d positive. background: the jr blood group system consists of jr a (jr1), a high frequency antigen expressed by the abcg2 gene. the individuals with jr(a-) phenotype are mainly found in the japanese population and may develop anti-jr a when stimulated by blood transfusion or pregnancy. anti-jr a is a dangerous antibody for pregnancy, but also could cause mild or moderate neonatal jaundice. aims: to conduct the antibody specificity identification of the high frequency antibody in a pregnant woman with history of pregnancy but no transfusion. methods: abo, rhd and some special blood group antigens were identified by tube method in saline. antibody screening and blood group specific antibody identification were performed by indirect antiglobulin test (iat) in gel column. the reagent cells treated with trypsin, chymotrypsin and papain, were used to test the antiserum to obtain the characteristic of antibody reaction. the antibody titer in the patient's serum was detected. dna sample was extracted and 16 exons and adjacent intronic sequence of the abcg2 gene were sequenced. the sample of one family member was collected for testing. results: the blood groups of the patient were b, rhd(+), lu b (+) and kp b (+). the negative reaction of the serum reacted with all reagent cells were tested in saline, but positive (2+) in iat test, while the self-control was negative. the antiserums reacted strongly (4+ in iat test in gel card) with the papain-treated cells, but kept the same reaction strength (2+) with trypsin-and chymotrypsin-treated cells, which indicated the possible existence of anti-jr a . the titer of igg antibody in serum was 2. in cross matching test, the red blood cell of the patient's brother with the same abo and rhd blood group with the patient was successfully matched with the serum of the patient. the sequencing analysis of the abcg2 gene in the patient and her brother revealed one homozygous nonsense mutation in exon 4 (c.376c>t, p.gln126x). after the delivery of the pregnant women, no pathological jaundice was seen in the newborn. summary/conclusions: in the condition of the anti-jr a reagent was unavailable for the identification of jr a antigen in the patient, having an indication with anti-jr a by serological test, the alternative genotyping method was used. the most common silencing jr allele reported in asian population, especially in japanese population, was identified to indicate jr(a-) phenotype. immunohemotherapy, centro hospitalar vila nova de gaia/espinho, vila nova de gaia, portugal background: if the investigation of irregular/unexpected antibodies reveals a pattern in which all or most screen and panel cells are positive, with reactions in the same phase and with the same strength, along with a negative autocontrol, an irregular antibody to a high-prevalence antigen may be suspected. high-prevalence antigens are those that are present in almost all individuals (98% or more). fortunately, because these antigens do occur so frequently, it is not common to find a patient with an antibody to one of them. however, when it happens, it may become a troubling situation. aims: clinical case report of panagglutination in assessment of irregular antibodies. methods: collection of clinical data in scl ınico â and sibas â applications. results: woman, 76 years old, o rhd+, previously transfused with 4 red blood cells concentrates in 2006, was proposed to a correction surgery of a periprosthetic hip fracture. pretransfusion serologic tests were requested and irregular antibodies were detected (2+ in all the 3 screening cells). in order to identify the specificity of the antibody, a panel of 11 cells was tested; the result was considered inconclusive, due to positive reactions (2+) with all test cells in liss/coombs and atypical positivity with dragging in all cells in enzymatic environment. autocontrol and direct antiglobulin test were negative. it was decided to send two blood samples to the reference laboratory for a more complete immunohematological study. compatibilization of red blood cells to this patient was also requested. during the waiting period, haematopoiesis was optimized. although the patient did not present anaemia at admission, the analytical study revealed iron deficiency; therefore, supplementation with intravenous iron was performed. the reference laboratory also obtained a panreactive panel (2+ with all cells) in liss/coombs and weak positivity in papain. after allo-adsorption, the search for irregular antibodies was negative. an anti-yt a , apparently without clinical significance (negative igg1 and igg3) was then identified. transfusion was not needed either during or after the surgery, with a good recovery of the haemoglobin value in the postoperative period. summary/conclusions: yt a , which belongs to cartwright system, is a high-prevalence antigen in all populations. anti-yt a , an igg antibody stimulated by pregnancy or transfusion, is not as uncommon as we may think, which suggests that it is reasonably immunogenic. these antibodies are not generally considered clinically significant, but there are reported cases of acute and delayed haemolytic transfusion reactions in which anti-yt a has been implicated. therefore, although the described pattern of panagglutination in assessment of irregular antibodies may suggest the presence of an alloantibody directed against a highfrequency antigen, it is very important to confirm that hypothesis, recurring to a reference laboratory if necessary, to identify the antibody and to determine its clinical relevance. even if the identified antibody is associated with rare haemolytic transfusion reactions, it is crucial to optimize haematopoiesis when it is not an emergent procedure, in order to minimize transfusion and its associated risks. both for emergent and elective procedures, the creation of a national database of patients with already identified irregular antibodies would facilitate the administration of red cells concentrates without the implicated antigen. aims: to investigate the frequency and explore the genomic characterization of jk (a-b-) phenotype in blood donors in harbin, china. methods: all samples were screened for jk(a-b-) phenotype using a direct urea lysis test. and the results were confirmed with by iat using anti-jka and anti-jkb with a standard tube test. additionally, polymerase chain reaction amplification and sequence analysis of the jk gene were performed. results: from 80865 blood samples, four donors with jk (a-b-) were selected, at a frequency of 0.0049%. among these four samples available for sequencing jk gene, a total of two genotypes were discovered: heterozygote of ivs5-1g>a combining with heterozygote of 359g>a (gly120glu) and heterozygote of 896g>a (gly299glu) combining with heterozygote of 956c>t(thr319met). summary/conclusions: the frequency of jk(a-b-) phenotype in blood donors in harbin area was lower than the reported data from the populations in other areas of china and in finland, but higher than that in japan. ivs5-1g>a, 896g>a and 956c>t were common mutations in the before reports, while 359g>a was reported first time. in addition, it is an effective measure which establish the jk(a-b-) phenotype donors in this region, to solve the blood transfusion problem in patients with anti-jk3. background: blood types, indicating the type of blood group antigen expressed in the red blood cells, is determined by the type of allele at the blood group gene locus. therefore, when allele frequency of each blood group gene is determined, it is possible to predict the frequency of a specific blood type donor with a homozygous allele. it is also possible to estimate the proportion of donors within a particular blood type through combination of specific alleles. and because the ratio of blood group allele differs between ethnicity and race, this can be used as basic data for population genetics and anthropology. therefore, we present a study that examined the allele frequencies of 10 blood group systems in the korean population through blood group genotyping. aims: the purpose of this study is to determine the frequencies of blood group alleles in the korean population, to predict the proportion of homozygous donors, and to obtain the basic data of population genetics. methods: 5,213 blood donors from age 19 to 54 were recruited at korean red cross blood centers located nationwide. acquired samples were examined by blood group genotyping methods using the rbc genotyping system id core xt (progenika biopharma). for each donor, genotypes of 10 blood group systems, excluding abo and rhd, were identified. calculation of the frequencies of blood group alleles in the korean population was done. results: we conducted molecular genotyping of the rhce, kell, kidd, duffy, mnss, diego, dombrock, colton, cartwright, and lutheran blood group systems. the allele frequencies of these blood group systems in the korean population were estimated as follows. -rhce*ce 0.3%, rhce*ce 65.0%, rhce*ce 28.8%, rhce*ce 5.9% -kel*k_kpb_jsb allele 100% -jk*a allele 49.1%, jk*b allele 50.8%, jk*b_null allele 0.1% -fy*a allele 92.8%, fy*b allele 7.2% -gypa*m allele 51.1%, gypa*n allele 48.9% -gypb*s allele 5.1%, gypb*s allele 94.8%, gypb*mur allele 0.1% -di*a allele 4.7%, di*b allele 95.3% -do*a allele 10.1%, do*b allele 89.9% -co*a allele 100% -yt*a allele 100% -lu*b allele 100% summary/conclusions: the significance of this study is accumulation of data on the allele frequencies of blood group genes through highly accurate genotyping method in the east asia region. this enables the prediction of the proportion of donors with a combination of specific blood group alleles in the korean population, which accounts for a decent percentage of the population in this region. background: in donors from arabian countries only little is known about blood groups other than abo and rhesus. during the last years increased migration to central europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. aims: blood group allele frequencies should be determined in individuals from syria, other arabian countries, and iran by molecular typing. methods: as most blood groups are defined by single nucleotide polymorphisms (snps) we introduced a maldi-tof ms assay to detect alleles encoding 16 blood groups including kk, fy (a/b), fy null , c w , jk(a/b), jo(a+/a-), lu(a/b), lu (8/14), ss, do (a/b), co(a/b), in(a/b), js(a/b), kp(a/b), rhce*c.697c>g, and rhce*c.733c>g. additional blood groups and polymorphisms like yt(a/b), s-s-u-, vel null , co null and rhce*c.667g>t were tested by pcr-ssp. a total of 1111 probands including 800 individuals from syria, 147 from iran, 123 from the arabian peninsula and 41 from northern african countries were included. results: 2% of the donors were homozygous for the fy null (fy*-67t>c, fy*02n.01) mutation, 16.2% carried the heterozygous mutation. 0.4% of the syrian probands were heterozygous for the do*350c>t mutation (both, do*jo1 and do*jo2; jo(a+/ a-)) that is virtually unknown in caucasian donors. 0.8% of the syrian donors heterozygously carried the kel*02.06 allele coding for js(a) (phenotype js(a+/ b+)) that is very rare in caucasians. however, no homozygous kel*02.06 carriers were identified. 1.8% of the syrian and 1.5% of all donors were negative for yt*a, which is definitely more frequent than in europeans. one donor from northern africa homozygously carried the gypb*c.251c>g, intron 5 + 5 g mutation, inducing the s-u+ w phenotype. 3.6% of all and 29.3% of northern african donors were heterozygous for the rhce*c.733c>g substitution, 0.3% of the syrian donors carried rhce*c.697c>g (heterozygously) and 0.004% of all donors were heterozygous for rhce*667g>t. heterozygosity for vel deficiency (vel*-01) was detected in 21 individuals (2%; 16 of them from syria) whereas only one syrian donor carried the homozygous mutation. none of the donors carried the aqp1*c.601delg (co*01n.06) mutation that induces the co null phenotype. summary/conclusions: the study provides a first overview on a number of different blood group alleles in blood donors from arabian countries. some blood group alleles that largely are lacking in europeans but had been described in african individuals are present in arabian populations at a somewhat lower frequency. in single cases it could be challenging to provide immunized arabian patients with compatible blood. methods: three unrelated individuals (2 blood donors and one pregnant woman) of polish origin who were typed as ab group with a very weak a antigen and normal b antigen expression were subjected to extended abo typing. in one case family studies were performed (blood samples from donor's mother, father and sister). sequencing analysis of this donor dna was performed three times (from two blood samples and buccal swab). serologic investigations were performed with standard methods: 1/gel cards diaclon id abo/d (anti-a: clone a5, anti-b: clone g1/2, anti-a,b: clone es131, es15+ birma 1+ es4; bio-rad) and diaclon id abd-confirmation for donors (anti-a: clone m297/628 = la-2; bio-rad); 2/tube techniques with: anti-a (birma 1; a-11h5, a1 s.pa1m095, c.9113d10), anti-b (lb-2, b-6f9, c.9621a8). genotyping was determined by rbc fluogene abo basic kit (inno-train, germany) and by sequencing of +7.21-kb site of abo gene to cover sequences ranging from the end of intron 1 to 3 0 utr of the abo gene. additionally sequence of exon 1 of the abo gene was analyzed. results: abo typing showed normal b and a very weak a antigens on rbcs of all three individuals (2 blood donors and one pregnant woman). the a antigen was detected by tube technique only using anti-a clones: birma 1 (1+ to 2+), a-11h5 (1+ to 2+) and c.9113d10 (weak+ to 1+); negative reaction of a antigen typing by gel cards was observed. the sera of all individuals contained anti-a1 antibodies. commercial pcr-ssp kit revealed three heterozygous a/b genotypes (absence of 1061delc typical for abo*a2 alleles). in all these individuals abo sequencing of 7.21-kb fragment confirmed the heterozygous genotype with 7 polymorphisms characteristic for abo*b.01 allele (297a>g; 526c>g; 657c>t; 703g>a; 796c>a; 803g>c; 930g>a) and detected a novel abo*a allele sequence with duplication-based insertion of 21 bp after 564 position (abo*a c.dup543_563; gcaggacgtgtccatgcgccg). as a consequence, the online protein translation predicts an in-frame duplication of seven amino acids after codon 188 (p.dup_182_188qdvsmrr), with synonymous change of the repeated codon 182 (cgc>cgg) and 188 (cgg>cgc) but both coding arginine (r). inheritance of abo*a c.dup543_563 allele was confirmed by family studies of one donor: his father and sister had a/b genotype associated with normal a and b antigens expression; his mother had normal a antigen expression. she carried abo*a1.01 allele and the same abo*a c.dup543_563 allele as a son. summary/conclusions: a novel a weak allele at the abo gene detected in three unrelated polish individuals is an in-frame insertion of seven amino acids to the wild-type glycosyltransferase a. the stability of the encoded protein may be affected causing the weak a phenotype. the inheritance of this mutation was confirmed in the family studies. background: since the cloning in 1990 of cdna corresponding to mrna transcribed at the blood group abo locus, polymorphisms and phenotype-genotype correlations have been reported by many investigators. although many subgroups have been explained at the genetic level, unresolved samples are still encountered in clinical practice. we report here the result of an abo investigation of a sample from a swedish blood donor that showed a very weak agglutination of rbcs with anti-a in routine forward typing. aims: to elucidate the genetic basis of the apparent weak a subgroup. methods: routine abo genotyping by pcr-asp and pcr-rflp including pcrbased analysis of the upstream cbf/nf-y-binding enhancer region was carried out. further genetic analysis was performed by dna sequencing of abo exons 1-7 (including 50 base pairs of the adjacent introns) and the proximal promoter. flow cytometric testing of rbcs was performed with monoclonal anti-a, anti-b and anti-h. results: the weak agglutination of erythrocytes with anti-a was accompanied by the expected lack of anti-a and anti-a1 in plasma. abo genotyping gave the genotype abo*a1.01/o2.01 usually consistent with normal expression of a antigen. enhancer analysis resulted in an amplification product corresponding to the expected single cbf/nf-y binding motif. flow cytometric testing of the sample showed a antigen expression with an almost chimeric pattern where the majority of the cells (approximately 85%) expressed the a antigen at a very low level, marginally distinguishable from the group o control. the remaining approximately 15% of the cells displayed an a antigen level ranging from normal to very weak. genomic abo sequencing showed an abo*a1.01-like allele except for a novel mutation located in intron 5, c.239+4g. the o 2 allele had an additional snp, c.689g>a, consistent with the abo*o2.03 allele variant summary/conclusions: a previously unreported variant, c.239+4a>g, likely effecting the 5 0 -donor splice site of intron 5 was found in an a weak sample. this type of mutations is expected to decrease mrna stability and/or cause skipping of the preceding exon in the mrna. however, small amounts of full-length enzyme might still be made, being able to give rise to the weak a antigen expression seen in this individual. interestingly, this mutation is very similar to the genetic variant underlying the weak a subgroup a finn . in this case, however, the c. 374+4a>g mutation is located in the 5 0 -end of intron 6 and is predicted to cause partial skipping of exon 6. strikingly, the a finn phenotype also results in a pseudochimeric pattern by flow cytometry but with only approximately 2% positively staining erythrocytes. due to the well documented lack of a-allele-derived mrna in peripheral blood, further transcript studies could not be undertaken. further studies are needed to investigate the exact mechanisms underlying the pseudochimeric pattern observed by flow cytometry in these two interesting genotypes/phenotypes abstract withdrawn. background: abo is the clinically most relevant blood group system in transfusion and transplantation medicine. abo genotyping is potentially useful in clarifying serologic blood grouping discrepancies. this scenario includes inherited subgroups alleles, temporary acquired variant abo phenotypes in disease or pregnancy, and chimerism due to exchange of progenitor cells early in fetal life or after blood progenitor cell transplantation. aims: to investigate the molecular basis for abo discrepancies detected in clinical samples, including donors and patients, sent to our reference laboratory during the past 3 years. methods: if routine abo grouping showed weak agglutination or forward vs reverse typing discrepancy, further abo typing studies were performed manually. adsorption-elution tests were also performed in some cases with polyclonal anti-a and anti-b to confirm whether a or b antigens were weakly expressed on the rbcs membrane. a pcr approach using sequence specific primers for a2, b, o1 and o2 alleles was used for initial genotype determination. the full abo coding region was analysed as previously described in selected samples for which abo discrepancy was still unexplained. allele specific fragments spanning exon 6, intron 6 and exon 7 were amplified using a forward primer targeting the 261g nucleotide (to exclude o1 alleles amplification) in combination with either b, a2 or a generic reverse primer. analysis was carried out by sanger sequencing. results: a total of 16 samples with suspected inherited abo subgroup alleles were selected for further molecular studies by sequence analysis. a subgroup alleles: in 8 out of 12 samples with suspected a subgroup alleles, the c.804insg insertion was detected corresponding to the abo*ael.01 allele. the abo*aw31.02-05 variant, a hybrid a1-o1v allele, was found in 2 cases. in 1 case we found the c.722g>c change, previously reported associated with weak a antigen expression. finally, a novel c.961c>g change was detected in an a2 allele. b(a) or cis-ab suspected alleles: the abo*b(a)04 variant carrying the c.640a>g change was found in 1 of 3 samples with bo1 genotype but a weak antigen expression. in the remaining 2 cases, a consensus b allele was detected, thus pointing to a potential chimerism as the cause of the results observed in abo grouping. finally, we have identified an abo*b01.02 allele carrying the nucleotidic change c.926a>g in the context of an abo phenotype vs genotype discrepancy. summary/conclusions: the sanger sequencing approach applied in this study have proved to be informative and helpful to determine the molecular basis of abo grouping discrepancies with suspected inherited subgroups. we found mutations, within exon 7 of the abo gene, in 14 out of 16 samples, including 2 novel alleles. chimerism was suspected in 2 cases of a antigen expression in samples with b1o1 genetic background carrying an apparent normal b allele. we are evaluating at the moment a deep sequencing approach by next generation sequencing to determine the presence of a small amount of a minor allele in the presence of a large surplus of the other two alleles. background: recently, the multiple pregnancy rate has been increasing due to advances in artificial fertilization including in vitro fertilization-embryo transfer. most dizygotic twins have dichorionic placenta, but 8% of them share the placenta. monochorionic dizygotic twins can have blood chimerism, leading to double rbc populations in routine abo serologic typing. recently, more sensitive and objective column agglutination tests with automated systems are being widely used. therefore, blood chimerisms in dizygotic twins can be detected more easily by routine abo blood typing. aims: we report congenital blood chimerism in monochorionic dizygotic twins of triplets, found incidentally during abo serological testing and confirmed by abo genotyping and str marker analysis. methods: a 20-year-old male (one of triplets) was admitted to the hospital for medical checkup. he did not have any history of transfusion or bone marrow transplantation. routine abo blood grouping test was performed using automated blood bank system ih-500; however, it showed abo discrepancy. the red blood cells showed double cell populations in a gel column with anti-a and anti-b. we carried out abo genotyping both from the blood and from a buccal swab. for the further evaluation, we performed abo serologic testing, abo genotyping, and str marker analysis in his family members. results: among the triplets, blood chimerism was demonstrated in the patient and his brother. they both showed a 3 b 3 phenotypes in the serologic test and tri-allelic abo genotypes in the blood, a102/b101/o01. however, in buccal swabs, the patient showed a102/o01 and his brother showed b101/o01. other members of the family (father, mother, and dizygotic sister) had regular abo blood types in the serologic test. we performed str analysis in the triplets and parents. eleven loci (d8s1179, d21s11, d7s820, csf1po, th01, d13s317, d16s539, d19s433, d18s51, d5s818, and fga) revealed more than one additional allele in the blood sample, apart from those in the buccal swabs. str marker analysis showed that his brother too had blood chimerism. summary/conclusions: we found blood chimerism in monochorionic dizygotic twins of triplets during routine abo blood typing, and this was confirmed by str analysis. as the application of assisted reproductive technology increases, the incidence of blood chimerism will also increase. blood chimerism can often create confusion during abo serologic typing and microchimerism can be overlooked in routine methods. therefore, it is helpful to use an automated blood bank system to improve sensitivity and blood chimerism should be considered if abo blood grouping reveals double populations. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial host. results: case #1 is a patient with an unclear abo phenotype: forward type b, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.29-5t>g in heterozygosity on an otherwise common a1 allele, and in trans an abo*b.01 allele. case #2 is a caucasian donor with an abo discrepancy: forward type aweak/o, reverse type a. sequencing also detected variant c.29-5t>g in heterozygosity on an a background, and in trans an abo*o.01.01 allele. given that this variant is located near the intron 1 splice acceptor site, abo*29-5g transcripts are postulated to undergo altered splicing, leading to an aweak phenotype. case #3 is a prenatal sickle-cell disease patient with an abo discrepancy: forward type aweak, reverse type a. dna sequencing detected variants c.467c>t (pro156leu) and c.709t>a (tyr237asn), both in heterozygosity on an otherwise common a1 allele, with an abo*o.01.01 allele in trans. thus, the data establish an association of allele abo*467t,709a with an aweak-like phenotype. case #4 is a donor with an abo typing discrepancy: forward type o, reverse type a. sequencing detected variant c.479c>g (pro160arg) in heterozygosity on an a2 background, and in trans an abo*o.01 allele. an interpretation of the data is that variant c.479c>g weakens the activity of the a2 transferase, with allele abo*a2(479g) encoding the aweak-like phenotype detected by serology. case #5 is a 9 year-old patient with an abo discrepancy: forward type o, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.803g>c (gly268ala) in heterozygosity on an a2 background, and in trans an abo*o.01.02 allele. the serology and molecular results suggest that allele abo*a2(803c) encodes a cisab weak phenotype. case #6 is a caucasian donor with an abo typing discrepancy: forward type o with a weak agglutination with anti-ab, reverse type o. dna sequencing detected variants c.739g>a (glu247lys) and c.871g>a (asp291asn), both in heterozygosity, in trans, and on a1 backgrounds. variant c.871g>a by itself constitutes allele abo*a3.01. the phenotype encoded by abo*739a is uncertain. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. results: case #1 is a 35 year-old pregnant female with an abo typing discrepancy: forward type o, reverse type a. pcr-rflp predicted abo*a1/abo*o1. sequencing detected variant c.107insg (val36gly>fs56ter) in heterozygosity on an otherwise common a1 allele, and in trans an abo*o.01.01 allele. it is unclear how the early truncation of the a1 transferase encoded by allele abo*107insg still allows for some residual enzyme activity, as suggested by the reverse a type. case #2 is a recently-transfused 72 year-old black patient with an unresolved abo type. sequencing detected variant c.297a>g (silent) in homozygosity and variant c.1049c>t (ala350val) in heterozygosity, both on an o1 background, with an abo*b.01 allele in trans. although variants c.297a>g and c.1049c>t are likely of no consequence to the abo phenotype of this patient, they are reported here as components of a new abo*o1(297g,1049t) allele. case #3 is a 40 year-old prenatal female with a rhd typing discrepancy. failure to yield an abo genotype on blood-chip (progenika), a genotyping microarray that interrogates polymorphic positions in rhd and abo, prompted dna sequencing. sequencing of genomic dna and cloned abo exon 7 detected variant c.436c>t (arg146cys) in heterozygosity on an abo*b allele background, and in trans an abo*o.01.01 allele. the phenotype encoded by allele abo*b(436t) is predicted to be b, as evidenced by forward typing on immucor neo and reverse manual typing. case #4 is a prenatal black patient with an abo typing discrepancy: forward type o in gel, a 2+ mixed field (mf) in tube. reverse type on a1 cells 1+ in gel, 0/1+ in tube. sequencing of genomic dna and cloned pcr products covering exons 6-7 detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*o.01.09 allele. case #5 is the newborn baby of case #4, with a forward type a 1+ mf in gel, a 3+ mf in tube. sequencing of the baby's dna detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*b.01 allele. from these results it is inferred that the phenotype encoded by allele abo*784c is a3-like. case #6 is an 18 year-old hispanic donor with an abo typing discrepancy: forward type a, reverse type o. sequencing of genomic dna and abo exons 5-6 and 6-7 detected variant c.979c>t (gln327ter) in heterozygosity, and in trans an abo*o.01.02 allele. the truncation of the a1 transferase at such a relatively late position is consistent with the retention of some enzyme activity, explaining the forward a type encoded by allele abo*979t. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. variants reported to date in the intron 1 enhancer include large deletions, small deletions and single-nucleotide substitutions. here we describe four new alleles with single-nucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. adsorption-elution by the heat elution method and testing for h and a substances in saliva were performed by following the procedures in the aabb technical manual. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. background: inactive alleles of the fut1 could be decreased or aborted the activity of the fucosyltransferase, which results in to form the bombay or para-bombay phenotype with weak or no h antigen expression on erythrocytes. now many para-bombay individuals have been found in the chinese population. according to names for h blood group alleles v5.1 170221 of red cell immunogenetics and blood group terminology working group of the isbt, 55 fut1 alleles were identified for bombay or para-bombay phenotype around the world. aims: the study was explored the distribution of fut1 alleles for the 19 chinese individuals with para-bombay phenotype. methods: the samples were come from the blood donors or the patients. the a, b, h antigens were determined using conventional serological method according to the manufacture's instruction. the sequences of the full coding region for fut1 was amplified, then amplicon was purified with enzymes digestion and used as template for sequencing bidirectionally. all nucleotide sequences obtained were analyzed and compared with standard fut1 sequence. results: nineteen chinese individuals with para-bombay phenotype were identified. ten of them were the donors and nine individuals were come from the hospital. the rbcs had a very weak agglutination reaction with anti-h in the most of the individuals. fut1 homozygous mutations were found in the 12 individuals and fut1 heterozygous changes were existed in 7 individuals after bidirectionally sequencing. .05%, 7.89%, 5.26%, 5.26%, 5.26%, 2.63%, 2.63%, 2.63% respectively in the 19 individuals with para-bombay phenotype. according to our previously reports, the fucosyltransferase activity of fut1*01n.06(c.551_552delag), fut1*01w.09 (c.658c>t) and fut1*01w.01 (c.293c>t) were abolished in vitro assay, while fut1 mrna levels of them had no effect compared with wild type. summary/conclusions: the fut1 mutations in the para-bombay individuals were various. the most common fut1 allele in the chinese individuals with para-bombay phenotype was fut1*01n.06(c.551_552delag). background: the regulatory mechanism of the abo gene is complicated and has been investigated extensively.variation in a antigen expression was recognized very early in the twentieth century and the a blood group was divided into a1 and a2. later the a blood group was subdivided further based on characteristic reactivity with human polyclonal antisera, i.e., strength of reactivity and presence of mixed field agglutination; presence of anti-a1, and whether a or h blood group substance was present in the saliva of secretor subjects. mutations critical for abo blood group phenotypes have predominantly been found in exons 6 and 7 of the abo gene, both of which encode the catalytic domain of abo glycosyltransferase. in our case report we show how mutation ranging from single nucleotide in the intron 1 enhancer element can alter the efficacy of enzyme and alter antigen expression. aims: this study aims to investigate the molecular basis of discrepant results of abo forward/reverse typing in blood donor. methods: the abo typing was performed using tube technique and column agglutination tests (bio-rad, grifols). standard tests were completed with adsorption-elution study using o plasma as a source of anti-a, and with saliva testing for presence of a and h substances. we performed quality control for these methods. abo group genotyping was performed using pcr with sequence-specific primer by commercial kit (abo-variant; bag healthcare, lich, germany). pcr-amplified exons and intron 1 enhancer were subject to bi-directional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results: standard serological forward tests identified blood group o, however, only anti-b iso-agglutinins were present. anti-a in adsorption-elution study was successfully adsorbed and eluted from the investigated cells. a and h substances were detected in saliva. abo genotyping using pcr-ssp indicated genotype o1v/a 1 . dna sequence analysis showed result abo*a01 (28+5859a), abo*o.01.02. the specimen was revealed as an a subgroup, probably a m with an unusual genetic variant in the intron region of the abo gene, the enhancer of the gene expression. summary/conclusions: we report the first case of abo*a01(28+5859a), the mutation located in the enhancer region of gene expression in allele a, that causes discrepant results not only in abo forward/reverse typing but also in molecular blood grouping tests. based on our serological findings, this subgroup is considered as a m . background: a chimera is a single organism composed of cells with distinct phenotypes and/or genotypes. several different types of chimeras are described: artificial, twin and dispermic. the artificial chimerism can be seen following hematopoietic stem cell transplantation, or more transiently following blood transfusion. the second type may also be inherited most commonly through blood exchange in utero between twins. dispermic chimerism is induced by the fertilization of two maternal eggs with two spermatozoa and their fusion into one body. this one is also called tetra-gametic chimerism. in transfusion medicine, chimeras are often detected when mixed field reactivity is observed in abo/d typing or, less commonly, when phenotyping for other blood group antigens. aims: this investigation was prompted by finding a double population of erythrocytes in a surgery patient with no transfusion history. our aim was to investigate the chimera and determine the underlying abo genotype of this patient. methods: routine blood grouping was performed by column agglutination. separation of the double cell populations was performed by differential agglutination with igm anti-d (immuclone, anti-d fast igm, clone: d175-2, immucor). initial abo genotyping was performed by pcr-ssp (fluogene; inno-train diagnostik gmbh); further resolution was performed using in-house pcr-asp and pcr-rflp methods. next generation sequencing (monotype abo; omixon using illumina sequencing platform) and sanger sequencing analysis were also performed. identification of reference alleles was investigated by fragment analysis of short tandem repeats (str) polymorphisms. results: double population was found in column agglutination in tests with anti-a and anti-ab, and subsequently when typing for d and c antigens, with approximately 85% of o d+c+ cells. the patient's genotype was identified as abo*o.01/*a by ce-certified pcr-ssp kit (fluogene). routine pcr-asp and pcr-rflp could not resolve the patient's genotype possible abo*a1/*o1 genotype was detected by pcr-rflp, but the pcr-asp analysis gave an apparent abo*a1 homozygote result. sanger sequencing of abo exons 6 and 7 also gave anomalous reactions: no abo*a allele was detected. homozygosity for c.261delg was observed as well as heterozygosity for c768c/a. this result therefore suggests the patient's genotype is abo*o.01/*o.01.26. next generation sequencing (omixon) revealed the same result. however, when pcr amplification of the cbf/nf-y enhancer vntr 3 0 -region was performed, possible heterozygosity was observed, i.e. a weak band representing a single copy, and one representing 4 copies of the enhancer region were present. presence of a single copy of the 43-bp cbf/nf-y enhancer vntr region is unique background: del is a very weak form of d antigen with low density expression of d antigen on the surface of red blood cell, which is generally typed as d-blood group as couldn't form agglutination in routine rhd blood group testing and could only be detected by the non-routine adsorption-elution test. in the east asian and southeast asian population, 9-30% of the individuals with serologically apparent d-phenotype are not these with truly d-phenotype, but del phenotype, which is very rare in caucasian and black ethic groups. and the rhd*01el.01 (rhd*1227a) is most prevalent (>99%) in del people in these regions, so the del carried this allele was commonly known as "asia type" del. in previous studies, no alloanti-d was observed in a large cohort of chinese "asia type" del pregnant women with d+ fetus to indicate no occurrence of alloanti-d immunization against d+ red cell in "asia type" del individuals. aims: to conduct genotyping analysis in the chinese patients having serologically apparent d-phenotype simultaneous with alloanti-d to confirm the existence of the "asia type" individuals to produce alloanti-d or not. methods: from 2017 to 2018, the blood sample of the patients or pregnant women identified with alloanti-d in our reference lab were collected. d antigen was confirmed again using the blend anti-d reagent (clone th-28/ms-26, igm/igg) by tube method in saline and indirect antiglobulin test (iat) in gel card. the zygosity of rhd gene was detected by hybrid rhesus box pcr with psti digestion. for the samples with d or dd genotypes obtained by rhd zygosity analysis, multiplex ligation-dependent probe amplification (mlpa) genotyping was conducted for rhd genotyping analysis. results: a total of 129 serologically apparent d-chinese patients (female, n = 128; male, n = 1) with alloanti-d were identified. different titers of alloanti-d from 1:2 to 1:4096 (≤1:16, n = 60; >1:16, n = 69) were detected including few cases with mixed antibodies (anti-d mixed with anti-c, n = 11; anti-d mixed anti-e, n = 5). serological rhd typing confirmed the serologically apparent d-phenotype. rhd*01n.01/01n.01 (homozygous rhd gene deletion) genotype was identified in majority of them (123/129, 95.3%) by rhd zygosity analysis, while rhd*01n.03/ 01n.01 genotype (n = 5) and rhd*01n.05/01n.01 genotype (n = 1) carried the rhd non-functional hybrid alleles were detected by mlpa. summary/conclusions: compared with the distribution of average 25% frequency of "asia type" del in serologically apparent dpopulation in guangzhou of china, no one case of "asia type" del was identified in the cohort of serologically apparent d-patients with alloanti-d in this study. this also provides evidence to confirm no occurrence of alloanti-d immunization in "asia type" del individuals. aims: a serologically rhd-negative donor was found to be rhd-positive in the routine rhd screen. to solve the discrepancy between serology and molecular screen, the sample was sequenced on dna and rna level. methods: phenotyping on id/iat-cards (bio-rad) was done using commercial anti-d antibodies. the adsorption-elution analysis was performed using an in-house pool of polyclonal anti-d antibodies. furthermore an antibody d-screen was performed (diagast). for rhd genotyping rh-type and partial d-type assays (bag health care) were carried out. the sample was further characterized by exon sequencing including flanking intronic regions. rna was extracted from whole blood, reverse transcribed and the cdna sequenced. for amplification and sequencing, both published (gassner, transfusion, 2005; legler, trans. med., 2001; richard, transfusion, 2007) and in-house primers were used. results: repeated phenotyping of the sample with commercial as well as, in-house anti-d antibodies confirmed the rhd negativity. in addition, the adsorption-elution analysis showed a negative result. however, genotyping, using commercially available kits, yielded a rhd positive result and no variants were detected. to investigate this discrepancy, all 10 rhd exons were sequenced. the sequencing data revealed the mutation c.148+2delt in the splice donor site of exon 1. to confirm the effect of the splice site mutation on transcription, rna from a fresh whole blood sample was analysed. as a positive control, gypb was amplified and sequenced from the same cdna. wild-type gypb (mns4) was found. with rhd specific primers, no product could be amplified. summary/conclusions: we present a serologically rhd negative case, that was identified as rhd positive by standard commercial genotyping kits. sequencing revealed the new splice site mutation c.148+2delt. rna sequencing yielded no detectable product. the donor was classified as rhd negative. this case of a discrepant result between serology and genetics shows the importance of a profound and highly sophisticated genetic investigation of conflicting laboratory results. j stettler, s lejon crottet, h hustinx, c von arx, f still, j graber, c niederhauser and c henny interregional blood transfusion src berne ltd., berne, switzerland background: one of the most immunogenic and clinically significant blood group antigens in transfusion medicine is the rhd antigen. variant rhd phenotypes with weakened or absent antigen expression pose a challenge for rhd status assignment in blood donors. to ensure patient safety, it is necessary to fully characterize these variants at the molecular level. aims: samples from two donors were investigated in our laboratory due to discrepancy in rhd typing. methods: rh blood group phenotyping was done by standard serological column agglutination testing (id-system, biorad). further rhd characterization was performed by an anti-d antibody panel containing 9 monoclonal antibodies (d-screen, diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-d antibodies. molecular investigation was initially performed by ssp-pcr detecting common rhd variants (rbc-ready gene cde inno-train; rh-type bag health care). rhd sequencing was done on either dna or rna using published and inhouse primers for amplification and sequencing. results: by tube testing, the rbcs of donor 1 were predicted to be rh:-1,-2,-3,4,5. however, all ten exons of the rhd gene could be detected by routine genotyping. sequencing of rhd revealed a homozygous mutation c>g at position 1151 which is the second last nucleotide of exon 8 and thus might have an influence on exon splicing. by cdna analysis a transcript with a correctly spliced exon 8 was identified. the mutation c.1151c>g leads to the amino acid substitution t384r located in the twelfth transmembrane domain of rhd using the model of flegel (transfus apher sci., 2011) as reference. adsorption-elution testing using a pool of polyclonal anti-d showed a weak positive reaction, re-classifying the donor as rhd positive. this novel allele, rhd*1151g, could thus be categorized as a del allele. serological results displayed an almost normal rhd antigen expression for donor 2. further serological determination of the rhd antigen with 9 different antisera, however, showed a reaction pattern typically observed with a weak d variant. with commercial available kits no rhd variant could be detected. rhd sequencing revealed a novel homozygous mutation c.526g>c in exon 4. this mutation causes a p.a176p exchange in the sixth membrane-spanning domain of rhd. based on serological data, the donor is rhd positive and in case of transfusion the patient would be treated as rhd negative. summary/conclusions: here we report two novel rhd missense mutations c.1151c>g and c.526g>c harbouring an amino acid substitution within a transmembrane segment. the c.1151c>g variation displayed an unusual low rhd antigen reactivity and would have been mistyped as rhd negative without extensive genotypic testing. molecular analysis of variant c.1151c>g suggests that the t384r exchange causes a del phenotype rather than a miss splicing event. this was also confirmed by adsorption-elution testing. interestingly, variant c.526g>c could only be detected due to comprehensive serological and genetically investigation. background: the rh blood group system is highly polymorphic and one of the most clinically relevant systems in transfusion. actually d antigen is of critical importance due to its involvement in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. rhd gene variants are common in africans and mostly related to partial d phenotype. aims: rhd gene sequence was investigated in two african brazilian samples. we further attempted to take advantage of combining the molecular data and the available in silico tools for the functional interpretation of the variations, in order to get insights into the clinical phenotype that may be predicted a priori from genotyping. methods: sample #id1 is a d-negative donor self-declared as african descent. sample #id2 is a patient with sickle cell disease (scd) typed as d-positive with anti-d in his serum. serologic d typing was determined by manual gel test and by microplate in an automated instrument. sample #id1 was also submitted to adsorption/elution test. after genomic dna extraction, all ten rhd exons and flanking intronic regions from sample #id1 were pcr-amplified with rhd-specific primers and analyzed by sanger sequencing. sample #id2 was investigated by next-generation sequencing on the miniseq platform (illumina) by using a previously published, custom (selected blood group genes) ampliseq panel. a reported three-dimensional (3d) structural model of the rhd-rhd-rhag heterotrimer was used to visualize the position of variations and predict their putative functional/clinical effect. results: in sample #id1, a single nucleotide missense change, i.e. c.1151c>g in exon 8, was identified. this transversion is thought to replace a threonine by an arginine residue at amino acid position 384 (p.thr384arg) of the rhd protein. analysis in the 3d model clearly suggests a dramatic impact of the p.thr384arg substitution occurring in a functionally-critical, conserved motif in terms of interhelix interaction, which is supposed to be highly deleterious to the stability of the protein, and potentially impairs totally its expression at the red blood cell plasma membrane. this predicted functional effect is definitely in accordance with the d-negative phenotype reported in sample #id1. in sample #id2, the single c.325a>g transition was found in exon 2 leading to a threonine-to-alanine substitution at amino acid position 109 (p.thr109ala). amino acid 109 is located in rhd protein extracellular loop 2, and is thus thought to alter d antigen structure, resulting in a partial d phenotype. this hypothesis is in accordance with anti-d found in the serum of sample #id2. summary/conclusions: for the past years, due to the advent of next-generation sequencing and the subsequent identification of numerous rare variants, bioinformatics prediction and modelling tools have evolved and currently help physicians in diagnostics, clinical management and genetic counselling. we took advantage of some of those in silico methods to predict retrospectively the effect of two novel variant rhd alleles, including one d-negative and one partial d alleles. although phenotype and clinical symptoms remain definitely the standard determinants to assess the effect of genetic variations, use of those approaches may soon become valuable for guiding subsequent investigations in immunohaematology. abstract withdrawn. alleles of the weak d type 4 and diva cluster. in africans, the 16 most frequent were typically associated with alleles of the weak d type 4 (including dol and rhdpsi), diva and dau clusters with f223v occurring in > 10% of alleles; in addition the key mutations of weak d type 1 and dii and two inactivating mutations (c.1056_1057inst and c.1060delg) not reported in rhb were among the first 20 polymorphisms. in east asians, rhd(1227g>a) at 0.8% was most frequent, followed by dfv, weak d type 33, dbo-3, key mutations of diva and weak d type 4 cluster as well as rhce-like substitutions and the mutations of weak d type 25, type 15, type 66, rhd(a85v), dvl-1, weak d 119 and rhd(n135s). weak d type 151 and rhd(t148r) were frequent in south asia but not elsewhere. summary/conclusions: data from tgp and gnomad add relevantly to the knowledge on rhd alleles; tgd discloses linked intron polymorphisms, gnomad frequency data not biased by the likelihood of serologic detection. current typing strategies usually start with serology later complemented by molecular typing. in the future, molecular methods will gain importance and frequent alleles currently not distinguished from "standard rhd" may need a rational transfusion strategy. in this respect, the high frequency of weak d type 45 and type 33 in europeans was surprising, might warrant confirmation by alternative methods and should trigger discussion on rational transfusion strategies for these alleles. consistent with an r 0 haplotype and 1 probable dc-. two siblings that were abo compatible including the dc-sibling were incompatible at iat phase. reactivity could be completely adsorbed from the serum using r 1 r 1 , r 2 r 2 , and rr rbcs indicating the antibody is probably a single specificity. the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. aims: the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. methods: serologic testing was performed by manual tube testing using ahg in the indirect antiglobulin phase. rbc phenotyping was performed by standard tube hemagglutination testing from edta anticoagulated blood. rhd and rhce exons were sequenced using genomic dna and standard sanger dideoxy method with the bigdye terminator v3.1 cycle sequencing kit. sequence data was aligned to rhd_ng_007494.1. rhd zygosity was performed using pcr-rflp with mspi. background: according to recent findings in molecular immuno-hematology, rhd genotyping is strongly indicated in rhc+ and rhe+ donors classified in routine as d-negative. among these, one could find a non-negligible share of entirely new genetic alterations or even del alleles, which are often not identifiable with routine serological methods due to the low number of antigenic sites. aims: the present study reports the genotyping data of rhd on 201 rhc+ and rhe+ caucasian donors classified serologically as d-negative, all enrolled by a single transfusion center in italy methods: rhd serological typing was carried out in microplate direct agglutination tests (iris, immucor) by using 2 different anti-d igm clones (clone 1, dvi+: ldm1+esd1m; clone 2, dvi-: rum-1, th28) and 2 different anti-d igg clones (clone 1: ms26; clone 2: d415 1e4). all donors with d-negative results (n = 201, divided into 153 subjects with rhc+, 45 with rhe+ and 3 with both rhc+ and rhe+) were addressed to genotype analysis with rhd beadchip molecular test (immucor), pcr-ssp (bagene, inno-train) and/or rbc-fluogene (inno-train). the discrepant results between serology (d-neg) and molecular biology (wild-type or full-length rhd gene) were further investigated by bi-directional sequencing of the rhd coding regions. results: one-hundred donors have been analyzed retrospectively, as part of a pilot study. following the data obtained in this first phase, the analysis methods described above have been implemented in routine, allowing to include further 101 donors, studied prospectively. in 92.5% of donors (n = 186), the molecular analyses showed the complete deletion of the rhd locus, while in 15 cases (7.5%) a genetic status was found with "non-deleted" rhd. over all, bi-directional sequencing on these 15 donors revealed the presence of 9 negative and 4 weak-d variants. the list of rhd alleles we have identified at the molecular level is as follows: rhd*01n.82 (2 cases), rhd*01n.83 (1), rhd*01n.80 (1), rhd*01n.61 (1) , rhd*01n.05 (3), rhd*01el.08 (1), rhd*01el.18 (1), rhd*01el.17 (1), rhd*01w.54 (1) . moreover we found a donor with a lack of signal encompassing exons 1-5 of the rhd sequence (bioarray rhd beadchip), while 2 additional cases are currently under investigation. summary/conclusions: our study confirms that a non-negligible number of caucasian subjects, classified serologically as d-negative, present rhd gene alterations that differ from the common total deletion. in line with the literature data, we also found a frequency of about 1 in 50 cases (4 subjects out of 201), in which a donor re-classification as d-positive (weak d type) was necessary. hence, a wider use of molecular typing methods is desirable in order to achieve the correct genetic characterization and the appropriate phenotypic classification of "apparently" d-negative donors. background: without evidence of abnormal serological d antigen expression there will be no quest for weak d, partial d or d variant on the red blood cells. according to our blood donor registry we found that out of 43960 serologically typed donors, 85.5% were d+, 14.0% d-and 0.5% weak d. aims: to compare different weak serological reactions of the d antigen to the rhd genotyping. methods: molecular rhd typing using isolated dna and rbc-ready gene cde and rbc-ready gene d weak kits was performed in 22 blood donors, who were serologically typed as weak d using monoclonal blended igm/igg and dvi-and dvi+ anti-d reagents by slide and microplate (mp) technique respectfully, as well as by the antiglobulin test (iat) in gel and with the set of monoclonal partial d typing reagents (biorad). in addition, rhccee phenotyping and genotyping was also performed. results: all of the donors with serologically weak reactions were confirmed to be weak d variants by genotyping except one donor whose iat was false positive due to rbc autoantibodies. the frequency of d variant genotypes was as follows: 62% weak d type 1, 28% weak d type 3, one donor was typed as weak d type 14 and another one as weak d type 11. these weak d types were associated with different degrees of serologically determined weakness ranging from negative to weak positive reactions concerning slide and mp. all of them gave positive reaction ranging from 2+ to 4+ with iat, except for the weak d type 11 with the score of <1+ which gave negative reaction by slide and mp and inconclusive result with the set of monoclonal anti-d reagents for partial d typing. the percentage of donors, who, at serological typing were only found to be d positive in the iat was 19%. one of the weak d type 1 donors was negative with dvi-and positive with dvi+ reagent in the mp. the additional rh phenotype (genotype) was ccee in all of the donors except in the one who was genotyped as weak d type 14, as well as in the d negative donor, being ccee. summary/conclusions: further rhd genotyping is required to estimate the actual frequency of d variants in our blood donors. in practice, current serological methods are sufficient to detect almost all variant d phenotypes. there is a consensus that routine molecular d antigen screening in d negative donors in order to detect del variant when ddccee phenotyped red blood cell transfusion is practiced in all d negative patients does not seem to be cost-effective. background: rh null or rh mod -the so-called rh-deficiency phenotypes-are characterized by a null or severely reduced rh antigen expression (including d, c/c and e/ e), respectively. molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. the former is caused by homozygosity for silent genes at rhd and rhce loci, caused by inactivating mutations in rhce and deletion of rhd. on the other hand, the regulator rh null type as well as the rh mod phenotype are attributed to mutations in rhag gene when in homozygous state or when in heterozygosity with another rhag allele containing an inactivating mutation. a functional rhag is essential both for the correct rh complex assembly and rh antigen expression in the erythrocyte membrane. aims: the aim of this study was to investigate the molecular genetic basis of an argentinean proband with no detectable d, c, c, e and e antigens by standard serological techniques. methods: blood samples were collected from the proband, her parents and sister. the proband was a 14 year-old young woman with parameters of hemolytic anemia: low hemoglobin level (10 g/dl), reticulocytosis (14%), hyperbilirubinemia, increased ldh and marked spherocytosis. the d, c, c, e and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. genomic dna was isolated using a modified salting-out method. dna samples were initially screened for the presence of intron 4 and the 3 0 untranslated region of the rhd gene using pcr strategies. rhc/c, and rhe/e alleles were studied by allele-specific pcrs to determine the rhce genotype. rhd zygosity was analyzed by pcr-rflp. rhd exon polymorphisms were studied by rhd exon scanning procedure based on pcr-ssp. rhag gene was investigated by exon-specific pcr amplification and sanger sequencing. results: no d, c, c, e and e antigens were detected in the proband's erythrocytes. the father and sister rh phenotype was: d+, c+, c+, e+, e+ whereas the mother rh phenotype was: d+, c+, c-, e-, e+. rh genotyping confirmed the rh phenotypes for all family members except for the proposita who genotyped rhd+, rhc+ and rhe+. all samples showed an homozygous status for the rhd gene and all rhd exons were detected by exon scanning. sequencing analysis revealed an homozygous c.920c>t mutation in rhag exon 6 in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. the c.920c>t mutation is responsible for the p.ser307phe amino acid substitution predicted to be in the 10 th rhag glycoprotein transmembrane segment. summary/conclusions: this study described the molecular background responsible for an rh-deficiency phenotype in an argentinean proband. we identified the novel missense mutation c.920c>t in the rhag gene which results in the ser to phe single amino acid substitution that shows to be critical for rh antigen complex assembly within the erythrocyte membrane. further studies are being performed in order to determine whether the proband is rh null or rh mod . background: rh blood group system is the most immunogenetic blood group system and blood donor typing should account for all expressing antigens in order to prevent anti-d alloimmunization. aims: the objective of this prospective study was to investigate rhd alleles among blood donors who typed d-by serologic methods and positive for c and/or e. for this reason we developed an easy-to-perform dna-based screening method for the detection of rhd gene and positive samples were further characterized by two commercial pcr-ssp kits. methods: of 15692 individual blood donors within a 15 month period, 1688 (10.76%) typed as d-with standardized immunohematologic methods including the indirect antiglobulin test (iat). residual edta-anticoagulated blood samples were used to isolate genomic dna using the qiaamp dna blood kit (qiagen, germany) from 112 out of 151 (8.95%) c/e+ and serologically d-donors. all dna samples were tested individually for the presence of rhd-specific dna sequences in the rhd promoter, intron 4, exon 7 and exon 10 by a multiplex pcr-ssp method. the reaction was conducted in a final volume of 20 ll with primers that were applied as described by f. wagner et al. (bmc genetics, 2001 ) except antisense primer for exon 10 and the two primers amplifying an hgh gene fragment as internal control, designed by our laboratory. pcr products were visualized by electrophoresis on a 4% agarose gel with ethidium bromide staining. in case of a positive reaction the sample was analyzed by pcr-ssp d weak and pcr-ssp cde (inno-train, germany). results: out of 112 d-individuals analyzed, 101 were ddccee, 8 ddccee, 2 ddccee and one had a ddccee phenotype. molecular analysis showed that 104 (92.86%) were negative for all four rhd dna regions. among the other 8 samples, all of ddccee phenotype, three were found to be positive for rhd promoter, intron 4, exon 7 and exon 10, three for rhd promoter and exon 10, and two for exon 10 alone. further genotyping revealed five hybrid rhd-ce-d alleles [3 rhd-ce(2-9)-d and 2 rhd-ce(3-7)-d], one allele represented the del(m295i) genotype, while the remaining two samples did not show an allele that could be determined with the pcr-ssp kits. summary/conclusions: serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples that show weak reactions in d. a rhd genotyping strategy is needed to confirm d-blood donors and thus to avoid anti-d immunizations. for these reasons we suggest the implementation of an easy and possible cost-effective method. background: more than 100 weak d types have been described to date. transfusion recipients with weak d type 1, 2, or 3 are not at risk for forming allo anti-d when exposed to conventional rh d-positive rbcs. molecular analysis of weak d offers a more reliable basis than serotyping to determine the prevalence of weak d types and optimal d transfusion strategies. background: the d antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. most people are either rhd-positive or rhdnegative, but there is a certain number of people who have a variation of the d antigen, which are called weak d, partial d and del phenotypes. aims: the objective is to use molecular methods to determine whether blood donors in republika srpska (with whom a serological weak d antigen has been detected) really have the weak d antigen. in addition, determine whether blood donors, who have been determined as persons who are rhd-negative, with the phenotypes c and/ or e, who have the rhd gene and d antigen on the erythrocyte membrane, so weak that it could not be determined by serological techniques. methods: blood samples were used from regular blood donors, who have been determined as persons with a weaker d antigen, as rhd-negative or as c and/or e positive (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. gp.mur was also modelled and shown to closely resemble the tertiary structure of glycophorin a. the predicted structure is 5 anti-parallel b sheets arranged in a "b barrel" also referred to as an ob-like-fold. the regions in which blood group antigens were identified in the predicted stable dimeric structure. summary/conclusions: ob-like-fold structures typically to bind oligonucleotides or oligosaccharides and are associated with cold shock proteins. further modelling is in progress to predict the structure of gpa/gpb heterodimers as a basis for understanding the presentation of blood group antigens. of interest, this finding is consistent with a previous report showing that this gpa binds to carbohydrates. this model serves as a foundation for future work regarding the properties of gpa, which includes identifying locations of specific interactions between gpa and other rbc surface proteins such as gpb and band 3, as well as identifying structural features of antigenic regions on gpa. . even though no significant differences were found among the groups studied, 4 haplotypes containing the mcc b and sl2 polymorphisms were identified in d samples but were not found in tb and l groups. summary/conclusions: this preliminary data obtained suggests that cr1 polymorphisms and haplotypes, especially those containing mcc b and sl2 snps, could be involved in the disease pathogenesis of tuberculosis and leprosy. the entrance of mycobacteria into macrophages is mediated by complement receptors that facilitate their uptake by host cells so the combined haplotypes could be enhancing parasite phagocytosis and inflammation. further studies are being carried out to establish whether cr1 polymorphisms are risk or protective factors and whether other genetic variations in this receptor are also involved. abstract withdrawn. background: the dombrock blood group system consists of two antithetical antigens, do a and do b , and three high-prevalence antigens, gregory (gy a ), holley (hy), and joseph (jo a ). the rare do null or gy(a-) phenotype lacks all dombrock antigens, and the do null alleles vary with both do*01 and do*02 backgrounds. here we report the molecular basis of a novel do null allele in a gy(a-) brazilian patient with anti-gy a . aims: case presentation: an alloantibody to a high-prevalence antigen was detected in the serum of a 42 year old woman from the northeast brazil with a history of 4 pregnancies but no history of previous transfusion. she required transfusion because of a schedule for total thyroidectomy surgery due to a large compressive nodular goiter. the antibody did not react with the autologous rbcs but reacted by the indirect antiglobulin test in liss with all panel rbcs and other rbc samples tested except with the gy(a-) phenotype. the corresponding antigen was resistant to treatment with papain but sensitive to dtt and trypsin. these results suggested that the antibody recognized an antigen in the dombrock blood group system. the purpose of this study was to identify the antibody specificity and to determine the molecular basis of the phenotype detected. methods: the red cells phenotype and the presence of the dombrock related antibody in the serum were detected by standard hemagglutination techniques. rbcs and antibodies were from our in-house collection of rare samples. genomic dna was prepared from peripheral blood of the patient. dombrock genotyping was performed by id-core xt platform (grifols, spain). the 3 exons of the do gene were amplified by pcr and directly sequenced. experimental immunohematology and diagnostic immunohematology 2 diagnostic immunohematology 3 experimental immunohematology, sanquin, amsterdam, netherlands background: typing of blood group antigens is essential to prevent transfusion reactions or haemolytic disease of the foetus and newborn. to date, the isbt recognises 360 blood group antigens. most antigens (322) belong to one of the 36 blood group systems. since the genetic basis of these systems is known, genotyping of these antigens is possible. the molecular background of 38 antigens is unknown and can only be determined serologically. one of these antigens is sd a (sid), first reported in 1967.~91% of the population carry sd a on erythrocytes, but this frequency might be higher since identification is difficult due to variability in expression. in 96% of individuals sd a is present in urine. cells with a high expression of sd a (cad/sda++) are used for detection of antibodies. recently, a 3-cells antibody detection panel of bio-rad contained a sda++ cell and many individuals with anti-sd a were detected. the b4galnt2 gene has been implicated in the synthesis of sd a . we collected individuals with and without anti-sd a to elucidate the genetic background of the antigen. aims: elucidation of the genetic basis responsible for loss of the sd a antigen on red blood cells. methods: routine diagnostics to identify antibodies in patients was performed using a bio-rad 3-cells panel, containing donor 626521 with high expression of sd a . additionally, 200 pregnant women were screened for anti-sd a . dna of eight samples with anti-sd a and eight samples without anti-sd a was isolated for further analysis. sanger sequencing was performed on b4galtnt2 exon 1-11. results: sequencing of b4galtnt2 revealed two homozygous mutations which are present in all eight individuals with anti-sd a , but not present in 6 controls. the remaining two controls are heterozygous for these mutations. the first mutation within exon 10, c.1396t>c (enst00000300404.2, rs7224888) changes a cysteine to arginine at position 466 of the protein. the second mutation in exon 11 c.1590a>g (rs16946912) does not change an amino acid. both snps have a maf of 0.11 and therefore we expect that 2.1% of the population is homozygous for the minor allele. genotyping of a large population of pregnant women and the serological detection of anti-sd a in women with a homozygous mutation is in progress. summary/conclusions: the high frequency antigen sd a has not been linked to a blood group system because the molecular basis for loss of the antigen has not been elucidated. the b4galtnt2 gene has been associated with sd a synthesis and therefore we analysed this gene for mutations in individuals with antibodies against sd a . a single homozygous mutation within exon 10 causing an amino acid change was found in all individuals with anti-sd a , and no individuals without antibodies were homozygous for this snp. from population studies we expected~4% sd a -negatives, but either this frequency is an overestimation because of difficulties to detect low expressed antigens or mutations in other genes are interfering with sd a synthesis. a larger study of individuals with homozygous mutations in b4galnt2 and linkage to sd a -negativity and presence of antibodies will be performed before sd a can be assigned to a new blood group system. abstract withdrawn. abstract withdrawn. background: erythrocyte duffy blood group antigen can scavenge chemokines in whole blood. duffy blood group gene consists of two major alleles: fy*a and fy*b. however, little is known regarding the association of duffy blood group polymorphisms with the red blood cell (rbc) chemokine scavenging. aims: the aim of this study was to determine the association of duffy blood group polymorphism with the rbc chemokine scavenging. methods: the duffy blood group were genotyped by 5ˊ-nuclease assay in healthy chinese han individuals, while erythrocyte chemokine scavenging function and duffy antigen expression from the same samples were measured using erythrocyte chemokine binding assays and quantitative flow cytometry respectively. results: rbc chemokine scavenging of cxcl8 was significantly lower in the individuals with the fy*a/fy*a genotype compared to those with fy*a/fy*b genotype (p = 0.016). similar result was also observed in rbc chemokine scavenging of ccl2 (p = 0.038). the expression of duffy antigen on rbc surface in the individuals with the fy*a/fy*a genotype was significantly higher compared to those with fy*a/ fy*b genotype (p = 0.021). summary/conclusions: duffy blood group polymorphism is associated with the differential rbc chemokine scavenging. it is probable that a change in duffy antigen structure caused by duffy blood group polymorphism is responsible for the differential rbc chemokine scavenging. background: individuals with p-phenotype can develop a naturally occurring anti-pp1pk and has clinical significance, causing hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. finding and procuring blood units of pphenotype is a challenge because of its rarity throughout the world. therefore, acute normovolemic hemodilution (anh) can be an on hand tool in the perioperative successful management of patient with rare blood group. however, this approach has not been commonly used aims: n/a. methods: n/a. results: a 66-year-old korean woman was referred to samsung medical center for surgical management for gallbladder malignancy. her blood type was group a, d-positive. the patient had no known history of transfusion. however, antibody screening and identification test using the column agglutination method (bio-rad, cressier, switzerland) showed panagglutination with negative reactions to autologous red blood cells, indicating the presence of alloantibodies to high frequency antigens. the specimen obtained from the patient was sent to the central laboratory of the swiss red cross (bern, switzerland) and confirmed as anti-pp1pk. at first, the transfusion team of our hospital recommended the surgical team to postpone the surgery. however, anh was planned because postponing surgery was not preferred and the patient's preoperative hemoglobin was 12.9 g/dl. 800 ml of blood was withdrawn through a radial arterial catheter in two 400 ml blood bags containing citrate-phosphate-dextrose-a solution after anesthetic induction. equal volume of 6% hydroxyethyl starch solution was infused during the procedure. the patient underwent radical cholecystectomy and liver wedge resection with lymph node dissection, and two units of autologous blood were returned to the patient during surgery. she was then discharged 48 h later with a hemoglobin level of 12.3 g/dl. later, the family study was performed with the standard serologic method using the proband's plasma containing anti-pp1pk and sequencing of the a4galt gene, which were conducted according to the protocols by koda et al.(transfusion. 2002) . the proband and her brother were homozygous for c.1029dupc, indicating a rare p phenotype. summary/conclusions: we experienced that autologous blood transfusions via anh is an alternative to allogenic rbc blood transfusion in patients who have no blood available because of high alloimmunization antibodies against rare blood groups. " and the third sample as "gypb*s_gyp*[140a], gypb*s_null(ivs5 + 5t)" with a predicted phenotype: s-s+ mi a + and s+s-mi a +, respectively. the gypa specific primers used for discrepancy resolution detected the nucleotide substitution, gyp.c.140c>a, in gypa-b-a hybrid associated to gp.hut allele, thus confirming the id core xt result. the expression of mi a for one of these samples was confirmed using non-commercial anti-sera. hence, these three samples were not gp.mur but gp.hut phenotype. both alleles codify for the expression of mi a antigen since it is expressed on several hybrids between the usual forms of glycophorin a and b. two of these three gp.hut samples are african-american donors. gp.hut was reported in white people with a frequency about 0.06% and in thais with 0.04%. these three gp.hut cases found by id core xt in this study point to a higher frequency of this glycophorin variant and also to the presence in african american population. summary/conclusions: id core xt was able to detect two glycophorin phenotypes, gp.mur and gp.hut, which codify for the expression of mi a antigen. standard molecular methods should be implemented in pre-transfusion testing and obstetrical care routine to detect the most clinically relevant glycophorin variants in mns system. background: serf(+) is a high prevalence antigen in the cromer blood group system, which is encoded by a crom*12 allele. the lack of the serf antigen, serf(à) on red cells is caused by a single nucleotide polymorphism, c.647c>t in exon 5 of the decay-accelerating factor, daf gene. alloanti-serf has been found in thai pregnant woman with serf(à) and a serf(à) individual was found among thai blood donors. anti-serf is not a marketed product; hence, a molecular technique has to be implemented to genotype for the crom*12 allele among blood donors. aims: this study aimed to identify the crom*12 allele among thai blood donors leading to predicted serf(+) and serf(à) phenotypes. methods: dna samples obtained from 1,512 central thai blood donors were genotyped for serf allele detection using in-house pcr with sequence-specific primer (pcr-ssp) and confirmed by dna sequencing. results: the allele frequencies of crom*12(+) and crom*12(à) among 1,512 central thais were 0.992 (3,001/3,024) and 0.008 (23/3,024), respectively. the homozygous of crom*12(à/à) alleles was not found in this study. additionally, the pcr-ssp technique was validated by dna sequencing using randomly chosen 100 samples together with 23 heterozygous crom*12(+/à) samples and the results were in agreement. summary/conclusions: our results confirm a high frequency of the crom*12(+) allele in the thai population and their frequencies were similar to those formerly reported among thai blood donors. this study would be beneficial to predict the serf antigen from genotyping results due to unavailability of commercial antiserum. background: there is increasing interest in the use of molecular methods for predicting abo grouping. though nextgen and sanger sequencing have both been used to predict abo type, predicting abo type from buccal swab-derived dna and from deceased donors benefits from a quick and reliable method. besides a pcr-rflp that has been used by many labs for more than 25 years, there is a commercially-available research use only (ruo) kit, and both interrogate nucleotides associated with o1, o2, a2 and b with a representing the ancestral allele. aims: the aim of this report is to compare two low-resolution polymerase chain reaction (pcr)-based methods, for investigation of samples submitted to a reference molecular immunohematology laboratory for abo typing discrepancies. fifty-six peripheral blood samples were tested, 31 from patients and 25 from blood donors. methods: genomic dna was isolated from peripheral blood mononuclear cells. background: del is the weakest known d positive phenotype in the rh blood group system and detectable only by adsorption and elution tests. the rhd1227g>a change is an important marker for del phenotype in east asians. a rapid and efficient pcr method for rhd gene 1227 g>a genotyping is useful in east asian countries. aims: the aim of this study was to develop a method for rhd 1227g>a genotyping by using single-tube pcr with melting temperature(t m )-shift primers. methods: two allele-specific primer for rhd 1227g>a and a common primer were designed and synthesized. two gc-rich tails of different lengths were attached to 5 0 ends of the allele-specific pcr primers. single-tube pcr with t m -shift primers was carried out with the three primers. after pcr, melting curve analysis was performed. rhd 1227g>a could be genotyped by differences of the t m s of the pcr products. all of genotyping results were compared with those obtained from conventional pcr-ssp. for the discordant results, rhd exon 9 sequencing was performed to determine rhd 1227g>a genotype. results: a total of 84 samples were genotyped for rhd 1227g>a by pcr with t mshift primers. 32 samples were typed as 1227a+/g-, 22 samples were typed as 1227a-/g+, 6 samples were typed as 1227a+/g+ and 24 samples were typed as 1227a-/g-. two samples typed as 1227a+/g+ by pcr-ssp but 1227a+/g-by pcr with t m -shift primers were confirmed as 1227a+/g-by rhd exon 9 sequencing. summary/conclusions: the single-tube pcr with t m -shift primers for rhd 1227g>a genotyping is simple, rapid, accurate, and it is superior to conventional pcr-ssp. abstract withdrawn. background: the rh blood group system has numerous variant alleles, which may affect rh antigen expression, including rhd-rhce (d-ce) hybrid genes. these variant alleles are frequently found in people of african descent, and typically result in either d-negative (d-) phenotype, or partial d antigen expression, including silencing of high-frequency antigens and/or expression of low-frequency antigens. patients carrying those alleles are particularly at risk of alloimmunization, suggesting that their identification is important in diagnostics. quantitative multiplex polymerase chain reaction (pcr) of short fluorescent fragments (qmpsf) has proven successful for genotyping those dna samples carrying d-ce hybrid genes by assessing both qualitatively and quantitatively rhd and rhce gene exons. aims: the aim of this project was to genotype both rh genes in a cohort of brazilian patients with sickle cell disease (scd), which are known to be of african descent, by using the qmpsf approach and report hybrid gene variability in this population. methods: one-hundred fifteen dna samples were selected for the study and analyzed prospectively by the rhd-qmpsf and rhce-qmpsf approaches to investigate the copy number of all exons in both rh genes. genotypes were further confirmed or investigated by sanger sequencing and conventional pcr-rflp assays. results: in the 115 dna samples, 75 (65.2%) exhibited a "wild-type" profile by qmpsf analysis. hybrid genes involving exon 8, which is functionally not relevant as reported before, was found in 28 samples, including 11 and 17 samples carrying respectively rhd-ce(8)-d and rhce-d(8)-ce (two homozygous each). except two samples that require additional studies (1.7%), rhd zygosity was resolved successfully: 60 (n = 2 rhd gene copies; 52.2%), 49 (1; 42.6%) and 4 (0; 3.5%). clinically relevant, i.e. partial d, genotypes were identified in four hemizygous samples (4/115, 3.5%) carrying rhd*dau5, rhd*dv.2, a rhd*diiia-like allele, and a novel rhd*d-ce(7:g329h-y330s-n331i)-d allele, as confirmed by sequencing. other hybrid alleles, such as rhd*03n.01 and rhd*diiic, were also found in trans with a normal rhd*01 allele. in rhce, c/c genotype could be resolved. the rhce*ce16 (rhce*ce (48c)-d(9)-ce) allele, which is commonly cis-associated with rhd*ψ, was observed in four samples. however the clinically relevant polymorphisms in variant rhce alleles, such as those involved in cemo, cear, ceag, and ceti, were mostly identified by other standard methods. summary/conclusions: although most of the brazilian patients with scd investigated in this study did not carry rhd-rhce hybrid genes, qmpsf analysis has been shown to be an efficient tool in the whole genotyping process to investigate rh gene variation. as previously reported, it has been conclusive for characterization of rhd zygosity and identification of rare, as well as novel, variant alleles. additionally, our results show a large diversity of hybrid genes among the brazilian patients with scd. therefore, we suggest that qmpsf may be used as a complementary screening approach for assessing rh genotype in selected patients and donors. = 19) vs. non-bleeding (n = 15) patients. platelet, pmp and cp phenotype and function were evaluated by flow cytometry: activation and granule release were examined by antibodies against granulphysin (cd63), p-selectin (cd62p), activated gpiib/iiia (pac-1) and phosphatidylserine (ps) (lactadherin) unstimulated and adp, trap or collagen stimulated. coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and gpiba (cd42b). normal healthy reference levels were available. results: the platelet count in bleeding (72 9 10 9 /l) and non-bleeding (68 9 10 9 /l) patients was comparable (p = 0,66). bleeding patients had a higher bat score compared to non-bleeding patients (10 vs. 2, p < 0,01). the proportion of cps was normal in all patients. however, in non-bleeding patients the proportion of ps+cps and per cell ps expression (mfi) (86,16% and 4,96mfi) were higher, compared to bleeding patients (64,08% and 2,44mfi, both p < 0,05), and the proportion of ps+cps correlated negatively with bat score (r 2 =0,26, p < 0,01). cd63 + cp was higher in non-bleeding (97,25% and 10,54mfi) compared to both bleeding patients (94,88% and 6,89mfi) and significantly higher than the reference level (88,44% and 5,36mfi, both p < 0,05). finally, the proportion of ps+pmps was normal in bleeding patients, but their pmps expressed higher than reference ps per cell, both unstimulated and for all agonist (134,12 mfi unstimulated vs 32,38 mfi reference, p < 0,01). summary/conclusions: patients with it exhibited different bleeding tendency despite comparable thrombocytopenia. in non-bleeding patients the proportion and per cell level of ps+ were higher, indicating that generation of cps with high ps expression is a critical factor determining bleeding phenotype. the finding of high pmp ps per cell level in bleeding patients could represent an inadequate compensation for lack of cp function, indicating that procoagulant pmps may be less important than cps for thrombocytopenic bleeding. quantification and characterization of cps may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in it and other conditions with bleeding diathesis and/or thrombocytopenia. more studies investigating this field are warranted. background: alloantibodies against human platelet antigens (hpas) and human leukocyte antigen (hla) are implicated in several immune-mediated platelet disorders. detection of these antibodies is crucial in the diagnosis and management of these disorders. aims: to establish a method detecting hpa-1, hpa-2, hpa-3, hpa-5 and hla antibodies using luminex bead technology. methods: monoclonal antibodies specific for platelet glycoproteins and hla class i molecules were separately coupled to the luminex microbeads. positive anti-hpa-1a, anti-hpa-2b, anti-hpa-3a, anti-hpa-5a samples were used to validate the specificities of the luminex assay. the anti-hpa-1a, anti-hpa-3a standard samples were used to evaluate the sensitivities of the luminex assay by serial dilutions (from neat to 1/1024). results: 44 samples collected from patients or isbt platelet workshop were tested by the luminex assay. the results showed that luminex assay could detect antibodies against hpa-1a, hpa-2b, hpa-3a, or hpa-5a successfully from the known samples. the sensitivities of the luminex assay detecting anti-hpa-1a, and anti-hpa-3a were 1:512 and 1:64, respectively, using the standard samples. no cross-reactivity was observed in the samples containing multi-platelet antibodies, or mixture antibodies against hpa and hla. the results of 44 samples with platelet disorders were agreement with those of monoclonal antibody immobilization of platelet antigens (maipa) assay. summary/conclusions: luminex beads coupled with monoclonal antibodies could be successfully used to detect hpa and hla antibodies with high sensitivity. background: platelet transfusion is important in clinical treatment. the expression of abo antigen on platelet surface is differential, so it is usually need to ensure the consistency of the abo antigen in clinical transfusion. but in many cases, it is difficult to find the platelets that the abo blood type matched between the recipient and donor, and abo-incompatible platelet infusion is required in these cases. to data, the expression of abo antigens on platelets in normal blood group individuals is rarely reported in chinese population. aims: to understand the differential expression of abo antigen on platelet surface in population of zhejiang province, china. methods: total of 358 individuals with normal abo groups (101 group a, 100 group b and 101 group ab individuals, and 56 group o as negative control of abo antigens on platelets) were analyzed. the expression of abo antigens on platelets was determined by flow cytometry using monoclonal antibodies: fluorescein isothiocyanate (fitc)-conjugated mouse antihuman blood group a and pe-conjugated murine igg1 anti-b antibody (9431pe bgrl1). flow cytometric parameters were statistically analyzed by the mann-whitney test or the kruskal-wallis test to observe the difference in two or more groups using graphpad software v5.01. the correlation and regression analysis between a and b antigen in the platelets and rbcs were also performed by the software. population studies were reported as the mean and standard deviation (sd), and p values less than 0.05 were considered statistically significant. results: according to mfi values of abo antigens expression on platelets, the samples were divided into three groups: low expression (le), high expression(he) and moderate expression (me) according to the background mfi observed in group o samples. it was found that about 66.22% of the individuals had a weak expression of abo antigen on the platelet surface in zhejiang province. there was a significant difference in the intensity of antigen expression between these three different groups of the same blood group. for each blood group, there was a positive correlation between the intensity of abo antigen expressed on the platelet membrane and red blood cells of the individuals. results: 40 cases were found with antibody positive. among them, 6 cases (15%) were only anti-hla-i positive, 4 cases (10%) were only anti-hpa positive, 30 cases (75%) were both anti-hla-i and anti-hpa positive. 8 cases were found without anti-hla-i or anti-hpa. among the 34 cases with anti-hpa positive, the distributions of anti-gpiib/iiia, anti-gpia/iia, anti-gpib/ix, anti-gpiv were 73.5%, 61.2%, 50%, 44.1%, respectively., hla antibody positive rate in the female patients was higher than that in the male and hpa antibody positive rate in the female was lower than that in male, but there was no significance difference between them (p > 0.05). summary/conclusions: in ptr patients, the platelet antibody was mainly hla-i antibody combined with hpa antibody. background: human neutrophil antigens (hna) are polymorphic structures located on surface membrane of human neutrophils. alloantibodies against hna are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. genotyping for human neutrophil antigen (hna) systems is an important in the diagnosis of disorders involving alloimmunization to hna. aims: the aim of this study was to investigate the hna allele frequencies among blood donors and hematological patients undergoing blood transfusions and to estimate possible hna incompatibilities and risk of hna alloimmunization. methods: a total of 303 blood donors and 302 hematological patients from the north-west region of the russian federation were recruited. dna samples were obtained and typed for hna-1, -3, -4 and -5 systems using polymerase chain reactions with sequence-specific primers (pcr-ssp). specific primers for hna were designed and the polymerase chain reaction amplification conditions were optimized. the v 2 test was used to test for the hardy-weinberg equilibrium for the hna systems. the probabilities of the incompatibility and the potential risk for alloimmunization against different hna systems after random transfusions were estimated based on the hna allele and genotype frequencies. results: in blood donors, the frequencies for the fcgr3b*01 (hna-1a), fcgr3b*02 (hna-1bd), and fcgr3b*03 (hna-1bc) alleles were 0.384, 0.584 and 0.032; for the slc44a2*1 (hna-3a) and slc44a2*2 (hna-3b) alleles, 0.804 and 0.196; for the itgam*1 (hna-4a) and itgam*2(hna-4b) alleles, 0.898 and 0.102; for the itgal*1 (hna-5a) and itgal*2 (hna-5b) alleles, 0.708 and 0.292, respectively. in hematological patients, the gene frequencies for hna-1a/1bd/bc, -3a/3b, -4a/4b, and -5a/5b were 0.376/0.588/0.036, 0.795/0.205, 0.887/0.113, and 0.699/0.301, respectively. no statistic significant difference between genotypes in these groups was observed. since the allele frequencies of hna -1, -3-5 for hematological patients and donors did not have statistically significant differences, possible hna incompatibilities and risk of hna alloimmunization were estimated based on the obtained data on the allele and genotype frequencies of hna in a group that combines donors and hematological patients (n = 605). the predicted risk of hna-1, -3, -4, -5 incompatibilities in this cohort were 34.9%, 26.9%, 19%, and 32.9%, respectively. the possible risk of hna-1a, -1bd, and -1bc alloimmunization were 0.233, 0.143, and 0.064, respectively; of hna-3a and -3b alloimmunization, 0.037 and 0.231; of hna-4a and -4b alloimmunization, 0.01 and 0.163; of hna-5a and -5b alloimmunization, 0.080 and 0.250, respectively. summary/conclusions: the information about hna gene frequencies can be used not only in blood services for detection and identification of hna alloantibodies in donors and assessment of alloimmunization risk but also for anthropological studies. background: non-invasive fetal rhd genotyping is performed using circulating cell-free fetal dna from maternal plasma sample and real-time polymerase chain reaction. this antenatal routine dna test is used to target rh-ig administration to prevent hemolytic disease of the newborn. aims: the aim of this study is to characterize maternal rhd variants responsible for indeterminate results during fetal rhd genotyping due to early amplification of at least one of the exons (5, 7 or 10) of the rhd gene. methods: 2004 samples were tested from 01/12/2017 to 01/12/2018 using free dna fetal kit â rhd. 44 samples (2,2%) yielded a premature signal for one or more exons of the rhd gene. after extraction of maternal cellular dna, the maternal rhd was characterized using rhd beadchip assay (immucor/bioarray). rhdiiia-ce(4-7)-d summary/conclusions: greater diversity is observed in the caucasian population rather than in the afro-caribbean. 65% of the identified variants are rhd negative alleles including alleles leading to partial rh2 antigen expression. unexpected alleles are found such as weak d type 1, 2, 5 or 11. these data underline the benefits of maternal rhd genotyping when abnormal early signals are detected during noninvasive fetal rhd genotyping. background: a considerable number of rhd alleles responsible for weak d phenotypes have been identified. serologic determination of these phenotypes is often doubtful and makes genetic analysis of rhd gene highly desirable in transfusion recipients and pregnant women. dna-based methods are useful for enhancing immunohematology typing in doubtful d phenotypes at pregnant women. aims: determination of the rhd gene in a cohort of pregnant women with doubtful d phenotypes. methods: determination of the rhd phenotyping was performed with microagglutination technique biorad and ortho diagnostic simultaneously. rhd genotyping was performed on 20 cases with d typing serological discrepancies with ready-to-use inno-train rbc-ready gene cde and rbc-ready gene d weak test kits based on polymerase chain reaction with sequence-specific priming (pcr-ssp) to unclear serologic findings. results: molecular analyses showed 16 of 20 (80%) pregnant women were rhd*weak d type 1 and not at risk for anti-d. rhd*weak d type 3 were typed in 3 cases (15%) and 1 case was rhd*weak partial 4.0 and potentially at risk for being alloimmunized producing anti-d allo-antibodies. summary/conclusions: appropriate classification of rhd phenotypes is recommended for correct indication of rhig in pregnant women. however, the serologic differences between rhd-negative and rhd-positive pregnant women is a real problem for unnecessary application of rhig prophylaxis in pregnant women with d variants. conclusion: antenatal rhig prophylaxis is useful in rhd negative pregnant women. with genotyping we found that 95% of serological doubtful rhd negative women was d variants that not produce anti d antibodies. in that cases those rhig prophylaxis was unnecessary and harmful as a product of human origin. on other hand there is a save up of a stock of rhig which is any way in deficit. is it time to think about cost benefit of rhig prophylaxis and genotyping in pregnant women. background: in may 2018, uk neqas (btlp) created an external quality assessment (eqa) sample designed to mimic a feto-maternal haemorrhage (fmh) bleed of 3 ml. all material used passed pre-acceptance serological testing; samples were dispatched to 298 participants in 17 countries. post-dispatch testing by flow cytometry (fc) using an anti-d marker showed a bleed volume of 1.1 ml so an investigation was initiated. aims: to determine the cause of the unexpectedly low bleed volume and what lessons could be learnt. methods: production methodology and results of pre-acceptance testing were reviewed. fc testing was repeated, plots examined, and the fmh scientific advisory group consulted for advice. further fc testing was performed at wbs using alternative markers, and the material used was investigated at ibgrl. participant results were examined to determine if the sample should be withdrawn from scoring. a questionnaire on how results were managed was sent to the 36 participants using fc with an anti-d marker. results: a material production methodology review showed no obvious cause of the erroneous in-house result. review of pre-acceptance testing images showed no issues, further d-typing of the cord showed 2 + reactions vs. two reagents by tube, cf. 4 + with two different reagents by column agglutination technology. repeat fc testing using the anti-d marker gave similar results; however, closer examination of the plots showed a left shift in the positive peak, indicating reduced fluorochrome binding, possibly due to reduced d antigen density on the cord cells. further fc testing at wbs demonstrated a marked reduction in fluorescence intensity with an anti-d marker. further investigation using an anti-hbf marker showed a bleed volume of 3.7 ml, indicating the correct proportion of cord material had been used during sample production. additional serology at ibgrl on the cord material showed reactions which were weaker than the control with 4/17 anti-d reagents. overall, the investigation supported the hypothesis that the cord material was d variant. a review of results submitted by participants mirrored the fc investigation and the sample was withdrawn from scoring, as the fc median result is used to calculate scores and the d variant cord was clearly affecting testing with an anti-d marker. the questionnaire showed that all 16 respondents examine fc plots and the gating used, but not all act on them before reporting results, and not all have a back-up plan for anti-d ig dosing in a similar situation. later sequencing of the d gene revealed the cord donor to be dvii which can have a lower than normal d antigen density. summary/conclusions: the use of a d variant cord in an eqa sample was not planned, but allowed uk neqas to highlight some important learning points: -thorough examination of fc plots is essential to avoid underestimation of fmh; a controlled procedure should be in place if modification of gates is required -access to cord/neonatal blood to allow serological investigation may be useful in a similar clinical situation -it is important to have a back-up plan for issuing anti-d ig in the event of an uninterpretable fmh result background: allo-antibodies against fetal blood group and platelet antigens produced by antigen-negative pregnant women can cause hemolytic disease of fetus and newborn (hdfn) and fetal and neonatal alloimmune thrombocytopenia (fnait). prediction of the fetus antigen status in immunized women is important for making decisions concerning further management of pregnancy. nipt is widely used for determination of fetal blood groups but determination of proper specificity in the real-time amplification of a single nucleotide polymorphism (snp), such as k or hpa-1a, requires modified protocols. droplet digital pcr (ddpcr) permits detection of low-grade fetal chimerism in maternal plasma dna with higher specificity using allelic discrimination pcr protocols. aims: to establish ddpcr protocols for non-invasive prenatal diagnostics (nipd) of clinically important blood group antigens. methods: dna was isolated from 133 plasma samples of pregnant women and donors with known genotypes (easymag, biomerieux). allelic discrimination protocols for determination of k/k (n = 29), s/s (n = 13), hpa-1a (n = 49), hpa-3 (n = 8), hpa-5 (n = 14), hpa-15 (n = 20) genotypes were performed using ddpcr method with droplet digital tm (biorad). the results of allelic discrimination performed using ddpcr were concordant with the already known phenotype/genotype of donors and pregnant women. ddpcr enabled the detection of 100-16,000 reads for total dna from plasma in tested samples. all fetal results were in agreement with antigen positive genotype of the neonates and the fetal chimerism was from 0,01% to 26,4% (one case was for advanced pregnancy -38 week of gestation). in 19/133 tested samples false positive results were detected at the level of 1 or 2 unspecific reads. summary/conclusions: the implementation of allelic discrimination protocols for ddpcr allowed detection of fetal-maternal incompatibility in k/k, s/s and hpa-1a, -3a/b, -5a/b, -15a/b antigens encoded by snp. background: in france, for pregnancies complicated by anti-d (rh1) and anti-c (rh4) allo-immunization, the tests currently used to quantitate maternal antibodies are tube method titration and continuous flow analysis determination of the antibodies concentration. recently, an automated assay was developed using the column agglutination technology on the ih-500 system (bio-rad â). aims: we wanted to evaluate the score, calculated from the agglutination profile of the antibodies on the ih-500 system, as a quantitative data to appreciate the level of maternal antibodies. methods: titers from 29 samples containing anti-d and 20 containing anti-c have been established using the semi-automated tube method performed since decades in our lab and the fully automated gel method on the ih-500 system. scores were calculated manually in both cases. antibodies concentrations were also determined for all samples by continuous flow analysis on our auto-analyzer device (evolution iii ams alliance). we looked for a possible correlation between anti-d and anti-c scores and the corresponding concentrations using the spearman correlation test. results: anti-d tube and gel scores were significantly correlated with the anti-d concentration values (p < 0.0001, r = 0.79 and p < 0.0001, r = 0.82 respectively). anti-c scores were also significantly correlated with anti-c concentration values (p < 0.0001) but gel scores have a better correlation coefficient than tube scores (r = 0.78 versus 0.55). it was easier to extrapolate gel score thresholds than tube score thresholds from the autoanalyzer values, with the aim of triggering fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery only for risk pregnancies. the determined gel score thresholds were 75 and 35, corresponding respectively to 5 ui/ml (250 uchp/ml) of anti-d and 7.5 ui/ml (500 uchp/ml) of anti-c. conclusions: calculating the score from the hemagglutination profile displayed by the ih-500 system provides added values compared to the sole reading of the titer. for anti-c immunization, gel scores are more discriminant than tube ones and better correlated to the concentration values established by continuous flow analysis. the proposed score thresholds to trigger fetal antenatal monitoring need, however, to be confirmed on more samples and to be clinically documented. background: hdnf is due to maternal igg alloantibodies directed against fetal antigens that cross the placenta during pregnancy, causing hemolysis in the fetus, anemia that can lead to edema, ascites, hydrops and, in some cases, death. the diagnosis and management of hdnf is based on maternal screening, and middle cerebral artery (mca) doppler monitoring. in severe hdnf intrauterine blood transfusions (iuts) and or exchange transfusion (et) after birth are necessary to correct anemia, to prevent and treat fetal hydrops. aims: we report eight years of experience in our immunohematology reference laboratory (irl) to highlight the importance of red cell antibody detection as a fundamental parameter to identify pregnancies with high fetal risk and to drive a correct treatment. methods: we report laboratory data from 250 pregnant women with a positive indirect antiglobulin test (iat) referred to our irl from january 2008 to december 2016. we performed antibody screening and identification by indirect antiglobulin test (iat) in microcolumn method with biovue system (ortho-clinical diagnostics, raritan, usa), and the title of antibodies in iat by tube method without additive. follow-up tests were also performed in the presence of significant red cell antibodies in order to check antibody title and begin clinical monitoring. threshold values were ≥ 1:8 for anti kell antibodies and ≥ 1:32 for other specificities. results: out of 250 women, 143 (57.2%) displayed clinically significant antibodies, 92 (36.8%) clinically insignificant antibodies and 15 (6%) natural antibodies of different specificities. among women with clinically significant antibodies the most frequent was anti-d (16.8%) also in combination with other rh antibodies (8.8%), while anti-k accounted for 10%, anti-e for 10% and antibodies against high-incidence antigens for 2.4%. anti-m and anti-le a antibodies were also found (16.8% and 8% respectively) but they were not clinically significant. among 143 women with clinically relevant antibodies, 37 showed a critic antibody title and they underwent gynecological and obstetric monitoring. 21 fetuses resulted affected by hdfn, displaying anti-d in 16 cases and anti-kell in 5. 11 fetuses with severe hdfn (anti-d in 7 and anti-kell in 4) required iuts, 2 were treated with et, 8 received red blood cells units at birth. summary/conclusions: the mother screening program led to important improvements in the outcomes of hdfn. the identification of women with clinically significant antibodies allowed an appropriate monitoring program and therapy. background: the hemolytic disease of the fetus and newborn (hdfn) is a severe disease, resulting from maternal erythrocyte alloantibodies directed against fetal erythrocytes. alloimmunization in pregnant women has been found to range from 0,4% to 2,7% worldwide. there are over 400 erythrocyte surface antigens, of which more than 43 have been reported to be associated with hdfn. although anti-rhesus d was once the major etiology of hdfn, the universal introduction of antenatal and postpartum rh immunoglobulin has resulted in a marked decrease in the prevalence of alloimmunization to the rhd antigen in pregnancy. consequently, alloantibodies other than anti-d emerged as an important cause of severe hdnf, in particular anti-k and anti-c. however, there are other antigens that have also been found to be associated with hdfn. aims: retrospective identification of erythrocyte antibodies in pregnant women in hospital de braga in 2016 and 2017. methods: this study was planned to assess the prevalence of erythrocyte antibodies responsible for alloimmunization, excluding abo-immunizations, in pregnant women attending the antenatal clinics of hospital braga during 2 years, from january 2016 to december 2017. in this study, we retrospectively evaluated the erythrocyte antibody screening results of pregnant women. women with positive erythrocyte antibody screening also underwent identification with gel card system following the manufacturer's instructions (diamed â ). the outcomes of infants, whose mother's indirect antiglobulin tests were found to be positive, were examined. direct antiglobulin tests, jaundice and phototherapy history, transfusion and mortality of the newborns were recorded. results: during the study period, 5982 pregnant women were attended in hospital de braga. the laboratory registered 100 positive erythrocyte antibody screening tests. the prevalence of positive erythrocyte antibody screening was 1,7%. anti-d was the most common antibody found (58,5%). anti-d prophylaxis given during pregnancy was responsible for 51 of 62 cases and maternal antibody titer levels did not exceed 8 among these cases. the prevalence of non-rhd immunization was 33%. anti-e (9,4%) was the most frequent alloantibody other than anti-d followed by anti-m (5,7%) and anti-c (4,7%). multiple maternal antibodies were found in 5 pregnant women. four women had 2 types of alloantibodies: anti-c and anti-e; anti-c and anti-d; anti-k and anti-cw; anti-e and a non-identified antibody. one pregnant had 3 types of alloantibodies: anti-d, anti-c and anti-e. of all cases of newborns whose mothers had a positive antibody screen tests, icterus occurred in 45% of them and phototherapy was given in 14%. summary/conclusions: the prevalence of positive erythrocyte antibody screening in hospital de braga was 1,7%. the erythrocyte antibody screening showed that anti-d was the most common antibody found (58,5%) in most of the cases because of anti-d prophylaxis. the prevalence of non-rhd immunization was 33%. the other most frequent alloantibodies were anti-e (9,4%), anti-m (5,7%) and anti-c (4,7%). an increasing prevalence of non-anti-d alloimmunization was found and there are currently no preventive strategies. in contrast to rhd alloimmunization, the main risk factor for non-anti-d alloimmunization is a previous transfusion therapy. thus, it is important to minimize the exposure of women to incompatible erythrocyte antigens through unnecessary transfusions when possible. background: the mns blood group system is one of the most complex blood group systems. although alloanti-m is a common antibody observed in pregnant women and could also be found in the serum of individuals who have not been exposed to m positive erythrocytes, it is rarely clinically significant and has been regarded as an unimportant antibody to cause hemolytic disease of the fetus and newborn (hdfn), especially in caucasian and black ethnic groups, for a long time. however, an increasing number of cases of severe hdfn resulting in fetal hydrops and recurrent abortion caused by alloanti-m have been reported mainly in the asian population, especially in the japanese and chinese populations. aims: to summarize the characters of serological testing in preterm twins newborns suffered with severe hdfn. methods: the blood sample of two newborns with severe hdfn and the mother, who had the history with three hydrops fetus, were collected. abo, rhd, rhce, and mn blood group typing of the twins newborn and their mother were performed in saline with tube or gel card. direct agglutination test (dat), elution test, antibody specificity identification and antibody titer detection were conducted by iat method in gel card. results: o, rhd(+), and ccee blood groups were identified both in the mother and the twins newborn. background: in france, since may 2018, the legislation does not promote anymore the use of the reference tube method for titration of anti-red blood cells antibodies. this opened the way to the use of newly developed automated anti-red blood cells antibodies quantitation by column agglutination technology. aims: we wanted to assess the performance of titration and scoring by the id-gel test on the ih-500 system (bio-radâ) and to compare it with the performance established for the reference tube method, used in our lab since decades. another objective of the study was to determine titer thresholds for the gel method, to trigger fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery. methods: an home-made internal quality control (iqc) prepared and calibrated using the international anti-d standard (01/572) was used to determine the intraassay and interassay imprecisions, regarding the score and the titer results. patients samples for testing were chosen during the 2-months assay period, regarding the specificity of the antibodies and the tube titer in order to cover a wide range of 2 have lower values. the highest differences (more than 2 to 3 dilutions higher) were seen for antibodies directed against rh system antigens. among the other specificities, anti-k (kel1) and anti-m (mns1) antibodies show the most samples with equal or lower titers compared to the tube method. conclusions: automated anti-red blood cell antibodies titration by column agglutination technology on ih-500 system shows better intra and interassay cvs compared to the tube method. it is explained by the fully automated process that includes the reading step. titer results are almost always higher with the gel technology. thus, it seems possible to safely extrapolate the titer thresholds defined for anti-red blood cells antibodies by the tube method to the gel method. however, based on future clinical studies and fetal/neonatal outcomes, it would probably be necessary to increase these thresholds, at least for anti-rh antibodies, in order to avoid heavy, expensive, stressful and useless monitoring of some pregnancies. results: the first case was a 1-day-old female infant, yellowish skin developed the next day after birth. her capillary bilirubin level was 13 mg/dl, the evidence favored neonatal hyperbilirubinemia and the clinical manifestation revealed hemolysis symptoms. her laboratory findings showed elevated reticulocytes (15.2%), ldh (1162 iu/l) and g6pd (25.3 u/ghb); dat (+/-), iat (-), anemia (hb 8.7 g/dl, hct 28%), and blood smear showed anisocytosis, spherocytes, and polychromatic rbc. her mother blood typed o, d positive, while her blood type was b, d positive and anti-b was found from her elution rbcs (3 + ). due to rarely severe anemia with abo incompatibility, maternal plasma was analysed for abo igg antibodies and showed high antibody a and b titre with 1:1024 and 1:2048. the female infant received one unit washed-prbcs for anemia and intensive phototherapy for hyperbilirubinemia. her clinical condition improved significantly, hb rose to 14.6, bilirubin level was within normal range, she was discharged. another 5-days-old male infant was our second case. on the third day after birth, yellowish skin discoloration developed and bilirubin level was 15 mg/dl. two days later, his transcutaneous bilirubin (tcb) measurement data was high and laboratory findings also showed raised reticulocytes (5.2%), dat (+/à), iat (à), hb 12. background: anti-indian b is a rare alloantibody against the high frequency antigen in b . individuals with the in:1,-2 phenotype (in(a+b-)) are observed with a frequency of < 0.1% in the indian population and have not been described in caucasians. the majority of anti-in b antibodies have been reported in individuals without previous transfusions, indicating the possibility of a naturally occurring antibody. anti-in b is considered clinically significant and haemolytic reactions after in b -incompatible transfusions have been reported. haemolytic disease of the foetus and newborn (hdfn) due to anti-in b has not been described. however, a positive direct antihuman globulin test (dat) may be observed. aims: to describe the challenges of managing a pregnancy and childbirth of a woman with an anti-in b . methods: serological investigations were performed by iat (tube and column agglutination). papain and trypsin treated cells were also utilised. soluble recombinant in blood group proteins (in-rbgp) (inno-train, germany) were used in neutralization tests. the clinical significance of the anti-in b antibody was determined by monocyte monolayer assay (mma). genomic dna was isolated from whole blood and the samples were further characterized by pcr amplification and sanger sequencing of exon 2 of cd44. results: in a 27-year-old pregnant (para 1) woman of indian origin without previous transfusions, an alloantibody of the specificity anti-in b with a titer of 1:2 was detected by iat (negative with papain-treated cells) at gestational week (gw) 32 and 36. the mma, performed in duplicate on samples taken at these dates, showed a mi of 0.5%/4.8% and 7.7%/6.3% respectively. the mi was interpreted as follows: 0-3% not relevant; 3-5% inconclusive; >5% clinical significant. the patient's parents were typed heterozygous, in:1,2 whereas her husband was homozygous, in:-1,2. due to the husbands phenotype, the fetus was predicted to be in b positive. doppler flow measurement of the peak systolic velocity in the middle cerebral artery of the foetus was normal. delivery took place at gw 41 without increased bleeding. the neonate presented no clinical manifestation of hdfn. neither the mother nor the baby required blood transfusions. summary/conclusions: we report the case of a pregnant woman of indian origin with an anti-in b alloantibody. the first mma, performed in gw 32, was inconclusive whereas the second mma, performed in gw 36, indicated that the antibody was clinically significant. if the mi-increase is only due to the pregnancy or has also a clinical significance, cannot be stated. in b negative blood components were not available and the patient's relatives were all in b positive. therefore, measures to avoid transfusions, including optimised peripartal management of haemostasis, was of utmost importance. with only few cases published, the risk of hdfn could not be excluded with certainty. an intrauterine investigation by doppler was performed to exclude relevant anaemia of the fetus. no transfusion was needed at delivery as there were no haemorrhagic complications. the neonate presented no clinical signs of hdfn. background: hemolytic disease of the fetus and newborn (hdfn) is a disease which if untreatedcan cause perinatal mortality and morbidity with a substantial risk for long-term sequela. in albania we lack of studies in this field. aims: the aim of this study is to determine the predictive value and the reliability of the "critical titre" during the evaluation of red cells alloantibodies ability to cause the hemolytic disease of fetus and newborn. methods: we conducted a descriptive, cross-sectional study. the data were collected in the university hospital for obstetrics and gynecology in albania. in the study were included 20 immunized pregnant woman for anti-d antibodies and their newborns which were affected from the hemolytic disease of fetus and newborn. the data belong to the period 2013 and 2018. results: the "critical titre" in our study was 8, meaning that this was the minimal value of the titre antibodies that could cause hemolytic disease of fetus and newborn. our study concluded that only 2 newborns were born without the hemolytic disease of fetus and newborn and the titre values were less than 4. moderate hemolytic disease of fetus and newborn were caused between the titre values 8-32. the summary/conclusions: the titre values of the mothers are a predictive option of the high risk of giving birth to a child with the hemolytic disease of fetus and newborn. it is recommended that in this cases the mother should be followed with doppler ultrasonography to measure the blood flow of the middle cerebral artery. also the doctors should recommend in pregnant women with positive coombs test not only the identification of the anti-d antibodies but also the identification of the other antibodies such as anti-e, anti-c, anti-k. background: rhd-negative pregnant women with allo-anti-d are at risk of having a fetus affected by haemolytic disease of the fetus and newborn (hdfn) where the fetus is rhd-positive. the rhd allele is highly polymorphic and many rhd variants give rise to an array of partial d phenotypes. the clinical significance for many partial d phenotypes is not well-established. rhd genotyping by non-invasive prenatal testing (nipt) to assess the fetal rhd status determines whether the fetus is at risk for hdfn. nipt tests also include strategies for detecting maternal rhd variants to provide for accurate reporting. however, the presence of a paternal rhd variant, while having the potential to confound nipt interpretation, is often not recognised. we report a "trio" family study triggered by a request for nipt for an rhd-negative pregnant mother, 16 weeks gestation, who presented with allo-anti-d and anti-jk a antibody. subsequent paternal and fetal rhd genotyping was conducted and revealed a novel variant rhd allele. aims: we aim to characterise the paternal rhd allele and review clinical case features. methods: rh phenotyping was performed by standard serological procedures. nipt tested for fetal rhd exons 4, 5 and 10. rhd genotyping on whole blood/cord blood dna was performed on the immucor bioarray rhd beadchip kit which predicts a rhd phenotypic variant of best fit. dna sequencing was performed using the illumina trusight one sequencing panel. copy number variation (cnv) analysis was used to assess the rhd exon structure and zygosity. results: the paternal red cells typed as group o rhd+c-c+e-e+, (ror). nipt genotyping detected fetal rhd signals for all 3 exons, predicting rhd-positive. no maternal rhd sequences were detected consistent with homozygosity for the rhd deleted haplotype. for both paternal and cord genomic dna (gdna), beadchip genotyping predicted a rhd variant "diiia/cehar". furthermore, signal drop out was observed at 3 nucleotide positions (c.1154, c.1193, c.1227) located in rhd exon 9 suggesting exon 9 was either deleted or rhce-replaced. paternal and cord gdna sequencing detected 4 out of 6 snps (c.186g>t, c.410c>t, c.455a>c, c.602c>g) associated with diiia phenotype plus 2 additional snps (c.604g>a, c.733g>c) on the rhd gene. both were rhd hemizygote by cnv analysis. no rhce variants were detected. clinical case features: the maternal anti-d quantitation increased from 5.3 iu/ml (16 weeks gestation) to 166 iu/ml (33 weeks gestation). the fetus required 3 intrauterine transfusions during the pregnancy to manage the hdfn. summary/conclusions: both father and fetus carry an rhd allele that does not align with alleles encoding diii phenotypes. this putative novel rhd variant allele comprises snps associated with diiia and with a possible exon 9 deletion/rhcereplaced. a similar allele was reported in literature, although based on sequence analysis only, with no phenotype data. the variant allele here encodes rhd-positive phenotype and we predict that there may be a loss of d-epitopes. notwithstanding, the clinical presentation shows that maternal anti-d against this rhd phenotype (presumed partial) is associated with a severe hdfn and that such rhd blood group phenotypes are of clinical significance for alloimmunised pregnancies. abstract withdrawn. background: cd109 is a glycosylphosphatidylinositol (gpi)-anchored protein with apparent molecular mass of 170 kda. in addition to being expressed on human plts, cd109 is expressed on activated t-cells, endothelial cells, cd34 + hematopoietic stem cells as well as on progenitor cells. in the chinese population, the calculated allele frequencies of hpa-15a and -15b are 0.505 and 0.495, respectively. based on these data, the risk of alloimmunization against hpa-15 alloantibodies due to incompatible plt transfusion or pregnancy is expected to occur in relatively high frequency. however, until today there is no report of hpa-15 alloimmunization in the chinese population. in this study, we analyzed sera from hydrop fetus cases by maipa technique and icfa. aims: to detect the anti-hpa 15b alloantibodies by maipa and icfa. methods: a 24-year-old mother, gravida 2/para 0. the mother in the first pregnancy was diagnosed hydrop fetus at pregnancy 33 weeks by ultrasound. in the second pregnancy, fetal hydrops was observed by ultrasound at pregnancy 24 weeks. the mother's irregular antibody test was negative. the maternal platelet specific antibodies and hla antibodies were negative. blood routine and morphological examination of fetal umbilical cord blood showed that plt count dropped to 4.7 9 10 10 /l, wbc count dropped to 2.96 9 10 10 /l, including neutrophil 37%, lymphocyte 16%, mononuclear 43%, eosinophil 2%, basophil 2%, red blood cells were normal, hb was 111 g/l. screening for hla and plt-specific antibodies was performed using a elisa-based plt antibody kit (pakplus, gti diagnostics) as recommended by the manufacturer. plt antibodies were detected by icfa and maipa.hpa genotyping was detected by cpr-ssp. results: the fetus's genotype was hpa-1a/a, -2a/a, -3a/a, -4a/a. -5a/a, 6a/a, 7a/a, 15a/b, naka (+) and the maternal was hpa-1a/a, -2a/b, -3a/a, -4a/a. -5a/a, 6a/a, 7a/ a, 15a/a, naka (+). the paternal genotype was hpa-1a/a, 2a/b, 3a/a, 4a/a, 5a/a, 6a/a, 7a/a, 15a/b, naka (+), which was the only incompatible antigen compared with the maternal hpa. samples were tested using the fresh plt panels consisting of hpa-15aa and -15bb homozygous donors. the reactivity of the negative control and the mother's sera with the plts from hpa-15a/a (donors 1), hpa-15a/b (donors 2) and hpa-15b/b (donors 3) donors by maipa. the mother's serum showed no reactivity against 15a/a plts, weak positive reactivity against 15a/b plts (od values 0.28), but strong reactivity against 15b/b plts (od values 0.38).this finding could be confirmed by one of the reference plt laboratories (japanese red cross kanto-koshinetsu block blood center, japan) using freshly isolated plts from hpa-15genotyped donors (anti-hpa-15b average value 9.8). summary/conclusions: in this study, we found anti-hpa-15b in a case of fnait (patient hpa-15aa, blood group o) using the maipa technique. we were able to detect the presence of hpa-15b alloantibody in one case of nait. background: fetal and neonatal alloimmune thrombocytopenia (fnait) occurs in 1:10000 live births in caucasians. serological and molecular human platelet antigens (hpa) genotyping tests are performed to investigate and conclude to fnait diagnosis. however, in few cases and particularly in case of suspicion of private platelet antigen, some specialized analyzes must be performed in the laboratory (lab). these analyzes can range from sanger or ngs sequencing to platelet serology with transfected cells. aims: the aim of our study was to explore where the frontier between research and care takes place in the field of platelet immunology through the prism of the fnait investigations carried out by the platelet immunology laboratories. methods: a two-part electronic survey have been sent to foreign platelet immunology experts (pie) from platelet immunobiology working party (piwp) members and espgi board members (n = 40). the first part focused on the lab practices and regulatory environment regarding to accreditation, contact with patient, informed consent and patient results. the second part stressed on the investigations performed to discover new platelet antigen and more precisely on the perceived status of these analyzes ( background: haemolytic disease of the fetus and newborn (hdfn) can occur when maternal red cell antibodies, directed against red cell antigens present on the fetal red blood cells, cross the placenta and enter the fetal circulation. in a "traditionally" conceived pregnancy, when hdfn occurs, it is as a result of maternal antibodies directed against fetal red cell antigens in the heterozygous state, whereby the antigen is inherited from the father only. with the advent of donor oocyte (do) in-vitro fertilisation (ivf), the addition of a third person into the reproductive equation allows for the possibility of a more severe form of hdfn when fetal red cell antigens are present in the homozygous state (one copy from father and one copy from donor) and maternal antibodies are directed against these. antigens expressed in the homozygous state will have more antigens sites per red blood cell and therefore are at an increased risk of red cell destruction from the maternal derived cognate antibodies. aims: to raise awareness of increased severity risk of hdfn in donor oocyte conceived pregnancies. methods: we describe two unusual cases of hdfn in our institution of two women whose pregnancy was induced using a donor oocyte and their offspring requiring transfusion support in the postnatal period to treat hdfn. results: the first is a case previously reported (doyle, quigley, fitzgerald et. al. transfusion medicine, 2014 ) of protracted hdfn due to anti-c, managed with phototherapy initially, then intervention with red cell top-up transfusion at 4 weeks post-delivery. the second is an unusual case of severe abo hdfn requiring exchange transfusion therapy (pre-publication). summary/conclusions: given the increased number of pregnancies conceived using do we recommend that antenatal guidelines are reviewed to create awareness regarding the potential increased risk of hdfn in do pregnancies complicated by allo-immunisation. critically, antenatal testing guidelines should highlight that the predicted outcomes associated with quantitation/titres can only be used when do has not been used to obtain the pregnancy. it is also essential that clinicians inform the blood transfusion laboratory when do has been used. abstract withdrawn. 19%) are deceased due to organ rejection, and 8/36 patients (22%) are deceased due to disease not related to rejection. summary/conclusions: the use of therapeutic plasma exchange for the treatment of antibody mediated rejection in solid organ transplant is safe and effective when used along with other treatment modalities. further studies will help determine whether it can be reproduced in larger cohorts and whether it is more effective in certain organs. background: extracorporeal photopheresis (ecp) is an important cellular therapy for the treatment of several (auto-)immune diseases such as graft-versus-host disease. the international standard for the ex vivo treatment of the leukapheresis product is the application of 8-methoxypsoralen (8-mop) and irradiation with uv-a light. however, the addition of 8-mop to the illumination bag is associated with a potential risk of contamination. aims: the basic principle of the ecp is the induction of apoptosis in the leukocytes. our aim was to find an alternative for the conventional apoptosis induction without the need of external substance application. the objective of the study was the investigation of the apoptosis levels and kinetics in leukocytes after treatment with 8-mop+uv-a compared to uv-c treatment without additional 8-mop. methods: we used an in vitro 72 h cell culture approach with human mononuclear cells from healthy blood donors. untreated control cells were compared with 0,2 lg/ ml 8-mop plus 2 j/cm 2 uv-a treated cells and 2 j/cm 2 (effective dose) uv-c treated cells. apoptosis in several leukocyte sub-populations was detected daily with annexin v and 7-aad flow cytometry standings. results: the apoptosis analysis of cd3 cd4 t-helper cells, cd3 cd8 cytotoxic tcells, cd19 b-cells, cd14 monocytes, cd3 neg cd56 nk-cells and cd3 cd56 nkt cells revealed no statistical differences in almost all of these cell types after treatment with 8-mop/uv-a or uv-c light. the apoptosis kinetic as well as the final apoptosis after 72 h were similar in both treatment groups. summary/conclusions: the addition of 8-mop to the photopheresis irradiation bag is a risk for potential infections. the main effect of the 8-mop/uv-a treatment is most probably the induction of apoptosis in the leukocytes. here, we provide information that this induction of apoptosis can also be achieved with uv-c irradiation without the need of 8-mop addition. the apoptosis patterns in most leukocyte subpopulations are very similar after treatment with uv-c compared with 8-mop/uv-a treatment. future in vivo studies are needed to prove the therapeutic effect of uv-c treated cells in the ecp setting. abstract withdrawn. background: therapeutic plasma exchange (tpe) is performed to remove the implicating substances from the plasma causing the disease. a periodic appraisal of tpe data is important to get insight into the procedural related effects and toxicities and overall outcome in order to have a guided future approach. aims: the purpose of this study is to observe the overall profile and outcome of the patients receiving the tpe in the medicine intensive care unit (micu) of a tertiary care hospital in south india. methods: a record based audit was conducted for the all the patients who were admitted to our tertiary care hospital of south india with 16 bedded micu and received tpe therapy between 1 june, 2016 and 31 december 2018. all the tpe procedures were performed using haemonetics multicomponents system (mcs) + ln9000 apheresis system based on intermittent flow centrifugation. we audited our tpe for: number of treatments, clinical indications, treatments prescribed and administered, any procedural or patient complications, and adherence to current best practice recommendations. results: sixty nine patients had undergone 269 tpe procedures. among them, thirty were female patients (43%). the median age 45 (13-75) years. guillain-barre syndrome (gbs) was the most common indication (72%) followed by cases of thrombotic thrombocytopenic purpura, diffuse alveolar hemorrhage, myasthenia gravis, autoimmune encephalitis and hypertriglyceridemia respectively. the tpe regimens received by patients in this icu were not always prescribed in accordance with current best practice recommendations. there were 48 (18%) episodes of patient related complications during the tpe treatments. in 8 (3%) procedures, technical error in the machine was encountered. summary/conclusions: the findings of this audit have identified differences between the current prescription recommendations for tpe and those applied. the infrequency of the therapy and the different indications may present a challenge for medicine intensive care clinicians to provide best care in all cases. background: microangiopathic hemolytic anaemia (maha) encompasses a spectrum of disorders characterised by widely disseminated thrombosis in small blood vessels resulting in formation of schistocytes and concomitant thrombocytopenia. plasma exchange (pe) needs to be considered as empirical and urgent life saving therapy in these disorders irrespective of waiting for specific testing like adamts 13 levels in thrombotic thrombocytopenic purpura (ttp) or complement levels or factor h antibodies in atypical hemolytic uremic syndrome (ahus). aims: to assess the efficacy and safety of plasma exchange in patients diagnosed as having microangiopathic hemolytic anaemias. methods: a retrospective analysis of all pe procedures performed in patients diagnosed as having maha was done over a period of 9 years (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . procedures were done on apheretic device (cobe spectra, terumo bct, lakewood co. usa). patients' pre and post procedural hematological and renal parameters were analyzed by applying paired t test. adverse event if any was recorded. results: pe was performed in 46 patients with diagnosis of maha (27-ahus, 16 -ttp, 1 each of post stem cell transplantation drug induced thrombotic microangiopathy (tma), post thyroidectomy tma and post-partum tma). the mean age of patient was 19.94 ae 19.58 years with m:f as 1.5:1. number of procedures per patient varied from 1 to 27. post pe recovery was observed within 10-14 days with statistically significant increase in mean platelet count from 40.05 ae 5.9 to 82.11 ae 12.10 9 10 9 /l (p = 0.000) and significant decline in mean lactate dehydrogenase level from 48.91 ae 34.73 to 10.98 ae 6.49 lkat/l (p = 0.000). there was also significant decline in mean percentage of schistocytes in peripheral smear from 5.44 ae 3.96% to 0.56 ae 0.89% (p = 0.000). the mean serum urea changed from 48.88 ae 24.56 to 24.50 ae 17.78 mmol/l and creatinine from 266.11 ae 142.59 to 173.09 ae 127.34 lmol/l (p = 0.000 and 0.001 respectively) with significant increase in urine output from 0.71 ae 0.53 to 1.06 ae 0.33 ml/kg/h (p = 0.000). adverse events were observed in 10 patients (21%), allergic reaction to replacement fluid (n = 6) being the commonest followed by hypotension (n = 2), rigors and chills (n = 2). overall survival rate at 6 months was 89%. summary/conclusions: pe had proven its safety and usefulness as life-saving first line treatment modality in maha. prompt and aggressive treatment helps in achieving early and complete remission in these patients. background: neuromyelitis optica (nmo) also known as devic's disease or devic's syndrome is a rare demyelinating disease of the central nervous system that most often results in selective involvement of the optic nerves (optic neuritis) and spinal cord (myelitis)and has female preponderance. neuromyelitis optica (nmo) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments. assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. aims: to study the effect of tpe in neuromyelitis optica. methods: a 17 year old female in the medicine department, civil hospital, ahmedabad admitted with chief complains of weakness and numbness in the arms and legs, blurred vision, reduced sensation, difficulty in controlling bladder and bowels, uncontrollable vomiting and hiccups since 2-3 days in the medicine department, civil hospital, ahmedabad. attacks were treated with short courses of high doses of intravenous corticosteroid -methylprednisolone intravenous. but there was no clinical improvement. results: clinician advised for the trial of tpe in this patient. the procedure was performed by automated device with continuous flow centrifuge machine fresenius kabi-com.tec using double lumen femoral catheter. after obtaining informed consent from the relative of the patient, 6 cycles of tpe were performed on daily basis. after 6 cycles, both subjective and objective clinical response to tpe was estimated by three different sources (the patient, a transfusion medicine physician, and the treating neurologist). [1] for motor performance, patient was assessed on a disability scale (0 = healthy; 1 = minor symptoms; 2 = able to walk 5 meters without support; 3 = able to walk 5 meters with support; 4 = confined to bed or wheelchair; 5 = requiring assisted ventilation; 6 = dead).patient's motor performance was increased to scale 1(upper limb) and 2(lower limb) from scale 5, deep tendon reflexes were normal. visual function began to improve 1 week after the treatment. visual acuity was 6/6 after 4 weeks. summary/conclusions: assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. this suggests that tpe is beneficial in nmo patients during acute attack if there is no response to corticosteroid treatment. background: babesiosis is a tick borne infectious disease caused by the protozoa babesia. while most infections with babesia are asymptomatic, some patients present with a symptomatic infections and rarely this can be a severe life threatening illness. treatment is primarily with antibiotics but red cell exchange (rce) has been used in the more severe cases which are characterized by high grade parasitemia, evidence of severe hemolysis and or multi-organ failure involving the kidney, lung or liver. a threshold parasite level of 10% has arbitrarily been applied as an indication for rce, however, this threshold is not evidence based. aims: to report on patients with babesiosis and high grade parasitemia who were treated with antibiotics only without rce methods: data were collected from july 2014 to july 2018. a case was defined as a patient diagnosed with babesiosis for whom rce was requested on the basis of a parasitemia of > 10% but on clinical evaluation it was considered that rce could be withheld and the patient monitored awaiting response to antibiotics. results: three cases of severe babesiosis in which the use of rce was requested on the basis of a parasite level of greater than 10%, but was not performed. the rce was deferred on account of the good clinical state of the patient and the absence of renal failure. levels of parasite at diagnosis were 10.6%, 11% and 31%. all patients were followed daily until discharge. two of these patients had been splenectomized and each received a single unit of red blood cells during the hospitalization. the third patient had a long history of refractory lymphoma and was pancytopenic requiring multiple transfusions during the years before the diagnosis of babesiosis. she had transfusion transmitted babesiosis from a red blood cell transfused 46 days prior to the diagnosis. all three patients responded well to antibiotics and were discharged between 9-16 days with undetectable parasites. summary/conclusions: this small case series suggests that requests for rce solely on the basis of an arbitrary level of parasitemia should be questioned and the clinical state and evidence of organ failure considered in the decision to perform rce. abstract withdrawn. chronic transfusion program (ctp) remains the gold standard therapy for stroke prevention and for patients with a severe disease who have inadequate response to hydroxyurea treatment. aims: to evaluate the safety, efficacy and cost between scd patients on ctp that underwent both aet and partial manual exchange transfusion (pmet) procedures. methods: retrospective observational cohort study of patients with scd on ctp that have switched between pmet and aet. this study was carried out from 01/01/2017 to 31/12/2018 in a hospital in portugal. data on patient history, haematological values, duration of the procedure, intervals between them, adverse events as well as the cost of material and working hours were collected and compared between both procedures. results: a total of 6 patients met the inclusion criteria described. however, 1 patient was excluded from our study because of the lack of attendance to the ctp. during the study, we recorded 88 exchange procedures (42 pmet and 46 aet), both on peripheral venous access. from all those procedures the major concern was the poor venous access, which was the reason why 2 patients had returned to pmet. no major complication or alloimmunization was observed. the indications for ctp were cerebral vasculopathy (n = 2), stroke (n = 1) and recurrent vaso-occlusive crisis with multiorgan failure (n = 2). for both procedures, target values were to obtain a pre-exchange hbs level ≤ 30% for stroke and cerebral vasculopathy and ≤ 50-60% for other indications. the median hbs level before pmet was 42,6% (31,3-59,1) and 38,4% (18, 9) before aet. we documented a higher hbs level prior to the next procedure in 11,4% of patients (n = 10). despite that all patients remained stable without any major scd related event. both procedures were well tolerated and iron overload was well controlled (median ferritin level pmet: 1356,1 vs. aet: 1314,7 ng/ml). the duration of the exchange procedure was longer and the intervals between procedures were shorter with pmet (median pmet: 360 vs. aet: 60 min and pmet: 21 vs. aet: 24 weeks, respectively). annual rbc requirements per procedure were superior (median 2 vs. 4 units) and the overall costs related with aet were 2,2 times higher -18.180,93€ and 8.174,79€ aet and pmet, respectively (estimated cost per session aet: 790,48€ and pmet: 389,28€). summary/conclusions: our study shows, that the hbs level before both procedures, performed during the same interval, was similar. we verified that pmet has a comparable efficacy with aet in terms of preventing the development or progression of chronic complications and that the cost per procedure is significantly higher with aet. however, in a clinical situation where it is important to rapidly reduce the hbs level, and/or where the control of the target hbs is stricter so that the patients are clinically controlled without an increase in hospital visits, aet is preferred. we conclude that aet is more effective in the rapid reduction of hbs and ferritin levels, as well as being less time consuming. despite this, for the reasons described above, it is more cost-effective to maintain both aet and pmet procedures. background: erythrocytapheresis/red blood cell (rbc) exchange, involves removing of a large number of rbcs from the patient and returning the patient's plasma and platelets along with compatible allogenic donor rbcs. typical indication for rbc exchange is sickle cell disease and its related complications. however, one of the miscellaneous indications of rbc exchange is for the patients of methemoglobinemia who are refractory to treatment by methylene blue. acquired methemoglobinemia is more common than any genetic causes. acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds. for patients failing to respond to standard treatment with methylene blue or in whom its use is contraindicated; hyperbaric oxygen or rbc exchange is indicated aims: case reports on use of rbc exchange in methemoglobinemia are few and indications are based on anecdotal reports. methods: exchange was performed on the cell separator machine, com tec by fresenius kabi. results: we report a case of acquired methemoglobinemia where patient was admitted with peripheral capillary oxygen saturation (spo2) of 87% on air. the patient did not show improvement in spo2 level with effective emergency treatment of methylene blue. since, the patient was refractory to treatment with methylene blue, the decision was made by clinician to proceed with rbc exchange. the patient improved significantly after two cycles of one rbc volume automated rbc exchange, and was discharged with spo2 of 97% on air. summary/conclusions: automated rbc exchange can be used in patients of acquired methemoglobinemia successfully when methylene blue is ineffective, and may be superior to manual one. background: therapeutic plasma exchange (tpe) is known to disturb the ph and electrolyte status. patients with compromised liver functions may be at a higher risk of electrolyte imbalance due to metabolic abnormalities. aims: the aim of this study was to analyze the variation in ph, ionized calcium, sodium, potassium, and bicarbonate in liver disease patients undergoing tpe. methods: patients with liver disease undergoing tpe during the period from july 2016 to august 2017 were included in the study. data on patient demographics, details of the tpe procedure, blood gas analysis report and adverse effects of tpe (if any) were collected and analyzed. results: one hundred and seven procedures were done during the study period; of these 46 (43%) were done on the mcs plus (haemonetics corporation) and rest 61 (57%) were done on the spectra optia (terumo bct). the percentage change in ionized calcium, sodium, and potassium due to the procedure was found to be statistically significant (p = 0.000). the systolic (p = 0.010) and diastolic (0.001) blood pressure also changed significantly with the procedure. the predictors for the change in ionized calcium were found to be pre-procedure ionized calcium (p < 0.001), the age of the patient (p < 0.001) and the pre-procedure ph (p = 0.002). procedurerelated complications occurred during 30 procedures of which 13 complications (12.15%) were categorized as features of hypocalcemia. no association was found between hypocalcemic manifestations and pre-procedure calcium, change in calcium, age or gender of the patient. summary/conclusions: the tpe procedure in liver disease patients causes remarkable changes in ionized calcium, sodium, potassium and bicarbonate ions. the decrease in ionized calcium during the procedure is predicted by pre-procedure ionized calcium levels, ph and age of the patient. monitoring of these parameters and appropriate corrective measures are imperative to patient safety. background: therapeutic plasma exchange (tpe) in pediatric age group is technically demanding because of low blood volume, difficult venous access and poor cooperation of the patient during the procedure. we here present our experience of tpe in pediatric patients from our centre. aims: to assess the challenges during tpe in pediatric patients and formulate appropriate strategies. methods: we did retrospective analysis of all tpe procedures performed in pediatric patients over a period of 16 years (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . tpe procedures were done on two different apheretic devices (cs 3000 plus, fenwal usa and cobe spectra, terumo bct lakewood, colorado) daily or on alternate days depending on clinical condition of the patient. for all procedures, kit was primed with compatible packed red cells. adverse events during the procedure were noted and analyzed. results: a total of 356 tpe (range 1-22/patient with mean of 6.2 procedures) were performed for 55 pediatric patients with different indications like atypical hus (category i as per american society for apheresis (asfa) in total 44 patients, neuromyelitis optica (category ii) in 4 patients, rapid proliferative glomerulonephritis (category i), c3 glomerulopathy in 3 patients each and one patient of infective hemophagocytosis. the average age of patient population was 7.8 yrs (1.2-13 years) . the male:female ratio was 3:1 with an average weight of 25.5 kgs. adverse events were observed during 20 (5.61%) procedures. most commonly observed adverse events were allergic reaction to replacement fluid (1.4%) followed by hypotension (1.1%), line occlusion (0.8%), vasovagal, endotracheal tube blockage and symptomatic hypocalcemia was observed in one procedure each (0.28%).there was no corelation observed between physical parameters of patient with adverse events. all adverse events were managed as per departmental standard operating procedures (sops) and procedures were completed successfully except in one where the procedure was abandoned. no mortality was observed during the procedures. background: the hemoglobin (hb) content of packed red blood cell (prbc) units is heterogenous. the patient's blood volume is also variable which can be calculated based on the weight, height and body surface area (bsa) of the patient. the efficacy of a transfusion episode can be assessed if the hb content of prbc is known and the patient's post-transfusion hb increment is determined. aims: this prospective study was performed to compare the efficacy of the transfusion of prbcs based on hb content versus the standard transfusion practice in thalassemia major patients. we also determined the correlation between hb increment and the hb content of prbc units transfused. methods: a total of 160 registered thalassemia major patients of our institute were included in the study after excluding the patients who had allo-or auto-antibodies. the study was approved by the institute ethics committee. the enrolled patients were randomly divided into two groups: group i (n = 80)they received abo/rhd identical prbcs suspended in additive solution (saline, adenine, glucose, mannitol: sagm-prbcs) after determining its hb content (units with hb content ≥ 50 g); and group ii (n = 80)they received randomly selected abo/rhd identical sagm-prbcs. the hb estimation of the randomly selected units in group ii was blinded. following tests were done on pre-transfusion sample: hb estimation using the hematology analyzer (orion 60, ocean medical technologies, india), blood grouping using tube technique, anti-human globulin (ahg) crossmatch and direct antiglobulin test (dat) using gel technique (biorad, switzerland), antibody screening (abs) using a fully automated immunohematology analyzer (neo, immucor, usa). on the posttransfusion sample collected 1 h after transfusion, hb estimation and dat were performed. results: there was no significant difference among the patient characteristics of the two groups. the mean hb content of the sagm-prbc units was significantly higher (p = 0.000) in group i (mean ae standard deviation: 67.86 ae 8.07 g; range: 50.80-92.13 g) than group ii (60.92 ae 8.29 g; range: 40.86-86.76 g). the mean hb increment in group i patients (3.26 ae 0.83 g/dl) was significantly higher (p = 0.04) than the group ii patients (3.00 ae 0.76 g/dl). in both the groups i and ii, there was a significant negative correlation between hb increment and weight (p = 0.000 in groups i and ii), age (p = 0.001 for group i; p = 0.032 for group ii), body surface area (bsa) (p = 0.002 for group i; p = 0.000 for group ii) and blood volume (p = 0.006 for group i; p = 0.000 in group ii). in both the groups i and ii, there was a significant positive correlation between hb increment and hb dose adjusted for bsa and the hb dose adjusted for blood volume (p = 0.000 in both groups i and ii for both the parameters). summary/conclusions: the efficacy of transfusion is more when patients are transfused with sagm-prbcs having hb content of 50 g or more as compared to those who are transfused with randomly selected units. for optimal hb increment in thalassemia major patients, the transfusion strategy should be based on the hb content of the sagm-prbcs. background: in male transfusion recipients under 50 years of age, receiving red blood cells (rbcs) from an ever-pregnant blood donor has been associated with increased mortality, compared to receiving a product from a male donor. although it has been suggested that older units of rbcs could be associated with increased mortality, there are significant methodological challenges in these studies. other studies indicated the freshest units of rbcs could be associated with increased mortality among transfusion recipients. we hypothesize both the association between ever-pregnant donors, and fresh units, with mortality could be caused by passenger leukocytes in the transfused rbc units, which decay during storage. aims: to quantify modification of the effect of ever-pregnant donors on mortality in young male rbc transfusion recipients, by storage time. methods: data on transfusion recipients receiving their first-ever rbc transfusion in one of six major dutch hospitals between 20/03/2004 and 01/09/2015 was collected. for the current study, male transfusion recipients under 50 years receiving only transfusions from one donor sex exposure category were selected and followup was censored at three years after transfusion. differences in storage time between groups were estimated by linear regression, adjusted for total number of transfusions, patient age, blood group, transfusion year and month. in a single-unit, single-transfusion cohort, cumulative mortality was estimated separately for patients receiving transfusions from ever-pregnant or male donors and for 'fresh' (<10 days storage) or 'old' (>24 up to 36 days storage) rbcs. results: for recipients of only blood from male donors, the storage time of the freshest unit was 0.47 day shorter when comparing the 221 patients who died, to 1,623 patients who survived (ci: à1.41 to 0.48). for recipients of only blood from ever-pregnant donors, the storage time of the freshest unit was 0.64 day longer when comparing the 27 patients who died, to 101 patients who survived (ci: à2.53 to 3.80). in the single-transfusion cohort, 1,280 patients received a fresh rbc transfusion from a male donor, 52 of whom died; 138 patients received a fresh transfusion from an ever-pregnant female donor, 9 of whom died. 193 patients received an old transfusion from a male donor, 7 of whom died; 16 patients received an old transfusion from an ever-pregnant female, 2 of whom died. the 3-years cumulative incidence of death among young male recipients was 4.7% (confidence interval (ci): 3.6% to 6.2%) after a fresh transfusion from a male donor and 4.8% (ci: 2.2% to 10.4%) after a fresh transfusion from an ever-pregnant female donor. the 3-years cumulative incidence of death was 7.2% (ci: 3.8% to 13.6%) after an old transfusion from a male donor and 15.4% (ci: 4.1% to 48.8%) after an old transfusion from an everpregnant female donor. summary/conclusions: prolonged storage of rbcs from ever-pregnant donors was not associated with decreased mortality at 3 years. contrary to our expectations, our results indicate older units may potentiate the effect of ever-pregnant donors. however, due to limited sample size the observed differences were not statistically significant. background: according to the literature review, there was limited impact of premedication (antipyretics, antihistamines and steroids) before transfusion on the prevention of adverse transfusion reactions (atrs). however, the necessity of premedication remains controversial. the premedication before transfusion is still a common clinical practice in pacific-asian countries, along with the premedication rate ranging from 50 to 80%. in our previous investigation, we found that premedication rate was 92.5% in the outpatients in 2017, which was much higher than the reported rate in asia. aims: to investigate the incidence of atrs and decrease premedication rate without increasing the rate of atrs via education and evidence-based clinical practice. methods: the incidence of atrs from april to december, 2017 was retrospectively surveyed. evidence-based clinical practice was initiated since january, 2018. clinical data of the outpatients receiving transfusion therapy were requested and analyzed from january to september, 2018. the incidences of atrs and premedication rates in 2017 and 2018 were compared using chi-square test. a p value less than 0.05 was statistically significant. besides, feedback of the incidence of atrs and premedication rate was given quarterly to the clinicians during the investigation. results: from april, 2017 to september, 2018, a total of 5,018 blood units were transfused in the outpatients with 2,453 transfusion events. of these, 25 cases of atrs, including febrile nonhemolytic transfusion reactions (fnhtr) and minor allergic reactions were reported. the overall premedication rate in the outpatients was 92.5% in 2017, and was significantly decreased to 77.9% in 2018 (p < 0.001). it was reported that the incidences of atrs in 2017 and 2018 were 0.48% and 0.52% per unit, respectively. there was no remarkable difference between the incidence of atrs in 2017 and 2018 (p = 0.642). summary/conclusions: via education and evidence-based clinical practice, we successfully reduced premedication rate without increasing the rate of atrs in the outpatients. furthermore, introduction of computerized provider order entry (cpoe) and clinical decision support system (cdss) could be considered and be expected to prevent unnecessary premedication before transfusion, increasing the compliance with optimized transfusion strategies in the future. methods: a retrospective analysis was done over a period of one year to evaluate clinical efficacy of 19 granulocyte transfusions in 15 hemato-oncology patients with febrile neutropenia. mobilization of granulocyte donors was done as per standard protocol, which included subcutaneous injection of granulocyte colony stimulating factor (g-csf) 5-10 lg/kg and tablet dexamethasone 8 mg, 10-12 h prior to granulocyte harvest by apheresis. all granulocyte products were gamma irradiated before transfusion. patient parameters like white blood count (wbc), absolute neutrophil count (anc), hemoglobin and platelet count were recorded pre-and post-granulocyte transfusion. infection related mortality (irm) within 30 days of granulocyte transfusion was also recorded. results: minimum adequate granulocyte yield of 1 9 10 10 per unit was fulfilled in 90% of granulocyte harvests. clinical indications for granulocyte transfusions were fever, an absolute neutrophil count (anc) < 500/ll, evidence of bacterial and/or fungal infections (i.e. clinical signs of infection, positive cultures and radiological evidence) and unresponsiveness to appropriate antimicrobial therapy for at least 48 h. effects of clinical, microbiological and granulocyte transfusion related variables on infection-related mortality were investigated. the post transfusion anc (within 24 h) increased significantly (median value: 350/ll) as compared to baseline levels (median value: 40/ll) (p < 0.05). infection related mortality was observed in only 20% (3 out of 15) of patients. patients became afebrile within 2-4 days and culture negative within 3-6 days after granulocyte transfusion. for analysis purpose granulocyte transfusion episodes were grouped according to doses of granulocyte transfusions, based on european guidelines (standard dose: 1.5-3.0 9 10 8 cells/kg and high dose: >3.0 9 10 8 cells/kg background: hsa's blood services group (bsg) is singapore's national blood service. in 2016, we conducted our pilot national pbm audit to promote pbm practices. it was agreed that the audit would be performed annually with incorporation of a new indicator to continue promotion of pbm and sharing of good practices. aims: to provide an update on the second national pbm audit for 2017. results are compared to the pilot audit and summarized below. methods: we collected data on 3 performance indicators from 7 acute public care hospitals for 4 weeks each in march and august 2017 (the pilot audit covered 2 weeks in 2016). the performance indicators were: 1). percentage compliance to documentation of red blood cell transfusion indications 2). percentage of patients screened for pre-operative anaemia, 14 to 45 days before surgery 3). peri-operative transfusion rates (3 days before to 3 days after surgery) for 6 commonly performed surgeries: coronary artery bypass graft surgery (cabg), total knee replacement (tkr), total hip replacement (thr), nephrectomy, colectomy and hysterectomy. the first two indicators assess pbm efforts and were measured in the pilot audit. indicator 3) was added to the second audit to assess impact of pbm practices on transfusion in surgical patients. it was an appropriate time to incorporate this indicator as the hospitals would have been familiar with pbm since its introduction in 2012. results and recommendations were shared with the senior management and hospital transfusion committees of the participating hospitals. results: for indicator 1), 2 hospitals had a compliance of 57-61%, the remaining 5 had a compliance of 90-100%. all 7 hospitals incorporated electronic blood ordering but the usage was not compulsory in some. hospitals which mandated electronic ordering performed better as doctors could only order blood products after entering the transfusion indication. we saw compliance increase from 75% in 2016 to 100% in a hospital that had newly mandated electronic ordering. for indicator 2), results ranged from 30% to 92%. 2 hospitals made notable improvements when compared to 2016, achieving 83% and 88% respectively. they had implemented pre-operative workflows screening all elective surgical cases for anaemia at least 2 weeks before surgery. one hospital also started an outpatient intravenous iron service which reduced pre-operative anaemia rates. for indicator 3), mean number of transfused units for each surgery ranged from 1.5 to 2.8 units per patient, lowest being thr and highest being cabg. this suggests that some transfusions were potentially avoidable with more robust pbm practices. the rate of perioperative transfusions was highest for cabg at 46% and lowest for tkr at 7%. summary/conclusions: the annual national pbm audit increases pbm awareness, allowing hospitals to share and learn good practices and implement measurable improvements. based on this audit, a recommendation to mandate electronic ordering of blood products to improve adherence to red cell transfusion indications and implementing pre-operative workflows with consideration for intravenous iron support was made. this audit was more representative than the pilot, with a longer duration of data collection and incorporation of indicator 3) showing impact of pbm practices. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to the diagnosis, investigation and management of patients with autoimmune haemolytic anaemia (aiha) in english nhs trusts. methods: we designed and distributed a survey to the clinical transfusion leads at all english nhs trusts between november 2017 and march 2018. the survey requested information on detailed, simulated clinical scenarios. the first simulated scenario described a young patient with active aiha 3 months after an allogeneic stem cell transplant, who has received multiple transfusions in the last 2 weeks and is hypotensive, tachycardic, with a falling haemoglobin (hb), currently 48 g/l. the second scenario describes a young man with a new diagnosis of warm aiha who has an initial hb of 104 g/l and returns to clinic at a 2-week interval with symptoms of fatigue. he is actively haemolysing and commenced on 1 mg/kg prednisolone. results: there was a 42% (58/137) response rate by trusts. faced with a 4-6 h delay for allo-adsorption studies, 68% (38/56) of respondents would instead transfuse acutely with abo, rh and k matched red cells negative for any previously detected alloantibodies, 7% (4/56) would transfuse with o rh d negative red cells and 25% (14/56) would wait for completion of allo-adsorption studies before transfusing. in this first scenario, a quarter of respondents appeared to delay a potentially lifesaving blood transfusion. 2017 british society of haematology guidelines recommend that when anaemia is life-threatening in the time required for full compatibility testing, abo, rh and k matched red cells should be transfused. in the 2017 serious hazards of transfusion (shot) report, the most serious and fatal of 95 cases of preventable delayed transfusion was a patient with aiha who died untransfused with an hb of 38 g/l, while awaiting alloadsorption studies. a key shot message was that if clinical harm to patients from withholding blood outweighs safety concerns over a possible delayed haemolytic transfusion reaction, emergency blood is essential and should be offered. the second scenario also identified considerable variation in transfusion practice. it can take several weeks for patients with aiha to respond to prednisolone so a transfusion threshold < 60 g/l after an hb fall of at least 40 g/l in the previous 2 weeks is perhaps overly conservative. summary/conclusions: the overall findings support a need for studies to explore barriers to uptake of guidelines, and to identify areas for further audit and research to guide safe and appropriate transfusion practice in aiha. background: balance between supply and demand of o d negative red cells remains a challenge for almost every blood service. with this re-audit, we wanted to collect objective and comprehensive information regarding usage of o d negative red cells supplied by nhs blood and transplant (nhsbt) to private and nhs hospitals in england. aims: the aim was to understand hospital practices, actual needs and possible avoidable usage of o d negative red cells. where possible, comparisons were made with two previous audits (2008) (2009) (2010) . methods: participating hospitals were asked to determine the fate of all group o d negative red cells they received between 14 th and 27 th may 2018 excluding substitutions and complete an organisational survey regarding activities, policies and stockholding practices with respect to o d negative blood. participating hospitals were asked to provide (if available) the prevalence (as a percentage) of o d negative patients in their population. this information, in conjunctions with hospital activities, will be used to estimate appropriate o d negative stockholding levels. background: o rhd-negative (neg) red blood cells (rbcs) are a precious resource, are often in short supply and transfusion of these units in emergency settings carries the potential risk of transfusion-related adverse outcomes such as haemolytic reaction due to minor blood group incompatibility. as such, their use should be closely monitored within health services. most recent australian guidelines (2008) for their use in emergency settings include pre-menopausal females of unknown blood group (mandatory indication) or while the blood group is being established; use should be limited to 2 or less units where possible before a switch to group-specific rbcs (acceptable indication). aims: audit of use of emergency uncrossmatched o rhd-neg rbcs against national guidelines in our institution (an australian tertiary metropolitan public hospital providing acute medical and surgical, emergency and critical care services). methods: use of emergency uncrossmatched o rhd-neg rbcs units over a six-year period was retrospectively reviewed. we collected information about rbcs transfused and discarded, adverse outcomes, patient characteristics, clinical indications and whether use met national guidelines or could have been avoided. results: 105 episodes of emergency uncrossmatched o rhd-neg rbcs were identified, encompassing transfusion of 241 rbc units to 103 patients and the discard of 2 rbcs (due to incorrect transport). of the 105 episodes, 78 episodes (74%) involved an eventual switch to group-specific rbcs (range of emergency units, 1-13 units). the main requester was the emergency department (53%). the most common clinical indication for transfusion was acute gastrointestinal bleeding (49%). of the 105 episodes, 28 episodes (27%) did not meet the guidelines for emergency use because > 2 units of emergency uncrossmatched o rhd-neg rbcs were issued. 2 episodes (2%) were flagged as potentially inappropriate as the patients were clinically stable according to documentation in the medical records. 30 episodes (29%) were identified as potentially preventable due to delay in pre-transfusion sample collection (defined as > 1 h elapsed between patient arrival and group and screen sample collection) in the setting of acute bleeding (6%), receipt of an unsuitable pretransfusion sample requiring sample recollection (5%), delay in pre-transfusion sample processing (4%), no valid pre-transfusion sample being available at the time of the bleeding episode despite having a planned elective procedure or being an inpatient with recent clinical bleeding (10%). only one patient was investigated for potential transfusion-related adverse outcome (1%) which was thought likely due to concurrent sepsis. summary/conclusions: over six years, 105 episodes utilising emergency uncrossmatched o rhd-neg rbcs were identified with 241 rbcs issued and 2 rbcs discarded. a significant proportion of episodes (29%) were potentially avoidable if there had been a valid pre-transfusion sample available in the transfusion laboratory at the time of the episode. efforts to minimise use of this precious resource are ongoing, and include feedback to clinical units regarding importance of valid pretransfusion samples prior to applicable invasive procedures and in bleeding patients, ongoing education to medical and nursing staff, and continuing audit of use of this blood component in the hospital haemovigilance programme. abstract withdrawn. abstract withdrawn. background: platelet transfusions are often given prophylactically to thrombocytopenic hematology patients. to which extent platelet function improves after transfusion, and how this improvement correlates with an increase in platelet count, is not well studied. flow cytometry has been used to evaluate platelet function after transfusion in a few studies and can be performed even at low platelet counts. rotational thromboelastometry (rotem) represents a more physiological measure of platelet function in whole blood that has not been extensively used in transfusion settings. we used these methods to investigate if platelet transfusion improves platelet function in hematology patients and if improvement correlates with increased platelet counts. aims: the aim was to evaluate the relationship between response to platelet transfusion, measured as corrected count increments (cci), and platelet function in thrombocytopenic patients with hematological disorders. methods: blood samples (sodium-citrate anticoagulated) were collected from unselected hematology patients receiving prophylactic platelet transfusions, after informed consent had been obtained. samples were taken at three time-points: within 1 h before transfusion, 1 h after and 14-24 h after transfusion (via a central venous catheter or a subcutaneous venous port). for each time-point, platelet response to adenosine diphosphate (adp) and thrombin receptor-activating peptide (trap-6) was assessed by flow cytometry by measuring p-selectin and pac-1 expression on single platelets. rotem analysis was also performed on all samples, using intem and extem reagents. results: an interim analysis was performed after inclusion of 22 patients. the mean platelet count before transfusion was 8 9 10 9 /l (range 2-34 9 10 9 /l). 1 h cci was 11 9 10 9 /l and 14-24 h cci was 6 9 10 9 /l, but response was highly variable. pselectin expression after stimulation with adp and trap was significantly higher at 1 h after and 14-24 h after transfusion compared to before transfusion (p < 0.05). pac-1 expression after stimulation with adp was significantly higher at 14-24 h after transfusion (p < 0.001), but not at 1 h after transfusion. in rotem, clot amplitude at 10 and 20 min (a10 and a20) as well as maximum clot firmness (mcf) improved after transfusion (p < 0.05). a significant correlation between absolute platelet count and p-selectin expression after trap and adp stimulation was found (r s =0.53 and 0.45 respectively, p < 0.001). absolute platelet count was also significantly correlated with mcf (r s =0.61, p < 0.001), where 84% of patients with a platelet count of more than 20 9 10 9 /l reached mcf values within the reference interval. summary/conclusions: platelet function generally improves after transfusion and was in our patient population correlated to the absolute platelet count, but was also seen at the single platelet level in flow cytometry. a post transfusion platelet count of more than 20 9 10 9 /l might be sufficient to significantly improve coagulation in heavily thrombocytopenic patients, but larger studies are needed to confirm this conclusion. abstract withdrawn. abstract withdrawn. background: sickle cell disease (scd) is a genetic disorder that is frequently referred to as a hypercoagulable state. hydroxyurea (hu) is known to decrease the frequency of vaso-occlusive complications and need for blood transfusions in severely affected individuals. although cross-sectional studies show that treatment with hu is associated with decreased coagulation activation, there are no prospective studies evaluating the effect of hu on coagulation activation. aims: to assess the effect of hu on markers of fibrinolysis (d-dimer) and endothelial activation (soluble vascular cell adhesion molecule-1 [soluble vcam-1]) in patients with scd in their non-crisis, "steady state." methods: patients, at least 10 years of age, with documented hbss or hbsb-thalassemia, eligible for treatment with hu were studied in this prospective, observational study. laboratory investigations were obtained at baseline, prior to commencement of therapy with hu, with repeat evaluations at three and six months of therapy. non-parametric test was applied to observe the association between hu therapy and the biomarkers of interest. results: twenty-five patients with scd (hbss: 15, hbsb thalassemia: 10) were enrolled (females: 15 [60%]), with a median age of 23 years (iqr: 11). following 6 months of hu, median values for wbc count (9.02 9 10 9 /l vs. 7.22 9 10 9 /l, p = 0.007) and d-dimer (1243.8 ng/ml vs. 830.2 ng/ml, p = 0.028) were significantly lower than baseline values, while the mean corpuscular volume (76.0 fl vs. 88.0 fl, p = 0.001) was significantly higher than the baseline value. no significant differences from baseline were observed in the median values for hemoglobin (9.1 g/ dl vs. 9.6 g/dl, p = 0.72), platelet count (250 10 9 /l vs. 196.5 10 9 /l, p = 0.71), lactate dehydrogenase (744 u/l vs. 567.5 u/l, p = 0.23) or soluble vcam-1 (567.8 ng/ ml vs. 526.4 ng/ml, p = 0.33) following 6 months of hu therapy. summary/conclusions: this exploratory study confirms that treatment with hu is associated with decreased coagulation activation in patients with scd, although no effect on endothelial activation was observed. by decreasing coagulation activation, hu may decrease the risk of thrombotic complications in scd. abstract withdrawn. abstract withdrawn. transfusion medicine, apollo gleneagles hospitals, kolkata, india background: reduction of immune responsiveness through blood transfusion has been documented by previous authors. breast cancer is considered as one of the commonest cancer globally and the second main cause of death in females transfusion of allogeneic blood in breast cancer surgery is variable and differences of transfusion incidence have been observed in the literature. where the maximum surgical blood ordering schedule (msbos) dictates cross matching and reservation of blood before surgery, factors deciding their utilization are varied and numerous. our hospital protocol guides that every patient planned for elective breast cancer surgery should routinely have a blood sample sent for reservation of one unit of compatible packed red blood cell (prbc) in the blood bank. aims: in this prospective study we aimed to audit the blood utilization in patients undergoing elective breast surgery and thereby optimize the blood ordering schedule, economic burden and loss of clinical resources. methods: the study included 478 confirmed breast cancer patients planned for elective breast surgeries from january 2012 to december 2017. patient and disease details like age, stage, tnm status, estrogen receptor (er) and progesterone receptor (pr) status, human epidermal growth factor receptor 2 (her -2) expression, triple negative breast cancer (tnbc) status, reproductive and treatment status were documented. patients were divided into younger group [≤40 years] and older group (>40 years). before surgery blood samples for compatibility testing were sent to blood bank for blood reservation. details of test, blood issue and blood transfusion were documented in the blood bank. approximate loss of time in minutes and wastage of resources in terms of money (inr) in the blood bank were noted. all results were calculated as mean ae sd and a 'p' value of < 0.05 was considered statistically significant. results: of the total 478 patients most underwent wide local excision of the breast and modified radical mastectomy. a total of 16 patients received 71 units of blood and blood components in all categories of surgeries. only 103 were younger women (≤40 years) with mean age of 31 years. non-transfused patients were significantly more than transfused ones (p < 0.05). frequency of blood transfusion was more in young patients (4.9%). seven (22.6%) of the total 31 stage iv patients received blood transfusions. frequency of blood transfusion was more in patients undergoing surgery after chemotherapy (8.8%). a significant loss of time and loss of revenue was observed. summary/conclusions: we conclude that routine compatibility test is not justified for all patients undergoing breast surgery. a more targeted approach is needed to reduce blood demand and associated cost to patient and blood transfusion services. background: blood transfusion guidelines are not only essential for the optimal use of blood products, but also help reduce transfusion-related adverse reactions and improve patient outcomes. the korean national transfusion guidelines were developed in 2009 and fully revised in 2016 by the korean centers for disease control and prevention and the korean society of blood transfusion. in our hospital, which is a 700-bed university hospital, a transfusion-indication data-entry program based on the national transfusion guidelines was developed in 2016. it was applied to the electronic medical record system and all transfusion orders, except emergencies, have been performed through this program since then. aims: we planned to record and analyze the reasons for transfusion in order to monitor blood product usage and provide feedback to clinicians. furthermore, we intended to contribute to patient safety through the appropriate use of blood products. methods: we classified transfusion-indications by the blood product requested and created a pop-up window listing these indications, which would appear at each regular transfusion order. indications for transfusion with each blood product were as follows: red blood cells (rbcs)acute blood loss, chronic disease (sub-classified as hb ≤ 7 g/dl, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, respiratory disease, age ≥ 65 years, age ≤ 6 months, chemotherapy), surgery/ procedure, transplantation and 'other'; platelets (plts)present bleeding, bleeding prevention (sub-classified as hematologic disease, solid tumor, peripheral blood stem cell transplantation, disseminated intravascular coagulopathy, infant), surgery/procedure, massive transfusion and 'other'; fresh frozen plasma (ffp)bleeding in coagulopathy, bleeding prevention in coagulopathy, massive transfusion, plasma exchange and 'other'. transfusion indications entered into the data-entry program from sep 2016 to feb 2018 were analyzed. results: the number of transfusion-indications analyzed was 16138 for rbcs, 11158 for plts and 6024 for ffps. the most common indications for transfusion were chronic disease for rbcs (7977/16138, 49.4%), bleeding prevention for plts (5726/11158, 51.3%) and 'other' for ffp (2180/6024, 36.2%). 'hb ≤ 7 g/dl' was the most frequent sub-indication of chronic disease (3570/7977, 44.8%), and hematologic disease was the most frequent sub-indication of bleeding prevention (3432/ 5726, 59.3%). many clinicians entered transfusion indication as 'other': rbcs (2866/ 16138, 17.5%), plts (856/11158, 7.7%) and ffp (2180/6024, 36.2%). however, the free-text supplied by the clinician when 'other' was selected, often corresponded to an indication already categorized in the transfusion-indication data-entry program; 82.9% of rbcs and 54% of plts. of the indications entered as 'other' in ffp, 80.3% were surgery/procedure-related. summary/conclusions: in our hospital, the release of blood products has been dependent on the data-entry of transfusion indications (except in emergencies) since sep 2016. transfusions of rbcs and plts were most common for chronic disease and bleeding prevention, respectively, but many cases entered as 'other' could have been categorized as existing indications in our data-entry program. therefore, we conclude that additional training is needed for clinicians regarding the determination of transfusion-indications and correct use of the transfusion-indication dataentry program, in order to use blood products more appropriately. methods: this was a prospective cohort designed study. subjects were children aged 1-18 years with indication of platelet transfusions in sardjito hospital yogyakarta indonesia. the patient samples were collected before and 1 h post-transfusion, the expression of cd62p on platelet was determined by flow cytometry method. results: there were 102 subjects who were divided into two groups. fifty-one subjects received non-leukodepleted pcs and the other fifty-one transfused by pre-storage leukodepleted pcs. the mean of pre-transfusion platelet cd62p for nonleukodepleted and leukodepleted groups were 26.2% and 27.7%, and the mean increase of post-transfusion platelet cd62p for non-leukodepleted was 10.1% and the mean decrease of leukodepleted groups was 3.3%. it was shown the increase of post-transfusion platelet cd62p for non-leukodepleted group, and it was significantly (p < 0.05) higher than in the leukodepleted groups. summary/conclusions: there was an increase of post-transfusion platelet cd62p expression in patients received non-leukodepleted, but a decrease in leukodepleted pc transfusions. background: preoperative anaemia is a common finding in patients undergoing surgery and often neglected in our country. aims: the objective of this study was to evaluate hb(values and the identification of cardiac patients who entered operation with anaemia. and also to study the correlation between hb values and the number of rbc (red blood cell) transfused unit methods: this is a retrospective, descriptive and analytical study. the data for this study was collected from the files in the statistic's service at qsut (university hospital center "mother teresa"). the object of our study were the files of 158 patients hospitalized in the period january -may 2018 in the cardiac surgery ward, which were subjected to cardiac surgery. from the files were collected data on age, gender, primary diagnosis, accompanying diseases. we also collected hb, rbc, htc (hematocrit), mcv (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration). from the transfusion service at qsut and from the files were pulled out the transfused patients and the number of transfused units. results: based on the who definition for anemia (females < 12 g/dl and males < 13 g/dl), from the 158 patients included in the study, 61 (39%) were anaemic. from 117 males in the study, 40 (34%) of them were anaemic based on hb lab values, whereas from 41 women in the study anaemic were found to be 21 (51%) of them. from the 61 anaemic patients in the study, 35 (57.4%) of them with mild anaemia, 23 (37.7%) with moderate anaemia and 3 (4.9%) with severe anaemia. in the total of anaemic female 38.1% are under 65, while 61.9% are over/or 65 years old. in the total of anaemic males, 35% are under 65, while 65% are over/or 65 years old. it is noticed that most of them are with normochromic normocytic 67.2%, normocytic hypochromic anaemia 16.4%, hyperchromic microcytic anaemia 8.2%, macrocytic normochromic anaemia and macrocytic hypochromic anaemia respectively 1.6% and microcytic normochromic anaemia 4.9%. the average value of preoperative hb decreased from 12.8 g/dl before surgery to 10.3 g/dl after surgery, so there is a decrease of approximately 2.5 g/dl of hb value. in our 158 patients, 48% (76) were transfused and the remaining 52% (82) were not transfused. from 76 transfused patients 41 (54%) patients were anaemic. the correlation between the values of hb, rbc, htc and the number of transfusions shows that with the decrease of these values the number of transfused units increases. summary/conclusions: the diagnose of anaemia is underestimated before surgical intervention in our country and investigation of hb low values do not take the proper importance to find probable cause and correct it before surgical intervention. the lower the hb values, the greater the chance to be transfused and the number of rbc transfused units. failure to correct hb values before surgery results in unnecessary transfusions for the patients or which could have been avoided, eliminating also the risk of transfusion complications. background: alloimmunization after red blood cell transfusion is affected by various factors. it is known that the incidence of alloimmunization increases in certain diseases. extended red blood cell matching can be used to prevent the development of alloimmunization in diseases which the rate of alloimmunization is increased. in asia, extended red blood cell matching is not actively implemented. aims: we tried to investigate whether there is a difference in the disease categories between unexpected red blood cell antibody positive and negative groups. methods: from january, 2003 to december, 2017, the diseases of the patients who had undergone unexpected red blood cell antibody identification test at dong-a university hospital was examined through medical records. from january 2008 to december 2009, the diagnosis was made on patients who had two or more unexpected antibody screening tests. we analyzed the frequency difference of disease category between two groups. results: a total of 988 patients were performed with unexpected antibody identification tests. of 1896 patients who underwent more than 2 screening tests, 25 (1.3%) were positive. 1871 were consistently unexpected antibody negative. the patients with solid tumors (n = 375, 56.3%) and those with hematologic diseases (n = 112, 16.8%) had a higher incidence in unexpected antibody positive group. the patients with myeloid malignancy had a significantly higher frequency than lymphoid malignancy (p = 0.0002). the frequency of patients with liver cirrhosis was significantly higher in the unexpected antibody positive group (65/988, 6.6%) than in the negative group (77/1871, 4.1%) (p = 0.006). the incidence of non-hodgkin lymphoma was significantly higher in the unexpected antibody negative group (28/1871, 1.5%) than in the positive group (5/988, 0.5%) (p = 0.019). summary/conclusions: there was a difference in the distribution of diseases between unexpected antibody positive group and negative group. the patients with liver cirrhosis were more frequent in unexpected antibody positive group, suggesting that extended red blood cell matching would be considered. background: in hematological patients with multiple platelet transfusions (pc) often develop immune response to human leukocyte associated antigens (hla-i) and human platelet-specific associated antigens (hpa). besides, platelet associated immunoglobulins (paig) and complement components (pac) are found on platelet. this leads to increased platelet destruction and development of refractoriness to transfusions of donors' platelets. transfusion therapy using an individual selection of platelets and plasmapheresis, contribute, in the majority of cases, to the realization of efficient transfusion by pc. but, in difficult cases, there is a need to use intravenous immunoglobulin, which may promote the efficient transfusion of pc. aims: evaluate the algorithms of using the complex therapy of refractoriness to transfusions of donors' platelets with additional application of intravenous immunoglobulin (ivig). methods: in 2018 there were three female patients in the clinics of the centre for observation, age between 29 and 51 years (me = 39) with the ineffectiveness of complex therapy for overcoming refractoriness to transfusions of donors' platelets due to selection and plasmapheresis. the diagnoses were as follows: aplastic anaemia (aa)-2, acute myeloid leukemia (aml)-1. individual selection of platelets was carried out by the adhesion method on the solid phase (immucor "galileo neo"). paig and pac3/4 were evaluated by the method of flow cytometry (bd facscanto ii) by the method of double staining with cd41a. the density of fixed paig, pac was © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 evaluated by the median fluorescence intensity (mfi). the two patients with aa received ivig-igg therapy in the standard dose 0,4 g/kg per day, for 3 days. one patient with aml received ivig-iggam therapy in the standard dose 5,0 ml/kg/day for 3. results: under pressure of the complex therapy with the use of ivig in the standard dose there was are decrease in mfi over time in the case of two patients: aa-1 mfi-paigg reduced from 926 to 65; while the patient with aml: paiga reduced from 5506 to 187, and paigm from 1971 to 302, pac3 from 691 to 31, pac4 from 404 to 103. the patient with aa-2 over time, regardless of the treatment, there was an increase of mfi, but the effect of pc transfusions was achieved under pressure of complex therapy. under pressure of complex therapy all the 3 patients also reduced the frequency of reaction of alloantibodies when resorting to an individual selection and increasing the frequency of compatible couples "donor-recipient". summary/conclusions: delivery of complex therapy and the additional application of ivig enables an adequate transfusion therapy of pc, neutralize hemorrhagic syndrome and continue the treatment of the main disease. detection and monitoring of paig/pac during the development of refractoriness to transfusions of donors' platelets are additional markers for prescription of ivig therapy. anaesthesia, tan tong seng hospital, singapore, singapore background: blood transfusion is quite prevalent in paediatric cardiac surgical procedures. we hypothesized that the routine use of rotational thromboelastography (rotem) to guide transfusion decisions would reduce the overall proportion of patients receiving transfusions in paediatric cardiac surgery aims: the aim of the study was to find if the use of blood and blood products in pediatric cardiac surgical cases in a single centre is affected due to rotem. methods: sixty paediatric cardiac surgical patients undergoing cpb were included in this study. thirty patients (study group) were prospectively included and compared with thirty procedure and age-matched control patients (control group). in the study group, rotem, performed during cpb guided intraoperative transfusions. perioperative transfusions of blood and blood products, postoperative blood loss and hemoglobin levels were compared between the two groups. results: the patients in the control group received fewer transfusions of packed cells (60% vs 78%) and fresh frozen plasma (36% vs 84% p<o.oo1). more patients in the study group received platelets (72% vs 60%) and cryoprecipitate (50% vs 36%). the postoperative blood loss was lower in study group compared to the control group. the postoperative hemoglobin did not differ between the two groups. summary/conclusions: the results suggest that the routine use of rotem in paediatric cardiac surgery to guide transfusions is associated with reduced proportions of patients receiving transfusions and is evidence based practice. abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mycoplasma pneumonia (mp) infection has often been implicated in respiratory tract infections. although this agent has also been associated with other non-respiratory diseases, hemolytic anemia is the most common hematologic complication of mp infection. episodes of sudden onset of severe anemia are rarely seen in beta thalassemia. we report a 33-years-old female with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection aims: to report a case of patient with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection and how it was managed with minimal transfusion. methods: a 33-year-old female case of beta thalassemia major who developed autoimmune hemolytic anemia following mycoplasma infection. the patient is on monthly blood transfusion since the age of 3 years at dubai thalassemia center. she was admitted to the hospital with 5 days history of dizziness, severe low back and joint pain and her travel history indicated a four-week visit to pakistan. initial investigations showed a hemoglobin level of 7.8 g/dl the direct and indirect coombs test were positive. patient was transfused with two units leuko-depleted compatible prbc (o, rh +, negative for e and kell antigens.) and discharged next day. three weeks later, the patient was still with lower limbs pain and the hemoglobin was 8.5 g/dl with a positive dat with both igg+4 and c3+4, patient was not transfused as it was difficult to find matching blood; therefore she was given one week follow up. four days later, patient reported to the center with fever, generalized body pain and fatigue. she had significant drop in hemoglobin 5.6 g/dl, mcv 85.0, platelets count 216000, crp 3.6, retic count 2.18, blood culture no growth, total bilirubin 1.5, malaria parasite smear was negative, urine culture and microscopy were normal. mycoplasma pneumonia antibody showed positive with igg 57.1 and igm 21.3, she was started on azithromycin for 5 days and received 2 units prbc least incompatible leuko-depleted (o, rh +, negative for e and kell antigens.). after which her hemoglobin increased to 9.3 g/dl. she did not show any improvement in her condition over the following 2 weeks and repeat mycoplasma pneumonia antibody showed an increased titer, which denoted resistance to azithromycin; hence, we started her with second line of treatment levofloxacin for 10 days. screening for collagen disease were all negative apart from high esr of 130 mm/hr. patient remained afebrile and transfused with least incompatible available blood, post transfusion hb was 11.8 g/dl. three weeks later, during her routine follow up, she was doing well with esr dropping to 85 mm/hr and hemoglobin 10.2 g/dl, which indicated gradual settling of hemolysis, although the last dat remained positive. subsequent visits showed hb drop of 1.2 g/dl/week which is acceptable for transfusion dependant thalassemia. results: with conservative management patient condition improved. summary/conclusions: hemolytic anemia associated with mycoplasma pneumonia is usually self-limited and resolves spontaneously after mycoplasma infection elimination. packed red cell transfusion in transfusion dependent patient may be troublesome and can aggravate the condition; therefore it should be utilized and limited to significant symptomatic anaemia background: early plasma transfusion in women with severe postpartum hemorrhage is often instituted to prevent and treat coagulopathy, but whether maternal outcomes improve with this treatment is unclear. aims: to quantify the association of plasma transfusion within the first 60 min after diagnosing persistent postpartum hemorrhage and adverse maternal outcome. methods: we performed a retrospective cohort study of women with persistent postpartum hemorrhage, defined as postpartum hemorrhage refractory to first-line measures to control bleeding. women were enrolled from january 1, 2011 to january 1, 2013 in 61 dutch hospitals. in a time-dependent propensity score-matched analysis, women with transfusion of fresh frozen plasma within 60 min following the diagnosis of persistent postpartum hemorrhage were matched to women without plasma transfusion or plasma transfusion at a later time point during hemorrhage. plasma transfusion was not guided by coagulation tests. the main outcome measure was adverse maternal outcome, defined as a composite of mortality, hysterectomy and arterial embolization to control bleeding. we performed sensitivity analyses with initiation of plasma transfusion within 120 and within 180 min following diagnosis persistent postpartum hemorrhage, compared with other plasma transfusion strategies. results results: the cohort consisted of 1216 women with persistent postpartum hemorrhage, with uterine atony as cause of hemorrhage in 780 women (64.1%). in this cohort, seven maternal deaths (0.6%), 62 hysterectomies (5.1%) and 159 arterial embolizations (13.1%) were observed. plasma transfusion within 60 min in 114 women was matched on time-dependent propensity scores with 114 women with no or later plasma transfusion. rate of adverse maternal outcome was similar between women with and without early plasma transfusion, 21.2% versus 19.9%, adjusted odds ratio 1.09 (95% confidence interval 0.57-2.08). results of sensitivity analyses were comparable to the primary results. summary/conclusions: among women with persistent postpartum hemorrhage, initiation of plasma transfusion within 60 min following this diagnosis was not associated with improved maternal outcomes, compared with no or later plasma transfusion. whether other transfusion strategies or identification of women with high risk of coagulopathy will improve maternal outcomes remains to be studied. background: preoperative evaluation of increased risk for perioperative bleeding is essential for cardiac surgery patients. platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also for assessment of the increased risk of postoperative bleeding and perioperative application of allogenic red blood cell transfusions. aims: the aim of this study was to establish whether preoperative platelet function testing for the presence of residual effect of acetylsalicylic acid (asa) on platelet aggregation can be correlated with an increased risk for intraoperative bleeding during cardiac interventions. methods: a retrospective study included 60 patients who underwent surgical procedures (cabg ii and cabg iii) at the department of cardiovascular surgery, clinical center in ni s in period november 2017-july 2018. patients were previously taken asa at a dose of 100 mg per day, but therapy was discontinued 5 days before surgery. platelet aggregation was measured on the day of surgery using impedance aggregometer multiplate (multiplate platelet function analyzer, roche), using trap and aspi tests. patients were divided into two groups: control group (cg), which included patients with preserved function of platelets (aspi 790-1410 au*min), and examined group (eg), which included patients with inhibited platelet aggregation in aspi test (<790 au* min background: hemophilia b is an x-chromosome-linked inherited bleeding disorder resulting from reduced levels or an absence of factor ix clinically represented by recurrent prolonged bleeding. mutations can be inherited or acquired de novo. de novo mutations arise in about one-third of patients with haemophilia. the disease affects primarily males, but approximately 30% of carrier females have factor ix clotting activity lower than 40% and are at risk for bleeding. aims: case report of a hemophilia b carrier. methods: the clinical history of the patient was thoroughly studied by analyzing medical records and analytical results using the software available in the health institution and paper records. results: 45 year old woman, with no history of abnormal haemorrhage, healthy, up to 2004 when, after a dystocic delivery, presents with subcutaneous and ocular haemorrhage and dispersed bruises. a basic coagulation study is performed and a level of 20% fix is found. genetic analysis reveals a mutation in fix gene and she is diagnosed as a carrier of hemophilia b. except for a son, no other direct family members carry the mutation. in 2008, after total hysterectomy with bilateral adnexectomy due to an invasive carcinoma in the uterine cervix, the patient suffers a haemorrhagic shock with multiple transfusion support and around six months later undergoes an ureteronephrectomy also with the need for post-surgery transfusion support. the patient is referred to the immunohemotherapy department where she has regular appointments as an outpatient since 2009. in 2012, she requires surgery for the removal of a thyroglossal duct cyst, which is performed under prophylactic treatment with 2000 iu recombinant fix (rfix) daily, for four days, and up to 48 h after discharge maintaining basal levels of fix of 60% and with no major complications reported. in 2013, the patient is admitted to the er in haemodynamic shock and a history of black stools and syncope. at admittance, she receives a bolus of 4000 iu of rfix and requires severe transfusion support due to active bleeding. she is diagnosed with colonic angiodysplasia and undergoes surgery. daily doses of 3000 iu rfix are administered from day 1 (d1) and up to d7 maintaining fix levels around 60-80%. the patient is operated at d8 under the cover of 2000 iu of rfix, twice per day, and up to d14 after surgery reaching fix levels of around 80-110%. at d15 the dose was diminished to 2000 iu of rfix daily and at d17 she is discharged with no rfix replacement therapy necessary in ambulatory. since then, no complications or new occurrences have been reported. the patient is under prophylactic administration of rfix prior to minor invasive procedures (nodule biopsy, dental extractions and colonoscopies). summary/conclusions: hemophilia b carriers usually do not require any major health care for their daily routine or invasive procedures. however, in the case reported, basal fix levels conditioned any minor intervention to be performed. thus, this carrier must be considered as a moderate hemophilia b patient and mandatory prophylaxis with rfix must be performed prior to any invasive procedure. background: blood transfusion is a life saving procedure but transfusion associated complications are equally important that need to be addressed. with limited resources of blood donations and screening, excessive transfusions must be avoided to prevent risk and economic burden on patients. on the other hand discussion on appropriate use of blood product is a matter of debate in medical literature. it is recommended to restrict the liberal use of transfusion. aims: in this regard we aimed to observe the frequency of genuine and non genuine transfusion at our hospital and also to compare the outcomes of liberal and restrictive transfusions. methods: it was a cross sectional study carried out at nibd and bmt, pechs campus, karachi, pakistan, from february 2018 to january 2019. the study was approved from institutional review board. pack red cells (prc) transfusion in case of < 7 g/dl hemoglobin (hb) with cardiac history and < 7 g/dl hb with symptomatic anemia was considered genuine. platelets transfusion in case of sepsis and count < 50 9 10 9 /l, sepsis with bleeding and < 100 9 10 9 /l, patients on active chemotherapy without bleeding but platelet count < 10 9 10 9 /l and in patients presented with bleeding was considered genuine. patients who were presented with bleeding, deranged coagulation profile and with sepsis + bleeding considered genuine for fresh frozen plasma (ffp) transfusion. spss version 23.0 was used for descriptive and inferential statistics. results: a total of 250 transfusions were evaluated for genuine and non genuine transfusion. male predominance was high in our data 194(78%). out of 250, 134 (54%), 100(40%) and 16(6%) prcs, platelets and ffp were transfused respectively. out of 100 platelets, 10(10%) were platelet apheresis. 80/134 (60%) prcs and 22/90 (24%) platelets were non genuinely transfused as per the criteria described in methods. however, ffp and platelet apheresis were genuinely transfused. in non genuine group of platelet transfusion, all patients received more than 1 unit platelet and the increment of platelet count post transfusion was found statistically non significant (p = 0.121). thirty six (45%) of transfusions were done with more than 1 units of prcs in non genuine group. the mean hb level of patients received 1 unit and more than 1 units prcs was statistically same (p = 0.868) and it was found out that there was same increment in hb in both the groups post transfusion. (p = 0.867). summary/conclusions: we concluded that although blood transfusion is an integral procedure for saving life yet correct use of blood products is necessary in the era where blood donations are scarce and blood transfusion has its potential associated hazards. the approach towards the appropriate use of blood products will not only minimize the risk of transmission of infectious diseases but also will reduce the economic burden. longitudinal studies on large sample size are needed to validate this study. uncertain. an ovine model enables comparisons of; (a) invasive and non-invasive methods to measure oxygen delivery and utilisation at a tissue level, and of (b) the efficacy of various resuscitation fluids and transfusion protocols in treating haemorrhagic shock. aims: to develop an ovine model of haemorrhagic shock. methods: sheep (n = 4) were anaesthetised, ventilated and instrumented. instrumentation included blood flow, perfusion, and microdialysis probes placed in the brain, kidney, liver and skeletal muscle. haemodynamic, tissue oxygenation, tissue flow and respiratory data were recorded continuously by data capture software. non-invasive assessments, including brain and muscle oxygen saturation by nirs, and visualisation of sub-lingual microvascular perfusion, as well as arterial blood gas measurements and plasma/serum/urine collections were made at specific timepoints. after a baseline period (1 h) sheep were bled~40% of their estimated blood volume (estimated at 65 ml/kg) to a mean arterial blood pressure (map) of 30-40 mmhg. sheep were then resuscitated with either plasmalyte â (up to 20 ml/kg/ hr; n = 3) or transfused with sheep red cells (n = 1) to achieve a map > 65 mmhg. sheep were euthanised 4 h after resuscitation. data are presented as mean ae standard deviation. results: sheep were haemorrhaged an average of 931.7 ae 135.3 ml blood which combined with iatrogenic blood loss (~300 ml) corresponded to an average 40.2 ae 2.4% blood loss. two out of the four sheep met clinical criteria for haemorrhagic shock (map = 30-40 mmhg, lactate > 4 mm, svo 2 < 60%). across all four sheep the nadir map averaged 40.5 ae 13.1 mmhg, lactate peaked at 3.9 ae 1 mmol/ l, and nadir svo 2 was 41.3 ae 17.9%. all sheep survived to the end of the experimental protocol. summary/conclusions: these data demonstrate the successful induction of haemorrhagic shock in an ovine model. further experiments are planned to improve the protocol and to achieve 100% incidence of haemorrhagic shock, and then to compare invasive and non-invasive measures of oxygen delivery and utilisation as well as the efficacy of different resuscitation fluids and red cell transfusion. adverse events, including trali p-515 bilirubin were recorded within the 28-day period. the clinical parameters were compared against the reaction strength of the antibody reactions. the automated strength was measured by solid phase. the manual testing consisted of a 15-min incubation using liss and adding monospecific igg. the dat was performed manually by adding poly-specific igg and then testing with monospecific igg and c3d. the rh group and non-rh group had 11 and 10 cases performed manually, and results were 2+ or weaker further indicating the manual strength did not correlate with the clinical hemolysis. likewise, in 31/44 (70%) the dat was negative, and did not show any correlation with clinical hemolysis. however, when ldh and bilirubin were measured, the two parameters increased as the automated strength of the antibodies increased. summary/conclusions: most of the dshtr investigation was not associated with overt accelerated red cell destruction. a strong correlation was observed only between the automated immunohematology testing results and other laboratory markers of hemolysis. in our experience, the direct antiglobulin test and manual strength showed no correlation. background: numerous transfused patients present severe, sometimes critical clinical conditions. the occurrence of adverse transfusion reactions (atr) may induce deterioration in the clinical condition with a worsened clinical course and a lifethreatening or fatal outcome as is the case with nervous system impairment. in france, in 2017, out of 7,276 notified atrs, 113 (1.5%) and 6 (0.1%) were life-threatening and death respectively. aims: our aim was to evaluate the notified atrs with neurological signs that occurred in transfused patients over a period of six years and six months in hospitals in the auvergne rhône alpes area. the study included patients with reported atrs in hospitals in this area from january 1 st 2010 to june 30 th 2018. each atr was registered in the national haemovigilance database system. two signs observed at the time of the atr were analyzed: unconsciousness and convulsions. stroke was excluded. the type of atr, its severity, the blood product involved and its imputability were studied. results: during the period under study, 9,544 atr were reported, of which 29 included unconsciousness and/or convulsions (0.3%). of these 29 patients, 13 were females (44.8%) and 16 males (55.2%). unconsciousness alone was frequently observed (21 reports, 72.4%). convulsions were notified in 8 reports (27.6%) and were associated with unconsciousness in 2 of them. the diagnosis of seizure, with no other clinical signs, was established in 2 cases (6.9%). unconsciousness and/or convulsions were present in 8 allergic reactions (27.6%), 4 cases of transfusion-associated circulating overload (13.8%), 3 cases of suspected transfusion-transmitted bacterial infections and 2 hypertensive reactions. in allergic atrs, unconsciousness was notified in 7 cases and unconsciousness associated with convulsions in one. twelve atrs were severe (41.4%), 10 were life-threatening (34.5%) and in 4 cases, they resulted in the death of the recipient (13.8%). of the 8 allergic atrs, 4 were severe and 4 life-threatening. red blood cell concentrate was involved in 15 atrs (51.7%) and platelet concentrate in 9 (31.1%), including 5 cases with apheresis platelet concentrate and 4 cases with pooled platelet concentrate. fresh frozen plasma was involved in 5 atrs (17.2%). nevertheless, the imputability of the blood product was excluded or unlikely in 11 atrs (37.9%). in the 3 suspected transfusion-transmitted bacterial infections, the imputability of the transfusion was ultimately excluded after a negative result was obtained in the bacterial culture of the blood product. the imputability of the blood product was probable or possible in 7 and 9 atrs respectively, but was certain in only 2 atrs. summary/conclusions: unconsciousness and/or convulsions were rarely observed in atrs notified in transfused patients. nevertheless, the presence of these signs highlights the seriousness of the atr (26 ars, 89.7%). lastly, the imputability of the blood product was often excluded or unlikely. in the multivariate cox model for the effect of lpi on overall survival, adjusted for age and ipss-r category, elevated lpi levels were associated with inferior overall survival (hr 3.0, 95% ci 1.5-5.7, p = 0.001). this effect was most pronounced in the td-rs subgroup (hr 6.0, 95% ci 2.2-16.2, p < 0.001). similarly, elevated lpi levels were associated with inferior pfs (hr 3.4, 95% ci 1.9-6.3, p < 0.001) for the whole study population and the td-rs subgroup (hr 8.2, 95% ci 3.4-21.0, p < 0.001). in total 16 patients received iron chelation during the sample collection period (11 patients deferasirox, 5 patients desferrioxamine). lpi levels were normal in 14 out of the 17 samples collected during deferasirox treatment and in 2 out of 5 samples collected during desferrioxamine treatment. summary/conclusions: transfusion dependency is associated with the presence of toxic iron species and inferior overall and progression-free survival in lower-risk mds patients. in td-rs patients the effects were most pronounced indicating ineffective erythropoiesis leading to additional iron toxicity. background: post-transfusion immunomodulation has been reported to contribute to poor patient outcomes. clinically relevant transfusion models are needed to improve our understanding of underlying mechanisms. sheep transfusion models are of increasing importance in blood transfusion research as they provide several advantages over small animals, including their size, anatomy, physiology and similar blood volume compared to human. a current limitation of sheep transfusion models is the lack of characterisation of the sheep immune system. understanding the sheep immunology is necessary to advance sheep transfusion models, identify mechanisms that contribute to post-transfusion immunomodulation and facilitate the translation of findings into clinical settings. aims: to characterise the sheep leukocyte inflammatory responses to in vitro lipopolysaccharide (lps) challenge in edta and heparinized whole blood. methods: edta and heparinized sheep whole blood (n = 4 of each) was cultured with rpmi media (37°c, 5% co 2 ) alone or with the addition of lps (1-100 lg/ml; derived from escherichia coli 055: b5). the inflammatory response was assessed after 2 h (h), 6 h, 12 h, 24 h, 36 h and 48 h. supernatant was harvested at each time point and stored at à80°c. inflammatory cytokine/chemokine production was determined using sheep specific in-house elisa (il-1b, il-6, il-8 and il-10). twoway analysis of variance with bonferroni's post-test was used to measure the effect of incubation time and concentration compared to no lps matched samples. results: when edta was used as an anticoagulant, addition of lps resulted in production of sheep il-1b and il-10 but not il-6 or il-8. il-1b production was significantly increased following stimulation of 100 lg/ml lps for 6 h (p = 0.043) and declined following 36 h incubation. release of il-10 was significant 12 h post-lps stimulation with 100 lg/ml (p = 0.030) and reached a maximum at 24 h. the use of heparinized blood resulted in a different immune profile as all inflammatory markers tested were detected following stimulation with much lower concentrations of lps (1 lg/ml), although the incubation times differed. il-1b was significantly increased following 2 h incubation (p = 0.002), with increasing levels observed up to 24 h post-lps stimulation. il-8 production was evident from 6 h and reached significance at 24 h post-lps stimulation (p = 0.009). il-10 was significantly increased following stimulation of 5 lg/ml lps for 6 hr (p = 0.043) with lower concentrations of lps resulting in il-10 production at 24 h (p = 0.008). release of il-6 was significant after 6 h of 50 lg/ml lps stimulation (p = 0.046), with lower concentrations of lps resulting in il-6 production at 24 h (p = 0.015). in heparinized whole blood an lps concentration-dependent effect was evident for all cytokines. summary/conclusions: using a time-and concentration-approach our findings indicate that sheep are more tolerant and have a delayed response to lps stimulation compared to previous research using similar human in vitro whole blood culture models. in addition, data suggest that sheep have greater immune responses using heparin as anticoagulant for the collection of blood samples. improving our understanding of sheep immunology and development of relevant sheep transfusion models will provide a bridge between sheep models of transfusion and clinical settings. 1. rhdig inappropriately administered (unnecessary exposure) (n = 17, 40%) administered to: -rhd positive woman (n = 9) -rhd negative mother with rhd negative neonate (n = 5) -woman with immune anti d (n = 1) -administered in error (instead of other ig) (n = 2) 2rhdig delayed/omitted/wrong dose (risk of sensitisation to the d antigen) (n = 17, 40%) -omitted (n = 14) -delayed (n = 1) -inadequate dose (n = 2) 3administration without correct patient identification (n = 6, 14%) 4storage & handling (n = 3, 7%) failure to check the maternal and neonatal blood groups prior to administration was identified as a source of error. misinterpretation of blood results also led to women receiving product inappropriately. e.g. reading a negative antibody screen as the mother being rhd negative. patient identification was raised as an issue. rhdig is often stored in satellite blood fridges for easy access. collection from these areas did not always require confirmation against patient identifiers and there was no register of women who received product or link to the batch number to ensure traceability. two incidents involved the administration of rhdig when the prescription for other immunoglobulin products was not clear, leading to a child and a baby receiving rhdig instead of the intended immunoglobulin. summary/conclusions: these incidents indicate problems with the processes of appropriate identification of women who need rhdig, the use and interpretation of pathology tests and requirements for prescription and administration. these resulted in omitted and inappropriate doses of rhdig. blood matters has made a number of recommendations regarding rhdig administration: -all health professionals involved in rhdig administration should be appropriately trained in the use of rhdig -confirmation of the maternal rhd status is essential prior to prescription or administration -positive patient identification must be used prior to administration of rhdig -health services should consider regular auditing to identify areas for improvement relating to rhdig blood matters continues to work with maternity care providers to improve practice. centro comunitario de sangre y tejidos de asturias, oviedo 2 agencia gallega de sangre, organos y tejidos, galicia 3 banco de sangre y tejidos de cantabria, cantabria 4 banco de sangre de la rioja, la rioja 5 banco de sangre y tejidos de navarra, navarra 6 banco de sangre y tejidos de arag on, aragon 7 fundaci on de hemoterapia y hemodonaci on de castilla y le on, castilla y leon 8 fundaci on banco de sangre y tejidos de las islas baleares, islas baleares, spain 9 terumo bct europe nv, zaventem, belgium background: hemovigilance, a long-term monitoring process made mandatory by national and supranational regulations, begins with a systematic whole blood or blood component collection and ends with an examining period after transfusion of blood components into the patients. in spain, organized in 17 autonomous regions, the hemovigilance system is structured in three levels: (1) the local level comprised of transfusion centers and hospital based transfusion services that monitor and collect all transfusion related adverse events (ae) and level them up to (2) the regional hemovigilance coordinator, who communicates all the region's data to the (3) spanish ministry of health which issues an annual report and corresponds with european institutions. to ensure safer blood supply, pathogen reduction technology (prt) was approved and implementation started in spain in 2008. the mirasol prt system for platelets and plasma was introduced in 2010 and is currently being used in 10 of the 17 spanish regions. aims: to monitor the safety of the system, a passive hemovigilance study on mirasol treated products was initiated in the region of asturias and collaboration was extended to other regions (baleares, galicia, la rioja, cantabria, navarra, castilla y leon and aragon). methods: collected data included allergic and febrile reactions, trali and all other adverse event observed. severity of the event and level on imputability of the transfusion were also assessed using the who grading scale. hemovigilance data of mirasol treated products (platelets or m-pc and plasma or m-p) are included from 2012 to 2017 as blood centers started to apply the technology in routine. results: increase adoption of the mirasol system is observed between 2012, when 4,196 mirasol treated blood products were issued to hospitals and 2017 with 56,229 mirasol products issued. due to low number of transfusions of mirasol-treated blood components in 2012 and 2013, notification rates began to be analyzed in 2014, showing ae rates of 0.2%, similar to reports at the national level. stable transfusion reaction rates were observed with m-pc (around 0.23). rate of ae after transfusion of m-p is fluctuant between 0.06 and 0.17. this fluctuation could be due to the inconsistent numbers of m-p transfused from one year to the other. most of transfusion reactions (around 80%) were of grade i severity and grade ii level of imputability. allergic reactions accounted for most of the adverse events, with g&i > 2 reactions in 2016 and 2017 of respectively 0.18 and 0.07 no bacterial nor viral transfusion transmission was recorded on mirasol products during the study period (2012) (2013) (2014) (2015) (2016) (2017) . at the national level, nine cases of bacterial transfusion transmission (with g&i > 2) were reported. these transmissions were probably due to transfusion of non-pathogen reduced products. summary/conclusions: the observed notification rate of ae is similar to the national rate but allergic reactions with g&i > 2 is inferior with mirasol treated products. also, we found no reports of transfusion transmission infections nor cases of transfusion associated graft-vs-host disease, demonstrating safety of mirasol treated products. were attributed to human error (75%) with the lowest frequency in equipment failure (10%), compared to 21% and 29%, respectively, in the following three years. root cause analysis demonstrated failures in the quality management system including failures in administration, inadequate staffing for blood collection as well as in distribution and processing, and failures arising from institutional constraints and system failures in hospital management. high numbers of "other" aes (25%) in distribution and whole blood collection call for further investigation to indicate measures necessary for prevention and correction. errors related to incorrect blood component transfused (ibct) in 2007-2017 were 80 in 8,385,448 blood units (1/104,818) issued for transfusion. these resulted in 27 serious reactions (34%) (1 fatal, 11 life-threatening) . another 21 (26%) were related with ibct that did not cause a reaction. near misses (component not transfused) were 24 (40%) summary/conclusions: our data demonstrate increasing compliance with reporting requirements. questions about the initial factors for deviations in certain activities specifying failures in equipment and materials due to system as well as human errors, highlight the need for further specifications beyond "other" and "human error". background: the weakest link in the transfusion chain currently is the handling of blood components after their issue and the bedside blood administration practices. aims: to evaluate compliance with standard procedures for bedside blood transfusion practices by analysis of the "transfusion feedback forms" in a tertiary care multi-specialty hospital setting. methods: during the study period of 24 months, the transfusion feedback forms received from various clinical areas of the hospital were studied with special reference to the transfusion times. the data was categorized based on the patient's location as well as the time of transfusion, whether done in routine or emergency hours. results: 42,403 blood components were issued during the study period, while transfusion feedback forms for 37,816 components (89.1%) were received in the transfusion medicine department. delay in starting the transfusion (more than 30 min after issue) was observed in 2965 transfusion events (6.9%). the component transfusion time exceeded the permissible limits for 930 component (2.1%).the overall total permissible time for completion of components exceeded permissible limit in 2217 (5.2%) of transfusion events. the pediatric ward (23.9%), icu and ot complex (25.4%) were found to be the most non-compliant delay in transfusion, transfusion time and total transfusion time. amongst the 2965 delayed transfusions after issue, 2120 (71.5%) were during the routine hours i.e. between 7 am to 7 pm and 845 (28.5%) were in the non routine hours i.e. between 7 pm to 7 am. summary/conclusions: the audit of bedside blood transfusion practices has given us a good insight into various areas of noncompliances as well as the predominant locations in the hospital where the practices need to strengthened further. focused training program on safe blood administration practices for all staff involved in handling and transfusion of blood components is now planned to combat this issue. background: the international surveillance of transfusion adverse reactions (ars) and events (aes) (istare) of the international haemovigilance network (ihn) collects aggregate data from member national haemovigilance systems (hvs) in order to estimate the morbidity and mortality of blood transfusion in a holistic approach. the ultimate goal is to contribute to improving the safety of transfusion by close monitoring throughout the chain "from vein to vein". aims: we analyse recent istare data on suspected transfusion transmitted infections (sttis) for 2013-2016 in comparison to previous years of surveillance, [2006] [2007] [2008] [2009] [2010] [2011] [2012] methods: annual aggregate data from ihn member hvs on transfusion associated bacterial, viral and parasitic infections collected online in istare are analyzed by incidence in blood components (bcs) issued for transfusion, by severity and imputability as well as by blood component. ars with definite, probable or possible imputability were included in the analysis. trend analysis is performed to allow comparisons and to collect information on established and newly emerging infectious threats of blood transfusion. results: for 2013-2016 89 sets of annual aggregated data from 25 countries covering 85,521,393 bcs issued were analyzed. all ars totalled 76,907 and infectious ars amounted to 285 (0.4%). the overall incidence of the infectious ars was 0.33/ 100,000 units of bcs issued. bacterial infections were the most frequent (188, 66%), next viral (84, 29.5%) and then parasitic (13, 4.5%). serious were 46% and there were 11 fatalities (3.9%, incidence 0.013/100,000). nine deaths were attributed to sepsis and the other two were associated with non-malarial parasitic pathogens. one geotrichum clavatum fungal infection associated with apheresis platelets was reported as a free text comment. this very rarely recognized fungal pathogen caused a very severe infection in a patient but the route of transmission is inconclusive. the 84 viral sttis included 17 hbv (20%), 10 hcv (12%) and 2 hiv (2.5%). the 55 recorded as "other" (65.5%) including 24 cases of hev, one case of parvovirus b19, one cmv and one ebv. no case of tt-malaria was reported. other stt-pi were 15 (two fatal). the prevailing bcs were in general rbcs followed by platelets. comparison with corresponding data for 2006-2012 shows a consistent overall incidence in total sttis (0.33 vs 0.4/100,000). however, considerable differences were seen in separate categories, such as bacterial infections (significantly increased rate in 2013-2016, p < 0.001) and an almost doubled rate of parasitic infections (p < 0.001). compared to the earlier period, there were many fewer hbv infections (17 vs 160) and many more hev. a similar reduction in the rate of hcv and hiv was observed in 2013-2016 in comparison with previous years. this may be explained by the fact that nat testing for hcv/hiv/hbv has been implemented in many countries in the last decade. summary/conclusions: the infectious risk of transfusion overall remains very low. the rate of bacterial cases has increased and among other viral sttis the frequency of hev is increasing. the mortality of transfusion due to sttis is lower than in the previous period of surveillance. abstract withdrawn. background: one of the main aspects of haemovigilance system in hospitals is following of adverse events and reactions related to blood transfusions. aims: it was intended to analyse the adverse reactions related to transfusion of blood components in pediatric patients. methods: over a four year period (january 2015-december 2018), the haemovigilance records of all patients receiving blood transfusions procedures were reviewed and transfusion reactions were analysed. statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). majority of blood components were provided by regional blood center organized by national red crescent society. but granulocytes collected by apheresis after donor mobilization and reconstituted whole blood for exchange transfusions were prepared in the transfusion center of the hospital. results: the median age of patients who developed transfusion reactions was 72 months (interquantile range-iqr 108). the median for the numbers of individual transfusions in children in a year was 12 (iqr 17). the median for the numbers of blood components individually transfused to patients was 18 (iqr 24). patients, anaphylactic transfusion reactions in 4 patients and transfusion-related lung injury (trali) in a patient. the overall incidence of transfusion reactions was estimated at a rate of 2.7 per 100000 units. summary/conclusions: it was reported that adverse effects related to blood transfusion, especially allergic reactions and fnhtrs are common in pediatric patients than adults. in a multinational study concerned about the transfusion reactions related with red cell concentrates, allergic transfusion reactions and fnhtrs were reported at a rate of 11 and in 100000 units and 26 in 100000 units, respectively. while the incidence of transfusion reactions in children was found 0.62% in a study from the u.s.a., the overall incidence of transfusion reactions in our study which was estimated at a rate of 2.7 per 100000 units represents a lower rate. hospital gran canaria dr. negr ın, gran canaria 6 hospital general universitario, ciudad real, spain 7 hospital nostra senior de meritxell, andorra, andorra 8 banco sangre y tejidos, santander 9 banc de sang i teixits, barcelona, spain 10 fundaci on hematol ogica colombia, bogot a, colombia 11 centro regional de transfusi on de almer ıa, almeria 12 complejo hospitalario de navarra, pamplona 13 fundaci on banc de sang i teixits illes balears, palma de mallorca 14 hospital de cabueñes, gij on, spain background: root analysis cause is defined as the cause of an error that, if it is treated, eliminates the repetition of the error aims: describe types of human and latent errors detected by a work group in the root analysis cause of transfusion incidents, analyze the concordance between the individual responses of the members and propose recommendations in order to improve transfusional safety. methods: in 2018 fifteen participants (nurses and hematologists dedicated to transfusion and component preparation) studied some incidents of administration of nonirradiated components and tried to approach the root causes by applying the classification of errors in mers-tm transfusion medicine. they transferred the answers to a questionnaire (simple or chain error, initial process affected, human and latent errors and measures derived from the analysis to correct the errors). the communication was made by mail and by the spanish transfusion society web forum, which contained the consultation documents. data and percentages are exposed for each type of error and the answers of the participants are tabulated. results: cases corresponded to 3 patients. two patients of 55 years of age diagnosed of acute myeloblastic leukemia (case 1 and 2) and chronic lymphatic leukemia (case 3). in one case, the hematologist of the transfusion service canceled an irradiation prescription; in another, a patient with fever was transfused in the emergency room without the irradiation requirement and it was later discovered that he had received a transplant of hemopoietic progenitors 1 month earlier; in the last case, neither the requesting doctor nor the laboratory technician nor the following doctor (prescriber) detected the alerts located in their respective computer applications. in all 3 cases, the story was judged as sufficient for analysis. the majority of reviewers (93%) diagnosed a chain of errors. there was agreement of 95% with respect to the initial process affected. the initial error was communication (42%), monitoring (45%) and compliance (62%), in cases 1, 2, and 3. 2-3 human errors were detected per case (average: 2.2, 2.7 and 2.4 errors respectively) and 2-3 latent errors per case (average: 1.8, 2.7 and 2.1, respectively). the latent errors most punctuated were: failures in the quality of the protocols (24%), in the transfer of important knowledge (22%), in the available technology (21%) and in the information to the patient (10%). all the participants contributed feasible measures of improvement according to root causes: 1) improve the quality and drafting of work procedures and their compliance, including procedures of effective communication between professionals, 2) train staff in knowledge important for safety, 3) communicate with computer application providers to improve the effectiveness and visibility of the alerts and 4) involve the patient with essential information to ensure transfusion safety. the measures were processed later as recommendations. summary/conclusions: the root analysis shows agreement between participants and allows the elaboration of useful recommendations to increase patient safety. this strategy can contribute to the comprehensive prevention of errors. background: in transfusion-associated circulatory overload (taco), pulmonary oedema develops primarily due to volume excess. data from the uk haemovigilance scheme, serious hazards of transfusion (shot) suggest that either the incidence of taco, or the recognition and reporting of taco, has increased over time. from 2007 to 2017, reports of taco increased from 6 to 92; deaths from 1 to 7, major morbidity from 3 to 20. known risk factors include pre-existing cardiac and/or renal dysfunction, low body weight, extremes of age (eg, <3 years, >60 years), concomitant fluid administration, positive fluid balance, peripheral oedema and hypoalbuminemia. in a small subset of cases reported to shot, taco developed following transfusion for severe anaemia in the absence of other risk factors. this may be an under-recognised independent risk-factor. aims: to raise awareness of severe anaemia as an under-recognised risk factor for taco and is potentially life-threatening transfusion. methods: cases of taco submitted to shot over the last 3 years were reviewed to identify cases where transfusion for severe anaemia was a key identifiable patient risk factor. results: the following are illustrative cases: -case 1: a patient in their 50s weighing 67 kg was prescribed six units of red cells for iron deficiency anaemia after being admitted with hb 37 g/l. the patient had no risk factors for taco except for profound anaemia. during transfusion of the fifth unit the patient became dyspnoeic, hypoxic and hypertensive. the patient recovered after diuretic therapy and had a post-transfusion hb level of 100 g/l. -case 2: a patient in their 50s presented with a 4-week history of weakness and dizziness and had felt unwell for 6 months. the hb was 34 g/l, ferritin 26 lg/l and ecg showed cardiac ischaemia. two units of red cells were transfused. after the second unit oxygen saturations fell despite supplemental oxygen, post-transfusion hb of 51 g/l. a third unit was transfused over 125 min and the hypoxia worsened with dyspnoea and crackles on chest auscultation. the chest x-ray showed an enlarged cardiac silhouette and pulmonary congestion. the patient improved with diuretics. -case 3: a patient in their 90s with severe megaloblastic anaemia, hb 41 g/l and peripheral oedema developed taco after transfusion of 3 units and recovered with diuretic therapy. summary/conclusions: chronic and acute anaemia are associated with compensatory cardiac changes irrespective of the aetiology of anaemia. this is further compounded by the underlying cause of anaemia particularly haematinic deficiencies (iron/b12 deficiency) that independently affect myocardial function. hyperdynamic circulation related to anaemia increases the load on the heart, causing myocardial ischaemia and hypoxia and if the anaemia is not corrected, eventually leads to heart failure. clinicians need to make an accurate diagnosis and avoid excessive transfusion in patients with severe anaemia with or without other additional risk factors. patients with chronic iron/folate/b12 deficiency without haemodynamic instability should be given the appropriate haematinic replacement. haematinic deficiency responds rapidly to appropriate vitamin/mineral. blood transfusions are to be given only when clinically indicated and even then, only the minimum volume needed for symptomatic relief transfused with consideration for diuretic therapy. methods: legal forms for reporting transfusion reactions were used in the retrospective analysis, which were adjusted by the department of quality assurance and quality control in the electronic form and distributed to clinics using blood components. clinicians were trained to report transfusion reactions through the hospital's transfusion board and through the "service for improvement of the quality and safety of health services" at the clinical center of sarajevo. analysis of the reported reactions in the institute include immunohematological and microbiological examination based on which the guidelines for further treatment with blood components are being made. users of registered services are obliged to report since 2015. results: total of 121,076 different blood components were applied in the period between 2015-2018. department for quality assurance and control has received 4 serious adverse reactions, 1 serious adverse event, 157 reports of transfusion reactions, of which 11 (7%) were inadequately filled, in the same period. from the above, 54 (34.39%) were transfusion reactions to erythrocyte blood components which were applied, 86 (54.77%) to platelet components and 17 (10.82%) were transfusion reactions after the application of fresh frozen plasma. the analysis has shown that the most frequent were febrile non-haemolysis reactions (88 or 56.05%), followed by allergic reactions (50 or 31.84%). two transfusion reactions (1.27%) were characterized as circulation overload. summary/conclusions: the frequency of serious adverse reactions and events was 0.004% (5 of 121,076) and 0.12% were reported transfusion reactions (157 of 121,076). with the establishment of the hospital transfusion board and with the increase of collaboration with the clinical center, significant progress has been made. it is necessary to increase awareness among clinicians in regards to the safe transfusion practice. reporting transfusion reactions should be a mandatory procedure, a path to the proper selection of blood components, monitoring adverse reactions, and for us, transfusiologist, guide to the safest, most efficient blood components. j garc ıa-gala, e martinez-revuelta, a caro-g omez, c castañ on-fern andez and i fern andez-rodriguez hospital universitario central de asturias, oviedo, spain background: elderly patients are the main group of transfusion recipients in our country. given their comorbidities are a risk group for the development complications related to transfusion. aims: analyze the incidence of adverse effects related to transfusion in the elderly population and to assess what factors may influence its appearance methods: transfusions were reviewed in patients > 70 years old. the variables analyzed were: sex, age, diagnosis/reason for transfusion, pre-transfusion hemoglobin (hb), number of transfused units, infusion rate and transfusion side effects, as well as the measures used to prevent or treat the transfusions. effects of circulatory overload results: a total of 93 patients were analyzed (48 women, 45 men), with a median age of 86 years (70-97). in total, 189 ch were transfused. 70 patients received 2 ch, 3 patients 3 ch, 10 patients received 1 ch. 10 patients were transfused at two different times. the median hb prior to transfusion was 7.6 g/dl. in the patients who received 2 ch was 7.5 g/dl, those who received 3 ch: 6 g/dl and those who received 1 ch: 7.75 g/dl. the infusion time could be estimated in 84% of the patients. in those who received 2 ch was 223.62 min; 249.33 min in those who received 3 ch and 109.75 min in those who received 1 ch. 13 patients (14% of the total) suffered some type of adverse effect related to the transfusion. in 9 patients there was an increase in posterior ta, in 2 an increase in hr, in 1 an episode of hypotension and in another one episode of acute respiratory failure. 70% of those who had an adverse effect were older than 85 years. patients with aht after transfusion, 75% received 2 ch and the remainder 1 ch. among their background, 66% had a history of ischemic heart disease. 33% also had a positive balance. the average previous bp was 137/65 mmhg and the subsequent one was 182/72 mmhg. 55% of patients did not receive diuretic treatment. in the case of the patient with acute respiratory failure was in oligoanuria, with positive balances. 2 ch was transfused in total. she was treated with oxygen therapy and with intensification of the diuretic treatment, recovering later. summary/conclusions: -patients > 85 years have a higher risk of suffering some type of adverse effect related to transfusion because they have pre-existing risk factors such as ischemic heart disease or heart failure. -we see that the risk of suffering some type of adverse effect in the elderly population is greater when we transfuse 2ch than 1ch. -we have appreciated that in those patients receiving 3ch, the infusion rate is higher. -the study highlights the lack of methods to prevent the development of circulatory overload. background: iron deficiency anemia is the commonest cause of anemia worldwide. weakness, fatigue, reduced physical activity and difficulty in concentration are the symptoms which are associated with its deficiency. the forefront treatment available is oral iron replacement therapy which is convenient, cost effective and has substantial outcome. another option is intravenous (i/v) iron when oral is not tolerable. despite of potential transfusion associated hazards and limited availability of blood due to shortage of voluntary blood donations, it is insisted by the patients prior to iron therapy. aims: the aim of conducting this study was to observe the impact of administration of oral iron, i/v iron and transfusion on hemoglobin levels in patients presented with iron deficiency anemia. methods: this was an observational study carried out at nibd and bmt, pechs campus, karachi, pakistan from february 2018 to december 2018. the study was approved by the institutional review board. diagnosed ida patients presented at our hospital were recruited for analysis who were given oral iron, i/v iron and transfusion for the correction of anemia. informed consent was taken from the participants. descriptive and inferential statistics was applied by using spss version 23.0. results: a total of 108 ida patients were analyzed in which 74(69%) were females and 34(31%) were males. the most common symptom in females and males was fatigue followed by body aches in females 12(16%) and pallor in males 10(29%). menorrhagia was present in 24(44%) of females of reproductive age. surgical history was present in 12(16%) of females while there was no surgical history in males. mean hemoglobin, mch and mcv of females at baseline was 8.0 ae 2.2, 60.5 ae 9.6, and 20 ae 8.3 while in males it was 7.8 ae 2.3, 63 ae 14.3 and 18.4 ae 6.1 respectively. sixty two (84%) females were advised oral and i/v iron and 12 (16%) received transfusion. however, in males 8(24%) received transfusion and 26 (76%) were advised oral and i/v iron therapy. it was observed that the increment of hemoglobin after oral/iv iron at average of 3 months follow up in males and females was same as that the transfusion (p > 0.05). summary/conclusions: in our society where blood donations are scarce especially voluntary blood donations that are considered to be the safest type of blood donation. we would like to draw attention towards the alternatives to correct anemia such as oral and i/v iron replacement therapy as our results revealed that there was no difference in the increment of hemoglobin between the two groups. we need to educate our society especially the older age adults and young women who are more vulnerable of getting ida to opt oral and i/v iron therapy. it will be cost effective, convenient and also has less risk than transfusion. cellular therapies -stem cell and tissue banking, including cord blood background: the differentiation of megakaryocytes plays an important role in the production of platelets. however, the underlying mechanisms regulating megakaryocytes differentiation have rarely been studied. aims: to identify candidate genes involved in megakaryocytes differentiation and investigate the potential regulatory mechanisms of megakaryocytes differentiation from human cord blood hematopoietic stem cells in vitro. methods: cb-derived cd34 + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (macs). cultures were stimulated with only recombinant human tpo (100 ng/ml). after 12, 14 and 18 days, the mk fraction was selected by immunomagnetic sorting from the non-mk fraction using an anti-cd41a monoclonal antibody. rna-seq-derived gene expression data was performed on uncultured samples (day 0), cultured but unselected samples (day 7), and cultured, selected samples (day 12, 14 and 18) by using the next-generation sequencing (ngs) platform, and rq-pcr technology was used to verify the expression of transcription factors. results: the comparison of the transcriptome profiles among the five stages showed that a massive gene expression change occurred in megakaryocytes differentiation. a total of 65140 genes were detected, of which 16612 showed up-regulation and 48528 down-regulation. among these genes, 8 differentially expressed genes (degs) (fold change ≥ 10; false discovery rate < 0.05) were selected were further validated with rq-pcr, including gabre, cdhr1, wasf3, pkhd1l1, thbs1, pf4v1, lrrc32 and lgals12. the rq-pcr result indicated that the mrna expression level increased with the prolongation of culture time. however, pf4v1 mrna expression level was highest at day 14, lgals12 was highest at day 18. summary/conclusions: conclusion: our study identified a series of genes that may participate in the regulation of megakaryocytes differentiation. these results should serve as an important resource revealing the molecular basis of megakaryocytes differentiation and thrombocytopoiesis. preoperative anemia and blood transfusion requirement during hip and knee surgery rambam health care campus, haifa, israel background: blood transfusion (bt) is independently associated with increased morbidity, mortality and hospitalization length across different patient populations. due to bt-related risks, the concept of "patient blood management" (pbm) has been introduced to clinical practice. the three pbm pillars are: optimizing red cell mass, minimizing blood loss and optimizing physiological reserve. bt indications during orthopedic surgery include excessive bleeding or hemodynamic instability and not the hemoglobin (hb) level. in most clinical scenarios, a restrictive transfusion threshold (hb level: 7-8 g/dl) appears to be non-inferior to the liberal transfusion strategy in terms of blood use, morbidity and mortality. similar results are observed in highrisk patients after hip surgery. we hypothesize that preoperative anemia may lead to blood product overuse and its complications. aims: evaluating potential correlation between preoperative anemia and bt requirement during hip or knee surgery. methods: we reviewed medical files of patients who underwent hip or knee replacement surgery at rambam between 2011-2018. patients with hb level measurement within 90 days pre-surgery were included. receiving > 1 blood unit was considered a surgery complication and such patients were excluded. patient demographic and clinical data, including comorbidities, surgery type, length of hospital stay, were collected. we created a synthetic data cohort using mdclone healthcare data sandbox, an environment enabling fast data extraction and producing synthetic data for analysis that does not require irb approval. results: during the evaluated period, 2591 patients underwent hip or knee surgery; 231 were excluded from the analysis due to receiving > 1 blood unit. hb measurement within 90 days pre-surgery was available for 1202 patients. hip or knee surgery was performed in 588 (49%) and 614 (51%) patients, respectively. women comprised 60% (n = 356) of patients who underwent hip surgery. in the hip-surgery group, 14.5% of patients required bt, with this need being slightly higher among women (31.5% vs. 26.2%; p-value=ns). only 50 (8%) patients were transfused during knee surgery and this cohort was not further analyzed. patients receiving bt had a significantly lower mean hb level than those who didn't require it (11.93 g/dl versus 12.8 g/dl for women and 12.3 g/dl vs. 13.8 g/dl for men; p-value < 0.001). hospitalization was longer in transfused patients compared to non-transfused ones (mean 7.52 vs. 6.91 days, p-value = 0.018) and in patients with a low hb level (female < 12, male < 13.5) than in those with a high hb level, irrespective of receiving bt (p-values < 0.00045). patients with at least one of the following diagnoses: diabetes, renal failure, ischemic heart disease, were significantly more likely to have a lower preoperative hb level (p-value < 0.05). no other factors (e.g., patient's weight, rdw value or blood pressure) were predictive of transfusion need. the probability of a need for 1 blood unit was 0.43 in the hb 11 g/dl group and 0.15 in hb 13 g/ dl group (35%>reduction). summary/conclusions: anemia presence before elective hip surgery is a risk factor for bt requirement and longer hospitalization. diagnosis and management of anemia using timely pre-surgery consultations may minimize intraoperative bt, particularly in women and patients with comorbidities. real-patient data and prospective trials are warranted. abstract withdrawn. abstract withdrawn. background: cd36, known as platelet glycoprotein iv, belongs to type b scavenger receptor and is related to the pathogenesis of many diseases. type i cd36 deficiency was cd36 not expressed on platelets and monocytes. individuals with type i deficiency can produce homologous antibodies and cause related immune diseases. in recent years, it has been reported that cd36 deficient individuals cause fetal immune thrombocytopenia with fetal edema syndrome in asia. cd36 is not expressed in mature rbc, but exists in hematopoietic stem (progenitor) cells. anemia is an important cause of edema. in view of the phenomena of clinical and animal experiments, cd34 + hematopoietic stem (progenitor) cells were cultured in vitro to observe the effect of anti-cd36 monoclonal antibody on cd34 + hematopoietic stem (progenitor) cells. aims: to investigate the effect of anti-cd36 monoclonal antibody on proliferation and differentiation of human cd34 + hematopoietic stem (progenitor) cells in vitro. methods: choose 3 healthy full-term maternal women without various obstetric complications, take cord blood 20 ml. after density gradient centrifugation of ficoll cell separation solution, cd34 + hematopoietic stem (progenitor) cells were sorted by flow cytometry and cultured for 2-3 generations. mtt was used to examine the effect of anti-cd36 monoclonal antibody on the growth of hematopoietic stem (progenitor) cells. flow cytometry analysis was used to detect the apoptosis and cell cycle of cd34 + hematopoietic stem (progenitor) cells. the effect of anti-cd36 monoclonal antibody on the formation of cfu-e/bfu-e in hematopoietic stem (progenitor) cells was analysis by cfu-e/bfu-e account after 10-14 days culture. results: after umbilical cord blood was isolated by ficoll to obtain mononuclear cells, the hematopoietic stem (progenitor) cells of cd34 + were sorted by flow cytometry, and about 0.44% of cd34 + hematopoietic stem (progenitor) cells were isolated. different concentrations of anti-cd36 monoclonal antibody and hematopoietic stem (progenitor) cells were cultured in vitro. the od value of value (0.9 ae 0.15) of anti-cd36 monoclonal antibody group (2 mg/ml) was decreased than normal group (1.05 ae 0.12) (p < 0.05), and the od value (0.81 ae 0.11) was significantly decreased at the cd36 monoclonal antibody concentration of 32 mg/ml (p < 0.01). there was no significant difference between the hematopoietic stem (progenitor) cells culture group and the igg control group (p > 0.05). in the annexin v flow detection, the apoptotic rate of anti-cd36 monoclonal antibody group (2 mg/ml) was statistically increased than the normal group (p < 0.05). anti-cd36 monoclonal antibody significantly induced hematopoietic stem (progenitor) cells to undergo s phase cell reduction, g1 phase cells increased, and g1/s phase cell arrest occurred. the number of cfu-e/bfu-e clones in the normal group was 169 ae 12, the number of cfu-e/bfu-e clones in the control group was 172 ae 12, and the number of cfu-e/bfu-e clones in the anti-cd36 monoclonal antibody culture group was 85 ae 6. the number of colonies formed by hematopoietic stem (progenitor) cells in the anti-cd36 monoclonal antibody culture group was significantly lower than that of the other groups (p < 0.05). summary/conclusions: anti-cd36 monoclonal antibody can reduce the proliferation of human cd34 + hematopoietic stem (progenitor) cells and reduce the ability of erythroid differentiation. background: recently the new modern collection techniques were introduced in the apheresis procedures. cobe spectra system was replaced with spectra optia, and it was necessary to verify the efficiency of spectra optia in pbpc collections. aims: the aim of the study was to evaluate and optimize the new cmnc protocol spectra optia v. 11 (terumo) for pbpc collections in patients with haemato-oncological malignant diseases. methods: the results of 159 autologous pbpc collections were evaluated in: (a) well mobilized patients with precollection cd 34+ cells concentration in blood higher than 20/ll, (b) from only the first collections, which were performed either by the use cmnc spectra optia v. 11 or cobe spectra v. 6, v. 7, terumo (c) collections were performed in the standard and large volume leukapheresis regimen, lvl. engraftment data in 56 transplanted patients were assessed. results: standard collections were performed in 52 patients. the yield of cd 34+ cells was high, and no significant differences were found between the numbers of cd 34+ cells prepared from spectra optia 8,6 (1,3-41) 9 10 6 and cobe spectra 10,9 (1, 6 ) 9 10 6 /kg b. w. (a = 0,05; pval 0,619). the dependence of cd 34+ cell yield on the precollection concentration of cd 34+ cells in blood can be considered as linear with high correlation coefficients in cmnc spectra optia r = 0,95, and cobe spectra r = 0,93. lvl collections were performed in 107 of patients, and there were no significant differences between the numbers of cd 34+ cells prepared by cmnc spectra optia 10,9 (2-61,2)9 10 6 and cobe spectra 9,3 (2,4-86) 9 10 6 /kg b.w. (a = 0,05; pval 0,35). the relations between the precollection cd 34+ cells concentration in blood and the numbers of cd 34+ cells from collections can also be considered as linear with the correlation coefficients in cmnc spectra optia r = 0,93, and cobe spectra r = 0,78, respectively. in lvl, the median platelet loss was significantly lower in cmnc spectra optia (45%) than in cobe spectra (57%). a group of 12 patients was transplanted by means of pbpc prepared in the standard regimen. median time in the neutrophil reconstitution was in cmnc spectra optia as well as cobe spectra 11 days, while in platelets from cmnc 14 days, and from cobe spectra 12 days, respectively. the number of 44 patients obtained pbpc from lvl. the median time in neutrophils and platelets reconstitution was in cmnc spectra optia as well as cobe spectra the same, and corresponded with 11 and 13 days, respectively. summary/conclusions: cmnc protocol spectra optia is a modern, efficient and the safe system, which can be used for both standard and lvl procedures. in well mobilized patients the sufficient dose of cd 34+ cells for transplantation could be prepared from one standard or one lvl procedure. no serious adverse reaction have been observed. background: peripheral hematopoietic stem cells are collected from patients/donors after mobilization with g-scf. the aim of the collection is a fixed number of cd34 + cells/kg. this number depends on the pre-apheresis cd34 + number, the blood processed and the collection efficiency of the procedure. the aim should be to collect all the requested cells in 1 day, whenever possible. this is in order to reduce the dose of g-csf given to donor/patient and the resources used in the collection centre. the only parameter that can be adjusted is the volume of blood processed, if this is increased, the likelihood of collecting the requested amount of cells is increased, but only if the pre-apheresis cd34 number is high enough. therefore, you need to know, when it is feasible to increase the volume and thereby increasing the time of the procedure with the intention to collect all the requested cells in 1 day. it can also show if it is possible to reduce the volume of blood processed, thereby reducing the time of the procedure. aims: to develop an easy tool to calculate the volume of blood processed in order to collect the requested cells in 1 day. methods: the mean collection efficiency (ce) was calculated. ce is calculated as cd34 + cells collected/(pre-apheresis cd34 + number*processed volume)*100%. based on the mean ce, an excel sheet was generated to calculate the volume of blood that should be processed in order to collect all the requested cells. the excel sheet is designed so the user enters the pre-apheresis cd34 + number, patient weight and the requested number of cd34 + cells. the ce is fixed according to the mean ce calculated. the result is the volume of blood processed in order to collect the requested yield. based on that result, the apheresis machine will provide time for the procedure, thereby it is possible to evaluate if the collection can be finished in 1 day or not, e.g. by increasing the volume of blood processed. spectra optia â was used for all collections, cmnc for allogeneic donors and mnc for autologous patients. results: mean ce = 64% (n = 348). a ce of 40% was chosen as the cut-off for the cd34 calculation tool. using the cd34 calculation tool: allogeneic donors (n = 61): mean ce = 61%, mean blood volume processed = 3.3 9 tbv, mean time: 253 min, 37 donors were finished in 1 day collection (61%) autologous patients (n = 205): mean ce = 65%, mean blood volume processed = 2.4 9 tbv, mean time = 218 min, 173 patients were finished in 1 day (83%). the calculation failed in only 1 case (0.4%). in this case the volume of blood processed was reduced according to the calculation, but because of unexpected low ce, the requested number of cells was not achieved. summary/conclusions: the cd34 calculation tool based on an excel sheet has shown to be simple and easy to use in order to personalize the stem cell collection. immunotherapy products: blood product, pharmaceutical, or a new category all together? from 2001 till 2019. all donors were hla typed and matched; they were fully informed on the donation procedure and signed an informed consent for donation. minimum dose required to ensure successful and sustained engraftment was 2 9 10 6 /kg cd34 + cells and 2 9 10 8 /kg mono-nucleated cells (mnc). pbsc harvesting was performed with continuous flow cell separator baxter c53000, cobe spectra and spectra optia using conventional-volume apheresis processing the 2-2.5 total blood volumes per apheresis. a femoral catheter was used for harvesting and acid citrate dextrose formula a is used for anticoagulation. recombinant human granulocyte colony-stimulating factor (g-csf) is used to mobilize pbpc for collection. harvesting of pbsc is usually performed after 4 to 5 days of g-csf subcutaneous administration at a dose of 10 lg/kg body weight. results: all the donors were siblings of the patients treated at the university hematology hospital. there were 159 apheresis procedures performed in 106 healthy sibling donors. there were 75 males and 31 females, aged 20-55. one to two apheresis procedures were required to collect adequate graft. the single procedure usually took 3-4 h and the volume of collected stem cells was 50-220 ml. the needed number of mnc and cd34 + cells was successfully collected by 1.5 apheresis. there were 29 abo incompatible donors. procedures for mobilization and collection of pbpc from healthy donors are generally well tolerated. the only adverse effects of the apheresis procedure were bone pain as reaction of g-csf and numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and were very mild. the collected pbsc were used in allogeneic stem cell transplantation in patients with: acute myeloid leukemia -57 patients (53.7%), acute lymphoblastic leukemia -14 patients (13.2%), chronic myeloid leukemia -9 patients (8.5%), myeloproliferative disorders -8 patients (7.5%), myelofibrosis -5 patients (4.7%), severe aplastic anemia -5 patient (4.7%), non-hodgkin lymphoma -3 patient (2.8%), multiple myeloma -3 patient (2.8%), chronic lymphoblastic leukemia -1 patients (0.9), hodgkin disease -1 patient (0.9%). summary/conclusions: the apheresis collection of pbsc in healthy donors is an effective and safe procedure. we are developing our national stem cell donors registry as a part of bone marrow donors worldwide. in that way we hope we will help widen the world network of stem cell donors and enlarge the possibility for each patient to find the right match. background: leukocyte-removing filters for blood are being used widely as a universal leukocyte reduction policy is being adopted progressively throughout the world. filtration is one of the most effective methods for preventing various adverse transfusion effects caused by leukocytes included in blood components, such as febrile reaction, alloimmunization, and transmission of leukotropic viruses. aims: the goal was to evaluate whether the new domestic blood filter finecell, developed by kolon industries, gumi, korea, is appropriately suited to the international standards. and to reveal its efficacy and safety in the settlement of leukocyte reduction system in korea. methods: thirty-two units of packed red blood cells obtained from 400 ml whole blood collected from 32 healthy donors were used. this was done by analyzing the filtration time, residual leukocyte count, rbc recovery, and hemolysis rate during a storage period of 35 days after leukoreduction. results: the standards commonly used for the evaluation of leukocyte-removing filters are set by the food and drug administration of the usa and the council of europe. the results of our study satisfied these international standards. summary/conclusions: the newly developed domestic leukoreduction filter was, thus, found to be efficient and will contribute to the improvement of the quality of isolated blood components used in korea. faculty of science, humanities and education, technical university of liberec, liberec, czech republic background: as polymeric fibrous scaffold fabrication techniques strive to create structures that more closely replicate tentative extracellular matrix form and function, the need for increased scaffold bioactivity becomes more pronounced. the fibrous structure made from biocompatible and nontoxic polymers ensures mechanical stability, however cell proliferation requires further stimulation. platelet-rich plasma, which has been shown to contain over 300 bioactive molecules, has the potential to deliver a combination of growth factors (gfs) and cytokines capable of stimulating cellular activity. aims: the presented work deals with the preparation of nanofibrous materials with platelet growth factors incorporated into the internal fiber structure. polyvinyl alcohol (pva) was used for the preparation a material providing a progressive release of native gfs without need of subsequent crosslinking. methods: materials were prepared from pva (mw 125,000, 98% of hydrolysis) using electrospinning technology (nanospider tm 1ws500u). platelet lysate (pl) was prepared from thrombocyte rich solution (obtained from regional hospital in liberec, the concentration of 700-900 x10 6 plt/ml, freeze-thaw method with subsequent centrifugation). nanofibers were electrospun from 10% pva solution using water: ethanol (8:2) solvent system. materials with proteins were electrospun from solution containing 10% of pva and 10% of pl. morphological analysis was performed by scanning electron microscopy. protein release was monitored using spectrophotometry (bradford method) and chromatography. results: the prepared fibrous materials consisted of random oriented end-less fiber with smooth surface with minimal defects in structure. the morphology of materials was not altered by the addition of proteins. the average fiber diameter was: 340 ae 120 nm for pristine pva fibers and 350 ae 148 nm for pva with incorporated proteins (pva/pl). pva/pl layers contain 5-10 mg of protein per gram of pva. 60% of the proteins are released during the first day (burst release) followed by a gradual release of up to 2 weeks. summary/conclusions: nanofibrous pva-based nanofiber materials were prepared with native growth factors. the process used for the preparation of solutions and subsequent spinning does not affect the activity of the incorporated proteins, which are being gradually released. therefore, we believe that the developed material has great potential for use in tissue engineering e.g. to promote healing of chronic wounds. acknowledgements: supported by the czech health research council, project no nv18-01-00332. background: human a-defensins are small cationic peptides with antimicrobial and anticancer activity. up till now, six a-defensins have been described in humans. they include the human neutrophil peptides (hnp) 1, 2 and 3 which present in large amounts in neutrophil azurophilic granules and differed from each other only in the first amino acid. a fourth defensin, termed hnp-4, comprises less than 1% of the total defensins in neutrophils and has a distinct sequence from hnp1-3. the other two, human defensin 5 and 6, are synthesized mostly by intestinal paneth cells. neutrophil defensins (hnp 1-3) are 3.4 kda peptides that are characterized by three disulfide bridges. the pattern of disulfide bonds in the mature forms is crucial for the functional properties. due to this structural feature, synthesis of defensins using the chemical and recombinant approach presents quite a challenge. moreover, purification from the natural source can be very difficult because the large number of neutrophils is needed to obtain a sufficient amount of protein. in blood banks, leukocyte reduction filters are used to remove leukocytes from blood components in order to prevent adverse transfusion reactions. leukofilters contain high numbers of normal human cells and discard after use. aims: the aim of this study was to purify a-defensins from neutrophils trapped in leukocyte reduction filters. methods: blood bags from healthy blood donors were collected after written consent. all donors were screened for infectious diseases (hbv, hcv and hiv) and negative samples were included in the study. blood bags were filtered at 22°c by leukoflex lst-1 filters. the cells were extracted from the filters by back-flushing with cold phosphate buffered saline (pbs), ph 7.4, without mgcl2 and cacl2, containing 5 mm edta and 2.5% sucrose. the pbs was homogenized with the filter content and then collected in a sterile tube. neutrophils were separated from mononuclear cells by ficoll. isolated neutrophils resuspended in pbs 1x at a concentration of 1 9 10 7 cells/ml. for degranulation, cells were stimulated with 100 nm of formylmethionyl-leucyl-phenylalanine (fmlp) for 5 min followed by stimulation with 10 lm of cytochalasin b for 5 min. supernatant was collected by centrifugation at 200 9 g for 6 min. supernatant was incubated with mouse monoclonal antibody to hnp 1-3 and purification of hnp 1-3 was performed by lmacs protein g microbeads system. the presence of protein was confirmed by western blot. results: the presence of the 3.4 kda band was confirmed by western blot, which corresponded to the size of the a-defensins. summary/conclusions: the development of defensins as therapeutic products requires access to a steady supply of neutrophils. our results indicated that lst-1 filters are economical source for purifying a-defensins. anatomical sciences, abadan school of medical sciences, abadan, iran background: epigenetic reprogramming of terminally differentiated cell can modify somatic cells to a pluripotential state. there are several approaches that induce pluripotency in somatic cells. exposure the cells with the embryonic stem cell extract is an easy way, and some investigations were done on fibroblast cell line. however, its efficiency was low aims: the purpose of this study was to increase the number of reprogrammed granulosa cell as a full differentiated cell into pluripotential state methods: the human granulosa cells were cultured in the medium containing 5-aza-deoxycytidine and trichostatin a. then, the cells were exposed to mouse escs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (lif). alkaline phosphatase test and also immunohistochemistry staining for oct4, sox2 and nanog were performed after 24 and 72 h and 1 week results: the results indicated that after 24 h the granulosa cells were revealed a round and expanded morphology. the cells in all groups except in negative control, were showed alkaline phosphatase activity. the cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a and exposed to the extract had the most numbers of alkaline phosphatase positive cells. immunocytochemistry showed the granulosa cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a with extract expressed oct4 with weak intensity after 24 h. no expression of oct4, sox2 and nanog were observed in other groups at the same time. after 72 h, oct4, sox2 and nanog were over expressed in the cells that were treated with 5-aza-deoxycytidine, trichostatin a and extract. furthermore, there was high expression of oct4 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract. after one week, the expression of oct4 and sox2 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract was continued while its expression ceased in the other groups. the expression of nanog were ceased in all groups after one week summary/conclusions: present study revealed that the inhibitors of the methyl transferase (5-aza-deoxycytidine) and histone deacetylase (trichostatin a) could delete the epigenetic markers and improved cells reprogramming by administration of the extract abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mesenchymal stem cells (mscs) are adherent spindle shape cells expressing different surface markers. they show special criteria including, paracrine effects, differentiation to several tissue cells, migration, immunomodulatory and regenerative potentialities. mscs are isolated from different sources and employed as therapeutic tools to treat several diseases and injuries. however, some of mscs properties including secretion of growth factors and migration ability are controversial especially during remission or in presence of tumor. interestingly, msc-derived compartment could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. exosomes are kind of extracellular vesicles (evs) characterized via their size and releasing mechanism. usually they defined as less than 150 nm in diameter vesicles. they secreted from different cells and are also found in urine, blood, breast milk, cerebrospinal fluid and other body fluids. exosomes contain genetic material including dna, mrnas, micrornas (mirnas) and other biomolecules. mirnas are single stranded non-coding rnas transcribed from dna. immature mirnas are subjected to two known cleavages to modify to mature mirna that involve to either mrna degradation and gene expression process or cell-cell interaction and communication via secretion as the part of exosomes. aims: this study was aimed to discuss some aspects of exosomal micrornas derived from mscs in progression, diagnosis and treatment of some diseases. methods: different scientific data bases including pubmed, google scholar and scopus were used to find and review related articles. results: evs play important role either in intercellular communication related to pathological and physiological situation or intracellular communication, angiogenesis, immune system modulation and metastasis progression. mirnas could regulate expression of multiple mrnas then they play important role in different biological processes and contribute cell-cell interaction as well as influence in the progression of different disease. exosomal mirna-derived mscs are involved in cancer procession, tumor growth, angiogenesis and metastasis. they are used as diagnosis and therapeutic tools to treat different diseases such as renal failure, liver fibrosis, myocardial infarction. summary/conclusions: due to controversial aspect of using of intact mscs especially during remission or in presence of tumor, msc-derived exosome could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. aims: the aim of study try to use sybr green i based real-time pcr to identify homozygous, heterozygous, gene deletion or wild type for rhd exon 1, 5, 10 and 1227a. methods: for this study, we used real-time pcr with high resolution melting curve mode, and matrix mix containing sybr-green i were used for sequence specific primers of 1227 g>a and rhd exon 1, 5, 10. samples with rhd gene deletion homozygous/heterozygous, 1227 g>a heterozygous with rhd gene deletion and normal rhd, normal rhd homozygous/heterozygous and rhd1-rhce(2-9)-rhd10 homozygous/heterozygous were enrolled in our study. all samples were screened using rhd exon genotyping, sanger sequencing and rhesus box analysis. concentration and mass of dna samples were 1 in alleles of normal rhd/rhd gene deletion. the tm ratio of rhd exon 1 (87°c) to internal control (77°c) were 2.33 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.09-2.21 in alleles of rhd gene deletion/normal rhd, 2.53-2.66 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 10 (81°c) to internal control (77°c) were 3.35 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 4.29-4.39 in alleles of rhd gene deletion/normal rhd, 4.67-6.10 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 5 (81°c) to internal control (77°c) were 3.64 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.49 in alleles of rhd gene deletion/rhd1-rhce(2-9)-rhd10 3.47-3.59 in alleles of rhd gene deletion/normal rhd, 3.97-4.77 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. summary/conclusions: using the tm ratio of sequence specific primers to internal control is an effective way to detect the rhd gene deletion or rhd weak d types 1, 2 and 3 not detected") were tested with a method based on next generation sequencing (ngs) using the illumina miseq platform to detect a possible rhd variant not interrogated by id rhd xt. results: in total 583 dna samples were tested in 87 pools. fifteen (15) pools (181 samples) gave rhd deletion genotype and seventy two (72) pools (402 samples) resulted to the presence of rhd gene. the 72 positive pools were also analyzed individually. the genotype results obtained were: rhd exon 1 no amplification (1), rhd exon 6 and the genotype results obtained with id rhd xt (in pools and individually) were concordant with the results provided by the centers. hence, 100% accuracy was obtained using id rhd xt with dna pooled samples. the results of rh ngs for the samples with inconclusive results by id rhd xt showed rhd variants previously described: 1 sample rhd*93-94inst (del), 1 sample rhd*ivs3+1g (del), 9 samples rhd*weak d type 11 (partial d), 1 sample rhd*weak d type 5 (weak d), 1 sample rhd*weak d type 61 (weak d) and not described: 1 sample rhd*del 1-5 (unknown) summary/conclusions: id rhd xt is a high accurate tool for genotyping the most common rhd alleles associated to weak d and d negative phenotype in up to 20 pooled dna samples. use of rhd genotyping improve rhd typing in blood donations variant rhd alleles generate qualitative/quantitative alteration in serological expression of d antigen such as weak and partial rhd phenotypes which are clinically important in transfusion setting. population studies have shown varied distribution of the variant d alleles in caucasians, africans, east asians and indians. many countries have developed their own population-specific strategy for identifying d variants. our previous study in indian population showed absence of weak d type 1, 2, and 3 which are commonly found in caucasians d variant individuals. instead, a novel population-specific pattern i.e.~12-kilobase duplication event, including exon 3, was predominantly identified in 58.3% d variant samples. functional analyses showed that this genetic variation results in the expression of several transcripts, including a wild-type product. commercial genotyping assays available, mainly detect common d variants found in caucasians and africans, thus limiting its usefulness in india. hence, based on our findings we have designed a multiplex pcr assay specific for indian population that can be easily implemented at the laboratory level for genotyping variant rhd. aims: to characterize rhd variants using "indian-specific, rhd genotyping assay". methods: seventy samples referred to our laboratory for molecular characterization of rhd variants were included in this study. all rhd variant samples were serologically typed for results: out of the 70 rhd variants included in this study, 49 samples (70%) showed presence of indian specific allele i.e. exon 3 duplication. seventeen rhd variants samples showed presence of both exon 5 and 10. qmpsf analysis of these samples excluded involvement of rhd-rhce-rhd hybrids. sixty of the seventy d variant individual had r 1 r genotype this assay thus can be used routinely in indian laboratories to identify and characterize rhd variants. -128 non-invasive fetal kel1 genotypes from allo-immunized anti-kel1 women were done (34 positive confirmed fetuses, 6 undetermined, 16 positive non-confirmed, 22 negative non-confirmed and 50 negative confirmed). -190 non-invasive fetal rhc genotypes from allo-immunized anti-rh4 women were done non-invasive fetal rhe genotypes from allo-immunized anti-rh3 women were done (66 positive foetuses, 1 undetermined for 21,5% of the allo-immunized women, the pregnancy was compatible and no specific antenatal monitoring was necessary summary/conclusions: non-invasive fetal red blood cell genotype is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring s purchla-szepioła 1 , m krzemienowska 2 , m pelc-kłopotowska 2 , m jurkowska 3 , m debska 4 , m uhrynowska 2 and e brojer the test developed by ihtm has been offered to clinicians and pregnant women since 2016 but it is not covered by the health care system. rhd nipt is not informative for mothers with rhd variant. in such cases further analysis of the molecular background is offered to exclude from immunoprophylaxis the women with weak rhd type 1, 2 and 3. aims: summary of a 3-year period of routine rhd nipt available at ihtm. methods: cffdna isolated using easymag, biomerieux from plasma of 366 pregnant women determined with standard serology as rhd-negative (in 8-38 week of gestation) was examined for the presence of exons 5 and 7 of rhd and ccr5 by realtime pcr using lc480ii (roche). maternal dna from whole blood was tested for identification of rhd variant using rbc fluogene rbc-dweak/variant (inno-train, germany) or the home-made protocol. results: in 123 cases the rhd gene was not detected in cffdna and the administration of rhig was not recommended. in seven cases ct-values for rhd and ccr5 indicated a maternal d variant (d ct ccr5-rhd >2); the genetic follow-up of six of them identified: rhd*01w.2 in 4 cases, rhd*01w.1 and rhd*15. in 235 cases the rhd nipd results indicated that a fetus is rhd positive and rhig administration was recommended it was recommended in the remaining 67% of mothers. in 1.9% cases with maternal d variant rhd nipt was not possible. however, in 5/6 such cases the test is unnecessary because follow up analysis revealed maternal rhd variant of the weak d type 1 and 2 in switzerland extended antigen-matching for duffy, kidd and mnsbesides rhesus and kell -is recommended for sickle cell disease (scd) patients. the ethnic diversity of red blood cell (rbc) antigen polymorphism engender that these patients are often transfused with rbcs from donors of african origin. this strategy, however, increases the likelihood of being exposed to certain low-prevalence antigens, such as rh23 (d w ), as these are almost exclusive to african populations. rh23 is encoded by several types of rhd*dv as well as by dau-5. anti-rh23 is associated with delayed hemolytic transfusion reactions (htr) aims: here we report a specific low-prevalence antibody newly formed by the same patient, meanwhile gravida 4, para 1, causing positive crossmatches with the rbcs of two of the four selected donors. subsequently, advanced serologic and genetic workup and close international collaboration enabled optimal patient care. methods: standard serological methods were used for antibody specification (biorad, cressier, ch and in-house). crossmatches were carried out by indirect antiglobulin test at 37°c. molecular typing of donors' and parental blood group antigens was performed by further serological analysis (institute national de transfusion sanguine, paris) revealed an anti-rh23 in addition to anti-fy 5 , anti-e and anti-jk a . genotyping of the two donors causing positive crossmatches presented heterozygosity for rhd*10.05 which encodes rh23. the newborn's phenotype was a r0r k-, fy(a-b+) and most likely rh23-and jk (a+b+), considering both maternal and paternal (a r0r, k-k+, fy(a-b+), jk(a+b-), rh23-) predicted phenotypes. the neonatal serum contained maternal anti-a1, anti-rh23 and anti-e. the direct antiglobulin test was positive but elution only showed nonspecific reactions with papain-treated cells. latter might have been caused by anti-fy 5 during her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-rh23, derived from several rh23 + rbc transfusions during the previous pregnancy. despite this challenging blood supply we were able to support the patient with a total of ten antigen compatible and crossmatch negative rbc units from french and swiss donors until delivery with increasing age, the relative number of women in the study population raise from 22% in the patients younger than 60 years to 58% in the patients older than 80 years. our study showed that cardiovascular diseases were the commonest indications for warfarin use in older patients with 59%. only 48,5% achieved target therapeutic range while the risk of thromboembolism and the subsequent need for proper anticoagulant therapy increases sharply with age, the bleeding risk rises as well. older patients are more sensitive to any given dose of warfarin and need a significantly lower total weekly dose. a well-informed patient provides one of the best defenses against bleeding complications. recent data demonstrate doacs advantages over warfarin, especially for older population: more predictable dosing, fewer drug interactions and reduced risk of intracranial bleeding although vast majority of fh cases are caused by mutations in ldl-r gene 17%-33% patients do not harbor genetic cause in the known loci. patients with homozygous/severe heterozygous fh are unresponsive (ldlc above 200 mg/dl with diet and drug therapy) and require additional extracorporeal therapy to reduce ldlc concentrations to prevent the development/progression of cad. ldl apheresis techniques remove apolipoprotein b-containing lipoproteins from blood and include heparin-induced extracorporeal ldl precipitation(help), immunoadsorption, dextran-sulfate adsorption methods: a 28y iraqi male visited cardiac-opd. ct coronary angiogram showed cad-dvd. he had multiple tendinous xanthomas and xanthelasmas. family history was significant for death of elder brother from coronary event at 30y, a sister with similar profile age 26y and one sister apparently normal. he was taking medical treatment for dyslipoproteinemia (ecosprin 75 mg od, ticagrelor 90 mg bd, rosuvastatin 40 mg od). despite dietary and medical treatment his dyslipoproteinemia was refractory. therefore cascade-filtration was planned with evaflux5a plasma fractionator. one procedure of cascade plasmapheresis was done on com.tec apheresis system (fresenius kabi, germany) separating patient's plasma as the first step and passing it through a pore sized based filter column 5a20 (evaflux, kawasumi, japan) as the second step. a total of 1.5x plasma volume(4470 ml) was processed. the patient was given continuous calcium infusion. the flow rate of 50 ml/min was maintained immunoglobulins) were not assessed. summary/conclusions: the procedure successfully met the requirement of reduction of cholesterol by 60%. the patient became responsive to the medical treatment. follow up of the patient up to a year has been uneventful with no additional procedure requirement actions included development of major haemorrhage protocols with improved communication and required instances of delayed transfusion to be reported to the uk national haemovigilance scheme (serious hazards of transfusion, shot) methods: delayed transfusion data have been collected from 2010. hospitals identify incidents and report them via an online database. mh may also result in avoidable or overtransfusion. reports are analysed and collated data published in the shot annual reports in july each year. results: the total number of reports of delayed transfusion has increased with time: 101, 95, 111 in the last 3 years. delayed transfusion in relation to mh was reported for 54 cases 2016-2018 contributing to death in 6 patients 7%) miscommunication was noted between clinical different teams, between emergency departments, porters and the transfusion laboratory, failure of bleeps, failure to communicate the urgency, failure to confirm the patient location. failures to follow mhp correctly occurred in 31/71 (43.7%): incorrect activation including failed alerts to porters, wrong contact telephone details and wrong components in the mhp packs most transfusions included red blood cells (median, 2 units); 14% of women were transfused with fresh frozen plasma (median, 2 units) and 2% with platelets. mean pre-and post-transfusion hemoglobin levels were 6.7 g/dl and 9.0 g/dl, respectively, representing an increment of 1.0 g/dl per rbc unit transfused (1.09 g/dl in soweto and 0.83 g/dl in durban). indications for transfusion included obstetric hemorrhage (31%), chronic anemia (29%), surgery or anesthesia (13%), other (9%) and not specified (17%). transfusion for chronic anemia (vs. hemorrhage) was associated with gestation ≥ 26 weeks (odds ratio = 7.07, 95% confidence interval 3.52-14.24). surgical blood loss was a common indication in trimester 1 (21%) that declined to 7% then 1% in trimesters 2 and 3. summary/conclusions: hemorrhagic complications accompanying spontaneous abortions and ectopic pregnancies in the first and second trimesters were the most common reasons for antenatal transfusion surveillance and analysis of blood transfusion reactions represents inseparable part of hemovigilance. aims: summarization of data on reported cases of transfusion reactions. methods: analysis of serious undesirable reactions to blood products administration in the czech republic (cr) during period 2016-2018. results: there were evaluated 1,281 688 of blood products administrations in 117,414 patients in the cr during defined three years period. announced 1,456 (0,11%) transfusion reactions including 50 severe transfusion reactions (20 9 adjudged with grade 3). the most frequent types of severe transfusion reactions: anaphylaxis 20, trali 7x, taco 5x, hcv 4x, hbc 2x, bacterial infection 1x, delayed hemolysis 1x. transfusion reactions incidence according to administered bp: red blood cells products: 882,017 administrations, 883 transfusion reactions (fnhtr 386x, allergy 227x, circulatory overload 66x, anaphylaxis 6x, trali 4x, hbv 2x, hcv 1x) platelets: 88,438 thrombocyte administrations, including 178 transfusion reactions (allergy 129x, fnhtr 30x, anaphylaxis 5x, circulatory overload 8x, delayed immune hemolysis 3x, acute circulatory overload 8x. granulocytes: 203 administrations, 2 transfusion reactions plasma: 293,801 administrations, 359 reactions reported (allergy 278, fnhr 25, circulatory overload 14, anaphylaxis 17x, trali 4x, hbv 3x, ards 1x. summary/conclusions: conclusions: comprehensive analysis and data processing help to appropriate prospective setting of blood products (bp) production and hemotherapy. concrete outputs from processed data triggered undermentioned changes in many departments in the cr: 1. plasma for clinical uses from male blood donors, 2. prestorage of leucocyte reduced blood products, 3. production of platelets in additive solutions, 4. implementation of pcr testing method for blood donors screening. background: skae's basic activities include epidemiological surveillance of all adverse events (aes) associated with deviations in the collecting, testing, processing, storage and distribution of blood and blood components that may affect quality and safety near misses" and "uneventful transfusion errors" are collected to identify preventable causes. incorrect blood component transfused (ibct) events are reported following ihn instructions. results: a total of 5 they were mainly associated with deviation in processing (22.4%) and attributed to equipment failure and materials (71%) whole blood collection, materials and distribution, as a result of product defect, equipment failure, human error and other. trend analysis showed a significantly increasing (p < .001) annual rate of total aes by 28% (95% confidence interval 26-30) ) 10% fibrinogen-depleted phpl or (3) 10% fibrinogen-depleted phpl plus heparin. internalization of fluoresceinamine-labeled heparin in stcs was investigated by flow cytometry and immunocytochemistry. all stromal cells were subjected to whole genome expression analysis (affymetrix human gene 2.1 st array) and data were analyzed using r/bioconductor and panther analysis tools. confirmative qrt-pcr was performed and protein levels of selected pathways were analyzed by a bead-based western blot system (digiwest â ). immunophenotyping, in vitro differentiation, longterm proliferation and colony forming units (cfu) assays were done for all cell types. results: in vitro exposure of heparin induced differential internalization and lysosomal accumulation in stromal cells, as well as regulation of distinct gene sets, both in a tissue-source dependent manner. affected signaling cascades were mainly involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis. the influence of heparin on protein expression and phosphorylation of four pathways (wnt, pdgf, notch and tgfbeta) was further analyzed, revealing most alterations in bm-stcs. independent of origin and medium composition, flow cytometry analysis revealed the characteristic fibroblastoid phenotype profile (cd73+/90+/105+ and cd14-/19-/34-/45-/hla-dr-). in addition a comparable osteogenic and adipogenic differentiation capacity was found summary/conclusions: internalization of heparin in lysosomes by stromal cells, differential gene and protein expression and phosphorylation changes were observed in a tissue-source dependent manner. however, stromal cell characteristics as immunophenotype pattern, long-term proliferation, clonogenicity and in vitro differentiation were unaffected, putatively by post-translational protein modifications. in this respect, application of porcine heparin is compatible with efficient manufacturing of stromal cell based medicinal products abo incompatibility may have no effect on the clinical outcome after allogeneic hematopoietic stem cell transplantation. however, it carries additional risks of hemolytic reactions, delayed red blood cell (rbc) engraftment and incidence of graft-versus patients were categorized according to abo compatible and mismatched groups; these were further sub-categorized into major, minor and bidirectional. direct coombs test (dct) was performed when hemolysis was suspected in the post-transplantation period along with serum lactate dehydrogenase (ldh) 5%) were male and 31(36.5%) female. mean age of abo matched and mismatched groups were (6.99 ae 4.6) years. most common indications for transplant included beta thalassemia major 53(60.2%), aplastic anemia in 25(28.4) and pure red cell aplasia 2(2.35%). source of stem cell was bone marrow in 45 and peripheral blood 40 patients abo matched while abo mismatched group comprised of 28(32.9%) patients with further subdivision into major (n = 13), minor (n = 13) and bidirectional in the post transplantation period, packed red blood cell and platelets were transfused in matched group (n = 401) and (n = 1837) comparably(n = 102) and (n = 480) in mismatched group. primary and secondary graft failure in matched group was 8.77% and 12.28% patients while in mismatched group graft failure was observed in 4(14.28%) patients respectively. positive dct in abo matched group in 1(1.75%) patient, whereas 2(7.14%) patients with major and minor abo mismatch group with raised ldh levels and deranged lfts were found. episodes of acute and chronic gvhd in abo compatible and incompatible groups were insignificant. overall survival in abo summary/conclusions: these results indicate that abo incompatibility does not seem to influence outcome of the patients undergoing allogeneic hematopoietic stem cell transplantation. careful monitoring of patients can help detect problems early and treat them efficiently, thus, reducing the number of life threatening events a picascia 1 , c sabia 1 , f cavalca 1 , g nicoletti 2 and c napoli 1 in our routine work with one lambda sab class ii reagents, we observed non-specific reactivity with some beads bearing dr4 and dr16 in patients without sensitizing events. this pattern was not confirmed by testing same sera with screening-and pra-beads suggesting non-specific reactions. aims: here, we sought to determine if fetal bovine serum (fbs) treatment would be effective in reducing/eliminating non-specific reactivity. methods: we tested 5 sera pre-treated with fbs from non-sensitized patients that showed the dr4/dr16 pattern. in particular, 5 ll of fbs was added to 95 ll of patient serum; incubated for 30 min at 37°c; centrifuged and subsequently tested in the sab assay. as controls, we treated sera from 5 patients with documented dsa including dr4/dr16 and 5 patients without hla antibodies. results: dr4/dr16 non-specific reactivity was eliminated or significantly reduced after fbs treatment. we found that patients with dr4 and dr16 dsa had no change in mfi values and additional reactivity was not observed in negative fbs treated sera transfusion medicine, national blood transfusion centre 2 transfusion medicine, national blood transfusion center 3 transfusion medicine, national blood transfusion centre, tirana, albania background: abo blood group, has been associated with many diseases, although the explanation for abo's blood group association and some illnesses is still unclear. aims: to find the distribution of cases by blood groups in patients with malignant pathology compared to donors in order to assess the presence of the abo blood group as an epidemiological indicator to identify populations exposed to different malignant pathologies methods: we conducted a case-control study. abo blood group and diagnosis of all patients have been studied. the control sample was collected from 17,992 healthy donors from which group a (36,8%), group o (41,7%), b (16,2%) and group ab (5,3%) resulted. the study was conducted in 3727 patients who have been transfused and submitted a request to determine the blood group at the blood bank at qsut during the period 2011-2017 results: among the 3727 patients, when all malignant pathologies were taken together, the highest frequency was seen in blood group a (39.7%), followed by 0 (39.5%), b (15.2%) and ab (5.4%). group a frequency was higher and o was lower compared to controls. a high incidence of blood group a is seen in: pancreatic cancer a (42%), in gastric cancer a (43%), colorectal cancer a (39, 6%), breast cancer a (41%), cervical cancer a (43%) and ovarian cancer a (42%) versus a (36.8%) in the control group. a high incidence of blood b is seen in multiple myeloma b (22%) and cervical cancer b (20%) versus b (16.2%) in the control group. blood group ab has a high incidence in malignant lymphoma ab (10%) versus (5.3%) in the control group summary/conclusions: it appears that individuals with blood groups a, b and ab are more at risk of developing malignant pathologies and individuals with blood group o are more protected. background: the high homology and opposite orientation of rh genes promote many rearrangements between them and generate a large number of rhd and rhce variants which can be inherited together. several studies have shown that those rh variants in patients with scd represent an additional risk for alloimmunization and delayed hemolytic transfusion reactions (dhtrs), but little clinical or biological evidence related to alloimmunization and dhtr are presented for all the rh variant alleles. it is well established that transfusion recipients with the most common weak d types 1, 2 and 3, are not at risk for forming alloanti-d when exposed to conventional rhd-positive rbcs. aims: we report here a case of a 12-year-old patient typed as weak d type 3, receiving d+ rbc units who presented anti-d in his plasma detected three weeks after the last transfusion. methods: rhd beadchip (immucor, nj, usa), was performed to identify the rhd variant allele associated with the weak expression of d. rhce genotyping was performed by laboratory developed tests. sequencing of rhd, rhce and rhag were performed to determine if there were other mutations that could explain the production of alloanti-d. serologic testing was by standard hemagglutination methods. the clinical significance of the antibody was evaluated by monocyte monolayer assay (mma). results: serological analysis showed a negative dat and the presence of anti-d in plasma (2+ by gel). anti-lw was ruled out. rhd genotyping revealed the patient was rhd*weak d type 3. rhce genotyping predicted the d+c+c+e-e+ phenotype. sequencing of rhd, rhce and rhag found no additional changes and confirmed the presence of rhd*weak d type 3. mma showed the anti-d was clinically significant (>5%). summary/conclusions: we report the production of alloanti-d in a scd patient with rhd*weak d type 3 allele. weak d type 3 patients are not considered to be at risk for allo anti-d but our results show that there are exceptions and that these anti-d can be associated with clinically significant rbc destruction. background: the mns blood group system is located on glycophorin a (gpa), glycophorin b (gpb) and hybrid glycophorins on the surface of the red blood cell (rbc). these glycoproteins are involved in complex structures interacting with other rbc surface proteins including the band 3/diego protein. the glycophorins are heavily glycosylated and contains multiple clinically significant blood group antigens. it has proved difficult to model the gpa extracellular structure due to its heavy glycosylation, and lack of homology with existing modelled proteins. aims: to develop an in silico model of gpa as a basis for improved predictions of structure function relationships methods: prediction of secondary structure and disorder: 1.1. predictprotein (https://predictprotein.org); 1.2. spider2 (http://sparks-lab.org/server/spider2/); 1.3. dsc (discrimination of protein secondary structure class): using an in-house implementation; 1.4. jpred4 (http://www.compbio.dundee.ac.uk/jpred4/); 1.5. raptorx (http://raptorx.uchicago.edu). prediction of secondary structure: 2.1. robetta (http://robetta.bakerlab.org/submit. jsp); 2.2. falcon (http://protein.ict.ac.cn/treethreader/); 2.3. itasser (https://zha nglab.ccmb.med.umich.edu/i-tasser/) threading methods to evaluate the quality of protein structures: 3.1. verify3d (http://servicesn.mbi.ucla.edu/verify3d/); 3.2. prosa (https://prosa.services.came.sb g.ac.at/prosa.php) protein-protein docking: 4.1. gramm-x protein-protein docking web server (http://vakser.compbio.ku.edu/resources/gramm/grammx/); 4.2. gramm (http://va kser.compbio.ku.edu/main/resources_gramm1.03.php) results: using in silico modelling we derived a stable tertiary glycosylated structure for gpa both as an individual protein and a homodimer. the hybrid glycophorin background: non-invasive prenatal testing of fetal antigen using cell-free fetal (cff) dna from maternal plasma of immunized women is widely implemented into clinical routine but the sensitivity and specificity of the method, especially for genotyping antigens encoded by single nucleotide polymorphisms such as k antigen, is limited by low proportion of cffdna in maternal plasma dna. according to literature reports detection of circulating tumour (ct)dna can be improved by selection of short ctdna fragments using automated electrophoresis methods. aims: the aim was to assess the feasibility for enrichment of cffdna fraction in maternal plasma dna by size selection using the pippin prep gel selection system. methods: plasma dna isolated using easymag (biomerieux) from rhd negative and k-negative pregnant women (n = 10) carrying fetuses with known genotype was loaded into 3% agarose gel casette (3% df marker q3, sage bioscience) and size selection of fraction was performed on a blue pippin tm (sage bioscience) with the elution from 45 min to 2 h 30 min of electrophoresis. results for real-time pcr detection of fetal rhd, kel*1 and ccr5 (as a marker of total plasma dna) in dna fraction after gel selection were compared to results obtained from non-processed plasma dna. results: the total dna level (measured by ccr5) was significantly lower in dna samples tested after gel selection (from 8.4 to 25.9geq/pcr) versus the level obtained from non-processed plasma dna (from 130 to 133geq/pcr). the results for fetal fraction (measured by rhd) from dna samples of rhd-negative pregnant women carrying rhd positive fetus tested after gel selection were from 1,5 to 6.6geq/pcr versus 10.8-11.2geq/pcr for non-processed plasma dna. results for kel*1 detection in plasma dna from k-negative pregnant women carrying k-negative fetus were kel*1-negative in dna samples tested after gel selection comparing to nonprocessed dna samples were false kel*1 positive amplification was observed. however, kel*1 detection in plasma dna from two k-negative pregnant women carrying k-positive fetus gave false kel*1-negative results in dna samples tested after gel selection comparing to non-processed dna samples were kel*1 positive genotype was obtained. the total dna level in samples from k-negative women was from 18.3 to 18.7geq ccr5/pcr after gel selection versus from 52 to 746geq ccr5/ pcr in non-processed dna samples. summary/conclusions: using the pippin prep gel selection system increases the proportion of cffdna fraction in total plasma dna by retaining long maternal dna fragments in the gel cassettes, but the protocol of gel separation dilutes the separated material decreasing the concentration of fetal dna and leading to false negative results of nipt. anti-rh1 quantification assay using ih-500 (bio-rad â ): promising results for monitoring rh:-1 pregnant women j beaud, h delaby, c toly-ndour, a mailloux and s huguet-jacquot centre national de r ef erence en h emobiologie p erinatale (cnrhp), hôpital saint-antoine, paris, francebackground: the generalization of immunoprophylaxis by anti-rh1 immunoglobulins since 1970 complicates the interpretation of the anti-red blood cell antibodies screening during pregnancy. to distinguish an alloantibody from a passive one, many laboratories in france use anti-rh1 microtitration. it is a column agglutination technology using red blood cells rh: 1, -2, -3,4,5 (r0r) . it permits to quantify low levels of anti-rh1 in comparison to a range of an anti-rh1 standard. performed since 1999 at the cnrhp and automated on evo clinical base tecan in 2008 (dilutions and distribution), anti-rh1 microtitration is well adapted to rh prophylaxis. aims: the aim of this study was to evaluate this technique on the ih-500 system from bio-rad â . methods: on ih-500, the reactivity of the bio-rad â reagents was compared with the cnrhp reagents (red blood cells r0r, anti-rh1 standard). the performances of the method were evaluated using three internal quality control (icq) (2 cnrhp home-made at 2 and 12 ng/ml and 1 bio-rad â at 12 ng/ml) on papainized r0r (plc) and native r0r (nlc). a comparison of results from patient sera ranging from 1.5 to 48 ng/ml was done between ih-500 and evo clinical base tecan. results: the results of the 3 qci are similar between the different reagents used. there is no significant difference between the 2 types of red blood cells except for the limit of detection: 1.5 ng/ml in plc -6 ng/ml in nlc. for the 3 qci, the intra and interassay imprecision based on the dilution degree show coefficients of variation between 0 and 15%, similar to those found with the evo clinical base. the correlation with the cnrhp technique performed on 50 samples in plc and 44 samples in nlc was satisfactory (deming plc: r2 = 0.80 y = 0.89x + 0.78 -nlc: r2 = 0.86 y = 1.08x-0.25). summary/conclusions: the anti-rh1 microtitration on the ih-500 offers similar performances to the method conducted at the cnrhp. the ih-500 allows automated reading of gel cards. however, it does not have a calculation or interpretation algorithm and does not directly give the concentration of anti-rh1. this final part remains manual and requires trained staff. background: haemolytic disease of the foetus and newborn (hdfn) due to maternal-foetal incompatibility has been perfectly framed for decades from the etiologic, pathogenetic and therapeutic point of view. the anti-d alloantibody is most frequently responsible for the most serious form of hdfn due to rhd incompatibility (rhdi hdfn). although immunoprophylaxis (ip) has reduced the number of cases of rhdi hdfn, this disease continues to occur and red blood cell alloimmunization still remains the most common cause of foetal anaemia. hdfn due to abo incompatibility (ab0i hdfn) is currently the most common neonatal haemolytic disease in the western world. however, only in about 1.5-2% of cases haemolytic disease demands transfusion support. aims: analysis hdfn from 2011 to 2018. methods: the hdfn's transfusional support is: intrauterine transfusion (iut) in the antenatal period; exchange transfusion (et) for severe hyperbilirubinaemia and neonatal transfusion of small volumes red cells for the newborn's late anaemia in the postnatal period. our policy for iut, for et and for the neonatal transfusion requires the use of a concentrated blood cells (ec) preferably group 0 rh negative (cde/cde) or negative for any red cell antigens if the mother has antibodies, fresh (<5 days), preferably cmv safe. for iut, the ec must be compatible with mother's plasma, must have hematocrit 70 + 5%, and irradiated. the unit for et must be compatible with the newborn's plasma, whit hematocrit 40% -60% and irradiated. the ec used in post-natal transfusions is usually divided into rates of 70 ml, hematocrit 55 ae 5%. results: in last 7 years, we calculated 59 neonates with hdfn (28 males and 31 females): 31 with rhdi hdfn, 19 with ab0i hdfn and 9 with hdfn due to incompatibility for other red blood cell antigens. we have produced 81 iut: 48 for our hospitalized patients and 33 for patients located in other hospitals. 25 of these 48 patients, who received iut, were immunized: 19 showed anti-d antibody and 6 antibodies different from anti-a and anti-b. 7, of the 31 infants with rhdi hdfn, were transfused in utero. 4 neonates on 59 (6.8%) have performed et: 1 with ab0i hdfn and 3 with rhdi hdfn; the latter had also been transfused in utero. 27 neonates on 59 were transfused after birth: 18 with rhdi hdfn, 3 with ab0i hdfn and 6 with hdfn due to incompatibility for other antigens. summary/conclusions: our case studies reflect the literature data. neonates with rhdi hdfn are the most numerous (52.7% of the total) and are those who have requested the highest blood supply both in the antenatal period (39.6%) that postnatal (9.6% performs et, 66.6% performs postnatal transfusions). neonates with aboi hdfn are 32.3%: nobody has received iut, only one has been subjected to et, and 11% has transfused after birth. patients with hdfn due to other antigens are 15%, have undergone iut 12.5% and were transfused after birth 22.2%. background: according to british guidelines on neonatal transfusion, since 2012 we shared with neonatologists a transfusion protocol for preterm babies. patients are anemic premature newborns with a gestational age ≤ 30 weeks and/or a birthweight lower than 1500 g, until 4 months of age. aims: reduce the incidence of iatrogenic anemia. methods: pre-transfusion tests were based on ab0 direct test, rh phenotype, direct and indirect antiglobulin test (dat, iat). a second blood sample was required for ab0/rh confirmation. blood transfusions were performed with 0 negative kell negative (0 cde/cde/kk) cmv negative irradiated erythrocyte concentrates (ec) of less than 5 days. ec were subdivided in 70 ml aliquots with a hematocrit of 55 ae 5%. according to the definition of "small volume transfusions", our protocol established that further four transfusions had to be delivered free of testing. after the fifth ec transfusion the supplementary release of ec was provided of type and screen (t&s) test with 72 h of validity. serological investigation and full compatibility testing were applied in the presence of a iat and/or dat positivity and in the case of mother alloimmunization. results: from october 2012 to the end of 2018, 694 premature newborns received 2328 ec transfusions within their first 4 months of life. in 51% of cases (n = 1186), transfusion requirement was comprised within the 'small volume transfusions'. another 44% of cases (n = 1035), requiring further ec administration, was requested of a blood sample for t&s determination and 5% (n = 107) for a cross-match test. in 79.4% of newborns (n = 551), being transfused within the " small volume transfusions", blood requirement of 1401 ec was fulfilled by the initial blood test (1102 blood samples). 13.3% of newborns (n = 92) received more than 5 transfusions (6-21; median = 8) accounting for 820 ec released and for this group 381 blood samples were required. summary/conclusions: with the exception of babies requiring crossmatch test, 381 blood tests were performed to sustain 643 infants transfused with 2221 units. the alternative option of omitting crossmatch test (otherwise suggested by italian directives), allowed a reduction of 75% of blood drawn without any adverse effect or incident reported. due to the relevance of anemia in premature babies, we suggest the application of this transfusion policy in all newborns in the first 4 months of life. background: glucose-6-phosphate dehydrogenase deficiency (g6pdd) is a sexlinked enzymopathy which is usually asymptomatic unless individuals are exposed to oxidative stress agents. the g6pd 202 genotype is the most common g6pd genotype in sub saharan africa (ssa). some studies have linked blood from g6pdd donors to poor outcome of a transfusion. however, there are no genetic screening programmes for blood donors in the region hence the contribution of g6pdd to the donor pool in the ssa setting had not been described.aims: this study aimed to describe the prevalence of g6pdd 202 genotype among donors in two regions in uganda. it also described the effect of g6pdd and the coinheritance of haemoglobin s and a-thalassaemia on the haematological quality of blood. methods: 3,255 blood samples from donor packs were utilized in a transfusion trial conducted in uganda, were genotyped for g6pd 202, haemoglobin s and a-thalassaemia. haemoglobin and haematocrit measurements for the donor units (packs) at the time of transfusion were used to describe the effect of g6pd 202 and co-inheritance with a-thalassaemia (n = 2,546) and haemoglobin s (n = 2,642) on the haematological quality of blood packs. a subset of 142 donor blood packs was utilized to determine the sensitivity and specificity of the carestarttm rapid diagnostic kit (rdt) for g6pdd. results: based on g6pd 202 genotyping, 10.3% (n = 274) of the blood samples used in the trial were deficient for g6pd enzyme while 5.3% (n = 142) were heterozygous. significant lower hemoglobin values were observed in red cell concentrates (p = 0.010) and whole blood (p = 0.009) donations of heterozygous g6pd 202 genotype. co-inheritance of g6pdd and a-thalassaemia resulted in significantly lower haemoglobin levels. the carestarttm rdt test was 83.3% sensitive and 0.8% specific for detecting donor blood packs with g6pdd. summary/conclusions: the prevalence of g6pdd among ugandan blood donors was similar to that in the general population. the heterozygous genotype resulted in lower haemoglobin concentration of the blood units. the use of carestarttm rdt for screening of stored blood units was not as efficient in this study hence further testing for the determination of g6pdd needs to be done on fresh samples from donors. transfusion medicine, jaypee hospital, noida, india background: during last two decade there has been a continuous remarkable improvement in desensitization therapy in high risk hla sensitized kidney recipients. in india there has been a tremendous increase in the number of kidney transplantations in patients having anti-hla antibodies (hla sensitized) with excellent success rate. aims: in present study, we are describing successful role of desensitization in 39 hla sensitized patients having preformed donor-specific hla antibody (dsa). methods: all patients were preconditioned with combined modality of a standard dose of rituximab, therapeutic plasma exchange (tpe) and low dose ivig. tpe was started using com. tec (fresenius kabi, germany) after 21 days of administration of rituximab. complement dependent cytotoxicity cross match (cdc-xm), luminex cross match with donor lysates (lm-xm, immucor inc., ga, usa) and flow cytometry cross match (fc-xm, bd facs canto ii).) was done in all cases. if any of the three tests was positive, single antigen bead assay (sab) was performed. desensitization therapy was given in all cases where dsa was detected. pretransplant tpe procedures were done until dsa (mfi < 1000) and cdc-xm became negative. cdcxm labeled positive at ≥ 10%. t-cell fcmx was considered positive above 42 mfi and b-cell fcmx was considered positive above 120 mfi. lmxm was considered positive above 500 mfi. sab was performed using lifecodes single antigen (lsa) class i and class ii kits (immucor, usa). if the specificity of anti-hla antibodies was against donor hla antigen(s) it was called as donor specific antibody (dsa). results: present study demonstrated the diagnostic and clinical superiority of adding fc-xm and lm-xm in pretransplant compatibility testing algorithm over cdc-xm. cdc-xm alone was not able to detect anti-hla antibody in 8 patients (20.5%). among the three pretransplant compatibility tests, fcxm demonstrated highest sensitivity. among the 39 cases initially screened 35 showed dsa positivity in sab. desensitization was done in those 35 cases only. in our study, sab was positive for class ii alone in 13(37%) while in remaining 22 (63%) cases it was positive for both class 1 and class ii. the number of pre transplant tpe procedures required was 6.7 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) . the mean number of post-transplant tpe sessions required was 0.7 (range, 1-6). during pretransplant and post transplant tpe procedures, five (14.3%) patients presented with allergic or hypotensive reactions which were managed conservatively. most of the patients were discharged after seven days of hospital stay whereas patients who required post-transplant tpe were discharged after a relatively longer hospital stay (mean-8.5, median-7 days). after three months, protocol biopsy was done in those cases only where post transplant tpe was required. protocol biopsy showed normal findings. in present study, the mean duration of follow up was approx 17 months with the longest duration of follow up of 36 months. summary/conclusions: in a country like india where there is a huge gap in the demand and supply of kidney and a large no. of patients waiting for a suitable organ, transplant across hla barrier could a good doable option. thorough pretransplant compatibility and tpe are essential tools to make these transplants program successful background: most transfusion-dependent chronically anemic patients are managed by simple red cell transfusions. however, some patients are not able to tolerate the additional volume associated with simple transfusions and are at a high risk of developing transfusion associated circulatory overload, if transfused with multiple red cell units. plasma-to red cell exchange (prx) is a modified procedure wherein an apheresis machine is used to remove patient's plasma, while simultaneously replacing with donor rbcs. this procedure allows for a rapid euvolemic transfusion of rbcs to patients that are severely anemic and intolerant to excess fluid volume. others as well as our group have previously described this procedure. we now summarize our institutions nearly seven years of experience performing this procedure on a routine basis. aims: to document patient experience with prx. methods: we performed a retrospective chart review of all patients that underwent prx at our institution in the last seven years. our protocol for prx has evolved during this period. currently, we perform the procedures using spectra optia (terumo bct, lakewood, co) machine using the plasma-exchange program and tubing set. if the patient's pre-procedure hematocrit (hct) is < 22%, we custom prime the tubing set with 5% albumin. the number of red cell units transfused to the patient depends on their pre-procedure hematocrit and is individualized to the patients. results: we have treated four patients with prx procedure. patient #1 is a 56-year-old transfusion-dependent male with beta-thalassemia major. the patient had experienced multiple congestive heart failure exacerbations secondary to simple transfusions and we consequently performed prx procedure, every 4 weeks, starting in 2012. the patient has completed 84 procedures with 3-4 units of washed red cells transfused to achieve a target hct goal of 45 to 57%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #2 was a 25-year-old female who had symptomatic anemia secondary to sickle cell disease (hb ss complicated by end-stage renal disease (esrd). she had progressively become intolerant to simple transfusions, including an episode of severe dyspnea, which required intubation. she underwent 9 prx procedures with 2-3 units of washed red cells. patient tolerated the procedures without any significant complications. however, during a different surgical procedure, she experienced cardiac arrest and subsequently passed away. patient #3 is a 33-year-old transfusion-dependent male with severe anemia secondary to sickle cell anemia (hb ss). he was intolerant to excess fluid because of esrd and congestive heart failure. he has undergone 38 prx procedures with 3-5 red cell units transfused to achieve a hct goal of 30%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #4 is a 48-year-old male with a sickle cell disorder (hb ss) complicated by esrd, heart failure and chronic hypoxemic respiratory failure. the patient has undergone two prx procedures with 4-5 red cell units. other than an episode of non-bloody emesis that was symptomatically treated, he tolerated both procedures. he continues to be managed on this regimen. however, the patient remains noncompliant with treatment. summary/conclusions: prx is a safe and efficient method to transfuse multiple red cell units to volume-intolerant anemic patients. background: transplanted organ failure due to antibody mediated rejection in abo-compatible organs is a serious complication with a bad prognosis. the goal treatment in these cases encompasses the following strategies: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. the 2016 american society for apheresis has assigned a category i to the use of therapeutic plasma exchange for the treatment of abo-compatible antibody mediated rejection in kidney, but a category iii to all other abo compatible organs: liver, lung, and heart. at our institution, a standardized approach for the use of therapeutic plasma exchange as a supportive intervention for abo-compatible immune mediated rejection, regardless of the organ type, has been in place since 2011. aims: a retrospective review was performed to evaluate our patient outcomes using therapeutic plasma exchange for the treatment of antibody mediated allograft rejection in abo-compatible solid organ transplantation. methods: patients used for the retrospective review were selected from an existing therapeutic apheresis list. the therapeutic plasma exchange protocol consists of: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. it is performed as follows: one plasma volume exchange is performed on days 1, 2, 3, 5, 7, 9 along with one or more of the above strategies followed by an ivig infusion. cases with allograft rejection in which plasmapheresis was not used were excluded. and t devos 7 aims: this study aimed to explore the possible causes of the decreased transfusion rate for all adult cardiac surgery patients. methods: data were collected from adult cardiac surgery patients during the mentioned time frame and were extracted from electronic patient files and a database of the department of cardiac surgery. a set of 63 variables was defined as possible confounders by a panel of experts. after discussion, 9 global variables (age, gender, duration of surgery, use of cpb (cardio-pulmonary bypass), american society of anesthesiologists (asa) risk score, type of surgery, urgency, attending cardiac surgeon and attending anesthesiologist) and 7 cpb-related variables (administration of cardioplegia yes/no (cpg), duration of cpb, circulatory arrest, hypothermia, duration of aortic cross-clamp, baseline hemoglobin and cpb-priming volume) were retained. negative binomial models for counts were used for data analysis. all analyses were performed with spss. results: 1018 patients were extracted from databases and further analyzed. the mean age of this group was 65,9 years (sd +/-14,1 years) and 67.3% of them were male. the mean duration of surgery was 256 min (sd +/-88,3 min). the decrease of perioperative rbc transfusion rate over four years was statistically significant (p < 0.001). in 2011, the mean use was 2,34 units per operation (sd +/-2,277), which changed to 1,38 units (sd +/-2,158) in 2014. three variables (urgency, attending cardiac surgeon, attending anesthesiologist) changed significantly over 4 years and were used in a multivariable model as confounders together with rbc transfusions and year. even after adjustment for these factors, the decrease in rbc transfusion rate was still statistically significant (p < 0.001). in the specific group of patients undergoing cardiac surgery with cpb (n = 640), the use of rbc was also significantly reduced (p < 0.001). in 2011, the mean use was 3,06 units per operation (sd +/-0,448) and this changed to 1,94 units (sd +/-0,167) in 2014. after correction for the 3 cpb variables that notably changed over the 4 years (cpg, priming volume and hypothermia) and the three previously defined confounders (urgency, attending cardiac surgeon and attending anesthesiologist) the reduction of rbc transfusions over 4 years still remained statistically significant (p < 0.001). summary/conclusions: our study shows evidence for a decreased rbc transfusion rate in adult patients undergoing cardiac surgery between 2011 and 2014. this tendency was also seen in the subgroup of patients undergoing surgery with cpb. possible explanations of the decrease are implementation of various established parts of patient blood management. however, a unique reason could not be identified in this study. background: growing worldwide demand for immunoglobulin products such as intravenous immunoglobulin (ivig) and subcutaneous immunoglobulin (scig) is driving plasma collection. patients with primary immunodeficiency (pid) or secondary immunodeficiency due to haematological malignancy or its treatment (sid) rely on these products to maintain therapeutic serum igg levels to minimise recurrent infection. efficacy of immunoglobulin replacement therapy (irt) in pid is well established but information on sid is limited. the different aetiologies of hypogammaglobulinaemia between pid and sid raised the question of whether sid patients on irt experience similar clinical and quality of life (qol) benefits as reported in pid patients. aims: to assess whether sid patients experience similar clinical and qol benefits while on irt as pid patients. methods: following ethics approval, data on dosage, serum igg trough levels and infection (bacterial, viral and fungal requiring treatment such as antibiotics) was collected from 14 adult pid and 13 adult sid patients from medical records and pathology reports, for their last 12 months of ivig and their first 12 months of scig. the starting and maintenance dose was 0.4 g/kg/month for ivig, transitioning immediately to 0.1 g/kg/week for scig without a washout period. a study specific questionnaire was developed to gather data on patient perceived side effects, treatment satisfaction and impact of irt on social/family life, work/study and their overall qol. paired t-test was used for parametric data and the wilcoxon signed-rank test for non-parametric data. results: sid patients were significantly older with a mean age of 62.9 years versus 43.4 years in pid patients (p = 0.007). a mean of three training session was required to reach competency in scig administration in both cohorts. there was a trend of reduced side effects on scig for pid and sid patients compared to ivig, with a significant reduction of headaches in the pid cohort (p = 0.041). the majority of patients experienced infusion site reactions, which were predominantly perceived as manageable. 55% of infections were respiratory tract infections. pid patients had slightly higher mean serum igg trough levels with scig (9.3 g/l) compared to ivig (8.4 g/l), and fewer infections on scig than ivig (mean annual infection rate of 1.64 vs 2.14 respectively). sid patients had higher mean serum igg trough levels on scig (8.4 g/l) than ivig (7.1 g/l) (p = 0.009) but experienced more infections while on scig versus ivig (mean annual infection rate of 2.15 vs 1.62 respectively). the number of hospitalisation due to infection decreased in both cohorts with scig. pid patients perceived that switching from ivig to scig improved their health and qol. in contrast sid patients perceived no improvements in health and qol. summary/conclusions: data from this pilot study suggests that the clinical and qol impact of irt in sid patients is different to that of pid patients. to support evidence based irt management and effective use of this limited and expensive blood product in sid, larger studies which account for different stages of malignancy and associated treatment regimes are required. background: there is an increasing platelet transfusion for treatment and prophylaxis of bleeding in patients with hematologic disorders and malignancies. because of limited resources, leukoreduced platelet concentrates is not yet implemented in most indonesian hospitals. in vitro platelet activation may cause morphology, functional, and ultrastructure changes. those changes will reduce the platelet viability, in vivo functions, and clinical efficacy. high platelet cd62p expression is the cause of faster platelet destruction in the reticuloendothelial systems. post-transfusion in vivo hemostatic efficacy can be determined by the measurement of corrected count increment (cci), recovery, and platelet cd62p expression. aims: to analyze the increase of platelet cd62p expression in patients of non-leukodepleted compared to pre-storage leukodepleted pc transfusion.background: haemorrhage is a leading cause of preventable death not only in the military trauma care, but also for civilian population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. resuscitation using blood products and limited infusion of normal saline improves survival for critically bleeding patients. nowadays there are hems programs (helicopter emergency medical system) carrying blood products around the world. the hems in castilla-la mancha, with physician and nurse, is the first out-of-hospital emergency service in spain that provides packed red blood cells (prbc) transfusion where the accidents happen without delaying the transport to the proper hospital for definitive treatment. this program has been developed between the blood center of ciudad real and the hems team ('gigante 2', emergency service castilla-la mancha). the goal of the designed protocol was to preserve the properties of the product to be transfused in out-of-hospital environment by hems teams. aims: to describe the process for out-of-hospital prbc transfusion in hems of ciudad-real. the protocol for out-of-hospital blood transfusion was developed according to criteria of medical indications and security, monitoring, and tracking of the product. methods: data for the observational retrospective study were collected from june 2014 to august 2018. the medical helicopter (ec 145t2) was provided with two prbc o rh(d) negative. the shock index was selected for the indication for transfusion according to the literature revised and as a simple rate to obtain out-of-hospital data. to achieve the feasibility and preservation of the prbc a prospective monitoring of volume was established, haematocrit, haemoglobin, leucocytes, coulter, hemolysis and microbiological culture. blood center established two groups: the case group for the prbc kept in the hems base and helicopter and the control group for the units remaining in the blood center with standardized blood conservation. for both groups, control and comparison of immediately obtained hematologic analyses, and 35 days after collection, were performed. the statistical analysis used spss 23.0 version (significance p < 0,05). results: 138 prbc samples were evaluated, 48,5% (67) from case group and 51,5% (71) from control group. analyses were tested day 1 and day 35 after collection. haemolysis was not observed. all cultures were negative. although significant differences were found between the parameter in the value of before-after in the value of the hematocrit, leukocytes and coulter, there are no differences between the prbc that flew and those conserved in the transfusion-service. all results comply with current legislation and blood transfusion standards. there have been administered 35 prbc transfusion to 28 patients during out-of-hospital advanced medical assistance. no post-transfusion reactions have been registered. prbc units have a 35-day rotation to allow the use of the units in the hospital after achieving their optimal status. summary/conclusions: the out-of-hospital transfusion protocol designed to transport blood (prbc) in the helicopter for hems has demonstrated to keep the standard conditions and properties of the product to be considered useful in the treatment for critically bleeding patients in the out-of-hospital. background: early and adequate treatment of major bleeding is important for survival and a good outcome. blood and plasma are given increasingly early including before hospital admission in trauma based on successes reported from combat experience. in 2010 the national patient safety agency issued a rapid response report requiring national health service hospitals in england to take actions to improve provision of blood in an emergency including provision of major haemorrhage protocols (mhp) and drills. the national reporting and learning scheme had identified reports of 11 deaths and 83 instances of harm due to delay over a 4-year period. aims: the aim of the study was to monitor the acid-base status of the patient by means of abg and to administer the blood component therapy based on teg results. methods: this study was a prospective observational study of 18 adult patients over a period of 7 months. serial monitoring of the abg in the intra-operative period was done. teg guided resuscitation was performed in all 18 cases. results: the abg analysis of all 18 patients showed decrease in the ph, increase in pco 2 , decrease in serum bicarbonate level and elevation in negative base excess. these components of metabolic acidosis can be attributed to massive transfusion. increased lactate, an independent parameter, which reflects poor tissue perfusion or shock and predicts need for massive transfusion was observed in all patients. all the 18 cases showed a decrease in ionized calcium levels which could be a result of citrate related toxicity. increased glucose was observed in all patients which may be due to increase in the catecholamine release as a response to haemorrhagic shock. electrolyte correction was given depending on results of the abg analysis wherever appropriate. two out of 18 cases showed an increase in r time indicating deficient coagulation factors, which was corrected with fresh frozen plasma (ffp). three cases showed elevation in k time indicating deficient fibrinogen levels, which was corrected by ffp. fresh frozen plasma was also given in 4 cases, which showed decrease in the alpha angle, indicating deficient fibrinogen, and cryoprecipitate was given in 2 cases. platelets were transfused in 5 patients showing a decrease in the maximum amplitude (ma), which indicates deficient platelets. summary/conclusions: teg as poc testing is an important tool in appropriate blood component therapy in massive transfusions. serial monitoring of abg helps in monitoring acid-base status of the patient and therefore is a guide in the correcting electrolyte level in patients undergoing massive transfusion. background: massive blood loss is encountered in various situations like trauma, major surgeries, gastrointestinal bleeds and obstetric haemorrhages. haemorrhage is an important cause of mortality and morbidity in massively bleeding patients. early recognition of haemorrhage and intervention is essential for survival. massive transfusion (mt) of blood is required to replenish blood losses and is a lifesaving treatment in these patients. a variety of haemostasis and pathophysiological changes occur during massive haemorrhage and massive transfusion. all of these changes contribute to the vicious cycle of progressive coagulopathy due to the 'lethal triad' of refractory coagulopathy, progressive hypothermia and persistent metabolic acidosis. aims: the aims of the study included understanding management of cases of massive blood transfusion in surgical patients, impact of mt of blood components on patient outcome, evaluating post-operative complications of massive transfusion and the development of institutional massive transfusion protocol (mtp).methods: this prospective observational study commenced after institutional ethics committee (iec) approval. it comprised of 40 adult surgical oncology patients who received massive transfusions and was conducted for a period of 7 months. every case of a massive transfusion was studied under the following headings (1) patient's details (2) patients base-line laboratory tests (3) resuscitation with transfusion (4) intra-operative laboratory tests (5) thromboelastography (teg) (6) post-operative complications (7) duration of stay in the hospital (8) 30 day mortality rate. results: complete blood count, serum electrolytes, arterial blood gases, coagulation profile and teg were used to monitor transfusion therapy in the intraoperative period. intraoperative laboratory parameters of patients showed dilutional coagulopathy, metabolic acidosis, hypocalcaemia, hypomagnesaemia, hyperkalaemia and hypokalaemia, increased lactates and glucose. electrolyte correction was done based on the derangement. the derangements were on a decreasing trend in the postoperative period and returned to baseline level by 2 nd or 3 rd post-operative day with no requirement of correction in the post-operative period. the post-operative outcomes were evaluated in terms of the surgical site infection (ssi) as per the centers for disease control (cdc) criteria, surgical complications as per modified clavien-dindo classification and respiratory complications. a total of 13 (32.5%) patients had ssi, 14 (35%) had surgical complications and 22 (55%) patients had respiratory complications. the length of the stay in the hospital was longer for patients who had postoperative complications. despite complications, owing to excellent peri-operative care, 38 (95%) patients were discharged alive. summary/conclusions: surgeries associated with massive transfusion are at an increased risk of ssi as well as morbidity and mortality. complications associated with rapid transfusions of blood, acute haemorrhage and associated risk of the surgery lead to a prolonged icu stay and increased length of stay in the hospital. a well-developed massive transfusion protocol optimizing the ratio and dose of the blood component therapy results in excellent patient outcome with minimal postoperative morbidity and mortality. background: despite the introduction of new oral anticoagulants (dabigatran, rivaroxaban, apixaban), vitamin k antagonists (vka), such as warfarin and acenocoumarol are still the most widely used oral anticoagulants for the treatment of non-valvular atrial fibrillation (nvaf). the use of vka must be regularly and often laboratory controlled in order to ensure the adequacy of therapy and to avoid subdosing or drug overdose. the most commonly used test for the control of oral anticoagulant therapy is the prothrombin time (pt), expressed in inr system, which provides an internationally standardized monitoring of the treatment. time in therapeutic range (ttr) represents a measure of the quality of the anticoagulant effect of vka and estimates a percentage of time a patient's inr is within the desired therapeutic. aims: the aim of this study was to evaluate of the effectiveness of vka therapy in patients with nvaf and to identify factors affecting the anticoagulation efficacy. methods: a retrospective study was conducted on a population of 725 outpatients with nvaf, treated with vka and followed in blood transfusion institute of ni s from january to december 2017. laboratory control of inr was done from capillary blood of patients on thrombotrack solo (axis shield, norway) and thrombostat (behnk elektronik, germany). targeted 15 ae 17.52%, but 49.72% of patients had a ttr less than 60%. patients were at high risk of thrombosis in 6.15% of time (inr < 1.5) and high risk of bleeding in 2.2% of time (inr > 4.5). the most significant independent factors affecting the quality of vka therapy are gender, arterial hypertension, diabetes mellitus and the use of amiodarone and antiplatelet drugs (aspirin, clopidogrel). summary/conclusions: the ttr is undoubtedly useful indicator of the effectiveness of vka treatment. the most important predictors of poorer efficacy of vka therapy are arterial hypertension, diabetes mellitus, patients' gender and the use of amiodarone and antiplatelet (aspirin, clopidogrel) drugs. to improve the quality of vka therapy, education of patient and better collaboration with them, as well as a successful teamwork with clinicians are also imperative. background: an estimated 1.9 million deaths per year result from haemorrhagic blood loss. at a cellular level, haemorrhagic shock develops when oxygen delivery is insufficient to meet oxygen requirements to maintain aerobic metabolism. successful resuscitation prevents further oxygen debt and repays the prior oxygen debt. this includes the administration of fluids and blood components (e.g. plasma, red cells and platelets). measurement of oxygen delivery and utilisation at a tissue level requires invasive monitoring not possible clinically, meaning that surrogate markers such as lactate and venous oxygen saturation (svo 2 ) are used instead. new technologies such as incident dark field imaging and near-infrared spectroscopy may offer a non-invasive alternative; however their utility in haemorrhagic shock remains background: transfusion-induced red cell alloimmunization is still a major challenge in transfusion practice. besides logistic problems for the transfusion laboratory, it may compromise available blood supply, and when undetected and unanticipated, it may risk haemolytic transfusion reactions. knowledge about risk factors can help to optimize preventive matching strategies. severe renal failure and subsequent renal replacement therapy influence the immune system and could therefore modulate the risk of alloantibody formation against foreign red cell antigens subsequent to transfusion. aims: this study aims to quantify the association between renal failure, according to its degree and its treatment with renal replacement modalities, and transfusioninduced red cell alloantibody formation. methods: we performed a multicenter case-control study within a source population of patients receiving their first and subsequent red cell transfusion between 2005 and 2015 in the netherlands (risk factors for alloimmunization after red cell transfusion, r-fact study). using a conditional multivariate logistic regression, we compared first-time transfusion-induced red cell alloantibody formers (n = 505) with two similarly exposed non-alloimmunized control recipients (n = 1010) during a five-week alloimmunization risk period. degree of renal function was categorized as: 'no renal failure' i.e. glomerular filtration rate (gfr) > 30 ml/min/1.73 m 2 , 'moderate renal failure' i.e. gfr ≥ 10-30 ml/min/1.73 m 2 during a continuous period of minimally seven days, 'severe renal failure' i.e. gfr < 10 ml/min/1.73 m 2 and/or use of any type of renal replacement therapy during at least one day of the alloimmunization risk period. odds ratios were interpreted and presented as relative risks (rr). adjusted rrs were conditioned on the matching variables and identified confounders. results: no renal failure was observed among 441 (87.3%) cases versus 838 (83.0%) controls; moderate renal failure among 24 (4.8%) cases versus 54 (5.3%) controls; and severe renal failure among 40 (7.9%) cases versus 108 (11.7%) controls. among the latter, 30 cases and 97 controls underwent renal replacement therapy. moderate renal failure and severe renal failure without renal replacement therapy were not significantly associated with red cell alloimmunization (adjusted rr 0.82, 95% ci 0.67-1.01 and adjusted rr 0.81, 95% ci 0.58-1.11, respectively). however, patients undergoing renal replacement therapy had a two-fold lower alloimmunization risk (adjusted rr 0.48, 95% ci 0.39-0.59) as compared to transfusion recipients without renal failure, unrelated to type and duration of renal replacement therapy. summary/conclusions: these findings suggest that patients undergoing renal replacement therapy have strongly diminished red cell alloimmunization risks. further research should confirm these results and elucidate the underlying pathophysiological protective mechanism. background: the ability of allogeneic hematopoietic stem cell transplantation(allo-hsct) to prevent relapse depends partly on donor natural killer (nk) cell alloreactivity. nk effector function depends on specific killer-cell immunoglobulin-like receptors (kir) and hla interactions. thus, it is important to identify optimal combinations of kir-hla genotypes in donors and recipients that could improve hematopoietic transplantation outcome. aims: to obtain the optimal combinations of inhibitory kir and its ligand between donor and recipient which is helpful for the guidance of selecting donors and recipients in hsct. methods: the pcr-sbt method was used for kir2dl1, kir2dl2/kir2dl3, kir3dl1, kir3dl2 and hla-a, -b, -c, -drb1, -dqb1 genotyping. 58 pairs of hla 10/10 identical donor/recipients matching samples were retrospectively analyzed. three different models of kir were established. there were kir-kir gene model, kirligand model and haploid model. in kir-ligand model, patients were divided into three groups: c1/c1 homozygote group (48 cases), c1/c2 heterozygote group (8 cases) and c2/c2 homozygote group (2 cases). according to the expression of 3dl1, 53 cases were 3dl1 positive and 5 cases were 3dl1 negative. there were 5 cases of bw4/bw4, 24 cases of bw4/bw6 and 24 cases of bw6/bw6 in the 3dl1 positive samples. according to the expression of a3/a11, they were divided into three groups: a3/a11 negative group (28 cases), a3/a11 heterozygous group (28 cases) and a11/ a11 homozygote (2 cases). according to kir genotyping, kir haploidentical group (38 cases) and kir haploid mismatched group (20 cases) were divided. the clinical data on neutrophil and platelet remodeling time, gvhd and os of 58 cases were statistically analyzed by the mann-whitney test or the kruskal-wallis test using graph-pad software v5.01. results: there was no significant difference in the time of neutrophil and platelet remodeling, the incidence of agvhd and the survival time after transplantation in the kir genotype model. in haplotype model, there was no significant difference in neutrophil and platelet remodeling time and survival time after transplantation. the incidence of agvhd was low when the kir haploid mismatched and kir3dl1 was positive. it was conducive to neutrophil and platelet remodeling when bw4/bw6 and a3/a11 was heterozygosity. summary/conclusions: it is important to establish the three different models of kir genotypes, haplotypes and receptor-ligand mismatches for analyzing the effect on the prognosis of allo-hsct. kir-ligand model plays an important role in hla unre-background: transfusion of platelet concentrates (pcs) has been associated with adverse outcomes including transfusion-related acute lung injury (trali). the underlying mechanism of trali has been related to the accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in pcs. current room temperature storage limits the shelf-life of conventional pcs to 5-7 days. alternative storage conditions, including cryopreservation, offers extended storage and a solution for blood banking logistics. cryopreservation of human pcs has been associated with higher concentrations of immunomodulatory mediators compared to room temperature stored pcs and it has been suggested that cryopreserved pcs may be immunomodulatory. to investigate the effects of cryopreserved pcs, a transfusion sheep model would be a beneficial approach. aims: to characterize immunomodulatory mediators in supernatants of sheep conventional and cryopreserved pc and to investigate whether storage duration and cryopreservation impacts the accumulation of these mediators. methods: buffy coat pooled sheep pcs (n = 3), prepared in 30% plasma/70% ssp+ with minor modifications to standard human procedures, were stored room temperature (rt) for 7 days (d) and sampled on d2, d5 and d7. cryopreserved sheep pcs (n = 3), prepared by the addition of 5-6% dimethyl sulfoxide, were stored at à80°c and sampled pre-freeze and post-thaw. supernatant was prepared at each time point with double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), anti-inflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferongamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of non-polar lipid mediators, such as arachidonic acid (aa), 12-hete and 15-hete were assessed in the stored sheep pc-and cryo-pc supernatant using commercial elisa. results shown as mean ae standard deviation. the effect of storage was determined at p < 0.05 using paired t-test. results: in rt stored sheep pc supernatant il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete were detected at d2, d5 and d7. storage duration significantly increased accumulation of ip-10 at d5 (253.4 ae 266.7 pg/ml compared to 569.7 ae 272.7 pg/ml, p = 0.008) and further increased at d7, and il-8 at d7 (3477 ae 937.4 pg/ml compared to 5092 ae 521.1 pg/ml, p = 0.0460). cryopreserved sheep pc supernatant pre-freeze and post-thaw contained equivalent or higher concentrations of il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete than rt stored d5 pcs. however, cryopreservation did not impact levels of any of the platelet derived mediators. summary/conclusions: several platelet-derived cytokines/chemokines, including high concentration of il-8 with neutrophil priming activity, and non-polar lipids were found in stored sheep pc supernatant. these immunomodulatory mediators may contribute to adverse outcomes associated with pc transfusion. storage at rt, but not cryopreservation was associated with increased accumulation of immunomodulatory mediators in sheep pcs. most importantly, similar to human pcs, sheep cryopreserved pcs contained at least if not higher concentrations of majority of cytokines as pcs stored at rt, therefore making sheep a suitable model in which to investigate immunomodulatory effects of cryopreserved pc transfusion. background: transfusion, despite being a lifesaving therapy, has been associated with adverse transfusion outcomes. transfusion related acute lung injury (trali) remains one of the leading causes of transfusion-related mortality. accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in blood products, including packed red blood cells (prbcs), have been implicated with the development of non-antibody mediated trali. however, how specific mediators contribute to the underlying mechanism is yet to be defined. during routine storage of human prbcs fewer than 10 cytokines/chemokines and several biologically active lipids have been identified. a sheep model of trali has successfully been developed using human prbc supernatant, however transfusing sheep prbc has not been investigated. to support the use of sheep prbc in the trali model and to better understand the precise mechanism, characterization of the potential mediators in sheep prbc is required. aims: to characterize immunomodulatory mediators in sheep prbc supernatants and to investigate whether storage duration impacts the accumulation of these mediators. methods: sheep prbcs (n = 5), prepared according to standard human procedures with minor modifications, were stored (2-6°c, 42 days (d) ) and sampled at d2 and d42. supernatant was prepared by double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), antiinflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferon-gamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of potential non-polar lipid mediators (arachidonic acid (aa), 12-hydroxyeicosatetraenoic acid (hete) and 15-hete) were assessed in the sheep prbc supernatant using commercial elisa. paired t-test was used to compare fresh and stored prbc supernatant (p < 0.05). results are mean ae standard deviation. results: at day 2, aa (75,142 ae 47,205 pg/ml), 12-hete (849.1 ae 235.6 pg/ml), 15-hete (87.6 ae 32.7 pg/ml) and il-1b (144.7 ae 198.9 pg/ml) were detectable in sheep prbcs supernatant. at day 42, storage duration significantly increased concentrations of aa (136,254 ae 60,433 pg/ml, p = 0.0425) and 15-hete (380.9 ae 116.3 pg/ml, p = 0.0022) in sheep prbcs supernatant. summary/conclusions: similar to reported findings of human prbcs, the predominant type of immunomodulatory mediators present in sheep prbcs were non-polar lipids. the concentration of these non-polar lipids increased during storage. these immunomodulatory mediators may contribute greatly to adverse outcomes associated with prbc transfusions. further investigation is required to determine whether stored sheep prbcs supernatant induce immunomodulation in sheep in vitro and in vivo transfusion models. background: dshtr incidence is reported as 1 in 2,500 transfusions, presenting days to months after the transfusion. the published data addressing the correlation between the strength of the antibodies detected after a dshtr has taken place and the corresponding clinical symptoms as measured by laboratory parameters that assess the presence of hemolysis is limited. aims: the aim of this study is to evaluate the correlation between the results of the dat, automated and manual antibody reactivity strength with the corresponding clinical parameters of hemoglobin, lactate dehydrogenase (ldh), bilirubin, and haptoglobin. methods: a dshtr is defined as discovering a new antibody within 28 days of a transfusion. for all positive antibody screens, a work-up is initiated consisting of identification panels, dats, antigen typing of the red cells transfused, and eluates at the discretion of the transfusion medicine physician. additional laboratory testing for hemolysis is requested when indicated. a retrospective review was conducted of patients who were identified as having a dshtr. levels of hemoglobin, ldh, and transfusion safety background: rhd immunoglobulin (rhdig) has been available for 50 years in australia. since its introduction for routine antenatal and postpartum prophylaxis, alloimmunisation has decreased from 16% to 0.2%, reducing the number of australian deaths from haemolytic disease of the newborn over a hundred-fold, to approximately 0.01 deaths per 1000. blood matters serious transfusion incident reporting (stir) system has been collecting transfusion incidents and adverse events across four australian jurisdictions since 2007. since january 2015, rhdig administration errors have been reported. aims: to understand incidents relating to the administration of rhdig and increase safety and awareness of risks. methods: health services registered with stir (n = 93) were notified of the inclusion of reporting rhdig incidents. when an incident is identified, the reporter sends an online notification to stir, prompting the appropriate investigation form to be sent for completion. the completed incident data are reviewed and validated by an expert group. data is de-identified and collated for reporting. results: during the period january 2015-december 2018, 45 reports were received; 43 reports were validated, with 2 reports excluded (reactions rather than administration errors). reports were categorised as below: background: following the nice transfusion guidelines, recommending offering iron before and after surgery to patients with iron-deficiency anaemia (ida), we worked collaboratively with the anaesthetic and pre-operative team to implement a clear and robust anaemia pathway for pre-operative haemoglobin (hb) optimisation. oral iron was started, where appropriate, and our anaemia pro-forma was sent for review in a virtual clinic to assess eligibility for intravenous iron. we performed a retrospective evaluation of the patients who received iv iron during the anaemia pathway. aims: the aim of this retrospective evaluation was to look at the cohort of patients who had received iv iron in 2017 and assess the effect of iv iron on haemoglobin levels for different defined groups. methods: we classified patients, as described in munting and klein, 2019, depending on their iron parameters as having either: -idaserum ferritin < 30 mg/l -chronic inflammation with idaserum ferritin 30-100 mg/l with transferrin % of < 20%/crp > 5 mg/l -anaemia of chronic inflammationserum ferritin > 100 with transferrin % of < 20%/crp > 5 mg/l patients were considered eligible for iv iron if the following criteria were met:1. an inadequate response to oral iron, or were unable to tolerate oral iron or the interval between diagnosis and surgery was short 2. the anaemia pro-forma was completed 3. hb was ≤ 120 g/l 4. they were classified as either having ida or chronic inflammation with ida or anaemia of chronic inflammation hb was measured prior and on average, 20 days following the iv iron infusion. we excluded patients who had their post iv iron follow up blood tests done after surgery. results: this retrospective evaluation included 80 patients. 48 patients were classified as having ida and 32 patients classified as having chronic inflammation with ida. those classified with ida had a mean hb of 96 g/l (55-120), a mean mcv of 77.4 fl and a mean serum ferritin of 16 lg/l. those with chronic inflammation with ida had a mean hb of 106 g/l (77-120), a mean mcv of 87.9 and a mean serum ferritin of 57 lg/l. follow-up hb was measured on average twenty days post iv iron infusion in both groups. the average hb post iv iron infusion in the ida group was 119 g/l (100-149) with an average increment of 23 g/l and in the group with chronic inflammation with ida the average post iv iron hb was 117 g/l (85-129) with an average increment of 11 g/l. summary/conclusions: in conclusion the group with ida had, on average, a lower starting hb that the group with chronic inflammation with ida and the average increment in hb 20 days post iv iron infusion was greater in the group with ida. however, the group with chronic inflammation with ida cases also responded to iv iron and therefore we strongly consider the use of iv iron in both groups. further studies to evaluate the ongoing effect of iv iron would help assess whether the same level of increment seen with ida can also been seen for the group with chronic inflammation with ida over a longer period and how long the increment was sustained. background: the expansion of personalized genomic medicine has led to the development of targeted therapeutic approaches for patients. one example is sipuleucel-t, an autologous cellular immunotherapy product used to treat prostate cancer manufactured from the patient's white blood cells. this study describes our experience with incorporating autologous cellular immunotherapy products into the workflow of the blood bank. the policies supporting the workflow are outlined and compliance with them is assessed. aims: this study aims to evaluate the process and method used to dispense and track the infusion of sipuleucel-t. methods: this is a retrospective analysis of the dispensation and administration of sipuleucel-t from january 2012-august 2018, which was handled exclusively by the blood bank. standard operating procedures and hospital policies were reviewed and compliance with these policies evaluated. included were patients who had the sipuleucel-t product dispensed and administered. information collected included the total number of products dispensed, patient age, adverse reactions/incidents, premedication, and patient outcome. descriptive statistics were used for data analysis. results: there were 154 products dispensed to 53 patients. the recipients were male patients diagnosed with prostate cancer with a mean age of 74 years. there were 3 doses (a complete course) administered to 50/53 (94%) recipients and a partial course (1-2 doses) administered to 3/50 (6%) recipients, for a total of 154 products. the blood bank workflow treated sipuleucel-t as a derivative in the computer system, listing the manufacturer (dendreon corporation) as the supplier. health care providers were instructed to follow the nursing policy for the administration of blood products and derivatives for the infusion of sipuleucel-t. this policy required documentation of the infusion in a transfusion nursing note and reporting adverse events to the blood bank as transfusion reactions. there were no adverse events reported to the blood bank, yet there were 5 adverse events described in provider notes; 2 of them necessitating transfer to the emergency department, and 1 requiring hospital admission. of the 154 infusions, 6 infusions were documented in a chemotherapy note rather than a transfusion note (4%), and 59 (38%) were documented as both a transfusion and a chemotherapy administration. there were 6 additional deviations from the blood product administration policy: 2 cases where the consent check was not performed, 1 case where the product was infused with ringer's lactate rather than normal saline, and 3 cases where the 2-person 3-way check erroneously indicated the product was irradiated. summary/conclusions: this study describes one approach to managing cellular therapy products as an extension to existing blood products dispensed by a blood bank. the findings demonstrate noncompliance with hospital policy with this new product as evidenced by failure to report adverse events and failure to follow hospital practices regarding administration. although sipuleucel-t is a product manufactured from an autologous blood product donation, the administration and perceptions of this product may be more similar to a pharmaceutical. as the field of immunotherapies derived from blood product donations continues to expand, these products may necessitate an entirely new approach to ensure proper management. abstract withdrawn. (ref 10310) . while the mnc procedure is fully automated, cmnc requires frequent interface checks to ensure the collection of the correct cell layer. at the rambam health care campus, a tertiary care center, solely the mnc procedure had been employed till 2017, at which point, the cmnc has been introduced for the use in patients with a white blood cell (wbc) count of ≥ 20,000/ll on the collection day. aims: the current study aimed to compare various parameters of peripheral blood stem cell (pbsc) collection, using the cmnc protocol in allogeneic donors and patients undergoing autologous stem cell (autosc) transplantation. additionally, data on autosc collection using mnc (n = 31) and cmnc (n = 88) procedures were compared. methods: data were retrospectively obtained from pbsc collection reports in 134 consecutive cmnc procedures, including 88 autologous and 46 allogeneic donors. the following comparisons were made: cmnc results of allogeneic versus autologous donors, a sub-analysis of cmnc results for autologous donors with a pb cd34 + count ≥20/ll versus allogeneic donors as well as mnc versus cmnc results in autologous donors. the collection efficiency-2 (ce-2) was defined as the total cd34 + amount in the collection bag divided by the amount of cd34 + cells in the pb processed by the collection apparatus. results: in the cmnc, the following parameters significantly differed between autologous and allogeneic donors: mean collection time (333 ae 59 and 289 ae 73 min, respectively; p = 0.001), the total blood volume processed (3.4 ae 0.8 and 2.4 ae 0.8, respectively; p = 0.001) and the final volume in the collection bag (337 ae 77 and 291 ae 84 ml; p = 0.001). the mean ce-2 in autologous versus allogeneic donors was 49 ae 24 and 66 ae 22, respectively (p = 0.003). using cmnc, the collection was effective in 94% of allogeneic and 63% of autologous donors. in autologous donors, a significantly lower collection bag volume (341 ae 84 and 396 ae 132, respectively; p < 0.01) and increased total wbc in the collection bag (263 ae 119 versus 149 ae 36, respectively; p < 0.000) were obtained using cmnc compared to mnc protocol. thirteen patients were treated with plerixafor due to a low pb cd34 + count following g-csf therapy; 7 of them achieved a cd34 count ≥20 and their collection was considered effective. summary/conclusions: the cmnc protocol is highly effective in terms of the cell yield in both allogeneic and autologous donors with a pb cd34 + count ≥ 20/ll. significantly superior collection results are obtained in allogeneic donors versus autologous ones. cmnc provides a significantly higher wbc and a lower final collection volume than mnc. similar total cd34 + cell counts are obtained with both methods. . tbv processed ranged from 1-4.8 tbv with mean of 2.6, average was 2.65 tbv for females and 2.74 tbv for males mean pre-apheresis cd34 + count was 92.89 cells/ll (range 33.14-152.64). mean postapheresis cd34 + count was 1402.3 cells/ll (559.02-2245.5). mean cd34 + cells x10 6 / kg recipients body weight was 7.5 (range: 2.77-12.33). our target yield was ≥3 9 10 6 cd34 + cells/kg body weight of the recipient and in only 2/34 (6%) cases, the yield was <3. 18/34 (52.9%) procedures were lvl and 16/34 (47.1%) were svl. summary/conclusions: most of our pbsc were done for haematological indications (85.3%) and the target dose was 3 9 10 6 cells/ll in single leukapheresis. in 32 cases (94%), target yield was achieved, only 2 cases had <3 but >2 yield. in our study donors <5 years have shown to mobilize better than the older children. hematocrit (hct) and weight showed correlation with cd34 + cell yield but they cannot be taken absolute predictors. wbc count cannot be taken as a predictor for cd34 yield as high wbc count did not convert into high cd34 yield or vice versa. high prepheresis cd34 + count gave higher postpheresis cd34 + count. large volume leukapheresis (lvl), >3 tbv gave higher yield as compared to standard volume leukapheresis (svl). blood volume processed related to prepheresis cd34 + count and/or the weight difference between the donor and recipient. other parameters like hematocrit, wbc count, age etc did not show correlation to the volume processed. in our study, younger age and prepheresis cd34 + count were found as the most relevant predictors for stem cell yield. background: allogeneic hematopoietic stem cell transplantation is an established therapy for many hematologic disorders. since the discoveries of the potential of peripheral blood stem cells (pbsc) in the hematopoietic reconstitution mid 1980s and early 1990s pbsc gradually replaced bone marrow as the preferred source of stem cells. the introduction of hematopoietic cytokines that can mobilize large number of progenitors into circulation accelerated pbsc usage. aims: the aim of our study is to present our 18 year experience with apheresis collecting of pbsc in donors. methods: this is a retrospective study performed in the institute for transfusion medicine of republic of macedonia and university hematology hospital for period background: obtaining unambiguous results of hla typing plays an important role in the transplantation of hematopoietic stem cells. appropriate selection of alleles in the level of hla between recipients and unrelated bone marrow donors reduces the risk of transplant rejection and graft-versus-host disease. new generation technology ensures the highest possible resolution and obtaining unambiguous genotyping results due to the high complexity of the hla system. currently, this is the selection method for obtaining hla test results at the high resolution level. aims: the aim of this study was to determine 11 hla loci (hla-a, -b, -c, drb1/3/ 4/5, dqb1, dpb1, dpa1, dqa1) in potential bone marrow donors from poland. the research included 18,500 potential bone marrow donors registered between 2017 and 2018. a novelty of this paper was that the amplification of all 11 hla loci was performed by using multiplex pcr primers in a single tube. that solution completely eliminated the need to pool amplicons. methods: the typing of the 11 hla loci (hla-a, -b, -c, drb1/3/4/5, dqb1, dpb1, dpa1, dqa1) of potential bone marrow donors was made by using the alltype tm ngs 11-loci amplification kit (one lambda). genomic dna was isolated from peripheral blood of 18,500 donors. hla genotypes were determined according to the manufacturer's protocol on the miseq illumina platform. the obtained sequencing data was evaluated by using the typestream tm visual ngs analysis software. results: the ngs method allowed to obtaining unambiguous results of genotyping of potential bone marrow donors, and also provided the identification of rare alleles, such as: c*12: 143, c*12: 30, c*05: 37, c*07: 151, drb1*11: 28, c*14: 04, b*51: 22, c*15: 13, dqb1*03: 12, drb1*14: 87, drb1*11:69. summary/conclusions: 1. new generation sequencing technology (ngs), which is based on pcr, ensures the highest possible resolution. 2. the ngs method allows to obtain more accurate sequencing results compared to the conventional methods. 3. the research has confirmed the superiority of the ngs method over conventional methods in obtaining unambiguous hla genotyping results at the high resolution level. background: the accurate results of hla typing are significant for ensuring the success rate of hematopoietic stem cell transplantation. currently, hla typing is mainly based on sanger sequencing, which has a high proportion of ambiguous combination results indicating potential errors for hla typing. it is necessary for finding a more accurate typing method to reduce the risk. next-generation sequencing (ngs) method could provide clonal sequencing of single molecules, which has been used for hla genotyping and improved the scope and precision of hla study. aims: to establish a full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology and be evaluated by classical sangersequencing method, which can effectively improve the accuracy of hla typing for donor and recipient in hematopoietic stem cell transplantation. methods: hla-i (hla-a, -b, -c) gene-specific primers were screened, and the amplification parameters were optimized to obtain full-length sequences of hla-i gene under the same condition. the sample library for the amplicon was prepared with transngs tn5 dna library prep kit and the sequencing step was carried out with illumina miseq platform according to the manufacturer' protocol. all the sequencing data in fastq format were analyzed by typestream visual software version 1.2.0(one lambda inc.)with the default setting. 94 cord blood samples were collected for hla typing with the mentioned above next-generation sequencing method in our study. in parallel, all the sample were also tested with the sanger sequencing method according to the previous study in our laboratory. results: 94 samples were successfully tested with two methods and the coincidence rate between two sequencing methods was 100%. with the next-generation sequencing method, the probability of ambiguous results among 94 samples in our study is 1.06%(1/94) for hla-a, 5.31% (5/94) for hla-b and 0% (0/94)for hla-c. however, the probability of ambiguous results with the sanger sequencing method is 96.8% for hla-a, 95.7% for hla-b, 100% for hla-c. summary/conclusions: the full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology was established, which could greatly reduce the probability of ambiguous results and effectively improve the accuracy of existing hla typing techniques. key: cord-010119-t1x9gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober 7‐10, 2017 date: 2017-09-04 journal: transfusion doi: 10.1111/trf.14286 sha: doc_id: 10119 cord_uid: t1x9gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna1 blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks 1, 3, 6, 12 and 24 following index donations from 50 donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna1 samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to 3 months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by 3 months. urine and saliva detection decreased significantly after 2 weeks and was undetectable by 3 months. of donors who were enrolled in the acute pre-seroconversion stage of infection 65% (15/23) developed multiple zikv related symptoms 1 week post index donation, compared to only 30% (7/27) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* 1 , yuelong guo 2 , ritchard g. cable 3 , joseph e. kiss 4 , michael paul busch 5 , grier page 2 , stacy endres-dighe 2 , steve kleinman 6 , simone glynn 7 , alan mast 8 and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) 9 . 1 american red cross, 2 rti international, 3 american red cross blood services, 4 blood systems inc., 5 blood systems research institute, 6 university of british columbia, 7 nih/ nhlbi, 8 blood research institute, 9 nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over 12,600 donors were enrolled from 4 us blood centers for ferritin testing. the study population was enriched for racial minorities [1600 african-american (aa), 1600 asian (as), 1000 hispanic (hisp)] and for "super donors" (1600, who had completed 101 donations in two years without low hemoglobin deferral). the minority donors and the remaining 6800 non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml). results/findings: across all subjects, 19% had ais and 42% had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all 4 groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > 50 years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed %20% decreased risk for ais compared to nhw, while hisp donors had 25% higher risk. daily use of exogenous iron reduced risk for lf and ais by 30 to 40%, respectively, while the estimated benefit from less-than-daily use was lower (5 to 19% protection). regular use of antacids was associated with a 20% or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by %15-20%, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean (6sd) *p < 0.05 compared to batf31/1, 200ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to 25% of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d1 and d14 of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration (62% on d14 vs 100% on d1 of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized (74%) and macrophage-depleted (79%) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than 50% at 2h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at 2h posttransfusion. at 24h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* 1 , amanda l richards 2 and krystalyn e hudson 2 . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod 1 otii 1 mice are predisposed to have autoreactive cd4 1 t cells. study design/method: four cohorts of hodxotii f1 mice (16-48 mice/ cohort) were bled monthly for 15 months to assess for autoab production. peripheral rbcs were stained with anti-complement (c3) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd71 and ter119 to assess for the presence of rbc progenitors. statistical analysis between hod 1 otii 1 autoab 1 vs. hod 1 otii 1 autoabvs. hod -otii 1 was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd4 1 t cells were not deleted in the thymus of hod 1 otii 1 mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod 1 otii 1 . however, as they aged, 15-50% of hod 1 otii 1 were positive for rbc autoab by 6 months. thereafter, $50% of the autoab 1 mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in 3 of the 4 cohorts, 60-100% of autoab 1 mice were female. hod 1 otii 1 autoab 1 mice also had enlarged spleens compared to hod 1 otii 1 autoaband hod -otii 1 mice (0.42g vs. 0.21g and 0.14g, resp., p<0.04). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd71 hi ter119 inter ) were observed in the spleens of hod 1 otii 1 autoab 1 mice but not in hod 1 otii 1 autoaband hod -otii 1 (2.86% vs. 0.06% and 0.05% resp., p<0.03). moreover, autoab and c3 deposition were found (0.1-2% and 3-10%, resp.) on ter119 1 rbcs in all of the hod 1 otii 1 autoab 1 mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b5-a01a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards 1 , christopher a tormey 2 and krystalyn e hudson* 1 . background/case studies: red blood cell (rbc) alloimmunization occurs in up to 10% of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c57bl/6 (b6) mice were treated with pbs, or anti-ly6g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at 18-24 hours post-transfusion. anti-hod alloantibody generation was assessed 14 days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n55), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n55). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls (3/3 experiments, p<0.01). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio1 leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l1), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p<0.05); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b9-a03b cxcr5 1 pd1 1 and ccr7 1 expressions characterize responders to rbc immunization benoît vingert* 1,2,3 , marie tamagne 1,2,3 , sadaf pakdaman 1,2,3 , anoosha habibi 2,3,4 , philippe bierling 1,2,3,4,5 , rachid djoudi 1 and france pirenne 1,2,3,5 . 1 efs ile de france, 2 laboratory of excellence gr-ex, 3 imrb u955 -eq2, 4 ap-hp, 5 universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd4 1 t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd4 1 t cells have a th17 profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd4 1 t cells with a cxcr5 1 pd1 1 phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr5 1 pd1 hi profile, with a differentiated expression of ccr7. ccr7 is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr6 and cxcr3 can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr5 1 pd1 1 lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr5 1 pd1 1 cells were compared in 2 groups of transfused sickle cell patients : alloimmunized (n514) and non-alloimmunized patients (n510). the analysis was also performed in non-transfused healthy controls (n512). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr5 1 pd1 hi subpopulation expression was identical between transfused groups and controls. ccr6 and cxcr3 expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr7 expression was very strong independently of the expression of pd1. in the aim to determine the help of the circulating cxcr5 1 pd1 1 cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for 5 days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr5 1 pd1 1 subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr5 1 pd1 1 cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr7 on circulating cxcr5 1 pd1 1 cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr5 1 pd1 1 profile and the ccr7 1 expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd4 t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* 1 , ashley bennett 1 , kathryn girard-pierce 1 , connie arthur 1 , amanda mener 1 , patty zerra 1 , christopher a tormey 2 , jeanne hendrickson 3 and sean stowell 4 . 1 emory university, 2 yale-new haven hospital, 3 yale university, 4 emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd4 t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd4 t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b6 recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod 1 kel). to examine the role of cd4 t cells, pic/kel primed b6 recipients were cd4 t cell depleted prior to transfusion. in addition, b6 recipients were adoptively transferred with cd4 t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd4 t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < 0.001); pic/kel primed recipients transfused with (hod 1 kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd4 t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < 0.0001) and transfer of cd4 t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < 0.001). conclusion: these results demonstrate that cd4 t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd8 t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within 24 hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd8 t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd8 t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h2kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c57bl/6) mice or oti mice, whose cd8 t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h2kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes 48 hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after 2, 4, 8, 16, and 24 hours and on days 2-5. results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after 8 hours and peaking at 24 hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is 28-35% versus >60% in wt recipients at 24 hours (p<0.05), whereas transfusion of wt platelets into either oti or wt recipients is approximately 60% at 24 hours after transfusion. adoptive transfer of oti cd8 t cells into wt mice recapitulates the effect, with significant mova platelet clearance at 24 hours compared to wt platelet clearance (p<0.05). conclusion: this work extends the ability of cd8 t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv1r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv1r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv1r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c57bl/6 mouse blood and treated with uv1r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv1r treated wbc-rich prp, or uv1r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c57bl/6 donor blood. a second transfusion of untreated wbc-rich c57bl/6 prp was given 2 weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv1r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p<0.01) or necrotic (p<0.05) wbcs, but not those given uv1r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c57bl/6 wbcs were reduced in recipients of either uv1r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv1r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv1r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so 2 ) of venous blood is generally assumed to be around 70-80% as measured from a central venous line. however, a recent investigation of so 2 levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so 2 distribution (mean 45.9%617.5% [yoshida et al. 2017; blood transfusion 15, 172] . the present study was undertaken to determine the distribution of so 2 in lr-rcc produced at a medium-size blood center using a novel non-invasive so 2 probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so 2 levels. study design/method: the so 2 from 977 units of lr-rcc were examined on five consecutive days representing 78% of the collected units during the period at a regional blood center where all the units were processed at room temperature within 8 hours of blood collection. so 2 was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a3u11; pendar technologies, cambridge ma). in addition to so 2 , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n54) from human volunteers were stored in as-3 under normoxic, hyperoxic, or hypoxic conditions for up to 42 days (so 2 ranging from <3 to >95%) prior to uhplc-ms metabolomics analyses in presence of 13 c, 15 n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so 2 carried out non-invasively at a blood center yielded a similar wide distribution as previous study from 497 units of lr-rcc procured and sampled invasively within 24 hours after blood collection [yoshida ibid]. the shape of the so 2 distribution appeared near normal with the mean of 47.0%621.0%, median 45.2%, range < 5% to > 95% and inter-quartile range (iqr) of 31.4%-61.9%. male donors showed higher so 2 compared to female donors (p<0.04). no correlations were observed between so 2 levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so 2 levels ameliorate the energy and oxidative metabolic lesion. lower so 2 levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp 1 ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so 2 levels was observed from lr-rcc manufactured at a blood center using 8-hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a 3-fold increase in the absolute lymphocyte count (ko 7.59 6 4.63 x10 9 /l vs. wt 2.90 6 1.32 x10 9 /l, p 5 0.0303), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko 6.00 6 0.29 fl vs. wt 5.24 6 0.56 fl, p50.0140). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd81foxp31 regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd8 1 foxp3 1 tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd8 1 foxp3 1 tregs derived exosomes and their functions involved in cd8 1 tregs mediated immune-modulation were seldom reported. study design/method: cd8 1 t cells were freshly purified from pbmcs, cultured with anti-cd3/cd28 antibody packaged beads and il-2, and then polarized with tgf-b and rapamycin into cd8 1 foxp3 1 treg cells. the harvest cells were co-cultured with cd3/cd28 beads stimulating cd41cd25effector cells in the transwell plate. the supernatant derived from cd81 tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd63, cd81, tsg101 and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir-155, let-7b, let-7d were measured by qpcr. the precipitated exosomes were further purified by cd63 immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd8 1 treg cells could suppress the proliferation of effector cells with a small decline (p>0.05), which means some non-contact factors involved in the cd8 1 treg mediated immune modulation. a total number of 4.57 6 0.52 3 10 8 /10 6 cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle 50-150nm in diameter (145.16 6.7nm by nta). cd63 and cd81 were expressed on these background/case studies: regulatory t cells (tregs), containing cd4 1 and cd8 1 subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd4 1 foxp3 1 regulatory t cells (ntregs) in inflammation conditions (including instability of foxp3, conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd81 regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd8 1 treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd8 1 regulatory t cells in inflammation and transfusion. study design/method: human cd8 1 tregs were induced with tgf-b1 and rapamycin from cd8 1 t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd8 1 tregs when encountering with inflammation were test by foxp3 expression, th1 and th17 cells conversion in inflammations conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6) on day3, day6 and day9. in vivo, cd8 1 tregs were transfused into cia mice and then their survival in mice and foxp3 express were evaluated to reveal the stability of cd8 1 tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd8 1 tregs when induced factor tgf-b1 and rapamycin were removed by testing the foxp3 expression on day3, day6 and day9. results/finding: ex vivo induced human cd8 1 treg were foxp3 1 (90.40 6 1.40%) and did not secret il17a (both in supernatant and % of cells). foxp3 express in cd8 1 tregs were maintained after induced factor tgf-b1 and rapamycin were removed on day3, day6 and day9. in vitro, foxp3, il2 and ifn-c expression has no significant difference when compared with controlled tregs on day3, day6 and day9 and did not secret il17a when encounter with inflammation conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6). in vivo, cd81 treg cells were transfused into cia mice on the peak of disease onset (35 days after the first collagen immunization, has inflammation condition in vivo) to test cd8 1 tregs survival. cd8 1 tregs were found in cia mice foot (27.4 6 2.03%), blood (4.55 6 1.03%) and spleen cells (1.90 6 0.05%) 72 hours after transfusion and their % of foxp3 1 were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd8 1 tregs are stable in inflammation and transfusion and can maintain foxp3 expression when induced factor were removed, these make cd8 1 treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s6k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd83/cd80/cd86 expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p70s6k and its downstreanm proteins, especially the protein s6, which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s6 related protein translation inhibition. xiaoyun fu* 1,2 , mikayla anderson 1 and james c zimring 1,2 . 1 bloodworksnw research institute, 2 university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in 405 leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day 43 (one day past their expiration). 35 bioactive lipids including 10 common fatty acids, 10 oxylipins, and 15 lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in 405 stored rbc units. for example, arachidonic acid (aa) ranges from 0.5 -10.7 mm, linoleic acid (la) (1.4-28 mm), dihomo-c-linolenic acid (dgla) (0.1-0.8 mm), eicosapentaenoic acid (epa) (0.03-3.1 mm), docosahexaenoic acid (dha) (0.2-3.0 mm), and alpha-linolenic acid (ala) (0.06-2.3 mm). ten oxylipins including hetes, hodes, and dihomes, and 15 lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of 35 analytes quantified, 25 showed a significant difference in concentration among different blood types by one-way anova testing (fdr<0.05). the ab rh1 blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh1). the fold increase of o rh-/o rh1 among pufas ranges from 1.3 to 2.1, suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among 405 stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna-223 was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna-223 targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps (49 6 6 nm) surrounded by a thick fluorescent silica shell (22 6 2 nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r 2 rnaprobe 5 0.96 and r 2 dnaprobe 5 0.98) with mirna-223 concentration, down to a 10-nm limit of detection. hybridization assays in 1% human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in 1% human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. 6.7 6 0.0 a 6.9 6 0.0 6.7 6 0.0 a 6.6 6 0.0 6.7 6 0.0 a 6.9 6 0.0 total atp,lm/ghb 7.6 6 0.3 a 5.2 6 0.3 7.3 6 0.2 a 5.5 6 0.3 7.3 6 0.4 a 4.9 6 0.5 extracellular lactate,mm 5 6 1 a 6 6 1 7 6 0 8 6 0 6 6 0 6 6 1 extracellular glucose,mm 32 6 1 a 50 6 3 2 9 6 1 a 38 6 1 2 5 6 0 a 28 6 1 extracellular na 1 ,mm 142 6 2 143 6 2 138 6 1 a 137 6 1 144 6 1 141 6 1 extracellular k 1 ,mm 1 6 0 a 4 6 0 1 6 0 a 5 6 0 1 6 0 a 4 6 0 a p<0.05, paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso100201600009c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n56) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by 50% (63/12,492) compared to the previous year without pathogen inactivation (42/12, 931, p50.030, chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased (2/15,286 vs 10/ 13,488). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5.0x10 11 ) than unaffected controls (3.5x10 11 ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above 5.0x10 11 . in vitro quality of single dose amotosalen/uva treated platelets in 35% plasma/65% pas-3 after 5 days of storage crystal stanley 1 , marguerite kelher 2 , nero evero 1 , melissa vongoetz 3 , betsy donnelly 3 and anna erickson* 3 . 1 belle bonfils memorial blood center, 2 university of colorado, 3 cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas-3 to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct02298842) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas-3 and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in 35% plasma/65% pas-3, collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, 7.5 6 0.6 310 11 platelets in 602 6 52 ml, were collected on the trima apheresis platform in 35% plasma/65% pas-3. a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n56, were 302 6 26ml (t) and 300 6 27ml (c) with doses of 3.8 6 0.3 3 10 11 (t) and 3.7 6 0.3 3 10 11 (c). all pc were stored under the same conditions and evaluated on day 5 and day 7 for physical/metabolic characteristics. results/finding: on days 5 and 7 all t and c pc had ph228c !6.2. the dose recovery for t was 87%63%. on day 5, t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table 1) . conclusion: trima pc in 35% plasma/65% pas-3 treated with the inter-cept blood system for platelets using the sv set and stored for 5 days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan 1,2 , yang yu 1 , li-ping sun 1 , shu-fang wang 1 , rui wang 1 , lei-ying zhang 1 and deqing wang* 1 . 1 the department of blood transfusion, the pla general hospital, 2 the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: 1 five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d0, d14 and d35. we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in 17% final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. 2 levels of k and lactic acid (la) were tested using automatic biochemical analyzer.3 k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. 4 treated hips-cms with d35 ssrbcs, d35 k and cell culture media for 48h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: 1 d0 ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d14 ssrbcs stop beating, but beating patterns restored at 48h. hips-cms treated with d35 ssrbcs stop beating, and beating patterns did not restored at 48h. 2 levels of k and la in ssrbcs changed most obviously. 3 only d35 k solution made hips-cms stop beating and can restore in 48h; d0 k, d14 k and la solution did not influence the beating pattern in at the end of the treatment for 24h, hips-cms treated with d35 ssrbcs show obvious shrinkage. at the end of the treatments for 48h, cells treated with d35 k and d35 ssrbcs both show obvious shrinkage, the shrinkage in d35 ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. 5 gene expression array results show a total of 140 genes were differentially expressed in d35 ssrbcs group compared with naive group. there was no consistent separation within the d35 k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. 2 in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. 3 further study should be applied to signal pathways on ssrbcs induced cytotoxicity. 4 large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as-3 as compared to sagm. the presence of citrate in as-3 seems to be necessary to prevent hemolysis of thawed cells. during storage in as-3, atp and 2,3-dpg levels rapidly decline. recently developed additive solutions like pag3m and as-7 have shown to better maintain 2,3-dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag3c in which the mannitol of pag3m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at 2-68c in pag3c. study design/method: leukoreduced rcc (n56) in pag3c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at 2-68c. on day 8, rccs were glycerolized using acp215 (haemonetics v r , braintree, ma) to a final concentration of 40% (w/v), frozen and stored for at least two weeks at -808c. after thawing and deglycerolization using acp215, rcc were resuspended in pag3c. during storage at 2-68c, stability (hemolysis), atp and 2,3-dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n58) resuspended in or sagm (n54). results/finding: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels at day 8 as compared to storage in sagm, resp. 9.1 6 7.6 mmol/g hb and 1.9 6 0.7 mmol/g hb. hemolysis during post-thaw storage in pag3c remained below 0.8% for 35 days and was comparable with storage in as-3. in sagm, hemolysis remained below 0.8% for 7 days. during the first 2 weeks of post-thaw storage in pag3c, both atp and 2,3-dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag3c showed significantly higher atp and 2,3-dpg levels compared to as-3 or sagm. while in sagm and as-3, 2,3-dpg levels were undetectable after 7 days post-thaw storage, in pag3c, 2,3-dpg levels only decreased to 6.1 lmol/g hb after 35 days of storage. conclusion: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels. as compared to as-3, post-thaw storage in pag3c showed comparable hemolysis while atp and 2,3-dpg levels were much better maintained. based on a maximum allowed hemolysis of 0.8% and an atp content of >2.7mmol/g hb, thawed rcc can be stored at 2-68c for 35 days in pag3c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold (48c, 4c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only 2 apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n54-5) in 65% iso using a trima or in 65% int using an amicus and stored for 15 days at rt and 4c. samples were tested on day 1 (baseline, bl), 5, 10, and 15 of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at 40% hct. labeled blood was perfused through microfluidic channels (fluxion) coated with 100 ug/ml type-1 collagen at 720s -1 shear rate. images were acquired every 30 sec for 10 min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p<0.05. results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day 5 of storage compared to bl (bl: 11.6 6 1.7%, rt: 4.9 6 1.2%; p<0.005). 4c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, 4c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day 5 (p50.03) and compared to 4c-int by day 10 (p<0.01). conclusion: our work suggests that 4c storage of plts collected with a trima ap system in iso for up to 15 days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at 4c. these results are surprising since both 4c-int and 4c-iso have been shown to express similar levels of cd62p, pac-1, and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at 4c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life 3x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table 1. comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after 2-week storage between unirradiated and irradiated groups (n 5 29) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone (10mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin (250mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th17 cells and increase in cd4 regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th1,th2,tfh and tfr cd4 subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd4 positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* 1 , sarah kloss 1 , sara crew 1 and sandra j nance 2 . 1 american red cross, 2 american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd109 have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to 28 unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only 510k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from 40 plasma and serum clinical specimens. group 1 contained a single hpa alloantibody specificity with or without hla antibodies (n526). group 2 included 5 specimens with hla antibodies alone and group 3 consisted of 9 patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa-1, hpa-2, hpa-3, hpa-4, hpa-5, gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is 100% concordance observed for hpa-1a, hpa-1b and hpa-5b antibodies. the pak-plus assay had difficulty discriminating hpa-5b from hpa-5a antibody when hpa-5a antibody was present (3 false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa-5a when compared to hpa-5b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within 10% of the cut-off for pakplus and <2.0 adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb231 cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac6 specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut1 overexpression on cell motility. results/findings: fut1 overexpression increases both ley expression and cell migration, while fut1 knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut1 overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac6. as tumor promoter, hdac6 becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac6 function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at 48c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n53) and stored at 48c for up to 10 days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and 30 minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd62p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means6sem, and paired student's t-tests were used to determine statistical significance (p<0.05). results/finding: on day 10, p-selectin levels were significantly higher in pre than bl (p50.03). mirasol treatment caused a significant increase in pac-1 expression compared to pre (pre: 10.5 6 3.1%, post: 28.1 6 4.7%; p50.04), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post-30 samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after 10 day storage at 48c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, 48c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day 10 4c-stored pas plts followed by incubation (30 minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* 1 , arthur p. bode 1 , anne s hale 2 , michael stanton 3 , mark johnson 4 and g. michael fitzpatrick 3 . 1 cellphire, 2 bodevet, inc, 3 cellphire, 4 background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to 33, 10, and 3.3% of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control (2-day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the 4 hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to 33% of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of 10% and 33% of the tcpc produced a significant decrease in blood loss. the lcps at 10% and 33% tcpc were as effective in mitigating blood loss as 2-day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to 33% of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of 10% and 33% of the tcpc reduced blood loss. these results suggest a starting dose above 3.3% tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as 10% tcpc had similar efficacy signals as 33% tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso100201300021c. the study on pcr-ssp technique for the genotyping of cd36 329-330del.ac mutation and the genetic polymorphism of cd36 329-330del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd36 (platelet glycoprotein iv, scarb3) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd36 is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd36 deficiency in china. cd36 gene mutation is the main reason that leads to cd36 deficiency. cd36 329-330del.ac (frameshift at aa110) mutation is one of the cd36 mutations that causes cd36 deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in 28 pediatric patients who received both doses of the mmr vaccine at 12 and 18 months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are !90% for all mmr components. results/finding: table 1 shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged 6.8 years (0.5 to 16.5 years). thirteen patients (46%) were chronically transfused at the time of serology. twenty-three patients (82%) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to 6 months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper 1,2 , franklyn cladis 2 , richard saladino 2 , darrell triulzi 3 , barbara a gaines 2 and mark yazer* 1 . 1 university of pittsburgh, 2 children's hospital of pittsburgh of upmc, 3 institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients !3 years old and !15 kg with evidence of hemorrhagic shock were eligible to receive up to 20 cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (<50) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately 11 months, 15 trauma patients received wb: 7 group o and 8 group a recipients, 53% male, median (iqr) age was 11 (4.5-14) and 73% blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of 36 (22-51) and 47% mortality rate. the median (iqr) quantity of wb transfused to group o recipients was 21.9 (14.8-24.3) ml/kg versus 13.4 (9-18) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean 6 standard deviation haptoglobin concentrations for non-group o recipients was 51.3 6 14.4 mg/dl on day 0, 86.3 6 36.8 mg/dl on day 1, and 126.9 6 45.8 mg/dl on day 2; the corresponding haptoglobin concentrations for group o recipients were 51.4 6 38.0 mg/dl, 84.7 6 61.5 mg/dl, and 134.8 6 68.3 mg/dl, respectively (p>0.42 for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n57) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n514). the mean 6 standard deviation platelet volume administered was 112 6 24 cc for whole blood recipients versus 147 6 68 cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to 30 ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) 41 with anti-human igg only, and a 31 positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a 60 minute 378c incubation, followed by 3 automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than 3 days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer 4096 and the breast milk showed anti-d with a titer between 16 and 64. the patient had a consistent plasma anti-d titer of 8. the patient's mother chose to stop breast feeding after 8 weeks, and the patient's hemoglobin was improved at 12 and 16 weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that 30% of c1 scd patients from the west indies and west and central africa are partial c and at (30%) risk for alloimmunization to the c antigen through transfusion of c1 rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce(733g,1006t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce(4-7)-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of 255 patients with genotype/rh phenotype data available, 78 (30.6%) were c antigen positive serologically. the allele frequency of rhce*ce(733g,1006t) was 0.071. in total, 15 (5.9%) patients possessed rhce*ce(733g, 1006t) in the absence of conventional c gene in trans. of the 78 c antigen positive patients, 15 individuals (19.2%) were predicted to be partial c based on four molecular profiles [rhce*ce(733g, 1006t)/rhce*ce:12; rhce*ce(733g, 1006t)/rh*ce:1; rhce*ce(733g, 1006t)/rh*ce(733g):1; rhce*ce(733g, 1006t)/rh*ce(733g, 1006t):1]. in these 15 partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after 60 transfusion exposures (57 c-antigen negative units; mean: 4, range: 0-36), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce(733g, 1006t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* 1 , megan l nguyen 1 , melanie c proctor 1 , david e krysztof 1 , gregory a foster 1 , erin k sash 1 , sandy s dickson 1 , joua yang 1 , jeffrey m linnen 2 , kui gao 3 , jaye p brodsky 4 and susan l stramer 1 . 1 american red cross, 2 grifols diagnostic solutions inc., 3 grifols diagnostic solutions, inc, 4 quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and 4 suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on 6/20/16 (fl, ga, sc, ms, al). following revised guidance on 8/26/16, testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on 12/12/16. travel history questions were discontinued on 1/23/17. confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of 4/8/17, 2,288,855 donations were tested including 393,713 (17%) in 24,611 mps. no reactive donations were identified by mp-nat. of the 1,895,142 id-nat donations, 72 were initial reactive (ir) of which 8 (11%) confirmed positive (cp) by subsequent testing (cp rate of 1:286,107; positive predictive value of 11%; specificity of 99.997%). five (62%) cp donations were id-nat repeat reactive (rr); 3 (38%) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and 3 in fl, 2 of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from 2 to 73 days prior to donation. two donors with a travel risk reported clinical symptoms; 6 cp donors (75%) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than 40 copies (c)/ml to about 8ê 5 c/ml. at the time of writing, the longest period of detection in rbcs was 91 days vs. 17 days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from 1 ir and all rr donors, ranging from 12 to 2000 c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat1 samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of 6 were prepared by diluting nat1 plasma 1:6 and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n5308) were sorted into 4 categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm1/high vl; igm1/low vl. results/finding: of 52,942 donations collected april 3-december 31, 352 were reactive for zikv rna. igm-index donations had higher vls (mean 1.1 x 10 6 vs 8.3 x 10 4 iu/ml) and higher proportions of simulated mp-detectable results (93% vs 23%) than igm1 donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm1 donations increased (table 1) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the 2016 pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r 6800/8800 systems lisa lee pate* 1 , phillip c williamson 2 , michael paul busch 3 , susan rossmann 4 , scott jones 5 , ann butcher 1 , john duncan 1 , jean stanley 1 and susan a galel 1 . 1 roche molecular systems, inc., 2 creative testing solutions, 3 blood systems research institute, 4 gulf coast regional blood center -sugar land, 5 qualtex laboratories background/case studies: in february 2016, the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march 30, 2016 and testing of puerto rico donations began on april 3, 2016. as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august 2016, the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april 3, 2016 -february 28, 2017 using the investigational cobasv r zika for use on the cobas v r 6800/8800 systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a 1:6 dilution to simulate mini-pool testing. results/findings: a total of 1,776,190 blood donations were screened using cobasv r zika. of 56 ir donations, 12 were repeat reactive (rr), 39 non-rr and 5 had no repeat testing. of the 12 rr donations, 7 were positive by altnat; 3 of these were igm positive. all 4 altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the 5 rr donors that were not igm positive on index, 3 enrolled in follow-up and all seroconverted. of 39 non-rr donations, 38 were altnat negative and 1 is pending supplemental testing. 8/38 donors were igm positive on index. 30 donors were igm negative on index; 15/30 enrolled in follow-up; 14 remained igm negative and 1 was 1gm inconclusive. of 5 donations without repeat testing results, 2 met criteria for positive (1 was altnat positive, igm negative and 1 altnat negative, igm positive). 1 donation is pending additional testing. altogether, 22/56 ir donations met the criteria for true positive on the index donation. 9/22 (41%) true positive donations were reactive when retested in a simulated minipool. 16/22 were igm positive. conclusion: 0.001% of the 1,776,190 donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* 1 , marc germain 2 , gilles delage 3 , maria esther lopes 1 and yves gr egoire 3 . 1 hemorio, 2 hemaquebec, 3 h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia (2013) , and in brazil (2015/2016), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since 80% of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january 1 st , 2016 through november, 26 th , 2016, from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: 5 days, with 99% of the values lower than 18 days); 20% of infected donors with symptoms lasting 2 days; 1.2 donation/donor/year for wb and 1.75 for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x (1 -proportion of refused donors) x (1proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain 1:13,598, for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at 4 large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from 6 donations). the residual volume of mp plasma, 0.35 -0.45 ml, is routinely discarded. beginning in april 2016 each blood center saved $67 mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april 3-15) 3 mp6 were combined into pools of 18 donations; thereafter mp6 were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and 95% confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity (95% limit of detection <20 copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first 6 months of samples is complete for 6,292 mp, comprised of 37,752 donations collected from april 3 to october 9, 2016. a total of 77 pools were positive, with 76 detected between april-june 2016. the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over 0.6% of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over 0.4% of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in 2016, zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* 1 , sze sze chua 1 , mars stone 2 , michael paul busch 2 and ai leen ang 1 . 1 health sciences authority, blood services group, 2 blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on 26 august 2016. the numbers rose rapidly to 386 cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since 1 october 2016. zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using 300 blinded frozen samples consisting of 25 replicates of 11 half log dilutions of the who international standard for zikv and 25 replicates of negative controls prepared by bsri. probit analysis was performed to determine the 50% and 95% limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a 14 member blinded zikv reference panel from the usa-fda. results/finding: a total of 63,144 donations were screened from 1 october 2016 to 31 march 2017, with 1 false positive case and 1 zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of 9.54x10 5 copies/ml. zika igm was negative in the index donation sample but present in the 10-day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be 2.1 copies/ml at 50% lod and 10.0 copies/ml at 95% lod. the procleix zikv assay detected rna in 6 out of 9 patient samples and provided 85.7% agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with 1 confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of 1 in 25,888 donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types 1, 2 and 3 express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to 21 reactivity on initial gel testing. if genotyping demonstrated weak d types 1, 2 or 3, the intent was to manage the patient as rhd-positive. if weak d types 1, 2 or 3 were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in 2 to 4 weeks. results/finding: rhdgenotyping was performed on 22 patient samples over 15 months. of these 22 patient samples, 13 (59%) were weak d types 1 or 2. the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type 1 required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types 1 and 2 patients have not received transfusions at this institution since they were genotyped. four of 4 obstetric weak d types 1 and 2 patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this 15 month study period 13 serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using 3 fda approved anti-d reagents. when reactivity with all 3 reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over 80 rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of 8 months we tested 509 rhd-negative blood donors. there were 3 (0.6%) partial-d, 1 weak d (0.2%), and 3 (0.6%) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs5-46_42deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these 8 donors showed that 6 rhd-negative recipients received rbcs from 4 of these donors. five of these recipients underwent antibody screening after an average follow-up period of 5 months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, 5 grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which 63% of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d1" with 31 reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october 2016 to march 2017, we performed routine d typing (neo, immucor) on 1875 obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of 31 using at least 1 antibody. solid phase and manual testing used the series 4 and series 5 reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh1) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all 13 samples. two of 13 (15.4%) were d1 with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs1-29c, rs2301153; ivs3 1 117c, rs28521909; and ivs3 1 124a, rs28562109). two (15.4%) were d1 and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four (30.8%) patients had rhd alleles with known potential to make anti-d (rhd*dol2, rhd*dar1.2, and 2 with weak d type 4.0). one had weak d type 96, which has uncertain susceptibility to alloimmunization and one was weak d type 1, which has not yet been associated with anti-d. interestingly, two (15.4%) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type 2 is a variant of the rhd protein that comprises an amino acid substitution located in the 12th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c.1154 g>c which is the first nucleotide of the exon 9 of the rhd gene and thus could be implicated in exon 9 skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v6 (agilent) and the nextseq500 platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon 9 skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type 2 rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of 10 patients previously characterized by beadchip technology. interestingly, 4 out of 10 carry the c.1154-31c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last 4 patients, one has been previously characterized as rhd weak type 2 carrying the c.1154g>c (p.gly385ala). independently, sanger sequencing on 50 unrelated rhd weak type 2 samples pinpoint to a linkage disequilibrium between c.1154g>c (exac, maf 5 0.001145) and the c.1154-31c>t (exac, maf 5 0.2496). in silico analysis of both mutation located close to the splice acceptor site of the exon 9 does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon 9, mutant rhd c.1154g>c, mutant rhd c.1154-31c>t and double rhd mutants c.1154g>c plus c.1154-31c>t, we showed no influence on skipping of exon 9 due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type 2 between ala385 (transmembrane helix 12) and val183 (transmembrane helix 6) hampering membrane insertion. conclusion: the c.1154-31c>t variation is always associated in cis with the missense mutation c.1154g>c on the allele rhd weak type 2. the c.1154-31c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type 2 red blood cells is due to the substitution of alanine at amino acid position 385 to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has 10 transmembrane (tm) and 2 tilted ureapore a-helices, a long extracellular connector segment, and 2 cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p.280. we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and 2010-2016 aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, 2012) . results/finding: seven snmvs located within 1 amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all 3 at the exofacial ends (p.a93t, p.w240r, p.v333d) are jk-weak; the two jkneg exceptions p.g298e and p.g299e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, 13 snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v10m, p.e44k, p.v76i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the 13 jk-neg variants are within 19 aa (p.270-p.299) of jk a/b at p.280. none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n289s and p.s291p are adjacent to p.288f and p.292l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among 13 jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc14a1 gene, which encodes the urea transporter ut-b1. the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in 1965. in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc14a1 gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within 1 aa from tm a-helix end v76i, a93t, w171r, w240r, g298e, g299e, v333d* cytoplasmic n-terminal v10m, g40s, e44k, l45p in membrane tm and urea-pore a-helices r64w, r64q, g65d, i117t, a183v, l246r, a248t ‡, a270a §, l272f, n289s, s291p, t319m 2/10 * second nucleotide variant in this allele is synonymous (p.p196p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a 5 mbp region in 19q13.11-13.2 with an lod score of 9.6. using deep sequencing, we identified a potential deleterious mutation in the znf850 gene, which deletes 84 bp resulting in loss of an entire zing finger domain. the identical del84-znf850 mutation is present in all affected individuals, and is absent from all controls tested (n>2000). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf850 locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf850del84. none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc14a1gene, is a urea transporter that has been associated with renal function, we found that people with the znf850del84 in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf850, prevalent in southern spain due to a founder mutation, leads to ut-b1 dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-(2-ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis(2-ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at 30 days and 1 year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the 12 pools included 5 group a, 6 group o and 1 group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than -208c within 8 hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day 0 (pool), day 30, and 1 year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day 0, day 30, and 1 year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp 5 2.9 ppm; mehp 5 0.3 ppm; deht 5 0.9 ppm; and meht 5 0.2 ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than 80% of its initial value. plasma stored in deht bags had an average plasticizer content 90% lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at 100 g to separate rbcs from platelet-rich plasma (prp). prp was diluted 3-fold in pipes-saline with 1.4mm pge1 and centrifuged at 1900 g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of 34-40% and 150,000-250,000 platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted 1:1 (spdp50%) with plasma from a patient with type 3 vw disease (t3vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of 1600 s -1 for 180 seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was 20% (spdp/ffp > 0.8). results/finding: six batches of spdp/ffp were evaluated using 17 subjects. there was no statistical difference between the spdp/ffp pairs (p50.7558). the mean ratio of spdp/ffp was 1.21 with a 95% ci of 0.84 -1.57. comparing spdp vs. spdp50%, there was no difference (median ratio 5 1.045, range: 0.95-1.14) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was 20% greater than in samples reconstituted with ffp. the lower limit of the 95 th % ci is a difference of 16%, which is less than the a priori determined margin of noninferiority of 20%. even with 50% dilution with t3vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* 1 , qiyong peter liu 2 , grantham c. peltier 1 , ryan c. carney 2 , ashley s. taylor 1 , colby s. mcintosh 1 , james a. bynum 1 and andrew p cap 1 . 1 u.s. army institute of surgical research, 2 velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: (1) ffp; (2) ffp with 70mm glycine; (3) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; (4) spdp pretreated with glycine-hcl (20mm); and (5) spdp pretreated with glycine-hcl:glycine (20mm:50mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood (40% hct with 200 platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < .05). fibrin polymerization density was slightly diminished in rspdp vs. ffp (0.879 vs. 0.742 o.d., p < .01), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < 0.001). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < .01) and an additional twofold in pretreated spdps vs. rspdp (p < .05). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < 0.01). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples (71.53% surface coverage vs. 30.26-43.87%, p < .05). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* 1 , anita tuip-de boer 1 , ruqayyah almizraq 2 , jason p. acker 3 , philip j. norris 4 , jennifer a muszynski 5 and nicole juffermans 1 . 1 academic medical center, 2 university of alberta, 3 canadian blood services, 4 blood systems research institute, 5 nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from 8 donors (blood type a or b). supernatants were prepared after 4-5 (fresh) and 41-42 days of storage (stored) for measurement of thrombin generation and ev analysis. a549 type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to 25% stretch using a cellstretcher. control cells were not stretched. after 24 hours, il-8 and il-6 production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il-6 and il-8 production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p<0.05) . incubation of stretched cells with stored wbf products resulted in higher il-8 production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by 4 different methods from 5 individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after 4-5 days (fresh) and 41-42 days (expiry). monocytes were co-cultured in media plus 20% rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in 5 replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean 6 sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table 1) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin-8 production was higher after exposure to fresh wbf (248 6 115 % control, p 5 0.02) or wbd at expiry (292 6 111 % control, p 5 0.0005). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* 1 , alessandro tocchio 1 , anita howell 2 , kaushik sridhar 1 , jason p. acker 3 and utkan demirci 1 . 1 stanford university, 2 canadian blood services, centre for innovation, 3 background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of 10 -4 g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at 7, 14, 21, 28, 35 and 42 days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from 24 volunteers with four different age and sex categories (male, 18-40 years; male, >60 years; female, 18-40 years; female, >60 years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young (1.098 g/ml) and older female donors (1.109 g/ml) (p < 0.01). moreover, rbcs from young males (1.096 g/ml) were significantly less dense compared to rbcs profiled from older female donors (1.109 g/ml) (p < 0.05). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il-6 (pg/ml) il-8 (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: 120 icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of 426 rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p<0.001). substantial correlations were also found between orp and free hemoglobin (p<0.05) and orp and free heme (p<0.05). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections 132 6 10 vs 127 6 13 (p<0.05). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than 35 days compared to rbcs stored for 7 days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of 1 to 42 days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to 7 days storage duration -reference group), medium age (at least 1 rbc of 8-35 days storage), and oldest (at least 1 rbc greater than 35 days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for 7 days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every 7 days, and b) a finer partition using cut-points every 3 days. results/finding: 24,726 patients receiving 90,530 rbcs were included in the analysis. exposure to rbcs stored for more than 35 days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for 7 days or less) after adjusting for several fixed and time-dependent potential confounders (hr 5 0.91; 95% ci: 0.72, 1.14; p 5 0.400). exposure to blood stored for at most 8-35 days yielded a similar hazard ratio (hr 5 0.90; 95% ci: 0.73, 1.10; p 5 0.295). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than 35 days compared to exclusive exposure to rbcs stored 7 days or less was not significant (hr 0.90; 95% ci 0.72, 1.14; p 5 0.381). the confidence intervals around the hazard ratios for the other 7-day intervals all include 1. similar findings were obtained with partitioning exposure data into 3 day intervals where exposure to rbcs stored for 40-42 days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for 1-3 days (hr 0.82; 95% ci 0.37, 1.83; p 5 0.635). the confidence intervals around the hazard ratios for the other 3-day intervals all include 1. conclusion: individuals exposed to rbcs stored for more than 35 days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for 7 days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* 1 , cynthia walser 1 , tatsuro yoshida 2 , andrew dunham 2 and pedro cabrales 1 . 1 university of california san diego, 2 new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o 2 ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o 2 carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o 2 saturation <10%) stored rbcs, or anaerobic/hypercapnic (o 2 saturation <10% and pco 2 (@378c) $70mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as-3 after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either 1) conventional; 2) anaerobic; or 3) anaerobic/hypercapnic conditions. rats (150-200g) were hemorrhaged to 50% of blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre-hemorrhage with prbc stored for either 1 or 3 weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. 1(11%) neg patient showed short term response and 6(67%) patients showed progressive disease. at the neg group standard eval 1(11%) patient showed response and 3(33%) had progressive disease. 1(11%) neg patient had long term response compared to 11(21%) pos patients. at the pos short term eval 22(42%) patients showed response and 20(38%) patients had progressive disease. at the pos group standard eval, 20(38%) patients showed response and 6(11%) patients had progressive disease. overall, 28(53%) pos patients responded compared to 2(22%) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd38 neutralizing substance could play a role in treatment response. alternatively, reduced cd38 expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a 24-hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april 2017 were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate 24% replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of 0.2m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* 1 , laurie sutor 1,2 , germ an leparc 3 , marjorie doty 3 and william crews 1 . 1 carter bloodcare, 2 ut southwestern medical center, 3 oneblood background/case studies: anti-cd38 drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with 0.2m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a 28 day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and 0.2m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual (18 th edition). each of the 12 plasma aliquots was further separated into 28 aliquots and stored at -208c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. (25) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than 11 with the untreated or dtt-treated cells during the study. conclusion: long term storage of 0.2m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque-10771), pooled, suspended in cryopreservation media (20% dmso; 1:1) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips (378c, 5% co 2 , 1 h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o1) rbcs sensitized with either anti-d (positive control), anti-scianna-2 (sc2) or anti-anwj or lipopolysaccharide stimulated for 2 h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/100 monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed 96.2 6 1% viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il-1b, il-6, il-8, mip-a (p < 0.01), mip-b and gro (p < 0.05) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc2-and anwj-sensitised rbcs resulted in a pi of 9.2 6 2% and 60.2 6 6.4% respectively vs anti-d sensitized rbcs (pi: 72 6 8.7%). a weak (11) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in 41 iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi>5%). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in 5 patients, involving 3 antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all 5 patients had 3-41 positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients 1 and 2 typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c1 by hea precise-type. ega-treated rbcs gave 31 reactions with the same anti-c reagent. patient 1 rbcs gave variable reactivity (vw-11) with bio-rad seraclone and ortho bioclone anti-c. patient 2 rbcs gave 11 reactivity with all 3 anti-c reagents when incubated for the maximum incubation time allowed. patient 3 rbcs were jk(a1) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat1rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients 4 and 5 tested s1 with bio-rad seraclone anti-s (3-41), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat1 rbcs but not all manufacturers include reagent limitations regarding testing of dat1 rbcs. we describe 2 cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and 3 cases with false positive tests with anti-s (n52) and anti-jk a (n51) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with 3-41 positive dat and supports testing to dissociate igg from rbcs strongly dat1 before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/2opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r :5 prob(ab|al-loexp), so that prob(ab) 5 prob(ab|alloexp)*prob(alloexp) and 0 r 1; rewriting prob(alloexp) 5 prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) 5 nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) 5 r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a 12 month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past 12 months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in 2 states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar 3 months before (2/ 2015 -4/2015) and 3 months after (2/2016 -4/2016) was selected; for state b, a similar 4 months before (12/2015 -3/2016) and 4 months after (12/2016 -3/2017) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (<12 months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a 13-and 3-fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from 13 to 567 (state a) and 151 to 1,496 (state b), which annualized, represents a potential gain of 2,216 (state a) and 4,035 (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the 2 states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from 2013 -2015 , there were 181 donors identified who had changed their gender from their birth gender; 121 female donors changed their gender to male and 60 male donors changed their gender to female. there were 7 (6%) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to 10.5 ml/kg or 15% of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the 15% limit. variable volume scales [vvs] can be programmed to vary unit volume (up to 550 ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by 10 ml at ebvs <3.5l in donors !23 yo, but increase by 5-40 ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the 18 mos. before a 6 mo. phased implementation of the vvs, and the subsequent 24 mos. multivariable analysis [mva] by 6-mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods 1 & 2, continued during impl and post-impl periods 1 & 2, returning to the baseline rate in post-impl periods 3 & 4 (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods 3 & 4. the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of 3.8 ml during post-impl periods 1 & 2 from the temporally matched baseline & pre-impl period 1. conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* 1 and yves gr egoire 2 . 1 hema-quebec, 2 h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, 2017. the vaccine is produced with live and attenuated yfv, which can circulate for at least 4 weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a 4 week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate 600 people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received 3,351 blood donors candidates; from those, 2,449 were accepted as a blood donor, after medical interview. the deferral rate was 26.9%. at the same period of the year 2016, there were 1,215 prospective donors, and 883 blood donations. the deferral rate was 27.3%. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, 1,566 additional donors, compared to 2016 same dates. that represents a 177.34% increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from 42.7% in 2016 to 45.8% in 2017. conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of !3.0x10 11 is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: 1,000 apheresis collections from 4 centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of 3.1 x10 11 for single (s), 6.3x1011 11 for double (d), and 9.5x10 11 for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or 100% plasma) assuming i) a minimum dose (allowing for production loss) of 3.5 x10 11 for s and 6.7x10 11 for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as 99%) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc 2rbc plt/p plt plt/rbc/p plt/rbc 2plt 2plt/rbc 2plt/p #donations 47990 63 1775 10832 968 476 67 1150 142 10252 citrate exposure (mls) 41-85 71 138 263 266 300 322 349 478 study design/method: a randomized (2:1), placebo-controlled, single blind, 15 subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into 5 cohorts, receiving increasing doses, ranging from 1/1,000 -1/10 of the lowest effective dose found in the above rabbit model. cohorts 4 and 5 received the 1/10th dose, but cohort 5 received two 1/20th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for 24hrs post infusion and followed for up to 60 days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of 68 aes: 40 were treatment emergent (teae), of which 8 were treatment-related (6 thrombosomes and 2 control). all teaes were mild or moderate in severity. in cohorts 4 and 5, 3/4 thrombosomes subjects had treatment related adverse events. one cohort 4 subject developed an upper respiratory infection and elevated wbcs within 8 hours post infusion, which resolved by 24 hours, and an elevated d-dimer at 24 hours post infusion, which resolved by day 7. this subject also had an elevation of prothrombin fragment 1 1 2 at baseline, which increased post transfusion and peaked at 24 hours with resolution by day 14. one cohort 5 subject developed non-specific t-wave changes at 1 and 2 hours following her 2nd infusion that resolved by day 21 without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort 5 subject developed an igg platelet autoantibody on days 7-21, which was undetectable on days 42-60; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days 7-14, and negative on days 21-60. background/case studies: cryopreservation of platelets (plts) could extend the shelf life from 5-7 days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with 5% dmso and stored at 2808c. after thawing, the unit was reconstituted in thawed ffp spiked with either 500 lm puromycin (pm) or 250 nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after 2, 4 and 24 hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd62p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x-100containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd62p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by 11-fold during 24 hour storage. immunoblot analyses of the plts showed a 2-and 4-fold increase in pm incorporation after 4 and 24 hours of storage, respectively. massspectrometry revealed 23 unique proteins that were synthesized after 4 hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac1, rap1 and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac1, rap1 and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in 2015, the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of 3 days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r 4-496 cooler with 2 units of ffp, 2 units of rbcs, and 1 unit of whole blood. three to 5ml of platelets were collected via syringe from each unit at 0 min (before storage in cooler or refrigerator) and after 0.5, 1, 3, 5, and 6 hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding) were measured by coulter counter, 2 channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p<0.05 deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for 6 hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c49-a03h molecular sieving: beyond genotyping ghazala hashmi 1 , reinhard klemm 2 and michael seul* 1,2 . 1 biomolecular analytics, 2 immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi2005 http://bit.ly/2ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding 401 rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of 30 rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, 4*4*96 samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for 135 sensitized sickle cell anemia ("sca") patients (tb1 in cas-tro2002, http://bit.ly/2oplxhr, excluding le and e(variant) and assuming 1 request per patient), presenting with up to 9 allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only 1 =2 plate holding 4*48 candidate units from actual black donors, followed by profiling of 44 samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for 127 of 135 requests (594.1%), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining 66 pooled samples produced 44 additional assignments to a second set of 135 requests, for a total of 171 assignments from only 92 wells. in another scenario, sieving of a full plate of 4*96 samples, produced $250 assignments for two successive batches of 271 requests from sca patients, a yield exceeding 2.5x. sieving alone typically fills 65-75% of requests of moderate complexity ( 5 ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez 1 , monica kalvelage 2 , ghazala hashmi* 1 and michael seul 1 . 1 biomolecular analytics, 2 lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou2013 http://bit. ly/2ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including 30 at the rhce locus) that encode 401 mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag2" (e.g. e2,c2,e2,c2) to specific combinations of "ag2" (e.g. c2e2k2fya2 and c2e2jsa2) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from 384 (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, 96 pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the 124 c2 samples, 24 that were also v2 and vs2 and, among the e2 samples, 12 that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select 6 specific "ee" pools of which 4 were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b (8 pools), co a|b (8) and others. conclusion: molecularsieving of a single 96-well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c2, e2 and jsa2. these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving 124 132 100 100 360 208 192 40 28 92 52 background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the 36 blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam4 (landsteiner-wiener) and ackr1 (duffy). for longer genes, such as abo of >20 kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of >21 kb each was used for all physically confirmed 48 ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least 4 clades representing clusters of 5 to 11 alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the 4 alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt1 genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors1) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons 266-268, and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors1. a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos1(b3galnt1) cells, and cell-surface expression of fors1 antigen was immunologically monitored with a monoclonal anti-fors1 antibody. results/findings: we found that met69thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon 3 or 4 of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors1 antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met69thr/ ser or exon 3/4 deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt1 genes is reminiscent of common ancestral origin of alpha 1,3-gal(nac) transferase genes. the finding that at can synthesize fors1 implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of 29,308 cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period 7/1/2008 -4/1/2017. abo genotyping targeting specific snps for groups a, a2, b, o1, and o2 and, if needed, gene sequencing was conducted in cases with indeterminate results, and in 4 cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two (0.21%) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in 53% the predicted abo phenotype was a rh neg (table 1a ). the predominant donor race was caucasian (65%). four cbu with abo discrepancy were also evaluated by genotyping (table 1b) . in 3 of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r 6800/8800 systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: 30,695 fresh and 20,029 frozen edta plasma samples from american red cross donors, collected from february 2015-2016, were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r 8800 system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of 50,724 valid results, a total of 3 donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a 65-year old male in indiana, a 21-year old male in california, and a 55-year old female in kentucky. all 3 donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna (1440 iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as 3a, the california donation genotype 3b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was 100% (95% exact ci: 99.993% to 100%). conclusion: based on the 3 confirmed-positive donations of 50,724 tested, the hev prevalence was 0.006% (95% exact ci: 0.001% to 0.017%) with a detection rate of 1:16,667 (95% ci, 1:588-1:100,000). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than 50% of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report 13 months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using 95% confidence intervals. this analysis contains data from 9/1/15-9/30/16. results/findings: among 7,578,462 donations reported (16.2% from firsttime and 83.8% from repeat donors), there were respectively 483, 1489 and 194 cp results for hbv, hcv and hiv with corresponding rates of 6.37,19.63 and 2.56 per 100,000 (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of 23:1, 24:1 and 5.4:1 for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv 8.3 vs 4.2; hcv 23.5 vs 15.2; hiv 3.9 vs 1.0). in general, higher rates for all markers were seen among minority donors, those in the 25-39-year age group (also 18-24 year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when 3-month periods were compared. conclusion: data from 4 major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* 1 , melanie c proctor 2 , deanna self 1 , monique portugal 1 , adrian gurrola 1 , laura tonnetti 2 , sonia bakkour 3 , cheryl lobo 4 , michael paul busch 3 , susan l stramer 2 and jeffrey m linnen 1 . 1 grifols diagnostic solutions inc., 2 american red cross, 3 blood systems research institute, 4 new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to 16 donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening 32,274 unlinked whole blood donations collected from august 25 th 2016 to april 7 th 2017 in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of 16. results/finding: the procleix babesia assay detected all four babesia species with a 95% lod ranging from 7.10-13.51 copies/ml. the preliminary 95% lod in parasites/ml ranged from 0.64-3.61 p/ml for b. microti (n59), from 0.92-1.52 p/ml for b. duncani (n52), and from 0.62-4.95 p/ml for b. divergens (n52). of the 32,274 donations screened, 17 initial reactive and 14 confirmed positive donations were identified for specificity of 99.991% (95%ci: 99.972-99.997%). of the confirmed positive specimens, 8 were reactive by both ifa and pcr, 5 by ifa only and 1 by pcr only. all confirmed positive samples were reactive in lysate pools of 16. donors of reactive donations resided in ct (11), nj (1), nh (1) and me (1) for an overall incidence of 1:2,305, and 1:1,433 in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of 16 thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* 1 , whitney r steele 1 , ed p notari 1 , james haynes 1 , roger y dodd 2 and susan l stramer 1 . 1 american red cross, 2 american red cross (retired) background/case studies: from 2004 -2012 , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from 2008-2015. study design/methods: prevalence was calculated in 2-year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of 18.5, 7.4 and 9.1 days for hbv, hcv and hiv, respectively. linear regressions were calculated with p<0.05 (*) as significant. results/findings: from 1/1/08-12/31/15, there were more than 48 million donations from 13,204,447 donors (51.4% female, 33% first-time (ft), 81.4% caucasian). there were significant decreases in donation prevalence for hbv and hcv (p50.014 and 0.044), but no significant decrease in hiv during the 8 years (see table for f and r 2 values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p50.026 and 0.042). prevalent ft donors were significantly more likely to be male (68.3% -hbv, 59.8% -hcv, 79.7% -hiv; p<0.001). incidence for all agents declined (significant only for hbv; p50.035). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last 2-year period (74 in 2012-2013 vs. 19 in 2014-2015) . hcv incident donors in 2014-2015 were more likely to be male (79.0% vs 46.0% in 2012-2013, p<0.001) and were younger (84.2% vs. 67.6% in 2012-2013 <40 years, p50.011). overall, incident donors were more likely to be caucasian males (p<0.01). rrs for all 3 agents decreased over time with rrs in 2014-2015 of 1 in 1,565,000; 1 in 2,680,000; and 1 in 2,435,000 for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the 8-year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in 2015, mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as-5 rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with 200lm amustaline, and incubated for 18hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the 3hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero76 cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, >6.9 log 10 , or >6.2 log 10 pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was >6.2 log 10 , or >5.5 log 10 pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts-13 inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts13 activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts13 deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts13 activity of <5% and high inhibitor (1.4-8). mean age of cohort 22.8 years (range 17-64). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean 15.2 x 10 9 /l, range 9 -27 x 10 9 /l) and low a-ipc (mean 1.5 x 10 9 /l, range 0.5 -3.6 x 10 9 /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab 375 mg/m 2 (4 patients) and cyclophosphamide 400 mg/m 2 (one patient). tpe continued until platelet count reached 150 x 10 9 /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of 2.4 days [range 1-4 days]) when they achieved a three-fold increase in a-ipc from baseline (mean 11.1 x 10 9 /l, range 2.2 -25.3 x 10 9 /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean 217.6 x 10 9 /l, range 200 -294 x 10 9 /l) and a-ipc (mean 19.4 x 10 9 /l, range 13 -28.5 x 10 9 /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of 11.6 days (range 8-14 days) mean platelet count was 65.4 x 10 9 /l (range 14 176 x 10 9 /l) and mean a-ipc 3.2 x 10 9 /l (range 0.7 -6.6 x 10 9 /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of 20.8 days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts13 inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* 1 , michelle n stram 1 , joan sevcik 2 , alesia kaplan 2,3 and joseph e. kiss 2,3 . 1 department of pathology, university of pittsburgh medical center, 2 blood systems inc., 3 university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within 4-8 hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january 1, 2013 to november 1, 2016 was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts-13 activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version 14 (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the 96 ttp patients identified, 22 were excluded due to missing temporal data for important variables. the majority (85%) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients (20%) had a prior history of ttp and 26% had severe adamts13 deficiency on admission. the median time from tpe request to initiation was 5.6 hours (interquartile range: 4.7-7.2 hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table 1) . treatment was not started within an 8-hour window in 13 patients; the median time to cv access was significantly longer in these patients (5.8 vs 2.47 hours, p<0.001). two of these patients had a prior history of ttp and only four patients had severe adamts-13 deficiency. the majority (more than 70%) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table 1) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus 4-8 hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi-48a transfusion 2017 vol. 57 supplement s3 hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, 2006 through january, 2017, we performed cytapheresis (cy) treatments (txs) for 123 pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. 84 pts (68%) had cml-at and received 319 leukapheresis (lp) txs; 39 pts (32%) had et and received 124 thrombocytapheresis (tc) txs. cml-at pts presented with median wbc 398 x 10 9 /l (range 193-689 x 10 9 /l), of which 63% had blast percent >75% or blast count >100 x 10 9 /l. median age was 42 years (8-79 years); 62% were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. 22% of cml-at pts had no sxs of lks; 40% pts had sxs of either cns or pulm lks (1 sxs), and 38% pts had sxs of both cns and pulm lks (2 sxs). et pts presented with median platelet (plt) count of: 1738 x 10 9 /l (642-3510 x 10 9 /l)and 71% pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was 66 years (31-89 years); 58% pts were male. results/finding: all pts received a course of cy tx with following objectives: 1) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and 2) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) <100 x 10 9 /l for cml-at pts, and plt ct <450 x 10 9 /l for symptomatic et pts and <750 x 10 9 /l for asymptomatic et pts. cml-at pts received median of 3 lp txs (mean 3.9 txs/pt; range 2-8 txs). et pts underwent median of 2 tc txs (mean 3.4 txs/pt; 1-7 txs). outcomes were evaluated by percentage of pts who: 1) reached wbc (or plt ct) tx goal, and 2) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved >50% reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, 76% pts improved, 21% pts stabilized; and 3% pts worsened. in et cohort, 85% improved, 14% stabilized, and 1% were unchanged. for cml-at pts, median final wbc ct 5 96 x 10 9 /l (range 66-307 x 10 9 /l); 94% pts received ind chemo. for et pts, median final plt ct 5 705 x 10 9 /l (263-1087 x 10 9 /l); 95% pts had resolution of thrombotic 49a transfusion 2017 vol. 57 supplement s3 symptoms. 4% of cml-at pts and 0% of et pts expired within 1-4 days after course of cy tx. of 3 expired pts, 2 pts had both blast crisis and sxs of cns/ pulm lks; 1 pt had intracranial hemorrhage or cva; and 2 pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median 2-3 txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used 100% albumin or 80% albumin-20% normal saline (80/20) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered (100% albumin vs 80/20), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where 100% albumin was used versus those that used 80/20. covariates included were fluid types, age and gender. odds ratios (or) and 95% confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < 0.05. results/finding: during the study period, 3650 procedures were documented for 414 subjects (46% female), age range 0-93 years, of which 2,470 (67.7%) received 80/20. the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with 100% albumin had a significantly lower risk of having either event than by using 80/20, [p50.002, or (ci): 0.40(0.22, 0.72)] , and also had a significantly lower risk of causing hypotension [p50.023, or (ci):0.45 (0.22, 0.89)] in addition to a lower risk of causing citrate toxicity [p50.042, or (ci): 0. 24 (0.06, 0.95)]. age had a significant effect on having a hypotensive event [p50.04, or (ci):1.1 (1.0, 1.1)] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a 10 year period and compared it to published literature. study design/method: we conducted a 10-year retrospective study of ta procedures performed and aes were classified according to criteria described in table 1 . during the study period, ta were performed using cobe spectra (software versions 4.7 and 6.1) and since 2013 the spectra optia apheresis system (version 8.0). literature search was conducted for data published on aes associated with ta. four studies from us and 13 non-us studies (canada, europe and japan) were analyzed. trend for ae rates from 2007-16 was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was 6.9% (396 of 5,684 procedures) during 10 year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher (8.5%, p<0.00001) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho 0.7, p50.002) over the 10 years and significant down trend of moderate and severe aes with a spearman rho of -0.64 (p50.04) and -0.83 (p50.003) respectively. there were no fatalities during the study period. majority of aes were grade i (60%) and grade ii (28%): 32/5684 (0.6%) procedures were not completed due to aes. comparison of aes [6.9% (396/5,684)] to both european [11.2% (n513, 12, 256/ 109, 842) ] and other us studies [13.6% (n54, 860/6,324)] showed a statistically significant difference (p<0.0001). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table 1) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether 200 platelet donors with a donation activity of up to 150 platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within 2 hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* 1 , jordy jurgens 1 , jacoline buchner-doeven 1 , joris roelofs 1 , philip spinella 2 , jennifer a muszynski 3 , carel goslings 1 and nicole juffermans 1 . 1 academic medical center, 2 washington university school of medicine, 3 nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells (14 days old) and platelets (5 days old) by washing. plasma was filtered through a 0.22um filter. rats ($350 grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $30% of their estimated blood volume, which was calculated to be 57ml/kg. hemorrhage continued until a mean arterial pressure of 40mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to 4h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of 17ml/kg of blood products in a 1:1:1 ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april 2013, which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and 30-day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january 2008 and july 2015 (n53535). because of missing data on patient characteristics, 257 patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january 2008 to march 2013, n51987) and after (april 2013 to july 2015, n51291) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of 18 baseline variable, generating 969 pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common (8.7% vs 3.7%, p<0.001) and ventilation time was longer (15 h vs 13 h, p50.04) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group (2000 ml vs 2200 ml, p50.009; and 1265 ml vs 1460 ml, p<0.001, respectively). however, 30-day mortality was not statistically different between the groups (1.6% vs 1.4%, p50.82). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april 2014, www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over 12,000 joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table 1 ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy16. length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of 4-factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: 4-factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in 2015. marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh5 rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p5.005) and ptt (p5.05) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p5.03) and plasma (p5.04) after off-label use was significantly greater than on-label use. 20 cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were 5.5 times (p5.0072) more with cell saver or anh, and 5.3 (p5.0130) times more with cpb. post-pcc thromboses were identified in 6 cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every 3 days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after 7 days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least 4 days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to 7 days. the transfusion service medical director reviews the case and gives final approval. we observed only 1 patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight (38) patients were in-patients continuously until delivery. five patients were discharged prior to delivery-1 moved to another state, 1 was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was 17 days (range 0-63). six (6) patients delivered within 3 days of approval. after approval, the mean number of additional specimens per patient was 2.1 (range, 0-9). no patient required transfusion prior to delivery. five patients received transfusion of at least 1 rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only 6 patients delivering within 3 days of approval for extended specimens, 37 patients avoided collection of at least 1 specimen each, and 16 patients avoided at least 4 collections each. since new antibodies are not detectable for at least 10 days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to 7 days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* 1,2 , jan m. binnekade 1 , benjamin nota 2 , pieter r tuinman 3 , kirsten van de groep 4 , olaf l cremer 4 , janneke horn 1 , marcus j schultz 1 , robin van bruggen 2 and nicole p juffermans 1 . 1 academic medical center, 2 sanquin research and landsteiner laboratory, 3 vu university medical center, 4 university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in 2 tertiary icus in the netherlands comparing 30 patients who developed ai during icu stay with 3 control groups: 30 non-anemic patients with sepsis, 30 non-anemic patients without sepsis, and 10 patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron (15.4 vs. 2.9 mmol/l, p<0.001) and transferrin saturation (53 vs. 9 %, p<0.001), and low ferritin (104 vs. 645 mg/l, p<0.001). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells4life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately 2.5 x 10 7 cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to 65%, whilst leaving 25% of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within 30 minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than 1% of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately 65% of the cd341 fraction post separation and freeze thaw (table 1) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table 1) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd451cd611) and early projenitor cells expressing oct4 and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: 1. routine recovery of the wcf at levels higher than current methods, independent of volume. 2. higher percentage recoveries of all cell types tested than can be achieved with existing methods. 3. markedly higher post-thaw recovery of viable nucleated cells than any current methodology. 4. almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd341 target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving >0.5x10 9 lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd341 cell target of 4.0x10 6 /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd341 yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf 1 plerixafor (g1pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< 0.05 considered significant. results/finding: 110 no alc and 159 alc collections occurred among the 50 patients. fenwal amicus was used for 91% of the no alc and 99% of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was 5 hodgkin's and 45 non-hodgkin's lymphoma (no alc); 7 hodgkin's and 43 non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc 49.3, lymph 2.0x10 9 /l) than alc (wbc 39.1, lymph 1.2x10 9 /l). equivalent whole blood (corrected for ac) was processed for no alc (16.4l) and alc (17.1l). for alc group, extra collections beyond cd341 target were: 0 days: 24%, 1 day: 36%, 2 days: 22%, 3 days: 16%, and 5 days: 2%. significantly more patients were mobilized with g1pl in no alc group (n581) than alc group (n560) and 42 collections in alc group had mobilization discontinued after cd341 cell target reached. there was no significant difference in g (13.2x10 9 lymph) compared to g1pl mobilized collections (13.0x10 9 lymph); both were significantly higher than the collections where mobilization had been discontinued (5.9 x10 9 lymph). days to wbc engraftment (13.5 no alc vs 13.0 alc) and platelet engraftment (13.0 no alc vs 12.0 alc) were not significantly different. median number of collections for no alc (2) and alc (3) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the 0.5x10 9 lymph/kg or even the 0.3x10 9 lymph/kg targets. implementation of a lymph target increased patients obtaining 0.5x10 9 lymph/kg from 40% to 54%. only 12% had <0.3x10 9 lymph/ kg. discontinuation of mobilization once cd341 cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* 1,2 and nicolas pineault 2,3 . 1 canadian blood services, 2 university ottawa, 3 canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost 48 hours at room temperature (rt) as long as units are cryopreserved by 48-hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n53) were split with one half processed immediately (baseline 8-12 hours) and the second after 43 hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd451 cells and cd341 cell (n53). primary nsg mice were transplanted with a ucb cell dose that contained a total of 7,500 annexinv neg viable cd341 cells. the latter was done to avoid any bias towards one group or another. short term platelets (190 vs. 140 hplt/ml, p50.06) and leucocytes (1.2% vs. 0.2% hcd451, p<0.02) engraftment at 4-weeks were significantly reduced in stored mice vs. baseline (n53), and similar results were observed long-term at 16-weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated 3 months post-transplant. strikingly, the frequency of human cd451 bm cells was 10-fold greater in baseline vs. stored mice (p<0.01, n52). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured 22-weeks post-transplants were reduced by 30% in unit 1, and by 80% in unit 2. conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($1mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to 97%, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of 1sec). total lymphocyte recovery was 43% and monocyte concentration was reduced 76%. furthermore, in a two-pass process platelets were reduced by 75%. in a 12-fold parallel system we tested rbc separation from plasma and achieved 90% separation at 72ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past 15 years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from 9 clinical protocols from 2008-2016. an infusion reaction was defined as any symptom from the time of nk cell infusion up to 4 hours afterwards. a severe reaction was defined as any symptom with grade 3 or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r 3.3.1. two major endpoints of interest were: 1) infusion reaction with any symptom and 2) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of 127 nk cell infusions. there were 119 (94%) patients with an infusion reaction of any symptom and there were 37 (29%) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median52.55, p50.42) and those with severe reaction (median52.52, p5 0.42). infusion rate (ml/min/kg) was also similar among those with any reaction (median50.03, p50.43) and those with severe reaction (median50.03, p50.15 respectively). incubation of nk cell product overnight in il-2 vs il-15 had similar reaction rates for those with any symptom (88% had reaction with il-2, 86% had reaction with il-15, p50.94) and those with severe reaction (28% had severe reaction with il-2, 24% had severe reaction with il-15, p50.80). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median52.44 x 107) versus those without (median51.92 x 107, p50.02). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade 3 or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan 1 , maryanne c herzig* 1 , barbara a christy 1 , james a. bynum 2 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at -80. mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at 50-60,000 cells/ well and cultured in 96 well plates for 4-48 h in their respective medias. on day 0, mscs were washed, resuspended in pbmc media and incubated with or without 150,000 freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, 0-5 lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by 72 h, with >6 fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was 10.0%. inter-assay variation of msc preps run under identical conditions was 7.5%. inhibition of pbmc proliferation was graded from 0-100% over the range msc concentrations therefore an ec50 of msc cell number resulting in 50% suppression of pbmc could be determined for each msc prep. this ec50 however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within 72 h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute 10% or more of the us blood supply. differences between donors 16-18 years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged 16-49 were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the 2015/16 academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml) were estimated for 16, 17, 18 and 19-49yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of 4265 donors contributed 6219 donations. donors were evenly split by gender, 66% were ft donors, and 87% were 16-18yo. ft and rpt 16-18yo donors had on average lower ferritin values at enrollment (p<.0001), and a greater percentage were iron-depleted than donors 19-49yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged 16-18 have sharply higher risk for iron depletion than donors 19-49yo. odds for lf were 4 to 6 times greater in the younger donors, and for ais were 3-to 4fold higher. preliminary statistical models indicate 16yo donors may have greater risk for lf than 17 or 18yo by 4 to 5 percentage points, controlling for other factors (p5.06). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in 16-18yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on 12/19/ 2016 by a large blood collector. testing was performed on successful 16-18 y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < 20 ng/ml in females (f) and < 30 ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations (12 months for f and 6 months for m) and counseled to take 18-28 mg of elemental iron daily for 60 days. for m and f, a ferritin < 12 ng/ml indicated absent iron stores (ais) and < 26 ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! 20 ng/ml in f and ! 30 ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior 24 months. an appreciable number of donors with no rbc donations in the prior 24 months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* 1 , joan williams 1 , michelle humphries 1 , nancy haubert 1 , ben reynolds 1 , michael phillips 1 , randall spizman 1 , ralph r vassallo 2 , hany kamel 2 , sally caglioti 1 , german leparc 1,3 and phillip c williamson 1 . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. 1 new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. 2 study design/methods: over 28,000 serum samples from donors aged 16, 17 and 18 years were analyzed for ferritin levels using the beckman coulter au680 instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. 3 results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba1c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among 21,007 adolescents (ages 16-19; 61.5% female) who donated blood from 2015 to 2016. study design/method: abnormal risk factor levels were defined as hba1c ! 5.7%, sbp/dbp ! 120/80 mm hg and tc !170mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of 2 or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table 1 shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, 11,283 (53.7%) adolescents had at least one abnormal risk factor (61.8% of males, 48.6% of females). of these, 8,709 adolescents had isolated abnormal risk factors, and 2,574 adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as !13.5 g/dl for men and !12.5 g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from 52 blood services worldwide and complete data were available for 25 blood services. deferral percentages for low hb varied from 0.01% to 8.81% among male donors and 0.03% to 46.73% among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with 53% lower hb deferral rates in men (95% confidence interval [ci] 11% to 75%) and 61% lower rates in women (95%ci 15% to 82%). iron supplementation was associated to 57% lower hb deferral rates among women (95%ci 22% to 76%) but there was no evidence of such an effect among men (p50.680). each one-week increase in minimum donation intervals resulted in 8% lower hb deferral rates among women (95%ci 1% to 14%) but not among men (p50.454). at the 5% level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the 5 previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: 9.15% of all candidates for wb donation were deferred in continental france in 2015. deferral was significantly more frequent in women (11.16%) than in men (7.29%), due to anemia in 24.41% of deferred women and 9.79% of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from 20 to 30 weeks. analysis (table) identified 3 main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the 5 previous years. conclusion: the 3 main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, 2 days stored or 35 days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, 2 ng/kg). blood was sampled every 2 hours up to 8 hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from 1.4e108 (iqr 8.3e107-1.9e108) /ml in the fresh product to 1.7e110 (iqr 7.9e109-2.3e110/ml; p<0.01) in the stored product (p <0.001), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within 6 hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: 200 cases of taco and 405 matched controls were enrolled from 20,845 transfused patients who received 128,263 blood components from may 2015 until july 2016. taco incidence was 1 case per 100 patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation (71% vs. 49%; p < 0.001), experienced longer intensive care (4 vs. 3 days; p50.04) and hospital length of stay following transfusion (10 vs. 7 days; p< 0.001), and had higher mortality (21% vs. 11%; p50.02). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march 2016 fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from 5 to 7 days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july 2008 and to routinely extend ap outdate to day 7 since february 2016. this study reports a 103 month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july 2008-january 2016, ap underwent rt on day 4. day 6 and 7 units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day 8 had a second rt performed. from february 2016-january 2017, ap underwent rt on day 5 with routine outdate extension to 7 days by performing a second rt on day 6 and a third rt on day 7, as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type 1) or repeat rt positive with negative confirmatory culture (type 2). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july 2008, 20,010 ap were entered into inventory. of these, 11,840 (59%) were transfused prior to rt testing. the remaining 8170 (41%) underwent rt on day 4 or day 5. of these 43 (0.5%) were rt positive (29 type 1 fp, returned to inventory; 14 type 2 fp, discarded), leaving a total available inventory of 8156 units tested by rt. of these, 5631 (28% of original inventory) were transfused before the end of day 5 and the remaining 2525 (13% of original inventory) reached a day 5 outdate. a total of 1561 (8% of original inventory) were transfused on day 6 or day 7. of these, 768 underwent a second rt on day 6 (2 rt positives; 1 fp type one and 1 fp type 2) and 233 underwent a third rt on day 7 (no positive results). a total of 964 (5% of original inventory) outdated on day 7. of these, 754 underwent a second rt on day 8 (no positive results). conclusion: to date we have performed 9925 rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted 13% to only 5%. a total of 1522 ap have been tested twice by rt (768 on day 5 and 6; 754 on day 4 and 8) with 2 (0.1%) positive results, both of which were deemed fp by repeat testing or culture. a total of 233 units have been tested 3 times (day 5, day 6 and day 7) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every 24 hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with 8 to 10 million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only 5 cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in 2007. contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between 10-10,000 parasites/ml. each parasite concentration in wb was tested x2. an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at 48c for up to 42 days; platelets were stored at 228c (rt) under agitation for 5 days and plasma was frozen at -208c. aliquots for culture were removed weekly from rbcs, daily from platelets and after 30 days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at 278c for detection of live parasites for up to 16 weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at 48c, rbcs from all units spiked with 10,000 parasites/ml were positive for up to 21 days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with 1000 parasites/ml were positive for up to 7 days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with 10,000 and 1000 parasites/ml were positive up to 5 days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at 48c for up to 3 weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* 1 , marion lanteri 2 and larry corash 1 . 1 cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom (2006 -2015 ), french (2006 -2015 , swiss (2011 -2015 ), and belgium(2009 -2015 hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately 1.35 million dlvbc-screened were issued with a 7 day outdate after release into inventory 3 days after collection, and $2.3 million amotosalen/uva-treated pc were released into inventory on day 1 or 2, with a 5 to 7 day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored >2.83 million conventional, non-dlvbc-screened pc and recorded 58 str and 9 fatalities. concurrently, zero definite and 2 possible str were reported with 607,871 amotosalen/uva-treated pc, significantly fewer than with conventional pc (table 1 ) (20.5 str per million vs. 0.0 per million, p<0.001). one definite, 1 possible, 7 undetermined/indeterminate non-fatal str and 5 contaminated "near miss" pc were reported with 1.35 million dlvbc-screened pc between 2010 and 2015, for a reduced falsenegative rate compared with the prior five years (3.7 str per million vs. 16.3 per million, p <0.05). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october 2016 for all platelets received at our institution. at time of receipt at the blood bank (day 3 post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, 10 ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at 35c for three days. results/finding: a total of 9473/11,066 (85.6%) platelet products were successfully cultured (934/1373 [68.03%] and 1842/1912 [96.3%] in october 2016 and march 2017 respectively). over the 6-month period, two true positive cultures were obtained (incidence of 1 in 4736 platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us16.83 per product tested. the cost per averted case was $us79,707. conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january 2009 and december 2016. the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the 7-year study period, a total of 3280 transfusions reactions were reported, 18 of which were bcptr (0.55% of transfusion reactions). of the 18 bcptr, 15 (83%) were associated with apheresis platelets, 2 (11%) with red blood cells, and 1 (6%) with plasma. recipient diagnoses spanned hematologic/oncology (n512), renal (n53), cardiac (n51), autoimmune (n51), and obstetrics (n51). an organism was identified in both the blood product and recipient in 10 (56%) cases; in 6 (33%) cases an organism was grown in the blood product but not the recipient; and in 2 (11%) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in 5 of the 6 cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever (83%), chills (67%), nausea and vomiting (50%), pain (27%) and dyspnea (22%). blood pressure (bp) decreased in 22%, increased in 17%; 61% of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie 1 , jenna lebedev 1 , linda kapp 1 , xiaohong wang 1 , meghan delaney 2 , lay see er 1 and james c zimring* 3 . 1 bloodworksnw research institute, 2 bloodworks nw, 3 university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg1-igg4), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least 29 natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all 29 known variants. study design/methods: the heavy and light chain variable regions of an anti-k1 monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known 29 igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k11 rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg2, igg3, and igg4 had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table 1). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg1 or for any of the monoclonal ahgs tested. monoclonal anti-igg3 had a blindspot for igg3-04, due to the shorter hinge region on igg3-04. no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* 1 , yves dominique pastore 1 and maryse st-louis 2 . 1 chu sainte-justine, 2 hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since 2008, our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of 2014, 205 scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september 2016, 117 (57%) patients had been transfused and 14 had antibodies with known blood group antigen specificity: anti-c, anti-e (2), anti-hrb, anti-fya, anti-jka, anti-jkb (2), anti-s, anti-m, anti-sc2, anti-leb (2). seventeen patients (8.3%) were either d2 or partial d. rhce results showed that 163 patients expressed a normal c antigen and 32 expressed partial c. as for e antigen, 163 had a normal antigen, 38 bore a partial antigen and 3 were weakly expressed. fy(a2b2) phenotype was found in 182 (89%) patients. a total of 2606 genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos 1 , emilia sippert 1 , mayra dorigan de macedo 1 , sheila fatima perecin menegati 1 and lilian castilho* 1,2 . 1 hemocentro unicamp, 2 university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa-308a, il1b-511t cytokine polymorphisms, rhag 808g>a and hla-drb1*15 alleles may predict a good responder phenotype (sippert et al, transfusion 2017) and that rhag 808a and hla-drb*15 alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included 96 non-alloimmunized patients with scd, homozygous for hbs, receiving a range of 5-289 rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa-308g>a, il1b-511c>t) and the rhag 808g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among 96 non-alloimmunized patients, 21 were homozygous or compound heterozygous for rh variant alleles. from those, 6 had rhag 808a and/or hla-drb*15 alleles and at least one cytokine polymorphism (tnfa-308a or ilb1-511t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other 15 patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and 2 were confirmed by sequencing. the third sample was found to be rhce*cevs.01,rhce*cebi on sequencing (predicted phenotype v1,vs1). the 3 samples were typed as v1 (or ce s ) and vs1 (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[712g]in 2 samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c1 by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the 2 methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising 80% of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies (20% of patients) bound young and old rbcs with no apparent prejudice. band-3 is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band-3 is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band-3 to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band-3 aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from 22 patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band-3 tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing 5 type i and 5 type ii patients, we found that type i is characterized by 5 percollv r fractions (similar to healthy storage-matched controls) but increased band-3 tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by 3-4 percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band-3 tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band-3 suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band 3 suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* 1 , burak bahar 1 , jeanne hendrickson 2 , krystalyn e hudson 3 and christopher a tormey 1 . 1 yale-new haven hospital, 2 yale university, 3 background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam30 abstract algorithm, e value51x10 -6 , word size5 6, gap costs: existence59 exten-sion51). search results were restricted to bacteria and fungi, with a selective threshold of >80% identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from 162 alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b5-0.0017, r 2 50.624 & p50.0197); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of 162 alloimmunized patients reviewed, 105 were culture-positive. of these, 76% of the anti-c/c group (13 of 17 patients) and 16% of the anti-k group (7 of 43 patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from 0 -11.1%. overall, 21.9% (23 of 105 patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of >80% sequence identity. while 27.6% (29 of 105) patients reviewed had positive cultures for klebsiella species, 62.1% of these (18 of 29 patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed 109 tegs performed on 76 patients undergoing cv surgeries at our institution from jan 1 to dec 31, 2016. no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the 56 tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at 30 minutes (min) all within reference range), "hypocoagulable" (r>10 min, k>3 min, a<53 degrees, ma<50 mm) and "hypercoagulable" (r<5 min, k<1 min, a>72 degrees, ma>70 mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of 56 tegs analyzed, 37 patients (66%) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients (8% vs. 32%, p50.02). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused (32% vs. 11%, p50.07). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused (100% versus 33%, p50.06). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from 670 liver transplants, performed from 2011 to 2015 in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, 670 olts were performed. a total of 345 patients was submitted to cs. the median age was 51 years (range 10-78 yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in 31,6% of the patients. the average meld score was 29,6 6 9,4 and it was slightly higher in the cs group (31,3 vs 27,9, p<0,001) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was 8856 6 4503 ml and mean reinfused blood volume was 914 6909 ml. allogeneic blood transfusion was required in 71,8% patients in the cs group, compared to 46,7% patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group (2,4 units vs 3,39 units, p<0,001 background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july 2015-december 2016 was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp (27 and 15 products, respectively) however, obp wastage occurred more frequently in the 18 month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations (23 versus 4 products). this is skewed by one month when 20 products were wasted due to expiration of product on the floor. cooler-related issues (6) and products dwelling too long out of a controlled environment (5) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were 1.7%, 0.3%, and 2.3%, respectively, with a total exsanguination protocol waste rate of 1.33%. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p50.176). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a 17 year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with 5 units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d1) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d(table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* 1 , edward smith 2 , thomas brown 2 , foeks jeremy 2 , metcalf suzanne 2 , james johnson 2 , peter davis 2 , karafa sw badjie 1 and abba zubair 1 . 1 department of laboratory medicine and pathology, transfusion medicine, mayo clinic, 2 department of anesthesia, mayo clinic background/case studies: our institution performs an average of 398 solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a 29 y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused 2 o(1) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused 10 more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the 2 o(1) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in 1% of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a 29 y.o. female patient should not have received o(1) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with 10 admissions during the 5 hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight (107 kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o(1) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a 50 y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o(1) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately 375,000 surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from 6-16%, whereas transfusion rates for vaginal and robotic pfd surgeries range from 0.2-1.6% and 0.3-1.4%, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately 15% of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may 2015 -may 2016 in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p<0.05. results/finding: we identified 66 patients for analysis, of whom 65 (98.5%) had a preoperative t&s ordered. two (3.1%) of these 65 patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine (90.8%) of the 65 patients required a second abo/rh specimen per hospital protocol; 51 (86.4%) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table 1) . no abo/rh discrepancies were identified. one patient received 1 unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution (90.8% vs. 15%, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n527) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of 1.05 g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs-1000i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant 1 removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), 5 units were subjected to the volume reduction while recording the time needed to process all 5 units. this was performed twice for a total of 10 units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of 72% (range 49%-87%). in units between 21 and 42 days (n510), the estimated mean residual k1 was 1.89 meq (range 0.61 to 2.21). in the two mock mtp trials, the time to complete the procedure was approximately 50 minutes and we estimate an additional 5-10 minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal (3) and caesarian (2) births. uc collections were divided into 3 segments to test 3 conditions. segment explants were placed on 0.1% gelatin-coated gridded tissue culture plates (32 explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of 21 days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the 2 remaining tissue segments were soaked in (ab/am) saline solution for 1 hr and 24 hrs at 48c, respectively. tissue segments were frozen in cryo bags with a proprietary 10% dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process 3 or 4 times the total blood volume (bv) of the patient, up to a maximum of 25 liters (l) per procedure, to obtain peripheral blood cd341 stem cells. as a consequence, a patient often would need to spend 6 hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd341 cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our 2016 collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd341 pre-count and cd341 yield, normalized per liter of blood processed, was derived utilizing the patient's cd341 pre-count, the patient's weight in kilograms (kg), and the target cd341 dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd341 stem cells. the initial equation was modified to add an additional 15% to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in 8 patients, representing both allogeneic and autologous donors, the average blood volume processed was 14.8 l. the range was 4.9 l -21.6 l. the target dose was achieved in all patients. our previous practice for these 8 patients would have required, assuming a standard 4 bv procedure, processing an average of up to 28 l per patient, with a range of 20-62 l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd341 yield. the result was a high correlation between these two ratios (r 2 5 0.92), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r 2 5 0.92, confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing 2 hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses (1-2) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd341 mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june 2015 and march 2016. patients (n525) evaluated were diagnosed with malignant lymphoma (n515), multiple myeloma (n59) and primary amyloidosis (n51) and were mobilized according to standard protocols. collection cd341 cellularity target was established ! 2x10e6/kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by >65 years old, previous fludarabine, lenalidomide, or bendamustine treatments or !2 previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd341 count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v4.0. results/finding: the media (range) general collection parameters were: cd341 (day 5) 27.50/ml (4.5-157.5/ml), blood volume processed 23204ml (9718-39618ml) and 4.96 (2-7.30) exchanged volemias. seventeen patients were considered bad mobilizers, 7 needed plerixafor and 5 had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p50.071]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n52), fever (n52) and flu syndrome; all grade 1]. two patients could not undergo hematopoietic stem cell transplantation due low cd341 cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd34) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with 20% fetal bovine serum. bmsc and amsc at passage 2-5 were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses (1.5 3 10 4 /ml, 7 3 10 4 /ml and 1.5 3 10 5 /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par4) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within 30min and 2hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: 334 6 35 seconds, versus low, medium and high doses of amsc (145 6 2, 111 6 6, and 75 6 12 seconds), and bmsc (155 6 2, 90 6 10, 80 6 7.0 seconds), p<0.05), clot formation time (cft, p<0.05) and increased alpha angle (p<0.05) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par4. no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* 1 , neil bagamasbad 2 , reynold dilag 2 , melissa nasser 2 , nicole bauer 2 , jennifer wheeler 3 and mary berg 1 . 1 department of pathology, university of colorado -anschutz medical campus, 2 department of medicine, division of hematology, university of colorado hospital, 3 scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from 120 collection procedures using the mnc protocol and 173 collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including 36 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd34)-positive (cd341) throughput, cd341 collection efficiency (ce%), platelet loss 71a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included 14 and 22 allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd341 throughput was significantly higher in the cmnc group than the mnc group. the cd341 ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded 20 ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient #1, originally typed as an a2, had 1 bone marrow donor and 2 cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient #1 is now typing as type o. patient #2 was originally typed as a2 and received a bone marrow transplant from a type b donor. patient #2 is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient #1 and patient #2 indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with 20% fetal bovine serum under either normoxia (20% o 2 ) or hypoxia (3.5% o 2 ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd90/cd29 and cd45 were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc (1.5 3 10 5 /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by 15%, but depressed the growth of amsc by 30% at day 5 in comparison to normoxia. both bmsc and amsc equally expressed cd90 and cd29 but not cd45 under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: 468 6 64 (control), versus 170 6 13 (bmsc), and 195 6 60 (amsc) seconds) by natem. hypoxia also significantly shortened ct (165 6 20 (bmsc), 169 6 50 (amsc) seconds, p<0.05 as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd341 cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd341 cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd341 cells. study design/method: cryopreserved cd341 cells from 2 healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef1-alpha-yfp lentivirus (2.5% concentration) and media (x-vivo-10, human serum albumin(hsa), 100 ng/ml each of cytokines (scf, tpo and flt3-l) over 2 days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of 5% dmso, 6% pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd34 1 cd38 -cd45ra -cd90 1 cd49f 1 cells) phenotyping and cfu assays were done following first thaw (pt1), post-transduction (ptxn) and second cryopreservation-thaw (pt2). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd34%, cfus were similar before and after pt2. hscs ranged from 824 to 1655 cells/10 6 cd341 cells in the pt2-tr arm compared to a range of 286 to 1416 /10 6 cd341 cells after pt1. viability, % cd341 and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt2 (table) . conclusion: dec of mpb human cd341 cells decreases tnc recovery, but has minimal effects on cd341 cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd34% in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd341 cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd341 cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of 20% hydroxyethyl starch, 18% human serum albumin and 10% dmso at final concentration. pbsc were cryopreserved by direct immersion on -808c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of 03 consecutive days of neutrophil count >0.5 x10 9 /l and platelet count >20 x10 9 /l after 07 days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd341 was below 10 x 10 6 cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd341 on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd341 on the day of the collection versus collected cd341 per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd341 was calculated. final laboratory count of cd341 per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss 23 software. results/findings: among patients collecting hpc for autologous transplantation, 69,23% needed only one day of hpc harvesting, while 25,64% needed two days and 5,13% needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was 49 1-2,91%. after comparing predicted values with cd341 collected in the final product, we found a very strong correlation of 0.873 (p<0.01) for patients and a strong correlation 0.653 for healthy donors (p<0.01). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; !95% cd90, cd105, cd73 and 2% cd14, cd19, cd34, cd45, hla-dr) study design/method: umbilical cord tissue (n510) was washed, blood vessels removed, cut into 0.5-3mm pieces, and washed twice in saline. fresh tissue was immersed in 0.9% saline for same day culture, while frozen tissue was cryopreserved for at least 24 hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a 25cm 2 tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for 10 minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures >80% confluence. all cells were tested on an msc flow panel at passage 2 just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of 8 days (fresh 5 7.8, frozen 5 8.1), and 13 days (total) for the msc's to reach passage 1 (fresh 5 12.6, frozen 13.4). all cells were ready for flow analysis in approximately 3 weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p 5 0.81), or their growth rates (p > 0.05 for all). flow cytometry showed average !95% for positive markers and 2% negative markers. there was no statistical difference between fresh and frozen flow result (p > 0.05). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of 2 up to 11 years (2004 to 2017) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with 10% concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a 378 c water bath and 0.5ml aliquots were diluted at a 1:1 proportion with 5% human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using 7-aad marker through flow cytometric analysis. results/finding: ucb storage period was 7.24 years (mean) and cell recovery was 86.31% (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p 5 0.11). post-thaw cell viability of 63.13% (mean) showed no statistically significant correlation with unit storage period (p 5 0.07). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for 5 years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln 2 vapor in a dmso-based cryoprotectant for 5yrs. (5.49 6 0.431; n54). units were rapidly thawed and rinsed in dpbs, then 25 pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a 5x5 grid pattern in msc-supportive medium and incubated for 7 days, after which the tissue was discarded and media exchanged. cells were isolated on the 14 th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a 100% success rate. cells were positive for the msc markers cd73, cd90, and cd105 (98.8 6 0.7%, 98.7 6 0.6%, and 97.8 6 0.6%, respectively) and negative for the hematopoietic markers cd34/45 (1.1 6 0.7%). passage 1 and passage 2 doubling times were 1.92 6 0.47 days and 2.07 6 0.43 days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical 75a transfusion 2017 vol. 57 supplement s3 research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd34 yield prediction algorithm ines bojanic* 1 , nelly besson 2 , ivana vidovic 1 and branka golubic cepulic 1 . 1 department of transfusion medicine and transplantation biology, university hospital centre zagreb, 2 terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd341cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version 11) in adult and pediatric lvl. a prediction algorithm for cd341cell yield was also tested. study design/method: we evaluated retrospectively 67 lvl performed in 46 adult patients, and 14 lvl in 11 pediatric patients treated in uhc zagreb from march 2016 till september 2016. mobilization regimen combined chemotherapy and filgrastim; 2 poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio 1:24). in patients weighting 25kg (n59), a rbc prime was performed. cd34, lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd341cell count and cd341cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd34 values to real cd34 yield. results are presented as median (iqr). results/finding: in both groups, cd34, ly and mo ces were high. target cd34 dose was successfully reached in 1 procedure in 30 (65,2%)adults and in 9 (81.8%) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in 5 (7.5%) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd341cells and cd341cells collected/ blood volume was observed in both groups (r 2 50.97 and 0.83 in adults and children respectively, p<0.0001) suggesting cd34 yield could be predicted based on precd341cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd34 yield and observed cd34 yield (r 2 50.95 and 0.82 in adults and children respectively, p<0.001) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of 10.1(8.9-12.9)l of blood in 20 adult procedures, and 5.9(3.5-7.8)l in 7 pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd34, ly and mo ce1 were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* 1 , aniko barta 2 , arpad batai 2 , zoltan csukly 2 , zita farkas 2 , laszlo gopcsa 2 , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) 4 times per case weekly at a dose of 1 million cells/kg. clinical response was assessed 28 days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all 12 patients had received 13 cycles of msctreatment (4 dose per cycle). the median age was 47 years old (19-56) with a male/female ratio of 1:2. distribution of the original malignancies (n): acute myeloid leukemia: 6; acute lymphoblastic leukemia: 2; myelofibrosis: 1; myelodysplastic syndrome: 1; multiple myeloma: 1; t-cell lymphoma: 1. nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median 63rd day (7-455). the involved organs were skin (2), gut (4), skin and gut combined (7) and even lung in 3 cases. the median time of msc's first infusion was 274 days after the stem cell transplantation (hsct) and 165 (19-1974) days after the first episode of gvhd. 4 of the 13 cycles of msc-treatment led to complete remission (30.8%) and 7 resulted inpartial remission (53.8%). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with 83% overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is 2 million cd341 cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd341 cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the 2 million cells/kg goal. the ideal minimum cd341 cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from 55 patients to evaluate the predictive value of the cd341 cells/ml level. data was collected over 6 months from every patient who underwent a stem cell collection. four patients were allogenic donors and 51 were autologous donors. the patients' weight, diagnosis, and pre-procedure cd341 cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd341 cells collected were recorded. the collection efficiency and the cd341 cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd341 cells/ml and post-procedure cd341 cells/kg (r50.95). any patient who had a pre-procedure cd341 cells/ml count of 29 or greater had a collection of at least 2 million cells/kg. any patient who had a pre-procedure cd341 cells/ml count of 16 or less collected less than 2 conclusion: the pre-procedure cd341 cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd341 cells/kg level. to confidently know that a patient will be able to produce the desired 2 million cells/kg, a pre-procedure cd341 cells/ml count of at least 29 should be obtained. for any patient with a count below 16, they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between 16 and 29 cd341 cells/ml should be conducted. heidi elmoazzen 1 , antonio giulivi 1 , michael halpenny* 1 , lisa martin 1 , donna perron 1 , chris bredeson 2 , lin yang 1 , locksley mcgann 1 , paul birch 1 and jason p. acker 1 . 1 canadian blood services, 2 ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso (5% final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of 3 phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd34, viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in 5% dmso and 1.7% hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of 12.6 days for anc500 with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current 5% dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using 5% dmso and 1.7% hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from 5% to 40%. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is 0.24 mg/kg, therefore patients weighing >100 kg would require a second vial, thus doubling the drug cost. in 2013 we implemented a policy of capping plerixafor at 24 mg for patients weighing >100 kg. this retrospective study compares the mobilization of patients >100kg who received capped doses (2013) (2014) (2015) (2016) , with historical control patients (2010-2013) who received full or uncapped doses. study design/method: patients weighing >100 kg with crcl >50ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of 47 and 40 consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd341/cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at 24 mg for patients >100 kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd34 in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (<30yrs) have a higher pre-apheresis %cd34 level than any of the other groups, reaching statistical significance when comparing the %cd34 pre-apheresis between the youngest group (<30 yrs) and the oldest group (>540 yrs). hispanic donors show statistically similar %cd34 pre-apheresis levels over all age groups. moreover, the hispanic older age group (>540yrs) had a statistically higher %cd34 pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of 121 sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd34 level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd34 level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last 3 years was analyzed to determine the 95 th percentile, median and 5 th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc (234 x 10 6 cell / ml) and three low wbc (114 x 10 6 cells / ml) concentrations, each at high (505 ml), low (265 ml) and median (355 ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax 2 (pericell protocol, cs.430.1 kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and 7-aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting 7-aad viability of 76 6 8% [range 64-85]% and a hematocrit of 14 6 5% [9-19] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the 6 mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was 97 6 8% [64-105] with a 3 6 3% [-2-11] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of 99 6 4 [92, 104] % and a change in 7-aad viability of 2 6 2 [0, 11] % from the input product. the method was found to have a cv of 2.0%. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating 78a transfusion clinical assessment. in the first phase, 2 cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd34 7-aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to 3 hours post thaw. the second in vivo phase included use of an infusion pump for 10 consecutive autologous patients, with comparison of infusion and transplant outcomes to 18 previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the 2 products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than 20% within 1 hour, while cd341 cell viability remained stable up to 3 hours post thaw. small aggregates appeared after 1 hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were 10.8 6 1.3 and 11.6 6 1.0, respectively (p-value50.075). platelet days to engraftment for pump and drip were 17.9 6 2.2 and 20.2 6 5.0, respectively (p-value50.207). infusion rates were slightly higher for the pump group. for control patients, 2 required transfer of products to syringes due to slow infusion rate and 2 others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the 278 zikv ineligible cbus was: caucasian 52%, asian 9%, black/aa 20%, and multi-race 21%. racial distribution of all clinical cbu donors was caucasian 49%, asian 15%, black/aa 20%, and multi-race 17%, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: 78% of all ineligible cbus and 21% of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete 2,3-diphosphoglycerate (2,3-dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring 2,3-dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a 10 ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n 5 20), which were stored in sagm for 22 days, to act as untreated controls. the remainder of each unit ($270 ml) underwent treatment with the rejuvenation solution (50ml, 60minutes at 37 o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from 39 random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p 1 , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of 22 day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp 215 (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros 60. pc were centrifuged at 1250g in a sorvall rc3c1 centrifuge (sorvall, usa) for 10 min. the combination cryoprotectant dmso1dextran (cryosure dex40, germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at 37 degrees c (barkey plasmatherm) for 10 min. cpc osmolality was measured with an osmomat 030 osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru 169287 u1). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso1dextran (cryosure dex40) , as a cryoprotectant, to obtain a final concentration of 5% dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m3 were instrumental in automating that phase. pc to be frozen had an osmolality of no less than 1500 mosm/l. prp and ppp were frozen at a cooling rate of 1-38c/min and stored at -85 0 in the chest freezer for up to 24 months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru 167874 u1). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to 380 mosm/l. freeze-thaw recovery of platelets was 80% or more of the original population. defrosted pc were stored at 20-24 0 with continuous gentle stirring from a helmer platelet agitator for no longer than 4 hours before transfusion. it took no more than 30 min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over 20 min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day 4 and day 5 evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to 7 days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day 4 for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day 5. a total of 60 lrap units were tested over a 3-month period: 50 were cultured and rapid tested on day 4; 10 were rapid tested on day 5. the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were 59 true negatives (tn) and 1 false positive (fp) on day 1 when tested by bact/alert, with 60 tns on day 4. bactx testing results showed 50 tns on day 4 and 10 tns on day 5. testing using the pgd kit showed 50 tns on day 4; and 8 tns and 2 fps on day 5. fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and 30 minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within 20 minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in 100% plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day 4 and day 5 during the night shift to be accomplished without additional staffing and allows to extend outdate to 7day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for 1 year jos lorinser 1 , pieter f van der meer 1 , hans van der heiden 2 and dirk de korte* 1 . 1 department of product and process development, sanquin blood bank, 2 mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers (140 ml) is unknown. if these products can be stored at -188c it will be feasible to store this product in 3-star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at -188c or <-25 to -358c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from 500 ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <-258c for 3-12 months and controlled thawing, six different sera were used to fill a large number of mini (140 ll) containers, which were refrozen and stored at either -188c or <-258c. during storage at 3 months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <-808c. growth factors tested were pdgf-aa&ab/bb, tgf-ß1/2/3, vegf, 80a transfusion 2017 vol. 57 supplement s3 egf, fgf2. the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß1 were the most abundant growth factors, on average 35, resp. 40 ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average 11 ng/ml. tgf-ß2, egf and vegf were detected at relatively low values, resp. 3 ng/ml, 0.5 ng/ml and 0.3 ng/ml. average levels of fgf2 and tgf-ß3 were close to detection limit (< 0.2 ng/ml). the controls stored at <-808c showed for all growth factors close to 100% of the initial values in samples at t50 (moment of filling mini containers). for serum stored at <-258c for up to 12 months, most factors showed less than 2 % decrease, except for pdgf-aa and tgf-ß2, showing 6% resp. 3% lower values. for serum stored at -188c the values for tgf-ß1, egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß2 showed a decrease of resp. 9, 17 and 3%. conclusion: human serum eye drops can be stored in the new micro dose device at -188c (3-star household freezers) or <-258c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at -188c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond 1 year. ruqayyah almizraq* 1 , heather inglis 2 , phillip norris 2,3 , jennifer a muszynski 4 , nicole juffermans 5 , jelena holovati 1 and jason p. acker 1,6 . 1 university of alberta, 2 blood systems research institute, 3 university of california, san francisco, 4 nationwide children's hospital, 5 academic medical center, 6 canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n58 per method). residual platelets and white blood cells (wbcs) were measured on day 5 using flow cytometer (fc). on storage day 5 and 42, number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day 5, apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p<0.01, wbd p<0.05) and wbf (vs: apheresis p<0.0001, wbd p<0.01) methods. while rcf units yielded the lowest count of platelet-evs (cd41a1) on day 5 and 42, the highest number of platelet-evs were in apheresis (day 5) and in wbd (day 42). similarly, there was significant difference among methods in the number of wbc-evs (cd31, cd141, cd161, cd191, cd66b1) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day 42 vs day 5 in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< 200 nm) was greater than large evs (! 200 nm) in all of the products on day 5 and 42, and the highest level of evs < 200 nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p<0.05). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at 48 centigrade maryanne c herzig* 1 , crystal lafleur 2 , chriselda g fedyk 1 , sherrill j. slichter 3 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research, 3 university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least 2 weeks of storage at 48c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at 48c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a2 (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for 10, 12, 15, or 22 days after collection. units were stored for 12 days without agitation. units stored for 10, 15 or 22 days were agitated during storage with a model 400 hybridization incubator at 48c set for end over end rotation at 2-3 rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at -808c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd40l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai-1) as another fibrinolytic measure; and complement activation markers c3a, c4d, c5a and c5b-9. data was analyzed by one way repeated measure anova. results/finding: only 49 6 12% of the platelets were recovered in units stored for 12 days without agitation. these levels did not meet fda requirements of 5.5 x 10 10 platelets per wb unit. subsequently, wb was agitated and platelet recovery was 71-76%. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t0 (day of collection) and t10, 12, 15, or 22 measurements. significant elevations of pai-1 and scd40l indicate activation of platelets and inhibition of fibrinolysis (p<0.001). activated complement peptides c3a, c5a, and c4d were all elevated over time (p<0.001) while sc5d-9 was not. however, only c3a and c4d levels at t22 were above normal reference ranges at 1.30 and 1.41 times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at 48c for 10-22 days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc5d-9 reported, wb showed elevation of c3a, 5a and c4d and not sc5d-9. complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* 1 and christian todd 2 . 1 cerus corporation, 2 community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly 1.1x10 10 6 5.6x10 9 9.8x10 10 6 5.6x10 10 310 6 330 430 6 440 900 6 260 28000 6 33000 3 6 3 186 31 30 6 22 110 6 97 rcf 1.9x10 10 6 7.4x10 9 4.2x10 10 6 1.1x10 10 13 6 4 316 14 530 6 160 5100 6 2000 3 6 3 96 7 176 7 346 11 apheresis 2.4x10 10 6 2.0x10 10 1.0x10 11 6 6.1x10 10 520 6 320 700 6 310 2200 6 1900 9800 6 4100 14 6 17 7 6 5 466 15 120 6 24 wbd 6.4x10 9 6 3.1x10 9 4.6x10 10 6 1.5x10 10 350 6 140 760 6 360 1000 6 180 4400 6 2400 3 6 2 426 23 57 6 24 120 6 56 platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following 4 collection targets: 4.4x10 11 in 350ml, 6.6x10 11 in 400ml, 6.8x10 11 in 400ml, and 7.0x10 11 in 400ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of 3.0x10 11 or 6.0x10 11 was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: 64% of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was 1.34. conclusion: it is possible to treat 64% of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward 100% while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* 1 , andrey skripchenko 1 , fei xu 1 , ying li 1 , stephen j wagner 2 , pamela h whitley 3 and jaroslav g vostal 1 . 1 fda/cber/ obrr/dbcd/lch, 2 american red cross holland laboratory, 3 american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature (4-6 o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts (11 hrs ct: 1 hr 37 o c [tc]). autologous apheresis plts stored for 7-days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n59) and the same non-labeled plts were also infused into mice (n590). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts <5% were considered background. results/finding: the mean recoveries of infused plts were 51.2 6 16.7% for rt, 37.7 6 12.3% for tc and 23.1 6 8.8% for ct in humans. in mice, mean recoveries of the same plts were 24.9 6 10.3% for rt, 19.1 6 9.8% for ct and 16.2 6 6.9 for ct (mean6sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $74% and ct was $45% of rt. in mice tc was $76% and ct was $64% of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to 100% for rt plts. human tc plts had 26% auc while ct plts had 11% auc compared to rt plts in humans. in comparison, the same tc plts had 39% auc and ct plts had 26% auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are 2.4 and 1.5, respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* 1 and geeta paranjape 1,2 . 1 coastal bend blood center, 2 carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g5 was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g5 with the compomaster net software for data management. implementation was planned for a november 2014 go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g5s and compomaster in june 2014. training and validations were successfully completed and a full launch occurred mid-march 2015. device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december 2014. validation was completed and signed off in march of 2015. manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g5 system. data points were collected from 210 units bi and 302 units ai. results/finding: upon initial implementation, staff training and use, the compomat g5 was found to be easy. plt weight spread was reduced from an average of 22gm to an average of 15 gm. actual plt weights were reduced from an average of 63gm to 59gm, resulting in an average increase in recovered plasma of 3.78ml per unit. plt count on average increased from a count of 1435 to 1506 (10 3 /mm 3 ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by 31.8% after implementation of the compomat g5 and our plt concentrations increased on average by 5%. we were able to consistently produce a smaller volume plt (average 59 gm), which gave us 3.78ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell 1 , angela hill 1 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: 13 abo/rh matched lr sagm rccs were pooled and split to produce 6 large (354 ml) and 6 small (244 ml) rccs. the rccs were stored to 14 d and glycerolized manually by mixing 400 ml of glycerol with the rcc in a 2000 ml freezing bag. units were frozen at -658c for ! 72 h before being removed from frozen storage and thawed in a 378c water bath. 3 large rccs and 3 small rccs were deglycerolized using the organization's current procedure on the cobe 2991 cell processor prior to re-suspension in 0.9% saline, 0.2% dextrose. the remaining rccs were transferred into a 1l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of 75 6 5%, and deglycerolized in a 275 ml centrifuge bowl on the acp-215 with re-suspension in as-3. rbc quality was tested at 24 6 2 h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p50.006, acp215: p50.007) and lower cell recovery (cobe: p50.002, acp215: p<0.001) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe 2991 had higher hemolysis (p<0.001) and supernatant potassium (p50.001) than did small volume rccs. large cobe 2991 rccs had higher hematocrits (p50.033), hemoglobin (p50.006), and recovery (p50.001) than did large acp-215 rccs. however, all cobe 2991 rccs had higher (p<0.001) hemolysis (0.99 6 0.24 %) levels than did acp-215 rccs (0.31 6 0.02 %). cobe 2991 rccs failed to meet regulatory hemolysis standards of 0.8%. conclusion: addition of a 400 ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as-3 and storage for 24 6 2 h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp-215 cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using 0.9% saline and centrifugation and the semi-automated washing method (sam) using the cobe 2991blood cell processor. study design/method: in this study, 20 units of single donor platelets were evaluated (10 washed using the mm and 10 washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas 6000. results/finding: table 1 shows that the average platelet recovery for the sam (92%) was significantly higher compared to the mm (82%). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took 10-15 minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas-3) for input platelet doses of 2.9 to 8.0 3 10 11 platelets in 255 to 420 ml of 47 to 68% plasma and 32-53% pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas-3 containing doses of 6.0 to 12.0 3 10 11 platelets in a volume of 420 to 650 ml. study design/methods: apheresis pcs (amicus v r ) were collected in 35% plasma and 65% pas-3. one study was performed at the nominal dose (9.2 -10.0 x10 11 platelets), volume (558 -629 ml) in 65% pas/35% plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition (9.7 -11.8 x 10 11 platelets in 593 -659 ml) using either single or pooled donations. input pcs (n520) were treated with the intercept ts set by the end of day 1 post collection; the incubation time in the compound adsorption device (cad) container ranged from 4 to 16 hours and the intercept treated pcs were stored in 3 containers (n560). day 5 and 7 post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas-3 treated in the intercept ts set demonstrated acceptable in vitro function (table 1 ). all intercept treated pcs had ph(228c) !6.2. platelet dose and volume recovery post-treatment ranged from 82% to 99% and 88% to 92%, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through 7 days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing 100% plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n56). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n56). numerous in-vitro quality markers (plt concentration, atp, po2, pco2, ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days 1, 3, 5 and 7 for apheresis pcs, and on days 2, 3, 5 and 7 for wb-derived pcs. two flow cytometry assays were used to evaluate cd62p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion 2017 vol. 57 supplement s3 results/finding: platelet recovery was 92 6 5% and 81 6 10% for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells (3% 6 1 (test), vs. 1.7% 6 0.5 (ctl) on day 5) and a higher rate of cd62p expression than control pc units (58% 6 7 (test), vs. 23% 6 6 (ctl)) on day 5). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* 1 , lorraine blagg 2 , christi e marshall 1 , herman woodson 1 , sean erony 1 , krishna patel 1 and eric gehrie 3 . 1 the johns hopkins hospital, 2 johns hopkins hospital transfusion medicine dept, 3 johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. 340 intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day 4. as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day 3. the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of 340 lrap were tested. 335 lraps initially tested negative by bactx, while 5 lraps initially tested positive by bactx. all 5 initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was 98.5%. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only 340 platelet units. the expected rate of bacterial contamination of platelets is less than 1 per 2000 units. the 1.5% initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as-1 and cp2d/as-3 rbc alan d. gray* 1 , matt landrigan 2 , pamela whitley 3 , michael wellington 3 , sherrie sawyer 3 , shalene hanley 3 , emily rondeau 4 , louise herschel 4 , neeta rugg 5 , patricia a.r. brunker 3 , shawnagay nestheide 5 , jose cancelas-perez 5 , larry dumont 6 and zbigniew m. szczepiorkowski 7 . and 2,3-dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for >24 hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood (530-550 ml) was collected and processed at 3 sites into leukocyte-reduced rbc (a total of n563 cpd/ as-1 and n564 cp2d/as-3). 50 ml of rejuvenation solution (citra labs) was added to each rbc on day 35 (d-35), incubated for 60 minutes with agitation at 378c water bath (helmer dh4), washed (haemonetics acp215), and stored in as-3 at 1-6 oc for 7 days (d-36 through d-42). in vitro recovery (%) was calculated and hemolysis, atp, and 2,3-dpg were determined on day 0, d-35, d-35 after rejuvenation and washing (postrjv), d-36, d-38, d-40, and d-42. all units were cultured on d-35 postrjv and on d-42, and then concentrated by centrifugation on d-42. results/finding: in vitro rbc recoveries were 95.7% and 95.5% (as-1 and as-3, respectively) and no bacterial growth was observed. hemolysis on d-42 was maintained <1% in 58/63 (92%) as-1 units and 63/64 (98.4%) as-3 units. all as-1 and as-3 units (100%) had hemolysis <1% following concentration by centrifugation. morphology score was reduced to 77% (as-1) and 74% (as-3) by d-35, restored after rejuvenation (91%, 92%, respectively) and maintained through d-42 (>90%). atp was restored and maintained above fresh levels after rejuvenation. 2,3-dpg was restored above fresh levels and was maintained !80% of fresh levels through d-38. all values were significantly different compared to d-35 except as noted (p<0.001, paired ttest) ( table 1) . conclusion: rejuvenation of stored rbc restores atp and 2,3-dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d-42 when compared to nonrejuvenated rbc on d-35. this study is funded by zimmer biomet. storage >24 hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante 1 , jason p. acker* 1,2 and jelena holovati 1 . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during 42-day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and 2,3-dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome 1 rejuvesol-treated (l1r). the prbcs were incubated for 1 h at 378c with hepes-nacl (sham), liposomes (dopc:chol, 7:3 mol%, 2 mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and 2,3-dpg at day 42 hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control (0.60 6 0.06%): l (0.53 6 0.01%, p50.042), r (0.43 6 0.02%, p50.004), l1r (0.48 6 0.06%, p50.020). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r (0.55 6 0.01, p50.010) and l1r (0.55 6 0.01, p50.010) treatments compared to s (0.53 6 0.01) but not l (0.53 6 0.01, p50.936). rbc rigidity (kei) increased in all treatments compared to sham (1.19 6 0.07): l (1.28 6 0.06, p50.025), r (1.44 6 0.17, p50.010) and r1l (1.44 6 0.06, p50.004). aggregation amplitude was significantly increased by r treatment only (24.07 6 1.67 au vs. 19.12 6 1.38 au, p50.004). atp levels were significantly higher in all treatments compared to sham (1.64 6 0.14 mmol/g hb): l (2.00 6 0.21 mmol/g hb, p50.010), r (4.70 6 1.20 mmol/g hb, p50.004), l1r (5.00 6 1.56 mmol/g hb, p50.004). the levels of 2,3-dpg were no longer detectable in s and l treatments at day 42. the combined treatment was comparable to r (2.38 6 3.26 mmol/g hb vs. 2.62 6 2.20 mmol/g hb, p50.868). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l1r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer 1 , eric ducas 1 , patricia landry 1 , nathalie dussault 1 , jacques bernier 1 , danny brouard* 1 and anne maltais 2 . 1 h ema-qu ebec, 2 institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the 500-ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < 108c) of one to six 500-ml whole blood units (wbu) within 8h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between 18c and 108c for 24h under extreme external conditions (-308c to 408c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned 58c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for 24h at 20-248c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, 500-ml wb bags were filled with 555 ml saline 0.9% at t 5 308c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (-308c) and summer (408c) conditions. shipping boxes were filled with either one or six bags (n5 2). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under 108c in 4.55 6 0.62h and maintain their internal temperature between 18c and 108c for 24h with final values ranging between 6.38c and 9.38c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the 108c threshold value in 2.4 6 0.2h and the bags' internal temperatures were within the acceptable range for 24h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than -138c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* 1 , christopher c. c silliman 2 , beth shaz 1 , marguerite kelher 2 and claudia s. cohn 3 . 1 new york blood center, 2 bonfils blood center, 3 department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either 100% donor plasma (n550) or 65% pas-3 / 35% donor plasma (n550). within 12 hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd40 ligand (scd40l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c (3/50 products) compared to plasma platelets (2/50 products); however, the hla-antibody screen-positive supernatants of pas-86a transfusion 2017 vol. 57 supplement s3 abstract c platelets had fewer hla specificities (2 specificities) compared to those of the plasma platelets (18 specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd40l levels were increased in the supernatant of pas-c compared to plasma platelets (table 1) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd40l and not bioactive lipids. although scd40l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd40l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below 5x10 6 wbc in us and 1x10 6 wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n52) and leukoreduced (lr) rbc units (n52) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of 1000 wbc/ul. the spiked samples of 5, 12.5, 5, 25, 50 and 100 wbc/ul were prepared from the source sample of 1000 wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations (0, 12.5, 5, 25, 50, 100 wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples (0, 5, 25, 50 wbc/ul). 10 tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of 20 samples (plt and rbc) were run on both analyzers, repeated for 5 days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt50.996, slope50.972), (r-rbc50.999, slope50.992). the %diff-plt at 5, 25, 50 wbc/ul were 7.8, 4.7 and 10, respectively. the %diff-rbc at 5, 25, 50 wbc/ul were 10.8, 3.2 and 14.7, respectively. the average total testing time was similar on both instruments; 89 min for the facsvia and 92 min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated 62% (56 of 89 min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of 56 minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* 1 and mickey koh 2 . 1 department of laboratory medicine and pathology, university of minnesota, 2 st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of 62 completed and partially completed surveys were received. results/findings: responses came from 13 countries, but the majority of responses came from the united states (us). of the respondents, 35% reported aprp use in their hospital. aprp was used predominantly for outpatients, though >40% of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by 1-5 mds; however, 3 hospitals had >10 mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures (97%); however, 3 respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the 3 hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, 2017) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n56) under blood bank conditions at day 3 (d3), at day 42 (d42), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from 2.1-8.8% at d3 to 8.3-68.1% at d42. rejuvenation markedly reduced this storage-induced spherocytic shift (1.7-29.3%) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell 1 , angela hill 1 , tracey turner 2 , april xu 2 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units (314 6 15) than in rcf units (275 6 16), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as-3 additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: 12 small rcf (252-263 ml) and 12 large wbf (322-353 ml) rccs were stored for 21 d before being glycerolized and frozen at -658c for !72 h. large rccs whose red cell mass exceeded the capacity of the 275 ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a 378c water bath, deglycerolized and re-suspended in as-3. rccs were stored 14 d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p<0.05) hematocrit, specific gravity, hemoglobin per unit, supernatant k 1 and na 1 concentration, deformability (ei max ), and higher (p<0.001) recovery than did large wbf units. no significant differences in hemolysis, atp, 2,3-dpg, p50, rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table 1) , however 8 of 12 large wbf units had rbc recoveries < 80% due to pre-glycerolization volume reduction, and 2 of the small rcf units had hemoglobin values < 35 g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above 80% and the hemoglobin failure rate would be below 10% of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. 0.03 6 .02 0.33 6 .28 0.83 6 .3 0.32 6 .14 elongation index (30pa) 0.602 6 .008 0.585 6 .017 0.580 6 .017 0.578 6 .017 this study is funded by zimmer biomet. (hasan 1994) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo 2 ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for 42 days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two (52) rbc units (leukocyte-reduced), cpd/as-1 or cp2d/as-3, on day 0, day 42, and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o 2 /g hb) and total releasable oxygen (tro) of the unit (ml o 2 ). orc was determined by assessing the change in % o 2 saturation from 100 mm hg po 2 (e.g., lung) to 40 mm hg po 2 (e.g., venous blood) multiplied by 1.34 ml o 2 /g hb (li 2016). a simulated baseline pretransfusion vo 2 of 115 ml o 2 /min was estimated using the day 0 orc and assuming a 7 g/dl transfusion trigger with a cardiac output of 5 l/min and 5 l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day 42 restored orc and tro to levels greater than day 0 ( table 1) . orc of the rejuvenated unit was 1.5 6 0.2 times and 3.4 6 0.5 times greater than rbc on day 0 and day 42, respectively (p<0.001). vo 2 increased after a simulated single unit transfusion of rbc (day 0, day 42, and pw) by 19.3%, 8.9%, and 28.8% over the pre transfusion vo 2 , respectively (p<0.001). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release 3.3 times the volume of o 2 compared to standard, untreated rbcs stored for 42 days. inferior oxygen delivery to tissues (vo 2 max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for 42 days vs 7 days which seem dependent on genetic variability and storage time (bennett-guerrero 2017). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* 1 , jay srinivasan 1 , gustaaf de ridder 2 , alan d. gray 3 , matt landrigan 4 , keaton charles stoner 5 , angela crabtree 6 , jessica poisson 7 and ian welsby 8 . 1 duke university school of medicine, 2 duke health pathology, 3 citra labs, a zimmer biomet company, 4 zimmer biomet, 5 duke university, 6 department of pathology, durham veterans affairs medical center, 7 duke university hospital, 8 duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted 2,3-diphosphoglycerate (2,3-dpg). the loss of 2,3-dpg increases the oxygen affinity of hemoglobin, resulting in lower p50 (partial pressure of oxygen at 50% hemoglobin saturation). decreased p50 may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and 2,3-dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at 378c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a1, leukoreduced prbc stored in as-1 were obtained from our local blood center. after 3 days of storage, units were divided into 4 separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution (50ml) was added to the cr group, and all groups were then stored for another 12 days at 1-68c. on day 15 of storage, the sr group was incubated for 1 hour at 378c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p50 was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps1) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a 5 0.05. results/finding: significant differences in p50 were noticed between all groups (table 1) . ei, ps1, and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p50) seen over 15 days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p50 that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k 1 , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc (300ml) units stored in as-1, obtained from a regional blood donor center at expiration (42 6 2 days), were passed by gravity through sorbent-devices containing 50 ml of multifunctional polymer bead, at a flow rate of 20 ml/min. supernatants were analyzed for k 1 removal as well as free hb, antibodies and cytokines (27-plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k 1 ] from 54.2 6 5.0 to 1.98 6 1.3 meq/l; equivalent to an 84.6% reduction. free hb was reduced by 96.3% from 2.5 6 1.0 to 0.39 6 0.2 mg/ml. antibodies, specifically igg, iga, and igm decreased from 9.91 6 3.1 to 2.40 6 1.1 mg/ml (77.7%), 0.48 6 0.1 to 0.25 6 0.01 mg/ml (48.9%), and 0.73 6 0.2 to 0.49 6 0.1 mg/ml (31.5%), respectively. inflammatory cytokines were significantly reduced, specifically: ip-10 from 144.27 6 16.2 to 18.43 6 2.7 pg/ml (87.2%), mip-1b from 37.37 6 5.7 to 7.23 6 2.5 pg/ml (80.7%), and pdgf from 1348.3 6 291.9 to 77.91 6 22 pg/ml (94.2%). filtration had no significant impact on cell surface markers of rbc viability (<0.1% decrease) or sensitivity to osmotic changes. values listed represent mean 6 sem (p < 0.01 for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k 1 as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k 1 along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency (1.5%) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in 50 apheresis and 50 whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia 2120 (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on 10000 permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and 2,3-dpg profiles to fresh levels. the objective was to compare 50% hemoglobin-oxygen saturation (p50) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was 97.2 6 2.2%. hemolysis (%) was similar on day 42 before and after dry-air incubation with the rejuvenation solution (0.34 6 0.14% vs 0.35 6 0.14%). percent hemolysis (%) decreased after washing (0.24 6 0.07%) and was maintained below <1% for all units during storage for 24hr (0.51 6 0.19%). average atp and 2,3-dpg were restored above the average fresh values. the morphology score decreased $25% by day 42, which was restored to near fresh values following rejuvenation and washing and storage 24hr (93.7% and 95.1%, respectively). rbc oxygen affinity, as assessed by p50, was restored above fresh values. all values were significantly different compared to day 42 (p<0.001, paired t-test) ( table 1) . conclusion: rbc morphology was restored to near fresh and average atp, 2,3-dpg, and p50 were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and 2,3-dpg were maintained during storage 24hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d9tetrahydrocannabinol (thc) and 11-oh-d9-tetrahydrocannabinol (11-oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and 11-oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and 11-oh-thc to produce samples for lc-ms/ms analysis. lc used a c18 column. post-column detection by ms/ms used positive ion electrospray with q1:q3 ion pairs of m/z 5 605.3:225.3 (internal standard (is), d3-thc), m/ z 5 602.2:225.2 (thc), and m/z 5 618.3:256.1 (11-oh-thc). quantitative results for thc and 11-oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from 0-50 ng/ ml for both thc and 11-oh-thc. limits of quantitation, defined as 5 standard deviations above background, were 0.7 ng/ml for thc and 7 ng/ml for 11-oh-thc. results/finding: a total of 424 donor plasma samples were tested for thc and 11-oh-thc. no samples tested positive for either thc or 11-oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a 50% probability of one or more positives at a prevalence of 0.16% positive samples, and a 95% probability of one or more positives at a prevalence of 0.71% positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than 1% among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than 12 hours for post-exposure detection of thc and/or 11-oh-thc in plasma. conclusion: testing of 424 donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than 1%. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* 1 , ruqayyah almizraq 2 , daniel millar 3 and jason p. acker 4 . 1 university of british columbia, 2 university of alberta, 3 lightintegra technology inc., 4 canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days 7, 21, and 42 of storage. one rcf rcc was tested on days 1, 5, 14, 21, and 43 and six 10 ml aliquots were stored in parallel and tested on days 14, 21, and 43. all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days 14, 21 and 43 (p<0.001) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the 6 aliquots were consistent at each time point but statistically higher than in the original rcc on and after day 21 of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow 100% screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n 5 60) and paired ffp aliquots were stored for 31-33 days at 2-68c and -188c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! 80% levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c1 esterase and alpha 1-proteniase inhibitors). the level of factor xiii in odp was slightly lower, about 70% of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i1ii and ddimer) and complement (c3a and c5a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion 2017 vol. 57 supplement s3 abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to 62% in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to 15 minutes. in addition, donations with collection times between 12 and 15 minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from 12-15 minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n56) were prepared from one 12-15 minutes bc and 60 ml of autologous plasma in a 600 ml pvc-dehp container. as a reference, spc from donations with collection times of <12 minutes were prepared (n55). in addition, pc were prepared from 5 bc, of which at least 4 bc were from 12-15 minutes donations (n55). after pooling of the bc, 300 ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for 8 days at 22 6 28c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean6 sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume (67 6 5 vs. 66 6 16 ml) and platelet content (74 6 11 vs. 71 6 15 x10 9 ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day 8, ph(378c): 6.84 6 0.16 vs 6.83 6 0.17, other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day 6 and/or 8 in 2/5 pc (possibly because sometimes ab0 incompatibility was accepted). on day 8, plt showed low cd62p expression (17.1 6 1.8%) and phosphatidylserine exposure (annexin v binding, 8.9 6 1.9%). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from 12-15 minutes whole blood donations had a normal composition and showed good in vitro quality during 8 day storage. to substantiate that the exclusion of 12-15 minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* 1 , jessie miller 1 , ranee marie wannarka-farlinger 1 , sandra bryant 1 , scott a hammel 1 , sherry stern 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as-3 rbcs, 20 irradiated and 21 non-irradiated, were selected for the study. the units varied in age, ranging from 2 to 42 days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< 0.05) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p50.02). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after 5 days of storage pei lun karen lim* 1 , erma sofia sumardi 1 , isamar eduardo ancheta 1 , susan lim 2 , christina yip 1 , lip kun tan 2 and shir ying lee 3 . 1 national university hospital singapore, 2 national university hospital, 3 national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within 8 hours of processing and stored at -188c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of 24 hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for 5 days and kept at 1 to 68c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than 50 iu/dl. study design/method: randomly selected units of ffp (n510) were measured for fviii concentration based on clotting assay (stav r -deficient 92a transfusion 2017 vol. 57 supplement s3 viii diagnostica stago). fviii levels were measured at five time points: prefreezing, 0, 24, 72 and 120 hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at 30 to 378c for 35 minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration (0 hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at 1 to 68c for 5 days for subsequent testing. results/finding: results obtained were listed in table 1 . units 7 to 9 were not tested for fviii at post thaw-24 hour due to operational issues. the overall fviii concentration decreased at an average of 13% from pre-freezing to post thaw 0 hour. after further storage of tp post thaw-24 hour and -72 hour, residual fviii level remain to be above 50 iu/dl except unit 10 which had a lower initial fviii concentration. at post thaw-120 hour, 7 out of 10 units tested had residual fviii activity within the pre-set standard of 50 iu/ dl. the average decline from 0-hour post-thaw to 24-hour, 72-hour and 120hour post-thaw was 36.5%, 42.7% and 47.9% respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least 50 iu/dl of fviii. typically patients with factor levels below 30 iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi 1 , ayman mohamad sabri 1 , ali abdullah alajeafi 1 , ashwaq hasan alhekri 1 , saleem bin mahfouz 1 , ali hasan alkhodari 1 , rawya saeed shealy 1 , marcus picard-maureau* 2 and hussain bana almalki 1 . 1 king abdulaziz hospital and oncology center, 2 cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in 100% plasma over a 5 day storage period and the new "test" pathogen-reduced, pooled (pools of 5) prp pc in 100% plasma over a 7 day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of 4 leucoreduced test pcs were assessed at day 7 of storage and compared to leucoreduced control pc at day 5 of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day 0; the process was completed by day 1 post-collection. samples were taken daily for quality analysis from test and control pc until day 5 and day 7, respectively. for bacterial spiking, additional pc were spiked with each receiving 4 ml of 1 mcfarland ($ 1.2x10 9 cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was 4.7% 6 2.0, the total average platelet loss at day 7 was 11.2% 62.8. the average platelet loss in the control units at day 5 was 9.5% 61.4. the average ph of the test units at day 7 was 6.64 60.04 and in the same range as the control pc, ph 5 6.89 60.09. glucose concentration in test pc at day 7 (13.8 63.0 mmol/l) was lower than in the day 5 control units (18.32 61.06 mmol/l). lactate levels increased during the course of storage; lactate levels at days 5 and 7 were outside the range of the assay (>15 mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after 7 days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* 1 , jay srinivasan 2 , jessica poisson 3 and ian welsby 4 . 1 duke university, 2 duke university school of medicine, 3 duke university hospital, 4 duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified 256 proteins in cryo; of the 10 most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf (4.44 mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial (0á2 lm) filtration. cryoprecipitate mini-pools (400 6 20 ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di(2-ethylhexyl) phthalate (dehp)] adsorption device and a 0á2 lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table 1) . kit ensured bacterial sterility (table 3 ) and most importantly, final product was free of hbv, hcv and hiv (table 2) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to 4 days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase-1, thereby blocking synthesis of thromboxane a 2 from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n518) were prepared from a nsaid-bc and 60 ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for 8 days at 22 6 28c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n55) were investigated as a reference. values are expressed as mean6sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume (69 6 4 vs. 66 6 16 ml) and plt content (67 6 14 vs. 71 6 15x10 9 ) were similar in both groups. on day 8, both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day 8 was significant higher in a subset of donors who had used ibuprofen (n55). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin (0,0-30, p<0.05), diclofenac (31,1-76) and naproxen (0,0-24, p<0.05), compared to normal controls (76, . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day 1 in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (<24 hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, 2014] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type 2 diabetes (t2d). because of the strong rise of people with t2d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t2d, but accepted as donor. study design/method: twelve whole blood donors with t2d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for 8 days at 22 6 28c and sampled on day 1, 4 or 5 and 8. the diabetic marker hba1c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 3 'good' (ph day8 >6.6) and 3 'poor' (ph day8 <6.3) storing spc were selected and analysed in more detail. results/finding: donors were of age 57 6 10 year and primarily men (75%). donors with t2d had a higher mean bmi (30.3 6 4.6 vs.25.4 6 3.4 kg/m 2 ) and higher hba1c than controls. the spc of both groups had the same volume (70 6 5 vs 726 2 ml) and plt content (71 6 9 vs 73 6 11x10 9 ) but on day 1 glucose concentration was higher in the diabetic group (20.5 6 1.7 vs 18.9 6 1.4 mm, p<0.05). on day 8, the average in vitro quality was comparable in both groups (data not shown). when combining 94a transfusion 2017 vol. 57 supplement s3 the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed (0.14 6 0.04 vs 0.36 6 0.03 mmol/ day/10 11 plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc-1) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day 1 ('poor':2.2 6 0.7 vs 'good':1.1 6 0.2, p<0.01). conclusion: bc from donors with t2d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t2d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of 30 minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of 2017 detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table 1). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to 60 minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* 1,2 , katharine a downes 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts13 are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy 12 month old unvaccinated girl presented with history of diarrhea for 5 days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: 32 x 10 9 /l, platelets: 62 x 10 9 /l, bun: 77mg/dl, creatinine: 2.4mg/dl, lactate dehydrogenase 1940 u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to 44 x 10 9 /l, adamts13 sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation (74 x 10 9 /l and a-ipc of 4.7 x 10 9 /l). two consecutive tpe were completed which resulted in a platelet count decrease to 54 x 10 9 /l and a-ipc of 5.1 x 10 9 /l. a-ipc ratio was 1.1 below the ratio of 3 which has been reported for ttp patients. similarly a-ipc count was not below 5 x 10 9 /l threshold reported in setting of ttp with severe adamts13 deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o157:h7 toxin. testing of c3, c4, factor h, factor h autoantibody, factor i and factor b were normal. adamts13 activity was 93%. patient was treated for the infection and platelet count improved within 10 days to 315 x 10 9 /l, with resolution of her renal failure: bun: 42 mg/dl, creatinine: 0.65 mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts13 testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts13 activity. many patients with severe autoantibody-mediated adamts13 deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts13 activity <5%) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value 2-3 days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column 1), increased peak a-ipc value (results shown as percent increase, column 2), delayed a-ipc peak, and delayed plt recovery (table 1) . moreover, recurrent episodes required more procedures compared to initial presentation (table 1) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect 3 cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february 2016 to march 2017 on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the 2 nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs1 separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version 5.0 after taking consent from the donors. the target collection of each procedure was a dose of 3 x 10 11 platelets in 200-250 ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at 2 consecutive donations within 7 days were considered. data was analyzed by stata 14. within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the 98 donors, 35 repeated the plateletpheresis within a week (group i) and 63 underwent 2 nd plateletpheresis within 8-30 days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the 2 groups (p50.025). though above the eligibility cutoff of 1.5 lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at 2 nd plateletpheresis in group ii donors. there were 49 donors who presented to us for the 3 rd time for plateletpheresis with a mean gap between 1 st and 3 rd plateletpheresis being 46 days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p50.000). plateletpheresis through all the 3 cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type 1 receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type-1 receptor antibody (at1rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at1rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at1r ab are reviewed. results/findings: case 1: the patient is a currently 43-year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age 22, and a second deceased donor transplant due to a rejection of the transplanted kidney at age 38. three years post-transplant, her creatinine (cr) started to rise from 0.7 to 1.35 mg/dl and a biopsy showed banff criteria grade 2 amr, grade 2a t-cell mediated rejection (tcmr) and grade 3 interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at1rab was identified at >40 u/ ml (high: >17 u/ml, intermediate: 12-17 u/ml, negative: <12 u/ml). she received 6 tpe treatments every other day and started losartan. after a course of tpe, at1rab decreased to 32 u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to 1.9 ml/dl. in one month, her at1rab increased again to >40 u/ml, therefore, she received 3 more tpe treatments with a decrease in her at1rab to 16 u/ml. although at1rab level increased slightly to 20 u/ml after 3 months, her cr has been stable at 1.3-1.6 ml/dl. case 2: the patient is a 25-year-old mean 1/-se -54.71/-12.9% * 183.1%1/-12.8%* * p<0.05 96a female with malignant hypertension who received a deceased donor kidney transplant at age 24. her cr started to rise 2 weeks post-transplant from 1.4 to 2.68 mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at1rab level at 18 u/ml. she received 6 tpe procedures every other day and at1rab decreased to 8 u/ml with a decrease of cr to 1.98 mg/dl and improved arteriopathy in histology. because her at1rab level slightly increased to 12 u/ml over the next 2 weeks, she started weekly tpe treatment. after 5 weekly tpe, tpe treatment was stopped because her at1rab level remained relatively unchanged. her cr has been stable at around 1.5 ml/dl to date. conclusion: we present 2 kidney transplant recipients who received tpe treatments for high at1rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at1rab levels; however, weekly tpe had no effect on reducing at1rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a 36 year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < 10%], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a 33 year old man (patient b) with a medical history of hypothyroidism (on synthroid for 2 years), end stage renal disease and non-ischemic cardiomyopathy (ef of 20-25%) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a 1-1.5 plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within 12 hours of the procedure completion. their total t4, t3 and free t4 levels trended to normal or near normal range within 24 hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy 3-4 weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* 1 , laura martínez molina 2 , cristina muniesa montserrat 2 , octavio servitje bedate 2 , silvia cosano navarro 1 and maria isabel gonz alez medina 1 . 1 banc de sang i teixits, 2 dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since 30 years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last 20 years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) 2016, as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category 1. since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of 10 patients diagnosed with ss and compare them in their first evaluation once the 20 th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( 8-methoxypsoralen, 8-mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from 1.5 to 2 total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in 2 where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in 7/10 patients) and with online system (therakos) just in 3. main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is 77'7% (partial remission 66.6% and complete remission 11.1% with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : 3835 u/l) requiring transfusions, mild thrombocytopenia (144 x 10 9 /l), acute kidney injury (bun 175 mg/dl, creatinine 2.51 mg/dl). by the third hospitalization day hgb improved to 10 g/dl, however with worsening thrombocytopenia (16 x 10 9 /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days 6-10). platelet count and a-ipc improved to 52 x 10 9 /l and 6.6 x 10 9 /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count (30 x 10 9 /l) and a-ipc 1.98 x 10 9 /l. these dynamics did not resemble those which had been described for ttp patients with adamts13 deficiency. adamts13 obtained prior to tpe initiation was resulted at this time and was 67%. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc (2.64 x 10 9 /l) that preceded platelet count increase to 80 x 10 9 /l three days later when patient was discharged. other laboratory values at this time were ldh of 635 u/l, hgb: 11.2 g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts13 deficiency eiman hussein* 1 and jun teruya 2 . 1 department of clinical pathology, cairo university, 2 texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts13. since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts13, the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts13. the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january 2008 through march 2017 were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts13 activity of less than 10%. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts13 deficiency. eight patients (25%) were associated with suspected bacterial infection. four of the 8 patients (50%) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in 3 patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than 10 kilograms using a single apheresis procedure. study design/method: in october 2015 and june 2016, two children with possible leukemia were submitted to tl procedure. they were 6 and 9 months old, and weighted 7,0 and 9,1 kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with 285 ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, 64% hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin (750 ml of acd-a and 7,500 units of heparin), at a blood to anticoagulant ratio of 25:1. a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every 30 minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were 120.000 and 150.000/ mm 3 . the formula "collection pump flow 5 0,0003 x inlet flow x preapheresis wbc count" was used with the goal of removing up to 3 x 10 9 leukocytes/ml. a single leukapheresis procedure was performed with 2 total blood volume processed per patient. immediately after the 2-hour procedures, wbc count were 74.000 and 92.000 wbc/mm3, and 12-hour post tl, wbc count were respectively 45.000 and 70.000/mm3. net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing 10 kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since 2006. we report the data from the year 2016. study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of 13,302 apheresis procedures were performed at 37 hospitals. therapeutic plasmapheresis was the most frequent procedure (50.4%) followed by autologous peripheral blood stem cell (pbsc) collection (23.9%), allogeneic pbsc collection (11.0%), donor leukapheresis (4.0%), and therapeutic leukapheresis (3.9%). cobe spectra (37.4%) and amicus (16.8%) were the most widely distributed instruments. centrifugation was the dominant technique (92.2%) for therapeutic plasmapheresis. detailed information was given for 4,199 therapeutic plasmapheresis procedures performed on 786 patients (some items were not completely filled out). spectra optia (42.7%) and cobe spectra (26.6%) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently (47.2%) as the replacement fluid followed by 5% albumin (26.3%), 4% albumin (13.3%), and 5% albumin 1 ffp (11.1%). most of the procedures were performed for 1 plasma volume (72.4%). acd (88.4%) and heparin (11.5%) were used for anticoagulation. central venous catheter (91.9%) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation (24.1%), antibody mediated rejection in renal transplantation (19.9%), thrombotic microangiopathy (11.5%), desensitization for abo compatible renal transplantation (4.7%), neuromyelitis optica spectrum disorders (4.6%), and hyperviscosity in monoclonal gammopathies (4.6%). adverse reactions were observed in 8.5% of the procedures. allergic reaction (55.2%), hypocalcemic symptom (20.4%), and hypotension (6.9%) were frequently reported. therapeutic effect was achieved in 86.5% of the patients. our apheresis registry has been well run for 10 years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride >1000-2000 mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $18 deaths/100,000 cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of 41 pts who were diagnosed with hp from january, 2009 through january, 2017, and referred for immunotherapy evaluation. 27/41 (66%) pts received conventional therapy (ct) and pe (pe group), and 14/41 (34%) pts received ct alone (ct group). mean age was 36 years (range 16-79), and 56% were female. baseline mean triglyceride level (normal <150 mg/dl) for pe group was 6,728 mg/dl (4,652-12,486) versus 3,142 mg/dl (1,697-5,120) for ct group. baseline mean lipase level (normal <393 u/l) for pe group was 1,798 u/l (797-2,745) versus 923 u/l (472-1,796) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving 2-3 medications. 24/27 (89%) of pe group and 11/14 (79%) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. 20/27 (75%) of pe group and 6/14 (43%) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of 2.85 pe treatments (txs) (median of 2, range 1-4 daily txs) using 5% albumin; 7/27 (26%) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were <3000-4000 mg/dl and lipase <950-1375 u/l (2.5-3.5 x upper limit of normal). mean triglyceride levels after 2 pe txs were 1,976 mg/dl (627-3,968) for pe group (mean decrease 72%); mean triglyceride levels after 48 additional hours of ongoing ct were 1,576 mg/dl (487-2,873) for ct group (mean decrease 50%). while the pe group achieved a greater mean decrease in triglyceride levels after 2 pe txs (compared to the ct group after 48 hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p>0.05). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a 47-year-old man with a chronic history of hypertriglyceridemia >1000 mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa 2016 guidelines, in a patient unresponsive to optimal medical management. asfa 2016 guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed 39 tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely 5% albumin for exchange fluid (100% albumin procedures) or partial plasma replacement (2-3 units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while 27 were performed with 100% albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* 1,2 , metha apiwattanakul 3 , sompis santipong 2 , jutaluk jaipian 2 , jettawan siriaksorn 2 and ponlapat rojnuckarin 1 . 1 chulalongkorn university, 2 king chulalongkorn memorial hospital, 3 prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june 2016 through february 2017 were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a 20% solution. before using, it was diluted to a 4% albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of 156 tpes in 38 patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was 3,000 (range 1,750-4,200) ml. although the corrected calcium level was low (<8 mg/dl) in 3.2% (5/156) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in 2 patients. the first patient had 2 events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was 1.9% (3/ 156). in 2014, the incidence of tpe adverse effects was 1.6% (2/125) when commercial albumin was used. the difference was not statistically different (p 5 1.000). median serum albumin levels pre-tpe and post-tpe were 3.6 (1.9-4.4) and 3.9 (2.4-5.0) g/dl. the increase in serum albumin after tpe was statistically significant (p<0.001). eighty-two percent of pre-tpe serum albumin levels were lower than 4.0 g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether 60 outpatients who underwent a total of 100 la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%). based on differences in the study protocols la was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, 3 out of 60 patients (5%) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was 1.4x10 10 wbc consisting of 1.1x10 10 mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg4-related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category 1 indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a 70 year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to 57,000 iu/mls (ref. range < 35) and anti-cyclic citrulline peptide antibody was elevated to 34,339 units (ref. range < 20) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were 4610, 2890 and 1320 mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was 1.74. peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be 8.5 centipoise (cp) at admission (ref. range 1.6 -1.9). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated -1 total plasma volume; replacement fluid -5% albumin and normal saline in a 50:50 ratio; replacement fluid volume: 110% of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to 2.4 cp. serum igg, igm and iga levels decreased to 2040, 1510 and 672 mg/dl respectively. her rf had decreased to 19,900 iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than 3 cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan 2013 -dec 2016). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with 5% human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between 4 to 35 years age (m: f; 1:2) underwent 62 tpe procedures with an average of 5.6 per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three (27%) patients had only visual symptoms, 5 (46%) had both visual as well as muscular symptoms whereas 3 (27%) patients had muscular symptoms only. three (27%) out of the seven tested, were positive for aqp4-igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in 8 patients with grade-1, in 1 patient, and by grade-2, in seven. adverse events were observed in 8% (5/62) of the procedures with allergic reactions to replacement fluid as most common event (n-3) followed by hypotension (n-2). follow up was available in 55% (6/11) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after 3 months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from 6.0 to 6.4 for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version 6.4 as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version 6.0. the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to 1/16/2016, plt collections were performed on nine trima accel machines operating with version 6.0. upgrading and validating all nine machines to version 6.4 occurred from 1/16/2016 to 4/30/ 2016. the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version 6.0 (5/1/2015 to 9/30/2015) was compared to version 6.4 (5/1/2016 to 9/30/ 2016). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version 6.0 and 6.4, adjusting for multiple visits per donor, with significance defined as p-value < 0.05. results/findings: following the upgrade to version 6.4, staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version 6.4 of the trima accel showed a statistically significant increase in possible leukocyte contamination from 3% to 5% of collections as compared with version 6.0. both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version 6.4. conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version 6.4 software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version 6.4 currently does not provide added value over version 6.0 for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in 32 yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is 30 weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of 500 ml 5% albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using 500 ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of 500 ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a 45 years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at 20 years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with 3 acute episodes requiring prolong hospital admission of approximately 2 months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of 4980 mg/dl, lipase 92 u/l, glucose 250 mg/dl, bicarbonate 24 mmol/l, anion gap 12. ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega 3 fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day 3 of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day 3 after admission. results/finding: the patient tg decreased by 52% (2365 mg/dl) with medical therapy, followed by additional 67% (767 mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day 6 after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below 1000 mg/dl at 20 days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from 2009 to 2013, a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected 5,129 cases whose ferritin levels have been monitored more than twice with an interval of detection in 150-160 days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over 50 lg/l. and the upper limit was set to be 244 lg/ l in male and 158 lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: 0 times, 1 to 3 times, 4 to 6 times, 7 to 9 times and more than 10 times. the high frequency (more than 10 times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were 5,129 donors included in the study, of which 4,944 were male (96.4%) and 185 were female (3.6%). the mean ferritin was 82.0 lg/l in male (95% ci: 80.7-83.2 lg/l) and 66.5 lg/l in female (95% ci: 60.9-72.0 lg/l). the result of anova indicates that the group with the highest frequency (more than 10 times) has the significant lowest ferritin level (p<0.05). the average change of ferritin if donation over 10 times would up to 13.4 and 14.1 lg/l in younger and elder 50 y/o male and 18 and 23 lg/l in female. and then for high frequency (half a year more than 10 times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over 10 times in 150$160 days) was reduced from 21.5 lg/l in the first period to 4.1 lg/l in the third period (1 period5150$160 days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* 1 , mary townsend 2 and lizabeth rosenbaum 3 . 1 university of new mexico hospital, 2 blood systems, inc., 3 blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a 22 year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately 10 minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within 24 hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* 1 , william korzun 1 , teresa nadder 1 , susan roseff 2 and elizabeth ripley 1 . 1 virginia commonwealth university, 2 virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for 1% of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a 1-hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of 142 subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from 2015 to 2016 could be a result of a change in the blood drive timing of the training schedule of that location. in 2015, basic trainees at site a were scheduled at day 57 of 70. in january 2016, the blood drive date changed to day 60 of 70. the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from 2015 to 2016 of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in 2016. these observations support the hypothesis that the increase in hemoglobin deferrals in 2016 resulted from the implementation of the male hemoglobin standard change from 12.5 to 13.0 g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional 24% of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* 1 , eva alonso 1 , oscar bascuñana 1 , monica romero 1 , teresa vich 1 , elena castaño 1 , laura carbonell 1 , eva palomas 1 , saray almerge 1 , francesc carpio 1 and xavier curia 2 . 1 banc de sang i teixits, 2 institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january 2015 to december 2016 we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t5 due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t5 and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, 219 donors came to give blood, of these, 15 (7%) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t5 excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t5 but his hemoglobin levels were lower than our selection criteria. of the 204 donors selected for donation 16 (7.8%) had sci lower than t5 and t6. adverse reactions to donation (1.4%) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t5 have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of 2013, automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an 11 month period from january 2013 to november 2013. the same information was assembled for the automated bp process for the 11 month period of january 2014 to november 2014. the automated bp process implemented in mid-december 2013; so the december data for both 2013 and 2014 has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < 0.05. results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p50.006). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < 0.03) with the automated bp while and reactions remained non-significantly lower (p 5 0.086). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below 12.5 g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < 0.05. results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under 12.5. statistically more visits with hgb less than 12.5 g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under 12.5 was 16.5% higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from 1 january to 31 march 2017. after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of 6680 donors who has donated blood in the blood bank's main branch were used as the baseline for this study. 85% of donors (n55678) accepted automatic appointment booking, whereas some donors (n51002) were not comfortable with it. 77% of those who declined still preferred walk-ins (n5771) based on their own time schedule, the rest decided that variable situations (n5112), donation frequency (n569) and choice of preferred donation locations (n550) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was 19%. a comparison was made and found that this study shown a significant increase of acceptance rate by 66%. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has 4 collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. 4) , 3.28 0.4940 4 1 poisson distribution, 2 normal distribution, 3 logistic distribution, 4 lognormal distribution 105a transfusion (p>0.05) in donor and reference populations except in younger (20-44 yrs) male donors (p<0.0021; donor 4.9%, reference 10.0%). mean donor sbp, dbp, and pulse were 125 6 14.7 mmhg, 75.1 6 9.6 mmhg, and 75.9 6 11.2 bpm, respectively. screening blood pressure levels consistent with hypertension (29.4% male; 16.6% female) in the 20-44 year donor group, significantly (p<0.0001) higher than the reference population (11.2% male; 8.7% female). no differences were observed in the 45-64 year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in 20-49 year old females. developing blood donor educational materials gay wehrli* 1 , susan rossmann 2 , louis m. katz 3 and dan a waxman 4 . 1 university of virginia health system, 2 gulf coast regional blood center -sugar land, 3 americas blood centers, 4 indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an 8 th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same 10 multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, 3.5 page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table 1. results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level (8 th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in 2011 for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has 4 major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from 2011 to 2016 after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from 5.6% to 21.4% in 2011 and 2016, respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from 26.8% in 2011 to 57% in 2016. moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to 24 times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately 1.3 million doses of ap transfused within 1.375 billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since 2006. firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than 180 days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than 4 times and had not donated for more than 90 days or less than 4 times with an interval of more than 60 days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than 8 times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by 7.46 times from 5550 to 41420 and the doses of ap increased by 7.41 times from 7363 to 54553 within 10 years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into 5 groups: those who donated ap once, those who donated 2-4 times, 5-9 times, 10-29 times, and those who donated more than 30 times, respectively. it was found that the number of permanent ap donors who donated ap more than 30 times was only 965 (2.1%), but they denoted a total of 76432 doses of ap (29.2%) from 2006 to 2016. conclusion: aps increased at a rapid and steady pace in wuhan blood center from 2006 to 2016, which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at 4 sites on 2 consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer <26 ng/ml and zpp levels >100 umol/mol heme) at 3 hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all 3 hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp 87.8% (r50.937) at first and 86.5% (r50.93) at second donations. at first donation when compared to fs hb, only 10.4% (r50.323) of variation could be explained by variation in fs zpp, 12.3 % (r50.35) by ven zpp and 9.4% (r50.307) by ven fer. at second donation, when compared to fs hb, only 9% (r50.30) of variation could be explained by variation in fs zpp, 14.4% (r50.38) by ven zpp and 20.1% (r50.448) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p<0.001) suggesting strong evidence against correlation. 55% (181) responded to the survey of which 4% (13) reported not feeling well after donation. it should be noted that noted that 1% (3) female study participants reported feeling unwell after the first donation and had ferritin levels below 26ng/ml but the zpp levels were less than 100 umol/mol heme. of the 3% (10) male participants that reported not feeling well none had ferritin levels below 26 ng/ml nor ven or fs zpp levels above 100 umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from 12.5 to 13.0 gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from 59 blood centers over two intervals, july-dec. 2015 and july-dec. 2016 (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male5m, female5f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab 17.0, chicago il). p <.05 was considered significant. results/findings: data were provided by 40 of 59 centers invited, representing 2,420,886 and 2,945,802 wb donations and 272,094 and 319,161 ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from 1.5% to 2.9% in the two intervals among aggregated donation attempts (p<.001), and for m ap from 1.8 to 3.5% (p<.001). the mean "by center" deferral rates (table) were similar to that and significant (p<.001). mean by center hb deferral rates among f donations during the two intervals were 11.6 and 11.9% (p50.241) for wb, 11.8 and 13.0% (p5.041) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only 12 centers could provide specific high vs. low vs. irregular pulse deferrals; 27 provided only a summary (i.e total pulse deferrals), and 1 could provide none. for bp, 8 provided detail (high vs. low), 28 summary and 4 none. p deferrals increased in the successive intervals among f wb donors from a center mean of 0.57 to 1.49% (p5.018) and for m wb donors from 0.78 to 1.16% (p5.006). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba1c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba1c levels among those with rbs >180 mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from 1 st march 2017 to 31 st march 2017. total of 1,861 blood donors were tested for rbs. those with rbs > 180 mg/dl were further tested for hba1c by gold standard hplc method using variant ii biorad. blood donors with >180 mg/dl rbs and hba1c > 6.5% were advised to consult a physician for further evaluation. results/findings: of the 1,861 donors tested, 44 (2.36%) donors showed a rbs of > 180 mg/dl. forty two (95.45%) were males and 2 (4.54%) females with a mean age of 40.55 years (26-56 years). of these, 14 (31.81%) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining 30, 8 (26.66%) of them had a family history of dm. of these 30 donors, 8 donors did not give a consent for testing for hba1c. among the 22 donors tested for hba1c levels, 16 (72.72%) had hba1c > 6.5%. all the 16 donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is 0.87% (16 of 1839 donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* 1 , hwee huang tan 1 and ai leen ang 2 . 1 health sciences authority blood services group, 2 health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from 12.5 to 13.0 g/dl last may 2016. the current minimum acceptable hemoglobin for male donors in singapore is 12.5 g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels (12.5-12.9) and in donors with hemoglobin 13 g/dl and above. study design/method: during a 4 month period, serum ferritin testing was performed on 350 regular male whole blood and 250 regular male apheresis donors who made at least 3 donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin 12.5-12.9) group b (whole blood with hemoglobin !13, group c (apheresis with hemoglobin 12.5-12.9) and group d (apheresis with hemoglobin !13). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below 30 ug/l is considered low and levels below <12 ug/l are considered having absent iron stores. results/findings: 55.1% of donors in the study have ferritin levels below 30 ug/l. there were more donors with low ferritin in group a compared to group b, 80% and 53% respectively (p<0.05). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, 49% and 30% respectively (p50.001876). ferritin results for the 4 groups can be seen in table 1. conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or 54.3% have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after 4 months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin 12.5-12.9 g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to 13.0 g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than 15 % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among 600 students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of 54% male and 46% female students in the age group of 18-28 years. only 65 % of the students have heard about voluntary blood donation and 28 % of the students have given blood once in their lifetime and among them 19 % are blood donors at the moment. 42 % of the participants believed that there is a specific reason why they don't donate blood and 59 % believed that there is a risk involved for the donors, when donating blood. 80 % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. (21); miscellaneous effects were reported in 23 courses. side effects led to interruption of supplementation in 55 instances. ferritin levels (mgt6sd) at entry into the program and at the last visit were 48.9 6 2 and 65.4 6 1.7 mg/l in participants, vs 64.1 6 2.2 and 56.3 6 2.2 mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took !75% of the tablets. ferritin levels<26mg/l were found in 4,8% of participants and 14.7% of controls. deferral for low hemoglobin was below 1% in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only 50% of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under 21cfr630.10 and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § 640.120 to make a collection under this provision if the requirements set forth in § 630.15(a)(2) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in 2001, an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of 50 -75 ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached 547, of whom 365 (67%) are c282y homozygotes. without active recruitment, accrual rate is about 7 per quarter, with 69% of subjects qualifying as allogeneic donors. the mean current age is 59.7 years, 65% male, 96% caucasian. the majority of hh donors (276 of an active cohort of 318) are in the maintenance phase of therapy with an average of 2.6 donations/year and a 4% deferral rate. over the last 5 years, hh donors contributed approximately 8-11% of the hospital's allogeneic blood supply, averaging 475 whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided 30-40% of blood for in vitro research at our institution with an average of 180 wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over 16 years. since 5/23/16, with an increase in male hgb deferral threshold to 13g/dl, there has been only 1 hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm-200 non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm-200 non-invasive occlusion spectroscopy device. over a span of 7 days, 200 eligible blood donors, both male and female, were first screened by the nbm-200 non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the 200 blood donors for the performance of hb measurement on the sysmex hematology analyzer within 1-3 hours of collecting the venous samples. results/finding: the sd of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed 1.1g/dl. the hb measurements obtained from the nbm-200 and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be 0.978 g/dl. the precision of the nbm-200 yielded a co-efficient of variation of .02 g/dl and a standard deviation of .33 g/dl. conclusion: the operators found the nbm-200 easy to install, maintain, and operate with minimal training. the nbm-200 non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder 1 , ravi reddy 1 , dhuly chowdhury 2 , don brambilla 2 and edward l. murphy* 3 . 1 sanbs, 2 rti international, 3 ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in 2014 and followed them for one year. within 56 days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used 4-point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. 2015) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included 2,902 first-time black donors with median age 23 and female predominance (59%). within one year, 1,786 donors (62%) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable (45 strongly agree to 15 strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) 5 1.16, 95% ci 1.06-1.28), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or51.11, 95% ci 1.00-1.23). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or50.83, 95% ci 0.72-0.96) and "i wasn't treated well by the <blood center> staff" (or50.85, 95% ci 0.74-0.97). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or51.19, 95% ci 1.03-1.37). a secondary analysis treating the likert scales as 4-level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard 1 , ramya ghantasala 1 , obhijit d hazarika 1 , nicole leonard 1 , cori a polonski 1 , zachary b wunrow 1 , michelle heleba 2 , jan k carney 1 and mark k fung* 1 . 1 university of vermont larner college of medicine, 2 american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous 16 question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of 292 surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" (27.5 %) and "traveling is a time for me to relax." (30.6 %). of the respondents who travel in the summer, very few reported donating while traveling (3.4 %). summer donation rates between summertime travelers (36.5 %) and non-travelers (36.4 %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website (45.6 %) and phone (28.4%). willingness to use a regional blood donation smartphone app was highest among respondents ages of 18 to 34 (45-55%) and lowest among ages 55 and older (13-15%). of respondents with no prior knowledge of summer seasonal shortages (22 %), 2/3rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv6, and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin-4(il-4) and il7. others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent 2 hard spins at 3800 rpm for 7 minutes with separation after each spin on a compomatev r g5. plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb1 loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers (13m [29%]: 32 [71%] f); median age 42 years (range 21-70) donated a unit (500 ml) blood from which buffy coats (average volume 56 ml) were processed. the buffy coat process was previously validated on 20 wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures 1 and 2. all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by 20% within 12 months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for 34 days. results/findings: there were 158 completed responses, of which 73 (46%) indicated that their hospital had an msbos and 85 (54%) did not. the majority of hospitals without an msbos were academic centers (36/85, 42%) from oceania (26/85, 31%) or europe (23/85, 27%), had between 500-999 beds (30/85, 35%); the majority of these hospitals transfused between 1,001-4,999 rbcs (21/85, 25%) per year. 15/85 (18%) are going to implement an msbos in 2017. of those with an msbos, the majority 23/73 (32%) were from north america. the majority were academic hospitals (39/ 73, 53%) with 500-999 beds (43/73, 59%) that transfused !20,000 rbc units per year (21/73, 29%) offering a wide range of surgical services. on average there were 207 6 577 procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of 30% of the procedures listed, a pre-operative type and screen for 38%, crossmatching rbc units for 28%, and for 4% of procedures a different recommendation was made. most (32/73, 44%) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only 5/73 (7%) of msbos' were created solely by using procedure-specific data, and most (35/73, 48%) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually (30/73, 41%), and the hospital transfusion committee is often (39/73, 53%) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents (30%) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, 23% of respondents felt that it was regularly used by all surgeons and anesthesiologists; 10% felt that it was not used at all at their hospital, 36% did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, 18% of the hospitals currently without one indicated that it would be implemented in 2017 suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a 1490-bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see 93,000 hospital admissions and nearly 300,000 emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in 2015 was approximately $15.8m. in nov. 2015, an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $1.2m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns (2014 and 2015), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee 1. development of evidence based transfusion triggers. 2. education on evidence based transfusion triggers across multiple campuses, specialties and resident programs 3. clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to "1" unit instead of "2" units. 4. updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in 2016, we were still able to reduced blood product expenditures by $933,874 when compared to 2015. conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* 1 , anna w rains 2 and christopher t clark 3 . 1 university of tennessee graduate school of medicine, 2 univeristy of tennessee medical center, 3 univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for 1 week post-delivery, with cost of $1.10 per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in 2016 was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc 3000 system. by using the blood volume values, and assigning a value of 1.5 ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in 2016, there was a total of 3,331 infants delivered at our facility. out of all the deliveries, 487 (14%) infants were transferred to the nicu. of those infants, 27% received at least one red blood cell transfusion and 7% received at least one platelet transfusion. of the 487 infants transferred to the nicu, 98 (20%) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to 1% (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from 1.0% all the way up to 3.9%. in those 98 infants, the birth weight ranged from 400-1650 grams, and the gestational age ranged from 22 weeks to 36 weeks and 4 days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than 2500 grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* 1 , shannon davis 2 , suzan new 2 , vaishali patel 2 and oren guttman 2 . 1 university of texas southwestern medical center, 2 ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable 1:1 communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to 50% of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july 2016; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of 79 randomized controlled trials (8, conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* 1 and stephanie rogers 2 . 1 dignity health st joseph's medical center, 2 dignity health background/case studies: over 12 million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across 39 hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels >5 7.0 g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of 7.0 g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which 4 or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of 7.0 g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to 21 hospitals including 16 cme presentations, online physician and nursing educational videos, communication tools including infographics and "7 is the new 10" buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb >5 7.0 g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy2015 to fytd2017, there was a 26% reduction in prbc units transfused to patients with hgb >5 7.0 g/dl, starting at a baseline of 67% down to 41%. this represents an fy17 annualized savings of $9.732m, from a baseline of 82 units per 1,000 patients days down to an average 71 units and approximately 2,000 fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels >5 7.0 g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm5-0.258) reduced rbc units and two studies decreased the percentage of patients transfused (or5 0.700). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or 50.264) and rbc units transfused (sdm5-0.553). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a-6 method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was 7g/dl/21% or 8g/dl/24%. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately 42% of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was 590 1 24 units/ month. after the first 5 months of buc activation the mean number of units was 439 1 50 units/month a reduction of 151 units/month or 26% of nonsurgical blood use (p50.003 by t-test). non-surgical rbc use now represents approximately 29% of the total rbc use hospital wide a 13% reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital -30 month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital (4001 beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first 30 months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating 115a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance (2q14 vs. 1q17): present and completed consents (66 vs. 94%), present and completed nursing flow sheets (19 vs. 96%), transfusion thresholds supported (73 vs. 100%), discharge instructions provided (17 vs. 86%); (3q15 vs.1q17) vital sign compliance (39% vs.71%). jwp increased from 27 to 225 (04/16-03/17). cost savings were realized by decreased utilization and implementation of bpa. (table 2016 -1q17) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately 30-40 trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about 8 minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only 10 percent of coolers fully used. it also consumed valuable staff time as technologists typically made 20-45 trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november 2016 implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level 1 trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated 6-10 hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains 2-4 units of group o rhd negative, 4 units of o rhd positive, and 4 units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in 2015, the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < 10 k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june 2015-october 2016 were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a 17-month period, 1,270 cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table 01 below. overall, 532 (42%) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." (325), "active bleeding" (303), "platelet count of . . ." (173), and "downtrend" (92), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, 618 (49%) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years 2014-2016. study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by 207 km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of 5,5g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> 9 g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey (12 questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of 32 complete responses (16%). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, 81% with physicians, and of these, 59% reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only 3% responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of 7-8 g/dl (56%), platelet count of 20-50,000 (38%), and inr of greater than 2.0 (69%). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per 1,000 inhabitants may vary 3 folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of 18 key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies (14.4% and 14.0%), for the remaining strategies weights varied between 7.0% and 10.6%. we estimate that 384,704 patients would be eligible for pbm strategies in one year time horizon, resulting in 594 premature death avoided (3.8% reduction) corresponding to a gain of approximately 1,500 life years and a reduction of 3,660 (6.0%) disability adjusted life years (daly) relative to the current clinical practice. a decrease of 233,141 in-hospital days is expected mainly due to a 8.4% reduction in hospital length of stay and a 37.3% reduction in 30-day readmission rate. in this population the overall transfusion rate could decrease to 4.3% from the current 8.7% (51.2% reduction) implying 17,202 blood transfusion avoided and 65,214 red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: 237 adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted <1250ml of rbcs, all cases with 0ml transfused were captured and only 7.8% of the time >1250ml were used. if 1250-2000ml rbcs were predicted to be transfused, >2000ml were used 25% of the time. if predicted usage was >2000ml, 53% of the time it exceeded 2000ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for >1250ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than 2.0 is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a 3 month period (jan to mar 2017). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the 3 month period, there were 274 patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of 494 units of blood were requested. 154 units were crossmatched, of which 138 units were sent to the operating theatre (ot). only 33.3% of blood issued to ot were transfused (n546) while the rest were unutilized. the observed ct ratio was 3.35. conclusion: although only 31% of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of !2.0, with almost 70% of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study 35 patients who received tranexamic acid (txa) (study group) and 31 patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa 1gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < 7.0 g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were 1732 unique alert encounters. of these, 1531 (88.4%) led to a crossmatch being ordered while 201 (11.6%) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds (7.0 g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds (8.0 or 9.0 g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at 4 academic hospitals with data-derived msbos. study design/method: the 4 hospitals were in 2 groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if 5% of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if 5-24% of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if !25% of the patients had been transfused. data were collected at each center over a 1 month period between january to march 2017 and included a maximum of 400 cases per hospital during that one month to ensure equal representation between centers results/finding: between these 4 centers there were a total of 1599 cases analyzed. some of the more frequently performed surgeries included orthopedics (23% of cases), general surgery (16%) and cardiac surgery (11%). there were 1362 t&s ordered for these cases, of which 5 were positive for antibodies on the day of surgery. of all the t&s ordered, 52% were ordered in accord with the msbos recommendation, 26% were ordered when the msbos did not recommend one, and in 0.2% a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio 5 6.12 for prenatal hemoglobin (hgb) 8-8.9). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of 29% among obstetric patients. study design/method: we studied a sample of anemic (hgb<10.0 g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of 301 women were enrolled, with median age 27 (interquartile range 23-32) years, median gravida 2 / para 1 and median gestational age 28 weeks. mean hgb before referral was 7.5 g/dl and most were already taking oral iron therapy. a total of 169 women were hiv positive with mean cd41 lymphocytes counts of 394 cells/ul; 29 (12%) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and 156 (92%) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in 292 (97%) of women. there was concurrent chronic disease (n52), infection (n52), vitamin b12 deficiency (n52) and antenatal hemorrhage (n56); 10 had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a 2 month old boy presented to our institution after a 1 month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige (15270 ku/l; rr: 0-2.9). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp3, while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp31 cd41 lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $1.8%. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of 8.2% over the last 5 years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from 2014 to 2016, with a peak of 11.6% (range 3.2%-11.6%). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for 2016 mtps. results/finding: in 2016, mtp was activated 28 times. in 6 cases the patient did not receive any blood product and in 11 cases plasma was already available at the time of rbc allocation/issue. this left 11 cases to evaluate. the median time to plasma availability was 29 minutes (range 4 minutes -61 minutes). the mean plasma wastage for mtp activations was 32% (range 0-100%). of the 9 cha replies, 3 were using thawed plasma and their wastage was </5 5%. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of 29 minutes. our implementation began with thawing a single unit of plasma for "level 1 neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are 9.5 %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over 40 years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated 5 cases of gtx in pediatric patients with age from 8 months to 16 years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of 56 granulocytes donations from 2015 to 2016 were collected from 43 health donors (21 male/ 22 female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, 5mcg/kg/dose (donors up to 70kg, maximum of 600mcg) and oral dexamethasone (8mg), 12h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with 40gy. results/finding s: granulocytes number increased 5 times from basal levels and the median of cells collected were 4.89 x10 10 (range 1.09-9.85). in general donors reported no significant adverse reactions. from 56 donations, only in 4 was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within 24 hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table 1 . adverse reactions for patients were minor and not frequent (4 in 66 gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* 1 , elena kurnikova 1 and pavel trakhtman 2 . 1 russian institute for paediatric haematology, oncology and immunology, 2 instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period 04/2012-02/2016. donors underwent granulocyte mobilization with g-csf in a dose 3-5 mg/kg s.c. 12-16 hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed 169 granulocytes collections in 153 donors (82 f, 71 m), ages 18 to 58. the mean increment of wbc after stimulation was 22,6x10 9/l (9,2-41,9 x10 9/l; f-23,2 x10 9/l; m-21.9 x10 9/l); in the age groups 18-39 years (n5121) and over 40 years (n532), the mean increment of wbc was respectively 22,2 x10 9/l (9,2-41,9x10 9/l) and 24,02x10 9/l (12,6-35 x10 9/l). the mean volume of treated blood was 566, a50,05) . the mean number of total wbc in the product was 38,43x10 9/l (13.38-77x10 9/ l; f-34,8x10 9/l, m-42,7x10 9/l). only three donors (1,96%) had apheresisrelated adverse effects the analysis included 244 granulocyte transfusions in 35 cases of infectious complications in 32 patients. the mean number of transfusions was 6.9 (1-60). each patient received transfusions from mean 5 donors (1-32); the granulocyte transfusions started at the mean 8 days (2-30) after infection; the mean dose of wbc on the transfusion was 11.6x10 8/kg (2-30.7x10 8/kg). wbc increment was able to estimate after 144 transfusions. wbc increment was recorded after 96 transfusions (66.7%); the mean increment was 1,12x10 9/l (0,01-11,38 x10 9/l). efficacy was determined by the number of patients who survived an infection episode and amounted to 80.6%. in the cases of severe sepsis, the efficacy of transfusions was 76.2%. the frequency of post-transfusion reactions was 22%, (n 5 8): febrile nonhemolytic reactions 8,3% (n 5 3); pulmonary complications 11,1% (n 5 4); the anti-hla antibodies formation 2,7% (n 5 1) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp150 hemolytic disease of the newborn due to anti-rh17 keegan barry-holson* 1 and jennifer andrews 2 . 1 stanford university, dept of pathology, 2 stanford university, dept of pathology & pediatrics background/case studies: anti-rh17 is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh17 has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh17 alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g3p1 > 2 mother with a negative 1 st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the 1 st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at 6 hours of life because he was found to have anemia (hemoglobin 12.0 g/dl), severe hyperbilirubinemia (total bilirubin (t bili) 9.0 mg/dl), reticulocytosis (8%) and a positive direct antiglobulin test (igg 21). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of 16.7mg/dl on day 8 of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin 6.3 g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh17 was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh17 sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d1 c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh17 antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* 1 , cyril jacquot 1 , valli criss 1 , philippe p pary 1 , jay greenberg 1 , naomi lc luban 1 and edward cc wong 2 . 1 children's national medical center, 2 quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as 4-11%. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a 2 month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of 1.6 g/dl/4.9%. wbc counts (19 x 10 9 /l) were mildly elevated and platelet counts (410 x 10 9 /l) were within normal limits. her history was notable for upper respiratory tract infection 6 days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total 7.1 mg/dl, direct 1.4 mg/dl), normal ldh (318 u/l), and undetectable haptoglobin (<7 mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin (3-41 reactivity) with positive autocontrol. dat was 41 positive for anti-igg and negative for c3 despite a positive cold antibody screen. the patient weighed 6.9 kg with an estimated total blood volume of 620 ml. she initially received simple transfusions totaling 20 ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with 463 ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of 40%, utilizing the central venous catheter. no adverse events took place over the course of the 2 hour exchange. her one hour post-exchange hb was 9.5 g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin (21). after initiation of steroid therapy (methylprednisolone, 2 mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of 11 g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels (3168 mg/dl). at a subsequent follow-up visit 3 months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a 24 or 16 gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, 1.431/-0.49 ml/ second) or with a mechanical syringe pump (slowly, 2 ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the 24g catheter, the mean change in hct was -3.531/-0.69% with the one-way valve and 0.221/-0.13% without (p<0.00001). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p<0.0001). during rapid manual transfusion with a 24g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse 4.5ml when using a one-way valve (change in hct versus time: r5-0.75, p<0.0001) compared to a significantly different (p50.0085) slight increase in hemolysis for samples that took less time to transfuse 4.5ml when not using a one-way valve (change in hct versus time: r50.58, p50.23). correlations between time and hemolysis were similar, but insignificant using 24g with washed rbcs and the 16g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in 99.9% of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge3. study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at 24 weeks gestation with passive anti-d and an anti-ge3 titer of 256. she was d-and ge:-2,-3, 4 by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at 37 weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge3. the birth hemoglobin (hgb) was 12.6 g/dl, reticulocyte (retic) was 8.6%, bilirubin (bili) was 2.8 mg/dl; the infant was discharged. on day 7 of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb 7.6 g/dl, retic 2.6%, and bili 6.6 mg/dl. ge3-blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh1. obstetrics had to authorize maternal blood donation due to her hgb of 10.9 g/dl. maternal blood collection and rbc washing was expedited and the infant received 40ml of maternal rbcs within 24 hours, at which time his hgb was 6.1 g/dl. post-transfusion hgb was 10.8 g/dl. one week later, the infant was symptomatic with hgb 7.1 g/dl, retic 1.0%, bili 2.1 mg/dl. a 2 nd aliquot of 60ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb 7.8 g/dl, retic 0.7%, anti-ge3 titer 8, and needed another transfusion. the maternal blood stored for just 3 weeks had hemolyzed necessitating a 2nd maternal donation for baby's 3 rd transfusion. at 6 weeks, the infant's anti-ge3 titer was 2, hgb 9.2 g/dl, retic 1.7%; no transfusion was necessary. at 8 weeks of life, hgb was 10.2 g/dl, retic was 3.3%, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge3 hdfn. molecular analysis revealed that the mother was homozygous ge3-negative ge*01.-03, the father had homozygous wild type ge*01, and the infant was heterozygous ge*01/ge*01.-03. conclusion: the infant had hdfn due to antibodies to the high prevalence ge3 antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge3. hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results (1) supporting a hemoglobin trigger of 7 g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in 2016, 14, 247 rbc orders occurred and the top three patient groups were: 34% in congenital heart disease patients, 25% in hematology/oncology patients and 14% in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was 9.85 g/dl as measured in the 72 hours prior to rbc order placement. in 2016, 3105 ffp orders occurred and the top three patient groups were: 46% in neonates in the nicu, 28% in congenital heart disease patients and 13% in pediatric intensive care patients. average inr of every patient was 2.09 as measured in the 72 hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day 5 bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates (0-4 months of age), infants (>4-12 months of age) and children (>12 months-18 years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after 4 months, pr-plt represented 30% of our platelet inventory (average daily plt inventory: 45 units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among 16 transfused neonates (0-4 m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased 2,3 dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of 60 pediatric patients receiving prbc transfusion over a 12 month period were retrospectively reviewed. a total of 44 patients were identified as receiving allogeneic prbc transfusion. 16 patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was 10.6 g/dl with post-transfusion hgb rising to 14.5 g/dl. the mean prbc volume transfused was 46.3 ml using a dose of 15ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of 10-15 ml/kg in a 2 kg patient, for instance, would translate into 3 full prbc units (about 1000 ml) in an average size adult. the current standard dose of 10-15 ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only 3 case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a 7month-old, previously healthy female infant who presented to the hospital with a 1-week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of 2.7 g/dl and 9.1%, respectively, platelets of 635,000, and a reticulocyte count of 10.3%. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of 4.9 mg/dl with a direct fraction of 0.43 mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens (19 total) were non-detectable. the patient was started on a 5-day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c3. an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her 8-day hospital course, the patient received 2 rbc transfusions on the day of admission and several rbc transfusions thereafter (see table 1 ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day 6. her hemoglobin rose to 8.4 g/dl on hospital day 7 and increased to 9.5 g/dl on hospital day 8. at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next 2 weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to 11.2 g/dl on day 57 after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is 3 or 41. platelet and leukocyte immunohematology, testing and genetics table 1 . of 53 pairs, 7 pairs were complete match (2/2), 26 pairs were partial match (1/2), 20 pairs were complete mismatch (0/2). the matching rate of hla-dpb1 in our study is 13%. conclusion: the matching rate of hla-dpb1 in 10/10 hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb1 in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb1 and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china (81401732) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts13. therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts13 with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a 56 year old male with a 7-year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts13 <5% and inhibitor of 3.6. on day of admission platelet count was 95 x 10 9 /l which decreased within five days to 14 x 10 9 /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were 1 x 10 9 /l and 14 x 10 9 /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to 3.2 x 10 9 /l achieving the ratio of 3 previously shown to be diagnostic of ttp. on day 5 his a-ipc and platelet counts had improved to 7.5 x 10 9 /l and 218 x 10 9 /l respectively. absence of anti-pf4 antibodies ruled out heparin-induced thrombocytopenia at this time. on day 6 he had an unexpected decrease in both a-ipc and platelet count to 4.8 x 10 9 /l and 132 x 10 9 /l respectively, worsening by day 8 to 1.7 x 10 9 /l and 40 x 10 9 /l respectively despite daily tpe. patient received 25 additional tpes that failed to improve a-ipc or platelets which on day 32 were 0.4 x 10 9 /l and 13 x 10 9 /l respectively. a-ipc had remained at this level for 16 days suggesting that the observed decrease was irreversible. adamts13 activity remained <5% low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa-1a, hpa-3a, and hpa-5a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa-1a, hpa-3a, and hpa-5a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p2 and gi9, specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa-1aa, hpa-3ab and hpa-5aa) were collected and reacted with anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex100. the hpa-1a serum was diluted to 10 serial dilutions (from neat to 1/502) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa-1a, hpa-3a, hpa-5a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( .08 vs 37.05), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa-1a, anti-hpa-3a, anti-hpa-5a. furthermore, because the platelet was hpa-5aa, the hpa-5b serum did not react with the coupled beads with mfi was comparable to negative control (286.59 vs 127.25). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa-1a was 1/128 (0.78iu/ml) and 1/64 (1.56iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa-1a, hpa-3a and hpa-5a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in 50% of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from 1 january 2014 to 31 december 2016. clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c3, c4bp, thbd, dgke, cfhr3, cfhr1, cfhr4 and cfhr5) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of 134 patients tested, pathogenic mutations were detected in 13% (18/134) and vus in 35% (47/134). 20% (27/134) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and 9 (33%) had vus. 31% (42/134) of patients had primary ahus; of these, 28% (12/42) had pathogenic mutations and 40% (17/42) had vus. 48% (65/134) of patients had secondary tma; of these, 9% (6/65) had pathogenic mutations and 32% (21/65) had vus. in patients with pathogenic mutations, 39% (7/18) were children, 22.5% (4/18) had a positive family history of ahus and 28% (5/18) had recurrent disease. patients with primary ahus had a significantly lower age at presentation (22 6 18 vs. 33 6 20 yrs; p-value: 0.005) and a higher proportion of pathogenic mutations (28% vs. 9% p-value: 0.009) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl80 and sysmex xn9000 compared with a flow cytometric method farshid ezligini 1 , kjersti roen eriksen 1 , annette vetlesen 1 , thomas larsen titze 1 and geir hetland* 1,2 . 1 oslo university hospital, 2 university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion 2012). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and 33 buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl80 (horiba abx, montpelier, france) and xn9000 sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd41a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x10 9 /l 6 sd were 819 6118, (<) 1106 6137, (<) and 1195 6176 for counting by sysmex toa, pentraxl80, and the gallios flow cytometer, respectively. sysmex count was the very lowest 129a transfusion 2017 vol. 57 supplement s3 abstract (31.4% less than for flow cytometry), but all plt counts were significantly different (p<0.001), although least so (7.4%) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl80 seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing 1 , arishma lata 1 , roland russnak 2 , zachary antovich 2 , heather dunckley 1 and thierry viard* 2 . 1 new zealand blood service, 2 linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of 24 reactions that identify both variants of 12 relevant snps located within hpa genes (hpa-1 through hpa-11, and hpa 15). genomic dna purified from 48 blood samples, previously genotyped for hpa-1,-2,-3,-4,-5 and -15 by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were 100% concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately 90 minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late 2016 and to date has tested 749 dna samples from 400 blood donors (349 donors were tested in duplicate). concordance between the sample replicates was 100%. there were 24 occasions where the assay had to be repeated, giving a repeat rate of 3.2%. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa-3 (4.7%) and hpa-5 (1.2%) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd36 antigen deficiency expression in jiangsu chinese han population qing chen* 1 , jianyu xiao 1 and chengyin huang 2 . 1 jiangsu province blood center, 2 jiangsu province blood center background/case studies: cd36 has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd36 deficiency varies widely among different ethnic populations, with the frequency of 3-11% in asians and 2.4% of african americans, respectively. however, there is little information on the molecular basis of individuals with cd36 deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd36 expression levels and to determine the molecular basis of cd36 deficiency on the platelet surface of the han population in jiangsu region. cd36 expression levels on platelets were detected by flow cytometry among 243 blood donors in jiangsu region. donors without cd36 antigen expression on their platelet surface were further to be determined the expression of cd36 antigen on their peripheral blood monocyte cells. the coding exons of cd36 gene and adjacent introns were amplified and sequenced in cd36 deficient individuals. results/finding: among these 243 blood donors, cd36-deficient and cd36-expression individuals were 2.47% (6/243) and 97.53% (237/243), respectively. the frequencies of type i and type ii cd36 deficiency among the study population were 0.41% (1/243) and 2.06 % (5/243), respectively. among 237 individual with platelet cd36 expression, according to mean fluorescence intensity (mfi) value, 45, 141 and 51 individuals showed low, moderate and high expression levels of cd36, respectively, and their mfis were 1725.9 6 343.6, 3876.1 6 788.5 and 8431.6 6 529.9 (p<0.05), respectively. the type i cd36 deficiency individual were heterozygous for 1200-13a>g and 430-14c>g, respectively. among type ii cd36 deficiency individuals, two harbored a t insertion at position 560 in exon 6 which caused frameshift at codon 187; one has a t>c exchange at position 538 in exon 6 which resulted in a tryptophan to arginine substitution at codon 180; one has a a insertion before the 17th bp of the start codon atg in the promoter region; one were heterozygous for 748 1 2t>c and 1006 1 2t>g, respectively. conclusion: platelet cd36 surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd36 deficiency was higher than that in type i. the study findings indicated that the frequency of cd36 deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd36-deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd36-deficient individuals widely varies among ethnic groups, with 3% to 11% in japanese, 8% in sub-saharan africans, 2.4% in african americans, and 0.3% in caucasians. although some studies of cd36 deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd36 expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from 1282 unrelated platelet-apheresis donors in the eastern china. the expression of cd36 antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd36 and peanti-cd41). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd36-nagtive phenotype. for those donors with cd36-negative platelets, cd36 antigen expression on monocytes was analyzed further to distinguish between cd36 type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china (81570170) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd36 in all 1282 samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd36 phenotypes using the (mean 1 3sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd36 deficiency on platelet, in which one sample was cd36 negative both on platelet and monocyte. the frequency of cd36 type i and type ii deficiency in the eastern chinese donors was 0.08% and 3.3%, respectively. the average mfi of cd36 deficiency samples was significantly lower than cd36 positive samples (15.2 6 7.9 vs 79.8 6 37.8, p< 0.0001). conclusion: the frequency of platelet cd36 deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd36 antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd36-deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd36 antibody. background/case studies: cd36 (gpiv, chromosome 7q11.2) is an 88 kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd36 deficiency (cd36-n) is observed in 3-10% of africans (t1264g) & is classified as either type i (cd36-n plt, cd36-n mono) or type ii (cd36-n plt, cd361 mono). an acquired type ii cd36-n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type 1 cd36-n individuals can develop anti-cd36 alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd36 in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd36 phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd36 staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd36 dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an 80 year-old, group o1 african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin 4.4 g/dl & plt count 5k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, 5-10% blasts & a complex karyotype with del(7)(q22q34) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < 5. hla antibody testing was negative (class i panel reactive antibody (pra)50%). the patient was plt-xm-incompatible with most donors (10/14). a trial of 4 group o, plt-xm-compatible plts was unsuccessful (cci 1). subsequent testing for plt-specific alloantibodies identified anti-cd36. fc-phenotyping showed no cd36 on patient's mono or plt, consistent with type i cd36-n. preliminary dna results show that the patient is heterozygous for t1264g. because cd36-n apheresis plt were unavailable from blood suppliers, the patient's 3 children & grandson were screened as possible donors: all showed normal cd36 expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day 16) demonstrated new class i alloantibodies (pra 5 55%) in response to transfusion (21 apheresis plts, 5 rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd36-n & severe plt refractoriness in the setting of new mds, and 7q-chromosomal abnormalities. the absence of cd36 on plt & mono support congenital type 1 cd36-n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel 1 , kristopher fernandez* 1 , eric senaldi 2 , pascal george 2 , michael seul 1 and ghazala hashmi 1 . 1 biomolecular analytics, 2 central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur1978 http://bit.ly/2q51heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $100 adult donors who had made ! 6 donations in the previous 12 months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for 2, 3, 4, 5, 6, 7, 8, 9, 11, 15 and for hla class 1 (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/2pdplf8) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a *26:82 chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are 16,429 hla alleles documented according to the imgt / hla sequence database in janury2017, and more than 80% of them were identified in the last 10 years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a *26:82 allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn72d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove1 nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a *26:01:01:01, but 1 nucleotide substitution in exon 4, by nt 746 c-a (codon225 acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a 13 year-old pregnant woman typed as ab1, who delivered a baby affected by severe hdfn. the newborn was typed as b1 and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted 41 with the four monoclonal anti-d used (igm clones p3x61 and rum 1 and the blends clones th281ms26 and d1751d415) and were typed as c-c1e-e1. an anti-d was identified in her serum. molecular analysis showed the 410c>t and 455a>c in exon 3, the snp 509t>c changes in exon 4 and the 667t>g nucleotide change in exon 5. the set of snps found is similar to the molecular background of dol3, except for 455a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of 1089 immuno hematological tests (465 abo/d grouping (including 33 newborn samples), 12 extended erythrocytic phenotype, 562 antibody screening, 14 antibody identification, 16 dat) and 20 crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in 99.2% of the abo/d tests (n5265), 99,7% of the antibody screening tests (n5377), 88,8% of the antibody identification tests (n59) and 100% of the dat tests (n510). there were 4 discrepancies (2 abo/d for the same patient, 1 for antibody screening and 1 antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than 10 min). detection threshold of the d antibody was assessed at 2.5 ng/ml (0.0125 ui/ml) whereas the french recommendations are 20 ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso 15189 accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a 31 year old g5p3 presented during her fifth pregnancy with anti-kpb with an initial titer measured of 64. by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at 16 during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams (1.7 moms) peaking at 27 weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at 35 weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was 3.4 mg/dl with 13.6 g/dl hemoglobin. the baby typed as o positive, kp (b1) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at 6 weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by 8 weeks of age. his hemoglobin recovered to 9.0 g/dl with an indirect bilirubin of 1.4 mg/dl at 9 weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c3d-specific dat may be too insensitive to detect low, but significant levels of c3d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a "1111" reaction corresponds to about 500 molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c3d have been published. however, these are mainly designed to quantify the fraction of rbcs with c3d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c3d. study design/method: ten microliters (ul) of 1:80 (after documenting experimentally that this amount ensured maximum binding of anti-c3d) mouse monoclonal anti-human anti-c3d (abcam, clone 7c10) were added to 5 ul of a 2.5% rbc suspension. after incubation for 60 minutes at 4c, samples were washed x3, and 25 ul of 1:10 diluted anti-mouse-f(ab)2-pe (ro480, dako) were added. after incubation at 4c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro480 was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c3d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a1 rbcs stained with 10 levels (2fold dilution, 1:1 -1:512) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c3d, edta-stabilized samples from 4 healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c3d (values ranging from 0 to 3,393 abc) with level of 0-serum dilution (used to sensitize a1 rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r2 5 0.97, p < 0.0001). compared with dc-screening 1, the sensitivity of the flow cytometric assay was superior. it detected c3d sensitization at least 4 dilution steps further. the median normal level of rbc-bound c3d was 11 abc (range 7-20 abc, n54). the assay enabled demonstration of specific c3d-sensitization in the patient; the level of rbc-bound c3d in the sample was significantly elevated (1,907 abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c3d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt1 gene. in humans, the abo and gbgt1 genetic loci are located on chromosome 9q34, and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors1) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt1 genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt1 genes have been constructed of 88 vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt1 genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k1 antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a 14 year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion 2017 vol. 57 supplement s3 the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c1, e-, e1 and k1-. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d1, c-, e-, partial c1, partial e1. the probable rhce genotype, rhce*ce-jal/rhce*ce733g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant woman and her newborn carolina bonet bub* 1 , maria giselda aravechia 1 , thiago costa 1 , marilia sirianni 1 , eduardo bastos 1 , leandro santos 1 , lilian castilho 2 and jos e kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp background/case studies: rhd*weak d type 2 is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion 2007) the c.1154g>c change (p.g385a), which characterizes the rhd*weak d type 2 allele is a splicing variant that induces skipping of the whole rhd exon 9. we report an altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d1 w c-c1e1e1. the samples showed weak hemagglutination reactions (11/21) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon 9 in both dna samples. sequencing showed the c.1154g>c change and the intronic c.1154-8t>a and c.1154-31t>c substitutions, which are associated to the rhd*weak d type 2 allele. conclusion: our results showed that c.1154g>c associated with c.1154-8t>a and c.1154-31t>c variations had probably a functional impact on splicing inducing exclusion of exon 9 in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type 2. background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than 50% of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per 100 transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with 1,057 patients and 3 studies from other regions (brazil, egypt and france) with 641 patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was 0 to 26 years and for the other countries 0 to 20 years. available data from 5 us studies included a total of 91 alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens (18.7%, 16.5%, 15.4%, 7.7%, 7.7% and 6.5% respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per 100 transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of 16.5 % (14.1 to 19.2, 95% ci) vs. 9.4% for non-us studies (7.3-11.8, 95% ci) (p50.0008) and 134a transfusion 2017 vol. 57 supplement s3 more alloantibodies per transfused patient (0.25 vs. 0.096, p50.0001). similarly, the number of alloantibodies per 100 transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort (0á68 vs. 0.33, p50á0005). average number of rbc units transfused per patient in the us was also higher compared to data from france (77 vs. 45, p50.0001). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* 1 , melissa grohotolsky 2 , lisa deblass 1 , bala carver 1 and kip kuttner 2 . 1 health network laboratories, 2 miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: 23 year old white mennonite female g1,p0 presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in 21 panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated 11 reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was 2 at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at 2. although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a 15 year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g1 (r g r) cells. both rhce*c and rhd genes encode ser103 which determines g expression. rare rhd variant antigens lacking ser103 are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e1c1e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r 2 r 2 , r'r, r g r and rr cells. this patient is predicted to be r 2 r 2 (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is 135a transfusion 2017 vol. 57 supplement s3 excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* 1 , christa voliva 1 , kathy fletcher 1 , heather vaught 2 and tracie ingle 1 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology 2011; 27:1-5) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than 7 days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were >21 dat positive, 2 were weakly dat positive and 16 were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman 6.0-8.1ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for 5 minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: 5 bb, 14 bg and 1 bp. the ph of all eluates ranged from 6.9-8.1 with the highest percentage of eluates at a ph of 8.1 (35%). sixteen of the 20 eluates tested yielded the same results in both automation platforms (concordance of 80%). four eluates with different results are summarized in table 1 . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results (3) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in 1900 led to the discovery of human blood groups. in the abo system >200 alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor (38 units over 17 years) who is actually a w . study design/methods: donations were tested with the pk7300 instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns 1,2 and 4 and exons 6 and 7. specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table 1 . tests with anti-a, -a1, -b anti-a,b were negative as were the a2 cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table 2 . the significant changes were found in exons 6 and 7. in exon 6 there was a nucleotide (nt) deletion of 261g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly117ala. mutations in exon 7 included a nt substitution causing a pro156-leu change and a nt deletion 1061c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a2 cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o.01.01/abo*aw.02] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a1 and b cells. this abo discrepancy was caused by the presence of anti-a1 in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a2 cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer >1:1000. (schmidt, nacarrow et al. 1959) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. 136a transfusion 2017 vol. 57 supplement s3 extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to 96 blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to 15 days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: 92 edta blood tubes collected from random blood donors were used to extract dna from 200 microliters of whole blood on day 5, 12 and 15 days post collection. blood samples were stored at 2-8c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a260/a280 using a nanodrop 2000 (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result50) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the 92 samples, 20 samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from 10.8 to 62.6 ng/ul. all readings with the exception of 1 (10.8ng/ ul) had concentrations >5 15ng/ul. interestingly, the one that was <15ng/ ul on day 5, yielded >515ng/ul on day 12 and 15 post collection. over the next 3 months, 67 sets of 92 samples were extracted and tested by hea. eighty-three (1.3%) failed extraction and 82 (1.3%) failed hea. none of the samples that failed extraction were 12 or 15 days post collection; none of those that failed hea were 15 days post collection; 3.7% were >10 <15 days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to 15 days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas 1 , carlos villa 1 , rachel davis-rauser* 1 , helen carpenter 1 and vrunda patel 2 . 1 university of pennsylvania, 2 hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd38 monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in 2015. communications suggest all patients receiving therapy would have a positive antibody screen because cd38 is a common antigen expressed on red blood cells. currently, 154 patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd38 antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for 1 hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at 1:1 dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for 2 negative patients was observed up to a 1:4 dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a 28 y/o g1p0 at approximately 23 weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [1, 2] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m1n-s1s1 phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency >99.9), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [1, 2] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [3] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays 137a transfusion 2017 vol. 57 supplement s3 that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during 3 years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in 67 dna samples from chronically transfused patients with scd, in 65 patients with thalassemia and in 3000 dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in 3 levels: (1) rh and k matching; (2) extended matching and (3) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of 2 donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for 100% of our thalassemic and scd patients at level 1, 90% for scd patients and 70% for patients with thalassemia at level 2 and 30% for patients with scd and 90% for patients with thalassemia at level 3. the patients were transfused with a median of 36.4 rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to 5-10% with c e k matching and <1% with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube (6 drops plasma, 30 minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to 10 minutes and specimen volume to 2 drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of 202 specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of 164 clinically significant antibodies were detected using sprca technique, as well as 9 warm autoantibodies and 97 nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being 100% specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions (30 versus 13), it identified more clinically significant antibodies (129) than liss (93). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd38 on rbcs patricia a arndt* 1 , anthony salazar 2 and regina m. leger 1 . 1 american red cross blood services, 2 long beach memorial medical center background/case studies: monoclonal anti-cd38, e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd38 on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd38 antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or 2aminoethylisothiouronium bromide (aet). chapuy et al described (2015) and validated (2016) 2), and 6% aet (ph 8.0) as per the aabb technical manual, 17th ed. these treated and untreated rbcs were stored in alsevers at 4c and tested on days 1, 2, 4, 5 and 8 by two methods: 1) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from 8 total dara patients were tested with reactivity 5 1-31), and 2) flow cytometry using phycoerythrin (pe)labeled anti-cd38. rbcs were also tested on days 1 and 5 or 8 with a serum containing anti-k by peg iat. results/findings: the 0.2m dtt in ph 8.0 pbs had a final ph of 7.3 and the ph of the commercial 0.2m dtt was 6.5. results are in table 1 ; flow cytometry results from days 2, 4 and 5 (data not shown) were similar to those from days 1 and 8. rbcs treated with 0.2m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd38 by flow cytometry for up to 8 days after treatment. rbcs treated with 0.01m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening (10-30%) of reactivity with pe anti-cd38. background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery 1997 of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week 12 were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon 5 and 10) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr5) in gestational week 25. the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of 1618 pregnant women were included. nip rhd was positive in 987/1618 (61%), negative in 582/1618 (36%) and inconclusive in 49/1618 (3.0%). compared to the postnatal rhd type, 9/987 (0.1%) of nip rhd results were false positive (fp) and 4/582 (0.7%) were false negative. in 5/49 (10%) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n51618) at gestational week 12 was 25.3 (10-and 90-percentiles: 20.0 -32.4). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p50,71). the fraction of ccfdna was calculated for 150 randomly selected nip rhd true positive cases. median ccfdna ratio was 5.47 (the distribution had a highly positive skew, 10-and 90-percentiles: 0.64 -27.2). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r2 50,012; p50.49). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the 25th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an 83 year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of 1, suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only 1 in 20,000 donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with 1 cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, 4 lan-rbc units were transfused over 4 days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day 2, the patient had symptomatic anemia with hemoglobin (hb) 5.3 g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan1 but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of 10.2 g/dl was maintained for 4 days. the antibody screen was negative on day 3 post-transfusion, but strongly panreactive on day 6, with a positive dat (igg 21, c3 11) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from 10.9 g/dl on day 5 to 7.4 g/dl by day 8 with no bleeding identified, and increase in total bilirubin and ldh (peak 2.4 mg/dl and 304 u/l on day 7) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day 8 with good response (hb 8.1 g/dl). the patient remained stable and was discharged to a skilled nursing facility 6 days later. conclusion: transfusion of lan1 rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction 6 days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan1 units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* 1 , andrea gerner 1 , lynne stewart 1 , carol sostok 1 , mollie bell 2 and gregory r halverson 2 . 1 st. elizabeth healthcare, 2 hoxworth blood center background/case studies: anti-f was first described in 1953 by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the 6th antigen assigned to the rh blood group system (isbt rh6). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a 56 year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused 1 unit o-rbcs. two weeks later the patient received an additional o-rbc. within 4 days the hgb had decreased from 8.3 to 7.1 g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused 3 r 1 r 1 (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c1e1f1 phenotype. the rh phenotype and as was repeated on a sample collected 18 days later. the c typing was micro positive, mixed field only after 5 minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post 24 hour hgb increment from the receipt of a standard unit of blood should be near 1 g/dl (or 3% hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a 0.9 g/dl increase, and the second unit was only 0.5 g/dl. the last transfusion of 3 units increased by only 1.2 g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of 3 r 1 r 1 units was nearly complete. in a case from 1989, ohto and kariyone (transf. 1989; vol29, no. 3) reported a 51 cr ásurvival study of f1 rbcs in a patient with anti-f. they showed that the initital survival of f1 cells was fairly normal, however, after 18 days, there was a sudden increase of red cell destruction, and by day 27 all f1 cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd38 drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to 9 days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with 0.2m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table 1 ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to 9 days following the dtt treatment of rrbc. reactions were graded using standard serological grading of 0 (negative) to 41 (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table 1 for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to 9 days. this suggests that dtttreated reagent red cells can be stored for at least 9 days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd38 therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd38 that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd38 is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with 0.2m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract 2-5c, and observed for hemolysis (none was seen) for up to 21 days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted 2-41 with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in 4 patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than 6 months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* 1 , monique scott 2 , garcia curtis 2 , ellice wong 2 , alexa j siddon 1 and christopher a tormey 1 . 1 yale-new haven hospital, 2 va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately 25% to 50% of persons of african descent is characterized by neutrophil count of <1.5x10 9 /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p<0.05. results/finding: subjects who were clinically identified as having probable ben included 7 patients (mean age 48.7; all self-identified as african-american; 6/7 were male) and controls included 50 patients (mean age 68.5; 10 self-identified as african american; (50/50 male). all of the cases (100%) diagnosed with ben had fy(a-b-) phenotype. mean anc (1.95x10 3 /ul) and wbc counts (4.04x10 3 /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p50.0008 and 0.001, respectively) compared with controls (mean anc 5 5.46x10 3 /ul ; mean wbc count 5 8.14x10 3 /ul). there was no significant difference in mean platelet counts (161x10 3 /ul vs 213x10 3 /ul; p50.2301) or mean hemoglobin levels (12.4 g/dl vs 11.7 g/dl; p50.6031) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, 18 subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c3d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih-1000 had 100% concordance for all blood grouping assays. for ahg assays, the ih-1000 detected an anti-jka1e, anti-fya 1 warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih-1000 identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih-1000 with anti-igg,-c3d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih-1000 analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih-1000 is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of 2944 tests were performed on 1,214 adult patient samples and 208 donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in 99.9% of samples tested. there were 4 discrepancies, all antibody screening (2 false positives, 1 failure to detect a very weak prophylactic anti d and 1 positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing 80-100 group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp201 evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* 1 , kathleen bensing 1 , michael schanen 1 , cindy piefer 1 , randall w. velliquette 2 , christine lomas-francis 2 and connie m. westhoff 2 . 1 immunohematology reference laboratory, versiti/bloodcenter of wisconsin, 2 immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new 7/0 7/0 7/0 cecf 1/1 2/0 2/0 rhce*ce or rhce*ce compound heterozygotes ce254g 1 ce733g or ce48c,733g or ces or ceti 6/0 6/0 6/0 ce733g 1 ce48c,712g or 48c,733g 4/0 4/0 4/0 ce733g 1 ces or cemo or ceek or ceek(var) or cern 8/0 8/0 8/0 ce48c,733g 1 ce48c,712g or cemo or ceti 2/0 2/0 2/0 ce48c,712g/ce254g/733g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi 7/0 7/0 7/0 total 106/8 110/4 113/2 142a transfusion 2017 vol. 57 supplement s3 reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd9/4 and rd12/2, and a licensed comparator anti-e (p3gd5121ms63), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n 5 42) or edta blood from donors (n 5 72) and were tested using a manual tube method or on a pk7300 automated platform. a score 6 (11) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table 1) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r 1 r 1 , r 2 r 2 , r 1 r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: 40 were rhce*ce that were in trans to rhce*ce; 16 were various rhce*ce plus rhce*ce48c compound heterozygotes; 31 were rhce*ce or rhce*ce homozygotes; 27 were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for 6 rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with 1 rhce*cear/rhce*ce48c compound heterozygote, and with 1 rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and 1 of 2 rhce*cecf homozygotes were detected using the comparator reagent. rd9/4 and rd12/2 failed with 4 and 1 e variants, respectively (table 1) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd9/4 clone. none of the reagents detected e antigen variant expressed on 1 example of rhce*cehar/rhce*ce. conclusion: rd9/4 and rd12/2 anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the 3 monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of 19 blood group genes associated with the expression of 56 blood group antigens from 17 blood group systems. we used the illumina's hiseq 2000/2500 system to perform next generation sequencing first on sureselect-enriched genes from 16 dna reference samples with average target design coverage of 97.5%, and then on haloplex-enriched genes from 32 dna reference samples with average target design coverage above 97.0%. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for 38 blood group genetic variants in these 19 genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads (80.54%) were mapped to the target regions relative to the sureselect reads (29.23%). the mean sequence coverage depth of the targeted bases was around 200x for sureselect method and 300x for haloplex method. some exons, such as rhd exons 4 and 8, 10, rhce exon 10, ermap exons 5 and 12, cd55 exons 10 and 11, cr1 gene (most exons) and gypb exon 5, are consistently covered with less than 10x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on 38 blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than 90% concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than 90% blood group genetic variants in 19 selected genes. evidence rhce*cehar does not encode for rh34 (hr b ) antigen debra j bailey* 1 , trina horn 2 , paul mansfield 2 , najmi qazi 1 , pamela nickle 2 , jessica keller 2 , margaret a keller 2 and jan r hamilton 1 . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in 1996 and has a phenotype of c2e2c1e1 w f1 w , g2, hr 0 1 w , hr2, hr s 2, rh:33, rh:50 with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh:234 (hr b 2) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c.254c>g and rhd c.1136c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d1c1e2c1e1. her plasma contained an alloanti-s and an antibody that reacted strongly with all random e2k2s2 reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d22 and dc2 red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e2s2k2 red cells homozygous for the rhd*diiia-ce(4-7)-d, rhce*-ce48c,733g,1006t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce48c,733g,1006t/ rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr1 (2 of 2 sources) and hr b 2 (2 of 3 sources) phenotype. conclusion: the rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t haplotype is one of the rh haplotypes expressed by the original hr b 2 individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between 1/1/2006 and 3/ 31/2017. all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately 11 years, 81 patients had htla established at least once by titration studies. serological investigations on a total of 118 samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on 20 samples was successful in rule out in 60% of cases. in an additional 12 patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only 40% of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for 71% of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c.1136c>t (p.thr379met). the dau0 allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion 2016, 56:2520) recently summarized serologic characteristics and associated anti-d alloimmunization for 18 dau family alleles. we investigated two samples with the c.1136c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample 1 was from a 17 yo multiracial female. her rbcs reacted 11 s at immediate spin (is) and 31 in iat with immucor gammaclone and series 4 and 5, and mi1 at is and 41 in iat with ortho bioclone anti-d. rbcs did not react with 2 of 12 (lhm 174/102 & 57/17) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c.1136c>t characteristic of dau. rhd sequencing confirmed c.1136c>t and identified two adjacent changes, c.787g>t and c.788g>t (c.787_788delinstt), in exon 5 encoding p.gly263leu. sample 2 rbcs reacted 1w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series 4 and 5, and quotient albaclone blend and alpha anti-d. papain treated rbcs were 11s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c.1136c>t. sequencing confirmed c.1136c>t and found a new c.761c>t change (p.ser254-leu) in exon 5. the c.761t has not been reported, but c.761g encodes a stop codon (p.ser254stop) in japanese (vox sang 2015, 109:359). conclusion: we report two new alleles: rhd with c.787_788delinstt (p.gly263leu) and rhd with c.761c>t (p.ser254leu), both also carrying the c.1136c>t (p.thr379met) characteristic of the african dau cluster. d antigen associated with p.263leu is a partial d antigen with a novel epitope pattern. the p.254leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by 5/7 commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to 21. the number and diversity of alleles in the dau cluster supports that the c.1136c>t change is a major ancestral african background allele (wagner et al, blood 2002,100:306). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september 2016 to present day we got 130 samples of repeated blood donors who are known to be d negative, c positive and/or e negative from 15 blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon 4, exon 7 and exon 10 in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon 9 to confirm the existence of c.1227g>a and c.1222t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the 130 sample, we identified 71 cases (54.6%) of total rhd deletion, 18 cases (13.8%) of rhd-ce-d hybrid, and 41 cases (31.5%) of rhd variant. 39 of rhd variant were determined to be asian type del with c.1227g>a variation. 2 cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was 10 % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b 3 phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b 3 phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from 30 taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n552) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon 6 and exon 7 of the abo gene were amplified and sequenced. the abo*b3.03 allele was confirmed by pcr-rflp analysis. results/finding: among 52 subjects with b 3 or ab 3 phenotypes, 47 were genotyped as abo*b3.03. the abo*b3.03 group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage (37.81 6 6.62) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other 5 subjects with b 3 or ab 3 subjects, genotyped as abo*b3.06(n51), abo*bw.03(n51), abo*bw.11(n51), abo*bw.12(n51) and abo*bw.29(n51), displayed flow patterns differed from the abo*b3.03 group. the abo*bw.03, abo*bw.11 and abo*b3.06 subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (<10% in abo*bw.03 and abo*bw.11 subjects and <20% in abo*b3.06 subject). both abo*bw.12 and abo*bw.29 displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b3.03 genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout 2016, the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of 398 patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in 12% (n549) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n511), 4% (n52), and 74% (n536) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n54), anti-jka (n53), anti-k (n52), anti-jkb (n51), both anti-e and anti-c (n51) (see table 1 ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr1 alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr1 gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr1 gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study 60 volunteers in the gambela region (nct01282021). the whole ackr1 gene was amplified in one reaction covering 12,125 base pairs (bp). this primary amplicon was re-amplified using nested primers covering 5782 nucleotides. nucleotide sequence was obtained by 14 sequencing reactions and manually annotated using ncbi refseq ng_011626.2. the sequencing covered 1008 bp of both exons, 480 bp of intron, 2101 bp of 5'-flanking region, 947 bp of 5'-utr, 53 bp of 3'-utr and 1092 bp of 3'-flanking region and encompassed all the 470 variations present in dbsnp and nhlbi esp databases. results/finding: among the 60 samples, a total of 15 snps, including one novel snp in 5'-utr were observed. 4 snps occurred in the exons, 5 in 5'and 3'flanking region, 4 in 5'-utr and 2 in the intron. all 60 individuals carried the snp indicative of the common fy:2 phenotype; while 58 individuals were homozygous and 1 was heterozygous for the gata box mutation. no splice site mutation was detected. as 46 individuals were observed as being homozygous or heterozygous for 1 snp, we could unambiguously assign 8 distinct alleles. in the remaining 14 individuals with 2 or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than 5.5 kb of the ackr1 gene and identified at least 8 different alleles. the present study found that the vast majority of alleles (117/120) in the gambela population as defined by 15 snps, were similar to the clinically relevant fy*02n.01 allele, which in turn is defined by only 2 snps at positions c.1-67t>c and c.125g>a. out of the remaining 3 alleles, 2 were similar to fy*02 with the fy(b1) phenotype and 1 was similar to fy*02w.01 with the fy x phenotype. the high frequency of fy*02n.01 (95%) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique (95%-100%). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce(4-7)-d is the most common hybrid and is found in african blacks. it arose by conversion of exons 4-7 of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample 1 (male) and sample 2 (multiracial female), both c1c2e2e1 (presumed r1r1), presented with weaker than expected d typing; 11 is and 31/41 at iat. rhd beadchip identified the common african rhd*diiia-ce(4-7)-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c.733c>g and c.1006g>t (heterozygous) was also detected. as rhce*ce with 733g and 1006t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons 2 and 3 replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce(4-7)-d were found in both samples. sample 3 (scd male), d1c2e2c1e1, by rh beadchip had one conventional rhd and rhd*diii type 8, and rhce*ce733g/ces. as rhd*diiia type 8 has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce(48c) exons 1 and 2 had replaced those exons in the common hybrid rhd*diiia-ce(4-7)-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky926711and ky926710. we report two different and novel complex rh rearrangements: two samples thought to be r1r1 had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia(2-3)-ce. in kind, a sample genotyped as diii type 8 rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce48c(1-2)-diiia(3)-ces(4-7)-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples 2 and 3 have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r1 haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a 81-year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at 81g/l. her pregnancy history was not provided. she had received 5 units of packed red blood cells (rbcs) in the past including 1 unit within the last 3 months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r 1 r 1 , r 2 r 2 , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse 29 polymorphisms which determine 37 antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a1) and lu(b1). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of 36 antigens encoded by the kel gene, is organized into 19 exons. there are approximately 30 kel alleles associated with a kell null phenotype (k 0 ) in which no kell antigens are expressed, and 12 alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a 53 year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons 10, 11, 12,13 and 14 and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k1 kp(a-b1) js(a-b1). kel-cdna sequence analysis was performed and detected a single transcript species with c.578c, c.841c, 1790t, and missing the sequences corresponding to exons 11, 12 and 13. amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after 37c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel*02 allele. this donor was presumed to have a k 0 phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the 11 variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel*02m.12. here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table 1 ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a1 and a2 cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c.261 deleted g, characteristic of o alleles, c.467t, characteristic of a2 and some uncommon o alleles, and c.703a and c.1096a, characteristic of b alleles. genomic sequencing of exons 6 and 7 confirmed the presence of an o allele, abo*o09 261del g, 318t, 467t), and the presence of a b allele (297g, 526g, 657t, 703a, 796a, 803c, and 930a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , diana gazito 2 , afonso cortez 2 , lilian castilho 4 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the 17-bp deletion in smim1 in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs1175550 located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of 400 blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting 21 and in samples with reactivity of 31. dna was isolated from peripheral blood and smim1 was sequenced. results/finding: from 400 donor samples studied, 4 were serologically vel negative by gel-iat but positive by adsorption-elution, 158 presented a 21 reaction and the remained samples showed a reactivity of 31. genotyping results showed that the 4 samples with negative results and 5 of 26 samples that presented 21 reaction were heterozygous for the 17 bp deletion and presented the a allele rs1175550 in homozygous status. from the 21 of 26 remaining samples with reactivity of 21, 19 (90%) had the a allele of rs1175550 and 14 (66.7%) had the a allele of rs6673829. in contrast, in the 16 samples with stronger reactions we found the a allele of rs1175550 in 5 (31.25%) samples and the a allele of rs6673829 in 3 (18.75%) samples. conclusion: the molecular changes rs6673829 and rs1175550 are located in intron 2 distancing 38 nucleotides. this study reinforces the association of the a allele of rs1175550 with reduction of vel expression and suggests the involvement of a new rs6673829 change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as 0 (rh negative), or !31 on the neo or !21 on the echo (rh positive) for both series 4 and series 5 anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series 4 and series 5 anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: 80 patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in 51 of 80 (63.7%) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in 16 of 80 (20%) of samples. 67 of 80 (83.8%) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. 40 of 80 (50%) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining 13 of 80 (17.3%) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but <31 on the galileoneo or positive but <21 on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , rosangela person 2 , lilian castilho 4 , afonso cortez 2 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of 108 samples were included in the study, being 69 previously genotyped as rhd*dar1.2, 37 rhd*dar3.1 and 2 rhd*dau6. the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from 0 -99 corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon 10. rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon 3 was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that 10 of 108 samples (5 dar1.2, 4 dar3.1 and 1 dau6) had 2 rhd genes, were phenotyped as c1e-c1e1 and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these 10 samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in 2 dar1.2 samples showed the rhce*-cear/ce s genotype, in 2 dar3.1 samples the rhce*cevs.02/ce s genotype and in the dau6 sample the rhce*ce s /ce genotype. table 1 describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized 5f9 antibody (hu5f9-g4) that binds human cd47 has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu5f9-g4 (anti-cd47) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a 69 year old female with progressive follicular lymphoma who was enrolled in phase 1b/2 trial of hu5f9 g4 in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a 48 year old male with refractory diffuse large b cell lymphoma enrolled in hu5f9-g4 clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd47 therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing (41 with anti-a, non-reactive with anti-b) and the reverse typing (31 with both a 1 cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase (11 to 41), at liss-37c (11 to 31), at liss-polyspecific ahg (m1), and at peg-anti-igg (m1 to 11). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and 37c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at 37 o c ahg phase. the abo typing of the second case performed after anti-cd47 administration showed a discrepancy between the forward (41 with anti-a) and the reverse (41 with both a 1 and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is (21), at 37c in liss (21), and liss-polyspecific ahg (m1). the dat and autocontrol were negative. his genotype was determined to be a1/o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd47 therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, 37c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd38 interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected 31 reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o.01.01/ o.01.01, consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n523) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt1 and a4galt. papain-treated patient rbcs were used to screen donor plasmas (n578) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with 3 polyclonal anti-a,b and a monoclonal anti-b (clone g1/2) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors1 or nor antigens. the patient was le(a-b1) and thus a secretor. a positive crossmatch was seen with 47% of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type 1 or 2) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type 1 chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $15 years earlier. the reactions are likely due to uptake of recipient-derived ble b (type 1) antigen (isbt no. 007006), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b1). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of 2017. shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive (11) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from 1/29/2017 to 3/31/2017. reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of 19,647 columns run as part of antibody screens, 1,633 (8.3%) columns generated "?" results. assuming 30 seconds of technologist time per "?", we estimate that 13.6 hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table 1) . in 26 cases, all three columns were visually negative but the analyzer reported 11 reactivity with 1 of 3 cells. all cases had mts-gel tm antibody identification panels performed, 25 of 26 also had a mts-gel tm ficin panel. the yield for the 51 panels performed was two routine panels with weak reactivity against hla1 cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; 13 were negative. one patient newly demonstrated anti-jka. fifty percent (13/26) of visually negative but analyzer positive samples were tested with gel card lot number 3, 38% (10/26) with lot 1, and 12% (3/ 26) with lot 2. conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from 28 patients (137 cross-match samples, 301 units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in 16/28 patients. further testing was performed in 9/16. eight were tested for the presence of antibodies at 188c and confirmed in 8/8. rouleaux formation was observed in 5/9 patients, 4/5 had reactivity detectable at 188c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict 378c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* 1 , sandra nance 2 , david moolten 3 and p. dayand borge 3 . (2):47-53). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this 2.5 year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from 150 random allogeneic and 20 autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing 0.6% bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed 50k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of 62 these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* 1 , anna burgos 1 , virginia lew 2 , sunitha vege 1 , susan veneman 3 , christopher j gresens 3 , jonathan hughes 3 and connie m. westhoff 1 . 1 immunohematology and genomics laboratory, new york blood center, 2 blood centers of the pacific, 3 bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons 1-6, and long range sequencing of exon 2-6 (5.4kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least 100 rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m1n-individuals (meyer et al. br j haematol. 2016; 174:624-36) . the st a allele, also described as gyp*401, is a hybrid gypb-gypatranscript with the crossover in intron 3. we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a 24 years old pregnant, african american female g1p0 was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a 1 antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a 3 like phenotype. genetic testing did not support the serological findings of a 3 subgroup and a new abo allele, abo*784c that has never been reported in correlation with an a 3 like subgroup was detected study design/method: the patient rbcs were typed with anti-a 1 (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a 1 antibody work up was performed using three different lots of a 1 cells and three lots of a 2 cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and 48c for 30min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at 35 weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a 3 b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo*784c found in the mother. the previously reported abo*784a allele encoded an aspartic acid to asparagine change at position p.256 consistent with an a weak phenotype. also, at least five other alleles encoding an a 3 phenoytpe consisted of polymorphisms at positions c.745 through c.871, giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a3/ aweak phenotype is encoded by the variant allele abo*784c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated biorobot m48 robot using the magattract dna mini m48 extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of 38.1ng/ml and 1.83 respectively. in the second phase of the study (n 5 39), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was 20-80ng/ll and the recommended purity was an absorbance ratio of 1.63-2.1 (a260/280) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was 100% concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano 1 , izaskun apraiz 1 , maría azcarate 2 , miguel angel vesga 2 , montserrat rubia 2 , mercedes piedrabuena 2 , fernando puente 3 , barbera veldhuisen 4 , ellen van der schoot 4 and m onica l opez* 1 . 1 progenika biopharma, a grifols company, 2 centro vasco de transfusi on y tejidos humanos, 3 banco de sangre y tejidos de arag on, 4 sanquin blood supply research background/case studies: it is well established that weak d 1, 2 and 3 phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type 1, rhd*weak d type 2, rhd*weak d type 3, rhd deletion, rhd*pseudogene and rhd*diiia-ce(3-7)-d and itgb3 gene: hpa1a and hpa1b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa-1 blood group typing. study design/method: a cohort of 1000 previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications 2009/108/ce for a ivd product of list a (!10% clinical samples, >2% neonatal specimens and !2% weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n5160, 16%). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa-1 predicted phenotype were used for comparison. transfusion results/finding: no system failure, 100% call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a 100% concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was 100%. the following id rhd xt predicted phenotype results were obtained: d negative (n5361), no amplification variant (n515), weak d type 1 (n522), weak d type 1 heterozygous (n51), weak d type 2 (n532), weak d type 2 heterozygous (n51), weak d type 3 (n534), weak d type 3 heterozygous (n51), weak d types 1, 2 or 3 not detected (n5533). regarding hpa-1 blood group, the predicted phenotype results obtained by id rhd xt were 100% concordant with bds results: hpa-1a positive (n5157) and hpa-1a negative (n56), hpa-1b positive (n546) and hpa-1b negative (n5117). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types (100% specificity and 100% sensitivity for d antigen, hpa-1a and hpa-1b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with 11 rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value <50.05 (clinical analysis) or <5x10 -8 (gwa) was considered statistically significant. results/finding: of the 2795 cohort patients, 2272 (81.3%) transfused subjects were included with 129 alloimmunized children <18 years (11.0% of 1172) and 224 alloimmunized adults (20.4% of 1100). in multivariable logistic regression models, age (or 4.2, p50.009, for age 501 compared to 0-4), gender (or 1.3, p50.04, for female compared to male), transfusion history (or 3.5, p<0.0001, for 811 transfusions compared to 1-5), site, hemolysis (or 1.3, p50.05, for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or 4.5, p<0.0001) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from 32 newborns less than 2,000 gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on 32 sample pairs. dat test was negative on 30 sample pairs and two were positive. there was 100% concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on 32 placental blood samples and 29 heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < 2,000 g birth weight newborns. o-(7.4%), ab1(6.8%), b-(2.7%), and a-(0.6%). among the tested donors, 89.2% were d positive with r1r being the most common rh phenotype. in the kell blood group system, 4.5% of the donors were k positive, while the k antigen was found to be 99.4%. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a1b1) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a1b1) and m1n-s1s1 at 47% and 22.6% respectively. the le a 1 and le b 1 alleles were seen in 21.7% and 67.3% of donors respectively, while lu b -phenotype was found in 3.3% of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a1b1) and lu(a-b-) were 0.3% , 0.3% and 2.7% respectively, while the m1n-s-s-and m-n1s-s-phenotypes were not found. the frequency of the p1 antigen was found to be at 78.9% similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph 7.2 and the supernatant was discharged. a dilution buffer containing 2% human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with 10ll of washed red cells. the cell suspensions were incubated for 30 min at 378c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional 15 min at 228c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for 515-548nm. events were recorded at a frequency of 1000 cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* 1 , mark yazer 2 , nancy m. dunbar 3 and biomedical excellence for safer transfusion (best) collaborative 1 . 1 university of vermont medical center, 2 university of pittsburgh, 3 dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of 50. study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a 1:50 dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a1 and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a 4-year and 5-year period, respectively. one center provided plasma and wb testing data for a 1-year period. results/findings: in total there were 7106 group a plasma units tested and 654 (9.2%) had a high titer anti-b. the range of high titer group a plasma units between these three centers was 7.4%-12.5%. of the 1778 wb units tested, 388 (21.8%) units had a high titer; 221/1778 (12.4%) of the units had a high titer anti-a, 61/1778 (3.4%) had a high titer anti-b, and 106/1778 (6%) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with 0.2m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd38 on reagent rbcs and render them free from plasma anti-cd38 drug interference. procedures for the preparation of 0.2m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on 0.2m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of 0.2m dtt, was adjusted to ph7.16, ph 7.56, ph 7.96 using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n53) were treated with the 0.2m dtt solutions in parallel by mixing 1:4 ratio of packed rbcs to 0.2m dtt solution followed by incubation at 378c. for up to 45 minutes during treatment, the expression of k antigens was measured every 5 minutes by tube method using 2 different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every 5 minutes for each ph level. the reduction in average scores between different phs were also calculated at every 5 minutes to measure the impact of 0.2m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by !11) after 15 minutes of dtt treatment at ph 7.96; 15 minutes at ph7.56 and 20 minutes at ph7.16. complete loss of k expression was seen after 25 minutes of dtt treatment at ph7.96; 35 minutes at ph7.56 and 35 minutes at ph 7.16. the reactivity patterns of k antigen tested with 2 sources of anti-k correlate with each other. the reductions in average scores were seen between 15 to 30 minutes range of dtt treatment time when ph 7.16 was raised to ph7.56; 15 to 30minutes range when ph7.56 was raised to ph7.96; and 15 to 30 minutes range when ph7.16 was raised to ph7.96. conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to 15 minutes and/or beyond 35 minutes of incubation. the ph of the 0.2m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of 0.2m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n 5 756,221) drawn between july 1, 2013 and june 30, 2014. these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, 11,647 patients were found to possess clinically significant red cell antibodies for an overall incidence of 1.5 percent. the three most commonly encountered antibodies were anti-d (n 5 7639) 63.1%, anti-m (n 5 1288) 0.6%, and anti-e (n 5 1227) with a frequency of 10.1%. a total of 455 (3.9%) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with 182 instances (40.0%) followed by anti-e and anti-c with 79 (17.4%), and anti-c, anti-e with 26 (5.7%). of the multiple antibodies identified, 435 (95.6%) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with 7474 or 61.8% of the total. the west had 2111 (17.4%), the midwest 1538 (12.7%) and the northeast with 979 (8.1%). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of 0.05 and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the 0.05 thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p 5 0.17). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* 1 , lindsy rich 1 , sherry stern 1 , sharon wangen 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous 4 days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was 1 to 2 days. results/finding: of the 30 ntd specimens from the immucor neo, 8 resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all 3 results, it was determined that there was no reactivity and a valid result was present. the other 19 samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the 23 absc specimens that were resulted out as positive on the immucor neo, 11 specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: 3-41 with ms24 (n515), 1-31 s with ms23 (n59), no reaction with ms273, dgc02, p3x255 (n514). 14 samples tested with a polyclonal anti-c showed a 1-31 reactivity. 3 d1c1e1c1e1 cases tested with a polyclonal and monoclonal anti-e (ms16, ms21, ms62, ms63) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c.286g>a mutation in exon 2, predicted to encode the p.gly96ser substitution. for 2 apparent r 1 r 2 donors, a f-negative type allowed the prediction of a rhce*ce286a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce286a allele (c and e in cis) in all samples. 3 d1c1e1c1e1 individuals were reactive 11 s with the original source of anti-rh55, slightly weaker when compared to rhce*ce286a/rhce*ce rbc samples available from our cryobank (21). conclusion: our results confirm that the c.286g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh55). the locr reactivity appears to be rather similar when coded by rhce*ce286a or rhce*ce286a alleles. this was quite an unexpected finding, since the p.gly96ser substitution is close to the critical amino-acid for c/c expression (p.pro103ser). none of our 15 cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce286a was reported to code for a partial c (rh:-26), we consider that rhce*ce286a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly96ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes 1, 2 or 3. the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d 1, 2, or 3 genotypes. study design/methods: between 9/2015 and 2/2017 50 samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types 1, 2, or 3 genotypes, but 156a transfusion 2017 vol. 57 supplement s3 had evidence of rhd genetic sequences in exon 7 and/or intron 4 in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons 1-10 to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients (62%) followed by transfusion patients (28%); 10% had no clinical indication provided. 34 samples (68%) were found to be weak d type 1, 2, or 3 (24, 6, and 4 samples, respectively). 5 samples (10%) appear to be genetically rhd negative. genetic sequencing was performed on 11 samples; 9 had rhd genetic variants that were not weak d types 1, 2, or 3 (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples (4%) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types 1-3. of the 11 samples that had evidence of an rhd gene and did not carry the known weak d types 1-3 polymorphisms, 9 (82%) of were found to have other rhd variants, and 2 (18%) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types 1-3 variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel 2006, curr opin hematol13:476) , that individuals with weak d types 1, 2, and 3 are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report 15 months experience with rhd genotyping on 352 samples referred with discrepant or weak d typing investigated from january 2016 to april 2017. study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for 153 samples (53.3% caucasian, 32.2% african american/african, 6.6% multiracial, 4.6% hispanic, 2% asian, and 1.3% other). results/finding: rhd genotyping identified weak d types 1, 2, and 3 in 155/352 (44%) and alleles known to encode partial d phenotypes in 168/ 352 (47.7%) (table) . uncommon or rare weak d alleles including types 6, 15, 40, 42, 45, 51, 57 (n52), 59, 61, 78, 91 , and 119 were found in 13 (3.7%). the partial d alleles found were diverse, but the largest number included partial rhd*d 4.0 (n562) and *dar ( conclusion: in a multiracial cohort of 352 individuals with weaker than expected d typing 44% were due to weak d types 1, 2, or 3 and would not be considered at risk of clinical significant anti-d, but for 56% there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp242 rhd*07.02 allele causes discrepant genotyping results for rhce small c sabine scholz* 1 , sandra schneider 1 , sabrina k€ onig 1 , susanne helmig 1 and vicky van sandt 2 . 1 inno-train diagnostik gmbh, 2 rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c (307c) and c (307t) is caused by the snp on position 307 on the rhce gene. the rhd*07.02 allele (also known as rhd cat vii type 2) carries the snp 307t>c on the rhd gene and additionally the snp 329t>c. this rhd*07.02 allele has been described to partially express rhc on the d polypeptide (faas, transfusion, 2001) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, 81938) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles (307t>c, 329t>c) confirming a rhd*07.02 and one rhd*01 allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series 4 and 5 anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series 4 and series 5, and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c.463a>g change in exon 3 encoding an amino acid change p.met155val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c.463a>g (p.met155val) change in exon 3. several snps, deletions, and insertions have been reported with changes in exon 3. phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c.463a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* 1 , juan merayo-rodriguez 2 , christopher lough 1 and nancy eckert 3 . 1 lifesouth community blood centers, 2 life south community blood centers, 3 lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is 100% in most populations and greater than 99% in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, 57 year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is 6.4/ 18.8 and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal 510(k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found 251 eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to 7.1/21. the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient 1 was a 29 yo female, c2e2c1e1, whose rbcs reacted 11 by echo and 31 by neo with anti-d4, and '?' with anti-d5. testing with d4 and d5 by tube gave 21 and 11 w on initial spin (is) respectively and 41 by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and 1 w with immucor gammaclone anti-d, and all were 21 at iat. rbcs did not react with two (lhm 174/102 & 57/17) of 12 anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c.780c>a change encoding p.his260gln. patient 2 was a 20 yo pregnant female, c2e2c1e1, whose rbc were 1 w at is and 31 at iat with immucor series 4 and 5 and gammaclone, and moderately reactive, 21 is and 41 iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm 174/102 & 170/45) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon 2 gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon 2 from c.150 to c.203 encoding amino acid changes p.ile60leu and ser68asn. conclusion: we found two previously reported rare alleles: rhd with a c.780c>a (p.his260gln), previously found in france (lefloch et al. genbank ku363612), and rhd*dar with part of exon 2 replaced by rhce, reported in sub-sahara africa (granier et al. transfusion 53:3009) designated rhd*dar(ce2:v505v-s68n) with an allele frequency of 0.002 to 0.016. blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu5f9-g4 is a human monoclonal igg4 antibody recognizing cd47 that is in clinical trials to treat hematologic or solid malignancies. cd47 is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd47 is thought to enhance phagocytosis and promote anti-tumor responses. cd47 is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd47 drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n57) from 2 patients were tested over the course of 1 month treatment. plasma was tested at immediate spin (is) and by iat with r2r2, rr, d--, rh mod and rh null rbcs, as cd47 expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg4) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd47 was observed in plasma as soon as 1 hour post infusion. plasma reacted 31 to 41 at is and 41 with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker (31 and 21) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was 31. in contrast, iat reactivity using gamma-clone anti-igg was only 1 w to 11, and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd47 titer was 1 at is and peg iat with gamma-clone anti-igg, but was ! 256 with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n54) were 31 reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after 4x allo-adsorption with papain treated rr cells, but in some samples low level (micro-11) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu5f9-g4 anti-cd47 therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd47 expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu5f9-g4 is igg4. reactivity was observed in all phases and with all test methods. cd47 is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd47 reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg4, can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd47 on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat1 rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in 1985. in august 2016, she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh1k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was 21 with untreated and 31 with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:-3 etc. had been excluded, k-phenotyping revealed a k 0 -phenotype. a total of 38 silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k 0 -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel*02n.19 with c.2023t encoding p.675ter (reported in an individual from austria in 2007). there are two known k 0 -patients in our country, both homozygous for c.2023t. the daughter was a c.2023t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k 0 -donors are available in our country. with the help of the isbt rare donor working party, a k 0 o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* 1 , kshitij srivastava 2 , houda romdhane 3 , saloua jemni yacoub 4 and willy albert flegel 1 . 1 nih, 2 dtm/cc/nih, 3 regional blood transfusion center sousse, 4 regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, 159a transfusion 2017 vol. 57 supplement s3 since 2009. the tunisian population has the largest known prevalence of weak d type 4.0 alleles, occurring in 1 of 105 rh haplotypes, compared to 1 in 6,060 or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type 4.0 in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of 13,431 random blood donors were serologically screened for the d antigen using 3 routine techniques. samples with weak reactivity were tested with a panel of 6 monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of 67 discrepant samples (0.5%) were observed and expressed the serologic weak d phenotype. among them, 60 carried an allele of the weak d type 4 cluster (89.6%), of which 53 samples (88.3%) showed the weak d type 4.0 allele. only 1 sample each was found for the weak d types 1, 3 and 100 and the dvii, while 3 samples showed the consensus rhd sequence. no mutation in any of the 10 rhd exons was detected in another 3 samples. the molecular analysis of the rhce gene showed that 59 out of 67 samples with serologic weak d phenotype (88.06%) had a variant rhce allele and the most common associations were: weak d type 4.0 linked to rhce*cevs.04.01; weak d type 4.2.2 with cear; and weak d type 4.1 to rhce*cevs.02, while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost 90% of the weak d phenotypes in tunisia were caused by alleles of the weak d type 4 cluster, of which 88% represented the weak d type 4.0 allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type 4.0 in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type 4.0 phenotype. there is a possibility that the rhce*cevs.04.01 allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* 1 , heather simmons 1 , christine lomas-francis 2 , gayane shakarian 2 , sunitha vege 2 , lauren hutelmyer 3 , sandra nance 4 , jessica poisson 1 , nicholas bandarenko 1 and connie m. westhoff 2 . 1 duke university hospital, 2 immunohematology and genomics laboratory, new york blood center, 3 arc pennjersey, 4 american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a1, 2 year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with 0.2m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c2, k2, fy(a2),s2]. reactivity was detected to a titer of 64; it was not removed by prewarm technique or by 4x peg alloadsorption. the adsorbed plasma reacted with 0.2m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k2, k2, js(b2), kp(a2b2) and sc:21,23. her plasma reacted with k o , mcleod, sc:21,22 rbc samples and dtt-treated sc:21 rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused 4 aliquots of crossmatch incompatible kp(b2), s2 rbcs. her post-transfusion dat was 21 with anti-igg, 11 with anti-c3d. the eluate reacted with all rbc samples except 1 kp(b2) sample. she tolerated additional aliquots from 4 phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k2, k1, kp(a2b1), js(a2b1) and sc:1,22, discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c.1481a>t (p.glu494val) (kel*02.10) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a2). sc sequencing found heterozygosity for a 5'-2g>a change (rs12124733, 24 to 30% prevalence) and conventional sc*01, predicting sc:1,22,3. kel and sc results on the mother were kel*02/kel*02.10, heterozygous for the sc change 5'-2g>a, and her rbcs typed k2k1 kp(a2b1), sc31, ula1, consistent with dna predictions. plasma collected 7 months later was nonreactive at rt and in peg iat. her rbcs were dat2 and now typed k1, kp(a2b1), ul(a1) sc11 and sc31,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel*02.10 homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc:21,23 rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b1). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a 55-year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron 5 polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs5-1a)genotype, associated with a jkb null phenotype. anti jk3 was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused 64 old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a1) 41 and jk(b1) 21. genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk3. however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk3 or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms 160a transfusion 2017 vol. 57 supplement s3 (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a1b-); kp(a1b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on 3 separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon 2 revealed a 287g>a mutation, fy01*n.04, known to silence fya. sequencing of kel exons 1-19 exposed a silent polymorphism in exon 8, 846g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy01*n.04 mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly 100% of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, 64 self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have 2 genetic variants not previously reported in those of african descent. only 1 was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient 1. study design/methods: patient 1 was a 7 year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at 3 years of age. anti-b changed from undetectable/weak to strong at the age of 7 years. patient 2 was a 17 month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was 0/11. both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table 1 . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient 1 with 0-11 reactions up to 7 years of age. thereafter, abo typing showed mainly strong anti-b. patient 2 had 0/11 anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for 2 children on long-term tpn. patient 1 had absent/weak anti-b since birth up to 7 years of age, then developed strong anti-b with no change in feeding regiment and medications. patient 2 had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used 28 times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* 1 and elizabeth hart 2 . 1 brigham and women's faulkner hospital, 2 university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title 42, cfr part 493.1271(a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in 2016. a total of 232 samples were evaluated. each abid was subcategorized; (1) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and (2) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of 83 abids were performed on new patient samples. of the new abid samples, 29 (35%) had microscopic absc results. for the previously known antibody patients, there were 35 which accounted for 15% of the total abids performed. when reviewing the total abid workups, a total of 64 (28%) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the 29 new antibody samples were: conclusion: a total of 86% of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: (1) discontinue the use of the microscope, (2) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or (3) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than 99% in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only 10 cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a 56-yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a 59-year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received 10 rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only (1-21), and negative with anti-c3b, c3d reagent. the antibody showed a peak gel-igg iat titer of 32. results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w1), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from 33 to 83%, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody 12 anti-p 1 4 anti-m 2 anti-sd a 6 anti-le b 1 anti-jk a 1 anti-k 1 anti-e 1 anti-c 1 results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of 21% (type 1), 5% (type 2), 9% (type 3), 25% (type 42) and 40% other than 1, 2, 3 or 42. further investigation was conducted to determine the molecular identity of the «others». out of 157 samples, 119 (75%) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon 5 or both exons 4 and 5. a surprising amount of 38 samples were discovered to be normal rhd. conclusion: along with sandler et al. (2015) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types 1, 2, 3 and 42 obtained in serological weak d, 45 years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was 0,6%. no trali happened in the period. prophylaxis were used in 98% of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred 3 times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, 358 rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p <0.05). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) (0.27% vs 0.11%, p <0.001) or aes with a non-allergic type inflammation etiology (0.30% vs 0.14%, p <0.001) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes (0.028% vs 0.024%, p 5 0.614) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for 1, 2, 3, and 4 weeks were 0.25%, 0.32%, 0.39% and 0.41%, respectively (p 5 0.084, logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week (0.25%) and longer than a week (0.35%) (p < 0.05). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than 1 week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd10 codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found 11 patients from 2011-2016, who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these 10 patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these 10 cases, only 2 of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those 10 cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as 1 out of 100 transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was 8.7 g/dl but declined to 7.3 g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days 2 and 3 with poor responses. on day 4, routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from 1.0 mg/dl to 1.8 mg/dl (reference 0.8-1.3 mg/dl), and lactate dehydrogenase was above reportable linearity, >2500 u/l (reference 122-222 u/l). testing revealed additional anti-e, anti-jkb, dat c31, plasma free hb 64.4 mg/dl (reference 1-15.2 mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b1), one of which was also e1. one volume tpe was performed to remove free hb on days 5, 6, and 7 using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at 3.7 mg/dl on day 13, decreased to 2.3 mg/dl before discharge on day results/findings: twenty three cases were identified, of which 20 had medical records available for analysis. ten (50%) patients were male, the mean age was 50.4 years (range 24-76 years), 15 (75%) had an underlying hematologic malignancy or bone marrow disorder, and 3 (15%) had a history of coronary artery disease (cad). the implicated units included 14 (70%) red blood cells and 6 (30%) platelets; 17 (85%) patients received a single unit, and 3 (15%) received two or more within the previous 6 hours; the mean volume transfused was 153.3 ml (range 20-280 ml). the mean time to onset of chest pain was 92.15 minutes (sd 85 minutes), with 90% of patients presenting within 2.5 hours and 100% within 6 hours of starting the transfusion. chest pain was present as the only symptom in 35% of the cases, and for the other cases the accompanying symptoms included dyspnea (30%), fever (25%), back pain (20%), and hypo-and hypertension (10%). a post-transfusion chest x-ray was performed in 65% of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in 70% of cases and showed no findings to suggest acute ischemia. three (15%) patients had a minimal increase in their troponin levels, although 1 had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen (70%) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than 10 minutes in the majority of patients (90%). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a 6 months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts 13. based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, 37 degree, or anti-human globulin phase. check cells were found to be 21. these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o1 to a1) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a 63-year-old o1 man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha-1 antitrypsin deficiency who presented for olt (donor o1). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving 46 units of o1 rbcs and 26 units of o1 plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a1 plasma were transfused to wash out anti-a antibody prior to transfusion of a1 rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for 7 hours post-transplant. the patient's total estimated blood loss was >20l. he received a total of 71 units of rbcs (including 23 a1), 63 units of plasma (including 37 a1), 6 units of cryoprecipitate, and 9 units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o1. on postoperative day (pod) 1, a ts showed predominantly a1 rbcs with trace o1 rbcs, as well as very low anti-a igm and igg titers (table 1) . he received two additional o1 rbc units (1 each on pod 4 and pod 12) with increasing o1 rbcs on ts and rising anti-a titers. his blood type was unequivocally o1 by pod 13. the patient showed recovery of liver synthetic function on pod 1 (factor 5 activity 5 58%) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod 13, the patient had reverted to o1 with recovery of anti-a titers. at 3 months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* 1 , alfred mingo 1 , maria isabel gonzalez 1 , antoni mena 2 and juan pedro benitez 2 . 1 bst, 2 at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h1) and an oncology center (h2). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of 2016 transfusion activity in both h1 and h2 shows 7970 pse and 12572 bca, out of 13163 and 20676 respectively, since the tss deployment in 2015. retrospective analysis and classification of 6700 security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h1, nm related to pse accounted for 42.39% of all, being the mistake in concordance between patient identification and prescription order the most frequent (52.03%). the nm detected in bcas were 12.1% of all and mostly (74.52%) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are 45.51% of all and mostly (36%) the systems detects a not assigned bracelet. for h2, nm related to pse accounted for the 47.49% of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent (65.84%). the nm detected in bcas accounts for 24.48% of all and in 69.08% occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are 28.02% of all and in 65.61% of them the blood components were assigned to another patient. (1, 3, 4, 5, 8, 9, 12, 14, 19, 23, 26, 51, 56, 68) were analyzed via a commercially available elisa. comparison of adequate response to ppv23, defined as ! 2 mcg/ml for >7 serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in 72 patients (alloimmunized, 15); pre-and post-vaccine titers were available for 19 patients (alloimmunized, 6). of the 72 patients, 25 were on chronic transfusions, 24 were on hydroxyurea, 11 were surgical splenectomized, 58 patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv23 in the previous 10 years; 9/44 also reported previous history 13-valent sp conjugate vaccine within the last 5 years. baseline pre-vaccination titers (n572) showed no difference between alloimmunized and non-alloimmunized patients (all p-values >0.13). in the group with pre-and post-vaccination (n519) titers available, 11 out of 13 (85%) non alloimmunized patients had an adequate response versus 4 out of 6 in the alloimmunized group (67%, p 5ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from 2012 to 2016). patient harm events recorded within the veritas system from january to july 2016 were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of 114 tres per month and 1300 per year were found over five years. 81% of tres are associated with pre-bb activities, 10% occur within bb, and 9% are post-bb events. sample collection and handling represent 80% of total tres. most tres (96%) were reported by bb staff, 4% were reported by non-bb staff. patient harm analysis revealed an average of four level 0 (near miss), three level 1 (no known harm), and 0.3 level 2 (patient harm) per month. no deaths related to tre were detected over the seven month january to july 2016 period. patient harm was associated with tres occurring in the bb (17%) and post-bb (83%). these events were reported externally (78%) and by bb staff (22%). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january 1, 2013 and march 28, 2017 was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within 6 hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by 5 and added to the number of individual whole blood plts; apheresis plasma units were multiplied by 2 and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was 31. the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the 4-hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions (18[10 9 /l vs. 14[10 9 /l, p50.029). there were no significant differences in the frequency of effective therapeutic (55% vs. 72%, p50.1) and prophylactic (63% vs. 54%, p50.09) transfusions between the prcs and 25gypcs. we did not find significant differences between prcs and 25gypcs in cci1 after prophylactic (16.0 6 7.1 vs. 19.2 6 8.7) and therapeutic (11.3 6 9.0 vs. 11.8 6 5.8) transfusions, in cc24 after prophylactic (20.0 6 9.2 vs. 22.5 6 12.8) and therapeutic (13.3 6 8.9 vs. 13.9 6 8.) transfusions. there were no significant differences between prcs and 25gypcs also in ma1 after prophylactic (62.2 6 8.5 vs. 60 6 8.5, p50.7) and therapeutic (61.3 6 9.9 vs. 60.9 6 12.7, p50.08) pc transfusions. reduction of the severity of bleeds was obtained in 78 (86%) of the 91 cases after prpc transfusions and in 51 (84%) of 61 cases after 25gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, 3 and 2 cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in 2006. it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in 2014 under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were 593 units administered using the emergency transfusion process in the 3 months before the change was implemented. it was found that 51/593 (9%) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month (2530 components administered), 109/2530 (4%) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: 1) a stable anemic inpatient, 2) a stable anemic inpatient to be discharged, and 3) an asymptomatic post-operative inpatient. results/finding: we identified 67 canadian tm experts: 48 (71.6%) provided a response and most had a primary place of practice in a laboratory setting (38/48; 79.2%). for a stable, non-bleeding, anemic inpatient, 87.5% of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with 31.2% generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period (1-2 hours), a repeat hemoglobin >18 hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse (38.1%) compared to an inpatient potentially symptomatic due to anemia (72.1%). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium-51 ( 51 cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n514) were divided into two 150ml aliquots, which were labeled with selected concentrations of s-nhsbiotin (3 and 30 lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of 38.4 6 1.6%). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately 2 million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that 561nm excitation of phycoerythrin (pe)-sa and high laser power (150mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with 3lg/ml of biotin resulted in $50,000 mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, 95% ci) of 1 in 380,000 (0.0003%). the lld95 for rbc labeled with biotin at 30lg/ ml was $ 1 in 1 million (0.0001%). biotinylation was not associated with increased levels of hemolysis (0.40 6 0.22% before labeling versus 0.34 6 0.12% after labeling; p50.09) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes (150ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer-250 (hboc-201) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: (1) wb, (2) wb110% hboc volume (model of two units in an adult), (3) wb110% fdp, and (4) wb110% hboc110% fdp. samples (5)-(8) simulated autoresuscitation by adding 25% plasmalyte to 1-4. susceptibility to lysis was tested with 75ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of 50%, 60%, 75%, and 100% volume replacement with hboc and/or fdp, with or without prior 25% plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb1hboc, and wb1hboc1fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb1hboc in autodilution simulation (mean lysis 4.79% vs. 16.36%; p<.05). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc (10%) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even 75% hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc-201, there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc1plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in 8/2013 and 2/2015. we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of 81 patients, 24 were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery (43%). pcc was given for warfarin reversal in 31% of cases. a subset of patients received plasma within 2 hours prior to pcc (40%) or 24 hours after (47%). pcc was most frequently ordered in the or/perioperative service (25%). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in 2016, several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; 1) simplify and expedite the process; 2) improve communication and expectations to decrease tat; 3) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood (4 units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of 61 er episodes were received. the average tat from order to delivery at the bedside was reduced by 50% (7.0 minutes compared to 14 minutes previously), while the compliance rate for er orders and physician documentation was 100% (61/61), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february 2016 -february 2017. mt was defined as the transfusion of ! 10 rbc units in a 24-hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at 30 days. results/findings: thirty mts occurred during a one year period. a total of 192,441 blood products were transfused during that time period. gender distribution was 21/30 (70%) males, and the average age of all patients was 68 with a range of 21 to 70 years of age. surgical patients accounted for 26/30 (86.7%) mts, and 4/30 (13.3%) were critical care patients. tumor categories included carcinomas (14/30), sarcomas (13/30), leukemias (2/30) and lymphoma (1/30). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer (6/30) was the most common disease seen followed by sacral chordoma (4/30). mtps were activated in only 8/30 (26.7%) cases. thirty-day survival was seen in 25/30 (83.3%) patients. only 1 of 5 mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage (3/ 5) or perisplenic hematoma (1/5). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a 54-year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a 26-year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a 70-year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* 1,2 , rebecca ross 2 , christopher a tormey 1,2,3 and amit gokhale 1,2 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: daratumumab (dara) is a igg1 monoclonal antibody therapy that specifically targets cd38, a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd38 expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, 54 subjects were identified for analysis. their mean age was 67.8 years, with 29 male and 25 female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of 0% (0/54) prior to administration of dara. of these patients, 22 were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these (0%; 0/22) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the 22 patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of 2015 for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september 1, 2015 through april 7, 2017 were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age 60 was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in 2016, 2,523 unique women receiving a total of 5,889 units of red cells (rc) in 2,906 transfusion episodes were identified. their median age was 45 (range 11 -60). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in 46 (1.6%) and 283 (9.7%). 635 (21.9%) transfusion episodes were associated with the use of 3 units or more rc. as a result, 1385 (47.7%) episodes resulted in a post transfusion hb ! 9g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given 2 (<0.1%) and 116 (4.6%) women respectively. upon discharge, 442 (15.0%), 84 (3.2%) and 1,994 (65.8%) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to 386 (16.0%) women. conclusion: in the present study, it is observed that 49.7% transfusion episodes were given at hb ! 7g/dl. a substantial number of episodes (71.7%) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! 9g/dl). for iron replenishment and bleeding control, up to 16.0% transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes (0.8-1.3x10 10 per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with 25 gy and transfused over 3-4 hours within 24 hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* 1 , lee grabner 1 , brenda herdman 2 , robert fallis 1 , amin kabani 3 and charles musuka 3 . 1 canadian blood services, 2 kenora rainy-river regional laboratory program, 3 diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over 50 years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between 2008 and 2011 before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and 38% were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels (8.7 g/dl; p50.94); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units (9.9 vs 9.8 g/dl; 1.2 vs. 1.1 g/dl; both p50.02). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients (1.3 g/dl vs. 1.0 g/dl; p<0.001). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p50.01). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p50.53 and p50.32, respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than 1% of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered 33 times in total, affecting 15 patients and 21 users. stratified by location, the majority of triggers occurred in the perioperative areas (18 times) and the liver icu (6 times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin 25% iv solution, human albumin 5% iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* 1,2 , mahmut akgul 1,2 , hollie m reeves 1,2 , robert w maitta 1,2 , marcie pokorny 2 , anne capetillo 2 and katharine a downes 1,2 . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a 7-month retrospective study (january-july 2016) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was 200-400 mg/dl with critical value of 50mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were 301 adult (>18 years) orders reviewed by tms out of which 299 were approved. of the 299 approved orders, 136 (45.5%) were in agreement with tms's estimated dose. of 163 (54.5%) orders that were not in agreement with the tms's estimate, 142 (47%) were underestimated and 21 (7%) were overestimated. seventeen of 299 orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of 23.6 mg/dl (range 0.3 to 124 mg/dl) and a median excess of 13.3 mg/dl (range 0.6 to 155 mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was 12 mg/dl above target, which is significantly higher with intervention than without (which could have been 12 mg/dl below the target; p<0.0001). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention (11 vs. 2.7 mg/dl, p50.07). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention (15 vs. -23.5 mg/dl, p<0.0001). seven of 299 (17) 40 (24) orders were for critically low fibrinogen (<50 mg/dl) and 4 of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were 10 and 5 units (59.5% and 25%) i.e. 2 and 1 pools and the most frequent orders in the disagreement group were 10, 1, 5 and 2 units (33%, 20%, 17% and 16%). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was 2.36 and posttransfusion was 1.91. only 22% of patients had their inr corrected to 1.5, while 28% had no change, or had increased inr. (table) . the majority (67%) of patients received 2 units of plasma. the mean plasma dose was 6 ml/kg. there were 4 transfusion reactions reported, 1 non-hemolytic and 3 transfusion associated circulatory overload reactions in which 1 required admission to the icu. two patients experienced bleeding during ir procedures (tips) and 1 developed a hematoma (tunneled central line). the median of inr correction in this study was 1.9 with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is 1.9. randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. 3 of the 111 patients experienced bleeding complications indicating that inr of 1.9 may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately 4% of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up 45 % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population (40%) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately 85% of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july 1, 2014 as follows: 2 units of group a plasma and 3 units of group ab plasma is provided for the massive transfusion protocol (mtp) along with 5 units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed 235 mtps at our institution between july 2014 and march 2017. twenty patients (8.5 %) were transfused with incompatible group a plasma (5 group ab and 15 group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining 15 patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april 2017, our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in 2013, bonfils immunohematology reference lab (irl) sent out approximately 245 special platelets for patients with hla antibodies. by 2016, hla platelet orders increased dramatically and the irl sent out over 650 special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over 10,000 donors in the database with historical hla typing. however, only approximately 3500 of those donors actively donate. in the denver area, one of the most common hla types is a1 a2 b7 b8. only 81 of the 10,000 donors have this type (0.81%). therefore, to fill an hla platelet order request for a common hla type, only 28 donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a9 a11 b17 b35, there is only 1 out of 10,000 donors (0.01%) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a1 for example, all of the a1 positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in 2012, approximately 27% of these special order platelets were pra matched and the remaining 73% were hla matched by donor recruitment. by 2017, approximately 59% of special platelets sent are pra matched. this change resulted in a 2.2 fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains 6 packed red blood cells (prbcs), 4 fresh frozen plasma (ffp) units, and 1 plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august 2013 to march 2017 mtps were activated at ucm, of which 251 orders could be traced to the origin: 118 on inpatient floor (including icus), 58 in the operating rooms, 46 in the emergency department, 25 in labor and delivery, and 4 in other procedure rooms. of the 2207 prbcs that were issued, 1406 were transfused (64% utilized); of the 1446 units of ffp that were issued, 901 were transfused (62% utilized); of the 359 platelet packs that were issued, 246 were transfused (69% utilized); of the 64 units of cryoprecipitate that were issued, 49 were transfused (77% utilized). since march 2016, the time of first product issue after the initiation of an mtp has also been tracked. of the 84 events that fall within this time period, 39 (46%), had the first product issued in 5 minutes or less. another 31 (37%) were issued between 5-10 minutes, resulting in over 80% of patients being issued their first blood product within the first 10 minutes. only 15 of 84 (17%) events had an initial time greater than 10 minutes and none were greater than 21 minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level 1 trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($60-70%). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within 10 minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* 1 , shailesh macwan 1 , arline stein 1 , jane fischman 1 , nancy nikolis 1 , matthew bank 1 , lennart logdberg 1 , alexander indrikovs 2 , sherry shariatmadar 1 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: massive bleeding is generally defined as any patient who requires 1 blood volume replacement within 24 hours and/or receives transfusion of greater than or equal to 4 units in one hour with 177a transfusion 2017 vol. 57 supplement s3 ongoing bleeding. our mtp was officially implemented in 2013 in preparation for an initial verification as a level 1 trauma center by acs. our mtp has the following packages: 1st pack has a ratio of 4:4:1 (rbcs, plasma & platelets) and subsequent packs a ratio of 6:6:1. our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march 2016 to add cryo and pcc at a defined point in the mtp (cryo is included in the 3rd pack and pcc in the 4th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received >20 rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had 8 patients who received >20 rbc in 2015 and 2016. mtp had been activated for all patients and all patients received between 0.5 to 1 unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt >16 seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of 200 mg/dl (table 1) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of >20 rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's 5-day and 114-day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective 10-year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of 63 published cases (31 reports) of ta-pls, 8 (4 reports) were stem cell and 55 (27 reports) were organ transplants. all 8(100%) stem cell transplants and 52 (95%) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the 4 reports of stem cell ta-pls, 3 actively screened for antibodies in the immediate post-transplant period, and of the 27 reports of organ ta-pls, 1 actively screened for antibodies. these screens detected 5 cases of stem cell ta-pls before hemolysis became apparent and 2 cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* 1 , rosario mallari 2 , marc de asis 2 , elaine shu 3 , jonathan hughes 4 and tho pham 1,3 . 1 stanford university, 2 stanford health care, 3 stanford blood center, 4 bloodsource background/case studies: mur antigen is present in 7-10% of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. 41 year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received 1-2 rbc units every 1-2 weeks to maintain a hemoglobin (hb) level of 8 g/dl. the patient remained stable for 5 months when his hb level acutely dropped to 6.6 g/dl. the antibody screen remained negative for an additional 2 months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for 30 of the 33 rbc units he received. 3 units were from an asian donor, and a unit transfused 13 days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of 64,495 donors at a hospital-associated blood center located in a region where asians comprise approximately 30% of the population. results/finding: 6.6% of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for 5245 of 37933 (13.8%) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over 10% of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* 1 , princess maynie 1 , carol chandler 1 , shelia garret 1 and pampee young 2 . 1 vanderbilt, 2 vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [3] [4] [5] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of 26 samples have been analyzed (table) , 17 rh and 9 non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being 2.77 (range 1-7) times higher. the average fold change for rhd/c/e antibody titers were 3.2, whereas the average fold change for non rh titers was 1.03 (range 1-2). the range for anti d titers was particularly variable, 2-7, whereas for c/e, it was 1-3. the overall reproducibility/precision of the automated analyzer was $90%. to correlate the 178a transfusion 2017 vol. 57 supplement s3 increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $3 times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of 250. units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to 33% of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately 65% of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix 0.8% suspension of a1 and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with 15 min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, 32 group a, and 16 group b. results/finding: of the 100 whole blood edta samples tested, 26 (25 group o and 1group b) exceeded a high titer threshold of 250. when the pas samples of these 26 donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of 250 when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of 250, a 96% decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from 2 main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year 2016 was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the 27(59%) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the 2 mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these 3 hospitals had a lower overall rate of wastage including their own donations than the other 13 hospitals that did not collect in-house plt. the other 13 hospitals had wastage rates ranging between 9-54%. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* 1,2 , christopher j gresens 3 , anne capetillo 2 , hollie m reeves 1,2 and katharine a downes 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center, 3 bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of <0.1%. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a 58-year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure (117/65 mm hg to 205/89 mm hg) followed by hematuria (500 ml). chills and rigors resolved; blood pressure stabilized after 15 min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of 256 (igm) and 1,024 (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* 1 , leila patricia de sousa fontenele 1 , isabel nagle reis 1 , carolina bonet bub 1 , araci sakashita 1 , raffael zamper 1 , cristiane nakazawa 1 , tatiane almeida omura paula 1 , patricia silva batista 1 , marcio dias almeida 1 , fernanda loureiro de andrade orsi 2 and jose mauro kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of 671 cases of olt performed between 2011 and 2015 in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of 671 consecutive patients submitted to liver transplantation between 2011 and 2016 were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or 1,726 -95% ci: 1,147-2,597, p:0,009), absence of hcc (or 0,295 -95% ci:0,199-0,437, p:0,0), cirrhosis of any cause (or 4,161 -95% ci 1,816-9,534 -p:0,001), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or 5,236 95%ic 2,212-12,394) and retransplantation due to primary non function of the graft (or 5,791 95%ci 1,33-4,25,206, p: 0,019) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a1/b in group o platelet products. charles k. childers* 1 , mark destree 2 , ashley rose 2 and theresa nester 3,4 . 1 madigan army medical center, 2 bloodworks northwest, 3 bloodworks nw, 4 dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a1 or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a1/b in group o platelet products is presented from a large regional blood center collected over 10-12 months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in 2 ml edta sample tubes. a single 1:150 dilution of plasma was prepared using a hamilton microlab 600 series dilutor using 2235.0 ml saline diluent and 15.0 ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a1 or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at 3175 rpm for 20 seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a1 cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a1 or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of 150 is used, approximately 3% of group o apheresis platelets will have a high titer, most commonly with anti-a1. less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their 5 day outdate. after 10 months of testing pspp units and verifying that the products rarely had a high titer (0.28%), the blood center stopped performing this testing for pspp units. rh1) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to 19% (from 24% on the previous day). his hematocrit did not increase (18%), and over the ensuing 12 hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of 8 in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c3 in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of 24% while reducing the number of a rbcs in his circulation by approximately 70%. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes 7-14 days for antibodies to develop and they are short-lived (3-5 weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the 2016 calendar year from 9 hospitals. an additional hospital* provided data for august-december 2016. rbc transfusions in patients <1 year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/5 70 years was also determined. results/findings: see table 1. the fraction of all transfused rbcs that were oneg ranged from 5-14% (row f). the percentage of oneg rbcs transfused to oneg patients ranged from 37-89% (row g); thus, non-oneg patients received 11-63% of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients (68%-100%; row i). overall use of oneg rbcs could have been reduced by 10%-39% if opos units had been given to all oneg patients >/ 5 70 years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* 1 , nehad mohammed 2 , marwa aly 2 and nashwa fahmy 2 . 1 national blood transfusion services, 2 nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on 206 multitransfused patients who received blood transfusion chronically at our central blood center. they were 129 thalassemia patients (128 bthalassemia patients, one patients with a thalassemia), 10 sickle cell anemia patients and 6 immune hemolytic anemia patients (4 auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). 29 oncology patients, 32 chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: 32 out of 48 (67%) alloimmunized patients and 16 out of48(33%) non alloimunized patients(p<0.001) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june 2016. screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified 136 articles for data abstraction, where 48 articles were transfusion guidelines. there were 12 guidelines (25%) that made a recommendation, 11 for a single unit and 1 for multiple unit transfusion strategy (table 1) . review b identified 3 retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or 9.4, 95% ci 5.02-17.60), although heterogeneity was high (i 2 597%). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december 2016 and february 2017 was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the 3 months, 1723 units of platelets were transfused to 238 recipients. over the 3 months, a median of 4 units was given to each patient with a range of 1 to 69. the overall distribution of products used was 58% plasma, 24% pr, 7% pas f and 11% pas c. thirty percent of patients (n572) received all of their products on a single day. single units were given to 54 patients while 14, 3 and 1 received 2, 3, and 4 units respectively. the distribution by product type was 56% plasma, 25% pr, 13% pas c and 4% pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the 3 month period (p5 1.00). the distribution by service was different for the groups receiving multiple units. for single units the distribution was 44% hematologic malignancy, 22% infusion clinic (nos), 13% solid tumor medicine, 11% surgery, and 9% pediatrics. for those receiving multiple units the distribution was 50% surgery and 16% each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of 0.022. conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the 3 month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* 1,2 , yvemarie b.o. somsen 2 , maike e. van hezel 2 , marleen straat 2 , robin van bruggen 1 and nicole p juffermans 2 . 1 sanquin research and landsteiner laboratory, 2 academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains 220 mg of iron and 25% of the rbcs are cleared by macrophages within 1 hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in 52 icu patients who received one rbc transfusion, different iron parameters were measured before and 24 hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il-6 levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion (4.1 vs. 4.3 mmol/l, p50.69). also, the transfusion had no effect on transferrin saturation (12 vs. 13 %, p50.13), ferritin (531.0 vs. 599.0 mg/l, p50.74) and il-6 levels (35.0 vs. 25.5 pg/ml, p50.09). hepcidin levels increased in these icu patients after rbc transfusion (223 vs 332 ng/ml, p50.01). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (-2.7 vs. 3.7 % change, p50.05). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il-6 or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since 2011 and pathogen reduced platelets have been available since 2016. in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between 2012 and 2016. during this time pas c, pas f and pr went from 13% to 40% of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in 2011 patients received an average of 5.41 units/recipient/month and in 2016 the average was 5.39 units/recipient/month. the intervening data points for 2013, 2014, and 2015 were 5.92, 5.66, and 5.92 respectively. the 5 year average was 5.66. the slope of the graph for all 5 points was y5 -0.004 15.672. the two sample t-test showed that the plt/recipient/month from 2012 to 2016 was not statistically different with a p value of 0.81. conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that 4% to 8% develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab 17 statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least 5 k/ul. the analysis revealed that the mean of 9.35 k/ul (n584) had a 95 percent lower bound confidence interval platelet increment of 7 k/ul (p<50.001) results/findings: 123 (median 4 range [1-43]) hla matched leucoreduced irradiated sda platelets were transfused to 17 (6m/11f) patients, median age 60 years (range 27-83). 15/17 (88%) patients showing broad alloimmunization to hla class i/class ii antigens. 2/17(12%) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority 16/17 (94%) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); 9/11 (81%) female patients had prior exposure via pregnancy and 4/11 (24%) had a history of hsct. 63 (51%) platelets were abo identical-platelet increment median 7 k/ul (range -14 to 61), 53 (43%) were abo compatible -platelet increment median of 2k/ul (range -10 to 46) and 7(6%) were abo incompatible with platelet increments median 8k/ul ( range -10 to 32). platelet counts were performed within 24 hours in 73 (57%) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* 1 , renee leblanc 2 , dongfu xie 2 , alice cabe 1 and yanyun wu 2 . 1 overlake hospital, 2 bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. 1 these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about 350 for 3 years (from 2014 to 2016) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and 68 % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than 0.25% of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a 32-year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of 6.4 g/dl (baseline 9-10 g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was 5.9 g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin (8.8 to 6.2 mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to 6.2 g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october 2015 to march 2017. patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were 139 rbc products transfused to 40 waiha patients. twenty-three (57.5%) patients received at least 1 incompatible unit. the mean age was 51.4 years (range 4-93 yrs) with 50% women. ethnic composition was 55% african-american, 40% caucasian, and 5% patients of mixed/other ethnicity. one hundred fourteen (82%) of these products were released as li products and 25 (18%) were compatible. ninetythree (81.6%) of the li product transfusions had a post-tfn hct change of <3% whereas only 14 (56%) of the compatible product transfusions resulted in a post-tfn hct change of <3% (p50.0092, v 2 (1), exact methods). the mean hct increase in the compatible group was 1.83% per unit vs. a slightly lesser per-unit increase of 1.71% in the li group (p50.82, t-test, 2-tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for 12 units. units that were 31 incompatible had a lower mean post-tfn hct rise compared to all other li units (1.49% vs. 2.15%); however, this difference was not statistically significant (p50.38). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected 3% per unit more frequently than it was for waiha patients who received compatible products (81.6% vs. 56%). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the 31 li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first 4-hours from mt onset) was calculated with 95% and 99.8% control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were 5482 mt cases from 25 hospitals (17 tertiarylevel, 6 smaller/medium sized acute-care and 2 specialist women's). number of mt cases per hospital ranged from 5 to 721. patient median age was 65 years (iqr 49, 76), 62% were male and 73% required admission to intensive care. the most common clinical groups were cardiac surgery (21% cases), trauma (20%) and gastrointestinal hemorrhage (13%); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the 17 tertiary-level hospitals was 21% (range 13% to 33%) and 16/17 (94%) were within the 95% control limit. cb that required !10 rbcs within 24-hours of mt onset occurred in 40% of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals (67% versus 78%; p50.03). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of 5 bcs and 280 ml of platelet additive solution iii (pasiii) were used to produce pcs (n55). pcs were stored on a flatbed agitator (60 cycles/min) in a temperature-controlled cabinet at 22 6 28c for 4-7 days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of 300 mm hg was applied. using clamps, a flow velocity of 90-120 ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted 10-30%, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration (0.8-1.6x10 11 /l) and number (>250x10 9 /unit). simulated transfusion had no effect on the percentages of cd62p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* 1,2 , joan sevcik 2 and joseph e. kiss 1,2 . 1 university of pittsburgh, 2 blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, 2 ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for 3 patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution 1:50 is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient 1 and 2 and 3 (control). results/finding: all 3 patients were ab blood type. patient 1, a 57 year old female with recurrent adamts13 deficient ttp, received 2 courses of tpe (total 12 tpe procedures) for relapse and exacerbation. ten out of 12 procedures were performed with ab and a plasma (average 916 ml of a plasma or 24% of total plasma volume for 10 tpe procedures). patient 2, a 27 year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of 12 tpe procedures. four out of 12 procedures were performed with ab and a plasma (average 1210.5 ml of a plasma or 48% of total plasma volume for 4 tpe procedures). patient 3, a 33 year old female with adamts13 deficient ttp who served as a control, received a total of 10 procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between 3 patients. the trends of hemolysis laboratory data for patient 1 and 2 were comparable with patient 3. all 3 patients had negative dat. only patient 3 received 2 rbc transfusions. all 3 patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to 48% of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd36 negative platelet unit is not available for a patient with anti-cd36 antibodies sameer khatri* 1 , charles harmon 1 , brian r curtis 2 and chisa yamada 1 . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd36) is one of the identified plt surface ags and deficiency is rare, but found in asians (3-11%), sub-saharan africans (7-8%) and also in some people from mediterranean descent. two types of cd36 deficiency have been described. type 1 deficiency is the complete lack of cd36 on both plts and monocyte-macrophages whereas type 2 deficiency lacks cd36 on plts with variable expression (12-99%) on monocytemacrophages. transfusing plts in a patient with cd36 deficiency is challenging given the rarity of cd36 negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd36 negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a 21 year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than 20 units of apheresis plt units over a 2 week period without any significant increase in plt count. cross-match compatible plt unit found in 1 of 32 units and hla matched units were tried without success. at that point, a cd36 ab was identified in the serum and the patient's type 1 cd36 deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was 95% due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd-36 negative (but blood type different and hla 187a transfusion 2017 vol. 57 supplement s3 unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd36 non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every 2 weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd36 antibody positive reactivity in serial dilutions has reduced from 1:32 to 1:2 dilutions and his hla class i pra has decreased to 37%. he is currently receiving 2 apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to 1.0 k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd36 abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct 2015 utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for 2016 to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec 2016, 101,854 blood donations were screened for b microti by immunetics elisa. of those, 267 (0.26%) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between 0.08% and 0.42%. no patient babesia transmission has been reported since implementing this test, but we only had 4 documented babesia ttd cases from 2007-2017. donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of 267 positive test results, 160 lookback investigations were initiated representing 59% of positive donations. lookbacks were only performed when there was a donation within 12 months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to 80% were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented 0.25% loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only 0.25% of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in >90% of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for 53 blood donors, 51 were positive with an average load of 1.49x10 2 copies/ml of plasma, a median value of 80.5 copies/ml of plasma, ranging from 0 to 1.87x10 3 copies/ml. pre-transplant viral loads were similar. for 41 transplant candidates, 40 were positive with an average of 3.70x10 2 copies/ml of plasma, a median value of 88 copies/ml of plasma, ranging from 0 to 1.18x10 5 copies/ml. post-transplant viral loads were remarkably different. for 94 transplant recipients, all were positive with an average of 3.14x10 5 copies/ml of plasma, a median value of 1.25x10 5 copies/ml of plasma, ranging from 0 to 4.6x10 7 copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around 100-200 copies were present in >90% of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least 2 orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt (17) 0.2 post-ivig: all plt (41) 5.5 post-ivig: cd36-negative plt from relative (1) 0.8 post-ivig: single donor apheresis (23) 4.3 post-ivig: cross-match compatible (15) 6.1 post-ivig: flow cross-match compatible plt (2) 12.6 188a transfusion 2017 vol. 57 supplement s3 detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv (1/2/3/4), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows 100% of specificity, with no false positive results on the 40 control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of 1 tcid 50 /ml for denv-1, denv-3 and chikv and of 10 tcid 50 /ml for denv-2, denv-4 and zikv. finally, the first results obtained on 110 denv(1), 69 zikv(1) and 50 chikv(1) clinical samples show 85%, 87% and 96% correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of 4, 8, 16 and 32 were prepared from 192 plasma or 192 serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from 1:128 to 1:1024 (2-fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of 16 or higher and was not eliminated by the addition of a blocking step. pools of 4 or 8 samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of 4 or 8 samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to 8 plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $100 cfu/ml of 12 organisms associated with platelet contamination and incubated at room temperature for 18-24 hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher (1 1 log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc 12924, were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into 4 ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta 3d and virtuo the organism was recovered 100% . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc 12924. however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of 4ml lrap demonstrated 100% recovery when loaded onto the virtuo and 3d ( table 1) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus 1-4, and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in 2014, a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of 150 mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as-5 rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final 200 mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and 3hrs after amustaline addition, respectively, for titration by plaque assay on vero76 cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, >5.1 log 10 or log 10 /ml of rrv was achieved, with >5.5 log 10 or >5.2 log 10 /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; 255-325 ml), large volume (lv; 300-390 ml) and dual storage containers (ds; 300-420 ml) designed to treat platelet doses between 2.9 and 8.0x10 11 . the new triple storage (ts) set was designed to expand the dose range to 12.0x10 11 and the maximum volume to 650 ml, generating either 2 or 3 doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or 100% plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in 47% plasma/53% pas or 100% plasma with a final volume of $650 ml and a dose of 9-12 3 10 11 platelets. these conditions represent inactivation using the lowest amotosalen concentration (135 mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log 10 titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of 6-10 logs in respiratory secretions of mers patients, and with lower genomic titers of 4-5 logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log 10 reduction/ml (log 10 /ml) 47%plasma/ 53% pas e .coli 6.0 <-1.0 >6.0 e. cloacae 6.4 <-1.0 >6.4 k. pneumoniae 6.6 <20.1 >6.5 s. aureus 6.7 <-1.0 >6.7 blue tongue virus 4.9 <-1.0 >4.9 bovine viral diarrhea virus 4.6 <-1.0 >4.6 adenovirus-5 1 3.9 <-0.6 >3.9 100%plasma k. pneumoniae 1 6.5 -0.5 >6.5 s. aureus 1 6.2 <-0.7 >6.2 adenovirus-5 1 4.5 <-0.6 >4.5 1 n53 190a transfusion 2017 vol. 57 supplement s3 abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged 3 times up to 9 days, assessing the infectious titer and genomic titer every 3 rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of !5.8 log infectious titer. no viral replication was observed after 9 days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above 5 logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* 1 , jen-wei chen 1 , chi-ling chen 2 , sheng-nan lu 3 and pei-jer chen 2 . 1 department of research, head office, taiwan blood services foundation, 2 graduate institute of clinical medicine, college of medicine, national taiwan university, 3 division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by 2030, and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since 1992) and 8-sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since 2013) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during 1999-2016 and 2013-2016, respectively. age-standardized prevalence and its 95% confidence interval (95% ci) were calculated with adjustment of who world standard population 2000-2025. for the incidence study, donors who have donated blood two or more times during 2013-2016 and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its 95% confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from 15.2 per 1,000 donors (95% ci: 14.8-15.7) to 4.0 per 1,000 (95% ci: 3.7-4.3) during 1999-2016, and the agestandardized prevalence was also decreased from 27.0 per 1,000 donors (95% ci: 25.6-28.4) to 7.7 per 1,000 (95% ci: 6.9-8.5). the agestandardized prevalence of anti-hcv was generally higher in female donors before 2015, but it was significantly higher in male donors at 2016 (p-value50.03). a total of 1,036 hcv rna positive cases, 1.9% of them were anti-hcv negative, identified from 579,286 first-time donors during 2013-2016, and the crude and age-standardized prevalence of hcv rna was 1.8 per 1,000 (95% ci: 1.7-1.9) and 5.0 per 1,000 (95% ci: 4.3-5.7), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value <0.0001), but no significant difference was found after age standardization (p value50.93). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend<0.0001). in the incidence study, a total of 68 new hcv rna positive cases, 23.5% of them were anti-hcv negative, found from 1,202,165 donors followed for 2,415,668 person-years. the incidence of hcv rna was 2.8 per 100,000 person-years (95% ci: 2.2-3.5), and no significant difference was observed between both genders (p-value50.41) and between age groups (p for trend 0.37). conclusion: the prevalence of hcv infection has been dramatically decreased by 71.5% during 1999-2013. it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* 1 , sahar muhmmad 2 and dalia el dewi 2 . 1 national blood transfusion services, 2 azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is 70-12 days, hiv from 22 to 11 days, and hbv from 25-30 days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from 2012 to 2015, the total number of donor samples to be screened is 178685, the age of the donors ranged from 18 to 50 years, and they were of both sexes (m: f 5 3:1).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v2) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of 75 nat yield donations among 178685 (0.04%) seronegative donors. among these 75 nat yields cases, 53 (0.03%) were reactive for hbv, 20 (0.011%) were reactive for hcv and 2 (0.001%) were reactive for hiv-1. we stratified the age of the donors into 3 groups; group a (18 -28 years), group b (29 -39 years) and group c (40 -50 years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p 5 0.0089; with 95% confidence interval (ci) 5 0.0085 -0.0520 & p 5 0.0247; with 95% ci 5 0.0025 -0.0534 respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p 5 0.0224; with 95% ci 5 0.0032 -0.0413). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p 5 0.0335; with 95% ci 5 0.0015 -0.0352). nat-hcv; did not differ significantly between the three groups (p 5 0.3222; with 95% ci 5-0.0089 to 0.0161 between groups a and b & p 5 0.1340; with 95% ci 5 -0.0055 to 0.0270 between groups a and c & p 5 0.4277; with 95% ci 5 -0.0080 to 0.0215 between groups b and c). nat-hiv; did not also differ significantly between the three groups (p 5 0.3801; with 95% ci 5-0.0077 to 0.0077 between groups a and b & p 5 0.3172; with 95% ci 5 -0.0056 to 0.0077 between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p 5 0.0013; with 95% ci 5 0.0136 to 0.0507). nat hbv was significantly higher in males (p 5 0.002; with 95% ci5 0.0103 -0.0413), but the prevalence of either hcv or hiv did not differ significantly between males and females (p 5 0.3835; with 95% ci 5 -0.0077 -0.0145 & p 5 0.2751; with 95% ci 5 -0.0044 -0.0066; respectively). conclusion: in this study the nat yield of 75 in 178685 assumes more significance when one considers the fact that single donation is used for generating 3 components that can be used by 3 recipients. hence, in effect the nat yield becomes 3 times that is, 225 in 178685. saving 225 recipients from tti out of 178685 (0.13%) is indeed very significant. results/finding: of the 303,569 donors who were tested by our donor center, 1,386 (0.460%) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at 1:128 titer. the screening elia s/co of this donor was 0.2782. both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were 6-10 transfusion transmitted babesia cases per year from 2008-2015 (table 2 ). in the 11 months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed (1 in 303,569 donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of 4 years of additional nat testing at blood bank, dmch, ludhiana from september 2012 to december 2016. results/finding: results 1.73% (2041 of 118021) units were initially nat reactive. these units were further tested, of which 90.98% were discriminated (70 hiv, 1051 hcv, 726 hbv and 10 co-infections). the remaining 6.71% (137) were repeat non-reactive and 1.91% (39) could not be discriminated. overall, nat yield rate was one in 837, whereas virus-specific nat yield rates were one in 59,010 for hiv, one in 1873 for hcv, one in 1639 for hbv and one in 29,505 for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past 4 years has increased the screening sensitivities to check viral load and prevented transmission of 141 probable transfusion transmitted viral infections. assuming 100 % component preparation it saved 423 transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu 1,2 , wei mao 3 , tao he 3 , yashan yang 1,2 , zhan gao 1,2 , chunhong zhang 3 , hongmei liao 3 , jingxing wang 1,2 and miao he* 1,2 . 1 institute of blood transfusion, chinese academy of medical sciences & peking union medical college, 2 sichuan blood safety and blood substitute international science and technology cooperation base, 3 chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from 5,000 voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a 250pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch37 human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as 1e -5 . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: 1.23 gb raw data with 2,450,046 reads were generated in the dna library. meanwhile, 1.98gb raw data with 3,967,242 reads were generated in the rna library. after cleaning the human background, 211 reads from bacteria, 98 reads from viruses, and 341 reads from parasites were identified (table 1) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table 1) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in 2015 most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of 1 february 2016 to 1 february 2017. thrombocyte concentrates are prepared out of 4 single donation units by the buffycoat method. results/finding: over the period 260 platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* 1 , germ an leparc 2 , phillip c williamson 1 , lani palmer 1 , ben reynolds 1 , maria noedel 1 and lindsey houghton 1 . 1 creative testing solutions, 2 oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february 16, 2016 recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march 1, 2016. with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february 16, 2016 and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february 16, 2016, the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within 6 weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in 2015, who reported $212 million new cases worldwide, resulting in >400,000 deaths. malaria prevalence is highest in sub-saharan africa, home to 90% of all infections accounting for 92% of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of 0.2 mm amustaline and 2 mm glutathione (gsh) and a 24h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to 7 days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed 24h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with 5% fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching >1% parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at >7.5 log 10 or >6.0 log 10 /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with 7 day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from 5 to 7 days using an fda cleared rapid test (rt). in february 2016, our hospital based transfusion service implemented the use of rt on day 5, 6 and 7 to routinely extend ap shelf life to 7 days. prior to this, we tested aps by rt on day 4 and transfused day 6 or day 7 units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of 7 day platelets. study design/methods: data were obtained for two 12-month study periods: october 2014-september 2015 (pre-implementation) and february 2016-january 2017 (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table 1 . the number of ap transfusions increased by 7% post-implementation, comparable to a 4% increase in inpatient admissions and an 11% increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different (3.16 pre; 3.12 post, p50.91). the number of rts performed increased by 130%. the percentage of transfused units tested at least once by rt prior to transfusion increased by 21% (p<0.0001). the outdate rate decreased from 5% to 2% (p<0.0001). ad-hoc ordering decreased from 21% to 9% (p<0.0001). conclusion: use of an approved rt for routine ap outdate extension to day 7 was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* 1 , vito scalia 1 , carla osiowy 2 , michael carpenter 2 , anton andonov 2 and margaret fearon 1 . 1 canadian blood services, 2 public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low (6.6 and 4.9 per 100,000 donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in 2011 the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of 6 units. hcv nat was in place since 1999 (using minipools of 24) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march 2016 all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at -20 o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the 5' ntr-e1 and ns5b regions. sanger sequencing of these regions represents approximately 15% of the genome. results/findings: all confirmed positive donations were whole blood donations. there were 42 hbv positive donations. of these, 37 had tested hbv nat positive. genotypes were 8 type a, 6 b, 4 c, 17 d and 2 e. there were 5 samples hbv nat negative but hbsag positive (2 were anti-hbc reactive). of these, 4 could not be sequenced and one was genotype a (also anti-hbc reactive). there were 30 samples considered hcv positive. of these, 17 samples were hcv nat positive. genotypes were 5 type 1a, 3 1b, 3 2c, 2 2b and 4 3a. there were also 13 samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first 8 months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert 3d microbial detection system (bta 3d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert 3d (bta 3d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels (1-20 cfu/ml) of 11 bacterial species commonly associated with platelet contamination, and 20 replicates (10 per instrument) of 4 ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta 3d and the other into virtuo and incubated until signaled positive by the instruments or for up to 7 days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally 98 bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august 2013. this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a 100% voluntary donor program since 2011 and is the only center in the country that has achieved this goal. results/findings: a total of 264,343blood donations were studied from august 2013 to december 2016. in the rbdc, where only voluntary blood donations are recruited, the prevalence was 18 per 100,000 donations for hiv (ic95% 8-34:100,000); 14 per 100,000 for hbv (ic95% 7-29:100,000) and 18 per 100,000 for hcv (ic95% 8-34:100,000). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was 89 per 100,000 donations for hiv (ic95% 77-103:100,000); 70 per 100,000 for hbv (ic95% 59-83:100,000) and 78 per 100,000 for hcv (ic95% 66-91:100,000). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of 1: 66,086 for hbv; 1: 132,172 for hiv and 1: 264,343 for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s201 system. hbv s region was also sequenced. results/finding: 234 obi were found in the 230,000 donations. in the viral loads assay, 43 samples were negative and 104 samples' viral loads were lower 20iu/ml. the mean viral loads was 1.85 6 0.41 (log10) iu/ml in other 87 samples,while the mean viral loads with hbsag1/hbv dna1 samples was 2.38 6 0.83 (log10) iu/ml. 60 samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype (69.0%) and the other was hbv c genotype(31.0%). compared the samples with hbsag1/hbv dna1 ,we found two obi samples carrying with 318t>c mutation, which could cause an amino acid s55f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag1/hbv dna1, and some unique variation was identified in the obi individuals. 195a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat 15 th days, 1month, 3 months& 6 months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may 2015, we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may 2015 to dec 2016 was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s201 platform using pools of 6 serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, 68547 seronegative donations were screened by nat and a total of 20 hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group 31-55 years old showed a large proportion, who accounted for 80% of reported infections. most of the hbv dna cases (about 80.0%) reached senior high school education. the average hbsag dna positive rate was 0.029% (20/ 68547). incidence among apheresis donors in this period for hbsag dna were 2.91/10000. these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf24 with 5 day stability at 228c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control (225 610 ml) and test components (625 ml 625 ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within 8 hr and wbd pf24 within 24 hr. cryo was manufactured according to site sops and frozen at -308c (test 62 6 2 ml, control 22 6 2 ml ). test and control cryos were thawed at 378c, and characterized immediately post thaw (t50), and after 5 d storage at 228c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over 5d at 228c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through 5d storage at 228c. conclusion: pr cryo can be processed from 3 plasma sources, including pf24, and stored at rt for 5 days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf24 with stability over 5 days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , lynne fleischmann 1 , mirjana sarac 3 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics, 3 abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects 6-8 million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over 20 days using a panel of positive and negative samples. sensitivity was evaluated on 407 presumed antibody positive specimens and specificity was evaluated on 7621 random blood donor samples. results/finding: precision was 7% cv or less for positive samples over 20 days. the overall specificity in a blood donor population was 99.99% (7620/ 7621). sensitivity was 100.00% for 407 presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , lynn martin 1 , daniela kaleve 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over 20 days. sensitivity was evaluated using 511 known positive samples, 30 commercially available seroconversion panels, the who standard, 23 hbsag mutants, and 94 hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.98% (7998/8000). sensitivity was 100% for 511 presumed positive samples. sensitivity was 100% for all genotypes. 100% of the mutants were detected vs 83% for the comparator assay. seroconversion detection was equivalent to the comparator assay with 157 reactive samples detected with the alinity s assay and 154 reactive samples detected by the comparator assay. analytical sensitivity ranged from 0.015 to 0.016 iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including 3 hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , kevin callear 1 , susan sullivan 1 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types 1 and 2 (anti-hiv-1/2). blood centers require very high throughput anti-hiv-1/2 assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen was evaluated on the abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv-1, hiv-2 and hiv group o antibodies and hiv-1 p24 antigen. seroconversion sensitivity was evaluated with 41 commercial seroconversion panels. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.96% (8082/8085). sensitivity was 100% for 813 presumed antibody positive samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. also, sensitivity was 100% for 102 antigen positive viral isolate samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 135 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , joyce siregar 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who 1st international standard. seroconversion sensitivity was evaluated using 10 commercial seroconversion panels. results/finding: precision was less than 6% cv for positive samples over 20 days. the blood donor specificity was 99.93% (6946/6951). sensitivity was 100% for 500 samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from 0.57 to 0.62 iu/ml. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 134 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* 1 , anton vanweert 2 , ed bakker 2 , mark paradowski 1 , jane bryant 1 , tuan bui 1 , joyce siregar 1 , george chen 1 , george schlauder 3 and gregg williams 1 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over 20 days using htlv i and htlv ii positive samples. specificity was evaluated using 8,001 blood donor specimens from europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 500 preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot 2.4. results/finding: imprecision was less than 7.0% for positive samples over 20 days. clinical sensitivity was 100.00% (500/500) on preselected htlv i and htlv ii positive samples. the specificity was 99.98% (7,999/8,001) on a blood donor population and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez 1 , michel garcia* 2 , fernando palomino 3 and guillermo orjuela-falla 1 . 1 national blood bank colombian red cross, 2 universidad del rosario, 3 fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, 387 anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than 1.0; abbott architect i2000sr) underwent supplemental testing by immunoblot (either chiron riba hcv 3.0 sia or hcv blot 3.0 test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: 1-4.99, 5-9.99, >10. band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in 57.9% (224/387) of samples, indeterminate in 30.7% (119/387) and were positive in 11.4% (44/ 387). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group 5-9.99 (63.2%) compared with the 1-4.99 (29.9%). in samples with indeterminate results, ns3_2 was the most frequent band detected (52,9%). in contrast, the most frequent band in the group of positive results was core (93,2%). only one sample from the indeterminate group (0.8%) had a strong band intensity (31), compared with 10 samples from the positive group (22.7%). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group (5-9.99) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (>5). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns3_1 and ns3_2 cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* 1 , ivanka mihaljevic 2 , manuela miletic 2 , miljana stojic vidovic 2 , irena jukic 2 , jane bryant 1 , mark paradowski 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 croatian institute of transfusion medicine, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over 20 days using positive samples. specificity was evaluated on samples obtained from 9,101 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 514 preselected positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with 3 confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than 6.0% cv for positive samples over 20 days. clinical sensitivity was 100.00% (514/514) on preselected syphilis positive samples. the specificity was 99.97% (9,063/9,066) for blood donor specimens and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* 1 , ed bakker 2 , anton vanweert 2 , jane bryant 1 , mark paradowski 1 , tuan bui 1 , lynn martin 1 , george chen 1 , gregg williams 1 and george schlauder 3 . 1 abbott laboratories, 2 sanguin diagnostics, 3 background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over 20 days evaluating positive samples. sensitivity was evaluated using 501 preselected positive samples and 30 seroconversion panels. specificity was evaluated on samples obtained from 8,113 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than 7.0% cv for positive samples over 20 days. overall clinical sensitivity was 100% on 501 preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying 5 more bleeds than the comparator assay. the specificity was 99.99% (8,111/8,112) for blood donor specimens and 98. 98% (194/196) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of 2015, was declared as the public health emergency of international concern by who in feb 2016. in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where 2.8% of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, 73.8% of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately 1:5 to 1:6. thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of 1lm and assayed after illumination with visible light from both sides for 5, 15, and 30min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was 4.5 log 50% tissue culture infectious dose (tcid 50 )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for 5min, 15min or 30min and the losses of the infectivity were further demonstrated by 3 repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial 5min of treatment whereby ct-value jumped from 18.25 (control) to 25.50 (mbpt for 5min) (table 1) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* 1 , margaret fearon 2 , susan l stramer 3 , megan l nguyen 3 , france bernier 1 , sheila o'brien 2 , vito scalia 2 , sakina smith 3 , yves gr egoire 1 and boris hogema 4 . 1 h ema-qu ebec, 2 canadian blood services, 3 american red cross, 4 sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in 14,000 canadian blood donors in 2013. in a subset of 4,000 donor samples the seroprevalence was 5.9%. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of 250 iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately 50,000 canadian whole blood donors including 30,000 from canadian blood services (cbs) and 20,000 from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all 199a transfusion 2017 vol. 57 supplement s3 donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test (95% lod 18.6 iu/ml, 95% ci 15.9-25.6) for use on the cobas v r 6800/8800 system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for 6 months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous 6 months are destroyed. recipients will be traced in the event of any products transfused in the previous 6 months. results/finding: as of april 10, 2017, 9 of 39,834 (19,395 cbs, 20,439 hq) tested samples with valid results have been found hev-nat reactive: 8 donors have been confirmed by further testing to date. confirmation is pending in 1 donor. of the 9 donors, 7 were from quebec, and one each from nova scotia and alberta (7 male, 2 female). ages ranged from 21 to 70 years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: 6 ate pork (including 3 who ate pork liver), 4 ate shellfish, 2 ate venison, and 3 drank well water. one donor had no identifiable risk factor. viral loads ranged from 3 to 151 iu/ml, of which 2 were <10, 3 were 10-50, and 3 were >50 iu/ml; 2 were anti-hev igm positive and 4 anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around 1/4400. the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about 300 to 500 million cases and 2 to 3 million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august 2014. both thin and thick glass stained blood smears of 417 blood samples with giemsa was examined using microscope. results/finding: of the 417 donated blood samples, 17 (4.1%) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p>0.05), majority of the blood donors that tested positive belonged to blood group o (64.71%). the prevalence of malaria parasite in the study was 4.1%. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert 3d (bta 3d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life (3, 4 and 5 days after collection). study design/method: pooled lrap were seeded with low levels of 6 organisms commonly associated with platelet contamination at 3, 4 and 5 days post collection. the seeded lrap were inoculated into bpa and bpn bottles on 10 different days (not consecutive) alternating between 2 teams of 2 people each. seeded bottles were loaded into a virtuo and a bta 3d and incubated until declared positive or negative (up to 7 days). additionally, bpa and bpn bottles inoculated with 4 ml of unseeded lrap were tested on the virtuo and the bta 3d (120 and 40 bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of 99.9% for the virtuo and 99.5% for the bta 3d. the virtuo demonstrated an average improved ttd of 3.2 hours, when compared to the bta 3d in the presence of 4 ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within 5 day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began 12 weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for 120 days barring continued zikv testing and nonreactive results. a total of 2,485 donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low (1.0%). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus 17d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn 1 , felicia santa maria 1 , yvette girard 1 , peter bringmann 1 , marion lanteri* 2 and adonis stassinopoulos 2 . 1 microbiology department, cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in 2015. the rapidly increasing number of infections in brazil, with hundreds of fatalities since december 2016, is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the 17d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a 2 weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate 17d-yfv using amotosalen (s-59) and uva light prt of platelet components (pc). study design/method: pc in 65%pas (n53) or 100% plasma (n51) were spiked with high titers of 17d-yfv and treated with s-59/uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero76 cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were 4.71 6 0.7 log 10 pfu/ml for pc in 65% plasma and 5.19 log 10 pfu/ml for pc in 100% plasma while titers in post-prt samples were <-0.7 6 0.0 log 10 pfu/ml for pc in 65% plasma and <-0.7 log 10 pfu/ml for pc in 100% plasma. inactivation to the limit of detection of >5.41 6 0.7 log 10 or inactivation of >4.71 6 0.7 log 10 pfu/ml was achieved for pc in 65% plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately 1000 ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples (1 hiv-1 antibody and 1 hiv-1 p24 antigen, and 1 htlv-i antibody and 1 htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were 0.00 for hcv, hbc, syphilis, cmv igg, and chagas, 0.01 for hiv ag/ab and htlv i/ii, and 0.03 for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were 0.00 % (antibody sample) and 0.36% (antigen sample) for hiv ag/ab combo; 0.90% (htlv i antibody sample) and 0.32% (htlv ii antibody sample); -1.43% for anti-hcv, -2.52% for chagas, -0.71% for hbsag, -0.37% for anti-hbc, -1.62% for syphilis, and -0.59% for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of 1000 ng/ml. robustness of the abbott prism methods to biotin interference c fischer 1 , r schneider 2 , w leonard 2 , m cobb 3 , g schlauder 3 , g williams 3 , m zuske 2 m janulis* 2 . 1 transfusion medicine, abbott diagnostics, wiesbaden, germany, 2 add diagnostics, 3 transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, 3 assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from 10 to 100 colony forming units (cfus)/bag (i.e. 0.03 to 0.3 cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of 10 8 -10 9 cfu/ml over the 5 days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day 2 after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, 24 hours (day 1) after collection a sampling volume of spiked platelets (0.1-1 cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of 25 bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate (7.5%). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by 2030 in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann 1 , frank tolksdorf 2 , wiebke handke 1 , thomas h. m€ uller 1 and axel seltsam* 1 . 1 german red cross blood service nstob, 2 maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from 5 buffy coats using the additive solution ssp1 (macopharma) with a residual plasma content of 35%. for inactivation kinetics, pcs (n53) were spiked with bacteria to a final concentration of approx.10 6 colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately 0.3 cfu/ml. bacteria were allowed to grow for 6 h in the pcs at 22 6 28c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log 10 reduction factors ranged from 6 to 7 for enterobacter cloacae (6.3 6 0.6, pei-b-p-43), pseudomonas fluorescens (7.1 6 0.4, pei-b-p-77), staphylococcus aureus (6.6 6 0.4, pei-b-p-63), and streptococcus bovis (7.0 6 0.3, pei-b-p-61). pcs (n512 for each species) spiked with these different bacteria species were efficiently sterilized (12 out of 12). treated pcs remained sterile during storage for 7 days, while bacteria in non-treated pcs grew to high titers of 10 6 -10 8 cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of 7 days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july 1, 2008 to july 30, 2013 was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version 16 software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than 0.05 were considered significant. results/finding: a total of 173, 207 consecutive blood donors were screened between 2008 and 2013. the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was 5.0%, 1.6%, 1.4% and 0.1% respectively. the hiv-hbv co-infection was higher among blood donors 135(41.79%) followed by hbv-hcv co-infection whish accounts about 103(31.89%). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of 26-35. in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was 746,773.9 ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a 14 month old infant patricia davenport* 1 , geeta paranjape 1 and laurie sutor 1,2 . 1 carter bloodcare, 2 ut southwestern medical center background/case studies: in 2017 at a large pediatric hospital, a 14 month old infant was supported for 31 days by extracorporeal membrane oxygenation (ecmo). over this time 113 blood products were transfused. about 10 days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified 27 donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed 3 years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october 2014 and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an 81 y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of 13 units of red blood cells. approximately 4 weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 20's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of 1:256. the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* 1 , andrea j linscott 2 and donny dumani 3 . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from 2011-2015, 8% of 173 transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a 27-year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day 1 of hospitalization showed no growth after five days. on day 3, the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the 318 ml unit had been aliquoted via sterile connecting device 12 days prior for a pediatric patient. all 26 ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was 37.18c. within 45 minutes of starting the transfusion, the patient's temperature increased to 39.38c and subsequently reached a maximum of 39.58c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at 4-68c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus 3 standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of 6 determinations of a low positive control in 1 run. inter-assay variability was determined by testing at least 3 representative negative production pool samples, at least 1 low positive sample (about 3 s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of 1.00 and were 0.72 and 0.48 respectively. the hbsag assay detection limit was 0.065 iu/ ml for source plasma and 0.120 iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at 15-25 0 c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between 1:10,000 to 1: 1,250,000 depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than 5% for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than 14%. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on 6 donors (3-17 days after the index donation) -3 donors in the follow up study and 3 tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all 6 donors. denv antibodies were negative in 9 donations and equivocal in 1. our initial reactive rate is higher than that reported to date for the procleix zikv tma of 1 per 23, 342 [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since 2014. over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late 2011, for which the program consisted of 2 types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june 2016, bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, 300 blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with 60 ml of plasma, vs. 30 ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, 2017; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation #1: updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation #2: implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over 120, 000 donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on 16-18 year olds and premenopausal women (ages 19-55) donors. on average, 16-18 year olds donate 1.3 times a year and premenopausal women donate 1.49 times a year. if both of these groups were limited to donating once a year, a total of 4,845 donations from 16-18 year olds and 9,272 donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin #17-02, the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for 16-18 year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a 2.5 hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: 97% (101/104) of the simulation group students improved their post-test scores and had an average likert scale rating of 4.1 (very good). 89% (63/71) of hybrid group students improved their post-test scores and had an average likert scale rating of 4.2 (very good). 89% (90/101) of online only students improved their post-test scores and had an average likert scale rating of 3.0 (good). the average changes in scores were statistically significant within all training groups (p value < 0.0001). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p<0.0001) and the hybrid group (p<0.0001). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in 2016. all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july 2016 through january 2017 were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a 7-month period, 504 cases of complex antibody identification workups (65%), transfusion reactions (2%), consultations for blood component utilization (6%), and deviations from standard operating procedures and massive transfusion protocols (27%) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $68,000 of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* 1 , david lancaster 2 , dianne geary 2 , robert scott 1 , anh thu nguyen 1 , adam garcia 2 , raina shankar 1 , leslie buchanan 1 and tho pham 2 . 1 stanford health care, 2 stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to (1) streamline the ordering process to accurately reflect inventory status and transfusion practices and (2) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over 50 product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a 5-month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a 3-month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: (1) the ratio of units transfused per week to the number stocked (t:s), (2) the number of products ordered as stat, and (3) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. 2486 lines of code were written for both programs, including 2 class modules and 34 distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were 1.03, 1.21, and 1.48 before the pilot period compared with 0.88, 1.17, and 1.40 during the pilot period. these differences did not reach statistical significance (p50.28). we also monitored the number of stat ordered products before and during the pilot period, which were 27 and 31 stat units per week, respectively (p50.86). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were 226 and 196 units, respectively (p 5 0.28). an estimated 7 hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to 0.175 fte and $18,200 per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over 360 hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* 1 , yulia lin 2 , troy thompson 1 , allison collins 1 and sheena scheuermann 1 . 1 ontario regional blood coordinating network, 2 sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in 2014 to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that 74 of the province's 158 hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in 2017 indicated that 93% plan to implement or already have implemented the ptqip and 43% of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october 2016 a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in 2017. publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* 1 , rana hajjeh 1 and cees th. smit sibinga 2 . 1 world health organization regional office for the eastern mediterranean, 2 international quality management (iqm) consulting background/case studies: more than 76 million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between 2006 and 2016; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may 2016 in tunisia. results/finding: we found 24 publications on disaster from five countries in the region and 16 publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries (54.5%) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated 10-85% of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. (63, 85, and 108 for 2014-2016) . the number of collections per registered trt donor varied significantly, ranging from 0 to 12 therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from 3.8 to 2.8 between 2014 and 2016. conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from 2014 through 2016. it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than 56 days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of 2010. after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march 2017 to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a 77% response rate (n533). of these, 78.8% have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. 87.9% of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although 93.9% of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only 48% of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate 3% (770 units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs1 to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in 2015, apheresis red cells (arc) represented 4.7 % of total red cell collections at our center. hae mcs1 ln8150 was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs1 operators first and then operators new to apheresis with a training goal of 30% of mobile staff. validation of the 12 alyx began 06/01/16 and took approximately 45 days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs1 machines were removed from service 07/08/16. alyx go-live occurred 07/13/16. additional operator training continued through september 2016. results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs1 and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $19.21 each providing an estimated annual savings of $239,000. conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately 2.5% and decrease our kit costs by 22%. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october 2016-march 2017. background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at 125g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! 130 g/l) and for females (minimum interdonation interval increased from 56 to 84 days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october 2016, changes in rebooking of donation appointments in december 2016 and culminating with eprogesa criteria changes on march 5, 2017. both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by 100. percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from 0.89% in the 3 weeks pre-implementation to 2.16% in the 3 weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were 12.6% in september, 12.0 % in october/november, and 9.9 % from december to march, 2017. conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to 130 g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past 8 years, 59,223 blood products, derived from 10,509 procedures, were distributed to 185 different investigators in over 200 laboratories. whole blood was the most common product (45.2%), followed by unmanipulated mononuclear cell collections (28.6%), and elutriated monocytes or lymphocytes (19.8%). less common requests included platelets (2.5%), plasma (2.5%) and granulocytes (0.8%). adverse donor reactions were infrequent (0.33% of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable 100% collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in 100% plasma. the corresponding pathogen reduction system used for the study has 3 kits with 3 different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than 6.8 x10 11 , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than 1800 x 10 3 /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of 1867 x 10 3 / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of 3.5 x10 11 and platelet concentration of 1167 x10 3 /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of 7.0 x10 11 and 6.8 x10 11 at the volume of 400 mls and platelet yield of 4.2 x10 11 , 4.0 x10 11 , and 3.5 x10 11 at the volume of 300 mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* 1 and steve cihura 2 . 1 bonfils blood center, 2 bbc / bsi background/case studies: in 2012, the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated 3 devices with the following criteria in mind: 1) device disposable costs, 2) reagents/controls/quality control, 3) objective hgb/hct measurement, 4) portability and durability for a mobile environment, 5) ease of use, 6) donor experience, 7) battery life, 8) validation requirements plans, 9) blood center suitability, and 10) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested 50 donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february 2013 with a targeted implementation date of july 2013. after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately 15%. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to 4.59% in 2014 and 4.29% in 2015. during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in 2016, the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may 2016. conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* 1 , ayumi araki 1 , hiromi sanyoshi 1 , hiromi kanai 1 , hiroya kikuchi 2 , katsushi tsukada 2 and kazuhide mure 2 . 1 hokkaido red cross blood center, 2 japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from 354 individuals who donated platelets during the 6-month period between february and august 2015, and data from the 30 donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the 156 men (5.1%) and 22 of the 198 women (11.1%) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the 30 donors in the vvr group was 64.7 6 13.7%, which was significantly higher than the 25.6 6 11.7% in the non-vvr group. at a maximum dbf threshold of 45%, sensitivity for discriminating between vvr and non-vvr donors was 93.3% and accuracy was 94.4%. when 45% dbf was used as the alert level, alerts were issued for 44 donors, including 25 in the vvr group. therefore sensitivity for predicting vvr was 83.3% and specificity was 94.1%. mean time from alert to diagnosis in the vvr group was 4.03 6 4.35 minutes, and accuracy of the alert was 56.8%. some of the vvr could not be predicted even the value of maximum dbf exceeded 45%. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* 1,2 , salam abdus 3 and shabrina shah 3 . 1 inova blood donor services, 2 inova fairfax medical campus, 3 inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of 10 scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every 4 to 8 weeks from december 2013 to march 2017. blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of 10 patients, 3 females and 7 males, who underwent a total of 178 rbcx from october 2013 to march 2017, using an average number of 7 rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for 8 patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the 178 rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of 168 healthy volunteers were collected before blood donation and after blood donation immediately, 1 day, 1 week, 4 weeks, and 12 weeks among men and 16 weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component 3 ( c3) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c3 decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at 12 weeks among men and 16 weeks among women, while c3 significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover 1 week after blood donated and reached their levels before blood donated within 12 weeks among men and 16 weeks among women. conclusion: the biomarkers mutually changed over the course of 12 weeks among men and 16 weeks among women. donating 400 ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low 40% split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate 15 percent and increased concurrent plasma collections by 24 percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than 62 percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below 3 percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october 2015. after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september 2016 for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in 2015 and 2016, a total number 1,097 rns and 965 rns completed the questionnaires, giving a response rate of 78.5% and 67.4% respectively. the overall mean score in 2015 was 6.24 points (range 0 to 8). the mean score in 2016 was 6.57 points (range 2 to 8). the percentage of rns having perfect scores of 8 increased from 8.8% in 2015 to 20.5% in 2016. table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; 264 purposively selected blood prescribing clinicians and nurses from 60 hospitals in 13 countries of the 4 human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at .05 level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r5 .137; p5.03; df5262) and accessibility (r5.184; p5.01; df5262) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f(3,260) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, 7 junior faculty co-investigators from 5 west coast institutions each had 2 months to create a 30 minute powerpoint presentation on a fundamental tm topic, after which 2 other members had 2 months to review and edit. therefore, each member created 1 and reviewed 2 presentations (three total steps). during each step, members wrote 2 multiple-choice questions for those particular topics. in the end, each topic would have 6 quiz questions to assess learning. at completion, 7 evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: 1) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; 2) pre and post-lecture 20 question validated examination (best collaborative) to assess learning; 3) resident in-service examination trends specific to tm. results/finding: six presentations were developed as 6 of the 7 abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables 1 and 2. abo leaders pre-test data could not be obtained for institution b, and 3 trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsõ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all 6 presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a 10 question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the 40 minute vignette performance and the 20 minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean 95 1 4%) when compared to the pre-test scores (mean 67 1 26%) ttest p<0.017. during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and 90% reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than 350 healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between 8.3 to 9.4 (in the range of 4-10). nps varies between 83 and 98. according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased 2011-2016 from 8.4 to 9.0. conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* 1 , anne eder 2 , beth a. dy 1 and mary o'neill 1 . 1 american red cross, 2 georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about 1 in 100,000 apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. 2015, a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to 2,300 hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the 12 months before and after launching the educational outreach. results/finding: the web based course was completed by more than 700 participants; 117 were physicians. based on a review of the evaluations, the course was highly valued with 93% of participants rating it excellent or very good. the blood center physicians gave over 200 presentations to hospital customers. reporting of suspected strs in 2016 increased by 23% compared to the prior year. the increased reporting came from 2 specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required 30-45 minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, 2014.) subscription-based e-learning utilizes 5-7 minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in 2016 by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june 2016, with a new equipment module assigned each month for the following five months. the series concluded in december 2016 with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of 3.5 on a 4-point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with 21 positive and 1 negative comment. level 2: learning the average score of users increased 13% from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of 1.647 and a t-stat value of 5.641. level 4: results while equipment-related errors decreased by 20% after training, there is not enough data to demonstrate a statistical significance. conclusion: our level 1 and 2 evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october 2013, a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n5151) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was 35%. of those, 96% endorse that fbc creates a climate of respect within our transfusion practice, 94% believe it has improved communication between work units, and 98% feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only 56% responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: 41 students of undergraduate semester 3 and 59 students of semester 4 participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with 86% of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, 12/7/16 from 9-11am. there were 17 attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, 4/20/17 from 7-11am. there were 14 attendees, including 2 regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and 11 surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) 50% as patients with fcr 50% may benefit from delaying the procedure for performance in the future. validation process included (1) a deming regression to globally assess the predicted vs. actual results and (2) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| 15%. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table 1 background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during 2 days (18 academic hours) the trainees can attend 6 lectures, discuss the methodical materials, participate in 3 seminars, 2 interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, 2016 with the group capacity 220a transfusion 2017 vol. 57 supplement s3 of up to 35 people the number of medical specialists who have attended the training is nearly 450. results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of 1-100; 1 being least satisfied/comfortable and 100 being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of 35 total technologists, 31 technologists took the pre esd survey and 25 technologists took the one year post esd implementation survey. table 1 shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, 19 (76.0%) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were 14 unplanned sop deviations; in the year after esd implementation there were only 5 deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from 0600 to 1600. the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in 100% plasma is broken up in to two days. on day 0 platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for 12-24 hours. on day 1 products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day 0 and day 1. in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: 13 of 18 employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was 21. for the two month training period the daily average rose to 23. conclusion: our "flip the switch" training plan for implementing prt platelets in 100% plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly 100% of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as 90% of an activity. in 2015 we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of 2015 we performed a supply inventory on all departments to plan future purchases and control residual stocks. in 2016, we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a 2016 cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter 2015 (q4/15) with last quarter 2016 (q4/16). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q4/15 1756 blood bags were used compared to 1998 in q4/16, demonstrating an activity "13.78%. price negotiation resulted in 12.58% readjustment. both indicated an estimated cost "28.10% with a possible impact of over us$ 35,000. we have identified a real cost #2.31% in q4/16, representing an overall #14.89% and us$ 3,716.72 (r$12,235.16) savings. conclusion: economy had deteriorated in our country in 2016 with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih-1000 tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17, statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in 2006. in the 2015-2018 nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on 60% individual and 40% for the other four levels (10% for each). the bonus (%) is calculated based on the iis as follows; category a: 100% (iis >575%), category b: 75% (iis: 50 -<75%), category c: 50% (iis: 25-<50%) and category d: 0% (iis < 25%). on the pilot implementation, the individual scores for 12 staff ranged from 71% to 100%. the iis were 76% to 81%. the number of staff in each bonus categories were 11, 92% (category a) and 1, 8% (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* 1 , deborah r fludd 1 and sandra j nance 2 . background/case studies: rare donors are defined as a blood type occurring in less than 1 in 1000 people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in 2016, there were 65,801 active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the 6398 (9.7% of total active donors) returned contact cards alerting ardp of changes in calendar year 2016, 355 (5.5%) were donors moving from one ardp facility to another, 1369 (21.4%) were donors no longer eligible to donate, and an additional 4324 (68.4%) were address changes. other changes were 115 (1.8%) reactivated donors and 235 (3.5%) donors who we were notified were deceased, or did not want to be listed in the ardp. in 2016, 5390 new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in 2016 was 4709 (355 1 4324), which would be 87.4% of the new donors submitted. conclusion: with nearly a 10% response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in 2016, 4709 donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* 1 , barbara j bachman 2 , mike leamy 2 , susan olson 2 and candace williams 2 . 1 university of florida college of medicine, 2 bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih-1000 (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two (22) runs of one to six (6) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing 153,000 types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. regardless of quality or speed metrics evaluated, the ih-1000 demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < 0.001). ih-1000 process steps and time studies addressed in the table below did not account for the ih-1000 reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih-1000 (77% reduction, a difference of 43 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* 1 , kimberly monnin 1 , barbara j bachman 2 , kyla warren 2 , susan olson 2 and candace williams 2 . 1 clinical pathology labs, 2 bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih-1000 tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of 12 separate test runs of 72 or 144 samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately 211,500 type & screens (t&s). the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih-1000 demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < 0.001). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih-1000 (difference of 120 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih-1000 tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih-1000 and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih-1000 with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17. statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/findings: using automated result verification, only 0.93% out of 6,339 samples evaluated for abo/rh testing would require visual verification, resulting in a 98% reduction in operator touchpoints (p < 0.001) and a labor saving of 444 minutes (7:01 hh:mm) for abo/rh testing. for 8,750 antibody screens, automatic validation of results would result in 99.5% reduction in operator touchpoints (p < 0.001) and a labor savings of 378 minutes (6:18 hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past 2 years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by 1% annually and peaked at 16% in mid 2016. to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an 8week period in late 2016. hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than 10 day shelf life remaining. units with tie tags attached were in hospital inventories for up to 3 months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was 100% of the 56 hospitals surveyed. mean percent o neg rbc gross sales for a 3 month period before, during, and after the survey was 16.6%, 16.0% and 16.5%, respectively. mean percent o neg net sales during the 3-month survey fell to 13.5% compared to an average of 15.4% in the 3 months prior. during the 3-month survey period o neg rbc monthly return rate increased to an average of 28.4% compared to an average of 23.0% in the 3 months prior. for the 3 months after the survey the average o neg rbc return rate further increased to 28.9% while mean percent o neg rbc net sales trended slightly upward to 14.1%. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* 1 , jeremie gachelin 1 , veronique ollivier 2 , thibaut mutin 1 , xavier telot 1 , benoit ho tin noe 2 and sandra sanfilippo 1 . 1 aenitis technologies, 2 hôpital bichat, inserm u1148 background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from 14 donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac1) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than 80 % (p< 0.001) and a purity of platelets close to 91.0 %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over 250.000 blood donations for an area with more than 7 million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in 2005, orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in 2007, when we moved from this equipment to atreus 2c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in 2011 we changed to atreus 3c (terumo bct) and finally in 2013, we moved to reveos system (terumo bct). since the changes in 2007, our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in 2009, bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during 2008 and 2016. conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of 140-160 patients and an average, round-trip travel time of approximately 15-minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about 30% of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november 2015, the hb has significantly improved the turnaround time of rbc issue -from 15-minutes to less than 60-seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately 750 rbc per month out of the window for non-surgical patients. this has been reduced to approximately 300 rbc per month, a 60% average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in 2015, they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the 131 st rescue squadron (131 rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the 131 rqs master sergeant in january 2016. we asked what 131 rqs's order and delivery expectations were. he said sporadic use and the blood order would be 2 rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the 131 rqs needs. staff was trained based on data from january 2016 meeting. we contacted the 131 rqs in september 2016 to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the 131 rqs association with a civilian blood center. based on his field experiences, he changed the blood order from 2 to 6 rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and 131 rqs performed a mock run on october 31, and we felt prepared for any future events. results/finding: on november 11, 2016, the 131 rqs was deployed to a civilian aeromedical evacuation. we anticipated a 6 rbc order. the actual order was 7 rbcs and 4 ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the 131 rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at 4:30am by the 131 rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november 28 th , we established a maximum blood order of 10 rbc and 4 liquid plasma, noting future orders may request fewer products, yet meet the preferred 2 rbc; 1 plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table 1 ). the highest mean fib concentration was 535 mg/donor unit; lowest mean fib concentration was 264 mg/donor unit. all sites had a mean fib concentration at least 100 mg/donor unit above the fda minimum requirement of 150 mg/donor unit. fifteen of 17 blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; 12 manufactured pooled donor cryoprecipitate. most froze plasma in a -188c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than 10 hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of 345 mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every 15 minutes for a 12 hour period or until the temperature exceeded 68c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached >68c in 75 minutes as shown in table 1 . the control thermometer recorded temperatures maintained at 1-68c for the entire 12 hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at 1-68c for more than 45 minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to 27 us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: 100% conventional (c-pc), 100% pr-pc, and mix of 75% c-pc/25% pr-pc. the model predicts a modest ($4%) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january 2011. a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august 2013, inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january 2015. a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from 2010 to 2016 (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in 2016, zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within 4 weeks (sept. phase 1) and nationwide within 12 weeks (nov. phase 2). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools (16-donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab 1 had 86% and lab 2 had 77% of clients requiring universal zkv testing. we evaluated a 12-month test result upload performance period to determine the impact of zkv test implementation. results/finding: during 2016, lab 1 upload time performance ranged from 92% to 94.2% from january to july; upload time performance fell between august through november, returning to 94.2% performance in december. lab 2 upload time performance ranged from 91.4% to 95.3% january to august. performance fell september through december 83.3% -88.5%. lab 1 experienced a low of 75% upload time performance during phase 1 when there was a rapid implementation; 69% clients required zkv nat. improved performance was observed during phase 2, with a 16% increase in zkv clients. for lab 2: phase 1 experienced a modest decline of upload performance ranging from 83.3% to 88.5% with 33.3% of clients implementing zkv nat. performance was 87.1% in phase 2, when an additional 43.3% of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* 1 and colleen a. aronson 2 . 1 advocate lutheran general hospital, 2 acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march 2016 regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in 6 of the 8 hospital transfusion services. the 2 sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the 6 sites which implemented the verax pgd test perform testing on all day 4 and day 5 platelets to be issued for transfusion. this abstract summarizes the data collected for the first 5 weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day 4, day 5, and those that were tested twice. inventory reports were reviewed for platelets issued on day 2 or day 3 that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february (1 week of performing the test), 48.1% of all platelets issued by the 6 participating transfusion services were day 2 or day 3 platelets. in march that number dropped to 29.9%. it is expected that this number will level off at some percentage at or below 29.0% with further data collection. in february 25.9% of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day 2 or day 3) will likely level off at some number at or below 29.9% due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, 5 of the 6 sites performing testing are level 1 trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed 100%. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from 400 ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n57). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd62p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days1, 3, 5 and 7 of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was (86.7 6 1.6)%, relative change rate of hsr was (3.87 6 12.75)%, the residual leukocytes were (0.15 6 0.15)310 6 . the ph, hsr, and the cd62p expression of pooled platelet concentrates before and after filtering were (7.00 6 0.17) vs (7.06 6 0.16), (66.96 6 12.35)% vs (63.22 6 8.26)% and (28.94 6 14.25) % vs (31.60 6 16.77)%. there is significant change for wbc after filtering (p<0.01). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table 1. conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb 18469-2012) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of 500ml and 1000ml. two users then evaluated the ss tubing segment types with 500ml or 1000ml samples for a total of 10 data points. samples were collected into the attached 1ml or 3ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of 10 ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the 5 day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january 2015, a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by 22%. the platelet outdate rates dropped after implementing the platelet inventory tool from 14% (1324 units) to 11% (874 units); a 21% decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of 7 fte to 6.2 fte, lowering fte by 11%. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* 1 , jaclyn mckay 1 , jennifer curnes 1 and rowena punzalan 1,2 . 1 bloodcenter of wisconsin, 2 children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a 3 month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: (1) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, (2) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july 2016 for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within 8-hours after collection. this tight 8hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight 8-hour completion time constraint for cryo production (capacity expansion). in particular, during the 4 th -quarter of 2016, a blood processing region was able to process about 1000 more cryo units/month (an increase of 20%) at a slightly lower collection cost (cost avoidance), resulting in an approximately 40% reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the 2017-the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after 7-day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from 5 to 7 days in the usa using an fda cleared rapid test (rt). in august 2016, our hospital based transfusion service began using a rt on day 6 and 7 to routinely extend ap shelf life to 7 days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of 7-day ap. study design/methods: data were obtained for two study periods: september 2015-february 2016 (pre-implementation) and september 2016-february 2017 (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from 5-day to 7-day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by 3% post-implementation while inpatient admissions and surgical volume increased by 1% and 3%, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by 36% post-implementation and the outdate rate decreased from 29% to 15% (p<0.0001). ad-hoc ordering was not statistically different between study periods (p50.10). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different (2.1 and 1.9, respectively, p50.65). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years (2012) (2013) (2014) (2015) (2016) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was 4892 units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was 8546, 24, and 2 units respectively. the mean number of discarded rbc units of the five years of the study exceeds 50% of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below 1000 mg/dl, and so the rejection threshold for lipaemia is level equal to or more than 1000 mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including 4892 rbcs, 8546 plasma products and 24 apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from 200 mg/dl to 1000 mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* 1,2 , rebecca ross 3 , debra mraz 3 , anne baker 3 , zenna neal 3 , melanie champion 3 and edward l. snyder 2,3 . 1 johns hopkins university school of medicine, 2 yale university, 3 yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first 4 months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, 777 pr-sdp were transfused at our hospital (out of a total of 3286 platelets transfused). after 4 months of scale-up, pr-sdp were approximately 30% of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($12 mg per 100g) to the content in pr-sdp (<1 ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september 2017, the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from 10 donors was collected in k 3 edta tubes. plt concentrations were determined at the qc department using the coulter act 5 diff hematology analyzer (beckman coulter). sample tubes were stored at 20-248c and measured at 24, 48 and 72 hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd (250 ml; n58) or dpd units (500 ml; n54). plt pools were stored at 20-248c under mild agitation for seven days except for dpd, which were split in two 250-ml bags after 18 6 1 h. samples were taken on days 1 and 7. ph, po 2 and pco 2 , hypotonic shock response (hsr), extent of shape change (esc), cd62p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at 24h (247 6 32 pltx10 9 /l), 48h (247 6 27 pltx10 9 /l) and 72h (247 6 32 pltx10 9 /l) postdonation. dpd can be stored in the same collection bag for 24h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits (1141-1526 pltx10 9 / l) before splitting. on day 1, lactate and pco 2 concentrations increased, and po 2 decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march 2017. there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted 12% increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* 1 , colleen vincent 2 and patricia kopko 1 . 1 university of california -san diego, 2 american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only 40-45% of double platelet collections meet requirements for pathogen reduction treatment. 1 blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: 9,500 apheresis platelets annually), which includes two hospitals (750 inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week 0) and the bone marrow transplant (bmt) ward (week 6). an e-mail communication explained the change to all physicians and nurses. in phase 1, we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase 2, we expanded usage to include the inpatient bmt ward. in phase 3, we lifted all restrictions so prp could be used throughout the health system, with the goal to reach 100% prp within 6 months. results/finding: in phase 1 (weeks 1-6), we requested 31 prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of 23 prp weekly (range 9-33), and prp constituted 44% of platelet transfusions in the cancer center. in week 2, excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase 1. in phase 2 (weeks 7-8), we formally expanded issuing of prp to include the inpatient bmt ward and requested 91 prp products weekly. our blood supplier provided an average of 57 prp weekly (range 44-69), and prp constituted 53% of platelet transfusions in the phased-in areas. in phase 3 (weeks 9-10), we began issuing prp throughout the health system. our supplier provided an average of 70 prp weekly (range 61-78), and prp constituted 43% of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises 21 hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of 2 apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, 2 ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a 48 hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table 1 ) resulting in a cost savings of $50k. an additional cost savings of approximately $25k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as 237a transfusion 2017 vol. 57 supplement s3 we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* 1 , james r stubbs 1 , scott a hammel 1 and manish gandhi 2 . 1 mayo clinic, 2 mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing 100% pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of 100 apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of 3.0, 3.1, and 3.3 x10 11 may end up below a 3.0 in the final storage bag and would need a post-processing sample to ensure the product met criteria at !3.0x10 11 platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a 3.4 yield but ended with a yield below 3.0. these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe-2100d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that 99 of 100 results were 0.00 or 0.01x10 3 / mcl with the exception being the wbc failure with a count of 0.29. further monitoring of the wbc counts discovered a result of 0.04 which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of 0.03 will be tested on the adam to confirm a leukoreduced product. we also discovered 2 of 4 (50%) of the 3.4 preprocessing yields products ended with a post processing yield <3.0. we decided to increase the yields requiring post processing samples to include the 3.4. conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of !3.0x10 6 platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from 9/16-12/16 were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need 1 6) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in 2017; the revised prediction method (estimated clinical need 1 11) was then evaluated retrospectively using the same data sources covering 1/17-4/17 and then compared to the prior method. results/finding: the average number of platelets transfused from 9/16-12/ 16 was 18.2 u/d with a standard deviation of 5.3 u/d; the predicted amount was 13.7 u/d. the difference between the predicted amount and the number of units used was -4.5 u/d. 79% days (23d/month) were under-predicted (average: 6 u/d). 17% of days (10) were under-predicted by !10 u (average: 12 u; max: 15 u (4x)). the average number of platelets used from 1/17-4/17 was 17.5 u/d with a standard deviation of 4.4 u/d; the predicted amount was 18.3 u/d. the difference between the predicted and units used was a 10.8 u/d. 38% days (11d/ month) were under-predicted (average: 3.5 u/d). one day (1%) over this period was under-predicted !10 u (11 u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages (17% a 1%), reduced the number of days under-predicted (79% a 38%), and decreased the discrepancy on those under-predicted days (5.9 u a 3.5 u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* 1 , maria noedel 1 , nancy haubert 1 , kenneth hudson 1 , larry morgan 1 , robert shaw 1 , tracy fickett 1 , jamie jue 1 , valerie winkelman 1 , sally caglioti 1 , german leparc 2,3 and phillip c williamson 1 . background/case studies: on 08/26/16, fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within 4 weeks; nationwide in 12 weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on 1 of 2 manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for 44 clients within 4 weeks, and an additional 21 clients within 12 weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the 4-week period but overcome during the 12-week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* 1 , hala samuel boules 1 , fatemah saleh al matroud 1 , rabab hussien ali dashti 1 , hanan alawadhi 2 and reem al radwan 3 . 1 kuwait central blood bank, 2 kuwait central blood bank, 3 kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of 3000 rbcs baby units were randomly chosen to be traced to their final deposition from the year 2012 till 2016. half of them (1500 units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb (60 units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is 100% efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (<5 days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march 2017. results/findings: a total of 339 products were tested. fifteen units (4%) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all 15 by the vendor and none were true positives by re-culture. of the 324 units that were successfully tested, 200 were tested again on day 6 for use on day 7(62%). there were 166 platelets transfused (51%) and 158 expired after day 7 (49%). the cost to test the products including controls was $12,970 and our calculated cost to produce 324 products would be $77,436. if we had needed to import products to meet needs, the cost would be roughly $91,300 without shipping costs which are estimated at $14,815.50. we averaged 40 expired platelet products per month (range 6-67) before verax testing and 26 (range 9-40) after implementation. conclusion: using verax point-of-care testing saved 166 platelet products from discard. the cost savings were $93,145.50 from importing and $64,466 from producing a replacement for those 324 products. the average discard rate per month went from 40 to 26 after verax implementation. extending platelet shelf life to 7 days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december 2016 were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april 2017 were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for 2016 data, 129 units were investigated without the use of secure texting. of these, 118 units were identified as preventable wastage, and 11 wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: 1) product not released after procedure/ or when patient stabilized (42) 2) product returned outside of appropriate temperature range (40) 3) clinician unaware product was assigned (36). thus far in 2017, wastage records have identified 31 units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, 11 responses thanked the transfusion service for the information, and in 8 instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: 99.9% dmso, plas-a, 25% hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) 99.9%dmso: plas-a : 25%hsa51:2:2. plas-a and hsa are kept at room temperature (20-258c, rt) and refrigerated at 48c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point 18.58c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total 14 tests. at least 10 minutes cooling after dmso, before adding the next reagent. see table: (1) after directly adding 99.9% dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. (2) in tube, autologous plasma first, dmso next, powder-like precipitates. (3) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. (4) & (5) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at 48c. (6)&(7) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the 48c group is slightly milder/slower than rt group. so hsa should not be added first. (8)&(9) trace of hsa(<1ml) was mixed into the plas-a bag (500ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below 10 in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5% to 3.2%. with an annual platelet usage of approximately 13,000 units, this reduction equates to approximately 950 units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $450,000 to $650,000, per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* 1 , russell thorsen 1 , rosaline ma 1 , antonio g insigne 1 , amy decourten 1 , florence panganiban 1 , patricia mckean 1 , cyril jacquot 2 , sara bakhtary 1 and ashok nambiar 1 . 1 ucsf health, 2 children's national medical center background/case studies: plasma (ffp, pf24, pf24rt24) stored at 1-6c outdates 24 hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to 5 days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (<4 months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited (24 hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf24 for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk 1 and richard gammon* 2 . 1 oneblood, 2 oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average 5-8 minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average 2-5 minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < 4 hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from 34% to 14%). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp-215 using the low glycerol (40%) freezing method and frozen at -658c within six days of collection. thawing occurred in a 328c water bath in the following order: 7 o positive and 1 o negative on 3 january 2017; 7 o positive and 1 o negative on 7 february 2017; and 8 o negative on 22 february 2017. deglycerolization occurred on site using the acp-215 with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for 13 days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $240-280 and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $400 per unit to produce and distribute. drbcs have a shorter shelf life, 14 days versus the 211 days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp-215's and deglycerolize four units at a time. in january and february 2017, it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of 10 years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c8000 izekial butler* 1 , karen leighton 1 , scott jones 1 and rachel beddard 2 . 1 qualtex laboratories, 2 biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c8000 instrument. precision of the new assay parameters was determined by testing 10 replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from 1 iu/ml to 60 iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of 2.0 to 45.0 iu/ml. stability of samples was determined by testing samples stored at 2-8 0 c and -20 0 c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from 1.2855 to 1.3142. so, precision was acceptable since the %cv for all samples tested was 5%. the mean values for the samples tested in the accuracy study were all 610% of the expected value which was much lower than the acceptance criteria which was 615% of the expected value. the linearity of the assay was acceptable with a r2 ! 99.0%. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to 14 days at 2-8 0 c and up to 1 month at -20 0 c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for 14 days at 2-8 0 c and stored up to one month at -20 0 c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in 2010 to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, 2017) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april 1, 2014 to march 31, 2015. local research ethics board approval was obtained. results/finding: 40 patients received ivig for itp at smh over the study period for a total of 76 unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding (13, 17%), pre-operative or antepartum care (22, 29%), a platelet count of less than 10 and contraindication to corticosteroids (8, 11%). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between 30-50 x 109/l. 6 patients received ivig for a likely diagnosis itp while 245a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. 18/76 (24%) of infusions consisted of 2g/kg over 2 days; the remainder of infusions consisted of 1g/kg. of those who received 2g/kg,3 of patients (17%) had evidence of partial remission after a first 1g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single 1g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our 2016 survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to 32 staff members in early february of 2017. twenty-two technologists responded for a 69% response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, 81% indicated that workspace size was insufficient and 71% that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* 1 , neil bangs 1 and kimberly sanford 2 . 1 vcu health system, 2 virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from 1/1/2015 to 6/30/2015 to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (<4 hours, > 4 hours < 24 hours), and if a second sample was required. results/finding: a total of 3144 t&s orders were initiated from the ed in this time period. 2787 (88.7%) patients were not subsequently transfused any type of blood product within 4 hours of tsd and 2584 (82.2%) patients were not subsequently transfused any type of blood product within 24 hours of tsd. a total of 2119 (67.3%) patients required a second sample. of these patients requiring a second sample, 1960 (92.5%) were not subsequently transfused any type of blood product within 4 hours of tsd and 1886 (89%) were not subsequently transfused any type of blood product within 24 hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within 24 hours of tsd amounted to an estimated $699,706 in unnecessary patient charges and approximately 628.7 nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* 1 , debra berry 1 , yunchuan delores mo 2 and gay wehrli 1 . 1 university of virginia health system, 2 children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* 1 , elizabeth halperin 2 , sharon breining 2 and mona papari 3 . 1 acl laboratories/ advocate hospitals, 2 advocate health care, 3 itxm background/case studies: a large midwest hospital system with 5 level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and 3 months of data were evaluated that contained 29 events. results/finding: there was an equal number of events that were initiated in the ed and the or (12). male patients were involved 69% of the time and 31% of time the patients expired. trauma of some type was the majority of the cause but 13.8% of the cases involved gi bleed and only 6.9% were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average 7.1 with the post hgb average of 9.7. ratios of 1:1 for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of 4:1. it was found that the rbc: plasma was 1.9:1, rbc: plt was 5.9:1 and rbc to cryo was 7.4:1. use of txa was only 24.1% and cacl was utilized in 58.6% of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* 1 , jody a barna 1 , donald e ulinski 1 and nancy m. dunbar 2 . 1 dartmouth hitchcock medical center, 2 dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to 2 weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july 2016, we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior 24 hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august 2015 and november 2015 (pre-implementation) , newly identified clinically significant antibodies were resulted for 56 patients compared to 51 patients between the months of august 2016 and november 2016 (post-implementation). the tat for allergy alert entry for both periods is shown in table 1 . we observed that 57% of allergy comments were performed within 24 hours in the post-implementation period versus only 30% pre-intervention (p50.0067). using the new process, nearly all of the alerts were entered into the emr within 72 hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* 1 , shailesh macwan 1 , nancy nikolis 1 , arline stein 1 , janelle richardson 1 , manju bagu 1 , lennart logdberg 1 , alexander indrikovs 2 , vishesh chhibber 1 and sherry shariatmadar 1 . 1 north shore university hospital, 2 northwell health background/case studies: our institution is a tertiary care facility performing over 1500 cardiovascular surgeries (cvs) in 2016, an increase of 117% after the healthcare system cvs integration in 2015. transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december 2016, the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after 2 reported events in q3 2016 that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: 1. open discussions and collaboration between blood bank and cvs nursing teams 2. mapping the process using flowcharts for additional blood orders from cvs. 3. identify bottlenecks and brainstorm solutions. 4. a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. 5. the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. 6. follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period 12/23/16-4/7/17 the blood bank has received 327 verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* 1 , tracie ingle 1 and heather vaught 2 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter 12-6 and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as 11 or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with 8-11 o, d negative red blood cells over 30 days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series 4, anti-d series 5, a1 cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table 1 summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion 2009; 49:1672 -1677 who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for 75% and 20% of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february 2014-june 2015 as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june 2015-october 2016. results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table 1) . this led to a reduction in observed overall sli (7.2 6 1.8 days vs 6.0 6 1.5 days, p<0.01) and odr (0.9% vs 0.5%). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. (6)) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january 2014 and december 2016 thirty-five (35) notifications were sent to physicians using epic letters and of those, fourteen (14) responded to the epic notification and five (5) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining 21 cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* 1 , denden benabdessadek 2 , annu george 2 and alexandra jimenez 2 . 1 westchester medical center, 2 new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of 941 orders were reviewed. approximately, 30% of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases (89%), but of the issued products, all were returned to the blood bank in 40% of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> 50 type and screens (t&s) per day] blood banks 1 , respectively. our laboratory which serves a large 1278-bed multispecialty academic hospital and receives 275-300 t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of 32.2 6 4.5 and 27.5 6 5.6 minutes 2 , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was 30% longer and had a larger standard deviation (s.d.) than the published trial result of 32.2 6 4.5. transfusion 2017 vol. 57 supplement s3 abstract during phase i visionv r 1 performed 263 panels. during phase ii visionv r 1 performed 351 of the 361 visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* 1 , john roback 2 , ronald arkin 1 , michael bartlett 1 , robert geiger 1 and jaxk reeves 1 . 1 university of georgia, 2 emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february 2016 guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august 2016 guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over 4,000 emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a 10% target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of 317 participants responded to the survey (7.94% response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n-15317-15316 df) of the null hypothesis that the mean50 vs. the alternative that the true mean is> 0. overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on 3 run cycles. ct was comprised of 3 metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of 2 components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table 1 provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc (2:50), xm (32:23), cb (13:18) and dat (10:00). by implementing the future state, an average $1.3 min. lt and vt is saved on each sample loaded for ts equating to a 73% labor reduction over the current state. a 19% improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a 59% lt reduction. on average, a 38 min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $100 type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing 8-column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on 100 patient samples for abo/d typing and antibody screening; of which at least 10 had a positive antibody screen. out of the 10, 5 had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which 17 were d(-) and 25 were d(1). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the 100 patient and 42 donor samples tested (100% concordance). all 10 samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave 100% sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for 5 samples at 3 different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over 3 months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* 1 , craig fletcher 2 and peter millward 1 . 1 beaumont hospital, 2 beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short 5-day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october 2014 and quality data was reviewed from august 2013 to december 2016. the collected data was then analyzed using descriptive statistical methods. results/finding: data from 2016 indicates platelet wastage comprised 1% of total received platelets and 79% of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units 12 months preimplementation of the report was 13 units, compared to 11 units 12 months post-implementation and 5 units 24 months post-implementation. wastage rates improved from 6% (wasted yearly platelets/total received yearly platelet units) in 2014, the year of report implementation, to post-implementation rates of 3% in 2015 and 1% in 2016 (see table) . importantly, this occurred despite a greater than 30% increase in platelet inventory between 2014 and 2016 and resulted in cost savings of over $60,000 in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all 4 participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within 2 years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* 1 , lisa marie button 2 , lori scanlan-hanson 2 , karen koch 2 , janet finley 2 , deepi goyal 2 and camille van buskirk 3 . 1 mayo clinic-rochester, 2 mayo clinic, 3 mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in 2014 to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in 2013 and 2014 were 63/1187 (events/ed transfusions 2013-2014). study design/method: electronic ordering for the ed was implemented march 31 st 2015. any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients <35kg and pediatric patients >35kg. 252a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for 2 years (april 2015 -march 2017), and during that time there was 1 instance of blood being ordered for an unintended patient 0.09% (1/1081). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in 2013, 17/651 (2.61%) units were transfused in the ed but not charted in the patient's medical record. in 2014, 18/536 (3.36%) transfusions were not charted. however, in 2016, the first full year of electronic transfusion order capability, only 4/462 (0.87%) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level 1 trauma center, with over 700 inpatient beds and over 50 operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option 1 (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option 2 (verbal) for verbal orders and coolers set up; and option 3 (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table 1 showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by 68% with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* 1 , steven zibrat 1 , geoffrey wool 2 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in 2015, blood bank accounted for 48% of all rejected clinical laboratory samples, yet comprised only 5% of total laboratory volume; 88% of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was 0.006%. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september 2016. results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of 0.128%, three times the national average of 0.043%. under the new system, rejected blood bank samples decreased from an average of 50% to 28% of all rejected laboratory samples, a 43% decrease. implementation of the new process produced a net savings of $55.8k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. 253a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured 19 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-78 days (mean 19 days, sd 20). in the postimplementation period, we cultured 22 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-12 days (mean 7 days, sd 2; p50.0082). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* 1 , marcus holme 1 , johnathan bakst 1 , gunta musa 1 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an 80 minute turnaround time (tat). in april of 2016, the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased 104%. tat analysis of a representative one week sampling per month showed an increase in outliers from 28 per month to 57 per month. average monthly tys samples performed is 2758. these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was 12/20/ 2016. results/finding: the average number of outliers decreased 61% from 57 per month to 22. further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $134,000 for fiscal year 2017. conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific 7 month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from 0-65 days with a mean of 13.64 days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for 7 months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of 0-7 days with a mean of 2 days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* 1 and randy levine 2 . 1 northwell health, 2 lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, 67 units were transfused on the oncology floor with 38 units (57%) requiring irradiation and only 22 of those 38 units (57%) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, 9 additional irradiated units were issued (31/67; 46%). post-intervention, 29 units were transfused on the oncology floor with 15 units (52%) requiring irradiation and all 15 of those units (100%) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus 23 of the 29 (79%) total units were ordered as irradiated. again, 4 additional irradiated units were issued (27/29;93%) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* 1 , charlotte van dyke 2 , dina garza van hoose 2 and rachel beddard 1 . 1 biobridge global, 2 south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics 8150s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january 1, 2016 to april 19, 2017, 2,097 total collections were flagged for additional qc by our trima accels and haemonetics 8150 instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in 2015, the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: 1) risk assessment, 2) quality control plan and 3) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* 1 , rachel jug 1 , kimberly ingersoll 1 , nicholas bandarenko 2 , nicole guinn 3 and jessica poisson 2 . 1 duke health pathology, 2 duke university hospital, 3 duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced 6:6:1 transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july 2015-december 2016. data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the 6:6:1 ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* 1 , nancy nikolis 1 , linda benison 1 , ruthmire thelusca 1 , renee liberty 1 , sherry shariatmadar 1 , alexander indrikovs 2 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: our blood bank (bb) processes approximately 60,000 specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june 2016, a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed 24/7. the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of 2016. the implementation of this new process has led to a decrease in the number of unacceptable specimens up to 30% quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from 75% to 96%, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* 1,2 , morgan rockwell 2 , joseph hagan 1 , jun teruya 1,2 and shiu-ki hui 2 . 1 texas children's hospital, 2 baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within 24 hours pre and post transfusion were evaluated. patients (0-18 years) receiving prophylactic ptx from july to december 2016 admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < 10ml/kg for patients < 35kg and one apheresis unit (au) for patients >35kg; therefore, patients were separated into 2 groups: < 35 kg and > 35 kg. a significant proportion of orders for both < 35 kg and > 35 kg did not meet patient platelet threshold criteria (p<0.001). conclusion: ptx threshold above ir for both groups were 31 ( 35 kg) and 48% (> 35 kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than 10% of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p>0.05) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to 56 days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding 3 months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only 2.1% of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated 3220 (805x4) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately 68 patients (17x4) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts (97.9%) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, 7 columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by 29% with the communication category average rpn having the greatest reduction of 72%. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* 1 , karen king 1 and joseph sweeney 2 . 1 rhode island hospital, 2 lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level 1 trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was 80 6 32 minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different (30 minutes for the provuev r and 28 for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over 90 minutes are shown in the table. the results show a reduction in tat by 14 minutes with a 20% reduction of tat greater than 90 minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* 1,2 , lorna orengo 3 , monique scott 3 and christopher a tormey 1,2,3 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as 1 in 19,000 blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as 25% prior to interventions, but may potentially be reduced to as little as 1.5%. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of <5%, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding 6 months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged 8.2%. reasons for specimen rejection were divided into 5 groups: 1) hemolysis, 2) blood bank witness collection form errors, 3) quantity not sufficient, abstract 4) duplicate sample, and 5) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table 1) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a 6.8% rejection rate with only 1 rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s 201 system christopher shahan* 1 , christina dejesus 1 , mosi mccall 1 , fallon hampton 1 , tangi herring 1 , judy davis 1 , anjali patel 1 , sonya gomillion 1 and bonnie maltby 2 . 1 qualtex laboratories, 2 qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s 201 system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual 10-12 hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently 42% of whole blood donor testing turnaround time delays are due to issues and failed runs on the s 201 system and we have 3 technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s 201 system. study design/method: the number of technician related failed runs on the s 201 system were tracked from 10/11/2015 thru 12/11/2016. a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the 5 whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s 201 system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s 201 system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s 201 system. after counter measures were implemented, the number of technician related failed runs decreased from 3 to 1.2 failures per week, which was a 58% decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s 201 system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by 58%. patients who were transfused for pre-transfusion hgb >7g/dl with resulting post-transfusion hgb >9g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by 11 volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: 265 patient charts were reviewed. 91 were excluded for bleeding and cardiovascular instability. 106/174 (60.9%) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors (76.19% vs 55.73%, p50.0181) and anemia of chronic disease (76.47% vs 54.1%, p50.006) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb 7.7 g/dl vs 7.3 g/dl, p<0.0001). inappropriately transfused patients also had higher median post-transfusion hemoglobin (9.9 g/dl vs 9.4 g/dl, p<0.0001). moreover, lab evalutions revealed association with lower folate levels (median 8.1 nmol/l vs 15.7 nmol/l, p50.029). 29/106 (27.3%) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. 7/64 providers were responsible for 32.3% of all inappropriate transfusions. 1/68 appropriately-transfused patients experienced an fnhtr. 3 deaths unrelated to transfusion occurred (1 in appropriate, 2 in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* 1 , willem martin smid 2 and ashley john duits 1 . 1 red cross blood bank foundation, 2 sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of 8 worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* 1 , dee dee cassidy 1 , jed b gorlin 1,2 and nancy l van buren 1,2 . 1 hennepin county medical center, 2 innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant 10 or more minutes may be required for transit of units often released in less than 5 minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july 2016. data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january 2015-june 2016, and post change included august 2016-december 2016. july 2016 data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of 99 rbc/month in coolers. post change this dropped to 62 rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of 54 rbc/month were returned (55%). post change, the average rbc/ month returned was 27 (44%), this represents an absolute 50% reduction in number of returned products. each rbc dispensed and returned takes approximately 20 minutes to complete paperwork and transport, therefore this change saved an average of 540 minutes per month. it was also noted that the average rbc/month transfused was 44 for baseline and 57 post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of 15-20 minutes (estimated) was reduced to 3-5 minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over 250 patent-applications have been filed related to "transfusion medicine" and over 400 related to "transfusion alarm", during the last 30 years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an 1/4 inch color cmos sensor, providing effectively 1.0 mp, a 3.6 mm lens, ir-cut, day/night minimum illumination 0.1 lux/f 1 and 808 viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a 24 hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood 260a transfusion 2017 vol. 57 supplement s3 abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h.264, video frame rate (fps) 1-30/s, refresh rate 50 hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/802.11/b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p2p is provided. typical 5v power-supply, sized 165x125x101mm and weighing 370 g. client software is required. the ir range is 10-15 m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of 10 m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a 32 gb sd-card. pan/tilt-horizontal 355 o and pan/tilt-vertical 90 o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo 2 , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than 6 3 %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under 20 $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to 4 participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot (30 cfu), multishot 550 cfu or highdose 10k organism preparations at a low level (< 50 cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a 14 day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at 36 c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < 1 days and the fungal organisms in < 2 days. the overall agreement was 99.8 % in 698 bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* 1 , diane schafer 2 , debra brown 1 , jesse cox 3 , scott koepsell 3 and sara shunkwiler 3 . 1 nebraska medicine, 2 the nebraska medical center, 3 university of nebraska medical center background/case studies: anticipating the implementation of the new (30 th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, 2 nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september 11, 2016), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling 4 in 6 months post implementation compared to 144 in the 6 months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential 2 nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for 2 nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late '90s and in early 2000, intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since 2010 have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january 1, 2005 to june 30, 2016. data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: 57% were female and 7% were less than 34 weeks of gestational age. none had co-existing g6pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn (41%) and rhesus-hdn (59%). antibodies most often implicated in rh-hdn were anti-d (22/57), anti-d and anti-c (22/57) and anti-c (5/57). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was 1 g/kg (range from 0,3 g/kg to 3,8 g/kg). neonates received one to four ivig administrations. table 1 shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, 14 required an et for rh-hdn and 3 for abo-hdn. forty-five (46%) patients needed top-up transfusions during hospitalisation and until three months of age: 8 with abo-hdn and 37 with rh-hdn. the mean number of transfusions was three (range:1 to 7). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over 90% case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a 4-part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include 3 specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than 4 months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, 30 coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate (2 each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and 45 different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than 1 hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on 2 separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to 10 hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, 2 georgia institute of technology ap72 reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with 2 o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add 2 type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to 1a) all adult males (am), 1b) women of non-childbearing age (wncba), and 1c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to 2a) women of childbearing age (wcba) and 2b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed 3%) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap73 saving apheresis platelets through use of verax point of care testing jennifer rhamy* 1 and rebecca wride 2 . 1 st. mary's regional blood donor center, 2 st. mary's regional medical center background/case studies: our rural hospital-based blood center serves 17 hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between 0 and 8 per day in 2017), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson 2 and jun teruya 1,2 . 1 texas children's hospital, 2 baylor college of medicine ap127 vision titers --easier or problematic? (table 1) . results/findings: post intercept, t had volumes of 261-320 ml, with 98 6 4% hemoglobin (hb) recovery. t had 10-fold less extracellular protein than c. after 35 days of storage t had higher atp and na 1 than c while lactate and hemolysis were lower. hct, ph, k 1 and glucose were equivalent between t and c on d35. d35 hemolysis for t was 0.08-0.31%, while for c it was 0.10-0.57%. t and c atp was >2mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table 1) . hematocrit (hct, %) 58.5 6 2.5* 56.3 6 1.7 60.8 6 2.7 61.8 6 2.9 hemoglobin (g/unit) 59 6 7 6 0 6 4 not measured hemolysis (%) 0.02 6 0.01* 0.06 6 0.04 0.16 6 0.07* 0.29 6 0.14 ph (378c) 6.9 6 0.1* 7.3 6 0.1 6.7 6 0.1 6.6 6 0.1 total atp (mmol/g hb) 7.7 6 0.5* 5.0 6 0.4 4.6 6 0.6* 3.9 6 0.5 k1 (mm) 1.5 6 0.3* 5.7 6 2.5 53. total tested total plts issued feb 87 121 64 48 24 185 181 mar 226 516 342 276 133 858 757 totals 352 730 471 375 174 1201 table: 2. resident reports to the intranet "drop box" increased from 53.7% to 69.3% to 100%, each over 2 month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from 17.4 per day or 10,370 dpmo to 6.3 per day or 3,539 dpmo. this is a statistically significant difference since the p-value calculated was 0.002. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately 4,400 specimens per month. since 1998, the requirement of having a second blood type on record was met by:1. utilizing the historical blood type and the current specimen, or 2. having second type performed on same specimen by different technologist, and 3. each type and screen specimen signed by 2 staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards 29 th edition, #5.16.2.2 a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april 2016, the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding:1. there was a minor adjustment period with more phone calls made to blood bank to explain the process. 2. there was minimal impact on turn around times for release of components. 3. aborh retype workload decreased from 1500 to 950 (35% to 20% of t&s volume) per month. 4. unnecessary blood draws minimized, improving patient experience. 5. no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as 15 to 48%. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our 926 bed hospital, a retrospective chart review was performed (07/01/15-12/31/15) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a 11 reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< 2 grade" or "> 2 grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table 1 demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples (48%) were > 2 titer results higher, while the majority was 2 titer results different (52%). the cost analysis is summarized in table 2 . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated 41% decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after 23 samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc.15 samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another 8 samples were measured with fc in ucc maribor. results/finding: 15 samples (6 rbc, 3 plt, 3 ffp-all leucocyte depleted and 3 non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from 44,10 -173,21; plt from 86,60 -100,00; ffp from 86,60 -173,21; and for non-leucocyte depleted ffp from 0,69 -8,85 (table 1) . 8 samples (2 rbc, 2 plt, 2 ffp -all leucocyte depleted and 2 nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from 91, 56; plt from 78, 21, ffp from 66, 21 ; and for non-leucocyte depleted ffp from 11,17 -15,91 (table 2) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than 1x10 9 /unit for leucocyte depleted or 1x10 6 / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january 2017 we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control.